Evaluation of Atypical FISH Findings by RNA Sequencing.

Fluorescence in situ hybridization (FISH) utilizing a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded (FFPE) tumors. While clinically validated BAP FISH assays usually yield unequivocal positive or negative results, rare tumors yield equivocal FISH results. This study had two aims: 1) to summarize typical and atypical BAP FISH results on 56,584 FFPE solid tumors over approximately one decade of clinical testing, and 2) to investigate the clinical utility of RNA-seq for tumors with equivocal FISH results. Of 8,586 (15.2%) cases with abnormal FISH results reported, 748 (8.7%) were equivocal. RNA sequencing (RNA-seq) was performed on 113 tumors, and oncogenic fusions involving the gene of interest were detected in 46/113 tumors (40.7%). Of the 106 tumors with equivocal FISH results, RNA-seq detected a fusion involving the expected gene target in 37/62 (59.7%) tumors with isolated probe signals corresponding to the active side of the gene region but only 4/44 (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.