High-throughput Clinical Cancer High-throughput Clinical Cancer GenotypingGenotyping

A. John Iafrate, MD/PhDA. John Iafrate, MD/PhD

Department of PathologyDepartment of PathologyDiagnostic Molecular Pathology LaboratoryDiagnostic Molecular Pathology Laboratory

Translational Research LaboratoryTranslational Research LaboratoryMassachusetts General HospitalMassachusetts General Hospital

Boston, MABoston, MAaiafrate@partners.orgaiafrate@partners.org

A New Paradigm in Cancer TreatmentA New Paradigm in Cancer Treatment

A New Paradigm in Cancer TreatmentA New Paradigm in Cancer Treatment

Haber, Gray, Baselga Cell 2011

BCR-ABL Imatinib100% CML

HER2 Trastuzumab20-30% IDC

EGFR Erlotinib/ Gefitinib20% Lung adenocarcinomas

ALK Crizotinib3-5% Lung adenocarcinoma

BRAF V600E PLX403250-60% Melanoma

BRAF1799 T>A

V600E

BCR-ABL Imatinib100% CML

HER2 Tastuzumab20-30% IDC

EGFR Erlotinib/ Gefitinib20% Lung adenocarcinomas

ALK Crizotinib3-5% Lung adenocarcinoma

BRAF V600E PLX403260% Melanoma

BRAF1799 T>A

V600E

Romond EH et al., Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer. NEJM 2005.

O’Brien et al., Imatinib Compared with Inter-feron and Low-Dose Cytarabine for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia, NEJM 2003

Mok et al., NEJM 2009

Comprehensive Genetic Characterization of Tumors for Personalized Cancer Care

DNA mutations

DNA chromosomal alterations

mRNA and miRNA profiling

Proteomics

DNA epigenetics

MGHTranslational Research Laboratory

Cancer Patients

Oncology Clinical trials

Improved Clinical Useof Genotyping

Basic Research CentersMGH Pathology Specimen Repository

Genotyping

• 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.

ProspectiveEnrollment

Clinical Genotyping in Guiding Therapeutic Decisions

• Platform and clinical validation• Archived specimen size and quality• Informatics• Turn-around time• Disease group customer support

– Phased roll-out– Lung, Colon, GBM, Breast

• Finances and billing

Challenges in Establishing a Clinical Genotyping Program

ddNTP

ddNTP

ddNTP

loci of interest

Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis

Electrophoretic Output

Increasing molecular weight

Rel

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luo

resc

en

ce

A B DC FE

SNAPSHOT Overview

Gene Amino Acid – cDNA Residue Gene Amino Acid – cDNA Residue AKT1 49G - E17 KRAS G12 - 34G KRAS G12 - 35G APC R1114 - 3340C KRAS G13 - 37G APC Q1338 - 4012C KRAS G13 - 38G APC R1450 - 4348C APC T1556fs* - 4666_4667insA NOTCH1 L1575 - 4724T NOTCH1 L1601 - 4802T BRAF V600 - 1798G BRAF V600 - 1799T NRAS G12 - 34G NRAS G12 - 35G CTNNB1 D32 - 94G NRAS G13 - 37G CTNNB1 D32 - 95A NRAS G13 - 38G CTNNB1 S33 - 98C NRAS Q61 - 181C CTNNB1 G34 - 101G NRAS Q61 - 182A CTNNB1 S37 - 109T NRAS Q61 - 183A CTNNB1 S37 - 110C CTNNB1 T41 - 121A PIK3CA R88 - 263G CTNNB1 T41 - 122C PIK3CA E542 - 1624G CTNNB1 S45 - 133T PIK3CA E545 - 1633G CTNNB1 S45 - 134C PIK3CA Q546 - 1636C PIK3CA Q546 - 1637A EGFR G719 - 2155G PIK3CA H1047 - 3139C EGFR T790 - 2369C PIK3CA H1047 - 3140A EGFR L858 - 2573T PIK3CA G1049 - 3145G EGFR E746_A750 - 2235_2249del EGFR E746_A750 - 2236_2250del PTEN R130 - 388C EGFR Exon 19 deletions PTEN R173 - 517C PTEN R233 - 697C FLT3 D835 - 2503G PTEN K267fs*- 800delA IDH1 R132 - 394C TP53 R175 - 524G IDH1 R132 - 395G TP53 G245 - 733G TP53 R248 - 742C JAK2 V617 - 1849G TP53 R248 - 743G TP53 R273 - 817C KIT D816 - 2447A TP53 R273 - 818G TP53 R306 - 916C

SNAPSHOT Genotyping Assay16 cancer genes – 120 described mutations

ERBB2 Exon 20 insertions

IDH1 R132 -394CIDH1 R132 -395G

AKT1 49G – E17

7-plexPanel 1

KRAS34

EGFR2235_49F

EGFR 2573NRAS181

PI3K1633bCat94

bCat121

8-plexPanel 2

EGFR2235_49R

NRAS38

BRAF1799

NRAS182

PI3K263 bCat122

bCat95

TP53.742

5-plexPanel 3

NRAS35

EGFR2236_50F

EGFR2369

bcat133

PI3K1624

8-plexPanel 4 KRAS35

EGFR2236_50R

PTEN517

FLT3.2503

PI3K3139

NOTCH1.4724

NOTCH1.4802TP53.733

SNAPSHOT v3

EGFR mutation Glu746_Ala750del(c.2235_2249del)

