Detection of BCL2-IGH Using Single-Round PCR Assays

Abstract

The translocation t(14;18) resulting in fusion of the BCL2 and the immunoglobulin heavy chain genes (BCL2-IGH) is present in 80% to 90% of follicular lymphomas and 20% to 30% of diffuse large B-cell lymphomas. Polymerase chain reaction (PCR) analysis for the translocation products suffers from low analytic specificity. As a result, either nested PCR or probe hybridization is required to aid in the identification of the specific translocation products. These added procedures are undesirable in clinical laboratories because nested procedures increase the possibility of contamination and probe hybridization increases assay turnaround time. To simplify the BCL2-IGH assay procedure, we attempted to eliminate the nonspecific PCR products by optimizing the annealing temperatures of the PCR assays using a gradient thermocycler. We showed that gradually increasing the annealing temperature from 55 degrees C to 67 degrees C significantly enhanced the intensity of the specific PCR products while eliminating the nonspecific ones. We compared the simplified procedure with a PCR-probe hybridization method on 68 patient specimens. The simplified procedure had increased analytic and diagnostic specificities with comparable sensitivities. With significantly improved analytic specificity, one round of PCR is sufficient to detect the BCL2-IGH gene rearrangements without further confirmation.