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 Fluorescent 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 approximately 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.

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice.

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly x4 with -0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8(+) T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens.

Presence and prognostic significance of melanoma-associated antigens CYT-MAA and HMW-MAA in serum of patients with melanoma.

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.

Melanoma vaccines: what we know so far.

Vaccines are a promising but still experimental treatment for melanoma. They are intended to stimulate immune responses against melanoma and by so doing, increase resistance against and slow the progression of this cancer. Key requirements for vaccines to be effective are that they contain antigens that can stimulate tumor-protective immune responses and that some of these antigens are present on the tumor to be treated. Unfortunately, these antigens are still not known. To circumvent this problem, polyvalent vaccines can be constructed containing a broad array of melanoma-associated antigens. Several strategies are available to construct such polyvalent vaccines; each has advantages and disadvantages. Clinical trials have shown that vaccines are safe to use and have much less toxicity than current therapy for melanoma. Vaccines can stimulate both antibody and T-cell responses against melanoma, with the type of response induced, its frequency, and its magnitude depending on the vaccine and the adjuvant agent used. A growing body of evidence suggests that vaccines can be clinically effective. This evidence includes correlations between vaccine-induced antibody or T-cell responses and improved clinical outcome, clearance of melanoma markers from the circulation, improved survival compared to historical controls, and most convincingly, two randomized trials in which the recurrence-free survival of vaccine-treated patients was significantly longer than that of control groups.

Changes in the presence of multiple markers of circulating melanoma cells correlate with clinical outcome in patients with melanoma.

PURPOSE: Melanoma cells can be found in the circulation of patients with melanoma. The following study was conducted to examine whether changes in their presence could provide an early marker of response to therapy. EXPERIMENTAL DESIGN: We measured the presence of several markers of melanoma cells in the peripheral blood of 118 patients with resected stage IIb, III, or IV melanoma before and after immunotherapy with a polyvalent, shed antigen, melanoma vaccine using reverse transcription-PCR assays for tyrosinase, gp100, MART-1, and MAGE-3. Assays were conducted at baseline and after 3, 5, and 11 months of therapy. RESULTS: Overall, 47% of patients were positive for at least one marker during the study. Before vaccine treatment, circulating melanoma cell markers were present in 23% of patients. After 5 and 7 months of vaccine therapy, the proportion of patients with circulating markers decreased by 27% and 55%, respectively (P for trend = 0.02). The recurrence-free survival of patients whose melanoma cell markers disappeared during vaccine treatment was significantly longer than that of patients in whom they increased, i.e., the percentage of patients who were recurrence free at 1 year was 80% versus 58% (P = 0.03). CONCLUSIONS: Therapy with a polyvalent melanoma vaccine was associated with clearance of melanoma cell markers from the circulation, and the clearance was associated with an improved prognosis. These findings suggest that the sequential assay of tumor cells in the circulation by reverse transcription-PCR may provide an early indication of the effectiveness of cancer therapy.

Vaccine-induced CD8+ T-cell responses to MAGE-3 correlate with clinical outcome in patients with melanoma.

PURPOSE: Vaccine-induced antitumor CD8+ T-cell responses are believed to play an important role in increasing resistance to melanoma. The following study was conducted to examine whether these responses are associated with improved clinical outcome in melanoma vaccine-treated patients. EXPERIMENTAL DESIGN: We measured CD8+ T-cell responses to gp100, MART-1, MAGE-3, and tyrosinase by enzyme-linked immunospot assay in peripheral blood of 131 HLA-A*01- or HLA-A*02-positive melanoma patients before and after immunization to a polyvalent, shed antigen, melanoma vaccine, and correlated the results with clinical outcome. RESULTS: Fifty-six percent of patients had a vaccine-induced CD8+ T-cell response to at least one of the four antigens. Recurrences were significantly reduced in patients with vaccine-induced responses to MAGE-3 (hazard ratio, 0.42; 95% confidence interval, 0.18-0.99; P = 0.03) by the Cox proportional hazard model but were unrelated to responses to the other three antigens. Patients with a preexisting response to any of the four antigens were significantly more likely to have a further vaccine-boosted response to that same antigen (P < 0.0001-0.036). CONCLUSIONS: There was a correlation between vaccine-induced CD8+ T-cell responses to melanoma-associated antigens and improved clinical outcome, but the correlation depended on the antigen against which the response is directed. The only significant correlation was with responses to MAGE-3.