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Molecular Pathology in Cancer Precision Medicine: the Heidelberg Experience

Albrecht Stenzinger, 20.06.2017

Evolution not Revolution

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Macroscopy Microscopy Molecular

The Morpho-Molecular Age

BASKET Trials

• NCI-MATCH (NCI Molecular Analysis for Therapy Choice) – NCI (4 central labs), >15 drugs-approx. 1,000 women with BC• TAPUR (The Targeted Agent and Profiling Utilization Registry) - ASCO (any lab)• Pharma: e.g. Novartis Signature Trial Program, Genentech My

Pathway Trial

Preliminary Best Response According to Cohort.

Hyman DM et al. N Engl J Med 2015;373:726-736

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CANCER IS NOT A SINGLE GENE DISEASE

What about benign tumors?

Burden and spectrum of mutations in normal human skin.

Martincorena et al. Science 2015;348:880-886

What about normal tissue?

Approx. 140 driver mutations

Current Major Predictive Tests in Cancer Precision Medicine

ColorectalCancer

NRAS

KRAS

MSI

Melanoma

NRAS

BRAF

PD-L1

NSCLC

EGFR

ALK

ROS

RET

BRAF

MET

HER2

Resistance Mutations

PD-L1

GIST

KIT

PDGFRA

Resistance Mutations

HGSOC

BRCA1/2

BreastCancer

Intrinsicsubtypes by IHC

or mRNAProfiling

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Clinical utility and exploitation ofmolecular findings is governed by

therapeutic response

Molecular Features Therapeutic options

This process is evolutinonary and not static

Case Load Increases

0500

100015002000250030003500400045005000

2007 2015 2017 2020

cancer precisionmedicinedifferential diagnosis

infectious diseases

*

* Estimated values

Quantity and Complexity Increases

Three Core Considerations

What do I want toAchieve?/What is

my Purpose?

Who is ReimbursingWhat and to Which

Extent?

Clinical Needs andDemands?

Research vs. Diagnostics

Single Entity vs. Many Cancer

Types

Validated vs. Non-Validated

Assay

Low vs. High Throughput

Single Gene Assay vs.

Scalable System

Broad Profiling vs. FocusedScreening

Input Material

What do I want toAchieve?/What is

my Purpose?

Who is ReimbursingWhat and to Which

Extent?

Clinical Needs andDemands?

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Test and Assay Requirements

GeneticTests/AssaysHigh Quality

Standardized

Validated

Scalable

High-throughput

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KEY POINTS: PARR+2xAS • Precision, Accuracy, Reportable range, Reference range• Analytical Sensitivity• Analytical Specificity

Quality parameters of a test

More Very Challenging Fields

• TAT is part of quality!• Variant annotation and interpretation

Clinical relevance and impact• Diagnostic reporting: level of granularity• Communication Oncologist – Pathologist• Training in both directions strongly needed• Ressources – Stakeholders? Payors?

Ideal world scenario:WGS + RNAseq of histology

controlled fresh tissue

•Low tumor cellularity•TAT•Throughput•Read depth/Coverage (Tumor heterogeneity)•>90% is FFPE! Data quality!•Financial toxicity•Ratio: Resources vs. Clinical Benefit/Therapeuticresponse

• Currently only feasible at very fewdedicated centers

• Unlikely to become a standard of care in a true dx outreach setting

• …10 years ahead? Let‘s discuss again!

My thinking is……

Parameters Focused Sequencing Comprehensive Sequencing

Validated Assay/Test Mandatory Mandatory

Drug Targets/Biomarkers Yes Yes

Discovery Limited (panel dep.) YesSeq Matching Normal Tissue Maybe Required (panel dep.) Required

Turn Around Time per Case ~5 WD ~30 WD

Scalability/High-Throughput Feasible Limited

Bioinformatics pipeline Straight-Forward SophisticatedManpower Less More

Seq Hardware (starts at: EUR) ~30.000 ~200.000

Raw Costs/Sample (EUR) ~150-300 ~700-1,500

Seq Matching Normal Tissue Maybe Required (panel dep.) Required

Key Purpose and Economic Parameters: Focused vs. ComprehensiveNext Generation Sequencing

