MART-1 and gp100 are prototypical melanoma antigen (Ag), but their clinical use as vaccines or as targets of cytotoxic lymphocytes achieved modest success. Possible explanations could be that as MART-1 and gp100 are melanocyte differentiation Ag, clonogenic Ag-non-expressing cells would be spared by immune effectors, or that clonogenic cells would be intrinsically resistant to cytotoxic lymphocytes. We therefore analyzed the proliferative status of MART-1/gp100-expressing and -non-expressing cells in biopsies, and the clonogenicity and sensitiveness to cytotoxic lymphocytes of the human cutaneous melanoma cell lines MEL-XY1 and MEL-XY3. Analysis of MART-1/gp100 and Ki-67 expression in 22 melanoma tumors revealed that MART-1/gp100-expressing and -non-expressing cells proliferated competitively. MART-1, gp100, tyrosinase, and CD271 expression were studied in MEL-XY1 and MEL-XY3 colonies. At 7 days, colonies displayed positive, negative, and mixed expression patterns. By 14 days, colonies of different sizes developed, showing cells with different clonogenic potential, and Ag were downregulated, suggesting Ag plasticity. Subcloning of MEL-XY1 colonies showed that Ag expression varied with time without interfering with clonogenicity. Finally, clonogenic, MART-1/gp100-expressing cells were lysed by specific CD8 lymphocytes. Thus, MART-1 and gp100 expression and plasticity would not interfere with proliferation or clonogenicity, and clonogenic cells may be lysed by cytotoxic lymphocytes.

Inasmuch as Scientific Criticism and Polemic are of the Greatest significance for the development of every branch of historical science, including, of course, the study of Latin America, I should like to offer a few thoughts on this subject.Soviet historians of Latin America, just as scholars in other fields, attach great significance to the views of specialists everywhere-be they their colleagues in the USSR or abroad; be they Marxists or non-Marxists. Even if we hold different philosophical and methodological positions than our opponents, we are always ready to lend an attentive ear to critical observations. Disagreement with the world view or general historical conception of one or another of our foreign critics by no means prevents us from recognizing the correctness of his views on given problems, provided that the viewpoint in question is convincingly argued and scientificallydemonstrated. Even though we reject any questioning of the basic conceptions advanced by us, we are still able to accept admonitions and to seek a kernel of reason in otherwise totally inadmissible criticisms.

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Background and objectives: Cutaneous melanoma (CM) is the neoplasia with the fastest growing incidence worldwide. When CM metastasizes, only a minority of patients may be cured. However, CM is an immunogenic tumor. MART-1 and gp100 are prototypical melanoma Ag, since in HLA-A0201 patients (40% Latin Americans), most tumor-infiltrating lymphocytes are directed against them. However, Ag-specific therapies, including vaccines or adoptive therapy with cytotoxic T lymphocytes (CTL), have achieved modest success. Therefore, mechanisms of tumor resistance still need to be addressed. One possibility is that clonogenic Ag-negative cells would be spared by immune effectors. Thus it is important to discern whether Ag heterogeneity is due to the presence of differentiated cells with limited proliferative potential (cancer stem cell, CSC model), or to Ag plasticity independent from proliferation (phenotypic plasticity model). Another possibility is that clonogenic cells would be intrinsically resistant to CTL. Therefore, we studied the relationship between MART-1 and gp100 expression and proliferation in CM cells in primary tumors and metastases. We analyzed Ag expression in clonogenic cells (CC), including early melanocyte differentiation Ag (MD-Ag) MART-1 and gp100, late MD-Ag tyrosinase, and CSC marker CD271; and CC sensitivity to specific CTL clones. Methods and Results: Analysis of MART-1/gp100 and the proliferation marker Ki-67 expression in 15 primary and seven metastatic CM biopsies by immunohistochemistry (IHC) revealed that MART-1/gp100 expressing and nonexpressing cells proliferated competitively, with no differences between primary and metastatic tumors (Wilcoxon tests, p<0.05). We studied colonies obtained from anchorage-independent growing clonogenic cells (CC) of two human CM cell lines, MEL-XY1 and MEL-XY3, as a model of in vitro proliferation. Two growth stages were analyzed: 7-day colonies, with most cells Ki-67+, and 14-day colonies, with colonies of different sizes, many of them with proliferative cells in the periphery and necrotic cores. MART-1, gp100, tyrosinase, and CD271 expression were studied in colonies by IHC. By 7 days, colonies displayed positive, negative, and mixed expression patterns. By 14 days, Ag were downregulated, suggesting Ag plasticity (t-test, p<0.05). Analysis of Ag expression in CC by FACS further supported these results (Kolgomorov-Smirnov test, p<0.05). Subcloning of MEL-XY1 colonies showed that MART-1 and gp100 expression varied with time without interfering with clonogenicity (t-test, p<0.05). We studied CpG methylation in Mart-1 promoter by MSP (methylation-specific PCR), and found that this mechanism would be involved in MART-1 heterogeneity in MEL-XY1 7-day CC (t-test, p<0.05). Finally MART-1/gp100 expressing CC were lysed by specific CTL (0.3% survived, t-test, p<0.05). Conclusions: The expression of MART-1 or gp100 is not associated to a diminished proliferative potential of CM cells. MART-1, gp100, tyrosinase, and CD271 expression do not interfere with clonogenic potential, and are endowed with considerable plasticity. Furthermore, CC expressing the proper Ag and HLA-class-l haplotype are not intrinsically resistant to lysis by CTL. Since MD-Ag-expressing and nonexpressing cells are proliferative and clonogenic, giving rise to colonies of several thousands of cells, both subpopulations should be taken into consideration as targets to eradicate tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B43.

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