1. 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 1 ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT 2 Department of Pathology, University of Utah Health Sciences Center and ARUP Laboratories, Salt Lake City, UT Detection of EML4-ALK variant 3a/b by IHC, break apart FISH and RT-PCR. All 3 methodologies are shown for one representative positive sample (#28). Two of two (100%) variant 3a/b positive samples displayed loss of the green probe by FISH. Introduction  52 FFPE lung adenocarcinomas were selected from the University of Utah surgical pathology files  The study population was enriched for wildtype EGFR status as determined by direct sequencing of exons 18-21 (WT: n = 46; Mutant: n = 6).  ALK IHC: monoclonal mouse CD246, clone ALK1 (Dako)  ALK FISH: Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe (Abbott Molecular)  Positive cutoff: ≥ 40% cells rearranged  RT-PCR:  Variant 1 primers (109 bp)  EML4 Ex13 Fwd: 5’-TAGAGCCCACACCTGGGAAA-3’ ALK Ex20 Rev: 5’-CGGAGCTTGCTCAGCTTGTA-3’  Variant 1 positive control: FFPE HEK293 cells transiently expressing pcDNA3-EML4-ALK variant 1  Variant 3a/b primers (105 bp & 138 bp)  EML4 Ex6 Fwd: 5’-GCATAAAGATGTCATCATCAACCAAG-3’ ALK Ex20 Rev: 5’-CGGAGCTTGCTCAGCTTGTA-3’  Variant 3a/b positive control: H2228 cells  MRPL19 primers (94 bp)  MRPL19 Ex4 Fwd:5’-GGAAGAGGACTTGGAGCTACT-3’ MRPL19 Ex5 Rev: 5’-TCCTGGACCCGAGGATTAT-3’  The use of human tissue for this analysis was approved by the University of Utah Institutional Review Board (#22487). Materials and Methods Lung cancer is the most common and deadly form of cancer in the United States with a 5-year survival rate of only 15.7%. Recently, it has become apparent that non-small cell lung cancer (NSCLC) can be further divided into clinically relevant subsets based on specific molecular alterations within the tumor. In 2007, Soda et al identified a small chromosome 2p inversion (~12 Mb) in a subset of Japanese NSCLC patients, which resulted in the fusion of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene. Multiple different EML4- ALK translocation variants have since been identified that contain various truncations of EML4 but always contain the tyrosine kinase domain of ALK, starting at exon 20. The most common (~70%) translocations in NSCLC involve EML4 exon 13 (variant 1) or EML4 exon 6 (variant 3a/b; 3b contains an additional 33-bp due to alternative splicing). The incidence of EML4-ALK translocations in NSCLC is low (2-7%) but can be higher (13%) if the population is enriched for adenocarcinoma, never or light smokers, younger age and lack of EGFR or KRAS mutations. Early-phase clinical trials of ALK inhibitors in patients with known EML4-ALK translocations have yielded promising results. Therefore, a need exists for molecular testing to identify the correct target patient population. We compared ALK immunohistochemistry (IHC), ALK break apart FISH and RT-PCR in a subset of lung adenocarcinomas for the detection of EML4-ALK variants 1 and 3a/b. V3a/b Pos V3a/b Neg #28 NTC EML4-ALK Variant 3a/b RT-PCR -500 bp- - 50 bp - V3b (138 bp)- V3a (105 bp)- V3a/b Pos V3a/b Neg #28 NTC MRPL19 RT-PCR -MRPL19 (94 bp) Results #28: IHC Positive (2+) #28: FISH Positive (Avg. 70% Rearranged) #30: IHC Negative (0) #30: FISH Negative (Avg. 18% Rearranged) #51: IHC Negative (0) #51: FISH Positive (Avg. 74% Rearranged) V1 Pos V1 Neg #51 #30 -500 bp - 50 bp EML4-ALK Variant 1 RT-PCR V1 (109 bp)- NTC#30 MRPL19 RT-PCR MRPL19 (94 bp)- -500 bp -50 bp V1 Pos V1 Neg #51 #30 NTC#30 SampleEGFRALK IHC ALK FISH Reader 1 ALK FISH Reader 2 ALK FISH Reader 3 