MAGE-A3 Expression Research Articles

Melanoma Antigen-A11 (MAGE-A11) Enhances Transcriptional Activity by Linking Androgen Receptor Dimers

Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.

Abstract P4-04-04: Immunohistochemical analysis of Cancer Testis antigens and Topoisomerase 2-alpha expression in triple negative breast carcinomas: a retrospective study

Abstract Aim. The aim of this study is to assess the expression of cancer testis (C/T) antigens in the TNBC group, expression of topoisomerase 2-alpha (TOPO2A), basal-like (BL) immunophenotype defined by basal markers (CK5/6, CK14, and EGFR), BL morphology and conventional clinicopathologic factors as well as to demonstrate their prognostic relevance with respect to this group of tumours. Methods. The study includes 83 patients who underwent surgery between January 2003 and December 2009. Slides were stained immunohistochemically with CK5/6, CK14, EGFR, Ki-67, TOPO2A, MAGE-A1, MAGE-A10, NY-ESO and multi-MAGE. Survival-time and multivariate survival analyses were performed for the purpose of identifying prognostic markers for tumours with more aggressive behaviour. Results. Of the 83 triple negative tumours, 55 (66.3%) have BL immunophenotype and 40 (48.2%) BL morphology. MAGE-A1 show the most frequent expression (69.0%), followed by multi-MAGE (58.0%), NY-ESO (27.1%) and MAGE-A10 (13, 2%). MAGE-A10 expression is significantly correlated with tumour size (p = 0.026). The expressions of MAGE-A1, MAGE-A10 and multi-MAGE are significantly correlated with clinical stage (p = 0.024, p = 0.041, p = 0.031, respectively). A significant correlation has been found between the expressions of MAGE-A10 and NY-ESO and the expression of TOPO2A (p = 0.005, p = 0.013). There is no significant correlation between the expression of C/T antigens and disease-free survival (DFS) or overall survival (OS). Conclusions. High expression levels of MAGE and NY-ESO observed in TNBC are of potential clinical relevance, especially in the adjuvant setting of treatment, and may be of therapeutic value as a vaccine-based treatment in the TN group of breast tumours for which therapeutic options are limited. Key words: triple negative breast cancer, basal-like breast cancer, cancer-testis antigen, topoisomerase 2-alpha, prognosis, survival analysis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-04.

MAGE‐A10 cancer/testis antigen is highly expressed in high‐grade non‐muscle‐invasive bladder carcinomas

Bladder cancer is a common urinary malignancy and a prevalent cause of cancer-related death. Current therapies of early stage non-muscle-invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen-specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer-testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE-A10 is probably the most immunogenic antigen of the MAGE-A family. We evaluated the expression of MAGE-A10 in NMIBC. Seventy-nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE-A10 gene expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed on paraffin-embedded sections. MAGE-A10 gene was specifically expressed in one-third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE-A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE-A10 protein was also more frequently detectable in high-grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE-A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE-A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers.

The Novel Anti-Myeloma Agent Chaetocin Enhances Tumor-Specific Dendritic Cell Response Via the Induction of Heat Shock Protein and Cancer Testis Antigens On Myeloma Cells

