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Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint‐flanking probes
Author(s) -
Vaandrager JanWillem,
Schuuring Ed,
Raap Ton,
Philippo Katja,
Kleiverda Karin,
Kluin Philip
Publication year - 2000
Publication title -
genes, chromosomes and cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.754
H-Index - 119
eISSN - 1098-2264
pISSN - 1045-2257
DOI - 10.1002/(sici)1098-2264(200001)27:1<85::aid-gcc11>3.0.co;2-9
Subject(s) - breakpoint , biology , microbiology and biotechnology , follicular lymphoma , gene rearrangement , fluorescence in situ hybridization , southern blot , interphase , cosmid , locus (genetics) , immunoglobulin heavy chain , genetics , gene , population , chromosome , lymphoma , immunology , demography , sociology
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. © 2000 Wiley‐Liss, Inc.