MART-1 (1-20)

Background: HLA-A2 tetramer flow cytometry, IFNgamma real time RT-PCR and IFNgamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods: Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126-35(27L), IFNgamma real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127-35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results: The validation process demonstrated that the HLA-A2 tetramer, IFNgamma real time RT-PCR, and IFNgamma ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNgamma response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Conclusion: Our results demonstrated the tetramer flow cytometry assay, IFNgamma real-time RT-PCR, and INFgamma ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.

Malignant melanomas of the vagina are rare tumors. In this study we present the clinicopathologic features and immunohistochemical analysis of 26 such cases seen in our institution over a period of 30 years. The patients' age ranged from 38 to 90 years (mean 60 years); three patients were premenopausal. Ethnicity was known in 24 patients: 20 white, 2 hispanic, 1 black, and 1 Asian. The most common presenting symptom was vaginal bleeding, followed by vaginal mass. Grossly, the tumor was polypoid-nodular in the majority of cases. The neoplastic cells were epithelioid in 15 cases and spindled in three cases; eight cases had both cell types. Vascular-lymphatic invasion was seen in six cases and perineural invasion was seen in four cases. S-100 was strongly and diffusely positive in 25 of 26 cases (96%). HMB-45 was strongly positive in 16 (62%), 3 (11%) were focally positive, 1 case showed a rare positive cell, and 6 (23%) were negative. With MART-1, 20 cases (77%) were strongly positive, 1 (4%) showed a rare weakly positive cell, and 5 (19%) were negative. Twenty-one cases (81%) expressed tyrosinase and 20 (77%) expressed microphthalmia transcription factor. Twenty cases were Chung's level IV, 3 were level III, and 2 were level II. The patients were treated as follows: anterior exenteration with or without lymph node dissection and with or without radiotherapy (RT) or chemotherapy (CT) (7 cases), wide local excision with or without lymph node dissection and RT/CT (10 cases), hysterectomy with vaginectomy with or without RT/CT (3 cases), vaginectomy with RT (1 case), RT (1 case), and RT and CT (1 case). One patient had palliative RT for the brain metastasis only. Follow-up was available in 23 patients ranging from 3 to 276 months (median 18 months). Local recurrence after primary treatment was seen in six patients and distant metastases in 11 patients. Fifteen patients died of the disease (3-83 months), 4 have no evidence of disease (5-24 months), and 4 are alive with disease (6-276 months). This study confirms the poor prognosis of patients with vaginal melanoma. S-100 remains the most sensitive marker for these tumors. HMB-45 is negative in 23% cases of vaginal melanoma. Tyrosinase and MART-1 are useful markers when S-100 is negative or only focally positive.

Objective: To assess the effects of the culture of melanoma cells in 3-dimensional (3D) architectures on their immunorecognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens. Summary background data: Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. We investigated the effects of 3D culture on the recognition of melanoma cells by antigen-specific HLA class I-restricted CTLs. Methods: Culture of HBL melanoma cells expressing Melan-A/Mart-1 tumor-associated antigen and HLA-A0201 on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates resulted in the generation of aggregates of 400- to 500-microm diameters containing on average 30,000 cells and characterized by slower proliferation, as compared with monolayer (2-dimensional) cultures. HLA-A0201 restricted Melan-A/Mart-127-35-specific CTL clones were used to evaluate tumor cell immunorecognition measured as specific IFN-gamma production. Comparative gene and protein expression in 2D and 3D cultures was studied by real-time PCR and flow cytometry, respectively. Overall differences in gene expression profiles between 2D and 3D cultures were evaluated by high-density oligonucleotide array hybridization. Results: HLA-A0201 restricted Melan-A/Mart-127-35 specific CTL clones produced high amounts of IFN-gamma upon short-term (4-24 hours) coincubation with HBL cells cultured in 2D but not in 3D, thus suggesting altered antigen recognition. Indeed, Melan-A/Mart-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures. Conclusions: Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL.