European Journal of Cancer Advance/journals/ejc/EJC... · European Journal of Cancer Aims and Scope The European Journal of Cancer (EJC) is an international multidisciplinary oncology

Volume 54, Supplement 1February 2016ISSN 0959-8049

European Journal of Cancer

Abstracts ofA Conference of New Ideas in Cancer - Challenging Dogmas

26 - 28 February 2016

Tata Memorial Centre Mumbai, India

Amsterdam • Boston • London • New York • Oxford • Paris • Philadelphia • San Diego • St Louis

European Journal of CancerEditor-in-Chief: Alexander M.M. Eggermont Institut Gustave Roussy Villejuif, FranceEditors:Basic Science and Preclinical Research: Richard Marais, Manchester, UK

Ulrich Keilholz, Berlin, GermanyDrug Development: Jordi Rodon, Barcelona, SpainEarly Breast Cancer: Kathleen I. Pritchard, Toronto, CanadaAdvanced Breast Cancer: David Cameron, Edinburgh, UKGastrointestinal Cancers: Volker Heinemann, Munich, Germany Michel Ducreux, Villejuif, FranceGenitourinary Cancers: Karim Fizazi, Villejuif, FranceLung Cancer: Mary O’Brien, London, UKGynaecological Cancers: Ignace Vergote, Leuven, BelgiumSarcomas: Jean-Yves Blay, Lyon, FranceMelanoma: Dirk Schadendorf, Essen, GermanyNeuro-oncology: Roger Stupp, Zurich, SwitzerlandEpidemiology and Prevention: Jan Willem Coebergh, Rotterdam, The NetherlandsTumour Immunology and Immunotherapy: Aurélien Marabelle, Villejuif, FrancePaediatric Oncology: Rob Pieters, Utrecht, The NetherlandsFounding Editor: Henri TagnonPast Editors: Michael Peckham, London, UK; Hans-Jörg Senn, St Gallen, Switzerland; John Smyth, Edinburgh, UKEditorial Office: Elsevier, The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK

Tel: +44 (0) 1865 843590, Email: [email protected]

EDITORIAL BOARD

CLINICAL ONCOLOGY

R. Baird (UK) N. Brünner (Denmark) R. Califano (UK) E. Calvo (Spain) F. Cardoso (Portugal) J. Cassidy (UK) H. Cody (USA) R. Coleman (UK) A. Costa (Italy) J. De Bono (UK) E. de Vries (The Netherlands) A. Dicker (USA) R. Dummer (Switzerland) F. Eisinger (France) S. Erridge (UK) G. Ferrandina (Italy)

H.Gabra (UK) H. Gelderblom (The Netherlands) B. Hasan (Belgium) J.C. Horiot (Switzerland) J. Jassem (Poland) A. Katz (Brazil) I. Kunkler (UK) C. Le Tourneau (France) C-C. Lin (Taiwan) P.E. Lønning (Norway) P. Lorigan (UK) C. Massard (France) K. McDonald (Australia) F. Meunier (Belgium) A. Miller (Canada) T. Mok (Hong Kong)

D. Nam (Korea) J. Overgaard (Denmark) J. Perry (Canada) J. Ringash (Canada) A. Rody (Germany) M. Schmidinger (Austria) S. Sleijfer (The Netherlands) S. Stacchiotti (Italy) M. van den Bent (The Netherlands) G. Velikova (UK) U. Veronesi (Italy) A. Voogd (The Netherlands) E. Winquist (Canada) T. Yap (UK)

BASIC SCIENCE, PRECLINICAL AND TRANSLATIONAL RESEARCH

P. Allavena (Italy) J. Anderson (UK) M. Barbacid (Spain) M. Broggini (Italy) C. Catapano (Switzerland) M. Esteller (Spain) E. Garattini (Italy) A. Gescher (UK)

R. Giavazzi (Italy) J.M. Irish (USA) H.E.K. Kohrt (USA) J. Lunec (UK) A.G. Papavassiliou (Greece) V. Rotter (Israel) V. Sanz-Moreno (UK) S. Singh (Canada)

J. Stagg (Canada) C.G.J. Sweep (The Netherlands) P. Vineis (UK) A. Virós (UK) B. Weigelt (USA) N. Zaffaroni (Italy)

EPIDEMIOLOGY AND PREVENTION

B. Armstrong (Australia) P. Autier (France) V. Bataille (UK) J.M. Borras (Spain) C. Bosetti (Italy) H. Brenner (Germany) L.E.M. Duijm (The Netherlands) J. Faivre (France) S. Franceschi (France)

D. Forman (France) A. Green (Australia) K. Hemminki (Germany) C. Johansen (Denmark) L.A. Kiemeney (The Netherlands) E. Lynge (Denmark) M. Maynadié (France) H. Møller (UK) P. Peeters (The Netherlands)

A.G. Renehan (UK) S. Sanjose (Spain) M.K. Schmidt (The Netherlands) I. Soerjomataram (France) H. Storm (Denmark) L.V. van de Poll-Franse (The Netherlands) H.M. Verkooijen (The Netherlands) E. de Vries (The Netherlands) R. Zanetti (Italy)

PAEDIATRIC ONCOLOGY

C. Bergeron (France) A. Biondi (Italy) E. Bouffet (Canada) M. Cairo (USA) H. Caron (The Netherlands)

G. Chantada (Argentina) F. Doz (France) A. Ferrari (Italy) M.A. Grootenhuis (The Netherlands) K. Pritchard-Jones (UK)

L. Sung (Canada) M. van den Heuvel-Eibrink (The Netherlands) M. van Noesel (The Netherlands)

European Journal of CancerAims and Scope

The European Journal of Cancer (EJC) is an international multidisciplinary oncology journal, which publishes original research, reviews, and editorial comments on basic and preclinical cancer research, translational oncology, clinical oncology – including medical oncology, paediatric oncology, radiation oncology, and surgical oncology, and cancer epidemiology and prevention. The EJC is the official journal of the European Organisation for Research and Treatment of Cancer (EORTC), the European CanCer Organisation (ECCO), European Association for Cancer Research (EACR) and the European Society of Breast Cancer Specialists (EUSOMA).

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P re fa ceThe Tata Memorial Centre, Mumbai, the premier tertiary cancer treatment and research centre in South Asia and one of the largest globally, is on the cusp of a momentous year, namely, the 75th year of its existence. To commemorate the event a conference entitled “A Conference of New Ideas in Cancer – Challenging Dogmas” is being organized at the National Centre for Performing Arts in Mumbai and the iconic Taj Mahal Palace Hotel.

The idea for the conference came from surveying the current landscape of cancer research and treatment and the disappointments that it has brought us. The last decade has been particularly frustrating as we have realized that the hype generated from molecular and genetic ‘breakthroughs’ are translating neither into a meaningful understanding of the malignant process nor to a clinically relevant relief to our patients. In our desperate quest to prove our scientific approach to be right, we are even modifying our time-tested methodologies of clinical research, and getting increasingly excited by meaningless gains of “progression free survival” by a few months. A 2%‒3% improvement in overall survival has become the outer limit of our intellectual expectation.

This platinum jubilee conference is devoted to challenging the current dogmas and to debate the ‘currently entrenched versus contrarian viewpoints’ in cancer research and treatment. Key international speakers and participants will deliberate on these issues critically over 3 days of the conference on carefully chosen themes in the form of symposia, debates and special lectures. Some of the emerging messages may lead to potentially paradigm shifting approaches to how we perceive and treat cancer. We are sure that the conference will be an academic feast and would appeal to all scientists, clinicians and health care professional involved in researching and treating cancer.

The Lancet Oncology, the US NCI and AACR are our partners.

Rajendra Badwe Director, TMC & Chairman

Indraneel Mittra Chair, Scientific Committee

Rakesh Jalali Organizing Secretary

Programme Advisory Committee

1. Hans-Olov Adami

Stockholm, Sweden

2. Rajendra Badwe

Mumbai, India

3. Michael Baum

London, UK

4. Michael Brada

Liverpool, UK

5. Amit Dutt

Navi Mumbai, India

6. Varsha Gandhi

Houston, USA

7. Rakesh Jalali

Mumbai, India

8. Indraneel Mittra

Mumbai, India

9. Michael Retsky

Boston, USA

10. Carlos Sonnenschein

Boston, USA

11. Ana Soto

Boston, USA

12. Ian Tannock

Toronto, Canada

Abstracts Core Scientific Committee

Coordinators

Raees Tonse

Manoj R Gorade

Pankaj K Panda

Mukul Godbole

Rosily Joseph

Meenakshi R Ganeshan

Rakesh JalaliIndraneel MittraAmit DuttManju Sengar

TATA MEMORIAL

CENTRE

A CONFERENCE OF

NEW IDEAS IN CANCER -

CHALLENGING DOGMAS

Friday, February 26, 2016

26 - 28 FEBRUARY 2016 TATA THEATRE, NCPA, Mumbai

Timings Topics / Sessions Faculty / Speaker

07.30 am Registration

08.30 - 08.40 am Welcome

Symposium 1: Has the molecular revolution reached the bedside? Chairpersons: Indraneel Mittra & Daniel Hayes

08.40 - 09.00 am Biological insights from the cancer genome Akhilesh Pandey

09.00 - 09.20 am How relevant are oncogenes in human cancer? Aruna Ramachandran / Mathew Meyerson

09.20 - 09.40 am How much of genomic data has translated into clinical practice? Fabien Calvo

09.40 - 10.00 am Discussion

Key note address Chairpersons: Mammen Chandy & Arnie Purushotham

10.00 - 10.30 am Cancer research in need of a scientific revolution Carlos Sonnenschein

10.30 - 11.00 am Break

Debate Moderators: Sudeep Gupta & Hemant Malhotra

11.00 - 11.40 am Debate 1: Has targeted therapy fulfilled its promise? Yes / For (20 min) Amit Oza No / Against (20 min) Tito Fojo

11.40 - 12.00 noon Discussion

Invited Lectures: Future of cancer research - US and European perspectives Chairpersons: P. B. Desai & G. K. Rath

12.00 - 12.30 pm Invited lecture 1: Thinking Outside the Box in Cancer Research - Edward Trimble Perspectives from the US NCI

12.30 - 01.00 pm Invited lecture 2: Future of Oncology - European perspective Alberto Costa

01.00 - 02.30 pm Lunch and Poster Viewing

Key note address Chairpersons: Ronald DePinho & S. V. S. Deo

02.30 - 03.00 pm Act of observation and its impact on cancer outcomes Rajendra Badwe / Priyanka Pandey / Amit Dutt

Symposium 2: New ideas in cancer research - I Chairpersons: Vinod Raina & Ramesh Nimmagadda

03.00 - 03.20 pm Peri operative NSAIDs in breast cancer Michael Retsky

03.20 - 03.40 pm Novel PI3 kinase inhibitors: Bench to bedside Varsha Gandhi

03.40 - 04.00 pm Biological principles of metronomic therapy Nicolas Andre

04.00 - 04.20 pm Precision medicine: Lessons from the SHIVA trial Christophe Le Tourneau

04.20 - 04.40 pm Discussion

04.40 - 05.00 pm Break

Key note address Chairpersons: Harit Chaturvedi & C. S. Pramesh

05.00 - 05.30 pm How relevant are the randomized controlled Ian Tannock trials in clinical practice?

Symposium 3: Large data sets and their relevance to the patient Chairpersons: Susan Shurin & Fabien Calvo

05.30 - 05.50 pm RCT’s and health outcome research Chris Booth

05.50 - 06.10 pm Omics and big data sets: Does statistical significance equal Rajiv Sarin clinical relevance?


06.45 - 08.00 pm Opening Ceremony Chief Guest: Dr. R. Chidambaram, Principal Scientific Advisor to the Govt. of India

08.00 -10.00 pm Inaugural Dinner

Saturday, February 27, 2016 Timings Topics / Sessions Faculty / Speaker

08.00 am Registration

Symposium 4: Public health issues in cancer Chairpersons: Preetha Rajaraman & Rajesh Dikshit

8.30 - 8.50 am Problems and pitfalls in public health research Matti Hakama

8.50 - 9.10 am GWAS: Are risk variants biologically and clinically relevant? Julian Peto

9.10 - 9.30 am Time trends in cervical cancer: does it justify national Freddie Bray vaccination / screening programmes in cervical cancer?

9.30 - 9.50 am Environmental determinants of neoplasia: Ana Soto Does cancer start in the womb?

9.50 - 10.10 am Discussion

Key note address Chairpersons: A. Nandakumar & R. A. Badwe

10.10 - 10.40 am Harms of cancer screening Michael Baum

10.40 - 11.10 am Break

Hospital Day Oration Function

11.10 - 11.20 am Welcome Address RA Badwe

11.20 - 12.20 pm Oration: The cure that does not come - Jan Vijg Stalling innovation in the 21st century

12.20 - 12.30 pm Valedictory function

12.30 - 02.00 pm Lunch and Poster Viewing

Key note address Chairpersons: Jan Vijg & J. V. Divatia

02.00 - 02.30 pm The role of dead cell chromatin in cancer initiation, Indraneel Mittra progression and metastasis

Symposium 5: Dead cell chromatin and cancer Chairpersons: Ian Tannock & Anita Borges

02.30 - 02.50 pm Cancer: But which cell is it anyway? Sen Pathak

02.50 - 03.10 pm Cancer metastasis: Which cell is it anyway? Akshita Singh

03.10 - 03.30 pm Is the metastatic tumour genetically similar to the primary? Nikolas Stoecklein

03.30 - 03.50 pm Clinical implications of apoptotic circulating tumour cells Daniel Hayes

03.50 - 04.10 pm Dead cell chromatin, tumour heterogeneity and therapy resistance I. Mittra / Amit Dutt


04.30 - 05.00 pm Break

Key note address Chairpersons: A. K. D’Cruz & Paul Sebastian

05.00 - 05.30 pm Cancer Moon Shot - Making Cancer History Ronald DePinho

Debate Moderators: Michael Brada & Vineet Gupta

05.30 - 06.10 pm Debate 2: Is precision medicine an achievable goal? Yes / For (20 Min) Hope Rugo No / Against (20 Min) Vinay Prasad / Tito Fojo


07.30 pm onwards Cultural programme and Conference Banquet at NCPA (Padma Vibhushan Pandit Shiv Kumar Sharma)

Sunday, February 28, 2016

Timings Topics / Sessions Faculty / Speaker

Top scoring oral abstracts session (Detailed information in the following pages) Chairpersons: Clinical Research - Tapan Saikia & Pankaj Chaturvedi

Basic Research – T. Rajkumar & Chitra Sarkar

08.00 -10.00 am Abstracts: 18 (2 parallel sessions) Platform presenters

10.00 - 10.30 am Break

Invited Lecture 3 Chairperson: TBA

10.30 - 11.00 am Fractal geometry in oncogenesis Michael Baum

Key note address Chairperson: Nagraj Huilgol & Somasundaram Subramanian

11.00 - 11.30 am Harnessing technology for cancer treatment - Who benefits? Michael Brada

Symposium 6: The lure and promise of technology Chairperson: Alberto Costa & Shaleen Kumar

11.30 - 11.50 am Advanced technologies and global cancer outcomes Mary Gospodarowicz

11.50 - 12.10 pm The politics and economics of novel technologies Richard Sullivan

12.10 - 12.30 pm Technologies around the corner; promise and risks David Jaffray


12.50 - 02.00 pm Lunch

Symposium 7: The promise of cancer immunotherapy Chairpersons: Subrata Sinha & S. V. Chiplunkar

02.00 - 02.20 pm Biological principles of immunotherapy Vijay Kuchroo

02.20 - 02.40 pm Applications of immunotherapy Gordon Freeman


Symposium 8: New ideas in cancer research - II Chairpersons: Soumya Swaminathan & Rakesh Kumar

03.00 - 03.20 pm Attenuated bacterium Listeria monocytogenes as a new delivery Claudia Gravekamp platform for anticancer agents

03.20 - 03.40 pm Copper-depletion using tetrathiomolybdate: A phase II clinical Linda Vahdat study in high risk for recurrent breast cancer

03.40 - 04.00 pm Metronomic maintenance therapy improves outcome in triple Sripad Banavali negative breast cancer

Invited lecture 4 Chairpersons: N. K. Ganguly & Arun Nanivadekar

04.00 - 04.30 pm Should the industry reconsider its approach B. Khamar to cancer drug discovery?

04.30 - 05.00 pm Break

05.00 - 06.30 pm Grand discussion and summing up

Moderators: Ian Tannock & David Collingridge Alberto Costa, RA Badwe, Ted Trimble, Neel Mittra, Daniel Hayes, Hope Rugo, Mike Brada, Tito Fojo, Soumya Swaminathan, Arnie Purushotham, N. K. Ganguly,

06.30 - 06.40 pm Closing and Valedictory Function Carlos Sonnenschein...

European Journal of Cancer

Con te n t sPrelims i-x

Abstracts

A. Clinical Oncology S1

B. Extracellular Nucleic Acids S10

C. DNA Damage Repair and Apoptosis S14

D. Novel Therapeutics S17

E. Cancer Stem Cells and EMT S24

F. Systemic Therapies S28

G. Novel Diagnostics S33

H. Therapy Resistance S41

I. Tumor Biology S44

J. Cancer Prevention and Screening S49

K. Omics and Technologies S54

L. Immuno Oncology S61

M. Cancer Metabolism S66

N. Cancer Metastasis S70

Author Index S73

Volume 54, Supplement 12016

Indexed/Abstracted in:Current Contents;EMBASE/Excerpta Medica;Index Medicus; MEDLINE;CABS, BIOSIS Database;PASCAL-CNRS Database; CINAHL®

ISSN 0959-8049

European Journal of Cancer 54 (2016) S1–S72

A. Clinical Oncology

OT56-005Head and neck cancer and oncologic emergencies

C. Reyes-Gibby1

1 University of Texas MD Anderson Cancer Center, Houston, TX, USA

Background: Major advances have been made in treatment of squamous cell carcinoma of the head and neck (HNSCC). However, HNSCC treatment has significant adverse effects, leading to visits to Emergency Departments (ED). In this study, we first determined the chief complaints for their ED visits, and assessed the clinical and epidemiological predictors of time to first ED visit after treatment initiation and frequency of ED visits.Methods: A prospective cohort study of newly diagnosed, histologically confirmed HNSCC patients being treated at MDACC. This study was approved by the Institutional Review board at MDACC.Results: As many as 32.5% (97/298) of our study participants presented to the ED for a total of 108 EC visits (median time of approximately 30 days). Within 14 days of treatment initiation, the most common chief complaints for the ED visit were GI (29.03%) and pain (19.35%). Pain persisted as a primary chief complaint associated with presentation to the ED for more than 180 days. Overweight BMI (HR 5 0.45, 95% CI: 0.26–0.77), severe pain (HR 5 1.72, 95% CI: 1.00–2.98), history of hypertension (HR 5 2.03, 95% CI: 1.24–3.33) and depression (HR 5 2.14, 95% CI: 1.21–3.80) were significant predictors of time to first ED visit from initiation of treatment, accounting for factors known to predict EC visits.Overweight BMI (P 5 0.01), treatment with chemotherapy or chemo-radiation (P 5 0.003), severe pain (P 5 0.002) and history of hypertension (P 5 0.02) were factors associated with frequency of ED visits in the multivariable analysis.Conclusions: Significant morbidity is associated with cancer treatment in this patient population. Efforts targeted at significant clinical problems including pain and GI distress in specific subgroups of the HNSCC patient population (those with pretreatment depression, hypertension, and low BMI) have the potential to improve overall quality of life and can potentially improve cancer-related outcomes by avoiding ED presentation and hospitalizations in these patients.

OF5-003Challenging the role of frozen section for margin assessment in oral squamous cell carcinoma

A. Mishra1, P. Chaturvedi1, S. Datta2, S.V. Kane3, D. Nair1, A. Garg1

1 Head & Neck Services, Department of Surgical Oncology, Tata Memorial Hospital, Mumbai, India2 Narayana Superspeciality Hospital, Kolkata, India3 Department of Pathology, Tata Memorial Hospital, Mumbai, India

Background: Frozen section (FS) is routinely used for margin assessment in oral squamous cell carcinoma. However, several studies question the significance of FS guided margin revision. We had conducted a prospective study to show that gross examination by surgeon predicted the margin status accurately. We analysed our findings on a larger data set to evaluate its implication. Our primary aim was to evaluate the role of FS in margin assessment.Method: This is a retrospective study done on basis of results of 1 prospective and 1 retrospective study conducted at our centre. In the retrospective study, patients operated from January 2007 to June 2010 we found that FS resulted in 50% reduction in positive/close margin. However we wanted to find an alternative for FS. We conducted a prospective study and found that gross examination (GE) by surgeon is equivalent to FS for margin assessment. GE predicts 88% of inadequate

margin at a margin cut off of 7 mm. To confirm our findings, we analysed 1310 patients operated from January 2012 to October 2013. The gross and microscopic margin status of each patient was extracted from electronic medical records. The cost estimates were performed to calculate the expenditure on FS.Results: Microscopic spread changed the gross margin status in 5.2% patients. FS was beneficial in 1 out of 43 patients (2.3%) to achieve tumor free margin and it affected the adjuvant treatment plan in 1 out of 137 (0.7%) patients. The cost benefit ratio of FS was 12:1. Gross examination alone could have identified majority of the inadequate margins.Conclusion: Meticulous gross examination of the entire surgical specimen is pivotal in identifying majority of inadequate margins. Achievement of 7 mm margin by surgeon obviates the need for FS. Use of FS for margin assessment bears a poor cost benefit ratio.

P17-A001Feasibilty of primary osseointegrated implants during free fibula microvascular reconstruction in locally advanced head neck cancers

R. Katna1, N. Kalyani1, A. Hamid1, A. Deshpande1, V. Jacobs1

Bombay Hospital and Medical Research Center New Marine Lines, Mumbai, India

Background: Free fibula microvascular reconstruction (FFOCF) is the preferred reconstruction following segmental mandibulectomy in locally advanced oral cancers. However, these patients lack complete functional and aesthetic rehabilitation affecting their quality of life. To study the feasibility of primary osteointegrated implants during free fibula microvascular reconstruction in locally advanced oral cancers, we did present analysis.Material and methods: The treatment charts of prospectively maintained data of 196 patients who underwent surgical resection for locally advanced oral cavity between March 2013 to April 2015 were evaluated. Of these, 60 patients had FFOCF reconstruction done. Eight patients had osteointegrated implants insertion during the microvascular reconstruction. All these patients received adjuvant radiotherapy/chemo-radiotherapy based on final histopathology report. All the patients were followed up every three monthly post completion of treatment with serial orthopantomogram (OPG).Results: The median age of the cohort was 52 years with 6 male and 2 female patients. The number of implants was decided based on extent of defect post mandibulectomy. The median number of implants inserted was 3 (Range 1–4). There were no post-operative complications related to implants or FFOCF. The median dose of adjuvant radiotherapy was 60 Gy (Range 56–60 Gy). The median and mean follow up was 4 and 6 months respectively. One of these eight patients had implant extrusion post radiotherapy while remaining 7 has no complications till date. None of the patients had dental restoration till date.Conclusion: Primary osseointegrated implants in free fibula microvascular reconstruction in patients with oral cancers is clinically feasible and will help in achieving absolute aesthetic and functional outcomes in these patients. A larger series with longer follow up will validate these encouraging results.

P22-A002Roots of cancer are deep and ancient

R.H. Austin1

1 Princeton University Princeton, NJ, USA

We propose that the roots of cancer are deep and ancient; stretching back billions of years (cancer is by definition a breakdown of the contract between germ line and soma). We see cancer or cancer-like processes across almost all multicellular species, including corals and sponges, suggesting very deep common roots. We are suggest that cancer is a reversion to an ancient ancestral phenotype, triggered by stress. This

S2 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 A. Clinical Oncology

stress response is similar to safe mode on a computer - a reversion to core functionality (and by implication, ancient basic functionality). The fact that 1.5 billion years of evolution has not eliminated cancer is because the coregenetic programs that drive cancer also drive critical multicellular processes. What are they? Oncologists have known the answer for decades: embryogenesis is a key one, wound healing another. We propose that cancer research should focus not on the mutations that evolve under stress, which is the current orthodoxy, but rather on the genes that NEVER mutate, we aim to show that cancer is primarily driven not by damaged genes but by undamaged ancient “safe mode” genes as a response to stress or damage, connected with embryogenesis and wound healing.

P23-A003Locally advanced cancer esophagus: New frontier for hypofractionation?

R.S. Kabre1, K.M. Kamble1, A.K. Diwan1, A.K. Mahoobia1

1 Government Medical College Nagpur, Nagpur, Maharashtra, India

Background: Concurrent chemo-radiotherapy is standard of care for locally advanced cancer esophagus (LACE) despite showing 50% local failures. This study prospectively evaluated for evidence better response in dose de-escalated Hypofractionated Radiotherapy (HRT) with concurrent chemotherapy which was lacking in literature.Aims: To compare therapeutic effect, survival and quality of life parameter ‘Dysphagia’ using EORTC OES 18 questionnaire, between two fractionation regimens in LACE.Methods: Sixty eligible patients with locally advanced thoracic esophageal squamous cell cancers were enrolled from February 2014 to November 2015. 30 patients received Conventional Radiotherapy (CRT) with total dose (including ILRT/EBRT Boost) of 63 Gy/35 fractions for 7 weeks and 30 patients received HRT with total dose of 48 Gy/16 fractions for 3½ weeks on Cobalt-60 unit. Concurrent chemotherapy comprised of weekly cisplatin.Results: Both study arms were comparable demographically and in clinical parameters. Improvement in weight, overall survival & disease free survival were significantly better in CRT arm at follow up period of ≈1½ years. However, overall (tumor) response, locoregionaland distant failure were similar in both arms. On assessment of quality of life parameter ‘Dysphagia’, significantly better results seen in CRT Arm. Grade 3 esophagitis and patients undergoing feeding gastrostomy/jejunostomy during treatment were significantly higher in HRT arm.Conclusions: Higher dose of conventional radiotherapy, altered fractionation regimens, boost techniques, neoadjuvant chemotherapy proved less efficacious in LACE. Present study anticipated equal or better response, improved quality of life and better resource utilization with HRT. Study showed HRT being less efficacious than CRT in locally advanced cancer esophagus. However verification of dose de-escalated Hypofractionated RT protocol with more number of patients, longer follow up & conformal radiation techniques is warranted.

P28-A004Nomogram for prediction of prognosis in patients with oral cavity squamous cell carcinoma

S. Bobdey1, B. Ganesh1, P.S. Mishra2

1 Department of Medical Records, Biostatistics & Epidemiology, TATA Memorial Hospital, Mumbai, India2 Department of Pathology, TATA Memorial Hospital, Mumbai, India

Background: Nomograms are visual predictive tools that are widely used for estimating cancer prognosis. However, in contrast to other cancers nomograms have been used only sparingly for head and neck tumors and more so in India, no nomogram has ever been developed for oral cavity tumors. Our study sought to develop a prognostic tools that will accurately predict overall survival in individual patients with oral cancer.Methods: Demographic, host and tumor characteristics of 609 patients with cancer of the oral cavity, who were residents of Mumbai, diagnosed and treated surgically at Tata Memorial Hospital between 01 Jan 2006 – 31 Dec 2008 were reviewed from hospital medical records. The following variables were analyzed as predictors of prognosis: age, sex, comorbidities, presence of lymph node, clinical stage of disease as well as microscopic features such as tumor thickness, differentiation, bone infiltration and presence of perineural invasion. The stepdown method was used to select the statistically most influential predictors for inclusion in the final nomogram for predicting overall survival (OS).

Results: The most influential predictors of mortality probability were age, comorbidities, clinical lymph node status, stage of disease, tumor thickness, differentiation and perineural invasion. Nomogram was generated for prediction of overall survival. The discrimination ability of the nomogram was internally validated using estimation of bootstrap-adjusted concordance index of 72%. In addition, based on the individual scores obtained from the nomogram patients were grouped into three categories namely Early, Moderate and Advanced disease. The survival rates for patients in these categories were found to be 70.5%, 61% and 32.1% respectively (p , 0.00).Conclusion: Nomogram has been widely used in number of cancers, but its application in oral cancer prognostication has not been explored. Here we have developed a nomgram which can reasonably estimate 5 year OS in oral cavity cancer patients.

P38-A005Minimally invasive supra-omohyoid neck dissection for oral squamous carcinomas

R. Raj1

1 Shalby Hospitals, Ahmedabad, India

Introduction: Various surgical techniques are being developed to improve aesthetic and functional outcomes in all surgical conditions including cases of malignancy. Neck dissection via a big cervical scar is questioned nowadays when majority of primary oral cancer lesions are addressed by intra-oral resection. Recently two papers have been published for robotic assisted neck dissection with the same philosophy. We present a small series of 5 cases operated by total endoscopic method.Objectives: To study feasibility of total (pure) endoscopic technique for selective neck dissection (using CO2 insufflation) in cases of early oral cancers.Methods: Consented patients (total of 5) were operated by a single surgeon using total (pure) endoscopic technique using conventional laparoscopic instruments. The parameters for assessment of results were: duration of surgery, amount of blood loss and complications for intra-operative outcome, hospital stay and cumulative length of incision for functional outcome and number of lymph nodes dissected in pathology specimen for oncological outcome.Results: Average duration of surgery was 99.2 minutes, average blood loss was 40 ml and there was no reported intra/post operative complication. Average hospital stay was 2.4 days and average cumulative length of incision was 4.1 cm. The average number of lymph nodes dissected in pathology specimen was 19.4.Conclusion: Minimally invasive supra-omohyoid neck dissection for oral squamous cancers by total endoscopic surgical technique is feasible with better aesthetic outcomes without compromising oncologic principles. It can become a cheaper option to robotic surgery.

P42-A006Identification of novel mutation in APE1 gene in head and neck cancer patient

D. Dutta2, R. Abarna1, V. Kannan3, Sreeprakash3, C. Rayappa3, M. Anbalagan1

1 School of Biosciences, VIT University, Vellore, India2 Radiation Oncology, Apollo Hospitals, Chennai, India3 Head & Neck Oncology, Apollo Hospitals, Chennai, India

Introduction: DNA repair enzymes play a major role in cause, disease progression and response to treatment. Present project is for identification of novel mutations in genomic DNA in Asian squamous cell head and neck cancer (SCCHNC) patients and understanding their role in cause, progression and response to treatment.Material and Methods: One hundred (n 5 100) head & neck cancer patients (SCC) treated with radical intent were prospectively recruited with consent from the patient. Tissue samples from the primary tumour site were collected during the operation. Fresh crushed samples were stored in Trizol and TNES solution for genetic mutation analysis (AA sequencing, RT-PCR and electrophoresis) in dry ice. Mutation and aber-ration for APEX-1, XRCC gene and other novel mutation were assessed. Intact fresh tissue stored in NBF solution for immunohistochemistry (IHC) and FISH test for EGFR, p53, HPV (p-16). Correlation with genetic mutations and survival functions were analyzed.Results: Demographic profile (n 5 100), treatment, molecular and genetic parameters were evaluated. Correlation with survival functions with molecular and genetic markers were done in Asian patients

A. Clinical Oncology European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S3

suffering from SCCHNC. Ape1 is DNA repair enzyme, in order to detect novel mutations in APE1 gene, several genomic DNA samples were isolated from head and neck cancer patients were sequenced.Conclusion: For the first time we report here a novel mutation in the coding region of APE1 gene. Studies are in progress to establish a relationship between this novel mutation in APE1 gene and cancer.

P44-A007Abstract withdrawn

P81-A010Doxper: Doctor friendly oncology information system

R. Singh1

1 InformDS Technologies (P) Ltd. Mumbai, India

Background: Most of the hospitals (in India) lack information systems to maintain patient data in electronic form. The medical reports consist of handwritten notes that are added in the patient’s file by the physician responsible for the treatment and for follow-up visits. The handwritten notes and other lab reports are consolidated into a single physical file and stored in the medical records department of the hospital. It takes time and effort to retrieve the patient’s file and deliver to the requesting physician for treatment and follow-up.Current trends and Issues: Hospitals maintain paper records but there is a strong drive for electronic records. The data in digital format will allow for immediate remote access by any physician who is authorized to review the patient information. Reports can be stored electronically and printed selectively as the need arises. However, there are few core issues in going forward from manual records to completely electronic records, 1) the doctors are not computer savvy and do not want to type the observation and treatment notes, 2) the cancer patient load in developing countriesis very high compared to western world and doctors get few minutes with the patients in which to have social interaction as well as clinical examination, and 3) in case of an unscheduled downtime of the IT system, the hospital returns to the manual system.Approach: We work on semi-automated approach for collectively transcribing the medical data. The doctor in hospital writes the observation and treatment notes with a digital pen on a digital paper and is transmitted digitally to the cloud. The data can be consumed via intuitive Android or iOS Apps. The advantage of this approach is that the patient information is duplicated on paper and electronic form and there is absolutely no disruption to the existing clinical work flow.

P119-A011Clinical validation of contouring consensus guidelines for the treatment of locally advanced carcinoma cervix with intensity modulated radiation therapy in the Indian scenario

I. Ahmad1, I. Bashir1, A. Bhatnagar1, A. Thakwani1, S. Kumar1, C. Bhatt1, A. Bhargava1, K. Chufal1

1 Department of Radiation Oncology, Batra Hospital, New Delhi, India

Background: To evaluate the clinical outcomes and patterns of failure in patients with Locally Advanced Cervical Cancer (LACC) treated definitively utilising Intensity Modulated Radiation Therapy (IMRT).Methods: This retrospective cohort review included 18 newly diagnosed and consecutively treated patients with LACC between January 2013 and December 2014. Consensus contouring guidelines were followed for all patients. Treatment consisted of External Beam Radiation Therapy (EBRT) via IMRT technique and concurrent chemotherapy followed by IntraCavitary Radiotherapy (ICRT) with Fletcher-Suit applicator. EBRT dose prescription was 50.4 Gy in 28 fractions, concurrent cisplatin was prescribed at 40 mg/m2 and ICRT dose was 3 fractions of 7 Gy each prescribed to Point A. The endpoints were overall survival (OS) & disease free survival (DFS). Acute haematological toxicity was evaluated using Radiation Therapy Oncology Group (RTOG) criteria.Results: 16 patients were either stage IIB or IIIB and the median follow up was 19 months. The overall survival at 18 months was 72.2% (SE 5 10.6) and disease free survival was 64.2% (SE 5 11.8). There were 7 treatment failures out of which 3 patients had persistent disease after completing treatment. The predominant pattern of failure (4/7) was located in the cervix. Acute haematological toxicity was low [RTOG Grade 2 or higher for Haemoglobin, Total Leukocyte Count (TLC) & Platelet count were 55.6%, 16.7% & 5.9% respectively].Conclusion: IMRT delivered following the consensus contouring guidelines is associated with favourable outcomes for patients with LACC, supporting its safety & efficacy in routine clinical practice. More prospective trials are needed to establish its comparative efficacy versus conventional techniques.

P122-A012Bowel and bone marrow sparing volumetric modulated arc therapy (BBMSVMAT): Series of first three successfully treated patients

A. Venkatesan1, G. Lavanya1, M. Rifayi1, K. Nithiyanantham1, R. Sambasivaselli1, V. Senniandavar1, S. Gowda2, P. Coca2, R. Krishnatry1

1 Department of Radiation Oncology, Mazumdar Shaw Cancer Center, Bangalore, India2 Department of Medical Oncology, Mazumdar Shaw Cancer Center, Bangalore, India

Background: Dosimetric and clinical data support sparing either small bowel or bone marrow separately. It is important to spare both in some cases, which is challenging in view of proximity to target volume.Aim: To report acute gastro-intestinal and haematological toxicities of first three patients treated with BBMS-VMAT at our center.Methods: Case 1: 46 year lady previously treated carcinoma cervix stage IIB (2007), developed pelvic lymph nodal recurrence in left internal iliac (II) lymph nodes. She was treated with chemoradiation for PET positive LN to a dose of 60 Gy/25 fractions, left II and common iliac (CI) LN (50 Gy/25#) followed by adjuvant chemotherapy. Case 2: 58 year old lady, post hysterectomy for DUB 1 year back, presented with bilateral II & CI LN (Adenoca). Treated with 3 cycles of three weekly chemotherapy, had complete functional response on PET CT, but residual morphological disease. Treated with 60 Gy/28 fractions to residual LN and Lymphatic PTV to 50.4 Gy/28#; followed by adjuvant chemotherapy. Case3: Ca cervix stage IIIB (FIGO) with small 1–2 cm paraaortic and left pelvic LN positive on PET CT, treated with pelvic and para aortic PTV to a dose of 45 Gy/25# and to PET positive LN to a dose of 55 Gy/25# and concurrent chemotherapy followed by local brachytherapy. Standard guidelines for contouring primary PTV and LN PTV was followed. Bowel was contoured as total bowel (small1large) 2 cm above the PTV. Bone marrow was contoured using PET uptake above the liver SUV.Results: Maximum acute RTOG bowel toxicity was in case 3: grade 2, others grade 0 and max acute CTCAE hematological toxicity was grade 1 neutropenia for case 3, grade 0 for others.Conclusion: BBMS-VMAT is feasible and valid for pelvic RT. Further prospective studies are needed for validation and to understand better dose constraints for extended field RT.

P129-A013Impact of infratemporal fossa clearance on clinical outcomes for T4b carcinoma of oral cavity: Time to change the paradigm?

R. Katna1, N. Kalyani1, B. Bhosale1

1 Bombay Hospital and Medical Research center, New Marine Lines, Mumbai, India

Aim: T4b carcinoma of gingivobuccal sulcus (GBS), buccal mucosa and alveolus are associated with poor outcomes. The present analysis is done with an aim to evaluate the outcomes with infratemporal fossa clearance in addition to bite composite resection for stage T4b carcinoma of oral cavity.Material and methods: The treatment charts of prospectively maintained data of 190 patients who underwent bite composite resection for carcinoma of GBS between March 2013 and October 2015 were evaluated. Of these, 51 patients had T4b disease i/v/o infratemporal fossa (ITF) and masticator space involvement on imaging. All these patients underwent bite composite resection with ITF clearance. Five of these had high ITF involvement. Thirty six of these underwent per primum surgery followed by adjuvant therapy as per histopathology report. Remaining 15 patients received neoadjuvant therapy [chemoradiotherapy (n 5 8)/ chemotherapy (n 5 7)].Results: The median age of the cohort was 55 years. At last follow up, 35 patients were alive (31 disease-free). Seven patients had local recurrence (Five patients had recurrence in ITF), 3 patients had nodal recurrence, 4 patients had distant metastasis and 4 patients had local-regional recurrence and distant metastasis. The median and mean follow up was 11 and 13 months respectively. The one year local control and loco-regional control was 70% and 64% respectively for whole group. The one year DFS and OS was 55% and 62% respectively. For initial 39 patients with low ITF involvement having median follow up of 15 months, one year DFS and OS was 61% and 66% respectively.Conclusion: The infratemporal fossa clearance is feasible in clinical practice and initial analysis indicates better clinical outcomes as compared to historical series. Our future work will include larger series with longer follow up of present cohort to validate these encouraging results.


P130-A014Innovative cancer research at the interface: Challenging dogmas

K.N. Shetty1

1 Shri Krishna Research Centre, Mumbai, India

Background: In spite of great strides made in research, diagnosis and treatment of cancer worldwide, cancer still remains to be an enigma. Cell division are of two types, mitosis and meiosis. Dogmas in meiosis were challenged, resulting in a new fundamental theory, which partly applies to cancer research as well. Similarly, challenging dogmas in mitosis and cancer with integrative research at the interface, may result in effective ways of preventing and curing cancer.Material and methods: An innovative theoretical research at the interface, integrating molecular biology models of meiosis and fertilization and the twin polarities Yin–Yang (2ve & 1ve) was carried out. It challenged the well established dogma that the father is responsible for the sex of the offspring because of the presence or absence of the Y sex chromosome, as the other 3X sex chromosomes were assumed to be similar. However, the 3X sex chromosomes could be differentiated.Result: Only 2 of the 3X sex chromosomes were found to be similar. What emerged was a fundamental theory that is attributed to serendipity, which opens new vistas of understanding meiosis, fertilization, inheritance of chromosomes, sex determination and genetics. According to this fundamental theory, both parents are equally responsible for sex of the offspring. The differentiation of 46 chromosomes as ancestral and parental chromosomes gives a new dimension to inheritance of chromosomes, and a new nomenclature for pedigree charts. This novel pattern of inheritance of ancestral and parental chromosomes may help future research in cancer genetics and in genetic counselling.Conclusion: Incidence of cancer can be reduced by addressing lifestyle issues and environment. Innovative integrative research at the interface may help in looking beyond the horizon, and challenge dogmas in mitosis and cancer, to find effective and affordable cures for all types of cancer.

P140-A015Renal Non-Hodgkin’s lymphoma confused with renal cell carcinoma: Case report and how to avoid misdiagnosis

R. Thawani1, A. Aamr1, J. Patowary1, S. Kaul2, P. Das1

1 Department of Medical Oncology, Indraprastha Apollo Hospitals, New Delhi, India2 Department of Pathology, Indraprastha Apollo Hospitals, New Delhi, India

Kidney is a rare site of lymphoma in the body. The diagnosis is often confused with renal cell carcinoma (RCC). Previous case reports usually have diagnosed the renal Non-Hodgkin’s Lymphoma (NHL) after surgery from the biopsy specimen.We present a case of a 64 year old lady who presented with symptoms of right flank pain, weight loss and loss of appetite. On examination, a right renal mass was palpated, and it was confirmed with an ultrasound. The patient also had a small node palpable in the supraclavicular area. The ultrasonography was suggestive of renal cell carcinoma. Instead of operating on the patient, we decided to rule out lymphoma. We did a FNAC from the renal mass, which showed high grade lymphoma cells. A PET MRI was done which showed metabolically active cervical, retroperitoneal and mesenteric nodes. An avid thyroid lesion was also found. The patient underwent excision biopsy of the supraclavicular node which confirmed CD20 positive Diffuse Large B Cell Lymphoma. She received chemotherapy with R-CHOP and her pain decreased and general condition improved.In conclusion, features like lymphadenopathy with a renal mass, should suggest a diagnosis of lymphoma. Cytological analysis of a renal mass may be done to rule out lymphoma as it is inexpensive and saves the patient from a surgery.

P145-A016Impact on quality of life with stereotactic RT in advanced hepatocellular carcinoma: Initial institutional experience

M. Rifayi1, Lavanya G1, K. Nithiyanantham1, B. Sirohi2, G. Gopalakrishnan3, R. Krishnatry1

1 Department of Radiation Oncology, Mazumdar Shaw Cancer Center, Bangalore, India2 Department of Medical Oncology, Mazumdar Shaw Cancer Center, Bangalore, India3 Department of Gastroenterology, Mazumdar Shaw Cancer Center, Bangalore, India

Background: Locally advanced hepatocellular carcinoma (La-HCC) is treated with palliative intent, and recently role of stereotactic RT (STRT)

is being explored in RTOG 1112 trial with or without sorafenib. There are no documented studies on safety, utility and impact on Quality of life (QoL) from India. We present initial experience for first two patients as a feasibility study.Methods: Two patients are reported here. Patient-1 was 54 year male with hepatitis-B associated chronic liver disease (CLD) and La-HCC (10 3 12 cm) with portal vein and IVC thrombus, Child-Pugh A. Patient-2 was 56 year male with alcohol induced CLD and La-HCC (10 3 15 cm) with portal vein thrombosis and Child-Pugh B. Both patients were treated with 6 fractions over 2–3 weeks of STRT using 2–4 arcs (VMAT), active breath control and on-board CBCT to highest achievable dose to PTV while respecting mean liver doses and other organs at risk as prescribed in RTOG1112 protocol. Acute toxicity during and immediately after treatment are recorded and graded using CTCAE v4. QoL scores were collected with FACT-Hep v4 and EQ-5D pre-treatment, post-treatment and 3 months post treatment.Results: Patient-1 had PTV-liver volume of 905 cc (75%) received 45 Gy/6#, patient-2 with PTV-Liver volume of 1000 cc (50%) received 36 Gy/6#. The maximum acute grade CTCAE toxicity was grade II thrombocytopenia (patient-1) which is in recovery phase at one month. QoL scores at post-treatment compared to pre-treatment improved in all subscales (FACTHepv4); ranging from 9% for social well being (SWB) to 666% for functional well being (FWB) and average improvement of 242% in patient-1; 0% for SWB to 228% in physical well being (PWB), average of 93.2% in patient-2. EQ-5D also almost doubled in both patients.Conclusion: STRT in LA-HCC is feasible at our limited setup safely with good tolerance and improvement in QoL. Long-term impact with more patients and further sorafenib therapy need to be studied.

P146-A017Skull base bony tumours: Management nuances. A retrospective analysis from a tertiary care centre

A.K. Singh1

1 Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Background: Skull base lesions are not uncommon, but their management has been challenging for surgeons. There are large no of bony tumours at the skull base which have not been studied in detail as a group. These tumours are difficult not only because of their location but also due to their variability in the involvement of important local structure. Through this retrospective analysis from a tertiary care centre we are summarising the details of skull base bony lesions and its management nuances.Materials and Methods: The histopathologically, radiologically, and surgically proven cases of skull base bony tumours or lesions involving bone were analyzed from the neurosurgery, neuropathology record of our tertiary care institute from Jan 2009–Jan 2014. All available preoperative and postoperative details were noted from their case files. Extent of excision was ascertained from operation records and postoperative MRI if available.Results: We have surgically managed 41 cases of skull base bony tumors. It includes 11 patients of anterior skull base, 13 middle skull base and 17 posterior skull base bony tumours. The commonest bony tumor was chordoma 15 (36.6%), followed by fibrous dysplasia 5 (12.2%), chondrosarcoma (12.2%) and pPNET-EWS 5 cases (12.2%) each. There were more malignant lesions (n 5 29, 70.7%) at skull base than benign (n 5 12, 29.3%) lesions. The surgical approach employed depended on location of tumour and pathology. Total mortality was 8 (20%) of whom 5 pts were of histological proven EWS-pPNET.Conclusions: Bony skull base lesion consists of wide variety of lesions, and require multispecialty management. The complex lesions required tailored approaches surgery of these lesions. With the advent of microsurgical and endoscopic techniques, and use of navigation better outcomes are being seen, but these lesions require further study for development of proper management plan.

P163-A018Correlation of distress score with Edmonton Symptom Assessment Scale (ESAS) score in patients referred to palliative care: Prospective correlational study

S. Ostwal1, J. Deodhar1, N. Salins1, M. Muckaden1

1 Department of Palliative Medicine, Tata Memorial Hospital, Mumbai, Maharashtra, India

Background: Distress is a multifactorial unpleasant emotional state that may affect how one feels, thinks, and acts as per National Comprehensive Cancer Network (NCCN), and includes feelings of unease, sadness, worry,


anger, helplessness and guilt. Patients with cancer have some distress at some point of time. The Distress Thermometer is a tool that can be used to assess distress; it is postulated that it’s correlation with ESAS will help us identify Factors causing distress and thus intervene at the appropriate time.Objectives: Assessment of Patient’s Distress And Correlation with ESAS Score.Methods: English version of NCCN Distress Themometer (available tool for measuring distress) was translated in Marathi and Hindi. This Scale was served to total of 40 referred patients, along with the ESAS scale (symptom burden), at first visit. Data was collected and analyzed using SPSS. We categorized the distress score into- none (0), mild (1–3), moderate (4–7) and severe (8–10) similar to ESAS scale.Results: Mean distress score was found to be 5.32. In patients with Mild distress, Negative correlation was found between symptoms and distress. In Moderate distress, Positive correlation was found with ESAS symptoms- pain, breathlessness, loss of appetite and loss of sleep, in descending frequency. While in Severe distress, Positive correlation with ESAS- anxiety, fatigue, nausea, pain in descending frequency was observed. Other factors in distress scale are not addressed with ESAS (spiritual, emotional, practical and physical).Conclusion: Our data suggest that we cannot rely totally on ESAS symptoms for determining distress. Other factors (problems) should also be taken into consideration. Timely diagnosis and Proper intervention for problems would help to alleviate the distress.

P174-A019A single lateral incision for all thyroid neoplasms – Horizontal lateral thyroidectomy, Thomas’ technique, the “minimally invasive open thyroidectomy” a novel approach

T. Varughese1

1 Surgical Oncology and Reconstructive Surgery, Renai Medicity Hospital, Cochin, India

Background: Kochers’ thyroidectomy is considered gold standard for thyroid neoplasm. Horizontal lateral thyroidectomy, minimally invasive open technique based on 3D digital volumetric interactive anatomy is a novel concept in minimizing reported complications to the Superior and Recurrent Laryngeal nerves, Parathyroids and vascular pedicles, which are lateral and posterior to the gland.Objectives: This prospective study was initiated to evaluate the impact of lateral approach for thyroid neoplasms.Methods: Pilot study conducted in August 2008 on 25 patients to test the new hypothesis showed superior results than historic controls, prospective study was undertaken for the next 5 years. Out of 566 subjects, from August 2008 August 2013, 462 were females and 104 males. 330 had cancer and 136 benign diseases. Out of 136 benign cases 96 were MNGs, 31 adenomas and 9 cysts. Out of 330 cancer cases 230 were papillary, 60 follicular, 35 mixed type and 5 medullary cancers. 18 patients had intra thoracic extensions. Using single ipsilateral incision, 90 total thyroidectomies for benign and 396 for cancers, were done. Bilateral approach used in 20 cases of carcinomas with bilateral nodal disease and in 8 cases of bilateral intra thoracic extensions. Parathyroids at stake were pulverized injected into sternomastoid.Results: Blood loss was 10–15 ml and hospital stay 24 hrs. There were no nerve injuries. Hypocalcaemia was reported in 20/566, 17 had temporary and 3 permanent. 546/566 had single lateral incision. Lateral scar disappeared within 6 months, giving excellent cosmesis and quality of life.Conclusion: “Thomas technique”, is a novel method applicable for all thyroid neoplasm providing direct vision of nerves, parathyroids, facilitating better lymph adenectomy, safest for large thyroids in neck or in mediastinum. Complications described are minimized, cosmetic results, much superior.

P213-A020Implant dosimetric parameters correlation to local control & critical organ toxicity – Lessons from 240 CT based interstitial implants in cervical cancer!

V. Shankar1, C. Haritha1, A.R. Gupta1, V. Kumar1, S. Kumar1, R. Rajesh1, J. Joseph1, P. Rajesh1, R. Kumar1

1 Geetanjali Cancer Center, Geetanjali Medicity, Udaipur, India

Purpose and Objective(s): To investigate the value of DVH parameters like Conformal index (CI), Uniformity index (UI), Natural dose ratio (NDR), Peak dose (PD) and percentage of volume of critical organs - bladder & rectum (MPD-rectum, MPD-bladder) for predicting local

control and long term critical organ toxicity in patients undergoing CT-guided Interstitial brachytherapy for cervical cancer.Materials/Methods: Study comprised of 120 pts. who underwent, following completion of external RT, afluoro-guided interstitial implant placement. Natural DVH of 240 implants were analysed. Dosimetry parameters derieved included UI, CI, NDR, and PD. For critical organ dosimetry, the entire volumes are divided into 12.5%, 25%, 50% & 100%. The mean prescribed dose delivered to respective volumes of the critical structures was calculated and the multinomial logistic regression model applied to check its relationship with manifested late toxicity.Results: At a median FU of 70 months, 81 patients achieved good local control. In 240 cervical interstitial implants, mean QI, UI, CI for all the implants is 1.58, 1.29 & 1.18 respectively. 73% of patients had grade I, 20% had grade II and 7% had grade III rectal toxicity. For rectal volumes (PVCD) V12.5%, V25%, V50% and V100% the mean doses delivered are 75%, 65%, 50% and 18% of the prescribed dose. While 70% of patients had no bladder toxicity, 20% developed grade 1 and 10% had grade 2 RTOG reactions. For bladder volumes V12.5%, V25%, V50% and V100% the mean doses delivered are 60%, 45%, 35% & 10% of the prescribed dose. Logistic regression analysis showed statistical significance for V12.5% & V25% for rectum (p , 0.052) & Bladder (p , 0.063). Geometrically optimized plan resulted in better protection for the bladder wall and rectum.Conclusions: This is largest study reported so far incorporating DVH based analysis of the dose distribution thus enabling a clinically realistic evaluation of the brachytherapy application.

P214-A021Effect of effortful swallow on oesphageal cancers motion – Implications for ITV margins



Objectives: Evaluate the deglutition induced motion of primary oesophageal cancers using 4-D CT image acquisition.Methods and Materials: 4D CT scans using external metric device were obtained to quantify the resting and deglutition induced motion of in 5 normal healthy volunteers and 15 oesophageal primary tumours located in the proximal, mid- or distal thoracic oesophagus. For patients, the gross tumour volume (GTV), lung and heart were delineated on the CT set for one phase. B-spline DIR algorithm was used to propagate the same volumes in all other image datasets of 10 bins. Respiratory motion was measured from changes in the isocenter of GTV in the superior–inferior (SI), anterior–posterior (AP), and left–right (LR) directions and was analysed for correlation with anatomic location. Structure motion was defined as change in COM position, with maximum 3D motion defined as the vector of largest magnitude between two individual phases. Recommended margin expansions were determined for both primary and nodal targets selected. Deformable image registration was used to map the full expiratory motion gross tumour volume (GTV) to the full-inspiratory CT image, allowing quantitative assessment of each voxel’s displacement. These displacements were correlated with patient tumor and respiratory characteristics.Results: The mean (SD) peak-to-peak displacements of all primary tumors in the SI, AP, and LR dimensions were 0.80 (0.45) cm, 0.28 (0.20) cm, and 0.22 (0.23) cm, respectively. Distal tumors were found to have significantly greater SI and AP motion than proximal or mid-esophageal tumors. The mean (SD) SI, AP, and LR peak-to-peak displacements of the celiac-region lymph nodes were 0.92 (0.56) cm, 0.46 (0.27) cm, and 0.19 (0.26) cm, respectively.Conclusions: The ITV based on all 10 phases is recommended for adequate converage of primary tumors as well as the draining lymph nodes.

P231-A022A cross sectional observation study regarding patients and their physician willingness to wait for driver mutation report in NSCLC

V. Noronha1, A. Joshi1, V.M. Patil1, J. Ghosh1, A. Bhattacharjee2, K. Prabhash1

1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India2 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India

Background: Palliative chemotherapy 1/2 targeted therapy in accordance with mutation profile is the norm in NSCLC. The objective of this audit was to determine the proportion of patients and physicians, who are unwilling to wait for the mutation report and the reasons thereof.


Methods: All newly diagnosed NSCLC patients, post biopsy, seen at our centre between Nov-2014 to Jan-2015 were included. The relationship between patient and physician decision and objective factors was explored by Fisher’s exact test. The factors considered were ECOG PS, presence of cough, haemoptysis, fatigue and breathlessness. The agreement between patients and physician decision was tested by contingency table.Results: Out of 168 patients, 57 were unwilling to wait for driver mutation report (33.9% 95% CI 27.2%–41.4%). The commonest reason provided by patients was symptomatic status (23, 40.1%). No other objective factor except PS (p 5 0.00) was associated with patient’s decision.In 56 patients (33.4% 95% CI 26.6%–40.7%) physicians were unwilling to wait. Among the tested factors ECOG PS (p 5 0.000), breathlessness (p 5 0.00) and fatigue (p 5 0.00) were associated with decision of not waiting for the report. The percentage corrected value of contingency between patients and physician decision was 78.74%.Conclusion: Nearly 1/3 of patients are unwilling to wait till the availability of mutation report and poor control of symptoms seem to be the predominant cause. There is a good level of agreement between patients and physicians decision.

P232-A023A tertiary care experience with paclitaxel and cetuximab as palliative chemotherapy in platinum sensitive and nonsensitive head and neck cancers

A. Joshi1, V. Noronha1, V.M. Patil1, A. Bhattacharjee2, D. Paul1, S. Dhuma1, S. Juvekar3, S. Arya3, K. Prabhash1

1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India2 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India3 Department of Radiodiagnosis, Tata Memorial Hospital, Mumbai, India

Background: The combination of paclitaxel and cetuximab has led to an encouraging response rate in the phase 2 setting with limited toxicity. The aim of our study was to assess the efficacy of this regimen in our setting in platinum sensitive and nonsensitive patients.Method: This was a retrospective analysis, of head and neck cancer patients treated with weekly paclitaxel and cetuximab as palliative chemotherapy between May 2010 and August 2014. The standard schedule of cetuximab along with 80 mg/m2 of weekly paclitaxel was administered till either disease progression or withdrawal of patient’s consent. The toxicity and response were noted in accordance with CTCAE version 4.02 and RECIST version 1.1 criteria respectively. The response rates between platinum sensitive and nonsensitive patients were compared by chi-square test. Overall and progression free survival were estimated by Kaplan-Meier survival method and log rank test was used for comparison. COX proportional hazard model was used for identification of factors affecting PFS and OS.Results: 100 patients with a median age of 52 years (IQR 46–56 years) were included. Forty five patients (45%) were platinum insensitive while 55 patients (55%) were platinum sensitive. In platinum insensitive patients and sensitive patients the response rates were 38.5% and 22.2% respectively (p 5 0.104) while the symptomatic benefit in pain was seen in 89.5% and 71.7% respectively (p 5 0.044). The median PFS in platinum insensitive and sensitive patients were 150 and 152 days respectively (p 5 0.932) while the median OS were 256 days (95% CI 168.2–343.8 days) and 314 days (95% CI 227.6–400.4 days) respectively (p 5 0.23). Nineteen patients (19%) had grade 3–4 adverse events during chemotherapy.Conclusion: Weekly paclitaxel combined with cetuximab has promising efficacy and good tolerability in the palliative setting in advanced head and neck cancer in both platinum sensitive and insensitive patients.

P234-A024Long term results and quality of life of unique drainless breast cancer surgery – Thomas’ technique, reported for the first time

T. Varughese1

1 Surgical Oncology and Reconstructive Surgery, Renai Medicity Hospital, Cochin, India

Background: Suction drains for seroma, after axillary dissection, increases hospital stay, morbidity, delays adjuvant treatment, and cannot obviate subsequent seromas. Normal Saline instillation into the dissected dead space in axilla and primary site, above central venous pressure to block lymphovenous ooze is a novel technique, described for first time. This reduces morbidity, hospital stay, total cost, improves quality of life.

Objectives: Since the randomized study by the author using this technique has shown to offer best quality of life with follow up for more than 5 years, long term benefits in terms of cosmesis, arm edema, local recurrences, psychological satisfaction and quality of life were analyzed.Methods: 762 patients were recruited from 1998–2007 in the randomized trial, 389 in study arm and 373 in conventional arm. Self evaluation questionnaire on cosmesis, arm edema, psychological satisfaction and quality of life was used. Clinical evaluation; tests for local and distant recurrences were conducted by surgeon.Results: Cosmetic outcome were excellent in 310/389 in study arm, 292/373 in the conventional, very good in 51/389 and 47/373, good in 28/389 and 34/373, mild to moderate arm edema was observed in 4/389 in study arm, 13/373 in conventional arm, local recurrence occurred in 0/389 in study arm, 2/373 in conventional arm, Quality of life, excellent 98% study arm and 90% in conventional arm. Total cost 60% more in conventional arm. Hospital Stay was 24 hrs and 7 days, respectively.Conclusion: This novel drain less technique, helps in faster rehabilitation in terms of psychological, occupational, and sexual aspects, reduces morbidity, shortens adjuvant treatment time interval, reduces hospital stay, costs and offers better cosmetic outcome, quality of life.

P237-A025Prospective study to determine the incidence and risk factors for ICU delirium in Indian cancer patients

Harish M.M.11 Tata Memorial Hospital, Parel, Mumbai, India

Background: Delirium in ICU associated with both short and long term adverse outcomes. Cancer patients admit to ICU with risk factors like hypertension, anemia, sleep deprivation, sepsis, need for mechanical ventilation and chemotherapy. Most published data on delirium was on general ICU patients and there were no large studies in cancer patients, data from Indian ICUs in cancer patients is not available, hence the study was planned.Methods: 428 adults included over 12 months. Data like demographics, type/stage of cancer, treatment details, hemodynamics, ventilation, presence of sepsis, patientst’s sleep and mobilization status were noted. Delirium was assessed 3 times a day by bedside nurses using Confusion Assessment Method, even one shift positive in a day was taken as delirious.Results: Of the 500 screened, 428 patients were finally included. There were 251 males and 177 females with a mean age 53.4 and APACHE II of 13.4. There were 363 solid and 65 liquid cancers among which 8 were under evaluation, 347 were curative treatment intent, 39 were metastatic and 34 were palliative. 223 underwent surgery, 195 received chemo and 62 received radiation. The incidence of delirium was 65.4%.

Delirium No Delirium

Age (yr) 54.3 (19–85) 51.9 (19–80)

APACHE II 13.6 (2–41) 12.99 (2–35)

Mean ICU Stay 9.47 6 8.9 (2–50) 3.72 (1–11)*

Mean Hospital Stay 34.96 (2–194) 19.34 (2–83)*

Mean Ventilation Days 6.67 (0–41) 1.78 (2–11)*

Hospital Mortality% 70 38.5*

Mean Midazolam, mg/kg/day 26.4 (0–96.9) 13.1 (0–96)*

*P, 0.05

On multivariate analysis following showed significant association with delirium.

RISK P OR

95% C.I

Lower Upper

Comorbidity 0.000 580.4 29.5 11410.1

Tobacco 0.000 214.5 16.6 2772.7

Midazolam 0.000 284.8 12.9 6283.0

Chemotherapy 0.000 70.5 8.0 616.9

Sleep Deprivation 0.002 14.3 2.7 76.4

Surgery 0.003 21.7 2.8 167.1


Conclusion: Incidence of ICU delirium in cancer patients was 65.4%. Chemotherapy, Midazolam, comorbidities, tobacco consumption, surgery and sleep deprivation were the independent predictors. Delirium increase both ICU and hospital stay with increase in mortality.

P238-A026Integrative oncology: Newer potential for immunotherapy and chemoprevention

M.S. Gundeti1, H. Rasane2

1 RRA Podar Ayurveda Cancer Research Institute (CCRAS, Ministry of AYUSH, GoI), Mumbai, India2 Ayurveda Research Centre, Department of Pharmacology and Therapeutics, S.G.S. Medical College and KEM Hospital, Mumbai, India

Background: Integrative oncology emphasizes on multitude of approaches accompanying the conventional therapy finally facilitating patient’s health. Ayurveda, Indian System of Medicine, possesses tremendous potential for filling various gaps in cancer care especially related to immune system. Various Ayurveda drugs have been sought for and documented with anti-cancer activity as well as immune enhancing activity. ‘Ayurveda Wellness’ is advised for prevention of disease. Ayurveda also offers personalized medication accompanying lifestyle and dietary modifications, which contribute, to patient’s sustenance. Currently, very less published literature is available with integrative approach for cancer management. This paper presents scientific approach for amalgamation of Ayurveda and Conventional cancer care for Immunotherapy and Chemoprevention.Methods: In literature review, various online resources, databases, journals and Ayurveda classical texts were accessed with focus on specific activities of Ayurveda drugs viz. anti-cancer, immune-modulatory, immune enhancement and anti-inflammatory. Clinical studies related to cancer that had intervened Ayurveda medicines as stand-alone or adjuvant therapy were also included.Results: Studies state that Ayurveda drugs exhibit anti-cancer, anti-inflammatory and immune-modulatory activities. These enhance the host defense and create sustenance for conventional therapy aiding to complete the entire regimen. Few studies also concluded ‘Ayurveda Wellness’ have positive effect on immune system further helping in chemoprevention. The dietary and lifestyle modifications aid in providing nutrition and favorable internal environment in the body helping in combating the disease and supporting the novel concept of ‘Living with the disease’ with any serious ailments.Conclusion: Traditional medicines are time-tested and experience-based. Integration of such therapy must be questioned and investigated without any bias. Synergy of Ayurveda medicine and Wellness with conventional cancer care forms the backbone of Integrative Oncology clearly achieving the motto of existence of medicine, i.e. betterment of health.

P239-A027The advanced buccal mucosa cancer involving masticator space (T4b): New classification and outcome of newer surgical technique (compartment resection)

N. Trivedi1

1 Narayana Health, Ahmedabad, Gujarat, India

Objective: The buccal-complex tumors with masticator space involvement (T4b) are treated with different protocols ranging from conventional surgery, NACT-surgery-CTRT, CTRT or palliative care.Methods: All buccal cancer patients with masticator space involvement (T4b) treated at Narayana Multi-specialty Hospital, India, from March 2009-January 2014. All these patients underwent compartment surgical resection and appropriate adjuvant therapy based on pathology report. Their charts were reviewed to evaluate oncological and functional outcome. The radiological findings were used to classify these tumors into three categories - Category I (low-medial pterygoid), Category II (intermediate-lateral pterygoid and plates) and Category III (high-pterygomaxillary fissure).Results: Total 85 cases had T4b buccal cancer. Fifty-nine (69.4%) patients were alive and disease free at median follow-up of 23.7 months (range 06–56 months). For entire group, 16 (18.8%) patient had local failure, 19 (22.4%) patient had regional failure and 12 (14.1%) patient had distant metastasis. 5 (5.8%) patients were lost to follow up. The median DFS for entire group was 23.7 months. Total 30 patients were classified in category I, 47 patients in category II and 8 patients in category III. Surgical margins at skull base were positive in 0 patients in category I, 6 (12.8%) patients in category II and 4 (50%) in category III (p 5 0.001). There were 2 local recurrence in category I, 10 from category II and

4 (50%) were from category III (p 5 0.017). There was no statistically significant difference in DFS between three categories (p 5 0.912) but category II and III had statistically significant poorer margin status and local failure (p 5 0.001, p 5 0.017) On multivariate analysis, involvement of neck and ECS contributed with negative DFS (p 5 0.01, p 5 0.01).Conclusions: All patients with masticator space involvement (T4b) should not be put in one group and category I & II have good oncological outcome. Compartment surgery concept has potential to improve outcome for this group.

P240-A028Compartment Surgical resection for truly advanced oral-oropharyngeal-paranasal sinus cancer: New concept and early outcome

N. Trivedi1 Narayana Health, Ahmedabad, Gujarat, India

Background: The truly advanced, infiltrative and posteriorly located head neck tumors have poor outcome with conventional methods. This study focuses on concept of compartment resection to achieve improved margin and local control rate for all truly advanced head neck cancers where surgery remains the primary treatment modality.Materials and Methods: All the truly advanced (T4) head neck cancer patients treated at Narayana Multispecialty Hospital, Ahmedabad and Trivedi Polyclinic and Nursing Home, Mehsana, from June 2012 to June 2014. All these patients underwent upfront surgical resection and adjuvant therapy and their charts were reviewed. The surgical approach consisted of compartment resection and was tailored for each subsite like buccal mucosa, tongue-floor of mouth, oropharynx and paranasal sinus.Results: Total 93 cases of advanced cancer were included in this study. There were 50 (53%) buccal mucosa patients, 21 (23%) tongue patients, 10 (11%) paranasal sinus cases and 12 (13%) oropharynx cases. All patients underwent upfront surgery by compartment approach and 85 (91%) patients received adjuvant radiotherapy. The median follow up is 23 months (range 16 to 40 months) and 7 patients are lost to follow up. Total 4 (5%) patients have soft tissue margins positive. Total 10 (11%) patients have developed local recurrence and 12 (13%) patients have developed regional or distant failure. Total 60 (64%) patients are alive and disease free. Majority of local recurrence (8/10) were out of field recurrence in intra-cranial compartment. This fact demonstrates efficacy of such approach in eradicating tumor locally. Total 9 (10%) patients had feeding tube while 6 (7%) had tracheostomy at last follow up.Conclusions: The approach has potential to improve margin control and loco-regional control for truly advanced cancers in head neck region. These are early results to draw significant conclusions but they show feasibility and future potential of such selective radical approach - needs further research.

P263-A029Zolpidem and fluoxetine in pediatric postoperative cerebellar mutism syndrome: A case report

R. Krishnatry1, A. Lassaletta2, E. Bouffet2, Lavanya G1, S. Pillai3

1 Department of Radiation Oncology, Mazumdar Shaw Cancer Center, Bangalore, India2 Department of Haematology and Oncology, Division of Pediatric NeuroOncology, The hospital for Sick Children, Toronto, Canada3 Department of NeuroSurgery, Mazumdar Shaw Cancer Center, Bangalore, India

Background: Post operative cerebellar mutism syndrome (PCMS) is a severe debilitating complication of surgery for posterior fossa tumors in kids and mechanism of injury or treatment is poorly understood. It usually recovers spontaneously over 8–12 weeks but severity and duration may be associated with poorer long term neurological rehabilitation/outcomes. Currently multi-institutional prospective study of zolpiden use is underway in North America.Case Report: 6-year-old male presented with 4th ventricular mass lesion, underwent subtotal resection and reoperated two days later (30th Sept) with GTR and EVD. Histology revealed a classic medulloblastoma. Post-operatively patient developed a PCMS with severe mutism (total absence of speech and any form of expression), lower cranial nerves deficits and severe hypotonia in all limbs and neck rigidity. He was started on Zolpidem 2.5 mg OD in afternoon on Oct 11th; one hour later he started snoring with eyes open, as he was sleeping for first time since surgery. This continued for 2–3 hours. His neck stiffness also considerably improved. After four days he started neck holding, and 7th day he started sleeping for 8–10 hours a day with spontaneously closing eyes. Further


he started blinking, started spontaneous smiling or crying with a loud unusual sound and flicker like movements in all four limbs over next 15 days. 9th Nov, dose was increased to 5 mg OD and developed aggressive limb movements and longer sleep hours. So the dose was adjusted to 2.5 mg BID but no further improvemt was noted for next 10 days. 26th Nov Fluoxetine 10 mg was added and he started to speak single vowels in his local language (haan for yes, na for no) after two days. Third day he started vomiting after the dose of zolpidem, so parents wanted to discontinue the drug. Although vomiting persisted after stopping zolpidem, family did not want to re start it. Fluoxetine dose was increased to 20 mg once daily. By Dec 5th he had started saying mama for mom and baba for father. At the time of reporting (15 days postfluoxetine) he was bisyllabic with faster uphill recovery of other neurological status (able to sit by himself for 10–15 minutes, give shake hand, appropriate emotional expression, eating orally safely). He is advised to continue same dose for one month and review.Findings/results: Few unusual obersvations include: zolpidem induced sleep, aggressive limbmovements after increase in dose, no dramatic speech recovery with continued mutism after 48 days of drug use which responded better to fluoxetine.Conclusion: In severe PFS, zolpidem and fluoxetine may be tried safely, and patients non responsive to zolpidem may respond to Fluoxetine.

P271-A030Dilemmas inherent in decision making – Changing face of supportive oncology in advanced cancer

R.D. Arora1

1 Department of Palliative Medicine, Tata Memorial Centre, Tata Memorial Hospital

Background: The setting of a Tertiary Cancer Hospital with a dedicated Palliative Medicine unit provides a unique opportunity where the traditional role of this subspeciality can be challenged and new inroads be made in Patient care which offers a relook into the stringent concept of disease trajectory.Case presentation: A 43 year old female type 2 DM, Metastatic Gall Bladder Cancer with Malignant Biliary stricture. FDG avid soft tissue mass observed at the confluence of the right and left hepatic ducts causing significant bilateral IHBR dilatation. ERCP suggestive of a Type iiia Hilar stricture managed by PTBD. Presented with symptoms of stent block and cholangitis, and managed conservatively, discharged with reference to Palliative Medicine. Attended by Resident on call in the ED where a decision to provide Inpatient admission under the department was necessitated. Managed as cholangitis with stent block, a liason with Interventional Radiology was initiated and she subsequently underwent bilateral PTBD. management of Hyperglycemia proved to be a vexing issue as the OHAs had been stopped earlier due to an episode of sepsis induced hypoglycaemia and she was started on regular Insulin. Uncorrected Hyperglycemia was managed by Inj NPH Insulin basal dose and Inj Regular Insulin as replacement therapy and corrective insulin dosage when required. The importance of improving quality of life by amelioration of jaundice by restenting was recognised and a discussion with IR facilitated. Biliary culture to facilitate Antibiotic titration is planned.Discussion: This particular case highlights the following issues managed by inpatient admission under a Palliative Medicine department after curative treatment was deemed unsuitable-Pain, Cholangitis, Diabetes mellitus, Hypoalbuminemia and Facilitation of decision regarding re-stenting. Holistic management involving personalisation of treatment goals and Life sustaining medical treatment is an integral part of Palliative Medicine.

P278-A031Randomized controlled trial of yoga among non small cell lung cancer patients: Effects on pulmonary function

I. Khan1, A. Manna1, S. Sinha1, S.K. Sarkar1

1 Medical College Kolkata, India

Background: Since time immemorial, yoga is being practised in India as a complete wellness schedule integrating the body and mind. The importance of yoga is being highlighted in recent times through World Yoga Day celebrated on June 21 across the world. While there are claims that yoga can help patients cope with cancer, there is little scientific research material to establish this. Herein it became imperative to study the effects of yoga on different types of cancer. As a first of its kind, through this randomized controlled trial, the effects of yoga on pulmonary function is being studied in non small cell lung cancer patients receiving chemoradiotherapy.

Aim: This study demonstrates the impact of yoga, including physical poses, breathing and meditation exercises on pulmonary function among non small cell lung cancer patients receiving chemoradiotherapy.Patients and Methods: From March 1, 2015 to June 20, 2015, 60 patients attending Radiotherapy department of a tertiary cancer centre in India with locally advanced non small cell lung cancer (Stage IIIA and IIIB) were randomly assigned (1:1 ratio) to a test group receiving concomitant yoga schedule alongwith chemoradiotherapy and a control group receiving only chemoradiotherapy. Pulmonary function testing was done 2 weeks before commencement and 6 months after completion of radiotherapy in both the groups; predictors of adherence were also assessed.Results: Regression analyses pointed out that the intervention group experienced more improvement in FEV1 (p 5 0.03) compared to the control group.Conclusion: The above data suggests yoga as a beneficial adjunct tool to improve pulmonary function in non small cell lung cancer patients receiving chemoradiotherapy.

P279-A032Peritoneal carcinomatosis in colorectal cancers – Time to change

M.S. Bhandare1, P. Patil1, V.D. Pai1, A.P. Saklani1

1 Tata Memorial Centre, Mumbai, India

Background: Peritoneal carcinomatosis (PC) from colorectal cancers (CRC) either at initial presentation or at subsequent recurrence presents a significant treatment challenge. Aim of our study was to find its incidence and analyse outcomes of patients PC from CRC origin managed by different treatment modalities.Patients & Methods: A retrospective analysis of patients, from Aug 2013 to July 2014, presenting as or diagnosed as metastatic peritoneal disease from colorectal cancer with or without metastasis to other sites was performed. PC was classified as limited (PCI , 10) and widespread (PCI . 10).Results: This study included 70 patients, 53 had synchronous presentation and 17 were metachronous. 45 patients had peritoneum as the only site of metastasis and the remaining 25 visceral metastasis with peritoneum. Resections were performed in 23 patients (19 underwent R0 resection and 4 were R1). All patients received systemic chemotherapy (SC) (FOLFOX/CAPOX). At median follow up of 11 months, the median OS was 14 months. Patient with PCI , 10 had significantly better survival (41 mths) as compared to those with PCI . 10 (15 mths). Patients undergoing R0 resection had better survival (27.8 mths) as against those with R1 resection (18 mths). Survival of patient receiving only SC was 11 mths.Conclusion: In conclusion, the incidence of peritoneal metastasis in colorectal cancers is about 10%. Select group of patients who have low PCI and who undergo R0 resection of only disease portion, without entire peritonectomy still do well. Where facilities for HIPEC are not available, in select group of patients, cytoreduction followed by systemic chemotherapy should be considered. Added role of HIPEC in this subgroup needs to be evaluated.

P284-A033Geriatric oncology: Are we there yet?

D.A. Thaker1

1 Northside Clinical School, Uni.of QLD, Australia

Traditionally Oncologists feel uncomfortable offering chemotherapy to geriatric population specifically patients older than 80 years of age. These patients are generally excluded from clinical trials leading to lack of data to make evidence based decisions. Older patients have higher incidences of comorbidities and end-organ dysfunction. Usual assessment of Eastern Cooperative Oncology Group (ECOG) status or Karnofsky Performance Scale (KPS) for fitness of chemotherapy doesn’t correlate directly in older patients. Various factors affect the tolerance of the treatment, which includes nutritional status, cognition, comorbidities, social support and psychological state and polypharmacy. Comprehensive Geriatric Assessment (CGA) is the most useful tool to assess the fitness of older patients for chemotherapy and to provide prognosis. CGA includes assessment of patient’s ability to complete activities of daily living (ADLs) and instrumental activities of daily living (IADLs). ADLs are basic self-care skills while IADLs are skills to maintain independence in the community. Multiple studies have confirmed that the correlation between CGA and ECOG/KPS is moderate. Studies on CGA in older patients with cancer have demonstrated better prediction on survival, chemotherapy toxicity, post-operative morbidity and mortality. Most studies on CGA are done for inpatients. Further studies are required for Oncology outpatients and day therapy unit. Pharmacokinetics and


pharmacodynamics of chemotherapy drugs are different in geriatric population. As an example, serum creatinine cannot be taken as direct measure of renal function as muscle mass of older patients is less leading to near normal creatinine levels despite underlying renal dysfunction. Choice of chemotherapy agents, the dose and use of single/multiple agents should be in accordance with associated comorbidities and list of current medications. Recent studies have shown that use of single agent vinorelbine in metastatic lung cancer in patient population of 80 years and older have shown better tolerance and longer survival compared to standard platinum doublets. Recent ASCO guidelines have suggested not keep age as exclusion criteria for clinical trials. We need more trials including only geriatric population to provide better evidence base to mange this vulnerable elderly patient population.

