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 recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30+ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30+ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens 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 results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells.