High-throughput Clinical Cancer
Genotyping
A. John Iafrate, MD/PhD
Department of Pathology
Diagnostic Molecular Pathology Laboratory
Translational Research Laboratory
Massachusetts General Hospital
Boston, MA
aiafrate@partners.org
A New Paradigm in Cancer Treatment
A New Paradigm in Cancer Treatment
Haber, Gray, Baselga Cell 2011
BCR-ABL Imatinib
100% CML
EGFR Erlotinib/ Gefitinib
20% Lung adenocarcinomas
HER2 Trastuzumab
20-30% IDC
BRAF V600E PLX4032
50-60% Melanoma
BRAF
1799 T>A
V600E
ALK Crizotinib
3-5% Lung adenocarcinoma
BCR-ABL Imatinib
100% CML
HER2 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 V600E PLX4032
Romond
EH
et
al.,
Trastuzumab
60% Melanoma
3-5% plus
Lung adenocarcinoma
Adjuvant Chemotherapy for Operable
HER2-Positive Breast Cancer. NEJM
2005.
BRAF
1799 T>A
V600E
Mok et al., NEJM 2009
Comprehensive Genetic Characterization of Tumors for Personalized Cancer Care
DNA epigenetics
Proteomics
DNA mutations
DNA chromosomal
alterations
mRNA and miRNA
profiling
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
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
ddNTP
ddNTP
loci of interest
ddNTP
Relative fluorescence
Electrophoretic Output
A
B
C
D
E
F
Increasing
molecular weight
SNAPSHOT Genotyping Assay
16 cancer genes – 120 described mutations
Gene
Amino Acid – cDNA Residue
Gene
Amino Acid – cDNA Residue
AKT1
AKT1
49G
49G- –E17
E17
APC
APC
APC
APC
R1114 - 3340C
Q1338 - 4012C
R1450 - 4348C
T1556fs* - 4666_4667insA
KRAS
KRAS
KRAS
KRAS
G12 - 34G
G12 - 35G
G13 - 37G
G13 - 38G
NOTCH1
NOTCH1
L1575 - 4724T
L1601 - 4802T
BRAF
BRAF
V600 - 1798G
V600 - 1799T
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
D32 - 94G
D32 - 95A
S33 - 98C
G34 - 101G
S37 - 109T
S37 - 110C
T41 - 121A
T41 - 122C
S45 - 133T
S45 - 134C
NRAS
NRAS
NRAS
NRAS
NRAS
NRAS
NRAS
G12 - 34G
G12 - 35G
G13 - 37G
G13 - 38G
Q61 - 181C
Q61 - 182A
Q61 - 183A
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
ERBB2
FLT3
G719 - 2155G
T790 - 2369C
L858 - 2573T
E746_A750 - 2235_2249del
E746_A750 - 2236_2250del
Exon 19 deletions
Exon 20 insertions
D835 - 2503G
PIK3CA
PIK3CA
PIK3CA
PIK3CA
PIK3CA
PIK3CA
PIK3CA
PIK3CA
R88 - 263G
E542 - 1624G
E545 - 1633G
Q546 - 1636C
Q546 - 1637A
H1047 - 3139C
H1047 - 3140A
G1049 - 3145G
PTEN
PTEN
PTEN
PTEN
R130 - 388C
R173 - 517C
R233 - 697C
K267fs*- 800delA
IDH1
IDH1
IDH1
IDH1
R132
R132 -394C
- 394C
R132
R132 -395G
- 395G
JAK2
V617 - 1849G
KIT
D816 - 2447A
TP53
TP53
TP53
TP53
TP53
TP53
TP53
R175 - 524G
G245 - 733G
R248 - 742C
R248 - 743G
R273 - 817C
R273 - 818G
R306 - 916C
SNAPSHOT v3
Panel 1
bCat121
EGFR2235_49F
PI3K1633
KRAS34
7-plex
bCat94
EGFR 2573
NRAS181
Panel 2
NRAS38
bCat122
PI3K263
NRAS182
BRAF1799
EGFR2235_49R
8-plex
bCat95
TP53.742
5-plex
EGFR2369
Panel 3
EGFR2236_50F
PI3K1624
NRAS35
bcat133
Panel 4
KRAS35
8-plex
PTEN517
FLT3.2503
EGFR2236_50R
TP53.733
NOTCH1.4724
PI3K3139
NOTCH1.4802
SNAPSHOT v3
Normal
Lung cancer
EGFR mutation
Glu746_Ala750del
(c.2235_2249del)
SNAPSHOT v3
Normal
Melanoma
BRAF mutation
Val600Glu
(c.1799T>A)
SNAPSHOT v3
Normal
Colorectal cancer
KRAS mutation
Gly13Asp
(c.38G>A)
SNAPSHOT v3
Normal
Breast cancer
PIK3CA mutation
His1047Arg
(c.3140A>G)
Mutational profiling in lung cancers
AKT 1%
BRAF 2%
NRAS 1%
IDH1 <1%
HER2 2%
CTNNB1 2%
ALK 3%
PIK3CA 4%
TP53 5%
No Mutation 42%
EGFR 15%
KRAS 23%
