Detection of clonal T cell receptor gamma gene rearrangements in cutaneousT cell lymphoma by LightCycler-polymerase chain reaction

Cutaneous T cell lymphoma is thought to be characterized by a monoclonal T cell infiltrate in the skin that can be detected by polymerase chain reacti on-based amplification of T cell receptor gamma gene rearrangements. We sou ght to establish a new, simple, and fast LightCycler-based real-time polyme rase chain reaction assay for the detection of monoclonality in cutaneous T cell lymphoma, which was suitable for routine laboratory application. Mono clonal T cell receptor gamma gene rearrangements were detected by polymeras e chain reaction with consensus primers using: (i) a thermocycler followed by polyacrylamide gel electrophoresis; (ii) a LightCycler followed by melti ng curve analysis; and (iii) a LightCycler and subsequent polyacrylamide ge l electrophoresis. The detection limit of monoclonal Jurkat T cells diluted in polyclonal peripheral blood mononuclear cells was: (i) 1-3% by thermocy cler-polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 10% by LightCycler-polymerase chain reaction and melting curve analysis; a nd (iii) 1% by LightCycler-polymerase chain reaction and polyacrylamide gel electrophoresis. In skin biopsies of 22 cutaneous T cell lymphoma patients , a monoclonal or biclonal T cell infiltrate was detected in: (i) 15 of 22 (68%) by thermocycler-polymerase chain reaction and polyacrylamide gel elec trophoresis; (ii) 13 of 22 (59%) by LightCycler-polymerase chain reaction a nd melting curve analysis; and (iii) 16 of 22 (72%) by LightCycler-polymera se chain reaction and polyacrylamide gel electrophoresis. All three techniq ues revealed negative results in skin biopsies from 26 patients with benign dermatitis. In conclusion, LightCycler-polymerase chain reaction and melti ng curve analysis is a fast, simple and specific method to detect monoclona l T cell infiltrates in cutaneous T cell lymphoma. Sensitivity of LightCycl er-polymerase chain reaction and polyacrylamide gel electrophoresis is slig htly higher compared with sensitivity of thermocycler-polymerase chain reac tion and polyacrylamide gel electrophoresis. Melting curve analysis, howeve r, is less sensitive compared with polyacrylamide gel electrophoresis, and in case of negative results of the melting curve analysis, it is recommende d to resolve LightCycler-polymerase chain reaction samples by gel electroph oresis.