Immunotherapies using cancer-testis (CT)antigensin 1 of these patients. These allogeneic immune responses were not detectable in pretransplantation samples and in the patients' stem cell donors, indicating that CT antigens might indeed represent natural targets for graft-versus-myeloma effects. Immune responses induced by alloSCT could be boosted by active CT antigen-specific immunotherapy, which might help to achieve long-lasting remissions in patients with MM.

Purpose: Reliable data on the persistence of tumor expression of cancer-testis (CT) antigens over time and consequent analyses of the effect of CT antigen expression on the clinical course of malignancies are crucial for their evaluation as diagnostic markers and immunotherapeutic targets. Experimental Design: Applying conventional reverse transcription-PCR, real-time PCR, and Western blot, we did the first longitudinal study of CT antigen expression in multiple myeloma analyzing 330 bone marrow samples from 129 patients for the expression of four CT antigens (MAGE-C1/CT7, MAGE-C2/CT10, MAGE-A3, and SSX-2). Results: CT antigens were frequently and surprisingly persistently expressed, indicating that down-regulation of these immunogenic targets does not represent a common tumor escape mechanism in myeloma. We observed strong correlations of CT antigen expression levels with the clinical course of myeloma patients as indicated by the number of bone marrow^residing plasma cells and peripheral paraprotein levels, suggesting a role for CT antigens as independent tumor markers. Investigating the prognostic value of CT antigen expression in myeloma patients after allogeneic stem cell transplantation, we found that expression of genes, such as MAGE-C1, represents an important indicator of early relapse and dramatically reduced survival. Conclusions: Our findings suggest that CTantigens might promote the progression of multiple myeloma and especially MAGE-C1/CT7, which seems to play the role of a ''gatekeeper'' gene for other CT antigens, might characterize a more malignant phenotype. Importantly, our study also strongly supports the usefulness of CTantigens as diagnostic and prognostic markers as well as therapeutic targets in myeloma.Cancer-testis (CT) antigens are a diverse group of genes of which more than 40 families have been identified during the past 15 years (1). CT antigens have been considered promising targets for immunotherapy of human malignancies based on their tumor-restricted expression and on their immunogenicity in cancer patients. Both of these characteristics could render CT antigens important diagnostic and prognostic markers; however, thus far, this aspect of the biology of CT antigens has not intensively been explored.Although an impressive number of studies have shown expression of CT antigens in a large variety of human tumor types on the RNA as well as on the protein level (2), there has not been a single study analyzing the expression of CT antigens in a human cancer over time. This seems surprising because reliable data on the persistence of tumor-related CT antigen expression are a prerequisite for the evaluation of these tumorspecific proteins as diagnostic markers and immunotherapeutic targets, especially considering data suggesting that immunoselection might lead to down-regulation or loss of CT antigen expression in cancer patients (3,4).We have recently shown that CT antigens are commonly expressed and are capable of inducing antibody-mediated and T-cell -mediated immunity in m...

BackgroundVery little is known about the number and function of immunosuppressive CD4