N=650
Lung Adenocarcinoma: Overlap of Mutations
PIK3CA
5
1
1
KRAS
56 isolated
(58 total)
1
EGFR
36 isolated
(50 total)
B-cat
3
1
1
TP53
2
1
T790M
5 1
4
APC
2
BRAF
ALK
13
1 NRAS
Belinda Waltman/ Lecia Sequist
Rapid integration of FISH : ALK Rearrangements in NSCLC
Telomere
2p23 region
Crizotinib: Potent & selective ATP
competitive oral inhibitor of MET and
ALK kinases and their oncogenic variants
Centromere
t(2;5) ALK gene
breakpoint region
3’
~250 kb
5’
~300 kb
Phase I Clinical Trial of ALK Inhibitor Crizotinib in ALK-rearranged Lung Adenocarcinoma
Timeline for Crizotinib and ALK in NSCLC
PF2341066 Inhibits
ALK activity
2005
Identification of
PF2341066
PF2341066 activity
in cells exhibiting
ALK fusion in broad
screen (MGHMcDermott)
PF2341066 FIP
May
2006
PF2341066
demonstrates
cytocidal activity in
cells exhibiting ALK
fusion (Pfizer in
house)
Slide Courtesy of Ross Camidge
2007
2008
Discovery of EML4ALK 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:
PF2341066 activity
in cells exhibiting
ALK enriched cohort of 82 subjects required FISH screening
of over 1200 NSCLCs
PF2341066 Inhibits
ALK fusion in broad
PF2341066 FIP
ALK activity
screen (MGHMay
McDermott)
2005
Identification of
PF2341066
2006
PF2341066
demonstrates
cytocidal activity in
cells exhibiting ALK
fusion (Pfizer in
house)
Slide Courtesy of Ross Camidge
2007
2008
Discovery of EML4ALK 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 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%
34%
PIK3CA 6%
KRAS
TP53
25%
21%
N=250
Colon Adenocarcinoma: Overlap of Mutations
BRAF
6 isolated
3
3
PIK3CA
4
1
1
1
KRAS
20 isolated
(36 total)
TP53
18 isolated
(28 total)
4
2
6
APC
1
1
NRAS
3
Genomic
TL-09-267
20 ng/panel DNA
TL-09-285 3.04ng/panel DNA
More Than Just Point Mutations
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
Next Generation Sequencing
First Generation Sequencing
Next Generation Sequencing
Roche 454
Illumina/Solexa
Life Technology SOLiD
Helicos
Next 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 Sequencing
Roche 454 GS Jr
Illumina MiSeq
Life Technology Ion Torrent
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
• 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 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!
Average
Coverage
Maximum
Coverage
Minimum
Coverage
%
Mapped
Total
Reads
Case
SOLiD Sequencing Pilot Results
1
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
2
3
4
5
6
7
8
9
SOLiD
Next Generation
Sequencing
Variant Calls
SNaPshot
Single Base Extension
Genotyping Results
KRAS c.34G>T (30.1%)
TP53 c.743G>T (26.0%)
KRAS c.34G>A (16.4%)
TP53 c.536A>T (10.4%)
NRAS c.182A>G
TP53 c.880G>T (63.3%)
KRAS c.34G>C
KRAS c.38G>A (22.6%)
PIK3CA c.1633G>A
(18.8%)
TP53 c.818G>A (39.9%)
KRAS c.34G>T
TP53 c.743G>T
BRAF c.1799T>A (22.1%)
PIK3CA c.1636C>A
(14.4%)
EGFR c.2264C>A (7.4%)
no mutations
KRAS c.35G>T (12.0%)
TP53 c.713G>T (20.9%)
KRAS c.34G>A
NRAS c.182A>G
KRAS c.34G>C
KRAS c.38G>A
PIK3CA c.1633G>A
TP53 c.818G>A
BRAF c.1799T>A
PIK3CA c.1636C>A
TP53 c.743G>A
KRAS c.35G>T
Clinical 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?
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
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 McDermott
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 SNaPshot assay.