P286-A034Addition of GCSF to antibiotics reduces morbidity & is cost effective in a low-income setting: A randomized open labeled parallel group phase III study

B. Arora1, A. Paradkar1, P.A. Kurkure1, M. Sengar1, S.K. Pai1, R. Hawaldar1, S. Talole1, S.D. Banavali1

Department of Medical Oncology, Tata Memorial Hospital, Parel, Mumbai, India

Purpose: Granulocyte colony-stimulating factor ([G-CSF] use in children with febrile neutropenia in high-income countries has been shown to reduce the duration of febrile neutropenia (FN), hospital admission, and antibiotics usage but has had no impact on prevention of infection related complications and cost of care. FN in low-income countries (LIC) is associated with higher degree of morbidity and mortality due to limitations in supportive care, less manpower and host comorbidities such as malnutrition. Hence, reduction in duration of FN by G-CSF in LIC setting may have much more impact on morbidity and mortality and would be potentially more cost-effective.Patients and Methods: In a prospective randomized study, 200 pediatric patients with FN were randomized to receive antibiotics with G-CSF (filgrastim; 5 microg/kg/d subcutaneously) or antibiotics alone. Children were stratiefd for their diagnosis and focus of infection. Patients with acute myelogenous leukemia and myelodysplastic syndrome were excluded GCSF was started within 24 hr of antibiotics. The study protocol required a resolution of fever and a neutrophil count . or 5 0.2 3 10 (9)/L for hospital discharge.Results: Patients randomized to G-CSF had a shorter hospital stay (median, 3 v 6 days; P 5 .01), shorter duration of febrile neutropenia (median, 5 v 6 days; P 5 .02), fewer days of antibiotic use (median, 6 v 8 days; P 5 .02), and rapid neutrophil recovery (median, 6 v 7 days; P 5 .04) but there was no difference in mean ANC nadir (mean, 0.12 vs 0.32 days; P 5 .07), ICU admissions, shock or infection related mortality. The 3-day reduction in hospital stay significantly reduced the cost per patient admission as well as solved the problem of shortage of hospital beds for admissions.Conclusion: The use of G-CSF in the treatment of established FN in LIC produces a significant reduction in duration of FN, antibiotic usage, and hospital admission, with significant cost savings and improved treatment delivery by reducing need for hospital beds.

P287-A035Urbanization and cancer: pattern of cancer in urban and rural cancer registries in Punjab in Northern India

J.S. Thakur1, A. Budukh2, R. Kapoor1, S.P.S. Bhatia1, P. Malhotra1, S. Kathirvel1, P. Arora1, R. Dikshit2, R.A. Badwe2

1 Post Graduate Institute of Medical Education and Research, Chandigarh, India2 Tata Memorial Centre, Mumbai, India

Background: India is experiencing epidemiological transition and Punjab has reported higher occurrence of cancer in Northern part of India. Two urban Population Based Cancer Registries, in Chandigarh Union Territory and Mohali district of Punjab and two rural PBCRs in Sangrur and Mansa districts of Punjab were initiated covering a population of 4.5 millions in 2013 to assess the magnitude, pattern, urban rural difference and comparison of cancer rates at National level.Methods: The registry staff undertakes home visits in the community and visit cancer diagnostic and treatments facilities to collect the information of cancer incidence and death cases. The data collected was checked for quality parameters and resident confirmed cancer cases were entered in the Canreg5 software. Data was analyzed to assess the magnitude and pattern of cancer in urban and rural registries. The data collection is still in progress in Sangrur.

Results: In the year 2013, 833, 767, 798 and 403 incidence cases were registered in Chandigarh, Mohali and Sangrur and Mansa respectively. The age adjusted incidence rates among males were 93.4 and 73.7 in urban registry of Chandigarh and Mohali as compared to 43.7 and 52.6 in rural registry of Sangrur and Mansa and among females as 105, 103.6 in urban registry of Chandigarh and Mohali and 52.6 and 55.8 in rural registry of Sangrur and Mansa per 100000 respectively. In males, leading cancer sites in urban area were lung and prostate as compared to esophagus, leukemia in rural registry of Sangrur and Mansa and in females, Breast, Cervix were leading sites in urban registries as compared to cervix, breast followed by esophagus in rural registries respectively.Conclusions: Within state, cancer incidence rates are significantly higher in urban registries as compared to rural registry with different pattern and broadly comparable rates with national level. Urbanization, life style and environmental risk factors may be responsible for this difference which needs further studies.

P296-A036Gemcitabine and carboplatin combination chemotherapy in treatment naïve patients with inoperable gall bladder cancer

V. Talwar1, S. Rajpurohit1, V. Goel1, S. Raina2, D. Chandragauda2

1 Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India2 Medical Oncology, BL Kapoor Memorial Hospital, Delhi, India

Background: The primary objective of this study was to determine the response rates of the gemcitabine and carboplatin combination chemotherapy in treatment naïve patients with inoperable gall bladder cancer. The secondary objectives were to evaluate the toxicity, progression free survival (PFS), and overall survival (OS).Methods: Treatment naïve patients with histologically proven inoperable gall bladder cancer treated with gemcitabine and carboplatin chemotherapy between January 2011 and October 2014 were included in this retrospective audit. The median dose of gemcitabine was 1 gm/m2 (range 1–2.2 gm/m2) on day 1 and 8, and carboplatin (target AUC of 5.0 mg/ml) on day 1, in a 21 day cycle. CT scan was used for response assessment.Results: There were 41 men and 107 women with a median age of 59 years (range 26–82 years). Of the 148 patients, 9 (6.1%) patients achieved a complete response and 52 (35.1%) patients achieved a partial response for an overall response rate of 41.2%. The median PFS was 5.1 months [95% confidence interval (CI) 1–27 months], with 1-year survival rate of 20.3%. CTC grade 3 anaemia was seen in 1 (0.7%) patient. Grade 4 neutropenia was observed in 1 (0.7%) patient whereas 4 (2.7%) patients experienced Grade 3 thrombocytopenia.Conclusion: The combination of gemcitabine and carboplatin is active in advanced gall bladder carcinoma with mild toxicity.

P298-A037Epstein-Barr virus infection in monoclonal gammopathies – a clue to pathogenesis?

Y. Kumar1, K. Sangam1, R. Minz1, N. Varma2, S. Varma3

1 Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India2 Department of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh, India3 Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Background: Monoclonal gammopathy (MG) is a group of B cell/plasma cell disorders. Multiple factors have been proposed in its etiology i.e. genetic, epidemiologic and environmental however, its exact cause remains unknown. Epstein-Barr virus (EBV) has its established role in the development of a wide range of B-cell lympho-proliferative disorders. MG also being a B cell disorder might have some association with EBV infection. With this hypothesis we aimed to determine the incidence of EBV infection in patients of MG using a robust and sophisticated method.Methods: Quantification of EBV load was done in the peripheral blood of 50 patients of M-band positive MG by quantitative Real time-PCR (qRT-PCR) and 50 age and sex matched healthy controls.Results: Majority of the patients were between 40–60 years of age. Body pain and backache were the commonest symptoms seen in 67% of patients. There were 35 cases of multiple myeloma, 6 cases of plasmacytoma, 1 case each of MGUS, POEM syndrome, lympho-plasmacytic lymphoma and amyloidosis. Five cases were either uncate-gorized or other than the MG hence were excluded. EBV infection was found in 37.8% (17/45) of cases of MG compared to 16% (8/50) control

S10 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 B. Extracellular Nucleic Acids

subjects (p 5 0.008). EBV copy number was also significantly higher in these patients (mean 5 1.8 3 105/ml) than in controls (1.7 3 104/ml) with a p value of 0.003.Conclusions: The study shows a significant association between EBV infection and MG. However role of EBV infection in pathogenesis of MG, if any, needs to be studied further in larger cohort.

P8-A038Would a structured research nurse-led consultation improve adherence to oral anticancer investigational medicinal products (IMP) in clinical research trials?

C. Glendon1, C. Pigden1, P. Sucindran1, S. Whelan1, N. Gomm1

1 Oncology and Hematology Clinical Trials (OHCT) Department, Guys and St. Thomas’ NHS Foundation Trust, London, UK

Background: The complexity of anti-cancer drug regimes is estimated to lead to a non-adherence rate between 16–100% in adults. Within the OHCT department over the last 12 months, four incidents directly related to inaccurate information provision or misunderstanding of information provided, have resulted in non-adherence. Non-adherence to IMP can significantly impact upon trial related outcomes thus interventions to enhance rates of adherence are required. This study aims to investigate if research participant adherence to oral IMP can be enhanced through the implementation of a research nurse-led IMP consultation.Method: A systematic literature review was conducted to inform the project and to identify how structured nurse-led IMP consultations could enhance research participants’ adherence. A semi-structured questionnaire was created to identify what information OHCT research nurses currently provide to trial participants during oral IMP consultations. All research nurses within the OHCT department were surveyed and the results analysed.Results: The survey identified inconsistencies in the quality and quantity of information provided to research participants. No structured approach to oral IMP consultations was identified. Despite an extensive systematic literature search, no current literature was identified on the effects of a nurse-led IMP consultation on research participants’ adherence.Discussion: Further research is required to establish current practice across the NHS. 20–25% of new anti-cancer drugs are expected to be in oral form. As research participants are becoming increasingly responsible for self-administration of IMP, interventions are required to promote adherence with the aim of improving clinical and trial related outcomes. Based on this research project the OHCT department intends to pilot a structured nurse-led IMP consultation in an attempt to improve participants’ adherence to IMP. Further research will be carried out to investigate the efficacy of this nurse-led IMP consultation on enhancing participants’ adherence and the capacity of implementing structured consultations throughout NHS investigator sites.

B. Extracellular Nucleic Acids

P249-B001Chromatin from dead cells spontaneously enter into nuclei of neighbouring cells: implications for tumour–microenvironment cross-talk

P. Tidke1, T. Saha1, A. Gaikwad1, P. Prasannan1, H. Nagare1, I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India

Introduction: We have reported that chromatin fragments (Cfs) that circulate in blood of cancer patients can freely enter into healthy cells and induce dsDNA breaks in the recipients by integrating into their genomes. Here we show that Cfs from dead cancer cells can spontaneously enter into nuclei of surrounding living cells and, similarly, Cfs from dead non-cancerous cells can spontaneously enter into nuclei of living cancer cells. This suggests that Cfs maybe the medium of cross-talk between tumour and the microenvironment.Material and methods: 1) B16-F10 (melanoma) or HeLa (cervical cancer) cells were labeled with BrdU and sparsely plated (1 3 103) on cover slips in 35 mm3 petri dishes. After 12 hr cells were irradiated with g-rays (15 Gy) and were immediately overlaid with NIH3T3 mouse fibroblast cells (5 3 105). After 12 hr, cover-slips were processed for immunofluorescence using anti-BrdU antibody. 2) In other experiments the reverse was done: NIH3T3 cells (1 3 105) were labeled with BrdU and plated on cover slips in 35 mm3 petri dishes. After 12 hr cells were irradiated with g-rays (15 Gy) and were immediately overlaid with

B16-F10 or HeLa cells (10 3 103). After 12 hr, cover-slips were processed for immunofluorescence using anti-BrdU antibody.Results: 1) BrdU labeled chromatin particles from dead cancer cells were clearly seen to have entered into nuclei of neighbouring NIH3T3 cells. 2) BrdU labeled chromatin particles from dead NIH3T3 cells were clearly seen to have entered into the nuclei of the living cancer cells.Discussion: Our results clearly show that chromatin fragments from dead cancer cells can spontaneously enter into nuclei of living non-cancerous cells and, conversely, that those from dead non-cancerous cells can enter into living cancerous cells. This suggests that Cfs maybe the medium of cross-talk between tumour and the microenvironment.

P260-B002Chromatin from dead cancer cells trigger bystander DNA damage and inflammation

P. Prasannan1, A. Gaikwad1, Raghuram G.V.1, R. Chaubal2, N. Gardi2, D. Gupta1, P. Tidke1, A. Dutt2, I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai2 Integrated Cancer Genomics Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India

Introduction: Cancer-related bystander effect is a phenomenon wherein non-cancerous cells of the tumour microenvironment and those of distant organs show evidence of DNA damage and inflammation. DNA damage and inflammation are hallmarks of cancer and acts synergistically to drive the oncogenic process. We show here that dead cancer cell-derived chromatin fragments (dcCfs) are key agents in inducing cancer-related local and systemic bystander effects.Material and methods: Jurkat cells (human lymphoblastic leukemia) were induced to undergo cell death by treatment with antibody against FAS-ligand. In some experiments the cells were pre-labeled with BrdU before inducing cell death. The dead Jurkat cells were co-cultured with NIH3T3 cells or injected intravenously into mice to investigate local and systemic bystander effects in the recipient cells.Results: When BrdU labled dead Jurkar cells were co-cultured with NIH3T3 cells, numerous dcCfs that emerged from the dead cells were seen to rapidly enter into nuclei of the recipients triggering a DNA-damage-repair response (DDR) which facilitated their incorporation into host cell genomes. Whole genome sequencing of single-cell clones derived from dead Jurkat cell treated NIH3T3 cells revealed presence of hundreds of thousands of human reads in the mouse-cell genomes. Genomic integration was associated with dsDNA breaks, global deregulation of transcription involving 1004 genes and an intense up-regulation of inflammatory cytokines. When injected intravenously into mice, dcCfs from dead Jurkat cells integrated into nuclei of cells of distant organs and activated DDR and inflammation.Discussion: Our results suggest that dcCfs-induced DDR and inflammation may create a pro-oncogenic milieu in cells of the tumour microenvironment and in those of distant organs which could promote their oncogenic transformation. These findings suggest a new mechanism for local and systemic spread of cancer.

P250-B003Chromatin from dead cancer cells triggers chromosomal instability, senescence and oncogenic transformation

A. Gaikwad1, P. Prasannan1, Raghuram G.V.1, T. Saha1, P. Tidke1, I. Mittra1


Introduction: We have shown that chromatin fragments from dead cancer cells (dcCfs) can freely enter into healthy cells, to integrate into their genomes and induce dsDNA breaks, global deregulation of transcription and intense up-regulation of inflammatory cytokines. Here we investigate other downstream effects induced by dcCfs that include chromosomal instability, senescence and oncogenic transformation.Material and methods: Several cancerous cells were induced to undergo cell death by treatment with antibody against FAS-ligand or by treatment with Adriamycin. The dead cancer cells were co-cultured with NIH3T3 cells to investigate downstream cellular events.Results: NIH3T3 cells which had been co-cultured with dead Jurkat (human lymphoblastic leukemia) cells were allowed to grow and metaphase spreads were prepared from them at 5th passage. Cytogenetic analysis revealed numerous chromosomal aberrations in the treated cells; the most prominent of these being centromeric fusion and dicentric chromosomes. Spectral karyotyping analysis revealed numerous chromosomal translocations as further evidence of severe genomic instability. The treated cells underwent rapid oncogenic

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transformation by 96 hr and/or acquired a senescent phenotype. The latter was confirmed by the expression of the senescence related protein HP1a. These features could be abrogated by concurrent treatment with anti-histone antibody complexed nanoparticles confirming the role of dcCfs in the induction of the above phenotypic changes. The transformed cells were tumourigenic in SCID mice and presence of human DNA could be detected in these tumours indicating that dcCfs derived from dead human cancer cells that had integrated into the genomes of NIH3T3 cells had contributed to neoplastic development.Discussion: Our results demonstrate that dcCfs can induce chromosomal instability and oncogenic transformation in neighbouring cells and suggest a new mechanism of cancer metastasis whereby cells of target organs are oncogenically transformed by dcCfs from dead cancer cells to form new tumours which masquerade as metastasis.

P251-B004Isolation of circulating chromatin fragments from serum of cancer patients and healthy volunteers

N.K. Khare1, N. Pandey1, I. Mittra1


Introduction: 109–1010 cells die in the adult human body daily and a considerable quantity of chromatin fragments are released into the blood stream in cancer patients and in healthy individuals. However, circulating chromatin has not previously been isolated from blood. Here we describe a method for isolating Cfs from blood of cancer patients and of healthy individuals.Material and methods: Informed written consent was obtained from human subjects recruited for the study as approved by the Institutional Review Board of ACTREC, TMC. Serum was separated from 6 ml of collected blood and centrifuged at 700,000 g for 16 hr at 4°C. The pellet obtained was lysed with lysis buffer and centrifuged for 16 hr at 700,000 g as above. The pellet was suspended in PBS and passed through an affinity column containing a mixture of biotinylated anti-histone antibodies (20 μg each of H1, H2A, H2B, H3, H4 in a volume of 1 mL) bound to Pierce® Streptavidin Plus Ultralink® Resin (1 mL). The column was eluted with low salt (0.25 M NaCl), and Cfs were recovered by ultra-centrifugation as described above. The final pellet was re-suspended in PBS in a volume corresponding to the initial serum volume from which the pellet had been obtained (1 mL).Results: The presence of chromatin in the isolated Cfs fractions was confirmed by the Cell Death Detection ELISAplus designed exclusively for the detection of nucleosomes/chromatin. Under electron microscope, Cfs were found to retain the typical beads-on-a-string appearance of nucleosomes and were heterogeneous in size ranging from 10 nm to several hundred nm. Western blot analysis of Cfs using polyclonal antibodies against histone confirmed the presence of histone proteins H2A, H2B, H3 and H4.Discussion: This is the first report of isolation and characterization of circulating chromatin fragments from blood.

P247-B005Circulating chromatin damages DNA of healthy cells by integrating into their genomes

N.K. Khare1, Raghuram G.V.1, R. Chaubal2, F. Khambatti1, D. Gupta1, A. Gaikwad1, P. Prasannan1, A. Singh2, P. Upadhyay2, A. Dutt2, I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India2 Integrated Cancer Genomics Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India

Introduction: It has been estimated that several hundred billion to a trillion cells die in the adult human body daily and a considerable amount of fragmented chromatin (Cfs) enters into the circulation in normal individuals, and in elevated levels in a multitude of acute and chronic human pathologies including cancer. Whether Cfs have any patho-physiological functions in the host have not been explored.Material and methods: We isolated Cfs from blood of cancer patients and healthy volunteers and tested for their biological activity in vitro and in vivo.Results: Cfs isolated from blood of cancer patients and healthy volunteers were readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear Cfs associated themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse

cells. Genomic incorporation of Cfs led to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, Cfs underwent genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs isolated from cancer patients were found to be more active with respect to all parameters examined compared to those from normal volunteers. All the pathological actions of Cfs described above could be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo.Discussion: Our results suggest that Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications for ageing and a multitude of human pathologies including initiation and metastasis of cancer.

P248-B006Circulating chromatin from cancer patients trigger inflammation, chromosomal instability and oncogenic transformation

N.K. Khare1, A. Gaikwad1, P. Prasannan1, Raghuram G.V.1, D. Gupta1, N. Gardi2, R. Chaubal2, A. Dutt2, I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India2 Integrated Cancer Genomics Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India

Introduction: We have reported that chromatin fragments (Cfs) that circulate in blood of cancer patients can freely enter into healthy cells and induce dsDNA breaks in the recipients by integrating into their genomes. Here we investigated whether the DNA damage inflicted by Cfs leads to deleterious downstream effects such as inflammation, chromosomal instability and cancerous transformation.Material and methods: We isolated Cfs from blood of cancer patients and healthy volunteers and tested for their biological activity in vitro and in vivo.Results: When metaphase spreads were prepared from NIH3T3 cells treated with Cfs from cancer patients and examined for cytogenetic abnormalities, multiple chromosomal abnormalities were apparent in the treated cells. Spectral karyotyping analysis revealed numerous chromosomal translocations as further evidence of severe genomic instability. Cfs treatment triggered a genome-wide deregulation of transcription and up-regulation of 21 cancer-related pathways encompassing 200 genes. We also observed intense up-regulation of inflammatory cytokines and microarray detected up-regulation of multiple inflammation-related pathways. In 26 out of 58 (45%) of our experiments with cancer Cfs, oncogenic transformation was observed in the treated cells within 96 hr while only 3 out of 36 Cfs samples from healthy volunteers showed any phenotypic change (p 5 0.0001). The transformed cells were tumourigenic in immune-deficient mice and FISH detected human DNA signals in nuclei of tumour cells indicating that Cfs that had integrated into the genomes of NIH3T3 cells had contributed to neoplastic development.Discussion: Our results demonstrate that Cfs from cancer patients can induce inflammation, chromosomal instability and oncogenic transformation in healthy cells and suggest a new mechanism of cancer metastasis whereby cells of target organs are oncogenically transformed by Cfs that circulate in blood of cancer patients. The ability of Cfs from healthy individuals to infrequently transform NIH3T3 cells suggests a new mechanism of cancer initiation.

OT244-B007Circulating chromatin from cancer patients and/or those derived from dead cancer cells imparts cancer stem-cell like properties to NIH3T3 cells

R. Chaubal2,4, N. Gardi2, E. Kaur3, A. Gaikwad1, P. Prasannan1, S. Dutt3, A. Dutt2, I. Mittra1

1 Translational Research Laboratory; Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India2 Integrated Cancer Genomics Laboratory; Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India3 DNA Repair and Chromatin Biology Laboratory; Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India4 Department of Surgical Oncology, Tata Memorial Centre, Mumbai, India

Introduction: We have reported that circulating chromatin from cancer patients and/or those derived from dead cancer cells can oncogenically transform NIH3T3 cells by integrating into the host cell genomes. Here we investigate whether the treated host cells acquire cancer stem-cell like properties?

S12 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 B. Extracellular Nucleic Acids

Material and methods: NIH3T3 cells were induced to undergo oncogenic transformation by treatment with circulating chromatin fragments (Cfs) from cancer patients and/or by co-culture with dead Jurkat (human lymphoblastic leukemia) cells. Two single cell clones each were generated from Cfs-treated (B2 and D5) and dead Jurkat cells-treated (B10 and E7) transformed NIH3T3 cells and examined for stem cell like properties using transcriptome sequencing.Results: The transcriptomes for these clones showed extensive de-regulation of cancer-related pathways and stem cell-related genes. When cultured under appropriate conditions, one each of the Cfs-treated (D5) and apoptotic Jurkat-treated (B10) clones formed spheroids typical of stem cells. Cells from clones D5 and B10 formed multiple large colonies in soft agar indicating their oncogenic potential.Discussion: The data demonstrates that Cfs derived from cancer patients and/or chromatin derived from dead cancer cells can impart cancer stem-cell like properties to NIH3T3 cells.

P252-B008A new theory of cancer metastasis involving dead-cell chromatin

I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer Tata Memorial Centre Navi Mumbai, India

The existing theory of cancer metastasis: The entrenched theory of cancer metastasis teaches that tumour cells detach from the primary tumour and intravasate into the blood stream to reach distant organs whereupon they extravasate into the target tissues and grow to form metastatic tumours. However, there are several problems with this prevailing theory: 1) How do tumour cells survive the shearing force of circulation? 2) How do tumour cells (diameter ~20 µm–25 µm) negotiate capillary vessels (diameter ~6 µm–8 µm)? 3) How do circulating tumour cells (CTCs) cross capillary walls to extravasate into target tissues? 4) How do CTCs cross the blood-brain barrier? 5) How do CTCs survive and grow in the hostile environment of the new host organ?The new theory of cancer metastasis: We have shown that chromatin derived from dead cancer cells can oncogenically transform neighbouring cells which are tumourigenic in immune-deficient mice. Similarly, circulating chromatin fragments isolated from blood of cancer patients can transform cells in culture. Based on these findings, I propose a new theory of cancer metastasis which posits that CTCs either die en route target organs or do so upon reaching them. The chromatin fragments that emerge from the dead CTCs oncogenically transform cells of host organs by integrating themselves into their genomes. Metastases are thus new cancers that arise from cells of host organs and are not derived from cells released from the primary tumour.Support for new theory:1. Majority of CTCs are apoptotic in nature.2. Patients whose CTCs are enriched in apoptotic cells have poor

prognosis.3. Metastases are histologically dissimilar to the primary.4. Metastases are antigenically related to cells of host organs rather

than those of primary tumours.

P253-B009Are metastatic tumours histologically similar to the primary? A blinded study

A. Singh1, T. Shet2, M. Ramadwar2, N. Jambhekar2, I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India2 Department of Pathology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India

Introduction: There is an entrenched belief among medical doctors and biologists that the histological features of metastatic tumour resemble the primary. The reification of this belief can be traced to the fact that it supports the prevailing theory of cancer metastasis. However, this belief has never been scientifically tested. We have recently proposed a new theory of cancer metastasis which posits that metastases arise as new cancers from cells of target organs that are oncogenically transformed by chromatin fragments released from dead circulating tumour cells derived from the primary tumour. If this theory is to be true, then metastatic tumours should not resemble the primary.Material and methods: Three highly experienced professor grade pathologists of Tata Memorial Hospital were asked to blindly examine 299 H&E stained slides from metastatic tumours and to correctly identify their primary sites of origin. The metastatic tumours comprised of those from 5 distant organs viz., bone, brain, skin, liver, lung and 4 lymph nodal regions viz., inguinal, mediastinal, mesenteric, and supraclavicular.

Results: The ability of the three pathologists to correctly identify the sites of primary origin stood at 40%, 46%, and 45%. We performed k statistics to assess inter-investigator agreement among the three pathologists. The k value proved to be 0.27 which is well below the k value of 0.4 which is required to indicate moderate agreement. Thus, the correct diagnosis of the primary tumour by the pathologists is likely to have been chance events.Discussion: The results of this study indicate that metastatic tumours are histologically dissimilar to the primary. The entrenched belief of histological similarities between primary tumours and their metastases needs to be revised.

P254-B010Do metastases arise from cells of the primary tumour or those of host organs? An immunohistochemical investigation

A. Singh1, T. Shet2, I. Mittra1

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India2 Department of Pathology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India

Introduction: We have recently proposed a new theory of cancer metastasis which posits that metastasis arise as new cancers from cells of target organs that have been oncogenically transformed by chromatin fragments released from dead circulating tumour cells released from the primary tumour. If this theory is to be true then metastases should express antigens of the target tissues more often than they are expressed in the primary tumour.Material and methods: 113 archival paraffin embedded samples of metastatic tissues from bone, brain, lung, liver and an equal number of primary tumours from various sites were selected for this study. Immunohistochemical analysis was performed using three antibodies each against normal host-tisssue-specific antigens (NHSA) of lung, liver, brain and bone. Their expression was compared between metastatic and primary tumours. The pathologist evaluating the slides was blinded to the tissue being examined.Results: Expression of NHSA was observed more frequently in the corresponding metastatic tissues than in primary tumours (42/318 vs 21/321, p 5 0.0052). This was particularly striking in case of lung and liver metastases (9/107 vs 1/108 and 24/103 vs 12/105, p 5 0.0098 and p 5 0.028 respectively). To rule out the possibility that the higher frequency of NHSA expression in metastatic tumours could have been due to a higher frequency of random aberrant expression of antigens in metastases, we examined the expression of 4 normal non-host-organ-cell-specific antigens (NNHSA) for each metastatic tissue. The frequency of expression of NNHSA was observed significantly less frequently than NHSA (19/423 vs 42/318, p , 0.0001).Discussion: Immunohistochemical analysis indicated that metastatic tumours frequently express antigens of the host organs suggesting a common cellular origin. This finding supports the alternative hypothesis that metastasis arise from cells of host organs.

P259-B011Degrading extracellular chromatin by resveratrol-Cu (II) prevents lung metastasis in mice

P. Bala1, M. Shetty1, K. Pal1, A. Shaikh1, P. Prasannan1, A. Gaikwad1, I. Mittra1

1 Translational Research Laboratory; Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India

Introduction: We have recently proposed a new theory of cancer metastasis which posits that metastases arise as new cancers from cells of target organs oncogenically transformed by chromatin fragments that are released from dead circulating tumour cells (CTCs). If this theory is to be true, then metastasis could be prevented by degrading/neutralizing chromatin fragments as they emerge from dead CTCs at target sites. Since we have demonstrated that Resveratrol-Cu (R-Cu) can degrade extracellular chromatin, we investigated whether R-Cu could prevent lung metastasis from B16-F10 melanoma cells.Material and methods: C57/Bl6 black mice were intravenously injected with 1 3 105 B16-F10 melanoma cells alone or were simultaneously injected i.p. with: 1) R alone (1 mM); 2) R-Cu in ratio of 1:1 mM; 3) R-Cu in ratio of 1:1 3 1024 mM; 4) R-Cu in ratio of 1:1 3 1026 mM. Animals were sacrificed on day 21 and the number of metastatic tumours in both the lungs of each animal were counted.Results: Treatment with R alone produced a non-significant reduction in number of lung metastasis. However, R-Cu in all three ratios reduced the number of metastasis to highly significant degrees (p 5 0.000 in all three

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groups). Reduction in lung metastasis in mice treated with R-Cu ratios of 1:1 3 1024 and 1:1 3 1026 were particularly striking.Discussion: This data provides support for the new theory that cancer metastases are generated from target cells that are oncogenically transformed by chromatin fragments released from dead CTCs upon reaching target organs.

P255-B012Chromatin from dead tumour cells alter radio- and chemo-sensitivity in neighbouring tumour cells

H. Nagare1, I. Mittra2

1 Translational Research Laboratory, Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India

Introduction: We have shown that chromatin fragments derived from dead cancer cells (dcCfs) can freely enter into surrounding cells to trigger DNA-damage-repair response (DDR) which facilitated their incorporation into host cell genomes. Genomic integration triggers dsDNA breaks, global deregulation of transcription and activation of inflammatory cytokines. We hypothesized that intra-tumoural cell-death resulting from hypoxia or chemo-/radiotherapy may release dcCfs which integrate into genomes of neighbouring tumour cells resulting in mutations and intra-tumoural genetic heterogeneity that could lead to alterations in their sensitivity to chemo- and radiotherapy.Material and methods: We induced cell death in 5 different cell lines, namely, 1) HeLa (cervical cancer), 2) A2780 (ovarian cancer), 3) T-24 (bladder cancer), 4) MDAMB-231 (breast cancer), 5) A549 (lung cancer) using Adriamycin treatment for 48 hr. The dead cell-pellets were co-cultured with the corresponding healthy cells. The recipient cells were allowed to grow and their chemo- and radio-sensitivity was assessed by MTT assay.Results: We made the following observations: 1) corresponding dead-cell treated HeLa, A549 and T-24 cells had become more resistant to both chemo- and radiotherapy compared to untreated controls, 2) corres-ponding dead-cell treated A2780 cells had become more sensitive to radiotherapy but more resistant to chemotherapy compared to untreated controls, 3) corresponding dead-cell treated MDAMB-231 cells had become more resistant to radiotherapy compared to untreated controls but had developed paradoxical sensitivities to Adriamycin (resistant) and Paclitaxel (sensitive) when compared with untreated controls.Discussion: dcCfs emanating from intra-tumoural dead cells can enter into surrounding living tumour cells to alter their sensitivity to chemo- and radiotherapy by inducing genetic mutations in them.

P256-B013Synthesis of pullulan-histone antibody nanoconjugates for neutralizing extracellular chromatin

M.R. Rekha2, K. Pal1, P. Bala1, M. Shetty1, I. Mittra1, G.S. Bhuvaneshwar3, C.P. Sharma2

1 Translational Research Laboratory; Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Navi Mumbai, India2 Biosurface Technology Division, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, Kerala, India3 Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, Kerala, India

Introduction: We have reported that chromatin fragments (Cfs) derived from dead cancer cells can spontaneously enter into healthy cells to damage their DNA and induce inflammation and genomic instability. Furthermore, Cfs isolated from cancer patients can induce oncogenic transformation in the recipient cells. Thus, it follows that if extracellular Cfs could be prevented from reaching other cells, it could potentially inhibit pathological conditions, including cancer.Material and methods: We prepared anti-histone antibody-complexed nanoparticles for neutralizing extracellular chromatin. Pullulan was complexed with anti-histone antibodies by a process which involves three steps: (1) activation of pullulan: typically Pullulan was dissolved in 20 mM borax buffer, mixed with Traut’s reagent (2-Iminothiolane) under continuous stirring (final pH 7.0) and dialyzed against 0.1 M sodium phosphate (pH 7.4) containing 0.15 M NaCl and 1 mM EDTA. (2) Activation of Histone antibodies: Antibodies against H1, H2A, H2B, H3 and H4 histones (200 µg/ml) were mixed with 3-maleimido benzoyl NHS (Sigma Aldrich™, USA) and kept for half an hour at 25°C for the reaction to be completed. (3) Conjugation of activated pullulan-histone antibody: 50 µl aliquots of each of these activated antibodies were immediately conjugated with 100 µl of activated pullulan, under vigorous stirring to form monodisperse nanoconjugates.

Results: The nanoconjugates were analyzed for various physico-chemical characteristics to confirm efficient conjugation. The nanoconjugates were capable of neutralizing chromatin released from apoptotic cancer cells in vitro. Experiments in mice revealed that the nanoconjugates could prevent the rise in circulating chromatin levels, prevent rise in inflammatory cytokines and prevent lethality in the animals following lipopolysaccharide challenge.Discussion: Anti-histone antibody-complexed nanoparticles may have therapeutic potential in multitude of human pathologies including ageing and cancer by its ability to neutralize extracellular chromatin.

P257-B014Resveratrol and copper (II) can degrade DNA/chromatin in vitro and in vivo

S. Subramaniam1, I. Vohra1, M. Nobre1, R. Basak1, K. Pal1, A. Shaikh1, I. Mittra1


Introduction: We have reported that chromatin fragments (Cfs) from dead cancer cells can enter into healthy cells to damage their DNA and induce inflammation and genomic instability. Furthermore, Cfs isolated from cancer patients can induce oncogenic transformation in NIH3T3 cells. It follows that if extracellular Cfs could be prevented from reaching other cells, it could inhibit pathological conditions, including cancer. Resveratrol is an anti-oxidant, but can have pro-oxidant properties in the presence of copper by its ability to reduce Cu (II) to Cu (I) thereby generating free radicals which can cleave plasmid DNA. We show here that a combination of Resveratrol and Cu can also degrade genomic DNA in vitro and extracellular chromatin in vivo.Material and methods: Stock solutions of Resveratrol (20 mM) and that of CuSO4.5H2O (20 mM) were prepared in 60% ethanol and water respectively. Reaction mixtures (20 µl) was prepared in microfuge tubes containing 500 ng of genomic DNA and a fixed concentration of Resveratrol (5 mM) and successively reducing concentrations of Cu (5 mM – 1 3 1023 mM). The mixtures were incubated at 37°C for 1 hr and electrophoresed on a 1% agarose gel at a constant voltage of 100 V. Activity of R-Cu was also tested in mice for its ability to degrade circulating chromatin.Results: Resveratrol – Cu could degrade genomic DNA in vitro as demonstrated by gel electrophoresis. Surprisingly, the DNA degrading activity increased when the Cu concentration was reduced keeping R concentration constant until such point that the Cu concentration was so low that the DNA degrading activity of R-Cu was lost. We found similar effects in vivo in that R-Cu could degrade circulating chromatin in mice even when the ratio of Resveratrol – Cu (II) was 1:1 3 1024 or 1:1 3 1026.Discussion: R-Cu may have therapeutic potential in multitude of human pathologies including ageing and cancer by its ability to degrade extracellular chromatin.

OF246-B015Chromatin from dead cells causes radiation-induced bystander effect

S. Kirolikar1, M. Nobre1, I. Mittra1


Introduction: Radiation-induced bystander effect (RIBE) is a pheno-menon associated with ionizing radiation wherein non-targeted cells show evidence of heritable DNA damage and genomic instability. RIBE is thought to be responsible for radiation-related local and systemic toxicity. Damaging substances released from irradiated cells are thought to be responsible for RIBE, although these “damaging agents” have not been identified. We show here that chromatin fragments that emerge from radiation-induced dead-cells are responsible for RIBE.Material and methods: NIH3T3 mouse fibroblasts cells and MDA-MB-231 human breast cancer cells were irradiated at 10 Gy and kept in a humidified incubator at 37°C for 6 hrs. The conditioned medium (CM) was collected, passed through 0.2 µm syringe filter and transferred to non-irradiated NIH3T3 or MDA-MB-231 recipient cells in presence or absence of anti-histone antibody complexed nanoparticles (CNPs), DNase I or Resveratrol-Cu (R-Cu), all of which can neutralize/degrade chromatin fragments. After 6 hrs, H2AX activation was estimated in the recipient cells by immunofluorescence. Four combinations were investigated as follows: 1) CM from irradiated NIH3T3 cells applied on NIH3T3 cells; 2) CM from irradiated MDA-MB-231 cells applied on

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NIH3T3 cells; 3) CM from irradiated NIH3T3 cells applied on MDA-MB-231 cells and 4) CM from irradiated MDA-MB-231 cells applied on MDA-MB-231 cells.Results: RIBE was evidenced by increase in g-H2AX levels (approximately 3-fold) in recipient cells in all 4 combinations. Addition of CNPs, DNase I and R-Cu resulted in a highly significant reduction of g-H2AX levels that were comparable to g-H2AX levels in untreated cells.Discussion: Our results show that chromatin from dead cells are responsible for RIBE. Prevention of RIBE by CNPs, DNase I or R-Cu might be of therapeutic value in reducing radiation-induced local and systemic toxic side-effects.

P177-B016Dynamics of circulating chromatin levels following surgery for stage IV head and neck squamous cell carcinoma

A. Garg1, P. Chaturvedi1, P. Pawar2, N.K. Khare3, D. Nair1, S. Nair1, I. Mittra1

1 Department of Surgical Oncology Tata Memorial Hospital, Mumbai, India2 Department of Head & Neck Surgery Fortis Hospital, Mumbai, India3 Translational Research Lab, ACTREC, Mumbai, India

Background: It has been reported that levels of circulating chromatin (CC) are associated with tumour urden. These levels have prognostic significance and help in assessing treatment response. Studies have shown dramatic rise in their levels following chemotherapy and/or radiotherapy. However, studies highlighting the effects of surgical removal of tumour on levels of CC are scarce. We studied the impact of surgical removal of tumour on the levels of CC in stage IV head & neck squamous cell carcinoma (HNSCC).Method: 5 ml blood was collected from 25 treatment nave patients of stage IV HNSCC. Blood was also collected from 5 patients undergoing surgery for benign conditions of thyroid & parotid glands who acted as controls in the study. Following time points were used for blood collection: 1) Pre-operative (after induction of anaesthesia); 2) Immediate Post-operative (during reversal of anaesthesia); 3) Post-operative day 1; 4) Post-operative day 4; and 5) Post-operative day 7. The serum CC levels were measured using Cell Death Detection ELISA plus kit (Roche) based on quantitative sandwich-enzyme-immunoassay principle.Results: In patients of HNSCC the pre-operative CC levels were high and decreased dramatically in the immediate post-operative period (p , 0.039). Controls showed low pre-operative CC levels and the change after surgery was not significant (p , 0.109). Patients with primary tumour in tongue had reduced pre-operative chromatin level as compared to other sub-sites of oral cavity (p , 0.020). Pre-operative CC did not show any statistically significant relation with grade of tumour differentiation (GTD) (p , 0.565), extra-capsular spread (ECS) (p , 0.853), lympho-vascular emboli (LVE) (p , 0.261) and peri-neural invasion (PNI) (p , 0.70).Conclusion: This study showed elevated level of pre-operative CC in HNSCC patients was largely derived from the tumour and its removal lead to a decline in their levels after surgery. Pre-operative CC level is not statistically related to GTD, ECS, LVE and PNI.

C. DNA Damage Repair and Apoptosis

P6-C001Reactive oxygen species mediated tumor cell targeting: Dr. Jekyll or Mr. Hyde?

B.V. Sajesh1, M.I. Vanan1,2

1 Research Institute of Oncology and Hematology and Cancer Care Manitoba, Winnipeg, Canada2 Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Manitoba, Canada

Medulloblastoma is the most common pediatric malignant brain tumor that is located in the cerebellum. Based on molecular profiling medulloblastomas are classified into 4 main subgroups; Group 3 account for ~30% of all medulloblastomas and have very poor prognosis. Additionally, they are highly resistant to radiation warranting studies to identify targets that sensitize these tumors. Due to the relative paucity of information available, no specific treatments to selectively target them are yet available. We looked at gene expression signature of Group 3 medulloblastoma and identified that PRDX1, a key molecular player in scavenging reactive oxygen species (ROS) was overexpressed. We hypothesized that targeting PRDX1 by

employing RNAi approaches or by a chemical inhibitor would result in an increase in ROS and lead to radio-sensitization of group 3 tumors. To this end, we have characterized the radiation sensitivity in Group 3 medulloblastoma cells with diminished expression of PRDX1 in vitro and ongoing in vivo experiments and have shown promising preliminary data. D425-med cells with endogenous PRDX1 were rendered sensitive to radiation when PRDX1 expression was diminished by RNAi or by using adenanthin. Further, tumor cells injected into the flank of nude mice were highly sensitive to targeting PRDX1 using Adenanthin. We are in the process of characterizing the radiation sensitization of D425 cells with diminishedPRDX1 expression that were implanted intracranially into nude mice. Results from all of these experiments will be presented identifying PRDX1 as a potential target to radiosensitize group 3 medulloblastoma. Owing to the association with genomic instability, endogenous ROS such as hydrogen peroxide have been recognized as a dangerous molecule and is viewed as a non-specific tumor-initiating event. Contrary to this centraldogma, we provide evidence that an increase in ROS may be exploited for a therapeutic benefit.

P31-C002Role of a novel ubiquitin ligase FBXO4 in pathogenesis of breast cancer

R. Dhar1, S. Karmakar1

1 Institute for Genomics and Systems Biology, University of Chicago Medical Center, Chicago, USA2 Department of Biochemistry, AIIMS, New Delhi, India

Background: Whole genome RNAi screen aimed to interrogate intracellular mediators for estrogen (E2) signaling in the pathogenesis of breast cancer identified a novel ubiquitin ligase FBXO4 necessary for proliferation. FBXO4 is a member of the Fboxprotein family which is characterized by 40 amino acid motif, the Fbox.Methods: Whole genome RNAi screen were performed using Thermo on Target plus siRNA library followed by cell proliferation (BrDU) assay in E2 responsive MCF7 breast cancer cells. HiSeq2000 was used for NGS. 10 nM E2 were used for stimulation.Results: FBXO4 has been identified as a potential hit in our RNAi screen showing a hyper-proliferative phenotype upon its knockdown. Western blot and ubiquitination assay identified cyclin D (CCND1) as a dedicated FBXO4 target for degradation. In genuity pathway analysis and chromatin immunoprecipitation assay revealed an exploitation of E2 signaling and enhanced recruitment of ER alpha to estrogen response element (ERE) in FBXO4 depleted cells. These effects were observed even in absence of E2. Further we observed a significant overlap of differentially expressed genes in E2 stimulated and FBXO4 knockdown cells that were nullified by CCND1 siRNA.Conclusions: Our study elucidated that CCND1 is spared from degradation following loss of FBXO4 which in turn leads to a hyperproliferative state. Our findings correlated with TCGA and METABRIC databases indicating a poor patient survival and relapse in FBXO4 low and CCND1 high cohorts of ER 1ve breast cancer thereby implying a diagnostic value. Along with traditional hormonal therapy, an effective targeted therapy in breast cancer should be aimed to restore FBXO4 levels.

P35-C003Programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC) and correlation with clinical variables and other oncogenic drivers: An immunohistochemical analysis

V. Singh1, D. Jain1, P.S. Malik2, S. Kumar3

1 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India2 Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India3 Department of Surgical Oncology, All India Institute of Medical sciences, New Delhi, India

Background: The field of immunotherapy is rapidly expanding with the recent successful early clinical studies targeting the PD1–PDL1 axis in lung cancer. Over-expression of these molecules detectable by immunohistochemical methods is considered to be a potential biomarker for treatment response.Objective: We aimed to analyze PD-L1 expression in NSCLC and its correlation with clinicopathologic and molecular biomarkers.Methods: Formalin-fixed paraffin embedded resected tumor samples from 50 patients of NSCLC were analyzed. PD-L1 expression was evaluated by immunohistochemistry (IHC) using a mouse monoclonal

C. DNA Damage Repair and Apoptosis European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S15

anti-PD-L1 antibody (Rabbit Anti-Human PD-L1/CD274 Monoclonal Antibody, Clone SP142). PD-L1 positivity on tumor cell membrane was defined as $5% of tumor cell membrane staining. Correlation with EGFR mutation and rearrangement of ALK, ROS 1 and FGFR gene was performed in addition to clinical and histopathological parameters.Results: Histopathologic analysis showed cases of adenocarcinoma (25), squamous cell carcinoma (23) and adenosquamous carcinoma (2). There were 32 males and 18 females, aged 42 to 78 years with mean age 65.5 years. Ten (20%) cases were positive for PD-L1. Expression was more in adenocarcinoma (8/10) than squamous histology PD-L1 expression was higher in smokers and with more lymph nodal involvement. There was no significant difference in recurrence rates and disease free survival in PDL1 positive versus negative tumors however the follow-up is short and numbers are less. PD-L1 expression was not correlated with any of the molecular markers.Conclusion: Our preliminary study showed higher rate of PD-L1 expression in adenocarcinoma than squamous cell carcinoma. Due to small number of cases relationship with survival was not statistically significant although a trend towards widespread disease was seen in PD-L1 positive tumors. Additional samples are being studied for a better understanding of the PD1-PD-L1 mechanisms in NSCLCs and their subtypes for therapeutic purposes.

P66-C004Tumor suppressor FBXO31 inhibits apoptosis by inactivating PARP1

S. Choppara1, M.K. Santra1

1 National Centre for Cell Science, University of Pune, Pune, Maharashtra, India

Tumor suppressor FBXO31, a SCF (SKP1-Cul1-F-box) ubiquitin ligase, functions as dedicated checkpoint protein in genotoxic stresses through proteasomal degradation of cyclin D1 and MDM2 in DNA damages. To better understand role of FBXO31 as tumor suppressor we performed mass spectrometry to identify its cellular substrates. We identify poly (ADP-ribose) polymerase 1 (PARP1) as one of the targets of FBXO31. Ectopic expression of FBXO31 degrades PARP1 through proteasome pathway. All the cleaved fragments of PARP1 by the activity of caspases and cathepsins i.e. 89 kDa and 24 kDa, 55 kDa and 42 kDa are associated with apoptosis and/or necrosis. We also check the role of FBXO31 on these cleaved fragments of PARP1 along with WT-PARP1 and observe that ectopic expression of FBXO31 decrease the protein level of all cleaved fragments of PARP1. Further, knockdown of FBXO31 leads to stabilization of full length as well as fragments of PARP1 suggesting that FBXO31 is regulating PARP at the physiological level. Co-immunoprecipitation data reveals that FBXO31 interacts with all the cleaved forms of PARP1. FBXO31 knockdown cells were found to be lethal upon exposure to genotoxic stress and our present data elucidate the molecular mechanism of lethality of the FBXO31 knockdown cells. So, FBXO31 plays important role in preventing the induction of cell death as a first choice and thereby allowing the cells repair their genome upon genotoxic stress. Our study reveals a novel role of tumor suppressor FBXO31 in the degradation of PARP1 to function as anti-apoptotic agent.

P79-C005Molecular basis of DISC assembly and procaspase-8 activation in a novel adaptor-independent cell death pathway

K. Bose1

1 Integrated Biophysics and Structural Biology (IBSB) Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai, India

Early protein E2 of human papilloma viruses (HPV), that are associated with cervical and anogenital cancers, regulates viral DNA replication and transactivation of essential viral oncogenes. Apart from these functions, E2 protein from high risk virus types such as HPV-16 and -18 triggers apoptosis in the host cell. Although the exact mechanism is unclear, recent literature suggests that in HPV-18 E2, the N-terminal transactivation domain directly interacts with procaspase-8, a component of Death Inducing Signaling Complex (DISC) in the extrinsic cell death pathway. This interaction bypasses the requirement of upstream adaptor proteins which are essentially required for DISC formation, thereby representing a novel adaptor-independent caspase activation pathway. However, the molecular events governing DISC assembly and the unique non-death-fold mediated E2-procaspase-8 interaction have largely remained elusive. In this work, we dissected the binding interface of E2-procaspase-8 and classical FADD-procaspase-8

complex using an interdisciplinary approach employing techniques such as in silico, mutational, biochemical, biophysical and ex vivo analyses. Based on our observations, we propose a new model for DISC formation which refines the current understanding of caspase-8 activation mechanism. Furthermore, our results provide a molecular basis of the novel E2-procaspase-8 interaction and help establish a model for E2-induced apoptosis in high risk HPV types. This critical information might provide lead toward designing E2 analogs so as to modulate procaspase-8 activation especially in cases where upstream classical extrinsic pathway might be non-functional, and hence promote apoptosis.

P83-C006Autophagy triggered by BA caused G2/M cell cycle arrest leading to non-apoptotic cell death in PANC-1 cells

F. Malik1, A. Singh1

1 Indian Institute of Integrative Medicine, Canal Road, Jammu and Kashmir, India

Autophagy in cancer cells behave either as cell survival mechanism or an alternative form of cell death (type-II). In the present study, we tried to explore the role of autophagy in the regulation of cell cycle in pancreatic cancer cell line PANC-1.We used mTOR kinase (IC50~1 µM) targeting natural product analogue of boswellic acid (BA) as an inducer of autophagy in PANC-1 cells. BA inhibited mTOR kinase pathways triggering robust autophagy in PANC-1 cells leading to G2/M arrest and cell death independent of apoptosis as was analysed through several pro-apoptotic parameters like single or double stranded DNA nicks by Apo BrdU assay, DNA fragmentation by DNA ladder pattern and cleavage of apoptotic proteins caspase-3 and PARP.BA triggered autophagy in Panc-1 cells instigated G2/M cell cycle arrest associated with decreased expression of cyclin A, cyclin B, cyclin E, cdc2, cdc25c and CDK-1. Inhibition of mTOR signalling by BA was followed by concomitant activation of Akt (pAkt) and its membrane translocation. Inhibition of Akt through various pharmacological inhibitors and siRNAs enhanced BA induced autophagy leading to G2/M arrest associated with further down regulation of cyclin A, cyclin B, cdc2, cdc25c and CDK-1 and Taking in consideration the possible roles of feedback loop of IGF-IR/PI3K and Mek/Erk pathways in the activation of Akt. Our results demonstrated that BA co-treatment with Erk inhibitors PD98059 and U0126 did not show any effect on BA induced Akt activation thus disregarding the role of this pathway. Intervention in IGFR-1/PI3K axis, by using various pharmacological inhibitors against PI3K, Akt and IGF-IR and siRNAs augmented BA mediated autophagic flux, G2/M arrest and death in PANC-1 cells. These results demonstrated that BA triggered autophagy caused G2/M cell cycle arrest and autophagic cells death in PANC-1 cells.

P125-C007Promising magnetic nanoparticles functionalized with diosgenin inducing apoptosis against breast cancer

S. Ghosh1, A. Derle1, R. Kitture2, S. Kale2, B.A. Chopade3

1 Institute of Bioinformatics and Biotechnology, University of Pune, Pune, India2 Department of Applied Physics, Defense Institute of Advanced Technology, Pune, India3 Department of Microbiology, University of Pune, Pune, India

A potent bioactive saponin, diosgenin from Dioscorea bulbifera was functionalized onto magnetic iron oxide nanoparticles (IONPs) core via citric acid linker molecule was synthesized. FTIR analysis of IONPs-D exhibited signature peaks of diosgenin at 2914, 1166 and 1444 cm-1, confirming the efficient functionalization of diosgenin. IONPs-D exhibited 51.08 6 0.37% antiproliferative activity against MCF7, which was found to be superior to free citric acid (17.71 6 0.58%) and diosgenin (33.31 6 0.37%). Least cell migration upto 40.83 6 2.91% on IONPs-D treatment supported its ability to inhibit motility of MCF7 cells as compared to bare IONPs (89.03 6 2.58%) and IONPs-CA (50.35 6 0.48%). IONPs-D and D induced apoptosis was exhibited by Alexa Fluor 488 annexin V/PI double-stained cells indicating extensive cell membrane damage coupled with PI influx leading to nuclear staining in treated cells. IONPs-D mediated selective superior apoptotic induction was indicated by PARP cleavage. In view of this background, IONPs-D can be considered as first diosgenin functionalized novel magnetic nanomaterial with antiproliferative potential against MCF-7 with inhibition of its migration and induction of apoptosis against breast cancer.

S16 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 C. DNA Damage Repair and Apoptosis

P136-C008Mechanistic insights into dynamics and substrate specificity of pro-apoptotic serine protease HtrA2

L.K. Chaganti1, K. Bose1

1 Integrated Biophysics & Structural Biology Lab, ACTREC, Tata Memorial Centre, Mumbai, India

Background: HtrA2 is a trimeric pyramidal serine protease involved in mitochondrial homeostasis and induction of apoptosis. HtrA2 expression is altered in ovarian, gastric, prostate and breast cancers etc. Thus, makes it as a potential drug target in cancer therapy. HtrA2 comprises a short N-terminal region, a serine protease domain and a C-terminal PDZ regulatory as well as protein-protein interaction domain. Although the crystal structure of the inactive protease was available in the literature, there had been ambiguity in its molecular mechanism and dynamics of action. Understanding this complex mechanism of action is important in delineating its role in various biological pathways.Methods: Using structure-guided design, molecular simulation and functional enzymology, we have characterized various combinations of HtrA2 domains and mutants. Further to understand the proapoptotic property of HtrA2, we characterized the interaction of HtrA2 with one of its known anti-apoptotic binding partner cum substrate Pea15 using in silico tools, in vitro binding experiments and enzymology studies.Results: Our findings highlight importance of N-terminal region, oligomerization, and intricate intermolecular PDZ-protease interaction in proper active-site formation. Our interaction studies with Pea15 provide first mechanistic insight into the substrate recognition and specificity of HtrA2. Binding studies with Pea15 define a bipartite mode of interaction between HtrA2 and Pea15. Enzymology studies suggest that allosteric activation of HtrA2 might be required by adaptor proteins in the cell to relieve the inhibitory effect of PDZ domain. Substrate specificity studies reveal that HtrA2 has broad substrate specificity toward the residues near the cleavage-site.Conclusions: Our advancement in understanding toward HtrA2 mechanism of action will help in developing tailored allosteric effectors that enhances the proteolytic activity of HtrA2 towards its substrates. This could be a promising tool in cancer treatment in combination with other chemotherapeutic agents such as cisplatin and paclitaxel.

P138-C009Designing allosteric activators of proapoptotic HtrA2 and their implication in cancer therapeutics

K. Raghupathi1, K. Bose1

1 Integrated Biophysics and Structural Biology Lab, ACTREC, Mumbai, India

Background: HtrA2, a proapoptotic serine protease, is known to be altered in many cancers such as chronic lymphocytic leukemia (CLL), renal, ovarian, breast and prostate cancers. It has basal proteolytic activity that is enhanced several folds during apoptosis through a complex allosteric mechanism. Therefore, allosteric activators might play a crucial role in HtrA2-mediated apoptosis. Developing a strategy that increases HtrA2 proteolytic activity toward its substrates could hence be a promising tool in cancer treatment in combination with conventional chemotherapeutic agents. It has been reported that interaction with GRIM-19, a tumor-suppressor, significantly increases HtrA2 activity resulting in an elevated rate of apoptosis. Therefore, in this study, we aim at delineating the driving force of HtrA2-GRIM-19 interaction as well as the mechanism of allosteric activation of the protease so as to design peptidic or small molecule analogs of GRIM-19. This might be important toward devising strategies for regulating HtrA2 for therapeutic intervention.Methods: A multidisciplinary approach has been envisioned for this study, which includes in silico tools (modelling, molecular dynamic simulation, docking), protein engineering, biophysical and enzymology studies.Results: GRIM-19 cDNA cloned in bacterial expression system and purified using standard chromatographic techniques. Molecular modelling of GRIM-19 using I-Tasser followed by docking with HtrA2 was done to identify putative critical interacting residues. Our enzymology studies not only pioneered in identifying GRIM-19 as an HtrA2 substrate but also confirmed it to be an activator of HtrA2. Currently, with leads from in silico studies, characterization of several deletion constructs of both the proteins is underway to identify the minimal binding regions.Conclusions and future perspectives: These studies, along with high resolution structural data will pave way towards peptidomimetics/small molecule design to modulate HtrA2 activity with desired characteristics.

P139-C010Characterization of interaction between serine protease HtrA2 and its novel binding partner: an apoptotic modulator

S. Acharya1, K. Bose1


Background: Apoptosis, an essential biological phenomenon required for tissue development and homeostasis is deregulated in various pathological conditions including cancer. Research in the past decade has identified several regulators involved in this complex phenomenon. Despite the plethora of information available on underlying mechanisms, an intense effort is required to uncover new regulators. In this light, the mammalian oligomeric serine proteases, HtrAs (High temperature requirement protease A) have recently taken centre stage as apoptotic modulators and for their association with a wide variety of diseases such as arthritis, neurodegeneration and cancer. HtrA2, the most extensively studied member of this family is differentially expressed in many cancers and has been found to be consistently down-regulated in lymphomas, ovarian and prostrate cancers. Albeit structural-functional insights gained into pro-apoptotic HtrA2, understanding its mechanism of activation has been limited by its few known substrates or binding partners. Therefore, identifying them as well as characterizing their interactions will further help understand its biological role.Methods: With this aim, using extensive in silico tools, a putative binding motif and stringent filtering parameters, a number of binding partners were narrowed down from the human proteome. These putative binding partners were further validated using biochemical studies.Results: The interaction between HtrA2 and its putative binding partner has been validated by our biochemical studies while characterization of the binding interface as well as ex-vivo studies to deduce its biological relevance are currently underway.Conclusions: Since protein-protein interactions play an important role in modulating the apoptotic function of HtrA2, the identification and characterization of its novel interacting partner might aid conventional cancer therapeutics. Interestingly, literature reports that both HtrA2 and this binding partner are involved in the p38 stress kinase pathway and also regulate apoptosis wherein we speculate them to crosstalk.

P165-C011Molecular mechanism of Fanconi anaemia complementation group I (FANCI) ARM repeat protein to understand DNA intercrosslink repair

M.Q. Siddiqui1, A.K. Varma1

1 ACTREC, Tata Memorial Centre, Mumbai, India

Proteins in Fanconi anemia pathway are well known for its DNA intercrosslink repair mechanisms. Fanconi anemia pathway comprises 15 complementation groups of proteins that act in a common pathway to repair the DNA damage. Fanconi Anaemia Complementation Group I (FANCI) is one of them, composed of DNA binding domain and Armadillo repeat region. Armadillo repeats are structurally conserved and has been found in different proteins with diverse function. To understand the behaviour, stability and dynamics, we have performed in-vitro and in-silico approach. It has been observed that ARM repeat is well folded and majorly composed of a-helices. Limited proteolysis and mass spectrometry based approach shows that N-terminal part of ARM repeat is more stable than the C-terminus. Interestingly, C-terminal region found to be structurally conserved. Looking at the folding pattern at C-terminus and protein stability at N-terminus, we have performed molecular dynamics. It has been observed that C-terminus of ARM repeat has more dynamicity compare to N-terminus. Our study concluded that stable region and dynamic region in ARM repeat might act as molecular recognition module in protein-protein interactions during intercrosslink DNA repair.

P166-C012Multidisciplinary approach to understand BARD1-CstF50 complex interface and evaluate functional implication of BARD1 Gln 564 His mutation

R.K. Choudhary1, A.K. Varma1


BRCA1-BARD1 E3 ubiquitin ligase and CstF50 ternary complex play an important role in transcriptional coupled DNA damage repair. CstF50 has been implicated in direct interaction with BARD1 (425–777) and RNA polymerase II CTD through its C-ter and N-ter region respectively.

D. Novel Therapeutics European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S17

However, mutation indentified in BARD1 linker region at Gln 564 His position impairs the complex formation between BARD1 and CstF50, which further leads to formation of premature transcript, uncontrolled mRNA regulation and reduced apoptosis. To investigate the effect of BARD1 Gln 564 His mutation, on the structure and function, we have purified BARD1BRCT (425–777) wild-type and the mutant proteins and further proceeded for FPLC, chemical crosslinking assay, DLS, mass spectrometry, CD and fluorescence spectroscopy analysis to unravel the changes in molecular behavior and domain integrity of proteins. To our conclusion, BARD1 BRCT (425–777) wild-type and mutants have well folded structural conformation. However, Molecular Dynamics simulation studies with wild-type and mutant protein predicted the highly dynamic nature of proteins. However, mutant proteins show higher dynamic behavior than wild-type protein. The complex between BARD1-CstF50 rigidifies the dynamic nature of the wild-type protein. Comparative docking study of wild-type and mutants with CstF50 suggests that mutation Gln 564 His abrogates the complex formation. This study will be helpful in understanding the role of BARD1 and CstF50 in cancer predisposition.

P173-C013Influence of DNA repair gene polymorphisms in acute myeloid leukemia

S. Vuree1, C. Anuradha1, D.N. Rao4, G. Manjula1, E.M. Prajitha1, A. Sandhya1, D. Raghunadharao3, S.Sinha2, S. Vishnupriya1

1 Department of Genetics, Osmania University, Hyderabad, Telangana, India2 MNJ Institute of Oncology, Red Hills, Hyderabad, Telangana, India3 HomiBhabha Cancer Hospital and Research Center, Visakhapatnam, Andhra Pradesh, India4 School of Chemical & Biotechnology, Sastra University, Thanjavur, Tamil Nadu, India

Purpose of the study: Deregulated DNA repair is one of the hallmarks of cancers including Acute Myeloid Leukemia (AML), as it results in genomic instability. ATM gene functions as a sensor, activates cascade of events leading to stimulation of multiple DNA damage- responsive signaling pathways. Principal DNA repair mechanism activated in the hematopoietic stem cells is the Non Homologous End Joining (NHEJ) pathway. However, this pathway was shown to be error prone. Functional SNPs in the genes involved in DNA repair might influence the gene expression leading to altered DNA repair which might confer the risk to AML.Methods: This hospital-based case-control study included 225 AML patients and 326 cancer-free controls from South Indian population. Six polymorphisms of XRCC5, XRCC6, XRCC7 and ATM were genotyped using polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Statistical analyses were performed by using SPSS (version 20v) and SNPSTAT online tool. Protein-Protein Interaction (PPI) analysis was also done to see the relationship between these genes.Results: We found that there was an elevated risk of AML associated with the XRCC5 VNTR 0R repeat and A allele of 2408G . A polymorphism (p-0.04 and p , 0.0001 respectively), the frequencies of G allele (p-,0.0001) of XRCC6 -1310C . G and T allele (p-0.003) of ATM -5144A . T polymorphisms were also significantly increased in AML cases. Further, analyses of the variant genotypes with epidemiological and clinical variables revealed a significant association of the risk genotypes with development and progression of AML.Conclusion: The XRCC5 0R repeat, 2408G . A, XRCC6 -1310 C . G and ATM-5144A . Tpolymorphisms, but not XRCC6 -61C . G and XRCC7 6721G . Tpolymorphisms, play an important role in the pathogenesis of AML.

P204-C014Mutant p53 alters regulation of survivin by p63

R. Mishra1, D. Mundhe1, R. Waghole1, T. Teni1

1 Teni Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Navi, Mumbai, India

Background: Tumor suppressor protein p53 is inactivated in half of the oral cancer cases. Previous studies from the lab demonstrate overexpression of the p53 repressed inhibitor of apoptosis protein, Survivin, in oral cancers. However current understanding implies a limited role for the survivin minor isoforms in oral cancer pathogenesis. We evaluated the expression of survivin isoforms in oral tumor tissues and its association with clinicopathological parameters. We also assessed the regulation of survivin and its

isoforms by ΔNp63 in the presence/absence of mutant p53 in oral cancer cell lines.Methods: The expression of the survivin isoforms was assessed by real time PCR. Binding of p63 to the survivin promoter was determined using ChIP assay. The role of p63 in proliferation, migration and invasive capabilities of the two oral cancer cell lines, AW8507 and SCC040 was also assessed.Results: Our studies demonstrate upregulation of all the survivin isoforms in oral tumors. Interestingly, a correlation of the antiapoptotic isoforms with higher tumor stage, with 64% stage IV tumors showing upregulation of atleast one antiapoptotic isoform, was demonstrated. We also showed the differential regulation of survivin wherein in the presence of the R273H mutant p53, p63 activates survivin expression whereas in the absence of functional p53, p63 represses the expression of survivin gene. The possible differential binding of p63-mutant p53 complex to the survivin promoter was also demonstrated.Conclusions: Our studies thus suggest that the minor isoforms of survivin play a significant role in oral tumorigenesis, possibly through interactions with the antiapoptotic wild type survivin isoform. Our studies indicate that R273H mutant p53 alters the regulation of survivin expression by p63 possibly through interaction with the latter. This study is the first to determine a gain of function role for the R273H p53 mutant in the regulation of survivin by p63.

P210-C015Genomic instability and DNA repair in BRCA1 associated breast cancers

S. Abbas1, P. Kowtal1, P. Gera1, R. Sarin1

1 Cancer Genetics Unit, Tata Memorial Centre-Advanced Centre for Treatment research and Education in Cancer, Navi Mumbai, India

Background: Germline BRCA1/2 mutations predispose to breast, ovarian, prostate and few other cancers. Genomic instability with Microsatellite Instability (MSI) classically seen in MMR gene associated HNPCC, has not been examined in any large cohort of BRCA1 associated breast cancers.Objectives: To investigate MSI and MMR protein expression in BRCA1 associated breast cancers and sporadic breast cancers and its correlation with DNA repair kinetics.Methods: Detailed family history, clinico-pathological features and blood was collected after consent from patients and at risk relatives at the TMH cancer genetics clinic. BRCA1/2 mutations were confirmed by Sanger sequencing. MSI and LOH was studied on DNA extracted from BRCA1 (1/2) breast tumours and matched genomic DNA from lymphocytes using the Bethesda panel of STR markers. gH2AX foci formation assay was performed for DNA repair kinetics.Results: In this ongoing study, germline deleterious mutations in BRCA1/2 genes have been identified in 168 probands and twice as many relatives so far. Of the matched tumour/blood samples analysed so far, microsatellite instability was observed in 8/24 (33%) breast tumours and LOH in 5/24 (21%) breast tumours. DNA repair kinetics was also impaired with residual gH2AX foci persisting till 48 hrs.Conclusion: This is the first report showing frequent occurrence of Microsatellite instability & LOH in tumours with deleterious germline BRCA1 mutations its correlation with DNA repair kinetics. This further confirms the role of BRCA1 in genomic instability. While genomic instability is known to be a direct effect of BRCA1, our study raises the possibility BRCA1 associated genomic instability is mediated through Mismatch Repair Pathway and could be help further improve molecular characterization of tumours and its therapy.

D. Novel Therapeutics

O-001Attenuated bacterium Listeria monocytogenes as a new delivery platform for anticancer agents

C. Gravekamp1, D. Chandra1, Z. Yuan2, S.K. Libutti2, E. Dadachova3

1 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA2 Department of Surgery, Albert Einstein College of Medicine, Bronx, New York, USA3 Department of Radiology, Albert Einstein College of Medicine, Bronx, New York, USA

Selective delivery of anticancer agents to the tumor and particularly to metastases is one of the most important problems in cancer therapies. Our

S18 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 D. Novel Therapeutics

laboratory has developed a novel delivery platform using an attenuated non-toxic and nonpathogenic bacterium. Listeria monocytogenes that selectively infects tumor cells in metastases and primary tumor. This is possible because Listeria infects myeloid-derived suppressor cells (MDSC), which are selectively attracted by the primary tumor through the production of attractants. Once at the tumor site Listeria spreads from MDSC into tumor cells through a mechanism specific for Listeria. Listeria bacteria are protected from immune clearance in both the tumor microenvironment (TME) and MDSC because of their strong immune suppression, which is absent in healthy tissues. We were the first to demonstrate that Listeria delivered radioisotopes like 188 Rhenium (188Re)(coupled to anti-Listeria antibodies) with high efficiency to the TME, resulting in a strong eradication of tumors and metastases without serious side effects in syngeneic mice with pancreatic cancer (Panc-02). In the study presented here we added radioisotope 32P as nutrition to the culture medium of Listeria, resulting in the incorporation of 32P into the Listeria (Listeria-32P). When tested in syngeneic and transgenic mouse models of pancreatic cancer (Panc-02 and KPC), we found that Listeria-32P was even more effective than Listeria-188Re and strongly reduced the growth of metastases primary tumors in both models, even at a later stage. The dramatic effect of Listeria-32P on metastases and tumors, without having serious side effects is remarkable and strongly suggest that Listeria-32P has great promise for the treatment of pancreatic cancer. This study shows again the great potential of Listeria as delivery platform for anticancer agents to the TME. New candidates have already been developed for selective delivery to the TME, i.e Listeria with adjuvants, RNA-aptamers, and highly immunogenic childhood recall antigens.

O-002Targeting the tumor microenvironment: A phase II study of copper-depletion (CD) using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence

E. Nackos1, A. Willis1, N. Kornhauser1, M. Ward1, M. Cobham1, T. Cigler1, A. Moore1, V. Fitzpatrick1, S. Schneider1, A. Wiener1, J.G. Abraham1, J.D. Warren1, A. Rubinchik1, M. Lane1, V. Mittal1, L. Vahdat1 Weill Cornell Medicine, New York, United States

Background: Bone marrow derived VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent pathways, including lysyl oxidase (LOX), are critical components to remodeling the tumor microenvironment and establishing the pre-metastatic niche. We hypothesized that TM-associated CD would reduce EPCs and other copper dependent processes in the pre-metastatic niche in BC pts.Methods: Phase II study in BC pts with node1 triple negative (TN), stage 3 and 4 with no evidence of disease (NED) received oral TM to maintain ceruloplasmin (Cp) between 5–17 mg/dl for 2 years (yrs) on the primary study (1 cycle 5 28 days). Selected pts continued on a series of extension studies in 2 yr increments. Primary endpoint: change in EPCs measured by flow cytometry before and during treatment with TM. Secondary endpoints: safety and effect of copper depletion on other markers including LOX, quantified by ELISA.Results: 75 pts received over 2344 cycles of TM on the primary and extension studies. Median age: 51 yrs (range 29–66). Stage 2/3 BC: 45 pts, Stage 4 NED: 30 pts. TNBC pts: 48%, Stage 4 NED 40%. Median Cp level decreased from 28 at baseline to 15.5 (p , 0.0001) after one cycle. Grade 3/4 toxicities: reversible neutropenia (2.3%), anemia (0.04%), leukopenia (1.2%) and fatigue (0.09%). CD was associated with a decrease in EPCs (p 5 0.0014) and serum LOX (p , 0.001). At a median follow-up of 5.9 years, the PFS for all 75 pts is 72%, including a PFS of 90% for all stage 2/3 pts with TNBC. The overall survival is 84%. Relapse after two years is a rare event.Conclusions: TM is safe, well tolerated and appears to affect multiple copper dependent biologic processes in the tumor microenvironment known to be important for tumor progression which is most striking in TNBC. Phase III trials in a high risk for relapse population are warranted.

O-003Oral metronomic maintenance therapy (MMT) prevents relapses in patients with triple negative breast cancer (TNBC)

S. Banavali1, A. Desai2, A. Kulkarni2, V. Ostwal1, N. Patil2, S. Desai1, R. Hawaldar1, S. Shastri1, S. Pimple1, S. Gupta1, R. Badwe1

1 Tata Memorial Center, Mumbai, India2 BKL Walawalkar Hospital, Chiplun, Maharashtra, India

Background: TNBC patients are more likely to achieve a pathologic complete remission (pCR) after neo-adjuvant chemotherapy compared to other breast cancer subtypes. However those who don’t, have a poor

prognosis. We did a retrospective analysis of consecutive evaluable TNBC patients treated at BKLWH, the rural outreach center of TMC, to test whether MMT given after completion of standard therapy could improve their DFS.Materials & Methods: All patients received standard therapy with CAF chemotherapy; surgery; 1/2 radiotherapy depending on the disease status, followed by either observation or MMT. MMT consisted of 12 to 15 months of oral daily metformin (500 mg BD), cyclophosphamide (50 mg OD) along with weekly methotrexate (12 mg/m2) [22 patients also initially received 6 to 12 doses of weekly IV cisplatin (25 mg/m2)].Results: There were 59 evaluable TNBC Pts: 37 received MMT and 22 did not. The mean age was 48 yrs (range 27 to 75). Majority (55.5%) had AJCC Stage III & IV disease; 57.9% were node- positive; pCR was achieved in 6/25 (24%) post 3 cycles of CAF. MMT was overall well tolerated. At median follow-up of 41.8 months (44.3 for observational group; 41.3 for MMT group), there were 4 relapses in the MMT group (AJCC 1 IIB; 2 IIIB; 1 IV) compared to 10 in observation group (AJCC 2 IIA; 1 IIB; 1 IIIA; 4 IIIB; I IIIC; 1 IV). The 5 yr DFS was 89.2% for MMT v/s 54.5% for observation group [HR 5 0.26; 95% CI 5 0.08 – 0.86; p 5 0.026]. On Cox-regression analysis after adjusting for age & stage, treatment group (MMT v/s observation) was found to be significant [HR 5 0.25; 95% CI 5 0.08 – 0.81; p 5 0.02].Conclusions: Our data shows that MMT prevents relapses and significantly improves the outcome of TNBC women. This needs to be explored further in Phase III studies. The low cost associated with MMT represents an opportunity for the utilization of this treatment in LMICs, where TNBC is seen in a higher percentage of women; in younger women & carries a particularly aggressive course and worse survival.

OT61-001KPT-330, an inhibitor of CRM-1, in combination with proteasome inhibition enhances tumor suppression through transcriptional down-regulation of survivin by inhibition of NF-kB through increased nuclear localization of ikB

J.S. Nair1, G.K. Schwartz1

1 Columbia University, New York, USA

KPT330 is a small molecule that irreversibly blocks the major nuclear export protein CRM1 (exportin 1, XPO1). The Chromosome Region Maintenance 1 (CRM1) protein is responsible for the nuclearexport of several tumor suppressor proteins and oncogenic snRNAs to cytosol. XPO1 is the only transporter for proteins such as ikB, the inhibitor of NF-kB, a transcription factor which plays an important role in carcinogenesis and apoptosis. In clinical trials KPT-330 has demonstrate defficacy in both solid tumors as well as in hematologic malignancies with evidence of clinical activity in sarcoma as a single agent. We investigated the effects of KPT-330 in sarcoma cell lines as both monotherapy as well as in combination with proteasome inhibitors. The intent was to increase apoptosis by inhibiting ikB nuclear export and increasing its nuclear fraction so as to inhibit NF-kB activity. Our data clearly demonstrates that KPT-330 in combination with the proteasome inhibitors carfilzomib and bortezomib enhanced apoptosis. Pre-treatment with the proteasome inhibit or resulted in increased intra-nuclear ikB which in turn became bound to and inhibited NFkB from its transcriptional activity. Results from chromatin immune precipitation assays showed decreased binding of NF-kB to the survivin promoter resulting in its decreased expression. Immuno-fluorescence assays showed that by virtue of KPT-330’s inhibitory effect on nuclear export, these stabilized proteins were trapped within the nucleus and were unable to enter the cytosol where their anti-apoptotic activity normally occurs. The growth inhibitory effects of KPT-330 were further confirmed in the malignant peripheral nerve sheath (MPNST) and dedifferentiated liposarcoma (LS141) xenograft models as both monotherapy as well as in combination therapy with proteasome inhibition. This mechanism based treatment strategy seems to be promising for the treatment of this rare but dangerous cancer and clinical trials with this combination are planned.

OF281-010A novel approach to the management of metastatic solid tumors – role of high dose radiation in inducing an immune response

A.K. Anand1, V. Goel1, A. Gulia1

1 Department of Radiation Oncology, Max Cancer Centre, Max Hospital, New Delhi, India

Cancer progression is mainly driven by the expansion of tumor cells, but tumor microenvironment and anti-tumor immunity may also play a role. In addition to the direct effects of radiation, the ensuing immune response


promotes the expression of inflammatory and immunostimulatory mediators, which act on neighboring, non-irradiated, cells called “Bystander” effects. Preclinical studies have suggested that high dose RT, can stimulate anti-tumor T cell immunity by promoting the cross-priming of antigen-specific dendritic cells (DCs). SBRT is now being explored within the broad concept of immunomodulation. We wish to report case histories of 3 patients where SBRT yielded encouraging results.Case 1 – A 50 years old man with non seminomatous germ cell tumor of the testis suffered 3 recurrences in left paraaortic nodes, following multiple lines of chemotherapy and 2 surgeries. SBRT (45 Gy/6 frs) was delivered in March’ 2010 and he is disease free since then (68 months).Case 2 – A 39 years old man with carcinoma lip, and subsequently carcinoma left buccal mucosa, post 2 surgeries and radiation (60 Gy/ 33 frs), recurred in March’ 2015 at two sites. One in suprasternal region (4 3 3 cm mass) and other in posterior margin of flap. SBRT was delivered (30 Gy/5 frs) and is currently disease free at 9 months post treatment.Case 3 – A 55 years old man with carcinoma hard palate (post surgery and radiation) suffered local recurrence 9 months later at two sites. SBRT was delivered (30 Gy/6 frs) to both the sites with complete response in CT – PET. He also received palliative chemotherapy subsequently. At present he is disease free at 15 months post SBRT.Conclusion: It seems possible that Bystander and or Abscopal effects of RT might have contributed to improved outcome which compares favorably with the expected outcome in locally recurrent or relapsed metastatic disease. However more clinical data and studies are needed to validate this hypothesis.

