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uef.fi
PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND
Dissertations in Health Sciences
ISBN 978-952-61-2069-0ISSN 1798-5706
Dissertations in Health Sciences
PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND
SAMI RAATIKAINEN
TWIST AND OXIDATIVE STRESS RELATED BIOMARKERS IN OUTCOME PREDICTION OF PROSTATE CANCER
PATIENTS TREATED WITH RADICAL PROSTATECTOMY
This retrospective study examined the predictive value of the EMT marker TWIST and oxidative stress related biomolecules in prostate cancer patients after radical prostatectomy. Increased expression of TWIST, Nrf-2 and Prx6 was associated
with biochemical recurrence and augmented Nrf-2 expression predicted worse survival
of the patients. These biomarkers could help in developing a more accurate cancer risk
evaluation for prostate cancer patients after radical prostate surgery.
SAMI RAATIKAINEN
SAMI RAATIKAINEN
TWIST and Oxidative Stress Related Biomarkers in Outcome Prediction of Prostate Cancer Patients Treated with
Radical Prostatectomy
To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Auditorium 2, Kuopio University Hospital, Kuopio, on Friday, May 13th
2016, at 12 noon
Publications of the University of Eastern Finland Dissertations in Health Sciences
Number 340
Departments of Surgery and Pathology and Forensic Medicine, Institute of Clinical Medicine, School of Medicine, University of Eastern Finland
Departments of Surgery and Clinical Pathology, Kuopio University Hospital Kuopio
2016
Grano Oy Kuopio, 2016
Series Editors:
Professor Veli-‐‑Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology
Faculty of Health Sciences
Professor Hannele Turunen, Ph.D. Department of Nursing Science
Faculty of Health Sciences
Professor Olli Gröhn, Ph.D. A.I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology
Faculty of Health Sciences
Lecturer Veli-‐‑Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy
Faculty of Health Sciences
Distributor: University of Eastern Finland
Kuopio Campus Library P.O.Box 1627
FI-‐‑70211 Kuopio, Finland http://www.uef.fi/kirjasto
ISBN (print): 978-‐‑952-‐‑61-‐‑2069-‐‑0 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2070-‐‑6
ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714
ISSN-‐‑L: 1798-‐‑5706
III
Author’s address: Department of Surgery Kuopio University Hospital KUOPIO FINLAND
Supervisors: Professor Ylermi Soini, M.D., Ph.D.
Institute of Clinical Medicine, Pathology and Forensic Medicine University of Eastern Finland KUOPIO FINLAND Docent Sirpa Aaltomaa, M.D., Ph.D. Department of Surgery Kuopio University Hospital KUOPIO FINLAND Docent Vesa Kärjä, M.D., Ph.D. Department of Clinical Pathology Kuopio University Hospital KUOPIO FINLAND
Reviewers: Docent Juha Koskimäki, MD., Ph.D.
Department of Urology Tampere University Hospital TAMPERE FINLAND
Docent Tuomas Mirtti, MD., Ph.D. Department of Pathology, Haartman Institute University of Helsinki HELSINKI FINLAND
Opponent: Docent Mika Matikainen, M.D., Ph.D.
Department of Urology Helsinki University Central Hospital HELSINKI FINLAND
Grano Oy Kuopio, 2016
Series Editors:
Professor Veli-‐‑Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology
Faculty of Health Sciences
Professor Hannele Turunen, Ph.D. Department of Nursing Science
Faculty of Health Sciences
Professor Olli Gröhn, Ph.D. A.I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology
Faculty of Health Sciences
Lecturer Veli-‐‑Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy
Faculty of Health Sciences
Distributor: University of Eastern Finland
Kuopio Campus Library P.O.Box 1627
FI-‐‑70211 Kuopio, Finland http://www.uef.fi/kirjasto
ISBN (print): 978-‐‑952-‐‑61-‐‑2069-‐‑0 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2070-‐‑6
ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714
ISSN-‐‑L: 1798-‐‑5706
III
Author’s address: Department of Surgery Kuopio University Hospital KUOPIO FINLAND
Supervisors: Professor Ylermi Soini, M.D., Ph.D.
Institute of Clinical Medicine, Pathology and Forensic Medicine University of Eastern Finland KUOPIO FINLAND Docent Sirpa Aaltomaa, M.D., Ph.D. Department of Surgery Kuopio University Hospital KUOPIO FINLAND Docent Vesa Kärjä, M.D., Ph.D. Department of Clinical Pathology Kuopio University Hospital KUOPIO FINLAND
Reviewers: Docent Juha Koskimäki, MD., Ph.D.
Department of Urology Tampere University Hospital TAMPERE FINLAND
Docent Tuomas Mirtti, MD., Ph.D. Department of Pathology, Haartman Institute University of Helsinki HELSINKI FINLAND
Opponent: Docent Mika Matikainen, M.D., Ph.D.
Department of Urology Helsinki University Central Hospital HELSINKI FINLAND
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Raatikainen, Sami TWIST and Oxidative Stress Related Biomarkers in Outcome Prediction of Prostate Cancer Patients Treated with Radical Prostatectomy University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 340. 2016. 59 p. ISBN (print): 978-‐‑952-‐‑61-‐‑2069-‐‑0 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2070-‐‑6 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706 ABSTRACT Prostate cancer (PC) is the most frequently diagnosed cancer in Finnish men due to the common practice of testing for the presence of prostate specific antigen. Most of the organ confined PC cases have an excellent prognosis and only a minority of the patients suffer from a life-‐‑threatening disease. The evaluation of the cancer risk is based on clinical factors and pathological analysis of a biopsy sample. Additional data for assessment of cancer aggressiveness are gathered from the analysis of a prostatectomy preparate after radical prostatectomy (RP). The accuracy of these parameters tends to be insufficient in cases of localised PC. There is still a need for new prognostic tools in the cancer risk assessment to assist in prediction of the patient’s outcome and to help in choosing optimal strategies for adjuvant therapies. It is known that the epithelial-‐‑mesenchymal transition (EMT) process and oxidative stress linked pathways are activated in many cancer types. Increased concentrations of cellular signalling molecules for these pathological processes associate with aggressive behaviour of tumours. In this study, the expressions of several proteins implicated in prostate cancer pathology, i.e. the EMT-‐‑marker TWIST, androgen receptor and oxidative stress related proteins 8-‐‑hydroxydeoxyguanosine (8-‐‑OHDG), nuclear factor erythroid 2-‐‑related factor 2 (Nrf-‐‑2), peroxiredoxins (Prx) 1, 2, 5 and 6 and sulfiredoxin were analysed in samples obtained from 240 PC patients after RP. The results were compared with important clinicopathological factors, such as pathological stage, Gleason score, surgical margin status, and the patient’s outcome. TWIST, 8-‐‑OHDG and Nrf-‐‑2 concentrations were found to be elevated in malignant tissue in comparison to benign tissue. Many conventional clinical prognosticators were associated with increased expression of TWIST, Nrf-‐‑2 and Prxs. Elevated TWIST, Nrf-‐‑2 and Prx6 expression predicted shortened biochemical recurrence free survival. The augmented expression of Nrf-‐‑2 was also an independent predictor of poor overall survival.
The results of the current study indicate that TWIST and oxidative stress related biomarkers are associated with aggressive behaviour in localised PC. In the future, these molecules could be developed into candidate indicators to aid in the cancer risk evaluation of PC patients. National Library of Medicine Classification: WJ 762, WJ 768, WB 142, QZ 180 Medical Subject Headings: Prostatic Neoplasms; Prostatectomy; Prognosis; Biomarkers, Tumor; Epithelial-‐‑Mesenchymal Transition; Receptors, Androgen; Oxidative Stress
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Raatikainen, Sami TWIST and Oxidative Stress Related Biomarkers in Outcome Prediction of Prostate Cancer Patients Treated with Radical Prostatectomy University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 340. 2016. 59 p. ISBN (print): 978-‐‑952-‐‑61-‐‑2069-‐‑0 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2070-‐‑6 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706 ABSTRACT Prostate cancer (PC) is the most frequently diagnosed cancer in Finnish men due to the common practice of testing for the presence of prostate specific antigen. Most of the organ confined PC cases have an excellent prognosis and only a minority of the patients suffer from a life-‐‑threatening disease. The evaluation of the cancer risk is based on clinical factors and pathological analysis of a biopsy sample. Additional data for assessment of cancer aggressiveness are gathered from the analysis of a prostatectomy preparate after radical prostatectomy (RP). The accuracy of these parameters tends to be insufficient in cases of localised PC. There is still a need for new prognostic tools in the cancer risk assessment to assist in prediction of the patient’s outcome and to help in choosing optimal strategies for adjuvant therapies. It is known that the epithelial-‐‑mesenchymal transition (EMT) process and oxidative stress linked pathways are activated in many cancer types. Increased concentrations of cellular signalling molecules for these pathological processes associate with aggressive behaviour of tumours. In this study, the expressions of several proteins implicated in prostate cancer pathology, i.e. the EMT-‐‑marker TWIST, androgen receptor and oxidative stress related proteins 8-‐‑hydroxydeoxyguanosine (8-‐‑OHDG), nuclear factor erythroid 2-‐‑related factor 2 (Nrf-‐‑2), peroxiredoxins (Prx) 1, 2, 5 and 6 and sulfiredoxin were analysed in samples obtained from 240 PC patients after RP. The results were compared with important clinicopathological factors, such as pathological stage, Gleason score, surgical margin status, and the patient’s outcome. TWIST, 8-‐‑OHDG and Nrf-‐‑2 concentrations were found to be elevated in malignant tissue in comparison to benign tissue. Many conventional clinical prognosticators were associated with increased expression of TWIST, Nrf-‐‑2 and Prxs. Elevated TWIST, Nrf-‐‑2 and Prx6 expression predicted shortened biochemical recurrence free survival. The augmented expression of Nrf-‐‑2 was also an independent predictor of poor overall survival.
The results of the current study indicate that TWIST and oxidative stress related biomarkers are associated with aggressive behaviour in localised PC. In the future, these molecules could be developed into candidate indicators to aid in the cancer risk evaluation of PC patients. National Library of Medicine Classification: WJ 762, WJ 768, WB 142, QZ 180 Medical Subject Headings: Prostatic Neoplasms; Prostatectomy; Prognosis; Biomarkers, Tumor; Epithelial-‐‑Mesenchymal Transition; Receptors, Androgen; Oxidative Stress
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Raatikainen, Sami TWIST:n ja oksidatiiviseen stressiin liittyvien biomarkkereiden merkitys radikaalileikkauksella hoidettujen eturauhassyöpäpotilaiden ennusteen arvioinnissa. Itä-‐‑Suomen yliopisto, terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero 340. 2016. 59 s. ISBN (print): 978-‐‑952-‐‑61-‐‑2069-‐‑0 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2070-‐‑6 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706 TIIVISTELMÄ Eturauhassyöpä on miesten yleisimmin diagnosoitu syöpätauti Suomessa lisääntyneen prostataspesifisenantigeenin testauksen vuoksi. Suurimmalla osalla potilasta, joilla syöpä on rajoittunut eturauhaseen, ennuste on erinomainen ja vain pienellä osalla potilasta tauti kehittyy henkeä uhkaavaksi. Syövän riskin arviointi perustuu kliinisiin tekijöihin ja eturauhasnäytteen patologisiin tietoihin. Radikaalin eturauhasen poistoleikkauksen jälkeen lisätietoa syövän aggressiivisuudesta saadaan prostatapreparaatin analyysistä. Näiden ennustetekijöiden tarkkuus on rajallinen paikallisen eturauhassyövän riskiarviossa. On edelleen tarvetta uusille menetelmille punnitessa syövän aggressiivisuutta ja tarvetta lisähoidoille. Aiemman tutkimustiedon perusteella, epiteliaaliseen-‐‑mesenkymaaliseen transitioon (EMT) ja oksidatiiviseen stressiin liittyvät säätelyjärjestelmät ovat aktivoituneet monissa syöpätyypeissä. Suurentuneita pitoisuuksia näihin prosesseihin liittyviä soluvälittäjäaineita on havaittu aggressiivisesti käyttäytyvissä syövissä. Tässä tutkimuksessa analysoitiin EMT-‐‑välittäjä TWIST:n, androgeenireseptorin, 8-‐‑hydroksideguanosiinin (8-‐‑OHDG), erythroid 2-‐‑related factor 2:n (Nrf-‐‑2), peroksiredoksiinien (Prx) 1, 2, 5 ja 6 ja sulfiredoksiinin ilmentymistä 240:n radikaalileikkauksella hoidetun eturauhassyöpäpotilaan kudosnäytteissä. Tuloksia verrattiin kliinispatologisiin tekijöihin, kuten patologiseen levinneisyysluokkaan, Gleason-‐‑pisteisiin, leikkausmarginaalin puhtauteen ja potilaiden tautivapaaseen aikaan. TWIST:n, 8-‐‑OHDG:n ja Nrf-‐‑2:n ilmentyminen oli lisääntynyt syöpäkudoksessa verrattuna hyvänlaatuiseen kudokseen. Monet kliinispatologiset tekijät olivat yhteydessä TWIST:n, Nrf-‐‑2:n ja Prx:n ilmentymiseen. Lisääntynyt TWIST-‐‑, Nrf-‐‑2-‐‑ ja Prx6-‐‑aktiivisuus ennustivat PSA:lla mitattua uusiutumaa. Korkea Nrf-‐‑2-‐‑ilmentyminen myös ennusti itsenäisesti yleistä kuolleisuutta. Tutkimuksen tulokset osoittavat, että TWIST ja oksidatiiviseen stressiin liittyvät markkerit ovat yhteydessä paikallisen eturauhassyövän aggressiiviseen käyttäytymiseen. Tulevaisuudessa nämä molekyylit voisivat soveltua eturauhassyöpäpotilaiden riskiarvioon. Luokitus: WJ 762, WJ 768, WB 142, QZ 180 Yleinen Suomalainen asiasanasto: Eturauhassyöpä; Leikkaushoito; Markkerit; Oksidatiivinen stressi
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Raatikainen, Sami TWIST:n ja oksidatiiviseen stressiin liittyvien biomarkkereiden merkitys radikaalileikkauksella hoidettujen eturauhassyöpäpotilaiden ennusteen arvioinnissa. Itä-‐‑Suomen yliopisto, terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero 340. 2016. 59 s. ISBN (print): 978-‐‑952-‐‑61-‐‑2069-‐‑0 ISBN (pdf): 978-‐‑952-‐‑61-‐‑2070-‐‑6 ISSN (print): 1798-‐‑5706 ISSN (pdf): 1798-‐‑5714 ISSN-‐‑L: 1798-‐‑5706 TIIVISTELMÄ Eturauhassyöpä on miesten yleisimmin diagnosoitu syöpätauti Suomessa lisääntyneen prostataspesifisenantigeenin testauksen vuoksi. Suurimmalla osalla potilasta, joilla syöpä on rajoittunut eturauhaseen, ennuste on erinomainen ja vain pienellä osalla potilasta tauti kehittyy henkeä uhkaavaksi. Syövän riskin arviointi perustuu kliinisiin tekijöihin ja eturauhasnäytteen patologisiin tietoihin. Radikaalin eturauhasen poistoleikkauksen jälkeen lisätietoa syövän aggressiivisuudesta saadaan prostatapreparaatin analyysistä. Näiden ennustetekijöiden tarkkuus on rajallinen paikallisen eturauhassyövän riskiarviossa. On edelleen tarvetta uusille menetelmille punnitessa syövän aggressiivisuutta ja tarvetta lisähoidoille. Aiemman tutkimustiedon perusteella, epiteliaaliseen-‐‑mesenkymaaliseen transitioon (EMT) ja oksidatiiviseen stressiin liittyvät säätelyjärjestelmät ovat aktivoituneet monissa syöpätyypeissä. Suurentuneita pitoisuuksia näihin prosesseihin liittyviä soluvälittäjäaineita on havaittu aggressiivisesti käyttäytyvissä syövissä. Tässä tutkimuksessa analysoitiin EMT-‐‑välittäjä TWIST:n, androgeenireseptorin, 8-‐‑hydroksideguanosiinin (8-‐‑OHDG), erythroid 2-‐‑related factor 2:n (Nrf-‐‑2), peroksiredoksiinien (Prx) 1, 2, 5 ja 6 ja sulfiredoksiinin ilmentymistä 240:n radikaalileikkauksella hoidetun eturauhassyöpäpotilaan kudosnäytteissä. Tuloksia verrattiin kliinispatologisiin tekijöihin, kuten patologiseen levinneisyysluokkaan, Gleason-‐‑pisteisiin, leikkausmarginaalin puhtauteen ja potilaiden tautivapaaseen aikaan. TWIST:n, 8-‐‑OHDG:n ja Nrf-‐‑2:n ilmentyminen oli lisääntynyt syöpäkudoksessa verrattuna hyvänlaatuiseen kudokseen. Monet kliinispatologiset tekijät olivat yhteydessä TWIST:n, Nrf-‐‑2:n ja Prx:n ilmentymiseen. Lisääntynyt TWIST-‐‑, Nrf-‐‑2-‐‑ ja Prx6-‐‑aktiivisuus ennustivat PSA:lla mitattua uusiutumaa. Korkea Nrf-‐‑2-‐‑ilmentyminen myös ennusti itsenäisesti yleistä kuolleisuutta. Tutkimuksen tulokset osoittavat, että TWIST ja oksidatiiviseen stressiin liittyvät markkerit ovat yhteydessä paikallisen eturauhassyövän aggressiiviseen käyttäytymiseen. Tulevaisuudessa nämä molekyylit voisivat soveltua eturauhassyöpäpotilaiden riskiarvioon. Luokitus: WJ 762, WJ 768, WB 142, QZ 180 Yleinen Suomalainen asiasanasto: Eturauhassyöpä; Leikkaushoito; Markkerit; Oksidatiivinen stressi
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to Kerttu
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to Kerttu
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Acknowledgements
This research was carried out in the Departments of Urology and Clinical Pathology in Kuopio University Hospital and Department of Pathology and Forensic Medicine in University of Eastern Finland. I want to express my gratitude and respect to my principal supervisor, Professor Ylermi Soini, for the opportunity to join this research team. You provided the best scientific knowledge of cancer research and supported me during this study. I owe my deepest thanks to my supervisor and Chief of the Urology Department, Docent Sirpa Aaltomaa, for sharing her experience and guidance. You taught me how to undertake research and developed my scientific writing skills, provided advice and encouragement, even in my darkest hours of despair.
I am grateful to my supervisor Docent Vesa Kärjä for his provision of such excellent expertise in pathology. Your guidance and support have been indispensable during these years. I would like to thank my official reviewers Docent Juha Koskimäki and Docent Tuomas Mirtti for their valuable criticism and encouraging comments. I wish to thank Professor Heikki Kröger for his support and making it possible that much of my research could be scheduled during the working hours of my main position at the University. I thank Mrs Aija Parkkinen for technical assistance in the laboratory, Professor Jorma J. Palvimo for providing androgen receptor antibody and Ewen MacDonald, Ph.D. for revising the English of this thesis. I wish to express my warmest gratitude to my friends and neighbours and colleagues in the Department of Urology for their encouragement during this study. I also want to thank my mother Elina, my brother Tommi and his family and my parents-‐‑in-‐‑law Maija-‐‑Leena and Ilkka for their love and support. Finally I owe my deepest love and thankfulness to my family. My lovely and precious daughter Kerttu, you have brought such joy into my life. My wife Kaisa, the love of my life, there are no words to express my gratitude for all the encouragement and patience during these years. Kuopio, February 2016 Sami Raatikainen
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Acknowledgements
This research was carried out in the Departments of Urology and Clinical Pathology in Kuopio University Hospital and Department of Pathology and Forensic Medicine in University of Eastern Finland. I want to express my gratitude and respect to my principal supervisor, Professor Ylermi Soini, for the opportunity to join this research team. You provided the best scientific knowledge of cancer research and supported me during this study. I owe my deepest thanks to my supervisor and Chief of the Urology Department, Docent Sirpa Aaltomaa, for sharing her experience and guidance. You taught me how to undertake research and developed my scientific writing skills, provided advice and encouragement, even in my darkest hours of despair.
I am grateful to my supervisor Docent Vesa Kärjä for his provision of such excellent expertise in pathology. Your guidance and support have been indispensable during these years. I would like to thank my official reviewers Docent Juha Koskimäki and Docent Tuomas Mirtti for their valuable criticism and encouraging comments. I wish to thank Professor Heikki Kröger for his support and making it possible that much of my research could be scheduled during the working hours of my main position at the University. I thank Mrs Aija Parkkinen for technical assistance in the laboratory, Professor Jorma J. Palvimo for providing androgen receptor antibody and Ewen MacDonald, Ph.D. for revising the English of this thesis. I wish to express my warmest gratitude to my friends and neighbours and colleagues in the Department of Urology for their encouragement during this study. I also want to thank my mother Elina, my brother Tommi and his family and my parents-‐‑in-‐‑law Maija-‐‑Leena and Ilkka for their love and support. Finally I owe my deepest love and thankfulness to my family. My lovely and precious daughter Kerttu, you have brought such joy into my life. My wife Kaisa, the love of my life, there are no words to express my gratitude for all the encouragement and patience during these years. Kuopio, February 2016 Sami Raatikainen
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This study was supported financially by the Finnish Urological Association, the Finnish Anti-‐‑tuberculosis Association and the Finnish Cancer Society.
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List of the original publications
This dissertation is based on the following original publications:
I Raatikainen S, Aaltomaa S, Palvimo JJ, Kärjä V, Soini Y. TWIST overexpression predicts biochemical recurrence-‐‑free survival in prostate cancer patients treated with radical prostatectomy. Scand J Urol 49(1): 51-‐‑7, 2015.
II Raatikainen S, Aaltomaa S, Kärjä V, Soini Y. Increased nuclear factor erythroid 2-‐‑related factor 2 expression predicts worse prognosis of prostate cancer patients treated with radical prostatectomy. Hum Pathol. 45(11): 2211-‐‑7, 2014.
III Raatikainen S, Aaltomaa S, Kärjä V, Soini Y. Increased peroxiredoxin 6 expression predicts biochemical recurrence in prostate cancer patients after radical prostatectomy. Anticancer Res. 35(12):6465-‐‑70, 2015.
The publications were adapted with the permission of the copyright owners.
XII
This study was supported financially by the Finnish Urological Association, the Finnish Anti-‐‑tuberculosis Association and the Finnish Cancer Society.
XIII
List of the original publications
This dissertation is based on the following original publications:
I Raatikainen S, Aaltomaa S, Palvimo JJ, Kärjä V, Soini Y. TWIST overexpression predicts biochemical recurrence-‐‑free survival in prostate cancer patients treated with radical prostatectomy. Scand J Urol 49(1): 51-‐‑7, 2015.
II Raatikainen S, Aaltomaa S, Kärjä V, Soini Y. Increased nuclear factor erythroid 2-‐‑related factor 2 expression predicts worse prognosis of prostate cancer patients treated with radical prostatectomy. Hum Pathol. 45(11): 2211-‐‑7, 2014.
III Raatikainen S, Aaltomaa S, Kärjä V, Soini Y. Increased peroxiredoxin 6 expression predicts biochemical recurrence in prostate cancer patients after radical prostatectomy. Anticancer Res. 35(12):6465-‐‑70, 2015.
The publications were adapted with the permission of the copyright owners.
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Contents
1 INTRODUCTION ............................................................................... 1 2 REVIEW OF THE LITERATURE ..................................................... 3
2.1 Epidemiology .................................................................................. 3 2.2 Risk factors ...................................................................................... 3 2.3 Prevention ....................................................................................... 4 2.4 Diagnosis ......................................................................................... 4
2.4.1 Clinical diagnosis ................................................................... 4 2.4.2 Tumor node metastasis (TNM) classification .................... 4 2.4.3 Prostate biopsy ....................................................................... 5 2.4.4 Prostate specific antigen (PSA) ............................................ 5 2.4.5 Histology and Gleason score ............................................... 6 2.4.6 Risk groups ............................................................................. 6
2.5 Treatments for localised prostate cancer (PC) ............................ 7 2.5.1 Active surveillance (AS) ....................................................... 7 2.5.2 Definitive radiotherapy (RT) ............................................... 7 2.5.3 Radical prostatectomy (RP) ................................................. 7 2.5.3.1 Clinicopathological prognosis factors ........................ 7
2.5.3.1.1 Capsule invasion ................................................... 8 2.5.3.1.2 Surgical margin status .......................................... 8 2.5.3.1.3 Seminal vesicle invasion ....................................... 8 2.5.3.2 Definition of biochemical recurrence (BCR) after RP 8 2.6 Biomolecular prognostic markers ................................................ 8
2.6.1 Apoptosis ................................................................................ 9 2.6.2 Signal transduction ................................................................ 9 2.6.3 Proliferation and cell cycle regulation ................................ 9 2.6.4 Angiogenesis .......................................................................... 10 2.6.5 Androgen receptor (AR) ....................................................... 10 2.6.6 Cell adhesion .......................................................................... 10
2.7 Epithelial-‐‑mesenchymal transition (EMT) .................................. 10 2.7.1 EMT-‐‑related transcription factor TWIST and cancer ....... 11 2.7.2 Role of TWIST in prostate cancer ........................................ 12
2.8 Oxidative damage .......................................................................... 13 2.8.1 Oxidative stress in carcinogenesis ....................................... 13 2.8.1.1 8-‐‑Hydroxydeoxyguanosine (8-‐‑OHDG) ...................... 14 2.8.1.2 Nuclear factor erythroid 2-‐‑related factor 2 (Nrf-‐‑2) ... 14 2.8.1.3 Peroxiredoxin (Prx) and sulfiredoxin (Srx) ................ 16
3 AIMS OF THE STUDY ...................................................................... 18
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Contents
1 INTRODUCTION ............................................................................... 1 2 REVIEW OF THE LITERATURE ..................................................... 3
2.1 Epidemiology .................................................................................. 3 2.2 Risk factors ...................................................................................... 3 2.3 Prevention ....................................................................................... 4 2.4 Diagnosis ......................................................................................... 4
2.4.1 Clinical diagnosis ................................................................... 4 2.4.2 Tumor node metastasis (TNM) classification .................... 4 2.4.3 Prostate biopsy ....................................................................... 5 2.4.4 Prostate specific antigen (PSA) ............................................ 5 2.4.5 Histology and Gleason score ............................................... 6 2.4.6 Risk groups ............................................................................. 6
2.5 Treatments for localised prostate cancer (PC) ............................ 7 2.5.1 Active surveillance (AS) ....................................................... 7 2.5.2 Definitive radiotherapy (RT) ............................................... 7 2.5.3 Radical prostatectomy (RP) ................................................. 7 2.5.3.1 Clinicopathological prognosis factors ........................ 7
2.5.3.1.1 Capsule invasion ................................................... 8 2.5.3.1.2 Surgical margin status .......................................... 8 2.5.3.1.3 Seminal vesicle invasion ....................................... 8 2.5.3.2 Definition of biochemical recurrence (BCR) after RP 8 2.6 Biomolecular prognostic markers ................................................ 8
2.6.1 Apoptosis ................................................................................ 9 2.6.2 Signal transduction ................................................................ 9 2.6.3 Proliferation and cell cycle regulation ................................ 9 2.6.4 Angiogenesis .......................................................................... 10 2.6.5 Androgen receptor (AR) ....................................................... 10 2.6.6 Cell adhesion .......................................................................... 10
2.7 Epithelial-‐‑mesenchymal transition (EMT) .................................. 10 2.7.1 EMT-‐‑related transcription factor TWIST and cancer ....... 11 2.7.2 Role of TWIST in prostate cancer ........................................ 12
2.8 Oxidative damage .......................................................................... 13 2.8.1 Oxidative stress in carcinogenesis ....................................... 13 2.8.1.1 8-‐‑Hydroxydeoxyguanosine (8-‐‑OHDG) ...................... 14 2.8.1.2 Nuclear factor erythroid 2-‐‑related factor 2 (Nrf-‐‑2) ... 14 2.8.1.3 Peroxiredoxin (Prx) and sulfiredoxin (Srx) ................ 16
3 AIMS OF THE STUDY ...................................................................... 18
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4 MATERIALS AND METHODS ....................................................... 19 4.1 Study population ............................................................................ 19 4.2 Histopathological analyses ........................................................... 21 4.3 Immunohistochemistry ................................................................. 22 4.4 Evaluation of the expression ........................................................ 23 4.5 Statistical analyses .......................................................................... 26 4.6 Ethical considerations .................................................................... 26
5 RESULTS .............................................................................................. 27
5.1 TWIST and AR expression and their association with clinicopathological prognostigators (I) ....................................... 27 5.2 TWIST and AR expressions in the prediction of BFS (I) .......... 28 5.3 8-‐‑OHDG and Nrf-‐‑2 expression and their association with clinicopathological prognostigators (II) ...................................... 29 5.4 Nrf-‐‑2 expression in survival analysis (II) ................................... 30 5.5 The association between Prxs and Srx and clinicopathlological prognostigators (III) ....................................................................... 32 5.6 Prx2 and Prx6 expression in survival analysis (III) ................... 32
6 DISCUSSION ...................................................................................... 34
6.1 TWIST and AR in Prognosis of PC (I) ......................................... 34 6.2 8-‐‑OHDG and Nrf-‐‑2 in prognosis of PC (II) ................................ 35 6.3 Prxs and Srx in prognosis of PC (III) ........................................... 36 6.4 Clinical implications ...................................................................... 37 6.5 Limitations ...................................................................................... 38 6.6 Future perspectives ........................................................................ 38
7 SUMMARY AND CONCLUSIONS ............................................... 39 8 REFERENCES ...................................................................................... 40 APPENDIX: ORIGINAL PUBLICATIONS I-‐‑III
XVII
Abbreviations
AKT2 V-‐‑akt murine thymoma viral
oncogene homolog 2
AR Androgen receptor
ARE Antioxidant response element
AS Active surveillance
Bax Bcl-‐‑2-‐‑like protein 4
Bcl-‐‑2 B-‐‑cell lymphoma 2
BCR Biochemical recurrence
BFS Biochemical recurrence free
survival
Bmi1 B-‐‑cell-‐‑specific murine
leukemia virus integration
site 1
CI Confidence interval
c-‐‑Nrf-‐‑2 Nrf-‐‑2 expression in cytoplasm
cT Clinical stage
DNA Deoxyribonucleic acid
DRE Digital rectal examination
EDTA Ethylendiaminetetraacetate
EMT Epithelial-‐‑mesenchymal
transition
HER-‐‑2 human epidermal growth
factor receptor 2
HR Hazard ratio
ISUP International Society of
Urological Pathology
Keap1 Kelch ECH associating
protein 1
Maf Musculoaponeurotic
fibrosarcoma oncogene
homolog
M-‐‑TWIST TWIST expression in the
margin area of tumour
n-‐‑Nrf-‐‑2 Nrf-‐‑2 expression in nucleus
Nrf-‐‑2 Nuclear factor erythroid 2-‐‑
related factor 2
8-‐‑OHDG 8-‐‑Hydroxydeoxyguanosine
OS Overall survivall
p53 Tumour suppressor gene,
phosphoprotein 53
PBS Phosphate buffered saline
PC Prostate cancer
PCS Prostate cancer specific
survival
Prx Peroxiredoxin
PSA Prostate specific antigen
pT Pathological stage
RALP Robotic assisted radical
prostatectomy
Ras Rat sarcoma viral oncogene
homolog
ROS Reactive oxygen species
RP Radical prostatectomy
RT Radiotherapy
SD Standard deviation
Srx Sulfiredoxin
XVI
4 MATERIALS AND METHODS ....................................................... 19 4.1 Study population ............................................................................ 19 4.2 Histopathological analyses ........................................................... 21 4.3 Immunohistochemistry ................................................................. 22 4.4 Evaluation of the expression ........................................................ 23 4.5 Statistical analyses .......................................................................... 26 4.6 Ethical considerations .................................................................... 26
5 RESULTS .............................................................................................. 27
5.1 TWIST and AR expression and their association with clinicopathological prognostigators (I) ....................................... 27 5.2 TWIST and AR expressions in the prediction of BFS (I) .......... 28 5.3 8-‐‑OHDG and Nrf-‐‑2 expression and their association with clinicopathological prognostigators (II) ...................................... 29 5.4 Nrf-‐‑2 expression in survival analysis (II) ................................... 30 5.5 The association between Prxs and Srx and clinicopathlological prognostigators (III) ....................................................................... 32 5.6 Prx2 and Prx6 expression in survival analysis (III) ................... 32
6 DISCUSSION ...................................................................................... 34
6.1 TWIST and AR in Prognosis of PC (I) ......................................... 34 6.2 8-‐‑OHDG and Nrf-‐‑2 in prognosis of PC (II) ................................ 35 6.3 Prxs and Srx in prognosis of PC (III) ........................................... 36 6.4 Clinical implications ...................................................................... 37 6.5 Limitations ...................................................................................... 38 6.6 Future perspectives ........................................................................ 38
7 SUMMARY AND CONCLUSIONS ............................................... 39 8 REFERENCES ...................................................................................... 40 APPENDIX: ORIGINAL PUBLICATIONS I-‐‑III
XVII
Abbreviations
AKT2 V-‐‑akt murine thymoma viral
oncogene homolog 2
AR Androgen receptor
ARE Antioxidant response element
AS Active surveillance
Bax Bcl-‐‑2-‐‑like protein 4
Bcl-‐‑2 B-‐‑cell lymphoma 2
BCR Biochemical recurrence
BFS Biochemical recurrence free
survival
Bmi1 B-‐‑cell-‐‑specific murine
leukemia virus integration
site 1
CI Confidence interval
c-‐‑Nrf-‐‑2 Nrf-‐‑2 expression in cytoplasm
cT Clinical stage
DNA Deoxyribonucleic acid
DRE Digital rectal examination
EDTA Ethylendiaminetetraacetate
EMT Epithelial-‐‑mesenchymal
transition
HER-‐‑2 human epidermal growth
factor receptor 2
HR Hazard ratio
ISUP International Society of
Urological Pathology
Keap1 Kelch ECH associating
protein 1
Maf Musculoaponeurotic
fibrosarcoma oncogene
homolog
M-‐‑TWIST TWIST expression in the
margin area of tumour
n-‐‑Nrf-‐‑2 Nrf-‐‑2 expression in nucleus
Nrf-‐‑2 Nuclear factor erythroid 2-‐‑
related factor 2
8-‐‑OHDG 8-‐‑Hydroxydeoxyguanosine
OS Overall survivall
p53 Tumour suppressor gene,
phosphoprotein 53
PBS Phosphate buffered saline
PC Prostate cancer
PCS Prostate cancer specific
survival
Prx Peroxiredoxin
PSA Prostate specific antigen
pT Pathological stage
RALP Robotic assisted radical
prostatectomy
Ras Rat sarcoma viral oncogene
homolog
ROS Reactive oxygen species
RP Radical prostatectomy
RT Radiotherapy
SD Standard deviation
Srx Sulfiredoxin
XVIII
Trx Thioredoxin
TNM Tumour node metastasis
UICC Union International Contre le
Cancer
VEGF Vascular endothelial growth
factor
1 Introduction
Prostate cancer (PC) is the most common neoplasm among elderly men in the western countries. Currently, it is diagnosed more often at an early stage even without clinical symptoms due to the increase in prostate specific antigen (PSA) testing and screening. Many of the patients have low risk disease with no impact on life expectancy (Boyle, Ferlay 2005, Siegel et al. 2014, Bosetti et al. 2011). As a result of the elevated incidence and continuously growing proportion of elderly men in the developing countries, the total cost of PC is substantial and increasing; in 2009, it was estimated to be 8.43 billion euros in the European Union (Luengo-‐‑Fernandez et al. 2013).
Radical prostatectomy (RP) is commonly offered as a standard curative treatment for localised PC. However, this surgical procedure is associated with adverse effects, such as erectile dysfunction and urinary incontinence that may decrease quality of life (Sanda et al. 2008). In order to avoid overtreatment and lessen the economic burden, patients with low risk PC can be alternatively recommended to be subjected to active surveillance (AS) instead of definite treatment (Bastian et al. 2009). The choice of either surveillance or radical treatment, or whether or not to provide secondary treatments after curative procedures can be challenging, if the evaluation is only based on conventional clinicopathological prognostic factors. In order to predict more accurately the behavior of PC, several biomarkers have been proposed for clinical use. Despite some promising finding in cancer research, no molecular biomarker has been demonstrated to be suitable and sufficiently reliable for use in the clinic (Heidenreich et al. 2011).
Epithelial-‐‑mesenchymal transition (EMT) is an important cellular process in embryogenesis causing epithelial cells to lose their cell contacts and exhibit mesenchymal characteristics after a remodelling of the cytoskeleton. In addition to the physiological role of EMT-‐‑phenomenon, this process is activated in carcinogenesis, allowing tumour cells to become invasive and seed metastases (Kang, Massague 2004, Thiery 2002). The transcription factor TWIST is an inducer of EMT and its overexpression facilitates tumour progression in many types of cancer including PC (Yang et al. 2004, Kwok et al. 2005). The regulatory role of TWIST has not only been confirmed in localised and advanced PC but also been linked to androgen receptor (AR) expression (Shiota et al. 2010).
Mammalian cells can become exposed to reactive oxygen species (ROS); these are generated either as a result of aerobic respiration under physiological conditions or produced by exogenous stressors, such as toxic chemicals and radiation. ROS are able to damage many cellular macromolecules not only proteins but also those in lipid layers and DNA. During conditions of oxidative stress, numerous protective pathways are activated, leading to augmented gene expression and increased levels of neutralizing enzymes. If the capacity of mechanisms combating against ROS is overwhelmed, the cell starts to suffer oxidative stress with the associated cellular damage. Oxidative injury has been found to be linked with many steps of carcinogenesis including cancer initiation, promotion and progression (Karihtala, Soini 2007).
In this thesis, the expression levels of TWIST, AR as well as those of oxidative stress related biomolecules (8-‐‑hydroxydeoxyguanosine (8-‐‑OHDG), nuclear factor erythroid 2-‐‑
XVIII
Trx Thioredoxin
TNM Tumour node metastasis
UICC Union International Contre le
Cancer
VEGF Vascular endothelial growth
factor
1 Introduction
Prostate cancer (PC) is the most common neoplasm among elderly men in the western countries. Currently, it is diagnosed more often at an early stage even without clinical symptoms due to the increase in prostate specific antigen (PSA) testing and screening. Many of the patients have low risk disease with no impact on life expectancy (Boyle, Ferlay 2005, Siegel et al. 2014, Bosetti et al. 2011). As a result of the elevated incidence and continuously growing proportion of elderly men in the developing countries, the total cost of PC is substantial and increasing; in 2009, it was estimated to be 8.43 billion euros in the European Union (Luengo-‐‑Fernandez et al. 2013).
Radical prostatectomy (RP) is commonly offered as a standard curative treatment for localised PC. However, this surgical procedure is associated with adverse effects, such as erectile dysfunction and urinary incontinence that may decrease quality of life (Sanda et al. 2008). In order to avoid overtreatment and lessen the economic burden, patients with low risk PC can be alternatively recommended to be subjected to active surveillance (AS) instead of definite treatment (Bastian et al. 2009). The choice of either surveillance or radical treatment, or whether or not to provide secondary treatments after curative procedures can be challenging, if the evaluation is only based on conventional clinicopathological prognostic factors. In order to predict more accurately the behavior of PC, several biomarkers have been proposed for clinical use. Despite some promising finding in cancer research, no molecular biomarker has been demonstrated to be suitable and sufficiently reliable for use in the clinic (Heidenreich et al. 2011).
Epithelial-‐‑mesenchymal transition (EMT) is an important cellular process in embryogenesis causing epithelial cells to lose their cell contacts and exhibit mesenchymal characteristics after a remodelling of the cytoskeleton. In addition to the physiological role of EMT-‐‑phenomenon, this process is activated in carcinogenesis, allowing tumour cells to become invasive and seed metastases (Kang, Massague 2004, Thiery 2002). The transcription factor TWIST is an inducer of EMT and its overexpression facilitates tumour progression in many types of cancer including PC (Yang et al. 2004, Kwok et al. 2005). The regulatory role of TWIST has not only been confirmed in localised and advanced PC but also been linked to androgen receptor (AR) expression (Shiota et al. 2010).
Mammalian cells can become exposed to reactive oxygen species (ROS); these are generated either as a result of aerobic respiration under physiological conditions or produced by exogenous stressors, such as toxic chemicals and radiation. ROS are able to damage many cellular macromolecules not only proteins but also those in lipid layers and DNA. During conditions of oxidative stress, numerous protective pathways are activated, leading to augmented gene expression and increased levels of neutralizing enzymes. If the capacity of mechanisms combating against ROS is overwhelmed, the cell starts to suffer oxidative stress with the associated cellular damage. Oxidative injury has been found to be linked with many steps of carcinogenesis including cancer initiation, promotion and progression (Karihtala, Soini 2007).
In this thesis, the expression levels of TWIST, AR as well as those of oxidative stress related biomolecules (8-‐‑hydroxydeoxyguanosine (8-‐‑OHDG), nuclear factor erythroid 2-‐‑
2
related factor 2 (Nrf-‐‑2), peroxiredoxins (Prx) 1, 2, 5 and 6 and sulfiredoxin (Srx)) were analysed by immunohistochemistry in samples of PC patients who had been treated by RP. By comparing the results with conventional clinicopathological factors, biochemical recurrence and survival, their role as biomarkers was evaluated in cancer prognosis.
3
2 Review of the Literature
2.1 EPIDEMIOLOGY
PC is the most frequent malignancy experienced by elderly men in Europe. In Finland, 4778 new cases were diagnosed in 2013. Due to good prognosis of the average patient, there were 45690 patients living with the disease during that same period. There were 854 PC-‐‑related deaths registered during the year 2013 in Finland which means that PC is still the second leading cause of cancer deaths. It is believed that PSA-‐‑screening and early detection have augmented the detection of localised PC compared to distant stage disease. During the last decades, relative survival for PC has increased slightly (Figure 1)(Finnish Cancer Registry 2015, Arnold et al. 2015, De Angelis et al. 2014, Schroder et al. 2012).
Figure 1. Number of new cases and age-adjusted mortality trends of the most common malignancies among Finnish men (Finnish Cancer Registry 2015).
2.2 RISK FACTORS
The mechanisms linked with the risk of development of PC are not well established. Based on epidemiological findings, increasing age, ethnic origin and heredity are some of the few well-‐‑known risk factors. In studies conducted with autopsy material, incidental PC prevalence increases with age from 3–8% at age <35 years to 48–71% at age >79 years (Bell et al. 2015). In the black-‐‑skinned races, PC incidence and mortality is higher compared to white-‐‑skinned populations (Siegel et al. 2014). It is estimated that only 9% of patients have a
2
related factor 2 (Nrf-‐‑2), peroxiredoxins (Prx) 1, 2, 5 and 6 and sulfiredoxin (Srx)) were analysed by immunohistochemistry in samples of PC patients who had been treated by RP. By comparing the results with conventional clinicopathological factors, biochemical recurrence and survival, their role as biomarkers was evaluated in cancer prognosis.
3
2 Review of the Literature
2.1 EPIDEMIOLOGY
PC is the most frequent malignancy experienced by elderly men in Europe. In Finland, 4778 new cases were diagnosed in 2013. Due to good prognosis of the average patient, there were 45690 patients living with the disease during that same period. There were 854 PC-‐‑related deaths registered during the year 2013 in Finland which means that PC is still the second leading cause of cancer deaths. It is believed that PSA-‐‑screening and early detection have augmented the detection of localised PC compared to distant stage disease. During the last decades, relative survival for PC has increased slightly (Figure 1)(Finnish Cancer Registry 2015, Arnold et al. 2015, De Angelis et al. 2014, Schroder et al. 2012).
Figure 1. Number of new cases and age-adjusted mortality trends of the most common malignancies among Finnish men (Finnish Cancer Registry 2015).
2.2 RISK FACTORS
The mechanisms linked with the risk of development of PC are not well established. Based on epidemiological findings, increasing age, ethnic origin and heredity are some of the few well-‐‑known risk factors. In studies conducted with autopsy material, incidental PC prevalence increases with age from 3–8% at age <35 years to 48–71% at age >79 years (Bell et al. 2015). In the black-‐‑skinned races, PC incidence and mortality is higher compared to white-‐‑skinned populations (Siegel et al. 2014). It is estimated that only 9% of patients have a
4
true hereditary disease. In these patients, PC develops usually six to seven years earlier than with the sporadic disease (Nelson, De Marzo & Isaacs 2003, Leitzmann, Rohrmann 2012). However, one first-‐‑line relative having PC at least doubles the risk and two or more first-‐‑line affected relatives elevates the risk by 5-‐‑11-‐‑fold (Hemminki 2012, Jansson et al. 2012). Smokers have around a 9% to 30% increased risk for PC compared to nonsmokers, with the heaviest cicarette smoking elevating the risk of death from PC by 24–30% (Hunchared M et al. 2010). Exogenous factors, such as smoking, food with high animal fat content, promiscuous sexual behavior, increased alcohol consumption and chronic prostate inflammation have been postulated to exert an influence on PC progression (Nelson, De Marzo & Isaacs 2003). 2.3 PREVENTION Studies conducted with 5-‐‑alpha-‐‑reductase inhibitors, have shown that administration of finasteride and dutasteride reduces the risk for PC by approximately 25% only in patients with Gleason 6 disease (Thompson et al. 2003, Andriole et al. 2010). However, officially, these drugs have not been approved for prevention of PC. Even although high physical activity and low meat consumption have been linked with decreased PC risk, there is still insufficient evidence to recommend any particular life style changes or dietary interventions as a form of PC prevention (Nelson, De Marzo & Isaacs 2003, Leitzmann & Rohrmann 2012). 2.4 DIAGNOSIS
2.4.1 Clinical diagnosis Most of PC cases are found in the peripheral zone of the prostate and may be palpated. Nowadays, only 18% of tumours are diagnosed by digital rectal examination (DRE) alone and the predictive value of a suspicious DRE alone has been estimated to be as low as 5-‐‑30% when PSA is ≤ 4ng/ml (Richie et al. 1993, Carvalhal et al. 1999). An abnormal DRE finding predicts the presence of a more aggressive tumour and a higher Gleason score (Gosselaar et al. 2008). 2.4.2 Tumour Node Metastasis (TNM) classification PC is staged by the TNM classification system (Table 1) according to Union International Contre le Cancer (UICC) guidelines. Tumours belonging to T-‐‑classes of 1 and 2 are confined within the prostate. Tumours in T-‐‑classes of 3 and 4 are defined as locally advanced. N-‐‑class defines the presence of node metastases and distant metastases are indicated by the M-‐‑class (Sobin et al. 2010). In the case of localised and locally advanced PC, a higher T-‐‑class has been shown to predict an increased cancer recurrence rate and poorer survival. If one takes a ten year perspective, then the risk for biochemical recurrence (BCR) is 23–31% in patients with clinical stage (cT) 1 but 85% in those with cT3 tumours after RP (Roehl et al. 2004). The corresponding five year BCR probabilities are 7 – 12 % and 86 – 89 % in patients with pathological stage (pT) 2 and pT3 tumous, respectively (Chun et al. 2006). In addition, pT-‐‑class is an independent predictor of prostate cancer specific survival (PCS) (Porter et al. 2006).
5
Table 1. Tumour Node Metastasis (TNM) classification of prostate cancer according to Union Internationale Contre le Cancer (UICC) (Sobin et al. 2010). T – Primary tumour TX Primary tumour cannot be assessed T0 No evidence of primary tumour T1 Clinically inapparent tumour not palpable or visible by imaging T1a Tumour incidental histological finding in 5% or less of tissue resected T1b Tumour incidental histological finding in more than 5% of tissue resected T1c Tumour identified by needle biopsy (e.g. because of elevated PSAlevel T2 Tumour confined within the prostate T2a Tumour involves one half of one lobe or less T2b Tumour involves more than half of one lobe, but not both lobes T2c Tumour involves both lobes T3 Tumour extends through the prostatic capsule T3a Extracapsular extension (unilateral or bilateral) including microscopic bladder neck
involvement T3b Tumour invades seminal vesicle(s) T4 Tumour is fixed or invades adjacent structures other than seminal vesicles: external sphincter,
rectum, levator muscles, and/or pelvic wall N - Regional lymph nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Regional lymph node metastasis M - Distant metastasis MX Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis 2.4.3 Prostate biopsy The diagnosis of PC is based on the histopathological evaluation of a biopsy sample. The indication for biopsies is abnormal DRE or an elevated PSA value. Usually, 10-‐‑12 transrectal or alternatively perineal ultrasound guided biopsies are taken according to the routine protocol (Hara et al. 2008, Shariat, Roehrborn 2008).
2.4.4 Prostate specific antigen (PSA) Under physiological conditions, PSA is excreted by the epithelial cells of the prostate (Armbruster 1993). Increased levels of serum PSA may be detected in benign prostatic hyperplasia, prostatitis, urinary retention, urinary tract infection and after prostate operations. Currently, an elevated level of serum PSA is the most important reason leading to PC diagnosis. However, PSA is not truly cancer specific but rather an organ specific marker, which makes PSA less specific in PC diagnosis (Nadler et al. 1995, Hagood, Parra &
4
true hereditary disease. In these patients, PC develops usually six to seven years earlier than with the sporadic disease (Nelson, De Marzo & Isaacs 2003, Leitzmann, Rohrmann 2012). However, one first-‐‑line relative having PC at least doubles the risk and two or more first-‐‑line affected relatives elevates the risk by 5-‐‑11-‐‑fold (Hemminki 2012, Jansson et al. 2012). Smokers have around a 9% to 30% increased risk for PC compared to nonsmokers, with the heaviest cicarette smoking elevating the risk of death from PC by 24–30% (Hunchared M et al. 2010). Exogenous factors, such as smoking, food with high animal fat content, promiscuous sexual behavior, increased alcohol consumption and chronic prostate inflammation have been postulated to exert an influence on PC progression (Nelson, De Marzo & Isaacs 2003). 2.3 PREVENTION Studies conducted with 5-‐‑alpha-‐‑reductase inhibitors, have shown that administration of finasteride and dutasteride reduces the risk for PC by approximately 25% only in patients with Gleason 6 disease (Thompson et al. 2003, Andriole et al. 2010). However, officially, these drugs have not been approved for prevention of PC. Even although high physical activity and low meat consumption have been linked with decreased PC risk, there is still insufficient evidence to recommend any particular life style changes or dietary interventions as a form of PC prevention (Nelson, De Marzo & Isaacs 2003, Leitzmann & Rohrmann 2012). 2.4 DIAGNOSIS
2.4.1 Clinical diagnosis Most of PC cases are found in the peripheral zone of the prostate and may be palpated. Nowadays, only 18% of tumours are diagnosed by digital rectal examination (DRE) alone and the predictive value of a suspicious DRE alone has been estimated to be as low as 5-‐‑30% when PSA is ≤ 4ng/ml (Richie et al. 1993, Carvalhal et al. 1999). An abnormal DRE finding predicts the presence of a more aggressive tumour and a higher Gleason score (Gosselaar et al. 2008). 2.4.2 Tumour Node Metastasis (TNM) classification PC is staged by the TNM classification system (Table 1) according to Union International Contre le Cancer (UICC) guidelines. Tumours belonging to T-‐‑classes of 1 and 2 are confined within the prostate. Tumours in T-‐‑classes of 3 and 4 are defined as locally advanced. N-‐‑class defines the presence of node metastases and distant metastases are indicated by the M-‐‑class (Sobin et al. 2010). In the case of localised and locally advanced PC, a higher T-‐‑class has been shown to predict an increased cancer recurrence rate and poorer survival. If one takes a ten year perspective, then the risk for biochemical recurrence (BCR) is 23–31% in patients with clinical stage (cT) 1 but 85% in those with cT3 tumours after RP (Roehl et al. 2004). The corresponding five year BCR probabilities are 7 – 12 % and 86 – 89 % in patients with pathological stage (pT) 2 and pT3 tumous, respectively (Chun et al. 2006). In addition, pT-‐‑class is an independent predictor of prostate cancer specific survival (PCS) (Porter et al. 2006).
