Massively Parallel Sequencing in NSCLC: Comparison to Traditional Hot Spot Analysis for Selection of Approved and Novel Targeted
Therapies
JS Ross, A Parker, M Jarosz, S Downing, R Yelensky, D Lipson, P Stephens, G Palmer,
M Cronin, CE Sheehan
Department of Pathology and Laboratory Medicine
Albany Medical CollegeAlbany, NY
Foundation Medicine, Inc.Cambridge, MA
Background (1)
• Next Generation DNA Sequencing (NGS) has recently been applied to FFPE cancer biopsies and major resections (Ross JS et al. J Clin Oncol 29: 2011)
• Current Hot-Spot Genotyping only detects:– Mutations restricted to specific exons and codons
• NGS detects:– Whole exome mutations in numerous cancer related genes– Insertions and deletions– Translocations and fusions– Copy number alterations (amplifications)
Background (2)• Recently, biomarker testing has emerged as a major driver of
the selection of therapy for non-small cell lung cancer (NSCLC)
• Currently, “hot-spot” DNA sequencing and FISH are used to select therapies for NSCLC:– EGFR genotyping for tyrosine kinase inhibitor (erlotinib)– EML4:ALK translocation testing for crizotinib
• The emergence of comprehensive genomic profiling by NGS has led investigators to question whether more thorough gene sequencing techniques could discover potential targets for the treatment of relapsed and metastatic NSCLC not currently searched for in current routine practice
Targeted Therapies for Cancer
Molecular profiling is driving many new targeted cancer therapeutics
Subset of analyzed targets listed; data from BioCentury Online Intelligence Database
• ~500 compounds hitting ~140 targets in development
• Growing number of newly identified potential targets
Design (1)
• DNA was extracted from 4 x 10 m FFPE sections from 49 primary NSCLC (28 female; 21 male; mean age 68 years; 24% Stage I; 13% Stage II; 5% Stage III; 16% Stage IV; 46% Stage unknown)
• The exons of 145 cancer-related genes were fully sequenced using the Illumina HiSeq 2000 (Illumina, San Diego, CA) and evaluated for point mutations, insertions/deletions (indels), specific genomic rearrangements and copy number alterations (CNA)
• A total of 606,676-bp content was sequenced and selected using solution phase hybridization, to an average coverage of 229×, with 84% of exons being sequenced at ≥100× coverage
• This assay captures and sequences 2,574 coding exons representing 145 cancer-relevant genes (genes that are associated with cancer-related pathways, targeted therapy or prognosis), plus 37 introns from 14 genes that are frequently rearranged in cancer
Design (2)
• To maximize mutation-detection sensitivity in heterogeneous NSCLC biopsies, the test was validated to detect base substitutions at a ≥10% mutant allele frequency with ≥99% sensitivity and to detect indels at a ≥20% mutant allele frequency with ≥95% sensitivity, with a false discovery rate of <1%
• Samples included 5% fluid cell blocks; 5% regional lymph nodes; 3% pericardial biopsy and 87% lung biopsies or resections
• There were 46 adenocarcinomas (34 acinar, 19 lepidic, 2 mucinous, 1 papillary), 1 large cell carcinoma, and 2 squamous cell carcinomas
• Results were compared with commercial laboratory allele-specific PCR genotyping on the same tissue blocks
Cancer Genome Profiling Workflow
<14-21 days
Increasing Coverage To 500x Allows For >99% Sensitivity To Detect Mutant Alleles >5%, With No False Positive Mutation Calls
Sensitivity vs Allele Frequency at 500X Coverage (1Mb panel)
0%
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100%
Sens
itivi
ty (P
roba
bilit
y of
Fin
ding
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e M
utati
ons
With
Zer
o Fa
lse
Posi
tives
)
Allele Frequency
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80%
100%
Sens
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ons
With
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Positi
ves)
Allele Frequency
80X 0% Error
80X 0.5% Error
500X 0.5% Error
Deep coverage is required for clinical grade samples
5% 10%
Lower Coverage Misses Relevant Mutations
Mutant Allele frequency spectrum of known mutations found in a series of clinical samples
Fraction of mutations <5%
Fraction of mutations <10%
Fraction of mutations <20%
Fraction of mutations <25%
Fraction of mutations <50%
Fraction of mutations <100%
11% 32% 55% 67% 93% 100%
0-5%6-10%
11-15%
16-20%
21-25%
26-30%
31-35%
36-40%
41-45%
46-50%
51-55%
56-60%
61-65%
66-70%
71-75%
76-80%
81-85%
86-90%
91-95%
96-100%
0
5
10
15
20
25
30
35
40
Mutant Allele Frequency
Num
ber o
f Mut
ation
s
Genomic Alteration Categories
Highly Actionable“Page 1”
Actionable in Principle“Page 2”
Prognostic“Page 3”
Biologically Significant“Page 4”
Category A: Approved / standard alterations that predict sensitivity or resistance to approved / standard therapiesCategory B: Alterations that are inclusion or exclusion criteria for specific experimental therapies
Category C: Alterations with limited evidence that predict sensitivity or resistance to standard or experimental therapies
Category D: Alterations with prognostic or diagnostic utility
Category E: Alterations with clear biological significance in cancer (i.e. driver mutations) without clear clinical implications
Initial Cohort Results (1)• For EGFR status, the NGS result was concordant with commercial
laboratory genotyping in 23/23 (100%) cases
• In 22/23 (96%) NSCLC samples, NGS revealed 53 total genomic alterations, including :– 14 (64%) base substitutions– 2 (9%) INDELs– 6 (27%) CNA– 0 (0%) rearrangements
• Genomic alterations associated with sensitivity or resistance to targeted therapies for NSCLC were found in 16/22 (73%) of cases including: 10 KRAS 4 STK11 3 JAK2 2 PIK3CA11 BRAF 2 EGFR 1 NF1 1 TSC11 TSC2 1 CCNE1 1 PTCH 1 CDK41 CCND1 1 BRCA2 1 CDKN2A 1 ATM
•
Initial Cohort Results (2)• In comparison with the COSMIC database,
NGS results were similar for most genes except for– a lower rate of EGFR mutations (9% vs. 21%)– a higher rate of KRAS mutations (41% vs. 16%) – an unprecedented rate of JAK2 mutations (14% vs.
0%)• 7/22 (32%) of the NSCLC had 2 or more
potentially actionable alterations after NGS
NSCLC: Actionable Genomic AlterationsKR
AS
TP53
EGFR
STK1
1
LRP1
B
PIK3
CA
CTNN
B1 NF1
MDM
2
JAK2
DNM
T3A
CDKN
2A
ATM
TSC1
CCNE
1
BRAF
SMAR
CA4
SMAD
4
RUNX
1
RB1
PTPR
D
NOTC
H1
MYC
MSH
6
MAP
2K1
MLH
1
MCL
1
GNAS
FGFR
2
CDKN
2B
CDK4
BRCA
1
APC
0%
10%
20%
30%
40%
50%
60%
Tumor Type
Perc
enta
ge o
f Cas
es w
ith M
utati
on
Cetuximab/Panitum resist.
Tubulin
s.
Vemuraf
enib se
ns./Cetu
ximab
resis
t.
CDK inhibito
rs
MEK in
hibitors
(sens.
and re
sist.)
/Vem
urafen
ib resis
t.
Nutlins
MEK/E
RK inhibito
rs
CDK4/6 in
hibitors
mTOR in
hibitors
Dasati
nib
DNMT inhibito
rs
FGFR
inhib
PARP in
hibitors
Gefitinib, E
rlotinib, o
thers
PI3 kinas
e, mTO
R inhibito
rsJA
K2 inhibito
rs
mTOR in
hibitors
Notch in
hibitors
CDK4/6 in
hibitors
PARP in
hib
Genes with Actionable AlterationsGenes with Alterations, Actionability Unknown
Multiple ‘Potentially Actionable Alterations in a Single Sample
NSCLC sample SM92
BRAF c.1397G>T p.G466V
PIK3CA c.1035T>A p.N345K
CDK4 Gene amplification
MDM2 Gene amplification
JAK2 c.1849G>T p.V617F
NSCLC sample SM87
EGFR Gene amplification
MDM2 Gene amplification
NSCLC sample SM51
BRAF c.1397G>C p.G466A
STK11 c. 493G>T p.E165*
KRAS c.34G>T p.G12C
Expanded Cohort Results: Initial Gene Rearrangement Detected
RETKIF5B
ATG
ATG
32,316,377 bps 43,611,118 bps
KIF5B-RETRET-KIF5B
ATG
ATG
Break Break
ATG
Translation
KIF5B (exons 1—15) RET (exons 12—20)
Kinesin Coiled coil Tyrosine kinase
KIF5B-RET
