1/20/2016
Practical Molecular Diagnostics in
Lung Cancer: Beyond the NCCN
Guidelines
Lynette M. Sholl, M.D.
Department of Pathology
Brigham and Women’s Hospital
Harvard Medical School
Boston, MA
Disclosures
• Genentech: Scientific Advisory Board
Objectives
• Established and emerging molecular targets in
lung cancer
– Applying advanced technologies in clinical practice
• Role of pathology in current lung cancer
management
– Optimizing histologic diagnoses on small biopsies
– Application of predictive immunohistochemistry
– Accurate reporting of molecular results
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Trends in lung cancer histology
Carcinoma,
NOS, 3.1
Sarcoma, 0.2
Other,
5.0
Small cell, 13.1
NSCLC, 10.6
Large Cell, 2.1
Squamous cell,
22.8
Adenocarcinoma,
43.2
Histologic Diagnoses,
SEER data 2008-2012
Meza et al. PLOSOne 2015.
Reasons for increased fraction of adenocarcinoma?
• Change in smoking habits
– Decline in tobacco use since
the 1960s
– Increased use of filters
requiring more vigorous
inhalation
Trends in lung cancer outcomes
• 27% of all cancer deaths
in 2015
• Significant downward
trends in death rates
among both men and
women diagnosed with
lung cancer between
2000-2010
Edwards et al. Cancer 2014.
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Reasons for decreasing mortality
rates?
Improved detection:
Reasons for decreasing mortality
rates?
Improved therapy:
Kris et al. JAMA 2014.
Lung adenocarcinoma
TARGETED THERAPIES
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1/20/2016
EGFR an attractive therapeutic target
• Important pro-growth
signaling protein in normal
and neoplastic tissues
• Commonly overexpressed in
multiple tumor types,
including lung cancer
• EGFR TKIs the new wonder
drugs in lung cancer??
– Poor outcomes from clinical
trials of erlotinib and gefitinib
in unselected patients
– Occasional patients with
exceptional response to
therapy
EGFR mutation is key biomarker
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1/20/2016
EGFR mutation
…vs. copy number?
What’s the relationship?
• In an EGFR-mutated tumor:
– Step 1: mutation
– Step 2: amplification and
protein overexpression
•Amplification/overexpression
may occur in the absence of
mutation.
ALK translocations in NSCLC:
3 years (!) from bench to bedside
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1/20/2016
CAP/AMP/IASLC
Clinical Practice Guidelines
• Any advanced stage (IV) patient
with ACA or patient with
progressive disease should
receive EGFR and ALK testing
– Reflex testing may be appropriate
in certain environments
– This testing is not recommended
for lung tumors lacking evidence
of ACA differentiation
– Avoid the term “non small cell
lung carcinoma” whenever
possible
• Prioritize tissue for EGFR and ALK
testing
ROS1 rearrangements in lung cancer
•Rare (1-2% of lung
ACA)
•More common in
never smokers
•Promising responses to
crizotinib therapy
Shaw AT et al. N Engl J Med 2014;371:1963-1971.
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1/20/2016
RET rearrangements in lung cancer
• Rare (1% of lung ACA)
• Results from small
intrachromosomal
rearrangement
(inv (10)(p11.22q11.2)
• More common in never
smokers
• Reported responses to:
– cabozantinib
• (c-Met, VEGFR2 inhibitor)
Takeuchi et al Nat Med 2012
– Vandetanib
• (VEGFR, EGFR, RET inhibitor)
67 year old woman with lung mass and
numerous subcutaneous metastases:
EGFR, ALK, ROS1 wild type
Sequencing reveals METex14 mutation &
MET amplification with Exon 14 skipping
Efficacy of crizotinib in MET ex 14 splice tumors
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1/20/2016
• 64yo M, ~15 pack-year
smoker, quit 1 year prior to
diagnosis
• May 2014: diagnosed with
stage IV lung
adenocarcinoma
• Standard testing negative
for EGFR, ALK, ROS1
• Started on first-line
carboplatin/pemetrexed,
with mixed response
• BRAF c.1799T>A (p.V600E)
detected by sequencing
Dabrafenib (BRAF inhibitor) +
Trametinib (MEK inhibitor)*
October 2014
March 2015
*60% overall response rate in the phase 2 trial for patients with BRAF V600E mutations.
Planchard et al. J Clin Oncol 33, 2015 (suppl; abstr 8006)
Oncogenic drivers in
non-squamous NSCLC (%)
KRAS
33.8
No/unknown driver
29.9
HRAS
0.1
MAP2K1
0.3 AKT1
0.6
RET
1.0
ROS1
1.1
ERBB2
2.5
NRAS
PIK3CA
1.0
2.9
MET
3.0
BRAF
3.8
ALK
3.9
EGFR
19.1
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Reality check.
“Non small cell lung carcinoma”
Adenocarcinoma
Most common subtype in
nonsmokers
Unique chemosensitivity
profile (pemetrexed)
~60% have a defined oncogenic
driver
-“Targetable”
Squamous cell carcinoma
Smokers
Use of antiangiogenic agents
associated with massive
pulmonary hemorrhage
Minority with defined
oncogenic driver
-Limited targetability
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IASLC algorithm for Small Biopsies
Subclassification of morphologic
NSCLC-NOS
IASLC classification of small biopsies,
take home points:
• Distinguish ACA and SQC whenever possible
• The molecular profile of an ACA will dictate
targeted therapy
• Judicious use of IHC is critical
– Two first-line markers:
• TTF1 and p63 or p40
• Less established/less specific markers (napsin, mucin,
CK7, CK5/6) should be considered second line
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1/20/2016
Make a diagnosis
+
EGFR, KRAS, HER2, BRAF, PIK3CA,
ALK, ROS1, RET, MET, etc. mutations,
copy number alterations, translocations
EGFR mutation-specific
antibodies
(clones 43B2 and 6B6)
Excellent specificity, limited sensitivity
Informative if positive- may be useful in
scant specimens
Study
Yu et al.
Kawahara et
al.
Kato et al.
Brevet et al.
Fan et al.
Bondgaard et
al.
Ex19del
L858R
Sensitivity
92%
(24 of 26)
83%
(19 of 23)
75%
(9 of 12)
95%
(20 of 21)
93%
(40 of 43)
80%
(8 of 10)
Specificity
99%
(193 of 195)
100%
(16 of 16)
97%
(56 of 58)
99%
(171 of 173)
100%
(126 of 126)
98%
(152 of 155)
Sensitivity
86%
(23 of 26)
62%
(13 of 21)
50%
(9 of 18)
74%
(23 of 31)
74%
(17 of 23)
63%
(12 of 19)
Specificity
100%
(196 of 196)
100%
(16 of 16)
100%
(52 of 52)
99%
(161 of 163)
99%
(145 of 146)
99%
(153 of 155)
ALK IHC
93-100% sensitive and specific as
compared to FISH
Ventana ALK (D5F3) CDx assay approved as
a companion diagnostic for crizotinib
Cutz et al. JTO 2014
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ROS1 IHC
(D4D6 antibody)
Ideal screening tool:
Excellent sensitivity,
good specificity
Sholl et al. AJSP 2013.
Reference
N
Sensitivity
Specificity
Notes
Sholl et al. 2013
210
100
92
Focal expression in a KRASmutated tumor
Strong expression in a FISH
negative tumor
Mescam-Mancini 221
et al. 2014
100
96.9
Expressed in two HER2mutated tumors
Cha et al. 2014
330
100
72.6-93.4*
ROS1 expression seen in ROS1
WT tumors from ever-smokers
Boyle et al. 2014
33
100
100
As compared to FISH or RT-PCR
RET IHC
• Variable antibody
performance
• Current evidence doesn’t
support routine clinical use
Tsuta et al. Br. J. Cancer 2014
Reference
Tsuta et al.
