Evaluation of Atypical Fluorescence in Situ Hybridization Findings by RNA Sequencing

Abstract

Fluorescence in situ hybridization (FISH) using a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded tumors. Although clinically validated BAP FISH assays usually yield unequivocal positive or negative results, rare tumors yield equivocal FISH results. This study had two aims: to summarize typical and atypical BAP FISH results on 56,584 formalin-fixed, paraffin-embedded solid tumors over approximately one decade of clinical testing; and to investigate the clinical utility of RNA sequencing (RNA-seq) for tumors with equivocal FISH results. Of 8586 (15.2%) cases with abnormal FISH results reported, 748 (8.7%) were equivocal. RNA-seq was performed on 113 tumors, and oncogenic fusions involving the gene of interest were detected in 46 of 113 tumors (40.7%). Of the 106 tumors with equivocal FISH results, RNA-seq detected a fusion involving the expected gene target in 37 of 62 (59.7%) tumors with isolated probe signals corresponding to the active side of the gene region but only 4 of 44 (9.1%) tumors with other atypical signal patterns. This study provides a useful framework for categorizing atypical BAP FISH results and demonstrates the clinical utility of follow-up RNA-seq testing on tumors with equivocal FISH results.