Real-Time t(14;18)(q32;q21) PCR Assay Combined with High-Resolution Capillary Electrophoresis: A Novel and Rapid Approach that Allows Accurate Quantitation and Size Determination of bcl-2/JH Fusion Sequences

Abstract

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation that juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain (IgH) locus at 14q32. We have previously shown that accurate quantitation of t(14;18)-carrying cells in follicular lymphoma patients can be achieved by non-gelbased real-time TaqMan polymerase chain reaction (PCR; Applied Biosystems, Foster City, CA). Since our report, several studies have demonstrated that real-time PCR is highly sensitive and a reliable tool for evaluating treatment effectiveness and for following minimal residual disease in follicular lymphoma patients. Unfortunately, currently available real-time PCR methods do not determine the size of the amplification product, which is useful for excluding contamination and is commonly used as presumptive evidence of clonal identity or disparity when multiple samples from the same patient are analyzed. We describe a modified real-time PCR assay that rapidly allows accurate quantitation and precise determination of the size of the t(14;18) fusion sequence without the need for gel electrophoresis. In this assay, a consensus immunoglobulin heavy chain-joining region gene (JH) primer labeled at its 5' end with the fluorescent dye NED (Applied Biosystems) is included in the real-time PCR assay and thus is incorporated into the bcl-2/JH fusion product. The JH-NED primer did not interfere with the TaqMan probe fluorescent signal or target detection and allowed subsequent amplicon size determination by semiautomated highresolution capillary electrophoresis.KEY WORDS: Follicular lymphoma, High-resolution capillary electrophoresis, Real-time polymerase chain reaction, t(14;18). Mod Pathol 2002;15(4):448 -453Real-time polymerase chain reaction (PCR) for detection and quantitation of bcl-2/JH fusion sequences characteristic of t(14;18) (q32;q21) is being used increasingly to monitor tumor burden and the effect of therapy in patients with follicular lymphoma (FL; 1-5). Real-time PCR methods enable t(14;18) detection without the need to open amplification tubes and are target specific, highly sensitive, and reproducible. Thus, real-time PCR methods have several advantages over conventional PCR techniques that require gel electrophoresis-based amplicon detection and quantitation techniques. Currently available real-time PCR methods to detect the t(14;18) do not allow amplicon size determination. This is a major drawback for a few reasons. First, one cannot easily exclude contamination. Second, size of PCR products from multiple samples (either simultaneous or sequential) is a valuable means of determining clonal relatedness, as the size of bcl-2/JH fusion sequences generally vary from clone to clone (6 -8). Undoubtedly, sequencing of the fusion sequences is the most accepted method for determining clonal relatedness, but sequencing methods can be tedious and time consuming. Third, as t(14;18)-positive cells are detected in normal healthy individuals (9 -12), the ability to quantitate low-level...

Quantitative PCR of bone marrow BCL2/IgH+ cells at diagnosis predicts treatment response and long-term outcome in follicular non-Hodgkin lymphoma

“…[7][8][9] During the last few years, different quantitative PCR assays have been developed. [10][11][12] These methods provide a more reliable tool for an accurate evaluation of BCL2/ IgH ϩ cells in the BM or peripheral blood (PB) 13 and allow a better molecular monitoring of minimal residual disease after different therapeutic protocols. Moreover, some studies have provided evidence that real-time quantitative PCR (RQ-PCR) evaluation before and after autologous transplantation may predict the clinical course of these patients.…”

Quantification of bcl-2/JH Fusion Sequences and a Control Gene by Multiplex Real-Time PCR Coupled with Automated Amplicon Sizing by Capillary Electrophoresis

“…We have recently shown that PCR products labeled during realtime PCR by incorporation of a fluorescent dye-labeled primer can be resolved by CE, allowing precise size determination of each bcl-2/JH fusion sequence. 15 This assay allowed us to recognize identically sized clones in sequential biopsy specimens from two patients. One patient (patient 19) had four sequential bone marrow samples in which we identified a clone of identical size, 108 bp, in each specimen during the course of the disease.…”

“…In addition, the amplified bcl-2/JH fusion sequences are labeled with a fluorescent dye, NED (Applied Biosystems), in this real-time PCR assay as we described previously. 15 The labeling of the amplification products allows rapid determination of the size of the bcl-2/JH fusion sequences following real-time PCR by capillary electrophoresis (CE). As amplicon size is helpful for excluding contamination and is also commonly used to demonstrate clonal identity between pre-and posttreatment specimens, multiplex real-time TaqMan PCR combined with CE provides a simple and rapid approach for monitoring minimal residual disease facilitating highthroughput analysis of multiple samples.…”

“…As we have described previously, 15 after PCR we resolved the amplification products by CE and analyzed by GS. Peaks representing a unique amplicon size for each patient specimen (Table 2 and Figure 5) were identified corroborating that the fluorescent signal detected by real-time PCR was due to patient-specific clones.…”

The role of molecular studies in lymphoma diagnosis: a review

“…174 (ii) t(14;18) PCR for MRD testing and monitoring Sensitive nested t(14;18) PCR assays with analytical sensitivities of 1 in 10 5 -10 6 cells are used for MRD detection and monitoring, and for assessing the efficacy of marrow purging prior to autologous transplantion. [177][178][179][180] Where available, RQ-PCR assays are now the preferred method of testing, using TaqMan and LightCycler systems, 166,[181][182][183][184][185] which are at least as sensitive as conventional nested assays. Their high analytical sensitivity mandates caution in interpreting 'molecular relapse' in treated patients, as translocation-positive cells in normal individuals can be detected at levels as high as 1 in 10 4 .…”