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With the goal of finding serological markers to monitor patients with early- as well as late-stage melanoma, we compared the levels of the cytoplasmic melanoma-associated antigens (CYT-MAA) and high-molecular-weight melanoma-associated antigen (HMW-MAA) in the sera of melanoma patients and controls. Using double-sandwich ELISA, we measured levels of both antigens in 117 patients and in 62 age- and sex-matched controls. Patients were stratified into four risk group based on stage of the disease. Serum levels of both markers were significantly higher in melanoma patients than in controls. CYT-MAA was the more sensitive marker, with 61% of patients showing elevated levels regardless of the stage of disease. HMW-MAA was elevated in 29%. Elevated CYT-MAA was also significantly correlated with poorer clinical outcome. By multivariate analysis (adjusting for stage and age), patients who had elevated CYT-MAA were 81% more likely to recur than patients with undetectable levels (hazard ratio=1.81, 95% CI=[1.07, 3.06], p-value=0.03). Elevated levels of HMW-MAA did not correlate with poor prognosis. These results suggest that both CYT-MAA and HMW-MAA are serum markers for residual melanoma in patients with resected disease. Furthermore, CYT-MAA appears to be a prognostic marker of clinical outcome in melanoma vaccine-treated patients.

Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000

Summary The cloning of genes encoding melanoma antigens has opened new possibilities for the treatment of patients with cancer; however, most tumor rejection antigens recognized by tumor infiltrating lymphocytes are the products of genes that are also expressed by normal melanocytes. Hence, a large set of antigenic determinants of the self have not induced self-tolerance and these peptide determinants furnish target structures for immune responses directed against tumors. The notion that the immunotherapeutic targets involved in cancer regression comprise normal differentiation antigens is stressed by the association between vitiligo-like leukoderma, due to destruction of normal melanocytes, and melanoma regression, due to destruction of cancer cells. Nevertheless, this is the first report to demonstrate by means of a new technique based on reverse transcription polymerase chain reaction and denaturing gradient gel electrophoresis, the presence of clonally expanded T cells with identical BV regions in areas of destruction of both normal and neoplastic cells.

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, complemetary-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 β-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.

104 Melanoma associated antigen A3 (MAGE-A3) influences proliferation of metastatic squamous cell carcinoma (SCC) in vitro A Santana, N Roudiani, J Therrien, JH Lee, D Felsen and J Carucci 1 Dept. of Derm., NYU Langone Med. Ctr., New York, NY, 2 Stiefel Laboratories, Research Triangle Park, NC and 3 Inst. for Ped. Urol., Weill Cornell Medicine, New York, NY In order to identify novel targets for cancer immune therapy, our lab has profiled the expression of cancer testis antigens (CTA) in primary human SCC. CTAs are immunogenic and may be suitable targets for immunotherapy, or as biomarkers for aggressive biological behavior. We recently showed that MAGE-A3 is highly expressed in aggressive SCC with poor prognosis. We hypothesized that MAGE-A3 may be a novel target for immunotherapy and/or a biomarker for metastatic SCCs. In this study we determined MAGE-A3 expression in and the effect of antibody treatment on growth of human (A431) and murine (Pam 212 and LY2, BALB/c) SCC cell lines. Pam 212 cells develop locally invasive SCC in syngeneic hosts and LY2 were isolated from a rare Pam 212 metastasis (Yuspa et al., JID, 1983; Dong et al., J Cell Biochem 1997). A431, Pam 212, and LY2 cell lines express MAGE-A3 at the transcript and protein level as measured by qRT-PCR and immunoblot, respectively. Moreover, A431 SCC growth and proliferation was significantly inhibited after MAGE-A3 antibody treatment measured by MTT (P 0.001) and crystal violet (P 0.001). We found that cell growth and proliferation of locally invasive Pam 212 cells was unaffected by anti-MAGE-A3 treatment. However, metastatic tumor derived LY2 cell growth and proliferation was significantly inhibited after MAGE-A3 antibody treatment measured by MTT (P 0.01) and crystal violet (P 0.01). MAGE-A3 expression could potentially serve both as a cancer biomarker and vaccine candidate due to its expression by SCC cells in vitro. Interestingly, MAGE-A3 antibody treatment inhibited the growth of A431 and LY2, SCCs that metastasize in vivo. These results suggest that metastatic SCC may rely on signaling events downstream of MAGE-A3 to support proliferation. Ongoing studies focus on elucidation of pathways involved in MAGE driven SCC proliferation in vivo using immune competent BALB/c mice. 105 Keratinocyte p38a loss results in increased tumor initiation, decreased malignant progression, and altered tumor type specification during two-stage chemical carcinogenesis in murine skin A Kiss, AC Koppel, C Cataisson, J Anders, S Yuspa, P Bible, M Kellett, MI Morasso and T Efimova 1 Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, 2 Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD and 3 Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD The p38a signaling pathway not only regulates the inflammation and stress responses, but also controls, in a context-specific manner, the homeostatic balance of cell proliferation, differentiation and survival, deregulation of which is known to contribute to tumor development. Studies in mouse models revealed both tumor-suppressing and tumor-promoting roles of p38a, depending on tissue and tumor type, and tumor stage. However, the in vivo functional contributions of p38a to the skin carcinogenesis remain incompletely understood. We explored the in vivo role of p38a in two-stage chemical skin carcinogenesis using mice with conditional epidermal keratinocyte-specific genetic ablation of p38a (p38a-cKO). Mutant mice exhibited a significantly increased tumor multiplicity, and, in addition to characteristic benign and malignant squamous tumors, developed benign well-differentiated sebaceous adenomas containing a signature activating Hras mutation. Only 4% of tumors in p38a-cKO mice underwent malignant conversion to squamous cell carcinomas (SCCs), compared with 38% of tumors in wild-type (WT) mice (p