5
Table 1. Tumour Node Metastasis (TNM) classification of prostate cancer according to Union Internationale Contre le Cancer (UICC) (Sobin et al. 2010). T – Primary tumour TX Primary tumour cannot be assessed T0 No evidence of primary tumour T1 Clinically inapparent tumour not palpable or visible by imaging T1a Tumour incidental histological finding in 5% or less of tissue resected T1b Tumour incidental histological finding in more than 5% of tissue resected T1c Tumour identified by needle biopsy (e.g. because of elevated PSAlevel T2 Tumour confined within the prostate T2a Tumour involves one half of one lobe or less T2b Tumour involves more than half of one lobe, but not both lobes T2c Tumour involves both lobes T3 Tumour extends through the prostatic capsule T3a Extracapsular extension (unilateral or bilateral) including microscopic bladder neck
involvement T3b Tumour invades seminal vesicle(s) T4 Tumour is fixed or invades adjacent structures other than seminal vesicles: external sphincter,
rectum, levator muscles, and/or pelvic wall N - Regional lymph nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Regional lymph node metastasis M - Distant metastasis MX Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis 2.4.3 Prostate biopsy The diagnosis of PC is based on the histopathological evaluation of a biopsy sample. The indication for biopsies is abnormal DRE or an elevated PSA value. Usually, 10-‐‑12 transrectal or alternatively perineal ultrasound guided biopsies are taken according to the routine protocol (Hara et al. 2008, Shariat, Roehrborn 2008).
2.4.4 Prostate specific antigen (PSA) Under physiological conditions, PSA is excreted by the epithelial cells of the prostate (Armbruster 1993). Increased levels of serum PSA may be detected in benign prostatic hyperplasia, prostatitis, urinary retention, urinary tract infection and after prostate operations. Currently, an elevated level of serum PSA is the most important reason leading to PC diagnosis. However, PSA is not truly cancer specific but rather an organ specific marker, which makes PSA less specific in PC diagnosis (Nadler et al. 1995, Hagood, Parra &
6
Rauscher 1994, Oesterling et al. 1993). PSA is a continuous parameter and there is no agreement about what should be the cut-‐‑off value in PC risk evaluation. Histological PC might be found in up to 27% of men with a PSA value ≤4.0 ng/ml (Thompson et al. 2004). In addition to diagnostic purposes, PSA measures are used in PC cancer risk assessment, especially in disease follow-‐‑up after treatment. The PSA value at the time of diagnosis is also a well-‐‑known prognosticator of treatment failure after radical procedures (Paul et al. 2010, Roehl et al. 2004, Porter et al. 2006). 2.4.5 Histology and Gleason score Approximately 95% of PC cases are referred to as conventional adenocarcinomas consisting of glandular structures. Other variants of carcinoma, such as ductal, sarcomatoid and squamous cell carcinoma are rare (Santoni et al. 2015, Fine 2012). The incidence of isolated urothelial carcinoma is 4% of all prostatic neoplasms (Esrig et al. 1996). The Gleason score grading system for PC is based on the tumour histology. Nowadays, the standard histopathological report is given according to the modifications issued by the International Society of Urological Pathology (ISUP). The Gleason score is the sum of the most common and the second-‐‑most common Gleason grade in terms of tumour volume and it has a range between two and ten, with the score of ten representing the most aggressive form. Tertiary Gleason grade 4–5 and its proportion of cancer volume have also been reported (Epstein et al. 2005). The presence of gribriform glands is usually assessed as pattern 4 in routine practice (Brimo et al. 2013). Gleason grade is considered to be the strongest prognostic factor for clinical behavior and treatment failure of PC (Partin et al. 2001). Five years BCR probabilities are 9%, 44% and 90% in Gleason groups 6, 7 and 8-‐‑10 after RP, respectively (Chun et al. 2006). Higher Gleason score is also associated with shortened PCS and OS (Porter et al. 2006). 2.4.6 Risk groups The risk groups for BCR are divided into three classes (low, intermediate and high risk) criteria according to the PSA value at diagnosis, clinical T-‐‑class and Gleason score (Table 2). The classification is traditionally based on the system developed by D’Amico (D'ʹAmico et al. 1998, Cooperberg et al. 2005). Table 2. Risk groups for BCR of localised and locally advanced prostate cancer. Low-risk
Intermediate-risk High-risk
Definition
PSA < 10 ng/ml
PSA 10-20 ng/ml PSA > 20 ng/ml any PSA
and Gleason < 7
or Gleason 7 or Gleason > 7 any Gleason cT3-4
and cT1-2a
or cT2b or cT2c or cN+
Localised
Locally advanced
7
2.5 TREATMENTS FOR LOCALISED PROSTATE CANCER (PC) 2.5.1 Active surveillance (AS) Approximately 40–50% cases of new PC diagnoses represent clinical stage T1c (Klotz 2008). In order to minimize the adverse effects of curative treatments, AS can be offered to patients with low risk disease and a life expectancy of 10–15 years. In this kind of surveillance, the patient’s evaluation is based on clinical examination, frequent PSA-‐‑monitoring and repeated biopsies. AS aims to diminish the loss of quality of life and to detect possible cancer progression from the organ confined stage. Curative treatments are provided to selected patients at risk of tumour progression (Welty, Cooperberg & Carroll 2014). During ten years of surveillance, just over half of the men (55%) will terminate the AS protocol. The majority of these will be treated with radical modalities and only a minority (8–10%) terminates the surveillance by their own request (Thomsen et al. 2014, van den Bergh et al. 2009). In large cohorts of AS patients, disease specific survival with men continuing surveillance has been found to be excellent i.e. from 96 to 100% over a ten year period (Klotz et al. 2010, Thomsen et al. 2014, van den Bergh et al. 2009). 2.5.2 Definitive radiotherapy (RT) External RT can be given to PC patients in all three risk groups with a curative intent. Patients suffering from low-‐‑risk disease can be offered external RT or alternatively low dose brachytherapy which procedures having similar outcome results (Morris et al. 2013). Neo-‐‑adjuvant and adjuvant hormone therapies are recommended for those patients with intermediate or high risk PC undergoing RT (Bolla et al. 2010). There are adverse effects, such as impotence and genito-‐‑urinary toxicity associated with both RT and brachytherapy (Robinson, Moritz & Fung 2002, Zelefsky et al. 2008, Kishan, Kupelian 2015). 2.5.3 Radical prostatectomy (RP) The standard surgical treatment of PC is radical retropubic prostatectomy. The procedure can be performed by open, laparoscopic or a robotic assisted technique (RALP). During the proecedure, the entire prostatic gland is removed and the seminal vesicles are resected to achieve total eradication of cancer tissue. The procedure is often accompanied by a bilateral dissection of obturatoric or pelvic lymph nodes in subjects in the intermediate and high risk groups. Nowadays, RALP is the most commonly used technique since it is associated with lower blood transfusion rates and shorter hospital stays compared to open RP. There seems to be no significant difference between the open technique and RALP with repect to the incidence of urinary incontinence after the operation. There is some evidence that potency rates might be better in patients treated with RALP (Haglind et al. 2015, Ficarra et al. 2012, Gandaglia et al. 2014, Ramsay et al. 2012). All the surgical procedures have the same impact on cancer control, and the clearest benefit of survival can be observed with intermediate risk PC patients under the age of 65 years. In studies with a follow-‐‑up period of 18 years, PCS rates of 84.9–94.2% were reported in the low and intermediate risk groups (Wilt et al. 2012, Bill-‐‑Axelson et al. 2014). 2.5.3.1 Clinicopathological prognosis factors In addition to the possibility of a curative treatment, RP offers the advantage of accurate local staging after the prostatectomy preparate has been removed and analysed. In clinical
6
Rauscher 1994, Oesterling et al. 1993). PSA is a continuous parameter and there is no agreement about what should be the cut-‐‑off value in PC risk evaluation. Histological PC might be found in up to 27% of men with a PSA value ≤4.0 ng/ml (Thompson et al. 2004). In addition to diagnostic purposes, PSA measures are used in PC cancer risk assessment, especially in disease follow-‐‑up after treatment. The PSA value at the time of diagnosis is also a well-‐‑known prognosticator of treatment failure after radical procedures (Paul et al. 2010, Roehl et al. 2004, Porter et al. 2006). 2.4.5 Histology and Gleason score Approximately 95% of PC cases are referred to as conventional adenocarcinomas consisting of glandular structures. Other variants of carcinoma, such as ductal, sarcomatoid and squamous cell carcinoma are rare (Santoni et al. 2015, Fine 2012). The incidence of isolated urothelial carcinoma is 4% of all prostatic neoplasms (Esrig et al. 1996). The Gleason score grading system for PC is based on the tumour histology. Nowadays, the standard histopathological report is given according to the modifications issued by the International Society of Urological Pathology (ISUP). The Gleason score is the sum of the most common and the second-‐‑most common Gleason grade in terms of tumour volume and it has a range between two and ten, with the score of ten representing the most aggressive form. Tertiary Gleason grade 4–5 and its proportion of cancer volume have also been reported (Epstein et al. 2005). The presence of gribriform glands is usually assessed as pattern 4 in routine practice (Brimo et al. 2013). Gleason grade is considered to be the strongest prognostic factor for clinical behavior and treatment failure of PC (Partin et al. 2001). Five years BCR probabilities are 9%, 44% and 90% in Gleason groups 6, 7 and 8-‐‑10 after RP, respectively (Chun et al. 2006). Higher Gleason score is also associated with shortened PCS and OS (Porter et al. 2006). 2.4.6 Risk groups The risk groups for BCR are divided into three classes (low, intermediate and high risk) criteria according to the PSA value at diagnosis, clinical T-‐‑class and Gleason score (Table 2). The classification is traditionally based on the system developed by D’Amico (D'ʹAmico et al. 1998, Cooperberg et al. 2005). Table 2. Risk groups for BCR of localised and locally advanced prostate cancer. Low-risk
Intermediate-risk High-risk
Definition
PSA < 10 ng/ml
PSA 10-20 ng/ml PSA > 20 ng/ml any PSA
and Gleason < 7
or Gleason 7 or Gleason > 7 any Gleason cT3-4
and cT1-2a
or cT2b or cT2c or cN+
Localised
Locally advanced
7
2.5 TREATMENTS FOR LOCALISED PROSTATE CANCER (PC) 2.5.1 Active surveillance (AS) Approximately 40–50% cases of new PC diagnoses represent clinical stage T1c (Klotz 2008). In order to minimize the adverse effects of curative treatments, AS can be offered to patients with low risk disease and a life expectancy of 10–15 years. In this kind of surveillance, the patient’s evaluation is based on clinical examination, frequent PSA-‐‑monitoring and repeated biopsies. AS aims to diminish the loss of quality of life and to detect possible cancer progression from the organ confined stage. Curative treatments are provided to selected patients at risk of tumour progression (Welty, Cooperberg & Carroll 2014). During ten years of surveillance, just over half of the men (55%) will terminate the AS protocol. The majority of these will be treated with radical modalities and only a minority (8–10%) terminates the surveillance by their own request (Thomsen et al. 2014, van den Bergh et al. 2009). In large cohorts of AS patients, disease specific survival with men continuing surveillance has been found to be excellent i.e. from 96 to 100% over a ten year period (Klotz et al. 2010, Thomsen et al. 2014, van den Bergh et al. 2009). 2.5.2 Definitive radiotherapy (RT) External RT can be given to PC patients in all three risk groups with a curative intent. Patients suffering from low-‐‑risk disease can be offered external RT or alternatively low dose brachytherapy which procedures having similar outcome results (Morris et al. 2013). Neo-‐‑adjuvant and adjuvant hormone therapies are recommended for those patients with intermediate or high risk PC undergoing RT (Bolla et al. 2010). There are adverse effects, such as impotence and genito-‐‑urinary toxicity associated with both RT and brachytherapy (Robinson, Moritz & Fung 2002, Zelefsky et al. 2008, Kishan, Kupelian 2015). 2.5.3 Radical prostatectomy (RP) The standard surgical treatment of PC is radical retropubic prostatectomy. The procedure can be performed by open, laparoscopic or a robotic assisted technique (RALP). During the proecedure, the entire prostatic gland is removed and the seminal vesicles are resected to achieve total eradication of cancer tissue. The procedure is often accompanied by a bilateral dissection of obturatoric or pelvic lymph nodes in subjects in the intermediate and high risk groups. Nowadays, RALP is the most commonly used technique since it is associated with lower blood transfusion rates and shorter hospital stays compared to open RP. There seems to be no significant difference between the open technique and RALP with repect to the incidence of urinary incontinence after the operation. There is some evidence that potency rates might be better in patients treated with RALP (Haglind et al. 2015, Ficarra et al. 2012, Gandaglia et al. 2014, Ramsay et al. 2012). All the surgical procedures have the same impact on cancer control, and the clearest benefit of survival can be observed with intermediate risk PC patients under the age of 65 years. In studies with a follow-‐‑up period of 18 years, PCS rates of 84.9–94.2% were reported in the low and intermediate risk groups (Wilt et al. 2012, Bill-‐‑Axelson et al. 2014). 2.5.3.1 Clinicopathological prognosis factors In addition to the possibility of a curative treatment, RP offers the advantage of accurate local staging after the prostatectomy preparate has been removed and analysed. In clinical
8
practice, standard parameters, such as Gleason score, pT-‐‑class, surgical margin status, capsule invasion and seminal vesicle invasion, as assessed by the pathologist, can all be used in the cancer risk assessment of PC patients (Adamis, Varkarakis 2014). 2.5.3.1.1 Capsule invasion If the cancer tissue has invaded the prostatic capsule, this is designated as capsule invasion. In those cases with extraprostatic extension, the tumour has penetrated beyond the capsule. An extraprostatic extension is associated with shortened biochemical recurrence free survival (BFS) and worse PCS (Tanaka et al. 2003, Chun et al. 2006, Porter et al. 2006). In patients with low or intermediate risk disease, the rate for BCR after RP is reported to be 10% (no capsule invasion) and 55% (extra-‐‑capsular extension), respectively during five years (D'ʹAmico et al. 2000). 2.5.3.1.2 Surgical margin status The surgical margin is positive when tumour cells are in contact with the inked border of the tissue specimen. In RP series, the rate of positive margin has varied from 11 to 38% (Yossepowitch et al. 2009). There are several reports demonstrating that a positive margin is an independent risk factor for cancer recurrence, however the relationship between margin extent and risk of recurrence is uncertain (Sammon et al. 2013, Marks et al. 2007). Furthermore, artefacts, such as tissue crushing or incomplete inking, can make the determination of the margin status unreliable (Evans et al. 2008). 2.5.3.1.3 Seminal vesicle invasion Seminal vesicle invasion is found in 5–10% of PC patients undergoing RP. Seminal vesicle invasion independently predicts a shortened BCR and the median time to recurrence is estimated to be 2 years after RP. Of these patients, 30–60% are likely to progress to a metastatic stage within 5 years (Ploussard et al. 2013, Carver et al. 2006, Ball, Partin & Epstein 2015, Kasibhatla, Peterson & Anscher 2005). 2.5.3.2 Definition of biochemical recurrence (BCR) after RP The PSA-‐‑value is expected to be immeasurable following RP with curative intent and therefore any PSA-‐‑rise may be due to cancer recurrence in local or distant sites. The definition of BCR has commonly been stated as two subsequent PSA rises above the cut-‐‑off value 0.2 ng/ml after RP (Moul 2000, Stephenson et al. 2006, Walz et al. 2009). PSA-‐‑only recurrence has been detected in 15–40% of men subjected to RP but only a minority (20–35%) of these men develop a clinical recurrence and only around 10% will actually die of PC (Pound et al. 1999, Boorjian et al. 2011, Boorjian et al. 2012). 2.6 BIOMOLECULAR PROGNOSTIC MARKERS There are a large number of studies searching for candidate markers to help in PC prognosis. The cancer progression can trigger the activation of regulatory pathways within prostate tissue leading to altered levels of detectable biomolecules as putative indicators of PC aggressiveness. Several pathways are activated; these can be divided into apoptosis, signal transduction, proliferation and cell cycle regulation, cell adhesion and angiogenesis (Quinn, Henshall & Sutherland 2005, Lopergolo, Zaffaroni 2009).
9
2.6.1 Apoptosis Apoptosis is induced by two main routes in cells; the mitochondrion dependent and the mitochondrion independent pathways. In the mitochondrion dependent pathway, B-‐‑cell lymphoma 2 (Bcl-‐‑2) family proteins play a significant role. These proteins change the electrical potential of mitochondrial membranes, leading to an efflux of apoptosis-‐‑inducing compounds into the cell’s cytoplasm, ultimately leading to the formation of apoptosomes and the activation of caspase enzymes. In the mitochondrial independent pathway, caspases are induced directly through membrane receptor activation after a ligand binds to this receptor (Zielinski, Eigl & Chi 2013).
In the mitochondrion dependent pathway, the levels of p53 and Bcl-‐‑2 have been shown to reflect abnormal function of PC progression. The p53 protein is a tumour suppressor, the so-‐‑called guardian of the genome. When there has been genetic damage, p53 halts the cell cycle at its first phase to allow time for the action of DNA repair enzymes. If this fails, p53 launches apoptosis by inducing Bax, a proapoptotic gene of the Bcl-‐‑2 group, which ultimately leads to a process called programmed cell death (Quinn, Henshall & Sutherland 2005, Zielinski, Eigl & Chi 2013).
There are a large number of reports suggesting that in metastatic and hormone refractory PC, mutations are present in p53 and Bcl-‐‑2 linked genes accompanied by alterations in the expression levels of apoptotic signaling proteins. Some studies have also found the overexpression of p53 to be a predictor of BCR concerning localised PC (Quinn, Henshall & Sutherland 2005). Bcl-‐‑2 positivity has been revealed to predict PSA-‐‑relapse and analyses of Bcl-‐‑2 polymorphisms have suggested that certain modulated genotypes may be linked with cancer recurrence (Revelos et al. 2005, Hirata et al. 2009). 2.6.2 Signal transduction Modifications of tyrosine kinase receptors belonging the epidermal growth factor family, such as human epidermal growth factor receptor 2 (HER-‐‑2), are associated with worse outcome of PC. Overexpression of HER-‐‑2 has been linked with reduced BFS and PCS in the radically treated patients (Ross et al. 1997, Fossa et al. 2002). Caveolins, which are integral membrane proteins of the caveole, are involved in endocytosis and also act as cell signal regulators under physiological conditions. In addition, an overexpression of these normal cell-‐‑signal transduction proteins has been demonstrated to correlate with shortened BFS and aggressive behavior of PC in patients with localised disease (Yang et al. 2005, Karam et al. 2007). 2.6.3 Proliferation and cell cycle regulation The cell proliferation marker, Ki67 protein, has been one of the most extensively studied molecules of this group. An elevated level of this biomarker is associated with tumour progression and it has been shown to be an independent predictor of BFS (Bubendorf et al. 1996, Halvorsen et al. 2001). The better understanding of the molecular basis of cell cycle regulatory cyclins has revealed changes in the proteins linked with many cancer types. In the case of PC patients, alterations of the cyclin-‐‑dependent kinase inhibitor proteins p16, p21 and p27 have been associated with PSA relapse after surgery (Halvorsen et al. 2000, Lacombe et al. 2001, Freedland et al. 2003).
8
practice, standard parameters, such as Gleason score, pT-‐‑class, surgical margin status, capsule invasion and seminal vesicle invasion, as assessed by the pathologist, can all be used in the cancer risk assessment of PC patients (Adamis, Varkarakis 2014). 2.5.3.1.1 Capsule invasion If the cancer tissue has invaded the prostatic capsule, this is designated as capsule invasion. In those cases with extraprostatic extension, the tumour has penetrated beyond the capsule. An extraprostatic extension is associated with shortened biochemical recurrence free survival (BFS) and worse PCS (Tanaka et al. 2003, Chun et al. 2006, Porter et al. 2006). In patients with low or intermediate risk disease, the rate for BCR after RP is reported to be 10% (no capsule invasion) and 55% (extra-‐‑capsular extension), respectively during five years (D'ʹAmico et al. 2000). 2.5.3.1.2 Surgical margin status The surgical margin is positive when tumour cells are in contact with the inked border of the tissue specimen. In RP series, the rate of positive margin has varied from 11 to 38% (Yossepowitch et al. 2009). There are several reports demonstrating that a positive margin is an independent risk factor for cancer recurrence, however the relationship between margin extent and risk of recurrence is uncertain (Sammon et al. 2013, Marks et al. 2007). Furthermore, artefacts, such as tissue crushing or incomplete inking, can make the determination of the margin status unreliable (Evans et al. 2008). 2.5.3.1.3 Seminal vesicle invasion Seminal vesicle invasion is found in 5–10% of PC patients undergoing RP. Seminal vesicle invasion independently predicts a shortened BCR and the median time to recurrence is estimated to be 2 years after RP. Of these patients, 30–60% are likely to progress to a metastatic stage within 5 years (Ploussard et al. 2013, Carver et al. 2006, Ball, Partin & Epstein 2015, Kasibhatla, Peterson & Anscher 2005). 2.5.3.2 Definition of biochemical recurrence (BCR) after RP The PSA-‐‑value is expected to be immeasurable following RP with curative intent and therefore any PSA-‐‑rise may be due to cancer recurrence in local or distant sites. The definition of BCR has commonly been stated as two subsequent PSA rises above the cut-‐‑off value 0.2 ng/ml after RP (Moul 2000, Stephenson et al. 2006, Walz et al. 2009). PSA-‐‑only recurrence has been detected in 15–40% of men subjected to RP but only a minority (20–35%) of these men develop a clinical recurrence and only around 10% will actually die of PC (Pound et al. 1999, Boorjian et al. 2011, Boorjian et al. 2012). 2.6 BIOMOLECULAR PROGNOSTIC MARKERS There are a large number of studies searching for candidate markers to help in PC prognosis. The cancer progression can trigger the activation of regulatory pathways within prostate tissue leading to altered levels of detectable biomolecules as putative indicators of PC aggressiveness. Several pathways are activated; these can be divided into apoptosis, signal transduction, proliferation and cell cycle regulation, cell adhesion and angiogenesis (Quinn, Henshall & Sutherland 2005, Lopergolo, Zaffaroni 2009).
9
2.6.1 Apoptosis Apoptosis is induced by two main routes in cells; the mitochondrion dependent and the mitochondrion independent pathways. In the mitochondrion dependent pathway, B-‐‑cell lymphoma 2 (Bcl-‐‑2) family proteins play a significant role. These proteins change the electrical potential of mitochondrial membranes, leading to an efflux of apoptosis-‐‑inducing compounds into the cell’s cytoplasm, ultimately leading to the formation of apoptosomes and the activation of caspase enzymes. In the mitochondrial independent pathway, caspases are induced directly through membrane receptor activation after a ligand binds to this receptor (Zielinski, Eigl & Chi 2013).
In the mitochondrion dependent pathway, the levels of p53 and Bcl-‐‑2 have been shown to reflect abnormal function of PC progression. The p53 protein is a tumour suppressor, the so-‐‑called guardian of the genome. When there has been genetic damage, p53 halts the cell cycle at its first phase to allow time for the action of DNA repair enzymes. If this fails, p53 launches apoptosis by inducing Bax, a proapoptotic gene of the Bcl-‐‑2 group, which ultimately leads to a process called programmed cell death (Quinn, Henshall & Sutherland 2005, Zielinski, Eigl & Chi 2013).
