MAGE-A3 Expression Research Articles

Gene and Protein Expression of MAGE and Associated Immune Landscape Elements in Non-Small-Cell Lung Carcinoma and Urothelial Carcinomas.

Melanoma-associated antigen-A (MAGE-A) is expressed in multiple cancers with restricted expression in normal tissue. We sought to assess the MAGE-A3/A6 expression profile as well as immune landscape in urothelial (UC) and non-small cell lung carcinoma (NSCLC). We also assessed co-expression of immune-associated markers, including programmed cell death ligand 1 (PD-L1) in tumor and/or immune cells, and assessed the effect of checkpoint inhibitor treatment on these markers in the context of urothelial carcinoma. We used formalin-fixed paraffin-embedded (FFPE) tissue sections from a variety of tumor types were screened by IHC for MAGE-A and PD-L1 expression. Gene expression analyses by RNA sequencing were performed on RNA extracted from serial tissue sections. UC tumor samples from patients treated with checkpoint inhibitors were assessed by IHC and NanoString gene expression analysis for MAGE-A and immune marker expression before and after treatment. Overall, 84 samples (57%) had any detectable MAGE-A expression. Detectable MAGE-A expression was present at similar frequencies in both tumor tissue types, with 41 (50%) NSCLC and 43 (64%) UC. MAGE-A expression was not significantly changed before and after checkpoint inhibitor therapy by both IHC and NanoString mRNA sequencing. Other immune markers were similarly unchanged post immune checkpoint inhibitor therapy. Stable expression of MAGE-A3/A6 pre and post checkpoint inhibitor treatment indicates that archival specimens harvested after checkpoint therapy are applicable to screening potential candidates for MAGE therapies.

MAGE-A4-Responsive Plasma Cells Promote Non-Small Cell Lung Cancer.

Adaptive immunity is critical to eliminate malignant cells, while multiple tumor-intrinsic factors can alter this protective function. Melanoma antigen-A4 (MAGE-A4), a cancer-testis antigen, is expressed in several solid tumors and correlates with poor survival in non-small cell lung cancer (NSCLC), but its role in altering antitumor immunity remains unclear. We found that expression of MAGE-A4 was highly associated with the loss of PTEN , a tumor suppressor, in human NSCLC. Here we show that constitutive expression of human MAGE-A4 combined with the loss of Pten in mouse airway epithelial cells results in metastatic adenocarcinoma enriched in CD138 + CXCR4 + plasma cells, predominantly expressing IgA. Consistently, human NSCLC expressing MAGE-A4 showed increased CD138 + IgA + plasma cell density surrounding tumors. The abrogation of MAGE-A4-responsive plasma cells (MARPs) decreased tumor burden, increased T cell infiltration and activation, and reduced CD163 + CD206 + macrophages in mouse lungs. These findings suggest MAGE-A4 promotes NSCLC tumorigenesis, in part, through the recruitment and retention of IgA + MARPs in the lungs.

Expression pattern analysis of the MAGE family genes in breast cancer patients and hypomethylation activation in the MCF-7 cells

Melanoma antigen gene (MAGE) families are cancer-testis genes that normally show expression in the testes. However, their expressions have been linked with various types of human cancers, including BC. Therefore, the primary purposes of the present research were to assess the expression of MAGE-A, -B, and –C genes in Saudi female patients with BC and determine their regulation via the epigenetic mechanism. Ten BC samples were analyzed for the expression levels of nine MAGE-A genes, six MAGE-B genes, and three MAGE-C genes using the RT-PCR technique. All 18 evaluated genes except for MAGE-A1, -A3, -A4, and -B5 showed weak band expressions in some BC specimens. MAGE-A6 and -B2 were expressed in 40 % of the BC tissue samples, and MAGE-A9, -A10, and -B6 were expressed in 30 %. The lowest expression levels were found for MAGE-A11, -B1, -B3, -B4, -C1, and -C2 in 10 % of the BC specimens and for MAGE-A9,--B2, and --C3 in 20 % of the samples. The most frequently expressed gene was MAGE-A8 (found in 70 % of the BC samples), which suggests that it may serve as - a marker for screening of BC. In vitro treatment, the 5-aza-2′-deoxycytidine agent led to a significant rise in mRNA expressions for all tested genes related to the MAGE-A family, except for MAGE-A10. By contrast, among the genes in the MAGE-B and -C families, only MAGE-B1 and -C2 exhibited detectable mRNA expression levels after treatment.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

