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

Abstract

Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay 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 colocalization of probes for BCL2 and the immunoglobulin heavy chain (IGH) locus. Intrinsically high rates of false positive nuclei and high interobserver variability make such assays unsuitable for use on lymphoma tissue samples, where tumor cells often form only a minority of the cell population. Using YAC end cloning techniques and screening of a PAC library, we have isolated PAC clones flanking the BCL2 gene. Using these PACs, and several cosmid clones in the second BCL2 intron, we developed a segregation-based interphase 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 follicular lymphomas. To evaluate the results, the same lymphomas were analyzed by DNA fiber FISH with a 600-kb set of BCL2 DNA clones labeled in alternating colors in combination with a color barcode covering the IGH locus. This approach allowed precise mapping of BCL2 breakpoints, and simultaneously showed juxtaposition of IGH genes to BCL2. Comparison of the results of interphase and fiber FISH showed complete correlation. Five cases were negative with both FISH techniques as well as with Southern blotting. Interestingly, all of these 5 cases lacked BCL2 overexpression as determined by immunohistochemistry, against 3 of 35 rearrangement-positive follicular lymphomas. Furthermore, absence of t(14;18) seemed to be correlated with a higher histologic grade (grades 2 and 3 according to Berard). These data indicate that the segregation-based interphase FISH assay detects 100% of BCL2 rearrangements. Because interpretation of the results is straightforward and requires no extensive experience, this assay may be the best available diagnostic test for BCL2 rearrangement. Genes Chromosomes Cancer 27:85-94, 2000.

Follicular lymphoma in young adults: a clinicopathological and molecular study of 200 patients

“…Only one extranodal At the molecular level, 70-95% of follicular lymphoma are reported to contain a translocation between chromosomes 14 (IGH region) and 18 (BCL2 region). 37,38 In the present study, BCL2 rearrangement was present in 84 cases (74%; n ¼ 112). Rearrangement of the BCL6 gene, initially thought to be specifically associated with diffuse large B-cell lymphoma, is observed in 6.4% up to 14.3% of follicular lymphoma at the time of diagnosis, and may be prone to subsequent highergrade transformation.…”

Trisomy 3 is a specific genomic aberration of t(14;18) negative follicular lymphoma

“…4 Horsman et al (2003) investigated the cytogenetic aberrations of t(14;18) negative FL and found that the cases showing chromosome 18 duplication and BCL2-protein expression may comprise a unique subgroup of FL. 3 Recently, Guo et al (2005) reported that the t(14;18) positive group is a single tumor entity, most of which is accounted for by grades 1 and 2, whereas t(14;18) negative-FL was found to consist of distinct subgroups, majority of which is accounted for by grades 3. 5 These reports suggest that t(14;18) negative FL comprise distinct subtypes.…”

Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci

“…Breaks affecting the IGL locus were ruled out in the other 3 cases (Nos. [22][23][24]. DISCUSSION The accurate detection of IG illegitimate rearrangements is important for the diagnostic process and the clinical management of patients with B-cell malignancies; it is also a useful tool for discovering new genes associated with these neoplasms.…”