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High-throughput Clinical Cancer Genotyping A. John Iafrate, MD/Ph. D Department of Pathology Diagnostic Molecular High-throughput Clinical Cancer Genotyping A. John Iafrate, MD/Ph. D Department of Pathology Diagnostic Molecular Pathology Laboratory Translational Research Laboratory Massachusetts General Hospital Boston, MA [email protected] org

A New Paradigm in Cancer Treatment Cancer Patient Clinical Information Targeted Therapy Surgical Resection A New Paradigm in Cancer Treatment Cancer Patient Clinical Information Targeted Therapy Surgical Resection Molecular Pathology Routine Pathology

A New Paradigm in Cancer Treatment Haber, Gray, Baselga Cell 2011 A New Paradigm in Cancer Treatment Haber, Gray, Baselga Cell 2011

BCR-ABL Imatinib 100% CML EGFR Erlotinib/ Gefitinib 20% Lung adenocarcinomas HER 2 Trastuzumab 20 BCR-ABL Imatinib 100% CML EGFR Erlotinib/ Gefitinib 20% Lung adenocarcinomas HER 2 Trastuzumab 20 -30% IDC BRAF V 600 E PLX 4032 50 -60% Melanoma BRAF 1799 T>A V 600 E ALK Crizotinib 3 -5% Lung adenocarcinoma

BCR-ABL Imatinib 100% CML HER 2 Tastuzumab 20 -30% IDC O’Brien et al. , BCR-ABL Imatinib 100% CML HER 2 Tastuzumab 20 -30% IDC O’Brien et al. , Imatinib Compared with Interferon and Low-Dose Cytarabine for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia, NEJM 2003 EGFR Erlotinib/ Gefitinib 20% Lung adenocarcinomas ALK Crizotinib BRAF V 600 E PLX 4032 Romond EH et al. , Trastuzumab plus 60% Melanoma 3 -5% Lung adenocarcinoma Adjuvant Chemotherapy for Operable HER 2 -Positive Breast Cancer. NEJM 2005. BRAF 1799 T>A V 600 E Mok et al. , NEJM 2009

Comprehensive Genetic Characterization of Tumors for Personalized Cancer Care DNA epigenetics Proteomics DNA mutations Comprehensive Genetic Characterization of Tumors for Personalized Cancer Care DNA epigenetics Proteomics DNA mutations DNA chromosomal alterations m. RNA and mi. RNA profiling

Clinical Genotyping in Guiding Therapeutic Decisions • Real-time screening of patient tumor samples for Clinical Genotyping in Guiding Therapeutic Decisions • Real-time screening of patient tumor samples for genetic alterations. Cancer Patients Prospective Enrollment • Employing high-throughput genotyping technologies. (>100 samples/week) • Directing patient therapy based on genetic fingerprint. Genotyping Oncology Clinical trials Improved Clinical Use of Genotyping MGH Translational Research Laboratory MGH Pathology Specimen Repository Basic Research Centers

Challenges in Establishing a Clinical Genotyping Program • • • Platform and clinical validation Challenges in Establishing a Clinical Genotyping Program • • • 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

SNAPSHOT Overview Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis dd. NTP loci of SNAPSHOT Overview Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis dd. NTP loci of interest dd. NTP Relative fluorescence Electrophoretic Output A B C D E F Increasing molecular weight

SNAPSHOT Genotyping Assay 16 cancer genes – 120 described mutations AKT 1 49 G SNAPSHOT Genotyping Assay 16 cancer genes – 120 described mutations AKT 1 49 G – E 17 ERBB 2 Exon 20 insertions IDH 1 R 132 -394 C R 132 -395 G

SNAPSHOT v 3 Panel 1 b. Cat 121 EGFR 2235_49 F b. Cat 94 SNAPSHOT v 3 Panel 1 b. Cat 121 EGFR 2235_49 F b. Cat 94 PI 3 K 1633 KRAS 34 7 -plex EGFR 2573 NRAS 181 Panel 2 NRAS 38 b. Cat 122 PI 3 K 263 NRAS 182 BRAF 1799 EGFR 2235_49 R 8 -plex b. Cat 95 TP 53. 742 5 -plex EGFR 2369 Panel 3 EGFR 2236_50 F PI 3 K 1624 NRAS 35 bcat 133 Panel 4 KRAS 35 8 -plex PTEN 517 FLT 3. 2503 EGFR 2236_50 R TP 53. 733 NOTCH 1. 4724 PI 3 K 3139 NOTCH 1. 4802

SNAPSHOT v 3 Normal Lung cancer EGFR mutation Glu 746_Ala 750 del (c. 2235_2249 SNAPSHOT v 3 Normal Lung cancer EGFR mutation Glu 746_Ala 750 del (c. 2235_2249 del)

