The application of fluorescence-based PCR and PCR-SSCP to monitor the clonal relationship of cells bearing the t(14;18)(q32;q21) in sequential biopsy specimens from patients with follicle center cell lymphoma.
This study evaluates the utility of fluorescence-based polymerase chain reaction (PCR) and PCR-SSCP methodologies to monitor the clonal relatedness of cells with bcl-2 major break point region (mbr)/JR fusion sequences in sequential samples from patients with follicular lymphoma (FL). Fluorescence-tagged PCR products from 2-4 sequential samples from seven FL patients were resolved in acrylamide gels and analyzed on an Applied Biosystems' automated DNA sequencer equipped with Genescan software. The amplicons were sequenced directly using automated DNA sequencing to obtain the precise amplicon size and base sequence. Fluorescence-based PCR-single-strand conformation polymorphism (SSCP) analysis performed to distinguish amplicons of similar size but of different base sequence. Amplification products differing by as few as 5 bp resolved clearly under fluorescent PCR assay conditions making possible by visual inspection alone the distinction of two products that otherwise appeared to be of similar size by conventional gel electrophoretic methods. The size of the amplicons as determined by Genescan software correlated exactly with the sizes generated by sequence analysis confirming the precision and accuracy of the fluorescent PCR assay. Under nondenaturing conditions, the mobility profiles of the amplicons from sequential samples with identical base sequence remained indistinguishable, whereas amplicons of similar size but of dissimilar base sequence from different patients exhibited distinct migration patterns. Thus, this study demonstrates that a combination of fluorescent PCR and PCR-SSCP assays for the detection of the t(14;18) provides an accurate measure of clonal relationship based on molecular size and sequence similarities without involving radiolabeling and sequencing strategies. Furthermore, the demonstrated preservation of junctional sequences across sequential biopsy specimens validates the use of PCR in the monitoring of minimal residual disease and eliminates concern about the detection of secondary, non-tumor-related translocations.
Luthra
R
R
Division of Pathology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
McBride
J A
JA
Hai
S
S
Cabanillas
F
F
Pugh
W C
WC
D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D002999:Clone Cells; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005500:Follow-Up Studies; D006801:Humans; D008224:Lymphoma, Follicular / Q000235:genetics* / Q000473:pathology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D014178:Translocation, Genetic
D005456:Fluorescent Dyes