High-throughput Clinical Cancer Genotyping

A. John Iafrate, MD/PhD

Department of PathologyDiagnostic Molecular Pathology LaboratoryTranslational Research LaboratoryMassachusetts General HospitalBoston, MAaiafrate@partners.org

A New Paradigm in Cancer Treatment

A New Paradigm in Cancer TreatmentHaber, Gray, Baselga Cell 2011

BCR-ABL Imatinib100% CMLHER2 Trastuzumab20-30% IDCEGFR Erlotinib/ Gefitinib20% Lung adenocarcinomasALK Crizotinib3-5% Lung adenocarcinomaBRAF V600E PLX403250-60% Melanoma

BCR-ABL Imatinib100% CMLHER2 Tastuzumab20-30% IDCEGFR Erlotinib/ Gefitinib20% Lung adenocarcinomasALK Crizotinib3-5% Lung adenocarcinomaBRAF V600E PLX403260% MelanomaRomond EH et al., Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer. NEJM 2005.OBrien et al., Imatinib Compared with Inter-feron and Low-Dose Cytarabine for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia, NEJM 2003Mok et al., NEJM 2009

Comprehensive Genetic Characterization of Tumors for Personalized Cancer Care

DNA mutationsDNA chromosomal alterations

mRNA and miRNA profilingProteomicsDNA epigenetics

MGHTranslational Research LaboratoryCancer PatientsOncology Clinical trialsImproved Clinical Useof GenotypingBasic Research CentersMGH Pathology Specimen RepositoryGenotyping Real-time screening of patient tumor samples for genetic alterations.

Employing high-throughput genotyping technologies.(>100 samples/week)

Directing patient therapy based on genetic fingerprint.

ProspectiveEnrollmentClinical Genotyping in Guiding Therapeutic Decisions

Platform and clinical validationArchived specimen size and qualityInformaticsTurn-around timeDisease group customer supportPhased roll-outLung, Colon, GBM, BreastFinances and billing

Challenges in Establishing a Clinical Genotyping Program

Multiplex PCRSingle Base Extension ReactionCapillary ElectrophoresisElectrophoretic OutputSNAPSHOT Overview

SNAPSHOT Genotyping Assay16 cancer genes 120 described mutations ERBB2 Exon 20 insertionsIDH1 R132 -394CIDH1 R132 -395GAKT1 49G E17

SNAPSHOT v3

EGFR mutation Glu746_Ala750del(c.2235_2249del)NormalLung cancer

SNAPSHOT v3

BRAF mutation Val600Glu(c.1799T>A)NormalMelanoma

SNAPSHOT v3

KRAS mutationGly13Asp(c.38G>A)NormalColorectal cancer

SNAPSHOT v3

PIK3CA mutation His1047Arg(c.3140A>G)NormalBreast cancer

SNAPSHOT v3

Mutational profiling in lung cancersN=650

12KRAS56 isolated(58 total)EGFR36 isolated(50 total)

ALK 13T790M5PIK3CA5TP53 1111142APCNRASBRAF113B-catLung Adenocarcinoma: Overlap of MutationsBelinda Waltman/ Lecia Sequist

Rapid integration of FISH : ALK Rearrangements in NSCLCCrizotinib: Potent & selective ATP competitive oral inhibitor of MET and ALK kinases and their oncogenic variants

Phase I Clinical Trial of ALK Inhibitor Crizotinib in ALK-rearranged Lung Adenocarcinoma

Timeline for Crizotinib and ALK in NSCLCIdentification of PF2341066 PF2341066 Inhibits ALK activityPF2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)PF2341066 activity in cells exhibiting ALK fusion in broad screen (MGH-McDermott)Discovery of EML4-ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)2007PF2341066 FIPMay2005200620082009Objective responses demonstrated in ALK fusion positive NSCLC and IMTPhase III study of Crizotinib in ALK positive NSCLC startsSlide Courtesy of Ross Camidge

Timeline for Crizotinib and ALK in NSCLCIdentification of PF2341066 PF2341066 Inhibits ALK activityPF2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)PF2341066 activity in cells exhibiting ALK fusion in broad screen (MGH-McDermott)Discovery of EML4-ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)2007PF2341066 FIPMay2005200620082009Objective responses demonstrated in ALK fusion positive NSCLC and IMTPhase III study of Crizotinib in ALK positive NSCLC startsSlide Courtesy of Ross CamidgeFor phase I trial:ALK enriched cohort of 82 subjects required FISH screening of over 1200 NSCLCs

Formation of Lung Cancer Mutation Consortium (LCMC)

NIH-funded multicenter genotyping trial with mission of cross-validating platforms and accelerating recruitment into clinical trials of targeted agents.

Close collaboration of oncologists, pathologists and molecular diagnosticians

Mutational profiling in colorectal cancersN=250

Colon Adenocarcinoma: Overlap of Mutations

GenomicTL-09-267 20 ng/panel DNATL-09-285 3.04ng/panel DNA

More Than Just Point Mutations

The Future of Clinical Cancer GenotypingBy Angela Canada HopkinsDo we have the technology?

Is it cost-effective?

What to genotype?

The challenges?

