Clinical Cancer GenotypingClinical Cancer Genotyping
Long Phi Le, MD/PhDLong Phi Le, MD/PhD
Department of PathologyDepartment of Pathology
Diagnostic Molecular Pathology LaboratoryDiagnostic Molecular Pathology Laboratory
Translational Research LaboratoryTranslational Research Laboratory
Massachusetts General HospitalMassachusetts General Hospital
Boston, MABoston, MA
[email protected]@partners.org
The case for broad cancer genotypingThe case for broad cancer genotyping
Our current approachOur current approach
Next generation sequencingNext generation sequencing
SOLiDSOLiD / Ion Torrent Pilot/ Ion Torrent Pilot
The Old Paradigm in Cancer TreatmentThe Old Paradigm in Cancer Treatment
The New Paradigm in Cancer TreatmentThe New Paradigm in Cancer Treatment
t(9;22) in CMLt(9;22) in CML
Blast crisis
Obrien et al., Imatinib Compared
with Interferon and Low-Dose
Cytarabine for Newly Diagnosed
Chronic-Phase Chronic Myeloid
Leukemia. N Engl J Med 2003
-100
0
100
200
300
400
500
600
700
800
900
Well
Cycle
4
Cycle
8
Cycle
12
Cycle
16
Cycle
20
Cycle
24
Cycle
28
Cycle
32
Cycle
36
Cycle
40
Cycle
44
FISH detects HER2 amplification in
15-25% of ductal carcinomas
Predicts responsiveness to
Trastuzumab
HER2 Amplification in Breast CancerHER2 Amplification in Breast Cancer
Romond EH et al., Trastuzumab
plus Adjuvant Chemotherapy for
Operable HER2-Positive Breast
Cancer. N Engl J Med 353:1673.
2005.
EGFR: Erlotinib/ Gefitinib
20% Lung adenocarcinomas
KRAS: Cetuximab resistance
36-50% Colon adenocarcinoma
Molecular Markers and Targeted TherapyMolecular Markers and Targeted Therapy
ALK: Crizotinib
3-5% Lung adenocarcinoma
BRAF V600E: PLX4032
60% Melanoma
BRAF
1799 T>A
V600E
DRUG MECHANISM OF ACTION ACTIVE MGH STUDIES
Axitinib PDGFR, VEGFR Inhibitor 1
AZD-2171/Cediranib VEGFR Inhibitor 4
AZD-6244 MEK Inhbitor 1
Bevacizumab Anti-VEGF 15
BI-2536 Plk-1 Inhibitor 1
Cetuximab Anti-EGFR 6
Dasatinib Bcr-Abl, Src Kinase Inhibitor 2
Erlotinib hydrochloride EGFR Inhibitor 6
Enzastaurin hydrochloride PKC beta inhibitor 1
Gefitinib EGFR Inhibitor 4
Lapatinib ditosylate EGFR, HER2 Inhibitor 1
Targeted Molecular Therapy in Active MGH TrialsTargeted Molecular Therapy in Active MGH Trials
Neratinib EGFR, HER2 Inhibitor 1
PF-02341066 MET Inhibitor 1
Rituximab Anti-CD20 3
Sirolimus mTOR Inhibitor 1
Temsirolimus mTOR Inhibitor 1
Trastuzumab Anti-HER2 1
CP-751871 Anti-IGF-1R 1
EXEL-7647 EGFR, HER2, EphB4, VEGFR Inhibitor 2
EXEL-2880 MET, VEGFR-2 Inhibitor 1
AZD-6244 MEK Inhibitor 1
Everolimus mTOR, FKBP12 Inhibitor 1
Tandutinib PDGFR, KIT, Flt3 Inhibitor 1
Sorafenib Multi-kinase Inhibitor 2
Sunitinib Flt3, VEGFR, KIT, PDGFR Inhibitor 11
Vatalanib VEGFR-2, KIT, PDGFR Inhibitor 2
Volociximab Integrin Inhibitor 1
Metastatic Colorectal Cancer & KRAS TestingMetastatic Colorectal Cancer & KRAS Testing
“Based on systematic reviews of the relevant literature, all patients
with metastatic colorectal carcinoma who are candidates for anti-
EGFR antibody therapy should have their tumor tested for KRAS
mutations in a CLIA-accredited laboratory. If KRAS mutation in
codon 12 or 13 is detected, then patients with metastatic colorectal
carcinoma should not receive anti-EGFR antibody therapy as part of carcinoma should not receive anti-EGFR antibody therapy as part of
their treatment.”
