Towards Precision Oncology Through Multidimensional Tumor Characterization

The NCT/DKTK MASTER Program

Stefan Fröhling

59th Heidelberg Grand Rounds | March 6, 2018

Precision Oncology

MD Anderson Cancer Center Institute for Personalized Cancer Therapy

https://pct.mdanderson.org

Common cancers as multiple, molecularly distinct diseases of the same organ • Lung adenocarcinoma Improved clinical outcome through genotype-directed therapy • Gastrointestinal stromal tumor Distinct mutations shared across multiple cancers • BRAF p.V600E/K More than 50 FDA-approved targeted therapies • Erlotinib, trastuzumab, vismodegib, etc. Increasing number of precision medicine trials • NCI-MATCH, TAPUR, MOSCATO 01, etc.

Clinical Impact of Genomics-Guided Precision Oncology

Genomic Landscape of Cancer

Gene mountains and hills Wood et al. Science 2007

Long tail of actionable cancer gene alterations Lawrence et al. Nature 2013

• Large-scale sequencing projects have completed exomes/genomes from >50,000 cases representing >80 cancer types

• Most common (present at >5% frequency per tumor type) cancer drivers are defined

Majority of cancer genes mutated at frequencies of <5% within any given

histologic tumor subtype

Rare Driver Mutations: NTRK Fusions

• 1,500-5,000 NTRK fusion-positive cancers in the US annually

• Constitutively active receptor tyrosine kinases

• Small-molecule inhibitors • Larotrectinib • Entrectinib

• Pooled analysis of 3 larotrectinib basket trials

• 55 NTRK fusion-positive patients (pediatric, adult)

• Overall response rate, 75% • Complete, 13% • Partial, 62%

• Efficacy across age groups and histologies

• Median response duration not reached (median follow-up, 9.4 months)

Drilon et al. N Engl J Med 2018

Overall mutational load and neoantigen burden Snyder et al. N Engl J Med 2014, Van Allen et al. Science 2015, Rizvi et al. Science 2015, Le et al. N Engl J Med 2015 Neoantigen intratumoral heterogeneity McGranahan et al. Science 2016 Immunogenic insertion/deletion mutations Turajlic et al. Lancet Oncol 2017 PDL1 amplification and/or overexpression Ansell et al. N Engl J Med 2015 Structural rearrangements of PDL1/2 Steidl et al. Nature 2011, Chong et al. Blood 2016 Disruption of PDL1 3’ untranslated region Kataoka et al. Nature 2016

Complex Biomarkers: Benefit from Immunotherapy

Loss-of-function PBRM1 mutations Miao et al. Science 2018, Pan et al. Science 2018

Complex Biomarkers: Benefit from Immunotherapy

Clinical Implementation

Genetics

Clinical care

Molecular diversity and genetic taxonomy of cancer

Actionability of molecular lesions

Individual, “private” patterns of molecular lesions

Comprehensive molecular stratification approaches that are able to capture the entire spectrum of • Rare driver mutations • Complex, multifactorial biomarkers • Newly emerging diagnostic, prognostic, and

predictive parameters

Molecularly Aided Stratification for Tumor Eradication Research

Genetics

Clinical care

Molecular diversity and genetic taxonomy of cancer

Actionability of molecular lesions

Individual, “private” patterns of molecular lesions

MASTER Registry Trial

Molecularly stratified clinical trials

• Feasibility • Diagnostic information • Therapeutic opportunities

• Young adults with advanced-stage cancer

• Patients with rare tumors • Fast-track exome and

RNA sequencing • ~100 external partners

Start: 06/2013

Since 10/2016: Genome sequencing (60x)

Horak et al. Int J Cancer 2017

Workflow and Current Results

February 17, 2018 Molecular tumor board 935 patients (DKTK: 216) Management recommendation (Level 1-4) 84% (05/2016: ~60%) Genomics-guided clinical management 32% (05/2016: ~25%) Clinical benefit (CR, PR, SD >10 weeks) 32% (all patients, n = 252) 46% (patients with complete information on all treatments, n = 176)

NCT/DKTK MASTER

PI3K-AKT-mTOR

RAF-MEK-ERK

Tyrosine Kinases

Dev. Pathways

DNA Damage

Immune Evasion

Cell Cycle

Intervention Baskets

Goal: 28 days

Diagnostic Implications

Reevaluation of clinical diagnosis in ~5% of cases • Close interaction with pathology essential

Diagnosis Mutation(s) Differential Diagnosis Potential Clinical Action

Sarcoma NOS CDK4/MDM2 amplification MYOD1 p.V125L/p.L122R PDGFRA p.D842V COL1A1-PDGFB TPM3-ALK

