Gastroesophageal adenocarcinoma poses a significant threat, as existing systemic treatment options are limited in their efficacy. Therefore, it is essential to identify new therapeutic targets and develop treatments that specifically target these novel findings. One potential avenue of exploration involves targeting cancer/testis antigens (CTAs).

Before undertaking this task, a comprehensive understanding of the expression and co-expression landscape of cancer/testis antigens (CTAs) is crucial for identifying ideal targets for treatment. Unfortunately, data on CTA expression in gastroesophageal adenocarcinoma (GEAC) is currently limited. This study aims to fill this knowledge gap by investigating the expression patterns in a cohort of sixty-three GEAC patients through tumor sample gene expression analysis. Furthermore, the findings from our primary cohort were validated in a separate set of 329 patients extracted from The Cancer Genome Atlas. To our knowledge, this is the first study looking at CTA expression in GEAC. Some studies have explored CTA expression in cell lines, but none have explored this at a population level.

Our findings revealed that CTA genes exhibited expression levels ranging from 19 to 58% across patients. The patterns of expression were consistent across both the primary and TCGA cohorts, with the MAGE family of genes positioned at the higher end of the spectrum, while genes like BAGE, LAGE1A, and NY.ESO.1 demonstrated lower expression levels. An interesting anomaly was observed in the primary cohort, where MLANA was scarcely expressed, whereas in the TCGA cohort, it exhibited near-universal expression. This discrepancy might be attributed to MLANA primarily serving as a melanoma antigen, resulting in highly variable expression within esophageal and stomach tumors. Furthermore, our investigation revealed a significant level of co-expression among CTA genes. In both cohorts, CTA genes exhibited a cohesive cluster of expression, excluding MLANA. The MAGE family of genes exhibited the most consistent co-expression both within their own group and with other CTA genes, with Spearman R2 values ranging from 0.53 to 0.73. Likewise, similar patterns of co-expression were noted among the GAGE family of genes. Other genes like NY-ESO-1 and LAGE-1A exhibited more variable co-expression, with R2 values ranging from 0.37 to 0.71, which are still relatively high. Although the biological mechanism is unclear, co-expression patterns of Cancer/Testis Antigens (CTAs) may arise from their common expression on the X chromosome, shared epigenetic regulatory mechanisms [27], or a transcriptional master switch [28]. These findings highlight that CTAs are fairly highly expressed in GEAC. Therefore, researchers can use this preliminary data to explore targeted interventions to address CTAs in GEAC. Moreover, despite the variability in CTA expression, many genes are significantly co-expressed. This suggests the feasibility of targeting multiple genes in individuals positive for a single gene, thereby enhancing the potential efficacy of therapeutic interventions. Notably, one of the most extensively studied CTAs in gastric and esophageal cancer includes the MAGE family of genes.

The Melanoma Antigen Gene family (MAGE) proteins were among the first identified members of cancer/testis antigens (CTAs) [29]. In 1995, Inoue et al. reported that at least one MAGE gene was expressed in 33 out of 42 esophageal tumor tissues, while none were expressed in 42 normal esophageal tissues [30]. Furthermore, Lian et al. found positive MAGE-A expression in 54.7% of resected gastric cancer patients, associating increased MAGE-A expression with poor differentiation, and a high clinical TNM stage [31]. In our analysis, we observed high expression and co-expression of MAGEA3, MAGEA12, MAGEA4, MAGEA10, and MAGEA1. Additionally, patients positive for MAGEA1, MAGEA12, MAGEA3, and MAGEA4 treated with immune checkpoint inhibitors (ICIs) demonstrated improved overall survival, suggesting that the MAGE family of genes presents an attractive target for developing targeted therapies. Targeted therapies against various MAGE antigens are still in phase I and II trials. A phase I trial investigated oncolytic virus therapy against MAGE-A3 in esophageal and gastric cancer patients, establishing safety and potential anti-tumor immune response [32]. This phase I trial established the safety of oncolytic virus therapy against MAGE-A3 as well as potential anti-tumor immune response. Another promising area of current research is T cell receptor therapy (TCR-T). In this, artificial expression of engineered T cell receptors (TCRs) in autologous T cells enables targeting of specific antigens [33]. Multiple trials targeting MAGE-A4 using TCR-T are in progress [11]. Preliminary results from a phase II trial (SURPASS) have shown an acceptable safety profile and evidence of antitumor activity in patients with gastroesophageal cancer after injection with next-generation ADP-A2M4CD8 SPEAR T-cells co-expressing the CD8α co-receptor with the engineered MAGE-A4c1032 T cell receptor (NCT04044859). Despite promising results from phase I and II trials, randomized controlled trials are required to translate these promising findings into clinical practice.

Shifting our focus to the use of CTAs as prognostic biomarkers, previous evidence has indicated that increased expression of CTAs is associated with worse overall survival in different cancer types [14, 34, 35]. In a cohort of 102 patients with intrahepatic cholangiocarcinoma, those with at least one CTA marker expression were significantly associated with worse overall survival [36]. Similarly, in esophageal squamous cell carcinoma, Sang et al. found that MAGE-A11 expression was an independent poor prognostic factor [37]. In gastric cancer, Lian et al. found that MAGE-A expression was associated with lymph node metastasis and poor 5-year overall survival but that it was not an independent prognostic factor [31]. In this study, we sought to understand the value of CTA expression as a prognostic biomarker. We did not see any consistently significant results in the two cohorts; however, we did see several trends when looking at individual CTAs. In the TCGA cohort, our analysis revealed that the overexpression of MAGE4 and MAGEA1 correlated with significantly reduced survival. Conversely, in our primary cohort, we observed that patients positive for MAGEC2 exhibited markedly better survival than those negative for MAGEC2. This discrepancy may be attributed to the composition of the primary cohort, which encompasses primarily metastatic cases, in contrast to the TCGA cohort, which comprises mostly non-metastatic tumors. This incongruity may also be attributed to the fact that 28% of the patients in the primary cohort received treatment with immune checkpoint inhibitors (ICIs), whereas no patients in the TCGA cohort underwent such treatment. We subsequently conducted a subgroup analysis of the primary cohort, stratifying patients based on ICI treatment status. In this analysis, we found that MAGEC2-positive patients not receiving ICI treatment experienced significantly reduced survival compared to MAGEC2-negative patients, similar to the TCGA cohort. Interestingly, among patients treated with ICIs, those positive for GAGE13, SSX2, MAGEA1, MAGEA12, MAGEA3, MAGEA4, and NY.ESO.1 all demonstrated significantly higher overall survival than those negative for the corresponding CTA genes. This aligns with the notion that CTA overexpression is associated with worse survival but also suggests that they present attractive targets for immunotherapy, potentially leading to increased treatment response. In a recent study, Seager et al. demonstrated a significantly improved overall survival rate in pembrolizumab-treated lung cancer patients with increased CTA burden [38].

A significant caveat to our results is that this is a single institution retrospective study, and further prospective studies are required to evaluate this association. Other limitations of our study include small patient numbers, and lack of information on disease stage (treated or untreated), stage at diagnosis, and biopsy site.