Normal

Lung cancer

SNAPSHOT v3

BRAF mutation Val600Glu

(c.1799T>A)

Normal

Melanoma

SNAPSHOT v3

KRAS mutationGly13Asp(c.38G>A)

Normal

Colorectal cancer

SNAPSHOT v3

PIK3CA mutation His1047Arg(c.3140A>G)

Normal

Breast cancer

SNAPSHOT v3

Mutational profiling in lung cancers

KRAS 23%

No Mutation 42%

EGFR 15%

TP53 5%

IDH1 <1%

NRAS 1%BRAF 2%

HER2 2%

PIK3CA 4%ALK 3%

CTNNB1 2%

AKT 1%

N=650

1

2

KRAS56 isolated(58 total)

EGFR36 isolated(50 total)

ALK 13

T790M5

PIK3CA5

TP53

1

11

1

1

4

2

APC

NRAS

BRAF

1

13

B-cat

Lung Adenocarcinoma: Overlap of Mutations

Belinda Waltman/ Lecia Sequist

Rapid integration of FISH : ALK Rearrangements in NSCLC

Crizotinib: Potent & selective ATP competitive oral inhibitor of MET and

ALK kinases and their oncogenic variants

~250 kb ~300 kb

t(2;5) ALK genebreakpoint region

2p23 regionTelomere Centromere

3’ 5’

~250 kb ~300 kb

t(2;5) ALK genebreakpoint region

2p23 regionTelomere Centromere

3’ 5’

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

Timeline for Crizotinib and ALK in NSCLC

Identification of PF2341066

PF2341066 Inhibits ALK activity

PF2341066 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)

2007

PF2341066 FIPMay

2005 2006 2008 2009

Objective responses demonstrated in ALK fusion positive NSCLC and IMT

Phase III study of “Crizotinib” in ALK positive NSCLC startsSlide Courtesy of Ross Camidge

Timeline for Crizotinib and ALK in NSCLC

Identification of PF2341066

PF2341066 Inhibits ALK activity

PF2341066 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)

2007

PF2341066 FIPMay

2005 2006 2008 2009

Objective responses demonstrated in ALK fusion positive NSCLC and IMT

Phase III study of “Crizotinib” in ALK positive NSCLC startsSlide Courtesy of Ross Camidge

For 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 cancers

No Mutation Identified

34%

KRAS25%TP53

21%

PIK3CA 6%

NRAS 3%

APC 4%

BRAF 7%

N=250

Colon Adenocarcinoma: Overlap of Mutations

KRAS 20 isolated

(36 total)

TP5318 isolated

(28 total)

APC 1 NRAS

3

PIK3CA

BRAF6 isolated

4

3

4

1

1

3

2

1

61

Genomic

TL-09-267 20 ng/panel DNA

TL-09-285 3.04ng/panel DNA

More Than Just Point MutationsMore Than Just Point Mutations

The Future of Clinical Cancer GenotypingThe Future of Clinical Cancer Genotyping

By Angela Canada Hopkins

Do we have the technology?

Is it cost-effective?

What to genotype?

The challenges?

Next Generation SequencingNext Generation Sequencing

Next Generation Sequencing

First Generation Sequencing

Next Generation SequencingNext Generation Sequencing

Roche 454 Illumina/Solexa

Life Technology SOLiD Helicos

Next Generation SequencingNext Generation Sequencing

Illumina 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 SequencingNext Generation Sequencing

Roche 454 GS Jr Illumina MiSeq

Life Technology Ion Torrent

Cancer Driver MutationsCancer Driver Mutations

Published 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 - Unpublished

Bert Vogelstein:AACR 2010 MeetingPlenary Session

Mut

ation

s pe

r Tum

or

Non-Silent Mutations in Pancreatic Cancer

Mut

ation

s pe

r Tum

or

Non-Silent Mutations in Different Tumors

Cancer Driver Mutations: How Many?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 Session

Mut

ation

s pe

r Tum

or

Genetic Alterations in Pancreatic Cancer

Mut

ation

s pe

r Tum

or

Driver Gene Alterations in Pancreatic Cancer

Somatic Mutations: How much to sequence?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 ResultsSOLiD Sequencing Pilot Results

SOLiDNext Generation Sequencing Variant Calls

SNaPshotSingle Base ExtensionGenotyping Results

KRAS 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>C KRAS 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>A

BRAF c.1799T>A (22.1%)PIK3CA c.1636C>A (14.4%)EGFR c.2264C>A (7.4%)

BRAF c.1799T>APIK3CA c.1636C>A

no mutations TP53 c.743G>AKRAS c.35G>T (12.0%)TP53 c.713G>T (20.9%) KRAS c.35G>T

Clinical Cancer Genotyping: On the HorizonClinical Cancer Genotyping: On the Horizon

Clinical 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

SummarySummary

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.