Liposarcoma Rhabdomyosarcoma GIST DFSP IMT

à CDK4/MDM2 inhibition à CWS Guidance à Crenolanib*, BLU-285* à Imatinib* à Crizotinib*

Synovial sarcoma FUS-CREB3L2 LGFMS àSurgery

Hidradenocarcinoma SS18-SSX2 Synovial sarcoma àDoxorubicin/ifosfamide

Neuroendocrine carcinoma

EWSR1-WT1 DSRCT àEWING 2008

Carcinoma of unknown primary site

MYB-NFIB FGFR2-WAC IDH1 p.R132H EWSR1-WT1 NUTM1-NSD3

ACC CCC CCC DSRCT NUT midline carcinoma

à Vorinostat* à FGFR inhibitor à IDH1 inhibitor à EWING 2008 à BET inhibitor

Thymic carcinoma NUTM1-BRD3 NUT midline carcinoma à BET inhibitor

Urothelial carcinoma NUTM1-BRD4 NUT midline carcinoma à BET inhibitor

*Not approved for this indication in Germany

Germline Predisposition

Clinical evaluation of 1700 rare germline variants in 618 patients • Variants of uncertain significance (ACMG Class 3) in 220 tumor predisposition genes and candidate

genes in nearly all patients • Pathogenic variants (ACMG Class 5) in 23 tumor predisposition genes (BRCA1/2, PALB2, ATM,

NF1, MEN1, RB1, APC, SDHB, PTEN, CDH1, MSH2, etc.) in 11% of cases • Carrier status for autosomal recessive disorders (Fanconi anemia, Bloom syndrome, xeroderma

pigmentosum, etc.) in 4% of cases • Implications for patients and family members (genetic counseling, predictive diagnostics,

surveillance, prevention) in 15% of cases • Entry points for targeted therapies in individual patients (e.g. PARP inhibition in patients with

pathogenic BRCA1/2 or PALB2 mutations)

BRCA1 BRCA2

Evelin Schröck

Barbara Klink

Laura Gieldon

Undifferentiated sinonasal carcinoma • Pulmonary and dural metastases

KIT exon 11 mutation (p.579del) • Outlier KIT mRNA expression • KIT protein expression by immunohistochemistry • Imatinib* (400 mg/day) à complete/near-complete resolution of pulmonary and dural lesions • Secondary resistance due to KIT exon 17 mutation (p.D820_S821delinsG)

Dieter et al. Ann Oncol 2017

Therapeutic Implications

*Not approved for this indication in Germany

Therapeutic Implications

• Loss of MAPK activity in tumor cells after RAF blockade

• Co-existence of intermediate-activity BRAF mutations and oncogenic RAS in multiple cancers

• Cross-institutional project (Freiburg, Heidelberg, Munich [TU], Würzburg)

Cooperative activity of BRAF p.F595L and HRAS p.Q61R in aggressive histiocytic sarcoma

Kordes, Röring, Heining et al. Leukemia 2016

Therapeutic Implications

Undifferentiated cancer of unknown primary • Initially categorized as soft-tissue sarcoma, no

response to doxorubicin/ifosfamide and trabectedin • Histology and immunohistochemistry suggestive of

triple-negative breast cancer

High mutational load and PDL1 amplification • 380 single-nucleotide variants and insertions/deletions • Outlier PDL1 mRNA expression • PDL1 protein expression by immunohistochemistry • Immune checkpoint blockade with pembrolizumab* • Complete remission for >24 months

Gröschel, Bommer et al. Cold Spring Harb Mol Case Stud 2016

Baseline 2 months 6 months

*Not approved for this indication in Germany

Unmet Clinical Need in Bone and Soft-Tissue Sarcoma

Multiple disease categories (n = 37) and histologic subentities Paucity of evidence-based standards for systemic treatment No clinically validated molecular drug targets • Alveolar soft part sarcoma • Chordoma • Clear cell sarcoma • Desmoplastic small round cell tumor • Epithelioid hemangioendothelioma • Follicular dendritic cell sarcoma • Interdigitating dendritic cell sarcoma • Intimal sarcoma • Malignant peripheral nerve sheath tumor • Solitary fibrous tumor

MASTER

Therapeutic Implications

FGFR1-amplified leiomyosarcoma Clinical benefit from small-molecule FGFR inhibitors (BGJ398, nintedanib)* for >12 months M. Scheffler & J. Wolf, Cologne

Chudasama et al. Clin Cancer Res 2017

FGFR1 amplification and/or overexpression in various soft-tissue sarcoma subtypes FISH, array-based CGH and transcriptome profiling, RNA sequencing

