Comparison of IHC, FISH and RT-PCR for the Detection of EML4-ALK Translocation Variants in Non-Small Cell Lung Cancer Michelle L. Wallander 1, Katherine B. Geiersbach 2, Sheryl Tripp 1 and Lester J. Layfield 2 1ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT 2Department of Pathology, University of Utah Health Sciences Center and ARUP Laboratories, Salt Lake City, UT Introduction Results Results #28: FISH Positive Lung cancer is the most common and deadly form of cancer in the #28: IHC Positive (2+) (Avg. 70% Rearranged) ALK protein expression was detectable by IHC in 9 of 52 (17%) United States with a 5-year survival rate of only 15.7%. Recently, it samples. has become apparent that non-small cell lung cancer (NSCLC) can ALK break apart FISH was positive in 4 of 52 (8%) samples. be further divided into clinically relevant subsets based on specific molecular alterations within the tumor. In 2007, Soda et al identified EML4-ALK variant 3a/b RT-PCR was positive in 2 of 52 (4%) of a small chromosome 2p inversion (~12 Mb) in a subset of Japanese samples. NSCLC patients, which resulted in the fusion of the echinoderm EML4-ALK variant 1 RT-PCR was positive in 9 of 52 (17%) of microtubule-associated protein-like 4 (EML4) gene and the samples. anaplastic lymphoma kinase (ALK) gene. Multiple different EML4- EML4-ALK Variant 3a/b RT-PCR MRPL19 RT-PCR ALK translocation variants have since been identified that contain The total incidence of EML4-ALK translocations in our NSCLC various truncations of EML4 but always contain the tyrosine kinase V3a/b V3a/b V3a/b V3a/b series, which was enriched for adenocarcinomas with wild-type Pos Neg #28 #28 NTC Pos Neg #28 #28 NTC domain of ALK, starting at exon 20. The most common (~70%) EGFR status, was 21.1% (11 of 52). translocations in NSCLC involve EML4 exon 13 (variant 1) or EML4 -500 bp- Concordance between IHC, FISH and RT-PCR methodologies exon 6 (variant 3a/b; 3b contains an additional 33-bp due to was 100% for EML4-ALK variant 3a/b (2 of 2 samples). alternative splicing). V3b (138 bp)- -MRPL19 (94 bp) No concordance was found between IHC, FISH and RT-PCR The incidence of EML4-ALK translocations in NSCLC is low (2-7%) V3a (105 bp)- - 50 bp - methodologies for EML4-ALK variant 1 (0 of 9 samples). but can be higher (13%) if the population is enriched for adenocarcinoma, never or light smokers, younger age and lack of FISH and V1 RT-PCR concordance: 11% (sample # 51) EGFR or KRAS mutations. Early-phase clinical trials of ALK Sample EGFR ALK IHC ALK FISH ALK FISH ALK FISH Avg FISH EML4-ALK IHC and V1 RT-PCR concordance: 11% (sample # 39) inhibitors in patients with known EML4-ALK translocations have Reader 1 Reader 2 Reader 3 V3a/b RT-PCR yielded promising results. Therefore, a need exists for molecular # 16 wildtype positive 86% 50% 74% 70% (positive) positive One EML4-ALK variant 1 positive case (sample # 18) had a testing to identify the correct target patient population. We # 28 wildtype positive 70% 73% 67% 70% (positive) positive concurrent EGFR mutation (L858R). compared ALK immunohistochemistry (IHC), ALK break apart FISH Detection of EML4-ALK variant 3a/b by IHC, break apart FISH and RT-PCR. All 3 methodologies are shown for one representative EML4-ALK was not detected by RT-PCR in normal tissue (lymph and RT-PCR in a subset of lung adenocarcinomas for the detection positive sample (#28). Two of two (100%) variant 3a/b positive samples displayed loss of the green probe by FISH. node or lung) from 4 RT-PCR positive samples that had sufficient of EML4-ALK variants 1 and 3a/b. tissue for analysis (data not shown). Materials and Methods Results Conclusions EML4-ALK Variant 1 RT-PCR 52 FFPE lung adenocarcinomas were selected from the University #51: FISH Positive