Expression of cancer/testis tumor associated antigens in cervical squamous cell carcinoma

We investigated the expression of tumor-associated antigens (TAA) of the cancer/testis (C/T) gene family in cervical squamous cell carcinomas. First, we focused on the HeLa cervical cancer derived cell line, and we found that it expresses MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A12, GAGE-3/6, LAGE-1, and PRAME genes, encoding defined C/T TAA. In contrast, no expression of MAGE-A10, BAGE, GAGE-1/2, or NY-ESO-1 genes was observed. Corresponding gene products could also be detected by immunoblotting and immunocytochemistry, taking advantage of monoclonal antibodies recognizing discrete TAA. Capitalizing on these data, a monoclonal antibody predominantly recognizing MAGE-A4 TAA in paraffin-embedded sections (57B) was used to investigate the C/T gene expression in clinical tumor samples. A group of 60 patients was studied, and 57B positivity was detectable to different extents in 33% of the cases (20/60). In 13 of them (21%), staining of over 50% of the tumor cells was evident, whereas healthy cells always scored negative. Remarkably, MAGE-A4 expression was significantly (p < 0.05) more frequently detectable in poorly differentiated tumors (8/13) than in well-differentiated or moderately differentiated cancers (3/15 and 9/32, respectively) and in stage FIGO II as compared with stage FIGO Ib tumors (12/23 and 5/24, respectively, p = 0.04). Interestingly, staining was mostly nuclear in well-differentiated tumors, but involved both nuclei and cytoplasm in less differentiated cancers. Positivities of comparable frequency were also detectable in a smaller series of specimens upon staining with MAGE-A1- or NY-ESO-1/LAGE-1-specific reagents. Considering the high tumor specificity of C/T TAA, our data provide the rationale for the design of immunotherapy procedures targeting these antigens in cervical cancers.     

Expression of multiple epigenetically regulated cancer/germline genes in nonsmall cell lung cancer

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized on the X-chromosome may provide effective treatment for an extended number of patients.     

Cancer-testis antigen expression in uterine malignancies with an emphasis on carcinosarcomas and papillary serous carcinomas

The cancer testis (CT) family of antigens are expressed in certain malignant neoplasms and are silent in normal adult tissues, except for the testis. Expression of 2 members of this family, MAGE-A4 and NY-ESO-1, has been described recently in germ cell tumors, malignant melanomas, certain carcinomas and sarcomas. Our study is the first to describe the expression pattern of CT antigens in uterine neoplasms. Ninety-eight cases of uterine neoplasms, including 41 endometrioid, 19 papillary serous and 7 clear cell carcinomas, 22 carcinosarcomas and 9 endometrial stromal sarcomas were studied. Immunohistochemistry was carried out with the 57B monoclonal antibody that recognizes predominantly the MAGE-A4 antigen in paraffinized tissues and the D8.38 antibody that recognizes NY-ESO-1. MAGE-A4 expression was found to be present in 12% of the endometrioid adenocarcinomas, 63% of the papillary serous carcinomas and 91% of the carcinosarcomas. Within the tumor population the extent of MAGE-A4 expression was highest in the carcinosarcomas. In 12 of 22 positively staining carcinosarcomas more than 50% of the tumor cells expressed MAGE-A4. NY-ESO-1 expression was seen in 19% of the endometrioid adenocarcinomas, 32% of the papillary serous carcinomas and in 45% of the carcinosarcomas. CT antigen immunoreactivity was observed in both the carcinomatous and sarcomatous components of the carcinosarcomas and strong correlation between MAGE-A4 and NY-ESO-1 expression was present in individual cases. In summary, strong MAGE-A4 expression and to a lesser degree NY-ESO-1 expression is characteristic of the vast majority of uterine carcinosarcomas and a major subset of papillary serous carcinomas. These results suggest that CT antigen expression by these tumors may represent a novel target for immunotherapy.     

Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms.

