Sandra R. Reynolds

Stimulation of CD8+ T cell responses to MAGE‐3 and Melan A/MART‐1 by immunization to a polyvalent melanoma vaccine

A critical requirement for cancer vaccines is that they stimulate CD8+ T cell responses. In this study, we tested the ability of a polyvalent melanoma vaccine to induce CD8+ T cell responses to the melanoma associated antigens MAGE‐3 and Melan A/MART‐1. Fifteen HLA‐A2+ patients with resected malignant melanoma were immunized with the vaccine s.c. every 2–3 weeks. CD8+ T cells in peripheral blood reacting to HLA‐A2 restricted epitopes on MAGE‐3 (FLWGPRALV) and Melan A/MART‐1/(AAGIGILTV) were quantitated using a filter spot assay at baseline and following 4 immunizations. Vaccine immunization induced CD8+ T cells reacting to one or both of these peptides in 9 of the 15 (60%) patients. These cells were CD8+ and HLA‐A2 restricted, as reactivity was abrogated by monoclonal antibodies (MAbs) to CD8 and class I HLA, but not by anti‐CD4. All responding patients remained recurrence‐free for at least 12 months (median 15 months, range 12 to >21 months), whereas melanoma recurred within 3–5 months in non‐responders. The differences in outcome were unrelated to differences in disease severity or overall immunological competence between responders and non‐responders. Our results demonstrate directly that MAGE‐3 and Melan A/MART‐1 can stimulate CD8+ T cell responses in humans, and suggest that these responses are protective and surrogate markers of vaccine efficacy. Int. J. Cancer 72:972–976, 1997.

Identification of HLA-A*03, A*11 and B*07-restricted melanoma-associated peptides that are immunogenic in vivo by vaccine-induced immune response (VIIR) analysis.

With the discovery of increasing numbers of tumor antigens, there is a need to rapidly determine whether these antigens and the individual peptides they express are able to stimulate immune responses in vivo and thus, can be used to construct cancer vaccines. In this study we used the method of vaccine-induced immune response (VIIR) analysis to identify multiple immunogenic peptide epitopes derived from several melanoma associated antigens and presented by HLA-A*03, A*11 and B*07. Thirty-one patients with melanoma were immunized to a polyvalent vaccine containing multiple antigens, including MAGE-3, Melan A/MART-1, gp100 and tyrosinase. Their peripheral blood was tested for peptide-specific, vaccine-induced CD8+ T cell responses before and after immunization using an enzyme-linked immune spot (ELISPOT) assay with panels of peptides restricted by these three alleles. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*03, A*11 or B*07. Overall, 60% of the 20 peptides studied were recognized by at least one patient and 50% of the patients showed a vaccine-induced CD8+ T cell response to at least one peptide that matched their HLA specificity. We conclude that VIIR analysis is an effective strategy to directly identify immunogenic peptides that are good candidates for vaccine construction.

Multiple Breast Cancer Cell-Lines Derived from a Single Tumor Differ in Their Molecular Characteristics and Tumorigenic Potential

Background Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient’s breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment. Methods Five breast cancer cell lines were derived from a single patient’s primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fuorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. Results We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. Conclusions All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer.

Cytoplasmic melanoma-associated antigen (CYT-MAA) serum level in patients with melanoma: a potential marker of response to immunotherapy?

Simple, noninvasive methods are needed to follow effectiveness of new treatments in patients with melanoma. In our study, we examined cytoplasmic melanoma‐associated antigen (CYT‐MAA) serum level in melanoma patients during immunotherapy. Sera of 117 patients were assayed for CYT‐MAA by double‐sandwich ELISA before and during treatment with a polyvalent, shed antigen, melanoma vaccine. Vaccine‐treated patients included 30 with American Joint Committee on Cancer (AJCC) stage IIb or IIIa, 30 with stage IIc, IIIb or IIIc, 30 with resected stage IV and 27 with measurable stage IV disease. Prior to vaccine therapy, 63% of patients had elevated serum CYT‐MAA with high levels of antigen in all disease stages. After initiation of therapy, the level declined in more than 90% of the positive patients and fell below the positive cut‐off in 56% of these patients within 5 months. By contrast, there was no decline in CYT‐MAA serum level in 11 patients who served as untreated controls with melanoma. Multivariate analysis of the treated patients using accelerated failure time Weibull models adjusted for stage and age showed that patients whose CYT‐MAA serum level remained elevated during treatment were ∼3 times more likely to recur or progress than patients who were consistently below the positive cut‐off (hazard ratio = 3.42, 95% CI [1.38, 8.47], p = 0.0079). Measurement of CYT‐MAA serum level appears to show potential as an early marker of prognosis in patients with stages IIb to IV melanoma. Measurement of CYT‐MAA serum level during therapy could provide an intermediate marker of response in these patients.

Decrease in circulating tumor cells as an early marker of therapy effectiveness.

As melanoma cells are present in the circulation of many patients with this cancer, decreases in their number could provide an early indication of therapy effectiveness. To evaluate this possibility, we examined the effect of treatment with a melanoma vaccine on the number of melanoma cells present in the circulation. PCR was used to detect melanoma cells that expressed the melanoma-associated antigens MART-1, MAGE-3, tyrosinase and/or gp100 in 91 patients with melanoma. Melanoma cells that expressed one or more of these markers were present more often in advanced disease, i.e. in 80% of patients with advanced stage IV compared to in less than one-third of patients with less advanced disease. We then measured circulating melanoma cells in a subset of 43 of these patients who were treated with a polyvalent, shed antigen, melanoma vaccine. The vaccine contains multiple melanoma-associated antigens including MART-1, MAGE-3, tyrosinase and gp100. Immunizations were given intradermally q2-3 weeks x4 and then monthly x3. Prior to vaccine treatment, circulating melanoma cells were detected in 14 (32%) patients. Following 4 and 7 months of vaccine treatment, melanoma cells that expressed any of these markers were present in only nine (21%) and three (7%) of patients, respectively. Thus, vaccine therapy was associated with clearance of melanoma cells from the circulation in 78% of initially positive patients. As the number of these cells declined steadily with increasing length of therapy, it is unlikely that this was due to a random change in their number. Rather it suggests that the decline was a result of the therapy. These observations suggest that the presence of melanoma cells in the circulation is related to the extent of the melanoma, and that their disappearance may provide an early marker of the efficacy of therapy. However, the practical utility of assaying circulating tumor cells as a guide to the effectiveness of therapy or of prognosis will need to be confirmed by correlations with clinical outcome.