Biomarker Assessment for Selection of
Therapy in NSCLC
Marileila Varella Garcia, PhD
UC SOM, Depts. Medicine and Pathology
Livorno, Italy
April 13, 2012
Disclosures
Research Grants/Contracts:
Boehringer Ingelheim
Genentech
Imclone
Consultant:
Abbott Molecular
Outline





NSCLC: Molecular Heterogeneity and
Targeted Therapy
Testing Platforms: Strengths and Limitations
Clinical Assays: ALK
Novel Targets: ROS1 and RET
Novel Multiplex FISH Reagents: 4-target
Enumeration and 4-target Break-Apart
Molecular Heterogeneity in NSCLC
KRAS
Unknown
PDGFR
VEGFR
MEK1
AKT1
PIK3CA
EGFR
FGFR1
HER2
MET
RET
ROS1
ALK
Testing Platforms

DNA-based


RNA-based


mutation analyses, FISH, BRISH, SNPs
RT-PCR, quantitative PCR, gene expression
arrays
Protein-based

IHC
Selecting Clinical Assays
Defined reagent, standardized
scoring system, validated cut-off
TEST A
ALK Break Apart FISH
TEST B
complete concordance
?
?
?
TEST C
Partial concordance
False negatives or accurate
detection of primary
refractory population?
e.g. RT-PCR
TEST D
Partial concordance
TEST E
Partial concordance
?
False positives or
missed true positives?
e.g. IHC
Accuracy, Precision, Cost, Feasibility
STANDARDIZATION
ALK Dual Color, Break Apart FISH assay
Abbott Molecular
2p23
3’normal 5’
inversion with EML4-ALK fusion
Rearrangement positive - split
Rearrangement positive - single 3’ ALK
Bright-Field In Situ Hybridization
CISH
ALK+ by split
ALK+ by single 3’ALK
Kim et al., JTO, 6(8):1359-1366, 2011
Few publications but already include DIFFERENT:
a) criteria for classification of split signal
b) cut-offs for classification as ALK+
ALK Protein Expression by IHC: Several Reagents
DAKO clone ALK1
Novocastra clone 5A4
Cell Signaling clone D5F3
Yi et al, JTO 2011
Paik et al, JTO 2011
Mino-Kenudson et al., CCR 2010
Antibody
Authors
Journal, Year Group
ALK1
DAKO
Boland
Yi
Yang
Popat
Rodig
Shaw
Mino-Kenudson
Takeuchi
Mino-Kenudson
Salido
Takeuchi
Paik
Koh
McLeen-Florin
HP 2009
JTO 2011
JTO 2012
LC 2011
CCR 2009
JCO 2009
CCR 2011
MP 2009
CCR 2011
JTO 2011
CCR 2009
JTO 2011
JTO 2011
JTO 2012
D5F3, Cell
Signaling Technology
5A4, AbCam
Novocastra
Mayo
Mayo
Mayo
London, Royal Marsden Hospital
MGH/BWH/DFCC
MGH/BWH/DFCC
MGH/BWH/DFCC
Japan, The Cancer Institute
MGH/BWH/DFCC
Barcelona/Colorado
Japan, The Cancer Institute
Seoul National University
Seoul National University
Grenoble, France
ALK IHC: Heterogeneity in Scoring Systems
Standardization is urgently needed!!!
But likely lack of standardization is not
the only reason for discrepancies…
Discrepancies Are Likely Also Biology-Based
Comparison FISH, PCR and IHC
Pt. ID
% cells FISH
positive (BA)
1
2
3
4
5
6
7
8
9
10
11
12
24%
33%
37%
43%
47%
53%
59%
77%
77%
80%
80%
93%
EML4-ALK
EML4-ALK Variant-specific
FISH
RT-PCR
POS
POS
POS
POS
POS
POS
POS
POS
POS
POS
POS
POS
E13;A20*
E13;A20
E6;A20
E13;A20
E6;A20
E13;A20
E18;A20
E6;A20
E13;A20*
E6;A20
E13;A20
E13;A20
Insight
Genetics
qPCR
ALK IHC
(D5F3)
ALK IHC
(D9E4)
NEG
NEG
POS
POS
POS
POS
POS
POS
NEG
NEG
POS
POS
NEG
NEG
POS
POS
POS
POS
POS
POS
NEG
NEG
POS
POS
NEG
POS
NEG
NEG
Expression-based
Genomic-based
*Positive after 2 rounds of PCR
Varella-Garcia et al., unpublished data
Acquired Resistance to Crizotinib in ALK FISH+




Multiple mutations in ALK tyrosine kinase
domain
New oncogenic driver: EGFR and KRAS
Loss of the ALK activation by fusion
Gain in copy number of the ALK rearrangement
Implications in selection of further therapies:


