Original Articles. Implications for Optimal Clinical Testing

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1 Original Articles Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4 Anaplastic Lymphoma Kinase Fusion Positive Non Small Cell Lung Carcinoma Implications for Optimal Clinical Testing Michelle L. Wallander, PhD; Katherine B. Geiersbach, MD; Sheryl R. Tripp, BS, MT (ASCP), QIHC (ASCP); Lester J. Layfield, MD N Context. Echinoderm microtubule associated proteinlike 4 anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection. Objective. To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions. Design. Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n = 42; mutant, n = 4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b). Results. EML4-ALK variant 3a/b was detectable by RT- PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was Accepted for publication October 27, From the Institute for Clinical and Experimental Pathology (Drs Wallander and Tripp), ARUP Laboratories, Salt Lake City, Utah; and the Department of Pathology, Huntsman Cancer Hospital, University of Utah School of Medicine, Salt Lake City and ARUP Laboratories, Salt Lake City, UT (Drs Geiersbach and Layfield). The authors have no relevant financial interest in the products or companies described in this article. Reprints: Lester J. Layfield, MD, Department of Pathology, Huntsman Cancer Hospital, University of Utah School of Medicine, 1950 Circle of Hope, Room 6755, Salt Lake City, UT ( layfiel@aruplab. com). obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation. Conclusions. The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions. (Arch Pathol Lab Med. 2012;136: ; doi: / arpa oa) The identification of specific driver mutations in cancer cells is useful for predicting patient response to targeted therapy with tyrosine kinase inhibitors. For example, in non small cell lung cancer (NSCLC), inhibitors of the epidermal growth factor receptor (EGFR) are most effective in a subset of patients harboring EGFR activating mutations. 1 Recently, another molecular subgroup of NSCLC tumors has emerged, which is defined by the presence of echinoderm microtubule associated proteinlike 4 (EML4) and anaplastic lymphoma kinase (ALK) gene fusions. 2 These gene fusions are due to small inversions on chromosome arm 2p. Multiple in-frame fusion variants have been reported to date, all of which contain the same breakpoint in ALK but different breakpoints in EML The resulting EML4-ALK fusion proteins display constitutive kinase activity and are responsive to ALK tyrosine kinase inhibitors in vitro and in vivo. 2,3,9 Thus, ALK has emerged as a therapeutic target for patients with NSCLC who have EML4-ALK gene fusions. Indeed, promising results are emerging from clinical trials investigating ALK inhibitors in patients with positive results for EML4-ALK. 10 The reported incidence of EML4-ALK fusions in NSCLC is low (5% 7%) but can be higher (13% 18%) if the patient population is selected based on specific clinicopathologic characteristics In western populations, patients with 796 Arch Pathol Lab Med Vol 136, July 2012 EML4-ALK Mutational Analysis Wallander et al

2 EML4-ALK fusions are more commonly male, young, and nonsmokers. 11,14 Moreover, EML4-ALK+ carcinoma is characterized by adenocarcinoma with a distinct histologic appearance. 14,15 These carcinomas often have a solid or sheetlike growth pattern and may have signet ring cells. 14 Less frequently, NSCLC with this fusion has an acinar appearance or sometimes has a minor squamous component. 16 Additionally, most EML4-ALK fusion carcinomas are wildtype for EGFR and v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). 13,16,17 Accurate identification of EML4-ALK + lung adenocarcinomas is essential for the selection of appropriate therapy. These tumors do not respond to EGFR antagonists but do respond to specific ALK kinase inhibitors. 6,10,8,19 Given the many EML4-ALK fusion variants identified to date, development of a sensitive and specific molecular diagnostic assay for formalin-fixed, paraffin-embedded tissue is not trivial. EML4-ALK fusion carcinomas can be identified by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), or reverse transcriptionpolymerase chain reaction (RT-PCR). Each analytical method has its advantages, but no agreement has yet been reached on the optimal technique for clinical testing. We studied the concordance among these 3 methods to determine the optimal methodology for EML4-ALK testing in a reference laboratory setting. We used a study population that was enriched for EGFR-mutation negative adenocarcinomas to determine whether these selection criteria substantially enhanced the incidence rate of EML4-ALK fusions in NSCLC. MATERIALS AND METHODS Sources of Tissue and Cell Lines Forty-six cases of formalin fixed, paraffin-embedded lung adenocarcinoma with unknown ALK status were selected from the surgical pathology files at the University of Utah. The study set was enriched for patients lacking EGFR-activating mutations in exons 18 to 21, as determined by EGFR-mutational analysis and Sanger sequencing. 20 Forty-two of 46 samples (91%) were wild type for EGFR. The use of deidentified human tissue for this study was approved by the University of Utah institutional review board (IRB 24487). Formalin-fixed, paraffin-embedded human NSCLC cell lines H2228 and A549 (American Type Culture Collection, Rockville, Maryland) were used as positive and negative controls, respectively, for EML4-ALK variant 3a/b. EML4-ALK variant 1 gene construct (pcdna3_eml4- ALK) was kindly provided by Brunangelo Falini, MD. 21 Formalin-fixed, paraffin-embedded HEK293 cells transfected with pcdna3_eml4-alk or pcdna3 were used as positive and negative controls, respectively, for EML4-ALK variant 1. Immunohistochemistry Immunohistochemical staining for ALK (CD246) was performed with the mouse monoclonal antibody (clone ALK1) from Dako (Carpinteria, Carpinteria). An ALK-rearranged anaplastic large cell lymphoma was used as a positive control. Automated immunohistochemical staining was performed on 4-mm-thick sections of formalin-fixed, paraffin-embedded tissues. Sections were air-dried and then melted in a 60uC oven for 30 minutes. Slides were loaded onto the Ventana ULTRA automated staining instrument (Tucson, Arizona), where deparaffinization with EZ Prep solution and antigen-retrieval with cell conditioning 1 (64 minutes at 100uC) were performed (Ventana). Primary antibody (1:50) was applied at 37uC for 40 minutes. Nonspecific staining was decreased, and signal amplification was performed with the Avidin/Biotin Blocking and Amplification kits (Ventana), respectively. The slides were detected using the iview DAB detection kit (Ventana) and counterstained with hematoxylin for 8 minutes. Slides were dehydrated in graded alcohols (70%, 95%, and 100%), cleared in xylene, and then protected with a coverslip. Interpretation of ALK staininguseda0to3 + scale. Staining was classified as 0 (none), 1 + (faint, in,10% of cells), 2 + (faint to moderate, in $10% of cells) or 3 + (strong). ALK FISH The FISH analysis was performed on formalin-fixed, paraffin-embedded tumor tissue using the LSI ALK Dual Color, Break-apart Rearrangement Probe (Abbott Molecular, Abbott Park, Illinois) according to the manufacturer s instructions. Cell line H2228 (positive for EML4-ALK variant 3a/b) and an ALKrearranged anaplastic large cell lymphoma were used as positive controls. Fused (yellow) or adjacent, touching, orange and green signals were classified as negative for ALK rearrangement. ALK rearrangement was concluded if the orange and green signals were not touching (split). Splits less than 2 signal distances apart were classified as rearranged, given the nature of the 2p23 inversions. The reference range for ALK rearrangement was established in normal lung tissue and indicated a cutoff of $10% split signals, using the criteria defined above. ALK FISH sensitivity and specificity were calculated using cutoffs of $10%, $15%, or $20% of tumor cells with split signals. When used in combination with the scoring criteria described above, the $20% cutoff yielded the best sensitivity and specificity. Unless otherwise indicated, the $20% cutoff was used in this study. Three viewers independently scored 100 tumor nuclei from each tissue to determine interobserver agreement. EML4-ALK RT-PCR Total RNA was extracted from formalin-fixed, paraffinembedded tissues