Concordance among the three methodologies (IHC, FISH and RT- #51: IHC Negative (0) (Avg. 74% Rearranged) V1 V1 of Utah surgical pathology files PCR) differed for the two tested EML4-ALK translocation variants. Pos Neg #51 #51 #30 #30 NTC Variant 3a/b was detectable by all three methods. Conversely, The study population was enriched for wildtype EGFR status as -500 bp limited ALK protein expression and subjectivity in FISH scoring determined by direct sequencing of exons 18-21 (WT: n = 46; resulted in no concordance between all three methods for the Mutant: n = 6). detection of EML4-ALK variant 1. Agreement among FISH readers ALK IHC: monoclonal mouse CD246, clone ALK1 (Dako) V1 (109 bp)- - 50 bp was poor for variant 1. This was likely due to the expected small split in probe signals. These results suggest that the detection of ALK FISH: Vysis LSI ALK Dual Color, Break Apart Rearrangement other EML4-ALK variants that involve the 3’ region of EML4 (exons Probe (Abbott Molecular) #30: FISH Negative MRPL19 RT-PCR 15-20) may also be challenging by FISH. #30: IHC Negative (0) (Avg. 18% Rearranged) Positive cutoff: ≥ 40% cells rearranged V1 V1 RT-PCR was the most sensitive and least subjective methodology Pos Neg #51 #51 #30 #30 NTC for EML4-ALK variant 1 detection. While multiple specific primer RT-PCR: -500 bp sets would need to be designed for the detection of all known Variant 1 primers (109 bp) EML4-ALK translocation variants from FFPE tissue by RT-PCR, this EML4 Ex13 Fwd: 5’-TAGAGCCCACACCTGGGAAA-3’ approach would likely yield the greatest assay sensitivity. ALK Ex20 Rev: 5’-CGGAGCTTGCTCAGCTTGTA-3’ MRPL19 (94 bp)- -50 bp Additionally, our results confirm that the incidence of EML4-ALK translocations in NSCLC is greater (21%) in enriched patient Variant 1 positive control: FFPE HEK293 cells transiently populations. expressing pcDNA3-EML4-ALK variant 1 ALK FISH ALK FISH ALK FISH EML4-ALK V1 Variant 3a/b primers (105 bp & 138 bp) Sample EGFR ALK IHC Reader 1 Reader 2 Reader 3 Avg. FISH RT-PCR References # 14 wildtype negative 7% 10% 25% 14% (negative) positive EML4 Ex6 Fwd: 5’-GCATAAAGATGTCATCATCAACCAAG-3’ # 18 L858R negative 16% 44% 20% 26% (negative) positive Soda M, Choi YL, Enomoto M et al. Identification of the transforming EML4-ALK fusion gene in ALK Ex20 Rev: 5’-CGGAGCTTGCTCAGCTTGTA-3’ # 30 wildtype negative 4% 32% 19% 18% (negative) positive non-small cell lung cancer. Nature, 2007; 448:561-566. weak patchy Shaw AT, Yeap BY, Mino-Kenudson M et al. Clinical features and outcome of patients with non- # 39 wildtype 6% 6% 23% 11% (negative) positive positive small-cell lung cancer who harbor EML4-ALK. J Clin Oncol, 2009; 27:4247-4253. Variant 3a/b positive control: H2228 cells # 42 wildtype negative 12% 50% 7% 23% (negative) positive Kwak EL, Bang YJ, Camidge DR et al. Anaplastic lymphoma kinase inhibition in non-small-cell MRPL19 primers (94 bp) # 47 wildtype negative 9% 35% 11% 18% (negative) positive lung cancer. N Engl J Med, 2010; 363:1693-1703. # 50 wildtype negative 11% 39% 19% 23% (negative) positive MRPL19 Ex4 Fwd:5’-GGAAGAGGACTTGGAGCTACT-3’ # 51 wildtype negative 84% 52% 87% 74% (positive) positive MRPL19 Ex5 Rev: 5’-TCCTGGACCCGAGGATTAT-3’ # 57 wildtype negative 17% 22% 30% 23% (negative) positive Acknowledgements . The use of human tissue for this analysis was approved by the Detection of EML4-ALK variant 1 by IHC, break apart FISH and RT-PCR. All 3 methodologies are shown for two variant 1 positive We graciously thank Maria Martelli and Brunangelo Falini for providing controls. University of Utah Institutional Review Board (#22487). samples, as determined by FISH/RT-PCR (#51) and RT-PCR (#30). The remaining variant 1 data is summarized in the table.
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