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Techniques to Diagnose ALKtranslocation
Pan-Chyr Yang MD, PhD
National Taiwan University
Institute of Biomedical Sciences
Academia Sinica
3rd ITOCD-2012
Techniques to Diagnose ALK-translocation
 ALK as an oncogenic driver for NSCLC
 Clinical characteristics of ALK-translocation in NSCLC
 Methods to detect ALK-translocation
 Pros and cons of different detection methods
 Proposed algorithm for molecular testing in NSCLC
 Summary
Human Anaplastic Lymphoma Kinase (ALK)
MAM
extracellular
LDLa
Location/expression of
normal ALK:

Transmembrane receptor
tyrosine kinase

Chromosome 2p23

1620 aa, 220KDa

Developmentally silenced
except in small intestine,
nervous system and testis
in adult human
MAM
G-rich
TM
intracellular

PTK
(Palmer et al., Biochem J 2009; Drexler et al., Leukemia 2000)
Discovery of ALK in lymphoma

ALK first discovered
in a subset of anaplastic
large-cell lymphoma
(ALCL), leading to the
name anaplastic
lymphoma kinase

ALK fused to the
N-terminal portion
of nucleophosmin
(NPM-ALK), leading to
constitutive activation
of ALK activity
(Morris et al, Science 1994; Mathew et al, Blood 1997)
Mechanisms of ALK Activation
Oncogenic ALK activation
Gene Fusions
 Promoter of 5’ gene-fusion partner drives
expression of developmentally silenced ALK
 5’ fusion-partner dimerization domains
mediate activation of ALK
Normal ALK activation
Ligands
ALK
ALK
P
P
Promoter
P
P
5’ partner
ALK
P
P
5’ partner
Ligand-induced dimerization
Developmentally regulated
PP PP
P
P
ALK
Activating mutations
Neuroblastoma
Anaplastic thyroid carcinoma
JAK/STAT
JAK/STAT
JAK/STAT
Proliferation, Differentiation, Anti-apoptosis
Modify from Camidge et al. Nat Rev Clin Oncol 2012
EML4-ALK Transgenetic Mice Develop
Lung Adenocarcinoma
Soda et al PNAS 2008
EML4–ALK fusion variants in NSCLC
 Several EML4–ALK fusion variants have been identified in NSCLC that
demonstrate gain-of-function properties
 ALK tyrosine kinase activity is required for transforming activity
 Evidence shows ALK inhibitors lead to tumor shrinkage in vivo, and suggests
oncogene addiction, and the potential target for therapeutic intervention
EML4–ALK
E13;A20
E6;A20
E20;A20
E14;A20
E18;A20
E15;A20
ALK
EML4
EML4
TFG–ALK
KIF5B–ALK
ALK
E17;20
ALK
EML4
E13;A20
Variant 1
33%
ALK
EML4
ALK
EML4
ALK
EML4
EML4
E2;A20
E17;A20
unknown
E6a/b;A20
Variant 3a/b
29%
ALK
ALK
EML4
TFG
KIF5B
Coiled-coil domain
E2;20
E15;20
E18;20
E14;20
E20;20
ALK
E13;20
ALK
E6a/b;20
Tyrosine kinase domain
(Adapted from Sasaki et al., Eur J Cancer 2010)`
Crizotinib for ALK Positive Advanced NSCLC
(Sado and Mano et al, Nature 2007; Kwak EL et al, NEJM 2010)
Crizotinib for ALK (+) Adenocarcinoma
49 y/o man, never smoker, diagnosed in 2009/10, 6th line crizotinib
2010-12-30
2011-02-17
Clinical Characteristics of ALK-positive NSCLC

1.6-8.6% in unselected NSCLC, 2.4-5.6% in adenocarcinoma





Younger age
Adenocarcinoma (signet-ring,
cribriform, mucinous)
Never/light smoking history
Wild type EGFR, K-RAS