Expression of immune-related genes and breast cancer recurrence in women with ductal carcinoma in situ

Background and aimDuctal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer with highly variable clinical behavior, but risk stratification is still challenging. We sought to identify immune-related gene expression signatures of pure DCIS associated with different risks of breast cancer recurrence. MethodsA retrospective nested case-control study of 143 pure DCIS was performed including 70 women with subsequent ipsilateral breast event (IBE, in situ or invasive; cases) and 73 DCIS women with no IBE and matched for age, tumor size, treatment, hormone receptors/HER2 status, and follow-up time (controls). RNA was extracted from DCIS samples and subjected to next-generation sequencing gene expression analysis of 395 immune-related genes. Correlations between DCIS immune-related gene expression and IBE were analyzed using weighted Cox regression for nested case-control data. ResultsEight immune-related genes were differentially expressed between cases and controls. MAGEA10 expression (present vs. absent) and high expression levels of IFNA17 and CBLB (Q4 vs. Q1) were observed more frequently in DCIS of women with subsequent IBE, mainly invasive (p-valueFDR < 0.05). Conversely, expression of IL3RA1, TAGAP, TNFAIP8, and high expression levels of CCL2 and LRP1 were associated with a lower risk of IBE (p-valueFDR < 0.05). ConclusionThis exploratory analysis of pure DCIS showed significant differences in immune-related gene expression profiles between women with and with no subsequent IBE, particularly as invasive IBE. These results, after additional validation, could improve risk stratification and management of DCIS patients.

Cancer testis antigen MAGEA3 in serum and serum-derived exosomes serves as a promising biomarker in lung adenocarcinoma

Cancer testis antigen (CTA) Melanoma Antigen Gene A3 (MAGEA3) were overexpressed in multiple tumor types, but the expression pattern of MAGEA3 in the serum of lung adenocarcinoma (LUAD) remains unclear. Clinically derived serum and serum exosome samples were used to assess the mRNA expression of MAGEA3 and MAGEA4 by qRT-PCR, and serum MAGEA3 and MAGEA4 protein expression were evaluated by ELISA in total 133 healthy volunteers’ and 289 LUAD patients’ serum samples. An analysis of the relationship of the mRNA and protein expression of MAGEA3 and MAGEA4 with clinicopathologic parameters was performed and the diagnostic value of MAGEA3 and MAGEA4 was plotted on an ROC curve. In addition, the correlation of MAGEA3 mRNA with infiltrating immune cells was investigated through TIMER, the CIBERSORT algorithm and the TISIDB database. Expression of serum and serum exosome MAGEA3 and MAGEA4 mRNA were significantly higher in LUAD patients than in healthy donors. MAGEA3 mRNA associated with tumor diameter, TMN stage, and NSE in LUAD serum samples, and MAGEA3 mRNA correlated with N stage in serum-derived exosomes, possessing areas under the curve (AUC) of 0.721 and 0.832, respectively. Besides, serum MAGEA3 protein levels were elevated in LUAD patients, and were closely related to stage and NSE levels, possessing AUC of 0.781. Further analysis signified that the expression of MAGEA3 mRNA was positive correlation with neutrophil, macrophages M2, dendritic cells resting, and eosinophilic, but negatively correlated with B cells, plasma cells, CD8 + T cells, CD4 + T cells, Th17 cells, macrophages and dendritic cells. Collectively, our results suggested that the MAGEA3 expression in mRNA and protein were upregulated in LUAD, and MAGEA3 could be used as a diagnostic biomarker and immunotherapy target for LUAD patients.