PURPOSE: The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms and is silent in normal tissues, except for the testis. Expression of two members of this family, MAGE-A4 and NY-ESO-1, has been described in melanomas, germ cell tumors, certain carcinomas and sarcomas, and more recently in uterine neoplasms. The objective of this study was to evaluate the extent and prognostic significance of CT antigen expression in ovarian serous neoplasms. EXPERIMENTAL DESIGN: Seventy-four patients with ovarian neoplasms, including 10 with serous cystadenomas, 11 with serous tumors of borderline malignancy, and 53 with serous carcinomas, were studied. Immunohistochemistry was performed with the 57B monoclonal antibody, which recognizes predominantly the MAGE-A4 antigen and the D8.38 antibody that recognizes NY-ESO-1. RESULTS: MAGE-A4 expression was found to be present in 57% of the serous carcinomas and only in 9% of the serous tumors of borderline malignancy. No staining was detected in serous cystadenomas or in the normal ovary. In 8 of 30 positively stained serous carcinomas, >50% of the tumor cells expressed MAGE-A4. NY-ESO-1 expression was seen in 19% of the serous carcinomas, whereas serous tumors of borderline malignancy and cystadenomas were negative. A significant inverse correlation was found between MAGE-A4 expression and patient survival (P = 0.016). Multivariate analysis revealed that both tumor stage and MAGE-A4 expression were independent predictors of patient survival (P = 0.022 and P = 0.013, respectively). CONCLUSIONS: Cancer-testis antigen expression in ovarian serous neoplasms correlates directly with their degree of malignancy. MAGE-A4 expression, and to a lesser degree NY-ESO-1 expression, is characteristic of the majority of serous carcinomas. Determining the degree of MAGE-A4 expression in these tumors may provide important prognostic information. Finally, MAGE-A4 may represent a novel target for immunotherapy in serous ovarian neoplasms.     

Cancer/testis antigen expression and specific cytotoxic T lymphocyte responses in non small cell lung cancer

Non small cell lung cancers (NSCLC) express cancer/testis antigens (CTA) genes and MAGE-A expression correlates with poor prognosis in squamous cell carcinomas. We addressed cytotoxic T lymphocytes (CTL) responses to HLA class I restricted CTA epitopes in TIL from NSCLC in an unselected group of 33 patients consecutively undergoing surgery. Expression of MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1 CTA genes was tested by quantitative RT-PCR. Monoclonal antibodies (MAb) recognizing MAGE-A and NY-ESO-1 CTA were used to detect CTA by immunohistochemistry. CD8(+) TIL obtained from tumors upon culture with anti CD3 and anti CD28 mAb and IL-2 were stimulated with autologous mature DC (mDC) and HLA-A*0101 restricted MAGE-A1(161-169) or MAGE-A3(168-176) peptides or HLA-A*0201 restricted MAGE-A4(230-239), MAGE-A10(254-262), NY-ESO-1(157-165) or multi-MAGE-A (YLEYRQVPV) peptides or a recombinant vaccinia virus (rVV) encoding MAGE-A and NY-ESO-1 HLA-A*0201 restricted epitopes and CD80 co-stimulatory molecule. Specificity was assessed by (51)Cr release and multimer staining. At least one CTA gene was expressed in tumors from 15/33 patients. In 10 specimens, at least 4 CTA genes were concomitantly expressed. These data were largely confirmed by immunohistochemistry. TIL were expanded from 26/33 specimens and CTA-specific CTL activity was detectable in 7/26 TIL. In 6, however, specific cytotoxicity was weak, (<40% lysis at a 50:1 E:T ratio) and multimer staining was undetectable. In one case, high (>60% lysis at 50:1 E:T ratio) MAGE-A10(254-262) specific, HLA-A*0201 restricted response was observed. Supportive evidence was provided by corresponding multimer staining. Although CTA genes are frequently expressed in NSCLC, detection of CTL reactivity against CTA epitopes in TIL from nonimmunized NSCLC patients represents a rare event.     