There are a large number of reports suggesting that in metastatic and hormone refractory PC, mutations are present in p53 and Bcl-‐‑2 linked genes accompanied by alterations in the expression levels of apoptotic signaling proteins. Some studies have also found the overexpression of p53 to be a predictor of BCR concerning localised PC (Quinn, Henshall & Sutherland 2005). Bcl-‐‑2 positivity has been revealed to predict PSA-‐‑relapse and analyses of Bcl-‐‑2 polymorphisms have suggested that certain modulated genotypes may be linked with cancer recurrence (Revelos et al. 2005, Hirata et al. 2009). 2.6.2 Signal transduction Modifications of tyrosine kinase receptors belonging the epidermal growth factor family, such as human epidermal growth factor receptor 2 (HER-‐‑2), are associated with worse outcome of PC. Overexpression of HER-‐‑2 has been linked with reduced BFS and PCS in the radically treated patients (Ross et al. 1997, Fossa et al. 2002). Caveolins, which are integral membrane proteins of the caveole, are involved in endocytosis and also act as cell signal regulators under physiological conditions. In addition, an overexpression of these normal cell-‐‑signal transduction proteins has been demonstrated to correlate with shortened BFS and aggressive behavior of PC in patients with localised disease (Yang et al. 2005, Karam et al. 2007). 2.6.3 Proliferation and cell cycle regulation The cell proliferation marker, Ki67 protein, has been one of the most extensively studied molecules of this group. An elevated level of this biomarker is associated with tumour progression and it has been shown to be an independent predictor of BFS (Bubendorf et al. 1996, Halvorsen et al. 2001). The better understanding of the molecular basis of cell cycle regulatory cyclins has revealed changes in the proteins linked with many cancer types. In the case of PC patients, alterations of the cyclin-‐‑dependent kinase inhibitor proteins p16, p21 and p27 have been associated with PSA relapse after surgery (Halvorsen et al. 2000, Lacombe et al. 2001, Freedland et al. 2003).
10
2.6.4 Angiogenesis Blood vessel formation is essential to allow tumour progression. Many cancer types, including PC are characterized by increased microvessel growth. In agreement with this hypothesis, an augmented microvessel density has been shown to predict PSA recurrence after RP (de la Taille et al. 2000). Furthermore, the process of neovascularization involves many regulatory molecules, e.g. vascular endothelial growth factor (VEGF). An overexpression of VEGF in cancer tissue has been demonstrated to correlate with PSA relapse following RP and cancer death in a cohort of patients that underwent observation for organ confined PC (Strohmeyer et al. 2000, Borre, Nerstrom & Overgaard 2000). 2.6.5 Androgen receptor (AR) Since PC is an androgen-‐‑dependent cancer, one might consider the AR to be an obvious marker to predict the outcome of PC patients. However, AR signaling pathways are known to be complex and are regulated at many levels. The heterogeneity in AR expression has been demonstrated to be increased with the progression of PC from organ confined to metastatic disease, with AR signaling being more prominent in the later stages of PC (Miyamoto et al. 1993, Magi-‐‑Galluzzi et al. 1997). In patients who have undergone RP, AR overexpression has been associated with differentiation status and BFS, although the link between this parameter and cancer survival is less convincing (Li et al. 2004, Theodoropoulos et al. 2005b, Sweat et al. 1999). 2.6.6 Cell adhesion E-‐‑cadherin plays an important role in the maintenance of cell-‐‑to-‐‑cell adhesion and other aspects of cell morphology. Furthermore, E-‐‑cadherin has direct effects on signaling pathways in EMT process and modulates the activities of several transcription factors linked with remodeling of the cytoskeleton (Kang, Massague 2004). In cancer progression, E-‐‑cadherin is usually down-‐‑regulated and a reduced expression has also been linked with PSA relapse and disease progression in RP patients (Brewster et al. 1999, Kuczyk et al. 1998). 2.7 EPITHELIAL-MESENCHYMAL TRANSITION (EMT) Normal epithelial cells are arranged with close contacts to their neighbors through adherent junctions, desmosomes and tight junctions whereas mesenchymal stromal cells can move more or less loosely within the extracellular matrix. The phenomenon that converts epithelial cells into migratory mesenchymal-‐‑like cells is known as the EMT process (Kang, Massague 2004). Based on its biological context, EMT can be divided into three subtypes. In normal development, type 1 EMT takes place in implantation, embryogenesis and organ formation. Type 2 EMT is encountered during wound healing and tissue regeneration. EMT mediated inflammatory signaling ceases when the tissue damage is repaired and restored to normal conditions. However, if there should be ongoing inflammation, the prolonged EMT activation may lead fibrotic organ destruction, such as kidney, liver and lung fibrosis. The type 3 EMT process is cancer associated and characterized by diminished cell adhesion. In this type of EMT, E-‐‑cadherin is downregulated and simultaneously, mesenchymal type genes, such as vimentin and alpha smooth muscle actin, are activated, resulting in the tumor cells acquiring mesenchymal-‐‑type features. This property confers on these tumor
11
cells the capability to invade other tissues and metastasize (Acloque et al. 2009, Thiery et al. 2009, Kalluri, Weinberg 2009) (Figure 2). The activation of the EMT program has been postulated as one of the critical mechanisms initiating epithelial cell derived malignancy (Thiery 2002).
EMT is orchestrated by several genes, such as Snail1, Slug, Zeb1, TWIST and SIP1 which influence the E-‐‑cadherin switch by down-‐‑regulating it and up-‐‑regulating mesenchymal type genes. Upstream of these several signaling pathways, other protein factors such as, transforming growth factor β (TGF-‐‑β), influence the expression of these EMT related transcription factors. It has been claimed that TGF-‐‑β is most important of these up-‐‑regulating factors (Zheng, Kang 2014).
Figure 2. EMT-process in cancer initiation and progression. Epithelial cells acquire mesenchymal features, allowing them to transform so that they are invasive and spread metastases to distant sites. 2.7.1 EMT-‐‑related transcription factor TWIST and cancer TWIST is a basic helix-‐‑loop-‐‑helix transcription factor first reported to mediate mesoderm formation in Drosophila and later found to be an important regulator of mammalian
EMT
Spread to circulation
Fragmentation of
basement membrane
and invasion
10
2.6.4 Angiogenesis Blood vessel formation is essential to allow tumour progression. Many cancer types, including PC are characterized by increased microvessel growth. In agreement with this hypothesis, an augmented microvessel density has been shown to predict PSA recurrence after RP (de la Taille et al. 2000). Furthermore, the process of neovascularization involves many regulatory molecules, e.g. vascular endothelial growth factor (VEGF). An overexpression of VEGF in cancer tissue has been demonstrated to correlate with PSA relapse following RP and cancer death in a cohort of patients that underwent observation for organ confined PC (Strohmeyer et al. 2000, Borre, Nerstrom & Overgaard 2000). 2.6.5 Androgen receptor (AR) Since PC is an androgen-‐‑dependent cancer, one might consider the AR to be an obvious marker to predict the outcome of PC patients. However, AR signaling pathways are known to be complex and are regulated at many levels. The heterogeneity in AR expression has been demonstrated to be increased with the progression of PC from organ confined to metastatic disease, with AR signaling being more prominent in the later stages of PC (Miyamoto et al. 1993, Magi-‐‑Galluzzi et al. 1997). In patients who have undergone RP, AR overexpression has been associated with differentiation status and BFS, although the link between this parameter and cancer survival is less convincing (Li et al. 2004, Theodoropoulos et al. 2005b, Sweat et al. 1999). 2.6.6 Cell adhesion E-‐‑cadherin plays an important role in the maintenance of cell-‐‑to-‐‑cell adhesion and other aspects of cell morphology. Furthermore, E-‐‑cadherin has direct effects on signaling pathways in EMT process and modulates the activities of several transcription factors linked with remodeling of the cytoskeleton (Kang, Massague 2004). In cancer progression, E-‐‑cadherin is usually down-‐‑regulated and a reduced expression has also been linked with PSA relapse and disease progression in RP patients (Brewster et al. 1999, Kuczyk et al. 1998). 2.7 EPITHELIAL-MESENCHYMAL TRANSITION (EMT) Normal epithelial cells are arranged with close contacts to their neighbors through adherent junctions, desmosomes and tight junctions whereas mesenchymal stromal cells can move more or less loosely within the extracellular matrix. The phenomenon that converts epithelial cells into migratory mesenchymal-‐‑like cells is known as the EMT process (Kang, Massague 2004). Based on its biological context, EMT can be divided into three subtypes. In normal development, type 1 EMT takes place in implantation, embryogenesis and organ formation. Type 2 EMT is encountered during wound healing and tissue regeneration. EMT mediated inflammatory signaling ceases when the tissue damage is repaired and restored to normal conditions. However, if there should be ongoing inflammation, the prolonged EMT activation may lead fibrotic organ destruction, such as kidney, liver and lung fibrosis. The type 3 EMT process is cancer associated and characterized by diminished cell adhesion. In this type of EMT, E-‐‑cadherin is downregulated and simultaneously, mesenchymal type genes, such as vimentin and alpha smooth muscle actin, are activated, resulting in the tumor cells acquiring mesenchymal-‐‑type features. This property confers on these tumor
11
cells the capability to invade other tissues and metastasize (Acloque et al. 2009, Thiery et al. 2009, Kalluri, Weinberg 2009) (Figure 2). The activation of the EMT program has been postulated as one of the critical mechanisms initiating epithelial cell derived malignancy (Thiery 2002).
EMT is orchestrated by several genes, such as Snail1, Slug, Zeb1, TWIST and SIP1 which influence the E-‐‑cadherin switch by down-‐‑regulating it and up-‐‑regulating mesenchymal type genes. Upstream of these several signaling pathways, other protein factors such as, transforming growth factor β (TGF-‐‑β), influence the expression of these EMT related transcription factors. It has been claimed that TGF-‐‑β is most important of these up-‐‑regulating factors (Zheng, Kang 2014).
Figure 2. EMT-process in cancer initiation and progression. Epithelial cells acquire mesenchymal features, allowing them to transform so that they are invasive and spread metastases to distant sites. 2.7.1 EMT-‐‑related transcription factor TWIST and cancer TWIST is a basic helix-‐‑loop-‐‑helix transcription factor first reported to mediate mesoderm formation in Drosophila and later found to be an important regulator of mammalian
EMT
Spread to circulation
Fragmentation of
basement membrane
and invasion
12
embryogenesis (Castanon, Baylies 2002). TWIST is one of the inducers of EMT, not only leading to down-‐‑regulation of E-‐‑cadherin but also acting by up-‐‑regulating several mesenchymal markers. In the progression of tumours, TWIST activation allows cells to become invasive and penetrate through the lymphatic and blood vessels so that they gain access to the systemic circulation. Finally, tumour cells form micrometastases and secondary malignant tumours. TWIST expression is a sign of the aggressive potential of cancer cells, with very high expression being observed in metastatic tissues (Yang et al. 2004). The activation of TWIST has been shown to be a sufficient trigger to lead carcinoma cells through the EMT process and gain access into the circulation (Tsai et al. 2012). TWIST has also been found to inhibit several apoptotic genes, such as p53, and thus arrests on-‐‑going attempts at tumour suppression (Maestro et al. 1999). Bmi1, a member of the polycomb-‐‑group repressor complex proteins, has been demonstrated to be frequently overexpressed in tumour-‐‑initiating cells and is believed to be a direct target for the TWIST gene. Furthermore, the activation of a known oncogene, AKT2, has been identified as being a direct up-‐‑regulator of TWIST and it confers resistance to anticancer drugs (Cheng, Chan 2007).
In addition to a well known role of TWIST in cancer progression in vitro in PC-‐‑derived cell-‐‑lines, there are reports suggesting that TWIST could be used as a biomarker for cancer prognosis. Higher levels of TWIST expression have been reported in samples of malignant tissues in comparison to normal tissues in many cancer types. Furthermore, several authors have found strong positive associations between increased TWIST expression and disease progression and survival rates in malignancies, such as breast cancer, bladder cancer, cervical cancer, oral and pharyngeal squamous cell carcinoma and renal cell cancer carcinoma (Soini et al. 2011, Riaz et al. 2012, Song et al. 2014, Shibata et al. 2008a, Fan et al. 2013, Jouppila-‐‑Mättö et al. 2011, Ohba et al. 2014). 2.7.2 Role of TWIST in prostate cancer In experiments conducted in both PC cell lines and tissue material, TWIST expression has been shown to be significantly augmented in PC cells in comparison to benign cells. Kwok et al observed that the degree of TWIST expression was dependent on the Gleason score and the state of metastases suggesting that TWIST could be utilized as an indicator of cancer aggressiveness (Kwok et al. 2005). The association of TWIST expression with Gleason score and probability to bone metastases has been also detected in tissue bank material of PC samples. Furthermore, increased TWIST expression has also been linked with suppressed E-‐‑cadherin expression, suggesting that the changes in the cadherin pathway could be mediated by TWIST (Yuen et al. 2007). In addition, the E-‐‑cadherin switch to N-‐‑cadherin, a phenomenon commonly encountered in invasive carcinomas, has been observed to be activated by TWIST signaling at the transcriptional level (Alexander et al. 2006).
Since TWIST signaling is associated with unfavorable behavior in PC, it is not surprising that several studies have indicated that TWIST activation may promote the formation of metastases. This kind of TWIST mediated effect has been observed in metastatic PC cells and there is also evidence that TWIST can modulate bone cell activity in a paracrine manner in cell culture settings (Yuen et al. 2008). In agreement with findings originating from other cancer types, TWIST mediated signaling has also been demonstrated to induce lung metastases in animal models of PC (Gajula et al. 2013).
13
The pathways linked with TWIST signaling, interact with the mechanisms of some anticancer drugs, such as docetaxel and enzalutamide. There are experiments which have examined the mechanisms linking TWIST with chemo-‐‑resistance but it has also been suggested that TWIST could be a therapeutic target for future drugs (Shiota et al. 2013, Shiota et al. 2014).
There is one earlier report evaluating EMT-‐‑markers, including TWIST, in PC prognosis of clinical patients. In that study, Behnsawy et al found increased TWIST expression to be associated with traditional clinicopathological prognostic factors and to predict independently BFS in PC patients after RP (Behnsawy et al. 2013).
One important link between AR and TWIST expression was revealed by Shiota et al. In a cell line study conducted with human PC cells, induction of TWIST led to AR overexpression and conversely, silencing of TWIST suppressed AR expression and triggered cellular apoptosis. The site of action of the modulation was identified as being in the promoter region of AR-‐‑gene where there was a TWIST binding site (Shiota et al. 2010). 2.8 OXIDATIVE DAMAGE ROS refer to a wide range of molecules and molecular fragments formed in aerobic organisms mostly as a consequence of aerobic respiration. Extracellular stressors, such as exposure to chemicals or radiation can also lead to the formation of ROS. Highly reactive free radicals, superoxide-‐‑, hydroxyl-‐‑ and nitric oxide radical contain unpaired electrons on their outermost orbital. Even though it possesses less reactivity than many other free radicals, hydrogen peroxide is still important in carcinogenesis since it has the capability to diffuse through membranes and thus can reach critical cellular targets and cause an oxidative injury. Hydrogen peroxide can be produced spontaneously from oxygen or as the result of catalytic activity from superoxide radicals by superoxide dismutases (Evans, Dizdaroglu & Cooke 2004, Karihtala, Soini 2007).
Under physiological conditions, ROS are participants in many cellular processes. For example, nitric oxide radicals have an effect on platelet adhesion and vascular tone and superoxide radicals and hydrogen peroxide are involved in cellular signaling (Rhee 1999). Human cells have numerous mechanisms for neutralizing ROS. However, if the capacity of cellular antioxidant defense system becomes overwhelmed, oxidative stress occurs (Ray, Huang & Tsuji 2012).
ROS-‐‑mediated damage has been linked with several benign diseases. In times of infection, elevated levels of ROS are generated in damaged tissues by neutrophils and macrophages to combat the invading microbes. However, there are also conditions of chronic inflammation, such as rheumatoid arthritis and inflammatory bowel disease, and in this case, oxidative damage can evoke connective tissue destruction and modifications of biomolecules (Evans, Dizdaroglu & Cooke 2004, Wiseman, Halliwell 1996). 2.8.1 Oxidative stress in carcinogenesis Persistent oxidative exposure and augmented ROS formation are characteristic changes detected in carcinoma cells. Macrophage infiltration and increased glycolytic metabolism are also linked with oxidative imbalance in the tumour tissue. DNA is the most important site of ROS related damage in carcinogenesis. There are many complex and inter-‐‑related mechanisms leading to cancer initiation involving the inactivation of tumour suppressor
12
embryogenesis (Castanon, Baylies 2002). TWIST is one of the inducers of EMT, not only leading to down-‐‑regulation of E-‐‑cadherin but also acting by up-‐‑regulating several mesenchymal markers. In the progression of tumours, TWIST activation allows cells to become invasive and penetrate through the lymphatic and blood vessels so that they gain access to the systemic circulation. Finally, tumour cells form micrometastases and secondary malignant tumours. TWIST expression is a sign of the aggressive potential of cancer cells, with very high expression being observed in metastatic tissues (Yang et al. 2004). The activation of TWIST has been shown to be a sufficient trigger to lead carcinoma cells through the EMT process and gain access into the circulation (Tsai et al. 2012). TWIST has also been found to inhibit several apoptotic genes, such as p53, and thus arrests on-‐‑going attempts at tumour suppression (Maestro et al. 1999). Bmi1, a member of the polycomb-‐‑group repressor complex proteins, has been demonstrated to be frequently overexpressed in tumour-‐‑initiating cells and is believed to be a direct target for the TWIST gene. Furthermore, the activation of a known oncogene, AKT2, has been identified as being a direct up-‐‑regulator of TWIST and it confers resistance to anticancer drugs (Cheng, Chan 2007).
In addition to a well known role of TWIST in cancer progression in vitro in PC-‐‑derived cell-‐‑lines, there are reports suggesting that TWIST could be used as a biomarker for cancer prognosis. Higher levels of TWIST expression have been reported in samples of malignant tissues in comparison to normal tissues in many cancer types. Furthermore, several authors have found strong positive associations between increased TWIST expression and disease progression and survival rates in malignancies, such as breast cancer, bladder cancer, cervical cancer, oral and pharyngeal squamous cell carcinoma and renal cell cancer carcinoma (Soini et al. 2011, Riaz et al. 2012, Song et al. 2014, Shibata et al. 2008a, Fan et al. 2013, Jouppila-‐‑Mättö et al. 2011, Ohba et al. 2014). 2.7.2 Role of TWIST in prostate cancer In experiments conducted in both PC cell lines and tissue material, TWIST expression has been shown to be significantly augmented in PC cells in comparison to benign cells. Kwok et al observed that the degree of TWIST expression was dependent on the Gleason score and the state of metastases suggesting that TWIST could be utilized as an indicator of cancer aggressiveness (Kwok et al. 2005). The association of TWIST expression with Gleason score and probability to bone metastases has been also detected in tissue bank material of PC samples. Furthermore, increased TWIST expression has also been linked with suppressed E-‐‑cadherin expression, suggesting that the changes in the cadherin pathway could be mediated by TWIST (Yuen et al. 2007). In addition, the E-‐‑cadherin switch to N-‐‑cadherin, a phenomenon commonly encountered in invasive carcinomas, has been observed to be activated by TWIST signaling at the transcriptional level (Alexander et al. 2006).
Since TWIST signaling is associated with unfavorable behavior in PC, it is not surprising that several studies have indicated that TWIST activation may promote the formation of metastases. This kind of TWIST mediated effect has been observed in metastatic PC cells and there is also evidence that TWIST can modulate bone cell activity in a paracrine manner in cell culture settings (Yuen et al. 2008). In agreement with findings originating from other cancer types, TWIST mediated signaling has also been demonstrated to induce lung metastases in animal models of PC (Gajula et al. 2013).
13
The pathways linked with TWIST signaling, interact with the mechanisms of some anticancer drugs, such as docetaxel and enzalutamide. There are experiments which have examined the mechanisms linking TWIST with chemo-‐‑resistance but it has also been suggested that TWIST could be a therapeutic target for future drugs (Shiota et al. 2013, Shiota et al. 2014).
There is one earlier report evaluating EMT-‐‑markers, including TWIST, in PC prognosis of clinical patients. In that study, Behnsawy et al found increased TWIST expression to be associated with traditional clinicopathological prognostic factors and to predict independently BFS in PC patients after RP (Behnsawy et al. 2013).
One important link between AR and TWIST expression was revealed by Shiota et al. In a cell line study conducted with human PC cells, induction of TWIST led to AR overexpression and conversely, silencing of TWIST suppressed AR expression and triggered cellular apoptosis. The site of action of the modulation was identified as being in the promoter region of AR-‐‑gene where there was a TWIST binding site (Shiota et al. 2010). 2.8 OXIDATIVE DAMAGE ROS refer to a wide range of molecules and molecular fragments formed in aerobic organisms mostly as a consequence of aerobic respiration. Extracellular stressors, such as exposure to chemicals or radiation can also lead to the formation of ROS. Highly reactive free radicals, superoxide-‐‑, hydroxyl-‐‑ and nitric oxide radical contain unpaired electrons on their outermost orbital. Even though it possesses less reactivity than many other free radicals, hydrogen peroxide is still important in carcinogenesis since it has the capability to diffuse through membranes and thus can reach critical cellular targets and cause an oxidative injury. Hydrogen peroxide can be produced spontaneously from oxygen or as the result of catalytic activity from superoxide radicals by superoxide dismutases (Evans, Dizdaroglu & Cooke 2004, Karihtala, Soini 2007).
Under physiological conditions, ROS are participants in many cellular processes. For example, nitric oxide radicals have an effect on platelet adhesion and vascular tone and superoxide radicals and hydrogen peroxide are involved in cellular signaling (Rhee 1999). Human cells have numerous mechanisms for neutralizing ROS. However, if the capacity of cellular antioxidant defense system becomes overwhelmed, oxidative stress occurs (Ray, Huang & Tsuji 2012).
ROS-‐‑mediated damage has been linked with several benign diseases. In times of infection, elevated levels of ROS are generated in damaged tissues by neutrophils and macrophages to combat the invading microbes. However, there are also conditions of chronic inflammation, such as rheumatoid arthritis and inflammatory bowel disease, and in this case, oxidative damage can evoke connective tissue destruction and modifications of biomolecules (Evans, Dizdaroglu & Cooke 2004, Wiseman, Halliwell 1996). 2.8.1 Oxidative stress in carcinogenesis Persistent oxidative exposure and augmented ROS formation are characteristic changes detected in carcinoma cells. Macrophage infiltration and increased glycolytic metabolism are also linked with oxidative imbalance in the tumour tissue. DNA is the most important site of ROS related damage in carcinogenesis. There are many complex and inter-‐‑related mechanisms leading to cancer initiation involving the inactivation of tumour suppressor
14
genes and the activation of oncogenes. In most cases, mutations are either irrelevant as they do not occur in critical parts of the DNA base sequences or quickly neutralized by cell defense systems; only the mutations in critical genes are capable of inititaing a cascade resulting in the formation of a clone of malignant cells. However, ROS can harm almost all of the macromolecules within the cell, such as membrane lipid structures, signaling proteins and enzymes causing alteration of cell signaling and regulation of critical pathways (Karihtala, Soini 2007, Paschos et al. 2013). 2.8.1.1 8-‐‑Hydroxydeoxyguanosine (8-‐‑OHDG) DNA damage originating from hydroxyl radical exposure yields 8-‐‑OHDG, which has been used as a classical fingerprint marker of oxidative damage in the DNA molecule (Marnett 2000). Many of the mutagenic features of 8-‐‑OHDG are believed to be attributable to the appearance of guanine to thymidine transversions in the nucleotide bases, which have been detected in p53 tumour suppressor gene and ras oncogenes (Dreher, Junod 1996). In many different kinds of cancer types, such as B-‐‑cell lymphomas, bladder cancer, breast cancer, renal cell cancer and ovarian cancer, higher concentrations of 8-‐‑OHDG have been measured in the tumour than in healthy tissue and this is thought to be a consequence of an oxidative burst in carcinogenesis (Pasanen et al. 2012, Soini et al. 2011, Musarrat, Arezina-‐‑Wilson & Wani 1996, Okamoto et al. 1994, Karihtala et al. 2009).
There are a few reports that have explored the role of 8-‐‑OHDG in PC. Decreased levels of 8-‐‑OHDG have been found in urine after hormonal therapy and interpreted as a sign of a treatment effect (Miyake et al. 2004) and augmented 8-‐‑OHDG expression has been observed in metastatic lesions (Oberley et al. 2000). In studies conducted with prostate biopsy samples and radical prostatectomy specimens, an oxidative stress related mechanism has been shown to be linked with the progression of PC. However, the relationship between 8-‐‑OHDG expression as a biomarker and outcome of clinical patients is still unclear (Bostwick et al. 2000, Richardson et al. 2009). 2.8.1.2 Nuclear factor erythroid 2-‐‑related factor 2 (Nrf-‐‑2) Under normal conditions, oxidative damage related genes are minimally expressed but they can be induced by endogenous or exogenous stressors. Nrf-‐‑2 is one of the most important mediators leading to signaling at the transcriptional level. This property was first described in humans in 1994 (Moi et al. 1994). Under quiescent conditions, Nrf-‐‑2 is mainly in close contact in cytoplasm with Kelch ECH associating protein 1 (Keap1). It is believed that the cysteine residues in Keap1 function as sensors for oxidative stress and mediate structural changes, allowing Nrf-‐‑2 to be released from Keap1. Once liberated, Nrf-‐‑2 is transferred to the nucleus, where it accumulates. In the nucleus, it associates with Maf proteins and interacts with a specific enhancer, antioxidant response element (ARE). The activation of ARE-‐‑depended gene transcription initiates cell defensive functions, such DNA damage recognition, free radical metabolism, production of antioxidants and protease functions (Figure 3)(Kensler, Wakabayashi 2010, Nguyen, Yang & Pickett 2004, Kensler, Wakabayashi & Biswal 2007, Itoh, Mimura & Yamamoto 2010).
