The detection of t(14;18) in archival lymph nodes: development of a fluorescence in situ hybridization (FISH)-based method and evaluation by comparison with polymerase chain reaction

Abstract

Fluorescence in situ hybridization (FISH) has been used to demonstrate the t(14;18) in up to 100% of follicular lymphoma (FL) cases, however, there is little reproducible data using fixed tissue. The aim was therefore to develop a robust FISH method for the demonstration of translocations in archival tissue. The technique was evaluated by comparison with multiplex polymerase chain reaction (PCR), capable of detecting the majority of known breakpoints. Twenty-eight paired frozen and fixed cases of FL and 20 reactive controls were analyzed. The t(14;18) was detected in 23 of 28 cases using PCR on frozen material and 8 of 20 in paraffin. Using FISH, 24 of 26 frozen and 26 of 28 paraffin cases had a demonstrable translocation. All 20 reactive nodes were negative for the t(14;18) by PCR. Using FISH, one of the reactive cases had occasional cells with a translocation FISH pattern, demonstrable in frozen and paraffin samples. This is consistent with the presence of the t(14;18), which is well described in normal individuals. Both PCR and FISH are highly effective for t(14;18) analysis in unfixed tissue. When only paraffin blocks are available, FISH is the method of choice, and a result was achieved in 100% of cases. The method is applicable to the retrospective analysis of a range of translocations.

Figure 1.

Representative FISH images of the pattern of staining of the BCL2/IgH probe set. a: Normal pattern of staining. Each cell nucleus has two red (BCL2) and two green (IgH) FISH signals, one for each copy of the chromosome. The counterstain is DAPI, which is visualized as blue fluorescence. b: A case with a t(14;18). Each nucleus has three signals with both BCL2 and IgH probe sets, indicating a “split” in one each of the copies of the genes. The reciprocal translocation, t(14;18), is demonstrated by the presence of two fusion signals in each cell (indicated by yellow arrows), along with a residual normal copy of each gene. The counterstain is DAPI.

Figure 2.

t(14;18) multiplex PCR analysis on DNA extracted from 28 frozen biopsies of FL. a: MBR multiplex (JH + MBR): 15 of 28 FL cases had a visible band and were classed as positive (+). b: mcr multiplex (JH + mcr, 5′mcr, 3′MBR): 8 of 28 FL cases had a visible band and were classed as positive (+).