Quantitative Real-Time Polymerase Chain Reaction for Detection of Circulating Cells with T(14;18) in Volunteer Blood Donors and Patients with Follicular Lymphoma

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Abstract
The chromosomal rearrangement t(14;18)(q32;21) involves the major (MBR) or minor (mcr) breakpoint cluster regions and the immunoglobulin heavy chain joining regions (JH) in most follicular lymphomas. As a first step towards determining the clinical significance of circulating cells with t(14;18), we detected and quantitated circulating cells in samples obtained from volunteer blood donors and follicular lymphoma patients. The t(14;18) was co-amplified with β -actin with real-time quantitative PCR (QRT-PCR) in reactions containing 1 µ g of DNA from peripheral blood or bone marrow aspirates. The cell number was quantitated using linear regression and an external standard of serially diluted DNA from cell lines with MBR/JH or mcr/JH rearrangements. At dilutions of 10 5 and 10 6, sensitivity was 100 and 55% for MBR/JH, and 100 and 10% for mcr/JH rearrangements. Among 102 volunteer blood donors MBR/JH vs. mcr/JH amplicons were detected in 22 vs. 4% with duplicate 1 µ g DNA reactions, and in 41 vs. 6% with a total 10 µ g DNA analyzed in multiple reactions. Among volunteer blood donors the mean number of circulating cells with MBR/JH vs. mcr/JH rearrangements were 0.8 vs. 0.1/ µ g DNA, and exceeded the upper normal limit (defined as the mean of all volunteer samples plus two standard deviations) in 3% vs. 2%, respectively. Analysis for MBR/JH rearrangements revealed that follicular lymphoma patients vs. volunteer blood donors were positive in 76% vs. 22% (p =0.008 by Fisher's exact test); that the mean number of MBR/JH cells per µ g of DNA was 91 vs. 0.8 (p =0.0002 by Mann-Whitney test); and the number of the MBR/JH cells exceeded the upper normal limit in 32% vs. 3% of subjects (p =0.0001 by Fisher's exact test). Circulating cells with mcr/JH were not detected among any of these 25 lymphoma patients. We conclude that patients with follicular lymphoma are more frequently positive, have higher numbers of circulating cells with t(14;18), which exceed upper normal limit more frequently than in volunteer blood donors.