and cell lines with the Agencourt FormaPure Kit (Beckman Coulter Genomics, Danvers, Massachusetts). Reverse transcription was performed using 500 ng total RNA, random hexamers, and Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, California). The PCR was performed using Platinum Taq DNA polymerase (Invitrogen) and uracil N- glycosylase (Applied Biosystems, Foster, City, California [now Life Technologies]). EML4-ALK variant 1 PCR primers were EML4 Ex13F 59-TAGAGCCCACACCTGGGAAA-39 and ALK Ex20R 59-CGGAGCTTGCTCAGCTTGTA-39 (109 base pairs [bp]). EML4-ALK variant 3a/b primers are: EML4 Ex6F 59- GCATAAAGATGTCATCATCAACCAAG-39 and ALK Ex20R 59- CGGAGCTTGCTCAGCTTGTA-39 (105bp; 3b:138 bp). Mitochondrial ribosomal protein L19 (MRPL19) primers to control for complementary DNA (cdna) integrity were MRPL19 Ex4F 59-GGAAGAGGACTTGGAGCTACT-39 and MRPL19 Ex5R 59-TCCTGGACCCGAGGATTAT-39 (94 bp). Following uracil N-glycosylase incubation (50uC for 2 minutes) and inactivation (95uC for 10 minutes), PCR was performed with the following parameters for 40 cycles: 94uC for 30 seconds, 58uC for 30 seconds, and 72uC for 30 seconds. PCR products were analyzed on the QIAxcel System (Qiagen, Valencia, California). RESULTS Study Design The study cohort of 46 formalin-fixed, paraffin embedded lung carcinoma samples was enriched for adenocarcinoma histology (46 of 46, 100%) and wild-type EGFR (42 of 46, 91%). All samples had unknown ALK status before screening for EML4-ALK fusions by RT-PCR, FISH, and IHC. Three viewers independently scored the FISH and IHC to determine interobserver variability. EML4-ALK Variant 1 and 3a/b Detection by RT-PCR The RT-PCR assay was designed to detect the 2 most commonly reported EML4-ALK variants in NSCLC. Arch Pathol Lab Med Vol 136, July 2012 EML4-ALK Mutational Analysis Wallander et al 797

3 detected by RT-PCR in 2 samples. Figure 3, A, shows a sample that was positive for both variant 3a (105 bp) and 3b (138 bp), whereas Figure 3, B, shows the corresponding MRPL19 cdna control. Six of 11 samples (55%) positive by RT-PCR had matched normal tissue available for analysis. None of the matched normal tissues (lung or lymph node) were positive for EML4-ALK by RT-PCR (data not shown). Figure 1. Schematic of the reverse transcription-polymerase chain reaction assay design for detection of echinoderm microtubuleassociated proteinlike 4 anaplastic lymphoma kinase (EML4-ALK) variants 1 and 3a/b. Black arrows indicate forward primers in EML4 and gray arrows indicate the reverse primer in ALK. Abbreviation: bp, base pair. Variant 1 and 3, which account for approximately 80% of reported cases, 6,7,10,13,21 23 fuse EML4 exons 13 or 6, respectively, to ALK exon 20 (Figure 1). Variants 3a and 3b, which differ by 33 bp because of alternative splicing in intron 6, were both detected by the assay. The RT-PCR assay detected EML4-ALK fusions in 24% (11 of 46) of the formalin-fixed, paraffin-embedded lung adenocarcinoma samples (Table 1). Nine of the EML4-ALK + samples were variant 1. Figure 2, A, shows the presence of the EML4- ALK variant 1 amplicon (109-bp) for 2 representative, positive samples. MRPL19 was also assayed to ensure cdna integrity (Figure 2, B). EML4-ALK variant 3a/b was EML4-ALK Detection by FISH The FISH assay was performed using break-apart probes targeting ALK at the 2p23 locus. Rearrangement of the ALK locus is usually defined by split green and orange signals located at least 2 signal-distances apart. Because of the nature of the small inversions that generate the EML4-ALK gene fusions, any split in the green and orange signals was considered positive for rearrangement in this study. Three viewers scored 100 tumor cells using a cutoff point of $20% break-apart probes. Unanimous positive assessment by FISH was achieved in 15% (7 of 46) of the cases (Table 1). Two of the FISH-positive cases were characterized by loss of one green signal. One such case is depicted in Figure 4, A. These 2 cases also tested positive by RT-PCR for EML4-ALK variant 3a/b. Agreement among FISH viewers was, therefore, excellent for variant 3a/b detection. Conversely, agreement among viewers for the detection of variant 1 was poor. Only 1 of 9 samples (11%) positive for variant 1 by RT-PCR was