Abstract 2746: MAGE-A3 co-expression with IDH1-R132H and IL-1β in colon cancer

Abstract Colorectal cancer (CRC) remains a significant cause of cancer death worldwide with an estimated 52,000 deaths in 2023. Over the past few decades, the incidence rate of CRC in individuals younger than 50 has been steadily increasing. Currently, diagnostic biomarkers play an important role in the detection and treatment of CRC. MAGE-A3 has been shown to be expressed in many tumor types, including colon cancer by RNA expression. Due to their high sequence homology, members of the MAGEA family have been difficult to evaluate and screen for their protein expression. However, in a previous study, highly specific antibodies were identified using CytoSections for MAGE-A3 immunohistochemistry. IL-1β is a cytokine that induces inflammatory responses and when localized to the nucleus in colon cancer cells it can promote stemness. IL-1β expression has been shown to be increased in wildtype IDH1 positive glioblastoma. However, the relationship between IL-1β and IDH1 has not been studied in colon cancer. In this study, the difference in the expression of MAGE-A3, IL-1β, and wildtype IDH1 compared to mutant IDH1-R132H in colon cancer tissues was evaluated. The results show that IL-1β, wildtype IDH1, and MAGE-A3 were co-expressed in 27 of the 38 colon cancer tumors. However, IDH1-R132H expression was only seen in 20% of the tumors. This study suggests that MAGE-A3, IL-1β, and IDH1 are candidate biomarkers for immunotherapy due to their high expression in colon cancer tissues. Citation Format: Andy/Xi Han, Rachel Gonzalez, Jina Yom, Zhaoying Guo, Bailey Gilmore, Eden Zewdu, Tianli Qu, Hailey/YiChen Guo, Qi Ren, Xiaomin Hu, Ranran Zhang, Zhaohui Wu, Xuan Liu, Wei Fu. MAGE-A3 co-expression with IDH1-R132H and IL-1β in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2746.

Abstract 6845: MAGEA3 expression and secretion in MMR-MSH6 and MMR-PMS2 microsatellite-stable colorectal cancer

Abstract Despite the breakthrough of PD-1/PD-L1 blockade therapy in the recent years, mixed responses have been shown across multiple tumor types, including in colorectal cancer (CRC), the second leading cause of death worldwide. This immunotherapy has proven to be successful for a subtype of CRC that carries mismatch repair deficiency (dMMR) and high microsatellite instability (MSI-H), despite a lack of response by microsatellite-stable CRC. For these types of non-responsive cancers, new immunotherapy targets are currently being investigated for alternative therapy such as the MAGE-A cancer testis antigen family whose expression is restricted to germline cells in normal tissue but is overexpressed in many cancer cells. In this study, we examine the relationship between MAGEA3 and MMR proteins MSH6 and PMS2. Immunohistochemical staining of CRC tissues demonstrated co-expression between MAGEA3 with both MSH6 and PMS2. Immune cells were observed to express MAGEA3 in CRC as well. To investigate the possibility of secreted MAGEA3 from tumor cells, we performed western blot analysis of CRC cell lines, ELISA, and binding assay on cell lines and the positive results suggest that MAGEA3 protein is secreted. The results suggest that MAGE-A3 may be a promising target for microsatellite-stable colorectal cancer immunotherapy. Citation Format: Jina Yom, Rachel Gonzalez, Bailey Gilmore, Zhaoying Guo, Andy Han, Eden Zewdu, Dehe Kong, Tianli Qu, Hailey Guo, Alex Strom, Zoe Zhao, Xiaomin Hu, Qi Ren, Yan Ma, Ranran Zhang, Zhaohui Wu, Xuan Liu, Wei Fu. MAGEA3 expression and secretion in MMR-MSH6 and MMR-PMS2 microsatellite-stable colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6845.