Liver metastases, pericardial
pleural effusion
Normal CEA
Solid pattern in CT
More sensitive to pemetrexed
Lung Cancer Mutation Consortium Analysis of Adenocarcinomas
N=643
Mean age
Smoking history
Current
Former
Never
ALK-positive
ALK-negative
p
52.3 yr
59.9 yr
< 0.0001
3%
33%
64%
8%
61%
31%
0.0001
(Scagliotti et al. EJC 2012; Camidge et al. Nat Rev Clin Oncol 2012; Fukui et al.
Lung Cancer 2012; Varella Garcia et al., IASLC 2011; Abs #O05.01)
ALK Status by Smoking History in Asians
8%
1.2%
EGFR
KRAS
Nonsmokers
Ever smokers
（N=127）
（N=82）
ALK
WT/WT/WT
(Wong et al. Cancer 2009; 115. 1723-33)
OS of MPE adenocarcinoma patients with
wild type EGFR
Factors
Sex
Female
Male
Age(65 vs.65)
65
65
Smoking
Non-/Light-smoker
Heavy-smoker
ECOG PS
0-1
2-4
EML4-ALK
Negative
Positive
Number of
Patients
Median OS
(months)
54
62
Multivariate analysis
HR
P value
11.3
12.8
1.31
0.336
52
64
14.1
9.6
0.97
0.899
81
35
11.6
12.3
0.76
0.367
99
17
13.8
1.4
5.88
<0.001
77
39
10.3
14.7
0.53
0.011
Wu SG et al JTO 2011
Methods for ALK translocation detection





Fluorescence in situ hybridization (FISH)
Immunohistochemistry (IHC)
RT-PCR and multiplex RT-PCR
Direct sequencing
DNA mass and others
Sequencing
1-13
20
EA13-20
AGGACCTAAAGTGTACCGCCGGA
FISH
IHC
RT-PCR
DNA mass
Mechanism of ALK Break-apart FISH
Negative
Distance between
two signals < 2
signal diameters
Fusion
ALK
Wild Type
EML-4
2p21
2p23
Normal
Positive
(break-apart)
Rearrangement
Fluorescence in situ hybridization (FISH)
 The current standard for detecting EML4–ALK fusion in NSCLC
 ‘Break-apart’ assay used, in which red and green probes are
separated after inversion and ALK rearrangement
Wild type
ALK rearranged
 Separate red and green signals in ≥15% of cells in the sample
means a positive result
(Varella Garcia, IASLC 2011; Abs #MTE36.1)
Sample preparation and staining for FISH
Preparation of FFPE specimens
 Cut ≥ 2 sections, 5 ± 1 mm thick, from tissue block
 Mount on positively-charged glass slides
Begin here if only slides available
Identification of tumor area
 Stain 1 slide with H&E
 Mark tumor area (excluding necrotic areas, in situ carcinoma, small-cell carcinoma)
 Use the marked H&E slide as a template to indicate tumor area on unstained slide*
(subsequent steps refer to the unstained slide)
DeparaffinizationPretreatmentProtease treatment
Hybridization with probe
Wash (same day of hybridization)
*Slide used for FISH should be from within
10 serial sections of the H&E slide.
DAPI counterstain
Vysis ALK Break Apart FISH Probe Kit Package Insert.
Standardized procedure for ALK FISH scoring
Record the signal pattern for 50 tumor cells
< 5 cells positive
ALK negative
> 25 cells positive
ALK positive
5–25 cells positive
Equivocal
Second reader
Average of 1st and 2nd reader:
< 15% positive
≥ 15% positive
ALK negative
ALK positive
Vysis ALK Break Apart Probe Kit Package Insert
Practical aspects of FISH testing for
ALK-translocation
 At least 50 tumor cells should be counted for accurate results1
 FISH signal is considered positive for inversion when red and
green probes are split by >2 signal diameters1
 ALK rearrangements likely to be generalized throughout the tumor,
not focal2
 A false positive split can occur in approxmately 5–11% of cells1,2
 Increasing percentage of ALK-rearranged cells does not appear to
correlate with extent of tumor shrinkage with crizotinib3
1. Varella Garcia et al., IASLC 2011;Abs #MTE36.1; 2. Camidge et al., Clin Cancer Res 2010;
3. Camidge., IASLC 2011;Abs #MO04.04.
‘Background noise’ of FISH
1. Nuclear truncation
2. Distance between split signals
Benign
cell
Positive
Negative
Negative
ALK
ALKfalse+
Image courtesy of Lukas Bubendorf
IHC for ALK-translocation
IHC score
0
1+
2+
3+
Summary of recent data correlating IHC and FISH results
IHC
FISH
0
Negative
1+
Equivocal – need confirmatory FISH
2+
Equivocal – need confirmatory FISH
3+
Positive
 Accumulating data using improved antibody and detection systems indicate
potential for IHC in screening for ALK positivity
 Standardization needed: antibody, scoring system, cut-offs
Mitsudomi et al., ASCO 2011;Abs #7534; Park et al., IASLC 2011;Abs #O05.07.
IHC for ALK expression
 With the use of high-sensitivity detection systems, there are
increasing data supporting the use of IHC as an initial screen