designated positive by all 3 FISH viewers (Figure 4, B). A discrepant FISH case that was positive for variant 1 is shown in Figure 4, C. Agreement between 2 of 3 observers (67%) was achieved in 2 additional variant 1 positive cases Table 1. Non Small Cell Lung Cancer Specimens With a Positive Result by EML4-ALK Reverse Transcription-Polymerase Chain Reaction (RT-PCR), ALK Fluorescence In Situ Hybridization (FISH), or ALK Immunohistochemistry (IHC) EML4-ALK RT-PCR, Variant a ALK FISH b Viewer 1 (%) ALK FISH b Viewer 2 (%) ALK FISH b Viewer 3 (%) Avg ALK b FISH (%) EGFR Mutation Sample ALK IHC RT-PCR a/b 2 + Positive (50) Positive (86) Positive (74) Positive (80) WT 28 3a/b 2 + Positive (73) Positive (70) Positive (67) Positive (70) WT Negative (10) Negative (7) Positive (25) Negative (14) WT Positive (44) Negative (16) Positive (20) Positive (27) L858R Positive (32) Negative (4) Negative (19) Negative (18) WT Negative (6) Negative (6) Positive (23) Negative (12) WT Positive (50) Negative (12) Negative (7) Positive (23) WT Positive (35) Negative (9) Negative (11) Negative (18) WT Positive (39) Negative (11) Negative (19) Positive (23) WT Positive (52) Positive (84) Positive (87) Positive (74) WT Positive (22) Negative (17) Positive (30) Positive (23) WT FISH +, RT-PCR 2 10 Negative 0 Positive (36) Positive (33) Positive (33) Positive (34) WT 24 Negative 0 Positive (49) Positive (43) Positive (47) Positive (46) WT 27 Negative 0 Positive (23) Positive (25) Positive (39) Positive (29) WT 55 Negative 0 Positive (25) Positive (42) Positive (44) Positive (37) WT 48 Negative 1 + Positive (27) Negative (12) Positive (21) Positive (20) WT IHC +, RT-PCR 2 20 Negative 1 + Negative (7) Negative (8) Negative (6) Negative (7) WT 25 Negative 1 + focal Negative (13) Negative (17) Negative (14) Negative (15) WT 33 Negative 1 + Negative (10) Negative (13) Positive (28) Negative (17) WT 34 Negative 2 + Negative (6) Negative (11) Positive (27) Negative (15) WT 44 Negative 1 + Negative (9) Negative (11) Negative (13) Negative (11) WT Abbreviations: ALK, anaplastic lymphoma kinase; Avg, average; EGFR, epidermal growth factor receptor; EML4, echinoderm microtubuleassociated proteinlike 4 gene; WT, wild type. a For variants 1 and 3a/b. b FISH cutoff point for positive was $20% rearranged. 798 Arch Pathol Lab Med Vol 136, July 2012 EML4-ALK Mutational Analysis Wallander et al

4 Figure 2. Detection of echinoderm microtubule-associated proteinlike 4 anaplastic lymphoma kinase (EML4-ALK) variant 1 by reverse transcription-polymerase chain reaction (RT-PCR). A, Analysis of 2 (nos. 51 and 30) adenocarcinomas positive for EML4-ALK variant 1. EML4-ALK variant 1 is 109 bp. B, Mitochondrial ribosomal protein L19 (MRPL19) complementary DNA control is 94 bp. HEK293 cells transfected with pcdna3_eml4-alk or pcdna3 were used as positive (Pos) and negative (Neg) controls, respectively, for variant 1. Abbreviation: bp, base pair; NTC, no template control. Figure 3. Detection of echinoderm microtubule-associated proteinlike 4 anaplastic lymphoma kinase (EML4-ALK) variant 3a/b by reverse transcription-polymerase chain reaction (RT-PCR). A, Analysis of one adenocarcinoma (no. 28) positive for EML4-ALK variant 3a/b (V3a; V3b). EML4-ALK variant 3a is 105 bp and variant 3b is 138 bp. B, Mitochondrial ribosomal protein L19 (MRPL19) complementary DNA control is 94 bp. H2228 and A549 cells were used as positive (Pos) and negative (Neg) controls, respectively, for variant 3a/b. Abbreviations: bp, base pair; NTC, no template control. (22%). Taking the average of all 3 FISH viewers scores resulted in 5 of 9 positives (56%) for variant 1. Four additional negative cases by RT-PCR were unanimously scored as positive by viewers using FISH. These likely represent other EML4-ALK variants because the MRPL19 RT-PCR control indicated the presence of sufficient RNA in those samples. The cutoff point chosen to determine results that were positive by FISH for EML4-ALK in lung adenocarcinoma significantly altered test specificity and sensitivity. The reference range that was established with normal lung tissue indicated a cutoff point of $10% rearrangements (break-apart probes) for positive results. Table 2 demonstrates the sensitivity and specificity