Catherine Wölfel

A mutated cyclin-dependent kinase 4 (CDK4) was identified as a tumor-specific antigen recognized by HLA-A2. 1-restricted autologous cytolytic T lymphocytes (CTLs) in a human melanoma. The mutated CDK4 allele was present in autologous cultured melanoma cells and metastasis tissue, but not in the patient's lymphocytes. The mutation, an arginine-to-cysteine exchange at residue 24, was part of the CDK4 peptide recognized by CTLs and prevented binding of the CDK4 inhibitor p16INK4a, but not of p21 or of p27KIP1. The same mutation was found in one additional melanoma among 28 melanomas analyzed. These results suggest that mutation of CDK4 can create a tumor-specific antigen and can disrupt the cell-cycle regulation exerted by the tumor suppressor p16INK4a.

Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.

Our understanding of pathways leading to antitumor immunity may depend on an undistorted knowledge of the primary antigenic targets of patients' autologous T cell responses. In the melanoma model derived from patient DT, we applied cryopreserved short-term autologous mixed lymphocyte-tumor cell cultures (MLTCs) in combination with an IFN-gamma enzyme-linked immunospot (ELISPOT) assay to cDNA expression screening. We identified three previously unknown peptides processed from melanosomal proteins tyrosinase (presented by HLA-A(*)2601 and -B(*)3801) and gp100 (presented by HLA-B(*)07021) and five neoantigens generated by somatic point mutations in the patient's melanoma. The mutations were found in the genes SIRT2, GPNMB, SNRP116, SNRPD1, and RBAF600. Peptides containing the mutated residues were presented by HLA-A(*)03011, -B(*)07021, and -B(*)3801. Mutation-induced functional impairment was so far demonstrated for SIRT2. Within MLTC responder populations that were independently expanded from the patient's peripheral blood lymphocytes of different years, T cells against mutated epitopes clearly predominated. These results document a high degree of individuality for the cellular antitumor response and support the need for individualizing the monitoring and therapeutic approaches to the primary targets of the autologous T cell response, which may finally lead to a more effective cancer immunotherapy.

We have undertaken a comparative study of the bioactivation of a panel of promutagens by V79 Chinese hamster cells genetically engineered to metabolic competence. In vitro micronucleus assays of the test agents in V79 cultures in the presence of an Aroclor induced rat S9 yielded positive results. In the genetically engineered cell lines, benzo[a]pyrene was metabolized specifically by the 3-methylcholanthrene inducible rat liver CYP1A1 (cell line XEM2) whereas cyclophosphamide increased the micronucleus frequency only in cultures expressing the phenobarbital inducible CYP2B1 (SD1). Following exposure to the mycotoxin sterigmatocystin, elevated frequencies of micronucleated cells were recorded in XEM2, SD1 and XEMd-MZ (expresses the isosafrole inducible CYP1A2) cells. The aromatic amine 2-amino-anthracene elicited a weak response in the cell line XEMd-MZ which expressed CYP1A2. This response was enhanced when this cDNA was expressed in a V79 variant cell strain which also possessed endogenous acetyltransferase activity. Upon exposure to tobacco particulate matter, a greater induction of micronuclei was observed in the XEM2 cell line compared to V79 cultures, implicating polycyclic aromatic hydrocarbons in addition to direct-acting compounds as causal agents in the genotoxicity of tobacco particulate matter. The cytokinesis blocked in vitro micronucleus assay provides a faster, simpler alternative to metaphase analysis, and kinetochore labelling techniques enable the discernment of both structural and numerical chromosome changes. The inclusion of metabolically competent test strains in the in vitro micronucleus assay therefore creates a powerful system for detecting genotoxins and may be extended to elucidate both mechanisms of bioactivation and modes of genotoxic insult.