Both sensitivity and specificity can reach 100%, although there is variability
between studies

Compared with FISH, IHC is a more routine technique, is less costly, and is
also less labor intensive
ALK IHC
FISH
Patient 1:
Strong ALK expression
by IHC and
rearrangement by FISH
Patient 2:
No ALK expression by
IHC and no
rearrangement by FISH
(Mino-Kenudson et al., Clin Cancer Res 2010)
ALK IHC with Intercalated Ab-enhanced
Polymer Method

Complete matches in 20 fusion positive and 304 negative tumors
(Takeuchi K et al. Nature Med 2012)
Published series using both ALK IHC and FISH for ALK-translocation detections
Study
Mino-Kenudson,
et al., 2010
IHC
antibody
FISH
No. of cases
IHC/FISH
Sensitivity
(%)
Specificity
(%)
ALK1,
Dako,
Cells counted: NR
Positive: >15%
153/153
67
97
D5F3,
Cells counted: NR
Positive: >15%
153/153
100
99
Cell Signaling,
Technology
Palk, et al.,
2011
5A4,
Novocastra
Cells counted: 100
Positive: >15% split signals
or an IRS in tumor cells
Signal distance > 2
640/640
100
100
98.4
95.8
Palk, et al.,
5A4,
Novocastra
Cells counted: 100
Positive: >15% split signals
or an IRS in tumor cells
Signal distance > 2
735/735
100
100
99.2
96.2
Yang, et al.,
2012
ALK1,
Dako
Cells counted: 100
Positive: >15%
Signal distance > 1
300/216
90.9
100
94.4
62.7
McLeer-Florin,
et al., 2012
5A4,
Abcam
Cells counted: 75-116
(mean 135)
Positive: >15%
100/100
100
98.3
(Modified from Yi ES et al., Mol Diagn Ther 2012)
Reverse transcription-polymerase
chain reaction (RT-PCR)
 Multiplex RT-PCR can detect all EML4–ALK variants1
 Technique developed to carry out RT-PCR even using RNA from
FFPE samples2
 RT-PCR can also be used to screen for other biomarkers
simultaneously3
1. Takeuchi et al., Clin Cancer Res 2008;14:6618–6624; 2. Dannenberg
et al., ASCO 2010;Abs #10535; 3. Li et al., ASCO 2011;Abs #10520.
Other testing approaches under investigation
for detection of ALK rearrangements