Whole-exome sequencing

FISH Immunohistochemistry

*Not approved for this indication in Germany

Therapeutic Implications

Determinants of response to FGFR inhibition • FGFR1 overexpression with or without

underlying FGFR1 amplification • Suppression of MAPK-ERK1/2 signaling

Chudasama et al. Clin Cancer Res 2017

Traits of “BRCAness” in Leiomyosarcoma

23 point mutations and 5 insertions/deletions, biallelic inactivation of TP53 and RB1 Highly rearranged genome with multiple genomic losses affecting DNA repair genes • Chromosome 6q: HDAC2 • Chromosome 7q: NAMPT • Chromosome 9q: FANCC • Chromosome 13q: FANCB, FANCD1 (BRCA2) • Chromosome 14q: RAD51B

Enrichment of Alexandrov-COSMIC mutational signature AC3 Genomic imprints of defective DNA repair via homologous recombination (“BRCAness”)

Genomic Analysis of Leiomyosarcoma

Locally recurrent and metastatic leiomyosarcoma (n = 49) Extra-uterine and uterine Whole-exome and RNA sequencing • Mutational heterogeneity • Near-universal inactivation of TP53 and RB1 • Chromothripsis (35%) • Whole-genome duplication (51%) • Alternative telomere lengthening (78%) • Imprints of defective homologous

recombination DNA repair (“BRCAness”) in >90% of cases

• Mutations in homologous DNA repair genes

• Widespread DNA copy number alterations

• Significant enrichment of Alexandrov-COSMIC mutational signature AC3

Chudasama, Mughal, Sanders, Hübschmann et al. Nat Commun 2018

Strategies for Clinical Translation

NCT/DKTK MASTER Biology-driven patient stratification

Therapeutic activity of pathway-targeted interventions

Clinical trials of genomics-guided treatments Individual-case basis Existing stratified trials

Precision Medicine Studies in “Long Tail” Patients

85% of all hotspot mutations affect <5% of any cancer type in

which they are found

Clinical trials of genomics-guided treatments

Chang et al. Nat Biotechnol 2016

Conclusions and Outlook

Molecular profiling based on whole-exome/genome and RNA sequencing in a multi-institutional clinical setting

ü Is feasible

ü Provides relevant diagnostic information

ü Creates therapeutic opportunities

ü May be particularly fruitful in bone and soft-tissue sarcoma

ü Needs to be evaluated within controlled clinical trials of genomics-guided therapies

ü Should be complemented by additional layers of patient characterization • Proteomics • Immunomonitoring • Functional imaging

ü Should be complemented by additional treatment modalities • Radiotherapy • Surgery

RPPA

NCT MASTER Investigators

NCT Heidelberg Translational Oncology Hanno Glimm, Stefan Gröschel, Christoph Heining, Peter Horak, Simon Kreutzfeldt, Richard Schlenk Medical Oncology Dirk Jäger and Team

DKFZ High-Throughput Sequencing Unit Stephan Wolf and Team Applied/Theoretical Bioinformatics Barbara Hutter, Martina Fröhlich, Prakash Balasubramanian, Daniel Hübschmann, Matthias Schlesner, Sebastian Uhrig, Benedikt Brors Radiology David Bonekamp, Heinz-Peter Schlemmer Molecular Genome Analysis Leonie Wahjudi, Khalid Abnaof, Stephan Bernhardt, Stefan Wiemann

NCT POP/DKFZ-HIPO Sample Processing/Coordination Christina Geörg, Bettina Meißburger, Katrin Pfütze, Tina Föry, Peggy Grille, Christiane Lautenschläger, Katja Oehme, Katja Beck, Karolin Willmund, Daniela Richter, Heike Seiffart Board of Directors Peter Lichter, Roland Eils, Christof von Kalle

Heidelberg University Hospital Pathology Martina Kirchner, Amelie Lier, Anna-Lena Volckmar, Volker Endris, Roland Penzel, Stephan Singer, Felix Lasitschka, Albrecht Stenzinger, Peter Schirmacher

Frankfurt/Mainz Marit Ahrens, Oliver Bähr, Sven Becker, Christian Brandts, Nicola Gökbuget, Kristina Ihrig, Thomas Kindler, Anne Oehler, Michael Ronellenfitsch, Hubert Serve, Joachim Steinbach, Matthias Theobald, Sebastian Wagner, Thomas Wölfel Munich Hana Algül, Katharina Götze, Wolfgang Hiddemann, Philipp Jost, Andreas Jung, Ulrich Keller, Thomas Kirchner, Angela Krackhardt, Lars Lindner, Barbara Mayer, Klaus Metzeler, Roland Rad, Katja Specht, Karsten Spiekermann, Wilko Weichert, Benedikt Westphalen Dresden Martin Bornhäuser, Gunnar Folprecht, Laura Gieldon, Barbara Klink, Joseph Porrmann, Stephan Richter, Evelin Schröck, Martin Wermke Essen/Düsseldorf Sebastian Bauer, Johanna Falkenhorst, Julia Ketzer, Martin Schuler, Jens Siveke