Tumor-specific antigens in cutaneous T-cell lymphoma: expression and sero-reactivity

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of extra-nodal non-Hodgkin lymphomas with primary manifestation in the skin with poor treatment options in the advanced stages. As basis for future immune-therapeutic strategies we have investigated the possible expression of tumor-specific targets in CTCL focusing mainly on so-called cancer-germline genes. cDNAs derived from 20 CTCL tissues and 4 CTCL cell lines were tested with 15 gene-specific and 4 gene family-specific primers by RT-PCR and confirmative Northern blotting. The most frequently detected mRNAs were LAGE-1 (55% with only partial coexpression of the splicing variants), cTAGE-1 (35%), MAGE-A9 (27%) and the GAGE-3-7 group (35%). Furthermore, we could detect NY-ESO-1 (21%) and a MAGE-A subgroup (15%), whereas sub-specification of the latter proved absence of MAGE-A1, -A2, -A3, -A6 and -A12. SCP-1 was found in only one specimen and a several antigens could not been detected in any tumor tissue or cell line (MAGE-B, GAGE-1,2,8 and all 4 RAGE genes). 90% of all CTCL samples were positive for at least 1 of the frequent mRNAs in RT-PCR (LAGE-1, NY-ESO-1, cTAGE-1, MAGE-A9, or GAGE-3to7). Using a secondary SEREX approach we could detect sero-reactivity in sera of CTCL patients against recombinant cTAGE-1 (10/29), GAGE (3/19), MAGE-A1 (1/18), -A3 (1/18), -A6 (2/18) and -A9 (4/18) protein, but not against LAGE-1a, MAGE-A4b or MAGE-A12 protein (n = 19). We conclude, that certain cancer-germline genes can be detected frequently in CTCL and are able to elicit a systemic immune response. These candidate genes might therefore be promising targets for immunotherapeutic interventions in CTCL.     

NY-ESO-1与MAGE-A1在乳腺癌组织中的表达及意义

目的探讨NY-ESO-1与MAGE-A1在乳腺癌组织中的表达及意义。方法  健康对照者乳腺组织20例,不同病理类型及分级的病例90例,标本进行免疫组织化学SP法染色;采用Tanaka改良定量记分法测阳性率。结果  NY-ESO-1与MAGE-A1在正常乳腺组织中不表达;乳腺癌组织中NY-ESO-1与MAGE-A1阳性表达率分别为37.78%和23.33%;阳性表达与临床病理各参数不具有相关性(P>0.05)。结论  NY-ESO-1和MAGE-A1在乳腺癌组织中呈高特异性表达,对乳腺癌的早期诊断有一定的临床意义。     

Cancer-germline gene expression in pediatric solid tumors using quantitative real-time PCR

Cancer-germline genes (CGGs) code for immunogenic antigens that are present on various human tumors but not on normal tissues. The importance of CGGs in cancer immunotherapy has led to detailed studies of their expression in a range of human tumors. We measured the levels of expression of 12 CGGs in various pediatric solid tumors to identify targets for therapeutic cancer vaccines. Quantitative real-time PCR (qPCR) was used to measure the expression of 8 MAGE genes and of genes LAGE-2/NY-ESO-1 and GAGE-1, 2, 8 in 9 osteosarcomas, 10 neuroblastomas, 12 rhabdomyosarcomas and 18 Ewing's sarcomas. Nine tumors were also examined by immunohistochemistry with monoclonal antibodies specific for the MAGE-A1, MAGE-A4 and NY-ESO-1 proteins. All osteosarcoma and 80% of neuroblastoma samples expressed several CGGs at high levels. Six of 12 rhabdomyosarcomas and 11 of 18 Ewing's sarcomas expressed at least one CGG. Immunohistochemistry data correlated well with qPCR results and showed a homogeneous protein distribution pattern in most positive tumors. No correlation was found between the levels of CGG expression in the tumors and clinicopathological parameters of the patients. Pediatric solid tumors express several CGGs, which encode antigens that could be targeted in therapeutic vaccination trials. Several CGGs of the MAGE, GAGE and LAGE families are coexpressed in a large proportion of osteosarcoma and neuroblastoma samples. Some rhabdomyosarcomas express several of these genes at high levels. Ewing's sarcomas have an overall low CGG expression.     

