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 (Blood 2007; 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.