OF1-010Impact of human papilloma virus status on the response of head and neck carcinomas cell lines to novel therapeutic agents

A. Mahajan1,2, B. Ayaz3, N. Raulf1, J. Bullenkamp4, Y. Suh1, S. Thavaraj3, M. Tavassoli1

1 Department of Molecular Oncology, King’s College London, London, UK2 Department of Radiodiagnosis, Tata Memorial Hospital, Parel, Mumbai, India3 Department of Oral Pathology, King’s College London, London, UK4 Centre for Cell Death, Cancer and Inflammation (CCCI), UCL Cancer Institute, London, UK

Background: When compared with site-matched HPV negative tumors, HPV-positive carcinomas have a distinct prognostic profile with better therapeutic response and survival rates.Objectives: Determine the molecular mechanisms that lead to differential therapeutic potential of novel therapeutic agents (NTAs) in HPV-positive and negative HNSCC cell lines:• Evaluation of cell viability in response to agents that induce the

intrinsic (SMAC) and extrinsic (TRAIL) pathways of apoptosis.• Asses the efficacy of combination targeted therapy and possible inter-

action in these cell lines.• Determine the relative protein expression of potential key mediators

to explain the variation in their efficacy.Material and Methods:Cell lines• HPV-Negative: HSC3, H357 HN30, UPCI-SCC089, UPCI-SCC072• HPV-Positive cell lines: UPCI: SCC090, UPCI-SCC152, UPCI-SCC154,

UD-SCC2, VU-147TThe anticancer activity of Iz-TRAIL, EGFR-TRAIL, SMAC Mimetic and Cetuximab was confirmed all cell lines.Results: Having confirmed the HPV status and the pathological characteristics, we assessed the growth rates of these cell lines which revealed that HPV-negative cell lines were faster growing than the HPV-positive cell lines. One of the most interesting finding which is under investigation and mostly unpublished in literature was that cetuximab pre-treatment led to blocking of the activity of the EGFR-TRAIL in all cell lines, confirming the response is mediated through EGFR receptor. Interestingly, most of the HPV-negative cell lines showed sensitivity to these NTAs and most of the HPV-positive cell lines showed resistance which was confirmed on baseline protein expression of these cell lines.Conclusion: Our in-vitro preclinical study demonstrated that all the NTAs assessed in our study induced rapid apoptosis in HNSCC cell lines, especially in the ones not harboring HPV oncogenes. HPV-positive tumors have favorable prognosis than HPV-negative tumors and it does not seem to be associated with intrinsic sensitivity of the tumoral cells to NTAs tested in-vitro in our study.

P19-D001Smart nanomaterials with microtubule binding drug targets against liver cancer

R. Poojari1, R. Srivastava1, D. Panda1

1 Department of Biosciences and Bioengineering, Indian Institute of Technology, Mumbai, India

Hepatic malignancies like liver cancer are a major global cause of mortality, third most Cancer-related deaths. Liver cancer is quite unresponsive to common chemotherapeutic agents well known for inefficiency of drug penetration, non-selectivity to solid tumors and multidrug resistance. Deregulation in the proliferative and apoptotic pathways and chromosome instability promotes liver cancer. Microtubules (MT) represent one of the most dynamic cytoskeletal structures comprising of a,b tubulinheterodimers. Proper assembly of mitotic spindle apparatus requires regulation of microtubule dynamics as well as assembly of these microtubules into complex structures. Another crucial aspect in cell division are spindled effects, chromosome loss and tumorigenesis. Microtubule binding drugs (MBD) that affect polymerization of microtubules constitutes an important class of anticancer chemotherapeutics, which has opened a new scenario for liver cancer treatment. MBDs suffer from many drawbacks such as poor water solubility, poor pharmacokinetics, short half life and adverse drug effects like systemic neurotoxicity which limits its therapeutic window.Hence, inadequate drug efficacy is an impediment in patient care treatment. Recent trends in engineering versatile polymeric nano-formulations for biomedical applications and cancer therapy is in great demand. We are working on incorporation of MBD targets into polymer based nano particles drug delivery systems (MTPNDDS) and its mechanistic role leveraged to potentiate the therapeutic effects against liver cancer with less toxicity. Here, we report the detailed biophysicochemical characterization along with systematic screening of optimally formulated MBD encapsulated targeted nanoparticles as a highly selective and effective therapeutic modality. Detailed intracellular analysis (tubulin and mitosis) of this delivery system in liver cancer cells may provide the basis for improved designing and promoting of these smart nano materials as liver cancer therapeutics.

P25-D002Combination therapy of natural products with conventional anticancer drug(s)

S. Pawar1, S. Roy2, S. Mukherjee2, A. Chawdhary2, D. Dasgupta3

1 Department of Biochemistry, The Institute of Science, Mumbai, Maharashtra, India2 Department of Virology, Haffkine Institute of Training, Research and Testing, Parel, Mumbai, Maharashtra, India3 School of Biotechnology and Bioinformatics, D. Y. Patil University, Belapur, Navi Mumbai, Maharashtra, India

Background: Use of botanicals alone and in combination with conventional anticancer agents has increased remarkably in cancer patients. However, lack of scientific evidence has limited the use of these cocktails. Thus it is necessary to evaluate the effect of these combinations alone and with anticancer drugs. This study investigates the chemotherapeutic potential of extracts of H. mystax and N. nimmoniana and their combinations with camptothecin and cisplatin against MCF – 7 cell line.Methods: The ethanol (95%) and aqueous plant extracts (by soxhletion) were quantified for the presence of phenolic acids by RP – HPLC method. In vitro cytotoxicity analysis of individual extracts and in combination was performed by MTT assay. Selective toxicity of plant extracts were checked using normal cell line. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate the interactions between extract(s) and drug. Dose reduction index (DRI) was calculated. Synergistic combinations induced cancer cell death was studied by Flow cytometry.Results: Ethanol and aqueous extracts have shown presence of caffeic acid, catechol, vanillin, gallic acid and p – coumaric acid. The ethanol extracts of both the plants have shown potent activity against MCF – 7 cells. These extracts were also studied in combination with anticancer agents. Five combinations were prepared using constant drug ratio of their median effect dose. Two combinations showed synergistic growth inhibitory effect at all dose levels (IC50 – IC90) after 24 hr exposure on breast cancer cell line. The dose reduction level was different for each combination.Conclusions: Herbal mixtures alone or in combination with anticancer agent(s) produced a synergistic effect in inhibiting breast cancer cells. Therefore, herbals in combination with anticancer drugs may help


Conclusions: This procedure to deliver the anti-glioma drugs may be a successfully strategy to cross BBB in vivo for the benefit of patient.

P40-D005Inhibition of ribosomal protein S6 potentiates prostate cancer radiation therapy

A.P. Kumar1,2,5,6,7, S.S. Hussain1,2, N. Papanikolaou3, D.C. Chan4, R. Ghosh1,2,5,6

1 Department of Urology, The University of Texas Health Science Center at San Antonio, South Texas Veterans, USA2 Department of Pharmacology, The University of Texas Health Science Center at San Antonio; South Texas Veterans, USA3 Department of Radiation Oncology, The University of Texas Health Science Center at San Antonio; South Texas Veterans, USA4 University of Colorado Denver, Broadway, Denver, USA5 Molecular Medicine, The University of Texas Health Science Center at San Antonio; South Texas Veterans, USA6 Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio; South Texas Veterans, USA7 Health Care System, San Antonio, TX, USA Division of Medical Oncology, USA

Radiation Therapy (RT) is an effective treatment modality for many patients with localized, early stage prostate cancer (PCA). However, disease relapse in high and intermediate risk patients and numerous side-effects that affect quality of life (QOL) of patients receiving RT are critical barriers for RT. We examined the potential of Nexrutine (Nx; an over the counter supplement) to potentiate RT-induced biological effects using in vitro cell culture and preclinical animal models. Further we also evaluated the safety and toxicity of Nx in patients receiving conventional prostate cancer treatment. Isobologram analyses show that Nx pretreatment potentiates the effect of low dose RT in multiple prostate cancer cells in a synergistic manner via modulation of mTORC1/NFkB signaling as evidenced by RNAi experiments. Moreover, Nx in combination with RT increased accumulation of cells in G2/M phase, which was accompanied by Wee1 mediated phosphorylation of cdc2 coupled with inhibition of Chk1 and induction of apoptosis. Nx administered for 6 weeks prior to RT inhibited progression of prostate lesions to poorly differentiated carcinoma in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Remarkably, p-mTOR, p70S6K, NFkB, Ki67 and Cyclin D1 decreased in the prostate tissue of mice receiving the combination compared to RT alone. Interestingly, tumors from patients undergoing RT enrolled in phase 0/1 clinical trial of Nx showed decreased rpS6 staining intensity compared to adjacent normal tissue. Collectively, our data suggests that Nx has a radiosensitizing effect in a range of PCA cell lines, can prevent progression to advanced PCA and is safe for use in humans. This agent has great potential for human clinical use.

P41-D006Modulation of microRNAs to overcome tumour hypoxia and improve radiotherapy response in head and neck cancers

M. Tavassoli1, Y. Suh1, N. Raulf1, J. Gäken1, T. Guerrero1, K. Lawler1, E. Odell1

1 Head and Neck Oncology Group, Guy’s Hospital, King’s College London, UK

Head and neck cancer is the 5th most common cancer worldwide with over 60% of cancers presenting at an advanced stage. Over 90% of these cancers are of squamous cell carcinoma type (HNSCC).Locally advanced HNSCC represents tumours with multiple acquired genetic aberrations that have allowed the tumour to proliferate, grow and locally metastasise. They have an aggressive phenotype and are characterised by complex molecular changes that lead to treatment resistance. Despite the progress in the improvement of current treatments and developing novel targeted therapies, there have only been modest advances in the outcome for this group of patients over the last 30 years. Therefore there is an urgent clinical need to understand the mechanisms of therapy resistance and identify those at risk of treatment failure.Current treatment modalities are surgery and radiotherapy or radiotherapy alone and in advanced cases in combination with chemotherapy. These therapeutic options are associated with considerable toxic side effects and cause crucial organ function impairments emphasising an urgent need for targeted, more specific treatments. Molecular profiling of large tumour cohorts has revealed a number of cancer specific aberrations that provide opportunities for the development of lead drugs to optimise treatment to the specific

to increase therapeutic index and minimize dose related toxicity of anticancer treatments. However, further in vivo studies are needed to check its efficacy and safety.

P32-D003The role of endocannabinoid system and therapeutic potential of cannabinoids in cancer

S. Nag1

1 Rajiv Gandhi Cancer Institute & Research Centre, New Delhi, India

Background: For many millennia, the plant genus Cannabis has been used medically and recreationally in different parts of the world. The renewed interest on pharmacological importance of cannabinoids (biologically active components in cannabis plant) in a plethora of human disease has led to the discovery of an endogenous molecular signalling system called endocannabinoid system. The endocannabinoid system comprises of cannabinoid receptors such as CB1 and CB2, the endogenous ligands and the enzymes responsible for their synthesis and degradation. Experimental data suggest that endocannabinoid system is intricately involved in several aspects of cancer pathophysiology and exerts their antitumor effects by a number of mechanisms, including induction of apoptosis, direct inhibition of transformed-cell growth, inhibition of tumor angiogenesis and metastasis. Although cannabinoids are used in treatment of chemotherapy induced nausea-vomiting and cancer related pain for last 2 decades, recent researches open a promising possibility as anti-cancer agents. The present review will focus on the alteration of endocannabinoid system in different cancer types and the potential therapeutic role of cannabinoids as anti-cancer agent in respect to growth, metastasis, energy metabolism and immune environment.Methods: Electronic bibliographic databases like PubMed and Google Scholar was searched using various combinations of terms “cannabis”, “cannabinoids”, “endocannabinoids system” and “cancer”. Articles identified were screened for their relevance to the field of cancer.Results: Data presented in this review suggest that cannabinoids derived from different sources exert a direct anti-proliferative effect on tumors of different origin and regulate different signalling pathways & host physiological system.Conclusion: Although medical use of cannabinoids is limited by their psychoactive effects, its unique pharmacology and favourable drug safety profile has the potential to open a new horizon in cancer therapeutics challenging the current and existing dogmas in cancer care.

P39-D004Targeted delivery of molecule across the blood brain barrier for glioma therapy

M.K. Ghosh1

1 CSIR-Indian Institute of Chemical Biology, Kolkata, India

Introduction: Treatment of Glioma is a therapeutic challenge due to the existence of blood brain barrier (BBB). More effective therapeutic options for glioma treatment are urgently needed. Recently, we designed a novel exosome-mediated delivery of the intrinsic PTENdomain, PTEN-CT into different cancer cells and observed reduced proliferation, migration, and colony forming ability. The delivery of exosome containing PTEN-CT to tumor region in mice model was found to result in significant regression in tumor size with the tumor sections showing increased apoptosis (Molecular Therapy - 2015). In this study, we prepared ananoparticle encapsulated indole derivative (3,3’diindolylmethane or DIM), tagged with apeptide against Somatostatin Receptor 2 (SSTR2) on the surface of the nanoparticles for effective glioma therapy.Objectives: The major objective is to find out the strategy for effective delivery of therapeutic agents to glioma.Methodology: DIM was encapsulated by poly-lactic-co-glycolic acid (PLGA). The encapsulation was confirmed by TEM, FTIR, DLS. The effect of nano particle encapsulated DIM was examined on C6 rat glioma cell line by MTT assay, FACS, fluroscence microscopy, immunoblotting etc. To achive the goal of target based delivery, a peptide against SSTR2 (overexpressed in glioma) was designed, synthesised and validated its target specificity in silico (by docking and simulation study) and in vitro (by fluroscence microscopy, IP and FACS). After tagging it chemically with the surface of DIM containing nanoparticles, passaging it through BBB was determined by both in vitro BBB model and in vivo rat intracranial glioma model.Results: Evaluation of the transportation of ‘peptide coated nanop-encapsulated DIM’ through the tight junction (BBB) showed prominent passaging of the compound that induces apoptosis of glioma cells, evidenced by microscopic imaging, Immunohistochemical analysis, tunnel assay etc.


and Ingenuity Pathway Analysis have shown the altered integrin signaling pathway and MAPK pathway in Pancreatic Carcinoma Cell lines. Further, the activation of NRF2 transcriptional factor in BxPC-3 treated cells shows that it may bind to the DNA at the location of the Antioxidant Response Element (ARE) or also called hARE (Human Antioxidant Response Element) which is the master regulator of the total antioxidant system. It seems likely that upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, and superoxide dismutase. The pathway analysis has demonstrated that at least seven signal transduction cascades were induced by ECM interaction with integrin heterodimers, which may trigger aberrant signaling and lead to pancreatic cancer adenocarcinoma. The data have clearly shown the activation of ILK-PT3K-ILKAP-AKT and Caveolin-GRB2-SOS-cRas-Raf-MEK cascade. These results may have some promise in therapeutic intervention in the treatment of pancreatic cancer adenocarcinoma.

P106-D009Evaluation of new inhibitors of heme and calcium binding regions of BKCa channels as potential anti-cancer drugs

J. Kumar2, P. Tiwari1, S. Samantha1, P. Chowdari1, A. Biswas1, U. Das1, A. Roy1, H.G. Nagendra2, D. Khaitan1, N. Ningaraj2

1 Department of Molecular Oncology Research, Scintilla Bio-MARC-Pvt. Ltd., Bangalore, India2 Department of Biotechnology, Sir M Visvesvaraya Institute of Technology, Bangalore, India

Background: KCNMA1 codes large conductance calcium and voltage activated potassium (BKCa) channels, which are over-expressed in several cancers. It is shown to function as an oncogene in several cancers. BKCa channels are amplified, alternatively spliced with increased protein function due to augmented sensitivity to intracellular Ca2+. Due to these modifications, cancer cells show greater migration, invasion and proliferation functions. Therefore, BKCa channels is a putative target for anticancer therapies. Known inhibitors of BKCa channels, Iberiotoxin and Charybdotoxin are toxic to humans, hence new and safer inhibitors should be developed.Material and Methods: We employed in silico and in vitro biological studies to screen potential BKCa channel inhibitors. First, we focused on in-depth structural analysis of a-subunit of BKCa channel containing the Heme binding region [-CKACH-] in the N-terminus of the RCK1-RCK2 loop. Based on detailed in silico virtual screening of the modulators/structural analogues, naturally-occurring BKCa channel inhibitors for Heme binding regions were critically evaluated. In addition, we targeted four pairs of Ca2+ bowls present in the RCK1 and RCK2 domains for identifying small molecule inhibitors. Docking analysis was carried out across these sites via FlexX software, and the results were validated by Discovery Studio.3.5.Results: Our studies indicate that several ligands bind strongly across conserved Heme-binding sequence motif [CKACH-(677–681)]. Likewise, small molecule inhibitors were analyzed for the Calcium binding site-Asp 892 and Gln 907, which exhibited potential binding capabilities. The results suggest that these molecules are capable of altering the BKCa channel activity in cancer cells. In vitro studies are conducted using the 3-D model of metastatic breast cancer cell line (MDA-MB 361), where BKCa channels are abundantly expressed, to validate our in silico findings.Conclusion: Rigorous research is required to advance their development as anticancer drugs in an adjuvant set up to control cancer growth in a clinical setting.

P110-D010Non-coding RNAs as antiviral agent in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC)

M.K. Sannigrahi1, V. Singh1, R. Sharma2, B.D. Radotra3, V. Rattan4, N.K. Panda1, M. Khullar2

1 Department of Otolaryngology, Post Graduate Institute of Medical Education and Research, Chandigarh, India2 Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India3 Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India4 Unit of Oral Health Sciences, Post Graduate Institute of Medical Education and Research, Chandigarh, India

genetic makeup of a patient’s tumour. However, so far effective targeted therapies for HNC are unavailable. In order to improve HNC survival we need strategies which would enable us to tailor the available therapies to individual patient’s need and de-intensify current treatments to reduce side-effects.Recent tissue expression profiling in our lab has led to the identification of several genes and miRNAs associated with the more aggressive tumour phenotypes and radiotherapy resistance tumours (Raulf et al., 2014 Suh et al., 2013 & 2015). In addition, our studies using genetic analysis by RNA-Seq combined with imaging studies has identified a gene and miRNA expression signature correlated to both the volume of the hypoxic region of the tumour and patient survival. Extensive functional analysis of some of the discovered microRNAs has identified their role in regulating genes involved in tumour hypoxia, angiogenesis and DNA Damage Response. In addition we have developed a non-invasive test based on circulating miRNAs in the serum of patients to predict response to therapy. Identification of specific targets of the candidate miRNAs responsible for inducing chemo-radiotherapy resistance will be used to develop radio-chemotherapy sensitising drugs.

P52-D007Targeting inflammation induced carcinogenesis by dietary administration of 2-deoxy-D-glucose (2-DG)

S. Pandey1,2, S. Singh1,2, V. Anang1,2, S. Seth1, A.N. Bhatt1, K. Manda1, K. Natarajan2, B.S. Dwarakanath1,3

1 Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi, India2 Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India3 Sri Ramachandra University, Porur, Chennai, India

Inflammation is a well-known promoter of carcinogenesis. Inflammatory events are also shown to induce genetic instability and transformation (by inducing DNA damage) and hence act as an initiator of carcinogenesis. Inflammation is generally associated with a metabolic switch to glycolysis, which influences inflammatory signaling and its outcomes. Inflammatory mediators like activated infiltrated macrophages, and chronically secreted pro-inflammatory cytokines can promote the pro-tumorigenic microenvironment. We have hypothesized that early neoplastic transformation and progression of carcinogenesis can be controlled by targeting metabolic changes associated with inflammation, using glycolytic inhibitors like 2-deoxy-D-glucose (2-DG), a well-known Energy restriction mimetic agent (ERMA) as a dietary component. Chronic administration of dietary 2-DG (0.2%) in drinking water reduced the incidence and number of tumor per mouse and tumor load in chronic inflammation (Radiation and chemical induced) induced carcinogenesis models. When administered chronically 2-DG reduced radiation induced papilloma (~50%) and leukemia (~75%) and prolonged the survival in mice. 2-DG reduced the inflammation induced cancer-promoting processes like tumor cell migration and angio-tropism. Dietary 2-DG reduced the inflammation induced activation of splenic macrophages, their migration and infiltration into the inflamed micro-environment suggesting that targeting glycolytic metabolism of macrophages can lead to the resolution of inflammation. In-vitro experiment on RAW 264.7 macrophage cell line showed that 2-DG reverses the inflammation induced glycolytic shift of metabolism to OXPHOS and concomitant decrease in the macrophage activation. Theses finding suggests that chronic dietary administration of 2-DG can help in there solution of inflammation and thus it can be used a potential cancer preventive strategy.

P57-D008The Nrf2 mediated antioxidant defense against oxidative stress in BxPC-3 cell lines and targets for therapeutic intervention

H.D. Shukla1, P. Vaitiekunas2

1 Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD, USA2 Department of Biology, Johns Hopkins University, Baltimore, MD, USA

Integrins are cell surface glycoproteins that are involved in cell-cell and cell-extracellular matrix (ECM) interactions. These interactions are the basis for a number of diverse effects that include cell migration and anchorage, cell growth and invasion. In the present investigation the proteomic analysis of oxidatively stressed BxPC3 human pancreatic cancer cells have shown the elevated level of both a6b4 integrin, caveolin-1, K-RAS, EGFR, annexin a4 and annexin a11 and as compared to HPDE control. We have also identified the presence of a number of gene products involved in integrin signaling pathway and MAPK pathway. The bioinformatic analysis by Protein Center


Moringa root water extract has been earlier shown to have attenuating effect on whole ovary and other female reproductive organs. It contains isothyocyanate which was found to inhibit the proliferation of ovarian cancer (OC) cells and induced apoptosis in OC cells. Apoptosis was induced by a strong activation of caspase-3 and -9, and cleavage of PARP-1. Compounds could be immensely useful in prevention/cure of epithelial ovarian cancer. Hence, a cell culture experiment was done to see effect of Moringa root water extract in ovarian cancer cell line OAW 42.Moringa roots were dried and extract s were obtained by traditional methods of water (sterile) extract. End products were lyophilized and stored in room temperature for future use. Cytotoxicity was studied using tetrazolium dye MTT by incubating for three hours after removing media from OAW 42 culture.Western Blot analysis was done on lysed cells seed in a 6 well culture plate for one day before treatment and then after 2 days incubation with IC 50 concentration of extract. Cytoxicity assay showed IC-50 value of Moringa water extract to be 1.5 mg/ml. Western Blot analysis showed not much effect on cleaved caspase – 8 but in case of caspase – 9 when it is addaed as an adjuvant to cisplatin and paclitaxel it showed visible effect.Our results corroborate with previous finding of isothiocyanate on ovarian cancer culture in terms of caspase activity. However it is not clear how/if antigonadotrophin action will also play here. We will futher investigate the effect of Moringa extract on other cellular pathways via cell cycle and in other cell line. CNS activity of Moringa extract through nerve growth (NGF) mediated follicle stimulating growth factor (FSHR) pathway inhibition will also be seen in vivo.

P126-D013Knockdown of Pinch-1 protein sensitizes the estrogen positive breast cancer cells to chemotherapy induced apoptosis

A. Bhatia1

1 Post Graduate Institute of Medical Education and Research, Chandigarh, India

Background: PINCH-1 is a ubiquitously expressed protein belonging to the focal adhesion protein group which has a role in cell survival, spreading, adhesion and migration. It has been implicated in pathogenesis of several cancers. In the present study we aimed to investigate the role of this protein in estrogen positive and negative breast cancer subtypes.Materials and Methods: PINCH-1 expression was studied in two estrogen positive (T47D and MCF-7) and one estrogen negative cell lines before and after treatment with six drugs (Cyclophosphamide 2 μg/ml, Celecoxib 50 μM, Doxorubicin 2 nM, Paclitaxel 50 nM, Etoposide 10 μM and Tamoxifen 10 nM) by immunocytochemistry. The protein was then knocked down using siRNA against PINCH-1 and change in percentage of apoptotic cells was analysed by flow cytometry.Results: We observed increased but differential expression of PINCH-1 in the three breast cancer cell lines with a higher expression in estrogen positive cell lines. Knocking down of PINCH-1 led to a significant (p-value , 0.05) enhancement in apoptosis in T47D cells in response to 4/6 (cyclophosphamide, celecoxib, paclitaxel, doxorubicin) drugs. Though an increase in apoptosis was observed in MCF-7 cells also, the increase was not found to be significant. The MDA-MB-231 cells, however, did not show significant apoptosis upon PINCH-1 knockdown.Conclusion: The results suggest that PINCH-1 may be playing an important role in etiopathogenesis of both subtypes breast cancer. An enhanced rate of apoptosis in response to chemotherapy observed in estrogen positive cells upon PINCH-1 knockdown suggests that the later can serve as an adjuvant to chemotherapy in the above malignancy. On the contrary, no increase in apoptosis on PINCH-1 knockdown in estrogen negative cells points towards the presence of some other protein with redundant functions in the later subtype which needs to be explored.

P134-D014Use of R191, potent and selective IRAK1/4 kinase inhibitor, in treatment of hematologic malignancies

V. Markovtsov1, C. Lamagna1, M. Chan1, S. Yi1, C. Young1, R. Forances1, S. Siu1, S. Braselmann1, H. Li1, R. Singh1, G. Park1, E. Masuda1, V. Taylor1, D.G. Payan1

1 Rigel Pharmaceuticals, San Francisco, USA

MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways play a key role in multiple hema-tologic malignancies. A third of activated B-cell–like diffuse large B-cell lymph oma (ABC DLBCL) and nearly 100% of Waldenstrom macroglo-bulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with

Background: HPV is an emerging risk factor for HNSCC, however, molecular mechanisms of pathophysiology in HPV mediated HNSCC remains poorly delineated. Recent studies suggest that non-coding RNAs may be playing an important role in the aetiology and pathogenesis of HNSCC. In the present study, we examined miRNAs (microRNAs) involved in HPV-16 induced HNSCC.Material and Method: In-silico approach was used to identify host miRNAs with HPV-16 mRNAs as putative target. Tissue samples from HNSCC patients (n 5 300) were screened for HPV16 using PCR based approach with consensus PCR primers (MY09/MY11, Gp5+/Gp6+), HPV-16-specific PCR, HPV-16 E7 mRNA, p16 immunohistochemistry and viral load determination. Differential expression of identified miRNAs was determined by qRT-PCR in HPV-16 positive (UPCI:SCC090, CaSki, SiHa), HPV negative (UPCI:SCC116, HaCat) and transfected with HPV-16 full genome plasmid cell lines, and, HPV 16-positive/ HPV-negative tissue samples. Promoter methylation and chromatin immune-precipitation was studied as epigenetic mechanisms regulating miRNA expression. Putative targets of identified miRNAs were identified by bioinformatic approach and their mRNA levels were determined by qRT-PCR in HPV positive and negative cell lines, and tissue samples. Over-expression and inhibition studies with miRNA mimic and inhibitor were done to identify affected viral and host oncogenic pathways.Results: Total 32 HNSCC patients were found to be HPV-16 positive. Hsa-miR-139-3p showed significant down-regulation in HPV-16 positive samples and cell lines compared to HPV negative controls. This might be due to promoter methylation. Using bioinformatics tools, hsa- miR-139-3p was found to target viral HPV16 E1 region and host HOXB6 gene. Transfection with hsa-miR-139-3p mimic resulted in significant down-regulation of HPV 16 early mRNA as well as its targeted genes in luciferase 3’-UTR assay. Moreover, mimic transfected HPV-positive cell lines showed dysregulated MEK/ERK signaling pathway.Conclusion: Our results suggest important role of miRNA hsa-miR- 139-3p in regulating virus-host interaction in HPV-16 positive HNSCC.

P112-D011Translational success of endogenous iodine pump protein (NIS) based targeted therapy in breast cancer may rely on its maneuverability by transcriptional modulations

M. Kelkar1, M. Rathod1, A. De1

1 Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Mumbai, India

Background: Human Sodium Iodide Symporter (NIS) mediated radioiodide accumulation is the basis of successful targeted radioiodine therapy in thyroid cancers. Recently, several groups including ours have reported aberrant NIS protein expression in breast cancer (BC) signifying its potential for targeted radioiodine therapy. For successful repositioning of this gene targeted therapy in BC, achievement of stronger NIS expression while protecting normal thyroid is regarded as a promising strategy. Here we present a comprehensive study reporting novel gene transcriptional regulation targets to improve NIS-based therapy in BC.Methods: To gain an insight into the NIS-regulatory mechanisms, we performed genome wide in silico analysis of the promoter sequence. NIS promoter-luciferase reporter sensor was designed to screen the various genetic and epigenetic regulators. Their effect on endogenous NIS expression was verified by real-time PCR, immunofluorescence and western blotting. Functional significance was validated by radioiodine uptake assay, in vivo bioluminescence and Cerenkov imaging in animal model.Results: In silico analysis reveals the presence of numerous transcription factor binding sites including p53 and CREB on human NIS promoter. We establish that p53 activation represses NIS promoter activity in BC cells and tumor xenograft model (p , 0.05), while abrogation of p53 binding sites results in significant increase in promoter activity (p , 0.05). In contrast, CREB activation results in significant upregulation of endogenous NIS mRNA and protein content in BC cells (p , 0.05). Direct binding of p53 and CREB to NIS promoter is also confirmed by chromatin immunoprecipitation. Further, functional relevance signifies substantial alterations (p , 0.05) following activation of p53 and CREB, establishing the importance of transcriptional modulation strategy.Conclusion: This study for the first time reveals p53 as a repressor and CREB as an inducer of functional NIS expression in BC, indicating necessity of p53 profiling or pretreatment with CREB activator drugs to enhance NIS therapeutic efficacy in breast cancer patients.

P116-D012Anti cancer effect of Moringa (Moringa oleifera Lam.) root extract in OAW 42 (ovarian cancer) cell line

C.K. Bose1

1 Netaji Subhas Chandra Bose Cancer Research Institute, Kolkata, India


P202-D017Effect of aspirin on induction of autophagy through calcium signalling in cervical cancer cells

S. Chowdhury1, A. Evered1

1 Cardiff School of Health Sciences, Cardiff Metropolitan University, Cardiff CF5 2YB, UK

Background and Aims: Aspirin is a widely used analgesic and antipyretic drug. In addition to its anti-inflammatory action, clinical and epidemiological studies concluded that regular use of aspirin has the potential to reduce the risk of certain cancers. However, there are limited reports about cervical cancer. Even though the chemopreventive effect of aspirin has been established, the molecular mechanisms underlying the anti-tumour activity remain elusive. An interesting emergent mechanism of tumour growth and a prospective target in cancer therapy is autophagy. It is well known that intracellular Ca2+ is one of the key regulators of autophagy. An elegant study (Din et al., 2012) demonstrated the inhibition of mTOR signalling and activation of AMPK by aspirin in colorectal cancer cells. Autophagy, a response characteristic of mTOR inhibition, was also induced by aspirin. The same approach was extended to cervical cancer HeLa cells. Thus, the current study explores the induction of autophagy through Ca2+ signalling by aspirin in cervical cancer cells.Methods: The effect of aspirin on HeLa cell viability was evaluated by the CellTiter-Blue® assay. Intracellular Ca2+ levels were quantified using fluo-4 AM as the fluorescent indicator. Autophagy induction was examined by LC3 antibody-based assay.Results: The CellTiter-Blue® assay showed a dose-and time-dependent decrease in cellular proliferation. The non-toxic concentrations of 1, 2 and 5 mM were selected for subsequent experiments. Aspirin induced a significant decrease in intracellular calcium levels after 72 h of treatment with 5 mM aspirin. While exposure to 5 mM aspirin for 24 h caused no significant effect on the activation of autophagy.Conclusions: This study demonstrated the anti-proliferative activity of aspirin on cervical cancer HeLa cells. It was suggestive of exploring autophagy signalling pathways, apart from calcium signaling, such as the LKB1/AMPK pathway and the Ras/Raf/ERK pathway that have been implicated in cervical cancer.

P221-D018Mucinous carcinoma of breast – a wolf in sheep clothing

M. Gurav1, T. Shet1

1 Department of Molecular Pathology and Breast DMG, Tata Memorial Hospital, Parel, Mumbai, India

Background: We have previously shown for the first time in literature that mucinous carcinoma is essentially mucinous variants of papillary carcinoma (solid papillary and micropapillary) with extracellular mucin. We have proved that mucinous variants relapse as non-mucinous variants and also impacts patient survival. Mucin forms a protective layer around the otherwise angioinvasive infiltrating micropapillary carcinoma (IMPC) reducing its aggressiveness. Our original study was based purely on morphologic observations which were extended further for immunohistochemical panel of MUC1 to MUC6.Material and Methods: Fifty cases each of micropapillary group (infiltrating micropapillary carcinoma/IMPC and its mucinous variant-MUMPC) and solid variant of papillary group (SVPC and mucinous variant SVPCMU) were analyzed. One sample of MUMPC and IMPC are being analyzed by next generation sequencing (NGS) to evaluate the differences at the genetic level.Results: Non-mucinous variant of SVPC and IMPC did show mucin. A repertoire of MUC protein was seen in mucinous micropapillary carcinomas which were MUC2 and MUC6 followed by MUC3, MUC4 and MUC5AC. Gel forming mucin MUC2 and MUC6 were common in mucinous variants of SVPC while uncommon in IMPC mucinous variants (MUMPC, MU, MPC and IMPC). Co-expression of MUC2 and MUC6 was associated with best prognosis in MUMPC and compared to expression of MUC2 alone (Log rank pair wise – 0.032). MUC5 significantly impacted both the DFS and OAS in MUMPC. NGS results will be discussed during the meeting.Conclusion: Mucinous carcinoma is not a unique entity but includes variants of papillary carcinoma with propensity to secrete mucin. While the solid variant of papillary carcinoma with mucin is a better behaving tumor, the behavior of mucinous micropapillary carcinomas is proportionate to amount of mucin. These mucinous and non-mucinous tumor share MUC proteins and a comparison at genetic level will help us understand and treat patients better.

deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we iden-tified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50 nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1b-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies.

P137-D015A tale of two apoptotic players: HtrA2 and HAX1

R.R. Kuppili1, K. Bose1


Background: HtrA2, a proapoptotic serine protease plays pivotal role in maintaining cellular homeostasis by mediating apoptosis via multiple pathways. It has been implicated in ovarian, breast and prostatic cancers thus making it a potential therapeutic target. It executes its physiological roles by alterations in its interaction network and by virtue of its protease activity. HAX1 on the other hand is a ubiquitously expressing anti-apoptotic protein upregulated in several epithelial cancers. Despite suggestions of their involvement in a novel alternate apoptotic pathway, the modus operandi of HtrA2-HAX-1 interaction and subsequent event in the mitochondria are yet to be elucidated. This interaction might prove to be a cornerstone in the intrinsic pathway of apoptosis and thus in cancer phenotype.Methods: This study was aimed at addressing these questions using a two pronged approach, firstly to deduce HtrA2-HAX-1 interaction, and secondly decipher the allosteric mechanism in HtrA2. The intricacy of the interaction was dissected using in silico leads and in vitro pull down assays. Protein modeling and molecular dynamics simulation were carried to predict the putative binding pocket, which together with enzymology and other biochemical studies helped us identify and demonstrate its role in allosteric regulation of HtrA2 activity.Result and conclusions: Our interaction studies furnished details of the domains, and critical residues involved in HtrA2-HAX1 interaction. Binding at the unique non-classical allosteric pocket unfolds a novel mechanism of HtrA2-mediated apoptosis. We also demonstrate for the first time that HAX1 is an activator of HtrA2, which could prove to be a critical step for driving early stages of apoptosis. Overall, these studies helped us elucidate the mechanism of intrinsic pathway of apoptosis in context of these two players. These significant findings were harnessed to derive interface peptides that can act as modulators and can be utilized for designing small molecule inhibitors in therapeutic strategies against cancer.

P185-D016Effect of methyl jasmonate on A-549 lung cancer cell line and docking with targeted protiens

S. Kansara1

1 Sardar Patel University, Gujarat, India

Recent evidences indicate that methyl jasmonate (MJ) is plant stress hormone exhibit anticancer activity in human cancer. The aim of this study is to determine anticancer activity against lung cancer cell line A-549. In which we increase the concentration of MJ then, gradually decrese the cell viability of A-549 cell. The concentration required to kill 50% of A-549 lung cancer cell (IC50) is 4.937 mM. Similarly, derived IC90 value is 7.822 mM, is needed to kill 90% cancerous cell. In silico docking resulted into docking with two proteins GLUT1 and hexokinase 2 (HK2). Docking is done by using CLC main work bench, autodock tool, and marvin view. GLUT1 is membrane transport protein for glucose channel and MJ inhibit glucose transformation. MJ also bind to HK2 and detach interaction with VDAC lead to release cytochrome-c, resulted into apoptosis of cancer cell.

S24 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 E. Cancer Stem Cells and EMT

transcript-levels ,10% or . 20%, gain of WHSC1, YWHAE and ABL1 gene was observed. Transcriptionally, these genes were upregulated and also presented various isoforms. Using genotyping array, we assessed associations between individual SNP and TKI-CML-resistance risk and identified and validated rsIDs, rsID239798 and rsID9475077 in linkage with FAM83B which may act as potential candidate for therapeutic-resistance to TKIs.Conclusions: Hence, omics-based signature with BCR-ABL transcript levels can help to predict early diagnosis and disease progression for subsequent superior/inferior clinical-outcomes.

E. Cancer Stem Cells and EMT

OF175-011Epithelial-to-mesenchymal transition in cerebral metastases of cancers: genes, proteins and targets of therapy

D. Jeevan1, J. Cooper1, A. Kwasnicki1, A. Braun1, R. Murali1, M.J. Uniyal1

1 Department of Neurosurgery, New York Medical College, New York, USA

Introduction: Cancer metastasis to brain is a leading cause of morbidity and mortality among patients with lung, breast and other cancers. The metastatic process evolves through an orderly sequence of genetic/epigenetic events enabling tumor cells to migrate from the primary tumor site and colonize at secondary locations. Brain Metastasis may involve a multi-step cascade which allows reprogramming of primary tumor cells to acquire a stem cell-like phenotype via epithelial-to-mesenchymal transition (EMT), which is not fully elucidated. Since, the mechanistic target of rapamycin (mTOR), which forms 2-multiprotien complexes (mTORC1 and mTORC2), is known to play an essential role in the regulation of tumor growth and dissemination. In this study we sought to explore the role of mTOR in coordinating the process of brain metastases via EMT.Methods and Results: Immunohistochemical staining of metastatic brain tumor samples displayed expression of epithelial markers (SNAIL and Twist-1), mesenchymal marker vimentin, and stem cell marker CD44 along with mTOR markers. In addition, immunofluorescence analysis revealed co-expression of the epithelial marker E-cadherin and the mesenchymal marker vimentin, suggesting a state of transition. Expression analysis of transcription factor genes in metastatic brain tumor samples demonstrated that genes associated with neurogenesis, differentiation, and reprogramming were altered. Immunoflourescence analysis showed that E-cadherin underwent forced nuclear localization following Rapamycin or siRNA treatments resulting in acute mTOR inhibition. Functional analysis demonstrated that primary tumor and brain tumor cells exist in a cordial manner, and that tumor cells migrated with high affinity toward an astrocytic media, which was inhibited by mTORC1/2 inhibitors. Similarly, cell cycle analysis showed enhanced S-phase entry in astrocytic media, which was suppressed by mTOR inhibitors.Conclusions: These findings provide molecular evidence of EMT/MET in brain metastases, and underscore the therapeutic value of mTOR inhibition in the treatment of patients with brain metastases.

OF50-003Pro-oncogenic role of NOTCH1 in early tongue squamous cell carcinoma

P. Upadhyay1, S. Nair2, E. Kaur3, J. Aich1, P. Dani1, V. Sethunath1, N. Gardi1, P. Chandrani1, M. Godbole1, K. Sonawane2, S. Kannan4, B. Agarwal5, S. Kane6, S. Dutt3, A. Dutt1

1 Integrated Genomics Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, India2 Division of Head and Neck Oncology, Department of Surgical Oncology, Tata Memorial Hospital, Tata Memorial Centre, Navi Mumbai, India3 Shilpee Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai, India4 Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai, India5 Department of Pathology, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial hospital, Tata Memorial Centre, Navi Mumbai, India6 Department of Pathology, Tata Memorial hospital, Tata Memorial Centre, Mumbai, India

Background: NOTCH1 has been identified as an oncogene in breast cancer and other cancers. Interestingly, inactivating mutations in

P227-D019Efficacy of second line erlotinib in patients post progression of first line chemotherapy in head and neck cancers

V. Patil1, A. Joshi1, A. Karpe1, V. Noronha1, V. Muddu1, A. Bhattacharjee2, S. Dhumal1, K. Prabhash1

1 Department of Medical Oncology Tata Memorial Hospital, Mumbai, India2 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India

Background: Oral TKI (gefitinib and erlotinib) have been used in palliative treatment of head and neck cancers with limited success. In this report we aim to quantify the symptomatic benefit, progression free survival and overall survival when erlotinib is given as second line treatment in Head and neck cancers.Methods: This was a post hoc retrospective analysis of a randomized study comparing metronomic chemotherapy with cisplatin. Patient who progressed on chemotherapy and had a PS0-2 were offered second line chemotherapy. Patients who had received erlotinib (150 mg PO OD) as second line treatment were selected for this analysis. Erlotinib was discontinued in case of either progression of disease or if the patient had intolerable side effects. Patients were monitored 1 week after start of erlotinib and subsequently at monthly intervals. The toxicity was recorded in accordance with CTCAE version 4.02 and the response was graded in accordance with RECIST version 1.1. All of these patients were followed up till death.Results: Twenty three patients were identified. The median age of these patients at the start of second line was 47 years (IQR 40.5 to 51.75 years). The primary site of distribution was oral cavity primary in 17 patients (77.3%) and non oral cavity primary in 05 (22.7%) patients. The immediate last chemotherapy regimen received was cisplatin in 9 patients (40.9%) and metronomic chemotherapy in 13 patients (59.1%). Symptomatic benefit post second line erlotinib was seen in 18 patients (81.8%). The most common adverse events (any grade) seen were anemia in 20 patients (90.9%), rash in 10 patients (45.5%) and diarrhea in 7 patients (31.8%). The best radiological response documented were PR in 04 patients (19.2%). The median estimated PFS and OS were 110 days (95% CI 61–175 days) and 156 days (95% CI 126–185 days) respectively.Conclusion: Erlotinib single agent has promising activity in second line and needs to be explored in future studies.

P270-D020Combination of multiomics approach and BCR-ABL based molecular-testing: A better way to monitor treatment response, resistance and disease-progression in chronic myeloid leukemia

N. Singh1, A.K. Tripathi2, D.K. Sahu3, A. Mishra4, R. Chowdhry5, R. Kant6

1 Center for Advanced Research (Advanced Molecular Science Research Center), King George’s Medical University, Lucknow, India2 Department of Clinical Hematology, King George’s Medical University, Lucknow, India3 Imperial Life Sciences, Phase City, Gurgaon, Haryana, India4 Department of Biochemistry, King George’s Medical University, Lucknow, India5 Department of Periodontics, King George’s Medical University, Lucknow, India6 Department of Surgical Oncology, King George’s Medical University, Lucknow, Uttar Pradesh, India

Background: In tyrosine kinase inhibitors (TKI)-treated Chronic Myeloid Leukemia (CML) cases categorization and transition of disease to advanced phase is not so easy. Hence, molecular-profiling through Molecular Inversion Probe (MIP) based-, transcriptomics- and Axiom Biobank genotyping-arrays accompanied with BCR-ABL-levels may give a better approach to understand the disease-progression. Hence, we molecularly profiled TKI-treated-CML-samples in different-phases based on , or .10% copies of BCR-ABL, undetected and control-samples.Methods: We screened TKI-treated-CML-samples in different-phases based on , or .10% copies of BCR-ABL, undetected and control-samples for evaluating the frequency of copy-number variations (CNVs), generating transcriptomics-profile and identifing SNPs which show linkage with neighbouring genes with direct relevance to treatment-susceptibility or - resistance in Imatinib-treated unrelated CML cases versus controls.Results: Transcriptionally, three-clusters were identified which showed correlation with BCR-ABL transcript-levels. Based on CNV profiling, we identified gains of three genes which correlated with BCR-ABL transcript-levels i.e. in BCR-ABL undetected/new cases, absence of these genes were observed; at 10–20% levels, gain of WHSC1 and YWHAE was observed; in high CNVs group (mostly disease in advanced phase) with BCR-ABL

E. Cancer Stem Cells and EMT European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S25

Our previous study firstly demonstrated that DSGOST treatment controls the contraction of endothelial cellsvia RhoA inactivation and FAK dephosphorylation. Moreover, our group has indicated the anti-cancer effect of DSGOST by the result that DSGOST inhibits pancreatic tumor growth by suppressing VEGF-dependent tumor angiogenesis (Choi et al, unpublished results). Furthermore, some constituents of DSGOST have been widely researched about its anti-cancer effect. Taken together, DSGOST may be effective for cancer treatment. However, DSGOST effect on cancer treatment is yet clearly defined. In present study, we evaluated the inhibitory effect of DSGOST on TNF-a-dependent EMT in colorectal cancer cell line, HCT116. Non-cytotoxic dose of DSGOST inhibited TNF-a-induced migratory and invasive phenotype of HCT116. Moreover, DSGOST reversed TNF-a-mediated EMT. Furthermore, DSGOST suppressed TNF-a-induced translocation of Snail into nucleus. Therefore, our present data demonstrate that DSGOST is beneficial for preventing metastasis of colorectal cancer.

P46-E002Impact of IGF1R/Akt signaling on cancer stem cell heterogeneity and tumorigenicity

R.K. Singh1, P. Ray1

1 Ray Lab, ACTREC, Tata Memorial Centre, Navi Mumbai, India

Background: Generation of chemoresistance and tumor relapse are the major therapeutic barriers for successful treatment of ovarian cancer. According to current notion, a small population of drug resistant cells (Cancer Stem Cells/CSC) residing in the tumor bulk aids in tumor relapse. To identify the driver signaling cascade in CSCs for tumor relapse, we developed in vitro drug resistant models and categorized them into early (ER) and late resistant (LR) stages. Using these models we have earlier shown existence of an oscillatory pattern in Insulin Like Growth Factor1 Receptor (IGF-1R) signalling at early and late resistant cells. In the present study we intended to investigate the role of IGF-1R signalling in controlling the tumorigenic ability of CSCs.Methods: Side population and spheroid formation assays were performed to isolate putative CSC population from early and late resistant cells. FACS analysis and western blot were done to validate CSC biomarkers (CD44 and CD133) and qPCR to measure stemness gene expression. Noninvasive bioluminescence imaging was performed to monitor the tumor growth kinetics of CSCs in NOD/SCID mice.Results: An enrichment of CSC phenotype (Biomarker-CD44 & CD133, self-renewal, and Side Population/SP) was observed with gradual increase in resistance towards cisplatin, paclitaxel and dual drug. Expression of Oct4, Sox2 and Nanog increased from sensitive to early stage of resistance, which further remained constant at late resistant stages. In-vivo bioluminescence imaging revealed that CSCs isolated from early resistant stages (high IGF-1R) possessed higher tumorigenic potential than CSCs isolated from the late resistant stages (high pAKT).Conclusion: Our finding suggests that presence of an active IGF-1R signalling imparts heterogeneity and differential tumorigenic property to cancer stem cells and could emerge as an important therapeutic candidate for targeting CSC population.

P95-E003Differential expression pattern of pancreatic cancer stem cells expressing epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET)

N.K. Chopra1, K. Jain1, R. Mirza1, K. Choudhury1, S. Wajid2, S. Choudhury1

1 Department Of Research, Sir Ganga Ram hospital, New Delhi, India2 Jamia Hamdard University, New Delhi, India

The molecular mechanism of metastasis in pancreatic cancer is far from being clarified. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) effectors play critical roles during embryonic development, postnatal growth and epithelial homeostasis, but also are involved in cancer progression and metastasis.Using human pancreatic (hPan) cell-line model, our study showed the distinctive differential expression pattern between un-induced cell-lines (MiaPaCa2 and PanC1) and their spheroid (growth factor-induced). In brief, activation of EMT was associated with spheroids (MiaPaCa2 and PanC-1) expressing CD44+/CD24-. On the other hand, PanC-1 spheroid and cell line expressing CD44+/CD24+ showed comparable Cancer Stem Cell (CSC) property.The cell lines and their spheroids were further evaluated for their EMT-MET mRNA expression pattern. PanC-1 spheroids expressed distinctively MET whereas MiaPaCa2 spheroids were more predisposed towards EMT.Oct4 and Nanog, the stem cells transcription factors were highly expressed by the hPan spheroids. Sox2, known to regulate the pluripotency in

NOTCH1 have been identified in head and neck cancer has been known to be associated with poor prognosis in head and neck cancer.Patients and methods: Sixty-eight early staged primary TSCC tumor samples were analyzed by whole-exome (n 5 22); whole transcriptome sequencing (n 5 10); real time PCR for copy number (n 5 41) along with transcript expression by (n 5 44); and, immuno histochemical analysis (n 5 50). HNSCC cells harboring alteration at NOTCH1 were characterized for cell survival, proliferation, migration assay.Results: We observed that NOTCH1 is somatically amplified and over expressed in 31% and 37% of early staged TSCC patients, respectively. Activation of Notch pathway positively correlates with node positive and non-smoking status of patients. Pearson correlation analysis revealed NOTCH1 and DLL4 high copy number and over expression was significantly positively correlated (r-0.51; P 5 0.018, r-0.674; P 5 0.0004, respectively). Immunohistochemical analysis of activated Notch1 (n 5 50) revealed upregulation. Clinical correlation analysis showed statistically significant correlation with nodal status for activated NOTCH1 (x² 5 7.1, P 5 0.029) and expression profile of Notch pathway target genes HES5 (x² 5 5.7, P 5 0.057) and HEY2 (x² 5 9.8, P 5 0.007). HNSCC cell lines found to be dependent for Notch pathway based on knockdown, inhibition using Notch pathway inhibitor. Furthermore, treatment of XXI and shRNA mediated knockdown leads to decrease in soft agar colony formation, cell migration capacity of HNSCC cells.Conclusion: Taken together above observations suggests Notch signaling pathway gene to be implicated in early stage tongue cancer development.

OF168-008Secretory phosphopholipase A2-IIA: Emerging roles in stem cells regulation and cellular differentiation

S.K. Waghmare1

1 Stem Cell Biology Group, Waghmare Lab, ACTREC, Tata Memorial Centre, Mumbai, India

Secretory phospholipase A2 Group-IIA (sPLA2-IIA) catalyzes the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. sPLA2-IIA is deregulated in various cancers; however, its role in hair follicle stem cell regulation and tissue homeostasis is unknown. Here we show a transgenic mice overexpressing sPLA2-IIA (K14-sPLA2-IIA) showed enlargement of epidermal compartments such as sebaceous gland, infundibulum and junctional zone with a loss of ortho-arakeratotic tail skin organization. Further, increased proliferation and higher differentiation led to a gradual loss of hair follicle stem cell pool with age. BrdU pulse-chase label retaining cell (LRC) study showed decrease in slow-cycling stem cell population that demonstrated loss of stem cells quiescence. The colony forming efficiency of transgenic keratinocytes was significantly reduced. Moreover, K14-PLA2-IIA keratinocytes showed enhanced activation of EGFR and JNK1/2 that led to c-Jun activation, which co-related with enhanced differentiation. Our results for the first time uncovered that overexpression of sPLA2-IIA lead to depletion of hair follicle stem cells and abnormal epidermal components associated with increased differentiation. Thus sPLA2-IIA is not only deregulated in various cancers but also regulates stem cells that suggest its prospective implications in cancer pathogenesis.

P14-E001Herbal mixture, Danggui-Sayuk-Ga-Osuyu-Senggang-Tang, inhibits tumor necrosis factor-alpha-induced epithelial to mesenchymal transition of colorectal cancer cell HCT116

K. Lee1, Y.K. Choi1, M.S. Kim1, C.Y. Jun2, S.G. Ko1

1 Department of Preventive Medicine, College of Korean Medicine, Kyung Hee University, Seoul, Korea2 Department of Korean Internal Medicine, College of Korean Medicine, Gachon University, Seongnam, Korea

Inflammatory microenvironment has an important role in colorectal tumor development and metastasis. Particularly, tumor necrosis factor a (TNF-a) progresses proliferation and metastasis of colorectal cancer cells and its increased expression is often negatively correlated with survival rate in colorectal cancer patients. Moreover, TNF-a-mediated metastasis of colorectal cancer cells is accompanied with induction of epithelial-to-mesenchymal transition (EMT), which process is prerequisite for metastasis in CRC progression. Traditional Chinese medicine (TCM) has been clinically used for cancer patients with its cancer preventive and therapeutic effect. One of TCM theory-based formula is Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST) treatment for several vascular disorder patients such as chilly hands or feet, vasculitis, but not cancer.

S26 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 E. Cancer Stem Cells and EMT

adherence junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor free medium. The results of histomorphometry, immunohistochemistry and electron microscopy of the three types of cultures showed that the stratified growth, cell proliferation and differentiation was comparable between co-cultures and their respective native tissues, however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins viz, desmoplakin, desmoglein, and plakoglobin was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome like structures were identified using immunogold labelling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts as compared to their co-culture counterparts. To conclude, highly reproducible in-vitro tissue models representing major stages of human tongue carcinogenesis have been established. Our findings indicate that although there is no direct interaction between stromal fibroblasts and epithelium, the growth factors and cytokines released by fibroblasts regulate desmosomal assembly indirectly. In-vitro reconstituted tongue tissue models will be useful tool in addressing a broad range of unanswered questions in oral pathogenesis and especially in the multistep process of tongue carcinogenesis. In future, this model system might be a useful biological standardized system for testing anticancer drugs.

P172-E006Role of SFRP1 in epidermal stem cell regulation

R.R. Sunkara1, R.M. Sarate1, N.P. Gawas1, S.K. Waghmare1

1 Stem Cells Biology Group, Waghmare lab, ACTREC, Mumbai, India

Stem cells are involved in tissue homeostasis and wound repair, through their indefinite potential to self renew and differentiate into specialized cells. Wnt signalling pathway is involved in regulation of various tissue stem cells and its deregulation was reported in multitude of cancers. Wnt signalling is regulated by different families of Wnt inhibitors such as SFRP family, Dickkopf family and WIF1 (Wnt Inhibitory Factor) etc. SFRP1 (Secretary Frizzled Related Protein 1) is involved in the regulation of hematopoietic, mesenchymal stem cells, and is deregulated in various cancers such as breast, cervical etc. However, its role in epidermal stem cells and cancer is still obscure. In the present study, Sfrp12/2 (homozygous) keratinocytes showed an increase in proliferation as compared to WT (wild type). It could be due to an observed decrease in the expression levels of p21 (cell cycle inhibitor) and up regulation of Cyclin-D1 in Sfrp12/2 keratinocytes. Further, Sfrp12/2 mice keratinocytes showed increase in inter follicular epidermal stem cell marker (Lrig1) expression compared to WT keratinocytes. Additionally, increase in hair follicle stem cell (HFSC) population was observed in vivo in Sfrp12/2 mice. Hence, these results showed that deletion of SFRP1 gene disturbs the proliferation dynamics of epidermal stem cells and their functional properties in vitro, thereby indicating the importance of SFRP1 in epidermal stem cell regulation.

P179-E007Wnt signalling in the regulation of human oral cancer stem cells

S. Dash1, S.K. Waghmare1


Oral cancer is the sixth most common cancer in the world. In India, about 60–80% of the oral cancer patients are diagnosed in the advanced stage. Reports have shown that few resistant cells within tumour have stem cell like properties that are termed as cancer stem cells. Recent evidences suggest that Wnt signalling pathway may be involved in the regulation of cancer stem cells. However, the detailed mechanism by which the Wnt signalling regulates and maintains cancer stem cells remain mostly undefined. In this study, we have used CD44 and ALDH1 as markers to isolate cancer stem cells from an advanced stage oral cancer cell line. Functional characterisation by sphere forming assay showed that ALDH1+/CD44+ and ALDH1+ cells formed higher number of orospheres as compared to ALDH12/CD442 (control) cells. In vivo tumorigenesis assay showed that significantly lower number of ALDH1+/CD44+ cells were able to give rise to tumours in NOD/SCID mice as compared to control. Further, qRT-PCR data showed that ALDH1+/CD44+ and ALDH1+ populations expressed higher levels of stem cells markers such as Oct4 and Nanog. In addition, Wnt pathway genes were differentially regulated in the cancer stem cells as compared to control.

human embryonic stem cells, was highly expressed in both the cell lines. In order to further depict the molecular association of pancreatic CSCs and pluripotent/embryonic genes, Sox-9, Ngn-3, Pdx-1 and c-Myc genes were evaluated. PanC-1 CSCs showing MET characteristics significantly expressed embryonic genes and MiaPaCa2 CSCs having EMT features had lower expression of embryonic genes than its cell line.Thus, our findings suggest that PanC spheroids not only augment various stemness-associated genes but also differentially undergo EMT-MET transition, i.e, the mesechymal stem cell phenotype appears to regulate EMT and non-mesenchymal stem cell phenotype regulates MET.Further, the progression towards pancreatic metastasis through EMT regulated by its post-translational process targeting the key players of developmental pathway (Notch/Wnt/TGF-b) can provide novel therapeutic approaches to treat pancreatic cancer.

P97-E004Autofluorescence remodelingin lung cancer cells due to epithelial mesenchymal transition; A multi-modal study

A. Sarkar1, A. Barui2, J. Chatterjee1

1 School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Bengal, India2 Centre for Healthcare Education, Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, India

Background: Epithelial mesenchymal transition (EMT), a fundamental biological process during metastasis, is significantly responsible for lung cancer related death. Since EMT involves shedding of epithelial attributes at different level to become migratory and mesenchymal, multi-modal characterization is required to define this complex phenomenon. Present study explores cellular metabolic status through autofluorescence imaging during EMT, in association with shape and cytoskeletal arrangement.Method: Transformation growth factor b1 (TGF b1) was employed inducing EMT on lung cancer cell (A549) with varying treatment time. Cellular autofluorescence intensity (blue, green and red emission) along with shape, F-Actin randomness and vimentin expression were quantified from 200 cells at each time point. These six variables were further subjected to Principal Component Analysis (PCA) to elucidate importance of each feature in illustrating cellular behavior during temporal evolution of EMT.Results: mRNA expression of EMT related cardinal genes were assessed to validate EMT and results corroborated with previous reports. Due to EMT induction, significant rise in cellular aspect ratio was recorded as epithelial cells gradually acquired elongated morphology, with concomitant increment in F-Actin randomness and vimentin expression. Remarkable increase in green and red autofluorescence intensity was further noticed with gradual depletion of blue autofluorescence during EMT progression. PCA was able to segregate the cells under EMT and red autofluorescence feature was obtained as the best contributory feature, exhibiting its possible applicability in cancer diagnostics.Conclusion: Present study identifies changes in different autofluorescence features with corresponding endogenous fluorescing compounds under EMT related metabolic modulation. It also attempts to integrate cellular shape and structural information with plausible metabolic remodeling during EMT. Future high-end spectroscopic studies may provide more local and global molecular information for meaningful integration of these attributes during EMT progression.

P108-E005Establishment of in-vitro 3D co-culture models from different stages of human tongue carcinogenesis

S. Sharada1, D. Harsh1, S.A. Kumari1, J. Shriya1, D.E. Costea2a,2b, M. Snehal1, M. Vidhi3, D. Prerana1, S. Shilpi4, D. Chaukar4, V. Milind1

1 Vaidya Laboratory, ACTREC, Tata Memorial Centre, Maharashtra, India2a Gade Laboratory for Pathology, University of Bergen, Bergen, Norway2b Department of Pathology, Haukeland University Hospital, Bergen, Norway3 Nups and Sumo biology group, Department of Biological Sciences, Madhya pradesh, India4 Oral Surgery, Head and Neck Unit, Tata Memorial Hospital, Parel, Mumbai, India

To study multistep carcinogenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue carcinogenesis, and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal

E. Cancer Stem Cells and EMT European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S27

Purpose/Objective(s): Use of human adipose-tissue-derived, insulin-producing mesenchymal stem cells (h-AD-MSC) transfused with unfractionated cultured bone marrow (CBM) in T1 DM patients.Materials & Methods: 19 consecutive insulinopenic T1DM patients (M:F, 14:5) of 0.6 to 10 yrs duration, aged between 14 to 28 yrs under treatment with insulin showing postprandial blood sugars between 156 to 470 mg%, glycated hemoglobin (HbA1C) 6.8% to 9.9% and c-peptide levels of 0.02 to 0.2 ng/mL were enrolled into this prospective phase 1/2 study, since Oct. 2012. Healthy willing non-diabetic donors of matching blood group from the family of recipients provided consent for the extraction of adipose tissue from anterior abdominal wall (day -14) and bone marrow aspiration following G-CSF stimulation (day -10 & -9). All recipients underwent (a) Non-myeloablative low intensity conditioning (day -8 to -1) which included (1) fractionated TLI 10 gy/ 5# delivered by 3-DCRT technique by 6 MV/15 MV photons to cervical, Mediastinal, Paraortic & Iliac regions (day -8 to -3) (2) Anti-T cell antibody (day -2) & anti-B-cell antibody (day -1) and (b) Intra-portal administration of xenogeneic-free h-AD-MSC and Hematopoitic stem cell (HSC) (day 0).Fasting & PP BGL, C-peptide levels checked weekly for 1 month & monthly thereupon. Insulin requirement was made on sliding scale as per patient requirements. Key endpoints of study are morbidity/mortality from MSC 1 HSC transplantation & temporal changes in exogenous insulin requirements. Secondary end points were C-peptide levels & Hb A1c following SC transplantation.Results: With a mean follow-up of 24 months, all patients were successfully infused h-AD-MSC plus CBM without any untoward effects (infective episodes or GVHD) & heavy immunosupression. There was a statistically significant (p 5 0.0001): decrease in the insulin requirements (30%-50%), increase in C-peptide levels (4–26 fold) and decrease in HbA1C.Conclusions: This report, first of its kind on human subjects, describes safe and cost-effective treatment of insulinopenic diabetics.

P200-E012Immortalizing renal allografts with stem cell trasplantation using low dose irradiation as an adjuvant to non-myeloablative conditioning

C. Haritha1, V. Shankar1, H.L. Trivedi2, A.V. Vanikar2

1 Geetanjali Cancer Center, Geetanjali Medicity, Udaipur, India2 Institute of Kidney Diseases & Research center and Institute of transplantation Sciences, Civil Hospital campus, Ahmedabad, Gujarat, India

Purpose/objective: Evaluate the efficacy of tolerance induction protocol (TIP) incorporating RT in achieving MLHC in living related donor (LRD) renal allograft recipients.Materials and Methods: 412 patients with End stage renal disease (ESRD) were randomized into 2 groups: Stem cell (SC) group [n 5 206] & control (CON) group [n 5 206]. SC group underwent renal transplantation (RTx) following TIP and negative LCM. TIP for SC group patients included (a) 2 DLI (b) low-intensity conditioning & (c) Hematopoietic stem cell transplantation (HSCT). The conditioning regimen included fractionated low dose TLI, CyA, Cyclophosphamide, anti T-cell antibody & Treosulfan. Unfractionated HSC procured from donor bone marrow was administered into the BM, portal & peripheral circulation, within 24 hours of achieving CD4+/CD8+ T cell count ,10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells. Donor specific cytotoxic antibodies were eliminated with iv immunoglobulins & plasmapharesis before RTx. Immunosuppression was withdrawn 6 mon after RTx for patients with consistently positive chimerism. Robust, prope and metastable clinical tolerance was appropriately defined. CON group underwent RTx directly after negative LCM followed by triple drug immunosuppression with CyA, Prednisolone & mycophenolate mofetil/azathioprine.Results: In SC group pts, at mean follow-up of 30 months 12.5%, 85.5% & 2% had robust, prope& metastable tolerance respectively. Graft rejection was observed in 8.7% of which 3.8% had graft loss. Mortality rate was 3%. Mean Sr. creatinine was 1.3 mg%. None of the pts had GVHD. In the CON group, at the mean follow-up of 28 months, 59.2% had rejection of which 30% had graft loss. Mortality rate was 18.4% (34 pts - 21 non-renal 1 13 renal causes). Mean Sr. creatinine was 2.7 mg%. SC group had statistically significant reduction in rejection, graft loss & mortality (p 5 0.0001).Conclusion: Tolerance Induction Protocol is safe, Effective in inducing robust & prope tolerance across MHC barriers in LRD renal Transplantation.

Hence, our results indicate that Wnt pathway may play a potential role in the regulation of oral cancer stem cells.

P181-E008sPLA2IIA, a growth modulator of EGFR: Role in cancer and stem cell regulation

R.M. Sarate1, G. Chovatiya1, V. Ravi1, S.K. Waghmare1

1 Stem Cell Biology Group, Waghmare Lab, ACTREC, Tata Memorial Centre, Mumbai, India

sPLA2IIA, a growth factor modulator, is the mouse homologue of human secretory group II phospholipase A2 (sPLA2). sPLA2IIA has been implicated in various cancers and modulate tumourigenesis.The over expression of the sPLA2 IIA in skin, K14-sPLA2 IIA mice showed increase sensitivity to chemical carcinogenesis. However, its role in tissue homeostasis and epidermal stem cell regulation is unknown. Tissue homeostasis is maintained throughout the adult life by stem cells that self-renew and also generate progeny that undergo terminal differentiation. Stem cells are regulated and maintained by various signalling pathways and deregulation in these pathways leads to cancer. Hence, it warrants understanding the role of sPLA2 IIA in epidermal homeostasis and stem cell regulation. In the transgenic mice overexpressing sPLA2 IIA, we have investigated the hair follicle cycle at various postnatal day (PD) ages by performing histological analysis, tail whole mount and immunofluorescence staining by using the stem cells, proliferation and differentiation markers. We performed the flow cytometry analysis to assess the percentage of hair follicle stem cell pool in K14-sPLA2 IIA mice. In addition, the BrdU pulse-chase was performed to investigate the slow-cycling Label Retaining Cells (LRCs) in the hair follicle stem cell niche. Here we show K14-sPLA2 IIA mice showed depletion of hair follicle stem cell pool and loss of long-term BrdU label retaining cells. It showed increased differentiation, loss of ortho-parakeratotic organization and enlargement of sebaceous gland, infundibulum and junctional zone. The colony forming efficiency of keratinocytes was significantly reduced and also showed increased differentiation. Our study for the first time unravels the role of sPLA2 IIA in epidermal stem cell regulation and homeostasis.

P183-E009Effect of secretory phospholipase A2-IIA on epidermal proliferation and differentiation

G. Chovatiya1, R. Sarate1, N. Gawas1, S.K. Waghmare1


Secretory phospholipase A2 IIA (sPLA2-IIA) catalyzes the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Lysophospholipids are potent biologically active lipid mediators that exert a wide range of cellular effects through specific G-protein coupled receptors. sPLA2-IIA modulates tumorogenesis in various cancer and overexpression of sPLA2-IIA is known to enhance tumorigenic process induced by chemical carcinogen in skin however, the molecular mechanism involved is yet be elucidated. sPLA2-IIA is also known to have catalytic independent activity that promotes binding of epidermal growth factor (EGF). Here we have observed transgenic mice overexpressing sPLA2-IIA (K14-sPLA2-IIA) showed increased phospho-EGFR (activated EGFR) in mice epidermis as compared to wild type control. Moreover, keratinocytes isolated from K14-sPLA2-IIA mice showed enhanced EGFR expression in calcium free condition, suggesting its growth modulatory activity is independent of its catalytic activity. In addition, K14-sPLA2-IIA keratinocytes and mouse epidermis showed enhanced activation of JNK induced by EGFR and further leads to the activation of AP1 transcription factors as assessed by phosphorytion of C-Jun and C-fos.Moreover, K14-sPLA2-IIA mice skin showed two fold increased production of lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA). Overall, our data suggest involvement of synergistic effect of both non-catalytic and catalytic function of secretory phospholipase A2 in keratinocytes growth and differentiation.

P199-E011Mesenchymal stem cells transplantation with non-myeloablative conditioning for type I diabetes mellitus: Lab to clinic

C. Haritha1, V. Shankar1, H.L. Trivedi2, A.V. Vanikar2

1 Geetanjali Cancer Center, Geetanjali Medicity, Udaipur, India2 Institute of Kidney Diseases & Research center and Institute of transplantation Sciences, Civil Hospital campus, Gujarat, India

S28 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 F. Systemic Therapies

OT266-F003Assessment of theranostic potential of Lu-177-trastuzumab in HER2 expressing breast cancer patients

J. Shukla1, P. Bhusari1, R. Vatsa1, G. Singh2, A. Bal3, M. Parmar1, D.K. Dhawan4, B.R. Mittal1

1 Department of Nuclear Medicine & PET, PGIMER Chandigarh, India2 Department of Surgery & PET, PGIMER Chandigarh, India3 Department of Histopathology, PGIMER Chandigarh, India4 Centre for Nuclear Medicine, Panjab University Chandigarh, India

Introduction: Radiolabeled monoclonal antibodies are the modern agents for targeting the tumor cells with high specificity. Trastuzumab is an FDA approved monoclonal antibody against HER2 receptors. The HER2 tumors are aggressive and associated with poor prognosis. Lu-177 labeled trastuzumab may increase the efficiency of treatment. We evaluated the theranostic potential of Lu-177-trastuzumab in HER2 positive breast cancer patients.Methods: Trastuzumab was conjugated to the bifunctional chelator DOTA and characterized for antibody integrity and number of chelator molecules. DOTA conjugated trastuzumab was purified using gel filtration. The DOTA-trastuzumab conjugate was radiolabeled with Lu-177 and free Lu-177 was removed by gel filtration followed by quality control and distribution in the rats. The approval from the Institutional Ethics Committee was obtained to assess its potential in breast cancer patients. After getting oral and written consent the patients (n 5 10), HER2 positive (n 5 6) and HER2 negative (n 5 4), were administered 185–370 MBq Lu-177-trastuzumab. Wholebody and SPECT/CT imaging was performed at Day 5/7 post administration to see the tracer uptake and specificity of Lu-177-trastuzumab for targeting HER2 receptors.Results: The good radiolabeling yield (91%) could be achieved for Lu-177-trastuzumab with .97% radiochemical purity. All formulations were found sterile and the endotoxin content was much less than the permissible levels. Lu-177-trastuzumab showed 72–75% immune-reactivity to purified HER2 receptor by radioimmunoassay. The highest uptake was seen in liver. Rest of the organs had minimal activity. The primary tumors showed tracer uptake of Lu-177-trastuzumab both on whole body and SPECT/CT images. Several metastatic sites were also visualized on whole body and SPECT/CT images. The immuno-histopathological correlation demonstrated localization of the tracer in only HER2 positive patients. No tracer uptake was seen in the HER2 negative subjects.Conclusion: The preliminary results showed promising potential of Lu-177-trastuzumab in targeting the HER2 breast tumors. However, liver is the dose limiting organ.

OF77-007Solid-tumor cancer as a systems-biology disease: Mechanistic and clinical evaluation of the therapeutic effectiveness of murine renal adenocarcinoma cells in agarose macrobeads. (FDA BB-IND 10091;NCT01053013, 02046174)

B.H. Smith1, L.S. Gazda2, N. Berman1, P.C. Martis2, M.A. Laramore2, Z.P. Andrada1, A. Nazarian1, J. Thomas1, E. Akahoho1, A. Dudley2, T. Parikh1, A.J. Ocean3, T.J. Fahey III4, D.J. Wolf1

1 The Rogosin Institute, New York, NY, USA2 The Rogosin Institute-Xenia Division, Xenia, OH, USA3 Weill Cornell Medical College, New York, NY, USA4 New York-Presbyterian Hospitals, New York, NY, USA

Current approaches to solid-tumors therapeutics emphasize the principles upon which “precision medicine” is based. These principles include 1) the tremendous genomic and immunological variability (“individuality”) of tumors within a given cell, tissue, or anatomical site-of-origin class; and 2) the need, therefore, for highly specific genomic or immunologic profiling to identify mutant “driver” genes and/or immunological targets. Specific therapy can then be directed at those targets or developed to do so. We believe that the “precision medicine” approach, while important, is not sufficient for more effective solid-tumor treatment because such cancer is not a static disease based on one or more gene mutations, but rather a complex, multilevel systems-biology disease characterized by adaptability and rapid (non-Darwinian) evolution. If this is so, then an effective anti-cancer treatment is likely to have to be an (implanted) 24-hour-7-day biological system that is capable of tapping into or restoring the growth-control mechanisms in the neoplastic target cells, but also adapting flexibly to their evolution. Consistent with this hypothesis, we have shown that agarose-agarose macrobeads (MB) containing mouse renal adenocarcinoma cells secrete a factor(s) that inhibits the growth of cancer cells outside the bead

P274-E013The epithelial mesenchymal transition and metastasis: Can you have one without the other?

S. Basu1, K. Raychaudhari1, N. Chaudhary2, M. Gurjar1, J. Limzerwalla1, R. Thorat1, C. Braggs1, A. Sawant1, M.S. Reddy2, S.N. Dalal1

1 ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, India2 Centre for DNA Fingerprinting and Diagnosis, Hyderabad, India

Background: Most deaths in patients with solid tumors occur due to the presence of metastatic disease. Therefore, it is important to understand the molecular mechanisms underlying metastatic progression in order to design novel therapeutic strategies to treat metastatic disease. A primary requirement for the initiation of metastasis is the dissolution of cell-cell contacts followed by the cells entering the blood stream and migrating to distant organs. The ability of the tumor cell to dissociate from its surrounding cells is often dependent on the dissolution of cell-cell junctions and acquisition of the Epithelial Mesenchymal Transition (EMT), which allow the tumor cells to migrate through the extra cellular matrix and enter the blood stream, survive in the blood stream till they reach the target organ and then invade and colonise the target organ. Previous work from our laboratory has demonstrated that loss of a desmosomal plaque protein, plakophilin3, leads to tumor progression and metastasis.Results: Our laboratory has recently examined the role of plakophilin3 loss and the acquisition of EMT in regulating tumor progression and metastasis in an isogenic cell system. Our work has identified multiple changes in gene expression upon plakophilin3 loss that seem to be required for tumor progression and metastasis in HCT116 cells. However, we see no activation of the Epithelial Mesenchymal Transition (EMT) upon plakophilin3 loss, a program that is often activated in metastatic tumors. Indeed, loss of another tumor suppressor gene, 14-3-3s, in these cells results in activation of an EMT program but does not lead to tumor progression and metastasis.Conclusions: Our results suggest that acquisition of the EMT phenotype is not sufficient for metastasis in HCT116 cells and that the ability of the cell to respond to stress, might be more important in terms of the acquisition of the metastatic phenotype.

F. Systemic Therapies

OT105-002Role of neo-adjuvant chemotherapy for organ preservation in oral cancer: a randomized controlled trial

D. Chaukar1, K. Prabhash1, S. Arya1, S. Sharma1, P. Rane1, P. Pai1, P. Chaturvedi1, S. Nair1, S. Kane1, G. Pantvaidya1, D. Nair1, A. Deshmukh1, A.K. D’Cruz1

1 Tata Memorial Hospital, Mumbai, India

Background: Surgery is the mainstay in treatment of oral cancers. Mandible resection is associated with significant morbidity. Majority of resections are in view of paramandibular soft tissue disease and only 40% of resected mandibles show tumor invasion. Neoadjuvant chemotherapy has been used as an organ preservation strategy in other cancers e.g. breast cancers. We explored the role of neoadjuvant chemotherapy in downstaging the disease permitting mandible preservation.Material and Methods: A phase II randomized controlled trial with mandible preservation as the primary end point. Oral cancer patients with no clinico-radiological bony involvement but necessitating mandible resection in view of paramandibular disease were included. A sample size of 68 patients was calculated considering 30% mandible preservation rate. Standard group underwent surgery with adjuvant therapy as indicated. Intervention group received 2 cycles of docetaxal (75 mg/m2), cisplatin (75 mg/m2) and 5 FU (750 mg/m2) followed by surgery and adjuvant therapy. Decision to preserve the mandible was taken by operating surgeon.Results: There were 64 buccal and 4 tongue cancers. Median age was 47 years. Forty patients were T4 and 14 T3 & T2 each. Of 34 patients in intervention arm, 32 received 2 cycles of chemotherapy as planned. One patient had complete response, 29.4% had partial response and 53% had stable disease as per RECIST criteria. Overall 73% patients suffered grade III/IV toxicity. Mandible preservation rate was 48% in the intervention group. Margin positivity rate was similar in both the groups. Median follow up was 31.8 months. 9 patients had local recurrence (5-standard group, 4-intervention group). Overall survival at two years was 53.5% in the standard and 69.7% in the intervention group (log rank test- p 5 0.46).Conclusion: Neoadjuvant chemotherapy is a feasible option for mandible preservation in select group of patients.

F. Systemic Therapies European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S29

Conclusion: Neoadjuvant chemotherapy is a viable option in advanced ovarian cancer to increase chances of optimal cytoreduction, decrease operative complications and decrease postoperative morbidity.

P76-F003Paclitaxel-cetuximab versus oral metronomic chemotherapy as palliative chemotherapy in head and neck cancers – A match pair analysis

V. Agarwala1, V.M. Patil1, V. Noronha1, A. Joshi1, S. Zanwar1, V. Muddu1, S. Dhumal1, K. Prabhash1

1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India

Background: Cetuximab based palliative chemotherapy is the standard of care in metastatic head and neck cancers. However, only few patients can afford Cetuximab in developing countries. A recent study showed the superiority of oral metronomic chemotherapy in palliative setting over maximum tolerable dose chemotherapy which did not include Cetuximab. The present match pair analysis was planned to compare the efficacy of Cetuximab based chemotherapy versus metronomic therapy.Method: 60 patients with metastatic/recurrent head and neck squamous cell cancer treated with weekly Paclitaxel (80 mg/m2) and Cetuximab were matched with 60 patients treated with oral metronomic chemotherapy. The matching was done on 1:1 basis for site of primary and event free period from prior treatment. The primary endpoint of the analysis was overall survival (OS). OS was estimated by Kaplan–Meier method. The cohorts were compared using the log-rank test. A multivariable Cox proportional regression model was used to identify independent factors affecting PFS and OS.Result: The median OS was 191 days (95% CI 122.2–259.8 days) in metronomic cohort and 256 days (95% CI 177.0–334.9 days) in cetuximab cohort. On Cox proportional hazard model, ECOG PS (0–1 versus 2) & therapy (cetuximab versus metronomic) had a statistically significant impact on OS. The hazard ratio was 0.58 in favour of cetuximab cohort (95% CI 0.35–0.95, p 5 0.031).Conclusion: Cetuximab based chemotherapy leads to a significant improvement in OS as compared to metronomic chemotherapy in palliative chemotherapy of head and neck cancers.Highlights of the study: 1) The response rates and clinical benefit rates were higher with cetuximab therapy. 2) The grade 3–4 adverse events were lower with metronomic therapy. 3) The median PFS and OS were higher with cetuximab therapy. 4) Patients with previous EFS . 6 months had similar OS with both therapies.