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Parameters Focused Sequencing Comprehensive Sequencing

Input Material FFPE and Fresh Tissue Preferably Fresh Tissue

Low Tumor Cellularity Yes (~5%) No

DNA Amount ~10 ng At least 100ng

RNA Amount ~15ng At least 1000ng (1µg)

SNV Yes YesCNV Limited (panel dep.) Yes

Gene Fusions Yes but Biased Yes (Biased with WES, Unbiased with WGS)

Matching Normal Tissue Maybe Required (panel dep.) Required

Read Depth (average) ~500-1000x (and more) ~100x

Coverage Amplicon Performance Can Vary

Key Technical Parameters: Focused vs. ComprehensiveNext Generation Sequencing

Heidelberg Cancer Precision Medicine

Focused GeneticProfiling

Targeted Panel Sequencing

DNA and RNA

ComprehensiveGenetic Profiling

WGSDNA and RNA

Single Gene Profiling

FISHSanger Sequencing

Non-GeneticProfiling Mainly on

Protein Level e.g. PD-L1 IHC, HER2

IHC

Breast CancerGene Signatures

e.g. PAM50

Immuno-Monitoring

MSI Mutational Load

Immune-Signatures

University Hospital Heidelberg /National Center for Tumor Diseases (NCT) /DKFZ

Center for Molecular Pathology at Institute of Pathology (CMP-IPH) NCT/DKFZ

Targeted Sequencing

Targeted Seq Program

CR

C

NSC

LC

Mel

anom

a

Can

cer o

fUnk

now

nPr

imar

y

HG

SOC

and

Pros

tate

Sarc

oma

Dia

gnos

ticTo

ol

Tria

ls

Tissue-baseddiagnosis

PathologicalStaging

Sample registrationDNA/RNAextractionDNA conc. (QuBit/RNAseP)Library preparationCreate sequencingpoolExport Run plan

SequencelibrariesSignal processingBase CallingAlignmentVariant CallingCoverageAnalysisUpload IR

Run fusionanalysis

Transfer data tolocal serverImport variantsAnnotateValidate variantsAnalyse CNVsAnalyse HPVisualize reads in IGVCompare Sample variantsCreate reports

One-Stop Shop Diagnostics from FFPEOne-Stop Shop Diagnostics from FFPE

Panels for Routine Dx and Research

Colorectal Cancercustomized gene panel30 genes, 180 Amplicons (mean size: 114bp)

Lung Cancercustomized gene panel (DNA and RNA sequencing)43 genes, 164 amplicons (mean size 116bp) and 159 gene fusions

Melanoma, GIST, tumor driverscommercially available gene panel50 genes, 207 amplicons (mean size 106bp)

Cancer of Unknown PrimaryNCI-Match Trial related gene panel143 genes (mean size 103bp) and 176 gene fusions?

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Panels for Routine Dx and ResearchBreast Cancercustomized gene panel (DNA sequencing)35 genes, 180 amplicons

Head and Neck Cancercustomized gene panel (DNA sequencing)27 genes, 187 amplicons

Pancreatic Cancercustomized gene panel (DNA sequencing)26 genes, 163 amplicons

HCC and Cholangiocarcinomacustomized gene panel (DNA sequencing)23 genes

cfDNAComercially available gene panel (DNA sequencing)11 genetic regions, 36 amplicons (mean size: 51bp)

Panels for Routine Dx and Reseach

BRCA 1/2commercially available gene panel (DNA sequencing)183 amplicons

Core DNA Repair Genescustomized gene panel (DNA sequencing)35 genes, 2000 amplicons

Fusion Genes (Sarcomas)commercially available and customized gene panels (RNA-sequencing)

Mutational Load and ImmunesignatureIn development

Drop-Out Rates

93%

3% 4%Cases (%)

Category 1(all quality benchmarks passed)

Category 2(tumor tissue exhaustion)

Category 3(critically low tumor cellularity)

Material1 H&E

+ 5 - 8 blank slides

TAT5 WT (Ø)

Pfarr et al., 2016

Gene Fusions COPA

hematologysarcoma

other solids

Metaphase,living cells!

