2. 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

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

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

5. EGFR Erlotinib/ Gefitinib 20% Lung adenocarcinomas ALK Crizotinib
BCR-ABL Imatinib
100% CML
HER2 Trastuzumab
20-30% IDC
EGFR Erlotinib/ Gefitinib
20% Lung adenocarcinomas
BRAF V600E PLX4032
50-60% Melanoma
ALK Crizotinib
3-5% Lung adenocarcinoma
BRAF
1799 T>A
V600E

6. EGFR Erlotinib/ Gefitinib 20% Lung adenocarcinomas ALK Crizotinib
BCR-ABL Imatinib
100% CML
HER2 Tastuzumab
20-30% IDC
O’Brien et al., Imatinib Compared with Inter-feron and Low-Dose Cytarabine for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia, NEJM 2003
EGFR Erlotinib/ Gefitinib
20% Lung adenocarcinomas
BRAF V600E PLX4032
60% Melanoma
ALK Crizotinib
3-5% Lung adenocarcinoma
Romond EH et al., Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer. NEJM 2005.
BRAF
1799 T>A
V600E
Mok et al., NEJM 2009

7. Comprehensive Genetic Characterization of Tumors for Personalized Cancer Care
DNA epigenetics
DNA mutations
DNA chromosomal alterations
Proteomics
mRNA and miRNA profiling

8. Clinical Genotyping in Guiding Therapeutic Decisions
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.
Cancer Patients
Prospective
Enrollment
Genotyping
Oncology Clinical trials
Improved Clinical Use
of Genotyping
MGH
Translational Research Laboratory
MGH Pathology Specimen Repository
Basic Research Centers 8

9. 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

10. SNAPSHOT Overview Multiplex PCR Single Base Extension Reaction
Capillary Electrophoresis
loci of interest
ddNTP
Electrophoretic Output
Increasing
molecular weight
Relative fluorescence A B D C F E
Applied Biosystems (ABI) Prism® SNaPshot® Multiplex system, originally developed to detect SNPs. The identity of the different loci is given by the position of each peak, which is dictated by the length of the extension primer

11. SNAPSHOT Genotyping Assay 16 cancer genes – 120 described mutations
AKT G – E17
ERBB Exon 20 insertions
IDH R C
IDH R G 11

12. SNAPSHOT v3 Panel 1 7-plex Panel 2 8-plex Panel 3 5-plex Panel 4
KRAS34
EGFR2235_49F
EGFR 2573
NRAS181
PI3K1633
bCat94
bCat121
8-plex
Panel 2
EGFR2235_49R
NRAS38
BRAF1799
NRAS182
PI3K263
bCat122
bCat95
TP53.742
5-plex
Panel 3
NRAS35
EGFR2236_50F
EGFR2369
bcat133
PI3K1624
8-plex
Panel 4
KRAS35
EGFR2236_50R
PTEN517
FLT3.2503
PI3K3139
NOTCH1.4724
NOTCH1.4802
TP53.733

13. SNAPSHOT v3 Normal Lung cancer EGFR mutation Glu746_Ala750del
(c.2235_2249del) 13

14. SNAPSHOT v3
Normal
Melanoma
BRAF mutation Val600Glu
(c.1799T>A) 14

15. SNAPSHOT v3 Normal Colorectal cancer KRAS mutation Gly13Asp
(c.38G>A) 15

16. SNAPSHOT v3 Normal Breast cancer PIK3CA mutation His1047Arg
(c.3140A>G) 16

17. 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%
Our laboratory has gone live with clinical genotyping, starting in March of During this1st year, we have genetically profiled more than 400 lung cancer patients and ~70 colorectal patients at the MGH using our SNaPshot genotyping platform. Mutations have been identified in a majority of cases, often helping direct therapeutic decisions in these patients.
N=650 17 17

18. Lung Adenocarcinoma: Overlap of Mutations
KRAS
56 isolated
(58 total)
PIK3CA 5
EGFR
36 isolated
(50 total) 1 1
B-cat 1 3 1
TP53
T790M 5 2 1 1 1 4
ALK 13
APC 2
BRAF 1
NRAS
Belinda Waltman/ Lecia Sequist

19. Rapid integration of FISH : ALK Rearrangements in NSCLC
Crizotinib: Potent & selective ATP competitive oral inhibitor of MET and ALK kinases and their oncogenic variants

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

22. Timeline for Crizotinib and ALK in NSCLC
PF activity in cells exhibiting ALK fusion in broad screen (MGH-McDermott)
PF Inhibits ALK activity
PF FIP
May
2005
2006
2007
2008
2009
Identification of PF
PF demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)
Discovery of EML4-ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)
Objective responses demonstrated in ALK fusion positive NSCLC and IMT
Phase III study of “Crizotinib” in ALK positive NSCLC starts
Slide Courtesy of Ross Camidge

23. Timeline for Crizotinib and ALK in NSCLC
For phase I trial:
ALK enriched cohort of 82 subjects required FISH screening of over 1200 NSCLCs
PF activity in cells exhibiting ALK fusion in broad screen (MGH-McDermott)
PF Inhibits ALK activity
PF FIP
May
2005
2006
2007
2008
2009
Identification of PF
PF demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)
Discovery of EML4-ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)
Objective responses demonstrated in ALK fusion positive NSCLC and IMT
Phase III study of “Crizotinib” in ALK positive NSCLC starts
Slide Courtesy of Ross Camidge

24. 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

25. No Mutation Identified
Mutational profiling in colorectal cancers
No Mutation Identified
34%
KRAS
25%
TP53
21%
PIK3CA 6%
NRAS 3%
APC 4%
BRAF 7%
Our laboratory has gone live with clinical genotyping, starting in March of During this1st year, we have genetically profiled more than 400 lung cancer patients and ~70 colorectal patients at the MGH using our SNaPshot genotyping platform. Mutations have been identified in a majority of cases, often helping direct therapeutic decisions in these patients.
N=250 25 25

26. Colon Adenocarcinoma: Overlap of Mutations
KRAS
20 isolated
(36 total)
TP53
18 isolated
(28 total)
APC 1
NRAS 3
PIK3CA
BRAF
6 isolated 4 2 6

27. Genomic
TL ng/panel DNA
TL ng/panel DNA

28. More Than Just Point Mutations

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

30. Next Generation Sequencing
First Generation Sequencing

31. Next Generation Sequencing
Roche 454
Illumina/Solexa
Life Technology SOLiD
Helicos

32. Next Generation Sequencing
Illumina HiSeq 2000
Up to 1 billion clusters
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

33. Next Generation Sequencing
Roche 454 GS Jr
Illumina MiSeq
Life Technology Ion Torrent

34. Cancer Driver Mutations
Non-Silent Mutations in Pancreatic Cancer
Bert Vogelstein:
AACR 2010 Meeting
Plenary Session
Mutations per Tumor
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

35. Cancer Driver Mutations: How Many?
Genetic Alterations in Pancreatic Cancer
Bert Vogelstein:
AACR 2010 Meeting
Plenary Session
Mutations per Tumor
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

36. 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 x
0-4 errors in 200 reads  1%-2% error
Set threshold at ≥5%
Whole Genome Sequencing at 200x
>$60,000!

37. SOLiD Sequencing Pilot Results
Case
Total
Reads
Mapped %
Minimum
Coverage
Maximum
Average 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 4
7,487,505
905
100000
24460 5
7,447,964
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
283
146000
30972
SOLiD
Next Generation Sequencing
Variant Calls
SNaPshot
Single Base Extension
Genotyping Results
KRAS c.34G>T (30.1%)
KRAS c.34G>T
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>A
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.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
no mutations
TP53 c.743G>A
KRAS c.35G>T (12.0%)
TP53 c.713G>T (20.9%)
KRAS c.35G>T

38. Clinical Cancer Genotyping: On the Horizon
Clinical targeted sequencing of FFPE DNA
initially 100 exons  >1000
X coverage
Mb data
3-4 week turnaround time
$500 raw reagent cost
Desired
Whole exon coverage
Tumor vs. normal?
Copy number?
Rearrangements?
Methylation?
Transcription?

39. 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

40. MGH Molecular Diagnostics Leif Ellisen Darrel Borger
Dora Dias-Santagata
Kathy Vernovsky
Arjola Cosper
Breton Roussel
Kristin Bergethon
Hannah Stubbs
Vanessa Scialabba
Sara Akhavanfard
MGH Cancer Center
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.