P131-F004Oral TKI in poor PS lung cancer patients

S. Jandyal1, A. Karpe1, V. Patil1, V. Noronha1, A. Joshi1, A. Bhattacharjee2, K. Prabhash1

1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India2 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India

Background: Use of these oral TKI in stage IV lung cancer patients with ECOG PS3–4 status seems a logical option. This analysis was planned to study the overall survival with such practice.Material and methods: This was a retrospective analysis, of lung cancer patients with poor PS as palliative chemotherapy between Jan 2012 and December 2013. These patients after discussion in multidisciplinary clinic are offered oral TKI. At the same time patients are counselled about poor prognosis. These patients are started on gefitinib 250 mg OD or erlotinib 150 mg OD and are also started on best supportive care. Patients are then followed up till death. Analysis of estimation of overall survival was done with Kaplan-Meier survival method.Results: 331 patients were included for this analysis subjected to above mentioned criteria. The median age of these patients was 61 years (IQR: 53–68 years). 197 patients were male (59.5%, N 5 331). The ECOG PS was 03 in 294 patients (88.8%) and 4 in 37 patients (11.2%). None of these patients had received previous treatment. The oral tki give was erlotinib in 187 patients (56.5%) while it was gefitinib in others. 50 patients (15.1%) had rash within one month of starting TKI. No incidence of TKI induced death seen. The median estimated survival was 185 days (95% CI 146–223 days). For a median follow duration for this cohort 207 patients had died. The median estimated survival in egfr negative patients 131 days (95% CI 108–181 days) and egfr positive patients was 392 days (95% CI 298–493 days) (p 5 0.000).Conclusion: Oral TKI administered in poor PS patients leads to a median survival of around 6 months in EGFR negative and of around 13 months in EGFR positive patients.

both in vitro and in vivo and that this anti-neoplastic effect is partly mediated via Akt regulation of MEF2D activity, with concurrent down-regulation of genes associated with proliferation and angiogenesis and up-regulation of apoptosis (CHOP, GADD45). Phase I and II clinical trials (FDA BB---IND 10091; NCT 01053013, 02046174) have demonstrated safety and a survival advantage in 66 MB-treated late-stage, treatment-resistant colorectal cancer patients (tumor-marker “responder” mean survival 10.7 vs 4.9 mos, non-responders). The Data presented here support the concept of a multifactorial cell-therapy, systems-biology-based approach to anti-cancer therapy that differs from, but may be Complementary to, that defined by “precision medicine.”

P21-F001Evaluation of concurrent chemoradiation with neoadjuvant and adjuvant chemotherapy versus concurrent chemoradiation alone in locally advanced carcinoma cervix

R. Singh1

1 King George Medical University Lucknow, Uttar Pradesh, India

Background: Concurrent chemoradiation is a standard treatment for locally advanced carcinoma cervix and has improved survival outcomes compared to radiation alone. Despite an improved survival rate with concurrent chemoradiation against radiation therapy alone, local and distant failures (17% and 18%, respectively) of locally advanced carcinoma cervix are encountered with overall survival rates ranging from 60 to 65%. One method of improving treatment outcomes among these patients is to give additional chemotherapy along with the main treatment of concurrent chemoradiation therapy.Aim: Aim of the study was to compare concurrent chemoradiation with neoadjuvant and adjuvant chemotherapy versus concurrent chemoradiation alone in locally advanced carcinoma cervix in terms of treatment response and toxicities.Material & Methods: A randomized control study was done on 116 patients of locally advanced carcinoma cervix (stage IIB to IIIB) registered in Department of Radiotherapy, King George’s Medical University, Lucknow, Uttar Pradesh between January 2014 to February 2015. Patients were randomly divided to receive either one cycle of cisplatin/5-FU neoadjuvant chemotherapy and two cycles of same adjuvant chemotherapy with concurrent chemoradiation (Arm A) or only concurrent chemoradiation (Arm B). All patients received three fractions of HDR intracavitary brachytherapy.Results: A higher proportion of patients of chemotherapy arm achieved complete local control as compared to the non-chemotherapy arm, and this was statistically significant. There was a trend toward more treatment related acute toxicity with chemotherapy.Conclusion: These results have corroborated the view that if neoadjuvant and adjuvant chemotherapy are added to concurrent chemoradiation, it could further the effects of concurrent chemoradiation for patients with locally advanced cancer of uterine cervix.

P74-F002Role of neoadjuvant chemotherapy in advanced ovarian cancer: Single surgeon’s experience in 122 cases

M.D. Ray1

1 All India Institute of Medical Sciences, New Delhi, India

Background: More than 70% of newly diagnosed ovarian cancer patients present with advanced stage disease requiring both surgery and chemotherapy for optimal treatment. While optimal primary debulking surgery (PDS) followed by adjuvant chemotherapy is the standard of care, role of Neoadjuvant chemotherapy is being explored in advanced ovarian cancer.Material and Methods: Retrospective analysis of a physician maintained prospective computerized database of ovarian cancer patients at a tertiary care cancer centre in North India was performed. Optimal cytoreduction was defined as completion of cytoreduction score (CC) 0 or 1.Result: A total of 122 patients of epithelial ovarian cancer were operated during May 2012 to July 2015. Primary Debulking Surgery (PDS), interval Debulking Surgery (IDS) and secondary cytoreduction (SC) were undertaken in 53 (43.44%), 34 (27.87%) and 35 (28.69%) patients during the study period. IDS was attempted after three cycles of platinum and taxane based Neoadjuvant chemotherapy. Optimal cytoreduction could be achieved in 79.24% among PDS group compared to 88.23% among IDS group. Postoperative complications were significantly less in IDS group (2.9%) compared to (22.6%) in PDS group.


was analysed for chemotherapy induced percentage necrosis and divided as , / . 90% necrosis. Post operative adjuvant radiotherapy was decided on a case to case basis irrespective of percentage necrosis.Results: One patient had involved margins. Necrosis was available in 80 patients. 25 had , 90% and 55 had . 90% necrosis. 23 of these patients received radiotherapy (9 , 90%, 14 . 90%). All patients were available for follow up. The OS of all patients was 68% at 5 years. Currently 62 patients are alive (follow up range 33 to 90 months, median 61 months). There was no difference in OS in patients who received radiotherapy (57%) and those who did not (75%) p 5 0.133. In the cases with , 90% necrosis the OS in patients who received radiotherapy was 56% as against 49% in those who did not p 5 0.760. In the cases with . 90% necrosis, the OS in patients who received radiotherapy was 57% as against 85% in those who did not p 5 0.043.Conclusion: Our data suggests that the decision to offer adjuvant radiotherapy after surgical excision in Ewing sarcoma is multifactorial and independent of percentage necrosis after chemotherapy.

P215-F008Cone beam CT-based 3D dose verification in head-and-neck cancers – Validation study



Purpose/Objective(s): Evaluate the dosimetric feasibility of CBCT for 3-D Dose verification of head and neck VMAT treatments and independently validate the same, at treatment delivery, with 3DVH software.Materials/Methods: Prior to treatment dose verification on the CBCT data set, a CT-ED curve was created in Monte-Carlo treatment planning system, using Catphan cylindrical QA phantom, for the purpose of heterogeneity corrections. Following CBCT-CT fusion, manually contoured planning CT volumes were mapped on the first fraction CBCT dataset of 15 head and neck cancer patients planned for treatment by VMAT. Original clinically executed treatment plan fluence was superimposed on CBCT dataset and dose volume statistics, thus generated, were compared. Treatment delivery fluence was recorded and dose was reconstructed on both CT and CBCT datasets using 3DVH software based on perturbed dose algorithm. 3DVH output was used to independently validate the dose volume statistics of plans generated on CT and CBCT images.

Results: Level Study

VMAT Dose Variation between CT-CBCT

RemarksTarget OAR

Monte CarloPlanningsystem Level

catphan Phantom

Max , 1% Max , 3% Validated the HounsfieldUnit to relative electrondensity curve

Head & neck Max , 2% Max , 10% Variations are wrt

Conclusions: Margins of 1.5 cm SI, 0.75 cm AP, and 0.75 cm LR would account for respiratory tumor motion of .95% of esophageal primary tumors in the dataset. All celiac-region lymph nodes would be adequately covered with SI, AP, and LR margins of 2.25 cm, 1.0 cm, and 0.75 cm, respectively. The ITV based on all 10 phases is recommended.

P216-F009Pain management in patients undergoing interstitial pelvic brachytherapy: A quest for better analgesia

V. Shankar1, C. Haritha1, U. Kharod2, B. Shrivastava2, H. Kamat2, A. Shanubhogue3

1 Geetanjali Cancer Center, Geetanjali University, Udaipur, India2 Department of Anaesthesiology, Shree Krishna Hospital & Pramukhswami Medical College, Karamsad, Gujarat, India3 Department of Statistics, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India

Purpose/Objective(s): The objective of the study was to compare the efficacy, safety & immediate side effects of Patient controlled epidural analgesia with background infusion (group P) & Continuous epidural infusion (group C) using bupivacaine-fentanyl in patients undergoing IPB for cervical cancer.Materials/Methods: For this study, 40 IPB applications were randomly assigned into two groups 20 each. Implant insertion was done under spinal anesthesia with bupivacaine & buprenorphine. Epidural catheter

P189-F005Entrapment of paclitaxel-cyclodextrin inclusion complex in liposomes: The role in improving loading efficiency and in vitro cytotoxicity

P. Bhatt1, A. Misra1, R. Mashru1

1 The Maharaja Sayajirao University of Baroda, Vadodara, India

Paclitaxel (PTX), an effective anticancer agent, is poorly water soluble and toxic solubilizer used in its commercial preparation is responsible for side effects associated with PTX therapy. Being a hydrophobic drug, researchers have developed liposomal formulation by entrapping PTX into lipid bilayer. However, with conventional liposomes loading efficiency was low and crystallization of PTX was a major problem. Concept of entrapment of drug-cyclodextrin inclusion complexes in liposomes would permit water insoluble drug to accommodate in aqueous core of vesicles. This would potentially increase the loading efficiency of drug when compared with those achieved by conventional drug incorporation into the lipid bilayer. Purpose of the study was preparation and characterization of cyclodextrin-paclitaxel (CD-PTX) complex encapsulated liposomes. CD-PTX complex was formulated using Heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) to improve aqueous solubility and hence liposomal loading of PTX. The complex was encapsulated in aqueous core of PEGylated liposomes (PLs) using reverse phase evaporation technique. Liposomes thus prepared were characterized for size, zeta potential, loading efficiency and in vitro drug release profile. They were also evaluated for in vitro cellular uptake and cytotoxicity in A549 cells. Increased aqueous solubility of 11 mg/ml was achieved for PTX using DMBCD, at molar ratio of 20. MTT studies on A549 cells revealed that cytotoxic property of PTX was retained after inclusion in DMBCD complex. The PLs were found to be nanosized and showed improved loading efficiency of PTX. The in vitro release profiles exhibited controlled release of PTX from PLs when studied for 48 h. The IC50 values from MTT assay suggested that PLs was 4.5 times more cytotoxic than PTX solution for A549 cells after 24 h period. To conclude, PLs displayed improved intracellular uptake and enhanced cytotoxic compared to PTX solution in A549 cells.

P191-F006Chemotherapy induced necrosis as a prognostic marker in osteosarcoma – Do we need to raise the bar?

A. Gulia1, A. Puri1, G. Chinnasawmy1


Introduction: The degree of chemotherapy induced histological necrosis has proven to be one of the most robust prognostic markers in osteosarcoma. Currently a cut off using , / . 90% necrosis is employed as the determinant for classifying the response as poor or good.Material: We reviewed the data for 192 patients of localized extremity osteosarcoma operated between January 2006 to June 2010. All patients underwent appropriate surgical resection after neoadjuvant chemotherapy. Patients were divided into 3 groups based on percentage necrosis as ,90%, 90–99% and 100%.Results: Of the 173 patients available for follow up, 84 patients are alive (range 31 to 88 months, median 50 months). The traditional cut off using 90% necrosis showed no difference in overall survival (OS) [50% vs. 46% (p 5 0.139)] between groups. However when patients were grouped into 100% necrosis or ,100% necrosis significant differences emerged [OS 73% vs. 41% (p 5 0.001)].Conclusion: In our study only patients with complete absence of any viable tumor in response to neoadjuvant chemotherapy have better OS. This could be important when there is need to tailor post operative adjuvant chemotherapy regimens in an attempt to improve survival.

P205-F007Should percentage necrosis influence the decision for adjuvant radiotherapy in operated cases of Ewing sarcoma

V. Verma1, A. Puri1, A. Gulia1, S. Laskar2

1 Bone and Soft tissue Disease Management Group (Surgical), Tata Memorial Hospital, Mumbai, India2 Bone and Soft tissue Disease Management Group (Radiotherapy), Tata Memorial Hospital, Mumbai, India

Aim: To determine the indications of adjuvant radiotherapy after surgical excision in Ewing sarcoma.Materials and Methods: 94 consecutive patients of non metastatic Ewing sarcoma were analysed. Patients underwent appropriate surgical resection after receiving neoadjuvant chemotherapy. Excised specimen

F. Systemic Therapies European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S31

issue. The null hypothesis was that margin positive rate in technically unresectable buccal mucosa cancers post NACT would not be different from upfront operated T4 buccal mucosa cancers.Method: This was a 1:1 match pair analysis of oral cancer patients treated at Tata Memorial Hospital between 2010 and 2013. We had matched 215 upfront operated T4 buccal mucosa cancer patients to 215 T4 buccal mucosa cancer patients operated after NACT. In the upfront operated group surgery was performed with a 1 cm gross margin around the tumor while in post NACT groupsurgery was performed with a wide margin around the post NACT tumor. Chi-square test was used for comparison of margin status.Result: Overall we had a young cohort of patients in both groups and predominant population was of male gender. In both groups the margin positive status was low. The unfavourable margin status (either positive or close margin) was seen in 5.1% of upfront operated patients while it was 3.3% in patients operated after NACT. It was interesting to note that perineural invasion was seen in quite high proportion of our upfront operated patients (23.3%) while it was not the case in post NACT patients (7.4%) (p 5 0.000).Conclusion: The result of this match pair upholds the null hypothesis. It’s heartening to know that post NACT operated patients though at a high risk for margin positive resection upfront undergo R0 resections and the margin status is similar to our upfront operated patients.

P229-F012Neoadjuvant chemotherapy in technically unresectable oral cancers: Does HPV make a difference?

K. Prabhash1, S. Kane2, V. Noronha1, V.M. Patil1, A. Joshi1, V. Muddu1, S. Dhumal1, S. Juvekar3, S. Arya3, A. D’Cruz4, A. Bhattacharjee5

1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India2 Department of Pathology, Tata Memorial Hospital, Mumbai, India3 Department of Radiodiagnosis, Tata Memorial Hospital, Mumbai, India4 Department of Surgical oncology, Tata Memorial Hospital, Mumbai, India5 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India

Background: HPV positive oropharyngeal cancers have favourable prognosis, though the impact of HPV positivity is dependent on level of tobacco exposure and on the tumour stage. We wanted to study the impact of HPV in locally advanced and technically inoperable oral cancers treated with neoadjuvant chemotherapy (NACT).Methods: We performed an analysis on 124 randomly selected patients of oral cancer who underwent NACT. They received 2 cycles of NACT followed by definitive local treatment. HPV positivity was identified by p16 immunohistochemistry on the pretreatment biopsy. The response rate after NACT (RECIST v. 1.1) in the HPV positive and negative cohorts were compared by proportion test. Difference in the overall survival (OS) was assessed by log rank (Mantel-Cox) test. Multivariate Cox proportional hazard regression model was applied to identify factors affecting OS.Results: 16 patients were HPV positive (12.9%). In HPV positive cohort, 58.3% patients had response while in HPV negative cohort 30.3% patients had response to NACT (p 5 0.51). The locoregional failure rate was 45.2% at 2 years. It was 31.3% in HPV positive and 47.2% in HPV negative patients (p 5 0.40). The median OS for HPV negative patients was 684 days (95% CI 431-NA days) while it was not reached for HPV positive patients. (p 5 0.0845).Conclusion: HPV positive technically unresectable oral cancers seem to have better os. Larger prospective study is required to confirm the impact of HPV on OS in advanced oral cancers.

P230-F013Long term results of neoadjuvant chemotherapy followed by definitive local treatment in locally advanced carcinoma maxillary sinus

K. Prabhash1, V. Patil1, V. Noronha1, A. Joshi1, A. Sahu1, A. Ramaswamy1, V. Muddu1, S. Zanwar1, S. Juvekar1, D. Chaukar1, P. Chaturvedi1, P.S. Pai1, A.K. D’Cruz1


Background: Locally advanced carcinoma of maxillary sinus has been historically reported to have poor prognosis. Neoadjuvant chemotherapy has shown promising results.Methods: 41 patients with locally advanced borderline resectable (stage IV) or unresectable maxillary carcinoma (Stage IVb) were treated with induction chemotherapy between 2008 and 2011. The protocol included 2 cycles of chemotherapy, response assessment and multidisciplinary clinic review for definitive local treatment.

(EC) inserted for continuous analgesia. Pain assessed using VAS. At VAS $3 a bolus of 1 μg/kg fentanyl administered through catheter in both the groups. Then, in group P patients were started with infusion of 0.125% bupivacaine & 2 μg/ml fentanyl through PCA pump via epidural catheter with a background infusion of 4–6 ml/hr. While, in group C patients were given 0.125% bupivacaine & 2 μg/ml fentanyl through infusion pump via EC delivering a continuous infusion of 8–12 ml/hr. Patient’s VAS score, side effects, hemodynamic & respiratory parameters were monitored. Independent sample T test was used to evaluate the mean dose & mean volume delivered. Mann Whitney-U test was used for analysis of VAS Score, hemodynamic & respiratory parameters.Results: The mean dose of fentanyl in group P (10.5 μg/hr) was significantly less compared to group C (18 μg/hr) (p 5 0). The mean volume of bupivacaine was 5.2 ml/hr in group P and 9 ml/hr in group C which was also statistically significant; p value 5 0. The mean number of VAS scores $3 in group P (number of demands) was 2.05 while in group C it was 2.4. The pain relief achieved in both the groups was adequate & comparable. Grade 2 nausea & vomiting was observed in 3 patients in each group.Conclusions: Patient controlled epidural analgesia with background infusion is a better technique for IPB.

P226-F010DPD mutation in neoadjuvant chemotherapy in head and neck cancers. Myth or reality?

V. Patil1, V. Noronha1, A. Joshi1, S. Zanwar1, A. Ramaswamy1, S. Arya3, A. Mahajan3, A. Bhattacharjee2, K. Prabhash1

1 Department of Medical Oncology Tata Memorial Hospital, Mumbai, India2 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India3 Department of Radiodiagnosis Tata Memorial Hospital, Mumbai, India

Purpose: The TPF regimen in India is associated with high percentages of grade 3–4 toxicity. This analysis was planned to evaluate the incidence of DPD mutation in patients with severe GI toxicity, to assess whether the mutation could be predicted by a set of clinical criteria and whether it has any impact on post NACT response.Methods: All consecutive patients who received TPF regimen in head and neck cancers between January 2015 and April 2015 were selected. Patients who had predefined set of toxicities in Cycle 1 were selected for DPD mutation testing. Depending upon the results, C2 doses were modified. Post completion of 2 cycles, patients underwent radiological response assessment. Descriptive statistics has been performed. The normally distributed continuous variables were compared by unpaired student t test while variables which were not normally distributed by wilcoxon sum rank test. For non continuous variables, comparison was performed by fisher’s exact test.Results: Out of thirty four patients who received TPF, 12 were selected for DPD testing and 11 (32.4%, 95% CI 19.1%-49.3%) had DPD mutation. The predictive accuracy of the criteria for the tested DPD mutations was 81.3% (95% CI 62.1–100%). Of the 11 DPD mutation positive patients, except for one patient, all others received second cycle of TPF. The dose adjustments done in 5 FU were 50% dose reduction in 09 patients and no dose reduction in 1 patient. The response rate in DPD mutated patients was 27.3% (3/11) and that in DPD nonmutated/non tested was 39.1% (9/23) (p 5 0.70).Conclusion: Nearly 1/3rd of patients with Head and neck cancers in India have DPD mutation. Clinical toxicity criteria can accurately predict for DPD mutation. Post dose adjustments of 5 FU from C2 onwards, TPF can safely be delivered in majority of patients with DPD heterozygous mutations without decrement in efficacy.

P228-F011Neoadjuvant chemotherapy and surgical margin in technically unresectable buccal mucosa cancers

V. Noronha1, P. Sahu3, V.M. Patil1, A. Joshi1, S. Dhumal1, S. Kane2, A. D’Cruz3, K. Prabhash1

1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India2 Department of Pathology, Tata Memorial Hospital, Mumbai, India3 Department of Surgical oncology, Tata Memorial Hospital, Mumbai, India

Background: T4 buccal mucosa cancers with peritumoral edema reaching up to or above the level of zygomatic arch are termed as technically unresectable cancer. The risk of margin positive resection is high in such tumors. Neoadjuvant chemotherapy (NACT) followed by surgery remains an option in this group. Whether the margin positive rates of this cohort is similar to our upfront operated T4 technically resectable tumors is not known. This audit was done to address this


Methods: An analysis of prospectively collected data of 120 cases of GCT operated during February 2014 to October 2015 was done. 9 cases were recurrent and 21 were primary at presentation. The indication to start Denosumab was on a case by case basis with the aim to reduce the morbidity. The intent was documented at the start of therapy: convert resection to curettage (R-C), facilitate ease of resection (FR) or facilitate ease of curettage (FC). Denosumab was given in a dose of 120 mg subcutaneously at 0, 8th, 15th and 30th day and subsequently at monthly intervals (max- 7 doses). The response was assessed radiologically and clinically; documented as desired response or no response/disease progression.Results: The mean number of doses received was 5.6 (3 to 7). 25 cases achieved the desired result (R-C: 12 cases, FR: 10 cases, FC: 4 cases). 3 cases did not show response to Denosumab. 1 case (R-C) had a good response but the patient sustained fracture neck femur and underwent resection. None of the patients reported any adverse effects while on treatment. At a mean follow up of 11 months (range 4 to 22 months) two patients developed local recurrence for which they were subsequently operated.Conclusion: Though a longer follow up is mandated to decide its effect on local disease control, Denosumab has the potential to reduce the morbidity of index surgery in GCT thus facilitating better functional outcomes.

P245-F016Comparative evaluation of radiotherapy with concurrent weekly cisplatin versus concurrent daily erlotinib and weekly cisplatin in locally advance carcinoma cervix

S.J. Rawat1, M.K. Advait1, P. Kirar1

1 Department of Radiation Oncology, NSCB Medical College Jabalpur, India

Background: Carcinoma cervix in most common malignancy in female in rural population. Several studies have shown the superiority of platinum based chemotherapy combined with radiation when compared with radiation alone in localy advanced ca. cervix. Based on this the concomitant Weekly Cisplatinplus radiotherapy is a reasonable standarad of care. However despite the benefits obtained with addition of platinum based therapy have reached a peak. It has been shown that EGFR pathways play a major role in cancer progression. 80% of cervical cancer exhibits EGFR receptors which make it a candidate for EGFR inhibitor based therapy. Erlotinib is an oral well tolerated EGFR inhibitor that binds to the intracellular catalytic domain of EGFR.Aim: To compare weekly cisplatin versus daily erlotinib plus weekly weekly cisplatin with concurrent radiotherapy in locally advanced carcinoma cervix, to asseses the efficacy, tolerability, compliance and feasibility.Materials and Methods: Histologically proven case of carcinoma cervix, after taking consent, normal hematological, Biochemical parameters. EBRT given upto 50 Gy. The tablet Erlotinib started 5 days before irradiation in order to attain stable level and continued upto the end of EBRT and the patient will undergo HDR ICBT of 700cGY. In the study arm patients will be given erlotinib and weekly cisplatin, whereas those in the control arm will receive weekly cisplatin only.Results: Of the 30 patients who completed treatment in the study arm, 28 patients achieved full response and in the control arm of the 30 patients 19 achieved complete response and remaining achieved only partial response. The major side effects in study arm were loose stools which was managed by anti-motility drugs.Conclusion: Erlotinib plus weekly cisplatin along with radiation is a better agent in treating advanced cases of carcinoma cervix and side effects were easily manageable proving erlotinib is a radiosensitiser.

P265-F017Evaluation of anti-estrogenic and estrogenic activity of HM for the potential treatment of estrogen withdrawal (menopausal) symptoms

M.S. Kim1, S. Kang1, J.H. Kim1, K. Lee1, C. Jun2, S. Ko1

1 Lab of Clinical Biology and Pharmacogenomics, Department of Preventive Medicine, College of Korean Medicine, Kyung hee University, Seoul, Republic of Korea2 Department of Korean Internal Medicine, College of Korean Medicine, Gachon University, Seongnam, Korea

Estrogen Withdrawal Symptoms (EWS) refer to symptoms such as hot flush, bone mass loss caused by abrupt cessation of estrogen. Hormone replacement therapy has used to relieve such symptoms; however, the therapy has serious adverse effects, then complementary therapies like as phytoestrogen treatment and homeopathy, have been trying to replace it. In the present work, we evaluated the estrogenic activity of

Thedemographic profile, response to induction therapy, toxicity of chemotherapy, definitive treatment received, time totreatment failure (TTF) and overall survival (OS) were reported by us. This analysis was done to see long term resultswith this strategy. Univariate and multivariate analysis was performed to determine factors associated with OS.Results: The cohort of 41 patients had a median age of 48 years (22–71) with male preponderance (80.5%). The chemotherapy included two drugs (platinum and taxane) in 34 patients (82.9%) and three drugs (platinum, taxane and 5 FU) in 7 (17.1%) The taxane utilized was docetaxel in 22 patients (53.7%) and paclitaxel in 19 patients (46.3%). All patients completed two cycles of chemotherapy, adequate dose intensity was maintained in 33 patients (78%) and there were no deaths. Post-induction, the treatment planned included surgery in 12 (29.3%), CT-RT in 24 (58.5%), radical RT in 1 (2.4%), palliative RT in 1 (2.4%) and palliative chemotherapy in 3 (7.3%) patients. The median OS was 37.03 months (95% CI 9.1–64.96 months). On Cox regression analysis among tested variables, number of drugs in regimen, Type of drugs, Response, baseline albumin level, baseline hb level, type of resection, only factor associated with OS was albumin level. A low albumin level (4 g/dl or below) was associated with poor OS. The median OS in this cohort was 15.0 months as opposed to 52 months in patients with albumin above 4 g/dl (HR: 5.3 95% CI 1.16–24.72, p-0.031).Conclusions: In unresectable maxillary carcinoma, induction chemo-therapy followed by local treatment gives a 5 year OS of 35% which is much better than our historical results.

P236-F014Aggressive downstaging in hepatocellular carcinoma with portal vein tumour thrombus: Initial results of SHORT 1 TACE as neoadjuvant treatment before surgery

S. Nangia1, S. Gupta1, S. Agarwal1, H. Rastogi1, S. Vohra1, N. Goyal1, A. Agarwal1, P. Balakrishnan1, R. Khosa1, S. Rout1, S. Oommen1

1 Indraprastha Apollo Hospital, New Delhi, India

Background: Vascular invasion/Portal vein tumour thrombus (PVTT) is a contraindication for surgery/liver transplant for hepatocellular carcinoma, as per Milan & UCSF criteria, due to poor outcomes. Trans-arterial chemoembolization (TACE) and/or EBRT, are known to improve survival in HCC.Aim: To determine outcome of aggressive downstaging using stereotactic hypofractionated radiotherapy (SHORT) and TACE followed by surgery in selected cases of HCC with PVTT.Material & Methods: Consecutive patients with non-metastatic HCC with PVTT and adequate liver reserve underwent an aggressive downstaging regimecomprising TACE followed by fiducial directed stereotactic hypofractionated IMRT or RapidArc. Patients with complete/partial response, sustained for at least 3 months, and AFP , 400 IU were advised hepatectomy or liver transplant.Results: Thirteen patients, all male, age 35–61 years, Child Pugh Class A/B underwent SHORT. Eleven patients underwent TACE. Baseline AFP value ranged from 12 IU to 102000 IU. Tumour thrombus involved sectoral branches in 3 and main or branch portal vein in 10 patients. Radiotherapy target volume was 51–804 cc and normal liver 1130–2370 cc. Commonest dosage schedules were 50 Gy/10 fractions (4 patients) and 50 Gy/8 fractions (3 patients). Other patients received 32–48 Gy/6–14 fractions secondary to poor liver reserves. Three patients underwent surgery following the above, orthotopic liver transplant in 2, partial hepatectomy in 1. One patient underwent re-irradiation and is awaiting assessment for surgery. Four of 12 evaluable patients are alive at one year and 2 of 10, at 2 years.Conclusion: Aggressive downstaging with SHORT & TACE, followed by surgery in selected patients, provides reasonable survival in patients with dismal prognosis.

P243-F015Denosumab – A new bullet in the armamentarium for giant cell tumor

A. Gulia1, V. Verma1, A. Puri1

1 Bone and Soft tissue Disease Management Group (Surgical), Tata Memorial Hospital, Mumbai, India

Introduction: In view of the benign nature of Giant cell tumors (GCT) there is generalized consensus that function preserving intralesional surgery (curettage) is preferable to more morbid resections. Denosumab a fully human monoclonal antibody that binds and inhibits RANKL has the possibility of reducing the morbidity of surgery in GCT. We share our experience of using Denosumab in 30 operated patients of GCT.

G. Novel Diagnostics European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S33

resulted in formation of PTX-HSA-NPs with average diameter 184.8 6 6.3 nm (mean 6 SD) and zeta potential -26.8 6 3.1 mV (mean 6 SD). We also performed scanning electron microscopy (SEM) to observe the surface morphology of the HSA-NPs with and without PTX. Results suggest that increasing the HSA concentration increases the HSA-NPs diameter whereas increasing chloroform volume, homogenization pressure and number of repetitions decreases the HSA-NPs diameter. These Paclitaxel bound HSA-NPs will be used further for in vitro characterization as well as in vivo studies. Therefore, using this new method, it is possible to develop PTX-HSA-NPs with controlled size and surface properties for use in cancer therapy.

G. Novel Diagnostics

OF70-005Can we predict molecular subtypes of medulloblastoma based on MRI? Encouraging data from a blinded study

A. Dasgupta1, T. Gupta1, S. Pungavkar2, A. Janu3, N. Shirsat4, G. Chinnaswamy5, R. Jalali1

1 Department of Radiation Oncology, Tata Memorial Centre, Mumbai, India2 Department of Radiodiagnosis, Global Hospital, Mumbai, India3 Department of Radiodiagnosis, Tata Memorial Centre, Mumbai, India4 Department of Neuro Oncology, Advanced Centre for Treatment, Research and Education in Cancer, India5 Department of Paediatric Oncology, Tata Memorial Centre, Mumbai, India

Background: Recent investigations on medulloblastoma has revealed at least 4 different subgroups with diverse clinical, demographic profiles rendering better insight in understanding the heterogenous entity disease.Aims and Objectives: The study deals with the radiomics as an attempt of unifying the molecular subgrouping obtained from biological material with the radiological features obtained through the MRI scans of the patients.Methods and Materials: After obtaining the IEC approval the pre-operative scans of 35 patients were collected with due consent or assent. Interpretation was done on a set of 22 prefixed radiological parameters based on our previous experience and few other recent studies. Molecular subgrouping into either of the four groups- WNT, SHH, group 3 or group 4 was done based on RT-PCR. The scans were discussed in the institutional joint meeting of neuro-oncology group comprising of specialists from each of neuroradiology, radiation oncology, neurosurgery, paediatric oncology, who were blinded to the results regarding the molecular groups. Based on the features studied and our previous experience a provisional diagnosis was given predicting the molecular subgroups. In cases of controversy, a second probable diagnosis was also noted. Statistical analysis was done using IBM SPSS software (V20).Results: Patients ranged from 2 years to 36 year (mean-13 years) and 22 of 30 were male. RT-PCR based miRNA profiling revealed 6 patients to be in WNT, 12 in SHH, 5 in group 3 and 7 in group 4 molecular subtypes. Cross tab analysis revealed four distinct radiological features, viz horizontal tumor location (p-0.001), involvement of dorsal brainstem (p-0.032), perifocal edema (p-0.02), intratumoral calcification (p-0.002) significantly predictors of accurate molecular subtype. Overall, the prediction was correct for 20/30 (66%) taking 2nd prediction into account with kappa correlation coefficient for agreement being 0.43 (moderate agreement).Conclusion: We have demonstrated significant correlation between sets of certain radiological parameters with the molecular subgroups in medulloblastoma, which can have important implications in our clinical practice. These features need to be confirmed in the ongoing study and in an independent cohort of patients.

OF262-009BRAF v600e mutation confers poor survival and lack of response to conventional therapies in childhood low-grade glioma. A report from the PLGG taskforce, Toronto Canada

R. Krishnatry1, A. Arnaldo2, M. Mistry2, C. Ling2, A. Lassaletta2, E. Bouffet2, C. Hawkins2, U. Tabori2

1 Consultant Radiation Oncologist, Mazumdar Shaw Cancer Center, Bangalore, India2 The Hospital for Sick Children, Toronto, Canada

Background: Pediatric low-grade gliomas (PLGG) are the most common CNS tumor in children. PLGG are histologically and clinically diverse and

10 herbal medicines (HM) and examined its molecular mechanism. ER-positive breast cancer cells (MCF-7, T47D), prostate cancer cells (PC-3, DU145, LNCaP) and ER-positive endometrial adenocarcinoma Ishikawa cells were used. Treatment of HM resulted in exhibited (a) binding to estrogen receptors a (ERa) and estrogen receptors b (ERb) (b) induction of alkaline phosphatase (AP) activity, (c) increment of progesterone receptor (PR) mRNA expression, and (d) regulation of the estrogen-regulated target gene, presenelin-2 (pS2) mRNA expression. These data suggest a potential use for HM in the treatment/relief of EWS.

P273-F018An offbeat approach using genetic targets of cellular H2O2 generator to improve radiation therapy in breast cancer

S. Dutta1, A. De1

1 Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, India

Background: Radiation therapy is one key treatment modality in breast cancer (BC), which often suffers with huge side effects due to damage of surrounding normal tissue. Radiation treatment is also complicated by cellular adaptations upon repeated radiation exposure, called radio-resistance. Here, outside the extant dogma, we focus on elucidating the role of cellular hydrogen-peroxide (H2O2) generator gene Duox1 and Duox2, revealing important results with potentials to improve radiation therapy.Methods: We used semi-quantitative RT-PCR to investigate changes in transcriptional expression of two core H2O2 generating genes - Duox1 and Duox2 in BC cell lines with or without exposure to fractionated cobalt60 radiation. DNA damage response was measured by gH2AX immonufluorescence followed by radiation exposure. Protein expression is checked by immunofluorescence and functional activity by measuring cellular H2O2 level with or without over expression of Duox1/Duox2.Results: Duox1 transcript is found absent in differentiated BC cell lines or even upon low or high dose radiation exposure of cells. However, fractionated radiation exposure of 0.2 Gy (3 doses; accumulated to 0.6 Gy) show 2 fold increase of Duox2 transcript than radiation unexposed cells. Further, when BC cell lines treated with radio sensitizers like 2-deoxy glucose (2DG) and metformin followed by radiation exposure of 0.2 Gy and 2 Gy, we observed 2–4 fold increase (p # 0.05) in Duox2 transcript, but not Duox1 transcript. Interestingly, we also observe that the level of Duox2 transcript is down-regulated in radio-resistance BC cell lines developed in our laboratory. Further confirmation to these observations is made by measuring the Duox2 protein expression and cellular H2O2 content in the BC cell lines tested.Conclusion: For the first time, this study reveals Duox2 as the key player to sensitize breast cancer cells to radiation injury by producing higher H2O2 inside the cells and thus impose to be an important target game changer for radiation therapy in BC.

P293-F019Preparation of protein nanoparticles for targeted delivery of paclitaxel for use in cancer

N. Lomis1,2, S. Westfall1, L. Farahdel1, D.S. Tim3, S. Prakash1,2*

1,2* Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Montreal, Quebec, Canada1* Division of Experimental Medicine, Montreal, Quebec, Canada3 Division of Cardiac Surgery and Surgical Research, Royal Victoria Hospital, Montréal, Quebec, Canada

Cancer is one of the most fatal diseases caused by an uncontrolled growth of cells which may spread in the body. Despite the advancements in medical technology, there is still no certain cure for this disease. Common treatments include radiation, surgery and chemotherapy. However, their limitations are: lack of drug bioavailability, renal toxicity, and toxicity due to drug accumulation in non-target organs apart from other side-effects. Therefore, our aim was to design and develop human serum albumin nanoparticles (HSA-NPs) containing the anti-cancer drug, Paclitaxel (PTX) for site-specific drug delivery. A high-pressure homogenizer was used to prepare PTX-HSA-NPs of less than 200 nm diameter. The size of NPs must be controlled in order for them to remain in circulation, when administered into the body. We identified and optimized four crucial parameters: HSA concentration (mg/mL), organic solvent volume (%), homogenization pressure (psi) and number of repetitions of homogenization. An emulsion prepared from HSA (1%–4% aq. solution) and chloroform (1%–10%) was forced through a small diameter funnel at high pressure of 10,000–20,000 psi between 3–12 times. This was followed by rotary evaporation of chloroform, which

S34 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 G. Novel Diagnostics

prognosis. Lately spindle cell/ sclerosing RMS has been recognized as it’s another subtype. Only two studies from western population have unraveled MYOD1 L122R as the underlying mutation of this subtype.Methods: Genomic DNA was isolated from formalin fixed paraffin embedded tumor tissue samples. Targeted PCR was performed to amplify the MYOD1 L122R and PIK3CA (E542/E545 and H1047) exons. Purified PCR products were sequenced by Sanger sequencing and mutations were analyzed using Mutation Surveyor software V4.0.9.Results: We identified 8 (25%) out of 32 RMS samples with MYOD1 L122R mutation, including spindle cell/sclerosing (2); predominantly spindle cell (3); predominantly sclerosing (2) and a single RMS that could not be subtyped further. Among 8 MYOD1 L122R mutated samples, 5 samples have heterozygous mutation while other 3 samples have homozygous mutation. None of the sample we analyzed showed PIK3CA (E542/E545 and H1047) mutations. None of the remaining cases, including embryonal, pleomorphic or alveolar RMSs revealed MYOD1 L122R mutations.Conclusions: Our study provides first evidence for the presence MYOD1 L122R mutations in spindle cell/sclerosing RMS among Indian cohort, reinforcing this as a distinct RMS subtype. In view of its high specificity, MYOD1 L122R could be targeted for treating spindle cell/sclerosing type of RMSs.

P4-G001Ensuring cGMP compliance in production and quality control of various diagnostic radiopharmaceuticals – used in cancer treatment and management

A. Mitra1, S. Lad1, N. Yuvaraj1, S. Kulkarni1, M.G.R. Rajan1

1 MCF, RMC, BRIT/BARC, TMH Annexe, Tata Memorial Hospital, Mumbai, India

Background: With the inclusion of Radiopharmaceuticals (RPs) in the Indian Pharmacopoeia, cGMP in the production and rapid quality control of various diagnostic radiopharmaceuticals in hospital radio-pharmacy is a necessity that has to be met. There is a growing demand for these RPs for cancer diagnosis and treatment. At our centre the automated production of various pharmaceutical grade 18F, 99mTc and 68Ga labelled radiopharmaceuticals under clean air room are carried out while rapid physicochemical, biological quality control tests are performed prior to administration in patients, thereby fulfilling pharmacopoeial, cGMP and cGRPP regulations. The data presented here shows the different RPs produced at our centre, which are approved by the DAE-Radiopharmaceuticals Committee (RPC) after seeing the production and QA manuals so that parametric release for short lived RPs is permitted.Materials: Automated radiochemistry module, precursors, reagents and solvents required for radiochemical chemical synthesis.Methods: [18F]FLT, [18F]FMISO, [18F]NaF and [68Ga]DOTATATE synthesized in automated indigenous radiochemistry module. 99mTcO4

- extracted from low specific activity 99MoO4

2- using 99Mo/99mTc-TCM-Autosolex generator. Extracted 99mTcO4

- labeled with various TCK. API’s used in radiochemical synthesis prepared inhouse and quality control tested. RCP of [18F]FLT, [18F]FMISO, [18F]NaF, 99mTcO4

-, [99mTc]MIBI, [99mTc]Hynictoc, [99mTc]HSA-NC and [99mTc]TRODAT determined by inhouse developed rapid TLC/PC whereas endotoxin limit (EL) quantified by rapid gel clot-BET assay prior injections. Sterility test performed post-facto. Residual solvents quantified by gas chromatography.Results and Conclusions: For PET, we have produced pharmacopoeia grade [18F]FLT (n 5 157 batches), [18F]FMISO (n 5 112 batches), [18F]NaF (n 5 521 batches) and [68Ga]DOTATATE (n 5 172 batches) using automated process developed indigenously. For SPECT, we have produced [99mTc]MIBI, [99mTc]Hynictoc, [99mTc]HSA-NC, [99mTc]TRODAT, [99mTc]MDP, [99mTc]EC, [99mTc]DTPA and [99mTc](III)DMSA labelled with 99mTcO4

- obtained from automated solvent extraction process. All these batches of PET and SPECT RPs fulfil the physicochemical, radiochemical and biological quality control (QC). Since the process are validated and DAE-RPC approved, parametric release is followed with QC compliance.

P12-G002Role of technology in cervical cancer diagnosis

H. Shah1, S. Noronha1

Indian Institute of Technology, Bombay, Powai, Mumbai, Maharashtra, India

A major problem when addressing cervical cancer in India is the absence of a community-level screening system. It is important to screen women at a young age, towards avoiding expensive treatment later. In this work, we seek to understand the role of technology in cervical cancer

molecular stratification is desperately needed. BRAFV600E mutations are highly targetable for therapy but their biological relevance and impact on PLGG outcome is unknown.Methods: We determined BRAF mutation status on a large population based cohort of 399 PLGG with long-term outcome data. Tumors tested for BRAF-V600E, BRAF fusion using the QX200™ Droplet Digital™ PCR and nanostring technologies. Long term clinical data and imaging response to chemotherapy and radiation were collected on all patients.Results: BRAF-V600E mutation was observed in 23% (90/399) of PLGGs tested. Mutations were observed in previously undescribed histological subtypes such as pilomyxoid, diffuse astrocytomas and DNETs, and in locations including spinalcord, brainstem and optic pathways which are notcommonly biopsied. BRAF-mutated PLGG had poor long term outcome, especially for nonresected midline tumors with 15 years PFS of 7 6 6% and OS of 57 6 16% vs. PFS of 64 6 12% and OS of 94 6 6% for gross totally resected tumors. PFS was poor post both chemotherapy and radiation with 10 year PFS of 29.5 6 11% and 31.0 6 12% respectively. Importantly, comparison of Multiple common alterations in PLGG revealed that at 10 years, PFS were 84%, 50% and 20% for NF1, BRAF fused and BRAF mutant PLGG respectively (p 5 0.001). Specific quantitative Imaging analysis revealed response to chemotherapy in only 20% of BRAF-mutant PLGG. In contrast a V600E mutant PLGG in a 3 months old baby which was refractory to conventional chemotherapy and blind had .85% response to targeted V600E inhibition within 2 months of therapy with visual recovery.Conclusions: BRAF-V600E mutations are common and stratify a unique group of PLGG with distinct biology and survival. Early biopsies, aggressive surgery and targeted therapies may transform the management and outcome of these children.

OF193-012Raman spectroscopy for classification of high grade glioma from adjacent normal tissue: Towards surgical demarcation

A. Hole1, S. Mohanan1, S. Chatterjee1, E. Sridhar1, A. Moiyadi1, C.M. Krishna1

1 ACTREC, Tata Memorial Centre (TMC), Mumbai, India

Background: Glioma accounts for about 45% of all brain cancers. While the current diagnosis of gliomas is carried out by a combination of CT scanning, magnetic resonance imaging and electroencephalogram, the standard treatment is surgical resection in conjunction with adjuvant radiotherapy and chemotherapy; extent of resection is known to correlate with prognosis. Incomplete resection may lead to recurrence, while radical excision may lead to cognitive deficits. Adjuncts to facilitate proper surgical resection are imperative. Raman spectroscopy, a vibrational spectroscopic approach, has shown potential in differentiating normal and malignant tissues, for several cancers. In the present study, potential of RS in classifying normal and tumor from surgically-resected glioma and normal tissues was investigated.Method: Spectra were acquired from 50 micron sections obtained from 23 tissues (21 high grade glioma, 2 normal) using a commercial HE-785 Raman instrument. Histopathological evaluation on two 5 micron sections, before and after the 50 micron section, was also carried out.Results: Principal component based linear discriminant analysis (PC-LDA) was employed for building of standard models and evaluation of test data, leave-one-out-cross-validation (LOOCV) was used for validation. Standard model was built using 120 spectra from 14 high-grade and 12 spectra from 2 normal tissues. LOOCV findings indicate 12/12 normal spectra and 111/120 were correctly classified. Test prediction carried out using 7 independent high-grade tissues yield 183/197 correct predictions for glioma.Conclusion: Thus, the present study demonstrates potential of Raman spectroscopy in delineating normal from tumor tissue. After prospective validation, this approach may be used as a useful adjunct for surgical demarcation.

OF16-001Molecular characterization of MYOD1 and PIK3CA mutations in rhabdomyosarcomas

M. Ramteke1, B. Rekhi2, P. Upadhyay1, A. Dutt1

1 Integrated Genomics Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Mumbai, India2 Department of Surgical Pathology, Tata Memorial Centre, Parel, Mumbai, Maharashtra, India

Background: Rhabdomyosarcoma (RMS) is a high-grade sarcoma of skeletal muscle origin and constitutes as one of the chemosensitive sarcomas. It has various clinicopathological subtypes with variable


immobilization of the eye. Aim: Develop an indigenous non-invasive eye gaze tracking system (ETS) to modulate STRT in eye tumors.Methods: The standard non-invasive Fraxion Stereotactic Frame (Elekta) front-piece was modified using Perspex plate with original attachments but additional platform to hold ETS device. The ETS comprised of an Infrared low illumination network camera and a light emitting diode (LED) system. The patient focused on LED light with non effected eye, while camera captured real-time image of iris which was displayed at treatment console using standard camera driver software. The ETS assembly is adjustable and fixable in X and Y plane, and camera focal length can be adjusted to 60 mm. The treatment can be gated manually according to iris motion. This was tested for time lag as well as interference in image quality arising with the head beam turned on.Results: The time lag was estimated to be 5 milliseconds. The image quality was in acceptable limit of interference with direct or scatter beam. The first patient treated with this technique is ocular surface neoplasm involving left half of left eye. As patient is ongoing treatment, further results will be presented at the conference.Conclusion: Indigenous ETS development for STRT in eye tumors is feasible with collaboration between radiation oncologist, ocular oncologist and engineers in the setting of developing countries.

P71-G005Anti-tumor activity of EGFR inhibitors in the NSCLC patient derived xenograft diseases: Classic vs non-canonical EGFR mutants

H. Qi-Xiang Li1,2, M. Yang1, X. Xu1, J. Cai1, J. Ning1, S. Guo1, J.P. Wery1

1 Crown Bioscience Inc., 3375 Scott Blvd, suite 108, Santa Clara, CA, USA2 State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China

Activating EGFR mutations, including the classic exon-19 deletion, exon-21 L858R, exon-18 T790M and the non-canonical exon-20 insertion/exon-18 G719A mutations, are oncogenic drivers for many non-small cell lung cancer (NSCLC). At least the classic mutations have been considered important targets for developing several EGFR inhibitors, TKIs of different generations or monoclonal antibodies. However, not all mutations, and/or not all inhibitors, are equal. One of the most important hurdles toward understanding this is the shortage of experimental systems truly predictive of patient diseases.The NSCLC patient derived experimental systems are the ideal tool for understanding the patient EGFR molecular pathogenesis and testing various EGFR inhibitors to guide precision treatment. To this end, we treated this panel of EGFR mutant PDXs with various EGFR inhibitors at conventionally used preclinical doses to assess their responses (%ΔT/ΔC). Interestingly, all the classic EGFR activating mutations are generally sensitive to certain EGFR inhibitors, both TKIs and antibody, except that L858R/T790M (to the first generation TKIs) or the ones with alternative driver mutation (e.g. c-met gene amplification) (to all). These are largely correlated to clinical observations. In contrast, PDX-trial seems to suggest that the canonical exon-20 insertions immediately following regulatory C-helix of the kinase domain, as well as the exon-18 G719A mutant, generally responded poorly to all the tested EGFR inhibitors, consistent with the available anecdote clinical observations. Although the PDXs responses could be broadly predicted according to the mutational type (classic vs. non-canonical), each individual mutant can still display differences in response to various inhibitors. In summary, our in vivo and in vitro data from patient derived experimental models confirmed a pronounced difference between classic and non-canonical EGFR mutations in terms of responses to available EGFR inhibitors: classic mutations confer sensitivity, non-canonical mutants resistance, underlining fundamental differences in structure and biochemical properties of these mutants.

P78-G006Test for HPV induced cancer in low resource settings via direct detection of elevated HPVE6 oncoprotein

J. Schweizer1, M. Belmares1, S. Charles1, E. Gaillard1, O. Petrauskene1, S. Reveilhe1, E. Tajan1, C. Tan1, P.E. Castle2, J. Jeronimo3, Y.L. Qiao4, K. Effah5, A. Kaufmann6, P. Lu1

1 Arbor Vita Corp., Fremont, CA, USA2 Global Coalition Against Cervical Cancer, Arlington, VA, USA3 PATH, Seattle, WA, USA4 Cancer Institute and Hospital, Beijing, China5 Catholic Hospital Battor, Ghana6 Charité Hospital, Berlin, Germany

The high mortality caused by cervical cancer in the developing world calls for screening technology that allows widespread implementation

diagnosis. We have developed an inspection device and a digital scanner to support the VIA-VILI and PAP smear test in the field and reduce the burden on tertiary care facilities.The inspection device is a portable solution which captures a printable/electronically transferable high-resolution image of the cervix. After the healthcare worker performs the VIA-VILI test on the patient, she can capture the magnified image of the cervix, store it in an EMR, print it out, or send it to a referral center for evaluation, avoiding re-testing at the referral center.The scanner digitizes cell slides collected in the field and sends image files to a referral center; replacing the physical transfer of slides between the centers. The digital slide is presented as a panoramic image; relevant fields of view can be zoomed into for closer inspection. The images, stored on a central server, can be accessed via a web-portal which facilitates interaction between cyto-technicians on special cases. In addition to PAP smear analysis, this device can be used for other tele-pathology applications. For e.g., a multiple eyepiece microscope in pathology classroom can be replaced with monitors, increasing the student-teacher interaction.We are in the process of evaluating the appropriateness of these technologies for preventive oncology in the field. A few solutions currently exist in the West, but are prohibitively expensive and lack all the required functionality needed for deployment in an Indian context. We expect that our solutions will be indigenously manufactured at a significantly lower cost.

P20-G003Is the cardiac PAGE warranted for a patient with a diagnosis of life limiting illness? Is a trial of intubation justified in the setting of advanced cancer?

R.D. Arora1

1 Department of Palliative Medicine, Tata Memorial Centre, Mumbai, Maharashtra

Case Description A 34 year old Female, a known case of NSCLC Lung with Cerebellar metastasis with history of Pulmonary Thromboembolism awaiting Ceratinib as targeted therapy presented with complaints of sudden onset of breathlessness. She was on oxygen at the rate of 5–6 litre per minute and underwent CBC, ECG and Chest Radiograph. A history of cessation of LMWH treatment one month ago was obtained. Rapid desaturation was noted whereby a Central cause of breathlessness (raised ICT) was considered and decision to provide a trial of intubation was taken. A Cardiac Page was initiated. Troponin Assay performed subsequent to poor r wave progression on ECG was negative. The resuscitation team did not want to intubate the patient as poor lung function meant requirement of a ventilator which was not available as part of the Hospital Protocol. Decision to transfuse was taken after review of reports.Discussion Reason for discontinuation of Low molecular weight heparin which has been associated with prolonged survival, was the diagnosis of HIT confirmed? Cause of trilineagecytopenia? Is D Dimer warranted in this setting. Does an inability to provide ICU Admission at this junction ignore evidence that curbing maladaptive physical responses may lead to an improvement in patients survival? Are we in danger of interpreting Comfort care as a cessation of all interventions and symptom directed therapy? Is Intubation not to be considered as a life prolonging measure in this situation? Are we ready for a setup where ventilators are a part of APCU s linked to the ICU?

P69-G004Indigenous eye gaze fixation and tracking system to help stereotactic radiotherapy for eye tumors: first report from an Indian center

R. Krishnatry1, H. Gupta2, K.K. Pramod3, S. Kumar3, K. Nithiyanantham1

1 Department of Radiation Oncology, Mazumdar Shaw Cancer center, Bangalore, India2 Department of Ocular Oncology & oculoplasty, Narayana Netralaya, Bangalore, India3 Kummaya Design Studio, Bangalore, India

Background: External radiation is used for various eye tumors like post chemotherapy residual retinoblastoma, juxtrapaillary melanoma, ocular surface neoplasia etc. It is crucial to reduce the treatment volume margins to spare critical normal structure & simultaneously achieve high precision for tumor. The eye moves independent of head, hence conventional stereotactic frames are not sufficient. Periocular or general anesthesia are invasive procedures and associated with uncontrolled eye rolling. Although stereotactic radiotherapy (STRT) is commonly at all sites, no commercial solution is available to achieve precise, reproducible


Materials and Methods: Over 40 patients with biopsy/histopathology proven prostatic adenocarcinoma (10 staging; 12 restaging/recurrence evaluation post-surgery, radiotherapy or hormonal therapy) with raised serum PSA underwent simultaneous PET/MRI using [68Ga] Ga-PSMA-HBED-CC tracer. Prostatic bed lesion (Residual/recurrent), nodes and metastases were evaluated on PET, MRI and PET/MRI for lesion count and diagnostic confidence (DC). Histopathology, clinical/imaging follow-up served as the reference standard.Results: Of 40 patients, 10 did not reveal any locoregional or distant metastatic disease on follow up; 15 showed features of local recurrence in prostatic bed, 13 had lymph nodal involvement with upto 28 distinct lymph nodal lesions and 22 had distant metastasis yielding more than 54 distinct bony, brain, hepatic and lung metastatic lesions. Fused PET-MRI showed highest diagnostic confidence score as compared to PET and MRI alone. Apart from distinctly detectable larger nodal and bone lesions, 68Ga-PSMA PET/MRI identified smaller metastases and small sized lymph nodal involvement.Conclusions: Our preliminary results indicate possible incremental benefit of 68Ga-PSMA PET/MRI compared to MRI alone. 68Ga-PSMA PET/MRI shows promising potential in staging/restaging men with biochemical failure post definitive treatment of prostate cancer.

P99-G009Is it time to abandon invasive bone marrow biopsy in Non-Hodgkin’s lymphoma (NHL) – A study assessing the correlation of bone marrow involvement on 18 FDG PET/CT imaging and histopathological bone marrow biopsy

A. Pai1

1 Sri Venkateswara Institute of Medical Sciences, Tirupati, India

Objective: Invasive bone marrow biopsy is a part of staging workup of Non-Hodgkin’s lymphoma (NHL). However bone marrow involvement on 18 FDG PET/CT imaging has a high sensitivity and specificity. Hence this study was undertaken to predict the correlation of the bone marrow involvement by 18 FDG PET/CT imaging and histopathological bone marrow biopsy.Methods: Thirty eight consecutive patients diagnosed as NHL based on lymph node biopsy and who underwent whole body 18 FDG PET/CT scanning and bone marrow biopsies were included. The demographic profile, the haemogram parameters, the bone marrow involvement based on biopsy and PET/CT were noted. The correlation between the bone marrow involvement in PET/CT and bone marrow biopsy was done using Pearson’s correlation analysis. A p value of less than 0.05 was considered significant.Results: The median age was 52.5 years. Males were 20 (52.6%) and females were 18 (47.4%). The commonest histology was diffuse large B cell (20, 52.6%). The stage wise distribution was 55.3% stage IV, 26.3% stage III, 5.3% stage II and 13.2% stage I. Bone marrow biopsy showed lymphomatous infiltration in 16 patients (42.1%) and bone marrow involvement in 18 FDG PET/CT was seen in 19 patients (50%). Concordance between bone marrow biopsy and bone marrow involvement on PET/CT was seen in 14 patients. PET/CT upstaged the disease in 5 patients and failed to detect bone marrow involvement in 2 patients. However by Pearson’s correlation bivariate analysis there was a positive correlation between bone marrow biopsy and bone marrow involvement by PET/CT (r 5 0.640, n 5 38, p 5 0.000).Conclusion: This study showed that PET/CT accurately and positively correlates with bone marrow biopsy for lymphomatous involvement. Hence invasive bone marrow biopsy may be considered to be abandoned in patients who are PET/CT positive for bone marrow involvement in NHL.

P111-G010Multiparametric assessment of glioma recurrence with simultaneous O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) positron emission tomography magnetic resonance imaging (PET/MRI): A novel technique

S.K. Sogani2, A. Jena1, S. Taneja1, A. Gambhir1, R. Sarin4, S. Verma3, P. Negi1, G. Jadhav3

1 Pet Suite, Department of Molecular Imaging and Nuclear Medicine, Indraprastha Apollo Hospitals, New Delhi, India2 Institute of Neuro Sciences, Indraprastha Apollo Hospitals, New Delhi, India3 Institute of Radiation Oncology, Indraprastha Apollo Hospitals, New Delhi, India4 Institute of Surgical Oncology, Indraprastha Apollo Hospitals, New Delhi, India

Purpose: To investigate the potential of simultaneous 18F-fluoroethyl-L-tyrosinePET/MRI in distinguishing recurrence from radiation necrosis using multiple structural and functional parameters.

while minimizing unnecessary over treatment. The Onco E6TM Cervical Test (E6 Test) has been developed to meet requirements specific to low and limited resource settings: it is robust and easy of use; the unique feature of direct detection of elevated levels of the cancer causing HPV viral oncoprotein E6 results in high clinical specificity and high positive predictive value (PPV).The E6 Test features lateral flow format and is void of cold chain requirements for storage and for sample collection. Various sample types can be applied, including self-collected samples. The test procedure allows adaptation of sample throughput depending on actual needs in a given setting from single specimen to several dozen specimens applied per operator and session. The time from sample collection to result is 2.5 hours, allowing for immediate action upon a positive result and reducing thus the risk of loss upon follow-up. The E6 Test only requires low complexity equipment representing a comparatively low capital investment for laboratory setup.In clinical studies, the E6 test resulted in specificity around 99% for CIN3+ and PPV of 50% for CIN2+ (HPV testing and VIA had PPV of 15% in the same population); these studies also demonstrate that a positive E6 Test in women with normal pathology informs a high risk of future disease, and that the E6 Test can detect cases of micro-invasive cancers where the matching cytology outcome is negative.A synopsis of E6 Test clinical performance in field studies and possible algorithms for use of the E6 Test in different settings will be given.

P82-G007VEGF expression and its correlation with histopathological parameters in GBC of North Indian population

K. Mishra1, P. Shukla1, R. Vishwakarma1, N. Kumari1, N. Krishnani1, A. Behari1, V.K. Kapoor1

1 Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Introduction: Gall bladder cancer (GBC) is relatively rare malignancy, and 5th most common cancer of the biliary tract. GBC remains asymptomatic unless it attains advance stage and has poor prognosis attributed to complex nature and slow pathogenesis. The limited knowledge of molecular changes in GB carcinogenesis has motivated us to study the role of VEGF expression in differentiation of tumors. We carried out VEGF expression analysis in GBC tumors by performing immunohistochemistry.Method: VEGF expression analysis was carried out by Immunohistochemical staining on formalin fixed paraffin embedded tissue samples (n 5 93) using anti-VEGF antibody.Results: Among 93 GBC cases included in the study, 22 are males and 71 females (M/F ratio 1:3.02) with median age 55 years (mean 53.02 year). Older patients (.40 years, n 5 77) had higher expression of VEGF than younger patients. Over all, we observed VEGF expression in 40 cases, most of these tumors were well differentiated (27/40) with II (14/27) and III (12/27) stage of the disease. Nodal metastasis, perineural invasion and lymphovascular invasion was observed in 14, 17 and 5 GBC cases respectively.Conclusion: In our study, poorly and moderately differentiated tumors showed low VEGF expression, however in several other studies poorly differentiated tumours showed high VEGF expression compared to well differentiated tumors. Well differentiated adenocarcinoma are associated with good prognosis but presentation with VEGF expression ultimately leads to bad prognosis. So in turn increases the complex nature of the disease thereby diagnosis demands clinical, histopathological and molecular data.

P84-G008Gallium 68 prostate-specific membrane antigen simultaneous positron emission tomography–magnetic resonance imaging: A novel approach in evaluation of carcinoma prostate

A. Gambhir1, A. Jena1, S. Taneja1

1 Department of Molecular Imaging and Nuclear Medicine, Indraprastha Apollo Hospital, New Delhi, India

Aim: At present, Positron emission tomography (PET) combined with computed tomography (CT) is considered as the most accurate method of oncologic staging of recurrent prostate cancer. However PET/CT is marred by the limited soft tissue contrast of CT which may be overcome using a fully integrated, whole body simultaneous PET/MRI system combining high-resolution simultaneous morphologic and functional data. The purpose of this study was to assess the feasibility and the utility of whole body simultaneous 68 Ga PSMA PET/MRI in restaging recurrent prostrate carcinoma.


squamous cell carcinoma (HNSCC). In the current study we have comprehensively analyzed prevalence of transcriptionally active HPV in Indian leukoplakia and HNSCC. In addition saliva samples from HNSCC patients were screened as non-invasive method for HPV detection.Study Design: HPV testing was performed in leukoplakia (n 5 121), HNSCC tissues (n 5 427) and (n 5 215) saliva using nested PCR for L1 region followed by sequencing of positive samples for subtyping. In addition, p16 overexpression was analyzed by Immunohistochemistry (IHC). Presence of transcriptionally active HPV was confirmed by RNA in-situ hybridization.Results and Conclusion: None of the leukoplakia’s were HPV-positive, while 7/427 HNSCC were positive for presence of HPV16 DNA. However, RNA-positivity was observed in 8 samples and majority of these tumors were from patients without any tobacco habit. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All the HPV positive saliva samples showed expression of E6/E7-mRNA in concomitant tumors. Nevertheless, presence of HPV DNA does not necessarily reflect transcriptionally active virus; hence, it is important to consider this fact while categorizing HPV associated tumors.

P157-G013Kimura disease of head and neck: MR imaging findings

M. Mundhe1, N. Merchant1, M. Thakur1

1 Department of Radiodiagnosis, Tata Memorial Hospital, Parel, Mumbai, India

Background: Kimura disease is a rare chronic lymphoproliferative disease commonly seen in adult Asian population. It is of unknown etiology, however considered to be due to allergic or autoimmune response.It is common in the head & neck region especially in the major salivary glands and lymph nodes. Clinically presents as painless swelling in head & neck with associated involvement of regional lymph nodes mimicking a malignant neoplasm. Only few reports are available on MR imaging findings in the literature. For the definitive diagnosis, histopathological confirmation is required. We present three cases of Kimura disease (histopathologically confirmed) in head & neck region with emphasis on MR imaging findings.Methods and Results: Three cases of Kimura disease involving parotid, lacrimal gland & neck nodes with MR imaging findings were reported.Clinically these presented as painless swelling with elevated serum globulin levels. In first two patients, neoplastic mass (parotid / lacrimal gland tumour) & last patient tuberculous or malignant adenopathy was suspected. On MR imaging lesion appeared hypointense on T1W, heterogeneously hyperintense on T2W/STIR with marked heterogeneous nodular type enhancement on post contrast T1W fat sat images. Adjacent subcutaneous tissue involvement is noted. In one patient, recurrence was noted after parotidectomy.Conclusions: Kimura disease is often clinically & on imaging difficult to diagnose & may mimic a neoplasm. It should be considered as one of differential diagnosis for head & neck masses. On MR imaging, well defined margin, nodular pattern enhancement with subcutaneous tissue involvement are the characteristic findings. In addition, peripheral blood eosinophilia, elevated serum globulin levels present. These features help to suspect Kimura disease which avoids unnecessary diagnostic tests and radical surgery.

P158-G014Prediction of facial nerve position in vestibular schwannoma using diffusion tensor imaging (DTI) tractography and its intra-operative correlation

S.A. Borkar1

1 Department of Neurosurgery & Gamma Knife, All India Institute of Medical Sciences & Jai Prakash Narayan Apex Trauma Center, New Delhi, India

Objective: Resection of large vestibular schwannomas (VS), can be associated with postoperative facial nerve injury. Diffusion-based tractography has emerged as a powerful tool for three-dimensional imaging and reconstruction of white matter fibers; however, tractography of the cranial nerves has not been well studied.Material & Methods: Twenty patients with large vestibular schwannomas (. 3 cm) undergoing surgery were subjected to preoperative DTI imaging to predict the position of facial nerve in relation to the tumor. Diffusion tensor imaging sequences (using 32 channel head coil, no intersection gap) was acquired with a 3-T MR imaging scanner. The automatic fusion algorithm of the software was used to merge

Methods: 32 patients (7 females, 25 males; mean age 6 SD: 51.58 6 15.97) with single contrast enhancing brain lesions (n 5 32) on MRI after surgery and radiation therapy were evaluated with simultaneously acquired Gd enhanced 18F-FET PET/MRI. They were then followed up with histopathology and/or clinical follow-up or MRI or PET/MRI based imaging follow up. PET/MRI images were analyzed using manually drawn regions of interest over areas of maximal contrast enhancement and/or FET uptake. TBRmax, TBRmean, Cho:Cr ratios, N rCBVmean and ADCmean were determined. Accuracy of each parameter individually and in various possible combinations for differentiating recurrence vs. radiation necrosis was evaluated using two tailed independent samples student t-test, MANOVA and multivariate receiver-operating-characteristic analysis. Positive histopathology and long term imaging/clinical follow up suggestive of disease progression served as gold standard.Results: 25 of 32 patients were classified as recurrence with 7 showing radiation necrosis. Individually, TBRmax, TBRmean, ADCmean and Cho:Cr ratios and N rCBVmean were significant in differentiating recurrence from radiation necrosis with accuracy of 93.8% for TBRmax, 87.5% for TBRmean 81.3% for ADCmean, 96.9% for Cho:Cr ratio and 90.6% for N rCBVmean. The accuracy of both normalized rCBVmean and ADCmean was improved in combination with TBRmax or Cho: Cr ratio. However, TBRmax (or TBRmean) with Cho: Cr ratio yielded the highest accuracy approaching upto 97%. Furthermore, maximum AUC is achieved with combination of TBRmean, CBV and Cho:Cr values.Conclusions: Our findings suggest that FET uptake with Cho: Cr ratio and N rCBVmean could be most useful to distinguish primary glioma recurrence from radiation necrosis. Hybrid simultaneous multiparametric 18F-FET PET/MRI might play a significant role in the evaluation of patients with suspected glioma recurrence.

P123-G011Can PET-CT based parameters replace traditional TNM based prognostication in carcinoma nasopharynx treated with definitive chemo radiotherapy?

A. Pilar1, S.G. Laskar1, A. Budrukkar1, T. Gupta1, V. Murthy1, J.P. Agarwal1


Background: Advent of IMRT in nasopharyngeal cancer (NPC) has seen excellent loco-regional control exceeding 90% even in advanced stages and in this era the use of traditional prognostic parameters alone, no longer seems to be sufficient for prognostication of NPC.Materials and methods: Between 2008 and 2013, 85 patients treated with chemo-radiotherapy, were evaluated. Gross tumour volumes (GTV) were contoured on the contrast enhanced CT of the pre-treatment FDG-PET-CT. SUV max was automatically generated. MTVs were delineated on the pre-treatment FDG-PET-CT. All parameters were generated separately for primary tumour (primary GTV/MTV) and nodal disease (nodal GTV/MTV) and combined to get a total volume (Total GTV/MTV).Results: Seventy-six percent of the patients had stage III/IV disease. At a median follow-up (FU) of 26 months (range 7–87 months), 3-year LRC, DFS, DMFS and OS were 84.0%, 70%, 82% and 90%, respectively. At last FU, 23 patients (27%) had failed, 10 had loco regional failure, 12 had distant metastases and 1 had a second primary. Seven patients died of disease. On univariate analysis, significant predictors of DFS, DMFS and OS were nodal GTV and total GTV, nodal MTV and nodal TLG (hazard ratio 1.1–1.2, p value ,0.05). A multivariate analysis to find an independent prognostic factor, showed nodal GTV at a cut off of 110 ml, as an independent predictor of EFS and DMFS and stage IV as an independent predictor of DMFS.Conclusions: Nodal volume, GTV, MTV and TLG node are predictors of DFS, DMFS and OS and Total MTV influences DMFS. Conventional TNM and PET-CT parameters are complementary in determining prognosis in NPC patients treated with definitive chemoradiotherapy.

P156-G012Challenges of HPV detection in head & neck squamous cell carcinoma: Redefining the algorithm

M. Pandey1, P. Bhosale1, R. Desai2, A. Patil3, S. Kane3, K. Prabhash4, M. Mahimkar1

1 Cancer Research Institute, ACTREC, Tata Memorial Centre, Mumbai, India2 Department of Oral Pathology & Microbiology, Nair Hospital Dental College, Mumbai, India3 Department of Pathology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India4 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India

Background: Increasing evidences suggest role of high-risk Human Papilloma Virus (HPV) in development of subset of head and neck


All studies were performed on 3 tesla magnet and included routine imaging, including DSC, which was used for comparison. DSC was not satisfactory in 41 studies (18%), due to technical limitations. CBF and CBV measurements were obtained on DSC, while CBF measurements were obtained from ASL study, in 165 patients. These were normalized to uninvolved contralateral white matter. Good correlation was found between CBF and CBV obtained from DSC and CBF obtained from ASL in 146 patients (89%). ASL scored over DSC in immediate post-operative setting for assessment of residual disease. In serial scans, ASL depicted response and recurrent disease earlier than DSC. Discordance between ASL and DSC was seen in 19 patients (11%), with ASL proving better. Good correlation was obtained with clinical examination as well as follow up, where DSC was unavailable. False positives with ASL occurred in lesions such as pilocytic astrocytoma. Limitations include large areas of hemorrhage. We conclude that ASL is a novel technique, which can be used instead of DSC, in routine neuro-oncology practice, particularly in follow up. ASL used in combination with other advanced MR techniques such as susceptibility weighted angiography (SWAN), allows possible elimination of contrast in index cases.

P186-G017Raman spectroscopy of hamster buccal pouch model: Investigating suitability of ex vivo models to evaluate in vivo spectra

P. Kumar1, C.M. Krishna1

1 Advanced Center for Treatment Research and Education in Cancer, Mumbai, India

Background: Raman spectroscopy (RS) has been shown to be useful in classification of many cancers using both ex vivo and in vivo approaches, with concordance in findings. While cancers of oral and cervix can be directly accessed and studied, internal organs like stomach can be analysed using endoscopy mediated spectroscopy. However, spectroscopy of organs like brain is complicated as obtaining tissue samples, especially healthy tissues is difficult. An alternative approach in such studies can be building standard models from biopsied tissues and use them to identify cancerous region in vivo. Thus in this study, we have explored suitability of ex vivo spectral models to evaluate in vivo spectra using well established hamster buccal pouch model of oral carcinogenesis.Methodology: Spectra were acquired from hamster buccal pouch control and tumor tissues using HE785 commercial Raman instrument. Principal Component based Linear Discriminant analysis (PC-LDA) findings was used to build standard model which consisted of ex vivo (25 control; 20 tumors) and in vivo (26 control; 26 tumor) spectra; and cross validated using leave one out cross-validation algorithms. Independently obtained ex vivo (216 control; 63 tumor) and in vivo (886 control; 40 tumor) spectra were test predicted against the standard model.Results and Discussion: Correct prediction was observed 193/216 controls and 53/63 tumors in case of ex vivo spectra. For in vivo spectra, 805/886 control and 53/63 tumor spectra were correctly predicted. Prediction of few tumors (2 in vivo and 10 ex vivo) with control groups can be attributed to tumor heterogeneity. Findings suggest suitability of such models in identifying healthy and cancerous regions in vivo. Such an approach may be useful for identification of cancerous regions in vivo. Further, such an approach may also be useful in in situ surgical demarcations in case of critical organs like brain.

P188-G018Detecting the spectrum of multigene mutations in NSCLC by SNaPShot assay: A pilot study

A. Choughule1, V. Trivedi1, P. Bagayatkar1, P. Chandrani2, A. Dutt2, V. Noronha3, A. Joshi3, V. Patil3, K. Prabhash3, S.D. Banavali4

1 Medical Oncology- Molecular Laboratory, Tata Memorial Centre, Mumbai, India2 The Dutt Lab ACTREC, Tata Memorial Centre, Mumbai, India3 Medical Oncology, Tata Memorial Centre, Mumbai, India4 HOD Medical Oncology Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India

Background: Taking the bench to the bedside is the crucial goal in today’s personalized medicine.Determining multiple driver gene mutations in NSCLC patient tumors is important for targeting therapy in clinical practice. Optimized SNaPShot assay was extended in 81 lung adenocarcinomas to detect 23 somatic recurrent point mutations in 10 genes with relevance to targeted therapy.Methods: Genomic DNA from FFPE and cytologic samples, having .10% of tumor burden were subjected to multiplex PCR for EGFR, KRAS, NRAS, BRAF, AKT1, PIK3CA, PTEN, MEK1, ERBB2 and FGFR3 biomarkers.

DT imaging and conventional MR images. Then the image fusion was verified for accuracy and the fiber tracking process was performed. The tracking algorithm was initiated using two regions: the internal auditory meatus and facial nerve entry area at the brainstem. The surgeon was blinded to the results of the preoperative DTI tractography.Results: Out of the twenty patients who underwent DTI tractography, it was not possible to preoperatively identify facial nerve in one patient. In one patient, although DTI tractography predicted the position of facial nerve, it was not identified intraoperatively. Out of the remaining 18 patients, DTI tractography predicted facial nerve position was in sync with intraoperative facial nerve position in 16 patients (89% concordance). It was discordant in two patients (11%), but this was not found to be statistically significant (p value -0.3679).Conclusion: This study validates the reliability of facial nerve diffusion tensor (DT) imaging–based fiber tracking for prediction of facial nerve position in patients with large vestibular schwannomas. The reliable preoperative visualization of facial nerve location in relation to VS would allow surgeons to plan tumor removal accordingly and may increase the safety of surgery.

P159-G015Raman spectroscopy and oral exfoliative cytology: Investigating misclassifications between contralateral normal and tumor sites

A. Sahu1, S. Tawde1, P. Gera2, S. Nair3, C. Krishna1

1 Chilakapati lab, ACTREC, Tata Memorial Center, Mumbai, India2 Biorepository lab, ACTREC, Tata Memorial Center, Mumbai, India3 Head and Neck Surgical Oncology, Tata Memorial Center, Mumbai, India

Oral cancers are associated with low disease-free survival rates, attributed mainly to delayed diagnosis and recurrence-locoregional, second primary and field tumors. Studies based on Raman spectroscopy (RS) have shown to classify healthy, premalignant, contralateral normal and tumor, and even detect early changes like cancer-field-effects (CFE) in vivo. Several misclassifications between contralateral normal with premalignant and tumor sites were encountered in these studies. The speculated reasons for misclassifications included, a) tumor heterogeneity, b) presence of inflammatory foci, and c) multiple transformation zones in oral cavity due to field cancerization. Due to ethical considerations, histopathological evidence for these misclassifications could not be obtained. Therefore, this study on RS of oral exfoliative cytology was undertaken to understand the origin of misclassifications between contralateral normal and tumor sites. Raman spectra were acquired from exfoliated cell pellets from contralateral normal and tumor sites of same oral cancer patients (n 5 16). Following spectral acquisition, the cell-pellet was subjected to Pap staining. Principal-component-linear-discriminant-analysis (PC-LDA) followed by Leave-one-out-cross-validation (LOOCV) indicate ~27% misclassifications between contralateral normal and tumor groups. Cytopathological evaluation was carried out by a certified pathologist. The cytological findings correlated with RS findings; spectra from contralateral samples which misclassified with tumor group harboured cytological changes like mild dysplasia, dyskeratosis and increased number of parakeratotic cells and anucleate squames. Similar cytological profile is reported for tumor Pap smears, only with higher degree of dysplasia. Thus, the present study demonstrates definite cytological changes in contralateral normal sites and the potential of RS in detecting these changes. The study highlights the need for routine check-up and follow-up of even contralateral normal sites in oral cancer patients for early identification of recurrence and to improve prognosis.

