Gastric cancer (GC) ranks the fifth most commonly diagnosed cancer type globally and the third leading cause for cancer-related death, which was attributed to its diagnosis usually made at an advanced stage. Although gastric cancer incidence has declined in most countries over the past century, the aging population may contribute to increased diagnosis of gastric cancer (Smyth et al., 2020).

Gastric cancer is a highly heterogeneous disease characterized by histopathologic and epidemiologic features based on molecular and phenotypic levels (Van Cutsem et al., 2016). Next-generation sequencing has showed new insights into the heterogeneity of gastric cancer, and subtyping systems have been proposed (Cancer Genome Atlas Research, 2014; Cristescu et al., 2015; Sohn et al., 2017; Kim et al., 2019; Zhang et al., 2020). The Lauren classification system categorizes gastric cancer into the intestinal and diffuse subtypes (Lauren, 1965), while the WHO system divides gastric cancer into four subtypes (papillary, tubular, mucinous, and poorly cohesive) (Hu et al., 2012). Apart from the aforementioned classification subtypes, researchers from TCGA proposed four subtypes for gastric cancer: Epstein–Barr virus (EBV) positive, microsatellite unstable (MSI), genomically stable (GS), and chromosomal instability (CIN). The EBV subtype has the best prognosis among these four subtypes. Patients with the CIN subtype experienced the greatest benefit from adjuvant chemotherapy (Sohn et al., 2017). The Asia Cancer Research Group (ACRG) proposed four molecular subtypes for GC based on microsatellite instability, epithelial–mesenchymal transition, and TP53 mutation: MSI, MSS/EMT, MSS/TP53⁺, and MSS/TP53^- (Cristescu et al., 2015). The EBV subtype and MSI subtype were reported to potentially benefit from immunotherapy (Pang et al., 2009; Le et al., 2017; Amatatsu et al., 2018; Sundar et al., 2018). However, few gastric cancer subtypes development is based on immune signature and can be used to predict the prognosis of gastric cancer patients.

Immune processes play critical roles in carcinogenesis and progression of solid tumors, and they also affect the treatment and prognosis of patients. Researchers are confused with the association between the immune environment and the prognosis, and much attention has been paid to the tumor immune environment (Cully, 2018; Lim et al., 2018; Kubota et al., 2021). All tumors are potentially immunogenic, and the new knowledge about the interactions between tumor cells, immune cells, and tumor microenvironment allowed for reversal of possible immune resistance (Refolo et al., 2020; Ceresoli and Pasello, 2021). The immune response is a complex multistep process that finely regulates the balance between the recognition of non-self and the prevention of autoimmunity. Cancer cells can use these pathways to suppress tumor immunity as a major mechanism of immune resistance. The recent molecular classifications of gastric cancer by The Cancer Genome Atlas (TCGA) and by the Asian Cancer Research Group (ACRG) networks, together with the identification of multiple biomarkers, open new perspectives for stratification of patients who might benefit from a long-term immune checkpoint therapy (Newman et al., 2015; Jiang et al., 2018; Thorsson et al., 2019; Ceresoli and Pasello, 2021).

The purposes of this study are to characterize different potential molecular classification systems operative in gastric cancer and to identify previously unreported significant immune environments and independent prognosis factors for patients with gastric cancer. We collected 1386 samples from five datasets and applied molecular subtyping on each dataset. We achieved two distinct molecular subtypes of gastric cancer (i.e., C1/C2). The C2 subtype has a better prognosis and more activated immune microenvironment than the C1 subtype.

The clinical significance of the molecular subtype has been demonstrated in many kinds of cancers. However, few researchers have used immune signatures to predict gastric cancer subtypes.

A major clinically relevant finding in this study is based on signature of 390 immune-related genes; we classify gastric cancer into two prognostically distinct subgroups, namely, C1 and C2. The prognostic significance of C1/C2 was independent of age, gender, and stage. The C1 subtype is featured by “Epithelial Mesenchymal Transition (EMT)” and “Angiogenesis,” which had poorer overall survival, whereas the C2 subtype is characterized by “MYC Target,” “Oxidative Phosphorylation,” and “E2F Target,” with better overall survival than those in C1. Notably, previous research studies reported that EMT was shown to strongly enhance cancer cell motility and dissemination; it plays an important role in cancer metastasis (Brabletz, 2012; Brabletz et al., 2018). Angiogenesis is essential for the late stages of carcinogenesis, allowing the tumor to grow beyond 1–2 mm in diameter; it is associated with the malignancy of tumor (Sharma et al., 2001; Albini et al., 2012). Such processes may cause poor survival in C1.

The results of CIBERSORT suggest that C1 and C2 have very different immune environments. In C2, such as CD4 memory activated, NK cells activated, mast cells activated, and dendritic cells activated, and the composition of all these four kinds of activated immune cells are significantly higher than C1. In contrast, in C1, such as B cells were naive, T cells memory resting, dendritic cells (DC) resting, and mast cells resting, and these kinds of immune resting cells significantly higher than C2. The microenvironment between C1 and C2 shows a marked difference. Previous research studies reported that memory CD4 T cells could make effector cytokines early in response and they could enhance B-cell and CD8 T-cell responses, which enhance immune response (MacLeod et al., 2009). NK cells are important immune cells; they could swiftly kill multiple adjacent cells which show surface markers associated with oncogenic transformation; and they could also magnify immune responses (Shimasaki et al., 2020). Mast cells are evolutionarily ancient cells, and they finely modulate not only immune responses but also the mechanism of several inflammatory disorders, including cancer, autoimmunity, and infection (Frossi et al., 2017). DCs are a diverse group of specialized antigen-presenting cells; they play key roles in the initiation and regulation of innate and adaptive immune responses (Wculek et al., 2020). The activation of these immune cells in C2 may indicate higher immune activity, which leads better prognosis. The resting of these immune cells may cause poor immune activity in C1, which leads to worse prognosis in C1.

The proportion of C1/C2 in four tumor stages demonstrates C1 has higher proportion in advanced gastric cancer, while C2 has higher proportion in early stages. The proportion of C1/C2 in the Lauren type shows that C1 has the highest proportion in the diffuse type, while C2 has higher proportion in the intestinal type. This indicates that C1 has some characteristics of malignant gastric cancer.

The somatic mutation event of SMGs shows significant differences between C1 and C2, and C2 has higher TMB than C1. Previous research reported that TMB can be used as an indicator to predict the response to immunotherapy, and patients with high TMB were observed to have better clinical outcomes (Gibney et al., 2016; Gandara et al., 2018; Mandal et al., 2019). It also reported that high TMB is associated with a better prognosis in gastric cancer (Cai et al., 2020; Wang et al., 2021). The differences in mutation characteristics may lead to different clinical outcomes of C1/C2, and it also could offer some new insights into immunotherapy in gastric cancer.

In total, in this research, we predict C1 and C2, two subtypes of gastric cancer. Much evidence has shown that there are many different biological characteristics between C1 and C2. It makes two subtypes that could predict prognosis in gastric cancer patients. However, this research still has some limitation. First, the sample size is not large enough; therefore, research may not cover all types of gastric cancer. Second, due to the lack of clinical data, the subtypes in this research could only be used to predict the survival of gastric cancer patients but could not predict their response to chemoradiotherapy and immunotherapy. If more gastric cancer chemotherapy and immunotherapy data could be combined, C1/C2 could be given more clinical significance and immune characteristics could provide more insights into gastric cancer treatment.

Our research has developed two molecular subtypes of gastric cancer, and we have analyzed their immune signature and biological function. These findings may offer some new knowledge of molecular mechanisms for study on treatment of gastric cancer.