Custom Gene Capture and Next-Generation Sequencing to Resolve DiscordantALKStatus by FISH and IHC in Lung Adenocarcinoma

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Abstract

Introduction

We performed a genomic study in lung adenocarcinoma cases with discordant anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) analysis.

Methods

DNA from formalin-fixed paraffin-embedded tissues of 16 discordant (four FISH-positive/IHC–negative and 12 FISH-negative/IHC–positive) cases by Vysis ALK Break Apart FISH and ALK IHC testing (ALK1 clone) were subjected to whole gene capture and next-generation sequencing (NGS) of nine genes, including ALK, echinoderm microtubule associated protein like 4 gene (EML4), kinesin family member 5B gene (KIF5B), staphylococcal nuclease and tudor domain containing 1 gene (SND1), BRAF, ret proto-oncogene (RET), ezrin gene (EZR), ROS1, and telomerase reverse transcriptase (TERT). All discordant cases (except one FISH-negative/IHC–positive case without sufficient tissue) were analyzed by IHC with D5F3 antibody. In one case with fresh frozen tissue, whole transcriptome sequencing was also performed. Twenty-six concordant (16 FISH-positive/IHC–positive and 10 FISH-negative/IHC–negative) cases were included as controls.

Results

In four ALK FISH-positive/IHC–negative cases, no EML4-ALK fusion gene was observed by NGS, but in one case using fresh frozen tissue, we identified EML4–baculoviral AIP repeat containing 6 gene (BIRC6) and AP2 associated kinase 1 gene (AAK1)-ALK fusion genes. Whole transcriptome sequencing revealed a highly expressed EML4-BIRC6 fusion transcript and a minimally expressed AAK1 transcript. Among the 12 FISH-negative/IHC–positive cases, no evidence of ALK gene rearrangement was detected by NGS. Eleven of 12 FISH-negative/IHC–positive cases detected by ALK1 clone were concordant by repeat ALK IHC with D5F3 antibody (i.e., FISH-negative/IHC–negative by D5F3 clone). Among the 16 ALK FISH-positive/IHC–positive positive controls, whole gene capture identified ALK gene fusion in 15 cases, including in one case with Huntington interacting protein 1 gene (HIP1)-ALK. No ALK fusion gene was observed in any of the 10 FISH-negative/IHC–negative cases. Other fusion genes involving ROS1, EZR, BRAF, and SND1 were also found.

Conclusions

ALK FISH results appeared to be false-positive in three of four FISH-positive/IHC–negative cases, whereas no false-negative ALK FISH case was identified among 12 ALK FISH-negative/IHC–positive cases by ALK1 clone, which was in keeping with the concordant FISH-negative/IHC–negative status by D5F3 clone. Our targeted whole gene capture approach using formalin-fixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.

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