associated with cutoff points of $10%, $15%, and $20% rearrangements (breakapart probes) for positivity. Although the $10% cutoff point gave 100% sensitivity, the specificity was poor (23%). The $15% cutoff point yielded 82% sensitivity and 43% specificity. The $20% cutoff point gave the best specificity (66%) with a sensitivity of 64%. One of the 3 observers (33%) had considerably greater experience with FISH interpretation than did the other 2. That individual had more than 5 years experience reading FISH probes, including break-apart probes, and read approximately 1500 cases/y. When only data from the most experienced observer was used to determine the presence or absence of the EML4-ALK rearrangement, the sensitivity and specificity using the $20% cutoff point were improved to 82% and 71%, respectively (Table 3). EML4-ALK Detection by IHC ALK IHC was performed with the ALK1 monoclonal antibody and was independently scored by 3 viewers. ALK staining was detected in 20% (9 of 46) of the cases (Table 1). Agreement among viewers was achieved in all cases (100%). Only 2 of 9 IHC-positive cases (22%) were also RT-PCR + /FISH + (unanimous). These 2 cases expressed EML4-ALK variant 3a/b and had moderate ALK staining (2 + ) as shown by a representative image in Figure 5, A. Only 1 of 9 cases (11%) positive for EML4- ALK variant 1 by RT-PCR was scored weakly positive (1 + ) by IHC (Table 1). The IHC results were negative for avariant1rt-pcr + /FISH + case in Figure 5, B, and a variant 1 RT-PCR + /FISH 2 case in Figure 5, C. Five additional cases negative by RT-PCR were scored as positive by IHC. However, none of those 5 cases (0%) were unanimously scored as positive by FISH. Although the specificity of IHC for the combined detection of EML4-ALK variants 1 and 3a/b was 83%, the sensitivity was poor (27%). Arch Pathol Lab Med Vol 136, July 2012 EML4-ALK Mutational Analysis Wallander et al 799

5 Figure 4. Fluorescence in situ hybridization (FISH) analysis of EML4-ALK fusions. A, The FISH analysis of a positive case (no. 28) of variant 3a/b (FISH +, 3 of 3 viewers [100%]). B, The FISH analysis of a positive case (no. 51) of variant 1 (FISH +, 3 of 3 viewers [100%]). C, The FISH analysis of a positive case (no. 30) of variant 1 (FISH +, 1 of 3 viewers [33%]) (original magnifications 3600 [A through C]). Figure 5. ALK immunohistochemistry (IHC). A, The IHC of a positive case (no. 28) of variant 3a/b (2+, positive). B, The IHC of a positive case (no. 51) of variant 1 (0, negative). C, The IHC of a positive case (no. 30) of variant 1 (0, negative) (original magnifications 3200 [A through C]). Clinical Characteristics of Patients Who Are Positive for EML4-ALK EML4-ALK rearrangements were found by RT-PCR or FISH analysis (average score) in 16 of 46 NSCLC adenocarcinomas (35%) tested. Table 4 documents the relationship of ALK rearrangements with patient gender and age. In our study population, most ALK-rearranged carcinomas were from men (P 5.046). No statistical difference in age of occurrence was found between patients with ALK-rearranged carcinomas and wild-type 800 Arch Pathol Lab Med Vol 136, July 2012 carcinomas (P 5.88). Even though the study cohort was enriched for wild-type EGFR (91%), one case positive for EML4-ALK variant 1 coexisted with an L858R EGFR mutation. Methodology Concordance Concordance among all 3 methodologies (RT-PCR, FISH, and IHC) was 100% for the detection of EML4ALK variant 3a/b. However, no concordance was achieved among all 3 methodologies for the detection of EML4-ALK Mutational Analysis Wallander et al

6 Table 2. Sensitivity and Specificity of the ALK Fluorescence In Situ Hybridization Assay for the Detection of EML4-ALK Variants 1 and 3a/b at Different Thresholds for Positivity $10% $15% $20% Sensitivity, % Specificity, % Abbreviations: ALK, anaplastic lymphoma kinase; EML4, echinoderm microtubule-associated proteinlike 4. EML4-ALK variant 1. Only 1 of 9 cases (11%) positive for variant 1 by RT-PCR had faint staining (1 + ) by IHC, and that case had negative results by FISH (average score). Pairwise comparisons of the methodologies indicated 80% agreement between RT-PCR and FISH (Table 5), 70% agreement between RT-PCR and IHC (Table 6), and 67% agreement