15
Figure 3. Nrf-2 is released from Keap1 in the cytoplasm and is transferred to the nucleus where it associates with Maf proteins and initiates transcription of oxidative damage combating genes by binding to the ARE element.
Several studies have established the important link between Nrf-‐‑2 mediated cell
protection mechanism and cancer initiation caused by oxidative damage. In experimental animals, Nrf-‐‑2 knockout mice have shown to be susceptible to chemical induced fore-‐‑stomach and bladder tumours (Iida et al. 2004, Ramos-‐‑Gomez et al. 2001). Nfr-‐‑2-‐‑dependent chemoresistance has also been observed after genetic manipulation of Nrf-‐‑2 activity in cancer cell lines (Lau et al. 2008). In addition, mutations repressing Keap1 or Nrf-‐‑2 gene functions have been found in malignant tissue samples of patients with lung cancer or head and neck cancer (Shibata et al. 2008b). Furthermore, modifications of expression of Nrf-‐‑2 regulating downstream genes have evoked carcinogenesis and endowed cancer cells with invasive properties (Lau et al. 2008).
There are many reports conducted with clinical cohorts indicating that Nrf-‐‑2 expression can predict outcome of patients in several cancer types. For example, in patients with breast cancer, gastric cancer, glioma, lung cancer and ovarian cancer, increased Nrf-‐‑2 expression has been shown to associate with unfavorable clinicopathological factors and poorer survival (Hartikainen et al. 2012, Kawasaki et al. 2015, Zhao et al. 2015, Merikallio et al. 2012, Liew et al. 2015).
Cytoplasmm
Nucleus
ARE
Nrf-2
Nrf-2
Keap1
SH SH
Maf gene transcription
Target gene functions
Cell survival
Stressors, inducers
14
genes and the activation of oncogenes. In most cases, mutations are either irrelevant as they do not occur in critical parts of the DNA base sequences or quickly neutralized by cell defense systems; only the mutations in critical genes are capable of inititaing a cascade resulting in the formation of a clone of malignant cells. However, ROS can harm almost all of the macromolecules within the cell, such as membrane lipid structures, signaling proteins and enzymes causing alteration of cell signaling and regulation of critical pathways (Karihtala, Soini 2007, Paschos et al. 2013). 2.8.1.1 8-‐‑Hydroxydeoxyguanosine (8-‐‑OHDG) DNA damage originating from hydroxyl radical exposure yields 8-‐‑OHDG, which has been used as a classical fingerprint marker of oxidative damage in the DNA molecule (Marnett 2000). Many of the mutagenic features of 8-‐‑OHDG are believed to be attributable to the appearance of guanine to thymidine transversions in the nucleotide bases, which have been detected in p53 tumour suppressor gene and ras oncogenes (Dreher, Junod 1996). In many different kinds of cancer types, such as B-‐‑cell lymphomas, bladder cancer, breast cancer, renal cell cancer and ovarian cancer, higher concentrations of 8-‐‑OHDG have been measured in the tumour than in healthy tissue and this is thought to be a consequence of an oxidative burst in carcinogenesis (Pasanen et al. 2012, Soini et al. 2011, Musarrat, Arezina-‐‑Wilson & Wani 1996, Okamoto et al. 1994, Karihtala et al. 2009).
There are a few reports that have explored the role of 8-‐‑OHDG in PC. Decreased levels of 8-‐‑OHDG have been found in urine after hormonal therapy and interpreted as a sign of a treatment effect (Miyake et al. 2004) and augmented 8-‐‑OHDG expression has been observed in metastatic lesions (Oberley et al. 2000). In studies conducted with prostate biopsy samples and radical prostatectomy specimens, an oxidative stress related mechanism has been shown to be linked with the progression of PC. However, the relationship between 8-‐‑OHDG expression as a biomarker and outcome of clinical patients is still unclear (Bostwick et al. 2000, Richardson et al. 2009). 2.8.1.2 Nuclear factor erythroid 2-‐‑related factor 2 (Nrf-‐‑2) Under normal conditions, oxidative damage related genes are minimally expressed but they can be induced by endogenous or exogenous stressors. Nrf-‐‑2 is one of the most important mediators leading to signaling at the transcriptional level. This property was first described in humans in 1994 (Moi et al. 1994). Under quiescent conditions, Nrf-‐‑2 is mainly in close contact in cytoplasm with Kelch ECH associating protein 1 (Keap1). It is believed that the cysteine residues in Keap1 function as sensors for oxidative stress and mediate structural changes, allowing Nrf-‐‑2 to be released from Keap1. Once liberated, Nrf-‐‑2 is transferred to the nucleus, where it accumulates. In the nucleus, it associates with Maf proteins and interacts with a specific enhancer, antioxidant response element (ARE). The activation of ARE-‐‑depended gene transcription initiates cell defensive functions, such DNA damage recognition, free radical metabolism, production of antioxidants and protease functions (Figure 3)(Kensler, Wakabayashi 2010, Nguyen, Yang & Pickett 2004, Kensler, Wakabayashi & Biswal 2007, Itoh, Mimura & Yamamoto 2010).
15
Figure 3. Nrf-2 is released from Keap1 in the cytoplasm and is transferred to the nucleus where it associates with Maf proteins and initiates transcription of oxidative damage combating genes by binding to the ARE element.
Several studies have established the important link between Nrf-‐‑2 mediated cell
protection mechanism and cancer initiation caused by oxidative damage. In experimental animals, Nrf-‐‑2 knockout mice have shown to be susceptible to chemical induced fore-‐‑stomach and bladder tumours (Iida et al. 2004, Ramos-‐‑Gomez et al. 2001). Nfr-‐‑2-‐‑dependent chemoresistance has also been observed after genetic manipulation of Nrf-‐‑2 activity in cancer cell lines (Lau et al. 2008). In addition, mutations repressing Keap1 or Nrf-‐‑2 gene functions have been found in malignant tissue samples of patients with lung cancer or head and neck cancer (Shibata et al. 2008b). Furthermore, modifications of expression of Nrf-‐‑2 regulating downstream genes have evoked carcinogenesis and endowed cancer cells with invasive properties (Lau et al. 2008).
There are many reports conducted with clinical cohorts indicating that Nrf-‐‑2 expression can predict outcome of patients in several cancer types. For example, in patients with breast cancer, gastric cancer, glioma, lung cancer and ovarian cancer, increased Nrf-‐‑2 expression has been shown to associate with unfavorable clinicopathological factors and poorer survival (Hartikainen et al. 2012, Kawasaki et al. 2015, Zhao et al. 2015, Merikallio et al. 2012, Liew et al. 2015).
Cytoplasmm
Nucleus
ARE
Nrf-2
Nrf-2
Keap1
SH SH
Maf gene transcription
Target gene functions
Cell survival
Stressors, inducers
16
The studies concerning Nrf-‐‑2 signaling in PC have been carried out using cancer tissues and cell lines. Xu et al revealed that an increase in the oxidative burst affects the expression of many antioxidant genes as well as with activation of the Nrf-‐‑2 pathway in human prostate cells (Xu et al. 2013). Changes in Nrf-‐‑2 activity have also been observed as a result of KEAP1 manipulation and administration of oxidative stress-‐‑inducing chemicals in PC tissue models and cell cultures (Frohlich et al. 2008, Pettazzoni et al. 2011). Furthermore, radioresistance, chemoresistance and tumour growth were enhanced after promoting Nrf-‐‑2 function by Keap1 inhibition in PC cell lines. In addition, a higher expression of Nrf-‐‑2 target genes has been detected in PC cells compared to normal prostate cells in this situation (Zhang et al. 2010). In an animal model of PC, suppression of Nrf-‐‑2 expression in transgenic mice with PC facilitated cancer progression, in contrast to the situation in non-‐‑tumourigenic mice which were not subjected to alterations in their Nrf-‐‑2 levels (Yu et al. 2010).
2.8.1.3 Peroxiredoxin (Prx) and sulfiredoxin (Srx) The Prx-‐‑family is widely distributed in human tissues being one of the most crucial sensors and regulators of redox balance. Prx enzymes are found in six isoforms located in cytosol and with some also being found in distinct cell organelles (Karihtala, Soini 2007). Prxs possess enzymatic properties and act to protect the cell against oxidative damage by converting alkyl hydroperoxides and hydrogen peroxide into alcohol and water. The structure of Prxs includes one or two reactive cysteines that are weakly oxidized when they come into contact with ROS, leading to a reduction and loss of activity of the ROS (Rabilloud et al. 2002). In order to regenerate Prxs back to their active form, oxidized Prxs are normally reduced by thioredoxin (Trx) during the catalytic cycle (Rhee et al. 2005). However, Prxs may be converted into hyperoxidized forms if there is intense oxidation and then Trx cannot restore their enzymatic activity. In the cases of hyperoxidation, Srx may be able to perform the reduction in a reaction requiring hydrolysis of adenosine triphosphate, thus preserving Prxs from destruction (Woo et al. 2005). Srx is also sensitive to signals present in the oxidative burst; this enzyme mediates responses through cytoprotective pathways and thus has the functions of a modulator molecule (Jeong et al. 2012). Prxs function also as chaperones. When exposed to a high peroxide concentration, they form oligomers such as decamers which prevent proteins from being misfolded (Saccoccia et al. 2012). Glutathionylation of Prxs, such as Prx1 at Cys83 reverts the complex back to its dimer form, inhibiting its chaperone function and affecting its ability to modulate some cell signaling pathways (Chae et al. 2012).
In addition to the protective function exerted by Prxs under physiological conditions against oxidative injury, they have been claimed to act as endogenous indicators of disrupted redox balance (Poynton, Hampton 2014). The role of an up-‐‑regulated Prx pathway in carcinogenesis has been demonstrated in cell line studies and in animal models. ROS may directly activate enzymatic detoxifying systems, leading to malignant transformation but they can also affect signal transduction at the transcriptional level (Immenschuh, Baumgart-‐‑Vogt 2005). Furthermore, augmented levels of Prxs have been shown to be linked with aggressive features of many cancer types in humans, such as B-‐‑cell lymphoma, breast cancer, gall bladder carcinoma, hepatocellular cancer and renal cell cancer (Kuusisto et al. 2015, Karihtala et al. 2003, Li et al. 2013, Sun et al. 2014, Soini et al. 2006).
17
The reports evaluating the Prx expression in PC have been mainly carried out using tissue samples and cell cultures. Increased Prx 1 -‐‑ 6 activity has been detected in malignant samples compared to benign tissue and the up-‐‑regulation of Prx 3 and 4 has led to cancer progression in PC cell lines (Valdman et al. 2009, Whitaker et al. 2013, Chaiswing, Zhong & Oberley 2014, Ummanni et al. 2012, He et al. 2012, Riddell et al. 2011). Furthermore, Prx2 mediated regulation has been demonstrated to be linked with AR-‐‑expressing PC-‐‑cells, under conditions where the silencing of Prx2 has decreased the AR-‐‑dependent gene expression and further reduced cell survival (Shiota et al. 2011). In a study conducted with tissue bank material, augmented Prx3 activity associated with tumour grade and predicted biochemical progression (Basu et al. 2011).
Several reports have indicated that disturbances in the Srx pathway are associated with the pathogenesis of oxidative stress mediated diseases, such as atherosclerosis and chronic obstructive pulmonary disease (Ramesh et al. 2014). In malignancies, up-‐‑regulation of Srx has been detected in lung cancer and skin tumours and it has been found to predict worse survival of melanoma patients (Merikallio et al. 2012, Wei et al. 2008, Hintsala et al. 2015). In contrast, there do not seem to be any studies conducted with PC material.
16
The studies concerning Nrf-‐‑2 signaling in PC have been carried out using cancer tissues and cell lines. Xu et al revealed that an increase in the oxidative burst affects the expression of many antioxidant genes as well as with activation of the Nrf-‐‑2 pathway in human prostate cells (Xu et al. 2013). Changes in Nrf-‐‑2 activity have also been observed as a result of KEAP1 manipulation and administration of oxidative stress-‐‑inducing chemicals in PC tissue models and cell cultures (Frohlich et al. 2008, Pettazzoni et al. 2011). Furthermore, radioresistance, chemoresistance and tumour growth were enhanced after promoting Nrf-‐‑2 function by Keap1 inhibition in PC cell lines. In addition, a higher expression of Nrf-‐‑2 target genes has been detected in PC cells compared to normal prostate cells in this situation (Zhang et al. 2010). In an animal model of PC, suppression of Nrf-‐‑2 expression in transgenic mice with PC facilitated cancer progression, in contrast to the situation in non-‐‑tumourigenic mice which were not subjected to alterations in their Nrf-‐‑2 levels (Yu et al. 2010).
2.8.1.3 Peroxiredoxin (Prx) and sulfiredoxin (Srx) The Prx-‐‑family is widely distributed in human tissues being one of the most crucial sensors and regulators of redox balance. Prx enzymes are found in six isoforms located in cytosol and with some also being found in distinct cell organelles (Karihtala, Soini 2007). Prxs possess enzymatic properties and act to protect the cell against oxidative damage by converting alkyl hydroperoxides and hydrogen peroxide into alcohol and water. The structure of Prxs includes one or two reactive cysteines that are weakly oxidized when they come into contact with ROS, leading to a reduction and loss of activity of the ROS (Rabilloud et al. 2002). In order to regenerate Prxs back to their active form, oxidized Prxs are normally reduced by thioredoxin (Trx) during the catalytic cycle (Rhee et al. 2005). However, Prxs may be converted into hyperoxidized forms if there is intense oxidation and then Trx cannot restore their enzymatic activity. In the cases of hyperoxidation, Srx may be able to perform the reduction in a reaction requiring hydrolysis of adenosine triphosphate, thus preserving Prxs from destruction (Woo et al. 2005). Srx is also sensitive to signals present in the oxidative burst; this enzyme mediates responses through cytoprotective pathways and thus has the functions of a modulator molecule (Jeong et al. 2012). Prxs function also as chaperones. When exposed to a high peroxide concentration, they form oligomers such as decamers which prevent proteins from being misfolded (Saccoccia et al. 2012). Glutathionylation of Prxs, such as Prx1 at Cys83 reverts the complex back to its dimer form, inhibiting its chaperone function and affecting its ability to modulate some cell signaling pathways (Chae et al. 2012).
In addition to the protective function exerted by Prxs under physiological conditions against oxidative injury, they have been claimed to act as endogenous indicators of disrupted redox balance (Poynton, Hampton 2014). The role of an up-‐‑regulated Prx pathway in carcinogenesis has been demonstrated in cell line studies and in animal models. ROS may directly activate enzymatic detoxifying systems, leading to malignant transformation but they can also affect signal transduction at the transcriptional level (Immenschuh, Baumgart-‐‑Vogt 2005). Furthermore, augmented levels of Prxs have been shown to be linked with aggressive features of many cancer types in humans, such as B-‐‑cell lymphoma, breast cancer, gall bladder carcinoma, hepatocellular cancer and renal cell cancer (Kuusisto et al. 2015, Karihtala et al. 2003, Li et al. 2013, Sun et al. 2014, Soini et al. 2006).
17
The reports evaluating the Prx expression in PC have been mainly carried out using tissue samples and cell cultures. Increased Prx 1 -‐‑ 6 activity has been detected in malignant samples compared to benign tissue and the up-‐‑regulation of Prx 3 and 4 has led to cancer progression in PC cell lines (Valdman et al. 2009, Whitaker et al. 2013, Chaiswing, Zhong & Oberley 2014, Ummanni et al. 2012, He et al. 2012, Riddell et al. 2011). Furthermore, Prx2 mediated regulation has been demonstrated to be linked with AR-‐‑expressing PC-‐‑cells, under conditions where the silencing of Prx2 has decreased the AR-‐‑dependent gene expression and further reduced cell survival (Shiota et al. 2011). In a study conducted with tissue bank material, augmented Prx3 activity associated with tumour grade and predicted biochemical progression (Basu et al. 2011).
Several reports have indicated that disturbances in the Srx pathway are associated with the pathogenesis of oxidative stress mediated diseases, such as atherosclerosis and chronic obstructive pulmonary disease (Ramesh et al. 2014). In malignancies, up-‐‑regulation of Srx has been detected in lung cancer and skin tumours and it has been found to predict worse survival of melanoma patients (Merikallio et al. 2012, Wei et al. 2008, Hintsala et al. 2015). In contrast, there do not seem to be any studies conducted with PC material.
18
3 Aims of the Study
The prognosis of localised PC after radical prostatectomy is mostly good. In only a minor proportion of the PC cases is the tumor aggressive, leading to metastatic disease and death. Traditionally, prognostic tools have been based on clinical and pathological parameters. However, there is a clear need for more specific markers in order to predict more precisely which PC patients need adjuvant treatment modalities after RP and which patients should simply be followed. In clinical practice, there are still no biomarkers available for predicting the prognosis of PC. The specific aims of the study were:
1. To examine the expressions of AR and the EMT-‐‑marker TWIST in tissue samples from PC patients treated by radical prostatectomy and to evaluate whether there was any correlation between the extent of the expression and conventional clinicopathological prognostigators and BFS.
2. To explore the levels of expression of oxidative stress markers 8-‐‑OHDG and Nrf-‐‑2 in
PC samples taken from radical prostatectomy patients and to examine whether they show any association with clinopathological prognostic factors and survival.
3. To evaluate the expression levels of oxidative stress induced proteins Prx 1, 2, 5, 6 and Srx in PC tissues of patients after radical prostatectomy and to elucidate their association with clinical factors and survival of the patients.
19
4 Materials and Methods
4.1 STUDY POPULATION
The current study was retrospective and population-‐‑based. RP had been performed in Kuopio University Hospital in 181 PC patients in study cohort I and in 240 PC patients in study cohorts II and III. The patients underwent the surgery between the years 1998 and 2009 (I) and between the years 1987 and 2009 (II, III). Prostatectomy tissues were used as the sample material. Patient records and the laboratory database were the sources of the clinical data and clinical follow-‐‑up data. According to DRE and transrectal ultrasonography, all the patients had a clinically localised tumour with no invasion to adjacent structures, such as rectum or pelvic wall. Lymphadenectomy was performed on those patients in the intermediate or high risk groups, but none of these patients had positive lymph nodes. Bone scans were used to exclude distant metastases when there was a need for confirmation of the clinical status; this procedure initiated at the discretion of the urologist. Therefore, all the cases fulfilled the definition of the clinical classification of localised tumours (T1-‐‑3N0M0) according to UICC guidelines (Sobin et al. 2010).
None of the patients had received hormone therapy prior to RP. The follow-‐‑up was conducted after 2, 6 and 12 months and then according to clinical practice. The median follow-‐‑up times were 7.6 (2.3-‐‑14.1) years (I) and 11.7 (3.3-‐‑25.8) years (II, III), respectively. During the observation period, BCR was observed in 67 (37.0%) (I) and 109 (45.4%) (II, III) patients. Of the 240 patients, 51 (21.2%) patients died, with PC being the cause of death in 19 (7.9%) cases (II, III). An elevation of the PSA value of 0.2 ng/ml or more was considered as a biochemical recurrence (Walz et al. 2009). Tables 3 and 4 summarise the demographic data of the patients.
18
3 Aims of the Study
The prognosis of localised PC after radical prostatectomy is mostly good. In only a minor proportion of the PC cases is the tumor aggressive, leading to metastatic disease and death. Traditionally, prognostic tools have been based on clinical and pathological parameters. However, there is a clear need for more specific markers in order to predict more precisely which PC patients need adjuvant treatment modalities after RP and which patients should simply be followed. In clinical practice, there are still no biomarkers available for predicting the prognosis of PC. The specific aims of the study were:
1. To examine the expressions of AR and the EMT-‐‑marker TWIST in tissue samples from PC patients treated by radical prostatectomy and to evaluate whether there was any correlation between the extent of the expression and conventional clinicopathological prognostigators and BFS.
2. To explore the levels of expression of oxidative stress markers 8-‐‑OHDG and Nrf-‐‑2 in
PC samples taken from radical prostatectomy patients and to examine whether they show any association with clinopathological prognostic factors and survival.
3. To evaluate the expression levels of oxidative stress induced proteins Prx 1, 2, 5, 6 and Srx in PC tissues of patients after radical prostatectomy and to elucidate their association with clinical factors and survival of the patients.
19
4 Materials and Methods
4.1 STUDY POPULATION
The current study was retrospective and population-‐‑based. RP had been performed in Kuopio University Hospital in 181 PC patients in study cohort I and in 240 PC patients in study cohorts II and III. The patients underwent the surgery between the years 1998 and 2009 (I) and between the years 1987 and 2009 (II, III). Prostatectomy tissues were used as the sample material. Patient records and the laboratory database were the sources of the clinical data and clinical follow-‐‑up data. According to DRE and transrectal ultrasonography, all the patients had a clinically localised tumour with no invasion to adjacent structures, such as rectum or pelvic wall. Lymphadenectomy was performed on those patients in the intermediate or high risk groups, but none of these patients had positive lymph nodes. Bone scans were used to exclude distant metastases when there was a need for confirmation of the clinical status; this procedure initiated at the discretion of the urologist. Therefore, all the cases fulfilled the definition of the clinical classification of localised tumours (T1-‐‑3N0M0) according to UICC guidelines (Sobin et al. 2010).
None of the patients had received hormone therapy prior to RP. The follow-‐‑up was conducted after 2, 6 and 12 months and then according to clinical practice. The median follow-‐‑up times were 7.6 (2.3-‐‑14.1) years (I) and 11.7 (3.3-‐‑25.8) years (II, III), respectively. During the observation period, BCR was observed in 67 (37.0%) (I) and 109 (45.4%) (II, III) patients. Of the 240 patients, 51 (21.2%) patients died, with PC being the cause of death in 19 (7.9%) cases (II, III). An elevation of the PSA value of 0.2 ng/ml or more was considered as a biochemical recurrence (Walz et al. 2009). Tables 3 and 4 summarise the demographic data of the patients.
20
Table 3. Demographic data of the patients (I), (n=181). Variable Median age, years (Standard deviation, SD) 62.3 (5.3) Median follow up, years (range) 7.6 (2.3-14.1) Median PSA at diagnosis, ng/ml (SD) 7.8 (6.7) PSA µg/l, n (%) <10 10-20 >20
122 (67.4) 49 (27.1) 10 (5.5)
pT category, n (%) 2 130 (71.8) 3 51 (28.2) Gleason score, n (%) 2-6 111 (61.4) 7 58 (32.0) 8-10 12 (6.6) Capsule invasion, n (%) No 131 (72.4) Yes 50 (27.6) Surgical margin status, n (%) Negative 115 (63.5) Positive 66 (36.5) Biochemical recurrence, n (%) Yes 67 (37.0) No 114 (63.0)
21
Table 4. Demografic data of the patients (II, III), (n=240). Variable
Median age, years (Standard deviation, SD) 63.0 (5.5) Median follow up, years (range) 11.7 (3.3-25.8) Median PSA at diagnosis, ng/ml (SD) 8.1 (12.3) PSAa ng/ml, n (%) <10 144 (60.0) ≥10 92 (38.3) pT category, n (%) 2 160 (66.7) 3a 49 (20.4) 3b 31 (12.9) Gleason score, n (%) 2-6 154 (64.2) 7-10 86 (35.8) Capsule invasion, n (%) No 157 (65.4) Yes 83 (34.6) Surgical margin status, n (%) Negative 141 (58.8) Positive 99 (41.2) Biochemical recurrence, n (%) Yes 109 (45.4) No 131 (54.6) Prostate cancer specific survival, n (%) Alive 221 (92.1) Dead 19 (7.9) Overall survival, n (%) Alive 189 (78.8) Dead 51 (21.2) aPSA value missing in four cases
4.2 HISTOPATHOLOGICAL ANALYSES
All the samples had been fixed in 10% neutral buffered formalin and embedded in paraffin. Two experienced pathologists (Ylermi Soini and Vesa Kärjä), blinded to the clinical data, re-‐‑evaluated the samples to determine pT-‐‑class, Gleason score, capsule invasion and surgical margin status. The clinical TNM-‐‑classification was conducted according to UICC guidelines and the Gleason score was calculated according to the ISUP 2005 modification (Sobin et al. 2010, Epstein et al. 2005).
