Fish Is the Best Method to Detect BCL2/IgH Translocation in Follicular Lymphoma at Diagnosis. A Comparative Study with Conventional Cytogenetics, Fish and PCR (Biomed-2 Primers) Techniques.

Introduction: Follicular lymphoma (FL) is a B-cell neoplasm characterized by the presence of the t(14;18)(q32;q21) in nearly 85% of cases. There are different methods to find such translocation. The aim of the present study was to test the efficiency of the different techniques: conventional cytogenetics, FISH and PCR, to detect t(14;18) in FL at diagnosis in fresh and paraffin embedded samples.

Patients and Methods: Fifty-four FL patients were analyzed. Twenty-two samples were fresh specimens from peripheral blood (1 case), bone marrow (6 cases) and lymph nodes (15 cases). The remaining 32 samples were paraffin embedded tissues: 25 lymph nodes, 2 spleens, 1 duodenum, 1 tonsil, 1 lung and 1 nasopharynx. Three different techniques were employed and compared: conventional cytogenetics only in fresh samples and FISH and PCR in fresh and paraffin embedded samples. Conventional cytogenetics cultures were established for 72 hours with TPA and chromosomes were identified by G-banding pattern. FISH was performed with BCL2/IgH dual color-dual fusion locus specific probes (VYSIS). PCR was designed using the BIOMED-2 primers covering the MBR, 3′MBR and mcr regions for the BCL2 gene and the consensus JH primer for the IgH gene (

Results: Regarding fresh samples, 7/22 cases (32%) had cytogenetic aberrations, in three t(14;18) was found and in one t(18;22) was detected, all in a complex karyotype. FISH was performed in 19/22 patients and 14/19 (74%) were positive for BCL2/IgH translocation. PCR technique was applied to 21 cases, and positivity was detected in 10/21 (48%) samples (six cases positive for MBR region, three cases for mcr region and one case for 3′MBR region). In five cases, FISH showed positivity not detected by PCR and in one case, PCR showed positivity not detected neither by FISH nor by conventional cytogenetics. Among 22 cases, in three (14%) BCL2/IgH was not detected by any of the three tested techniques. In paraffin embedded samples, 29/32 cases (90.6%) showed t(14;18) by FISH, 8 of them not detected by PCR. Twenty-nine cases were studied by PCR and in 18 (62%) t(14;18) was detected (17 MBR, 1 mcr and no cases 3′MBR). All positive cases were detected by FISH. Ten percent of cases were negative both by FISH and PCR techniques.

Conclusions: 1. FISH becomes the most effective technique to detect BCL2/IgH at diagnosis in fresh and paraffin embedded tissues, probably because BCL2 locus specific FISH probe covers all the possible breakpoints in BCL2 gene. 2. The percentage of detection of t(14;18) by FISH is higher in paraffin embedded tissues because the analysis is made in the previously identified tumoral cells by hematoxilin-eosin staining and specific immunohistochemistry. 3. PCR is useful to define molecular breakpoints that will allow the detection of minimal residual disease. 4. In fresh bone marrow samples, PCR is the most sensible technique due to the heterogeneous infiltration.