High performance of multiplex fluorescence in situ hybridization to simultaneous detection of BCL2 and BCL6 rearrangements: useful application in the characterization of DLBCLs.

Chromosomal rearrangements involving BCL2, BCL6 and MYC are commonly found in the most frequent B cell lymphomas, namely follicular lymphomas (FLs) and diffuse large B-cell lymphomas (DLBCLs). Particularly, BCL2-rearrangement represents a diagnostic hallmark in FLs, whereas MYC translocation can occur simultaneously with BCL2 and/or BCL6 rearrangements, defining a specific category of DLBCLs with a poorer prognosis. In this study, we aim to validate the diagnostic performance of multiplex BCL2/BCL6 FISH approach in formalin-fixed paraffin-embedded FLs and DBCLs and cytological samples of DLBCL comparing to the classic set of single break-apart probes. We collected a series of lymphomas, including 85 DLBCLs, 45 FLs and 36 other B-cell lymphoma histotypes and 16 cytological samples of DLBCLs. MYC, BCL2 and BCL6 rearrangements were previously assessed by a classic FISH test using single break-apart probes. All samples were analysed by a multiplex FISH assay. In the FL series, 38 cases showed BCL2-R; in the DLBCLs series, MYC-R was detected in 21 out of 85 DLBCL patients, BCL2-R in 10 out of 85 and BCL6-R in 33 out of 85. In the DLBCL cytological series, MYC-R was detected in 4 out of 16, BCL2-R in 4 out of 16 and BCL6-R in 1 out of 16. Notably, in FFPE, 13 double-hit lymphomas (DHLs) and 3 triple-hit lymphomas (THLs) were detected; in the cytological series, only 3 DHL cases were observed. The dual BCL2/BCL6 FISH probe test results were fully concordant with the results obtained using classic BCL2 and BCL6 single break apart. Particularly, multiplex FISH to simultaneously detect BCL2-R and BCL6-R on a single slide could find a wide application in the characterisation of double- and triple-hit DLBCLs.