P170-G0163D arterial spin labeling – A novel, non-invasive technique to assess perfusion in brain tumors – experience of over 200 cases

S. Pungavkar1, T. Gupta2, A. Moiyadi2, P. Shetty2, E. Shridhar2, G. Chinnaswamy2, J. Godashastri2, R. Jalali2

1 Global Hospital, Mumbai, India2 Tata Memorial Hospital, Mumbai, India

Substances secreted by brain tumors, such as VEGF, EGFR etc. induce neoangiogenesis. This, in turn, increases blood flow within the tumor, which is exploited in dynamic contrast enhanced susceptibility weighted perfusion (DSC) and three dimensional arterial spin labeling (3D-ASL) to assess cerebral perfusion. DSC uses contrast as tracer. 3D-ASL is a novel technique, which uses an intrinsic tracer, representing water in flowing blood, without need for exogeneous contrast, rendering it non-invasive. It has potential to quantify tissue perfusion. We present our experience over one year in assessing pseudocontinous ASL in index and follow up cases of brain tumors in 203 consecutive studies.


a 6DOF couch. Then the static plan dose is further deformed on to first set-up corrected timeweighted Avg. derived static series from 4D-CBCT dataset. Using deformable image registration, subsequent fraction doses on reference geometry (First Time weighted avg. series) were added up to get accumulated doses. The contribution of different geometric errors to changes between the accumulated and predicted breathing dose were quantified and Changes greater than 1 gy, reported as percent change (normalized to prescription dose) were considered significant.Results: All patients had accumulated dose deviations relative to the planned static prescription dose .5%, ranging 3–18% in tumors and 5–25% in normal tissues. Residual setup errors were most often the largest cause of the deviations, followed by deformations and breathing variations. Gold seed markers, while producing delineation artifacts and deformation errors, were good surrogate for treatment verification when 4D CBCT was used (with acquisition time of 3 mts). These deformation errors were minimized with proper deformation QA using registration refining tools & alignment locks.Conclusion: Accumulated dose deviations .5% relative to the static plan are observed in all pts undergoing liver SABR. Modalities which impede breathing motion like diaphragmatic compression will produce more deformation errors leading to erroneous prediction of 4-D dose & accumulated dose.

P195-G021Deformable dose accumulation on 4-D CBCT static derived series in oligo-fractionated lung SABR – Trigger tool for adaptive re-planning?



Purpose/Objective(s): To investigate the accumulated dose deviations to tumor & normal tissues in Lung SABR.Materials/Methods: 12 NSCLC patients following 4D image acquisition, received underwent SABR under free breathing conditions. 4*12 gy/fr. was prescribed to 95% of the PTV. Cubic B-splines deformable registration used to accumulate doses on all the breathing phases thus giving predicted 4-D dose. Final midV static dose was subtracted from predicted 4-D dose & deviations quantified. MidV static dose is further deformed on to first setup corrected time weighted Average 4D-CBCT dataset (TWA) dataset. Fraction doses on reference geometry were added up to get accumulated doses on all TWCBCT datasets. The contribution of different geometric errors to changes between deformable accumulated dose on TWA CBCT & predicted 4-D dose were quantified for tumor & OAR. Changes .1 gy reported as % change considered significant.Results: When comparing 4-D CT deformable predicted dose to midV static plan, using dose subtraction, changes of atleast 1 gy to maximum prescription dose were observed in all but 2 pts (83%). Compared to MidV plan, using deformable accumulation based on 4-D CT, 77% & 88% pts had deviations .1 gy in PTV (max.7 gy) and .1 gy to normal tissue respectively. With full deformable dose accumulation on TW-4DCBCT frames, 35% & 100% pts had deviations .1 gy (max-5 gy) in PTV & normal tissue max dose .1 gy respectively. Baseline shifts & deformations when accounted, reduced the magnitude of deviations (max 3 gy in PTV & 4 gy in normal tissue) but 50% of pts still had dose deviations .1 gy in PTV & OAR. One factor common to all these pts included a statistically significant decrease in the size upto average of 60% (30–70%) of the original lesion after 2–3 treatment sessions.Conclusions: Dose accumulation revealed significant dose changes to either tumor or OAR & should always be considered for evaluation & dose escalation in moving targets.

P196-G022Dosimetric impact of intrafraction motion in spine SABR using deformable dose tracking

V. Shankar1, A.R. Gupta1, K. Vinoth1, P. Sathish1, R. Rajesh1, K. Ranjan1, J. Jijo1, C. Haritha1, J. Sachin1, S. Das1, D. Jain1

1 Geetanjali Cancer Centre, Geetanjali Medicity, Udaipur, India

Purpose: To assess dosimetric impact of intrafraction motion for spine SABR patients.Materials/Methods: SABR data of 48 spinal metastases in 37 patients was retrospectively analyzed. A median dose of 18 gy/fr was delivered. Following initial set-up, four CBCT’s were acquired (localization, pretreatment, mid-treatment, End-treatment). For Intrafraction motion (IFM) dosimetric analysis, CBCT data of patients with more than 0.50 & 0.5 mm mm vector shifts between localisation scan & postRx scan was considered. Cubic B-spline deform algorithm registered original plan, structures & dose with

Purified PCR products were then cycle sequenced using 23 allele specific SBE primers. Further, fragment analysis was performed on ABI 3500 Capillary Electrophoresis. Data analysis was conducted using ABI Gene Mapper software.Results: We reviewed a cohort of 81 patients studied for this assay. Out of 81, male:female ratio was 1.8:1 (52/29). All genotypes has been detected and distinguished from each other with significant signal intensity and very low noise. Overall mutation rate in this spectrum was 47% (38/81). Out of 38, 36 tumors DNA expressed for single gene alteration from different 23 genotypes. Of which, Exon19 and exon21 were predominantly high, 2 patients showed coexistence, in which one patient was exon21 L858R along with KRASG12C, another patient expressed for multiple biomarkers like Exon 21L858R, Exon20T790M and PIK3CAE542K has been further clinically correlated.Conclusion: We chose this highly sensitive and robust platform for multiple reasons: familiarity with capillary electrophoresis, ease of data interpretation and considering limited infrastructure demand of Sequenom and NGS. Mutation results of these 23 genotypes will definitely accelerate the treatment algorithm of lung cancer patient and can be part of standard care or clinical trial. This In-house developed SNaPShot diagnostic assay is very cost effective, robust and can also be extended as a mutation profiling panel in different solid tumours other than lung cancer.

P192-G019Prospective non randomized study which correlates the size and marrow extent of bone sarcomas as determined by MRI with that of final histopathology report

S. Byregowda1, S. Subi1, A. Gulia1, A. Puri1

1 Bone & Soft Tissue Services, Department of Surgical Oncology, Tata Memorial Hospital, Mumbai, India

Background: For the limb salvage procedure in bone tumors, preoperative accurate measurement of the extent of the tumor is mandatory for oncological clearance. MRI has been used as the gold standard to measure the extent of disease.Purposes: To analyse how well MRI correlates with histopathology in assessing extent of disease and hence accurately determining the osteotomy level during limb salvage procedures.Method: It is a prospective non randomized study, involving 100 consecutive patients of bone sarcomas, over a period of 6 months. Tumor extent in prechemotherapy and post chemotherapy MRI is evaluated by radiologist and the investigator. The craniocaudal intramedullay and extramedullary extent of disease was recorded on the resected specimen.Results: 100 cases were analysed (73 osteosarcomas, 20 Ewings and 7 chondrosarcomas). In 79 cases where the size in MRI was more than specimen size, mean difference was 1.9 cm with a SD of 1.5. In cases where marrow extent was more than extramedullary, mean difference was 1.8 cm with a SD of 1.5, and in cases where extramedullary dimension was more, these were 2.4 cm and 1.2, respectively. In the 14 cases where specimen size was more than MRI, mean difference was 0.54 with a SD of 0.44. In cases where marrow extent was more than extramedullary, mean difference in size was 0.43 cm with a SD of 0.4 cm. In cases where extramedullary extent was larger, mean difference was 0.93 cm with a SD of 0.4.Conclusion: 1) MRI is accurate in delineating the extent of bone sarcomas. 2) A margin of 2 to 2.5 cm on MRI may be sufficient enough for adequate oncological clearance while preserving normal tissue. 3) Post chemotherapy MRI correlates better with histopathology than pre chemotherapy MRI, even though the correlation of both pre and post chemo MRI with pathology are significant.

P194-G0204-D predicted dose vs accumulated dose variations in liver SABR



Purpose: Quantify the variations to tumor & normal tissues between 4D predicted dose & accumulated dose on time weighted average 4D-CBCT static derived series in liver SABR.Methods/Materials: 5 pts with 6 liver metastatic lesions underwent gold seed fiducial placement, triphasic liver scan, respiratory correlated 4D CT. A dose of 39 gy/6f delivered using step & shoot IMRT. 4D dose accumulation on the remaining frames was done by intensity-based-free-form deformable registration & final 4-D predicted breathing dose was derived. Treatment was executed using 4D-CBCT image guidance on


indices compared were conformity index, homogeneity index and R50% for PTV. For Lung-PTV and Heart- V20, V5, mean dose were evaluated and for other OARs (Esophagus, trachea and spinal cord), Dmax was compared. Dosimetric accuracy was assessed using gamma index (3% dose difference, 3 mm DTA, 10% threshold) and point dose measurement for HT plans (3% tolerance).Results: Mean volume of PTV was 129 cc (range 90–158 cc). Mean CI, HI, R50% for PTV with HT plan were1.06 (SD 0.07), 1.02 (SD 0.003) and 5.4 (SD 0.97) respectively. With Rapid Arc, mean CI, HI and R50% were found to be 0.658 (SD 0.48), 1.129 (SD 0.07) and 4.2 (SD 1.27) respectively. D2cm and R50 were slightly on higher side in HT plan. Delivery of a 7.5 Gy fraction required an average of 13 min (HT) and 3.7 min (Rapid Arc). All verification plans were well within tolerance dosimetrically.Conclusions: Both high precision radiotherapy techniques with HT and Linac were capable of achieving the desired goals. Shorter treatment time with Rapid Arc may benefit the patient to reduce intra-fraction uncertainty in dose delivery.

P275-G025EGFR exon 20 insertion mutations in lung adenocarcinoma: Prevalence, molecular heterogeneity, and clinicopathologic characteristics in our lung cancer patients

A. Choughule1, D.S.J. Philip2, N. Jambhekar3, V. Patil2, A. Joshi2, V. Naronha2, K. Prabhash2

1 Medical Oncology-Molecular laboratory, Tata Memorial Hospital2 Medical Oncology, Tata Memorial Hospital3 Department of Pathology, Tata Memorial Hospital, Tata Memorial Centre Mumbai/India

Background: EGFR Exon20 mutations have been considered to be markers of acquired resistance to Tyrosine Kinase Inhibitors. The association between Oral TKI response and Baseline Exon20 Mutations has not been addressed in many studies and remains to be evaluated.Methods: We conducted a retrospective audit of our prospectively maintained Lung cancer audit database in our institute in the year 2014. We reviewed data related to EGFR mutation testing by RQ-PCR assay and by direct sequencing for EXON 18, 19, 20, 21. We also reviewed data relating to baseline demographics, clinical profile, patient treatment and outcome measures in terms of response and survival.Results: We reviewed 807 sequentially tested lung cancer patients, who underwent molecular testing using RQ-PCR and Direct sequencing. Overall mutation rate was 23. 4% and 19 (2%) had baseline EGFR EXON20 mutation. Median age of patients was 56 yrs [range: 29–81 yrs], with 7 patients being females. 7 patients had past history of smoking. The most common site of metastasis was pleural effusion in 8, followed by Bone in 6, Brain in 5 and Liver metastasis in 2 patients. Histology was Adenocarcinoma in majority [16 patients]. Among the types of EXON20 Mutations, 7 patients had S7681, 4 patients had insGGT, 5 patients had insGCCAGCGTG and 4 patients had T790M mutation. All patients received chemotherapy as first line treatment. Documented a response assessment at 2 months in 8 patients, with progressive disease in 5 [63%], stable disease in 2 and partial response in 1 patient. Second line therapy with Oral TKI was given to 9 patients, in whom we have documented response assessment in 6, all of whom had progressed. The median OS of Exon-20 mutation positive patients were 5.5 months. [Range of 3.8–7.2 months], in comparison with other types of EGFR mutations which showed median OS of 16.3 months [range: 12.7–19.4 months].Conclusion: EXON-20 Mutations in general proclaim grave prognosis, diversity in biologic behavior, predicting limited benefit of chemotherapy and marked TKI resistance.

P276-G026Abstract withdrawn

P289-G027The T-box transcription factor 3: A diagonostic biomarker and therapeutic target for a diverse range of sarcoma subtypes

S. Prince1, T. Willmer1, D. Smuts1, J. Peres1, J. Parkes2, D. Govender3

1 Division of Cell Biology, Department of Human Biology, Cape Town, South Africa2 Department of Radiation Oncology, Cape Town, South Africa3 Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

Background: Sarcomas are heterogeneous neoplasms of mesenchymal origin whose clinical management is compromised due to inadequate diagnostic markers and limited therapeutic options. There is therefore

setup uncorrected Verification, midtreatment& post treatment CBCT Scan. Clinical relevance was measured as the percentage underdosage of target & max dose increase of dose to adjacent OAR.Results: Mean residual TE & RE recorded in localization scans were 4 mm (SD:0.6) & 1.80 (SD:0.9) respectively. Mean residual TE & RE on verification scans were 0.3 mm (SD:0.1 mm) & 0.20 (SD:0.30) respectively. Mean IFM averaged over all patients and directions was 0.3 (60.2) mm for translations and 0.40 (60.90) degrees for the rotations, in the interval between mid Rx & post Rx scan 17 patients with lesions in lower dorsal/lumbar spine had RE .0.50 but ,10 shift & 7 had .10 shift. For dosimetric analysis, the same errors were replicated for CBCT-DIR using the shift editor & doses were thus deformed. For non-spinous process target, a one degree shift in the roll, pitch and yaw directions revealed approximately 1.8% drop in tumor coverage and OAR dose increase of upto 100 cgy. For lesions where spinous process is included there was 2.8% drop in tumor coverage.Conclusion: Majority of errors were rotational occurring at target locations where evacuated cushions were used. Every half degree error translates into aprrox. 1% drop in tumor coverage or 50 cgy increase in OAR. DIR is simple fast tool to understand the clinical relevance of IFM errors.

P201-G023Deglutition induced dose deformations in head and neck cancer IMRT treatments - Volume shuttle imaging analysis

V. Shankar1, A.R. Gupta1, K. Vinoth1, P. Sathish1, R. Rajesh1, K. Ranjan1, J. Jijo1, C. Haritha1, J.A. Sachin1, S. Das1, D. Jain1


Purpose/Objective(s): Apneic phase during swallowing leads to sorting errors & artefacts deterring 4-D Image acquisition from being used to track motion displacements in head and neck region. DVHS fills this void. Present study aims to assess the deglutition induced displacement and dose deformations in patients with head and neck cancer using DHVS imaging.Materials/Methods: 20 patients oforopharyngeal cancers in treatment position underwent DVHS imaging. Image data for single patient was acquired during voluntary effortfull swallowing act (once in 20 sec) at 2 alternating table positions with the table shuttling back and forth. 3000 images were reformatted using Retro Recon& cine looped to quantify the range of motion. Deglutition-induced GTV & cord displacements were quantified based on position change during deglutition relative to pre-swallow structure location for anterior (A), posterior (P), superior (S), and inferior (I) directions. The image datasets in extreme bins were backprojected on to the original plan in QA mode and dose deformations analyzed.Results: Deglutition-induced maximal GTV displacements ranged 3 mm to 18 mm with mean and SD of 3.5 1/2 2, 2.50 1/2 1.51, 1.75 1/2 4.0, and 9.60 1/27.7 mm in the A, P, I, and S directions respectively. The SC displacement ranged from 2.8 mm 1/2 .2 mm, 2.2 mm 1/2 1.4 mm in A, P direction. Dosimetric analysis showed that 92% of the prescribed dose would still be delivered to the tumor even if the tumor partly vacated the PTV during the entire swallowing event for 15 of the 20 patients during the VHS scan. However, for the other 5 patients, cohort which had frequent swallowing, the target coverage failed to reach 80%.Conclusions: This is the first study reported in literature using DHVS for deglutition displacements quantification. Asymmetric IM’s & PRV margins, derived from directional displacement, using DVS imaging should be used for high precision treatments in HNC.

P211-G024Tomo IMRT vs Rapid Arc for lung SBRT – Dosimetry and delivery comparison

P. Sahoo1, R. Kinhikar1, S. Jain1, V. Manik1, D. Deshpande1, S. Shrivastava1, J.P. Agarwal1

1 Department of Radiation Oncology and Medical Physics, Tata Memorial Hospital, Mumbai

Background: Stereotactic body radiation therapy (SBRT) for lung demands high precision dose delivery technique to minimize the doses to organs at risk (OARs), while simultaneously enabling dose escalation to target. These techniques employ either intensity modulated radiation therapy (IMRT) or volumetric modulated arc therapy (VMAT).Methods: Four Patients (stage I NSCLC with peripherally located lesion) treated with Rapid Arc SBRT using 6 MV photons with one/ two half-arc plan (180°) in Linac were included. To determine the optimal treatment modality, a new treatment plan was generated using IMRT with HT for each patient for comparative purpose. For HT plans, the field width of 2.5 cm, pitch as 0.1 and modulation factor as 2.5 was used. Dosimetric

H. Therapy Resistance European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S41

genes that drive tumor progression or metastasis. Many inflammatory mediators produced in the tumor milieu can support tumor growth, cell survival, and promote metastatic competence.Using genomic and proteomic approaches, we identified TGM2 as one of the abundantly overexpressed genes in multiple drug-resistant and metastatic tumors. TGM2 is a stress response gene, which encodes a functionally and structurally complex protein, called transglutaminase type II (TG2). Stable expression of TG2 conferred cancer cells with ability to invade and survive the cytotoxic effects of radiation/chemotherapy by activating inflammatory signaling networks that play fundamental roles in drug resistance and metastasis. TG2 over expression induced epithelial-to-mesenchymal transition (EMT) and conferred stem cell traits in epithelial cancer cells. At molecular level, TG2 expression resulted in constitutive activation of NF-kB via a non-canonical pathway. Hypoxia-induced factor-alpha (HIF-1a) was identified as one of the direct downstream targets for TG2 activated NF-kB. The resultant increase in HIF-1 accumulation led to transcriptional regulation of Snail, Zeb-1, and Twist repressors. Importantly, elevated expression of TG2 in tumor samples is associated with poor disease outcome, increased incidence of metastasis, and early relapses. Taken together, these results suggested that inhibition of TG2 represents a promising therapeutic approach to treat refractory and metastatic tumors. As a proof-of-concept, our studies provided compelling evidence that in vivo silencing of TG2 by liposomal-TG2siRNA (LSRNA) could effectively inhibit the dissemination of orthotopically growing tumors (pancreatic and ovarian) and rendered them sensitive to chemotherapeutic drugs. In view of the usefulness and efficacy of LSRNA to inhibit TG2 in growing tumors, efforts are underway to develop it for treating patients with refractory and recurrent tumors.

OF161-004Understanding therapy resistance in GBM using in vitro transcriptome analysis and orthotopic mouse models

J. Nair1, E. Kaur1, R. Thorat2, N. Gardi3, K. Patkar1, R. Chaubal3, P. Chaudhari4, A. Dutt3, S. Dutt1

1 Shilpee Dutt Laboratory, ACTREC, Tata Memorial Centre, Mumbai, India2 Laboratory Animal Facility, ACTREC, Tata Memorial Centre, Mumbai, India3 Amit Dutt Laboratory, ACTREC, Tata Memorial Centre, Mumbai, India4 Small Animal Imaging Facility, ACTREC, Tata Memorial Centre, Mumbai, India

Background: Glioblastoma is the most invasive type of glial tumors, with 14.6 months median survival time of patients contributed partly due to inherent resistance to conventional therapies. Using an in vitro radiation survival models from Glioblastoma cell lines and primary patient samples, we had previously shown that a subset of cells, termed as radiation resistant (RR) cells, survive upon exposure to lethal and fractionated doses of radiation. Although these RR cells are transiently non-proliferative, they resume growth and give rise to recurrent population of GBM cells.Methods and Results: In order to understand the differential signalling mechanisms involved in radiation resistance and survival, transcriptome analysis was carried out on RR and recurrent cells from 2 cell lines and 2 patient samples and compared with the parent cells. Co-expression analysis of the significantly altered genes revealed pathways related to ECM receptor interaction, focal adhesion and cytokine-cytokine-receptor interactions to be deregulated in radiation resistant and recurrent population. Further, functional assays will help us decipher the role of these pathways in conferring therapy resistance. Additionally, we have established orthotopic mouse models of GBM by intracranial injections of grade IV glioblastoma cell lines in order to validate our in vitro findings. Analogous to patients, mice with tumours expressing luciferase will be subjected to a daily dosage regimen of radiation with and without concomitant temozolomide. These cells will be monitored using bioluminescence imaging and micro-CT till the time detection of residual cells will not be detected in micro-CT scans.Conclusion: This study, for the first time, will emphasize the existence of the residual therapy resistant cells, which are virtually inaccessible in patients as they cannot be detected in MRI. Overall, the study will aid in devising strategies that selectively eliminate these cells, thereby preventing tumour recurrence and improving patient survival.

OF11-011A novel mechanism of homotypic cell fusion promote formation of glioma resistance cells facilitated by NHEJ driven repair

E. Kaur1, J. Nair1, A. Kushwaha1, A. Shetty1, A. Srivastava1, A. Moiyadi1, S. Dutt1

1 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India

a need to elucidate the molecular mechanisms underpinning the pathogenesis of the more than 70 distinguishable sarcoma subtypes. The transcription factor, TBX3, is overexpressed in several epithelial derived cancers where it plays a direct oncogenic role and it has been proposed as a novel target for their treatments. However, the status of TBX3 in sarcomas is not known and whether it has an oncogenic function in these cancers has not been reported. The objective of this study was thus to identify the status and role of TBX3 in a range of sarcoma subtypes.Methods: TBX3 expression was analysed in a panel of sarcoma cell lines (fibro-, rhabdomyo-, chondro-, lipo- and synovial- sarcomas) by western blotting and in patient-derived sarcoma sections by immunohistochemistry. TBX3 was either silenced by shRNA or overexpressed in sarcoma cells and the impact on the cancer phenotype assessed using growth curves, soft agar and motility assays and injection into nude mice. To determine the mechanism by which TBX3 promotes sarcoma cell proliferation, cells were co-transfected with a TBX3 expression vector and a p21 reporter and luciferase activity measured.Results: We show that TBX3 (1) is overexpressed in all sarcoma cell lines and tissues tested; (2) contributes directly to sarcomagenesis; (3) promotes proliferation through a mechanism involving its ability to repress p21.Conclusions: TBX3 may be (1) a biomarker for the diagnosis of heterogeneous sarcoma subtypes and (2) useful in single targeted therapies to treat these aggressive cancers.

H. Therapy Resistance

OT26-002Disrupting protein: Protein interactions to overcome drug resistance

S.V. Malhotra1

1 Department of Radiation Oncology, Stanford University, Palo Alto, CA, USA

Ovarian cancer is the most lethal gynecological malignancy and the fourth leading cause of cancer related deaths with a drastic 5-year survival prognosis of ,25%. Approximately 80–90% of patients show relapse after chemotherapy. Resistance to current chemotherapeutic agents is a major cause of therapy failure in patients with ovarian cancer, but the exact mechanisms leading to the development of drug resistance remain unclear. Our research on tumor samples from ninety eight patients has identified a new functional circuit involving the cytoskeletal overexpression of a tubulinisotype (class III b-tubuli, TUBB3) and one G-protein i.e. (guanylate-binding protein 1). Study showed that once cancer cells are stressed in a compelling microenvironment featured by hypoxia and low nutrient supply, they increase the expression of TUBB3 and enhance the incorporation of GBP1 into cytoskeleton. GBP1 then recruits prosurvival kinases such as PIM1 and initiates a signaling pathway that induces resistance to paclitaxel.Therefore, we hypothesized that the inhibition of the GBP1: PIM1interaction could potentially revert resistance to paclitaxel. Subsequently, we designed and screened a library of natural product basedsmall molecules and identified inhibitors of this protein:protein interaction. We also found that this activity is maintained in vivo in ovarian cancer cells resistant to paclitaxel.Similarly, it is reported that a the Keap1-Nrf2 system is the major regulatory pathway ofcytoprotective gene expression against oxidative and/or electrophilic stresses. We have recently created a library of about 150 flavonoid-based compounds and identified compounds showing Nrf2 inhibition effect. Subsequent studies also showed that the potent inhibitors of Nrf2 also enhanced the apoptotic effect of Doxorubicin. Taken together, our studies have demonstrated that disruption of protein: protein interactions is a promising approach to overcome current problem of drug resistance and improve therapies for cancer patients.

OF43-001Drug resistance and metastasis: New perspectives of an old problem

K. Mehta1

1 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston TX, USA

Resistance to systemic therapy (refractory tumors) and metastasis (recurrent tumors) pose major clinical challenge in successful treatment of cancer and account for more than 90% of cancer-associated deaths. Although, tumor genome sequencing has offered powerful tool for cataloging cancer driver-mutating genes, it cannot distinguish those

S42 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 H. Therapy Resistance

P98-H002GCN5 assisted ATM hyperactivation a novel resistance mechanism that if targeted during early induction therapy eliminates the formation of MRD

S.J. Salunkhe1, E. Kaur1, S. Shah1, J. Nair1, V. Devanand2, S. Patkar1, S. Hasan2, N. Khattry2, S. Dutt1

1 Shilpee Lab, Advanced Centre for Treatment Research and Education in Cancer. Tata Memorial Centre, Navi Mumbai, India2 Hematology Lab, Advanced Centre for Treatment Research and Education in Cancer. Tata Memorial Centre, Navi Mumbai, India

Background: After induction therapy minimal residual (MRD) cells progress to more aggressive state of recurrent leukemia, which is a major problem in treating leukemia. Therefore, we hypothesize that elimination of MRD during early stages of resistance would abrogate relapse.Methods: We modeled acquire resistance of leukemia in vitro that help us to capture and monitor molecular changes in MRD cells at different stages of resistance. Cell that grow after surviving drug treatment we refer as DTEP (Drug Tolerant Expanded Population).Results: As early as two rounds of doxorubicin treatment to leukemic cells, small percentage of cells (DTEP-2) acquires survival advantage. We show that known mechanisms of acquired resistance: limited drug uptake, reduced DNA damage or loss of target (TOP2) are not operative in DTEP-2 population, instead DTEP-2 cells over express GCN5 (histone acetyl transferase) which acetylates H4K16 leading to hyperactivation of ATM (apical kinase of DDR). ATM then activates CHK-2, BRCA1 and anti-apoptotic protein MCL-1 leading to faster DNA repair and survival of DTEP-2 cells. Importantly, GCN5 is significantly upregulated in MRD positive (n 5 17) but not in MRD negative (n 5 17) AML patient samples. However, by 10 rounds of drug treatment (DTEP10) cells show less drug uptake, upregulation of ABCB1 transporters and downregulation of TOP2. Accordingly, ATM inhibitor in combination with doxorubicin completely kills DTEP-2 cells at much lower concentrations of doxorubicin while DTEP10 cells remain unaffected.Conclusion: We provide mechanistic explanation to clinical observation. This is the first report that demonstrates detection of GCN5 expression during induction therapy could be used as marker to detect acquired resistance in MRD cells, accordingly intervention with ATM inhibitor during induction therapy but not in recurrent leukemia eliminate the formation of MRD and prevent relapse. These data also provides explanation for failure of ATM kinase inhibitors in recurrent leukemia.

P100-H003Identification of cancer resistance pathways in healthy nonagenarians using NGS methods

A. Khanna-Gupta1, S.K. Vishwanathan1, S. Murugan1, R. Gupta1, R.S. Haojam1, S. Santosh1, A. Chaudhry1, V.L. Ramprasad1

1 MedGenome Labs, Pvt Ltd, Bangalore, Karnataka, India

Background: The prevailing dogma in cancer studies today is that mutations in two or more cardinal genes leads to the malignant phenotype. Earlier this year Tomasetti and Vogelstein argued that 70% of sporadic cancers arise as a result of mutations in the DNA of individuals by chance events and that simple “bad luck” may be the cause of these cancers. It is also known that mutations in DNA accumulate over the lifespan of individuals. Yet healthy nonagenarians do not develop cancer. Could they be the lucky few? We argue against this notion. Since longevity seems to run in families, we hypothesize that healthy women/men in the 90s have likely evaded cancers over their lifetimes due to their genetic predisposition. We further hypothesize that healthy elderly individuals harbor cancer-resistant pathways which function to evade the development of cancer. Identification of these pathways forms the basis of this study.Methodology: Blood samples from consented healthy nonagenarians along with their habits, lifestyle and medical histories have been collected. DNA from these samples is being interrogated for mutations using an NGS based cancer panel (20 MB Nimblegen panel). We will also interrogate expression levels of genes involved in DNA repair pathways using RNA-seq methods. The sequence and expression data obtained will be analyzed and compared to 500 samples collected from elderly Caucasians as part of the Wellderly project (Scripps Translational Science Institute).Results: DNA sequencing will identify the mutational burden in cancer causing genes while RNA-Seq expression data, especially associated with the expression of DNA-repair genes, will shed light on pathways that are likely specific to healthy nonagenarians compared to the general population.

Background: Here we address a fundamental problem of therapy resistance in Glioblastoma, most lethal form of brain tumor. Elucidating mechanisms of resistance is essential for developing more effective therapeutics. Therefore, we developed a cellular radiation resistance model from naïve primary GBM tumors and cell lines that recapitulate clinical scenario and provide us accessibility to innately resistant cells.Results: We have shown that a small percentage of inherently resistant GBM cells (RR cells) survive exposure to lethal (acute or fractionated) dose of radiation. RR cells are non-apoptotic, non-proliferative and undergo homotypic cell fusions forming multinucleated and giant cells (MNGCs). Interestingly, cell fusion led to senescence which reversed back forming recurrent population. Since DNA damage repair (DDR) pathway plays a significant role in maintaining genomic stability, we wanted to understand how MNGCs modulate their DDR pathway. We show that MNGCs activate either ATM-Chk2 or ATR-Chk1-RPA2 axis eventually repairing their DNA by Non homologous End Joining (NHEJ) pathway and not by Homologous Recombination. Modulation of resistance associated DNA repair pathway was seen to predict the patient’s survival outcome. Given the association of DDR and histone methylations, we screened for histone methylation in RR cells and found up-regulation of H3K36me2. H3K36me2 is known to enhance recruitment of NHEJ repair components including Ku80 and Mre11. Accordingly, we demonstrate that mutating H3.3 at lysine 36 abolished NHEJ repair response by decreasing association of early NHEJ proteins at DSBs. Additionally, inhibition of NHEJ pathway using DNA-Pk inhibitor NU7286 ablated the survival of resistant cells.Conclusions: Our data provides novel insights into a multifactorial process of radiation survival affecting the prognosis of Glioblastoma. We identified homotypic cell fusions of resistant Glioma cells undergoing NHEJ-driven repair of DSBsaided by histone methylation. These mechanisms facilitate survivaland recurrence of RR cells, thereby generating a rationale for developing combinatorial therapies.

P7-H001P53 dependent PIK3CA promoter modulation in chemoresistance: Mechanism beyond genomic alterations

B. Thakur1, P. Ray1

1 Advance Centre for Treatment, Research and Education in Cancer, TMC, Kharghar, Mumbai, India

Background: Apart from PIK3CA amplification, genetic alterations in components of PI3K/AKT signalling are rare in high-grade serous ovarian carcinoma. Yet therapy resistant ovarian cancer displays 50–70% escalated AKT activity. Such resistance-associated escalated AKT activity cannot be explained by genome-wide studies. p53 regulates PIK3CA promoter activity in sensitive cells/ tumors but its ordinance during resistance is unknown. In the present study, we investigated the mechanism involved in p53 mediated PIK3CA regulation during acquired chemoresistance.Methods: PIK3CA expression was assessed by qPCR and western blot and promoter activity by luciferase assay in ovarian and breast cancer cell lines. Site-specific ChIP was used to understand p53-PIK3CA promoter interaction. Simultaneous p53 induction and PIK3CA promoter modulation were studied using dual reporter imaging strategy in cellular models and in vivo tumor xenografts. Posttranslational modifications of p53 were investigated by western blot and levels of various molecular determinants were examined by qPCR and western blot analysis.Results: Our study demonstrates that cisplatin attenuates PIK3CA promoterin sensitive cells and upregulates in resistant cells. Utilizing PIK3CA-promoter sensor constructs and site-specific ChIP assay, we show that cisplatin induces p53 occupancy at specific binding sequences in PIK3CA promoter in sensitive cells. Such occupancy is altered in resistant scenario which was governed by hypo-phosphorylation at S15, S20 and loss of S46 phosphorylation in p53. Real time imaging of p53-protein and PIK3CA promoter in vivo confirms the outcome of our in vitro data. Elevated expression of p27, BCl2 and cFLIP indicated a growth arrested anti-apoptotic state in resistant cells. TCGA data revealed that 90% of high-grade serous ovarian carcinoma bear p53 mutation of which 37% hold complete loss of p53 functionality and DNA binding ability.Conclusion: Post-translational modifications and altered functionality of p53 is the probable cause of increased PIK3CA expression that aids cells to survive against chemotherapeutic agent.

H. Therapy Resistance European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S43

Experimental Design: In silico experiments will disclose the effects exerted by the “hot-spot” BCR-ABL1 mutations on the protein structure, thermodynamic stability, and ability to interfere with BCR-ABL1 binding to specific TKIs. Structural biology evidences of wild type and mutant SH2-linker-SH3-KD constructs (per se and in complex with TKIs) will discussed to support in silico prediction. Molecular biology assays will show the effects exerted by these mutations on the kinase ability to TKI binding, and the main downstream pathways normally/abnormally activated by the new BCR-ABL1 mutants in comparison with those stimulated by the native oncogene. “Prototypical” BCR-ABL1 KD mutations will be analyzed in parallel for comparison and to confirm/dispute the current, classical view of TKI resistance.

P144-H006Modulation of microRNAs to overcome tumour hypoxia and improve radiotherapy response in head and neck cancers

M. Tavassoli1, Y. Suh1, N. Raulf1, J. Gäken1, T. Guerrero1, K. Lawler1, E. Odell1

1 Head and Neck Oncology Group, Guy’s Hospital, King’s College London

Head and neck cancer is the 5th most common cancer worldwide with over 60% of cancers presenting at an advanced stage. Locally advanced HNSCC represents tumours with multiple acquired genetic aberrations that have allowed the tumour to proliferate, grow and locally metastasise. They have an aggressive phenotype and are characterised by complex molecular changes that lead to treatment resistance. Therefore there is an urgent clinical need to understand the mechanisms of therapy resistance and identify those at risk of treatment failure.Current treatment modalities are surgery and radiotherapy or radio-therapy alone and in advanced cases in combination with chemotherapy. These therapeutic options are associated with considerable toxic side effects and cause crucial organ function impairments emphasising an urgent need for targeted, more specific treatments. Molecular profiling of large tumour cohorts has revealed a number of cancer specific aberrations that provide opportunities for the development of lead drugs to optimise treatment to the specific genetic makeup of a patient’s tumour.Recent tissue expression profiling in our lab has led to the identification of several genes and miRNAs associated with the more aggressive tumour phenotypes and radiotherapy resistance tumours (Raulf et al., 2014 Suh et al., 2013 & 2015). In addition, our studies using genetic analysis by RNA-Seq combined with imaging studies has identified a gene and miRNA expression signature correlated to both the volume of the hypoxic region of the tumour and patient survival. Identification of specific targets of the candidate miRNAs responsible for inducing chemo-radiotherapy resistance will be used to develop radio-chemotherapy sensitising drugs.

P187-H007HLA associated susceptibility and resistance to AML patients in an Indian cohort: a case control study

A.G. Bodade1, M. Singh1, S.B. Rajadhyaksha1


Background: Various studies have suggested the association of HLA molecules in the development of malignancies. The present retrospective study was initiated to analyse the association of HLA A, B, C, DRB1, DQB1 alleles among AML patients and controls in Indian population.Materials and Methods: A total of 453 patients with AML and 2630 healthy donors which included the sibling and parents referred to our HLA and Immunogenetics laboratory were selected for the study. The allele frequency of HLA A, B, C, DRB1, DQB1 alleles was compared between patients and controls to understand the susceptibility and protective allele. Further, the patients were segregated on the basis of age and gender. The odds ratio (OR) and 95% confidence intervals limits (CI) (SPSS version 20.0) was calculated and values with p , 0.05 was considered to be significant.Results: Among the AML patients, overall frequencies of HLA-C*14, DQB1*05 and DQB1*06 alleles were significantly increased (OR 5.83, 1.81 and 1.65, respectively). Among females also HLA-C*14, DQB1*05 showed statistically significant increased frequencies with odds ratios of 31.52 and 3.22 respectively. Therefore, in Indian cohort among females HLA-C*14 and DQB1 may be a risk factor for AML. On the other hand DRB1*04 (p 5 0. 36, OR 0.58) may be a protective allele among females, albeit

Conclusions: Our study is designed to identify pathways that have evolved in nonagenarians to evade the development of cancer, i.e. cancer resistant pathways. Data will be presented to support our hypothesis.

P113-H004Differential proteomic analysis reveals role of a novel serine threonine kinase DCLK3 and 14-3-3 zeta in innately radiation resistant and relapse cells of glioblastoma

J. Rajendra1, K. Datta2, R. Reddy3, N. Gardi4, E. Kaur1, K. Patkar1, A. Moiyadi4, K. Bose3, A. Dutt4, H. Gowda2, S. Dutt1

1 Shilpee Dutt laboratory, Tata Memorial Centre, ACTREC, Mumbai, India2 Institute of Bioinformatics, Bangalore, Karnataka, India3 Kakoli Bose laboratory, Tata Memorial Centre, ACTREC, Mumbai, India4 Amit Dutt laboratory, Tata Memorial Centre, ACTREC, Mumbai, India

Background: Innately Radiation Resistant (RR)- the major cause of Glioblastoma recurrence have not been possible to target due to incomplete understanding of their biology. Therefore, to gain access to RR cells we developed an in vitro radiation resistance model from primary patient samples and GBM cell lines. The innately radiation resistant cells which are ,10% of the heterogenous parent population were then subjected to a quantitative proteomic analysis using iTRAQ.Results: We identified 34 differential proteins in the RR population in two biological independent experiments. A novel serine threonine kinase DCLK3 – Doublecortin like Kinase 3 was found to be upregulated in RR cells which was further confirmed by western blot in the RR population of Patient Samples and Cell lines. Interestingly, it is overexpressed in 232 tumor samples out of 242 in COSMIC database. Furthermore, it harbours 8 missense deleterious mutations, 6 of which were in the kinase domain of different tumor types in COSMIC database. Since the functional role and the interacting partners of DCLK3 are not known, a Gene String analysis of the differential proteins predicted that DCLK3 putatively binds with 14-3-3 zeta, which is also upregulated in the RR population. To further confirm the potential interaction of DCLK3 with these proteins we first generated the Homology model of DCLK3 since its crystal structure is not known. We are now in the process of doing in silico docking of DCLK3 with 14-3-3 zeta to identify the functionally important sites which will be further confirmed and characterized by functional studies. shRNA mediated knockdown studies of DCLK3 and 14-3-3 zeta are also being carried to depict the importance of these proteins in the formation of Radiation Resistant population.Conclusion: We have identified a novel serine-threonine kinase DCLK3 upregulated in Radiation Resistant Glioblastoma with a potential therapeutic relevance.

P127-H005Are we really looking the right way at resistant BCR-ABL1 kinase domain mutations in chronic myeloid leukemia?

S. Pricl1, E. Laurini1, M. Fermeglia1, N.J. Donato2, A.Q. Cardama3

1 University of Trieste, MOSE - DEA, Trieste, Italy2 University of Michigan Comprehensive Cancer Center, Division of Hematology-Oncology- Department of Pharmacology, Ann Arbor, USA3 Cell & Gene Therapies Unit, Novartis Pharma A.G., Basel, Switzerland

Background: Acquisition of mutations in the BCR-ABL1 kinase domain (KD) is frequently associated with tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia (CML). Recently, we revealed a novel mutation “hot-spot” in the BCR-ABL1 KD region (residues 295 – 312), associated with high resistance and poor clinical outcomes (Pricl et al, PNAS 2014).Hypothesis: Some of these new BCR-ABL1 variants locate in a protein region, which might not be directly involved in TKI interaction. Therefore we hypothesize that some of these mutations might be linked to TKI resistance via a direct mechanism while others might exert an indirect effect on drug resistance. Thus, we also wonder: “Do the majority of the BCR-ABL1 KD mutations determined so far, studied and characterized by considering just the BCR-ABL KD domain, exert their resistance mechanism by directly interfering with TKI binding, as often claimed?”Aims: The present contribution integrates structural, computational, and molecular biology techniques to understand the eventual role of the newly reported hot-spot mutations of the BCR-ABL1 KD in TKI resistance observed in CML patients.

S44 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 I. Tumor Biology

comprised of 4 HPV positive and 4 HPV negative freshly frozen primary HNSCC. For methylation confirmation, the targeted sequencing (Illumina MiSeq) cohort (n 5 18) consisted of an additional sample set of 10 HNSCC, 7 HPV positive and 3 HPV negative cases. HPV was measured using real-time quantitative PCR (qPCR). Assessment for biologic significance was performed using Ingenuity Pathway Analysis (IPA). Methylation status of all 11 genes as either hypo- or hypermethylated was in agreement with the results of the Lechner et al., study. IPA’s enriched networks analysis produced one network with MSX2 as a central node. Locally dense interactions between genes in networks tend to reflect significant biology throwing the spotlight on to the MSX2 gene. Our study further supports MSX2 together with PCDHB11, C14orf162, and MEI1 as potential epigenetic drivers in HPV-associated HNSCC.

OT30-003BCR-ABL mediated repression of miR-223 results in the activation of MEF2C and PTBP2 in chronic myeloid leukemia

S. Chakraborty1

1 Institute of Life Sciences, Nalco square, Bhubaneswar, Odisha, India

Background: BCR-ABL, the molecular hallmark of chronic myeloid leukemia (CML), promotes cell survival and proliferation through several intracellular signal transduction pathways, and is responsible for malignant transformation of the disease. Given the extensive regulation of normal hematopoietic development by miRNAs, it is of no surprise that miRNAs are extensively involved in the pathogenesis of hematopoietic malignancies. Since CML is characterized by chronic neutrophilic maturation arrest and the transcription factors involved in miR-223 expression are deregulated, we predicted that miR-223 may play a role in CML disease.Methods: RNA was isolated and quantified by RQ-PCR. Mononuclear cells were isolated from CML patients and cultured in a defined media. Bioinformatics and statistical analysis was done by using Genespring software.Results: RQ-PCR data showed that miR-223 expression is significantly down regulated in CML patients. Imatinib treated mono-nuclear cells isolated from CML bone marrow samples showed an up regulation of miR-223 with concomitant down regulation of BCR-ABL. Hybridization of two different CML mRNA datasets with the miR-223 target scan data base revealed eight genes that are possibly targeted by miRNA-223 in CML. Out of them two genes, transcription factor MEF2C and RNA binding protein PTBP2 and were found to be upregulated with CML disease progression. MEF2C targets a set of myeloid genes including the myeloid transcription factor CEBPA. MEF2C and CEBPA expression patterns are negatively correlated in CML patient samples. Imatinib, the drug of choice for CML, abrogates MEF2C expression and reverses CEBPA repression both in the cell line and the primary cells. Thus, we propose that MEF2C-CEBPAmay contribute to CML disease progression.Conclusion: Our findings establish the fact that miR-223 can be used as a biomarker for CML disease progression and MEF2C may be targeted along with BCR-ABL for efficiently treating CML.

OF68-005Evolution by tumor neofunctionalization: The role of heritable tumors in progressive evolution

A.P. Kozlov1

1 Biomedical Center and Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia

Earlier I formulated the hypothesis of the possible evolutionary role of tumors (A.P. Kozlov, “Evolution by Tumor Neofunctionalization”, Elsevier/Academic Press, 2014). This hypothesis suggests that tumors supply evolving multicellular organisms with extra cell masses for the expression of newly evolving genes. After expression of novel genes in tumor cells, tumors differentiate in new directions and give rise to new cell types, tissues and organs.In the presentation, the bulk of data supporting the positive role of tumors in progressive evolution will be reviewed, obtained both in the lab of the author and from the literature sources.The following issues will be addressed: the widespread occurrence of tumors in multicellular organisms; features of tumors that could be used in evolution; the relationship of tumors to evo-devo; examples of recapitulation of some tumor features in recently evolved organs; the types of tumors that might play the role in evolution; examples of tumors that have played the role in evolution.The discussion of experimental confirmation of nontrivial predictions of the hypothesis will include the analysis of evolutionary novelty of

not significant. Among the adults (age $15 years) the frequency of DQB1*05 was significantly increased and DRB1*11 showed decrement (OR 1.7, 0.064). In paediatric age group (age ,15 years) HLA-C*04,*15 and DQB1*06 showed significantly higher frequencies (OR 5.23, 3.46 and 4 respectively).Conclusion: This study highlights the importance of HLA and its association in with AML, its role in different age groups, genders, and their diagnostic and prognostic importance in malignancies to improvise our diagnostic and therapeutic streams for Indian population.

P241-H008Can energy balance strategies that target redox status augment cancer therapy, prevent drug resistance and reduce late effects of treatment?

R. Li1, M. Irwin1, L. Grasse1, B. Johnson1, H. Donnella1, P. Knouse1, J. Chandra1

Division of Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX

Background: Oxidative stress has been linked to the mechanism of action of cancer treatment modalities, suggesting that altering redox could enhance therapy efficacy. Cancer progression and emergence of drug resistance requires oxidative stress in many models, highlighting a role for reduction of oxidative stress. In cancer survivorship, reduction of oxidative stress similarly is desirable to prevent late effects that are linked to increased oxidative stress. Energy balance (use of diet and exercise) represents a low cost and holistic approach to modulating oxidative stress is cancer patients and survivors. Here we present data delineating analyses of redox status and energy balance interventions in models of cancer therapy, prevention of drug resistance and survivorship.Methods: Human cancer cell lines and animal models were used to study oxidative stress signaling in drug resistant leukemia and brain tumor models. Tissue microarrays and in silico analysis was conducted from leukemia and glioblastoma patient specimens. In leukemia patients undergoing treatment, redox and diet were tracked. In a randomized clinical trial, an energy balance intervention was conducted in leukemia patients.Results: In acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and glioblastoma (GBM), we identified a novel pathway that can be targeted to overcome drug resistance or sensitize cells to tyrosine kinase inhibitors. This pathway is transcriptionally upregulated in both leukemia and GBM and is dependent upon oxidative stress stemming from the NADPH oxidase and knockdown of the p47phox subunit reduces activation of the pathway. In a clinical setting, we have identified consumption of antioxidant micronutrients that track with increased oxidative stress. A nutrition counseling intervention was delivered to leukemia patients, and caloric intake was reduced in the intervention groups.Conclusions: Antioxidant strategies are applicable to improving cancer treatment and survivorship, however specific interventions must be tailored to accommodate the biological differences in each setting.

I. Tumor Biology

OT72-004Integrating epigenetics into personalized medicine

M.J. Worsham1, K.M. Chen1, J.K. Stephen1, I. Datta1, G. Divine1

1 Henry Ford Health System, Detroit, USA

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has yet to configure in epigenetics. It is well known that epigenetic silencing of driver genes leads to various genomic alterations, including mismatch repair deficiency, altered DNA repair and loss of chromosomal stability. In human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC), recent studies are beginning to establish a mechanistic role for promoter methylation with potential to impact improved survival outcomes. The purpose of this study was to assess the ‘driver’ potential of 11 genes identified by Lechner et al 2013 as significantly differentially methylated between HPV positive and HPV negative HNSCC tumor samples using the Illumina 450 K platform in two independent sample sets. Our independent Illumina 450 K cohort

I. Tumor Biology European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S45

was developed, wherein anchorage-independent state of growth was considered as the endpoint. Remarkably vimentin expressing clones showed more number of soft agar colonies as compared to vector control clones. Also molecular changes associated with transformation like downregulation of p21, E cadherin, involucrin, etc and upregulation of stemness related markers, were more prominent in benzopyrene treated vimentin overexpressing as compared to vector control clones. In conclusion, vimentin overexpression by itself is not enough to bring about transformation. Nevertheless, its forced expression in the initial stage proves to be advantageous to the cell in order to push itself towards transformation in presence of a carcinogenic stimulus. Also this benzopyrene induced in-vitro carcinogenesis model resembles the events in oral cancer development, hence can be used for validation of biomarkers in future.

P18-I003Abstract withdrawn

P34-I004Atomic insight into the altered O6-methylguanine-DNA methyltransferase protein architecture in gastric cancer

T. Patel1

1 VIT University, Vellore, Tamil Nadu, India

O6-methylguanine DNA methyltransferase (MGMT) is one of the major DNA repair protein that counteracts the alkalyting agent induced DNA damage by replacing m6G back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations due to SNP may result in altered protein structure which in turn can lead to the functional instability. In the present study, we focused on the population exposed to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon151 resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS). The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein.

P45-I005Vimentin regulates differentiation switch via modulation of keratin 14 levels and their expression together correlates with poor prognosis in oral cancer patients

M. Vaidya1, C. Dmello1, S. Sawant1, P. Gangadaran2, R. Thorat1, D. Chaukar2, S. Kane3

1 Advanced Centre for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India2 Department of Nuclear Medicine, Kyunpook National University school of Medicine, N101, Dongin-Dong, Jung-Gu, Daegu, Republic of Korea3 Head and Neck Unit, Tata Memorial Hospital (TMH), Parel, Mumbai, India4 Department of Pathology, Tata Memorial Hospital (TMH), Parel, Mumbai, India

Vimentin is a mesenchymal predominant intermediate filament protein, although its aberrant expression is seen in many carcinomas during epithelial mesenchymal transition. Expression of vimentin is associated with the withdrawal from a more differentiated epithelial phenotype to a dedifferentiated state. Keratins being markers and recently perceived as regulators of differentiation in epithelia, it was important to understand whether vimentin modulates differentiation through reprogramming keratins. Vimentin was stably downregulated in oral cancer derived cell line. Further, global differential keratin profiling was performed using high salt keratin extraction. Keratin 5/14 pair was found to be significantly downregulated both at protein and mRNA levels in vimentin knockdown clones. Subsequent investigations on K14 knockdown clones in the same system showed no difference in vimentin levels, indicating

tumor-specifically expressed EST sequences; ELFNI – AS1, a human gene with possible microRNA function expressed predominantly in tumors and originated in primates; PBOV1, a human gene of the recent de novo origin with predicted highly tumor-specific expression profile; and the evolutionary novelty of human cancer/testis antigen genes and other classes of genes; the expression of evolutionarily novel genes encoding progressive traits in transgenic fish tumors.The conclusion is made that expression of protogenes, evolutionarily young and/or novel genes in tumors might be a new biological phenomenon, a phenomenon of TSEEN (Tumor Specifically Expressed, Evolutionarily New) genes, predicted by the hypothesis of evolution by tumor neofunctionalization.

P2-I001Oncogenic Y641 mutations in EZH2 prevent Jak2/b-Trcp- mediated degradation in germinal center-derived B-cell lymphomas

A. Sahasrabuddhe1,2, X. Chen1, F. Chung1, T. Velusamy1, M.S. Lim1,3, K.S.J. Elenitoba-Johnson1,3

1 University of Michigan Medical School, Department of Pathology, USA2 Pt. Ravishankar Shukla University, Department of Biotechnology, India3 Universities of Pennsylvania, Department of Pathology and Laboratory Medicine, USA

Background: EZH2 is a critical enzymatic subunit of the PRC2 which trimethylates histone H3 (H3K27me3) to mediate gene repression. The recurrent gain-of-function somatic mutations in EZH2, at Y641 residue have been recently reported in germinal center B-cell (GCB) derived lymphomas. Consequently, B-cell lymphoma cell lines and lymphoma primary samples harboring heterozygous EZH2Y641 mutations exhibit increased levels of H3K27me3 and promote a lymphoproliferative phenotype. The EZH2 regulation at post-translational level in a cancer specific context is not known.Hypothesis: Dysregulated proteolysis of EZH2 contributes to cancer progression.Experimental design and Conclusion: Cullin-ring ubiquitin ligases (CRLs) form the largest known class of multicomponent E3 ubiqutin ligase family. Screening of a panel of Cullin family E3 ligases (Cul 1,2,3,4A,4B,5 and 7) by co-immunoprecipitation approach demonstrate that Cullin1 exclusively interact with EZH2. To investigate the F-box protein that recruit EZH2 to Cullin1 mediated SCF complex, we screened 8 different F-box proteins and using co-immunoprecipitation approach, observed that F-box protein b-TrCP (FBXW1) specifically interacted with EZH2. Further, RNAi-mediated silencing of b-TrCP resulted in EZH2 stabilization with attendant increase in H3K27me3 activity. Importantly, the Y641 mutants implicated in lymphoma pathogenesis were unable to bind b-TrCP. Further, cycloheximide chase experiments showed that Y641 mutants endogenously expressed in lymphoma-derived cells exhibit increased EZH2 stability and activity. We further show that Y641 is phosphorylated by Jak2 tyrosine kinase and Jak2 pharmacologic inhibition abrogates EZH2- b-TrCP interaction.Result: Our findings delineate a novel b-TrCP/EZH2 axis requiring an intact Y641 residue and oncogenic mutations in EZH2 at Y641 in GCB-cell derived lymphomas confer increased stability to EZH2 and render it resistant to b-TrCP-mediated degradation. This newly identified mechanism might help in design novel therapeutics against B cell neoplasms with increased activity of EZH2.

P9-I002Development of an in vitro carcinogenesis model to test the transformation potential of vimentin

C. Dmello1, S. Sawant1, M. Vaidya1

1 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India

Vimentin is an intermediate filament protein predominantly expressed in mesenchymal cells. In our earlier study, we had seen the expression of vimentin in oral leukoplakia and submucous fibrosis which are known premalignant lesions for human oral cancer. In our study, we saw sequential increase in aberrant expression of vimentin from early stages itself in 4NQO-model of rat lingual carcinogenesis. Hence we overexpressed vimentin in human oral premalignant derived cell line to ask if vimentin is one of the causes or just a consequence of the process of neoplastic development. Critical epithelial mesenchymal transition (EMT) and stemness related molecular changes were observed upon vimentin overexpression. However no transformation related changes were detected which suggested the need for an additional carcinogenic stimulus. Hence, benzopyrene induced in vitro carcinogenesis model


been implicated in a plethora of human cancers, although its role in MDS pathogenesis remains unexplored.Methods: CD341 HSPCs were isolated, using magnetic cell separation from bone marrow of patients presenting with MDS/AML or age matched healthy individuals. Total RNA was isolated and gene expression analysis was performed using quantitative RT-PCR, Flow-cytometric studies were also performed to check protein expression. Nuclear lysates from primary leukemic progenitors were subjected to biochemical, functional and chromatin immunoprecipitation (ChIP) analysis. Loss or gain of function studies were performed, using shRNA expressing or cDNA expressing retro/lenti virus particles, to check survival, proliferation and clonogenicity of the leukemic progenitors.Results: We demonstrate that there is no significant change in the mRNA expression of the individual BAF subunits in MDS HSPCs, irrespective of the disease phenotype, compared to normal HSPCs. Sub cellular fractionation studies indicate that BAFs retain functional chromatin remodeling activity. To understand the mechanistic aspect of the BAF functioning in leukemic progenitors ChIP analysis were performed which indicate aberrant recruitment of BAF on the promoters of selected genes commonly associated with MDS/AML development. We also provide evidence that leukemic BAFs can cooperate with few other established histone modifiers in HSPC transformation.Conclusion: Collectively these data highlight to a putative oncogenic role of BAF in MDS pathogenesis.

P109-I008A novel molecular imaging sensor to elucidate functional role of non-canonical STAT3 activation in breast cancer

S. Dimri1, R. Arora1, A. De1

1 Molecular Functional Imaging Lab, Tata Memorial Centre- ACTREC, Mumbai, India

Background: Activated STAT3 mediated signaling is known to be a cause in variety of cancers generally associated with poor prognosis. Activation of Stat3 oncogene is primarily characterized by Y705 phosphorylation (canonical pathway). However, less studied modifications like S727 phosphorylation and K685 acetylation are now emerging as key mediators (non-canonical pathway) of the pathway. We report here development of a molecular imaging sensor that can identify the kinetics and biology of non-canonical STAT3 activation in vivo.Method: Bioluminescence Resonance Energy Transfer (BRET)-based STAT3 dimerization sensor was developed using NanoLuc (donor luciferase) and TurboFP635 (acceptor fluorophore). Stat3 fused to either donor or acceptor was developed using recombinant DNA technology and confirmed for their sequence integrity. Further, these vectors were tested for protein expression and activity by immunoblotting, confocal microscopy, and spectral in vivo imaging. Various cancer cell line e.g. HT1080 (fibrosarcoma), PC3 (prostate cancer), MCF7 (breast cancer) were used to screen out the optimal BRET sensor and elucidate the role of key non-canonical PTMs in STAT3 dimerization.Results: Out of all four possible orientations tested, C-terminal STAT3 fusions for both donor and acceptor was found to form best orientation showing significant increase in BRET ratio (P , 0.05). The STAT3-BRET sensor showed definite activation followed by STAT3 dimer formation tested using EGF and IL6 (STAT3 activators) in live cell condition. The functionality of the model was also confirmed in different cancer cell lines to firmly establish the activation sensing efficiency (p , 0.05) in presence or absence of specific PTMs and recorded the time kinetics in vivo.Conclusions: We have successfully developed a Stat3 dimerization sensor that can capture the dynamics of STAT3 homodimerization in response to specific ligands in a concentration dependent manner. The set sensor will serve as a screening platform to identify novel drugs for STAT3 post-translational modifications S727p, K685Ac and Y705p for breast and other cancers.

P121-I009Reduced DOCK4 expression leads to erythroid dysplasia in -7/del (7q) myelodysplastic syndromes

S. Karmakar1

1 Department of Biochemistry, AlIMS, New Delhi, India

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders characterized by cytopenias caused by ineffective hematopoiesis. Even though a third of patients with MDS may transform to acute leukemia, cytopenias drive morbidity for most patients. Cytogenetic studies have shown that stem and progenitor cells in MDS contain deletions in chromosomes 5, 7, 20, and others. Deletions

that vimentin may lie upstream in the pathway that regulates expression of K14. Previous study from our laboratory has shown that K5/K14 pair plays a role in proliferation and maintenance of dedifferentiation. Our data demonstrated that, depletion of vimentin led to increase in the degree of differentiation marked by increase in levels of K1, involucrin, filaggrin and loricrin while proliferation remained unaffected. Rescue experiments with K5/K14 pair overexpressed in vimentin knockdown background resulted in decreased level of differentiation. Our data shows cross-regulation between notch and ΔNp63 to decide the differentiation fate, with respect to vimentin expression. Further, immunohistochemical analysis showed positive relationship between vimentin and K14 expression in oral tumor tissues which corroborated with our in vitro findings. Interestingly, significant correlation was seen between expression levels of these proteins and clinical parameters like stage, differentiation, tumor size and poor survival. Thus in conclusion, vimentin regulates the differentiation switch via modulation of K5/K14 expression possibly through crosstalk between ΔNp63a and notch. In addition, vimentin-K14 together may prove to be novel markers for prognostication of human oral cancer.

P65-I006Identification of Notch regulator for the growth of human brain tumor initiating cells

S. Sarkar1,2, F.J. Zemp3, D. Senger3, S.M. Robbins3, V. Wee Yong1,2

1 Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada2 Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada3 The Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada

Background: Intracellular signaling pathways in malignant glioma significantly deviate from normal preset. A methodical evaluation of this deviation can provide significant insights into the physiological function as well as etiology of specific malignant glioma abnormalities. Increased Notch signaling has been implicated in brain tumor initiating cells (BTIC), which are resistant to chemo- and radio-therapies. Not known is whether the Notch signaling pathway within BTIC constitutes a mechanism that promotes tumor growth.Methods and Results: We found that tenascin-C (TNC), an extracellular matrix molecule, and the Notch ligand, Jagged1 (Jag1) are over expressed in malignant glioma. Elevated levels of Notch ligand were correlated proportionally with the sphere forming capacity of BTICs in culture. Exogenous and intrinsic TNC increased BTIC growth through an a2b1 integrin-mediated mechanism that elevated Jag1. Microarray data and pathway analysis supported the activation of Notch signaling in BTICs shortly following TNC treatment. Moreover, an inhibitor of the g-secretase complex, DAPT, decreased BTIC sphere formation. Addition of the metzincin metalloproteinase inhibitors BB94 and GM6001 also reduced TNC-stimulated BTIC growth. Furthermore, Notch activation was evidenced by the increase of NICD (Notch intracellular domain) 1 and NICD2 in BTICs exposed to TNC for 6 h. TNC-stimulated Notch activity and elevated Jag1 expression was confirmed using lentiviral transduction-mediated knockdown of TNC in BTICs. Importantly, TNC and its activation of Notch signaling was validated using resected glioblastoma specimens and orthotopic human glioblastoma xenografts.Conclusions: Collectively, the current work identifies a new factor in Notch signaling for glioma growth. Identification of the Notch activator should help us understand the critical mechanisms involved in BTIC growth and can open up novel therapeutic avenues.

P96-I007Deciphering chromatin remodeling in myelodysplastic syndrome

S.S. Chatterjee1,4, M. Biswas1,4, L.D. Boila1, S. Sinha1, S. Chakraborty1, D. Banerjee2, A. Sengupta1,3

1 Stem Cell & Leukemia Lab, Cancer Biology & Inflammatory Disorder Division, CSIR – Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India2 Park Clinic, 4, Gorky Terrace, Kolkata, West Bengal, India

Background: Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders that originate in the hematopoietic stem and progenitor cell (HSPC) compartment of the aged population leading to impaired blood cell formation. MDS HSPC compartment is heterogeneous and includes refractory anemia (RA/RARS), refractory cytopenia with multilineage dysplasia (RCMD) or refractory anemia with excess blasts (RAEB) that may eventually transform into acute myeloid leukemia (AML). The BRG1/BRM associated factor (BAF) is a multi-subunit chromatin remodeling complex (CRC) whose loss of function has

I. Tumor Biology European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S47

chaperone HIRA. Strikingly, the alteration in the histone PTM profile shows similar pattern with decrease in gene activation marks and increase in repressive marks. We further show that with the decrease in the activating variant (H3.3) and PTM levels there is a global chromatin condensation leading to transcription repression in cancer cells. Our report suggests that there is co-operative interplay between histone modifiers, variants, chaperones and their transcriptional regulation which results in the stable maintenance of the highly dynamic histone marksrequired for maintaining the deregulated epigenetic landscape found in cancer cells.

P171-I012Histone H2A.1 isoform: The emerging regulator of the epigenetic landscape

S. Bhattacharya1, D. Reddy1, S. Gupta1

1 Gupta Lab, Epigenetics and Chromatin Biology Group, Cancer Research Institute, ACTREC, Tata Memorial Centre, Mumbai, India

The complex epigenetic regulatory mechanisms in the cell are now well recognized to be key players governing the physiology and phenotype of an organism besides the inherent genetic code in the DNA sequence. Not surprisingly aberrant alterations in the epigenetic landscape have been observed in all cancers. Unlike the genetic alterations though, the epigenetic alterations are more amenable to reversal. However, to discover promising therapeutic targets and possible biomarkers much remains to be understood about how epigenetic regulation of gene expression takes place in the cell. Here, we discuss the specific functional contribution of the major histone H2A isoform H2A.1 which was previously thought to be functionally redundant. We found that H2A.1, which exhibits drastically altered expression pattern in different normal tissues and human cancer cell lines (H2A1C in humans), promotes cell proliferation in a context dependent manner. To uncover the molecular basis of the non-redundancy, we used in silico, in vitro and in cellulo methods to show that H2A.1-containing nucleosomes are less stable compared to nucleosomes containing another major H2A isoform, H2A.2. Interestingly, we also found that the nucleosome stability is intimately linked with the physiological effects observed. We demonstrate that H2A isoforms modulate chromatin stability which leads to contextual alteration in gene expression pattern which may promote attainment/persistence of pathological states like cancer.

P180-I013Dual regulation of SGK1 and PR expression by progesterone via microRNAs and progesterone receptor (PR)

M. Godbole1, P. Chandrani1, H. Dhamne1, K. Patel1, N. Gardi1, K. Tiwary1, S. Gupta3, R. Badwe2, A. Dutt1

1 Integrated Genomics Laboratory, ACTREC, Tata Memorial Centre, Mumbai, India2 Department of Surgical Oncology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India3 ACTREC and Department of Medical Oncology, Tata Memorial Centre, Mumbai, India

Background: Breast cancer is the 3rd leading cause of death by cancer. Earlier observations indicate that surgery in menstrual phase of opposed estrogen showed better outcome in pre-menopausal breast cancer patients. Moreover, a recent randomized clinical trial with preoperative progesterone intervention in node-positive breast cancer patients showed an increase in survival of patients independent of progesterone receptor (PR) status. However, the mechanism through which progesterone acts on breast cancer is largely unknown.Methods: In the present study we are trying to understand the effect of progesterone through an integrated analysis of progesterone treated breast primary tumor derived cell lines. Small RNA sequencing and gene expression analysis was performed to identify the progesterone regulated microRNAs and genomic transcripts in four breast cancer cell lines. Real-time PCR and luciferase assay were performed for checking expression of microRNA and target genes and for validation purposes.Results: Targets of microRNAs de-regulated by progesterone were identified using microRNA target prediction databases. An overlap of microRNA target genes with differentially expressed genes identified Serum and glucocorticoid –regulated kinase 1, SGK1 and the Progesterone receptor, PR, to be predicted targets of three differentially expressed microRNAs. Further we validated the transcript and protein expression of these two genes and their corresponding microRNAs which showed anti-correlation for expression of genes and microRNAs. Luciferase assay with these microRNAs and 3’UTR region of SGK1 and PR indicated that these novel microRNAs can regulate the expression of their corresponding target genes.

of chromosomal 7q region are seen in 10% of cases and associated with significantly worse survival. These genomic deletions are usually large, and it is not clear which of the deleted genes contribute to the pathogenesis of hematopoietic defects in MDS. In a previous study, we had observed that a 7q gene, DOCK4, was deleted or epigenetically silenced in MDS, thus prompting an examination of its role in erythropoiesis in the present study.Methods: Human CD34+ progenitor cells were expanded and differentiated along erythroid lineage by adding EPO. Lentiviral particles carrying DOCkK4 shRNA were used for gene silencing.Results: Depletion of DOCK4 leads to erythroid cells with morphological defects in vitro using a human primary MDS erythroblasts. Reduced expression of DOCK4 leads to disruption of the F-actin network causing erythroid dysplasia that phenocopies the red cell defects seen in samples from MDS patients with a -7/del (7q).Conclusion: Anemia is the predominant clinical manifestation of MDS. Even though deletion of the 7q chromosomal region is commonly seen in MDS the pathogenic genes on this region have not been identified. DOCK4 is a GTPase exchange factor that is present on the commonly deleted 7q 31 region; is found to be underexpressed in MDS bone marrow samples, and the reduced expression is associated with decreased survival in patients. These data identify DOCK4 as a 7q gene whose haploinsufficiency can lead to erythroid dysplasia.

P132-I010Identifying mechanisms regulating tumor progression upon plakophilin3 loss

S. Basu1, C. Braggs1, A. Sawant1, S. Vaz1, S. Dalal1


Background: Desmosomes are adherens like junctions present in epithelial cells that anchor intermediate filaments at membrane associated plaques in adjoining cells allowing the formation of an intercellular network that promotes tissue organization and rigidity. Plakophilin3, a desmosomal plaque protein binds to a broad repertoire of proteins like the desmosomal cadherins, cytokeratin 18, desmoplakin and plakoglobin. Previous results from our laboratory demonstrate that plakophilin3 loss leads to alterations in the desmosome size, decrease in cell-cell adhesion, increased cell migration, anchorage independent growth, growth to high density in culture, accelerated tumor formation in nude mice and increased metastasis to lung. Plakophilin3 expression is known to be lost in poorly differentiated oropharyngeal cancer, colon cancer, gastric cancer and bladder cancers. The mechanism underlying tumor progression in plakophilin3 knockdown clones remained unclear.Methods: Microarray analysis, Real time PCR, shRNA mediated mRNA knockdown, Soft agar colony formation assay, scratch wound healing assay, tumour formation in nude mice, luciferase reporter assay.Results: Gene expression analysis demonstrated that plakophilin3 loss leads to an increase in the expression of Lipocalin2 (LCN2). Knockdown of LCN2 in plakophilin3 knock-down clone leads to decrease in cell migration, invasion and abolishes the ability of cells to form soft agar colonies. LCN2 knockdown also inhibits tumor formation in nude mice. Luciferase reporter assays using different LCN2 promoter fragments confirmed that LCN2 expression is regulated by plakophilin3 loss at the transcriptional level. The increase in LCN2 expression upon PKP3 loss was found to be regulated independently by both ELK1 and p38b MAPK pathways.Conclusion: Plakophilin3 loss leads to up-regulation of LCN2 expression. LCN2 regulates neoplastic progression observed upon plakophilin3 loss via ELK1 and p38b MAPK signaling.

P169-I011Histone H3 variants: Regulators of cancer epigenome?

D. Reddy1, S. Bhattacharya1, S. Gupta1

1 Gupta Lab, Epigenetics and Chromatin Biology Group, Cancer Research Institute, ACTREC, Tata Memorial Centre, Mumbai, India

Epigenetic regulatory network has become a matter of intense investigation over the years from perspective of disease pathology because unlike the genetic changes they are more amenable to reversal and are better targets for therapeutic intervention. To understand and increase the knowledge of epigenetics in context of cancer, in the present study, we have screened for histone pattern changes between the different stages of cancer and have identified that there is sequential downregulation in the histone H3 variant H3.3 with HCC development and concomitant increase in level of H3.2 variant both at the protein and transcript level. The same is observed even for histone chaperones, with increase in H3.2 chaperones CAF-1 complex and decrease in H3.3


P242-I016ER negative-specific differentially methylated genes identify master regulators to expose potential epigenetic drivers of aggressive disease

K.M. Chen1, I. Datta1, J.K. Stephen1, D. Chitale1, G. Divine1, M.J. Worsham1

1 Henry Ford Health System, Detroit, USA

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has yet to configure in epigenetics. There has been relatively little advancement in changing the management of women with ER negative BC, mainly due to a dearth of actionable therapeutic targets. Our drill-down approach identified an ER negative-specific 16 gene methylation signature (AHNAK, ALPL, ANXA2R, CCND1, CIRBP, CPQ, DST, EGFR, ESR1, GPRC5B, HERC5, IL22RA2, MITF, OBSL1, POU3F3, RB1CC1) starting from a discovery approach (Illumina Infinium HumanMethylation 450 BeadChip, 40 ER negative vs. 40 ER positive BC) followed by expression verification, significant rankings in biologicalpathways (Ingenuity Pathway Analysis), and confirmation by targeted sequencing using Illumina MiSeq. Causal Networks are small hierarchical networks of regulators that control the expression/methylation of dataset targets. They can enhance understanding of the effect of master regulators on disease or function. The objective of this study was to identify regulatory networks utilizing IPA’s Causal Network Analysis (CNA) in order to illuminate possible causes and mechanisms underlying the biological activities of the 16 candidate gene signature differentiating ER negative from ER positive BC. To reflect expected gene expression direction implied by methylation changes for the 16 candidate genes, the inverse of the methylation ratio from ER negative vs. ER positive tissue was used for CNA. CNA software identified 4 hierarchical networks and their corresponding master regulatory molecules, diethylstilbestrol, MSH2, 15-ketoprotaglandin E2, and transcription regulator SP1. Diethylstilbestrol and SP1 had direct regulatory influence (depth level 1) to the candidate molecules ALPL, CCND1, EGFR, ESR1 and CCND1, CIRBP, EGFR, ESR1, respectively. CNA raised the profile of ALPL, CCND1, CIRBP, EGFR, ESR1 (5/16 candidate genes) for further consideration as potential epigenetic drivers of ER negative BC.

P292-I017Study of the regulation of nucleus and nucleolus size: A prequel of organelle directed therapy for cancers

A. Ganguly1, D. Bhattacharyya1

1 ACTREC-TMC, Navi Mumbai, India

Size regulation of human cell nucleus and nucleolus are still poorly understood subjects. The nuclear to cell volume ratio (N/C ratio) or “Karyoplasmic ratio” remains a roughly constant value in cells with widely different DNA contents, ranging from single-celled eukaryote to mammalian cells. However this ratio does not remain constant in cancer cells. In fact, one of the primary mode of diagnosis of cancer requires light microscopic analysis of the biopsy sample in which a pathologist looks normally for abnormal organelle architecture, primarily altered nuclear size as a diagnostic for cancer. In spite of such an early knowledge of association with several forms of cancers, the regulation of nuclear or nucleolar size are still poorly understood field. Knowledge of mechanism of such size regulations of these vital intracellular sub compartments has the potential to develop organelle directed therapy. In the present study we have determined the range of variation of nuclear size in terms of N/C ratio and nucleolus size with respect to nucleus size in different immortalized and transformed cell lines of same tissue origin. Advanced imaging and consequent 3D reconstruction of the live images shows that the N/C ratio increases by 30–80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the N/C ratio value to increase by 30–50% in immortalized cell lines. Nucleolus volumes are significantly increased as well in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a dramatic increase in the nucleolus volume of immortalized cell lines. A cytosolic and nuclear fraction exchange experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

Conclusion: Our results indicate that progesterone regulates SGK1 and PR levels via the progesterone response elements in the promoter region and via microRNAs binding to 3’UTR region of the gene in a dual-regulated manner. Identifying more such unique associations in response to progesterone could help in better understanding of action of progesterone in breast cancer.

P203-I014Nucleolar clusterin in oral cancer cells – A new challenge to the old enigma

R. Kadam1, T. Teni1

1 Teni Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India

Background: Clusterin (CLU) is a stress induced chaperonic glycoprotein associated with various physiological functions like cell cycle regulation, apoptosis, DNA repair etc. The secretory and nuclear forms of CLU serve anti- and pro-apoptotic functions respectively. Recently we have demonstrated high expression of CLU protein in oral cancer cell lines. In the present study, we report for the first time the nucleolar localization of CLU in oral cancer cell lines. This study is an attempt to understand the novel function of CLU residing in the nucleolus.Methods: Immunofluorescence based localization studies were carried out to determine the CLU localization pattern in oral cancer cell lines. To confirm the nucleolar localization, cells transfected with pEGFP-C1-Fibrillarin construct were taken for colocalization studies of both proteins. Further the effect of radiation on CLU localization pattern was also evaluated. Studies are ongoing to mutate the predicted nucleolar localization sequence (NoLS) of CLU and to evaluate the localization pattern of CLU in the cells expressing NoLS mutated CLU under stressed versus unstressed condition.Results: Our preliminary data demonstrated the nucleolar localization of CLU, which was also supported by the presence of NoLS at the N-terminus of CLU and its co-localization with Fibrillarin. Also 2–6 hrs post-radiation, changed localization of CLU from nucleolus to nucleus and cytoplasm and back to nucleolus at 8 hrs post radiation was observed.Conclusion: This is the first report to demonstrate the presence of CLU in nucleolus of oral cancer cells. Based on the preliminary observation, it is possible that the excess of endogenous CLU translocate to nucleolus which upon radiation treatment changes its localization pattern. Studies to assess the possible interaction of CLU with DNA repair proteins residing in nucleolus which may translocate to the nucleus and cytoplasm in response to DNA damage response pathway have been initiated.

P217-I015Dynamic alteration in histone modification profile during ionization induced DNA damage response and radio-resistance

A.K. Sharma1, A. Sharda*1, S. Bhattacharya1, S.A. Khan1, D. Reddy1, S. Gupta1

1 Epigenetics and Chromatin Biology Group, Gupta Lab, Advanced centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial centre, Kharghar, Navi Mumbai, India

Chromatin acts as a natural barrier in DNA damage recognition and repair. Histones undergo differential Post Translational Modifications (PTMs) to facilitate DNA repair. Further, acquired or inherent radio-resistance is an enigmatic phenomenon and whether resistant cells have different epigenetic profiles compared to non-resistant cells needs to be addressed. In the present study, we have investigated the alteration in levels of different histone PTMs in response to DNA damage and radio-resistance. We show that the highly dynamic mark H3S10P decreases specifically from irradiated G1 enriched cells irrespective of the damaging agent or cell line used. Interestingly, the loss occurs predominantly from H3.3 variant suggesting that the alteration might be implicated in transcriptional repression. Further, we show that the MAPK Phosphatase MKP-1 and Kinase MSK1 are responsible for maintaining the dynamic phosphorylation status of H3S10. Also, profiling of histone modifications in radio-resistant v/s parental MCF7 breast cancer cells shows global hypo acetylation in radio-resistant cells. Collectively, our data proposes a pathway regulating reversible reduction of H3S10P on IR induced DNA damage in G1 phase cells and also raises the possibility of exploring epi-drugs against G1 phase and radio-resistant cells.

J. Cancer Prevention and Screening European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S49

P13-J003Trends of breast cancer in Aden Cancer Registry, Yemen (1997–2011)

H. Basaleem1, K. Al-Sakkaf1

1 Aden Cancer Registry and Research Center, Department of Community Medicine and Public Health, University of Aden, Yemen

Background: According to GLOBOCAN 2012; breast cancer is the second most common cancer in the world and the most frequent cancer among women. In Aden Cancer Registry, breast cancer is a frequently reported cancer. The present paper aimed at determining the trend of breast cancer over 15 years period (1997–2011) based on in Aden Cancer Registry data; the only population-based cancer registry in Yemen.Methods: Data were collected from the main public and private hospitals, abroad treatment registry at Ministry of Aden Health Office and diagnostic centers in the governorates covered by the Registry. The collected data were entered and analyzed using Canreg-4 software with duplicate entry checking by name, age, sex, morphology, topography and residence. The World Standard Population and the local population of the four Governorates were used in the analysis.Results: There were 1227 registered breast cancer patients; 95.5% of them were females. They represent 16% of all cancers and 35% of female cancers. The median age was 47 years (minimum 18; maximum 88) with 21.8% of cancers reported in those younger than 40 years. Around half of registered patients were from Aden (55.5%). Carcinoma not otherwise specified was the diagnosis found in 48.6% whereas 39.9% was infiltrating duct carcinoma. There is a rising trend in the registered patients and age standardized incidence rate per 100,000 inhabitants during 1997–2011.Conclusion: We concluded that breast cancer is the most incident cancer among all cancers in both sexes. The steady rising incidence indicates that efficient breast cancer control strategy is mandatory.