Avg FISH EML4-ALK V3a/b RT-PCR # 16wildtypepositive86%50%74%70% (positive)positive # 28wildtypepositive70%73%67%70% (positive)positive SampleEGFRALK IHC ALK FISH Reader 1 ALK FISH Reader 2 ALK FISH Reader 3 Avg. FISH EML4-ALK V1 RT-PCR # 14wildtypenegative7%10%25%14% (negative)positive # 18L858Rnegative16%44%20%26% (negative)positive # 30wildtypenegative4%32%19%18% (negative)positive # 39wildtype weak patchy positive 6% 23%11% (negative)positive # 42wildtypenegative12%50%7%23% (negative)positive # 47wildtypenegative9%35%11%18% (negative)positive # 50wildtypenegative11%39%19%23% (negative)positive # 51wildtypenegative84%52%87%74% (positive)positive # 57wildtypenegative17%22%30%23% (negative)positive Results Detection of EML4-ALK variant 1 by IHC, break apart FISH and RT-PCR. All 3 methodologies are shown for two variant 1 positive samples, as determined by FISH/RT-PCR (#51) and RT-PCR (#30). The remaining variant 1 data is summarized in the table. Conclusions  ALK protein expression was detectable by IHC in 9 of 52 (17%) samples.  ALK break apart FISH was positive in 4 of 52 (8%) samples.  EML4-ALK variant 3a/b RT-PCR was positive in 2 of 52 (4%) of samples.  EML4-ALK variant 1 RT-PCR was positive in 9 of 52 (17%) of samples.  The total incidence of EML4-ALK translocations in our NSCLC series, which was enriched for adenocarcinomas with wild-type EGFR status, was 21.1% (11 of 52).  Concordance between IHC, FISH and RT-PCR methodologies was 100% for EML4-ALK variant 3a/b (2 of 2 samples).  No concordance was found between IHC, FISH and RT-PCR methodologies for EML4-ALK variant 1 (0 of 9 samples).  FISH and V1 RT-PCR concordance: 11% (sample # 51)  IHC and V1 RT-PCR concordance: 11% (sample # 39)  One EML4-ALK variant 1 positive case (sample # 18) had a concurrent EGFR mutation (L858R).  EML4-ALK was not detected by RT-PCR in normal tissue (lymph node or lung) from 4 RT-PCR positive samples that had sufficient tissue for analysis (data not shown). Concordance among the three methodologies (IHC, FISH and RT- PCR) differed for the two tested EML4-ALK translocation variants. Variant 3a/b was detectable by all three methods. Conversely, limited ALK protein expression and subjectivity in FISH scoring resulted in no concordance between all three methods for the detection of EML4-ALK variant 1. Agreement among FISH readers was poor for variant 1. This was likely due to the expected small split in probe signals. These results suggest that the detection of other EML4-ALK variants that involve the 3’ region of EML4 (exons 15-20) may also be challenging by FISH. RT-PCR was the most sensitive and least subjective methodology for EML4-ALK variant 1 detection. While multiple specific primer sets would need to be designed for the detection of all known EML4-ALK translocation variants from FFPE tissue by RT-PCR, this approach would likely yield the greatest assay sensitivity. Additionally, our results confirm that the incidence of EML4-ALK translocations in NSCLC is greater (21%) in enriched patient populations. References Soda M, Choi YL, Enomoto M et al. Identification of the transforming EML4-ALK fusion gene in non-small cell lung cancer. Nature, 2007; 448:561-566. Shaw AT, Yeap BY, Mino-Kenudson M et al. Clinical features and outcome of patients with non- small-cell lung cancer who harbor EML4-ALK. J Clin Oncol, 2009; 27:4247-4253. Kwak EL, Bang YJ, Camidge DR et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med, 2010; 363:1693-1703. Results. We graciously thank Maria Martelli and Brunangelo Falini for providing controls. Acknowledgements