In the immunohistochemical analyses of TWIST and AR expressions, microarray samples were taken from four representative regions (high Gleason, low Gleason, central and margin) of the PC tissues and benign prostate tissue, respectively. The samples for assessing the expression of Nrf-‐‑2, 8-‐‑OHDG, Prx, Srx were taken from four representative regions of the PC tissues. In the analyses of Nrf-‐‑2 and 8-‐‑OHDG expressions, the samples of benign prostate tissue were taken for comparison. In all analyses, one microarray puncture of each tumour region was considered as one sample unit. All blocks from the resected prostate were used for selection of the array samples. The samples were taken from the
20
Table 3. Demographic data of the patients (I), (n=181). Variable Median age, years (Standard deviation, SD) 62.3 (5.3) Median follow up, years (range) 7.6 (2.3-14.1) Median PSA at diagnosis, ng/ml (SD) 7.8 (6.7) PSA µg/l, n (%) <10 10-20 >20
122 (67.4) 49 (27.1) 10 (5.5)
pT category, n (%) 2 130 (71.8) 3 51 (28.2) Gleason score, n (%) 2-6 111 (61.4) 7 58 (32.0) 8-10 12 (6.6) Capsule invasion, n (%) No 131 (72.4) Yes 50 (27.6) Surgical margin status, n (%) Negative 115 (63.5) Positive 66 (36.5) Biochemical recurrence, n (%) Yes 67 (37.0) No 114 (63.0)
21
Table 4. Demografic data of the patients (II, III), (n=240). Variable
Median age, years (Standard deviation, SD) 63.0 (5.5) Median follow up, years (range) 11.7 (3.3-25.8) Median PSA at diagnosis, ng/ml (SD) 8.1 (12.3) PSAa ng/ml, n (%) <10 144 (60.0) ≥10 92 (38.3) pT category, n (%) 2 160 (66.7) 3a 49 (20.4) 3b 31 (12.9) Gleason score, n (%) 2-6 154 (64.2) 7-10 86 (35.8) Capsule invasion, n (%) No 157 (65.4) Yes 83 (34.6) Surgical margin status, n (%) Negative 141 (58.8) Positive 99 (41.2) Biochemical recurrence, n (%) Yes 109 (45.4) No 131 (54.6) Prostate cancer specific survival, n (%) Alive 221 (92.1) Dead 19 (7.9) Overall survival, n (%) Alive 189 (78.8) Dead 51 (21.2) aPSA value missing in four cases
4.2 HISTOPATHOLOGICAL ANALYSES
All the samples had been fixed in 10% neutral buffered formalin and embedded in paraffin. Two experienced pathologists (Ylermi Soini and Vesa Kärjä), blinded to the clinical data, re-‐‑evaluated the samples to determine pT-‐‑class, Gleason score, capsule invasion and surgical margin status. The clinical TNM-‐‑classification was conducted according to UICC guidelines and the Gleason score was calculated according to the ISUP 2005 modification (Sobin et al. 2010, Epstein et al. 2005).
In the immunohistochemical analyses of TWIST and AR expressions, microarray samples were taken from four representative regions (high Gleason, low Gleason, central and margin) of the PC tissues and benign prostate tissue, respectively. The samples for assessing the expression of Nrf-‐‑2, 8-‐‑OHDG, Prx, Srx were taken from four representative regions of the PC tissues. In the analyses of Nrf-‐‑2 and 8-‐‑OHDG expressions, the samples of benign prostate tissue were taken for comparison. In all analyses, one microarray puncture of each tumour region was considered as one sample unit. All blocks from the resected prostate were used for selection of the array samples. The samples were taken from the
22
dominant tumour nodule and the benign tissue was collected from sites away from the cancer zone, usually from a different prostatic zone. The margin area or invasive front was defined as the boundary area between histological tumor mass and adjacent benign prostate tissue as visualized in haematoxylin-‐‑eosin stained slides. The samples were placed into multitissue microarray blocks with Beecher Instruments Manual Tissue Arrayer (Beecher Instruments, Silver Spring, MD, USA). The microarray sample diameter was 1300µμm. 4.3 IMMUNOHISTOCHEMISTRY Four-‐‑micrometer-‐‑thick tissue sections were cut from the paraffin-‐‑embedded microarray blocks. The sections were deparaffinised in xylene and rehydrated in descending ethanol series in the routine manner. In the analysis of TWIST, AR, 8-‐‑OHDG and Nrf-‐‑2, the sections were heated in a microwave oven for 2 x 5 min in Tris-‐‑ethylendiaminetetraacetate (EDTA) buffer (pH 9.0), incubated in a Tris-‐‑EDTA buffer for 20 min and washed twice for 5 min in phosphate buffered saline (PBS). Hydrogen peroxide (5%, 5 min) was used to block endogenous peroxidase. Non-‐‑specific binding was blocked with 1.5% normal serum in PBS for 35 min at room temperature. In the analysis of Prxs and Srx, the sections were incubated in 10 mM citrate buffer (pH 6.0) in a microwave oven for 2 minutes at 850 W followed by 8 minutes at 350 W. Endogenous peroxidase activity was blocked by incubation in 0.1% hydrogen peroxide in absolute methanol for 10 minutes. All the sections were incubated overnight at 4°C with specific antibodies (Table 5). The slides were then incubated with a biotinylated secondary antibody and avidin-‐‑biotin-‐‑peroxidase complex (ABC Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA) to reveal the primary antibodies in the analysis of TWIST, AR, 8-‐‑OHDG and Nrf-‐‑2. The primary antibodies for Prxs and Srx were revealed using the Histostain-‐‑Plus Kit (Zymed Laboratories Inc, South San Francisco, CA). Several rinses were performed with PBS at each step of the immunostaining procedure. The color was developed with diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA). The slides were counterstained with Mayer'ʹs haematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). In the negative controls, the primary antibody was omitted.
23
Table 5. Specific antibodies used for immunohistochemical stainings. Marker
Dilution
Primary antibody
Manufacturer
TWIST
1:500
mouse monoclonal anti-TWIST
Abcam, Cambridge, UK
AR
1:500 mouse monoclonal anti-AR non-commercial (Karvonen et al. 1997)
8-OHDG
1:1000 mouse monoclonal anti-8-OHDG
JaICA, Nikken SEIL Co., Ltd, Fukuroi, Shizuoka, Japan
Nrf-2
1:50 mouse monoclonal anti-Nrf-2
Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA
Prx1
1:1500 rabbit polyclonal anti-Prx1 LabFrontier, York, UK
Prx2
1:1000 rabbit polyclonal anti-Prx2 LabFrontier, York, UK
Prx5
1:2000 rabbit polyclonal anti-Prx5 LabFrontier, York, UK
Prx6
1:500 rabbit polyclonal anti-Prx6 LabFrontier, York, UK
Srx
1:500 rabbit polyclonal anti-Srx LabFrontier, York, UK
4.4 EVALUATION OF THE EXPRESSION The microscopic evaluation of biomarker expression was performed by two pathologists (Ylermi Soini and Vesa Kärjä) blinded to the clinical data. The pathologists made a consensus assessment in each case. In the analysis of all biomarkers, the grade of positive expression was evaluated as a percentage of positive cells.
The immunoreactivity for TWIST and AR was first analysed in tumor cells as follows; 0-‐‑5% = negative, 6-‐‑50% = weak positive, 51-‐‑100% = strong positive. The data were then dichotomized into two groups; negative = 0-‐‑5% and positive = 5-‐‑100%. It was determined that adequate immunostainings of AR expression were available in 165 cases (91.2%). TWIST expression in different tumour areas was obtained in the samples of the PC cases as follows: high Gleason (170, 93.9%); low Gleason (169, 93.4%); central (169, 93.4%); margin (167, 92.2%)(Figure 4). In the samples, TWIST expression was nuclear and cytoplasmic whereas AR expression was nuclear.
Nrf-‐‑2 was expressed both in the nucleus and cytoplasm and was assessed separately in these two locations, whereas the expression of 8-‐‑OHDG was exclusively nuclear. The immunoreactivity for 8-‐‑OHDG, Nrf-‐‑2 in nucleus (n-‐‑Nrf-‐‑2) and Nrf-‐‑2 in cytoplasm (c-‐‑Nrf-‐‑2) was initially analysed in tumour cells as follows; 8-‐‑OHDG: 0-‐‑5% = negative, 6-‐‑50% = weak positive, 51-‐‑100% strong positive; n-‐‑Nrf-‐‑2: 0-‐‑50% = negative, 51-‐‑100% = positive; c-‐‑Nrf-‐‑2: 0% = negative, 1-‐‑5% = weak positive, 6-‐‑50% = moderately positive, 51-‐‑100% = strong positive. The mean value of the sum from the four malignant areas was considered as the
22
dominant tumour nodule and the benign tissue was collected from sites away from the cancer zone, usually from a different prostatic zone. The margin area or invasive front was defined as the boundary area between histological tumor mass and adjacent benign prostate tissue as visualized in haematoxylin-‐‑eosin stained slides. The samples were placed into multitissue microarray blocks with Beecher Instruments Manual Tissue Arrayer (Beecher Instruments, Silver Spring, MD, USA). The microarray sample diameter was 1300µμm. 4.3 IMMUNOHISTOCHEMISTRY Four-‐‑micrometer-‐‑thick tissue sections were cut from the paraffin-‐‑embedded microarray blocks. The sections were deparaffinised in xylene and rehydrated in descending ethanol series in the routine manner. In the analysis of TWIST, AR, 8-‐‑OHDG and Nrf-‐‑2, the sections were heated in a microwave oven for 2 x 5 min in Tris-‐‑ethylendiaminetetraacetate (EDTA) buffer (pH 9.0), incubated in a Tris-‐‑EDTA buffer for 20 min and washed twice for 5 min in phosphate buffered saline (PBS). Hydrogen peroxide (5%, 5 min) was used to block endogenous peroxidase. Non-‐‑specific binding was blocked with 1.5% normal serum in PBS for 35 min at room temperature. In the analysis of Prxs and Srx, the sections were incubated in 10 mM citrate buffer (pH 6.0) in a microwave oven for 2 minutes at 850 W followed by 8 minutes at 350 W. Endogenous peroxidase activity was blocked by incubation in 0.1% hydrogen peroxide in absolute methanol for 10 minutes. All the sections were incubated overnight at 4°C with specific antibodies (Table 5). The slides were then incubated with a biotinylated secondary antibody and avidin-‐‑biotin-‐‑peroxidase complex (ABC Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA) to reveal the primary antibodies in the analysis of TWIST, AR, 8-‐‑OHDG and Nrf-‐‑2. The primary antibodies for Prxs and Srx were revealed using the Histostain-‐‑Plus Kit (Zymed Laboratories Inc, South San Francisco, CA). Several rinses were performed with PBS at each step of the immunostaining procedure. The color was developed with diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA). The slides were counterstained with Mayer'ʹs haematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). In the negative controls, the primary antibody was omitted.
23
Table 5. Specific antibodies used for immunohistochemical stainings. Marker
Dilution
Primary antibody
Manufacturer
TWIST
1:500
mouse monoclonal anti-TWIST
Abcam, Cambridge, UK
AR
1:500 mouse monoclonal anti-AR non-commercial (Karvonen et al. 1997)
8-OHDG
1:1000 mouse monoclonal anti-8-OHDG
JaICA, Nikken SEIL Co., Ltd, Fukuroi, Shizuoka, Japan
Nrf-2
1:50 mouse monoclonal anti-Nrf-2
Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA
Prx1
1:1500 rabbit polyclonal anti-Prx1 LabFrontier, York, UK
Prx2
1:1000 rabbit polyclonal anti-Prx2 LabFrontier, York, UK
Prx5
1:2000 rabbit polyclonal anti-Prx5 LabFrontier, York, UK
Prx6
1:500 rabbit polyclonal anti-Prx6 LabFrontier, York, UK
Srx
1:500 rabbit polyclonal anti-Srx LabFrontier, York, UK
4.4 EVALUATION OF THE EXPRESSION The microscopic evaluation of biomarker expression was performed by two pathologists (Ylermi Soini and Vesa Kärjä) blinded to the clinical data. The pathologists made a consensus assessment in each case. In the analysis of all biomarkers, the grade of positive expression was evaluated as a percentage of positive cells.
The immunoreactivity for TWIST and AR was first analysed in tumor cells as follows; 0-‐‑5% = negative, 6-‐‑50% = weak positive, 51-‐‑100% = strong positive. The data were then dichotomized into two groups; negative = 0-‐‑5% and positive = 5-‐‑100%. It was determined that adequate immunostainings of AR expression were available in 165 cases (91.2%). TWIST expression in different tumour areas was obtained in the samples of the PC cases as follows: high Gleason (170, 93.9%); low Gleason (169, 93.4%); central (169, 93.4%); margin (167, 92.2%)(Figure 4). In the samples, TWIST expression was nuclear and cytoplasmic whereas AR expression was nuclear.
Nrf-‐‑2 was expressed both in the nucleus and cytoplasm and was assessed separately in these two locations, whereas the expression of 8-‐‑OHDG was exclusively nuclear. The immunoreactivity for 8-‐‑OHDG, Nrf-‐‑2 in nucleus (n-‐‑Nrf-‐‑2) and Nrf-‐‑2 in cytoplasm (c-‐‑Nrf-‐‑2) was initially analysed in tumour cells as follows; 8-‐‑OHDG: 0-‐‑5% = negative, 6-‐‑50% = weak positive, 51-‐‑100% strong positive; n-‐‑Nrf-‐‑2: 0-‐‑50% = negative, 51-‐‑100% = positive; c-‐‑Nrf-‐‑2: 0% = negative, 1-‐‑5% = weak positive, 6-‐‑50% = moderately positive, 51-‐‑100% = strong positive. The mean value of the sum from the four malignant areas was considered as the
24
representative score. The data were then dichotomized into two groups; 0-‐‑50% = negative and 51-‐‑100% = positive.
The immunostainings revealing the expressions of 8-‐‑OHDG, c-‐‑Nrf-‐‑2 and n-‐‑Nrf-‐‑2 in 208 (86.7%), 204 (85.0%) and 205 (85.4%) were obtained from the cases of the PC samples. There were available adequate immunostainings of 8-‐‑OHDG, c-‐‑Nrf-‐‑2 expression and n-‐‑Nrf-‐‑2 expression in 159 (66.3%), 152 (63.3%) and 153 (63.8%) cases from the samples from the adjacent benign area, respectively (Figure 5).
In the samples, the expression levels of Prx1, Prx2, Prx5, Prx6 and Srx were partly nuclear but mainly cytoplasmic. At first, the immunoreactivity for these five biomarkers was analysed in the tumour cells as follows; Prx1 and Prx2: 0% = negative, 1-‐‑5% = weak positive, 6-‐‑50% = moderately positive, 51-‐‑100% = strong positive; Prx5, Prx6 and Srx: 0-‐‑5% = negative, 6-‐‑50% = weak positive, 51-‐‑100% strong positive. The mean value of the sum from the four malignant areas was considered as the representative score. The data were then dichotomized into two groups; 0-‐‑50% = negative and 51-‐‑100% = positive.
The expressions of the biomarkers were obtained in the samples of the PC cases as follows: Prx1 (233, 97.1%), Prx2 (233, 97.1%), Prx5 (232, 96.7%), Prx6 (231, 96.3%) and Srx (229, 95.4%)(Figure 6).
25
Figure 4. Positive nuclear TWIST expression in PC tissue taken from the margin part of the tumour (A) In benign prostatic hyperplasia, no TWIST expression could be detected (B).
Figure 5. Cytoplasmic (A) and nuclear (B) Nrf-2 positivity in a PC sample. Negative Nrf-2 expression in PC tissue (C). 8-OHDG expression in the nuclei of prostate adenocarcinoma (D).
Figure 6. Positive (A) and negative (B) Prx6 expression in PC tissue.
A B
24
representative score. The data were then dichotomized into two groups; 0-‐‑50% = negative and 51-‐‑100% = positive.
The immunostainings revealing the expressions of 8-‐‑OHDG, c-‐‑Nrf-‐‑2 and n-‐‑Nrf-‐‑2 in 208 (86.7%), 204 (85.0%) and 205 (85.4%) were obtained from the cases of the PC samples. There were available adequate immunostainings of 8-‐‑OHDG, c-‐‑Nrf-‐‑2 expression and n-‐‑Nrf-‐‑2 expression in 159 (66.3%), 152 (63.3%) and 153 (63.8%) cases from the samples from the adjacent benign area, respectively (Figure 5).
In the samples, the expression levels of Prx1, Prx2, Prx5, Prx6 and Srx were partly nuclear but mainly cytoplasmic. At first, the immunoreactivity for these five biomarkers was analysed in the tumour cells as follows; Prx1 and Prx2: 0% = negative, 1-‐‑5% = weak positive, 6-‐‑50% = moderately positive, 51-‐‑100% = strong positive; Prx5, Prx6 and Srx: 0-‐‑5% = negative, 6-‐‑50% = weak positive, 51-‐‑100% strong positive. The mean value of the sum from the four malignant areas was considered as the representative score. The data were then dichotomized into two groups; 0-‐‑50% = negative and 51-‐‑100% = positive.
The expressions of the biomarkers were obtained in the samples of the PC cases as follows: Prx1 (233, 97.1%), Prx2 (233, 97.1%), Prx5 (232, 96.7%), Prx6 (231, 96.3%) and Srx (229, 95.4%)(Figure 6).
25
Figure 4. Positive nuclear TWIST expression in PC tissue taken from the margin part of the tumour (A) In benign prostatic hyperplasia, no TWIST expression could be detected (B).
Figure 5. Cytoplasmic (A) and nuclear (B) Nrf-2 positivity in a PC sample. Negative Nrf-2 expression in PC tissue (C). 8-OHDG expression in the nuclei of prostate adenocarcinoma (D).
Figure 6. Positive (A) and negative (B) Prx6 expression in PC tissue.
A B
26
4.5 STATISTICAL ANALYSES The statistical analyses were performed with the SPSS 19.0 program package. Differences between groups were evaluated with t-‐‑test. The Chi-‐‑square test was used to determine the association between clinicopathological prognostic factors and expression of biomarkers. BFS, PCS and OS were analysed by the Kaplan Meier method. The univariate and multivariate analyses were done according to Cox’s method. P values <0.05 were considered statistically significant. 4.6 ETHICAL CONSIDERATIONS This study was approved by Research Ethical Committee of Kuopio University Hospital. All the procedures have been performed according to the institutional guidelines of Kuopio University Hospital and University of Eastern Finland.
27
5 Results
5.1 TWIST AND AR EXPRESSION AND THEIR ASSOCIATION WITH CLINICOPATHOLOCIGAL PROGNOSTIGATORS (I) TWIST expression was observed in all samples taken from different tumour areas but with a variable frequency (high Gleason 8.2%, low Gleason 3.0%, central 4.7% and margin 11.4%). In contrast to the situation with malignant samples, there was no evidence of TWIST expression in benign tissue. The difference between TWIST expression in the malignant parts of tumour was observed between the following tumour areas: high Gleason and low Gleason (P=0.029), low Gleason and margin (P=0.002), central and margin regions (P=0.016)(Table 6). Table 6. TWIST expression in benign samples and in different parts of the tumour. Tumour area positive
expression negative expression
benign vs malignant
high Gleason vs other parts of tumour
low Gleason vs central and margin
central vs margin
n (%) n (%) P-value P-value P-value P-value benign 0 (0) 169 (100) high Gleason 14 (8.2) 156 (91.8) ˂0.0001 low Gleason 5 (3.0) 164 (97.0) 0.025 0.029 central 8 (4.7) 161 (95.3) 0.004 ns. ns. margin 19 (11.4) 148 (88.6) ˂0.0001 ns. 0.002 0.016
TWIST overexpression in the invasive, margin front of the tumour (M-‐‑TWIST) was associated with a positive surgical margin status (P=0.047), capsule invasion (P=0.006) and BCR (P=0.004). The analysis was continued with the samples from the margin area, because of the significant findings found there for M-‐‑TWIST. The AR expression in the margin area of the tumour (M-‐‑AR) was associated with a higher Gleason score (P=0.004), positive surgical margin status (P=0.004) and BCR (P=0.05) (Table 7). The association between M-‐‑TWIST and M-‐‑AR expression was explored in order to test the possibility that TWIST could be regulating AR expression. The association was highly significant (P<0.0001).
26
4.5 STATISTICAL ANALYSES The statistical analyses were performed with the SPSS 19.0 program package. Differences between groups were evaluated with t-‐‑test. The Chi-‐‑square test was used to determine the association between clinicopathological prognostic factors and expression of biomarkers. BFS, PCS and OS were analysed by the Kaplan Meier method. The univariate and multivariate analyses were done according to Cox’s method. P values <0.05 were considered statistically significant. 4.6 ETHICAL CONSIDERATIONS This study was approved by Research Ethical Committee of Kuopio University Hospital. All the procedures have been performed according to the institutional guidelines of Kuopio University Hospital and University of Eastern Finland.
27
5 Results
5.1 TWIST AND AR EXPRESSION AND THEIR ASSOCIATION WITH CLINICOPATHOLOCIGAL PROGNOSTIGATORS (I) TWIST expression was observed in all samples taken from different tumour areas but with a variable frequency (high Gleason 8.2%, low Gleason 3.0%, central 4.7% and margin 11.4%). In contrast to the situation with malignant samples, there was no evidence of TWIST expression in benign tissue. The difference between TWIST expression in the malignant parts of tumour was observed between the following tumour areas: high Gleason and low Gleason (P=0.029), low Gleason and margin (P=0.002), central and margin regions (P=0.016)(Table 6). Table 6. TWIST expression in benign samples and in different parts of the tumour. Tumour area positive
expression negative expression
benign vs malignant
high Gleason vs other parts of tumour
low Gleason vs central and margin
central vs margin
n (%) n (%) P-value P-value P-value P-value benign 0 (0) 169 (100) high Gleason 14 (8.2) 156 (91.8) ˂0.0001 low Gleason 5 (3.0) 164 (97.0) 0.025 0.029 central 8 (4.7) 161 (95.3) 0.004 ns. ns. margin 19 (11.4) 148 (88.6) ˂0.0001 ns. 0.002 0.016
TWIST overexpression in the invasive, margin front of the tumour (M-‐‑TWIST) was associated with a positive surgical margin status (P=0.047), capsule invasion (P=0.006) and BCR (P=0.004). The analysis was continued with the samples from the margin area, because of the significant findings found there for M-‐‑TWIST. The AR expression in the margin area of the tumour (M-‐‑AR) was associated with a higher Gleason score (P=0.004), positive surgical margin status (P=0.004) and BCR (P=0.05) (Table 7). The association between M-‐‑TWIST and M-‐‑AR expression was explored in order to test the possibility that TWIST could be regulating AR expression. The association was highly significant (P<0.0001).
28
Table 7. Summary of the significant findings for the expression of biomarkers associating with clinical and pathological prognosis factors. Bio-marker
pT class
PSA at diagnosis
Gleason score
Margin status
Capsule invasion
BCR PCS OS
TWIST n=167 * ** **
AR n=165 ** ** *
c-Nrf-2 n=204 ** * * **
n-Nrf-2 n=205 *** * * * ***
Prx1 n=233 ***
Prx2 n=233 * ** *** * * ***
Prx5 n=232 ***
Prx6 n=231 ** ** ** *** ***
Srx n=229 *** *
*Association is statistically significant at the level P-value 0.05 **Association is statistically significant at the level P-value 0.01 ***Association is statistically significant at the level P-value 0.001 Srx expression has inverse association with clinical parameters
5.2 TWIST AND AR EXPRESSION IN THE PREDICTION OF BFS (I) M-‐‑TWIST overexpression was clearly associated with shortened BFS (P<0.0001) (Figure 7). An association with BFS was also found with a positive surgical margin status (P=0.003), Gleason score (P<0.0001) and M-‐‑AR expression (P=0.008).
When these factors (positive surgical margin status, Gleason score and M-‐‑AR expression) were included into a multivariate analysis, M-‐‑TWIST overexpression (Hazard ratio (HR): 2.516, 95% confidence interval (95% CI): 1.235-‐‑5.125, P=0.011) and Gleason score (HR: 1.483, 95% CI: 1.155-‐‑1.904, P=0.002) were found to be the only independent predictors of BFS.
29
Figure 7. Kaplan Meier curve demonstrating the association between positive M-TWIST expression and decreased BFS (log-rank: P<0.0001). 5.3 8-OHDG AND NRF-2 EXPRESSIONS AND THEIR ASSOCIATION WITH CLINICOPATHOLOCIGAL PROGNOSTIGATORS (II) The positive expression 8-‐‑OHDG (P<0.001), c-‐‑Nrf-‐‑2 (P=0.015) and n-‐‑Nrf-‐‑2 (P=0.016) was more abundant in the malignant samples as compared to benign tissues, respectively (Figure 8). The following associations were detected between c-‐‑Nrf-‐‑2 expression and clinicopathologial factors; positive surgical margin (P=0.005), capsule invasion (P=0.031), BCR (P=0.030) and OS (P=0.002). N-‐‑Nrf-‐‑2 expression was associated with pT class (P=0.001), Gleason score (P=0.026), capsule invasion (P=0.027), BCR (P=0.037) and OS (P<0.0001) (Table 7). The expression of 8-‐‑OHDG did not exhibit an association with any of the clinicopathological prognostic factors or outcome.
28
Table 7. Summary of the significant findings for the expression of biomarkers associating with clinical and pathological prognosis factors. Bio-marker
pT class
PSA at diagnosis
Gleason score
Margin status
Capsule invasion
BCR PCS OS
TWIST n=167 * ** **
AR n=165 ** ** *
c-Nrf-2 n=204 ** * * **
n-Nrf-2 n=205 *** * * * ***
Prx1 n=233 ***
Prx2 n=233 * ** *** * * ***
Prx5 n=232 ***
Prx6 n=231 ** ** ** *** ***
Srx n=229 *** *
*Association is statistically significant at the level P-value 0.05 **Association is statistically significant at the level P-value 0.01 ***Association is statistically significant at the level P-value 0.001 Srx expression has inverse association with clinical parameters
5.2 TWIST AND AR EXPRESSION IN THE PREDICTION OF BFS (I) M-‐‑TWIST overexpression was clearly associated with shortened BFS (P<0.0001) (Figure 7). An association with BFS was also found with a positive surgical margin status (P=0.003), Gleason score (P<0.0001) and M-‐‑AR expression (P=0.008).
