Histopathology 2014, 65, 539–548. DOI: 10.1111/his.12414
Detection of ALK expression in non-small-cell lung cancer with ALK gene rearrangements – comparison of multiple immunohistochemical methods Karen Zwaenepoel,1 Amber Van Dongen,1 Suzan Lambin,1 Christine Weyn1 & Patrick Pauwels1,2 1
Department of Pathology, Antwerp University Hospital, Edegem, Belgium, and 2Center for Oncological Research (CORE), University of Antwerp, Edegem, Belgium
Date of submission 15 October 2013 Accepted for publication 8 March 2014 Published online Article Accepted 12 March 2014
Zwaenepoel K, Van Dongen A, Lambin S, Weyn C, Pauwels P (2014) Histopathology 65, 539–548
Detection of ALK expression in non-small-cell lung cancer with ALK gene rearrangements – comparison of multiple immunohistochemical methods Aim: Testing for ALK rearrangements in advanced, non-squamous non-small-cell lung cancers that are wild-type for activating EGFR mutation has become standard care. Fluorescence in-situ hybridization is considered the gold standard for this evaluation. Pre-screening with immunohistochemistry has been suggested, to reduce testing costs and to make testing more widely available. By analysing the sensitivity and specificity of different ALK immunohistochemical assays, we aimed to identify the most reliable assay to detect ALK rearrangement. Methods and results: ALK screening performed by FISH analysis was compared with three different immunohistochemical assays, in which two ALK antibody clones (5A4 and D5F3) were used on two detec-
tion platforms (Dako AutostainerLink 48 and Ventana Benchmark GX). Data from 30 ALK FISHpositive cases show that the sensitivity of the immunohistochemical assays varies from 93.3% to 96.6%. Head-to-head comparison of the 5A4 and D5F3 ALK antibody clones demonstrates similar staining potency. In general, homogeneous, intermediate to strong staining of the ALK-positive samples was obtained. Conclusions: ALK immunohistochemistry can be considered as a pre-screen method if one accepts a sensitivity of 93.3–96.6%. Because ALK immunohistochemical staining needs to be performed close to the detection limit of the assay, vigilant quality control monitoring is required to guarantee trustworthy results.
Keywords: anaplastic lymphoma kinase, fluorescence in-situ hybridization, immunohistochemistry, non-small cell lung cancer
Introduction Identification of oncogenic drivers and accompanying therapeutic agents is dramatically changing treatment options in lung cancer.1 Cytotoxic chemotherapy remains the most suitable treatment option for the Address for correspondence: K Zwaenepoel, Department of Pathology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
majority of patients with advanced non-small-cell lung cancer (NSCLC). However, a subset of patients whose tumours contain EGFR activating mutations or ALK gene rearrangements benefits from the administration of EGFR or ALK tyrosine kinase inhibitors (TKI), respectively.2–5 Recently, the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) published recommendations for detecting lung cancer patients eligible for EGFR- and
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ALK-directed therapies. These guidelines recommend that all patients with advanced, non-squamous NSCLC should be tested for these genetic lesions. Selection of patients with ALK gene rearrangements for TKI therapy should be performed with an ALK fluorescence insitu hybridization (FISH) assay using dual-labelled break-apart probes. If validated carefully, preselection using an ALK immunohistochemical assay can be considered according to CAP, IASLC and AMP.6 The Vysis LSI ALK break-apart FISH test, which was used during the Phase II and III trials with crizotinib in NSCLC is approved by the US Food and Drug Administration and is considered the gold standard for ALK testing. Other methodologies, such as multiplex reverse transcription PCR and immunohistochemistry (IHC), have also been described.7–9 Reverse transcription PCR does not hold much promise as alternative detection method, as at least 25 different ALK fusion proteins have been described in NSCLC.10 Taking into consideration the fact that the incidence of ALK rearrangements in non-squamous NSCLC is between 3% and 6%, IHC could become an promising pre-screen method.11 As the expression of de-novo ALK fusion proteins in NSCLC is low, there are potential problems with the sensitivity of ALK IHC. Consequently, the method requires highly specific and sensitive primary ALK antibodies (e.g. 5A4 and D5F3) combined with an ultra-sensitive detection system containing a potent signal enhancement step.8,9,12–18 As not many laboratories have multiple ALK-positive samples available for testing, few publications describe the sensitivity of their IHC protocol to a satisfactory degree.8,12,17–19 As many laboratories will find it difficult to test this parameter properly prior to routine use, it is crucial that the ALK IHC assay becomes more standardized between different laboratories, and that the complete design of ALK IHC protocols which produce acceptable sensitivity is described in great detail. In this study, a set of 30 ALK FISH-positive and 23 ALK FISH-negative cases of NSCLC was tested with three IHC protocols: (i) the 5A4 clone with EnVision Flex+ on the AutostainerLink 48, (ii) the D5F3 clone with EnVision Flex+ on the AutostainerLink 48 and (iii) the D5F3 clone with OptiView on the BenchMark GX. In addition, ALK FISH testing using two commercially available break-apart probes was compared for signal intensity and signal pattern production.