Abstract 3194: Discovery of T cell receptor mimic antibody against MAGE-A4 with single B-cell cloning platform

Abstract Antibody therapeutics has been proven clinically against various cancer types and have achieved great success. Majority of the therapeutic antibodies target cell surface or extracellular proteins. Most of the tumor-specific antigens that control cell growth, proliferation, and death are intracellular, and traditional antibody therapies fail to recognize intracellular antigen targets. Such tumor specific intracellular targets consist of overexpressed self antigens, oncofetal protein, cancer testis antigens, viral protein and neo-antigens of mutant proteins. Recently, an emerging approach to target these neo-epitopes has been developed called T-cell receptor-like/TCR-mimic antibodies as they recognize similar epitopes to those of T cell receptors which target specific peptide-MHC complexes(pMHC). TCR mimic antibodies open a new avenue for targeting intracellular antigens yet there are lots of challenges for generating highly selective and potent TCR mimic antibodies. MAGE-A4, melanoma-associated antigen A4, is a member of the MAGE protein family of cancer-testis antigen (CTA). In healthy adult, MAGE-A4 expression is restricted to immune-privileged sites. But MAGE-A4 is widely expressed on many cancers such as lung cancer, head and neck squamous cell cancer, synovial sarcoma(SS), ovarian cancer, urothelial cancer and melanoma. The decapeptide GVYDGREHTV(amino acids 230-239) derived from MAGE-A4, can be presented by HLA-A*02:01 molecule, elicits specific cytotoxicity T cell response. Afami-cel, a genetically modified autologous T cell therapy against this pMHC complex, showed promising potency for patients with metastatic SS. So MAGE-A4/HLA-A*02:01 may be a good target for TCR mimic antibodies and cancer immunotherapy. In the present study, we utilized a newly established single cell cloning platform LyTARS to generate TCR mimic antibodies against HLA restricted MAGE-A4 peptide. Recombinant HLA-A*02:01 MAGE-A4 protein was used to immunize mice and plasma B cells from immunized mice were sorted and loaded onto LyTARS system. Clones that showed reactivity to recombinant HLA-A*02:01 MAGE-A4 but not to irrelevant pMHC (WT-1) were exported and sequenced. Purified antibodies were extensively characterized by the specificity, affinity, and potency by ADCC. Results demonstrated that the TCR mimic antibodies identified have sub-nano molar affinity by protein based ELISA, bind to peptide pulsed T2 cells that endogenously expressing HLA with nano-Molar apparent affinity. TCR mimic antibodies also demonstrated antibody dependent cell killing with the potency of sub-nanomolar. In summary, we have generated and extensively characterized a panel of high affinity and selective TCR mimic antibodies against HLA-A*02:01 MAGE-A4 using a newly established single cell cloning platform. Citation Format: Hao Cheng, You Wu, Tianfeng Shi, Yang Yu, Min Wang, Chenjun Jia, Wenfang Xin, Hu Liu, Teddy Yang, Danmei Yao. Discovery of T cell receptor mimic antibody against MAGE-A4 with single B-cell cloning platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3194.