When these factors (positive surgical margin status, Gleason score and M-‐‑AR expression) were included into a multivariate analysis, M-‐‑TWIST overexpression (Hazard ratio (HR): 2.516, 95% confidence interval (95% CI): 1.235-‐‑5.125, P=0.011) and Gleason score (HR: 1.483, 95% CI: 1.155-‐‑1.904, P=0.002) were found to be the only independent predictors of BFS.
29
Figure 7. Kaplan Meier curve demonstrating the association between positive M-TWIST expression and decreased BFS (log-rank: P<0.0001). 5.3 8-OHDG AND NRF-2 EXPRESSIONS AND THEIR ASSOCIATION WITH CLINICOPATHOLOCIGAL PROGNOSTIGATORS (II) The positive expression 8-‐‑OHDG (P<0.001), c-‐‑Nrf-‐‑2 (P=0.015) and n-‐‑Nrf-‐‑2 (P=0.016) was more abundant in the malignant samples as compared to benign tissues, respectively (Figure 8). The following associations were detected between c-‐‑Nrf-‐‑2 expression and clinicopathologial factors; positive surgical margin (P=0.005), capsule invasion (P=0.031), BCR (P=0.030) and OS (P=0.002). N-‐‑Nrf-‐‑2 expression was associated with pT class (P=0.001), Gleason score (P=0.026), capsule invasion (P=0.027), BCR (P=0.037) and OS (P<0.0001) (Table 7). The expression of 8-‐‑OHDG did not exhibit an association with any of the clinicopathological prognostic factors or outcome.
30
Figure 8. Positive expression of biomarkers (8-OHDG, P<0.0001; c-Nrf-2, P=0.015 and n-Nrf-2, P=0.016) in benign and malignant samples, respectively. 5.4 NRF-2 EXPRESSION IN SURVIVAL ANALYSIS (II) In the Kaplan Meier analysis, increased c-‐‑Nrf-‐‑2 expression (P=0.034) (Figure 9A) and n-‐‑Nrf-‐‑2 expression (P=0.024) was associated with a shortened BFS. An association with shortened BFS was also demonstrated for the positive surgical margin status (P<0.0001), high Gleason score (P<0.0001) and pT class (P<0.0001). None of the markers analysed predicted shortened PCS. An increase in the expressions of c-‐‑Nrf-‐‑2 (P=0.017) (Figure 9B) and n-‐‑Nrf-‐‑2 (P=0.006) was related to worse OS, as well as the clinicopathological factors high Gleason score (P<0.0001), pT class (P<0.0001) and higher PSA levels at diagnosis (P=0.037).
In the multivariate analysis, c-‐‑Nrf-‐‑2 expression (Hazard ratio (HR): 1.664, 95% confidence interval (95% CI): 1.056-‐‑2.621, P=0.028 and HR: 2.462, 95% CI: 1.162-‐‑5.216, P=0.019) and Gleason score (HR: 2.156, 95% CI: 1.373-‐‑3.386, P=0.001 and HR: 2.166, 95% CI: 1.066-‐‑4.401, P=0.033) proved to be independent prognostic predictors of BFS and OS respectively, when the other factors (PSA at diagnosis, surgical margin status, capsule invasion, pT class, age, n-‐‑Nrf-‐‑expression and 8-‐‑OHDG expression) were included in the analysis.
31
Figure 9. Positive Nrf-2 expression in cytoplasm is associated shortened (A) biochemical recurrence free survival (BFS) (log-rank: P=0.034) and (B) poor overall survival (log-rank: P=0.017) in Kaplan Meier survival analysis.
30
Figure 8. Positive expression of biomarkers (8-OHDG, P<0.0001; c-Nrf-2, P=0.015 and n-Nrf-2, P=0.016) in benign and malignant samples, respectively. 5.4 NRF-2 EXPRESSION IN SURVIVAL ANALYSIS (II) In the Kaplan Meier analysis, increased c-‐‑Nrf-‐‑2 expression (P=0.034) (Figure 9A) and n-‐‑Nrf-‐‑2 expression (P=0.024) was associated with a shortened BFS. An association with shortened BFS was also demonstrated for the positive surgical margin status (P<0.0001), high Gleason score (P<0.0001) and pT class (P<0.0001). None of the markers analysed predicted shortened PCS. An increase in the expressions of c-‐‑Nrf-‐‑2 (P=0.017) (Figure 9B) and n-‐‑Nrf-‐‑2 (P=0.006) was related to worse OS, as well as the clinicopathological factors high Gleason score (P<0.0001), pT class (P<0.0001) and higher PSA levels at diagnosis (P=0.037).
In the multivariate analysis, c-‐‑Nrf-‐‑2 expression (Hazard ratio (HR): 1.664, 95% confidence interval (95% CI): 1.056-‐‑2.621, P=0.028 and HR: 2.462, 95% CI: 1.162-‐‑5.216, P=0.019) and Gleason score (HR: 2.156, 95% CI: 1.373-‐‑3.386, P=0.001 and HR: 2.166, 95% CI: 1.066-‐‑4.401, P=0.033) proved to be independent prognostic predictors of BFS and OS respectively, when the other factors (PSA at diagnosis, surgical margin status, capsule invasion, pT class, age, n-‐‑Nrf-‐‑expression and 8-‐‑OHDG expression) were included in the analysis.
31
Figure 9. Positive Nrf-2 expression in cytoplasm is associated shortened (A) biochemical recurrence free survival (BFS) (log-rank: P=0.034) and (B) poor overall survival (log-rank: P=0.017) in Kaplan Meier survival analysis.
32
5.5 THE ASSOCIATION BETWEEN PRXS AND SRX AND CLINICOPATHOLOGICAL PROGNOSTIGATORS (III) The following associations were found between clinicopathological prognosis factors and positive Prxs expression: Positive Prx1 expression was related with capsule invasion (P=0.001); Prx2 with pT class (P= 0.037), positive surgical margin (P=0.003), capsule invasion (P<0.001), BCR (P=0.043), PCS (P=0.036) and OS (P<0.001); Prx5 with OS (P=0.001); Prx6 with pT class (P=0.006), capsule invasion (P=0.009), BCR (P=0.004), PCS (P<0.001) and OS (P<0.001). Furthermore, there was an inverse association between positive Srx expression and a high PSA level at diagnosis (P=0.001) and OS (P=0.026) (Table 7). 5.6 PRX2 AND PRX6 EXPRESSION IN SURVIVAL ANALYSIS (III) The Kaplan Meier analysis revealed that the shortened BFS was associated with positive expressions of Prx2 (P=0.027) and Prx6 (P= 0.020) (Figure 10). A positive surgical margin status (P<0.001), high Gleason score (P<0.001) and pT class (P<0.001) also displayed an association with shortened BFS. A positive expression of Prx6 (P=0.037) was associated with PCS. Furthermore, several clinicopathological factors, such as capsule invasion (P=0.008), high Gleason score (P<0.001) and pT class (P<0.001) were related with PCS. Positive Prx2 (P=0.045) and Prx6 expression (P=0.033) predicted worse OS, as well as the important clinicopathological prognosis factors i.e. high Gleason score (P<0.001), pT class (P<0.001) and higher PSA levels at diagnosis (P=0.037).
Figure 10. Kaplan Meier estimates of BFS according to Prx6 expression. (Log rank P=0.020).
33
The multivariate analysis revealed that positive Prx6 expression (P=0.030), pT class (P=0.020), positive surgical margin status (P=0.025) and Gleason score (P<0.001) were independent predictors of BFS. Those factors (Prx2 expression, surgical margin status, Gleason score and pT class) found to be statistically significant in the univariate analysis were included in the analysis. Subsequently, Prx2 and Prx6 expression displayed no independent predictive value for PCS or OS according to the results of the multivariate analysis.
32
5.5 THE ASSOCIATION BETWEEN PRXS AND SRX AND CLINICOPATHOLOGICAL PROGNOSTIGATORS (III) The following associations were found between clinicopathological prognosis factors and positive Prxs expression: Positive Prx1 expression was related with capsule invasion (P=0.001); Prx2 with pT class (P= 0.037), positive surgical margin (P=0.003), capsule invasion (P<0.001), BCR (P=0.043), PCS (P=0.036) and OS (P<0.001); Prx5 with OS (P=0.001); Prx6 with pT class (P=0.006), capsule invasion (P=0.009), BCR (P=0.004), PCS (P<0.001) and OS (P<0.001). Furthermore, there was an inverse association between positive Srx expression and a high PSA level at diagnosis (P=0.001) and OS (P=0.026) (Table 7). 5.6 PRX2 AND PRX6 EXPRESSION IN SURVIVAL ANALYSIS (III) The Kaplan Meier analysis revealed that the shortened BFS was associated with positive expressions of Prx2 (P=0.027) and Prx6 (P= 0.020) (Figure 10). A positive surgical margin status (P<0.001), high Gleason score (P<0.001) and pT class (P<0.001) also displayed an association with shortened BFS. A positive expression of Prx6 (P=0.037) was associated with PCS. Furthermore, several clinicopathological factors, such as capsule invasion (P=0.008), high Gleason score (P<0.001) and pT class (P<0.001) were related with PCS. Positive Prx2 (P=0.045) and Prx6 expression (P=0.033) predicted worse OS, as well as the important clinicopathological prognosis factors i.e. high Gleason score (P<0.001), pT class (P<0.001) and higher PSA levels at diagnosis (P=0.037).
Figure 10. Kaplan Meier estimates of BFS according to Prx6 expression. (Log rank P=0.020).
33
The multivariate analysis revealed that positive Prx6 expression (P=0.030), pT class (P=0.020), positive surgical margin status (P=0.025) and Gleason score (P<0.001) were independent predictors of BFS. Those factors (Prx2 expression, surgical margin status, Gleason score and pT class) found to be statistically significant in the univariate analysis were included in the analysis. Subsequently, Prx2 and Prx6 expression displayed no independent predictive value for PCS or OS according to the results of the multivariate analysis.
34
6 Discussion
6.1 TWIST AND AR IN PROGNOSIS OF PC (I) Augmented TWIST expression has been found in malignant tissue in comparison with benign tissue in other publications examining PC and other malignancies, such as breast cancer and squamous cell carcinoma (Kwok et al. 2005, Soini et al. 2011, Fan et al. 2013). In the present study, there was no TWIST expression detected in benign samples whereas positive expression was found in all PC samples taken from different malignant parts of the tumours, in agreement with earlier reports. The expression in a high Gleason area was more abundant than in a low Gleason area, and furthermore the expression in the margin part of the tumour was higher than in the tumour’s central part, evidence of the more aggressive behavior of the cancer cells in the invasive front. Furthermore, the associations between TWIST overexpression and clinical factors, positive surgical margin status, capsule invasion and BCR, were observed in the samples of margin area. In contrast, TWIST expression in the samples of the other tumour areas displayed no association with clinical prognostic factors or survival. This finding is in accordance with the hypothesis that generally in cancer biology, the most active area is the invasive front of the tumour since this is the region where one finds cells with the highest proliferation and the clearest signs of the EMT process.
Recently, Behnsawy et al. published a study conducted with almost identical methodology and sample processing as in the present study. They found a similar kind of association between TWIST overexpression and clinical prognostic factors, such as capsule invasion and surgical margin status, in agreement with this study. In contrast to the present results, these investigators observed also an association between TWIST overexpression and pT-‐‑class, Gleason score and PSA value at diagnosis (Behnsawy et al. 2013). One probable explanation is that in that other study, more aggressive PC material was used. In their report, tumours belonging to T-‐‑classes 3 and 4 accounted for 40% of the cohort compared to 28% of cases in the present study. In addition, 73% of the tissues samples displayed Gleason grade 7 or higher in comparison of 38% of the cases in this study. Furthermore, the discordant results might be explained by the difference in the incidence and mortality rates of PC between European and Asian countries (Center et al. 2012). The main observation in both studies was that TWIST overexpression was an independent predictor of shortened BFS in localised PC after RP. The strongly significant predictive potential was confirmed in the present study even though it assessed less aggressive PC material. The finding emphasizes that increased TWIST activity could be used as a sign of progression in the early stage of organ confined disease.
AR overexpression associated with several clinicopathological factors and predicted shortened BFS, in accordance with many other publications (Mori et al. 2008, Theodoropoulos et al. 2005, Takeda et al. 1996, Shukla-‐‑Dave et al. 2009, Li et al. 2004a). In the multivariate analysis, AR expression was not observed to be an independent predictor of BFS, probably due the stronger effect of TWIST expression. In an investigation conducted with human PC cells, TWIST activation has been shown to up-‐‑regulate AR. The authors
35
speculated that TWIST would be functioning as an AR gene promoter, modulating AR activity (Shiota et al. 2010). In support of this observation, in the present study, a significant association was also detected between TWIST and AR expressions. 6.2 8-OHDG AND NRF-2 IN PROGNOSIS OF PC (II) Higher 8-‐‑OHDG expression was found in malignant samples compared to benign tissues evidence that oxidative DNA-‐‑damage is associated with a malignant process also in PC, as observed in the case of other malignancies, such as ovarian cancer (Karihtala et al. 2009). In agreement with earlier reports evaluating 8-‐‑OHDG as a marker of the cancer aggressiveness in patients with low risk PC, the present study failed to detect any link between 8-‐‑OHDG expression and clinical parameters or survival (Richardson et al. 2009). In localised PC with a low malignant potential, 8-‐‑OHDG has been assessed as having insufficient reliability as a prognostic marker alone, due to the multistep nature of the oxidative injury in carcinogenesis (Bostwick et al. 2000). However, 8-‐‑OHDG might have predictive value in higher risk PC, since Miyake et al. have found augmented 8-‐‑OHDG concentrations in the samples of advanced PC cases in comparison to the samples of localised disease (Miyake et al. 2004).
The activation of Nrf-‐‑2-‐‑mediated oxidative stress as an adaptive mechanism has been demonstrated previously in experiments conducted with cell cultures, animal models and the samples of clinical patients in many cancer types (Iida et al. 2004, Ramos-‐‑Gomez et al. 2001, Lau et al. 2008). In addition, in the case of PC, augmented Nrf-‐‑2 expression has been detected in malignant tissue compared to benign samples, in agreement with findings in the present study (Pettazzoni et al. 2011, Zhang et al. 2010). In contrast to earlier publications, this is the first study comparing Nrf-‐‑2 expression with conventional prognosis factors in a clinical PC patient cohort. In the present study, increased levels of both cytoplasmic and nuclear Nrf-‐‑2 expressions were associated with several clinical prognostic factors and predicted shortened BFS and OS.
According to previous research, under normal conditions, Nrf-‐‑2 is located in cytoplasm bound to Keap1. When a cell is exposed to oxidative stress, Nrf-‐‑2 migrates to the nucleus and activates numerous protecting genes (Nguyen, Yang & Pickett 2004, Kensler, Wakabayashi & Biswal 2007, Itoh, Mimura & Yamamoto 2010). Based on previous evidence, one could theoretically expect nuclear expression to be more abundant and more relevant for comparison with the clinical parameters. However, only cytoplasmic expression, in contrast to the situation with nuclear expression, was an independent predictor of shortened BFS and OS in the multivariate analysis. The significance of this finding based on clinical outcome is still unclear and must be interpreted with caution.
This study could not find any evidence that Nrf-‐‑2 expression would be predictive of cancer specific survival in contrast to the situation with numerous other malignancies, such as lung cancer and breast cancer (Merikallio et al. 2012, Hartikainen et al. 2012). The much lower cancer specific mortality of organ confined PC compared to these other more aggressive cancers might explain this result. For example, the PC specific mortality was only 7.9% in this cohort, in comparison to the much more dismal, 87%, five year mortality of lung cancer (De Angelis et al. 2014). On the other hand, Nrf-‐‑2 expression exhibited a strong association with OS. Possibly, this observation is evidence that the Nrf-‐‑2 modulated mechanism plays a multimodal role in the cell survival process and the changes occurring
34
6 Discussion
6.1 TWIST AND AR IN PROGNOSIS OF PC (I) Augmented TWIST expression has been found in malignant tissue in comparison with benign tissue in other publications examining PC and other malignancies, such as breast cancer and squamous cell carcinoma (Kwok et al. 2005, Soini et al. 2011, Fan et al. 2013). In the present study, there was no TWIST expression detected in benign samples whereas positive expression was found in all PC samples taken from different malignant parts of the tumours, in agreement with earlier reports. The expression in a high Gleason area was more abundant than in a low Gleason area, and furthermore the expression in the margin part of the tumour was higher than in the tumour’s central part, evidence of the more aggressive behavior of the cancer cells in the invasive front. Furthermore, the associations between TWIST overexpression and clinical factors, positive surgical margin status, capsule invasion and BCR, were observed in the samples of margin area. In contrast, TWIST expression in the samples of the other tumour areas displayed no association with clinical prognostic factors or survival. This finding is in accordance with the hypothesis that generally in cancer biology, the most active area is the invasive front of the tumour since this is the region where one finds cells with the highest proliferation and the clearest signs of the EMT process.
Recently, Behnsawy et al. published a study conducted with almost identical methodology and sample processing as in the present study. They found a similar kind of association between TWIST overexpression and clinical prognostic factors, such as capsule invasion and surgical margin status, in agreement with this study. In contrast to the present results, these investigators observed also an association between TWIST overexpression and pT-‐‑class, Gleason score and PSA value at diagnosis (Behnsawy et al. 2013). One probable explanation is that in that other study, more aggressive PC material was used. In their report, tumours belonging to T-‐‑classes 3 and 4 accounted for 40% of the cohort compared to 28% of cases in the present study. In addition, 73% of the tissues samples displayed Gleason grade 7 or higher in comparison of 38% of the cases in this study. Furthermore, the discordant results might be explained by the difference in the incidence and mortality rates of PC between European and Asian countries (Center et al. 2012). The main observation in both studies was that TWIST overexpression was an independent predictor of shortened BFS in localised PC after RP. The strongly significant predictive potential was confirmed in the present study even though it assessed less aggressive PC material. The finding emphasizes that increased TWIST activity could be used as a sign of progression in the early stage of organ confined disease.
AR overexpression associated with several clinicopathological factors and predicted shortened BFS, in accordance with many other publications (Mori et al. 2008, Theodoropoulos et al. 2005, Takeda et al. 1996, Shukla-‐‑Dave et al. 2009, Li et al. 2004a). In the multivariate analysis, AR expression was not observed to be an independent predictor of BFS, probably due the stronger effect of TWIST expression. In an investigation conducted with human PC cells, TWIST activation has been shown to up-‐‑regulate AR. The authors
35
speculated that TWIST would be functioning as an AR gene promoter, modulating AR activity (Shiota et al. 2010). In support of this observation, in the present study, a significant association was also detected between TWIST and AR expressions. 6.2 8-OHDG AND NRF-2 IN PROGNOSIS OF PC (II) Higher 8-‐‑OHDG expression was found in malignant samples compared to benign tissues evidence that oxidative DNA-‐‑damage is associated with a malignant process also in PC, as observed in the case of other malignancies, such as ovarian cancer (Karihtala et al. 2009). In agreement with earlier reports evaluating 8-‐‑OHDG as a marker of the cancer aggressiveness in patients with low risk PC, the present study failed to detect any link between 8-‐‑OHDG expression and clinical parameters or survival (Richardson et al. 2009). In localised PC with a low malignant potential, 8-‐‑OHDG has been assessed as having insufficient reliability as a prognostic marker alone, due to the multistep nature of the oxidative injury in carcinogenesis (Bostwick et al. 2000). However, 8-‐‑OHDG might have predictive value in higher risk PC, since Miyake et al. have found augmented 8-‐‑OHDG concentrations in the samples of advanced PC cases in comparison to the samples of localised disease (Miyake et al. 2004).
The activation of Nrf-‐‑2-‐‑mediated oxidative stress as an adaptive mechanism has been demonstrated previously in experiments conducted with cell cultures, animal models and the samples of clinical patients in many cancer types (Iida et al. 2004, Ramos-‐‑Gomez et al. 2001, Lau et al. 2008). In addition, in the case of PC, augmented Nrf-‐‑2 expression has been detected in malignant tissue compared to benign samples, in agreement with findings in the present study (Pettazzoni et al. 2011, Zhang et al. 2010). In contrast to earlier publications, this is the first study comparing Nrf-‐‑2 expression with conventional prognosis factors in a clinical PC patient cohort. In the present study, increased levels of both cytoplasmic and nuclear Nrf-‐‑2 expressions were associated with several clinical prognostic factors and predicted shortened BFS and OS.
According to previous research, under normal conditions, Nrf-‐‑2 is located in cytoplasm bound to Keap1. When a cell is exposed to oxidative stress, Nrf-‐‑2 migrates to the nucleus and activates numerous protecting genes (Nguyen, Yang & Pickett 2004, Kensler, Wakabayashi & Biswal 2007, Itoh, Mimura & Yamamoto 2010). Based on previous evidence, one could theoretically expect nuclear expression to be more abundant and more relevant for comparison with the clinical parameters. However, only cytoplasmic expression, in contrast to the situation with nuclear expression, was an independent predictor of shortened BFS and OS in the multivariate analysis. The significance of this finding based on clinical outcome is still unclear and must be interpreted with caution.
This study could not find any evidence that Nrf-‐‑2 expression would be predictive of cancer specific survival in contrast to the situation with numerous other malignancies, such as lung cancer and breast cancer (Merikallio et al. 2012, Hartikainen et al. 2012). The much lower cancer specific mortality of organ confined PC compared to these other more aggressive cancers might explain this result. For example, the PC specific mortality was only 7.9% in this cohort, in comparison to the much more dismal, 87%, five year mortality of lung cancer (De Angelis et al. 2014). On the other hand, Nrf-‐‑2 expression exhibited a strong association with OS. Possibly, this observation is evidence that the Nrf-‐‑2 modulated mechanism plays a multimodal role in the cell survival process and the changes occurring
36
in PC may be one way to induce Nrf-‐‑2 expression. In support of this speculation, it has been proposed that over 200 genes are linked with ROS activated signalling through the Nrf-‐‑2 mediated pathway (Lewis et al. 2010).
In summary, the data suggest that increased Nrf-‐‑2 expression is associated with conventional prognosticators and predicts worse outcome of patients with localised PC. 6.3 PRXS AND SRX IN PROGNOSIS OF PC (III) In mammalian cells, Prxs have been found to exist in six isoforms which are distributed throughout tissues. The functions of these enzymes are to act as neutralizing enzymes in combatting an ROS attack. As a consequence of normal aerobic respiration, free radicals are continuously formed and should be detoxified. While this is the situation in physiological conditions, an augmented oxidative burst is involved in the pathogenesis of many diseases, such as malignancies (Karihtala, Soini 2007, Poynton, Hampton 2014). Since a clear association has been observed earlier between Prx 3 and 4 expression and cancer aggressiveness in PC cells, this present study explored Prx 1, 2, 5 and 6 expression in samples from PC patients (Basu et al. 2011, Whitaker et al. 2013). In previous publications, augmented Prxs expression has been found in malignant samples in comparison to benign samples (Basu et al. 2011, Chaiswing, Zhong & Oberley 2014, Valdman et al. 2009). The association between higher Gleason score and increased Prx expression has been detected with Prx 2, 3 and 4 (Basu et al. 2011, Chaiswing, Zhong & Oberley 2014).
In the present study, Prx 2 and 6 displayed the strongest association with the clinicopathological prognostic factors. Basu et al. reported augmented Prx6 expression to be linked with pT class, in agreement with this study. These investigators also revealed the association between the Gleason score and increased Prx2 expression in contrast to the findings in this study (Basu et al. 2011). The difference might be explained with the samples of more advanced and aggressive tumours compared to the material examined here. Indeed, in the study of Basu et al., the vast majority (95%) of tumours had a Gleason score of 7 or higher, in comparison with 36% in present study. On the other hand, an association was found here between Prx2 expression with positive surgical margin status and capsule invasion. There are no reports which have examined the association between expression of Prxs and surgical pathological parameters, such as capsule invasion and margin status.
There are a few publications which have evaluated Prx expression in PC progression but none of them have been conducted with a clinical patient cohort. The activity of Prx 3 and 4 has been shown to be increased in PC cell cultures after exposure to oxidative stress and the levels of protecting gene functions have been found be augmented, promoting cancer cell survival (Ummanni et al. 2012, Whitaker et al. 2013). Basu et al. analysed 150 samples of tissue bank material and found that Prx3 overexpression did predict BFS. However, these workers reported very little clinical data e.g. there was no information available on BCR, detailed follow-‐‑up routines or observation time. Furthermore, no multivariate analysis was performed (Basu et al. 2011). The present study is the first to demonstrate that increased Prx 2 and 6 expressions in PC samples with organ confined disease is connected with worse clinical outcome. Elevated expression of Prx 2 and 6 predicted shortened BFS and Prx6 expression remained an independent factor in the multivariate analysis. The finding is important in the context of PC patients after RP, since at present, PSA-‐‑relapse is the only
37
biochemical indicator of disease recurrence in clinical use (Oefelein et al. 1995, Stephenson et al. 2006).