AbstractAbstract 4066 Background:Dendritic cell (DC) based immunotherapy is emerging as a useful tool to treat multiple myeloma (MM). Although several tumor target antigens have been used, the clinical effectiveness of the vaccination is still limited. Chaetocin is a small molecule thiodioxopiperazine natural-product produced by Chaetomium species fungi currently in development as a candidate antimyeloma therapeutic, and has potent in vitro and in vivo activity conveyed by its ability to impose increased levels of cellular oxidative stress. Chaetocin has also been found to be used as histone methyl-transferase inhibitor with interest in the compound sufficient to kill various cancer cells. We investigated whether chaetocin could be used to induce apoptotic bodies for loading into DCs to enhance myeloma specific antitumor responses. Methods:Chaetocin was used to induce the apoptotic U266 myeloma cells, which were used as the sources of tumor antigens to loading onto DCs to generate myeloma-specific cytotoxic T lymphocytes (CTLs). Results:Chaetocin showed to induce apoptotic cells mostly from tumor cells and led to increase the expression of heat shock protein 90 (HSP90) at a level comparable to bortezomib. The transfer of activating signal from chaetocin-induced tumor cells to induce DCs maturation is mediated by the exposure of HSP90 on the surface of apoptotic cells. The HSP90 inhibitor geldanamycin can block this immunogenicity. Chaetocin up-regulate the expression of cancer testis antigens MAGEA3 and MAGEC1/CT7, which are commonly expressed in MM and the potent targets for active immunotherapies. The cytokines production during uptake tumor antigens and maturation was comparable in DCs loaded with either irradiated MM cells or chaetocin-induced MM cells. However, DCs loaded with chaetocin-induced MM cells can reduce the production of an inhibitory cytokine (IL-10) after stimulation with CD40 ligand. CTLs stimulated by chaetocin-induced MM cells-loaded DCs showed the increasing of CD8+T cells and displayed a greater number of IFN-g-secreting cells than did those stimulated by other DCs loaded with MM cell made by other conditions. Conclusion:These results indicate that anti-myeloma drug-induced apoptotic cells can be used as the source of myeloma antigens to loading onto DCs that could elicit potent anti-myeloma activity of CTLs due to the expression of heat shock proteins and cancer testis antigens as a mechanism of immunogenic death of human MM cells. Disclosures:No relevant conflicts of interest to declare.

Clinical significance of human leukocyte antigen loss and melanoma-associated antigen 4 expression in smokers of non-small cell lung cancer patients

Melanoma-associated antigen-A4 (MAGE-A4) is one of the candidates for a target of immunotherapy and is expressed in non-small cell lung cancer (NSCLC). However, tumors sometimes lose human leukocyte antigen (HLA) class I expression, and tumor-specific T cells cannot eliminate the tumor with loss of HLA. However, the relationship between MAGE-A4 expression and HLA loss has remained unclear. Among 363 NSCLC patients who consecutively underwent curative surgery, 187 cases whose material could be analyzed were reviewed. The expression of HLA class I molecules was assessed by immunohistochemical staining. The expression of MAGE-A4 was analyzed by RT-PCR. Seventy-seven tumors expressed HLA normally; however, 110 tumors lost HLA. The proportion of patients with a smoking habit and expressing the MAGE-A4 gene in patients with HLA loss was higher than those with HLA expression (p=0.04 and 0.028, respectively). Five-year overall survival (OS) rate in the patients expressing MAGE-A4 but with loss of HLA was 52.4%, and OS was significantly poorer than their counterparts (74.0%, p=0.036). Multivariate analysis indicated that advanced stage or history of smoking and HLA loss was an independently poor prognostic predictor of OS in NSCLC (p<0.01 and p=0.04, respectively). HLA class I loss in NSCLC was related to smoking history and MAGE-A4 expression of tumors. HLA class I loss in smokers or patients with the MAGE-A4 gene was a prognostic factors in NSCLC.

Expression of MAGE-A11 in breast cancer tissues and its effects on the proliferation of breast cancer cells

The melanoma-associated antigen (MAGE) genes are commonly expressed in tumors, but the function of many of these genes remains unknown. Here, we investigated the expression of one family member, MAGE-A11, in breast cancer tissues and corresponding adjacent normal tissues. MAGE-A11 expression was assessed in breast cancer tissues and adjacent normal tissues from 100 women by immunohistochemistry. MAGE-A11 was significantly more highly expressed in breast tumors (56% of samples) compared to normal tissues (0%; P<0.05). Additionally, its expression in breast tumors was investigated in relation to various clinicopathological features, including patient age, tumor stage and volume, and lymph node metastasis. MAGE-A11 protein expression was correlated with expression of the human epidermal growth factor receptor-2 (HER-2) and estrogen receptor (ER)-β (P<0.05), but not with age, pathological type, histological grade, clinical stage, tumor size or lymph node metastasis, or ER-α, progesterone receptor (PR) or amplified in breast cancer 1 (AIB-1) expression. To determine how its expression affects cancer cell proliferation invitro, MCF-7 human breast cancer cells were transfected with pCMV-AC-MAGE-A11-GFP. MTT colorimetry and colony-forming assays indicated that MAGE-A11 overexpression significantly increased breast cancer cell proliferation and the ability to form colonies (P<0.05). These findings indicate that MAGE-A11, similarly to HER-2 and ER-β, may be an important diagnostic or prognostic indicator in breast cancer and potentially promotes tumor proliferation.