2nd generation ALK inhibitor
Other options
Doebele et al., Clin Cancer Res 2011
Novel Target in NSCLC: ROS1
Fusion
CD74-ROS1
Chromosomal
Rearrangement
t(5;6)(q32;q22)
EZR-ROS1
inv(6)(q22q25.3)
240 kb interstitial
GOPC(FIG)-ROS2
del(6)(q21q21)
likely deletion
likely deletion
242.5 kb interstitial
deletion
LRIG3-ROS1
t(6;12)(q22;q14.1)
SLC34A2-ROS1
t(4;6)(p15.2;q22)
SDC4-ROS1
t(6;20)(q22;q12)
TPM3-ROS1
Unknown
t(1;6)(q21.2;q22)
Tumor type
NSCLC
Lung adenocarcinoma
Lung adenocarcinoma
Lung adenocarcinoma
NSCLC
Lung adenocarcinoma
Glioblastoma
1st ROS1 Partner Number of
exon
Exon
Cases
35
34
NR
32-34
34
34
6
6
NR
6
6
10
36
36
35
7
3
7
Cholangiocarcinoma
Cholangiocarcinoma
Low Malignant Potential Serous
Ovarian Carcinoma
35
Lung adenocarcinoma
35
NSCLC
33-35
Lung adenocarcinoma
Lung adenocarcinoma
NSCLC
Lung adenocarcinoma
NSCLC
Lung adenocarcinoma
Lung adenocarcinoma
Lung adenocarcinoma
NR
32-34
32-34
32
32-34
35
NR
NR
7
16
4
NR
12
4
2
2
8
NR
NR
1
2
5
3
2
2
1-cell line
U118MG
1
1
1
1
1-cell line
HCC78
1
1
2
3
1
2
12
1
Reference
Rikova et al., 2007
Li et al., 2011
Bergethon et al., 2012
Takeuchi et al., 2012
Doebele 2012
Takeuchi et al., 2012
Charest et al., 2003
Gu et al., 2011 PLOS
Gu et al., 2011 PLOS
Birsch et al., 2011
Takeuchi et al., 2012
Rikova et al., 2007
Bergethon et al., 2012
Takeuchi et al., 2012
Doebele 2012
Takeuchi et al., 2012
Doebele 2012
Takeuchi et al., 2012
Bergethon et al., 2012
Takeuchi et al., 2012
39 lung cancer clinical cases + HCC78 cell line
7 distinct fusions (in 26) + unknown (in 13)
ROS1 FISH Positive = Split 3’-5’ or Single 3’
3’
5’
ROS1 FISH probe for NSCLC TMA
ROS1 FISH+ are likely to benefit from Crizotinib
Bergethon et al, 2012; Doebele et al., under review
Novel Target in NSCLC: RET
KIF5BReference RET
Fusion
K16R12
Ju et al,
K15R12
2012
K23R12
K15R12
Kohno et K16R12
K23R12
al, 2012
K24R8
K15R12
K16R12
Takeuchi et
K22R12
al, 2012
K23R22
K24R11
K15R12
Lipson et K16R12
K22R12
al, 2012
K15R11
Total Patients
Number of Frequency in Tested
Patients
Cohorts
1
1
1
3
1
1
1
8
1
1
1
1
8*
3
1
1*
33
proband
1 /5 ADC (20%)
1 /15 ADC (6.7%)
6/319 ADC (1.9%)
12/1121 ADC (1.0%)
12/585 ADC (2.0%)
Activated RET
is a target for
Vandetanib
Ponatinib
Sorafinib
KIF5B-RET fusion
detected by FISH
Negative
Positive
Novel FISH Reagents
FGFR1/FGFR2 4-target Enumeration
FGFR1/CEP8 FGFR2/CEP10
Novel FISH Reagents
FGFR1/FGFR2 Enumeration
FGFR1/CEP8 FGFR2/CEP10
FGFR1/FGFR2
Novel FISH Reagents
Multiplex FGFR1/FGFR2 Enumeration
FGFR1/CEP8
FGFR1/CEP8 FGFR2/CEP10
FGFR2/CEP10
Novel FISH Reagents
Multiplex ALK-ROS1 Break-Apart (~10% ADC)
3’ALK/5’ALK 3’ROS1/5’ROS1
Positive for ALK
Negative for ROS1
Novel FISH Reagents
Multiplex ALK-ROS1 Break-Apart (~10% ADC)
normal
Single 3’
Single 3’
3’ALK/5’ALK
Positive for ALK
rearrangement
3’ROS1/5’ROS1
Negative for ROS1
rearrangement
Novel FISH Reagents
Multiplex ALK-ROS1 Break-Apart
normal
Single 3’
Single 3’
3’ALK/5’ALK
Negative for ALK
rearrangement
3’ALK/5’ALK 3’ROS1/5’ROS1
Negative for ALK
Positive for ROS1
Time is over, for updates follow us on
3’ROS1/5’ROS1
Positive for ROS1
rearrangement
and
CONCLUSIONS
• Discovery of molecular markers for sensitivity and
resistance to targeted therapy agents has brought new
excitement to the NSCLC field
• Greater understanding of molecular pathways will make
biomarker design more efficient
• It is unlikely that any single technical platform will reach
100% diagnostic specificity and sensitivity for any given
marker
• For each marker, a panel of assays should be optimized,
standardized and validated
• Multiplexing is highly recommended due to scarcity of
tumor material in advanced NSCLC
THANKS
Wilbur Franklin, MD
Dara Aisner, MD, PhD
Robert Doebele, MD, PhD
Ross Camidge, MD, PhD
Paul Bunn Jr, MD
Anna Baron, PhD
Margaret Skokan
Mariana Theodoro
Adelita Mendoza
Yong Gon Cho
Antonella Flacco
Severine Kako
Tara O’Brien
Liang-Guo Xu
Nathan Schulte
Abbott Molecular, for novel probe sets