Materials and methods TISSUE SPECIMENS
This study was approved by the Antwerp University Hospital Bioethical Committee and includes samples
from 73 patients with advanced NSCLC who were referred for ALK gene rearrangement testing at our institute (UZA) between 2011 and 2013. ALK FLUORESCENCE IN-SITU HYBRIDIZATION
FISH was performed on 5-lm formalin-fixed, paraffinembedded (FFPE) tissue sections using the Vysis LSI ALK dual-colour, break-apart rearrangement probe in combination with the Vysis pre- and post-treatment kit IV (Abbott Molecular, Des Plaines, IL, USA), according to the manufacturer’s instructions. Results were analysed using a fluorescence BX41 microscope (Olympus, Center Valley, PA, USA) and evaluated according to the Vysis LSI ALK Probe manufacturer’s enumeration guidelines. To compare the Vysis LSI ALK probe with the Kreatech ON ALK probe (Kreatech, Amsterdam, the Netherlands), samples were pre-treated in duplicate using the Vysis pre- and post-treatment kit IV. After probe mix application, the samples were denatured, hybridized and post-treated according the instructions of the provider of the probe. The slides were analysed independently by two experienced FISH evaluators (K.Z. and A.V.D.). Using the appropriate single filters, three images were obtained for each sample (Olympus DP50 camera and DP-Soft version 3.2 software) using equal camera settings. The intensities of 60 signals, 60 intranuclear non-signal spots and 30 extracellular spots) were determined using ImageJ 1.45s software upon image grey scaling. The ratio of signal intensity versus background was calculated using the following formula: (average signal intensity average extranuclear background)/(average intranuclear background average extranuclear background). Statistical analysis of differences was calculated using the paired two-sided Student’s t-test. ALK IMMUNOHISTOCHEMISTRY
FFPE sections (5 lm thickness) were stained using three different IHC protocols: (i) clone 5A4 (Novocastra, Newcastle upon Tyne, UK) with EnVision Flex+, mouse high pH detection reagents on an AutostainerLink 48 instrument (Dako, Glostrup, Denmark) (referred to as 5A4 EnVF); (ii) clone D5F3 (Cell Signaling Technology, Danvers, MA, USA) with EnVision Flex+, rabbit high pH Flex detection reagents on an AutostainerLink 48 platform (Dako) (referred to as D5F3 EnVF); and (iii) D5F3 with the Ventana Optiview DAB IHC Detection Kit and OptiView Amplification Kit on a Bench© 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
ALK IHC pre-screening in lung cancer
Mark GX (Cell Signaling Technology) (referred to as D5F3 OV). For the 5A4 and D5F3 EnVF assays, sections were subsequently incubated in high pH buffer (20 min, 97°C; PT-Link, Dako), peroxidase blocking buffer (5 min), primary antibody (1:50, 30 min), mouse- or rabbit-enhanced polymer-based linker (30 min), mouse or rabbit secondary antibody (20 min), diaminobenzidene (5 min) and haematoxylin (5 min). The D5F3 OV assay comprised the following incubation steps: cell conditioning 1 (92 min, 100°C), peroxidase blocking buffer, primary antibody (16 min, 37°C), HQ Universal Linker (12 min), HRP multimer (12 min), H2O2, amplifier (8 min), incubation with a multimer (8 min) and haematoxylin II (4 min). ALK expression was assessed independently by one pathologist (P.P.) and one scientist (K.Z.) without knowledge of the FISH results. For the 5A4 EnVF and D5F3 EnVF assay, samples were deemed IHC-positive if tumour-specific staining of any intensity was present (regardless of percentage extent and/or subcellular localization). The Ventana D5F3 OV assay was evaluated according the supplier’s guidelines; in short, only strong granular cytoplasmic staining of tumour cells (any percentage) was considered positive.