This study elucidates the nature of antigens recognized by monoclonal antibody (MoAb) HMSA-2, which was developed against human melanosome-associated antigen (HMSA) of malignant melanoma (Maeda and Jimbow, Cancer 59:415–423, 1987). Through flow cytometry analyses, indirect immunoprecipitation of antigen biosynthetically labeled with 35 S-methionine, enzyme-linked immunosorbent assays and immunoelectron microscopy, we found that a) the antigens recognized by MoAb HMSA-2 were melanosomal matrix glycoproteins; b) these antigens were expressed mainly in the cytoplasm, although they could also be detected on the cell surface; c) the cytoplasmic expression of MoAb HMSA-2 was cell-cycle dependent; d) large amounts of these antigens were released into culture supernatants; e) MoAb HMSA-2 immunoprecipitated two major glycoproteins with molecular weights of 94 and 53 kDa from culture supernatants, and f) both components have complex N-linked oligosaccharide chains with sialic acid, suggesting that these melanosomal proteins are derived from the transcisternae of the Golgi. These human melanosome-associated antigens may prove useful not only for studying the immunobiology of melanogenesis, but also for the immunodiagnosis of melanocytic disorders.

Fifty-five primary and 33 metastatic surgically removed melanoma lesions were stained in indirect immunoperoxidase with anti HLA-DR, DQ, and DP monoclonal antibodies and with the monoclonal antibody CL203.4 to a 96-K melanoma associated antigen (MAA). The latter antigen may represent a marker to monitor susceptibility of melanoma cells to modulation by IFN-gamma, because it is highly susceptible to induction by IFN-gamma. In primary melanomas 44%, 29%, 10%, and 55% of the lesions tested were evidently stained by anti HLA-DR, DQ, DP, and 96-K MAA monoclonal antibodies, respectively. A statistically significant association (P less than 0.01) was demonstrated between the degree of intratumoral lymphocytic infiltrate and the expression of HLA-DR and HLA-DQ antigens. In addition, a high degree of concordance in the reactivity pattern of individual lesions stained for HLA-DR antigens and for the 96-K MAA was found. In metastases 64%, 33%, 47%, and 100% of the lesions tested were evidently stained by anti HLA-DR, DQ, DP, and 96-K MAA monoclonal antibodies, respectively. This study indicates that HLA-DR and HLA-DP antigens are expressed in a higher percentage of metastatic than of primary melanomas and that there is no marked difference in the expression of HLA-DQ antigens between primary and metastatic melanomas. The data suggest that the regulatory mechanisms which control the expression of HLA-DR and DP antigens in primary and metastatic melanoma lesions are different. Locally produced IFN-gamma may play a role in the regulation of HLA Class II antigens in primary melanomas.

The species-specific and the interspecies cross-reactive melanoma antigenic determinants are defined by the monoclonal antibodies raised by syngeneic immunizations. The two types of monoclonal antibodies (M562 or M622 and M2590) were obtained by the fusion of P3U1 murine myeloma cell lines and spleen cells of C57BL/6 mice hyperimmunized with MMC-treated syngeneic B16 melanoma cells. The M2590 antibody recognizes the cross-species melanoma determinant commonly shared among at least mouse, hamster, and human, while the M562 or M622 antibody reacts with the mouse (B16) melanoma antigenic determinant. The immunochemical and physiochemical characteristics of the melanoma antigens on SDS-PAGE analyses show that these two characteristic determinants are present on the same molecule (molecular weight of 31,000) of a glycoprotein. Furthermore, the interspecies cross-reactive melanoma antigenic determinants are possibly composed of the sugar moiety, whereas the species-specific determinants seem to be proteinaceous in nature.