ISH,interphase

1956

46 chromosomes

NGSFISH GEA, aCGHG-band karyo

The hematology paradigm isbiased

• Samples easy to obtain• Near diploid genome

• Few secondary aberrations• Large chromosomal segments

• Methods available at time

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~10.000 genefusions

described so far90% in the last 5

years~5% recurrent

NTRK gene fusions in NSCLC

0.1%!

Farago et al., 2015

Results ALK/ROS1/RET Panel

Sample Barcode preTest Method preTest Result Archer Fusion Total Reads Unique Reads DNA Reads RNA ReadsSample 1 ION_MBC_1 Lung Fusion Panel negativ negativ 339508 25681 7988 114644Sample 2 ION_MBC_2 Lung Fusion Panel negativ negativ 362563 68860 8226 250795Sample 3 ION_MBC_3 Lung Fusion Panel negativ negativ 306008 19223 9354 162236Sample 4 ION_MBC_4 Lung Fusion Panel EML-ALK (E20A20) EML-ALK (E20A20) 375494 44947 19971 264907LC2/ad ION_MBC_5 Lung Fusion Panel CCDC6-RET (C1R12) CCDC6-RET (C6R12) 396678 128994 4952 311491Sample 5 ION_MBC_6 Lung Fusion Panel EML4-ALK(E6A20) EML4-ALK(E6A19) 376886 15567 13388 254023ROS1 positive Control ION_MBC_7 Lung Fusion Panel SLC34A2-ROS1 (S4R32) SLC34A2-ROS1 (S4R32) 392609 59559 4212 284892Lung Normal Control ION_MBC_8 Lung Fusion Panel negativ negativ 350409 58559 2056 232368

1 single run on IonTorrent PGM

(318v2 chip)

70 yr old male Caucasian patient• History of Colon Cancer

• History of Breast Cancer (Luminal B)

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• No asbestos exposure• Never smoker• Healthy lifestyle

Epitheloid mesothelioma, type A

Variant ReportingErgebnisbericht

Comprehensive Profiling

Genomic Landscape of Cancer

Gene “mountains” and “hills”Wood et al. Science 2007

“Long tail” pattern of actionable cancer gene alterations

TCGA Pan-Cancer AnalysisLawrence et al. Nature 2013

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

histologic tumor subtype

• Large-scale sequencing projects (TCGA, ICGC) have completed exomes/genomes

from >10,000 cancers• All common (present at >5% frequency

per tumor type) cancer drivers are defined

Unraveling the Unknowns

• More exome sequencing• Identify all mutations present at >1% frequency per tumor type

• Whole-genome sequencing• Discover mutations in non-coding regions, e.g. enhancers

• Broad diagnostic approaches that capture the “private” patterns of genetic alterations in individual patients

• Screening for genotype-selective dependencies

Knowndriver

Unknowndriver

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Molecularly Aided Stratification for Tumor Eradication Research

Genetics

Clinical care

Molecular diversity andgenetic taxonomy of cancer

Actionability of molecular lesions

Individual, “private” patterns of molecular lesions

NCT/DKTK MASTERRegistry Trial

NCT/DKTK MASTERRegistry 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• >60 external partners,

including all DKTK sites

• Young adults with advanced-stage cancer

• Patients with rare tumors• Fast-track exome and

RNA sequencing• >60 external partners,

including all DKTK sitesStart: 06/2013

Since 10/2016:Genome sequencing (60x)

Genomics-Driven Oncology Within DKTK

Institutional Review Board approval8/8 Partner Sites

Internet-based clinical data repository (ONKOSTAR system)8/8 Partner Sites

Access to sequencing data (FASTQ, BAM, VCF)8/8 Partner Sites

DKTK MASTER Molecular Tumor BoardWeekly videoconference

DKTK MASTER Scientific BoardMonthly videoconference

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

Bochtler et al. Cold Spring Harb Mol Case Stud 2016Kordes, Röring, Heining et al. Leukemia 2016

