Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR gamma-chain gene sequencing

Molecular analysis of T-cell receptor (TCR) chain rearrangement has recentl y become an attractive tool for demonstrating the clonal origin of cutaneon s T-cell lymphoma (CTCL) and for identifying the malignant clone at the mol ecular level. Over the past decade a number of attempts have been made to c ulture malignant CTCL cells using standard procedures and these attempts ha ve resulted in several cell Lines from the peripheral blood of Sezary syndr ome, mycosis fungoides and CD30(+) lymphoma patients. However, so far it ha s not been proven by sequence analysis that the cultured T cells truly repr esent the malignant cells. Aiming to functionally analyze the malignant T c ells at a clonal level, we generated a total of 150 T-cell clones (TCC) fro m lesional skin and peripheral blood of three patients with mycosis fungoid es and one patient with a CD30(+) lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using vario us conditions for T-cell stimulation by direct outgrowth or from skin speci mens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR gamma -chain sequences from clones of lesional skin with in vitro-generated TCC, With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin, Our resul ts indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrati ng T cells.