between FISH and IHC (Table 7). The RT-PCR process was the most sensitive test for the detection of EML4-ALK variants 1 and 3a/b, followed by FISH analysis, especially when performed by a highly experienced observer. The IHC methodology using the Dako antibody was the least reliable, with both a high percentage of false-negatives (17%) and false-positives (13%) when using RT-PCR as the definitive result. DISCUSSION Although carcinomas positive for EML4-ALK fusions make up only 5% to 7% of all NSCLC cases, they represent a therapeutically important subcategory. Non small cell carcinomas with EML4-ALK fusions have demonstrated an impressive responsiveness to the ALK tyrosine kinase inhibitor crizotinib in an early phase clinical trial. 10 Accurate identification of this molecular subgroup of NSCLC tumors will require a sensitive and specific testing modality. Currently, several testing methodologies exist; namely IHC, FISH, and RT-PCR. Although FISH has been used to enroll patients in clinical trials, the optimal technique for large-scale screening in a clinical setting has not yet been determined. ALK immunohistochemistry has several desirable characteristics, including low cost, relative ease, timeliness of performance, and theoretically, the ability to identify all EML4-ALK variants. However, commercially available ALK antibodies have been reported to lack the sensitivity to detect the EML4-ALK fusion protein. 21 In a study by Martelli et al, 21 no specific ALK staining was detected in NSCLC cases positive for EML4-ALK variant 1 or 3a/b when the Dako ALK1 antibody was used. We demonstrated moderate staining (2 + ) with the identical ALK1 antibody in 2 of 2 cases (100%) positive for EML4-ALK Table 3. Sensitivity and Specificity of the ALK Fluorescence In Situ Hybridization Assay for the Detection of EML4-ALK Variants 1 and 3a/b at Different Thresholds for Positivity (by the Most Experienced Observer) $10% $15% $20% Sensitivity, % Specificity, % Abbreviations: ALK, anaplastic lymphoma kinase; EML4, echinoderm microtubule-associated proteinlike 4. Table 4. Clinical Characteristics of ALK- Non Small Cell Lung Cancer Positive for Fluorescence In Situ Hybridization and Reverse Transcription-Polymerase Chain Reaction Characteristic ALK Wild-Type ALK Cases, No. (%), n (35) 30 (65) Sex, No. (%) Male 11 (69) 10 (33) Female 5 (31) 19 (63) Unknown 0 1 (3) Age, y Median Range Abbreviation: ALK, anaplastic lymphoma kinase. a Fisher exact test. b Mann-Whitney U test. P Value.046 a.88 b variant 3a/b. However, like Martelli et al, 21 detection of EML4-ALK variant 1 by immunohistochemistry in our study was poor. We identified only 1 of 9 RT-PCR + variant 1 cases (11%) by IHC, and the staining intensity for that case was weak (1 + ). Our results suggest that the accessibility of the ALK epitope differs between EML4- ALK variants 1 and 3a/b. Variant 1 involves a larger portion of EML4 (exons 1 13) than does variant 3a/b (exons 1 6), which may preclude ALK antibody binding because of alterations in the structure of the fusion protein. Alternatively, variant 1 expression may be truly weaker than that of variant 3a/b. However, that explanation is unlikely given that patients harboring EML4-ALK variant 1 have responded favorably to crizotinib. 10 In our study, sensitivity of IHC for the detection of variants 1 and 3a/b was only 27% when compared with RT-PCR. Six additional RT-PCR 2 cases were scored as either 1 + or 2 + by IHC, which may represent other EML4- ALK variants. However, none of those 6 cases (0%) were unanimously scored positive by FISH, a technique that should identify other variants. Therefore, these results suggest that the specificity of IHC is also poor. Another ALK antibody is currently in development and appears to have higher sensitivity for EML4-ALK detection but is not widely available. 10,24,25 Future studies with this antibody or other antibodies may demonstrate an improvement in the immunohistochemical recognition of EML4-ALK fusion proteins. Screening for EML4-ALK fusions by FISH requires specialized equipment and highly trained, skilled readers for interpretation. In theory, ALK break-apart FISH probes are capable of picking up most, if not all, EML4-ALK gene fusions. However, the EML4 and ALK genes are separated Table 5. Concordance Between Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Fluorescence In Situ Hybridization (FISH) a FISH RT-PCR WT 7 4 WT 5 b 30 Abbreviation: WT, wild type. a Pairwise comparison for percentage of agreement was 80%. b Likely other EML4-ALK variants. Arch Pathol Lab Med Vol 136, July 2012 EML4-ALK Mutational Analysis Wallander et al 801