Results ALK FISH ANALYSIS IN NON-SMALL-CELL LUNG CANCER USING THE VYSIS LSI ABBOTT PROTOCOL
Using FISH, we identified 30 NSCLC specimens positive for ALK gene rearrangement (Table 1). These samples consisted of surgical resection specimens (46.6%), bronchial biopsies (46.6%) and pleural aspirates or bronchial fragments embedded in agar cell blocks (6.6%). The observed ALK FISH rearrangement pattern was homogeneous in all tumour specimens. Almost two-thirds of the ALK-positive specimens displayed a break-apart pattern, while one-third displayed an isolated single red (ISR) pattern. Only 6.7% (n = 2) of the ALK-positive cases were categorized in the equivocal range after enumeration of 50 nuclei by a first reader (Table 2). COMPARISON OF VYSIS LSI AND KREATECH ON ALK PROBES FOR DETECTION OF ALK TRANSLOCATION IN NON-SMALL-CELL LUNG CANCER
Multiple break-apart ALK FISH probes with a design similar to the Vysis LSI ALK probe are currently © 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
541
available. Theoretically, one would expect that similar evaluation rules are applicable for these probe mixes. To test this hypothesis, 20 samples (10 Vysis ALK-positive and 10 Vysis ALK-negative cases) were selected. Enrichment of old archival FFPE blocks and borderline ALK-positive samples was applied to enlarge potential staining differences. The samples were pretreated in parallel and incubated with either the Vysis LSI or the Kreatech ON ALK break-apart probe. Manual analysis revealed that the diagnostic category, the percentage of positive cells and the observed ALK pattern is similar in all cases for which informative results with both probe mixes were obtained (Table 3). A remarkably higher percentage of uninformative results was observed using the Kreatech ON probe (50% for Kreatech versus 15% for Vysis). Digital analysis of 10 informative samples demonstrated a significantly higher signal versus background ratio (P < 0.05) with the Vysis ALK probe mix for both probe colours (Table 4). COMPARISON OF ALK ANTIBODY CLONES 5A4 AND D5F3 FOR THE DETECTION OF ALK EXPRESSION IN NON-SMALL-CELL LUNG CANCER
Fifty-three FFPE NSCLC samples were stained in duplicate for ALK expression using either the ALK 5A4 or the ALK D5F3 antibody at a dilution of 1:50, combined with the EnVision Flex+ mouse or rabbit detection kit on a Dako AutostainerLink 48 instrument (referred to as 5A4 EnVF and D5F3 EnVF, respectively). Analysis of 30 ALK fusion-positive NSCLC (Table 1) revealed strong cytoplasmic staining with both IHC protocols in 63.3% of these specimens (Table 5). In 93.3% of the cases, ALK IHC staining was visible in more than half the tumour cells. As FISH analysis did not reveal any focal events, this homogeneous ALK fusion expression was expected. With both antibody clones, two