2014
Clone
EPR2871
IHC pattern
vs.
RET
N
WT
N
Sensitivity
Specificity
Any
FISH or
RTPCR
21
1774
66.7
86.1
15
79
100
87.3
70.3
Lee et al. 2014
ab134100
Any
Transcript
profiling or
FISH
3F8
Any
RTPCR
3
154
100
Sasaki et al.
2014
EPR2871
Any
RTPCR
3
75
100
33.3
EPR2871
Mod to
strong only
RTPCR
3
75
66.7
77.3
How has next gen sequencing (NGS)
changed the game?
• Increased target coverage with decreased
tissue requirements
POPv2 GENE LIST
SNV, INDELS, COPY NUMBER ALTERATIONS
ABL1 BMPR1A CDKN1B EP300
FAS
HRAS
MDM2
ID3
MDM4
AKT1
BRAF CDKN1C EPHA3 FBXW7
AKT2
BRCA1 CDKN2A EPHA5 FGFR1
IDH1
MECOM
AKT3
BRCA2 CDK N2B EPHA7
FGFR2
IDH2
MEF2B
ALK
BRD4 CDK N2C ERBB2
FGFR3
IGF1R
MEN1
ALOX12B BRIP1
CEBP A
ERBB3
FGFR4
IKZF1
MET
APC
BUB1B
CHEK2
ERBB4
FH
IKZF3
MITF
AR
CADM2
CIITA
ERCC2
FK BP9 INSIG1
MLH1
ARAF
CARD11 CREBBP ERCC3
FLCN
JAK2
MLL
ARID1A
CBL
CRKL
ERCC4
FLT1
JAK3
MLL2
ARID1B
CBLB
CRLF2
ERCC5
FLT3
KCNIP1
MPL
ARID2
CCND1 CRTC1
ESR1
FLT4
KDM5C
MSH2
KDM6A
ASXL1
CCND2 CRTC2
ETV1
FUS
MSH6
ATM
CCND3 CSF1R
ETV4
GATA3 KDM6B
MTOR
ATRX
CCNE1 CSF3R
ETV5
GATA4
KDR
MUTYH
AURKA
CD274 CTNNB1
ETV6
GATA6
KEAP1
MYB
AURKB
CD58
CUX1
EWSR1
GLI1
K IT
MYBL1
MYC
AXL
CD79B
CYLD
EXT1
GLI2
KRAS
B2M
CDC73
DDB2
EXT2
GLI3 LINC00894 MYCL1
BAP1
CDH1
DDR2
EZH2
GNA11
LMO1
MYCN
BCL2
CDK 1 DEPDC5 FAM46C GNAQ
LMO2
MYD88
BCL2L1
CDK 2
DICER1 FANCA
GNAS
LMO3
NBN
BCL2L12
CDK 4
DIS3
FANCC GNB2L1 MAP2K1 NEGR1
BCL6
CDK 5
DMD
FANCD2 GPC3 MAP2K4
NF1
BCOR
CDK 6 DNMT3A FANCE
GSTM5 MAP3K1
NF2
MAPK1
BCORL1
CDK 9
EED
FANCF
H3F3A
NFE2L2
EGFR
BLM
CDKN1A
FANCG HNF1A
MCL1
NFKBIA
NFKBIZ PRAME
RPL26
NKX2-1 PRDM1 RUNX1
NOTCH1
PRF1
SBDS
NOTCH2 PRKAR1A SDHA
NPM1
PRKCI
SDHAF2
NPRL2 PRKCZ
SDHB
NPRL3
PRKDC
SDHC
NRAS PRPF40B SDHD
NTRK1
PRPF8 SETBP1
NTRK2 PSMD13 SETD2
NTRK3
P TCH1
SF1
PALB2
PTEN
SF3B1
PARK2
PTK2
SH2B3
PAX5
PTPN11 SL ITRK6
PBRM1 PTPRD SMAD2
PDCD1LG2 QKI
SMAD4
PDGFRA RAD21 SMARCA4
P DGFRB
RAF1 SMARCB1
PHF6
RARA
SMC1A
P HOX2B
RB1
SMC3
P IK3C2B
RBL2
SMO
PIK3CA RECQL4 SOCS1
PIK3R1
REL
SOX2
PIM1
RET
SOX9
PMS1
RFWD2 SQSTM1
RHEB
PMS2
SRC
PNRC1 RHPN2
SRSF2
ROS1
STAG1
STAG2
STAT3
STAT6
STK11
SUFU
SUZ12
SYK
TCF3
TCF7L1
TCF7L2
TERC
TERT
TET2
T LR4
TNFAIP3
TP53
TSC1
TSC2
U2AF1
VHL
WRN
WT1
X PA
XPC
XPO1
ZNF217
ZNF708
ZRSR2
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How has next gen sequencing (NGS)
changed the game?