In previous studies the monoclonal antibody (MoAb) M2590 elicited in C57/BL6 mice with the syngeneic melanoma cell line B16 has been shown to recognize a 31K glycoprotein expressed by human melanoma cell lines. The present study has shown that the MoAb M2590 cross-reacts with surgically removed benign and malignant lesions of melanocyte origin. The reactivity pattern of the MoAb M2590 with these lesions is different from that of the anti-high-molecular weight-melanoma associated antigen (HMW-MAA) MoAb 225.28S, of the anti-115K MAA MoAb 345.134S, and of the anti-100K MAA MoAb 376.96S, which were elicited with human melanoma cell lines. In particular, the MoAb M2590 reacts with blue nevi. The MoAb M2590-defined MAA, like other types of MAA, is heterogeneous in lesions removed from different patients, in autologous lesions removed from different anatomic sites, and in cells within a lesion. The distribution of the MoAb M2590-defined MAA in normal tissues and in tumors of nonmelanocyte origin is broader than that of the HMW-MAA, but is similar to that of the 115K MAA and of the 100K MAA. The results of this investigation suggest that immunization with xenogeneic melanoma cells may broaden the range of specificity of antihuman MAA MoAbs and provide information about the phylogenetic evolution of MAAs.

Using monoclonal antibodies, recombinant human gamma-interferon, and fluorescence-activated cell sorter, 2 human melanoma cell lines (KHm-1/4 and A101D) were examined quantitatively for HLA-DR and 97-kD melanoma-associated antigen (p97) expression throughout the cell cycle. Two-color flow cytometric analysis showed that the mean cell volume increased (KHm-1/4, 2.6 times; A101D, 3.6 times) during the progression of the cell cycle, and that fluorescence intensity of HLA-DR and p97 correlated well with cell volume, i.e., both antigens were maximally detected during the G2-M phase. The density of HLA-DR and p97 on the cell surface remained relatively constant throughout the cell cycle with the exception that cells in S phase showed a slightly lower density compared with those in G0/G1 and G2-M phases. gamma-Interferon treatment (500 IU/ml, 72 h) increased HLA-DR+ cells (KHm-1/4, 65% to 89%; A101D, 34% to 84%) and p97+ cells (KHm-1/4, 8% to 12%; A101D, 19% to 35%). Increased antigen densities were also relatively constant throughout the cell cycle as in nontreated cells. Cells treated with gamma-interferon tended to accumulate at G0/G1 phase (KHm-1/4, 21% to 37%; A101D, 17% to 53%), and had a reduced cell volume (0.82-0.95 times) throughout cell cycle. This study revealed that both melanoma cell lines showed heterogeneity in the expression of HLA-DR and p97, and that this heterogeneity was influenced, at least in part, by cell cycle and immunologic events such as gamma-interferon treatment.

Based on melanoma pathogenesis, phenotypic dynamics in pigment cell tumor progression detected with 11 MoAb have been defined. Anti-melanosomal A4F11 antibody reacts with every type of pigment cell tumor tested except for a few specimens. TNKH1 and anti-K.1.2 antibodies recognize nevocytic benign to premalignant tumors. HLA-DR, A.1.43, and A.10.33 antigens are expressed in advanced melanomas. Staining with anti-ganglioside GM3 and GD3 antibodies, M2590 and 4.2, respectively, reveals that most pigment cell tumors express gangliosides GM3 and GD3. But A2B5 antibody, which detects some polysialogangliosides such as GQ1C, reacts with highly progressed melanoma cells. Anti-ras p21 antibodies, RASK-3 and RASK-4, react with malignant melanomas and their premalignant lesions. These findings suggest the following: A4F11 is a universal marker of pigment cell tumors. TNKH1 and anti-K.1.2 antibodies might not be markers of melanocytic tumors but of nevocytic benign to premalignant tumors. Melanoma cells express gangliosides GM3 and GD3 as common pigment cell antigens and synthesize aberrant polysialogangliosides. Anti-ganglioside MoAb, including A2B5, are possible markers of the level of malignancy in melanoma cells like anti-A.1.43 and anti-A.10.33 antibodies. Enhanced ras p21 expression already appears on premalignant pigment cells.