Pitfalls and Challenges in the Detection of ZNF384 Rearrangements by Fluorescence in Situ Hybridization

Fusion genes involving ZNF384 define a rare subtype of B-cell acute lymphoblastic leukemia with favorable to intermediate outcomes. ZNF384 rearrangements (ZNF384-r) with diverse partner genes are often cryptic, making whole-transcriptome sequencing (RNA-seq) the most sensitive technique for their detection. For patient screening and routine diagnostics, fluorescence in situ hybridization (FISH) using a dual-color break-apart probe is considered a reliable alternative. However, the sensitivity and specificity of FISH for detecting ZNF384-r have not been systematically evaluated. In this study, we assessed the performance of FISH by comparing its results with RNA-seq data from the same patient cohort. We also performed single-nucleotide polymorphism array analysis to identify copy-number alterations indicative of ZNF384-r and the respective partner genes. While FISH proved highly specific, its sensitivity was lower than anticipated, potentially leading to misdiagnosis in a substantial proportion of patients.