Cancer/testis antigen expression as a predictor for epidermal growth factor receptor mutation and prognosis in lung adenocarcinoma

Immune therapy targeting cancer/testis (CT) antigens improve the survival in several types of solid tumours. The expression of CT antigens is related to poor survival in non-small-cell lung cancer (NSCLC). The epidermal growth factor receptor (EGFR) mutation is the best predictive factor for the sensitivity to tyrosine kinase inhibitors in lung adenocarcinoma. The aim of this study was to elucidate the correlation between the expression of CT antigens and clinicopathological factors, including the EGFR mutation, and to analyse the prognosis in lung adenocarcinoma. Data were collected from a total of 281 lung adenocarcinoma patients who underwent surgery. Among them, 125 cases, whose specimens were too small to extract sufficient DNA and/or RNA, and 2 cases with the coexistence of another histological lung cancer were excluded. A total of 154 patients were reviewed. The expression of CT antigens (melanoma-associated antigen gene [MAGE]-A4 and KK-LC-1) and the EGFR-activating mutation (L858R point mutation in exon 21 and inframe deletion in exon 19) was evaluated by using polymerase chain reaction amplification. The expression of MAGE-A4 and KK-LC-1 was detected in 14 (9%) and 54 patients (35%) with adenocarcinoma. The EGFR-activating mutation was found in 64 patients (42%). Univariate and multivariate analyses demonstrated that tumours expressing at least one CT antigen were associated with no EGFR mutation (odds ratio = 0.3; 95% confidence interval, 0.14-0.71; P < 0.01). A survival analysis was performed in 135 patients who underwent complete resection and the 5-year overall survival rate was 71.1% in those with any expression of CT antigens and 83.2% in those without expression of the genes (P < 0.04). Two different therapeutic targets, EGFR-activating mutation and CT antigen, have a negative relationship with each other.

Detection of MAGE-A Expression Predicts Malignant Transformation of Oral Leukoplakia

The study aimed at checking if MAGE-A expression in oral leukoplakia (OLP) lesions is related to malignant transformation. The 48 samples of OLP that transformed to oral squamous cell carcinoma (OSCC) (group 1) and 50 samples of OLP that did not transform to OSCC (group 2) were included in the study. The expression of MAGE-A was restricted to group 1. The correlation between malignant transformation and MAGE-A occurrence in OLP was statistically significant (p < .0001). Detection of MAGE-A may allow identifying OLP with a high risk of malignant transformation giving a view to a new approach to prevention of oral cancer.

MAGE-A3 expression in patients screened for the DERMA trial: A phase III trial testing MAGE-A3 immunotherapeutic in the adjuvant setting for stage IIIB-C-Tx melanoma.

