Detection of T-cell receptor-gamma gene rearrangement in lymphoproliferative disorders by temperature gradient gel electrophoresis.
OBJECTIVE
Polymerase chain reaction amplification of DNA for T-cell receptor (TCR) gene rearrangement analysis is helpful in the evaluation of T-cell lymphoproliferative disorders. Detection of polymerase chain reaction products is limited by the poor resolution of bands analyzed by agarose or polyacrylamide gel electrophoresis. To improve the detection of a clonal T-cell population, we used temperature gradient gel electrophoresis (TGGE) as an alternative method for analysis of TCR gene rearrangement.


METHODS
One hundred eighteen archival DNA samples were randomly selected based on previous Southern blot analysis results. Samples included 58 T-cell neoplasms with positivity for TCR beta gene rearrangement, 22 cases of reactive hyperplasia with germline pattern for both TCR beta and J(H), and 38 patients with B-cell lymphoma. MOLT-16, a T-cell lymphoblastic cell line, was used for the sensitivity assay. Polymerase chain reaction was performed using GC-clamped multiplex primers to amplify the TCR gamma locus and was analyzed by TGGE. The range of temperature gradients was empirically determined for optimal resolution of bands.


RESULTS
The sensitivity of TGGE was 0.1% when DNA from the MOLT-16 cell line was serially diluted with DNA from reactive lymphoid tissue. Fifty-four (93%) of 58 T-cell neoplasms with TCR beta gene rearrangements showed rearrangement patterns by TCR gamma TGGE, and only 1 of 60 samples (reactive or B-cell lymphomas) showed evidence of gene rearrangement by TGGE. Patients with T-cell neoplasm and involvement of multiple sites showed an identical migration pattern by TGGE analysis.


CONCLUSIONS
We demonstrate that TGGE is an effective method for analysis of TCR gene rearrangement in the evaluation of nodal and extranodal lymphoid lesions.
Alkan
S
S
Department of Pathology, Loyola University Medical Center, Maywood, IL 60153, USA.
Cosar
E
E
Ergin
M
M
Hsi
E
E
D001202:Ascitic Fluid / Q000737:chemistry; D015139:Blotting, Southern; D001853:Bone Marrow / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000235:genetics; D016399:Lymphoma, T-Cell / Q000235:genetics; D008232:Lymphoproliferative Disorders / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm