Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes

Rearrangement of the BCL2 gene is an important parameter for the differenti al diagnosis of non-Hodgkin lymphomas. Although a relatively large proporti on of breakpoints is clustered, many are missed by standard PCR. A FISH ass ay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene has limited the possibilities for a FISH assay to an approach based on col ocalization of probes for BCL2 and the immunoglobulin heavy chain (IGH) loc us. Intrinsically high rates of false positive nuclei and high interobserve r variability make such assays unsuitable for use on lymphoma tissue sample s, where tumor cells often form only a minority of the cell population. Usi ng YAC end cloning techniques and screening of a PAC library, we have isola ted PAC clones flanking the BCL2 gene. Using these PACs, and several cosmid clones in the second BCL2 intron, we developed a segregation-based interph ase FISH assay with two probe combinations enabling separate detection of 5 ' and 3' (mbr/mcr) breakpoints. The assay was applied to a series of 40 fol licular lymphomas. To evaluate the results, the same lymphomas were analyze d by DNA fiber FISH with a 600-kb set of BCL2 DNA clones labeled in alterna ting colors in combination with a color barcode covering the IGH locus. Thi s approach allowed precise mapping of BCL2 breakpoints, and simultaneously showed juxtaposition of IGH genes to BCL2. Comparison of the results of int erphase and fiber FISH showed complete correlation. Five cases were negativ e with both FISH techniques as well as with Southern blotting. Interestingl y, all of these 5 cases lacked BCL2 overexpression as determined by immunoh istochemistry, against 3 of 35 rearrangement-positive follicular lymphomas. Furthermore, absence of t(14; 18) seemed to be correlated with a higher hi stologic grade (grades 2 and 3 according to Berard). These data indicate th at the segregation-based interphase FISH assay detects 100% of BCL2 rearran gements. Because interpretation of the results is straightforward and requi res no extensive experience, this assay may be the best available diagnosti c test for BCL2 rearrangement Genes Chromosomes Cancer 27:85-94 2000. (C) 2 000 Wiley-Liss, Inc.