• Increased target coverage with
decreased tissue requirements
• Less biased genomic analysis
– Detect novel variants in known
oncogenes
– Detect variants outside of coding
regions
– Detect variants in genes not
typically included on clinical test
menus
– Detect common variants in known
oncogenes in unusual contexts
NGS vs. IHC vs. FISH for
ALK translocation detection
ALK translocations in Lung Adenocarcinoma
Clinical FISH and/or IHC
ALK +
Oncopanel
ALK
Fusion +
ALK
Fusion Total
ALK -
25
0
1*
190
26
190
Sensitivity
96%
Specificity
100%
* 20% tumor in this specimen
NGS as an arbiter in discrepant cases:
FISH negative
IHC positive
ALK IHC (5A4 clone)
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CLIP4
Fused
Red only
Green only
From Vysis ALK FISH probe product insert
NGS showed: CLIP4-ALK fusion AND EML4-ALK fusion
FISH cannot detect cryptic EML4-ALK fusions.
Unusual FISH results are considered “negative” but should be followed up.
MET in tumorigenesis
• Exon 14 deletion removes the juxtamembrane
domain of MET
• Tyr1003 phosphorylation site necessary for
Cbl binding
• Decreased ubiquitination and impaired
downregulation of the activated receptor
Lai AZ, et al, Trends in Cell Bio, 2009
Identification of tumor-specific, intronic mutations in Met leading to exon 14 splicing.
Monica Kong-Beltran et al. Cancer Res 2006;66:283-289
©2006 by American Association for Cancer Research
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MET: Diverse Deletions & Point Mutations
Awad et al. J Clin Oncol, 2016.
MET juxtamembrane splice mutations:
not uncommon in lung adenocarcinomas
and predict response to MET inhibitors
•
•
•
•
•
•
2.4% in Kong-Beltran et al. Cancer Res, 2006
3.3% in Onazato et al. J Thor Oncol, 2009
~4.5% in TCGA Nature, 2014
3% in Frampton et al. Cancer Discovery, 2015
4% in Paik et al. Cancer Discovery, 2015
3% in Awad et al. J Clin Oncol, 2016
Clinical Characteristics
Clinical Characteristic
Median age (range), years
Sex, N (%)
Male
Female
Smoking history, N (%)‡
Never-smoker
≤10 pack-years
>10 pack-years
Race, N (%)
White, non-Hispanic
Asian
Black
White, Hispanic
Unknown
Histology, N (%)
Adenocarcinoma
Pleomorphic w/ adenocarcinoma component
NSCLC, poorly-differentiated
Squamous
Adenosquamous
Stage at diagnosis, N (%)
I
II
III
IV
MET ex14
(N = 28)
72.5 (59-84)
EGFR
(N = 99)
61 (30-93)
KRAS
(N = 169)
65 (42-93)
9 (32%)
19 (68%)
30 (30%)
69 (70%)
62 (37%)
107 (63%)
10 (36%)
3 (11%)
15 (53%)
57 (58%)
10 (10%)
28 (28%)
6 (4%)
11 (7%)
152 (90%)
28 (100%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
79
15
1
3
1
(80%)
(15%)
(1%)
(3%)
(1%)
157 (93%)
0 (0%)
5 (3%)
3 (2%)
4 (2%)
18 (64%)
4 (14%)
5 (18%)
0 (0%)
1 (4%)
92
0
4
2
1
(93%)
(0%)
(4%)
(2%)
(1%)
150 (89%)
3 (2%)
10 (6%)
5 (3%)
1 (1%)
13 (46%)
2 (7%)
4 (14%)
9 (32%)
9
3
9
78
(9%)
(3%)
(9%)
(79%)
12 (7%)
12 (7%)
44 (26%)
101 (60%)
Awad et al. J Clin Oncol 2016.