P59-J004Surviving cancer and its treatment

M. Alexander1

1 Widener University, University Pl, Chester, PA, United States

Cancer survivors face a wide variety of problems as a result of having cancer and its subsequent treatment. Chemotherapy-induced cognitive dysfunction is the result of immunological inflammation and leads to psychological cognitive problems. It is not clear how clinician/scientists trained in immunology and psychology will treat this disorder. This work seeks to examine the immunological and psychological aspects of chemotherapy-induced cognitive dysfunction. It will discuss ways to treat the condition or prevent it from happening using newly developed biological treatments and psychotherapies. This is an intersection of immunology and psychology, which should yield valuable information applicable to other neurodegenerative diseases.

P67-J005Abstract withdrawn

J. Cancer Prevention and Screening

P3-J001Prevalence and intensity of dyspnea in advanced cancer and its impact on quality of life

A. Damani1, N. Salins1, J. Deodhar1, M.A. Muckaden1

1 Department of Palliative Medicine, Tata Memorial Hospital, Mumbai, India

Background: Dyspnea is a subjective, multidimensional experience of breathing discomfort, commonly seen in patients with advanced cancer that influences all aspects of patient’s life. To find the impact of dyspnea on the quality of life in this population, it is important to understand the prevalence and factors influencing dyspnea.Aim: The aim of this study was to determine the prevalence, intensity and factors influencing dyspnea in advanced cancer and determine its impact on overall quality of life.Methods: The study was a prospective cross sectional study. Prevalence of dyspnea and its impact on quality of life was determined on 500 patients registered with Palliative Medicine OPD. The patients were asked to fill a set of questionnaires which included the Cancer Dyspnea Scale (translated and validated Hindi and Marathi versions), Visual Analogue Scale for dyspnea and EORTC QLQ C 15 PAL. Other details of symptoms, disease, treatment and the demographics were collected from the case record form of the patient. Descriptive statistics, univariate and multiple regression analysis were used to calculate results.Results: 44.37% of the patients experienced dyspnea. The factors of dyspnea increased with increase in anxiety, depression, fatigue, loss of appetite, loss of wellbeing, pain, lung involvement by primary or metastatic disease, performance status and deteriorating overall quality of life and emotional well being on EORTC QLQ C15 PAL.Conclusions: Prevalence of dyspnea in advanced cancer population is as high as 44.37% and it causes a negative impact on overall quality of life of patients.

P10-J002Analysis of prostaglandin-endoperoxide synthase gene polymorphisms and risk of cervical cancer in an East Indian population: A case control study

D. Sur1, R. Chakravorty1

1 Department of Obstetrics & Gynecology, MAGS Medical & Research Center, West Bengal, Kolkata, India

Background: The Prostaglandin Endoperoxide Synthase (PTGS)2 gene appears to play a role inflammation or tumour and mitogenesis. Genetic polymorphisms in the (PTGS)2 might contribute to differential PTGS2 expression and subsequent inter-individual variability in susceptibility to cancer.Aim: The goal of this study is to identify genetic variants of PTGS2 gene in women of East India, which may associate with risk of cervical cancer.Methods: We enrolled 200 histopathologically confirmed patients with cervical cancer (age 18–60 years) (cases) and their corresponding sex and age matched 200 normal individuals (controls). To identify genetic variants responsible for cervical cancer, we performed sequence analysis of PTGS2 genes. Questionnaire survey was conducted to comprehend the demographic data, smoking status, and cancer stage of patients.Results: PTGS2 genotype rs689466:21195A/G, a functional variant of PTGS2 gene is strongly associated with cervical cancer disease in our study population. The genotype frequency of rs689466 polymorphism was significantly different between case and control groups (p , 0.001). Compared with the wild type genotype AA, the variant genotype GG was associated with 20 fold increased risk (p , 0.001; Odds ratio 5 20.76; 95% CI: 2.86–160.73) for cancer patients. The rs5275: exon11 837T.C polymorphism was not associated with cancer risk although this allele was correlated with decreased risk (p 5 0.701; Odds ratio 5 0.71; 95% CI: 0.26–1.90). C C genotype was more frequently found in controls as compared with cases and showed an inverse association with the development of cervical cancer, thus suggesting a possible protective effect.Conclusions: PTGS2 genotype rs689466:21195A/G gene polymorphism demonstrated strongly associated with cervical cancer disease. But exon11837T.C polymorphism was not associated with cancer risk in East Indian women. Further studies evaluating the role of PTGS2 gene polymorphisms in ethnically diverse populations and a larger cohort may help in understanding the etiopathogenesis of cervical cancer in women worldwide.

S50 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 J. Cancer Prevention and Screening

Conclusions: Central obesity is the single most factor for prevention of BC irrespective menopausal, rural-urban and receptor status.

P118-J008Early detection of cancer diseases in rural population using local resources

Ajaya K.T.11 Govt Medical College, Kerala, India

Background: Majority of cancer patients coming for treatment presents in advanced stages in India. The reasons for late presentations are lack of awareness and certain myths like incurability labelled with the disease. A rural outreach program to dispel these myths through awareness and helping the poor through early detection of cancer diseases is the need of the hour.Methods: One gramapanchayath near the medical college was chosen for the study. The total population covered was 35,937. After thorough training of ASHA workers and NGOs all the houses in the area were screened by the volunteers for picking persons with symptoms of early malignancy. The household are also sensitised towards the importance of early detection of malignant diseases. Four Medical camps were conducted in the near vicinity of the area. Suspected cases were taken to medical college for further assessment and tests.Results: 6548 households were visited and total four camps were held.832 people suspected to have cancer were brought to the medical camps. Four persons were diagnosed as having malignancy.Conclusion: Outreach program have a large impact on the cancer detection and cure rates on general population if done state wise or country wise.

P133-J009Early experience in laparoscopic single port onco-surgery in Nepal

P. Kharbuja1, U.M. Shrestha1, K.S. Amatya1, P. Neupane1

Department of Surgical Oncology, Bhaktapur Cancer Hospital, Bhaktapur, Nepal

Purpose: The aim of this study is to compare the Conventional Laparoscopic Surgery (CLS) and homemade Transumbilical Single Port Laparoscopic Surgery (SPLS).Study design: From 2013-01-01 to 2015-07-30, 28 cases of recto-sigmoid cancer underwent Transumbilical Single Port Laparoscopic Surgery (SPLS) and 34 cases underwent of Conventional Laparoscopic Surgery (CLS), and retrospective analytical study was done. Homemade Trocar Inserter was made by surgical glove, cutting a plastic suction tube to make a circle and inserting trocars in cut fingers of glove. In both SPLS and CLS radical resection was done as Dixon’s Procedure. The difference in SPLS group was the incisions 1–2 cm below the umbilicus to insert the laparoscopic instruments.Results: In SPLS group, mean operating time 117.8 min, mean blood loss 62.7 ml and in CLS group, 145.9 min, 40.6 ml respectively. In SPLS group, mean first flatus passage time 2.6 days, mean first defecation time 4.4 days, mean hospital stay 10.5 days and In CLS group, 3.0 days, 5.4 days, 9.7 days respectively. In SPLS group, 5 patients and in CLS group 6 patients had postoperative complications. In SPLS group the average distance of tumor from proximal/distal resected margin is respectively 5/6 cm and mean 15.85 lymph nodes harvested and in CLS group 5.6 cm/5.97 cm and 13.88 respectively. In CLS the drains are kept, but in SPLS no drains are kept so had better cosmetic appearance.Conclusion: Transumbilical Single Port Laparoscopic Surgery for recot-sigmoid cancer can be successfully and safely performed, and has better benefits over Conventional Laparoscopic Surgery for reduced incision, less postoperative pain and improved cosmetics and can be alternative to traditional laparoscopic surgery.

P149-J010Human papilloma virus and survival of patients with oral cavity squamous cell carcinoma

V. Palve1, J. Bagwan1, A. Suresh2, G. Siddappa2, U. Chandola1, B.L. James2, V. Kekatpure3, M.A. Kuriakose2,3, B. Panda1

1 Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied Biotechnology, Bangalore, India2 Mazumdar Shaw Centre for Translational Cancer Research, Bangalore, India3 Mazumdar Shaw Medical Centre, Bangalore, India4 Strand Life Sciences, Bangalore, India

The incidence of HPV in oral cavity tumors is relatively an unexplored area of research compared to oropharyngeal tumors. Several methods are

P102-J006Menstrual pad a cervical cancer screening tool for rural Indian women

A. Budukh1, V. Palayekar2, A. Maheshwari1, K. Deodhar1, P. Purvar1, S. Bagal1, A. Vadigoppula2, P. Meherji2, M. Lokhande3, N.S. Panse3, R. Dikshit1, K.M. Mohandas4

1 Tata Memorial Centre, Mumbai, India2 National Institute for Research in Reproductive Health, Mumbai, India 3 Nargis Dutt Memorial Cancer Hospital, Barshi, India4 Ex–Director, Centre for Cancer Epidemiology, Tata Memorial Centre, Mumbai, India

Objective: To test the DNA extraction from the menstrual blood is good enough in quality and quantity for HPV testing by PCR technique and further it can be used as a cervical cancer screening tool.Method: In phase I we have collected menstrual pad (cloth) from the consented women from the rural population. Method for extracting DNA from menstrual pad was standardized. The menstrual pads were tested by PCR technique. The women who have provided menstrual pad were also underwent HC2 testing. The HPV positive women based on PCR/HC2 testing were underwent colposcopy. In Phase II we have collected menstrual pad from the consented women in the different population. The HPV positive women based on PCR testing were underwent colposcopy and HC2 testing. Few randomly selected HPV negative women based on PCR technique were also underwent HC2 testing and Colposcopy.Results: In Phase I, 164 women provided menstrual pad as well they attended HC2 screening test. 6 cases (3.2%) reported as HPV positive. In PHASE II, 346 consented women provided menstrual pad for HPV testing. 13 (3.8%) cases were diagnosed as HPV positive. 49 randomly selected HPV negative cases based on menstrual pad PCR testing underwent colposcopy and HC2 testing. The concordance rate of PCR test and HC2 test was 98%, 86%, provisonal senisitivity 86%, 60%, specificity 99%, 88.7% respectively in Phase I and Phase II. Three CIN II cases were diagnosed and 2 were treated by LEEP, one CIN II case refused treatmet. One CIN I case was treated by Cryotherapy and 1 CIN 1 case was under follow up Detection rate of precancerous lesion was 0.9%.Conclusion: Based on the moderate level of sensitivity and high level of specificity of the test menstrual pad (cloth) can be used as a cervical cancer screening tool.

P103-J007Central obesity and not reproductive or genetic susceptibility is the most important factor for reducing breast cancer burden in India

R. Nagrani1, S. Mhatre1, R. Badwe1, R. Dikshit1

1 Centre for Cancer Epidemiology, Tata Memorial Centre, Mumbai, India

Background: In absence of suitable screening program and no contribution of hereditary or common genetic variants towards primary prevention of Breast Cancer (BC), search for a single most important factor is necessary which could reduce the burden of entire spectrum of heterogeneous BC.Methods: Questionnaire data was collected on 3152 case-control pairs. Genotyping was performed on a-priori 384 single nucleotide polymorphisms (SNPs) related to BC or obesity. Odds Ratios (OR) and Polygenic risk score (PRS) for SNPs identified from BC genome wide association studies were calculated using unconditional logistic regression from recently concluded hospital based case-control study. To estimate the contribution of central obesity, reproductive factors and heritability component in development of BC, population attributable fraction (PAF) was calculated.Results: Waist-to-hip ratio increased the risk of BC in women across the strata (ORpremenopausal 5 4.11, 95% CI: 2.78–6.08, ORpostmenopausal 5 3.43, 95% CI: 2.42–4.85, ORER+/PR+ 5 3.92, 95% CI: 2.79–5.49, OR ER-/PR- 5 2.24, 95% CI: 1.93–2.60, ORTNBC 5 2.36, 95% CI: 1.98–2.81) whereas BMI .30 kg/m2 increased the risk only in postmenopausal (OR 5 1.85, 95% CI: 1.05–3.28) and showed protective association in premenopausal women (OR 5 0.52, 95% CI: 0.36–0.76). Among reproductive factors age at first full-term pregnancy increased the risk across all strata of BC. In our population where breast feeding is still a common practice, it did not show any association with BC. PAF estimated for waist circumference in rural and urban women was observed to be 10% and 18% respectively. A higher difference in PAF was observed for age at first full-term pregnancy with women in urban areas showing 14% whereas rural women showing 3.5%. Only 3.2% of postmenopausal BCs in Indian women could be attributed to heritability over and above the familial risk of BC.


based screening for breast cancer by Clinical breast examination (CBE) by trained health workers and referral of screen-positive women to higher centre, Tata Memorial Hospital (TMH) for further diagnostic work-up and treatment. Women between 30–64 years of age with no history of breast cancer were included in this programme. Data was analysed using univariate and multivariate logistic regression in SPSS version 16.Results: The compliance to screening, referral and treatment were 81.42%, 73.83% and 100% respectively. Multivariate logistic regression analysis demonstrated that literacy, having family history of cancer, having tobacco habit, being treated for breast abnormalities, being ever pregnant and having history of contraceptive use were positive predictors of participation in screening. While, women belonging to Muslim religion or speaking mother-tongue other than Marathi or Hindi were negative predictors of participation to screening. Educational status was the only significant predictor of compliance to referral. Screen-positive women with education of secondary school level or more were more likely to comply with referral.Conclusions: The programme ensured good compliance with screening, referral and treatment indicating feasibility and acceptability of CBE based breast cancer screening programme by the community.

P206-J013Higher specificity of high risk HPV transcript versus the standard DNA based test in triage setting in primary cervical cancer screening

S. Pawar1, S. Pimple2, S. Chavan2, A. Pathania1, T. Teni1

1 Teni Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Navi Mumbai, India2 Department of Preventive Oncology, Tata Memorial Hospital, Mumbai, India

Background: The cervical cancer screening with 4% acetic acid (VIA) displays low specificity & poor predictive value, therefore a secondary HPV DNA based screening test viz; Hybrid capture II (HC II) is done for VIA positive women. However, only a fraction of the women infected with HR HPV types progress to cancer, often resulting in follow-up of women with clinically insignificant infection. Therefore detection of HPV E6/E7 mRNA may be a better indicator of active HPV infection. The aim of the present study is to develop and standardize a cost effective methodology for detection of E6/E7 mRNA for HR HPV 16 and 18 to triage false positives from primary cervical cancer screening.Methods: So far 288 women in age group 30–65 years who were found to be positive on VIA testing were enrolled in the proposed study. The exfoliated cells were collected by using DNA PAP cervical sampler and stored in RNA later. RNA was isolated by Masterpure kit and cDNA was prepared using High capacity kit. These women were tested for oncogenic HR HPV types by Hybrid capture II and also for HPV 16 & 18 E6/E7 mRNA by Taqman real time PCR, with histology as reference standard.Results: Out of total 288 samples analyzed 58 were positive for HR HPV types detected by HC II and 34 (59%) among these demonstrated HPV 16 and 18 E6/E7 mRNA expression. The E6/E7 mRNA detection test exhibited 93% specificity with 44% PPV while HPV DNA by HCII exhibited 86% specificity and 36% PPV.Conclusion: Our data shows that only a subset of samples which are HC II positive exhibit E6/E7 transcripts, indicating an active HPV infection. A higher specificity in triage setting may reduce the false positive burden eliminating the number of women subjected to unnecessary and expensive diagnostic follow-up.

P207-J014Low utility of p16 as surrogate marker in Indian HNSCC patients with high tobacco burden

P. Kalkar1, S. Pawar1, As. Patil2, V. Murthy3, T. Teni1*1 Teni Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India2 Department of Pathology, Tata Memorial Hospital, Dr. E Borges Marg, Mumbai, India3 Department of Radiation Oncology, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India

Background: The high incidence of Head and Neck Squamous Cell Carcinoma (HNSCC) in India is attributed to the prevalent tobacco chewing habit in the country, however recently the association of Human Papilloma Virus (HPV) with HNSCC is also emerging. Limited data is available on prevalence and prognostic significance of HPV in Indian HNSCC population. Also in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnosis in HNSCC.

available for the detection of HPV. However, they differ in their sensitivity and specificity. Sensitive DNA/RNA-based quantitative techniques are better suited to circumvent false positive and low sensi tive methods like immunohistochemistry (IHC) and in situ hybridization (ISH), those which are especially unsuitable to detect low copy number viral nucleic acids in tumor DNA. In the past, expression of the aberrantly expressed surrogate protein markers like p16 have been used to link outcome stratification with HPV infection. Past studies in oropharynx have shown that patients with HPV-positive tumors are not only more responsive to the treatment, but also show much better overall survival (OS) and disease-specific survival (DSS) than the HPV-negative patients. Unlike oropharyngeal tumors, the relationship between HPV and survival in oral cavity tumors is not unequivocal. Here, by using oral cavity tumors (n 5 157) and multiple sensitive and precise detection techniques, we established incidence of HPV in oral cavity tumors and linked HPV to survival. Depending on the method used, we found 24–54% HPV DNA in oral cavity primary tumors. Most of the tumors had very low copy HPV DNA and there was variation in incidence among different subsites in oral cavity. By using HPV calls from individal sensitive assays and also concensus calls from several methos, our data suggest that presence of HPV DNA is not linked with both OS and DFS in oral cavity tumors.

P160-J011Effective collaboration and new addition of cancer institutes under NCG-model to provide highest quality of care for cancer patients in India

S. Sanchetee1

1 Sanchetee Hospital and Cancer Institute Unit of Rajasthan Cancer Cure Hospital Private Limited, Jodhpur, India

Introduction: There has been an increase in the onset of early, aggressive cancers in the twenty first century. Health professionals are not providing the detailed information to the patients due to either lack of knowledge on the subject or conscious manipulation for vested interest. The money, human and resources spent by government, public and private organizations are not being properly utilised. There is an urgent need to manage the existing and newly diagnosed patients to get the proper treatment, which will enhance the credibility of our system and restore faith. The objectives is to have a well-informed policy on cancer control by provisioning state of art existing or new small well organized comprehensive cancer units under the umbrella of all major regional cancer centres like TMH, GCRI, ADIYAR, KIDWAI, AIIMS etc which are throughout India.Methods: NCG leadership through its major cancer centres will develop and train more than 40,000 health workers and surveillance assistants at each government, private and charitable institution for uniform code of preventive, early diagnosis, palliative and end of life care treatment. NCG will also develop and train more than 5000 doctors including oncologists to provide standardized care across India by adopting evidence-based management guidelines. Creation of National Cancer Library, e-resources like Ecancer and OERC-India planning is part of these training programs.Result: Effective organization and management under NCG includes adhering to preventive, early diagnosis, and treatment guidelines including palliative care, ensuring patients are treated by multidisciplinary team, having proper systems in place for monitoring –and of course ensuring that all patients have equal access. This result will not be in numbers but is needed to strengthen the cancer care system of the country.Conclusion: Team of leadership under NCG under above plan will improve cancer outcomes in India considerably, parallel with patients getting the highest quality of cancer care at affordable price.

P178-J012Determinants of compliance to breast cancer screening and referral among women from urban slums in India

S. Kulkarni1, G. Mishra1, R. Dussane1, S. Shastri1


Introduction: Breast cancer is the leading cancer worldwide. Early detection for better outcome and improved survival rates is the basis of breast cancer control.Objectives: To study socio-demographic determinants influencing screening and referral uptake behaviour of women in community-based breast cancer screening programme, down-staging breast cancer by clinical breast examination (CBE), and increasing awareness about breast cancer through community-based health-education sessions.Material and Methods: The Mobile Outreach Programme being implemented in urban slums of Mumbai, India, involves community-

S52 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 J. Cancer Prevention and Screening

Results: Of the 146 Sarcoma cases, 105 were bone sarcomas (64 Osteo sarcomas, 41 others) and 41 were STS. Osteosarcomas occurred at a significantly younger age - mean age 20y118.6 yrs; as compared to other bone sarcoma - 29.5110.1 yrs and STS- 40118.5 yrs (,0.0001). We identified deleterious germline TP53 mutations in 2/146 sarcoma. None of three TP53 polymorphisms studied showed an association with the gender and type of sarcomas, but in the patients with osteosarcomas, the CC and CG genotype in rs1642785 and Arg72Pro was associated with significantly younger age as compared to the GG genotype (T-Test p 5 0.041).Conclusion: Rarity of germline deleterious TP53 mutation (2/146) identified in this largest cohort of sporadic sarcomas studied for TP53 should be taken into account for genetic counselling and limited utility of genetic testing in these cases. TP53 polymorphisms rs1642785 and Arg72Pro show modifier effect on age at diagnosis of osteosarcoma. Studies on larger cohorts with more extensive genetic analysis could show other genetic alteration in sporadic sarcomas.

P218-J017Understanding the perceptions of Indian women regarding cervical cancer screening based on the health belief model

P. Adsul1

1 Florida International University/Public Health Research Institute of India, India

Introduction: Cervical cancer is the second most common cancer among Indian women. Screening tests such as visual inspection of the cervix using acetic acid (VIA) or Lugols Iodine (VILI) have the potential to reduce mortality due to cervical cancer. This study describes women’s perceptions towards cervical cancer and screening among women attending community based screening camps.Methods: This study was a cross sectional survey of 101 women attending community based cervical cancer screening camps. The paper-based survey was adapted from previous reliable and valid scales and translated to the local language. A total of 30 items were included that were based on the constructs of the Health Belief Model and included perceived benefits, barriers, susceptibility, severity and health motivations.Results: The mean age of the respondents was 42.5 years, 58.4% had an education level of 10th grade or more and most (83.4%) were employed. Women in the study sample were motivated towards maintaining a healthy lifestyle and regularly participated in preventive health practices (64–97%). Only 47.1% of the women felt that they would get cervical cancer sometime during their life. Most women (32.7–49.5%) neither agreed not disagreed on perceived susceptibility to cervical cancer. The most frequently reported barriers were the lack of female health workers to conduct VIA exams (89.1%), feeling shy to lie on the gynaec table for a pelvic exam (66.3%) and not having a health center close to their home to get a VIA exam (54.5%) or having to pay for the VIA exam (54.5%).Conclusion: Further research into exploring the barriers towards cervical cancer screening is required. Findings also suggest that there is a need for one on one counselling approaches to increase the perceived susceptibility and severity of cervical cancer among women to improve the uptake of cervical cancer screening at the population level.

P233-J018Agreement analysis between 3 different short geriatric screening scales in patients undergoing chemotherapy for solid tumors

A. Joshi1, N. Tandon1, V.M. Patil1, V. Noronha1, S. Gupta1, A. Bhattacharjee2, K. Prabhash1

1 Department of Medical Oncology Tata Memorial Hospital, Mumbai, India2 Division of Clinical Research and Biostatistics, Malabar Cancer Centre, Kerala, India

Background: Comprehensive Geriatric Assessment (CGA) in routine practice is not logistically feasible. Short geriatric screening tools are available for selecting patients for CGA. However none of them is validated in India. In this analysis we aim to compare the level of agreement between three commonly used short screening tools (Flemish version of TRST (fTRST), G8 and VES-13).Methods: Patients $ 65 years with a solid tumor malignancy undergoing cancer directed treatment were interviewed between March 2013 and July 2014. Geriatric screening with G8, fTRST and VES-13 tools was performed in these patients. G8 score # 14, fTRST score $ 1 and VES-13 score $ 3 were taken as indicators for the presence of a high risk geriatric profile respectively. R version 3.1.2 was used for analysis. Cohen kappa agreement statistics was used to compare the agreement between the 3 tools. p value of 0.05 was taken as significant.

Methods: The prevalence of HPV, its genotypes and p16 protein expression of 205 patients treated with curative chemo radiotherapy was determined using PCR, DNA hybridization, and IHC. Survival outcomes were analyzed and compared for both p16 positive (p161ve) and p16 negative (p162ve) population. The influence of tobacco use on outcomes was also studied.Results: This study demonstrates 39.4% prevalence of HPV, of which type 16 is the commonest subtype (91%). Oncogenic HPV as determined by p16 positivity was detected in 20% of study population. Statistically significant difference was not observed in 5 year Cause-Specific Survival (CSS) (79% vs. 72.2%, p 5 0.4), Disease Free Survival (DFS) (78.3% vs. 68.3%, p 5 0.5) and Loco-Regional Control (LRC) (82.9% vs. 71.5%, p 5 0.4) between p161ve and p162ve groups respectively. Hence p16 positivity was correlated with tobacco users and non users. In tobacco non users p161ve group showed large improvement in CSS (P 5 0.08) as compared to p162ve group.Conclusion: Although a number of studies from western countries have demonstrated the correlation between HPV and p16 expression status in HNSCC, our studies demonstrate a low prevalence of p16 positivity and no significant correlation with HPV. Thus our results indicate that p16 expression is less useful as a surrogate marker in the presence of high tobacco burden since prevalence of p16 positive HNSCC is comparatively low (20%). Studies to assess the methylation status of p16 in these samples are on-going in the lab.

P208-J015“A MANUSCRIPT OF NEW IDEAS IN ONCO-EDUCATION” TNM cancer staging app – Way to go!!

Sheela M.C1, P.P. Popat1, M.H. Thakur1, N. Sable1, S. Singh1, S. Kumar G.V.11 Tata Memorial Hospital/ HBNI University, Mumbai, India

Background and Aim: With the advent of technology, the books were compressed to be carried in laptops, then pendrive, note and iPads. Yet the voluminous remain voluminous in the view of matter to be studied. With respect to oncology management, the first step is staging of the tumor to plan further treatment. TMH radiology department has launched afree TNM app which gives an offline all time access for staging. The aim is to enlighten an innovative idea improving the quality and method of education in oncology. We share the reaping benefits of our TNM Cancer Staging app launched recently. We have also proposed promising areas for going digital in other areas of onco-education.Material and Methods: Receiving funds under the aegis of IAEA, radiologists from TMH, have conceptualized, and designed flowcharts to generate a unique interactive questionnaire to derive at the TNM stage of the cancer. The TNM app lists nearly 65 cancer types. The app is based on 7th edition of AJCC manual. The app had a global launch towards the end of 2015, and is being updated regularly. Approximately, 5,000 to 10,000 people have downloaded the app with an average review of more than 4 stars.Conclusion: We simply conclude by saying, that to understand our expression of words just download the App and feel the difference in onco-education!! Promising proposals:

— Stage based treatment algorithm. — An imaging algorithm for diseases, by modalities and specifications. — Synoptic reporting.

P209-J016Contribution of germline TP53 mutation in sporadic sarcoma cases

Md. M. Haque1, A. Puri2, R. Sarin1,2*1 Cancer Genetics Lab, Advanced Centre for Treatment Research and Education in Cancer, Navi Mumbai, India2 Bone and soft tissue department. Tata Memorial Hospital, Mumbai, India

Background: Sarcomas when occurring with family history of cancer or as multiple primary tumour suggest inherited cancer predisposition Li-Fraumeni Syndrome (LFS) due to germline TP53 mutation. For LFS, there are established criteria for genetic counselling, testing and risk management. However for sporadic sarcomas (no family history), the genetic basis and guidelines for genetic counselling / testing is unknown. The study aims to establish the frequency, spectrum and effect of germline mutations and polymorphisms in TP53 in a large cohort of Indian sporadic sarcoma cases.Material & Methods: 146 sarcoma cases enrolled in the TMC International Sarcoma Kindred Study provided consent and details of family history, phenotype, exposure and blood sample. From genomic DNA, TP53 exons with flanking regions were PCR amplified followed by Sanger sequencing.


from India as compared to 931 from US and 212 from Canada. 26 (40%) case reports, 17 (26%) pathology, genetics and translational reports, 7 (10.5%) institution retrospective outcome or epidemiological studies, 14 others and only one clinical trial. Arora et al, 2009 estimated 5-yr OS for CNS tumors in Chennai 27% (1990–2001) comparing to 67% (1993–1997) in Europe and 71% (1996–2004) in US. The OS for MB ranges in various retrospective series from 25% in infants (NIMHANS), 3-yr 40% (NIZAM) to 5-yr 73% (KEM). The prospective clinical trial (TMH Gupta et al) with 20 patients showed 3-yr OS 83.2% comparable to western countries.Conclusion: There is a large lacuna for estimation of pediatric BT population, appropriate facility for their treatment and data on clinical outcomes and trials in India. Development of first PNOFC is needed and suggested.

P290-J021Exploring the feasbility of mobile technology to improve the management of clients with cervical cancer precursor lesions

J. Moodley1,2, H. Botha3, D. Constant1, H.D. Merwe3, M. Momberg2, K. Daskilewicz2

1 Women’s Health Research Unit, School of Public Health and Family Medicine, University of Cape Town, South Africa2 Cancer Research Initiative, Faculty of Health Sciences, University of Cape Town, South Africa3 Unit of Gynaecological Oncology, Stellenbosch University and Tygerberg Academic Hospital, Cape Town, South Africa

Background: Cervical cancer is an important women’s health problem in low- and middle-income countries (LMIC). A challenge facing screening programs is loss to follow-up of women with cervical abnormalities. The mobile phone, one of the fastest growing technologies, could offer a potential solution, however little is known whether this would be feasible in a public sector LMIC context. The objectives of this study were to: (1) determine cell phone use by clients eligible for Pap smears, and interest in receiving results and appointment reminders via short messaging services (SMS) and (2) develop and pilot test SMS messages.Methods: Phase 1 included: a cross-sectional survey of 364 women attending primary care clinics; in-depth interviews with 10 primary level providers and focus group discussions with 27 colposcopy clinic clients. Results were used to develop SMS-text interventions (content, number of messages and timing) which were pilot tested in Phase 2.Results: The majority of women at primary care clinics owned a cell phone (98%), however 48% reporting cell phone loss in the past year. Interest in SMS interventions were as follows: 72% interested in receiving Pap results, 77% in receiving appointment reminders and 93% in receiving health messages. Women expressed concerns about privacy and HIV associated stigma. HIV positive women were significantly less likely to want an SMS appointment reminder (OR 0.3, 95% CI 0.13–0.72). Providers had concerns about messages reaching intended recipients. The pilot SMS intervention highlighted challenges in integrating mhealth interventions into a health system with a weak information and communication environment.Conclusion: Despite significant client interest, the potential for mobile health to improve follow-up of clients with abnormal Pap smears can only be realized if key health systems and privacy issues are addressed. Findings will be useful to inform Health decision-making in LMIC countries.

P291-J022Challenging perioperative surgical dogmas after elective colorectal cancer surgery

R. Oodit1, N. Moodley1, R. Baigrie1, A. Boutall1, F. Pieterse1, J. Moodley1

1 University of Cape Town, South Africa

Objectives: Colorectal cancer is the 3rd leading cause of cancer worldwide. Surgery is the first line of treatment. Perioperative care is largely based on surgical dogma. Less than 15% of traditional care is evidence based, with patient care fragmented, not patient centered and little quality outcome data. Enhanced Recovery After Surgery (ERAS) is an evidence based, patient centered integrated team approach combined with an interactive database to measure compliance and outcomes. Together with regular team meetings, change management skills and a commitment to the Plan – Do- Study- Act cycle, complication rates and hospital stay have been reduced by 50% in developed countries. The aim of this project is to determine the feasibility of implementing ERAS in South Africa, a low middle-income country.Methods: The study is being conducted at a public sector tertiary hospital (Department of Surgery) and a private sector general surgery

Results: The kappa statistics value for agreement between G8 score and fTRST, between VES-13 and fTRST and between VES-13 and G8 were 0.12 (p 5 0.04), 0.16 (p 5 0.07) and 0.05 (p 5 0.45) respectively. It was found that maximum agreement was observed for VES-13 and fTRST. The agreement value of VES-13 and fTRST observed was 59.44% (39.63% for high risk profile and 19.81% for low risk profile). The agreement value of G-8 and fTRST was 39.62% (2.83% only for high risk profile and 36.79% for low risk profile). The lowest agreement was between G8 and VES-13, 35.84% (7.54% for high risk detection and 28.30% for low risk detection).Conclusion: There was poor agreement (in view of kappa value been below 0.2) between the 3 short geriatric screening tools. Research needs to be directed to compare the agreement level between these 3 scales and CGA, so that the appropriate short screening tool can be selected for routine use.

P261-J019Current challenges on the breast cancer control strategy in Yemen

H. Basaleem1, K. Al-Sakkaf1

1 Aden Cancer Registry and Research Center, Faculty of Medicine and Health Sciences, University of Aden, Yemen

Background: In Yemen, breast cancer is a leading cancer. Nevertheless, the current efforts in approaching its burden appear to be patchy and fragmented due to the lack of a comprehensive national breast cancer control strategy (NBCCS). The present paper aimed at describing the actions needed to develop the NBCCS based on registry data and portraying the challenges for such strategy.Methods: Breast cancer data from the population based Aden Cancer Registry (2002–2013) were used to estimate the needed resources regarding raising awareness, early detection, diagnosis, treatment, rehabilitation and palliative care services. A qualitative participatory approach for the roles and responsibilities of the developing partners and the needs of the development process were critically analyzed.Results: The paper details the context through which the strategy could be developed starting from the purpose of providing a framework for an integrated, comprehensive set of activities covering all aspects related to breast cancer control, passing through the characteristics of the NBCCS, the guiding principles and steps of development, the process of development and the possible related challenges. Finally, the need for NBCCS to guide the existing and future actions with a set of evidence–based, cost–effective priority actions while being sensitive and realistic to resource opportunities and constraints was highly focused.Conclusion: To conclude, population based registry data are important resources to develop cancer control strategies. NBCCS cannot be achieved successfully by any single organization and its development should be consortium–based. The necessary partnerships of many partners and their responsibilities were indicated within a comprehensive NBCCS.

P264-J020Does India needs dedicated pediatric neuro-oncology services: A comparative study of available data for India and western countries

R. Krishnatry1, E. Bouffet2

1 Consultant Radiation Oncology, Mazumdar Shaw Cancer center, Bangalore; Member Indian Society of NeuroOncology, India2 Head and Professor of Division of Pediatric NeuroOncology, the Hospital for Sick Children, Toronto, Canada

Background: Children are the future of any nation and it is imperative to safeguard their wellbeing. Brain tumors (BT) are the second most common cancers and commonest solid tumors in children and need quite different approach from adult BT or other solid or haematological malignancies of children. One out of every kid on earth is in India.Methods: We reviewed the Indian census data (2011) and National Cancer Registry dat (NCR, 2011) and compared with numbers in Canada, US. We also searched for number of publications on [medulloblastoma](surrogate of pediatric neurooncology research) and [India] from 2006–2015. We also reviewed the Indian publications to assess research facility, trend and outcome information.Results: India has the second largest human population of 1,210,190,000 (March 2011), comprising 45% ,19 years (544,585,500) and no dedicated pediatric neurooncology facility center (PNOFC). US had only 43,532,795 children (8% of India) with .20 PNOFC, Canada had 5,356,270 children (1% of India) and 16 PNOFC. The annual incidence of BT varied from 6.6–19.8 (median 11.7) per million, projecting a median of 6365 (3590–10,880) new BT/year. As per Sarkar et al (combined data from 7 major tertiary centers), 710 new cases/year (11.2%; 710/6365) are seen of which medulloblastomas and PNET are second most common group (22.4%; 159). Pubmed search showed 65 publications

S54 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 K. Omics and Technologies

Methodology: DNA samples from Tissue and blood were sequenced for exon sequencing of 412 cancer related genes (NIMBLGNE panel, Custom). Raw fastq files processed using GATK tools and variants identified by Varscan2, & MuTect. Altered pathways identified by KEEG.Result: A total 105 Somatic exonic Single Nucleotide Variants (SNV’s), 9 somatic exonic Indels, 22 LOH mutations (both Indel and SNV’s) were identified in our sample set. Among the candidate genes of PDAC somatic SNV’s were found in TP53, SMAD4 in some patient whereas frequency of KRAS mutation was less frequent. 14 SNV’s were reported in cosmic which includes 5 pathogenic variants, whereas 91 SNV’s were novel. A total 75 non-synonymous, 6 stopgain, and 24 synonymous SNV’s were observed among 105 SNV’s. Eighty seven percent of transversion was observed from all SNVs, where fifty five percent SNV’s were C.T, G.T. PI3-Akt, Proteoglycans in cancer, Transcription misregulation, Rap1, ErbB, Neurotrophin pathways were found to be most commonly enriched in our patient population. Our data strongly suggest PI3-Akt, Proteoglycans in cancer pathways play a critical role in the disease.Conclusion: This study implicates an overview of somatic SNV’s in PDAC’s and PAC’s and alteration of biological pathways in our patient population. We also suggest PDAC’s and PAC’s of pancreatic head origin contribute very similar mutational pattern.

OT143-005Collateral lethality: Genomic passenger deletions as targetable vulnerabilities in glioblastoma

Y.H. Lin1, N. Satani1, N. Hammoudi1, P. Deng3, S. Colla4, P. Wu3, Y.A. Wang3, R. DePinho3, F. Muller1,2

1 Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, Texas, USA2 Department of Neurooncology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA3 Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA4 Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

Genomic deletions are an early and critical driver event in diverse cancer types. Since the first description of chromosomal band deletions over 40 years ago, extensive genomic studies have focused on identifying specific tumor suppressor genes (TSG) targeted by genomic deletions. The pace of knowledge has dramatically accelerated with the advent of economically feasible mass-genetic characterization of tumors and has made the prospect to true genetically personalized cancer therapies a realistic possibility. However, relatively little attention has been paid to the fact that besides TSG “targeted” by genomic deletions, a very large number of non-TSG, playing no role in tumor promotion are often “collaterally” deleted as a result of chromosomal proximity to the “intended” target. This is especially true in the case of large heterozygous scale chromosomal deletions (loss-of-heterozygosity, LOH) but also with homozygous deletions. In the present poster, we discuss the therapeutic possibility offered by collateral genomic deletions for personalized medicine and present proof-of-concept data, both in vitro and in vivo, for multiple different deleted genes as pharmacologically targetable vulnerabilities.

OF283-013Expanding the ‘druggable genome’ using structural constraints and dynamics – Innovative use of big and smart data

P. Venkatraman1

1 Protein Interacome Lab for Structural and Functional Biology ACTREC, TMC

Cancer is a genetic disease that manifests in the altered landscape of structure and the functions of the human proteome. Capitalizing on these observations, kinases, proteases and receptors have been targeted for drug development and drugs such as Gleevac, Herceptin and Velcade that block function of such proteins are used in cancer treatment today. Partly due to the bias of the initial successes and mostly due to challenges in the shape and structure of protein surfaces, the so called ‘druggable’ singular targets have become stereotypes and stand exposed in terms of their limitations. It is in this context that the evolving big data on multiple modifications in a vast number of proteins captured in an unbiased manner and strategies based on general rules that dictate protein interactions provide the necessary gateway to innovations. Here, we present two innovative but intricate and provocative strategies that can unmask novel, vulnerable targets in cancer networks. Both strategies rely on the rules that govern protein structure, dynamics and function. One example will demonstrate the identification and application of

specialist practice in Cape Town, South Africa. Pre-operative assessment by an ERAS team including the patient, surgeon, anesthetist, nurse coordinator, a dietician, and a physiotherapist are conducted for each patient. Baseline POSSUM and MUST scores are recorded. Anesthetic and surgical procedures and patient discharge plans are influenced by the assessment. Process measures include adherence to ERAS guidelines. Complication rates, length of hospital stay and date the discharge criteria are metis measured.Results: Participant enrollment commenced April 2015. 88 patients have been recruited and early results show a significant reduction in complications and hospital stay (50%) Discharge planning is influenced by patient’s social circumstances.Conclusion: The results are comparable to that achieved by dedicated ERAS centers in developed countries. ERAS has the potential to significantly improve the management of colorectal cancer patients in developing countries.

P294-J023Quality assessment of four new population based cancer registries (PBCRS) in Chandigarh & Punjab

M.A. Bashar1, J.S. Thakur1, A. Budukh2, R. Kapoor1

1 Department of Community Medicine, PGIMER, Chandigarh, India2 Tata Memorial Centre, Mumbai, India

Background: Population based Cancer Registries are hallmark of cancer surveillance activity. The value of cancer registries rely heavily on underlying quality of its data. Current study assessed data of four new PBCRs of Chandigarh, Ajitgarh, Mansa and Sangrur covering a total population of 4.5 millions on three quality indicators i.e. comparability, validity and completeness as recommended by IARC, Lyon, France.Methods: For assessing comparability, data of the registries were reviewed in terms of system of classification and coding, definition of incidence date and rule for multiple primaries. For assessing validity four different methods i.e. re-abstraction and recoding, percentage morphologically verified cases (MV%), death certificate only (DCO) cases and percentage of cases with other and unspecified sites (O & U%) were used. For assessing completeness of coverage, different semi-quantitative methods were used.Results: Re-abstraction done for 10% of the total incident cases yielded overall percentage agreement of 97.4%, 97.2%, 95.4% and 94.9% for PBCR Chandigarh, Ajitgarh, Mansa and Sangrur respectively. MV% was found to be 96.3% for PBCR Chandigarh, 92.8% for PBCR Ajitgarh, 89.3% for PBCR Mansa and 82.9% for PBCR Sangrur. Percentage of DCO cases and O & U cases were 1.4% and 2.8% for PBCR Chandigarh, 3.9% and 5.3% for Ajitgarh, 6.4% and 16.4% for Mansa and 6.3% and 8.3% for Sangrur. Completeness assessed through the various methods showed good level of completeness at PBCR Chandigarh and Ajitgarh and somewhat lower but acceptable level of completeness at PBCR Mansa & Sangrur.Conclusions: All the four PBCRs are comparable internationally. PBCR Chandigarh and Ajitgarh, predominantly urban registries, have higher validity of their data and good completeness level as compared to rural registries of Mansa and Sangrur. Cancer estimates given by all the four registries are reliable.

K. Omics and Technologies

OT104-006Somatic mutational profile of 412 cancer targeted genes of pancreatic ductal and periampullary adenocarcinoma in Indian patient population

G. Saha1, R. Singh1, U. Chakravartti1, D. Anchlia2, S. Ghatak3, S. Gulati3, S. Ghosh2, B. Roy1, N. Sikdar1

1 Human Genetics Unit, Indian Statistical Institute, Kolkata, India2 Department of Surgery, Calcutta Medical Research Institute, Kolkata, India3 Department of General Surgery, Calcutta Medical College and Hospital, Kolkata, India

Introduction: Pancreatic Ductal Adenocarcinoma (PDAC), the most common primary malignant disease of the pancreas and the periampullary region, accounts for about 75% of all nonendocrine tumors arising in this region. It is 4th most lethal malignancies, with a global mortality rate of 98%. The 5 year survivability rate is below 5%.Aims & Objectives: To identify the somatic single nucleotide variants (SNV’s) in 412 cancer related genes in PDAC & periampullary (pancreatic head origin) adenocarinoma (PAC’s). To identify possible altered biological pathways enriched in these cancers.

K. Omics and Technologies European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S55

expression in GBC group (p 5 ,.0001). In GBC group, statistically significant expression of Her2/neu (p 5 ,.0001), EGFR1 (p 5 ,.0001) and p53 (p 5 0.024) was observed in subgroup of patients amenable to curative surgery compared to palliative treatment/inoperable subgroup. No consistent correlation was observed between biomarker expression and T stage, lymph node status and histological subtype. Positive VEGF expression signified decreased Disease Specific Survival in Radical Cholecystectomy subgroup (p 5 .003). Increased co-expression of biomarkers correlated poorly with Disease Control Rate (D.C.R.) (p 5 .024) and mean overall survival in palliative treatment arm.Conclusions: GBC is associated with late diagnosis, unsatisfactory treatment and poor prognosis. Clearly, new therapeutic regimens are the need of the hour, based on our evolving understanding of molecular biology. Her2/neu, EGFR1and VEGF expression in GBC could be predictive of disease biology and response to chemotherapy. Co-marker expression could be indicative of aggressive disease biology and poor survival. Role of targeted therapy needs further evaluation and could prove to be the Holy Grail in treatment of GBC.

P48-K002Discovery of somatic ERBB2 mutations in human gallbladder cancer

P. Iyer1, N. Gardi1, M. Ranjan1, P. Chandrani1, P. Upadhyay1, M.R. Ramadwar2, S.V. Shrikhande3, A. Dutt1

1 Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, India2 Department of Pathology, Tata Memorial Centre, Parel, Mumbai, India3 Department of Gastrointestinal and Hepato-Pancreato-Biliary Surgical Oncology, Tata Memorial Centre, Mumbai, India

Background: Gall bladder cancer is relatively an uncommon disease worldwide with age standardized rates of 2/100000. In India, gall bladder cancer is a major problem with majority of patients with advanced disease (3.9–8.6/100000). The uncommonness of gall bladder cancer has led to poor understanding of the disease. Despite the good performance of surgical resection, there is a need for new molecular therapeutic targets in gall bladder cancer. The advent of next generation sequencing technologies has played a significant role in reducing the gap between basic research and clinical application in many human cancers. Here, we characterize the landscape of somatic alterations in gall bladder cancer genome.Methods: In this study we interrogated the coding region of 27 Indian gall bladder cancer samples using whole exome sequencing technologies at an average coverage of 100X and above. We further validated the findings using an additional set of 27 FFPE samples. The functional characterization of significant findings were carried out using primary tumor derived gall bladder cancer cell lines.Results: Using various steps of filtering in bioinformatics pipeline, we identify 383 somatic alterations across 17 tumors, which includes an average 112 synonymous, 245 missense, 8 nonsense, 8 indels and 8 splice site changes. The average mutation rate considering the paired tumors is about 14 mutations/mb. We found TP53 (35.2%), ERBB2 (17.6%), SF3B1 (17.6%), ATM (17.6%) and AKAP11 (17.6%) mutations in more than two samples by exome sequencing analysis. Sensitivity of 6 gall bladder cell lines was interrogated with commercially available ERBB2 inhibitors. Our results suggest 2 of 6 gall bladder cell lines were sensitive to ERBB2 inhibitors by MTT, western blot and anchorage independent assays.Conclusion: The profiling of somatic alterations in gall bladder cancer and identification of recurrent ERBB2 alterations may expand the current treatment modalities in gall bladder cancer.

P49-K003Discovery of actionable alterations in lung adenocarcinoma

P. Chandrani1, R. Prasad1, A. Chougule2, V. Sethunath1, J. Aich1, H. Dhamne1, P. Upadhyay1, D.N. Iyer1, A. Thavamani1, B. Mohanty3, R. Thorat3, P. Chaudhari3, R. Sindhi4, P. Chandna5, K. Prabhash2, A. Dutt1

1 Dutt Laboratory, ACTREC, Tata Memorial Centre, Navi Mumbai, India2 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India3 Laboratory animal facility, ACTREC - Tata Memorial Centre, Navi Mumbai, India4 Sandor Proteomics Pvt. Ltd., Hyderabad, India5 AceProbe technologies Pvt. Ltd., New Delhi, India

Objective: Lung cancer remains a major unmet medical need in India with ~63,000 new cases diagnosed each year. In this study we aim to identify therapeutically relevant genetic alterations in Indian lung adenocarcinoma patients.Methods: In a discovery set, we sequenced ~200 cancer genes in 125 Indian lung adenocarcinoma samples (FFPE blocks) at 1,000x coverage

conserved ‘hot spot’ sites in protein interfaces to map an oncogenic network. We argue that the shared interface on otherwise dissimilar proteins engaged in oncogenesis constitute the much needed poly pharmacologic drug target. In the second example, we will see how analysis of Big Data on phosphoproteins identified by highthroughput mass spectrometry reveals opportunities for new strategies that can phenocopy kinase inactivation.

OF295-025Straticyte: A proteomic signature image analysis based test for predicting risk of cancer development in oral lesions with dysplasia

R. Ralhan1,2,4,5, R. Gu5, P.G. Walfish1,2,3,5, K. Pritzker5

1 Alex and Simona Shnaider Research Laboratory in Molecular Oncology2 Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Department of Otolaryngology-Head and Neck Surgery3 Department of Medicine, Endocrine Division, Mount Sinai Hospital and University of Toronto4 Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, Ontario, Canada5 Proteocyte Diagnostics Inc., Toronto, Ontario, Canada

Background: Early detection of oral lesions (OLs) at high risk of cancer development poses a major clinical challenge. Dysplasia grading often does not correlate with cancer development. Biomarkers that allow identification of high-risk OLs are urgently needed. Using proteomics, we identified and verified a biomarker signature comprising of five proteins that distinguishes OLs with dysplasia and oral cancers from normal tissues. The potential of this signature for identification of oral dysplastic lesions at high risk of cancer development was evaluated using immunohistochemistry and clinical outcome. Based on these findings, Proteocyte developed a protein biomarker-based prognostic test, Straticyte, to better stratify OLs for predicting the risk of cancer development.Methods: FFPE tissue sections from 180 oral dysplasia cases with up to 14 years of follow-up from Mount Sinai Hospital were immunostained for these proteins using specific antibodies and evaluated by two observers blinded to clinical outcomes, image analyses performed and data analyzed using R package.Results: Our protein signature was a significant predictor of cancer progression (p , 0.001). Straticyte test stratified the high risk OLs with PPV 92.3% and NPV 87% as compared to 82.8% and 60% respectively with histopathologic dysplasia grading. Straticyte classified OLs into three categories measured by mean progression survival months (Log-Rank test p 5 0.003), while histopathologic grading failed to separate severe and moderate dysplasia (p . 0.1).Conclusions: Straticyte improves risk prediction beyond current histopathologic grading. For the low risk group, 87% NPV provides confidence to clinicians in monitoring the lesions at long intervals. For high risk group, 92.3% PPV enables clinicians to perform surgical excision with confidence. Straticyte could enter clinical practice through oral pathology labs. Oral surgeons can order Straticyte to Proteocyte. Straticyte will find utility in clinical practice for predicting patients with oral lesions at high risk of cancer development.

P24-K001Evaluation of biomolecular expression profile in carcinoma gallbladder and its clinicopathological correlation – Is it the way ahead?

S. Gupta1, S. Misra1, C. Kori1, V. Kumar1, N. Husain1


Background: North India reports one of the highest incidence of gallbladder cancer (GBC) in the world. There are very few studies evaluating biomolecular expression profile in GBC and study its clinic-pathological correlation.Methods: This prospective study was carried out to immunohisto-chemically determine growth factor receptors (Her2/neu, EGFR1and VEGF) and p53 expression in patients of GBC and the control (cholelithiasis) group and to study their clinic-pathological correlation. IHC expression was quantified using standard protocol based on percentage and the intensity of cell staining. Biomarker expression was correlated with various clinical and pathological parameters, Disease specific survival and overall survival.Results: Study comprised of 209 patients of GBC and 50 control group. Amongst 209 GBC patients, 76 patients (36%) underwent curative resection while 133 patients (64%) with locally advanced/metastatic disease received palliative chemotherapy. Histology was predominantly Adenocarcinoma. Compared to control group, all biomarkers demonstrated significant


volumes were expanded uniformly by 5 mm to create the high risk CTV and this expanded by 5 mm to create the PTV1. Similarly the involved nodal level was considered as intermediate risk (PTV2) and remaining nodal levels as low risk (PTV3). SIB-IMRT was delivered to a dose 70, 63 and 56 Gy in 35 fractions to high, intermediate and low risk volumes respectively.Results: Median age was 58 years (32–76). Hypopharyngeal (47%), oropharyngeal (27%) and laryngeal (26%). 72% stage IVA. Median overall treatment time was 49 (40–70) days. 24% of the patients received 6 fractions per week. Weekly Cisplatin (40 mg/sqm) was given concurrently, 90% received a minimum of 4 cycles. Toxicity of Grade 3 mucositis in 40%, grade 3 dysphagia in 6%, radiation dermatitis grade 2, Xerostomia grade 1 in 93%, 10% required placement of nasogastric tube and treatment interruption. Myelosuppression of grade 3 in 24%. A complete response of 90% and 86% was seen in primary and nodal disease at the end of the treatment and eventually in 100% and 94% in first three months. 3 patients needed neck dissection and showed residual disease. At 2 years median follow up, 62 patients are controlled with overall survival of 74.7%. Deaths are due to distant metastasis in 4% and 8% medical causes. Conclusion: Reduction of CTV margin to 5 mm is appropriate, with good loco-regional control, better compliance, reduced toxicity and superior outcomes. This study forms basis for a prospective controlled randomised study to generate further evidence.

P85-K006Clinical genomicist workstation collaborative knowledge base as a framework for identification of clinically actionable mutations

N.A. Sahasrabuddhe1, A. Bredemeyer2, K. Seibert3, R. Nagarajan2, A. Phatak1

1 PierianDx India Pvt. Ltd, Pune, India2 PierianDx Inc., USA3 Genomics and Pathology Services, Washington University School of Medicine, USA

Background: Next generation sequencing (NGS) based tumor gene profiling is now in clinical use for identification of actionable variants. Though there are a number of open source algorithms available for calling variants from NGS data, determining clinical importance of the many variants and crafting interpretations for clinical reporting remains a challenge.Methods: The Clinical Genomicist Workstation (CGW) is a comprehensive software platform that enables clinical labs interpret DNA sequence into clinically meaningful information. The CGW knowledgebase and learning algorithm were developed by querying clinically relevant genes to retrieve relevant research articles, clinical trials, FDA drug labels, and therapy guidelines from NCCN and ASCO and data is manually curated. Public variant databases like COSMIC, ClinVar and ClinVitae were integrated. Clinical interpretations created by CGW users at healthcare centers were shared across sites, linking thousands of prior medical interpretations to specific patient variants for constitutional disorders and somatic diseases. Sequence data from two lung cancer cases were used to test CGW’s collaborative knowledgebase. The PDx score algorithm was employed to prioritize the identified variants by clinical relevance.Results: By applying this CGW collaborative knowledgebase, actionable variants present in the lung cancer test cases including an EGFR exon 19 deletion and a MET splice site alteration were identified, classified, and annotated to enable review of source data and potential clinical trial options. Clinical interpretations from collaborating labs were presented for consideration in the draft clinical report, and the PDx score further facilitated quick prioritization among clinically relevant variants. The CGW represents a mature cloud-based big data informatics and interpretation solution from seamless management of data and workflow to clinical report generation.Conclusions: The key features of CGW include automated variant classification and Collaborative Knowledgebase which enable robust and quick support to oncologist in diagnostic and therapeutic decisions for personalized medicine.

P88-K007Association of CDH-1 (CADHERIN) gene polymorphism with metastatic breast cancer

G.K. Siddhartha1


Background: Mortality in breast cancer is mostly due to metastasis to distant organs. Metastatic breast cancer is progressive stage of the disease (stage-IV) and marked by expansion of cancerous cells

per sample per base. Selected mutations were genotyped in an additional validation set of ~350 lung adenocarcinoma samples using Sequenom Mass Array platform. Novel somatic mutations predicted to be activating were functionally validated using in-vitro and in-vivo models.Results: This is the first report of most comprehensive spectrum of alterations in ~350 Indian lung adenocarcinoma patients which includes mutation in: TP53, EGFR, KRAS, PIK3CA, NRAS, AKT1, NF2 and additional novel mutations in FGFR3. NIH-3T3 cells expressing mutant but not wild type FGFR3 constitutively activate MAP kinase pathway and form significant higher number of colonies in soft-agar assay, that are sensitive to FGFR pan inhibitor PD173074. Tumor xenografts developed by transformed NIH-3T3 cells responds to FGFR3 inhibitor BGJ398.Conclusion: We present the first landscape of actionable alterations in Indian lung cancer genome. This study also establishes FGFR3 as novel targetable proto-oncogene in lung adenocarcinoma as observed by in-vitro and in-vivo experiments.

P58-K004Genomic characterization of somatic alterations in cervical adenocarcinoma

T. Togar1, N. Gardi1, P. Upadhyay1, S. Chopra2, A. Dutt1

1 Integrated Genomics Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai, India2 Departments of Radiation Oncology, Tata Memorial Centre, Mumbai, India

Introduction: Cervical cancer is the second common cancer among women in India and accounts for 25% of the world incidence. There are two major histological subtypes-squamous carcinoma (85–90 %) and adenocarcinoma (10–15%). Despite such high incidence, there is no targeted therapy to cure cervical cancer. Here, we attempt to characterize the underlying genomic alterations using whole exome sequencing with hope that identified genomic alterations will help to design effective therapies in cervical cancer.Material and Methods: Thirty seven samples (18 matched normal tumors and one orphan tumor) of cervical adenocarcinoma were subjected to whole exome sequencing at 100X coverage. Somatic mutation analysis with stringent filter was carried out to identify significantly altered and biologically relevant alterations. Identified alterations were verified using Sanger sequencing and variants were further prioritized for functional analysis in cancer cell lines.Results: We present the somatic mutational spectrum of cervical adenocarcinoma and identified known and novel mutations in oncogenes such: PIK3CA, ERBB2, FGFR2, FGFR3 and inactivating mutations in tumor suppressors ARID1A, PTEN, TSC1 and TSC2 by preliminary data analysis. We observed PIK3CA and ARID1A to be recurrently mutated in our cohort. Selected mutations were verified using Sanger sequencing mutations with 86% validation rate. We find therapeutically relevant mutations in ERBB2 (D769Y) and FGFR2 (K659E) that could be targeted by inhibitors Nerafenib and PD compounds, respectively. Most frequently mutated genes were members ofPI3K/AKT pathway. Interestingly, we observed co-occurrence of both ARID1A and PIK3CAmutations in 2 of 19 patients and tumors harbouring such alterations are proposed to be sensitive to Akt inhibitors. Further, in depth analysis of mutation data will be presented.Conclusions: We for the first time characterize somatic genomic alterations of cervical adenocarcinoma in Indian patients and identified several therapeutically targetable mutations.

P73-K005Can reduced CTV margin for IMRT in head and neck squamous cell cancers (HNSCC) improve therapeutic outcomes?

T. Pasha1, U. Krishna1, V. Chandraraj4, A. Shenoy3, L. Jacob2, N. Thimmiah1, S. Palled1, L. Vishwanath1, K.P.R. Parmod1

1 Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India2 Department of Medical Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India3 Department of Head and Neck Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India4 Department of Radiation Physics, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India

Objective: To assess efficacy & toxicity of reduced CTV margins in the IMRT of HNSCC.Material and Methods: Between 2010–2015, 83 patients with locally advanced HNSCC, treated with chemoradiation by IMRT, with reduced CTV margins were analysed for local control, toxicity, compliance & survival. CECT based delineation of the involved primary and nodal


with at-risk relatives about the family’s inherited mutation so that the relatives can be pro-actively tested and managed according to their risk profile. However, this communication rarely extends beyond first degree relatives, leaving distant at-risk relatives unaware. Aim: To develop and test a web-based platform for communicating risk information directly to potentially at-risk relatives in LS families.Methods: Under current standard of care, LS patients undergoing genetic counseling, i) enter family data into existing online clinical tool to create a pedigree; ii) are provided with a sample letter, copy of mutation report and educational material about LS to facilitate communication of genetic cancer risk with relatives. With the expanded online approach, i) patients provide online informed consent to contact relatives, and provide relatives’ email addresses; ii) an email is sent to relatives with link to online registry that provides for authentication of identity, consent to participate, access to family’s mutation report, and ability to add new relatives and their contact information (with the overall goal to expand the pedigree well beyond the first degree relatives).Results: A working interactive model of the web-based program is ready for usability, and acceptability testing and assessment of barriers prior to full implementation of the program.Conclusion: The novel web-based family outreach approach will allow families to collaboratively collect and share family health information in a structured format and is expected to exponentially increase the number of at-risk relatives reached with significant potential for cancer prevention. Once fully validated, our model should be useful in other adult onset cancer susceptibility states.

P94-K010Next generation DNA sequencing of targeted regions for comprehensive genome profiling of cancer-causing genes using FFPE-derived DNA

D. Vishwanath1, A.K. Hariharan1, S. Parchuru1, K. Kumari1, K. Dhanuskodi1, M. Sen1, P. Agrawal1, V. Vittal P1, N.S.N. Swetha1, V. Pathak1, G. Deshpande1, V. Veeramachaneni1, S. Katragadda1, M. Parulekar1, K. Subramanian1, V. Gupta1

1 Strand Life Sciences, Bengaluru, Karnataka, India

Background: The advent of high throughput genomic technologies has led to a deeper understanding of the molecular mechanisms of cancer and genetic variations in a tumor profile that impact clinical outcomes or drug responses. Next-generation sequencing (NGS) with its high throughput, speed, scalability and resolution, can be integrated into clinical diagnostics to enable personalized treatment decisions. For genome profiling tests, the factors playing a crucial role are DNA input amount and quality. A majority of cancer tissues available in the clinic are Formalin Fixed Paraffin Embedded (FFPE) tissue samples which are compromised in quality during the fixation process. The test is designed for solid tumors for ultra-deep sequencing of known and novel mutations, copy number variations and translocations spanning 152 key cancer genes in FFPE samples, with the initial input amount being in the range of 50–200 ng of DNA.Methods: The method involves preparation of ~250 bp libraries, enrichment with 120-mer RNA probes targeting 152 cancer genes and sequencing on Illumina MiSeq Platform. During library preparation, randomly sheared DNA fragments are linked to adaptors specific to the Illumina sequencing chemistry. The resultant precap libraries are then pulled down in solution using biotinylated probes specific to the target regions. The final sequencing is done on the enriched library using Illumina MiSeq V3 Sequencing Chemistry.Results: The unique average coverage is about 200X with a % low coverage less than 5% when filtered at 100x over the 500 kb target region in the NGS assay. The reproducibility, robustness, and accuracy will be presented along with the limit of detection.Conclusion: This protocol allows us to use samples compromised both in terms of DNA quality and DNA amount, thus increasing the range of samples that can undergo testing.

P107-K011A novel in silico approach to identify significant genes in serous adenocarcinoma of ovary

Manasa P1, T.S. Ganesan1

1 Department of Medical Oncology and Clinical Research, Cancer Institute, Chennai, India

Background: The overall survival of advanced high grade serous ovarian cancer (HGS-OvCa) has not altered significantly over the past 3 decades despite advances in treatment. Whole exome sequence, copy number variations (CNV’s) and expression analysis by the TCGA project has identified very few mutated genes (except p53 and BRCA) at sufficient

beyond the tissue origin. At cellular and tissue level, the process of distant metastasis involves primarily differentiation from epithelial phenotype to mesenchymal phenotype, called as epithelial-mesenchymal transition (EMT). E-Cadherin (epithelial) or Cadherin (CDH1) gene (q16q22.1) provides information for making a protein called E-Cadherin, which is responsible for calcium dependent cell-cell adhesion. It has been seen that selective loss/decrease expression of E-Cadherin can cause de-differentiation and increased susceptibility of invasiveness of human carcinoma cells.Aim of this study was to screen candidate polymorphism of CDH-1 gene in metastatic and non-metastatic breast cancer patients and to study association of CDH-1 polymorphic variants with metastatic breast cancer and other clinical parameters.Methods: DNA was extracted from whole blood using a commercial DNA blood kit (GenElute Blood Genomic DNA Kit; SIGMA) and stored until processing for genotyping. Selected polymorphism of CDH-1 gene (SNP -616 G.C) were screened with RFLP or DNA sequencing.Result: In our study, total number of subjects studied was 51, which included 12 cases (metastatic breast carcinoma) and 39 controls (non metastatic breast carcinoma). Out of 12 cases studied, 3 tested positive for-616snp CDH-1 gene mutation, with genotypes GC, CC and CC. On the other hand GG was the wild genotype. Decreased E-Cadherin expression was significantly found in high-grade and metastatic (stage IV), in contrast to low and intermediate grade cancer. This seems to be in concordance with the fact that E-Cadherin alterations (lack of expression) occurs in advanced tumor stages.Conclusion: -Cadherin alterations occurs in patients with age $50 years, high grade tumor and advanced tumor stages (metastatic breast cancer), thereby rendering it as a tumor invasion suppressor gene.

P92-K008Identification of deleterious somatic and germline variants in GBC by targeted sequencing

S. Yadav1

1 Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Background: Gallbladder carcinoma (GBC) is a common gastrointes-tinal malignancy with specific geographical and ethnic variation. North India has one of the highest incidences of GBC in the world. NGS approaches have significantly advanced our understanding of somatic mutation landscape in common cancers. However, somatic landscape of GBC remains largely ambiguous. Here, we have performed somatic mutation profiling of GBC using targeted sequencing through NGS platform.Methods: We performed targeted amplicon sequencing of all exons of 409 genes of 12 well characterized tumor samples with blood samples as normal from GBC cases matched on Ion Torrent proton platform using AmpliSeq comprehensive cancer panel. These 409 genes encompass over 50% of the Wellcome Trust Sanger Institute cancer gene census.Results: We used Ion reporter, an ion torrent specific data analysis pipeline to identify somatic mutations. We identified ~200 probable deleterious somatic mutations in all the 12 GBC cases. These mutations have been annotated using the data from databases including 1000 genome, dbSNP, Cosmic, Drug bank etc. Our mutation list includes both known and novel somatic mutations in known cancer driver genes. Many of these somatic mutations have not been reported in GBC yet. In addition, we have also identified clinically relevant somatic mutations in cancer driver genes including platelet-derived growth factor receptor (PDGFRA), SMAD Family Member 4 (SMAD4), KIT etc. Further, we have used a different pipeline for analysis having additional filters and identified deleterious germline variants in GATA2, THBS1, KEAP1 and FGR3 that are very rare in public databases (DbSNP, 1000 genome and cosmic) while present in most of our cases suggesting their role in predisposition to GBC. These promising genetic alterations may be further helpful in assessing disease prognosis and therapeutic interventions.

P93-K009A structured web-based family outreach approach for communicating genetic cancer risk in extended families with Lynch syndrome

M. Pande1, S.K. Peterson1, S. Bannon1, M. Mork1, P.M. Lynch1

1 The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Background: Lynch syndrome (LS), caused by mutations in the DNA mismatch repair genes accounts for about 3% of all colorectal cancers and many other cancers like endometrial, ovarian, gastric, etc. Problem: Current practice relies on the index cancer patient with LS to communicate


Methods: HPA was meticulously contoured in 51 patients with low-grade brain tumours treated with stereotactic conformal RT (SCRT). Dose volume histograms were studied in patients with or without endocrine deficits at 2 and 3 year follow up.Results: Dosimetric correlation with 2–3 year clinical data showed post RT endocrine dysfunction (new deficit or additional deficit over preexisting one) in 27/51 patients, of which 74% had HPA within 1 cm of PTV and 66% inside PTV. GH axis was the most commonly impaired (66%), followed by steroid axis (48%), pubertal axis (40%) and thyroid axis (22%). Average Dmax and Dmin to HPA were 46.6 Gy and 40.5 Gy respectively. For patients who did not develop post RT new endocrine dysfunction, only 54% patients had HPA within 1 cm of their PTV and 50% within PTV. Average Dmax and Dmin for this group were 36.5 Gy and 29.6 Gy respectively. Detailed analysis is ongoing to see the possible doses to the entire or partial HPA at different dose levels and a linear regression model will be employed to correlate the doses with already available endocrine function data at 2–3 years follow up.Conclusion: There seems to be a direct dose relationship with endocrine dysfunction at post RT follow-up. A detailed analysis with HPA as a conformal avoidance model to minimize neuroendocrine deficits will be presented.

P148-K014Integrated analysis links TP53, NOTCH, SLC38A and 11p with survival in patients with oral tongue squamous cell carcinoma

N. Krishnan1, S. Gupta1, C. Khyriem1, V. Palve1, A. Suresh2, G. Siddappa2, V. Kekatpure3, M.A. Kuriakose2,3, B. Panda1

1 Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied Biotechnology, Bangalore, India2 Mazumdar Shaw Centre for Translational Cancer Research, Bangalore, India3 Strand Life Sciences, Bangalore, India

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of malignancies with different incidences, mortalities and prognosis for different subsites and are the sixth leading cause of cancer worldwide. Studies on molecular biology of HNSCC in the past 5 yrs have largely been concentrated on cataloging various genetic changes in many cancer types using high-throughput sequencing assays and computational methods. Unlike other oral cavity subsites, squamous cell carcinomas of oral/anterior tongue (OTSCC) tend to be associated more with younger patients, early spread to lymph nodes and a higher regional failure compared to gingivo-buccal cases. We generated data, on somatic mutations in genes from exome sequencing, immediate upstream and downstream flanking nucleotides of the somatic mutations, DNA methylation, loss of heterozygosity, copy number variations, gene expression, significant pathways altered and HPV infection, from 50 OTSCC patients and carried out an ensemble learning and prediction method with multiple parameters classifying survival in OTSCC. Results of our analysis identified somatic mutations in NOTCH2 and/or TP53, and/or LOH in 11p to associate with better disease free survival in HPV positive patients (P 5 0.0254) and not in HPV negative patients (P 5 0.414). We validated our results using data from TCGA cohort. Integrated analysis, including pathways, linked survival in our patient cohort with apoptosis and aberrant methylation in SLC38A8 (P 5 0.0129).

P162-K015Role of structure biology & bioinformatics to categorize the pathogenicity of mutations identified in BRCA1 gene for clinical management

A.K. Varma1

1 Tata Memorial Centre, ACTREC, Mumbai, India

BRCA1 (Breast Cancer Susceptibility gene 1) is one of the most studied tumor suppressor gene, expressed in breast and ovarian cancer. It has been observed that familial inheritance of breast and ovarian cancer is due to germ-line mutations in BRCA1 gene. Number of mutations have been indentified in large cohort of patients and are reported in Breast Cancer Information Core (BIC) also. Now, it is challenge for scientists/clinicians to explore the possibilities to evaluate the pathogenicity of mutations discovered in different breast cancer genes. Considering the role of structure biology & bioinformatics in clinical management, we have decided to evaluate pathogenicity of different mutations discovered throughout of 1863 amino acids of BRCA1. BRCA1 play a major role in cell-cycle check point control, cell-signaling and DNA repair mechanism. BRCA1 has different reported functional domains like N-terminal RING domain, tandem repeats of C-terminal and intrinsically unstructured regions at the central part. N-terminus, RING domain of BRCA1 interacts

frequency. Identifying genes that are important for further functional analysis from this data remains a formidable task. Using the data from TCGA project we have devised an innovative in silico method to identify new genes.Methods: All the data for 316 patients with HGS-OvCa were retrieved from the TCGA database. The parameters selected for analysis include frequency of amplification or deletion, size of the amplicon or deleted segment, number of genes within the amplicon or deletion, level of expression in the tumor compared to normal by both microarray and RNA sequencing, presence or absence of mutation, effect of alterations in the gene on overall survival. Each individual parameter was graded according to a score (maximum 24). Individual genes were then scored using this scale for all the parameters.Results: We identified using the above approach 33 oncogenes and 17 tumor suppressor genes that may have a role in the pathogenesis of ovarian cancer. Remarkably we also identified many known oncogenes like CCNE1, Myc and tumor suppressor genes like NF1, WWOX as the top ranking genes substantiating this approach. In addition, many novel genes previously unknown that are implicated in a number of pathways including cell cycle, histone modification, ubiquitin ligase, MAP kinase and DNA /RNA binding etc. were identified. We are in the process of validating the relevance of these genes in ovarian cancer.Conclusion: This in silico analysis has identified genes for future functional studies in the pathogenesis of the HGS-OvCa.

P115-K012Dosimetric comparison of variations in dose heterogeneity among three techniques of total body irradiation

Lavanya G1, V. Amirthalingam1, M. Rifayi1, K. Nithiyanantham1, S. Bhat2, S. Damodar2, R. Krishnatry1

1 Department of Radiation Oncology, Mazumdar Shaw cancer Center, Bangalore, India2 Department of Haematology, Mazumdar Shaw cancer Center, Bangalore, India

Background: Total body irradiation (TBI) as a conditioning regimen in bone marrow transplants with extended source to surface distance (SSD) technique with patient in standing position has been common practice. This technique has several limitations. We conducted dosimetric study comparing heterogeneity in doses in lying down TBI with two positional techniques (supine-prone & supine only) and standing technique.Materials & Methods: The CT dataset of 1 patient treated with TBI was used for dosimetric comparision. In supine-prone technique, 2 CT scans were acquired, 1 in supine and the other in prone position. Two separate plans (Anterior beam for supine and posterior beam for prone position) were generated with 3 fields matched for divergence. In supine only method, 4 fields (Anterior & Posterior) with moving junctions were used. In the above 2 methods, plans were done at SSD of 135 cm and points of prescription were in mid-plane of patient after optimization. In standing position, plan was generated at an SSD of 360 cm with umbilicus as prescription point. Doses at 12 points (occipital protruberance, optic nerves, sternal notch, base of heart, main bronchi bifurcation, L1 spine transverse process, femoral greater trochanters, patellas) were calculated and percent deviations from planned dose calculated and compared.Results: The average deviation in mean doses at various points measured in standing technique was 6.93% (SD: 3.64, range: 1.4 to 14.7), supine-prone was -1 (SD: 5.85, range: -6.2 to 9.3) and in supine only was -1.3 (SD: 3.97, range: -7 to 4.2). Dose deviation above 7% was observed in 3 regions in supine-prone, 1 region in supine only and 5 regions in standing technique.Conclusion: Lying down TBI with CT based planning should be considered in all patients planned for TBI in view of superiority of dose homogeneity, reproducibility of the set up and patient comfort.

P124-K013Dose constraint model to predict neuroendocrine dysfunction in young patients with benign and low grade brain tumours treated with high precision conformal radiotherapy

M. Maitre1, T. Gupta1, J. Swamidas1, J. Sastri1, U. Krishna1, D. Dutta1, N. Shah1, R. Jalali1


Background: Dose threshold to hypothalamic-pituitary axis (HPA) to predict neuroendocrine dysfunction is poorly defined. We report a possible dose constraint model for HPA, based on a prospective study of high-precision conformal radiotherapy to residual/recurrent brain tumours.


(p , 0.05). The max brainstem dose with 4-DOF correction was 9% more than 6 DOF correction (p , 0.05). A loss of prescribed isodose coverage of 18% was found with the 4DOF positioning (p , 0.05). Using CBCT DIR for IFM analysis, 0.3 mm vector translation error and 0.3 degree rotation error about each axis should be used as the maximum tolerated misalignment before initiation of functional SRS treatment.Conclusions: Functional radiosurgeries, with sub-cm target, should only be contemplated with 6-DOF patient positioning systems only.

P198-K018DVH metrics, serial MR imaging & outcomes in biologically based frameless image guided trigeminal radiosurgery

V. Shankar1, A.R. Gupta1, K. Vinoth1, P. Sathish1, R. Rajesh1, K. Ranjan1, J. Jijo1, C. Haritha1, A. Sachin1, S. Das1, D. Jain1


Purpose: To study the relationship between DVH metrics, serial MR imaging & pain relief after biologically based frameless image guided trigeminal radiosurgery.Materials & Methods: 10 pts. of medically refractory primary TGN having BNI Class IIIB & Class IV pain level & history of prior intervention underwent frameless Image guided radiosurgery. Median dose of 76 gray delivered on DREZ/Petrous dural ridge. Dosimetric parameters analyzed included dose to DREZ/PDR max. pons dose and cisternal length. All patients underwent serial MR imaging @ time frames 24 hrs, 1 week, 1 mo, 3 mo, 6 mo, & 12 mo post SRS. Target accuracy was assessed from deviation of the coordinates of the intended target compared with the center of enhancement on 24 hr post-SRS MRI.Radiation dose delivered at the borders of contrast enhancement was evaluated. “Favorable” responders were patients rendered free of pain, either with or without medications. Complications were defined as lasting neurologic deficits attributable to treatment, and included anesthesia, paresthesia or dysethesia.Results: At median follow-up of 12 mo. pain relief was complete in 6 patients, partial in 2 & minimal 2 pt. The actuarial rate of obtaining complete pain relief was 67.7%. Improved pain relief correlated with max brainstem dose .24 gy, fewer prior procedures (p , 0.04) and complications. Focal enhancement corresponding to SRS shot is seen in all the patients 24 hrs post SRS. The median deviation of the coordinates between the intended target and the center of contrast enhancement was 0.2 mm in Euclidean space. The radiation doses fitting within the borders of the contrast enhancement of the trigeminal nerve root ranged from 52 to 70 Gy.Conclusion: Biologically based Monte Carlo Optimization gives better clinical outcomes at lower doses compared to gamma knife procedures. Focal enhancement of the Vth nerve after SRS occurred in all pts within 24 hrs and was not associated with clinical outcome.

P212-K019Intrafraction needle displacement quantification by serial CT Scans during HDR MUPIT interstitial brachytherapy in cervical cancers



Purpose / Objective: To quantify the displacement of implant needles between the HDR fractions in cervical cancer patients undergoing interstitial brachytherapy boost as a part of definitive treatment.Material & Methods: Study evaluated 210 DRR films data in 70 consecutive interstitial brachy implants performed in 35 cervical cancer patients. All the patients underwent treatment under concurrent Chemo-radiation protocol using Inj. Cisplatin 40 mg/m2/week & a combination of 3DCRT to a dose of 5040cGy/28# (MLB at 3960cGy) followed by 2 applications of MUPIT interstitial brachytherapy, 1 week apart. In each application a dose of 13–15 Gy was delivered in 3 fractions over 24 hours. Following flouro-guided needle placement, all patients underwent initial CT scan, 3 mm axial images of the implant volume were acquired and all structures contoured. AP & Lateral DRR’s were generated with rendering of ischial tuberosity, sacrum, needle tips and perineal template.Results: The Mean/maximum X, Y, Z axis needle displacement between fractions in each application were 3 mm/12 mm & 4 mm/6 mm, 8 mm/ 22 mm. Majority of the displacement occurred in the craniocaudal direction with at least 13 mm caudal displacement occurring in 30% of the implants between the 1st and 2nd treatment fractions. Mean adjustment was 12 mm in 19% applications at 2nd fraction and 4 mm in 8% applications for 3rd fraction. In 13% applications needles slipped within the template due to caudal pull while disconnecting them

with BARD1 and performs the functions associated to protein-protein interactions. Furthermore, C-terminal region of BRCA1BRCT has been reported for transactivation function by recognising consensus sequences phosphorylated serine (pSer at 0 position) and phenylalanine (13 position) containing motif. However, mutations reported at the complex interface of BRCA1 and cellular partners abrogate the complex formation and associated functions. Different regions of BRCA1 have been evaluated to categorise pathogenic mutations. The protein-protein interactions (PPIs) with the binding partners of BRCA1 like RAP80, MERIT40, ABRAXAS have been explored. To our conclusion, the pathogenic mutations discovered in different regions of BRCA1 are impairing the PPIs and also further abolishing the signalling pathways. Furthermore, germ-line mutations screening in BRCA1 has helped in predisposing someone at the high risk of breast and ovarian cancer.