Expression of Melanoma Antigen Genes A11 and A12 in Non-Small Cell Lung Cancer

Highlights:1. In this study, new primers designed using the semi-nested polymerase chain reaction (PCR) method were utilized to identify MAGE-A11 and MAGE-A12 expressions in specimens collected from core biopsy, forcep biopsy, and bronchoalveolar lavage.2. The histopathological analysis revealed positive expressions of MAGE-A11 and MAGE-A12 in specimens diagnosed with non-small cell lung cancer (NSCLC) as well as in specimens with no malignant cells. AbstractThe melanoma antigen gene (MAGE) belongs to the group of cancer testis antigens that are exclusively expressed in germ cells but may be re-expressed in cancer cells. The highly expressed MAGE-A subfamily in lung cancer may potentially be a diagnostic and prognostic biomarker. This study aimed to identify MAGE-A11 and MAGE-A12 expressions in lung tumors obtained from core biopsy, forceps biopsy, and bronchoalveolar lavage specimens. A cross-sectional observational study was conducted on 90 patients clinically diagnosed with lung tumors. These patients received core biopsy, forceps biopsy, and bronchoalveolar lavage interventions after ethical approval was obtained. The complementary deoxyribonucleic acid (cDNA) quality was assessed by the polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assessment was performed to ascertain if all specimens exhibited positive PCR amplification of the GAPDH gene. MAGE-A11 and MAGE-A12 were identified through a semi-nested reverse transcription PCR. The positive results were detected by measuring the PCR products, with MAGE-A11 and MAGE-A12 measuring at base pairs (bp) of 858 and 496 in the first and second rounds, respectively. The expressions of MAGE-A11 and MAGE-A12 were observed in 3 (3.33%) and 40 (44.44%) out of 90 specimens, respectively. The prevalence rate of non-small cell lung cancer (NSCLC) was 31.11% (28/90). Among these cases, 3.57% (1/28) showed the expression of MAGE-A11, while 32.14% (9/28) exhibited the expression of MAGE-A12. Sixty-two (68.89%) out of 90 patients were diagnosed with no tumor cell malignancy. Out of 62 cases, 2 (3.23%) exhibited the expression of MAGE-A11, while 31 (50%) demonstrated the expression of MAGE-A12. MAGE-A11 and MAGE-A12 were detected in NSCLC and in certain specimens with a pathological diagnosis that indicated the absence of malignant cells. In conclusion, MAGE A11 and MAGE A12 have potential markers that can improve the pathological diagnosis of lung cancer. Further investigation is necessary to explore the expression of MAGE-A in correlation with lung cancer progression.

How the Intrinsically Disordered N-Terminus of Cancer/Testis Antigen MAGEA10 Is Responsible for Its Expression, Nuclear Localisation and Aberrant Migration.

Melanoma-associated antigen A (MAGEA) subfamily proteins are normally expressed in testis and/or placenta. However, aberrant expression is detected in the tumour cells of multiple types of human cancer. MAGEA expression is mainly observed in cancers that have acquired malignant phenotypes, invasiveness and metastasis, and the expression of MAGEA family proteins has been linked to poor prognosis in cancer patients. All MAGE proteins share the common MAGE homology domain (MHD) which encompasses up to 70% of the protein; however, the areas flanking the MHD region vary between family members and are poorly conserved. To investigate the molecular basis of MAGEA10 expression and anomalous mobility in gel, deletion and point-mutation, analyses of the MAGEA10 protein were performed. Our data show that the intrinsically disordered N-terminal domain and, specifically, the first seven amino acids containing a unique linear motif, PRAPKR, are responsible for its expression, aberrant migration in SDS-PAGE and nuclear localisation. The aberrant migration in gel and nuclear localisation are not related to each other. Hiding the N-terminus with an epitope tag strongly affected its mobility in gel and expression in cells. Our results suggest that the intrinsically disordered domains flanking the MHD determine the unique properties of individual MAGEA proteins.

Expression Pattern of Tumor-associated Antigens in Neuroblastoma: Association With Cytogenetic Features and Survival.