ALK FISH-positive cases (cases 12 and 20) displayed weak ALK expression. These sections were from agar cell blocks; during preparation, these samples came into contact with alcohol formalin-acetic acid fixative (AFA), which is a more aggressive fixative then neutral buffered formalin and seems to diminish the antigenicity of samples. As both IHC assays failed to stain case 20, a sensitivity of 96.6% to detect ALK fusions was achieved (Figure 1). Analysis of 23 ALK fusion-negative NSCLC samples revealed ALK staining in one sample using the 5A4 EnVF assay, and in three samples using the D5F3
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Table 1. Comparison of fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) results with three different protocols FISH Vysis LSI
IHC 5A4 EnVF
D5F3 EnVF
D5F3 OV
Case
%
Result
Intensity%
%
Intensity%
%
Intensity%
%
01
76
Pos (ISR)
S
100
S
100
S
100
02
44
Pos (mixed)
S
51–100
S
51–100
S
51–100
03
54
Pos (break)
S
100
S
100
S
100
04
58
Pos (ISR)
S
100
S
100
S
100
05
86
Pos (break)
I
100
I
100
S
100
06
60
Pos (break)
I
100
I
100
S
100
07
90
Pos (break)
S
100
S
100
S
100
08
90
Pos (break)
I
100
S
100
S
100
09
56
Pos (ISR)
S
26–50
S
26–50
S
26–50
10
52
Pos (break)
I
100
I
100
S
100
11
94
Pos (break)
S
100
S
100
S
100
12
54
Pos (break)
W
51–100
W
51–100
I>S
51–100
13
70
Pos (ISR)
S
100
I
100
S
100
14
90
Pos (ISR)
S
100
S
100
S
100
15
96
Pos (break)
S
100
S
100
S
100
16
58
Pos (break)
S
100
S
100
S
100
17
100
Pos (ISR)
S
100
S
100
S
100
18
70
Pos (break)
S
100
S
100
S
100
19
60
Pos (break)
S
100
S
100
S
100
20
54
Pos (break)
–
0
–
0
–
0
21
56
Pos (break)
S
100
S
100
S
100
22
54
Pos (ISR)
S
100
S
100
S
100
23
60
Pos (break)
W
51–100
W
51–100
S
100
24
60
Pos (break)
S
100
S
100
S
100
25
78
Pos (ISR)
I
100
I
100
S
100
26
40
Pos (break)
S
100
S
100
S
100
27
90
Pos (break)
I
100
I
100
S
100
28
58
Pos (ISR)
I
100
I
100
S
100
29
94
Pos (ISR)
I
100
I
100
–
0
30
78
Pos (break)
S
100
S
100
S
100
© 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
ALK IHC pre-screening in lung cancer
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Table 1. (Continued) FISH Vysis LSI
IHC 5A4 EnVF
D5F3 EnVF
D5F3 OV
Case
%
Result
Intensity%
%
Intensity%
%
Intensity%
%
31
0
Neg
–
0
–
0
–
0
32
0
Neg
–
0
I
26–50
–
0
33
8
Neg
–
0
–
0
–
0
34
6
Neg
–
0
–
0
–
0
35
0
Neg
–
0
–
0
–
0
36
6
Neg
–
0
–
0
–
0
37
0
Neg
–
0
–
0
–
0
38
6
Neg
–
0
–
0
–
0
39
0
Neg
–
0
–
0
–
0
40
0
Neg
–
0
–
0
–
0
41
6
Neg
–
0
–
0
–
0
42
10
Neg
–
0
–
0
–
0
43
6
Neg
–
0
–
0
–
0
44
0
Neg
–
0
–
0
–
0
45
0
Neg
–
0
S
51–100
W
50
46
8
Neg
–
0
–
0
–
0
47
4
Neg
–
0
–
0
–
0
48
4
Neg
–
0
–
0
–
0
49
6
Neg
–
0
–
0
–
0
50
12
Neg
–
0
–
0
–
0
51
4
Neg
–
0
–
0
–
0
52
0
Neg
W
51–100
W
51–100
W
51–100
53
0
Neg
–
0
–
0
–
0
ISR = isolated single red pattern; S = strong; I = intermediate; W = weak.
