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

Abstract

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 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.

A Case of Crizotinib-Resistant Lung Adenocarcinoma Harboring a KRAS Mutation and an EML4-ALK Fusion Gene

“…The frequency of EML4-ALK translocation variant 1 is approximately 30% [1]. Comparison of FISH, IHC, and RT-PCR methodologies for detecting EML4-ALK translocation variant 1 showed RT-PCR to be the most sensitive [2], as in the current case. The presence of the EML4-ALK fusion gene in lung cancer is mutually exclusive with EGFR and KRAS mutations [1], and patients with EGFR or KRAS gene mutations are unlikely to also harbor the EML4-ALK fusion gene.…”

Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization

“…Furthermore, it has been reported that the subtle signal separation because of the chromosomal inversion producing the EML4-ALK translocation variant 1, confirmed by reverse transcription-polymerase chain reaction, is more likely to be missed by FISH. 46 Wallander et al 46 reported that only 1/9 (11%) of variant 1 cases were designated positive by three FISH viewers. Sensitivity and specificity was increased following a combination of scoring FISH break-apart signals that were less than two signal distances apart, and increasing the cutoff to 420% of cells with positive FISH signal patterns.…”

Therapeutic management of ALK+nonsmall cell lung cancer patients

“…Its limited use to date is due to the requirement of a quality DNA sample from fresh or frozen tumour tissue. However, new platforms allowing use of routine tissue samples, including paraffin-embedded samples, have been reported [38,39].…”