Cancer testis antigen expression in primary and recurrent vulvar cancer: association with prognostic factors

Cancer testis tumour associated antigens (C/T-TAAs) were investigated in several gynaecologic and non-gynaecologic neoplasms as possible prognostic markers and targets for immunotherapy. The objective of the present study was to evaluate C/T-TAA expression patterns and prognostic significance in patients affected by vulvar cancer. Melanoma antigen E (MAGE)-A1, MAGE-A4 and NY-ESO-1 expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 45 primary and 14 recurrent vulvar carcinomas treated with surgery. MAGE-A1, MAGE-A4 and NY-ESO-1 were expressed in 25 (42%), 38 (64%) and 40 (68%) of the 59 samples, respectively. MAGE-A4 was significantly more frequently expressed in tumours with lymph node metastases (p<0.002) and in recurrent tumours (p<0.02). NY-ESO-1 was more highly expressed by moderately or poorly differentiated tumours (p<0.01). This study demonstrates that vulvar cancer frequently expresses C/T-TAAs. Antigen expression correlates with the presence of lymph node metastases and poor tumour differentiation.     

Frequent expression of the novel cancer testis antigen MAGE-C2/CT-10 in hepatocellular carcinoma

Cancer testis (CT) antigens are attractive targets for immunotherapy in cancer patients. Immunohistochemistry was used to study the expression of the CT antigens MAGE-C2/CT-10, MAGE-C1/CT-7, GAGE, MAGE-A4 and NY-ESO-1 in 146 hepatocellular carcinomas, 13 intrahepatic cholangiocarcinomas, 37 extrahepatic cholangiocarcinomas and 32 gallbladder carcinomas. Immunopositivity was correlated with clinicopathological parameters, MHC Class 1 expression, intratumoral CD4+, CD8+ and FOXP3+ T cells and CD163+ antigen-presenting cells. Of the 146 hepatocellular carcinomas, 34% were positive for MAGE-C2/CT-10, 12% for MAGE-C1/CT-7, 11% for GAGE and 2% for NY-ESO-1, respectively. MHC Class 1 coexpression was identified in almost all CT antigen-positive tumors. The number of intratumoral FOXP3+ regulatory T cells was increased in CT antigen-positive hepatocellular carcinomas (p<0.004), suggesting inhibition of immune response in such tumors. Furthermore, MAGE-C1/CT-7 and GAGE positivity was correlated with reduced overall survival in patients with hepatocellular carcinoma (p=0.03 and 0.01, respectively). Four (13%) gallbladder carcinomas stained positive for MAGE-C2/CT-10, of which 1 tumor (3%) was also positive for NY-ESO-1 and GAGE. CT antigens were not expressed in intra- and extrahepatic cholangiocarcinomas. Our results suggest that MAGE-C2/CT-10 may be a good candidate for peptide vaccination in patients with hepatocellular carcinoma.     

Expression of MAGE-A3, NY-ESO-1, LAGE-1 and PRAME in urothelial carcinoma

Background:

The potential for cancer-testis (CT) antigens as targets for immunotherapy in cancer patients has been heavily investigated, and currently cancer vaccine trials based on the CT antigens, MAGE-A3 and NY-ESO-1, are being carried out.

Methods:

We used specific q-RT-PCR assays to analyse the expression of the CT genes MAGE-A3, NY-ESO-1 (CTAG1B), LAGE-1 (CTAG2) and PRAME in a panel of bladder tumours from 350 patients with long-term follow-up and detailed treatment information.

Results:

Overall, 43% of the tumours expressed MAGE-A3, 35% expressed NY-ESO-1, 27% expressed LAGE-1 and 20% expressed PRAME. In all, 56% of the tumours expressed at least one of the CT genes analysed. Univariate Cox regression analysis of CT gene expression in non-muscle-invasive tumours showed that expression of MAGE-A3 (P=0.026), LAGE-1 (P=0.001) and NY-ESO-1 (P=0.040) was significantly associated with a shorter progression-free survival. In addition, we found that patients with tumours expressing PRAME responded poorly to chemotherapy (P=0.02, χ²-test).