EnVF assay (Table 1). Hence, in a pre-screen set-up, the 5A4 EnVF and D5F3 EnVF assay have a potency to eliminate 95.7% and 87% of the ALK native specimens, respectively. Evaluation of ALK staining in non-tumoral cells revealed weak macrophage staining and some weak general non-specific staining with both assays. In conclusion, a high concordance in staining intensity, subcellular distribution and staining distribution within tumours could be observed with both © 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
ALK antibody clones. Some minor variations in nonspecific staining of ALK FISH-negative tumours were present. DETECTION OF ALK EXPRESSION WITH D5F3 ALK ANTIBODY APPLYING DIFFERENT DETECTION PLATFORMS
Ventana has produced a CE-marked ALK IHC protocol for NSCLC with predefined staining protocol and
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Table 2. Distribution of ALK FISH rearranged cells in ALKpositive and -negative specimens 23 ALK specimens
30 ALK+ specimens
0% ≤ ALK+ cells
Detection of ALK expression in non-small-cell lung cancer with ALK gene rearrangements – comparison of multiple immunohistochemical methods Karen Zwaenepoel,1 Amber Van Dongen,1 Suzan Lambin,1 Christine Weyn1 & Patrick Pauwels1,2 1
Department of Pathology, Antwerp University Hospital, Edegem, Belgium, and 2Center for Oncological Research (CORE), University of Antwerp, Edegem, Belgium
Date of submission 15 October 2013 Accepted for publication 8 March 2014 Published online Article Accepted 12 March 2014
Zwaenepoel K, Van Dongen A, Lambin S, Weyn C, Pauwels P (2014) Histopathology 65, 539–548
Detection of ALK expression in non-small-cell lung cancer with ALK gene rearrangements – comparison of multiple immunohistochemical methods Aim: Testing for ALK rearrangements in advanced, non-squamous non-small-cell lung cancers that are wild-type for activating EGFR mutation has become standard care. Fluorescence in-situ hybridization is considered the gold standard for this evaluation. Pre-screening with immunohistochemistry has been suggested, to reduce testing costs and to make testing more widely available. By analysing the sensitivity and specificity of different ALK immunohistochemical assays, we aimed to identify the most reliable assay to detect ALK rearrangement. Methods and results: ALK screening performed by FISH analysis was compared with three different immunohistochemical assays, in which two ALK antibody clones (5A4 and D5F3) were used on two detec-
tion platforms (Dako AutostainerLink 48 and Ventana Benchmark GX). Data from 30 ALK FISHpositive cases show that the sensitivity of the immunohistochemical assays varies from 93.3% to 96.6%. Head-to-head comparison of the 5A4 and D5F3 ALK antibody clones demonstrates similar staining potency. In general, homogeneous, intermediate to strong staining of the ALK-positive samples was obtained. Conclusions: ALK immunohistochemistry can be considered as a pre-screen method if one accepts a sensitivity of 93.3–96.6%. Because ALK immunohistochemical staining needs to be performed close to the detection limit of the assay, vigilant quality control monitoring is required to guarantee trustworthy results.
Keywords: anaplastic lymphoma kinase, fluorescence in-situ hybridization, immunohistochemistry, non-small cell lung cancer
Introduction Identification of oncogenic drivers and accompanying therapeutic agents is dramatically changing treatment options in lung cancer.1 Cytotoxic chemotherapy remains the most suitable treatment option for the Address for correspondence: K Zwaenepoel, Department of Pathology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
majority of patients with advanced non-small-cell lung cancer (NSCLC). However, a subset of patients whose tumours contain EGFR activating mutations or ALK gene rearrangements benefits from the administration of EGFR or ALK tyrosine kinase inhibitors (TKI), respectively.2–5 Recently, the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) published recommendations for detecting lung cancer patients eligible for EGFR- and
540
K Zwaenepoel et al.
ALK-directed therapies. These guidelines recommend that all patients with advanced, non-squamous NSCLC should be tested for these genetic lesions. Selection of patients with ALK gene rearrangements for TKI therapy should be performed with an ALK fluorescence insitu hybridization (FISH) assay using dual-labelled break-apart probes. If validated carefully, preselection using an ALK immunohistochemical assay can be considered according to CAP, IASLC and AMP.6 The Vysis LSI ALK break-apart FISH test, which was used during the Phase II and III trials with crizotinib in NSCLC is approved by the US Food and Drug Administration and is considered the gold standard for ALK testing. Other methodologies, such as multiplex reverse transcription PCR and immunohistochemistry (IHC), have also been described.7–9 Reverse transcription PCR does not hold much promise as alternative detection method, as at least 25 different ALK fusion proteins have been described in NSCLC.10 Taking into consideration the fact that the incidence of ALK rearrangements in non-squamous NSCLC is between 3% and 6%, IHC could become an promising pre-screen method.11 As the expression of de-novo ALK fusion proteins in NSCLC is low, there are potential problems with the sensitivity of ALK IHC. Consequently, the method requires highly specific and sensitive primary ALK antibodies (e.g. 5A4 and D5F3) combined with an ultra-sensitive detection system containing a potent signal enhancement step.8,9,12–18 As not many laboratories have multiple ALK-positive samples available for testing, few publications describe the sensitivity of their IHC protocol to a satisfactory degree.8,12,17–19 As many laboratories will find it difficult to test this parameter properly prior to routine use, it is crucial that the ALK IHC assay becomes more standardized between different laboratories, and that the complete design of ALK IHC protocols which produce acceptable sensitivity is described in great detail. In this study, a set of 30 ALK FISH-positive and 23 ALK FISH-negative cases of NSCLC was tested with three IHC protocols: (i) the 5A4 clone with EnVision Flex+ on the AutostainerLink 48, (ii) the D5F3 clone with EnVision Flex+ on the AutostainerLink 48 and (iii) the D5F3 clone with OptiView on the BenchMark GX. In addition, ALK FISH testing using two commercially available break-apart probes was compared for signal intensity and signal pattern production.