Multiplex RT-PCR developed
to screen for all ALK fusion
variants1,2

Chromogenic in situ
hybridization (CISH)3

Uses standard bright-field
microscope; achievable

In 465 samples, high
correspondence with FISH (κ=0.92)
and IHC (κ=0.82)
RT-PCR results of newly discovered
EML4–ALK variant 4 reported by
Takeuchi et al.
1. Takeuchi et al., Clin Cancer Res 2008;14:6618–6624; 2. Li et al., ASCO
2011;Abs #10520; 3. Kim et al., IASLC 2011;Abs #O05.02.
Break-apart CISH probe
showing ALK rearrangement
Comparison between assessment results of EML4ALK fusion genes by FISH and RT-PCR
FISH
Positive
Negative
EML4-ALK(+)
10
2
12
EML4-ALK(-)
1
7
8
Total
11
9
20
RT-PCR
Wu et al JTO 2011
DNA Mass for EML4-ALK Variant 1: E13;A20
EML4
2
6
ALK
13
1-13
Positive signal
20
20
EA13-20
AGGACCTAAAGTGTACCGCCGGA
EML4
Internal control
21
22
23
1-23
24
24
control
TATCCCTGCTCCAAAGCAAAGGCT
Su KY et al 2012
DNA Mass Detection Coverage for EML4-ALK Variants
Variants
V1
Translocation (EML4-ALK)
E13;A20
Mutation rate
33% (with V6)
V2
V3a
V3b
E20;A20
E6a;A20
E6b;A20
9%
V4
V5a
V5b
E14;ins11del19A20
E2;A20
E2;ins117A20
3%
V6
V7
E13;ins69A20
E14;del12A20
33% (with V1)
3%
Control
E17;A20
E23-E24
1%
NA
29%
2%
Su KY et al 2012
Pros and Cons for Different ALK Detection Methods
− FISH −
 Companion diagnostic test for ‘On-Label” use of Crizotinib
 Biologic point of view, FISH may not necessarily be superior
to IHC, PCR or sequencing based methods
 More expensive, require special equipment, technique and
expertise for interpretation
 High false negative for intrachromosomal deletion or
inversion involving very few DNA base pairs
 Cost-effectiveness is the major concern, considering the high
cost (~$US1,000) and low prevalence of ALK+ NSCLC
 Tissue issues, FISH requires at least 50 tumor cells
Pros and Cons for Different ALK Detection Methods
− IHC −
 Less expensive, readily available, and stronger negative
predictive value, potentially useful for screening
 Not a gold standard, need confirmatory ALK FISH for
‘On-Label” use of Crizotinib in NSCLC
 IHC score system correlates well with ALK FISH
 Inter-observer variability can be an issue, no accepted
standard for the definition of IHC scores
 May be interpretable for smaller number of cancer cells
 Need multicenter studies to develop as a screening tool
for ALK+ NSCLC
Pros and Cons for Different ALK Detection Methods
− PCR based methods −
 Sensitive, less expensive and rapid
 May require high-quality RNA
 Can not detect unknown fusion partners
 Unable to confirm the presence of cancer cells
 Can detect multiple biomarkers simultaneously
by multiplex RT-PCR
Proposed algorithms for molecular testing in NSCLC
Sequential
Concurrent
Horn and Pao, J Clin Oncol 2009;27:4232-4235.
Mitsudomi et al. ASCO 2011;Abs #7534
Kon et al ., J Thorac Oncol 2011;6:905-912.
NCCN Guidelines Version 1.2012 – NSCLC
THERAPY FOR RECURRENCE OR METASTASES
FIRST-LINE THERAPY
EGFR mutation
or ALK negative
or unknown
Adenocarcinoma
 Large Cell
 NSCLC NOS

EGFR mutation
testinga
(category 1)
 ALK testinga

ALK
positive


EGFR mutation
testing not
routinely
recommendedb
EGFR mutation
discovered prior
to first-line
chemotherapy
Erlotinibc,d,e
Progression
See Second-line
Therapy
(NSCL-16)
EGFR mutation
discovered during
first-line
chemotherapy
Switch
maintenance:
erlotinib or may
add erlotinibf,g to
current
chemotherapy
(category 2B)
Progression
See Second-line
Therapy
(NSCL-16)
Progression
See Secondline Therapy
(NSCL-16)
EGFR
mutation
positive
Establish
histologic
subtypea
Squamous cell
carcinoma
See First-line Therapy
(NSCL-14)
Crizotinib
See First-line Therapy
(NSCL-15)
All non-squamous cell carcinoma should be tested for EGFR and ALK
Crizotinib is indicated as first-line treatment in ALK-positive patients
Adapted from: NCCN. http://www.nccn.org/professionals/physician_gls/f_guidelines.asp.
Key considerations for optimal ALK testing
Comments
Time frame
Upfront testing is optimal
Sequential or parallel
Simultaneous testing of multiple biomarkers or use of
panel is recommended
Which patients
Any patient can harbor ALK translocations
Patients with non-squamous histology are strong
candidates for testing
Patients should not be selected based on age,
ethnicity, sex, or smoking history
Concurrent ALK and EGFR mutation

1-6%, may have good response to Gefitinib in patients with
coexisting EML4-ALK and EGFR mutations
72 y/o female non-smoker
Adenocarcinoma
Kuo et al JTO 2010
Summary
 ALK FISH is the gold standard for detecting ALK+ and is required
for “on label” use of crizotinib in NSCLC
 FISH may be not appropriate for screening of non-selective cases
 Clinical characteristics and cell types are useful to select patient
for ALK testing
 IHC may be a practical and reliable screening method and can
reduce the need for ALK FISH. Large-scale multicenter
prospective validation of this approach is needed
 For crizotinib resistance NSCLC, test for secondary ALK mutations
and other oncogenic drivers are necessary