The prognosis of high-risk and relapsed neuroblastoma (NB) patients remains poor. The identification of tumor-associated markers is important for differential diagnosis, prognosis, and the development of targeted therapies. The aim of the study was to determine the expression profile of nine most common NB antigens and assess their association with clinicopathological characteristics and patient survival. Tumor samples from 86 patients with NB were evaluated for the expression of tumor-associated antigen (TAA) using quantitative PCR. Twenty-one patients with benign tumors and 17 healthy donors were assigned as controls. Overexpression of tyrosine hydroxylase (TH), PHOX2B, PRAME, GPC2, B7-H3, and Survivin is the most typical for NB. Positive expression of MAGEA3, MAGEA1, and NY-ESO-1 at low levels was detected in 54%, 48%, and 52%, respectively, and was not NB specific. Higher TH expression was observed in samples without MYCN-amplification, while higher expression of Survivin, PHOX2B, and GPC2 was significantly associated with the presence of 1p.36 deletion. Overexpression of TH, PHOX2B, and MAGEA1 was associated with better event-free (EFS) and overall survival (OS). Survivin overexpression was associated with poor EFS but had no impact on OS. Multivariate analysis confirmed Survivin as independent marker for poor survival, and PHOX2B and MAGEA1 for better survival. High expression of TH, PHOX2B, and MAGEA1 genes are favorable prognostic factors for OS and EFS, whereas high expression of Survivin is associated with an increased risk of relapse or progression.

Immunization with a multi-antigen targeted DNA vaccine eliminates chemoresistant pancreatic cancer by disrupting tumor-stromal cell crosstalk

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance.MethodsWe used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth.ResultsWe establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors.ConclusionsThese findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

MAGE-A10 Protein Expression in Advanced High Grade Serous Ovarian Cancer Is Associated with Resistance to First-Line Platinum-Based Chemotherapy.

Ovarian cancer has a dismal prognosis. Standard treatment following surgery relies on platinum-based chemotherapy. However, sizeable percentages of patients are unresponsive. Identification of markers predicting the response to chemotherapy might help select eligible patients and spare non-responding patients from treatment-associated toxicity. Cancer/testis antigens (CTAs) are expressed by healthy germ cells and malignant cells of diverse histological origin. This expression profile identifies them as attractive targets for cancer immunotherapies. We analyzed the correlations between expression of MAGE-A10 and New York esophageal-1 cancer (NY-ESO-1) CTAs at the protein level and the effectiveness of platinum-based chemotherapy in patients with advanced-stage high-grade serous ovarian carcinoma (HGSOC). MAGE-A10 and NY-ESO-1 protein expression was analyzed by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded samples from 93 patients with advanced-stage HGSOC treated at our institutions between January 1996 and December 2013. The correlation between the expression of these markers and response to platinum-based chemotherapy, evaluated according to RECIST 1.1 criteria and platinum sensitivity, measured as platinum-free interval (PFI), progression free (PFS), and overall survival (OS) was explored. The MAGE-A10 protein expression predicted unresponsiveness to platinum-based chemotherapy (p = 0.005), poor platinum sensitivity (p < 0.001), poor PFS (p < 0.001), and OS (p < 0.001). Multivariate analysis identified MAGE-A10 protein expression as an independent predictor of poor platinum sensitivity (p = 0.005) and shorter OS (p < 0.001). Instead, no correlation was observed between the NY-ESO-1 protein expression and response to platinum-based chemotherapy (p = 0.832), platinum sensitivity (p = 0.168), PFS (p = 0.126), and OS (p = 0.335). The MAGE-A10 protein expression reliably identified advanced-stage HGSOC unresponsive to platinum-based chemotherapy. Targeted immunotherapy could represent an important alternative therapeutic option in these cancers.

Expression of four cancer-testis antigens in TNBC indicating potential universal immunotherapeutic targets