Materials and methods TISSUE SPECIMENS
This study was approved by the Antwerp University Hospital Bioethical Committee and includes samples
from 73 patients with advanced NSCLC who were referred for ALK gene rearrangement testing at our institute (UZA) between 2011 and 2013. ALK FLUORESCENCE IN-SITU HYBRIDIZATION
FISH was performed on 5-lm formalin-fixed, paraffinembedded (FFPE) tissue sections using the Vysis LSI ALK dual-colour, break-apart rearrangement probe in combination with the Vysis pre- and post-treatment kit IV (Abbott Molecular, Des Plaines, IL, USA), according to the manufacturer’s instructions. Results were analysed using a fluorescence BX41 microscope (Olympus, Center Valley, PA, USA) and evaluated according to the Vysis LSI ALK Probe manufacturer’s enumeration guidelines. To compare the Vysis LSI ALK probe with the Kreatech ON ALK probe (Kreatech, Amsterdam, the Netherlands), samples were pre-treated in duplicate using the Vysis pre- and post-treatment kit IV. After probe mix application, the samples were denatured, hybridized and post-treated according the instructions of the provider of the probe. The slides were analysed independently by two experienced FISH evaluators (K.Z. and A.V.D.). Using the appropriate single filters, three images were obtained for each sample (Olympus DP50 camera and DP-Soft version 3.2 software) using equal camera settings. The intensities of 60 signals, 60 intranuclear non-signal spots and 30 extracellular spots) were determined using ImageJ 1.45s software upon image grey scaling. The ratio of signal intensity versus background was calculated using the following formula: (average signal intensity average extranuclear background)/(average intranuclear background average extranuclear background). Statistical analysis of differences was calculated using the paired two-sided Student’s t-test. ALK IMMUNOHISTOCHEMISTRY
FFPE sections (5 lm thickness) were stained using three different IHC protocols: (i) clone 5A4 (Novocastra, Newcastle upon Tyne, UK) with EnVision Flex+, mouse high pH detection reagents on an AutostainerLink 48 instrument (Dako, Glostrup, Denmark) (referred to as 5A4 EnVF); (ii) clone D5F3 (Cell Signaling Technology, Danvers, MA, USA) with EnVision Flex+, rabbit high pH Flex detection reagents on an AutostainerLink 48 platform (Dako) (referred to as D5F3 EnVF); and (iii) D5F3 with the Ventana Optiview DAB IHC Detection Kit and OptiView Amplification Kit on a Bench© 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
ALK IHC pre-screening in lung cancer
Mark GX (Cell Signaling Technology) (referred to as D5F3 OV). For the 5A4 and D5F3 EnVF assays, sections were subsequently incubated in high pH buffer (20 min, 97°C; PT-Link, Dako), peroxidase blocking buffer (5 min), primary antibody (1:50, 30 min), mouse- or rabbit-enhanced polymer-based linker (30 min), mouse or rabbit secondary antibody (20 min), diaminobenzidene (5 min) and haematoxylin (5 min). The D5F3 OV assay comprised the following incubation steps: cell conditioning 1 (92 min, 100°C), peroxidase blocking buffer, primary antibody (16 min, 37°C), HQ Universal Linker (12 min), HRP multimer (12 min), H2O2, amplifier (8 min), incubation with a multimer (8 min) and haematoxylin II (4 min). ALK expression was assessed independently by one pathologist (P.P.) and one scientist (K.Z.) without knowledge of the FISH results. For the 5A4 EnVF and D5F3 EnVF assay, samples were deemed IHC-positive if tumour-specific staining of any intensity was present (regardless of percentage extent and/or subcellular localization). The Ventana D5F3 OV assay was evaluated according the supplier’s guidelines; in short, only strong granular cytoplasmic staining of tumour cells (any percentage) was considered positive.
