Rapid detection of IgH/BCL2 rearrangement in follicular lymphoma by interphase fluorescence in situ hybridization with bacterial artificial chromosome probes

Abstract

Follicular lymphomas (FLs) can be difficult to diagnose on aspirated specimens since the architectural pattern is not present. FLs characteristically have rearrangements in the IgH and BCL2 genes resulting from the reciprocal t(14;18) (q32; q21) translocation. Because of the dispersed distribution of breakpoints, fluorescence in situ hybridization (FISH) using genomic probes that span or flank the breakpoints is ideal for detecting this rearrangement in fine-needle aspiration (FNA) biopsies. To develop a set of probes, a bacterial artificial chromosome library was screened and the clones were mapped by fiber FISH. The probes were produced by the direct incorporation of fluorochrome-labeled nucleotides. The colocalization base FISH assay was applied to Cytospin preparations from FNA biopsies of lymph nodes from 26 patients with FL and 10 patients without FL. In those with FL, the percentage of cells with at least one IgH/BCL2 fusion signal ranged from 22% to 100% (mean, 63%), which was statistically significantly higher than that in FL-negative samples (mean, 2.7%). The probes demonstrated a significantly lower cutoff value (7%) in normal controls and effectively reduced the false-positive rate in FL-negative cases. These results were confirmed with fiber FISH assays on the same specimens. This interphase FISH assay is rapid and reliable for detecting rearrangements in the IGH/BCL2 gene, thereby aiding in the diagnosis of FL on FNA biopsy specimens.

Figure 1.

Mapping of IgH and BCL2 probes by fiber FISH assay on DNA fibers of normal peripheral blood leukocytes. The top fiber shows the position of bacterial artificial chromosomes 442F20 (orange bar) and dJ998D24 (green bar) relative to the IgH locus. 442F20 was mapped on the constant and joining regions of IgH; it spans 220 kb and overlaps with the 110-kb dJ998D24, which covers the joining and diversity regions. The overlapping region is 20 kb long, indicated by the yellow barcode. The total size of the IgH probe is 310 kb. The bottom fiber shows the position of BACs 260D23 (orange bar) and 253F8 (green bar) relative to the BCL2 gene. 260D23 was mapped on exons II and III and on the major breakpoint and minor cluster regions, spanning 170 kb. 253F8 is 180 kb long, covering exons I, II, and III. The clones are connected to each other with approximately 50 kb of overlap (yellow). The total length of the BCL2 probe is 300 kb, spanning the entire 225 kb of BCL2 and its 5′ and 3′ flanking genomic regions.

Figure 2.

A: FISH on normal metaphase shows the chromosomal locations of the IgH probe on 14q32 (green) and the BCL2 probe on 18q21 (orange). B: Normal interphase nuclei show two IgH probe signals (green) and two BCL2 probe signals (orange). C: Detection of t(14;18) (14q32;18q21) in interphase nuclei of a specimen from a patient with follicular lymphoma shows yellow or touching signals of two different color probes, indicating colocalization of IgH/BCL2 fusion. D: Detection of t(14;18) (14q32;18q21) with fiber FISH on a specimen from a patient with follicular lymphoma; a single linear DNA fiber with two juxtaposed color barcode signals indicates IgH (green) and BCL2 (orange) fusion.