Next Generation SequencingNext Generation SequencingFirst Generation Sequencing

Next Generation SequencingRoche 454Illumina/SolexaLife Technology SOLiDHelicos

Next Generation SequencingIllumina HiSeq 2000 Up to 1 billion clusters 150-200 Gb with 8 day run time $690K, ~$10000 per human genome sequencing 4 cameras, 50 MB/s of imaging, 4 x 625 MB images every 30 seconds 32 TB if raw images stored

Next Generation SequencingRoche 454 GS JrIllumina MiSeqLife Technology Ion Torrent

Cancer Driver MutationsPublished Cancer Exomes 11 Colorectal Science 2007 11 Breast Science 2007 24 Pancreas Science 2008 22 Gliomas Science 2008 2 Leukemias NEJM, Nature 2008 1 Breast Nature 2010 1 Breast Nature 2009 4 Granulosa Cell NEJM 2009 1 Lung Nature 2010 1 Melanoma Nature 2010 22 Medulloblastomas - UnpublishedBert Vogelstein:AACR 2010 MeetingPlenary SessionMutations per TumorNon-Silent Mutations in Pancreatic CancerMutations per TumorNon-Silent Mutations in Different Tumors

Cancer Driver Mutations: How Many?Review of Literature/Databases 116,432 human cancers 353 histopathologic subtypes 130,072 intragenic somatic mutations 3142 mutated genes

Potential Driver Genes 286 tumor suppressor genes (>15% of mutations are truncating)

33 oncogenes (same codon mutated in at least 2 tumors)Bert Vogelstein:AACR 2010 MeetingPlenary SessionMutations per TumorGenetic Alterations in Pancreatic CancerMutations per TumorDriver Gene Alterations in Pancreatic Cancer

Somatic Mutations: How much to sequence?Desired Analytical Sensitivity 1-5%

Typical NGS Error Rate 1-2%

Whole Genome Sequencing 30x 1 error >3.3% sensitivity

Targeted Sequencing 200-500x 0-4 errors in 200 reads 1%-2% error Set threshold at 5%

Whole Genome Sequencing at 200x >$60,000!

SOLiD Sequencing Pilot Results

SOLiDNext Generation Sequencing Variant CallsSNaPshotSingle Base ExtensionGenotyping ResultsKRAS c.34G>T (30.1%)KRAS c.34G>TTP53 c.743G>T (26.0%)TP53 c.743G>TKRAS c.34G>A (16.4%)TP53 c.536A>T (10.4%)KRAS c.34G>ANRAS c.182A>GTP53 c.880G>T (63.3%)NRAS c.182A>G KRAS c.34G>CKRAS c.34G>CKRAS c.38G>A (22.6%)PIK3CA c.1633G>A (18.8%)TP53 c.818G>A (39.9%)KRAS c.38G>APIK3CA c.1633G>ATP53 c.818G>ABRAF c.1799T>A (22.1%)PIK3CA c.1636C>A (14.4%)EGFR c.2264C>A (7.4%)BRAF c.1799T>APIK3CA c.1636C>Ano mutationsTP53 c.743G>AKRAS c.35G>T (12.0%)TP53 c.713G>T (20.9%)KRAS c.35G>T

Clinical Cancer Genotyping: On the HorizonClinical targeted sequencing of FFPE DNA

initially 100 exons >1000 200-500X coverage 100-150+ Mb data 3-4 week turnaround time $500 raw reagent cost

Desired Whole exon coverage Tumor vs. normal? Copy number? Rearrangements? Methylation? Transcription?

Cancer genetics is rapidly expanding with high complexity

Molecular profiling will drive cancer management

Continued need for higher-throughput cancer genotyping

Clinical next generation sequencing is coming

Collaborative efforts such as genotyping consortium will be key to addressing problem of cancers with rare genotypes

Summary

MGH Molecular Diagnostics

Leif EllisenDarrel BorgerDora Dias-SantagataKathy VernovskyArjola CosperBreton RousselKristin BergethonHannah StubbsVanessa ScialabbaSara Akhavanfard

MGH Cancer Center

Daniel HaberDavid LouisEunice KwakJeff ClarkMari Mino-KenudsonEugene MarkJeff EngelmanUltan McDermottJeff SettlemanLecia SequistBelinda WaltmanAlice Shaw

COI Disclosure: AJI has a paid consulting relationship with Pfizer Inc. and has a provisional patentfor SNaPshot assay.

***Applied Biosystems (ABI) Prism SNaPshot Multiplex system, originally developed to detect SNPs. The identity of the different loci is given by the position of each peak, which is dictated by the length of the extension primer********Our laboratory has gone live with clinical genotyping, starting in March of 2009. During this1st year, we have genetically profiled more than 400 lung cancer patients and ~70 colorectal patients at the MGH using our SNaPshot genotyping platform. Mutations have been identified in a majority of cases, often helping direct therapeutic decisions in these patients.***Our laboratory has gone live with clinical genotyping, starting in March of 2009. During this1st year, we have genetically profiled more than 400 lung cancer patients and ~70 colorectal patients at the MGH using our SNaPshot genotyping platform. Mutations have been identified in a majority of cases, often helping direct therapeutic decisions in these patients.***********