Rapid detection of clonal T-cell receptor-beta gene rearrangements in T-Cell lymphomas using the LightCycler-polymerase chain reaction with DNA melting curve analysis.
Various molecular methods have been developed to diagnose clonal T-cell receptor (TCR) gene rearrangements in clinical samples. Most polymerase chain reaction strategies for detecting clonal TCR gene rearrangements rely on either gel or capillary electrophoresis. However, a cumbersome manual transfer step separates amplification from analysis. Recently, we developed a novel polymerase chain reaction assay using the LightCycler system to detect clonal immunoglobulin heavy chain gene rearrangement. In the current study, we extend this work to include the TCR. We report that clonal TCR-beta (TCR-beta) gene rearrangements can be detected in less than 1 hour after preparing the DNA by measuring DNA melting immediately after amplification in a single closed capillary tube. We retrospectively studied 52 fresh-frozen tissue samples from patients clinically suspected of T-cell malignancy. A clonal TCR-beta gene rearrangement was detected in 14 samples by DNA melting curve analysis. When DNA melting was compared to the gold standard methods of Southern blot or denaturing gradient gel electrophoresis, it achieved a sensitivity equal to 71% and a specificity equal to 94%. We also compared melting curve analysis and polyacrylamide gel electrophoresis: melting curve analysis reached a sensitivity equal to 100% and a specificity equal to 97%. We conclude that DNA melting curve analysis in the LightCycler system has potential for clinical use as a new, ultra-fast method for the initial diagnosis of clonal TCR-beta gene rearrangements.
Yang
Xiao Yan
XY
Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, North 4, 111 East 210th St., Bronx, NY 10467, USA.
Xu
Dongsheng
D
Du
Juan
J
Kamino
Hideko
H
Rakeman
Jennifer
J
Ratech
Howard
H
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D005260:Female; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / Q000235:genetics*; D006801:Humans; D016399:Lymphoma, T-Cell / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
D004247:DNA