5′ → 3′ Exonuclease-Based Real-Time PCR Assays for Detecting the t(14;18)(q32;21): A Survey of 162 Malignant Lymphomas and Reactive Specimens

Abstract

We describe our experience using two real-time polymerase chain reaction (PCR) assays for detecting the t(14;18)(q32;q21) in a large series of non-Hodgkin's lymphomas (NHLs). These assays utilize the 5′ → 3′ exonuclease activity of Taq polymerase, which cleaves a probe labeled with a fluorescent reporter dye at its 5′ end and a quencher dye at its 3′ end during the extension phase of PCR. In a previous study, Luthra and colleagues developed these real-time PCR assays for detecting the t(14;18) involving the major and minor breakpoint cluster regions of the bcl-2 gene and assessed a small number of NHLs. In this larger study, we analyzed 135 NHLs, 6 Hodgkin's disease, 10 reactive biopsy specimens, and 11 peripheral blood specimens. The NHL group included 46 of 70 (65.7%) follicular NHLs, 1 of 2 (50%) diffuse small cleaved cell NHLs, and 13 of 24 (54.2%) diffuse large B-cell NHLs with the t(14;18) detected by conventional PCR methods. There was excellent agreement between the real-time and conventional PCR assays with overall concordance in 160 of 162 (98.8%) specimens. For the NHLs, concordance was found in 134 of 135 (99.3%) specimens. Disagreement was observed in one case of follicular NHL in which the real-time PCR assay detected bcl-2 minor breakpoint cluster region/JH DNA fusion sequences and the conventional method was negative. The overall concordance for 10 benign biopsy specimens and 11 normal peripheral blood samples was 20 of 21 (95.2%). One lymph node biopsy specimen that showed reactive follicular hyperplasia was positive for the bcl-2 minor breakpoint cluster region/JH DNA fusion sequences detected by the real-time PCR assay but was negative by conventional PCR methods. This patient had no clinical evidence of NHL. We conclude that real-time PCR assays for detecting the t(14;18) are sensitive, specific, and more convenient than conventional PCR methods.