7 Table 6. Concordance Between Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Immunohistochemistry (IHC) a IHC RT-PCR Positive ($1 + ) Negative 3 8 WT 6 29 Abbreviation: WT, wild type. a Pairwise comparison for percentage of agreement was 70%. Table 7. Concordance Between Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC) a IHC FISH Positive ($1 + ) Negative 3 9 WT 6 28 Abbreviation: WT, wild type. a Pairwise comparison for percentage of agreement was 67%. by a distance of only 12.5 Mb on the short arm of chromosome 2. Potential mechanisms for fusion gene generation include simple 2p inversion or complex interchromosomal translocations involving both chromosome 2 homologs. Theoretically, translocations should be uncommon because they would involve at least 3 chromosome breaks to align EML4 and ALK sequences in the same reading frame, whereas simple inversion would require only 2 chromosome breaks on 2p. Given that an inversion results in the separation of 59 and 39 ALK probes by only 12.5 Mb, the EML4-ALK fusions resulting from simple inversion are likely to be more challenging to interpret than are translocations involving a fusion partner on a different chromosome, which yield a wide split of 59 and 39 ALK probes. Additionally, because of reports of tumor heterogeneity, 12,26,27 many nuclei should be scored from multiple, distinct tumor areas. In our study, 3 independent viewers blindly scored the ALK break-apart FISH to determine viewer variability. Interobserver agreement and sensitivity were 100% for detection of variant 3a/b by FISH because of the associated loss of the green probe signal that comprises the 59 portion of the dual-color ALK probe. Although we describe only 2 variant 3a/b positive cases, our results suggest that the chromosomal rearrangement that generates variant 3a/b involves a partial deletion of the 59 portion of the ALK gene. Unlike variant 3a/b, interobserver agreement using the ALK break-apart probe was poor for recognition of EML4- ALK variant 1. Only 1 of 9 samples (11%) positive for variant 1 by RT-PCR was designated as positive by all 3 FISH reviewers. In that case, most tumor cells had a wide split between the orange and green probe signals (Figure 4, B). When agreement among only 2 of 3 observers (67%) was required for designating a case as positive by FISH, 2 additional variant 1 cases were identified for a sensitivity of 33%. Although the perceived distances between split probe signals did vary among some of the variant 1 positive cases, we did not detect any gross loss of the green probe (59 portion of ALK). This is in agreement with a study by Lin et al 27 that used bacterial artificial chromosome probes to demonstrate no loss of the 59 portion of ALK or the 39 portion of EML4 in variant 1 positive NSCLC. Our data, in conjunction with the data from Lin et al, 27 suggest that the mechanism for EML4- ALK variant 1 generation involves a simple 2p inversion. As with IHC, FISH appeared to identify some EML4-ALK variants that were not identifiable by our RT-PCR primer sets. Four RT-PCR 2 cases were unanimously scored as positive by FISH and likely represent other EML4-ALK variants because the MRPL19 RT-PCR control indicated the presence of sufficient RNA in these samples. The cutoff point to assess a sample as positive by FISH for EML4-ALK fusions significantly affected test specificity and sensitivity. Reference ranges were determined using normal lung tissue, which established a $10% cutoff point for positivity. However, in tumor tissue, a $10% cutoff point gave a poor specificity at 23%. This is likely due, in part, to our scoring criteria, which defined splits of less than 2 probe signal distances apart as rearranged. Although these scoring criteria identified EML4-ALK variant 1 more easily, it was necessary to increase the cutoff point for