-Allegra et al. J Clin Oncol 2009
US Growth in Genetic TestingUS Growth in Genetic Testing
MolecularMolecular
CytogeneticsCytogeneticsMicroarraysMicroarrays
Molecular Diagnostic TechniquesMolecular Diagnostic Techniques
PCR and QPCRPCR and QPCR SequencingSequencing
Genomic
TL-09-267 20 ng/panel DNA
More With LessMore With Less
TL-09-267 20 ng/panel DNA
TL-09-285 3.04ng/panel DNA
Considerations in Clinical Genotyping PlatformConsiderations in Clinical Genotyping Platform
Clinical Test
• Archived FFPE tissue
• Analytical sensitivity
• 2 week turnaround time
• Report in patient’s record
• Performed in a CLIA lab
Actionable Targets
• Predicts response/resistance
• Clinches diagnosis
• Yields prognosis
• Stratify patients for trials
• Adaptability for new targets
Logistics
• Clinical patient coordinator
• Accessioning
• Automation
• Eventually test all tumors
Other
• Economics
• Finances and billing
• Bioinformatics
• Potential for research
Distribution of KRAS MutationsDistribution of KRAS Mutations
COSMIC, Wellcome Trust Sanger Institute, 2010
ddNTP
ddNTP
ddNTP
loci of interest
Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis
SNaPshot GenotypingSNaPshot Genotyping
Electrophoretic Output
Increasing
molecular weight
Re
lative
flu
ore
sce
nce
A B DC FE
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
AKT1 49G – E17
MGH SNaPshot AssayMGH SNaPshot Assay
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
ERBB2 Exon 20 insertions
IDH1 R132 -394C
IDH1 R132 -395G
EGFR2235_49R
NRAS38
NRAS182
PI3K263 bCat95bCat122
TP53.742
Genomic DNA
BRAF1799
MGH SNaPshot v1.0MGH SNaPshot v1.0
Melanoma
BRAF
1799 T>A
V600E
0.44
TP53 5%
IDH1 <1%
NRAS 1%BRAF 2%
HER2 2%
PIK3CA 4%
ALK 3%
CTNNB1 2%
AKT 1%
Lung Cancers: SNaPshotLung Cancers: SNaPshot
KRAS 23%
No Mutation 42%
EGFR 15%
TP53 5%
N=650
KRAS
56 isolated
(58 total)
EGFR
36 isolated
(50 total)
PIK3CA
51 1
13
B-cat
Lung Cancers: Mutation OverlapLung Cancers: Mutation Overlap
1
2
(58 total)
ALK
13
T790M
5TP53 11
14
2
APC
NRAS
BRAF
13
No Mutation Identified34%
PIK3CA 6%
NRAS 3%
APC 4%
BRAF 7%
Colorectal Cancers: SNaPshotColorectal Cancers: SNaPshot
KRAS25%TP53
21%
N=250
PIK3CA
BRAF
6 isolated
4
3
1
1
3
1
Colorectal Cancers: Mutation OverlapColorectal Cancers: Mutation Overlap
KRAS
20 isolated
(36 total)
TP53
18 isolated
(28 total)
APC
1 NRAS
3
4
261
More Than Just Point MutationsMore Than Just Point Mutations
The Future of Clinical Cancer GenotypingThe 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: The Big BrothersNext Generation Sequencing: The Big Brothers
Roche 454 Illumina/Solexa
Life Technology SOLiD Helicos
Next Generation Sequencing: Uno Tube, Mucho DataNext Generation Sequencing: Uno Tube, Mucho Data
Next Generation Sequencing
First Generation Sequencing
Ten Years of SequencingTen Years of Sequencing
Adapted from Nature 1 April 2010
Next Generation Sequencing: The Little BrothersNext Generation Sequencing: The Little Brothers
Roche 454 GS Jr Illumina GA IIe
Life Technology SOLiD PI Life Technology Ion Torrent
The Future of Clinical Cancer GenotypingThe Future of Clinical Cancer Genotyping
Do we have the technology?