BACKGROUND: Based on their immunogenicity and restricted tissue expression, cancer-testis (CT) antigens seem ideal targets for active immunotherapies. We have recently reported a frequent expression of CT antigens in multiple myeloma (Blood2007;109:1103–12). However, CT antigen expression has not been examined over time in patients with multiple myeloma (MM) or other malignancies. This seems surprising, since data on the persistence of CT antigen expression are needed in order to evaluate their usefulness as diagnostic markers and targets of immunotherapeutic approaches, especially in the case of minimal residual disease (MRD). METHODS: We analyzed 336 bone marrow (BM) samples obtained from 130 myeloma patients for expression of CT antigens. Samples of 41 healthy BM donors were used as controls. Expression of MAGEC1/CT7, MAGEC2/CT10, MAGEA3, and SSX2 was examined using qualitative RT-PCR. Real-time PCR was applied to quantify MAGEC1/CT7 expression over time. RESULTS: In MM patients with significant tumor load (>= 10% BM plasma cells), MAGEC1/CT7 was expressed in 69%, MAGEA3 in 55%, MAGEC2/CT10 in 44%, and SSX2 in 14% of samples. CT antigens were not expressed in healthy BM. Since expression of the remaining CT antigens was rarely observed without expression of MAGEC1/CT7, this CT antigen seemed to fulfill a ‘gatekeeper’ function. Expression of CT antigens correlated positively with clinical stage and was increased in recurrent disease compared to newly diagnosed MM. Noticeably, 76% of samples from patients who had not responded to therapy, 28% of samples from patients in partial remission, and only 8% of patients in complete remission expressed at least one CT antigen. Samples of patients who had received chemotherapy alone more frequently expressed CT antigens than samples of patients post autologous stem cell transplantation. The lowest frequency of CT antigen expression was observed in patients post allogeneic stem cell transplantation. Remarkably, in case a patient with significant tumor load had expressed a CT antigen once, 97% (MAGEC1/CT7), 88% (SSX2), 81% (MAGEA3), 67% (MAGEC2/CT10) of the subsequent BM samples of the same patient were positive for the respective antigen. When we analyzed 22 MM patients with at least three consecutive BM samples (median follow-up 21 months [range 4–35 months]) longitudinally and quantitatively for CT antigen expression, we observed a correlation between the BM expression of CT7/MAGEC1 mRNA and the clinical course of the disease as indicated by BM plasma cell infiltration (r=0.51, p<0.01) and, even more significantly, serum paraprotein levels (r=0.73, p<0.01). CONCLUSIONS: Performing the first longitudinal analysis of CT antigen expression in a human cancer, we demonstrate that in myeloma patients expression of MAGEC1/CT7, SSX2, MAGEA3, and MAGEC2/CT10 persists over time and represents an independent tumor marker. These findings suggest that downregulation of CT antigen expression is not a common tumor escape mechanism in myeloma and that CT antigens might, therefore, serve as diagnostic markers and targets for active immunotherapy, i.e. in the clinical setting of MRD.

Background: The introduction of autologous and allogeneic stem cell transplantation has resulted in improved responses to treatment in patients with multiple myeloma (MM). However, despite high remission rates, recurrences are frequent due to remaining minimal residual disease. Myeloma-specific immunotherapies would represent a potentially useful treatment option in these patients. Cancer/testis (CT) antigens are highly attractive targets for T cell-mediated immunotherapy of cancer, due to their immunogenicity and restricted tissue expression. We investigated the expression of a larger number of CT antigens, the expression of which is characteristically restricted to cancer and normal testis, in patients with MM. Methods: We analyzed the expression of 9 different CT antigens (SSX-1 to SSX-5, CT10/MAGE-E1, BAGE, MAGE-3, NY-ESO-1) in 10 myeloma cell lines, 54 bone marrow samples from patients with stage II-III myeloma (plasma cell infiltration 10–100 %), and 18 bone marrow samples from healthy donors using rtPCR. Results: Myeloma cell lines showed a remarkably high expression of the majority of CT antigens. All antigens, with the exception of SSX-3, were expressed in 80–100% of cell lines. SSX-3 was not expressed in any cell line. Analyzing bone marrow samples from MM patients, we observed that CT10 showed a very high expression level with 55% of all malignant samples expressing this antigen. The SSX gene family evidenced high expression levels with 40% (SSX-1), 17% (SSX-2), 13% (SSX-4), and 15% (SSX-5) of patient samples expressing the given SSX family member. SSX-3 was not expressed in any samples. In agreement with previous findings we observed a high expression of MAGE-3 (61%), an intermediate expression of BAGE (18%), and a comparably low expression of NY-ESO-1 (7%). With the exception of SSX-4, which was expressed in 18% of healthy bone marrow samples, none of the CT antigens were expressed in non-malignant samples. Conclusions: We show here for the first time that CT10 and members of the SSX gene family are strongly and specifically expressed in multiple myeloma. Importantly, both CT10 and SSX have been shown to elicit spontaneous immune responses in patients with solid tumors. Therefore, CT10 and SSX-1, SSX-2, and SSX-5 might represent valuable targets for antigen-specific immunotherapy of multiple myeloma.