Conclusion:

Cancer-testis genes are frequently expressed in bladder cancer and especially in tumours of high stage and grade. In addition, the CT gene expression may have both prognostic and predictive value. Development of specific immunotherapy against the CT antigens in bladder cancer may ultimately increase patient survival.     

Case-Control Study: Smoking History Affects the Production of Tumor Antigen–Specific Antibodies NY-ESO-1 in Patients with Lung Cancer in Comparison with Cancer Disease–Free Group

IntroductionLung cancer is the leading cause of cancer mortality worldwide; therefore, understanding the biological or clinical role of tumor-associated antigens and autoantibodies is of eminent interest for designing antitumor immunotherapeutic strategies.MethodsHere we prospectively analyzed the serum frequencies of New York esophageal squamous cell carcinoma 1 (NY-ESO-1), human epidermal growth factor 2/neu, and melanoma-associated antigen A4 (MAGE-A4) antibodies and expression of the corresponding antigens in tumors of 121 patients with NSCLC undergoing an operation without prior neoadjuvant chemotherapy and compared them with those in 57 control age-matched patients with no history of a malignant disease.ResultsWe found that only antibodies specific for NY-ESO-1 (19.8% [n = 24 of 121]) were significantly increased in the group of patients with NSCLC compared with in the controls. NY-ESO-1 seropositivity was significantly positively associated with an active smoking history in patients with NSCLC but not in smokers from the control group. In tumors, the frequency of NY-ESO-1 mRNA expression was 6.3% (in four of 64 patients), the frequency of human epidermal growth factor 2/neu (HER 2/neu) expression was 11.9% (five of 42), and the frequency of MAGE-A4 expression was 35.1% (20 of 57). MAGE-A4 expression in tumors correlated with smoking status and male sex in patients with NSCLC. Patients with squamous cell carcinoma displayed higher expression of NY-ESO-1 and MAGE-A4 in tumors than did patients with adenocarcinoma. On the other hand, 94.7% of nonsmoking patients in our study had adenocarcinoma (of whom 73.7% were women).ConclusionThese results confirm the reported high immunogenicity of NY-ESO-1 and suggest that a smoking-induced chronic inflammatory state might potentiate the development of NY-ESO-1–specific immune responses. Moreover, smoking might contribute to the expression of other cancer/testis antigens such as MAGE-A4 at early stages of NSCLC development.     

Decitabine facilitates immune recognition of sarcoma cells by upregulating CT antigens,MHC molecules,and ICAM-1

Rhabdomyosarcoma, osteosarcoma, and Ewing’s sarcoma are the most common types of sarcoma in children. Despite standard therapy, nearly one third of the patients with Ewing’s sarcoma relapse, and there are limited options with curative potential. Immunotherapy is a promising approach as it can target tumor-specific antigens that are specifically expressed on tumors while sparing non-malignant cells. We have demonstrated that a demethylating chemotherapeutic drug, 5-aza-2’-deoxycytidine (decitabine, DAC) can upregulate the expression of cancer-testis (CT) antigens, MHC molecules, and intracellular cell adhesion molecule-1 on pediatric sarcoma cell lines, resulting in enhanced killing of tumor cells by CT antigen-specific cytotoxic T lymphocytes derived from pediatric sarcoma patients. A significant increase in the mRNA expression levels of MAGE-A1 and MAGE-A3 were found in 70 %, and NY-ESO-1 in 80 % of the sarcoma lines following exposure to pharmacological levels of DAC. The high expression levels of MAGE-A1, MAGE-A3, and NY-ESO-1 were sustained in sarcoma lines and primary tumor lines over 30 days after the cessation of DAC. Furthermore, DAC treatment induced upregulation of MAGE-A1, MAGE-A3, or NY-ESO-1 protein expression in seven of nine lines studied. These studies show that demethylating chemotherapy could be combined with CT antigen-directed immunotherapy for treating pediatric sarcoma.