SNAPSHOT v 3 Normal Melanoma BRAF mutation Val 600 Glu (c. 1799 T>A) SNAPSHOT v 3 Normal Melanoma BRAF mutation Val 600 Glu (c. 1799 T>A)

SNAPSHOT v 3 Normal Colorectal cancer KRAS mutation Gly 13 Asp (c. 38 G>A) SNAPSHOT v 3 Normal Colorectal cancer KRAS mutation Gly 13 Asp (c. 38 G>A)

SNAPSHOT v 3 Normal Breast cancer PIK 3 CA mutation His 1047 Arg (c. SNAPSHOT v 3 Normal Breast cancer PIK 3 CA mutation His 1047 Arg (c. 3140 A>G)

Mutational profiling in lung cancers AKT 1% BRAF 2% HER 2 2% CTNNB 1 Mutational profiling in lung cancers AKT 1% BRAF 2% HER 2 2% CTNNB 1 2% ALK 3% PIK 3 CA 4% NRAS 1% IDH 1 <1% TP 53 5% No Mutation 42% EGFR 15% KRAS 23% N=650

Lung Adenocarcinoma: Overlap of Mutations PIK 3 CA 5 1 1 KRAS 56 isolated Lung Adenocarcinoma: Overlap of Mutations PIK 3 CA 5 1 1 KRAS 56 isolated (58 total) 1 EGFR 36 isolated (50 total) 1 B-cat 3 1 2 TP 53 1 T 790 M 5 1 4 2 APC BRAF 1 NRAS ALK 13 Belinda Waltman/ Lecia Sequist

Rapid integration of FISH : ALK Rearrangements in NSCLC Crizotinib: Potent & selective ATP Rapid integration of FISH : ALK Rearrangements in NSCLC Crizotinib: 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 Phase I Clinical Trial of ALK Inhibitor Crizotinib in ALK-rearranged Lung Adenocarcinoma

Timeline for Crizotinib and ALK in NSCLC PF 2341066 Inhibits ALK activity 2005 Identification Timeline for Crizotinib and ALK in NSCLC PF 2341066 Inhibits ALK activity 2005 Identification of PF 2341066 activity in cells exhibiting ALK fusion in broad screen (MGHMc. Dermott) PF 2341066 FIP May 2006 PF 2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house) Slide Courtesy of Ross Camidge 2007 2008 Discovery of EML 4 ALK fusions in NSCLC (CREST) Japan Science & Technology Agency) 2009 Objective responses demonstrated in ALK fusion positive NSCLC and IMT Phase III study of “Crizotinib” in ALK positive NSCLC starts

Timeline for Crizotinib and ALK in NSCLC For phase I trial: PF 2341066 activity Timeline for Crizotinib and ALK in NSCLC For phase I trial: PF 2341066 activity in cells exhibiting ALK enriched cohort of 82 subjects required FISH screening of over 1200 NSCLCs PF 2341066 Inhibits ALK fusion in broad PF 2341066 FIP ALK activity screen (MGHMay Mc. Dermott) 2005 Identification of PF 2341066 2006 PF 2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house) Slide Courtesy of Ross Camidge 2007 2008 Discovery of EML 4 ALK fusions in NSCLC (CREST) Japan Science & Technology Agency) 2009 Objective responses demonstrated in ALK fusion positive NSCLC and IMT Phase III study of “Crizotinib” in ALK positive NSCLC starts

Formation of Lung Cancer Mutation Consortium (LCMC) NIH-funded multicenter genotyping trial with mission of 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 APC 4% NRAS 3% No Mutation Identified BRAF 7% Mutational profiling in colorectal cancers APC 4% NRAS 3% No Mutation Identified BRAF 7% 34% PIK 3 CA 6% KRAS TP 53 25% 21% N=250

Colon Adenocarcinoma: Overlap of Mutations BRAF 6 isolated 3 3 PIK 3 CA 4 Colon Adenocarcinoma: Overlap of Mutations BRAF 6 isolated 3 3 PIK 3 CA 4 1 1 1 KRAS 20 isolated (36 total) TP 53 18 isolated (28 total) 4 6 2 APC 1 1 NRAS 3

Genomic TL-09 -267 20 ng/panel DNA TL-09 -285 3. 04 ng/panel DNA Genomic TL-09 -267 20 ng/panel DNA TL-09 -285 3. 04 ng/panel DNA

More Than Just Point Mutations More Than Just Point Mutations

The Future of Clinical Cancer Genotyping Do we have the technology? Is it cost-effective? The Future of Clinical Cancer Genotyping Do we have the technology? Is it cost-effective? What to genotype? The challenges? By Angela Canada Hopkins

Next Generation Sequencing First Generation Sequencing Next Generation Sequencing First Generation Sequencing

Next Generation Sequencing Roche 454 Illumina/Solexa Life Technology SOLi. D Helicos Next Generation Sequencing Roche 454 Illumina/Solexa Life Technology SOLi. D Helicos