ObjectiveImmunotherapy is an attractive treatment for breast cancer. Cancer-testis antigens (CTAs) are potential targets for immunotherapy for their restricted expression. Here, we investigate the expression of CTAs in breast cancer and their value for prognosis. So as to hunt for a potential panel of CTAs for universal immunotherapeutic targets.Material and methodsA total of 137 breast cancer tissue specimens including 51 triple-negative breast cancer (TNBC) were assessed for MAGE-A4, MAGEA1, NY-ESO-1, KK-LC-1 and PRAME expression by immunohistochemistry. The expression of PD-L1 and TILs was also calculated and correlated with the five CTAs. Clinical data were collected to evaluate the CTA’s value for prognosis. Data from the K-M plotter were used as a validation cohort.ResultsThe expression of MAGE-A4, NY-ESO-1 and KK-LC-1 in TNBC was significantly higher than in non-TNBC (P = 0.012, P = 0.005, P < 0.001 respectively). 76.47% of TNBC expressed at least one of the five CTAs. Patients with positive expression of either MAGE-A4 or PRAME had a significantly extended disease-free survival (DFS). Data from the Kaplan–Meier plotter confirm our findings.ConclusionsMAGE-A4, NY-ESO-1, PRAME and KK-LC-1 are overexpressed in breast cancer, especially in TNBC. Positive expression of MAGE-A4 or PARME may be associated with prolonged DFS. A panel of CTAs is attractive universal targets for immunotherapy.

Expression of cancer-testis antigens in the immune microenvironment of non-small cell lung cancer.

The antigenic repertoire of tumors is critical for successful anti-cancer immune response and the efficacy of immunotherapy. Cancer-testis antigens (CTAs) are targets of humoral and cellular immune reactions. We aimed to characterize CTA expression in non-small cell lung cancer (NSCLC) in the context of the immune microenvironment. Of 90 CTAs validated by RNA sequencing, eight CTAs (DPEP3, EZHIP, MAGEA4, MAGEB2, MAGEC2, PAGE1, PRAME, and TKTL1) were selected for immunohistochemical profiling in cancer tissues from 328 NSCLC patients. CTA expression was compared with immune cell densities in the tumor environment and with genomic, transcriptomic, and clinical data. Most NSCLC cases (79%) expressed at least one of the analyzed CTAs, and CTA protein expression correlated generally with RNA expression. CTA profiles were associated with immune profiles: high MAGEA4 expression was related to M2 macrophages (CD163) and regulatory T cells (FOXP3), low MAGEA4 was associated with T cells (CD3), and high EZHIP was associated with plasma cell infiltration (adj. P-value < 0.05). None of the CTAs correlated with clinical outcomes. The current study provides a comprehensive evaluation of CTAs and suggests that their association with immune cells may indicate in situ immunogenic effects. The findings support the rationale to harness CTAs as targets for immunotherapy.

Abstract 5948: MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer

Abstract Colorectal Cancer (CRC) is the second leading cause of death worldwide. Recent developments in cancer immunotherapy have obtained encouraging clinical responses, including PD-1/PD-L1 blockade therapy, although response rates vary depending on tumor type. For example, despite the lack of response by microsatellite-stable CRC, a subset of CRC with deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H) responds especially well to the PD-1/PD-L1 blockade therapy. For other types of CRC which do not respond to PD-L1 therapy, new targets are being investigated. One family of targets is the cancer testis antigen MAGE-A whose expression is restricted to germline cells in normal tissue but is overexpressed in many cancer cells. In this study, immunohistochemical analysis of MAGE-A3 expression in colon cancer along with IHC screening of mismatch protein MSH6 was performed. Many of the cases in this study observed a co-expression in the tissue. In the tumors, MAGE-A3 was observed to have weak cytoplasmic expression while MSH6 had strong nuclear expression in the epithelial cells. The immune cells adjacent to the epithelial cells were also positive for MAGE-A3. This result suggests that MAGE-A3 may be a promising target for microsatellite-stable colorectal cancer immunotherapy. Citation Format: Jina Yom, Rachel Gonzalez, Eden Zewdu, Aubrey Su, Patrick Yin, Bailey Gilmore, Dehe Kong, Andy Han, Zhaoying Guo, Tianli Qu, Zoe Zhao, Eric Christenson, Yan Ma, Hailey Guo, Xiaomin Hu, Qi Ren, Zhaohui Wu, Xuan Liu, Wei Fu. MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5948.