8559 Background: Administration of MAGE-A3 immunotherapeutic involves the active immunization of patients (pts) with tumors expressing the MAGE-A3 protein. This new investigational approach has been previously assessed in two phase II trials, one in pts with metastatic melanoma (NCT00086866) and another in pts with completely resected non-small cell lung cancer (NCT00290355). Based on the positive responses observed in both studies, a randomized phase III placebo controlled trial assessing MAGE-A3 immunotherapeutic as adjuvant treatment in pts with resected, regionally advanced melanoma (stage IIIB-C-Tx AJCC/UICC 2010) is currently ongoing (DERMA Phase III trial, NCT00796445). Methods: Formalin-fixed, paraffin-embedded tumor tissues were prepared from surgically removed metastatic lymph nodes and tested for MAGE-A3 expression by qRT-PCR. Other baseline patient and tumor characteristics were collected during screening to further investigate factors that could potentially influence MAGE-A3 expression. Results: Between Dec 1, 2008 and Oct 25, 2011, 3917 pts were screened. Of the 3,183 valid samples (excluding the 513 inconclusive tests [66% for poor quality, 27% out-of range, 7% miscellaneous]) and the 221 missing samples, 2,092 (65.7%) were positive for MAGE-A3 expression. In these stage III melanoma pts, no difference in MAGE-A3 expression levels were identified with regard to (1) disease stage - IIIB (62.6%), IIIC (66.5%) and IIITx (66.2%); (2) gender - male (64.2%), female (68.1%); (3) age group - 18 to 39 years (63.2%), 40 to 49 years (65.3%), 50 to 59 years (66.8%), 60 to 69 years (68.1%) and 70 years and over (63.4%) and (4) region: Europe (66.1%), North America (63.0%) and rest of the world including Argentina, Brazil, Australia, Mexico, Taiwan, Japan, Korea (67.9%). Conclusions: Expression of the MAGE-A3 gene in the DERMA trial population (stage IIIB-IIIC-IIITx) was 66%. It was not correlated with age, gender, disease stage or geographic region. This expression frequency is consistent with published data in metastatic melanoma (Van den Eynde, 1997; Vourc’h-Jourdain et al, 2009) and has potentially important clinical implications.

Expression of PRAME and MAGE-A3 in Asian and European patients with non-small cell lung cancer (NSCLC), bladder cancer, or hepatocellular carcinoma (HCC).

e13061 Background: Antigen (Ag)-specific cancer immunotherapy is based on induction of immune responses (humoral and cell-mediated) specifically targeting Ags expressed by tumor cells. This may be achieved by delivering the Ag as a recombinant protein combined with potent immunostimulants. PRAME and MAGE-A3 are tumor Ags with distinct tumor expression patterns, both under investigation as targets for immunotherapy in NSCLC and malignant melanoma. For additional clinical development of these immunotherapies in other cancer types, expression of PRAME and MAGE-A3 in these cancers must be investigated taking into account patient ethnicity and tumor characteristics. Methods: 4 retrospective studies were performed, each including all tumor stages: NSCLC pts (in Asia and Europe); bladder cancer pts in Europe; HCC pts in Asia. Formalin-fixed paraffin embedded tumor tissues were tested for PRAME and MAGE-A3 expression by quantitative RT-PCR using an expression cut-off value for positivity. Exploratory analyses of possible associations with patient and tumor characteristics were done to identify factors that may impact Ag expression. Results: The overall Ag expression rates are shown in the table below. In NSCLC, both Ags were expressed less frequently in Asian than in European patients (the histological distribution was the same). NSCLC tumors more frequently expressed PRAME than MAGE-A3 while the reverse tendency was seen for bladder tumors and HCC. In the NSCLC studies, squamous cell carcinomas expressed both Ags more frequently than adenocarcinomas. No study found any association between tumor stage and Ag expression. Some 60% of the tumors express at least one of the Ags. Conclusions: The expression rates of PRAME and MAGE-A3 are sufficiently high to consider these Ags as targets for immunotherapy against NSCLC, bladder cancer and HCC. [Table: see text]

Prognostic significance of MAGE-A10 and NY-ESO-1 cancer-testis antigen in breast cancer.

