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Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of cancer cell-specific CTL epitopes. Despite reported evidence for tumor-reactive cytotoxicity in CTCL patients, the nature of the recognized determinants remains elusive. The clonotypic TCR of CTCL cells is a promising candidate tumor-specific Ag. In this study, we report that the clonotypic and framework regions of the TCRs expressed in the malignant T cell clones of six CTCL patients contain multiple peptides with anchor residues fitting the patients’ MHC class I molecules. We demonstrate that TCR peptide-specific T cells from the blood of healthy donors and patients can be induced to become cytotoxic effectors after repeated stimulation with 6 of 11 selected peptides with experimentally proven affinity for HLA-A*0201. Importantly, 4 of these 6 CTL lines reproducibly recognize and lyse autologous primary CTCL cells in MHC class I/CD8-dependent fashion. These tumoricidal CTL lines are directed against epitopes from V, hypervariable, and C regions of TCRα. We therefore conclude that recombined as well as V framework regions of the tumor cell TCRs contain predictable epitopes for CTL-mediated attack of CTCL cells. Our data further suggest that such peptides represent valuable tools for future anti-CTCL vaccination approaches.

The use of tyrosinase-based polymerase chain reaction (PCR) tests for the detection of circulating tumour cells in the blood of melanoma patients has led to highly controversial results. We here report on the analysis of 120 blood samples from 76 stage I to IV melanoma patients using a new MART-1/Melan-A PCR system in conjunction with the tyrosinase-specific assay reported in the literature. While there were no positive results in localized disease (stages I and II), identification of specific PCR products in stage III melanoma patients was restricted to the MART-1/Melan-A tests, with positive results in 11% (two out of 19) of the blood specimens analysed. Stage IV melanoma patients presented with the highest incidence of detectable mRNA levels, with positive results for tyrosinase in 38% (12 out of 32) and for MART-1/Melan-A in 22% (seven out of 32). By delineating 64 follow-up specimens covering sampling periods of up to 33 weeks, stable mRNA expression profiles were identified in nearly 95%. Four patients, however, showed PCR changes towards positive MART-1/Melan-A expression that were linked to metastatic melanoma progression. Taken together, PCR tests for tyrosinase and MART-1/Melan-A seem to lack sufficient detection frequencies for the routine monitoring of melanoma disease. Regarding the link between MART-1/Melan-A seroconversion and the development of metastatic disease, further studies are needed to clarify the clinical value of this observation.

At present, very little is known about the types and heterogeneity of T cell responses and immunodominant epitopes of melanoma-associated antigens at coexisting sites of primary melanoma and metastatic lesions. To address this issue, we compared the T cell receptor (TCR) gene usage, complementary-determining region 3 diversity, and melanoma-associated antigens expression patterns of primary and metastatic melanoma specimens from three patients with partially homologous HLA class-1 types. Results obtained showed an overall predominance of a very limited number of TCRV regions with AV13 and BV14 being most frequently overexpressed. Sequencing of the dominating TCR transcripts confirmed the restricted usage of certain TCR specificities and, in two of the three patients, identified several identical TCR clonotypes at more than one metastatic site. Nevertheless, we failed to detect TCR transcripts that were common to all tumor deposits in a given patient and, within the majority of coexisting metastases, tumor-infiltrating lymphocytes preferentially used individual site-specifically expanded TCR beta-chain VJ segment combinations. This occurrence of individual responses simultaneously executed at and influenced in their specificity by the different sites of tumor growth, has important implications for the type of strategies chosen in the development of efficacious vaccines for patients with metastatic melanoma.

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