Novel RET:KIF 5B Rearrangement in NSCLC (11.3Mb Pericentric Inversion)
Lipson et al. Nature Med, Feb, 2012
Novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET in lung adenocarcinoma
KIF5B-RET Transformed Cells are sensitive to Multi-Targeted Kinase Inhibitors
• KIF5B-RET expression in Ba/F3 cells led to oncogenic transformation
• Cells were sensitive to sunitinib, sorafenib, and vandetanib
• Not sensitive to gefitinib
• Sunitinib, but not gefitinib inhibited RET phosphorylation
Hypothesis: RET kinase inhibitors should be tested in prospective trials for therapeutic benefit in NSCLC patients with KIF5B-RET rearrangements
Expanded Cohort NSCLC Gene Rearrangements Identified by NGS
• EML4:ALK– In FISH + tumors– In FISH – tumors
• RET:KIF5B
Percentage Of Samples With Actionable Alterations Across Major Tissue Types (224 Total Cases)
N=94 N=76 N=31 N=29 N=24
Colorectal Lung(NSCLC) Prostate Breast Melanoma0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
No Mutations FoundUnknown ActionabilityActionable Mutations
Tumor Type
Perc
ent o
f Cas
es
71% cases carried ≥1 plausibly actionable alterations32 % cases carried ≥2 plausibly actionable alterations
N = 111
INDELs; 13%
Copy Number Al-
terations (CNAs),
28%
Re-arrange-
ments, 3%
Substitu-tions; 28%
Covered by available targeted panels; 29%
Only Detected by NGS
Comparison of NGS with Traditional Hot-Spot Genotyping in NSCLC, CRC, Breast Cancer and Melanoma
Also Detected by Hot-Spot Genotyping
Novel Genomic Alterations* Discovered in NSCLC by NGS in an Expanded Cohort
Total Number of Sequenced NSCLC
Total Number of Novel Alterations
Types of Novel Alterations
249 6 (2%) - Chromosomal Inversions (2)- Chromosomal Rearrangements (2)- Gene Substitutions (2)
* Novel alterations discovered in tumor cell (somatic) sequence only as determined by comparison with the COSMIC database. Gene variants of undetermined significance which may represent germline variants are not included in this list.
NSCLC EGFR Activating Mutation
• Sample: SM58• Mutation: EGFR_c.2573T>G_p.L858R• Freq=32%, depth=53• 79 year old white female non-smoker• FNA of lung mass: NSCLC
• FNA sample cytocentrifuged and converted to an FFPE section• Very small numbers of viable tumor cells• Extensive tumor cell necrosis• Genotyping by allele-specific PCR showed identical activating EGFR mutation
Acquired Resistance to EGFR-TKI
By NGS, the resistance clone was seen in 6% of cells and the
sensitizing mutation in 25%
Sensitivity to gefitinib and erlotinib
Resistance to gefitinib and erlotinib
Nutlins
NSCLC: JAK2 Mutation Detected by NGS
• Sample: SM86• Mutation: JAK2_c.1849G>T_p.V617F• Freq=4%, depth=205• 77 year old white female• Lung adenocarcinoma diagnosed by pleural biopsy• Patient diagnosed with polycythemia vera
Low power of pleural biopsy positive for adenocarcinoma
High power view shows adenocarcinoma of the lung. Rare capillaries not blood filled. No nucleated RBC or blasts seen.
G T A T G T G T C T G T G G A
Val Cys Val Cys Gly
c.1849G>T p.V617F
Multiple CNAs in Adenosquamous Carcinoma• Sample: SM92• Mutation: CDK4 amp (6.6x), MDM2 amp (3.3x)• 77 year-old white male• Left lower lobe• Adenosquamous Carcinoma (composite tumor)• pT2 pN0 pMx
CDK4MDM2
Low power view of lobectomy
specimenHigh power view of tumor with adenocarcinoma glands to the left and squamous carcinoma
to the right
Conclusions• Deep sequencing (NGS) of clinical NSCLC samples is
completely concordant with traditional hot-spot genotyping
• NGS uncovers an unexpected number of genomic alterations that could influence therapy selection for NSCLC
• Broad-based, deep sequencing of cancer-related genes results in sensitive detection of all classes of genomic alterations in NSCLC and can reveal actionable genomic abnormalities that inform treatment decisions