Chudasama et al. Clin Cancer Res 2017Dieter, Heining et al. Ann Oncol 2016

Czink et al. Z Gastroenterol 2016

Joint DKTK activity since March 2016

Workflow, Patient Accrual, and Current Results

March 2017Molecular tumor board 567 patients

Reevaluation of clinical diagnosis ~5%Treatment recommendation (Level 1-4) ~75% (05/2016: ~60%)Genomics-guided clinical management ~40% (05/2016: ~25%)

NCT/DKTKMASTER

NCT/DKTKMASTER

PI3K-AKT-mTOR

RAF-MEK-ERK

Tyrosine Kinases

Dev. Pathways

DNA Damage

Immune Evasion

Cell Cycle

Intervention Baskets

28-42 days

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

https://pct.mdanderson.org

Level 1A: 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 2A: 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 3Preclinical data (in vitro or in vivo models and functional genomics) demonstrate that the

biomarker predicts response of cells to drug treatment.______________________________________________________________________

Level 4Biological 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

Technical Validation and Molecular Tumor Board Report

Metastatic gallbladder carcinoma• Peritoneal and cutaneous metastasis during adjuvant chemotherapy with oxaliplatin/gemcitabine

Amplification of chromosome 17q12, including ERBB2• Outlier ERBB2 mRNA expression

• ERBB2 protein expression by immunohistochemistry (3+ according to ASCO guidelines)

H&E ERBB2

Dual ERBB2 blockade:trastuzumab/pertuzumab*

Complete remission for>12 months

Czink et al. Z Gastroenterol 2016

Therapeutic Implications

*Not approved for this indication in Germany

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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 2016

Therapeutic Implications

*Not approved for this indication in Germany

Therapeutic Implications

Undifferentiated cancer of unknown primary• Initially categorized as soft-tissue sarcoma, no response to doxorubicin/ifosfamide and trabectedin

• Histology and immunohistochemistry consistent with triple-negative breast cancer

PDL1 (CD274, B7-H1) amplification• Outlier PDL1 mRNA expression

• PDL1 protein expression by immunohistochemistry• Immune checkpoint blockade with pembrolizumab*

• Near-complete remission for >12 months

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

Baseline 2 months 6 months

*Not approved for this indication in Germany

Therapeutic Implications

Undifferentiated cancer of unknown primary• Initially categorized as soft-tissue sarcoma, no response to doxorubicin/ifosfamide and trabectedin

• Histology and immunohistochemistry consistent with triple-negative breast cancer

PDL1 (CD274, B7-H1) amplification• Outlier PDL1 mRNA expression

• PDL1 protein expression by immunohistochemistry• Immune checkpoint blockade with pembrolizumab*

• Near-complete remission for >12 months

Cold Spring Harb Mol Case Stud 2016

Baseline 2 months 6 months

*Not approved for this indication in Germany

PD-L1 Amplifications in Relapsed or RefractoryHodgkin Lymhoma: Response to Nivolumab

Ansell et al., 2015 Roemer et al., 2016

Shell Model of Molecular Diagnostics –Subsidiarity is the key principle

Rare CommonSimple Complex

Com

pple

xity

ofge

netic

even

ts

Prevalenceof

geneticevents

GranularityCoarse

Fine

What else?

• Be technology agnostic• Think beyond NGS: Genes are only part of the full story• Stay tuned: Clincial Trials and Tech

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What we need

• Quantitative Pathology

• BiologicalPathology

• ClinicalPathology

Many Thanks!

Peter SchirmacherVolker EndrisRoland Penzel Amelie Lier Anna-Lena VolckmarMartina Kirchner Jonas Leichsenring Fabian StögbauerRegine Brandt Cristiano Moraisde OliveiraIvo BuchhalterAngelika BönischAngelika BrüntgensWaltraud Schmitz Meike Viole Marie-Sofie KaridipisMechthild Samer Christina Hofherr Natascha HorbachKatja Lorenz Kathrin Ridinger

Christof von KalleHanno Glimm

Stefan FröhlingStefan GröschelHolger Sültmann

Alwin KrämerTilmann Bochtler

John Iafrate

Stefan DuensingCarsten Gruellich