Is it cost-effective?
What to genotype?
The challenges?
By Angela Canada Hopkins
How Many Genes?How Many Genes?
Pre-Human Genome Project
• >100,000 genes
Post-Human Genome Project
• 30,000 genes• 30,000 genes
• 20,000-25,000
Human exome
• ~180,000 exons � 1% of the
genome � ~30Mb
Clinical Cancer Genotyping: A Mountain to Climb?Clinical Cancer Genotyping: A Mountain to Climb?
Published Cancer Exomes
• 11 Colorectal – Science 2007
Bert Vogelstein:
AACR 2010 Meeting
Plenary Session
Mu
tati
on
s p
er
Tum
or
Non-Silent Mutations in Pancreatic Cancer
• 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
Mu
tati
on
s p
er
Tum
or
Mu
tati
on
s p
er
Tum
or
Non-Silent Mutations in Different Tumors
Clinical Cancer Genotyping: Focus! Focus!Clinical Cancer Genotyping: Focus! Focus!
Review of Literature/Databases
• 116,432 human cancers
Bert Vogelstein:
AACR 2010 Meeting
Plenary Session
Mu
tati
on
s p
er
Tum
or
Genetic Alterations in Pancreatic Cancer
• 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)
Mu
tati
on
s p
er
Tum
or
Driver Gene Alterations in Pancreatic Cancer
A Little MathA Little Math
Desired Analytical Sensitivity
• 1-5%
Typical NGS Error Rate
• 1-2%
Whole Genome Sequencing
• 30x• 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!
Target Exon CaptureTarget Exon Capture
Microdroplet PCR Solid-phase Capture Solution-phase Capture
Metzker ML, Nature Reviews Genetics 2010
Reaction Array
Cost of Next Generation SequencingCost of Next Generation Sequencing
Adapted from Nature 1 April 2010
Library Preparation Cost
The Future of Clinical Cancer GenotypingThe Future of Clinical Cancer Genotyping
Do we have the technology?
Is it cost-effective?
What to genotype?
The challenges?
By Angela Canada Hopkins
SOLiD Sequencing Pilot Study on SNaPshot SamplesSOLiD Sequencing Pilot Study on SNaPshot Samples
• Emulsion PCR used to template
each sample on to magnetic beads.
• Beads modified at 3’ end to allow
for deposition on to sequencing slide.
• PCR amplicons concatenated and
sheared for barcoded fragment library
construction.
•Samples pooled prior to emulsion PCR
Courtesy of Life Technologies
50 bp
• 5 mer barcode sequenced first to parse each library
• Sequence 50 bases sequenced in the forward direction.
• Mapping and SNP calling performed in CLC Bio Genomics
Workbench (ungapped local alignment in colorspace, SNP
cutoff at 5%)
• Deposited slide loaded into one of two
available flow cells on SOLiD instrument.