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Pulmonary sarcomatoid carcinoma
• Sarcomatoid carcinoma
– Pleomorphic carcinoma
• Giant cells or spindled cells
comprise ≥ 10% of tumor
• Admixed adenocarcinoma,
squamous, or undifferentiated
carcinoma
– Carcinosarcoma
• Carcinoma + sarcoma with
heterologous elements
– Pulmonary blastoma
• Fetal adenocarcinoma+
mesenchymal stroma
• 2-3% of lung cancers
• Predominantly smokers
MET mutations in pulmonary
pleomorphic/sarcomatoid carcinoma
• Lui et al, JCO 2015:
• MET exon 14 splice
mutations in 8/36 (22%)
of pulmonary
sarcomatoid carcinomas
undergoing WES and
targeted MET analysis
• BWH/DFCI:
• MET exon 14 splice
mutations in 4/15 (27%)
pulmonary pleomorphic
carcinoma sequenced by
NGS
• Other alterations include:
– KRAS (13%)
– NRAS (7%)
– no known driver (53%).
Potential for NGS in diagnosis
• Distinguishing multiple primary lung tumors
from metastases
• Defining site of origin for poorly differentiated
carcinomas
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74 year old woman, former smoker,
incidental RUL mass and multiple GGOs
RUL
MET c.3028+2T>C (p.D1010_splice) - in 34% of 449 reads
RLL
EGFR c.2573T>G (p.L858R), in 2.8% of 285 reads
KRAS c.35G>A (p.G12D), in 15% of 438 reads
Breast vs. Lung?
• 58 year old woman, h/o
well differentiated
T1aN0 invasive ductal
carcinoma (ER+, PR+,
HER2-) 5 years prior
• Now with lung left
upper lobe mass and
diaphragmatic implants
Lung tumor: CK7+, TTF1-, GATA3 weak,
ER weak, mammoglobin -
“The carcinoma in the lung is poorly differentiated with high grade nuclei, abundant
cytoplasm, mucin production, and necrosis. The carcinoma does not resemble the
breast carcinoma in the excision from 2010. The possibility that the patient has a
different breast primary carcinoma should be considered.”
Back of the envelope:
Gene
Frequency
in breast
Frequency
in Lung
PPV
KRAS
mutation
1%
25%
98%
(for lung)
EGFR
mutation
0
13%
100%
(for lung)
ERBB2
amp
16%
0.5%
91%
(for breast)
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1/20/2016
Breast vs. Lung?