Freiburg Melanie Börries, Tilman Brummer, Nikolas von Bubnoff, Justus Duyster, Silke Laßmann, Christoph Peters, Julius Wehrle, Martin Werner Berlin Ulrich Keilholz, Frederick Klauschen, Mario Lamping, Sebastian Ochsenreither, Damian Rieke, Christine Sers, Reinhold Schäfer Tübingen Michael Bitzer, Hans-Georg Kopp, Yvonne Möller, Klaus Schulze-Osthoff Heidelberg Benedikt Brors, Martina Fröhlich, Hanno Glimm, Stefan Gröschel, Christoph Heining, Peter Horak, Daniel Hübschmann, Barbara Hutter, Simon Kreutzfeldt, Peter Schirmacher, Richard Schlenk, Albrecht Stenzinger, Sebastian Uhrig Sample Processing & Coordination Tina Föry, Christina Geörg, Peggy Grille, Christiane Lautenschläger, Bettina Meißburger, Katja Oehme, Katrin Pfütze, Daniela Richter, Heike Seiffart

All Partner Sites Many others

DKTK MASTER Investigators

Thank you for the attention!

Genomics-Driven Oncology Within DKTK

Joint DKTK activity since March 2016 Institutional Review Board approval 8 Partner Sites (11 Comprehensive Cancer Centers)

Internet-based clinical data repository 8 Partner Sites (11 Comprehensive Cancer Centers)

Access to sequencing data 8 Partner Sites (11 Comprehensive Cancer Centers)

DKTK MASTER Molecular Tumor Board Weekly videoconference

DKTK MASTER Scientific Board Monthly videoconference

Joint publications Chudasama, Mughal et al. Nat Commun 2018 Forschner et al. Clin Cancer Res 2017 Ugurel et al. Eur J Cancer 2017 Czink et al. Cold Spring Harb Mol Case Stud 2017 Dieter, Heining et al. Ann Oncol 2017 Chudasama et al. Clin Cancer Res 2017 Gröschel, Bommer et al. Cold Spring Harb Mol Case Stud 2016 Bochtler et al. Cold Spring Harb Mol Case Stud 2016 Czink et al. Z Gastroenterol 2016 Kordes, Röring, Heining et al. Leukemia 2016

Decrease in Turnaround Time as Critical Challenge

DNA/RNA sequencing, data processing and annotation

15 days

Validation, clinical interpretation, treatment recommendation

Variable

Biopsy, shipping, frozen section, DNA/RNA isolation, QC

Variable

ü Highly automated pipelines for DNA/RNA sequencing, data processing, and data annotation ü Unmet need for resources supporting front and back ends of workflow

• Conventional and molecular pathology • Evaluation of tumor histology and cellularity • On-demand validation of actionable genetic alterations

• Sample processing • Translational oncology

• Clinical evaluation of sequencing data • Literature search and screening of clinical trial databases • Multi-institutional tumor boards • Reporting of results

Adapted from: MD Anderson Cancer Center Institute for Personalized Cancer Therapy

https://pct.mdanderson.org

Level 1

A: Drug is approved for the same tumor type harboring the specific biomarker.

B: Predictive value of the biomarker or clinical effectiveness of the corresponding drug in a molecularly stratified cohort was demonstrated in an adequately powered prospective study or a meta-analysis. ______________________________________________________________________ Level 2

A: Predictive value of the biomarker or clinical effectiveness of the drug in a molecularly stratified cohort was demonstrated in a prospective trial with biomarkers as a secondary objective or an adequately powered retrospective cohort or case-control study in the same tumor type.

B: Predictive value of the biomarker or clinical effectiveness of the drug in a molecularly stratified cohort was demonstrated by clinical data in a different tumor type.

C: Case study or single unusual responder indicates the biomarker is associated with response to the drug, supported by scientific rationale. ______________________________________________________________________ Level 3

Preclinical data (in vitro or in vivo models and functional genomics) demonstrate that the biomarker predicts response of cells to drug treatment. ______________________________________________________________________ Level 4

Biological rationale exists that links the drug to the altered signaling pathway or relevant basket. No reported clinical or preclinical data on the response to the drug.

High

Low