P167-K016Studies of protein–protein interactions in the MAPK pathway to understand transcription activity

B.P. Jagilinki1, A.K. Varma1

1 TMC-Advanced Centre for Treatment, Research and Education in Cancer, Mumbai, India

Mitogen-Activated Protein Kinase (MAPK) pathway is a major signalling pathway within the Eukaryotes. Upon activation by cell surface receptors, the downstream MAPK members activate a number of substrates both in the nucleus and in the cytoplasm which are responsible for various cellular processes such as translation, transcription and cell cycle regulation. MAPK pathway is highly unregulated in most of the reported cancer cells due to accumulation of mutations within the MAPK members. Deciphering this pathway both structurally and functionally at atomic level and understanding the Protein-Protein Interactions between the members of MAPK pathway will help researchers to develop new small molecule inhibitors. Considering the role of extracellular signal regulated kinase (ERK) and Ribosomal S6 kinases (RSKs) which constitutes the bottom end of the MAPK pathway in transmitting the extracellular signal into the nucleus, ERK 2 and RSK 3 had been purified and characterized for its biophysical properties using various techniques such as CD, Fluorescence spectroscopy, thermal denaturation, limited proteolysis and Dynamic Light Scattering (DLS). ERK 2 was purified as soluble protein from E. coli whereas RSK 3 C-Terminal Kinase domain (CTKD) was purified from bacterial inclusion bodies under denaturing conditions Furthermore, RSK 3 CTKD was successfully refolded and verified by CD and fluorescence spectroscopy. It has been observed that RSK 3 CTKD exists as homo-trimer in solution. Protein-Protein Interactions between ERK and RSK had been established both by using in-silico docking tools and in-vitro experiments by performing Isothermal Titration Calorimetry (ITC). The important residues critical for interaction between the MAPK pathways and responsible for transactivation function had been established.

P197-K017Alignment accuracy & intrafraction motion analysis in frameless functional image guided radiosurgery

V. Shankar1, A.R. Gupta1, K. Vinoth1, P. Sathish1, R. Rajesh1, K. Ranjan1, J. Jijo1, C. Haritha1, J. Sachin1, S. Das1, D. Jain1


Purpose: Analyze the alignment accuracy and intrafraction motion (IFM) for frameless image-guided functional radiosurgery.Materials/Methods: 10 patients with Trigeminal neuralgia underwent frameless SRS using DCA 16DOF technique. Median dose of 80 gy/fr was delivered. CBCT Positioning results were acquired midway of treatment and post-treatment completion to analyse IFM. The patient data were simulated for 4-DOF positioning on the TPS & the mapped dose compared with 6 DOF positioning. The clinical relevance was analyzed by means of the Maximum brainstem dose, conformity index and ratio of prescribed isodose volume covered with 4 DOF to that obtained with 6 DOF. Original plan, structures & dose were deform registered with 3 setup uncorrected CBCT datasets to understand the dosimetric impact of the IFM& to determine what should be the max. tolerated misalignment.Results: The mean 3D setup error from CBCT after post isocenter localization using localizer box but before 6-DOF correction for treatment was 0.89 mm. The rotational errors were larger in the longitudinal & vertical direction when compared to lateral directions (p , 0.05). The mean 3D IFM was 0.26 mm & mean Intrafraction rotational errors for the vertical, longitudinal, and lateral directions: 0.30, 0.160, and 0.030 respectively. The mean conformity index decreased from 0.86 (SD, 0.07) with 6DOF correction to 0.73 with 4 DOF correction


Results: 444 platforms identified a total of 1,797 and 2,655 genes uniquely expressed in normal and cancer tissues, respectively with 2,466 genes expressed in both tissues. Among the genes expressed in both tissues, 1,842 and 624 were up-regulated & down-regulated respectively. The RT qPCR of selected transcripts demonstrated reproducibility of differential expression profiling by RNASeq analysis. Further analysis of the deregulated transcripts by KEGG pathway and gene ontology revealed significant enrichment of genes in pathways commonly found to be altered in cancer with gene ontology suggesting suppression of genes involved in the actin mediated cell contraction process in cancer.Conclusion: This study led to identification of novel transcripts, with significantly altered expression in buccal cancer.

P288-K022Relevance rank platform (RRP) for functional filtering of high content protein-protein interaction data

Y.R. Pokharel2, J. Westermarck1

1 Centre for Biotechnology, University of Turku and Åbo Akademi, Turku, Finland2 Faculty of Life Science and Biotechnology, South Asian University, New Delhi, India

High content protein interaction screens have revolutionized our understanding of protein complex assembly. However, one of the major challenges for translation of high content protein interaction data is identification of those interactions that are functionally relevant for a particular biological question. To address this challenge, we developed a Relevance Ranking Platform (RRP), that combines both functional and bioinformatic filters to provide a relevance rank among the interactome proteins. We demonstrate the versatility of RRP to enable a systematic prioritization of the most relevant interaction partners from high content data; highlighted by the analysis of cancer relevant protein interactions for oncoproteins Pin1 and PME-1. We validate importance of selected interactions by demonstration of PTOV1 and CSKN2B as novel regulators of Pin1 target c-Jun phosphorylation, and reveal previously unknown interacting proteins that may mediate PME-1 effects via PP2A-inhibition.In summary, the data presented here introduces a novel approach to address one of the major challenges in functional translation of high content protein interaction data. The RRP framework presented in this work can supposedly be modified to answer versatile research questions depending on the nature of the biological question under study. Importantly, the data indicate that RRP has clearly added value especially for analysis of protein interactions for which there is no sufficient prior knowledge available for using existing filtering tools.

P54-K023 Minimally invasive reconstruction of orbital and maxillofacial defects using autologous fat

H. Gupta1, H. Khazaei1

1 Ocular Oncology, Orbit and Oculoplasty Services, Narayana Nethralaya, Bangalore, India

Background: Orbital and maxillofacial defects post-tumor resection are often challenging. They impart facial disfigurement and may not be amenable for immediate reconstruction. Post chemo/radiotherapy, poor general condition and financial constraints preclude an extensive reconstructive procedure. Majority of ocular defects occur in pediatric patients with retinoblastoma, and some need multiple reconstructive procedures since tissue contraction is accelerated post irradiation. Repeat surgery itself, perpetuates soft tissue contracture. Autologous fat injections are routinely used in aesthetic practice for soft tissue augmentation. Mesenchymal stem cells of fat have a potential to grow and hence prove to be a safe, inexpensive and abundant soft tissue substitute. Cryopreservation enables storage of fat upto 1 year. Minimally invasive fat injections can be utilized for immediate and subsequent soft tissue reconstructions, minimizing the need for extensive re-surgeries.Aim: To describe a novel concept of harvesting free fat during primary surgery for immediate and subsequent use in orbital and maxillofacial defects, post tumor resection.Methods: Patients of sino-orbital cancer undergoing resection or reconstruction underwent intra-op autologous fat harvest from gluteal or periumblical region. Fat harvested by Cole technique using a blunt-tip cannula. Manual fat harvesting was preferred for patients undergoing dermis fat graft. Fat was decanted and supernatant separated by wick

from the transfer tubes. There was no statistically significant needle displacement noticed between the first and second application in the same patient.Conclusion: The present study, to the best of our knowledge, is the first of its kind addressing the implant motion in cervical cancer interstitial brachytherapy. There is significant caudal displacement of the interstitial implant needles between the HDR fractions. Obtaining verification films/CT scan prior to treatments and making necessary adjustments would avoid subtle inaccuracies in treatment delivery.

P220-K020Integrated genomics analysis of early stage tongue squamous cell carcinoma patients

P. Upadhyay1, N. Gardi1, P. Chandrani1, S. Desai1, A. Joshi, S. Nair2, A. Dutt1

1 Integrated Genomics Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai, India2 Division of Head and Neck Oncology, Department of Surgical Oncology, Tata Memorial Hospital, Tata Memorial Centre, Navi Mumbai, India

Background: Of the multiple anatomical sites represented in oral cancer, squamous cell carcinoma of the tongue (TSCC) shows the highest incidence among younger age group. Chewing betel leaf, areca nut & slaked lime and smoking tobacco are common practises in India which have direct clinical implication in TSCC carcinogenesis. Here, for the first time we define the landscape of genomic alterations in TSCC from the Indian diaspora which would help to identify novel therapeutic targets for clinical intervention and define the genetic basis for TSCC.Methods: We performed high throughput sequencing of fifty four tongue samples using whole exome sequencing (n 5 47, 23 paired normal tumor and 1 unpaired) and transcriptome sequencing (n 5 17, 11 tumor and 6 normal). Mutation, copy number analysis were carried out using exome sequencing data and transcriptome analysis provided expressed genes and transcript fusions in tongue cancer patients. Further, integrated analysis was performed to identify biologically relevant alterations.Results: Our preliminary analysis revealed presence of most frequently altered mutations in TSCC which includes mutations in TP53, NOTCH1, CDKN2A, USP6, KMT2D etc, consistent with literature. We observed high frequency of C G/T (G C/A) transversions in non-CpG islands, a signature associated with tobacco exposure. Somatic copy number analysis revealed copy number gain in known hallmarks such as CCND1, MYC, and ORAOV1 genes along with copy number alteration in novel genes. Significant positive correlation was observed in the genes harbouring copy number gains and showing increased expression. Integrated analysis of somatic mutations, copy number changes and transcript fusions will be presented.Conclusion: Our study identified alteration in known cancer causing genes along with novel and exclusive set of somatic mutations in Indian TSCC cohort. This is the first most comprehensive landscape of TSCC tumors of Indian origin.

P225-K021Transcriptome analysis of human buccal mucosal cancer

C.G. Joshi1, V. Shankar2, C. Haritha2, S. Shah2, M.R. Sajnani1, A.K. Patel1, V.D. Bhatt1, A.K. Tripathi1, S.J. Jakhesara1, V.B. Ahir1

1 Department of Animal Biotechnology, Agricultural University, Anand, Gujarat, India2 Geetanjali Cancer center, Geetanjali University, Udaipur, India

Purpose/Objective(s): Transcriptome analysis of BM cancers in tobacco (smokeless) users, with special emphasis on differential expression to identify novel biomarkers for future therapeutic intervention.Materials/Methods: Samples of cancerous and normal mucosa were collected from patients of cheek cancer. Transcriptome sequencing was performed by a nexgen sequencer following manufacturer’s protocol. To calculate in silicogene expression level RNA-Seq analysis using human RNA database as reference was performed. The RNA-Seq analysis was carried out for sequence reads obtained from normal and cancer tissues separately using default parameters. Genes expressed either in normal or cancer tissues or in both tissues were grouped based on Reads/kb of exon model per Million mapped reads (RPKM) values. Validation of the results was carried out by RT qPCR of selected transcripts. The differential expression between normal and cancerous tissue was derived using the RPKM values of the corresponding transcripts. Enrichment of the genes belonging to specific pathways analysed. To increase the reproducibility, genes were selected with RPKM values _ 5 in at least one of the samples and the genes which were detected in both the samples were further selected with fold change.

L. Immuno Oncology European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S61

the generation of comprehensive variant profiles and their phenotypic correlation still remain challenging. Cross-validation of NGS results by orthogonal molecular techniques has been considered essential, but there have only been a few publications that address the need to carry out such orthogonal testing. Here we evaluate the accuracy of NGS for variant detection using two distinct sample preparation approaches. In the first approach, an in-house developed targeted NGS protocol for 13 genes (both germline and somatic variants) implicated in solid tumors, hematological malignancies and other genetic disorders was used to analyze 213 samples. In the second approach, 24 samples were tested with a commercial germline cancer panel covering 94 genes associated with both common and rare cancers. The Illumina MiSeq instrument was used to generate the sequence data. In all samples, coverage exceeded 100x with a sequence quality (. Q30) of more than 80% in all the targeted regions. Higher sequencing depth on the order of 1000X or higher was used to interrogate the somatic mutations. Orthogonal testing (by ARMS PCR/ capillary sequencing/NGS on a different platform) was performed on all 237 samples demonstrating 100% concordance (both positive and negative). Clinically relevant mutations were detected in 66 samples, indicating a diagnostic yield of 28%. Based on our findings, we propose that confirmatory analysis through orthogonal testing of variants need not be carried out considering the cost and time involved, if the NGS data meet appropriate quality thresholds and there is optimum coverage in and around the targeted regions.

L. Immuno Oncology

OF152-008Cytotoxic microenvironment in angioimmunoblastic T cell lymphoma – When good comes from the bad

T. Shet1, S. Epari1, M. Sengar1, H. Menon1

1 Department of Pathology and Adult hematolymphoid DMG, Tata Memorial Hospital, Parel, Mumbai, India

Background: AITL is a subtype of PTCL with uniformly aggressive behavior and abysmal prognosis. Pathologically AITL is a polymorphous tumor with lot o background cells and the neoplastic follicular helper T cells (FTH) forming only a proportion of cells. A diagnosis of AITL was made on a conglomerate of criteria- FTH phenotype and dendritic cell proliferation being chief among that. However we have seen AITL with lesser number of FTH and large amount of cytotoxic T cells in background. Gene profiling studies have shown that microneviromental signature impacts prognosis in AITL. How does this translate to change in pathology and is there a simpler way of assessing microenvironment?Material and Methods: These 115 patients were part of a group of 260 peripheral T cell lymphomas (PTCL) accessioned at our institute from 2003 to 2010. A total of 39 cases of AITL were selected for analysis. For the diagnosis of AITL an extrafollicular dendritic cell proliferation was a must. A panel of T cell markers and CD4:CD8 ratio was calculated in these cases. Quantification of T cells was done manually by one author.Results: After histological evaluation AITL were divided into a) AITL with dominance of cytotoxic cells (AITL Cytotoxic) (n 5 21) and Typical AITL (n 5 18). Clinical data was retrieved for survival analysis and it was observed that AITL Cytotoxic has better DFS (p -0.009) than typical AITL with DFS at 2 year of 65% Vs 10% for typical AITL. The OAS was also significantly different (p – 0.01). Also few AITL relapsed as PTCL and vice versa.Conclusion: AITL is not a unique entity but a fluctuating tumor with microenvironment playing distinct role in behavior. Till date there is no study evaluating cytotoxic T cells in AITL and this microenvironment can be targeted for therapy and improving prognosis.

OF75-006The immunoregulatory protein B7-H3 regulates reprogramming of cancer cell glucose metabolism through reactive oxygen species mediated stabilization of HIF-1a

S. Lim1, H. Liu1, L. Barnes1, M. Tan1

1 Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA

B7-H3 is a transmembrane glycoprotein and a member of the B7 family of immunoregulatory proteins. Extensive studies have shown that B7-H3 is important in regulating the activity of immune cells. Overexpression of B7-H3 is associated with tumor progression and poor patient outcome in multiple types of cancers. However, studies

technique. It was centrifuged and used as volumizer. Rest of fat was cryopreserved under sterile conditions at -80 deg C for subsequent use.Results: Three patients of retinoblastoma (two post-irradiation), undergoing secondary socket reconstruction were included. All underwent fat harvesting and cryopreservation. Two underwent fat transfer to socket for immediate soft tissue augmentation. Patients were doing well in terms of donor site and socket outcomes at the end of 1.83 months (average).Conclusion: Autologous fat transfer represents a versatile, safe and inexpensive volumizer for maxillofacial soft tissue defects post resection and radiation. Cryopreservation enables subsequent soft tissue augmentation.

P55-K024Reconstruction of orbital and maxillofacial defects using PCL mesh (osteomesh) implant

H. Khazaei1, B. Khurana1, H. Gupta1

1 Ocular Oncology, Orbit and Oculoplasty Services, Narayana Nethralaya (NN2), Bangalore, India

Background: Reconstruction of orbital and maxillofacial bony defects following tumor resection is challenging. The defect may not be amenable for immediate reconstruction and imparts facial disfigurement. Post chemo/radiotherapy, poor general condition may render patients unfit for extensive grafts. Osteo mesh is a bioresorbable polycaprolactone (PCL) implant used in craniofacial surgery to repair fractures and fill surgical defects. When placed subperiosteally, its interconnected pores allow saturation with marrow, blood and nutrients, providing triggers needed for bone growth and remodeling. The mesh can be trimmed, layered, is malleable, and offers a rigid yet flexible scaffold with sufficient mechanical strength. It degrades as bone regeneration occurs. Being thermoplastic, minor shape adjustment can be done using warm water.Aim: To describe a novel technique of using osteomesfor orbital and maxillofacial defects post tumor resection.Methods: Bioresorbable osteomesh was used for orbital floor (56) and lateral wall (6) fracture repair in 62 patients. Per operatively, innovative acryilicorbital floor dummy was used for accurate sizing of implant. Osteomesh was customised. In large defects, where periosteum was not available, osteomesh wrapped in collagen sheet was used. In addition a autologus fat was utilized as volumiser on either side of the collagen wrapped PCL. Post operative analysis done. One patient of recurrent lacrimal adenoidcystic carcinoma who underwent eyeball sparing cranio facial resection is scheduled for a PCL implant reconstruction, outcomes pending.Results: All patients showed resolution of enophthalmos. Imaging showed early bone formation. One 12 year old had recurrence of diplopia and motility restriction post-operatively. He developed adhesions between implant and soft tissue needed osteomesh removal.Conclusion: Customized PCL mesh implant is a safe, effective and versatile option in orbital and craniofacial bony defects and can be used to address post tumor resection maxillofacial bony defects as well. Further studies are needed to establish its efficacy in oncological reconstruction.

P297-K026Confirmation of variants identified by next generation sequencing: Is orthogonal testing essential?

N. Gogate1, T. Ragte-Wathare1, P. Gangodkar1, S. Ranade1, A. Pilankar1, K. Patil1, M. Agarwal1, K. Khatod1, K. Kelkar1,7, P. Gadgil5,6, V. Khadilkar3,4, V. Ramanan2,7,8, N. Phadke1

1 GenePath Dx, Causeway Healthcare, Phadke Hospital, J.M. Road, Shivajinagar, Pune, India2 Ruby Hall Clinic, Pune, India3 Department of Pediatric Endocrinology, Jehangir Hospital, Pune, India4 Hirabai Cowasji Jehangir Medical Research Institute, Pune, India5 Breast Center, Jehangir Hospital, Pune, India6 Prime Surgical Centers, Pune, India7 KEM Hospital, Pune, India8 Yashoda HematologyClinic, Pune, India

Next-generation sequencing (NGS) provides the ability to analyse multiple genes in multiple samples in a highly parallel manner. Its ability to detect single nucleotide variants (SNVs) and insertion/deletion variants (InDels) with high sensitivity and cost efficacy makes it highly suited for use in a clinical setting for many genetic conditions. Although, there have been advances in the core NGS sequencing platforms, library preparation techniques and bioinformatic analysis pipelines,

S62 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 L. Immuno Oncology

with metastatic disease, yet predicting such responses remains a nascent science. More importantly, the toxicity and financial burden of receiving such therapy warrants a personalized, or precision medicine, approach to appropriate selection of treatment, thus molecular predictors of response may become as important as biomarkers such as PDL1 expression. Recently the high mutational burden of malignant melanoma was shown to be related to the degree of response to the anti-CTLA4 antibodyipilimumab, but mutational burden alone was not sufficient to predict benefit. Another study has shown NRAS-mutant had a trend to superior outcomes compared with other cohorts in terms of response to therapy with anti-PD1 antibodies and progression-free survival. Moreover, insight into the molecular evolution of melanoma was recently established, wherein somatic mutations in oncogenes such as BRAF, NRAS, GNAQ, or GNA11 occur early during progression and additional mutations of TERT, CDKN2A, TP53, and PTEN are seen in late-stage disease. To better evaluate the relationship of these mutations to immunotherapy response, our institution has profiled 95 patients with melanoma with next-generation sequencing (NGS). Of the 95 patients profiled, the most common mutations identified were BRAF (38%), NRAS (21%), TP53 (18%), PTEN (14%), TERT (9%), and CDKN2A (8%). We are presently conducting a retrospective analysis of these selected oncogenes as they relate to clinical responses, and intend to report disease outcomes (duration of response, progression free survival, and overall survival) as they relate to checkpoint inhibition.

P62-L002Targeting myeloid-derived suppressor cells (MDSCs) to rejuvenate the antitumor immunity in oral cancer

A.A. Dar1, A. D’Cruz2, D. Chaukar2, S. Chiplunkar1

1 Chiplunkar Lab, ACTREC, Tata Memorial Centre, Navi Mumbai-410210, India2 Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India

The immune system has the greatest potential for the specific destruction of tumors and has a long-term memory that can prevent cancer recurrence. However, tumors subvert normal immune regulation to their advantage and prevent the expansion of tumor antigen-specific helper and cytotoxic T cells. The oral-tumor microenvironment is characterized by chronic inflammation represented by infiltrating leukocytes and soluble mediators which lead to immune suppression. Earlier we have shown that T cells from oral cancer patients have functional abnormalities and are ineffective in generating effective immune response. In the present study, we investigated how MDSCs and regulatory T cells (Tregs) maintain the immune suppressive tumor microenvironment of oral cancer (OC) patients and dampen the T cell function. OC patients have significantly higher MDSCs than healthy individuals (HI) and correlated with cancer stage. Non-classical MDSCs were prevalent in the periphery while granulocytic subset dominated the tumor compartment. MDSCs suppressed lymphocyte proliferation, decreased CD3-z chain expression and IFN-g production. The levels of MDSCs correlated inversely with CD3-z chain expression in T cells. MDSCs expressed higher levels of COX-2, Arginase 1, NF-kB, STAT3 and pSTAT3. IL-6 induced pSTAT3 in MDSCs regulates PDL1, C/EBPa and IL-10 expression required for its generation and function. MDSCs secrete IL-10 which might facilitate crosstalk between MDSCs and Tregs. Increased Tregs observed in OC patients correlated with stage of the tumor and IL-10 levels produced by MDSCs. However in vitro experiments showed that MDSCs inhibit TGF-b induced generation of Tregs in a dose-dependent fashion. MDSCs secrete higher levels of IL1-b which is known to regulate splicing of Foxp3 and induce TH17 differentiation. Increased frequency of MDSCs, Tregs and decreased expression of CD3-z chain results in T cell tolerance and induces a chronic inflammatory state facilitating tumor growth. These cells could be used as potential targets to modulate antitumor immunity.

P63-L003T/NK cell lymphoma: Case report and review of literature

R. Thawani1, J. Patowary1, P.K. Das1, K. Singh2

1 Department of Medical Oncology, Indraprastha Apollo Hospitals, New Delhi, India2 Department of Burns and Plastics, Indraprastha Apollo Hospitals, New Delhi, India

NK cells are cytotoxic cells that lyse virus infected and tumorigenic cells. Malignancy of these cells is called T/NK cell lymphoma, which is an extremely rare entity. These are of three kinds, out of which nasal lymphoma is the commonest. We report a case of a T/NK cell lymphoma where the patient presented with sinusitis. After surgery and biopsy,

on how B7-H3 regulates cancer growth and metastasis are rare and the mechanisms underlying the role of B7-H3 in cancer malignancy are almost unknown. We and others have shown that B7-H3 mediates breast cancer invasion, metastasis and drug resistance and that silencing B7-H3 delays tumor growth and sensitizes cancer cells to chemotherapeutic agents in immune-independent in vitro and in vivo assays, indicating immunoregulatory B7-H3 also has immune-independent function.In this study, we investigated the role of B7-H3 in cancer cell metabolic reprograming in vitro and in two independent xenograft mouse models. We found that B7-H3 increased glucose uptake and lactate production while it suppressed oxygen consumption in B7-H3 high-expressing cells, indicating that B7-H3 promotes Warburg effect. B7-H3 increased the protein levels of HIF-1a and its downstream targets LDHA and PDK1, which are key enzymes in glycolytic metabolism. Moreover, B7-H3 promotes HIF-1a stability via enhanced reactive oxygen species (ROS) by suppressing the activity of transcription factor Nrf2 and mitochondrial Nrf2 target genes, including antioxidant enzymes SOD1, SOD2 and PRX3. B7-H3 mediated HIF-1a stability is suppressed by ROS scavenger treatment including N-acetylcysteine and PEG-catalase. Metabolic imaging of human breast cancer xenograft in mice further demonstrated that B7-H3 enhances tumor glucose uptake and tumor growth. Together, our results show that B7-H3regulates cancer glucose metabolism via ROS mediated-HIF1a pathway. These results provide novel insights into the function of B7-H3, which give us a new perspective to understand the role of B7 family of immunoregulatory proteins in cancer progression.

OF51-007Single-cell metrics of the efficacy of CAR1 T cells

G. Romain1, H. Singh2, I. Liadi1, J.R. Adolacion1, B. Roysam3, L. Cooper2, N. Varadarajan1

1 Department of Chemical and Biomolecular Engineering, University of Houston, USA2 MD Anderson Cancer Center, University of Texas, Houston, USA3 Department of Electrical Engineering, University of Houston, USA

T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR) have shown considerable promise in clinical trials even in tumors refractory to all other treatment methods. Despite the clinical promise of CAR therapy in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address this question and a priori define the therapeutic potential of T-cell based therapies.Using our novel single cell multiparametric assay, Timelapse Imaging Microscopy In Nanowell Grids (TIMING), we compared the efficacy of two second generation (CD28 endodomain) CD19-specific CAR constructs – bearing CD8a and IgG4 hinge respectively - by tracking their interaction with NALM-6 tumor cells in vitro. Superior efficacy was confirmed in a mouse model in which CAR1 T cells containing the CD8a extracellular domain were superior in controlling the disease.In order to identify biomarkers those distinguish between killer (and serial killer) CAR1 T cells and non-killers, we performed single-cell multiplexed gene expression profiling (96 genes) subsequent to a TIMING assay. Across three separate donors, killer CART cells consistently expressed higher transcript levels of the cytotoxic molecule Granzyme B, the costimulatory receptor CD137 (4-1BB) and the regulatory receptor TIM-3 (HAVCR2). By utilizing multi-color flow-cytometry, we confirmed that CD137 was overexpressed in cytotoxic CAR1 T cells and that its stimulation was associated with higher cytotoxicity and lower expression level of the immunoregulatory receptors CTLA4 and PD1and 2B4. By comparison, TIM-3 was not enriched in killer CAR1 T cells but rather was a likely marker for effector status since blocking TIM-3 boosted cytotoxic responses.In aggregate, these results demonstrate the utility of our TIMING single-cell methodology in uncovering not only the dynamic profile of T-cell behavior but in also uncovering the phenotypic biomarkers of CAR1 T cells with superior functional efficacy.

P53-L001Is it What’s On the Inside That Counts? Melanoma mutation profiling and outcomes with immunotherapy

M. Freeman1, H. McLeod2

1 H Lee Moffitt Cancer Center, Tampa, FL 33607, USA2 DeBartolo Family Personalized Medicine Institute, Petersburg, Florida Area, USA

Extraordinary advances have been made in the treatment of melanoma over the last few years; immune checkpoint blockade has provided durable responses and meaningful survival outcomes for patients


high grade) may depend on the potency of the immune system. Tumor calcification (macro or micro) or its any surrogate at molecular level may serve as useful biomarker and even an endpoint in cancer treatment.

P87-L006Association of cytokine profile with different established prognostic markers in breast cancer patients

S. Nigam1


Background: Comprehensive overview of breast cancer development and progression suggests that the process is influenced by intrinsic properties of the tumor cells, as well as by micro environmental factors. If a significant relationship could be established between such markers and prognosis of breast cancer, it would aid in classifying the patients based on prognosis of the disease.Objectives: The purpose of this study was to trace correlation between level of cytokine markers and established prognostic markers of breast cancer. The study had the following objectives.Methods: 1) Profiling a panel of serum cytokine (IL-2, IL-4, IL-6, IL-10, IL-17a, TNF-a, IFN-g) 2) Profiling of genetic risk marker, BRCA1. 3) Analyzing association of cytokine levels with established prognostic and genetic risk markers (ER/PR/Her-2 neu/BRCA1).Results: The mean age of breast cancer patients in our study was 44.7. On applying unpaired t-test, difference in serum levels of IL-6, IL-10, TNF-a and IL-17a between case and control groups was statistically significant (p value , 0.05). In our study tissue incidence of estrogen receptor positivity in breast cancer patients was 40%, of progesterone receptor positivity was 45% and for Her-2/neu positive status was 87.5%. BRCA1 mutation was seen in 10% of the cases included in this study. Data for IL-6, IL-10 and TNF-a is statistically significant for all the four established prognostic markers, however correlation for BRCA1 is in opposite direction (negative correlation) to that of other three markers. We could not find any explanation for this inverse relationship between BRCA1 mutation and ER/PR/Her-2neu.Conclusions: Thus to summarize, trend for IL-6, IL-10 and TNF-a is statistically significant for all the four established prognostic markers, however correlation for BRCA1 is in opposite direction (negative correlation) to that of other three markers.

P89-L007Abstract withdrawn

P90-L008Zoledronate induced gd T cells activation: A potential cell based therapy

S. Phalke1, R. Badwe2, V. Parmar2, S. Chiplunkar1

1 Chiplunkar Lab, ACTREC, Tata Memorial Centre, Navi Mumbai, India2 Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India

Antiresorptive bisphosphonates are used in the treatment of osteoporosis and bone metastasis. Zoledronic acid is the current standard of care for treating bone metastases from hormone-refractory advanced breast cancer. Randomized trials have demonstrated zoledronic acid to significantly delay the onset and decrease the incidence of SREs when used in conjunction with chemotherapy or hormonal therapy. Zoledronic acid inhibits fernesyl pyrophosphate synthase in the mevalonate pathway that upregulates intracellular levels of isopentenyl pyrophosphate that is known to be a potent activator of gamma delta T cells. We rationalized that Zoledronate treatment may also activate Vg9Vd21T cells in advanced breast cancer patients in whom all other common treatments have been exhausted. Immune profiles of breast cancer patients with bone metastasis, undergoing Zoledronate treatment (MET) were compared to breast cancer patients without bone metastasis (NMET) and healthy individuals (HI). MET patients showed reduced T-helper cells (CD4+), regulatory-T cells (immunosuppressive cells), but showed significant increase in cytotoxic T cells (CD8+), natural killer cells compared to NMET and HI. Gd T cell Percentages in peripheral blood were reduced but these cells were in highly activated state (high CD25, RANKL and increased IFNg production). gd T cells exhibited effector memory phenotype (highly cytotoxic) in MET compared to NMET and HI.Activated gd T cells showed increased anti-osteoclastogenic and antitumor properties. Zoledronate activated gd T cells showed increased RANKL expression and IFNg secretion. gd T cells isolated from MET patients showed reduced IL6 (pro-osteoclastogenic) levels compared to NMET and HI. In summary, the study demonstrates Zoledronate treatment to MET patients enhances the activation of gd

T/NK cell lymphoma was confirmed and the patient was given sequential chemotherapy and radiotherapy. The patient developed progressive destruction of the nose, palate and paranasal sinuses. After debridement, biogdpsy was sent which showed necrosis. The patient received supportive medications and required reconstruction. The patient underwent reconstruction and is no asymptomatic with no cosmetic anomaly.

P64-L004Pro-tumor role of IL17 producing gd T cells in gallbladder cancer

R.S. Patil1, S.U. Shah1, S.V. Shrikhande2, M. Goel2, R. Dikshit2, S.V. Chiplunkar1

1 Chiplunkar Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, Maharashtra, India2 Tata Memorial Hospital, Parel, Mumbai, Maharashtra, India

Cancer development is strongly influenced by the dynamic crosstalk of immune cells present in the tumor microenvironment. Immunotherapeutic approaches exploiting antitumor properties of T cells have shown improved efficacy of conventional treatment. Human T cells expressing gd-TCR exhibit potent anti-tumor activity in vitro. In recent past, cell based anti-cancer therapies using gd T cells have gained importance. However, the role of gd T cells in cancer promoting inflammation is not well explored. In the current prospective study of gallbladder cancer (GBC) patients, we report that the IL17 producing inflammatory subtypes of gd (Tgd17), CD4 (Th17) and CD8 (Tc17) T cells were increased in peripheral blood (PBL) and tumor infiltrating lymphocytes (TIL) of GBC patients compared to healthy individuals (HI). Regulatory T cells (Tregs) were decreased in PBLs and increased in TILs of GBC patients but their suppressive potential was comparable to HI. The ratios of Th17/Treg, Tgd17/Treg and Tc17/Treg were increased but CD8+IFNg+ and gd+IFNg+ cells were decreased in TILs. Serum cytokines profile of GBC patients showed elevated levels of cytokines (IL6, IL23 and IL1b) required for differentiation of IL17 producing cells. We demonstrated that Tgd17 cells migrate towards tumor bed through CXCL9-CXCR3 axis. IL17 secreted by Tgd17 induced VEGF production and other angiogenesis related factors in GBC cells. Tgd17 cells promote vasculogenesis as studied by chick chorioallantoic membrane assay. Tgd17, Th17, Treg and serum IL17 were associated with poor survival of GBC patients. Taken together, our data strongly suggests that Tgd17 is a pro-tumorigenic subtype of gd T cells, which may contribute to the negative clinical outcome of GBC patients. Thus, future immunotherapeutic treatment strategies for GBC may use a combined approach to block the trafficking of Tgd17 cells to the tumor and inhibit the functions of IL17.

P86-L005Is calcification a surrogate biomarker of effective immune response during carcinogenesis and anti-cancer treatment?

A. Jain1

1 Valentis Cancer Hospital, Mawana Rd, Meerut, Uttar Pradesh, India

Cancer is a multistage process and often develops on chronic inflammation. It seems that cancer is the outcome of final tussle between procarcinogenic and anticarcinogenic components of the immune system. We see that calcification is often present in low grade cancers with better prognosis as compared to higher grade ones. There is evidence that Receptor Activator of NF-[kappa]B ligand (RANKL) /RANK system is involved in the epithelial-mesenchymal transition, aggressive phenotype and hence poor prognosis of some cancers and even in progestin-induced mammary carcinogenesis besides its established role in bone remodelling. Zoledronic acid, an inhibitor of RANKL/RANK axis has anti-tumour activity against visceral metastases and has shown efficacy even in adjuvant treatment and prevention of breast cancer. I present 8 cases in which dense calcification occurred in very advanced cancers after radiotherapy/chemotherapy treatment with dramatic improvement and discuss whether calcification could be an important marker of an effective host immune response against the malignant cells. Careful observation over years suggests to me that the patients in whom even tiny calcification develops in the nodes or the primary tumor post treatment is a favorable prognostic sign. The limited published data also supports the same. From the literature, it’s clear that the calcification post treatment is not characteristic of some particular cancers. Though it’s infrequent but has been observed in variety of cancers. Further research in this direction may help to uncover the processes during carcinogenesis and during treatment. I hypothesize that cancer may not be the outcome of accumulation of random mutations developed over time; however, the development of cancer (benign, low grade or

S64 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 L. Immuno Oncology

of COX-2 in a different manner in different cells. We studied the mechanism by which NO regulates the expression of COX-2 in LPS stimulated murine macrophage cell line RAW264.7. Treatment of the cells with NOS inhibitor L-Nv-Nitroarginine (L-NNA) in the presence of LPS (1 μg/ml) reduced the production of NO in cultured macrophages. Moreover, significant inhibition of COX-2 was observed when the cells were treated with L-NNA (8.1 μM) for 18 hours in the presence of LPS. Reporter gene analysis and mRNA analysis by RT-PCR showed that NO regulates COX-2 expression transcription level also. Additional experiments suggested that L-NNA mediated inhibition of COX-2 is due to blocking of NF-kB, ERK, C-jun, AKT as well as proinflammatory cytokines like TNF-a and IL-1b.

P151-L012Granulomas in nodes draining cancer are tuberculosis in a country like India where Tb is rampant

O. Shetty1, A. Uttarkar1, T. Pai1, T. Shet1

1 Department of Pathology, Tata Memorial Hospital, Mumbai, India

Background: It is not uncommon to see granulomas in lymph nodes draining a carcinoma and in a population where tuberculosis is common this is automatically treated as tuberculosis.Material and Methods: We analyzed 63 node excisions (FFPE) from breast cancer patients with granulomas in draining nodes.Nested PCR using IS6110 was used for Mycobacterium tuberculosis (MTB) and CK19 PCR was performed to look for occult metastatic tumor cells within these granulomas. Granulomas were selected excluding metastatic tumor using macrodissection. Given the complex nature of CK19 PCR, the band densities of CK19 in reactive nodes and nodal granulomas from tubercular lymphadenitis was analyzed by gel densitometry with a cut off of 15ng band intensity as the endogenous level of CK19 expression. Molecular analysis was correlated with clinical and histological data.Results: The nodal group showing granulomas were axillary in 46 patients, while in 13 supraclavicular node and one patient each neck node and mediastinal node showed granulomas.Of the 63 patients, 26 had received chemotherapy; 29 did not receive chemotherapy and 8 patients history of chemotherapy was unavailable. Of the 63 patients the draining nodes showed metastases in 42 patients and 21 had purely granulomatous lesions in nodes. Eleven cases showed CK19 positivity and 6 cases were positive for MTB. One case was positive for both CK19 and MTB.In the 6 MTB positive cases, 3 patients had nodal metastasis while 3 patients were node negative. In CK19 positive cases 9 patients had nodal metastasis while 3 patients had no nodal metastasis. Presence of subcapsular or perisinusoidal granulomas was significantly associated with CK19 PCR positivity.Conclusion: Mycobacterial granulomas and granulomas as part of chemotherapy related tumor damage are seen in nodes draining cancer. Presence of sinusoidal granulomas is a feature that suggests a granuloma associated with metastatic tumor rather than tuberculosis.

P154-L013Tumors infiltrating natural killer T (NKT) cells express reduced Th1 and increased Th17 cytokine, and contribute to the tumor growth

S. Paul1, G. Lal1

1 National Centre for Cell Science, Pune India

NKT cells express T cell receptor and have ability to rapidly produce Th1, Th2 and Th17 cytokines, and shown to have inflammatory and protective function. The cytolytic function of NKT cells were explored in the adoptive cellular therapy to treat cancer patients, but showed limited clinical benefits. Cellular and functional phenotype of NKT cells in the tumor microenvironment and its interaction with adaptive immune cells and stromal cells are not clearly understood.Using B16F10 cell (1 3 106 cells/mouse) induced melanoma model in C57BL/6 mice, we show that CD31 NK1.11 NKT cells (18 6 9%, n 5 10 mice) were selectively recruited into the tumor compared to its frequency in spleen (1.6 6 0.5%, n 5 10 mice) at as early as day 5. Phenotypic analysis of tumor infiltrating NKT cells showed significantly increased expression of cell adhesion molecule CD62L (51 6 12%, n 5 8 mice) compared to splenic population (23 6 3%, n 5 8 mice). BrdU staining suggested that most of tumor infiltrating NK cells were recruited from the secondary lymphoid tissues than their local proliferation. Intratumoral NK cells showed increased CD27 expression with significantly decreased T-bet, and IFN-g and GM-CSF expression; and increased IL-6 and IL-21 cytokines compared

T cells that may offer a survival advantage to these patients through their anti-tumor, anti resorptive properties. Such treatments might be complemented by adoptive transfer of autologous gd T cells and will pave a way for immunotherapeutic treatment modality for advanced breast cancer.

P135-L009Notch and TCR signaling modulate the effector functions of human gd T cells through transcriptional and epigenetic mechanisms

S.A. Bhat1, S.V. Chiplunkar1

1 Chiplunkar Lab, ACTREC, Tata Memorial Centre, Mumbai, India

Background: Evolutionally-conserved Notch proteins play an impor-tant role in deciding cell fate from insects to mammals. The function of Notch signaling in T cell development has been intensively studied. However the role of Notch signaling in the effector function of T cells remains to be elucidated. The present study focuses how Notch signaling regulates the anti-tumor functions of gd T cells and how these functions of gd T cells are regulated through transcription and epigenetic mechanisms.gd T cells account for 5–10% of CD3+ peripheral blood T cells. gd T cells have been implicated in innate response against various tumors and pathogens and are rich source of IFN-g. Activated gd T cells exihibit cytotoxic potential and express higher levels of perforin and granzyme.Methods: gd T cells were sorted immuno-magnetically from peripheral blood of healthy individuals. Sorted gd T cells were treated with the GSI (Gamma secreatease inhibitor) and HDAC (Histone deacetylase) inhibitors. Flow cytometry and western blotting was used to analyze expression of molecules associated with effector functions of gd T cells and TCR signaling molecules. Chromatin immunoprecipitation was used to check the expression of histone modifications. Cytotoxic potential of gd T cells against tumor targets was checked by chromium release assay.Results: Inhibition of notch signaling alters the expression of transcriptional factors Eomes, Tbet, p-STAT5 and expression of key effector molecules perforin and granzyme. Inhibition of notch signaling also alters the histone modification on the promoter region of perforin and Granzyme B. The ability of gd T cells to lyse the tumors significantly increased in presence of HDAC inhibitors.Conclusion: These results insinuate that the notch signaling and TCR signaling pathways modulate the anti-tumor functions of gd T cells through transcriptional and epigenetic mechanisms. The present study will provide new avenues for cell based immune-therapy using gd T cells.

P142-L010Atypical presentation of angioimmunoblastic T cell NHL

K. Kishor1

1 Meherbai Tata Memorial Hospital, Jamshedpur, India

A 54 year gentleman, c/o-low grade fever, generalised itching since last 1 year. He developed neck swelling from last 20 days. O/e-dysphagia, low grade fever & weight loss present. LDH-917 U/L, b.m.examination –uninvolved. Usg abdomen-gbcalculi, B/L renal cysts, splenomegaly. CECT neck-enlarged palatine, nasopharynx & lingual tonsils, B/L cervical ln at all level, B/L axillary & superior mediastinal ln. Neck node biopsy & IHC- angioimmunoblastic T cell NHL, CD3+, CD4+, CD8+, Ki67–75%, PD1+, CD201, CD102, CD21 expanded dendritic network positive. 18F-FDG PET-CT SCAN reveal: low grade metabolic activity in bilateral cervical and axillary nodes, no active disease elsewhere in the body. Diagnosis - Angioimmunoblastic T cell NHL, stage 2 disease. Received 4 cycles of chemotherapy with CHOEP regimen 3 weekly. Post 4 CHOEP, PET-CT show post 4 CHOEP, PET-CT show no any metabolic activity received 2 more cycles of CHOEP. After 1 month he developed generalised itching. PET -CT reveals recurrence of disease. Advised to undergo SMILE regimen chemotherapy followed by bone marrow transplantation.

P150-L011Nitric oxide mediated cyclooxigenase-2 (COX-2) regulation in LPS activated mouse macrophages RAW264.7 cell

S.K. Karna1, F. Ahamad1, O. Faruq1, Y. Pokharel1

1 Faculty of Life Science and Biotechnology, South Asian University, New Delhi, India

Increased production of nitric oxide (NO) by iNOS (inducible nitric oxide synthase) and PGE2 (prostaglandin E2) by COX-2 (Cyclooxygenase -2) are the key molecular events that occur during the inflammatory process. Previous studies have shown that NO influence the expression


mutation in a daughter cell. During pregnancy, mothers’ immune cells must interact with highly mitotic fetal tissues surveying for the integrity of the progeny. Because maternal cells are hemizygous to the embryo, both share the classical ABC, restricting molecules, and the non-classical MHC antigens, the Qa/Tla equivalents in the mouse. Brickell (1983) wrote “Activation of Qa/Tla class I MHC genes is a general feature of oncogenesis in the mouse” These genes are restricted to developmentally immature stem cells and also are the ligands of the NK receptors (KIRs, CD94-NKG2A). The embryo clearly behave like a tumor, full of undifferentiated and rapidly proliferating cells prone to casual mutations, what keeps them reasonably under control? Haploidentical stem cell transplantation from mothers to sons have significantly stronger graft antileukemic effect (GVL) than from others donors (Stern). VanRood using cord blood (CB) transplants, showed indirect evidence that maternal microquimeric cells present in the CB were responsible for the anti-leukemia effect.Conclusion: Mothers should have cytolytic and memory immune cells, capable to strongly reject undifferentiated tumor cells if they expressed the classical & non-classical IPAs. Therefore mothers’ cells could be adoptively transferred to an allogeneic o semiallogeneic receptor (husband or sons) for cancer immunotherapy, after typing for classical and non-classical antigens, and their expression in the tumor.

P235-L018Association of the programmed cell death 1 (PD-1) receptor, programmed death ligand 1 (PD-L1), and androgen receptor (AR) with PARPi-7 and DNA damage repair gene expression in primary breast cancer (BC)

N. Vidula1, D. Wolf1, C. Yau1, H.S. Rugo1

1 University of California, San Francisco (UCSF)

Background: Although BC is classified based on hormone and HER2 receptors, it now appears that extensive biologic heterogeneity corresponds to a variety of activated pathways. Targeting PD-1, PD-L1, and AR has demonstrated efficacy in BC. DNA damage repair gene expression may predict response to DNA damaging agents. Understanding gene expression in these pathways may inform effective combination therapy.Methods: We studied the gene expression of PD-1, PD-L1, and AR in primary BC using microarray data from METABRIC (n 5 1992) and TCGA (n 5 817). Gene associations between PD-1, PD-L1, AR, PARPi-7 (7 gene DNA repair deficiency signature), and DNA repair genes, TDG and CHEK2 were assessed using Pearson correlations.Results: Significant (p , 0.05) gene expression correlations are shown by BC subtype (including triple negative (TN) BC when significant) and database (r: correlation coefficient, NS: non-significant).

METABRIC TCGA

(n 5 1992) (n 5 817)

INVERSE correlations

AR and PD-1 all (r 5 -0.20) all (r 5 -0.28)

AR and PD-L1 all (r 5 -0.16) all (NS)

TNBC (r 5 -0.15)

AR and PARPi-7 all (r 5 -0.21) all (r 5 -0.42)

TNBC (r 5 -0.16) TNBC (r 5 -0.22)

POSITIVE correlations

PD-1 and PARPi-7 all (r 5 0.082) all (r 5 0.29)

PD-L1 and PARPi-7 all (r 5 0.16) all (NS)

PD-1 and CHEK2 all (r 5 0.17) all (r 5 0.22)

PD-L1 and CHEK2 all (r 5 0.17) all (NS)

PD-1 and TDG all (r 5 0.21) all (NS)

PD-L1 and TDG all (r 5 0.086) all (r 5 0.11)

Conclusions: In large expression databases, PD-1 gene expression significantly correlated with DNA repair gene expression, and was inversely correlated with AR gene expression, consistent with the respective disease phenotypes with immune response to DNA damage. This data suggests that combining immunotherapy and DNA damaging agents may be an effective therapeutic strategy.

to the splenic subsets. Furthermore, intratumoral NKT cells showed significantly reduced IL-2Rb (CD122), increased IL-6Ra and IL-21R while no change in IL-2Ra (CD25) and IFN-gR expression compared to splenic population. Injection of a-GalCer a ligand that activate NKT cells through CD1d molecules significantly reduces tumor growth, and the effect of a-GelCer were lost by removing this population using anti-NK1.1 mAb.These results suggest that inflamed tumor microenvironment recruits NKT cells, and suppress the anti-tumor activity of NKT cells. Understanding cellular and molecular mechanism of intratumoral NKT cell function will help design better anti-tumor therapeutic strategies.

P155-L014Development and characterization of murine model of graft versus host disease (GVHD) based on high dose myeloablative chemo-conditioning (HDMCC)

M. Mehta1,3, A. Majumdar1, R. Kaushal2, V. Gota3

1 Department of Pharmacology and Toxicology, Bombay College of Pharmacy, Mumbai, India2 Department of Pathology, Tata Memorial Hospital, Mumbai, India3 Department of Clinical Pharmacology, ACTREC, Tata Memorial Center, Mumbai, India

Background: GVHD accounts for 15–30% of deaths and is a major cause of morbidity in 50–60% of transplant recipients following alloBMT. Available GVHD models are based on radio-conditioning and/or immune deficient mice, despite 50% of all conditioning in clinical practice being chemotherapy based. We have developed and characterized a GVHD model which recapitulates the clinical scenario of HDMCC based alloBMT.Methods: Recipient Balb/c mice were treated with Busulfan (80 mg/kg/4 days) followed by Cyclophosphamide (100 mg/kg/2 days). On “Day 0” recipient mice were transplanted with 20*106 bone marrow and 30*106 spleen cells from MHC mismatched donor C57BL/6 mice. Non transplanted mice served as chemo-controls. At pre-defined time interval, mice were sacrificed and major internal organs were dissected out for pathological evaluation. Serum was subjected for cytokine analysis.Results: Histopathological investigation of transplanted mice’s liver indicated foci of lobular inflammation, peri-portal vein chronic inflammation, neutrophilic infiltration around the central vein. Gut was characterized by chronic active colitis with surface ulceration, mucodepletion, moderate to severe lamina propria inflammation with crypt abscess, presence of apoptotic cells and sloughing of gut lining. Hyperkeratosis with minimal periappendicular and perivascular chronic inflammation was reported in skin. Serum IFN-Gamma level was significantly higher (250 folds) in transplanted group at Day 13. Transplanted mice died within 11 days, having a median survival of 8 days compared to 14 days in chemo-control group.Conclusion: The MHC mismatched and HDMCC based GVHD model has demonstrated 1) clinically and histologically relevant characteristics of GVHD, 2) strong and acute immunogenic response (cytokine storm) that resulted from disparity between MHCs of recipient and donor strain, and 3) an ability to serve as an in-vivo system for preclinical investigations related to GVHD. We believe that it will be a good pre-clinical system for screening anti-inflammatory and Immuno-modulatory compounds for anti-GVHD effect.

P184-L015Abstract withdrawn

P222-L016Adoptive transfer of mother’s memory immune cells to patients with malignancies

C. Giraudo1, A. Giraudo1

1 Fundacion Progreso de la Medicina. Cordoba-Argentina

Background and Objectives: Pregnancy represents a state similar to organ transplantation, in which inherited paternal antigens (IPAs) are foreign to the mother’s immune system, a semiallogeneic event. The immune system of eutherian mammals, therefore, had to evolve mechanisms to both prevent an immune rejection, and through the placental passage of antibodies & cells, keep the embryo free of infections or malignancies. Tomasetti & Vogelstein, showed “strong correlation between the frequency of tumors and the number of stem-cell divisions”. With every division, there’s a risk of a cancer-causing

S66 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 M. Cancer Metabolism

hematopoietic stem cell transplantation (HSCT). Current efforts are being focused on identification of markers that serve as potential predictors of early engraftment. We had reported for the first time that increased CD26/dipeptidylpeptidase-IV expression on cells of human mobilized PBSC harvest from cancer patients/allogeneic related donors leads to early WBC engraftment in transplanted cancer patients (Prabhash et al, Ann. Oncol., 2010). This study has given important leads suggesting that CD26 expression may be an independent predictor of engraftment as there was no correlation between CD26 and CD34. Further detailed study of CD26 signaling network molecules on cells and plasma of PBSC grafts may add valuable information towards better understanding of strategies for enhancing engraftment post-HSCT. CD26/DPPIV is an immunoregulatory enzyme which is mainly expressed on stem cells, immune cells and epithelial cells. Its secretary form is also functional as extracellular domain of CD26 (sCD26) carries catalytic activity. The other immunoenzymes in CD26 network viz. Adenosine deaminase (ADA), CD39 and CD73 regulate the extracellular levels of adenosine. Adenosine is known to be a potent suppressor of immune response and no study has reported its levels in PBSC harvests. Thus it is proposed to analyze CD26 and adenosine signalling molecule levels in plasma and as surface molecules on various immune cell types (TH1/TH2/TH17 cells, macrophages, NK cells, HSPC, CD271 1 MSC) in PBSC donor harvest before and at various time points post-HSCT and correlate them with immune recovery. The potential predictors of early engraftment can help in improving treatment outcome, reduce hospital stay and prevent complications like GvHD, fungal infections, etc in patients treated with autologous/allogeneic HSCT.

M. Cancer Metabolism

OT176-001Preventing and treating hepatic metastatic colon and pancreatic cancers by targeting cell metabolism

S.V. Jin1, A. Al-sadi1

1 Pharmacology Department, Rutgers Univ.-Robert Wood Johnson Med. Sch., New Jersey, USA

One fundamental change in cancer cells is the alteration of glucose metabolism. With few exceptions, cancer cells exhibit aerobic glycolysis (the Warburg effect). A majority of pyruvate derived from glycolysis is converted to lactate instead of entering mitochondria for oxidative phosphorylation. The functional significance of the inefficient metabolic mode to oncogenesis is to prevent complete oxidation of glucose. Instead, glycolytic metabolic intermediates are shunt to pentose phosphate pathway required for biomass production. We propose to target this mode of cell metabolism for treating cancer. Pyruvate flux into mitochondria can be dramatically enhanced through mitochondrial uncoupling, a process that allows protons cross the mitochondrial inner membrane without producing ATP. As a result, mitochondrial uncoupler promotes “futile” oxidation of acetyl-CoA, leading to complete glucose oxidation, which effectively annihilating the Warburg effect. We tested this novel cancer chemotherapeutic strategy with niclosamide ethanolamine (NEN) and one of its derivative (OXY-1) in culture cell models and a mouse model. Mouse colon and pancreaticcancer cells (MC38 and Panc02) were treated with NEN or OXY-1. At effective concentrations, NEN and OXY-1 reduced cells viability, induced cell cycle arrest, and reduced clonegenicity. These effects were associated with activation of AMPK and reduction of NADPH/NADP ratio. When MC38 or Panc02 cells were injected into the liver of syngenic mice, oral treatment of OXY-1 significantly reduced tumor size and tumor incidence. When MC38 or Panc02 cells were intrasplenically injected into NOD mice, oral NEN or OXY-1 either totally prevented tumor metastasis to liver or drastically reduced metastatic tumor numbers and tumor volume. Taken together, our results suggest that the safe mitochondrial uncouplers NEN and OXY-1 could be effective in preventing and treating hepatic metastasis of cancers through targeting cancer cell metabolism. Our study may provide a novel anti-cancer strategy for a wide spectrum of tumors.

OF117-002The clockwork of cancer metabolism reveals novel anticancer strategies

B. Grimaldi1

1 Istituto Italiano di Tecnologia, Genoa, Italy

Background: We recently obtained the first evidence that the pharmacological targeting of a circadian regulator may be a suitable anticancer strategy: the beta-variant of circadian nuclear receptor REV-

P267-L019Targeting inflammatory pathways for prevention and treatment of cancer

B.B. Aggarwal1

1 Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA

Chronic infections, obesity, alcohol, tobacco, radiation, environmental pollutants, and high-calorie diet have been recognized as major risk factors for the most common types of cancer. All these risk factors are linked to cancer through inflammation. While acute inflammation that persists for short-term mediates host defense against infections, chronic inflammation that lasts for long-term can predispose the host to various chronic illnesses, including cancer. Linkage between cancer and inflammation is indicated by numerous lines of evidence; first, transcription factors NF-kB and STAT3, two major pathways for inflammation, are activated by most cancer risk factors; second, an inflammatory condition precedes most cancers; third, NF-kB and STAT3 are constitutively active in most cancers; fourth, hypoxia and acidic conditions found in solid tumors activate NF-kB; fifth, chemotherapeutic agents and gamma irradiation activate NF-kB and lead to chemoresistance and radioresistance; sixth, most gene products linked to inflammation, survival, proliferation, invasion, angiogenesis, and metastasis are regulated by NF-kB and STAT3; seventh, suppression of NF-kB and STAT3 inhibits the proliferation and invasion of tumors; and eighth, most chemopreventive agents mediate their effects through inhibition of NF-kB and STAT3 activation pathways. Thus suppression of these proinflammatory pathways may provide opportunities for both prevention and treatment of cancer. We will discuss the potential of nutraceuticals derived from spices and from traditional Indian medicine in suppression of inflammatory pathways and their role in prevention and therapy of cancer.

P272-L020Anti-leukemic activity of gd T cells in T-acute lymphoblastic leukemia patients

G. Mirji1, S. Banavali2, M. Sengar2, S. Chiplunkar1

1 Chiplunkar Lab, Advanced Centre for Treatment Research & Education in Cancer, Kharghar, Navi Mumbai, India2 Tata Memorial Hospital, Dr. E. Borges Road, Parel, Mumbai, India

Background: gd T cells are involved in the immune defence against various solid tumors, but their role in T-Acute Lymphoblastic Leukaemia is not clear. In present study, we aimed at characterising the anti-leukemic functions of gd T cells in T-ALL patients.Methods: Multiparametric flow cytometry was used for studying gd T cell subsets, activation markers and memory markers in T-ALL patients at diagnosis and remission. Expansion of gd T cells from peripheral blood mononuclear cells (PBMCs) was monitored by co-culture with leukemic blasts. Cytotoxic potential of gd T lymphocytes against leukemic blasts was analysed using 51Cr release assay. Immune synapse formation between gd T cells and leukemic targets was studied using confocal microscopy.Results: Co-culture of leukemic blasts with PBMCs led to expansion of Vd2 T cells. LFA-1, Dynamin-2, CD166 and phosphotyrosine were involved in immune synapse formation between leukemic blasts and gd T cells. Increased F-actin polarization was observed at the immune synapse after zoledronate treatment. gd T cells exhibited potent cytotoxicity against zoledronate treated leukemic blasts. Patients at remission showed increased effector memory gd T cells, increased Vd2 and Vd1 T cells and higher expression of activation markers (CD25, CD69) and perforin.Conclusion: gd T cells exhibit robust anti-leukemic activity by killing the leukemic blasts through efficient immune synapse formation. Adoptive transfer of gd T cells may be a promising immunotherapeutic approach for treatment of T-ALL patients.

P190-L021CD26 and adenosine signaling pathway molecules as predictors of early engraftment in human haematopoietic stem cell transplantation

J. Kode1, N. Khattry2

1 Chiplunkar Lab, Mumbai, India2 Bone Marrow Transplantation Unit, Department of Medical Oncology, ACTREC, Tata Memorial Centre, Mumbai, India

The success of hematopoietic recovery post-transplantation depends upon the content of donor peripheral blood stem cell (PBSC) harvest and efficiency of hematopoietic stem/progenitor cells (HSPC). Stable white blood cell (WBC) and platelet engraftment is crucial for outcome of

M. Cancer Metabolism European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S67

Background: A better understanding of the biological pathways that determine the ability of cancer cells to adapt and survive to metabolic stress may lead to the identification of targetable elements for novel cancer therapeutic strategies. In this scenario, the sigma-1 receptor (Sig1R) could play a new and pivotal role as it is often up-regulated in cancer cell lines although detailed functional studies are missing. Aim of this study was to evaluate the expression profile of Sig1R and to address its potential as therapeutic target in human neoplasia.Material and methods: We examined whether Sig1R sustains proliferation, survival and tumorigenic properties of human cancer cells through: screening of tumors and tumor cell lines for expression of Sig1R; assessment of the effects of Sigma-1R gene silencing and antagonists on the proliferation, clonogenic and sphere-forming capacity of cancer cell lines; evaluating the effects of the SigR1 silencing and antagonists on the mitochondrial health.Results: Knockdown of Sig1R, using siRNA in human prostate and lung cancer cells, had profound effect on proliferation, clonogenic capability and tumor sphere formation, indicating reversal of tumorigenicity and stem-like phenotype in absence of Sig1R. Next, we identified various Sig1R ligands that behaved as antagonists of the receptor functions and at low micromolar concentration, these ligands almost completely suppressed clonogenic and tumor sphere forming capability of cancer cells. Summarily, both Sig1R knockdown and pharmacological antagonists led to impaired mitochondrial function, which was more evident under condition of metabolic stress (glucose starvation).Conclusions: Our data indicates the Sig1R has a pilot role in regulation proliferation, clonogenicity and sphere forming ability of various cancer cell lines. The role played by Sig1R appears to be dependent by the metabolic conditions. Importantly, two novel Sigma1R ligands can mimic the biological effect of specific siRNA. These data strongly support Sig1R as novel target for cancer therapy.

P15-M001PH domain regulates the stability and oncogenic activity of AKT

M.K. Santra1, R. Manne1, G. Sankaran1

1 National Centre for Cell Science, Pune, Maharashtra, India

Background: Activation of AKT pathway is highly associated with malignancy. The present strategy to inactivate this pathway is relying on by targeting either PIP3 or kinase domain of AKT using small molecule inhibitors. Inhibition of PIP3 and AKT activity through small molecule inhibitors induces significant cytotoxicity. Further, inactivation of PIP3 may lead to affect many cellular processes. Thus, PIP3 inhibitor may not be superior for therapeutic purpose. In addition, these inhibitors have potent side effects. Alternatively developing small molecule against PH domain of AKT will be better choice since it is crucial for its membrane localization and there by activation. Therefore, we aim to develop small molecule against the PH domain of AKT with minimal cytotoxicity.Method: Through docking study we designed the small molecule against the PH domain of AKT. We synthesize the molecules and tested their inhibition of AKT activity through immune blotting of mammalian cell lysates.Results: Our results demonstrated that synthesized molecules have minimal cytotoxic effect on multiple mammalian cell lines. For example, 50% inhibitory concentration (IC50) is found to .100 μM for all the synthesized compounds. Interestingly, we found that most of these compounds promote the proteasomal degradation of phosphorylated (active) as well as nonphosphorylated (Inactive) form of AKT. Interestingly, though the IC50 of compound MS10 is .100 μM, however; it promotes proteasomal degradation of AKT even at 1 μM. We have identified the proteasomal regulators of AKT and are in the process of elucidating molecular mechanism of proteasomal degradation of AKT in the presence of these small molecules.Conclusion: Commercially available small molecule inhibitors against AKT induce significant apoptosis mediated cell death and thereby have potential side effect. Our findings reveal that AKT pathway can be efficiently inactivated through proteasomal pathway with minimum side effect and me be beneficial for the chemotherapeutic purpose.

P27-M002Involvement of a specific phospholipase A2 isoform in cigarette smoke condensate induced changes in HCT-15 and HT-29 colon cells

S.K. Shrama1, S.K. Yadava1, G. Kaushik2, C.M. Pathaka1, S. Rana1, K.L. Khanduja1

1 Postgraduate Institute of Medical Education & Research, Chandigarh, India2 School of Medicine, KU Medical Center, Kansas City, USA

ERB, REV-ERBb, functions as an unpredicted major regulator of clock gene expression in different human tumor tissues cells, where it plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified a novel class of compounds with a dual inhibitory activity against both REV-ERBb and autophagy, which decreased the viability of different tumor tissue cells at concentrations from 5 to 50 times lower than the singular clinically relevant autophagy inhibitor, chloroquine. The crucial position of REV-ERB proteins in the regulation of cellular metabolism suggests the provocative hypothesis in which the inhibition of both REV-ERBb and autophagy cooperate to induce a metabolic dysfunction that is incompatible with cancer cell viability.Materials & Methods: Nuclear Magnetic Resonance (NMR) and Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS/MS)-based metabolic studies have been performed in cancer cells in which REV-ERBb and autophagy activity has been pharmacologically or genetically inhibited. In addition, RNA deep-sequence analysis has been conducted to identify REV-ERBb-specific metabolic genes.Results and conclusions: Our analyses indicate that REV-ERBb controls both glucose and lipid metabolism, two processes that play a prominent role in supporting cancer cell viability and that have been shown to be altered upon autophagy inhibition. Overall, our data suggest that REV-ERBb may be an unpredicted crucial regulator of cancer metabolism and support a scenario in which REV-ERBb inhibition may generate a metabolic stress that sensitizes tumor cells to a lethal “second hit” when the autophagy flux is compromised. Implicit in this hypothesis is the concept that the altered circadian regulation found in several tumors may be triggered by the special metabolic needs of the cancer cell.

OF182-009Therapeutic targeting of tumour cell specific metabolic reprograming through the dynamic stabilization of native and misfolded state of nuclear receptor co-repressor (N-CoR)

M. Khan1

1 Department of Pathology, School of Medicine, AIMST University, Kedah, Malaysia

Nuclear receptor co-repressor (N-CoR) is a generic transcriptional co-repressor essential for the suppression of cellular growth and self-renewal potentials during differentiation and cell maturation. We have shown that tumour cell specific misfolding and loss of N-CoR protein promotes malignant growth and transformation through the ectopic reactivation of cellular growth potentials in relatively matured cells. Here, we report that ATP generated from the catabolic recycling of misfolded N-CoR protein contributes to the survival and growth of tumour cells in some specific subtypes of leukaemia and solid tumours. Tumour cells harbouring the misfolded N-CoR displayed distinct metabolic profile as demonstrated by the selective activation of mammalian target of rapamycin (mTOR), the master regulator of cellular nutrients and energy balance. Moreover, ATP derived from the recycling of misfolded N-CoR was linked to the survival and growth of tumour cells in nutrient depleted tumour microenvironment. Abrogation of N-CoR degradation by Kaletra, a clinical grade protease inhibitor; or by Rapamycin, a clinical grade inhibitor of protein misfolding and mTOR signalling, promoted selective apoptosis or differentiation of tumour cells harbouring the misfolded N-CoR protein. While Kaletra promoted UPR (unfolded protein response)-induced apoptosis of tumour cells through the stabilization of misfolded state of N-CoR protein, Rapamycin, on the other hand, stabilized the native N-CoR state by blocking its aberrant phosphorylation. Treatment of tumour cells with Rapamycin or Kaletra, as well as siRNA-induced ablation of N-CoR transcript, resulted in significant down regulation of ATP level and abrogation of mTOR signalling. These finding define the destabilization of native and misfolded N-CoR state as a generic mechanism of malignant transformation in phenotypically diverse tumour cells and provides novel mechanistic insights into the design and development of protein conformation based therapeutic approach for a subgroup of cancer caused by misfolded N-CoR.

OF128-012Could be sigma-1 receptor a leading actor in the cancer pathway? A multidisciplinary approach to define its role in blocking metabolic stress response in cancer cells

E. Laurini1, G. Civenni2, C. Monte2, F. Sereni2, G. Carbone2, B. Wünsch3, M. Fermeglia1, C. Catapano2, S. Pricl1

1 MOSE-DEA, University of Trieste, Italy2 Institute of Oncology Research (IOR), Bellinzona, Switzerland3 Department of Pharmaceutical and Medicinal Chemistry, University of Münster, Germany

S68 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 M. Cancer Metabolism

cigarette smoke on the viability of cervical cancer cells and proteomically explored its effect on the expression of various proteins in cervical cancer cells. Exposure of cervical cancer HeLa cells to soluble reference-research cigarette smoke extract caused an increase in expression of a number of proteins, as identified by proteomic analysis. Alpha-Enolase, a glycolytic enzyme, known to be involved in tumorigenesis and metastasis, was significantly higher in cells exposed to soluble cigarette smoke extract when compared to untreated control cells. Alpha-enolase is a hypoxia-responsive gene, hence we explored the HIF pathway. We found a concomitant increase in Hif-1 Alpha expression in HeLa cells exposed to cigarette smoke extract through suppression of prolyl-4-hydroxylase activity. We find that cigarette smoke is inhibiting prolyl-4-hydroxylase activity to stabilize and enrich Hif-1a which in turn, is activating Alpha-enolase. Pathway enrichment analysis of the interacting proteins of enolase resulted in glycolytic pathways, supporting enolase function in a cell. Induction of ENO1 genes might provide a survival advantage to cells under hypoxia and acidic environment generated by cigarette smoke. Simultaneously, we analyzed E-cigarettes (nicotine) effect on cervical cancer cells. E-cigarettes are being used as an alternative to traditional cigarettes to facilitate smoking cessation. Our study also reveals that E-cigarettes induce alpha-enolase levels in HeLa cells to the same extent as traditional cigarettes, thereby facilitating tumorigenesis. Further studies on the role of Alpha-enolase in smoking-induced progression of cervical tumorigenesis needs to be evaluated.

P37-M005Deleted in breast cancer 1 (DBC1): A novel mitotic regulator coordinating metabolism with mitosis

Z. Sarwar1, I. Reshi1, S. Bhat1, Q. Geelani1, M. Shah1, S. Andrabi1

1 Department of Biochemistry, University of Kashmir, Srinagar, India

Background: DBC1 is an important regulator of cellular stress response. Current reports about the role of DBC1 in tumorigenesis are paradoxical and designating DBC1 as a tumor suppressor or a tumor promoter is still controversial. Therefore, the role of DBC1 in cells needs to be thoroughly investigated.Methods: Effects of DBC1 overexpression on cell growth, cell cycle and tumorigenesis were studied by crystal violet staining, FACS and soft-agar assays respectively. Interaction between Polyoma Small T antigen (PyST) and DBC1 was confirmed by immunoprecipitation. Knockdown and overexpression of genes were confirmed by RT-PCR and western blotting.Results: To explore the possible role of DBC1 in cell cycle regulation and tumorigenesis, we used PyST as a tool. Using tandem affinity purification followed by Mass Spectrometry (LC-MS-MS), DBC1 was found to be one of the many interacting partners of PyST. Later, immunoprecipitation results confirmed that PyST does indeed interact with DBC1. Here, we report that PyST significantly downregulates DBC1 protein levels. Further, when co-expressed with PyST, DBC1 markedly decreases the ability of PyST to cause a G2/M arrest in the host cells. Our studies also revealed that LKB1 may be an important upstream regulator of DBC1. LKB1 over-expression leads to the downregulation DBC1 protein levels, as was seen by western blotting as well as RT-PCR.Conclusion: Our studies suggest that DBC1 might have an important role in the spindle activation checkpoint (SAC). We propose that DBC1 may be a novel target of LKB1, and this pathway may have a significant role in mitotic regulation. As LKB1 is a major energy sensor of cell, a link between DBC1 and LKB1 may couple cell division with energy metabolism. Since reprogramming of energy metabolism is an emerging dimension of carcinogenesis, therefore, studies on the functions of DBC1 may provide some new insights into cancer cell metabolism.

P60-M006Implications of cell-to-cell dynamic baseline variation on drug sensitivity measurements

J.M. Greene1, D. Levy2, S.P. Herrada3, M.M. Gottesman3, O. Lavi3

1 Department of Mathematics, Rutgers University, New Brunswick, New Jersey, USA2 Department of Mathematics and Center for Scientific Computation and Mathematical Modeling, University of Maryland, College Park, MD, USA3 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

One of the challenges in cancer research is to identify and characterize important cell-to-cell variations based on their contribution in tumor dynamics. To address the multifaceted heterogeneity within a population of tumor cells, several experimental techniques have been developed, including genome sequencing, flow cytometry, and cell

Background: Four different phospholipase A2 (PLA2) isoforms namely secretory PLA2 (sPLA2), cytosolic PLA2s (cPLA2), Ca21 independent PLA2 (iPLA2) and platelet activating factor- acetyl hydrolase (PAF-AH) activated PLA2, which are involved in remodeling of membrane phospholipids exist in the mammalian cells.Materials and Methods: Effect of cigarette smoke condensate on cell viability by MTT assay, reactive oxygen species (ROS) by DFCHDA dye, mitochondrial reactive oxygen species (mtROS) by DHR123 dye, superoxideradicals (SOR) by DHE dye, mRNA expression and after knockdown the specific PLA2 groups with siRNA by RT-PCR, was studied in these cell lines were evaluated in HCT-15 and HT-29 cells.Results and Discussion: The cell survival decreased significantly after exposure with 50 μg CSC/ml in these cells. Total ROS and mtROS and SOR production were significantly increased in both cells after exposure with CSC. However, induction of ROS and SOR was more in HT-29 cells as compared to HCT-15 cells. Whereas, mtROS induction was found to be more in HCT-15 compare in HT-29 cells. The gene expression of PLA2 groups such as IB, IID, III, IVA, IVB, IVC, VI, X, AiPLA2 and iPLA2 were expressed in HCT-15 cells. However in HT-29 cells, all these PLA2 groups except IVC were observed. After exposure with CSC, sPLA2 IID group was up regulated significantly in HCT-15 cells only. HCT-15 cells transfected with IIDsiRNA did not respond to CSC in inducing ROS, mt ROS and SOR, and affecting viability.Conclusion: Therefore, it seems that specific PLA2 siRNA in the cells are involved in controlling the cascades related to reactive oxygen species.

P29-M003Impact of anticancer treatment on nutritional and functional status in cancer patients: A case control study

S. Gogna1

1 Pandit Bhagwat Dayal Sharma Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India

Background and Aims: Etiology of cachexia is multifactorial and is related to local effects of tumor and anticancer therapies, reduced food intake due to systemic and psychological effects of the disease. The indicators of nutritional status are anthropometric variables such as body weight, BMI, hand grip dynamometry, mid arm circumference and biochemical variables such as hemoglobin, serum albumin. These patients have poor nutritional and performance status. We aimed to establish improvement in both nutritional and performance status of patients after definitive anticancer treatment.Methods: We did a case control study with the 100 disease and site specific cancer patients treated by surgery followed by adjuvant chemo/radiotherapy. Control group was formed by age and sex matched healthy volunteers. The nutritional and performance status were assessed before start of anticancer therapy and at 6 & 12 weeks after completion of the therapy in study group. Variables assessed pre and post treatment were hemoglobin levels, serum albumin, mid arm circumference, body weight, Body mass index (BMI) and hand grip dynamometry. Functional status was assessed by using Karnofsky performance scale (KPS). We also assessed the interrelationship among nutritional status and functional status.Results: There was statistical significant increase in Body weight (p , 0.01), BMI (p , 0.01), hand grip dynamometry (p , 0.01), improvement in midarm circumference and triceps thickness was present but not statistically significant. Hb, serum Albumin. KPS also improved statistically. On studying the interrelationship between nutritional and performance status we found early and linearly increasing improvement in KPS as compared to nutritional status.Conclusion: We concluded that anticancer treatment does improves nutritional and performance status of cancer patient thus there is improvement in cachexia, fatigue and improvement in quality of life. Performance status improves earlier and helps to better the nutritional status also.

P36-M004E-cigarettes upregulate alpha-enolase in cervical cancer cells, similar to conventional cigarettes, potentially promoting tumorigenesis

W. Rizwani1, D. Jyothi1, D. Perumal1, R. Eslavath1, S.S. Singh1

1 Department of Biochemistry, Osmania University, Hyderabad, India

Cervical cancer is the third most common cancer in women worldwide. Cigarette smoking is considered to be a common risk factor for developing cancer of the cervix. A recent meta-analysis showed the risk of squamous cell cervical cancer doubling in women who currently smoke. However, because of the complex composition of cigarette smoke, the detailed mechanism is not fully understood. We analyzed the effects of soluble

M. Cancer Metabolism European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S69

development of ADR-induced cardiotoxic mouse model and effect of NAD+/NADH modulation by NQO1 action.Materials and Methods: ADR was intraperitoneally injected on C57BL/6 male mice and WK0202 was orally administrated with 20 mg/kg body weight of mice. Cardiac biomarkers (CPK, Trop I, LDH and SGOT) in plasma levels, oxidative biomarkers and mRNA levels of pro-inflammatory cytokines were assayed.Results: WK0202 administration was significantly alleviated ADR-treated mice, including cardiomyocyte lesions. In addition, ADR induced significant elevations of cardiac biomarkers, indicating its acute cardiotoxicity which is diminished by WK0202 administration. Notably, the intracellular NAD+/NADH ratio, whichever targets of NQO1 in myocardium, was signi-ficantly increased in ADR and WK0202 co-treated group compared to ADR alone. Pro-inflammatory cytokines and oxidative biomarkers were significantly attenuated by WK0202 co-treatment compared with those in the ADR alone. Furthermore, we verified that the downfall in SIRT1 and SIRT3 activities aggravates heart failure with concurrent elevated ROS generation by increasing PARP-1 activation and acetylation of NF-kB p65 and p53, suggesting cardiotoxicity may occur through the induction of inflammatory responses and oxidative stress as well as the apoptotic cell death, which, however, was abrogated by WK0202.Conclusions: Our results suggest the evidence that WK0202 has a protective effect against ADR-induced acute cardiotoxicity by direct modulation of the intracellular NAD+ levels through NQO1 action and it may support the concept that WK0202 can be beneficial for cancer patients with ADR treatment through alleviating the various organs toxicity.