e21126 Background: Cancer testis antigens (CTAs) are expressed in a variety of malignant tumors. In normal adult tissues CTA expression is restricted predominantly to germs cells of the adult testis and placenta. Based on their tumor-restricted expression pattern, CTAs are regarded as valuable targets for cancer immunotherapy. The prognostic significance of CTAs in breast cancer has not been analyzed previously. Methods: To evaluate the potential prognostic significance of two member of this family, MAGE-A10 and NY-ESO-1 antigens, we examined their expression in breast cancer patients who underwent curative surgery at our hospital between 2002 and 2003. Paraffin embedded tumor sections were collected retrospectively from 165 breast cancer patients and immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. Impact of MAGE-A10 and NY-ESO-1 expression on disease free survival (DFS) and overall survival (OS) was analyzed by the Kaplan-Meier method using 8-year follow up data. Results: MAGE-A10 expression (score ≥2+) was detected in 105/164 (64%) and NY-ESO-1 expression (score ≥2+) was observed in 14/164 (8.5%) patients. 8-year DFS for MAGE-A10 positive patients was 69% and 73% for MAGE-A10 negative (p=0.452). 8-year OS for MAGE-A10 positive patients was 80% and 90% for MAGE-A10 negative (p=0.134). For NY-ESO-1 positive patients 8-year DFS was 67% and 70% for NY-ESO-1 negative patients (p=0.837). 8-year OS for NY-ESO-1 positive patients was 83% and 84% for NY-ESO-1 negative patients. (p=0.991) Conclusions: To our knowledge this is the first study analyzing prognostic significance CTAs in breast cancer. In this retrospective study we did not show statistically significant correlation between MAGE-A10 and NY-ESO-1 expression and clinical outcome. Additional studies are warranted to determine weather this antigens might have prognostic value in breast cancer patients.

Prognostic value of MAGE-A3 and PRAME gene expression in non-small-cell lung cancer (NSCLC).

7056 Background: We investigated the expression of some cancer–testis genes and their association with disease prognosis. Here we report our results for the expression, in resected NSCLC samples, of two tumor-specific antigens (Ags): MAGE‑A3 and PRAME, both under evaluation in clinical trials. Methods: We conducted a single-center, uncontrolled, retrospective study of tissue from resected stage I–III NSCLC patients (pts): 650 were in stage I, 215 in stage II and 395 in stage III. 1260 FFPE tumor tissue samples from the tumor tissue bank of the Department of Pathology, Lungenklinik Hemer (Germany), were tested for MAGE‑A3 and PRAME expression by specific qRT-PCR assays. The prognostic value of these Ags was determined by estimating the median overall survival (OS), disease-free interval (DFI) and disease-free survival (DFS). Results: Expression rates were 36% for MAGE‑A3 and 66% for PRAME. The co‑expression rate was 30%. Almost all tumors expressing MAGE‑A3 also expressed PRAME. We observed no difference overall in Ag expression according to stage, tumor size and pts’ age. Squamous tumors expressed MAGE‑A3 and PRAME more frequently than did adenocarcinomas (43 vs. 27 % and 80 vs. 44% respectively), and PRAME expression rates were higher in males than in females (70 vs. 53%). In the overall population, which included stages IA–IIIB, no prognostic value was detected for the expression of either Ag. In subset analyses, we found in 83 stage IIIB pts a trend to worse OS linked to MAGE‑A3 expression (HR=1.977, p=0.0312). In 395 stage IB pts, PRAME expression was associated with worse OS (HR=1.483, p=0.0344) and DFS (HR=1.492, p=0.0167). Conclusions: The MAGE‑A3 expression rate found is consistent with the results observed in the phase III MAGRIT trial (J‑H Kim et al., 2011). No prognostic value for either PRAME or MAGE‑A3 was observed in the overall population, in contrast to previous reports with smaller sample sizes (Gure et al., 2005, Boli et al., 2002) and in other tumors (Vourch’jourdain et al., 2009, Epping et al., 2008). We observed a trend toward poor prognostic value of MAGE‑A3 in stage IIIB pts and of PRAME in stage IB pts.

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.

