title: Quantitative real-time polymerase chain reaction for detection of circulating cells with t(14;18) in volunteer blood donors and patients with follicular lymphoma.
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 beta-actin with real-time quantitative PCR (QRT-PCR) in reactions containing 1 microg 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 106, 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 microg DNA reactions, and in 41 vs. 6% with a total 10 microg 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/microg 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 microg 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.
authors.affiliation: Department of Lymphoma and Myeloma, The University of Texas M.D. Anderson Cancer Center, Houston, USA.
authors.forename: Apostolia-Maria
authors.initials: AM
authors.lastname: Tsimberidou
authors.forename: Yunfang
authors.initials: Y
authors.lastname: Jiang
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ford
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lichtiger
authors.forename: L Jeffrey
authors.initials: LJ
authors.lastname: Medeiros
authors.forename: Peter
authors.initials: P
authors.lastname: McLaughlin
authors.forename: Fernando
authors.initials: F
authors.lastname: Cabanillas
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Sarris
mesh_terms: D001773:Blood Cells / Q000378:metabolism*; D001782:Blood Donors; D001853:Bone Marrow / Q000378:metabolism; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D004247:DNA / Q000032:analysis; D006801:Humans; D008224:Lymphoma, Follicular / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014178:Translocation, Genetic
chemical_list: D004247:DNA