5 bp BC
SOLiD Sequencing Pilot ResultsSOLiD Sequencing Pilot ResultsC
ase
Tota
l
Re
ad
s
% Ma
pp
ed
Min
imu
m
Co
vera
ge
Ma
xim
um
Co
vera
ge
Ave
rag
e
Co
vera
ge
1
8,551,464 35.2 804 113000 28691
2
8,380,102 35.9 851 126000 29282
3
9,700,737 35.7 1270 123000 32229
SOLiD
Next Generation
Sequencing
Variant Calls
SNaPshot
Single Base Extension
Genotyping Results
KRAS c.34G>T (30.1%)
EGFR c.2235_2249del15
KRAS c.34G>T
EGFR 15 bp del
TP53 c.743G>T (26.0%)
TP53 c.743G>T
KRAS c.34G>A (16.4%)
TP53 c.536A>T (10.4%) KRAS c.34G>A9,700,737 35.7 1270 123000 32229
4
7,487,505 35.2 905 100000 24460
57,447,964 34.7 913 84008 24020
6
7,424,530 35.1 189 116000 25268
7
7,788,914 34.9 185 135000 26097
8
7,748,550 35.3 281 130000 25881
9
9,260,386 34.7 283 146000 30972
TP53 c.536A>T (10.4%) KRAS c.34G>A
NRAS c.182A>G (47.7%)
TP53 c.880G>T (63.3%)
EGFR c.2240_2257del18
NRAS c.182A>G
EGFR 18 bp del
KRAS c.34G>C (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.38G>A
PIK3CA c.1633G>A
TP53 c.818G>A
BRAF c.1799T>A (22.1%)
PIK3CA c.1636C>A (14.4%)
EGFR c.2264C>A (7.4%)
BRAF c.1799T>A
PIK3CA c.1636C>A
TP53 c.743G>A (2.3%)
TP53 c.752T>G (1.9%) TP53 c.743G>A
KRAS c.35G>T (12.0%)
TP53 c.713G>T (20.9%) KRAS c.35G>T
KRAS Exon 2: Per Base Error & CoverageKRAS Exon 2: Per Base Error & Coverage
Per Base CoveragePer Base Coverage
Average Per Base Error
+/- Standard Deviation
Average Per Base Error
+/- Standard Deviation
Per Base Error
95% Confidence Interval
Per Base Error
95% Confidence Interval
Circles = Outliers Outside
Per Base Error 95% CI
Circles = Outliers Outside
Per Base Error 95% CI
KRAS Exon 2: Per Base Error & CoverageKRAS Exon 2: Per Base Error & Coverage
KRAS Exon 2: Per Base Error & CoverageKRAS Exon 2: Per Base Error & Coverage
#1
#5
#4
#3
#2
#9
TP53 Exon 7: Detection of Compound HeterozygosityTP53 Exon 7: Detection of Compound Heterozygosity
#8
Detection of Compound HeterozygosityDetection of Compound Heterozygosity
Detection of Compound HeterozygosityDetection of Compound Heterozygosity
468 mutations
10 mutations
Sanger COSMIC Website
Sample #1 Sample #4
EGFR Deletion: MappingEGFR Deletion: Mapping
Digital Genotyping and Structured DataDigital Genotyping and Structured Data
CDS Variant aa Variant Confidence
Score
Allele
Frequency
Relevance Prevalence Literature
BRAF
c.1799G>T
BRAF V600E 95 45% (1) Oncogenic
(2) Response
to PLX4032
2% Lung ACA
7% Colon ACA
60% Melanoma
Flaherty et al.
NEJM 2010
Laboratory (genotype):
Variant Confidence Score = f(Quality Value, Coverage, Error, Bi-directionality)
Patient Diagnosis Stage Genotype Treatment Response Literature
Jane Doe Metastatic
melanoma
III BRAF
c.1799G>T
Plexxikon
PLX4032
81% Melanoma Flaherty et al.
NEJM 2010
Clinical (phenotype):
Clinical Cancer Genotyping: At The Tipping PointClinical Cancer Genotyping: At The Tipping Point
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• $500 raw reagent cost
Desired
• Whole exon coverage
• Tumor vs. normal?
• Copy number?
• RNA expression?
• Rearrangements?
• Methylation?
• Cancer genetics is rapidly expanding with high complexity
• Molecular profiling will drive cancer management
• Continued need for higher-throughput cancer genotyping
SummarySummary
• Next gen sequencing poses a huge informatics challenge
• Clinical next generation sequencing is comingis already here
^
MGH Molecular Diagnostics
Dora Dias-Santagata
Kathy Vernovsky
Arjola Cosper
Breton Roussel
Kristin Bergethon
Hannah Stubbs
Vanessa Scialabba
Sara Akhavanfard
Quynh Lam
MGH Cancer Center
Daniel Haber
David Louis
Leif Ellisen
Darrell Borger
ABI/LifeTech
Kevin McKernan
Rosemary Obrien
Steve McLaughlin
Clarence Lee
Nancy Gangemi
Timothy Harkins
Jeremy Stuart
Eric Tsung
Ion Torrent/LifeTechKenny Fan
Jae Han
Ion Torrent/LifeTech
Andrew Felton
Jason Meyers
Maneesh Jain
Simon Cawley
Stuart Davidson
Mike Lelivelt
Questions?Questions?
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