Granulocyte-colony stimulating factor does not prevent in vitro cisplatin-induced germ cell reduction in immature human and mouse testis

BackgroundCurrently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse (fetal and pre-pubertal) testicular tissues.MethodsOrganotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal (n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium (including groups receiving G-CSF before, during and after cisplatin exposure). In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified.ResultsExposure to cisplatin resulted in a reduced germ cell number in human fetal and mouse pre-pubertal testicular tissues at 240 hrs post-exposure. Germ cell number was not preserved by combined exposure with G-CSF using any of the exposure regimens (prior to, during or after cisplatin exposure). Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues.ConclusionsThis study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.

Safety and efficacy from the phase 1 SURPASS trial of ADP-A2M4CD8, a next-generation T-cell receptor T-cell therapy, in patients with advanced esophageal, esophagogastric junction, or gastric cancer.

349 Background: ADP-A2M4CD8 is a specific peptide enhanced affinity receptor mixed CD4+ and CD8+ T-cell therapy targeting the cancer testis antigen MAGE-A4 and modified with addition of a CD8α co-receptor designed to provide additional functionality to CD4+ T-cells. ADP-A2M4CD8 has demonstrated a favorable benefit to risk profile in the Phase 1 SURPASS trial (NCT04044859) in HLA A*02–eligible patients (pts) with unresectable or metastatic tumors positive for MAGE-A4 (Hong DS, et al. E-poster 540P: ESMO 2021; Virtual). Here we report updated clinical outcomes in pts with esophageal, esophagogastric junction (EGJ), or gastric cancer. Methods: SURPASS is a first-in-human trial consisting of a modified 3+3 dose-escalation design and an expansion cohort. Autologous T-cells are obtained by leukapheresis, transduced with a self-inactivating lentiviral vector expressing the MAGE-A4-specific T-cell receptor and the CD8α co-receptor, and infused back to the pts as ADP-A2M4CD8 following lymphodepleting chemotherapy. Primary and secondary objectives are safety and anti-tumor activity, respectively. Results: As September 6, 2022, 13 pts (11 male, 2 female) with esophageal (3), EGJ (8), and gastric cancer (2) received ADP-A2M4CD8 (range: 1.02–9.9x109 transduced T-cells). All pts had adenocarcinoma, median age was 55 years (range: 31–71), median MAGE-A4 expression H-score was 245 (range: 160–300), and pts received median of 2 lines of prior therapy (range 1–5). Adverse events (AEs) were consistent with those typically associated with lymphodepleting chemotherapy, cellular therapy, and/or disease. One pt had a Grade 5 (fatal) AE of pancytopenia. Overall response rate per RECIST v1.1 by investigator review was 15% (2 partial response [PR]). Disease control rate was 77% (2 PR+8 stable disease). This trial is ongoing, and additional data will be presented. Conclusions: Results indicate an acceptable benefit to risk profile and encouraging anti-tumor activity of ADP-A2M4CD8. An additional treatment cohort has been included in the updated SURPASS trial protocol to evaluate ADP-A2M4CD8 combined with nivolumab. A Phase 2, open-label trial in advanced esophageal and EGJ cancers has been initiated (SURPASS-2; NCT04752358). Clinical trial information: NCT04044859 .

157P Phase I expansion of IMC-C103C, a MAGE-A4×CD3 ImmTAC bispecific protein, in ovarian carcinoma

ImmTAC® bispecific proteins redirect polyclonal T cells to target intra/extracellular proteins using a T cell receptor as targeting domain. IMC-C103C (MAGE-A4 × CD3) is an ImmTAC against MAGE-A4, which is expressed in several tumors including ovarian carcinoma (OC). ≥ 90 μg IMC-C103C was demonstrated as the clinically active range (Davar 2021). Pre-selection for MAGE-A4 expression was not required as a majority of OC express MAGE-A4 (H score ≥ 1) and tebentafusp demonstrated OS benefit and ctDNA reductions regardless of H score, although RECIST responses were enriched at higher H scores (Leach 2021).