Results ALK FISH ANALYSIS IN NON-SMALL-CELL LUNG CANCER USING THE VYSIS LSI ABBOTT PROTOCOL
Using FISH, we identified 30 NSCLC specimens positive for ALK gene rearrangement (Table 1). These samples consisted of surgical resection specimens (46.6%), bronchial biopsies (46.6%) and pleural aspirates or bronchial fragments embedded in agar cell blocks (6.6%). The observed ALK FISH rearrangement pattern was homogeneous in all tumour specimens. Almost two-thirds of the ALK-positive specimens displayed a break-apart pattern, while one-third displayed an isolated single red (ISR) pattern. Only 6.7% (n = 2) of the ALK-positive cases were categorized in the equivocal range after enumeration of 50 nuclei by a first reader (Table 2). COMPARISON OF VYSIS LSI AND KREATECH ON ALK PROBES FOR DETECTION OF ALK TRANSLOCATION IN NON-SMALL-CELL LUNG CANCER
Multiple break-apart ALK FISH probes with a design similar to the Vysis LSI ALK probe are currently © 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
541
available. Theoretically, one would expect that similar evaluation rules are applicable for these probe mixes. To test this hypothesis, 20 samples (10 Vysis ALK-positive and 10 Vysis ALK-negative cases) were selected. Enrichment of old archival FFPE blocks and borderline ALK-positive samples was applied to enlarge potential staining differences. The samples were pretreated in parallel and incubated with either the Vysis LSI or the Kreatech ON ALK break-apart probe. Manual analysis revealed that the diagnostic category, the percentage of positive cells and the observed ALK pattern is similar in all cases for which informative results with both probe mixes were obtained (Table 3). A remarkably higher percentage of uninformative results was observed using the Kreatech ON probe (50% for Kreatech versus 15% for Vysis). Digital analysis of 10 informative samples demonstrated a significantly higher signal versus background ratio (P < 0.05) with the Vysis ALK probe mix for both probe colours (Table 4). COMPARISON OF ALK ANTIBODY CLONES 5A4 AND D5F3 FOR THE DETECTION OF ALK EXPRESSION IN NON-SMALL-CELL LUNG CANCER
Fifty-three FFPE NSCLC samples were stained in duplicate for ALK expression using either the ALK 5A4 or the ALK D5F3 antibody at a dilution of 1:50, combined with the EnVision Flex+ mouse or rabbit detection kit on a Dako AutostainerLink 48 instrument (referred to as 5A4 EnVF and D5F3 EnVF, respectively). Analysis of 30 ALK fusion-positive NSCLC (Table 1) revealed strong cytoplasmic staining with both IHC protocols in 63.3% of these specimens (Table 5). In 93.3% of the cases, ALK IHC staining was visible in more than half the tumour cells. As FISH analysis did not reveal any focal events, this homogeneous ALK fusion expression was expected. With both antibody clones, two ALK FISH-positive cases (cases 12 and 20) displayed weak ALK expression. These sections were from agar cell blocks; during preparation, these samples came into contact with alcohol formalin-acetic acid fixative (AFA), which is a more aggressive fixative then neutral buffered formalin and seems to diminish the antigenicity of samples. As both IHC assays failed to stain case 20, a sensitivity of 96.6% to detect ALK fusions was achieved (Figure 1). Analysis of 23 ALK fusion-negative NSCLC samples revealed ALK staining in one sample using the 5A4 EnVF assay, and in three samples using the D5F3
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Table 1. Comparison of fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) results with three different protocols FISH Vysis LSI
IHC 5A4 EnVF
D5F3 EnVF
D5F3 OV
Case
%
Result
Intensity%
%
Intensity%
%
Intensity%
%
01
76
Pos (ISR)
S
100
S
100
S
100
02
44
Pos (mixed)
S
51–100
S
51–100
S
51–100
03
54
Pos (break)
S
100
S
100
S
100
04
58
Pos (ISR)
S
100
S
100
S
100
05
86
Pos (break)
I
100
I
100
S
100
06
60
Pos (break)
I
100
I
100
S
100
07
90
Pos (break)
S
100
S
100
S
100
08
90
Pos (break)
I
100
S
100
S
100
09
56
Pos (ISR)
S
26–50
S
26–50
S
26–50
10
52
Pos (break)
I
100
I
100
S
100
11
94
Pos (break)
S
100
S
100
S
100
12
54
Pos (break)
W
51–100
W
51–100
I>S
51–100
13
70
Pos (ISR)
S
100
I
100
S
100
14
90
Pos (ISR)
S
100
S
100
S
100
15
96
Pos (break)
S
100
S
100
S
100
16
58
Pos (break)
S
100
S
100
S
100
17
100
Pos (ISR)
S
100
S
100
S
100
18
70
Pos (break)
S
100
S
100
S
100
19
60
Pos (break)
S
100
S
100
S
100
20
54
Pos (break)
–
0
–
0
–
0
21
56
Pos (break)
S
100
S
100
S
100
22
54
Pos (ISR)
S
100
S
100
S
100
23
60
Pos (break)
W
51–100
W
51–100
S
100
24
60
Pos (break)
S
100
S
100
S
100
25
78
Pos (ISR)
I
100
I
100
S
100
26
40
Pos (break)
S
100
S
100
S
100
27
90
Pos (break)
I
100
I
100
S
100
28
58
Pos (ISR)
I
100
I
100
S
100
29
94
Pos (ISR)
I
100
I
100
–
0
30
78
Pos (break)
S
100
S
100
S
100
© 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
ALK IHC pre-screening in lung cancer
543
Table 1. (Continued) FISH Vysis LSI
IHC 5A4 EnVF
D5F3 EnVF
D5F3 OV
Case
%
Result
Intensity%
%
Intensity%
%
Intensity%
%
31
0
Neg
–
0
–
0
–
0
32
0
Neg
–
0
I
26–50
–
0
33
8
Neg
–
0
–
0
–
0
34
6
Neg
–
0
–
0
–
0
35
0
Neg
–
0
–
0
–
0
36
6
Neg
–
0
–
0
–
0
37
0
Neg
–
0
–
0
–
0
38
6
Neg
–
0
–
0
–
0
39
0
Neg
–
0
–
0
–
0
40
0
Neg
–
0
–
0
–
0
41
6
Neg
–
0
–
0
–
0
42
10
Neg
–
0
–
0
–
0
43
6
Neg
–
0
–
0
–
0
44
0
Neg
–
0
–
0
–
0
45
0
Neg
–
0
S
51–100
W
50
46
8
Neg
–
0
–
0
–
0
47
4
Neg
–
0
–
0
–
0
48
4
Neg
–
0
–
0
–
0
49
6
Neg
–
0
–
0
–
0
50
12
Neg
–
0
–
0
–
0
51
4
Neg
–
0
–
0
–
0
52
0
Neg
W
51–100
W
51–100
W
51–100
53
0
Neg
–
0
–
0
–
0
ISR = isolated single red pattern; S = strong; I = intermediate; W = weak.
EnVF assay (Table 1). Hence, in a pre-screen set-up, the 5A4 EnVF and D5F3 EnVF assay have a potency to eliminate 95.7% and 87% of the ALK native specimens, respectively. Evaluation of ALK staining in non-tumoral cells revealed weak macrophage staining and some weak general non-specific staining with both assays. In conclusion, a high concordance in staining intensity, subcellular distribution and staining distribution within tumours could be observed with both © 2014 John Wiley & Sons Ltd, Histopathology, 65, 539–548.
ALK antibody clones. Some minor variations in nonspecific staining of ALK FISH-negative tumours were present. DETECTION OF ALK EXPRESSION WITH D5F3 ALK ANTIBODY APPLYING DIFFERENT DETECTION PLATFORMS
Ventana has produced a CE-marked ALK IHC protocol for NSCLC with predefined staining protocol and
544
K Zwaenepoel et al.
Table 2. Distribution of ALK FISH rearranged cells in ALKpositive and -negative specimens 23 ALK specimens
30 ALK+ specimens
0% ≤ ALK+ cells