FGFR2 Isoforms Support Epithelial–Stromal Interactions in Thyroid Cancer Progression

Alternate splicing yields two distinct isoforms of the fibroblast growth factor (FGF) receptor FGFR2-IIIb and FGFR2-IIIc varying their extracellular structure in human thyroid cancer, in which FGFR expression is commonly dysregulated. In this study, we characterized the function of these variants in modulating thyroid cancer behavior. Enforced expression of either FGFR2-IIIb or FGFR2-IIIc in thyroid epithelial cancer cells reduced expression of fibronectin, MAGE-A3 and MMP9, while increasing p21 and enhancing Rb dephosphorylation. Consistent with these tumor-suppressive properties, FGFR2-IIIb and FGFR2-IIIc each diminished invasive behavior in vitro and reduced tumor growth and metastasis in vivo. Notably, these effects contrasted with those produced by expression of these FGFR isoforms in fibroblasts, in which they both stimulated cell growth. Moreover, in xenograft tumors generated by coimplantation of epithelial and fibroblast cells expressing that same isoform, there was no significant effect on tumor progression. Conversely, FGFR2-IIIb expression in epithelial cells yielded higher FGF4/FGF7 expression that, in the presence of FGFR2-IIIc-expressing fibroblasts, enhanced tumor progression. Together, our findings highlight the importance of cellular context in assigning growth properties to growth factor receptor isoforms. More specifically, they show how alternative splicing of FGFR2 yields heteroisoforms critical to the growth-promoting actions of FGFs that exert distinct epithelial-stromal effects in thyroid cancer.

Evaluation of MAGE A1 in oral squamous cell carcinoma

MAGE A1 is a cancer testis antigen (CTA) described in a variety of human cancers. CTAs exhibit a highly restricted tissue expression and by virtue of their immunogenic potential, these genes are promising target molecules for cancer vaccines. DNA hypomethylation is associated with gene regulation in several types of tumours. The aim of this project was to identify the presence of MAGE A1 in oral squamous cell carcinoma (OSCC) samples and to investigate the hypomethylation profile of CpG islands situated in the promoter region of this gene. The expression of MAGE A1 in OSCC and healthy oral mucosal samples was determined by real-time quantitative and conventional endpoint PCR and also by immunohistochemistry staining. In addition, to investigate the hypomethylation profile of promoter MAGE A1 CpG islands, we performed bisulphite sequencing. Real-time quantitative and endpoint PCR assays demonstrated a lower level of MAGEA1 transcription. Endpoint PCR showed expression of MAGEA1 in 10% (2/20) of OSCCs. Sodium bisulphite sequencing analysis of MAGE A1 CpG islands did not reveal a difference between OSCC and normal oral mucosal samples. We further assessed MAGE A1 protein immunoexpression and found 80% (16/20) of immunopositivity in OSCCs. We did not observe a correlation between the presence of MAGE A1 protein and lower levels of transcripts. Identification of MAGE A1 protein in OSCCs and absence of immunoexpression in normal oral mucosa support the idea that this protein can be used as a biomarker for detection of OSCC; however, it is not associated with hypomethylation or high expression of the MAGE A1 gene.

Assessment of Prognostic Circulating Tumor Cells in a Phase III Trial of Adjuvant Immunotherapy After Complete Resection of Stage IV Melanoma

To verify circulating tumor cell (CTC) prognostic utility in stage IV resected melanoma patients in a prospective international phase III clinical trial. Our studies of melanoma patients in phase II clinical trials demonstrated prognostic significance for CTCs in patients with AJCC stage IV melanoma. CTCs were assessed to determine prognostic utility in follow-up of disease-free stage IV patients pre- and during treatment. After complete metastasectomy, patients were prospectively enrolled in a randomized trial of adjuvant therapy with a whole-cell melanoma vaccine, Canvaxin, plus Bacille Calmette-Guerin (BCG) versus placebo plus BCG. Blood specimens obtained pretreatment (n = 244) and during treatment (n = 214) were evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction (qPCR) for expression of MART-1, MAGE-A3, and PAX3 mRNA biomarkers. Univariate and multivariate Cox analyses examined CTC biomarker expression with respect to clinicopathological variables. CTC biomarker(s) (≥ 1) was detected in 54% of patients pretreatment and in 86% of patients over the first 3 months. With a median follow-up of 21.9 months, 71% of patients recurred and 48% expired. CTC levels were not associated with known prognostic factors or treatment arm. In multivariate analysis, pretreatment CTC (> 0 vs. 0 biomarker) status was significantly associated with disease-free survival (DFS; HR 1.64, P = 0.002) and overall survival (OS; HR 1.53, P = 0.028). Serial CTC (>0 vs. 0 biomarker) status was also significantly associated with DFS (HR 1.91, P = 0.02) and OS (HR 2.57, P = 0.012). CTC assessment can provide prognostic discrimination before and during adjuvant treatment for resected stage IV melanoma patients.