Next Generation Sequencing Illumina Hi. Seq 2000 • Up to 1 billion clusters • Next Generation Sequencing Illumina Hi. Seq 2000 • Up to 1 billion clusters • 150 -200 Gb with 8 day run time • $690 K, ~$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 Sequencing Roche 454 GS Jr Illumina Mi. Seq Life Technology Ion Torrent Next Generation Sequencing Roche 454 GS Jr Illumina Mi. Seq Life Technology Ion Torrent

Cancer Driver Mutations Published Cancer Exomes • 11 Colorectal – Science 2007 • 11 Cancer 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 Non-Silent Mutations in Different Tumors Mutations per Tumor Bert Vogelstein: AACR 2010 Meeting Plenary Session Mutations per Tumor Non-Silent Mutations in Pancreatic Cancer

Cancer Driver Mutations: How Many? Review of Literature/Databases • 116, 432 human cancers • 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) Driver Gene Alterations in Pancreatic Cancer Mutations per Tumor Bert Vogelstein: AACR 2010 Meeting Plenary Session Mutations per Tumor Genetic Alterations in Pancreatic Cancer

Somatic Mutations: How much to sequence? Desired Analytical Sensitivity • 1 -5% Typical NGS Somatic Mutations: How much to sequence? Desired Analytical Sensitivity • 1 -5% Typical NGS Error Rate • 1 -2% Whole Genome Sequencing • 30 x • 1 error >3. 3% sensitivity Targeted Sequencing • 200 -500 x • 0 -4 errors in 200 reads 1%-2% error • Set threshold at ≥ 5% Whole Genome Sequencing at 200 x • >$60, 000!

Average Coverage Maximum Coverage Minimum Coverage % Mapped Total Reads Case SOLi. D Sequencing Average Coverage Maximum Coverage Minimum Coverage % Mapped Total Reads Case SOLi. D Sequencing Pilot Results 1 2 3 4 5 6 7 8 9 8, 551, 464 35. 2 804 113000 28691 8, 380, 102 35. 9 851 126000 29282 9, 700, 737 35. 7 1270 123000 32229 7, 487, 505 7, 447, 964 35. 2 34. 7 905 913 100000 84008 24460 24020 7, 424, 530 35. 1 189 116000 25268 7, 788, 914 34. 9 185 135000 26097 7, 748, 550 35. 3 281 130000 25881 9, 260, 386 34. 7 283 146000 30972 SOLi. D Next Generation Sequencing Variant Calls SNa. Pshot Single Base Extension Genotyping Results KRAS c. 34 G>T (30. 1%) TP 53 c. 743 G>T (26. 0%) KRAS c. 34 G>A (16. 4%) TP 53 c. 536 A>T (10. 4%) NRAS c. 182 A>G TP 53 c. 880 G>T (63. 3%) KRAS c. 34 G>C KRAS c. 38 G>A (22. 6%) PIK 3 CA c. 1633 G>A (18. 8%) TP 53 c. 818 G>A (39. 9%) KRAS c. 34 G>T TP 53 c. 743 G>T BRAF c. 1799 T>A (22. 1%) PIK 3 CA c. 1636 C>A (14. 4%) EGFR c. 2264 C>A (7. 4%) no mutations KRAS c. 35 G>T (12. 0%) TP 53 c. 713 G>T (20. 9%) KRAS c. 34 G>A NRAS c. 182 A>G KRAS c. 34 G>C KRAS c. 38 G>A PIK 3 CA c. 1633 G>A TP 53 c. 818 G>A BRAF c. 1799 T>A PIK 3 CA c. 1636 C>A TP 53 c. 743 G>A KRAS c. 35 G>T

Clinical Cancer Genotyping: On the Horizon Clinical targeted sequencing of FFPE DNA • • Clinical Cancer Genotyping: On the Horizon Clinical targeted sequencing of FFPE DNA • • • initially 100 exons >1000 200 -500 X 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?

Summary • Cancer genetics is rapidly expanding with high complexity • Molecular profiling will Summary • 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

MGH Molecular Diagnostics MGH Cancer Center Leif Ellisen Darrel Borger Dora Dias-Santagata Kathy Vernovsky MGH Molecular Diagnostics MGH Cancer Center Leif Ellisen Darrel Borger Dora Dias-Santagata Kathy Vernovsky Arjola Cosper Breton Roussel Kristin Bergethon Hannah Stubbs Vanessa Scialabba Sara Akhavanfard Daniel Haber David Louis Eunice Kwak Jeff Clark Mari Mino-Kenudson Eugene Mark Jeff Engelman Ultan Mc. Dermott Jeff Settleman Lecia Sequist Belinda Waltman Alice Shaw COI Disclosure: AJI has a paid consulting relationship with Pfizer Inc. and has a provisional patent for SNa. Pshot assay.