DETECTION OF THE T(2-5)(P23-Q35) AND NPM-ALK FUSION IN NON-HODGKINS-LYMPHOMA BY 2-COLOR FLUORESCENCE IN-SITU HYBRIDIZATION

The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by its marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2;5)(p23;q35), occurs in 25 % to 30% of anaplastic LCLs and is also found in cases with diffuse la rge cell or immunoblastic morphology. We recently identified nucleopho smin (NPM) and anaplastic lymphoma kinase (ALK) as the genes on chromo somes 5 and 2, respectively, that are juxtaposed by this translocation . To provide a complementary approach to the use of classical cytogene tics or polymerase chain reaction-based methods for the detection of t his abnormality, we have developed a two-color fluorescent in situ hyb ridization (FISH) assay for the t(2;5) that may be used for the analys is of both interphase nuclei and metaphase chromosomes. Three overlapp ing chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, a nd used to visualize the derivative chromosome 5 produced by the t(2;5 ), evident as juxtaposed or overlapping red and green fluorescent sign als. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2; 5). The assay was then applied in a blinded fashi on to a series of eight cytogenetically t(2;5)positive clinical specim ens and seven known t(2;5)-negative cases, including three NHL and fou r Hodgkin's disease biopsy samples. Whereas the t(2; 5)-negative cases were negative by FISH, all eight t(2;5)-positive cases were positive. One additional case, initially thought to be positive for the translo cation by cytogenetics, was proven to not be a classic t(2;5) by inter phase and metaphase FISH. These data indicate that the FISH assay desc ribed is a highly specific and rapid test that should prove to be a us eful adjunct to the currently available methods for detection of the t (2;5). (C) 1997 by The American Society of Hematology.