Expressions of MAGE-A10 and MAGE-A11 in breast cancers and their prognostic significance: a retrospective clinical study

Melanoma-associated antigens-A (MAGE-A) family is a group of well-characterized cancer/testis antigens (CTA), because they are strictly tumor specific and are shared by many kinds of tumors. However, the expression pattern of MAGE-A10 and MAGE-A11 in breast cancer patients is still unclear. The purpose of our study is to investigate the expression pattern and prognostic significance of MAGE-A10 and MAGE-A11 in breast cancer patients. Formalin-fixed and paraffin-embedded tissues and the clinicopathological parameters from 75 primary breast cancer patients were collected. The expressions of MAGE-A10 and MAGE-A11 proteins were immunohistochemically detected, and the association of MAGE-A10 and MAGE-A11 expressions with the clinicopathological parameters and the survival of breast cancer patients were analyzed. The expression rates of MAGE-A10 and MAGE-A11 in breast cancer specimens were 73.3 and 52.0%, respectively. MAGE-A11 expression was more frequent in estrogen-receptor (ER)-positive breast carcinomas compared with ER-negative breast carcinomas (P = 0.004). MAGE-A11 expression was positively associated with HER-2 expression (P = 0.003). Overall survival of patients with MAGE-A11-negative expression was significantly longer than those patients with positive MAGE-A11 expression (P = 0.030), but no difference of overall survival was found between patients with MAGE-A10-negative and -positive expression (P = 0.881). MAGE-A10 and MAGE-A11 are tumor-specific antigens, and MAGE-A11 expression probably is a potential poor prognostic factor for breast cancer patients.

Adriamycin Enhances Proteasome-Mediated Generation of the Proapoptotic Processed Form of MAGE-A4 in Hepatoma Cells

Background: Melanoma antigen (MAGE)-A4 is processed to generate a C-terminal fragment with proapoptotic activity. Here we demonstrate that Adriamycin promotes generation of the processed MAGE-A4 by activating the proteasome. The proteasome is known to prevent accumulation of toxic proteins to maintain cellular homeostasis. Methods and Results: Treatment of hepatoma cells expressing MAGE-A4 with a sublethal dose of Adriamycin increased the MAGE-A4 processing and sensitized the cells to Adriamycin-induced apoptosis. The processing of MAGE-A4 was inhibited by the proteasome inhibitors MG115, MG132, lactacystin and epoxamicin. MAGE-A4 was coimmunoprecipitated with the S6 proteasomal ATPase, and present in the fractions containing the proteasome during glycerol gradient centrifugation. Consistent with the notion that the proteasome cleaves MAGE-A4, the 26S proteasome, ubiquitin, and cell lysates were necessary for efficient in vitrocleavage of MAGE-A4. Conclusions: The present study suggests that a low dose of Adriamycin increases the proteasome activity, which either maintains cellular homeostasis or leads to apoptosis depending, at least under the present conditions, on the expression of MAGE-A4.

High expression of MAGE-A10 cancer-testis antigen in triple-negative breast cancer