P153-M010Chemoprevention of lung cancer by polymeric black tea polyphenols (PBPs): Preclinical evaluation of potential molecular targets

R. Hudlikar1, V. Venkadakrishnan1, R. Kaushal1, R. Thorat1, S. Kannan1, A. Ingle1, S. Desai1, G. Maru1, M. Mahimkar1


Background: Lung cancer is the leading causes of deaths in smokers. Although smoking cessation is important for lung cancer prevention, ex-smokers still exhibit higher risk. Chemoprevention with phytochemicals is evolving approach for management of lung cancer. In our study we evaluated chemopreventive efficacy and possible mechanism of most abundant polyphenolic fraction in black tea, polymeric black tea polyphenols (PBPs), in experimental lung carcinogenesis model.Methods: Effect of 1.5% black tea derived PBPs on benzo(a) pyrene [B(a)P] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced lung lesions were studied over 28 weeks. Chemopreventive efficacy was studied using decrease in tumor incidence and/or multiplicity and/or delay in the latency period in A/J mice. Microscopic lung lesions were evaluated using histopathology in post carcinogen treatment weeks. Inflammation, cell proliferation and apoptosis markers along with signaling kinases like p38, Akt and their phosphorylated forms were studied using immunoblotting and immunohistochemical staining at 4th, 10th and 18th week post-carcinogen treatment.Results: Administration of PBPs throughout the treatment period significantly decreased the multiplicity of surface tumors as well as microscopic lung lesions, including adenomas. Although, tumor incidence and latency period remains unaffected, histopathological evaluation of lung at 6, 10 and 18 weeks post-carcinogen treatment showed decrease in tumor multiplicity which was also correlated with different molecular markers. Anti-inflammatory action of PBPs was demonstrated by reduced Cox-2 expression. PBPs down-regulated the cell proliferation (diminished PCNA and Bcl-2 expression) and enhanced apoptosis (increased Bax expression) potentially through phosphorylation of p38 and Akt.Conclusions: PBPs, most abundant polyphenolic component in the black tea, have chemopreventive effect through inhibition of carcinogen induced inflammation, cellular proliferation and induction of apoptosis possibly via modulation of signaling kinases. Emphasis on changes in life-style (mainly smoking and diet), along with evaluation of various doses of such compounds in multiple organs with circulating levels will further help in monitoring the chemopreventive clinical trials.

P164-M011Preliminary study of anti-cancer activity of semi-synthetic artemisinin derivatives in different cancer cell lines

F. Ahmad1, S. Karna1, S. Gupta1, P. Arora1, N. Shazad1, Y. Pokharel1

1 Faculty of Life Science and Biotechnology, South Asian University, New-Delhi, India

microscopy. Estimates of cell-to-cell variation resulting from those experiments typically ignore the expected spatiotemporal variations of the entire process of cell growth. To fully capture the extent of such dynamic variations, we developed a mechanistic mathematical model supported with in vitro experiments using the ovarian cancer OVCAR-8 cell line model. We introduce the notion of dynamic baseline variation, and show how the emerging spatiotemporal heterogeneity of one cell population can be attributed to differences in local cell density and cell cycle. Given a fixed global initial cell density, we demonstrate significant differences in growth, proliferation, and apoptosis rates that are based solely on cell movement and local conditions. We conclude that any statistical estimate of changes in the level of heterogeneity must be integrated with the baseline system’s dynamics and spatial effects. This approach should be considered as an underlying reference model for all cell biology studies that incorporate dynamic processes.

P91-M007AMP kinase: Evolving lessons about its context-dependent opposite roles in cancer

R.R. Chhipa1, J. Anderson1, Q. Fan1, L.M.L. Chow1, I. Nakano2, C. McPherson3, B. Dasgupta1

1 Division of Oncology, Cincinnati Childrens Hospital Medical Center, Cincinnati, OH, USA2 Department of Neurological Surgery, James Comprehensive Cancer Center, The Ohio State University, OH, USA3 Department of Neurosurgery, Brain Tumor Center, University of Cincinnati Neuroscience Institute and Mayfield Clinic, Cincinnati, OH, USA

A common theme entailing tumor evolution is that tumors undergo metabolic state transitions in response to flux of nutrients, O2, and therapy. Dynamic changes in cancer metabolism are also a direct cause of the evolving cancer-genome that undergoes oncogene amplifications, insertions and deletion of tumor suppressors. Many genomic regions undergo unintended suppression or deletion and regardless of the mutational landscape, these dynamic changes create vulnerability and impose a great burden on cancer cells. To thrive, evolving cancer cells acquire a remarkably altered metabolic and signalling flexibility. Identifying genes that protect tumor cells against cell-intrinsic vulnerabilities and the hostile tumor environment may provide novel therapy in human cancer. The multifunctional kinase AMPK is an evolutionarily conserved energy/stress sensor that plays important yet undefined role during tumor evolution. AMPK activated by the tumor suppressor LKB1 or the calcium-regulated kinase CAMKK2. AMPK inhibits mTOR and lipogenesis and therefore not surprisingly, many laboratories found that agents (e.g., metformin, now in several clinical trials) that activate AMPK suppress tumor growth. However, recent studies questioned this over-simplistic model and reported that the LKB1-AMPK may in fact be required for optimal growth and drug-resistance of certain tumors. Using glioblastoma multiforme (GBM) as a model, we found that this pathway is constitutively active in human GBM and in clinically relevant genetically engineered glioma mouse model. Surprisingly, we observed that AMPK and mTOR are co-active in this cancer. AMPK silencing suppressed tumor growth and was synergistic with mTOR inhibition. Biochemical, genetic and metabolic experiments uncovered that AMPK regulates a transcription program in GBM that allows optimal energy metabolism and survival. Unfortunately, no specific AMPK inhibitors are available. An important goal of our laboratory is to test if repurposing stress-inducing agents (including FDA-approved drugs) combined with inhibition of stress-response pathways leads to cancer meltdown.

P101-M008Enzymatic action of NQO1 attenuates Adriamycin induced cardiotoxicity in mice

S. Yang2, D. Khadka1, G.S. Oh1, H.J. Kim1, S.B. Lee1, A. Pandit1, A. Shen1, S. Lee1, H. So1

1 Center for Metabolic Function Regulation and Department of Microbiology, Korea2 Department of Internal Medicine School of Medicine, Wonkwang University, Iksan, Republic of Korea

Background: Adriamycin (ADR), a potent anticancer chemotherapeutic agent, used to treat divergent human neoplasms. Its clinical use is limited by various side effects. Various studies have demonstrated that ADR-induced cardiotoxicity is narrated to myocardial oxidative stress, disruption of cellular and mitochondrial Ca2+ homeostasis and DNA damage. Nevertheless, the clear-cut mechanism underlying ADR cardiotoxicity is still not well-defined. Here we describe that

S70 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 N. Cancer Metastasis

Result: The study included 137 patients; 85 were cN0 and 52 patients were cN1. Level Ib was involved in 8.2% of the cases with N0 and in 30.8% with N1 necks. Metastasis in N0 necks in the area deep to the submandibular gland was seen in only 1 case (9%). This was the only case where submandibular gland mobilization would have been required to take out the metastatic node. Even in cases with N1 neck deep metastasis was seen in 4 cases only (14.8%). None of them had primary in tongue.Conclusion: Involvement of level Ib in early tongue cancers is not common and direct metastases to submandibular gland are rare. Even when metastasis is present in level Ib it can be excised without affecting the submandibular gland. In tongue lesions, sub-mandibular gland mobilization for dissection at level Ib is not required as no metastasis deep to the sub-mandibular gland were seen.

OT33-006The role of Asap1 in physiological processes and tumor metastasis

S. Saraswati1, C. Schreiber1, W. Thiele2, M. Rothley2, N. Cremers1, C. Korn3, H. Augustin3, J.P. Sleeman1,2

1 Centre for Biomedicine and Medical Technology Mannheim, Universitätsmedizin Mannheim, University of Heidelberg, Germany2 Institute for Toxicology and Genetics, KIT Campus North, Karlsruhe, Germany3 German Cancer Research Center (DKFZ) Heidelberg, Germany

This study highlights the importance of information exchange between clinical and mouse based research as very often the results from one study model do not translate into another. Asap1 (Arf- GAP with SH3-domain, Ankyrin-repeats and PH-domain 1) was identified by our lab in an unbiased screen to identify genes whose expression is associated with the metastatic phenotype. Targeted deletion of the Asap1 gene has been used to generate Asap1 knockout (Asap1GT/GT) mice in our lab. We have been able to define the phenotype of the Asap1GT/GT mice - loss of Asap1 leads to bradypnea and significant growth retardation in neonatal Asap1GT/GT pups. Retinal angiogenesis and lymphangiogenesis were studied and we found that these processes do not involve Asap1 mediated cell motility. To understand the role of Asap1 in tumor development and metastasis, Asap11/GT mice were crossed with MMTV-PyMT mice to have mouse models of spontaneous mammary tumors. Loss of Asap1 did not significantly alter tumor onset and there was no difference in overall tumor growth in the MMTV-PyMT Asap11/1 and MMTV-PyMT Asap1GT/GT mice. Lung surface metastasis count revealed that the metastasis was significantly increased in the absence of Asap1.The contradicting results from mice models and patients highlight the importance of studying tumor progression in the context of tumor microenvironment in a more physiologically relevant setting in comparison to only in vitro studies. We have established tumor-derived cell lines from MMTV-PyMTAsap11/1 and MMTV-PyMT Asap1GT/GT tumors and are currently studying molecular interactions of Asap1and changes in the cell cytoskeleton in the absence of Asap1. We conclude that Asap1 is an important regulator of tumor metastasis and it is therefore crucial to further study its role at cellular level so as to obtain clinically meaningful results.

P47-N001Progesterone up-regulates and activates a tumor metastasis suppressor gene NDRG1 in human breast cancer cells

M. Godbole1, R. Badwe2, N. Gardi1, K. Patel1, K. Tiwary1, R. Chaubal1, K. Karve1, V. Parmar2, N. Nair2, S. Gupta2, A. Dutt1

1 Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Navi Mumbai, India2 Tata Memorial Hospital, Parel, Mumbai, India

Background: Breast cancer patients undergoing surgery in the luteal (progestogenic) phase of menstrual cycle are known to show better survival. According to a clinical trial conducted with pre-operative progesterone intervention better survival in operable breast cancer patients was observed as opposed to patients receiving only surgery. The effect was pronounced in women with lymph node metastasis but independent of their PR/ER/Her2 receptor expression. However the underlying mechanism is not known.Methods: The aim of this study is to understand the effect of progesterone on human breast cancer cells using primary tumor-derived cell lines. Expression array analysis of a panel of breast cancer cells treated with progesterone was performed to identify genomic targets of progesterone. Ectopic expression and knockdown of SGK1 was performed in breast cancer cell lines. Further we studied breast cancer cell line cellular migration in vitro using time lapse wound-scratch assay.

Artemisinin and its derivatives which are already approved anti-malarial drugs have been reported in recent past years having anti-cancer activity. The property of being anti-malarial and anti-cancer for these drugs has been shown to be associated with its endoperoxide moiety which when activated in the presence of ferrous ions lead to the production of reactive oxygen species which can further lead to DNA damage, oxidative stress and ultimately apoptosis, but the exact mechanism following its activation and causing cell death in the parasite as well as in cancer cells is still obscure. Cancer cells express high number of transferrin receptors which make them good target for artemisinin and its derivatives. Our primary screening of some semi-synthetic derivatives of artemisinin showed that at lower doses (up to nano-molar concentration) significantly suppressed the proliferation of most of cancer cells and some of the over-expressed transcription factor such as c-jun, Erk, NF-kB have been found significantly down regulated which are required for their inhibition of cell proliferation. Annexin V/PI staining by FACS and caspase activity by western blot showed apoptosis mediated cell death by these derivatives. Further study on these derivatives may prove them to be a potent drug for future cancer therapy.

P219-M012Investigation of protein dynamics using genetically encoded unnatural amino acids

A.K. Srivastava1,2,3, K. Halder1, J. Kok2, H. Neumann1*, S. Dutt3

1 Applied Synthetic biology, University of Gottingen, Germany2 Molecular Genetics, University of Groningen, Netherlands3 Advanced Center for Treatment, Education and Research in Cancer, Tata Memorial Center, Mumbai, India

Background: Protein engineering has become an extensively used tool in many fields which allow us to study protein functions and characterize proteins using range of available biophysical methods. With the genetic code expansion, it is now possible to incorporate functional groups and other properties in the proteins which are naturally not present. Site specific incorporation of unnatural amino acid (uAA) is widely used in protein engineering. However, incorporation of multiple uAAs remains a significant challenge. Tunability of protein expression for multiple uAAs incorporation using orthogonal ribosome remains another important challenge.Methods: In this study we try to investigate the role of inducible promoter in tunability ofprotein expression using orthogonal ribosome and orthogonal ribosome binding sites along with tRNA/amino acyl RNA synthetase pair.Results: Six different o-RBS were designed based on the work of Rackham and Chin (Rackham.O & Chin, J.W. 2005 Nat. Chem. Biol. 1, 159–166) during the study on an inducible promoter. 4 best fit o-RBS were selected after screening experiment. Expression of protein incorporatinguAA was performed with orthogonal ribosome. Unfortunately, the experiment failed. Furtheranalysis on failure concluded that the functionality of orthogonal ribosome was lost due to someunexplained reason. One of the possible hypotheses for loss of functionality is due to theconversion of orthogonal ribosome to natural ribosome.Conclusion: Six different o-RBS was successfully designed and the best fit was screened. Genetic code expansion system failed due to the loss of functionality/orthogonality of orthogonalribosome.

N. Cancer Metastasis

OF141-006A prospective study to evaluate the pattern of lymphatic metastasis in relation to submandibular gland in the patients with carcinoma of the oral cavity

A. Malik1, S. Nair1, D. Nair1, P. Chaturvedi1

1 Department of Head & Neck Oncology, Tata Memorial Centre, Mumbai, India

Background: Excision of sub-mandibular gland causes decrease in basal salivary flow resulting in subjective xerostomia and decreased QOL. In this study, we have tried to assess the pattern of nodal metastasis in relation to the submandibular gland to assess whether submandibular gland preservation is a viable option in patients with oral cancer.Methods: This was a prospective study conducted in a tertiary care cancer centre. The fibrofatty tissue surrounding the submandibular gland was divided into six parts depending upon its location with the submandibular gland. These were separately sent for histopathological analysis. Metastasis pattern in level Ib region was noted.

N. Cancer Metastasis European Journal of Cancer 54, suppl. 1 (2016) S1–S72 S71

experiments” for the search of new therapies of cancer and offer an opportunity for physics community to make fundamental contribution to biology and medicine.

P147-N004Breast cancer stem cells: Is it the only cell population responsible for recurrence and metastasis, and predictive and preventive markers and treatment?

J. Jeyapaul1

1 Jeyapaul Labs, Chennai, Tamil Nadu, India

This paper explores five important questions1. What factors can predict early and late recurrences / distant

metastasis?2. Is the recurrent or metastasis causing cell within the cancer stem cell

population or in cells that have progressed in disease state?3. What treatment can be advocated earlier on itself, across the board,

at the time of diagnosis of primary tumour, to prevent recurrences and distant metastasis?

4. What treatment options are open to the patients once metastasis has set in?

5. Do cancer stem cells in primary tumor, at time of migration or metas-tasis n during growth of metastatic site different in gene expression and drug sensitivities?

We also present data that demonstrates that terminal differentiation may be a very strong signal in the cell, hitherto not emphasized, which overrides either or both oncogenic and stem cell signals in the cell, The highlight of the study are:• 48K Keratin was found to be a marker of initiated cells, expressed

in initiated skin exposed to a single application of tumor promoter (TPA), and in papillomas and carcinomas but absent in adjoining skin. multiple applications of TPA.

• A single application of TPA to initiated mouse skin results in expres-sion of oncogenic phenotype in all cells stem cells and differentiated cells facilitating their visualization by SDS PAGE Further applications of TPA leads to terminal differentiation and loss of oncogenic pheno-type in all cells except stem cells which are few and difficult to detect by SDS PAGE.

• However in the clonally expanded initiated cells, it is maintained till seen again in papillomas and carcinomas.

P277-N006Tumour initiating cells and correlation with treatment response in locally advanced cervical cancer

S. Chopra1, K. Deodhar1, J.S. Goda1, V. Pai1, N. Rathod1, N. Sudhalkar1, S. Waghmare1, P. Ray1, R. Engineer1, U. Mahantshetty1, A. Maheshwari1, S. Gupta1, S.K. Shrivastava1

1 Tata Memorial Centre, Mumbai, India

Background: While tumour-initiating cells (TIC) have been reported across solid tumours, however there is dearth of data regarding TICs and its impact on radiation response in cervical cancer.Methods: From October, 2013-July, 2015 patients with locally advanced cervical cancer were included. Pretreatment biopsy was obtained and immunohistochemistry (IHC) was performed for Embryonic Stem cells (ESC) and TIC. IHC was performed for SOX-2, OCT-4, Nanog (ESC), CD44 and Podoplanin (TIC). Semiquantitative scoring was used for IHC. Protein expression was considered positive if there was nuclear staining for ESC and membranous or cytoplasmic staining for TICs. All patients received uniform concurrent chemoradiation and brachytherapy. On follow up, local control and distant relapse was recorded and confirmed with biopsy. Correlation was evaluated between ESC and TIC expression and local control and distant relapse. p , 0.05 was considered statistically significant.Results: Overall 102 patients were accrued in stage I in which 87 patients received treatment at TMC. IHC scoring was available in 73/87 patients and clinical outcome in 84/87 patients respectively. Moderate to intense expression of Sox-2, Oct-4 and Nanog was recorded in 20.4%, 7.9% and 29.6% patients respectively. Expression of TICs, CD44 and Podoplanin was observed in 35.2% and 10.2% of samples respectively. Strong co-expression was observed between all ESCs (Nanog, Oct-4, Sox-2) and TICs (p , 0.0001). Overall 9% and 12.6% patients had local and/or distant relapse. Moderate to strong expression of ESC Nanog correlated with local relapse and distant metastasis (p , 0.0001 and 0.0001, respectively). Low podoplanin expression was however associated with reduced local control and increased distant metastasis rate (p , 0.0001 and 0.0001, respectively).

Results: Our analysis indicates Serum- and Glucocorticoid-regulated Kinase 1 gene, SGK1 and N Myc Downstream Regulated Gene-1, NDRG1, to be up-regulated in breast cancer cellsin response to progesterone. We show that NDRG1, a known tumor metastasis suppressor, is phosphorylated at Thr346 upon progesterone treatment. Furthermore various components of the AP-1 transcription factor network, that are known to regulate the expression of NDRG1, are up-regulated upon progesterone treatment. Ectopic expression and knockdown of SGK1 revealed that SGK1 regulates the expression of the AP-1 network genes and phosphorylation of NDRG1 in breast cancer cells. We further show that progesterone treatment and SGK1 over-expression regulate the breast cancer cell migration in vitro.Conclusions: This study helps establish the link between SGK1 and NDRG1 in response to progesterone. We show that SGK1 functions downstream to progesterone to activate an important transcriptional factor network and can play a key role in the regulation of breast cancer cell migration in response to progesterone.

P114-N002A chemical carcinogen: N,N’Dinitrosopiperazine targeting clusterin mediates nasopharyngeal carcinoma metastasis

J. Lu1, Y. Li1, Z. Zhang1, Z. Dong2, T. Kang3, F. Tang1

1 Clinical Laboratory and Medical Research Center, Jinan University, Guangdong, China2 Hormel Institute, University of Minnesota, Minnesota, USA3 State Key Laboratory of Oncology in South China, University Cancer Center, Guangdong, China

Nasopharyngeal carcinoma (NPC) is common malignant cancer in southern China it has a high metastatic clinicopathological feature. The majority of NPC death is attributable to tumor invasion and distant metastasis. N,N’Dinitrosopiperazine (DNP), a specific carcinogen factor for NPC, is involved in not only NPC development but also metastasis, but its precise mechanism has not been fully elucidated. In this study, we showed that DNP promotes NPC metastasis through upregulating anterior clusterin (CLU). DNP was found to increase Clu gene expression through binding to ciselements of Clu gene promoter, DNP also increased matrix metalloproteinases (MMP) 9 and vascular endothelial growth factor (VEGF) expression and activity, further found that DNP increased MMP9 and VEGF expression was through upregulating CLU. We also found that DNP increased the binding of CLU with MMP9 or VEGF. DNP induced the motility and invasion of NPC cell, which was inhibited by siRNACLU. The clinical investigation showed that CLU, MMP9 and VEGF were positively correlated with the tumornode metastasis (TNM) classification. CLU is associated with a metastatic phenotype, exerts its function though regulating MMP2, MMP9, VEGF and Ecadherin expression. These results indicate that DNP may promote NPC tumor metastasis through upregulating CLU, MMP9 and VEGF expression. Therefore, DNP increased CLU expression may be an important factor of NPC high metastasis, and CLU may serve as a biomarker for NPC metastasis. This provides a novel therapeutic strategy for NPC metastasis.

P120-N003Endogenous network hypothesis for cancer genesis and progression

P. Ao1

1 Shanghai Jiao Tong University, Shanghai, China

Cancer is a prototypical complex disease such that the pathology cannot be properly understood by individual players-genes, proteins as well as molecular pathways. The accumulating large “omics” data on cancer also require a manageable framework to integrate for rationalizing cancer complexity and identify collective and individual roles to these players. We proposed a theory that cancer is a robust of the endogenous molecular-cellular network evolutionary built for the developmental process and physiological functions, but not optimized for the whole organism. The crucial individual players found in turn suggest the existence of a hierarchical structure in biological system, which enables a core network to be constructed. We demonstrated that by examining the non linear stochastic dynamics of the core network in the phase space, robust states corresponding to normal physiology and cancer are emerged. The theory establishes a more encompassing framework integrating existing biological knowledge than linear-additive thinking prevailing in the field. We apply this theory to study prostate, liver and gastric cancers and surprising find the models constructed from individual molecular biology experiments recapitulate high throughput microarray profiles. Our framework may serve as a platform as “dry

S72 European Journal of Cancer 54, suppl. 1 (2016) S1–S72 N. Cancer Metastasis

P282-N008Androgen receptor: Does it regulate metastasis in prostate cancer cells?

S. Jacob1, S. Nayak1, G. Fernandes2, R.S. Barai1, S. Menon3, U.K. Chaudhari1, S.D. Kholkute1, G. Sachdeva1

1 National Institute for Research in Reproductive Health (ICMR), Mumbai, India2 G.S. Medical College and KEM Hospital, Mumbai, India3 Department of Pathology, Tata Memorial Hospital, Parel, Mumbai, India

Introduction: Metastasis has been a major challenge in the treatment of androgen-independent Prostate Cancer (PCa). TGF-b, PDGF, TMPRSS2/ERG, SLUG and miRNAs have been implicated in the regulation of metastasis. While the role of androgen signaling in prostate cancer is well established, relevance of androgen receptor (AR) in the metastasis of PCa cells remains undefined. The present study was undertaken to delineate the role of AR in regulating the metastatic characteristics of androgen-independent PCa cell lines.Methods: Androgen-independent PC3 and DU145 cells were transfected with AR and Zinc Finger E-box binding protein 2 (ZEB2) cDNA constructs. Expression of AR and ZEB2 was determined using Q-RTPCR and immunoblotting. In vitro migration and invasion of AR expressing PC3 & DU145 cells were also investigated. Expression of E-cadherin and human Telomerase Reverse Transcriptase (hTERT) protein was determined using immunocytochemistry. Telomerase activity was analyzed using TRAP assay. In androgen-dependent LNCaP cells, levels of ZEB2 and Early Growth Response 1 (EGR1) expression were determined after DHT stimulation and siRNA based AR modulation.Results: AR expression led to a significant decrease in the migration and invasiveness of androgen-independent cells. Further, a significant decrease in the expression of ZEB2 and a significant increase in the expression of E-cadherin were observed in these cells. Restoration of ZEB2 levels in AR expressing androgen independent cells restored their abilities to migrate and invade. Further, a significant decrease was observed in the expression of EGR1 and hTERT in these cells. All these observations hint that AR regulates metastasis in androgen-independent PCa cells. This is in contrast to the pro-tumorigenesis role of AR in androgen-dependent LNCaP cells.Conclusion: Sustenance of AR levels in androgen-independent cells may help in regulating PCa metastasis. The study also suggests that the development of novel hormone based therapies for PCa should target only the proliferative role of AR.

Conclusions: Coexpression of ESC and TIC highlights the possibility of these markers being used to isolate cervical cancer stem cells. This first report of correlation of Nanog and Podoplanin with outcomes suggests the need to develop and investigate the “cancer stem cell” hypothesis for cervical cancer.

P280-N007Incidence and predictive factors for distant metastasis in carcinoma oral cavity: Do we need intense adjuvant systemic therapy?

N. Kalyani1, R. Katna1, B. Bhosale1

1 Bombay Hospital and Medical Research center, New Marine Lines, Mumbai

Aim: Predominant pattern of failure for carcinoma oral cavity following primary treatment is loco-regional failure. The incidence of distant metastasis has been reported to be 10–15 percent of patients. With an aim to define incidence and predictive factors for distant metastasis in carcinoma oral cavity, present analysis was carried out.Material and methods: Treatment charts of prospectively maintained data of 210 patients who underwent surgery for carcinoma of oral cavity between August 2013 to October 2015 were evaluated. All patients underwent surgery as per stage of disease. Of these, 190 patients underwent per primum surgery followed by adjuvant therapy as per histopathology report. Remaining 20 patients received neoadjuvant therapy.Results: Median age of cohort was 50 years. Thirty five patients had early disease (stage I and II), while remaining patients had stage III and IV disease. At last follow up, 161 patients were alive (140 disease-free). Twenty six patients had loco-regional recurrence, 20 patients had distant metastasis and 37 patients had both local-regional recurrence and distant metastasis.Incidence of distant metastasis was 27% (57 patients). Nine patients had non salvageable loco-regional disease recurrence. No metastatic work up was done for them to identify distant metastasis. Sites of distant metastasis were: Lung- 23 patients, bone- 4 patients, skin nodules- 5 patients and multiple sites in 25 patients. Median time for distant metastasis was 6 months post completion of primary treatment. Stage of primary disease, thickness of tumour and advanced nodal stage (N2 and N3) and perinodal extension were associated with higher incidence of distance metastasis.Conclusion: Incidence of distant metastasis in our cohort is higher as compared to published literature. The predominant site of distant metastasis was lung. Possibility of adding adjuvant systemic therapy may be explored in view of high incidence of distance metastasis in carcinoma of oral cavity.

Author Index

A

Aamr, A., S4 (P140-A015)Abarna, R., S2 (P42-A006)Abbas, S., S17 (P210-C015)Abraham, J.G., S18 (O-002)Acharya, S., S16 (P139-C010)Adolacion, J.R., S62 (OF51-007)Adsul, P., S52 (P218-J017)Advait, M.K., S32 (P245-F016)Agarwal, A., S32 (P236-F014)Agarwal, B., S24 (OF50-003)Agarwal, J.P., S37 (P123-G011),

S40 (P211-G024)Agarwal, M., S61 (P297-K026)Agarwal, S., S32 (P236-F014)Agarwala, V., S29 (P76-F003)Aggarwal, B.B., S66 (P267-L019)

Agrawal, P., S57 (P94-K010)Ahamad, F., S64 (P150-L011)Ahir, V.B., S60 (P225-K021)Ahmad, F., S69 (P164-M011)Ahmad, I., S3 (P119-A011)Aich, J., S24 (OF50-003), S55 (P49-K003) Ajaya, K.T., S50 (P118-J008)Akahoho, E., S28 (OF77-007)Alexander, M., S49 (P59-J004)Al-sadi, A., S66 (OT176-001)Al-Sakkaf, K., S49 (P13-J003), S53 (P261-J019) Amatya, K.S., S50 (P133-J009) Amirthalingam, V., S58 (P115-K012)Anand, A.K., S18 (OF281-010)Anang, V., S21 (P52-D007)Anbalagan, M., S2 (P42-A006)Anchlia, D., S54 (OT104-006)Anderson, J., S69 (P91-M007)Andrabi, S., S68 (P37-M005)Andrada, Z.P., S28 (OF77-007)Anuradha, C., S17 (P173-C013)Ao, P., S71 (P120-N003)Arnaldo, A., S33 (OF262-009)Arora, B., S9 (P286-A034)Arora, P., S9 (P287-A035), S69 (P164-M011) Arora, R., S46 (P109-I008)Arora, R.D., S8 (P271-A030), S35 (P20-G003) Arya, S., S6 (P232-A023), S28 (OT105-002),

S31 (P226-F010, P229-F012)Augustin, H., S70 (OT33-006)Austin, R.H., S1 (P22-A002)Ayaz, B., S19 (OF1-010)

B

Badwe, R., S18 (O-003), S47 (P180-I013), S50 (P103-J007), S63 (P90-L008), S70 (P47-N001)

Badwe, R.A., S9 (P287-A035)Bagal, S., S50 (P102-J006)Bagayatkar, P., S38 (P188-G018)Bagwan, J., S50 (P149-J010)Baigrie, R., S53 (P291-J022)Bal, A., S28 (OT266-F003)Bala, P., S12 (P259-B011), S13 (P256-B013)Balakrishnan, P., S32 (P236-F014)Banavali, S., S18 (O-003), S66 (P272-L020)Banavali, S.D., S9 (P286-A034), S38

(P188-G018)Banerjee, D., S46 (P96-I007)Bannon, S., S57 (P93-K009)Barai, R.S., S72 (P282-N008)Barnes, L., S61 (OF75-006)Barui, A., S26 (P97-E004)

Basak, R., S13 (P257-B014)Basaleem, H., S49 (P13-J003), S53 (P261-J019)Bashar, M.A., S54 (P294-J023)Bashir, I., S3 (P119-A011)Basu, S., S28 (P274-E013), S47 (P132-I010)Behari, A., S36 (P82-G007)Belmares, M., S35 (P78-G006)Berman, N., S28 (OF77-007)Bhandare, M.S., S8 (P279-A032)Bhargava, A., S3 (P119-A011)Bhat, S., S58 (P115-K012), S68 (P37-M005)Bhat, S.A., S64 (P135-L009)Bhatia, A., S22 (P126-D013)Bhatia, S.P.S., S9 (P287-A035)Bhatnagar, A., S3 (P119-A011)Bhatt, A.N., S21 (P52-D007)Bhatt, C., S3 (P119-A011)Bhatt, P., S30 (P189-F005)Bhatt, V.D., S60 (P225-K021)Bhattacharjee, A., S5 (P231-A022),

S6 (P232-A023), S24 (P227-D019), S29 (P131-F004), S31 (P226-F010, P229-F012), S52 (P233-J018)

Bhattacharya, S., S47 (P169-I011, P171-I012), S48 (P217-I015)

Bhattacharyya, D., S48 (P292-I017)Bhosale, B., S3 (P129-A013), S72 (P280-N007)Bhosale, P., S37 (P156-G012)Bhusari, P., S28 (OT266-F003)Bhuvaneshwar, G.S., S13 (P256-B013)Biswas, A., S21 (P106-D009)Biswas, M., S46 (P96-I007)Bobdey, S., S2 (P28-A004)Bodade, A.G., S43 (P187-H007)Boila, L.D., S46 (P96-I007)Borkar, S.A., S37 (P158-G014)Bose, C.K., S22 (P116-D012)Bose, K., S15 (P79-C005), S16 (P136-C008,

P138-C009, P139-C010), S23 (P137-D015), S43 (P113-H004)

Botha, H., S53 (P290-J021)Bouffet, E., S7 (P263-A029), S33 (OF262-009),

S53 (P264-J020)Boutall, A., S53 (P291-J022)Braggs, C., S28 (P274-E013), S47 (P132-I010)Braselmann, S., S22 (P134-D014)Braun, A., S24 (OF175-011)Bredemeyer, A., S56 (P85-K006)Budrukkar, A., S37 (P123-G011)Budukh, A., S9 (P287-A035), S50 (P102-J006),

S54 (P294-J023)Bullenkamp, J., S19 (OF1-010)Byregowda, S., S39 (P192-G019)

C

Cai, J., S35 (P71-G005)Carbone, G., S67 (OF128-012)Cardama, A.Q., S43 (P127-H005)Castle, P.E., S35 (P78-G006)Catapano, C., S67 (OF128-012)Chaganti, L.K., S16 (P136-C008)Chakraborty, S., S44 (OT30-003),

S46 (P96-I007)Chakravartti, U., S54 (OT104-006)Chakravorty, R., S49 (P10-J002)Chan, D.C., S20 (P40-D005)Chan, M., S22 (P134-D014)Chandna, P., S55 (P49-K003)Chandola, U., S50 (P149-J010)Chandra, D., S17 (O-001)Chandra, J., S44 (P241-H008)

Chandragauda, D., S9 (P296-A036)Chandrani, P., S24 (OF50-003),

S38 (P188-G018), S47 (P180-I013), S55 (P48-K002, P49-K003), S60 (P220-K020)

Chandraraj, V., S56 (P73-K005)Charles, S., S35 (P78-G006)Chatterjee, J., S26 (P97-E004)Chatterjee, S., S34 (OF193-012)Chatterjee, S.S., S46 (P96-I007)Chaturvedi, P., S1 (OF5-003), S14 (P177-B016),

S28 (OT105-002), S31 (P230-F013), S70 (OF141-006)

Chaubal, R., S10 (P260-B002), S11 (OT244-B007, P247-B005, P248-B006), S41 (OF161-004), S70 (P47-N001)

Chaudhari, P., S41 (OF161-004), S55 (P49-K003)

Chaudhari, U.K., S72 (P282-N008)Chaudhary, N., S28 (P274-E013)Chaudhry, A., S42 (P100-H003)Chaukar, D., S26 (P108-E005),

S28 (OT105-002), S31 (P230-F013), S45 (P45-I005), S62 (P62-L002)

Chavan, S., S51 (P206-J013)Chawdhary, A., S19 (P25-D002)Chen, K.M., S44 (OT72-004), S48 (P242-I016)Chen, X., S45 (P2-I001)Chhipa, R.R., S69 (P91-M007)Chinnasawmy, G., S30 (P191-F006)Chinnaswamy, G., S33 (OF70-005),

S38 (P170-G016)Chiplunkar, S., S62 (P62-L002),

S63 (P90-L008), S66 (P272-L020)Chiplunkar, S.V., S63 (P64-L004),

S64 (P135-L009)Chitale, D., S48 (P242-I016)Choi, Y.K., S25 (P14-E001)Chopade, B.A., S15 (P125-C007)Choppara, S., S15 (P66-C004)Chopra, N.K., S25 (P95-E003)Chopra, S., S56 (P58-K004), S71 (P277-N006)Choudhary, R.K., S16 (P166-C012)Choudhury, K., S25 (P95-E003)Choudhury, S., S25 (P95-E003)Choughule, A., S38 (P188-G018),

S40 (P275-G025)Chougule, A., S55 (P49-K003)Chovatiya, G., S27 (P181-E008, P183-E009)Chow, L.M.L., S69 (P91-M007)Chowdari, P., S21 (P106-D009)Chowdhry, R., S24 (P270-D020)Chowdhury, S., S23 (P202-D017)Chufal, K., S3 (P119-A011)Chung, F., S45 (P2-I001)Cigler, T., S18 (O-002)Civenni, G., S67 (OF128-012)Cobham, M., S18 (O-002)Coca, P., S3 (P122-A012)Colla, S., S54 (OT143-005)Constant, D., S53 (P290-J021)Cooper, J., S24 (OF175-011)Cooper, L., S62 (OF51-007)Costea, D.E., S26 (P108-E005)Cremers, N., S70 (OT33-006)

D

D’Cruz, A., S31 (P228-F011, P229-F012), S62 (P62-L002)

D’Cruz, A.K., S28 (OT105-002), S31 (P230-F013)

S74 Author Index

Dadachova, E., S17 (O-001)Dalal, S., S47 (P132-I010)Dalal, S.N., S28 (P274-E013)Damani, A., S49 (P3-J001)Damodar, S., S58 (P115-K012)Dani, P., S24 (OF50-003)Dar, A.A., S62 (P62-L002)Das, P., S4 (P140-A015)Das, P.K., S62 (P63-L003)Das, S., S39 (P196-G022), S40 (P201-G023),

S59 (P197-K017, P198-K018)Das, U., S21 (P106-D009)Dasgupta, A., S33 (OF70-005)Dasgupta, B., S69 (P91-M007)Dasgupta, D., S19 (P25-D002)Dash, S., S26 (P179-E007)Daskilewicz, K., S53 (P290-J021)Datta, I., S44 (OT72-004), S48 (P242-I016)Datta, K., S43 (P113-H004)Datta, S., S1 (OF5-003)De, A., S22 (P112-D011), S33 (P273-F018),

S46 (P109-I008)Deng, P., S54 (OT143-005)Deodhar, J., S4 (P163-A018), S49 (P3-J001)Deodhar, K., S50 (P102-J006),

S71 (P277-N006)DePinho, R., S54 (OT143-005)Derle, A., S15 (P125-C007)Desai, A., S18 (O-003)Desai, R., S37 (P156-G012)Desai, S., S18 (O-003), S60 (P220-K020),

S69 (P153-M010)Deshmukh, A., S28 (OT105-002)Deshpande, A., S1 (P17-A001)Deshpande, D., S40 (P211-G024)Deshpande, G., S57 (P94-K010)Devanand, V., S42 (P98-H002)Dhamne, H., S47 (P180-I013), S55 (P49-K003)Dhanuskodi, K., S57 (P94-K010)Dhar, R., S14 (P31-C002)Dhawan, D.K., S28 (OT266-F003)Dhuma, S., S6 (P232-A023)Dhumal, S., S24 (P227-D019), S29 (P76-F003),

S31 (P228-F011, P229-F012)Dikshit, R., S9 (P287-A035), S50 (P102-J006,

P103-J007), S63 (P64-L004)Dimri, S., S46 (P109-I008)Divine, G., S44 (OT72-004), S48 (P242-I016)Diwan, A.K., S2 (P23-A003)Dmello, C., S45 (P45-I005, P9-I002)Donato, N.J., S43 (P127-H005)Dong, Z., S71 (P114-N002)Donnella, H., S44 (P241-H008)Dudley, A., S28 (OF77-007)Dussane, R., S51 (P178-J012)Dutt, A., S10 (P260-B002),

S11 (OT244-B007, P247-B005, P248-B006), S24 (OF50-003), S34 (OF16-001), S38 (P188-G018), S41 (OF161-004), S43 (P113-H004), S47 (P180-I013), S55 (P48-K002, P49-K003), S56 (P58-K004), S60 (P220-K020), S70 (P47-N001)

Dutt, S., S11 (OT244-B007), S24 (OF50-003), S41 (OF11-011, OF161-004), S42 (P98-H002), S43 (P113-H004), S70 (P219-M012)

Dutta, D., S2 (P42-A006), S58 (P124-K013)Dutta, S., S33 (P273-F018)Dwarakanath, B.S., S21 (P52-D007)

E

Effah, K., S35 (P78-G006)Elenitoba-Johnson, K.S.J., S45 (P2-I001)Engineer, R., S71 (P277-N006)Epari, S., S61 (OF152-008)Eslavath, R., S68 (P36-M004)Evered, A., S23 (P202-D017)

F

Fahey III, T.J., S28 (OF77-007)Fan, Q., S69 (P91-M007)Farahdel, L., S33 (P293-F019)Faruq, O., S64 (P150-L011)Fermeglia, M., S43 (P127-H005),

S67 (OF128-012)Fernandes, G., S72 (P282-N008)Fitzpatrick, V., S18 (O-002)Forances, R., S22 (P134-D014)Freeman, M., S62 (P53-L001)

G

Gadgil, P., S61 (P297-K026)Gaikwad, A., S10 (P249-B001, P250-B003,

P260-B002), S11 (OT244-B007, P247-B005, P248-B006), S12 (P259-B011)

Gaillard, E., S35 (P78-G006)Gäken, J., S20 (P41-D006), S43 (P144-H006)Gambhir, A., S36 (P111-G010, P84-G008)Ganesan, T.S., S57 (P107-K011)Ganesh, B., S2 (P28-A004)Gangadaran, P., S45 (P45-I005)Gangodkar, P., S61 (P297-K026)Ganguly, A., S48 (P292-I017)Gardi, N., S10 (P260-B002), S11 (OT244-B007,

P248-B006), S24 (OF50-003), S41 (OF161-004), S43 (P113-H004), S47 (P180-I013), S55 (P48-K002), S56 (P58-K004), S60 (P220-K020), S70 (P47-N001)

Garg, A., S1 (OF5-003), S14 (P177-B016)Gawas, N., S27 (P183-E009)Gawas, N.P., S26 (P172-E006)Gazda, L.S., S28 (OF77-007)Geelani, Q., S68 (P37-M005)Gera, P., S17 (P210-C015), S38 (P159-G015)Ghatak, S., S54 (OT104-006)Ghosh, J., S5 (P231-A022)Ghosh, M.K., S20 (P39-D004)Ghosh, R., S20 (P40-D005)Ghosh, S., S15 (P125-C007), S54 (OT104-006)Giraudo, A., S65 (P222-L016)Giraudo, C., S65 (P222-L016)Glendon, C., S10 (P8-A038)Goda, J.S., S71 (P277-N006)Godashastri, J., S38 (P170-G016)Godbole, M., S24 (OF50-003), S47 (P180-I013),

S70 (P47-N001)Goel, M., S63 (P64-L004)Goel, V., S9 (P296-A036), S18 (OF281-010)Gogate, N., S61 (P297-K026)Gogna, S., S68 (P29-M003)Gomm, N., S10 (P8-A038)Gopalakrishnan, G., S4 (P145-A016)Gota, V., S65 (P155-L014)Gottesman, M.M., S68 (P60-M006)Govender, D., S40 (P289-G027)Gowda, H., S43 (P113-H004)Gowda, S., S3 (P122-A012)Goyal, N., S32 (P236-F014)Grasse, L., S44 (P241-H008)Gravekamp, C., S17 (O-001)Greene, J.M., S68 (P60-M006)Grimaldi, B., S66 (OF117-002)Gu, R., S55 (OF295-025)Guerrero, T., S20 (P41-D006),

S43 (P144-H006)Gulati, S., S54 (OT104-006)Gulia, A., S18 (OF281-010), S30 (P191-F006,

P205-F007), S32 (P243-F015), S39 (P192-G019)

Gundeti, M.S., S7 (P238-A026)Guo, S., S35 (P71-G005)Gupta, A.R., S5 (P213-A020, P214-A021),

S30 (P215-F008), S39 (P194-G020, P195-G021, P196-G022), S40 (P201-G023), S59 (P197-K017, P198-K018, P212-K019)

Gupta, D., S10 (P260-B002), S11 (P247-B005, P248-B006)

Gupta, H., S35 (P69-G004), S60 (P54-K023), S61 (P55-K024)

Gupta, R., S42 (P100-H003)Gupta, S., S18 (O-003), S32 (P236-F014),

S47 (P169-I011, P171-I012, P180-I013), S48 (P217-I015), S52 (P233-J018), S55 (P24-K001), S58 (P148-K014), S69 (P164-M011), S70 (P47-N001), S71 (P277-N006)

Gupta, T., S33 (OF70-005), S37 (P123-G011), S38 (P170-G016), S58 (P124-K013)

Gupta, V., S57 (P94-K010)Gurav, M., S23 (P221-D018)Gurjar, M., S28 (P274-E013)

H

Halder, K., S70 (P219-M012)Hamid, A., S1 (P17-A001)Hammoudi, N., S54 (OT143-005)Haojam, R.S., S42 (P100-H003)Haque, M. Md., S52 (P209-J016)Hariharan, A.K., S57 (P94-K010)Harish, M.M., S6 (P237-A025)Haritha, C., S5 (P213-A020, P214-A021),

S27 (P199-E011, P200-E012), S30 (P215-F008, P216-F009), S39 (P194-G020, P195-G021, P196-G022), S40 (P201-G023), S59 (P197-K017, P198-K018, P212-K019), S60 (P225-K021)

Harsh, D., S26 (P108-E005)Hasan, S., S42 (P98-H002)Hawaldar, R., S9 (P286-A034), S18 (O-003)Hawkins, C., S33 (OF262-009)Herrada, S.P., S68 (P60-M006)Hole, A., S34 (OF193-012)Hudlikar, R., S69 (P153-M010)Husain, N., S55 (P24-K001)Hussain, S.S., S20 (P40-D005)

I

Ingle, A., S69 (P153-M010)Irwin, M., S44 (P241-H008)Iyer, D.N., S55 (P49-K003)Iyer, P., S55 (P48-K002)

J

Jacob, L., S56 (P73-K005)Jacob, S., S72 (P282-N008)Jacobs, V., S1 (P17-A001)Jadhav, G., S36 (P111-G010)Jagilinki, B.P., S59 (P167-K016)Jain, A., S63 (P86-L005)Jain, D., S14 (P35-C003), S39 (P196-G022),

S40 (P201-G023), S59 (P197-K017, P198-K018)Jain, K., S25 (P95-E003)Jain, S., S40 (P211-G024)Jakhesara, S.J., S60 (P225-K021)Jalali, R., S33 (OF70-005), S38 (P170-G016),

S58 (P124-K013)Jambhekar, N., S12 (P253-B009),

S40 (P275-G025)James, B.L., S50 (P149-J010)Jandyal, S., S29 (P131-F004)Janu, A., S33 (OF70-005)Jeevan, D., S24 (OF175-011)Jena, A., S36 (P111-G010, P84-G008)Jeronimo, J., S35 (P78-G006)Jeyapaul, J., S71 (P147-N004)Jijo, J., S39 (P196-G022), S40 (P201-G023),

S59 (P197-K017, P198-K018)Jin, S.V., S66 (OT176-001)Johnson, B., S44 (P241-H008)Joseph, J., S5 (P213-A020, P214-A021), S30

(P215-F008), S39 (P194-G020, P195-G021), S59 (P212-K019)

Author Index S75

Joshi, A., S5 (P231-A022), S6 (P232-A023), S24 (P227-D019), S29 (P76-F003, P131-F004), S31 (P226-F010, P228-F011, P229-F012, P230-F013), S38 (P188-G018), S40 (P275-G025), S52 (P233-J018), S60 (P220-K020)

Joshi, C.G., S60 (P225-K021)Jun, C., S32 (P265-F017)Jun, C.Y., S25 (P14-E001)Juvekar, S., S6 (P232-A023), S31 (P229-F012,

P230-F013)Jyothi, D., S68 (P36-M004)

K

Kabre, R.S., S2 (P23-A003)Kadam, R., S48 (P203-I014)Kale, S., S15 (P125-C007)Kalkar, P., S51 (P207-J014)Kalyani, N., S1 (P17-A001), S3 (P129-A013),

S72 (P280-N007)Kamat, H., S30 (P216-F009)Kamble, K.M., S2 (P23-A003)Kane, S., S24 (OF50-003), S28 (OT105-002),

S31 (P228-F011, P229-F012), S37 (P156-G012), S45 (P45-I005)

Kane, S.V., S1 (OF5-003)Kang, S., S32 (P265-F017)Kang, T., S71 (P114-N002)Kannan, S., S24 (OF50-003), S69 (P153-M010)Kannan, V., S2 (P42-A006)Kansara, S., S23 (P185-D016)Kant, R., S24 (P270-D020)Kapoor, R., S9 (P287-A035), S54 (P294-J023)Kapoor, V.K., S36 (P82-G007)Karmakar, S., S14 (P31-C002), S46 (P121-I009)Karna, S., S69 (P164-M011)Karna, S.K., S64 (P150-L011)Karpe, A., S24 (P227-D019), S29 (P131-F004)Karve, K., S70 (P47-N001)Kathirvel, S., S9 (P287-A035)Katna, R., S1 (P17-A001), S3 (P129-A013),

S72 (P280-N007)Katragadda, S., S57 (P94-K010)Kaufmann, A., S35 (P78-G006)Kaul, S., S4 (P140-A015)Kaur, E., S11 (OT244-B007), S24 (OF50-003),

S41 (OF11-011, OF161-004), S42 (P98-H002), S43 (P113-H004)

Kaushal, R., S65 (P155-L014), S69 (P153-M010)

Kaushik, G., S67 (P27-M002)Kekatpure, V., S50 (P149-J010),

S58 (P148-K014)Kelkar, K., S61 (P297-K026)Kelkar, M., S22 (P112-D011)Khadilkar, V., S61 (P297-K026)Khadka, D., S69 (P101-M008)Khaitan, D., S21 (P106-D009)Khambatti, F., S11 (P247-B005)Khan, I., S8 (P278-A031)Khan, M., S67 (OF182-009)Khan, S.A., S48 (P217-I015)Khanduja, K.L., S67 (P27-M002)Khanna-Gupta, A., S42 (P100-H003)Kharbuja, P., S50 (P133-J009)Khare, N.K., S11 (P247-B005, P248-B006,

P251-B004), S14 (P177-B016)Kharod, U., S30 (P216-F009)Khatod, K., S61 (P297-K026)Khattry, N., S42 (P98-H002), S66 (P190-L021)Khazaei, H., S60 (P54-K023), S61 (P55-K024)Kholkute, S.D., S72 (P282-N008)Khosa, R., S32 (P236-F014)Khullar, M., S21 (P110-D010)Khurana, B., S61 (P55-K024)Khyriem, C., S58 (P148-K014)Kim, H.J., S69 (P101-M008)Kim, J.H., S32 (P265-F017)

Kim, M.S., S25 (P14-E001), S32 (P265-F017)Kinhikar, R., S40 (P211-G024)Kirar, P., S32 (P245-F016)Kirolikar, S., S13 (OF246-B015)Kishor, K., S64 (P142-L010)Kitture, R., S15 (P125-C007)Knouse, P., S44 (P241-H008)Ko, S., S32 (P265-F017)Ko, S.G., S25 (P14-E001)Kode, J., S66 (P190-L021)Kok, J., S70 (P219-M012)Kori, C., S55 (P24-K001)Korn, C., S70 (OT33-006)Kornhauser, N., S18 (O-002)Kowtal, P., S17 (P210-C015)Kozlov, A.P., S44 (OF68-005)Krishna, C., S38 (P159-G015)Krishna, C.M., S34 (OF193-012),

S38 (P186-G017)Krishna, U., S56 (P73-K005), S58 (P124-K013)Krishnan, N., S58 (P148-K014)Krishnani, N., S36 (P82-G007)Krishnatry, R., S3 (P122-A012),

S4 (P145-A016), S7 (P263-A029), S33 (OF262-009), S35 (P69-G004), S53 (P264-J020), S58 (P115-K012)

Kulkarni, A., S18 (O-003)Kulkarni, S., S34 (P4-G001), S51 (P178-J012)Kumar G.V. S., S52 (P208-J015)Kumar, A.P., S20 (P40-D005)Kumar, J., S21 (P106-D009)Kumar, P., S38 (P186-G017)Kumar, R., S5 (P213-A020, P214-A021),

S30 (P215-F008), S39 (P194-G020, P195-G021), S59 (P212-K019)

Kumar, S., S3 (P119-A011), S5 (P213-A020, P214-A021), S14 (P35-C003), S30 (P215-F008), S35 (P69-G004), S39 (P194-G020, P195-G021), S59 (P212-K019)

Kumar, V., S5 (P213-A020, P214-A021), S30 (P215-F008), S39 (P194-G020, P195-G021), S55 (P24-K001), S59 (P212-K019)

Kumar, Y., S9 (P298-A037)Kumari, K., S57 (P94-K010)Kumari, N., S36 (P82-G007)Kumari, S.A., S26 (P108-E005)Kuppili, R.R., S23 (P137-D015)Kuriakose, M.A., S50 (P149-J010),

S58 (P148-K014)Kurkure, P.A., S9 (P286-A034)Kushwaha, A., S41 (OF11-011)Kwasnicki, A., S24 (OF175-011)

L

Lad, S., S34 (P4-G001)Lal, G., S64 (P154-L013)Lamagna, C., S22 (P134-D014)Lane, M., S18 (O-002)Laramore, M.A., S28 (OF77-007)Laskar, S., S30 (P205-F007)Laskar, S.G., S37 (P123-G011)Lassaletta, A., S7 (P263-A029),

S33 (OF262-009)Laurini, E., S43 (P127-H005), S67 (OF128-012)Lavanya, G., S3 (P122-A012), S4 (P145-A016),

S7 (P263-A029), S58 (P115-K012)Lavi, O., S68 (P60-M006)Lawler, K., S20 (P41-D006), S43 (P144-H006)Lee, K., S25 (P14-E001), S32 (P265-F017)Lee, S., S69 (P101-M008)Lee, S.B., S69 (P101-M008)Levy, D., S68 (P60-M006)Li, H., S22 (P134-D014)Li, R., S44 (P241-H008)Li, Y., S71 (P114-N002)Liadi, I., S62 (OF51-007)Libutti, S.K., S17 (O-001)Lim, M.S., S45 (P2-I001)

Lim, S., S61 (OF75-006)Limzerwalla, J., S28 (P274-E013)Lin, Y.H., S54 (OT143-005)Ling, C., S33 (OF262-009)Liu, H., S61 (OF75-006)Lokhande, M., S50 (P102-J006)Lomis, N., S33 (P293-F019)Lu, J., S71 (P114-N002)Lu, P., S35 (P78-G006)Lynch, P.M., S57 (P93-K009)

M

Mahajan, A., S19 (OF1-010), S31 (P226-F010)Mahantshetty, U., S71 (P277-N006)Maheshwari, A., S50 (P102-J006),

S71 (P277-N006)Mahimkar, M., S37 (P156-G012),

S69 (P153-M010)Mahoobia, A.K., S2 (P23-A003)Maitre, M., S58 (P124-K013)Majumdar, A., S65 (P155-L014)Malhotra, P., S9 (P287-A035)Malhotra, S.V., S41 (OT26-002)Malik, A., S70 (OF141-006)Malik, F., S15 (P83-C006)Malik, P.S., S14 (P35-C003)Manasa, P., S57 (P107-K011)Manda, K., S21 (P52-D007)Manik, V., S40 (P211-G024)Manjula, G., S17 (P173-C013)Manna, A., S8 (P278-A031)Manne, R., S67 (P15-M001)Markovtsov, V., S22 (P134-D014)Martis, P.C., S28 (OF77-007)Maru, G., S69 (P153-M010)Mashru, R., S30 (P189-F005)Masuda, E., S22 (P134-D014)McLeod, H., S62 (P53-L001)McPherson, C., S69 (P91-M007)Meherji, P., S50 (P102-J006)Mehta, K., S41 (OF43-001)Mehta, M., S65 (P155-L014)Menon, H., S61 (OF152-008)Menon, S., S72 (P282-N008)Merchant, N., S37 (P157-G013)Merwe, H.D., S53 (P290-J021)Mhatre, S., S50 (P103-J007)Milind, V., S26 (P108-E005)Minz, R., S9 (P298-A037)Mirji, G., S66 (P272-L020)Mirza, R., S25 (P95-E003)Mishra, A., S1 (OF5-003), S24 (P270-D020)Mishra, G., S51 (P178-J012)Mishra, K., S36 (P82-G007)Mishra, P.S., S2 (P28-A004)Mishra, R., S17 (P204-C014)Misra, A., S30 (P189-F005)Misra, S., S55 (P24-K001)Mistry, M., S33 (OF262-009)Mitra, A., S34 (P4-G001)Mittal, B.R., S28 (OT266-F003)Mittal, V., S18 (O-002)Mittra, I., S10 (P249-B001, P250-B003,

P260-B002), S11 (OT244-B007, P247-B005, P248-B006, P251-B004), S12 (P252-B008, P253-B009, P254-B010, P259-B011), S13 (OF246-B015, P255-B012, P256-B013, P257-B014), S14 (P177-B016)

Mohanan, S., S34 (OF193-012)Mohandas, K.M., S50 (P102-J006)Mohanty, B., S55 (P49-K003)Moiyadi, A., S34 (OF193-012),

S38 (P170-G016), S41 (OF11-011), S43 (P113-H004)

Momberg, M., S53 (P290-J021)Monte, C., S67 (OF128-012)Moodley, J., S53 (P290-J021, P291-J022)Moodley, N., S53 (P291-J022)

S76 Author Index

Moore, A., S18 (O-002)Mork, M., S57 (P93-K009)Muckaden, M., S4 (P163-A018)Muckaden, M.A., S49 (P3-J001)Muddu, V., S24 (P227-D019), S29 (P76-F003),

S31 (P229-F012, P230-F013)Mukherjee, S., S19 (P25-D002)Muller, F., S54 (OT143-005)Mundhe, D., S17 (P204-C014)Mundhe, M., S37 (P157-G013)Murali, R., S24 (OF175-011)Murthy, V., S37 (P123-G011), S51 (P207-J014)Murugan, S., S42 (P100-H003)

N

Nackos, E., S18 (O-002)Nag, S., S20 (P32-D003)Nagarajan, R., S56 (P85-K006)Nagare, H., S10 (P249-B001), S13 (P255-B012)Nagendra, H.G., S21 (P106-D009)Nagrani, R., S50 (P103-J007)Nair, D., S1 (OF5-003), S14 (P177-B016),

S28 (OT105-002), S70 (OF141-006)Nair, J., S41 (OF11-011, OF161-004),

S42 (P98-H002)Nair, J.S., S18 (OT61-001)Nair, N., S70 (P47-N001)Nair, S., S14 (P177-B016), S24 (OF50-003),

S28 (OT105-002), S38 (P159-G015), S60 (P220-K020), S70 (OF141-006)

Nakano, I., S69 (P91-M007)Nangia, S., S32 (P236-F014)Naronha, V., S40 (P275-G025)Natarajan, K., S21 (P52-D007)Nayak, S., S72 (P282-N008)Nazarian, A., S28 (OF77-007)Negi, P., S36 (P111-G010)Neumann, H., S70 (P219-M012)Neupane, P., S50 (P133-J009)Nigam, S., S63 (P87-L006)Ning, J., S35 (P71-G005)Ningaraj, N., S21 (P106-D009)Nithiyanantham, K., S3 (P122-A012),

S4 (P145-A016), S35 (P69-G004), S58 (P115-K012)

Nobre, M., S13 (OF246-B015, P257-B014)Noronha, S., S34 (P12-G002)Noronha, V., S5 (P231-A022), S6 (P232-A023),

S24 (P227-D019), S29 (P76-F003, P131-F004), S31 (P226-F010, P228-F011, P229-F012, P230-F013), S38 (P188-G018), S52 (P233-J018)

O

Ocean, A.J., S28 (OF77-007)Odell, E., S20 (P41-D006), S43 (P144-H006)Oh, G.S., S69 (P101-M008)Oodit, R., S53 (P291-J022)Oommen, S., S32 (P236-F014)Ostwal, S., S4 (P163-A018)Ostwal, V., S18 (O-003)

P

Pai, A., S36 (P99-G009)Pai, P., S28 (OT105-002)Pai, P.S., S31 (P230-F013)Pai, S.K., S9 (P286-A034)Pai, T., S64 (P151-L012)Pai, V., S71 (P277-N006)Pai, V.D., S8 (P279-A032)Pal, K., S12 (P259-B011), S13 (P256-B013,

P257-B014)Palayekar, V., S50 (P102-J006)Palled, S., S56 (P73-K005)Palve, V., S50 (P149-J010), S58 (P148-K014)Panda, B., S50 (P149-J010), S58 (P148-K014)Panda, D., S19 (P19-D001)

Panda, N.K., S21 (P110-D010)Pande, M., S57 (P93-K009)Pandey, M., S37 (P156-G012)Pandey, N., S11 (P251-B004)Pandey, S., S21 (P52-D007)Pandit, A., S69 (P101-M008)Panse, N.S., S50 (P102-J006)Pantvaidya, G., S28 (OT105-002)Papanikolaou, N., S20 (P40-D005)Paradkar, A., S9 (P286-A034)Parchuru, S., S57 (P94-K010)Parikh, T., S28 (OF77-007)Park, G., S22 (P134-D014)Parkes, J., S40 (P289-G027)Parmar, M., S28 (OT266-F003)Parmar, V., S63 (P90-L008), S70 (P47-N001)Parmod, K.P.R., S56 (P73-K005)Parulekar, M., S57 (P94-K010)Pasha, T., S56 (P73-K005)Patel, A.K., S60 (P225-K021)Patel, K., S47 (P180-I013), S70 (P47-N001)Patel, T., S45 (P34-I004)Pathak, V., S57 (P94-K010)Pathaka, C.M., S67 (P27-M002)Pathania, A., S51 (P206-J013)Patil, A., S37 (P156-G012)Patil, As., S51 (P207-J014)Patil, K., S61 (P297-K026)Patil, N., S18 (O-003)Patil, P., S8 (P279-A032)Patil, R.S., S63 (P64-L004)Patil, V., S24 (P227-D019), S29 (P131-F004),

S31 (P226-F010, P230-F013), S38 (P188-G018), S40 (P275-G025)

Patil, V.M., S5 (P231-A022), S6 (P232-A023), S29 (P76-F003), S31 (P228-F011, P229-F012), S52 (P233-J018)

Patkar, K., S41 (OF161-004), S43 (P113-H004)Patkar, S., S42 (P98-H002)Patowary, J., S4 (P140-A015), S62 (P63-L003)Paul, D., S6 (P232-A023)Paul, S., S64 (P154-L013)Pawar, P., S14 (P177-B016)Pawar, S., S19 (P25-D002), S51 (P206-J013,

P207-J014)Payan, D.G., S22 (P134-D014)Peres, J., S40 (P289-G027)Perumal, D., S68 (P36-M004)Peterson, S.K., S57 (P93-K009)Petrauskene, O., S35 (P78-G006)Phadke, N., S61 (P297-K026)Phalke, S., S63 (P90-L008)Phatak, A., S56 (P85-K006)Philip, D.S.J., S40 (P275-G025)Pieterse, F., S53 (P291-J022)Pigden, C., S10 (P8-A038)Pilankar, A., S61 (P297-K026)Pilar, A., S37 (P123-G011)Pillai, S., S7 (P263-A029)Pimple, S., S18 (O-003), S51 (P206-J013)Pokharel, Y., S64 (P150-L011), S69

(P164-M011)Pokharel, Y.R., S60 (P288-K022)Poojari, R., S19 (P19-D001)Popat, P.P., S52 (P208-J015)Prabhash, K., S5 (P231-A022), S6 (P232-A023),

S24 (P227-D019), S28 (OT105-002), S29 (P76-F003, P131-F004), S31 (P226-F010, P228-F011, P229-F012, P230-F013), S37 (P156-G012), S38 (P188-G018), S40 (P275-G025), S52 (P233-J018), S55 (P49-K003)

Prajitha, E.M., S17 (P173-C013)Prakash, S., S33 (P293-F019)Pramod, K.K., S35 (P69-G004)Prasad, R., S55 (P49-K003)Prasannan, P., S10 (P249-B001, P250-B003,

P260-B002), S11 (OT244-B007, P247-B005, P248-B006), S12 (P259-B011)

Prerana, D., S26 (P108-E005)Pricl, S., S43 (P127-H005), S67 (OF128-012)Prince, S., S40 (P289-G027)Pritzker, K., S55 (OF295-025)Pungavkar, S., S33 (OF70-005), S38

(P170-G016)Puri, A., S30 (P191-F006, P205-F007),

S32 (P243-F015), S39 (P192-G019), S52 (P209-J016)

Purvar, P., S50 (P102-J006)

Q

Qiao, Y.L., S35 (P78-G006)Qi-Xiang Li, H., S35 (P71-G005)

R

Radotra, B.D., S21 (P110-D010)Raghunadharao, D., S17 (P173-C013)Raghupathi, K., S16 (P138-C009)Raghuram, G.V., S10 (P250-B003, P260-B002),

S11 (P247-B005, P248-B006)Ragte-Wathare, T., S61 (P297-K026)Raina, S., S9 (P296-A036)Raj, R., S2 (P38-A005)Rajadhyaksha, S.B., S43 (P187-H007)Rajan, M.G.R., S34 (P4-G001)Rajendra, J., S43 (P113-H004)Rajesh, P., S5 (P213-A020, P214-A021),

S30 (P215-F008), S39 (P194-G020, P195-G021), S59 (P212-K019)

Rajesh, R., S5 (P213-A020, P214-A021), S30 (P215-F008), S39 (P194-G020, P195-G021, P196-G022), S40 (P201-G023), S59 (P197-K017, P198-K018, P212-K019)

Rajpurohit, S., S9 (P296-A036)Ralhan, R., S55 (OF295-025)Ramadwar, M., S12 (P253-B009)Ramadwar, M.R., S55 (P48-K002)Ramanan, V., S61 (P297-K026)Ramaswamy, A., S31 (P226-F010, P230-F013)Ramprasad, V.L., S42 (P100-H003)Ramteke, M., S34 (OF16-001)Rana, S., S67 (P27-M002)Ranade, S., S61 (P297-K026)Rane, P., S28 (OT105-002)Ranjan, K., S39 (P196-G022),

S40 (P201-G023), S59 (P197-K017, P198-K018)

Ranjan, M., S55 (P48-K002)Rao, D.N., S17 (P173-C013)Rasane, H., S7 (P238-A026)Rastogi, H., S32 (P236-F014)Rathod, M., S22 (P112-D011)Rathod, N., S71 (P277-N006)Rattan, V., S21 (P110-D010)Raulf, N., S19 (OF1-010), S20 (P41-D006),

S43 (P144-H006)Ravi, V., S27 (P181-E008)Rawat, S.J., S32 (P245-F016)Ray, M.D., S29 (P74-F002)Ray, P., S25 (P46-E002), S42 (P7-H001),

S71 (P277-N006)Rayappa, C., S2 (P42-A006)Raychaudhari, K., S28 (P274-E013)Reddy, D., S47 (P169-I011, P171-I012),

S48 (P217-I015)Reddy, M.S., S28 (P274-E013)Reddy, R., S43 (P113-H004)Rekha, M.R., S13 (P256-B013)Rekhi, B., S34 (OF16-001)Reshi, I., S68 (P37-M005)Reveilhe, S., S35 (P78-G006)Reyes-Gibby, C., S1 (OT56-005)Rifayi, M., S3 (P122-A012), S4 (P145-A016),

S58 (P115-K012)Rizwani, W., S68 (P36-M004)Robbins, S.M., S46 (P65-I006)Romain, G., S62 (OF51-007)

Author Index S77

Rothley, M., S70 (OT33-006)Rout, S., S32 (P236-F014)Roy, A., S21 (P106-D009)Roy, B., S54 (OT104-006)Roy, S., S19 (P25-D002)Roysam, B., S62 (OF51-007)Rubinchik, A., S18 (O-002)Rugo, H.S., S65 (P235-L018)

S

Sable, N., S52 (P208-J015)Sachdeva, G., S72 (P282-N008)Sachin, A., S59 (P198-K018)Sachin, J., S39 (P196-G022), S59 (P197-K017)Sachin, J.A., S40 (P201-G023)Saha, G., S54 (OT104-006)Saha, T., S10 (P249-B001, P250-B003)Sahasrabuddhe, A., S45 (P2-I001)Sahasrabuddhe, N.A., S56 (P85-K006)Sahoo, P., S40 (P211-G024)Sahu, A., S31 (P230-F013), S38 (P159-G015)Sahu, D.K., S24 (P270-D020)Sahu, P., S31 (P228-F011)Sajesh, B.V., S14 (P6-C001)Sajnani, M.R., S60 (P225-K021)Saklani, A.P., S8 (P279-A032)Salins, N., S4 (P163-A018), S49 (P3-J001)Salunkhe, S.J., S42 (P98-H002)Samantha, S., S21 (P106-D009)Sambasivaselli, R., S3 (P122-A012)Sanchetee, S., S51 (P160-J011)Sandhya, A., S17 (P173-C013)Sangam, K., S9 (P298-A037)Sankaran, G., S67 (P15-M001)Sannigrahi, M.K., S21 (P110-D010)Santosh, S., S42 (P100-H003)Santra, M.K., S15 (P66-C004), S67 (P15-M001)Saraswati, S., S70 (OT33-006)Sarate, R., S27 (P183-E009)Sarate, R.M., S26 (P172-E006), S27 (P181-E008)Sarin, R., S17 (P210-C015), S36 (P111-G010),

S52 (P209-J016)Sarkar, A., S26 (P97-E004)Sarkar, S., S46 (P65-I006)Sarkar, S.K., S8 (P278-A031)Sarwar, Z., S68 (P37-M005)Sastri, J., S58 (P124-K013)Satani, N., S54 (OT143-005)Sathish, P., S39 (P196-G022), S40 (P201-G023),

S59 (P197-K017, P198-K018)Sawant, A., S28 (P274-E013), S47 (P132-I010)Sawant, S., S45 (P45-I005, P9-I002)Schneider, S., S18 (O-002)Schreiber, C., S70 (OT33-006)Schwartz, G.K., S18 (OT61-001)Schweizer, J., S35 (P78-G006)Seibert, K., S56 (P85-K006)Sen, M., S57 (P94-K010)Sengar, M., S9 (P286-A034), S61 (OF152-008),

S66 (P272-L020)Senger, D., S46 (P65-I006)Sengupta, A., S46 (P96-I007)Senniandavar, V., S3 (P122-A012)Sereni, F., S67 (OF128-012)Seth, S., S21 (P52-D007)Sethunath, V., S24 (OF50-003), S55 (P49-K003)Shah, H., S34 (P12-G002)Shah, M., S68 (P37-M005)Shah, N., S58 (P124-K013)Shah, S., S42 (P98-H002), S60 (P225-K021)Shah, S.U., S63 (P64-L004)Shaikh, A., S12 (P259-B011), S13 (P257-B014)Shankar, V., S5 (P213-A020, P214-A021),

S27 (P199-E011, P200-E012), S30 (P215-F008, P216-F009), S39 (P194-G020, P195-G021, P196-G022), S40 (P201-G023), S59 (P197-K017, P198-K018, P212-K019), S60 (P225-K021)

Shanubhogue, A., S30 (P216-F009)Sharada, S., S26 (P108-E005)Sharda, A., S48 (P217-I015)Sharma, A.K., S48 (P217-I015)Sharma, C.P., S13 (P256-B013)Sharma, R., S21 (P110-D010)Sharma, S., S28 (OT105-002)Shastri, S., S18 (O-003), S51 (P178-J012)Shazad, N., S69 (P164-M011)Sheela, M.C., S52 (P208-J015)Shen, A., S69 (P101-M008)Shenoy, A., S56 (P73-K005)Shet, T., S12 (P253-B009, P254-B010),

S23 (P221-D018), S61 (OF152-008), S64 (P151-L012)

Shetty, A., S41 (OF11-011)Shetty, K.N., S4 (P130-A014)Shetty, M., S12 (P259-B011), S13 (P256-B013)Shetty, O., S64 (P151-L012)Shetty, P., S38 (P170-G016)Shilpi, S., S26 (P108-E005)Shirsat, N., S33 (OF70-005)Shrama, S.K., S67 (P27-M002)Shrestha, U.M., S50 (P133-J009)Shridhar, E., S38 (P170-G016)Shrikhande, S.V., S55 (P48-K002),

S63 (P64-L004)Shrivastava, B., S30 (P216-F009)Shrivastava, S., S40 (P211-G024)Shrivastava, S.K., S71 (P277-N006)Shriya, J., S26 (P108-E005)Shukla, H.D., S21 (P57-D008)Shukla, J., S28 (OT266-F003)Shukla, P., S36 (P82-G007)Siddappa, G., S50 (P149-J010),

S58 (P148-K014)Siddhartha, G.K., S56 (P88-K007)Siddiqui, M.Q., S16 (P165-C011)Sikdar, N., S54 (OT104-006)Sindhi, R., S55 (P49-K003)Singh, A., S11 (P247-B005), S12 (P253-B009,

P254-B010), S15 (P83-C006)Singh, A.K., S4 (P146-A017)Singh, G., S28 (OT266-F003)Singh, H., S62 (OF51-007)Singh, K., S62 (P63-L003)Singh, M., S43 (P187-H007)Singh, N., S24 (P270-D020)Singh, R., S3 (P81-A010), S22 (P134-D014),

S29 (P21-F001), S54 (OT104-006)Singh, R.K., S25 (P46-E002)Singh, S., S21 (P52-D007), S52 (P208-J015)Singh, S.S., S68 (P36-M004)Singh, V., S14 (P35-C003), S21 (P110-D010)Sinha, S., S8 (P278-A031), S17 (P173-C013),

S46 (P96-I007)Sirohi, B., S4 (P145-A016)Siu, S., S22 (P134-D014)Sleeman, J.P., S70 (OT33-006)Smith, B.H., S28 (OF77-007)Smuts, D., S40 (P289-G027)Snehal, M., S26 (P108-E005)So, H., S69 (P101-M008)Sogani, S.K., S36 (P111-G010)Sonawane, K., S24 (OF50-003)Sreeprakash, S2 (P42-A006)Sridhar, E., S34 (OF193-012)Srivastava, A., S41 (OF11-011)Srivastava, A.K., S70 (P219-M012)Srivastava, R., S19 (P19-D001)Stephen, J.K., S44 (OT72-004),

S48 (P242-I016)Subi, S., S39 (P192-G019)Subramaniam, S., S13 (P257-B014)Subramanian, K., S57 (P94-K010)Sucindran, P., S10 (P8-A038)Sudhalkar, N., S71 (P277-N006)Suh, Y., S19 (OF1-010), S20 (P41-D006),

S43 (P144-H006)

Sunkara, R.R., S26 (P172-E006)Sur, D., S49 (P10-J002)Suresh, A., S50 (P149-J010), S58 (P148-K014)Swamidas, J., S58 (P124-K013)Swetha, N.S.N., S57 (P94-K010)

T

Tabori, U., S33 (OF262-009)Tajan, E., S35 (P78-G006)Talole, S., S9 (P286-A034)Talwar, V., S9 (P296-A036)Tan, C., S35 (P78-G006)Tan, M., S61 (OF75-006)Tandon, N., S52 (P233-J018)Taneja, S., S36 (P84-G008, P111-G010)Tang, F., S71 (P114-N002)Tavassoli, M., S19 (OF1-010), S20 (P41-D006),

S43 (P144-H006)Tawde, S., S38 (P159-G015)Taylor, V., S22 (P134-D014)Teni, T., S17 (P204-C014), S48 (P203-I014),

S51 (P206-J013, P207-J014)Thaker, D.A., S8 (P284-A033)Thakur, B., S42 (P7-H001)Thakur, J.S., S9 (P287-A035), S54 (P294-J023)Thakur, M., S37 (P157-G013)Thakur, M.H., S52 (P208-J015)Thakwani, A., S3 (P119-A011)Thavamani, A., S55 (P49-K003)Thavaraj, S., S19 (OF1-010)Thawani, R., S4 (P140-A015), S62 (P63-L003)Thiele, W., S70 (OT33-006)Thimmiah, N., S56 (P73-K005)Thomas, J., S28 (OF77-007)Thorat, R., S28 (P274-E013), S41 (OF161-004),

S45 (P45-I005), S55 (P49-K003), S69 (P153-M010)

Tidke, P., S10 (P249-B001, P250-B003, P260-B002)

Tim, D.S., S33 (P293-F019)Tiwari, P., S21 (P106-D009)Tiwary, K., S47 (P180-I013), S70 (P47-N001)Togar, T., S56 (P58-K004)Tripathi, A.K., S24 (P270-D020), S60 (P225-K021)Trivedi, H.L., S27 (P199-E011, P200-E012)Trivedi, N., S7 (P239-A027, P240-A028)Trivedi, V., S38 (P188-G018)

U

Uniyal, M.J., S24 (OF175-011)Upadhyay, P., S11 (P247-B005),

S24 (OF50-003), S34 (OF16-001), S55 (P48-K002, P49-K003), S56 (P58-K004), S60 (P220-K020)

Uttarkar, A., S64 (P151-L012)

V

Vadigoppula, A., S50 (P102-J006)Vahdat, L., S18 (O-002)Vaidya, M., S45 (P9-I002, P45-I005)Vaitiekunas, P., S21 (P57-D008)Vanan, M.I., S14 (P6-C001)Vanikar, A.V., S27 (P199-E011, P200-E012)Varadarajan, N., S62 (OF51-007)Varma, A.K., S16 (P165-C011, P166-C012),

S58 (P162-K015), S59 (P167-K016)Varma, N., S9 (P298-A037)Varma, S., S9 (P298-A037)Varughese, T., S5 (P174-A019), S6 (P234-A024)Vatsa, R., S28 (OT266-F003)Vaz, S., S47 (P132-I010)Veeramachaneni, V., S57 (P94-K010)Velusamy, T., S45 (P2-I001)Venkadakrishnan, V., S69 (P153-M010)Venkatesan, A., S3 (P122-A012)Venkatraman, P., S54 (OF283-013)Verma, S., S36 (P111-G010)

S78 Author Index

Verma, V., S30 (P205-F007), S32 (P243-F015)Vidhi, M., S26 (P108-E005)Vidula, N., S65 (P235-L018)Vinoth, K., S39 (P196-G022), S40

(P201-G023), S59 (P197-K017, P198-K018)Vishnupriya, S., S17 (P173-C013)Vishwakarma, R., S36 (P82-G007)Vishwanath, D., S57 (P94-K010)Vishwanath, L., S56 (P73-K005)Vishwanathan, S.K., S42 (P100-H003)Vittal P, V., S57 (P94-K010)Vohra, I., S13 (P257-B014)Vohra, S., S32 (P236-F014)Vuree, S., S17 (P173-C013)

W

Waghmare, S., S71 (P277-N006)Waghmare, S.K., S25 (OF168-008),

S26 (P172-E006, P179-E007), S27 (P181-E008, P183-E009)

Waghole, R., S17 (P204-C014)

Wajid, S., S25 (P95-E003)Walfish, P.G., S55 (OF295-025)Wang, Y.A., S54 (OT143-005)Ward, M., S18 (O-002)Warren, J.D., S18 (O-002)Wee Yong, V.,

S46 (P65-I006)Wery, J.P., S35 (P71-G005)Westermarck, J.,

S60 (P288-K022)Westfall, S., S33 (P293-F019)Whelan, S., S10 (P8-A038)Wiener, A., S18 (O-002)Willis, A., S18 (O-002)Willmer, T., S40 (P289-G027)Wolf, D., S65 (P235-L018)Wolf, D.J., S28 (OF77-007)Worsham, M.J., S44 (OT72-004),

S48 (P242-I016)Wu, P., S54 (OT143-005)Wünsch, B., S67 (OF128-012)

X

Xu, X., S35 (P71-G005)Yadav, S., S57 (P92-K008)Yadava, S.K., S67 (P27-M002)Yang, M., S35 (P71-G005)Yang, S., S69 (P101-M008)Yau, C., S65 (P235-L018)

Y

Yi, S., S22 (P134-D014)Young, C., S22 (P134-D014)Yuan, Z., S17 (O-001)Yuvaraj, N., S34 (P4-G001)

Z

Zanwar, S., S29 (P76-F003), S31 (P226-F010, P230-F013)

Zemp, F.J., S46 (P65-I006)Zhang, Z., S71 (P114-N002)

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