KRAS c.34G>T (p.G12C), exon 1
STK11 c.206C>A (p.S69*), exon 1
TP53 c.499_501CAG>G (p.Q167fs), exon 5
KEAP1 c.1016T>C (p.L339P), exon 3
KEAP1 c.382A>T (p.I128F), exon 2
JAK2 c.3214C>T (p.Q1072*), exon 24
BRIP1 c.434C>G (p.S145C), exon 5
CDC73 c.26G>T (p.R9L), exon 1
CHEK2 c.349A>G (p.R117G), exon 3
DMD c.10567G>A (p.E3523K), exon 75
JAK2 c.3214C>T (p.Q1072*), exon 24
SMARCA4 c.2439_splice (p.S813_splice)
TCF3 c.136G>A (p.G46R), exon 3
Genomic evidence supports a lung primary.
Dogan et al. Clin Cancer Res, 2012.
Calles et al. Clin Cancer Res, 2015.
NGS to explore new targets
No/unknown
dri ver
29.9
Jamie Wyeth
KRAS
33.8
HRAS
0.1
MAP2K1
0.3
AKT1
0.6 RET
1.0
ROS1
1.1
NRAS
1.0
ERBB2
2.5
PIK3CA
2.9
MET
3.0
BRAF
3.8
ALK
3.9
EGFR
19.1
“Targeted therapy is dead.”
-Anonymous
PD-1/PD-L1 inhibitors
• Applicable across most
tumor types
• 20-30% response rate on
average
• 18 month response
durability
Mutation-targeted inhibitors
• Applicable to few tumors
(lung, melanoma, GIST)
• 50-70% response rate
• Response durability 7-12
months
From a population standpoint, immune checkpoint blockade outperforms
mutation-targeted therapies.
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1/20/2016
PD-L1 IHC in practice
Dako 28-8
pembrolizumab
Roche SP142
nivolumab
E1L3N
atezolizumab
22C3 pharmDx
tremelimumab
Roche SP263
??
PD-L1 IHC: mass confusion.
Drug
Company
FDA approval
mAb/Platform
Scoring criteria
Comment
Pembroluzimab
(Keytruda)
Merck
FDA approved for
NSCLC
22C3 (DAKO
pharmDx)/
≥50% tumor cells
Companion
diagnostic1(as of
Oct 2015)
Nivolumab
(Opdivo)
Bristol- Myers
Squibb
FDA approved for
squamous and non
squamous NSCLC
≥1% tumor cells
Complementary
diagnostic (as of
Oct 2015)
Link 48 Autostainer
28-8 (DAKO
pharmDx)/
Link 48 Autostainer
Atezolizumab
(MPDL3280)
Roche
Expected in 2016
SP142 (Ventana)
Durvalumab
(MEDI4736)
Astra Zeneca
Expected in 2016
SP263 (Ventana)
Tumor cells and/or
tumor infiltrating
immune cells
≥25% tumor cells
Predictive only in
non-squamous
carcinomas
In development
In development
Perhaps other biomarkers will be better…
Characteristics of immune infiltrate?
Expression of other checkpoint inhibitors?
Mutational load?
Mutational signature?
Biomarker reporting
• http://www.cap.org/web/home/resources/cancerreporting-tools/cancer-protocol-templates
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1/20/2016
Biomarker reporting
• Optional reporting template
• Available as CAP electronic cancer
checklist (eCC)
• Focused on targets with accepted
clinical significance
– Alteration type
– Methodology
• Updated version available in 2016
covers:
–
–
–
–
–
–
–
–
EGFR
KRAS
BRAF
ERBB2
ALK
RET
ROS1
MET
Important topics given short shrift:
• Tumor types beyond
adenocarcinoma
Mechanisms of EGFR TKI resistance
• Mechanisms and
significance of acquired
resistance in patients
receiving EGFR TKIs,
MET/ALK/ROS1 inhibitors
• “Liquid biopsy” in solid
tumor clinical management
Sequist et al. Sci Trans Med. 2011
Thanks… Questions?
Acknowledgements:
Mark Awad, DFCI
Phil Cagle
Pasi Janne, DFCI
Neal Lindeman, BWH
Geoff Oxnard, DFCI
Members of the
Pulmonary Pathology
Society
20