Recent studies indicate that ER/PR/HER-2-negative (triple-negative, TN) breast cancers may be "CTA-rich" tumors, suggesting the possibility of CTA-based cancer vaccines as a treatment option for patients bearing these tumors. MAGE-A10 together with NY-ESO-1 is probably the most immunogenic CTA, representing a potentially highly attractive target of active specific immunotherapies. Paraffin-embedded tumor sections were collected retrospectively from 165 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. The expression of MAGE-A10 and NY-ESO-1 was correlated with other clinicopathological variables. MAGE-A10 expression (score ≥ 2+) was detected in 105/164 (64%), and NY-ESO-1 expression (score ≥ 2+) was observed in 14/164 (8.5%) patients. No correlation between MAGE-A10 and NY-ESO-1 expression and tumor size, tumor grade, Ki-67 and lymph nodes status was detectable. MAGE-A10 expression was significantly associated with ER-negative (P = 0.002), PR-negative (P = 0.002) and HER-2-negative (P = 0.044) tumors. We clearly showed that MAGE-A10 is frequently expressed in the group of TN patients, where the majority (85.7%) of tumors express this CTA. Because of limited therapeutic options for the triple-negative breast cancer, the frequent expression of MAGE-A10 CTA in these cancers may offer the opportunity for a much needed additional treatment for this group of patients.

Conventional Dose Hypomethylating Agents Induce CG Antigen Genes In Vivo

Abstract 2441 Background:Cancer testis or cancer germline (CG) antigens are a group of genes whose usual expression is limited to embryonic or germ cell tissues and which can also be aberrantly expressed in a variety of malignancies (Fratta E et al. Mol Oncol. 2011;5:164). The CG antigen genes MAGE-A1, XAGE-1, NY-ESO-1, and PRAME (among others) are epigenetically regulated. Most adult non-germline tissues demonstrate dense CpG island promoter methylation, with no gene expression. Endogenous expression of CG genes in epithelial malignancy can be immunogenic and a number of vaccine strategies are currently underway, however studies in hematologic malignancy have shown limited baseline expression, and thus there has been limited interest in them as a target in AML. Recently, several groups have shown that treatment with the hypomethylating agents, azacitidine(Aza) and decitabine(Dac) are sufficient to re-induce gene expression in myeloid cell lines, prompting interest in these genes as a therapeutic target in myeloid malignancy (Almstedt M et al. Leuk Res 2010;34:899). Re-expression of these genes has also been observed in MDS/AML patients treated with Dac, albeit at higher doses (15–270mg/m2) than those currently in standard use (Sigalotti L et al. Blood 2003;101:4644). Hypothesis:We hypothesized that conventional therapy with Aza(75mg/m2) and Dac(20mg/m2) induces CG promoter hypomethylation and gene expression, and that this may be sufficient to induce anti-CG antigen directed antibody responses. Methods:We obtained samples every 2–4 days from 2 AML patients during the first cycle of Aza or Dac treatment, and analyzed baseline and post-treatment samples for methylation (by pyrosequencing) and CG gene expression (by RT-PCR) of the above genes. We further tested serum from 10 patients for the presence of baseline and post treatment (following 1–10 cycles of Aza or Dac) anti-CG antibodies using an ELISA-based method. Results:Both AML patients treated with hypomethylating agents demonstrated a time dependent decrease in global methylation (by LINE-1 pyrosequencing), and CG antigen promoter methylation which nadired at day 6 post treatment. Baseline methylation of MAGE-A1 (90%), XAGE-1 (85%), NY-ESO-1 (92%) and PRAME (50–60%) was similar and high in both patients pre-treatment. Both Dac and Aza induced hypomethylation of all the CG genes studies, and this hypomethylation correlated with changes in LINE-1 methylation. Low level expression of MAGE-A1, XAGE-1, and PRAME were induced in a time dependent fashion following treatment with both Aza and Dac, however only limited re-expression of NY-ESO-1 was observed. ELISA testing for antibodies against a panel of 25 CG antigens (including MAGE-A1, XAGE-1 and NY-ESO-1) was performed on 10 patients with either MDS or AML. Following hypomethylating agents there was evidence for increased antibody levels, most notably against MAGE proteins, compared to baseline. Conclusion:Conventional hypomethylating agent therapy can induce CG gene expression, which may be sufficient to induce anti-CG antigen antibody responses. Augmentation of anti-tumor immune responses may be possible in myeloid malignancies using a combination of hypomethylating agents and CG antigen-directed vaccines. Disclosures:Odunsi:Ludwig Institute for Cancer Reserach: Membership on an entity's Board of Directors or advisory committees, Research Funding.