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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, [email protected]@partners.org
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
ativ
e f
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
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’
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
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.