There are no earlier studies exploring the expression of Prxs in predicting the survival of PC patients. In this study, the augmented expressions Prx 2 and 6 were found to be predictors of worse OS, but these biomarkers failed to show any independent value in the multivariate analysis. Furthermore, Prx6 expression predicted PCS in the univariate analysis. The observations are in line with the earlier findings that Prxs function as neutralizing enzymes, combatting ROS to promote cell survival. On the other hand, the progression of localised PC is slow and disease specific mortality low and these are factors that complicate conducting a survival analysis.
Srx expression displayed an inverse association with clinical factors such as high PSA at diagnosis and OS. One might presume Srx expression to be up-‐‑regulated, since Srx reduces hyperoxidized Prx enzymes into their active state (Woo et al. 2005). Based on this study conducted with clinical material, the relevance of this observation remains unclear. In addition, there are no previous publications examining Srx expression in PC. 6.4 CLINICAL IMPLICATIONS Nowadays, an ever-‐‑increasing number of PC cases are being diagnosed at an early stage. Most of these men suffering from PC have a slow-‐‑growing tumour with excellent prognosis and only a small number of patients are at risk of suffering a life-‐‑threatening disease (Johansson et al. 2004, Soloway et al. 2010).
Clinicopathological factors, such as Gleason score, pT class and PSA-‐‑value at the diagnosis have been used in cancer risk assessment with patients after RP. These have been simplified for clinicians by developing nomograms to quantify the aggressiveness of PC at the diagnosis or after curative treatment, but their accuracy is still as low as 70-‐‑80% (Capitanio et al. 2010). Several attempts have been made to improve the precision of the evaluation of the cancer risk by introducing biomarkers for clinical practice. Although there has been active research, at present, no biomolecules are available which are either suitable or sufficiently reliable for clinical use (Heidenreich et al. 2011).
The curative treatments may cause undesirable adverse effects and decrease the quality of life. Depending on the study design, after RP, as many as 72% of patients suffer from urinary incontinence and 69 % from erectile dysfunction, (Boorjian et al. 2012, Sanda et al. 2008). Since it is desirable to avoid overtreatment, active surveillance should be offered to patients with low risk PC. If the disease of a patient under active surveillance displays signs of progression, he should be treated at a sufficiently early stage to achieve a curative outcome (Welty, Cooperberg & Carroll 2014). Currently, monitoring is based on clinical examination, frequent PSA-‐‑testing and repeated prostate biopsy samples. During active follow-‐‑up, approximately 10% of the patients themselves request that they discontinue surveillance due to anxiety (Thomsen et al. 2014, van den Bergh et al. 2009). There is a clear need for new prognostic tools, such as biomarkers, which could be beneficial for counselling patients under surveillance in order to achieve a more accurate evaluation of cancer behaviour (Fleshner, Lawrentschuk 2009).
This study showed that TWIST is overexpressed in PC samples and predicts shortened BFS. In agreement with earlier reports, processes involved in EMT-‐‑transition and TWIST-‐‑mediated signalling are involved with carcinogenesis and tumour progression (Thiery
36
in PC may be one way to induce Nrf-‐‑2 expression. In support of this speculation, it has been proposed that over 200 genes are linked with ROS activated signalling through the Nrf-‐‑2 mediated pathway (Lewis et al. 2010).
In summary, the data suggest that increased Nrf-‐‑2 expression is associated with conventional prognosticators and predicts worse outcome of patients with localised PC. 6.3 PRXS AND SRX IN PROGNOSIS OF PC (III) In mammalian cells, Prxs have been found to exist in six isoforms which are distributed throughout tissues. The functions of these enzymes are to act as neutralizing enzymes in combatting an ROS attack. As a consequence of normal aerobic respiration, free radicals are continuously formed and should be detoxified. While this is the situation in physiological conditions, an augmented oxidative burst is involved in the pathogenesis of many diseases, such as malignancies (Karihtala, Soini 2007, Poynton, Hampton 2014). Since a clear association has been observed earlier between Prx 3 and 4 expression and cancer aggressiveness in PC cells, this present study explored Prx 1, 2, 5 and 6 expression in samples from PC patients (Basu et al. 2011, Whitaker et al. 2013). In previous publications, augmented Prxs expression has been found in malignant samples in comparison to benign samples (Basu et al. 2011, Chaiswing, Zhong & Oberley 2014, Valdman et al. 2009). The association between higher Gleason score and increased Prx expression has been detected with Prx 2, 3 and 4 (Basu et al. 2011, Chaiswing, Zhong & Oberley 2014).
In the present study, Prx 2 and 6 displayed the strongest association with the clinicopathological prognostic factors. Basu et al. reported augmented Prx6 expression to be linked with pT class, in agreement with this study. These investigators also revealed the association between the Gleason score and increased Prx2 expression in contrast to the findings in this study (Basu et al. 2011). The difference might be explained with the samples of more advanced and aggressive tumours compared to the material examined here. Indeed, in the study of Basu et al., the vast majority (95%) of tumours had a Gleason score of 7 or higher, in comparison with 36% in present study. On the other hand, an association was found here between Prx2 expression with positive surgical margin status and capsule invasion. There are no reports which have examined the association between expression of Prxs and surgical pathological parameters, such as capsule invasion and margin status.
There are a few publications which have evaluated Prx expression in PC progression but none of them have been conducted with a clinical patient cohort. The activity of Prx 3 and 4 has been shown to be increased in PC cell cultures after exposure to oxidative stress and the levels of protecting gene functions have been found be augmented, promoting cancer cell survival (Ummanni et al. 2012, Whitaker et al. 2013). Basu et al. analysed 150 samples of tissue bank material and found that Prx3 overexpression did predict BFS. However, these workers reported very little clinical data e.g. there was no information available on BCR, detailed follow-‐‑up routines or observation time. Furthermore, no multivariate analysis was performed (Basu et al. 2011). The present study is the first to demonstrate that increased Prx 2 and 6 expressions in PC samples with organ confined disease is connected with worse clinical outcome. Elevated expression of Prx 2 and 6 predicted shortened BFS and Prx6 expression remained an independent factor in the multivariate analysis. The finding is important in the context of PC patients after RP, since at present, PSA-‐‑relapse is the only
37
biochemical indicator of disease recurrence in clinical use (Oefelein et al. 1995, Stephenson et al. 2006).
There are no earlier studies exploring the expression of Prxs in predicting the survival of PC patients. In this study, the augmented expressions Prx 2 and 6 were found to be predictors of worse OS, but these biomarkers failed to show any independent value in the multivariate analysis. Furthermore, Prx6 expression predicted PCS in the univariate analysis. The observations are in line with the earlier findings that Prxs function as neutralizing enzymes, combatting ROS to promote cell survival. On the other hand, the progression of localised PC is slow and disease specific mortality low and these are factors that complicate conducting a survival analysis.
Srx expression displayed an inverse association with clinical factors such as high PSA at diagnosis and OS. One might presume Srx expression to be up-‐‑regulated, since Srx reduces hyperoxidized Prx enzymes into their active state (Woo et al. 2005). Based on this study conducted with clinical material, the relevance of this observation remains unclear. In addition, there are no previous publications examining Srx expression in PC. 6.4 CLINICAL IMPLICATIONS Nowadays, an ever-‐‑increasing number of PC cases are being diagnosed at an early stage. Most of these men suffering from PC have a slow-‐‑growing tumour with excellent prognosis and only a small number of patients are at risk of suffering a life-‐‑threatening disease (Johansson et al. 2004, Soloway et al. 2010).
Clinicopathological factors, such as Gleason score, pT class and PSA-‐‑value at the diagnosis have been used in cancer risk assessment with patients after RP. These have been simplified for clinicians by developing nomograms to quantify the aggressiveness of PC at the diagnosis or after curative treatment, but their accuracy is still as low as 70-‐‑80% (Capitanio et al. 2010). Several attempts have been made to improve the precision of the evaluation of the cancer risk by introducing biomarkers for clinical practice. Although there has been active research, at present, no biomolecules are available which are either suitable or sufficiently reliable for clinical use (Heidenreich et al. 2011).
The curative treatments may cause undesirable adverse effects and decrease the quality of life. Depending on the study design, after RP, as many as 72% of patients suffer from urinary incontinence and 69 % from erectile dysfunction, (Boorjian et al. 2012, Sanda et al. 2008). Since it is desirable to avoid overtreatment, active surveillance should be offered to patients with low risk PC. If the disease of a patient under active surveillance displays signs of progression, he should be treated at a sufficiently early stage to achieve a curative outcome (Welty, Cooperberg & Carroll 2014). Currently, monitoring is based on clinical examination, frequent PSA-‐‑testing and repeated prostate biopsy samples. During active follow-‐‑up, approximately 10% of the patients themselves request that they discontinue surveillance due to anxiety (Thomsen et al. 2014, van den Bergh et al. 2009). There is a clear need for new prognostic tools, such as biomarkers, which could be beneficial for counselling patients under surveillance in order to achieve a more accurate evaluation of cancer behaviour (Fleshner, Lawrentschuk 2009).
This study showed that TWIST is overexpressed in PC samples and predicts shortened BFS. In agreement with earlier reports, processes involved in EMT-‐‑transition and TWIST-‐‑mediated signalling are involved with carcinogenesis and tumour progression (Thiery
38
2002). Furthermore, it has been demonstrated that mechanisms linked with oxidative stress are activated in cancer cells supporting the findings of the present study in which increased expression Nrf-‐‑2 and Prx6 predicted worse outcome of PC patients (Karihtala, Soini 2007). Augmented expressions of TWIST, Nrf-‐‑2 and Prx6 were independent predictors of shortened BFS in organ confined PC. In the previous reports, the same biomarkers have been found to be linked to malignancies with poorer survival expectations, such as breast cancer and B-‐‑cell lymphoma (Soini et al. 2011, Hartikainen et al. 2012, Kuusisto et al. 2015). Since an elevation in the PSA value is the first sign of clinical progression in PC, it is important to note that these markers have the same predictive value in the PC patients examined here with long life expectancies as in those more aggressive malignancies (Stephenson et al. 2006). In this context, these markers could serve indicators for early cancer recurrence. 6.5 LIMITATIONS This study is retrospective and was conducted with PC samples collected after RP. In addition, the data were collated from patient records and the laboratory database and naturally, an exact control protocol could not be followed. Even although the samples were re-‐‑evaluated to ensure standardization of histopathological parameters, the treatment decisions were made individually for each patient according to clinical practice. In order to validate biomarkers for clinical use, a prospective study with biopsy samples would be needed. Since the natural history of PC is slow, a longer follow-‐‑up time and a larger patient cohort would be essential to evaluate prognostic potential of biomarkers for PCS. 6.6 FUTURE PERSPECTIVES Since TWIST, Nrf-‐‑2 and Prx6 exhibited independent prognostic value for BFS in localised PC, they could serve as candidate molecules for future research in developing more accurate surveillance protocols for PC patients after RP. In addition, it might be reasonable to analyse expression of these biomarkers in prostate biopsy samples in a prospective pre-‐‑treatment setting since this could help in selecting the optimal strategy between surveillance and curative treatment. Furthermore, a clarification of molecular targets in disturbed regulatory pathways in cancer cells could provide new targets for drug development and the possibility of personalized cancer treatment in the future.
39
7 Summary and conclusions
The prognosis of patients with localised PC is normally excellent and only a small proportion of PC cases progress to a metastatic stage. Currently, the evaluation of cancer aggressiveness is based on clinicopathological factors and the data from the pathological report of the prostatectomy specimen after RP. Biomarkers are needed to predict more accurately the behavior of PC and to pinpoint those patients with aggressive tumours for multimodal treatments and careful follow-‐‑up. TWIST and oxidative stress related biomolecules could provide possibilities for counselling of the PC patient about his cancer risk. Based on the results of the present study, the following conclusions can be drawn:
1. High AR and TWIST expression associated with several clinicopathological factors and augmented TWIST expression predicted shortened BFS in the margin area of tumour.
2. The levels of expression of 8-‐‑OHDG, n-‐‑Nrf-‐‑2 and c-‐‑Nrf-‐‑2 were abundant in
malignant tissue in comparison to benign tissue. Elevated n-‐‑Nrf-‐‑2 and c-‐‑Nrf-‐‑2 expressions were linked with conventional prognosticators and shortened BFS and OS. Increased c-‐‑Nrf-‐‑2 expression independently predicted shortened BFS and poor OS.
3. Augmented Prxs expression was associated with clinical and pathological
prognosticators. Elevated Prx6 expression proved to be an independent predictor of shortened BFS.
38
2002). Furthermore, it has been demonstrated that mechanisms linked with oxidative stress are activated in cancer cells supporting the findings of the present study in which increased expression Nrf-‐‑2 and Prx6 predicted worse outcome of PC patients (Karihtala, Soini 2007). Augmented expressions of TWIST, Nrf-‐‑2 and Prx6 were independent predictors of shortened BFS in organ confined PC. In the previous reports, the same biomarkers have been found to be linked to malignancies with poorer survival expectations, such as breast cancer and B-‐‑cell lymphoma (Soini et al. 2011, Hartikainen et al. 2012, Kuusisto et al. 2015). Since an elevation in the PSA value is the first sign of clinical progression in PC, it is important to note that these markers have the same predictive value in the PC patients examined here with long life expectancies as in those more aggressive malignancies (Stephenson et al. 2006). In this context, these markers could serve indicators for early cancer recurrence. 6.5 LIMITATIONS This study is retrospective and was conducted with PC samples collected after RP. In addition, the data were collated from patient records and the laboratory database and naturally, an exact control protocol could not be followed. Even although the samples were re-‐‑evaluated to ensure standardization of histopathological parameters, the treatment decisions were made individually for each patient according to clinical practice. In order to validate biomarkers for clinical use, a prospective study with biopsy samples would be needed. Since the natural history of PC is slow, a longer follow-‐‑up time and a larger patient cohort would be essential to evaluate prognostic potential of biomarkers for PCS. 6.6 FUTURE PERSPECTIVES Since TWIST, Nrf-‐‑2 and Prx6 exhibited independent prognostic value for BFS in localised PC, they could serve as candidate molecules for future research in developing more accurate surveillance protocols for PC patients after RP. In addition, it might be reasonable to analyse expression of these biomarkers in prostate biopsy samples in a prospective pre-‐‑treatment setting since this could help in selecting the optimal strategy between surveillance and curative treatment. Furthermore, a clarification of molecular targets in disturbed regulatory pathways in cancer cells could provide new targets for drug development and the possibility of personalized cancer treatment in the future.
39
7 Summary and conclusions
The prognosis of patients with localised PC is normally excellent and only a small proportion of PC cases progress to a metastatic stage. Currently, the evaluation of cancer aggressiveness is based on clinicopathological factors and the data from the pathological report of the prostatectomy specimen after RP. Biomarkers are needed to predict more accurately the behavior of PC and to pinpoint those patients with aggressive tumours for multimodal treatments and careful follow-‐‑up. TWIST and oxidative stress related biomolecules could provide possibilities for counselling of the PC patient about his cancer risk. Based on the results of the present study, the following conclusions can be drawn:
1. High AR and TWIST expression associated with several clinicopathological factors and augmented TWIST expression predicted shortened BFS in the margin area of tumour.
2. The levels of expression of 8-‐‑OHDG, n-‐‑Nrf-‐‑2 and c-‐‑Nrf-‐‑2 were abundant in
malignant tissue in comparison to benign tissue. Elevated n-‐‑Nrf-‐‑2 and c-‐‑Nrf-‐‑2 expressions were linked with conventional prognosticators and shortened BFS and OS. Increased c-‐‑Nrf-‐‑2 expression independently predicted shortened BFS and poor OS.
3. Augmented Prxs expression was associated with clinical and pathological
prognosticators. Elevated Prx6 expression proved to be an independent predictor of shortened BFS.
40
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44
D.G., Egevad, L., Epstein, J.I., Grignon, D.J., Jones, E.C., Montironi, R., Moussa, M.,Sweet, J.M., Trpkov, K., Wheeler, T.M. & Srigley, J.R. 2008, "ʺInterobserver variabilitybetween expert urologic pathologists for extraprostatic extension and surgical marginstatus in radical prostatectomy specimens"ʺ, The American Journal of Surgical Pathology,vol. 32, no. 10, pp. 1503-‐‑1512.
Evans, M.D., Dizdaroglu, M. & Cooke, M.S. 2004, "ʺOxidative DNA damage and disease: induction, repair and significance"ʺ, Mutation research, vol. 567, no. 1, pp. 1-‐‑61.
Fan, C.C., Wang, T.Y., Cheng, Y.A., Jiang, S.S., Cheng, C.W., Lee, A.Y. & Kao, T.Y. 2013, "ʺExpression of E-‐‑cadherin, Twist, and p53 and their prognostic value in patients with oral squamous cell carcinoma"ʺ, Journal of cancer research and clinical oncology, vol. 139, no. 10, pp. 1735-‐‑1744.
Ficarra, V., Novara, G., Ahlering, T.E., Costello, A., Eastham, J.A., Graefen, M., Guazzoni, G., Menon, M., Mottrie, A., Patel, V.R., Van der Poel, H., Rosen, R.C., Tewari, A.K., Wilson, T.G., Zattoni, F. & Montorsi, F. 2012, "ʺSystematic review and meta-‐‑analysis of studies reporting potency rates after robot-‐‑assisted radical prostatectomy"ʺ, European urology, vol. 62, no. 3, pp. 418-‐‑430.
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Finnish Cancer Registry 2015. Available: http://www.cancer.fi/syoparekisteri/en/statistics/
Fleshner, N.E. & Lawrentschuk, N. 2009, "ʺRisk of developing prostate cancer in the future: overview of prognostic biomarkers"ʺ, Urology, vol. 73, no. 5 Suppl, pp. S21-‐‑7.
Fossa, A., Lilleby, W., Fossa, S.D., Gaudernack, G., Torlakovic, G. & Berner, A. 2002, "ʺIndependent prognostic significance of HER-‐‑2 oncoprotein expression in pN0 prostate cancer undergoing curative radiotherapy"ʺ, International journal of cancer.Journal international du cancer, vol. 99, no. 1, pp. 100-‐‑105.
Freedland, S.J., deGregorio, F., Sacoolidge, J.C., Elshimali, Y.I., Csathy, G.S., Dorey, F., Reiter, R.E. & Aronson, W.J. 2003, "ʺPreoperative p27 status is an independent predictor of prostate specific antigen failure following radical prostatectomy"ʺ, The Journal of urology, vol. 169, no. 4, pp. 1325-‐‑1330.
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Gajula, R.P., Chettiar, S.T., Williams, R.D., Thiyagarajan, S., Kato, Y., Aziz, K., Wang, R., Gandhi, N., Wild, A.T., Vesuna, F., Ma, J., Salih, T., Cades, J., Fertig, E., Biswal, S., Burns, T.F., Chung, C.H., Rudin, C.M., Herman, J.M., Hales, R.K., Raman, V., An, S.S. &
45
Tran, P.T. 2013, "ʺThe twist box domain is required for Twist1-‐‑induced prostate cancer metastasis"ʺ, Molecular cancer research : MCR, vol. 11, no. 11, pp. 1387-‐‑1400.
Gandaglia, G., Sammon, J.D., Chang, S.L., Choueiri, T.K., Hu, J.C., Karakiewicz, P.I., Kibel,
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Gosselaar, C., Roobol, M.J., Roemeling, S. & Schroder, F.H. 2008, "ʺThe role of the digital
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Haglind, E., Carlsson, S., Stranne, J., Wallerstedt, A., Wilderang, U., Thorsteinsdottir, T.,
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47
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47
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48
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49
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49
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Experimental & molecular medicine, vol. 31, no. 2, pp. 53-‐‑59.
53
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A.K., Zhao, Y., Kiningham, K.K., Clair, D.K. & Clair, W.H. 2013, "ʺKEAP1 is a redox sensitive target that arbitrates the opposing radiosensitive effects of parthenolide in normal and cancer cells"ʺ, Cancer research, vol. 73, no. 14, pp. 4406-‐‑4417.
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59
radiotherapy and intensity-‐‑modulated radiotherapy for localized prostate cancer"ʺ, International journal of radiation oncology, biology, physics, vol. 70, no. 4, pp. 1124-‐‑1129.
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"ʺReduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-‐‑cys peroxiredoxins"ʺ, The Journal of biological chemistry, vol. 280, no. 5, pp. 3125-‐‑3128.
Xu, Y., Fang, F., Miriyala, S., Crooks, P.A., Oberley, T.D., Chaiswing, L., Noel, T., Holley,
A.K., Zhao, Y., Kiningham, K.K., Clair, D.K. & Clair, W.H. 2013, "ʺKEAP1 is a redox sensitive target that arbitrates the opposing radiosensitive effects of parthenolide in normal and cancer cells"ʺ, Cancer research, vol. 73, no. 14, pp. 4406-‐‑4417.
Yang, G., Timme, T.L., Frolov, A., Wheeler, T.M. & Thompson, T.C. 2005, "ʺCombined c-‐‑Myc
and caveolin-‐‑1 expression in human prostate carcinoma predicts prostate carcinoma progression"ʺ, Cancer, vol. 103, no. 6, pp. 1186-‐‑1194.
Yang, J., Mani, S.A., Donaher, J.L., Ramaswamy, S., Itzykson, R.A., Come, C., Savagner, P.,
Gitelman, I., Richardson, A. & Weinberg, R.A. 2004, "ʺTwist, a master regulator of morphogenesis, plays an essential role in tumor metastasis"ʺ, Cell, vol. 117, no. 7, pp. 927-‐‑939.
Yossepowitch, O., Bjartell, A., Eastham, J.A., Graefen, M., Guillonneau, B.D., Karakiewicz, P.I., Montironi, R. & Montorsi, F. 2009, "ʺPositive surgical margins in radical prostatectomy: outlining the problem and its long-‐‑term consequences"ʺ, European urology, vol. 55, no. 1, pp. 87-‐‑99.
Yu, S., Khor, T.O., Cheung, K.L., Li, W., Wu, T.Y., Huang, Y., Foster, B.A., Kan, Y.W. &
Kong, A.N. 2010, "ʺNrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice"ʺ, PloS one, vol. 5, no. 1, pp. e8579.
Yuen, H.F., Chua, C.W., Chan, Y.P., Wong, Y.C., Wang, X. & Chan, K.W. 2007, "ʺSignificance
of TWIST and E-‐‑cadherin expression in the metastatic progression of prostatic cancer"ʺ, Histopathology, vol. 50, no. 5, pp. 648-‐‑658.
Yuen, H.F., Kwok, W.K., Chan, K.K., Chua, C.W., Chan, Y.P., Chu, Y.Y., Wong, Y.C., Wang,
X. & Chan, K.W. 2008, "ʺTWIST modulates prostate cancer cell-‐‑mediated bone cell activity and is upregulated by osteogenic induction"ʺ, Carcinogenesis, vol. 29, no. 8, pp. 1509-‐‑1518.
Zelefsky, M.J., Levin, E.J., Hunt, M., Yamada, Y., Shippy, A.M., Jackson, A. & Amols, H.I.
2008, "ʺIncidence of late rectal and urinary toxicities after three-‐‑dimensional conformal
59
radiotherapy and intensity-‐‑modulated radiotherapy for localized prostate cancer"ʺ, International journal of radiation oncology, biology, physics, vol. 70, no. 4, pp. 1124-‐‑1129.
Zhang, P., Singh, A., Yegnasubramanian, S., Esopi, D., Kombairaju, P., Bodas, M., Wu, H.,
Bova, S.G. & Biswal, S. 2010, "ʺLoss of Kelch-‐‑like ECH-‐‑associated protein 1 function in prostate cancer cells causes chemoresistance and radioresistance and promotes tumor growth"ʺ, Molecular cancer therapeutics, vol. 9, no. 2, pp. 336-‐‑346.
Zhao, M., Xu, H., Zhang, B., Hong, B., Yan, W. & Zhang, J. 2015, "ʺImpact of nuclear factor
erythroid-‐‑derived 2-‐‑like 2 and p62/sequestosome expression on prognosis of patients with gliomas"ʺ, Human pathology, vol. 46, no. 6, pp. 843-‐‑849.
Zheng, H. & Kang, Y. 2014, "ʺMultilayer control of the EMT master regulators"ʺ, Oncogene,
vol. 33, no. 14, pp. 1755-‐‑1763. Zielinski, R.R., Eigl, B.J. & Chi, K.N. 2013, "ʺTargeting the apoptosis pathway in prostate
cancer"ʺ, Cancer journal (Sudbury, Mass.), vol. 19, no.
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PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND
Dissertations in Health Sciences
ISBN 978-952-61-2069-0ISSN 1798-5706
Dissertations in Health Sciences
PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND
SAMI RAATIKAINEN
TWIST AND OXIDATIVE STRESS RELATED BIOMARKERS IN OUTCOME PREDICTION OF PROSTATE CANCER
PATIENTS TREATED WITH RADICAL PROSTATECTOMY
This retrospective study examined the predictive value of the EMT marker TWIST and oxidative stress related biomolecules in prostate cancer patients after radical prostatectomy. Increased expression of TWIST, Nrf-2 and Prx6 was associated
with biochemical recurrence and augmented Nrf-2 expression predicted worse survival
of the patients. These biomarkers could help in developing a more accurate cancer risk
evaluation for prostate cancer patients after radical prostate surgery.
SAMI RAATIKAINEN