positivity to $20% to achieve a specificity of 66%. Unfortunately, that was associated with a sensitivity of only 64%. When the most experienced FISH observer determined the presence or absence of the EML4- ALK fusion using the scoring criteria defined above, the sensitivity and specificity improved significantly to 82% and 71%, respectively (Table 3). With a high level of experience and frequent examination of FISH samples with break-apart probes, it appears that the sensitivity and specificity of the FISH assay can reach approximately 80% and 70%, respectively. In the future, the development of new FISH probes specifically designed for the EML4-ALK fusions that are characteristic of NSCLCs may improve overall assay sensitivity and specificity. The third methodology we evaluated, RT-PCR, should be highly sensitive and specific but requires multiple primer sets to detect all EML4-ALK variants when using formalin-fixed, paraffin-embedded tissue. Because variants 1 and 3a/b account for approximately 80% of reported positive cases, 6,7,10,17,21 23 testing for only those 2 variants does appear to be clinically practical. In our study, RT-PCR was the most sensitive technique, especially for detection of EML4-ALK variant 1. Nine of 46 cases (20%) tested positive for variant 1 by RT-PCR; of those 9, only 1 (11%) was unanimously positive by FISH. The remaining 8 variant 1 positive cases (89%) were called FISH + by at least one viewer, which not only validates the RT-PCR result but indicates that FISH can achieve the same sensitivity as RT-PCR with multiple viewers. However, RT-PCR achieves the same result with much less subjectivity in the interpretation. In total, RT- PCR detected EML4-ALK variant 1 or 3a/b in 11 of 46 cases (24%) of lung adenocarcinomas that were enriched for wild-type EGFR status. Our frequency of detection compares favorably with other studies that enriched their study population based on associated clinical and histologic characteristics Most similar is a study by Zhang et al 13 that detected EML4-ALK fusions in 43% (9 of 21) of adenocarcinomas that lacked EGFR and KRAS mutations. Inclusion of our 5 FISH + /RT-PCR 2 cases, which likely represent other EML4-ALK variants, increases our total detection rate to 35% (16 of 46). In our study, concordance among all 3 methodologies was 100% for the detection of EML4-ALK variant 3a/b. No 802 Arch Pathol Lab Med Vol 136, July 2012 EML4-ALK Mutational Analysis Wallander et al

8 concordance was achieved among the 3 methodologies for the detection of EML4-ALK variant 1. Intertechnique concordance between any 2 of the 3 methodologies was variable, ranging from 80% (RT-PCR versus FISH) to 67% (FISH versus IHC). The RT-PCR methodology was the most sensitive, followed by FISH analysis. Some of the reduced sensitivity of RT-PCR is undoubtedly due to our use of primers directed against only 2 of the variants. Those 2 variants account for 80% of EML4-ALK fusions, but the remaining 20% could not be detected by our RT- PCR technique. That may explain some of the observed disagreement between FISH and RT-PCR. The varying sensitivities of these 3 techniques have some important implications. The FISH process is currently used to enroll patients in clinical trials, but that method may miss some, if not many, variant 1 cases. The FISH technique demonstrates considerable interobserver variability and experienced FISH reviewers are necessary for the technique to obtain adequate sensitivity. However, even when experienced reviewers are used, results may vary because of subjectivity about what constitutes a recognizably true break apart. Although clinical trials employ experienced FISH reviewers, we are unaware of published reports of interobserver reproducibility of FISH interpretation in those clinical trials. Our study indicates that currently, RT-PCR is the most sensitive method for detecting both EML4-ALK variant 1 and 3a/b. An additional advantage of RT-PCR is that it is free from subjectivity in analysis, unlike IHC and FISH. Identification of the specific variant can only be determined using RT-PCR, which may become important in the future for predicting patient response to therapy. 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