To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohistology in selected RT-PCR-positive cases. Antibody responses against 7 CT antigens were analyzed using recombinant antigen expression on yeast surface. In 98 evaluable cases, SCP-1 and SSX-4 were expressed most frequently (both 65%), followed by HOM-TES-85/CT-8 (47%), GAGE (26%), SSX-1 (20%), NY-ESO-1 (13%), MAGE-3 (11%), SSX-2 (8%), CT-10 (7%), MAGE-4 (4%) and CT-7 (1%). One CT gene was expressed by 90% of the cases; 79% expressed > or =2, 48% > or =3, 29% > or =4, 12% > or =5, 6% > or =6, 3% > or =7, 2% > or =8 and one case coexpressed 9 antigens. Of 100 serum samples screened for CT antigen-specific antibodies, antibodies against NY-ESO-1 were detected in 4 patients, against SCP-1 in 6 patients and against SSX-2 in 1 patient, while no antibodies were detected against MAGE-3, CT-7 and CT-10. Expression of CT genes or antibody responses was not correlated with clinical parameters (menopausal status, tumor size, nodal involvement, grading, histology and estrogen receptor status) or the demonstration of CT gene expression at the protein level, by immunohistology. Our results show that breast carcinomas are among the tumors with the most frequent expression of CT antigens, rendering many patients potential candidates for vaccine trials.

A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high-through-put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild-type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies. (c) 2005 Wiley-Liss. Inc.

The SSX2 gene encodes the tumor‐specific antigen HOM‐MEL‐40/SSX2 expressed in a broad spectrum of tumors of different origin, against which humoral and CD8+ T‐cell‐mediated MHC‐I‐restricted responses have been demonstrated. Searching for promiscuous MHC‐II‐restricted peptides that might be suitable as a CD4+ stimulating vaccine for many patients, we used the SYFPEITHI algorithm and identified a HOM‐MEL‐40/SSX2‐derived pentadecamer epitope (p45–59) that induced specific CD4+ T‐cell responses restricted by the HLA‐DRB1 subtypes *0701, *1101 and *1302 that have a cumulative prevalence of ∼ 25% in the Caucasian population. The CD4+‐mediated response against p45–59 and its DR restriction was demonstrated by inhibition with anti‐CD4 and HLA‐DR antibodies, respectively, and by blocking experiments using HLA‐specific antibodies. The natural processing and presentation of p45–59 was demonstrated by recognition of the SSX2+ melanoma cell line Me 275 as well as autologous and allogeneic dendritic cells pulsed with whole‐protein SSX2 by T cells with specificity for p45–59. p45–59 was able to induce responses in 3/6 breast cancer patients and 1/5 healthy controls. No correlation was found between CD4+ T‐cell responses against p45–59 reactivity and anti‐SSX2 antibody titers in the serum of patients, suggesting that CD4+ and B‐cell responses are regulated independently. p45–59 holds promise as a broadly applicable peptide vaccine for patients with SSX2‐positive neoplasms. © 2004 Wiley‐Liss, Inc.