A Prospective PCR-Based Screening for the EML4-ALK Oncogene in Non–Small Cell Lung Cancer

Abstract

Purpose: EML4-ALK is a lung cancer oncogene, and ALK inhibitors show marked therapeutic efficacy for tumors harboring this fusion gene. It remains unsettled, however, how the fusion gene should be detected in specimens other than formalin-fixed, paraffin-embedded tissue. We here tested whether reverse transcription PCR (RT-PCR)-based detection of EML4-ALK is a sensitive and reliable approach.Experimental Design: We developed a multiplex RT-PCR system to capture ALK fusion transcripts and applied this technique to our prospective, nationwide cohort of non-small cell lung cancer (NSCLC) in Japan.Results: During February to December 2009, we collected 916 specimens from 853 patients, quality filtering of which yielded 808 specimens of primary NSCLC from 754 individuals. Screening for EML4-ALK and KIF5B-ALK with our RT-PCR system identified EML4-ALK transcripts in 36 samples (4.46%) from 32 individuals (4.24%). The RT-PCR products were detected in specimens including bronchial washing fluid (n ¼ 11), tumor biopsy (n ¼ 8), resected tumor (n ¼ 7), pleural effusion (n ¼ 5), sputum (n ¼ 4), and metastatic lymph node (n ¼ 1). The results of RT-PCR were concordant with those of sensitive immunohistochemistry with ALK antibodies.Conclusions: Multiplex RT-PCR was confirmed to be a reliable technique for detection of ALK fusion transcripts. We propose that diagnostic tools for EML4-ALK should be selected in a manner dependent on the available specimen types. FISH and sensitive immunohistochemistry should be applied to formalinfixed, paraffin-embedded tissue, but multiplex RT-PCR is appropriate for other specimen types.

Pre-analytical issues in effusion cytology

“…Cytological lung carcinoma specimens frozen at -80°C were informative for fusion between the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes [67]. Specimens were mixed with a buffer preventing RNA degradation before freezing in the latter study.…”

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

“…156,166 Although at the time of writing RT-PCR and NGS are not approved by FDA in the United States as first-line methods for determining ALK status in selection of patients for ALK inhibitor therapy, these approaches have shown comparable performance with IHC 163e165 when designed to detect the majority of fusions, and are standard practice in many other countries. 163e165,167 These methods are highly specific for most fusions, 97,168,169 and patients with positive results should be treated with an ALK inhibitor, although patients with negative results may benefit from a more sensitive method to exclude the possibility of a variant fusion. Similarly, amplicon-based NGS assays of DNA may likewise fail to detect all fusion variants, and therefore a capture-based DNA or RNA approach is preferred for NGS testing for ALK fusions.…”

Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

“…This fusion results from the rearrangement within chromosome 2 [inv (2)(p21p23)] and fusion of the 5' portion of EML4 with the 3' portion of ALK (6). At least 14 variants of the EML4-ALK fusion gene have been reported thus far, encoding for the cytoplasmic portion of ALK protein and containing varying lengths of EML4 (19). Variants v1, v2, v3a and v3b of EML4-ALK fusion gene are the most commonly detected, together accounting for more than 90% of variants in some series (20).…”

“…Most of the current guidelines do not include it ALK RT-PCR in their diagnostic algorithms, as the rate of failures and falsenegative tests can be significant. Nevertheless, RT-PCR can find its best utility in samples not suitable for tissue blocking, mainly represented by bronchial washing fluid and pleural effusions (19,58). RT-PCR could represent a useful tool to detect fusion variants of the EML4-ALK fusion gene which otherwise, besides harboring unclear clinical significance (see above), can be currently revealed by nextgeneration genomic and transcriptomic analyses (see next paragraph).…”