In this study, we detected the microsatellite phenotype, mucin phenotype and PD-L1 expression of INADA by PCR and IHC. Among 54 patients, 19 showed the MSI phenotype (35.2%), which is lower than the rate of 51.6% reported by Watari et al. (Watari et al. 2019). This discrepancy may be attributed to different microsatellite markers used, different detection methods, or patients with different malignancies. In our study, all patients were classified as having INADA, which invaded the submucosa (T1) and deeper tissues (T2, T3 and T4), whereas the study reported by Watari et al included all stages of NADA from stage 0 to stage IV. Nonetheless, the MSI rate reported by us was similar to other reported rates in small bowel cancer (Overman et al. 2010; Warth et al. 2011). According to our analysis, there was no significant correlation between microsatellite status and clinicopathological factors. In general, immune checkpoint ligands, including PD-L1, are strongly expressed in the MSI tumor microenvironment to counterbalance the antitumor immune activity of cytotoxic T lymphocytes. Several studies have reported a correlation between MSI and PD-L1 expression in gastrointestinal tumors, some of which showed that the MSI phenotype correlates positively with PD-L1 expression (Choi et al. 2020; Giuffrida et al. 2020; Liu et al. 2020). The lack of a positive correlation between MSI and PD-L1 expression in tumor/immune cells in our study is consistent with some recent reports (Klose et al. 2020; Salem et al. 2018; Watari et al. 2019). In general, expression of PD-L1 in tumor cells is variable and not always accompanied by MSI. PD-L1 expression may also be affected by other factors, such as the tumor mutational burden and number of CD8 + tumor-infiltrating lymphocytes. MSI-L/low tumor mutational burden but high PD-L1 positive expression have been observed in gastrointestinal stromal tumors, anal cancer and esophageal squamous cell carcinoma (Salem et al. 2018).
Through Kaplan-Meier survival analysis, we found that INADA patients with MSI had better survival than did patients with MSS (P = 0.013), which was consistent with the results of previous studies in gastric cancer, small bowel cancer and CRC (Giuffrida et al. 2020; Polom et al. 2018; Yang et al. 2019; Yang et al. 2020). The detailed mechanism by which MSI-positive patients have a better prognosis has been explored, and it appears that this phenomenon may be due to a strong antitumor immune response triggered in the tumor microenvironment. MSI patients tend to present a higher tumor mutational burden and strong ability to produce novel, nonself neoantigens, such that many more cytotoxic T lymphocytes are attracted to infiltrate into the tumor microenvironment. These high-density tumor-infiltrating lymphocytes inhibit the invasion and infiltration of cancer cells. Therefore, MSI patients often have a favorable prognosis and good survival time (Badalamenti et al. 2019). Because MSI patients have a higher tumor mutational burden, MSI in coding regions predisposes generation of truncated proteins as novel antigens, also known as frameshift peptides (FSPs), as a result of frameshift mutations. Extensive studies related to FSP vaccines have been conducted (Roudko et al. 2020). For example, through basic research, Leoni G et al (Leoni et al. 2020) found that the FSP vaccine can induce the optimal extent of the immune response, which might result in clinical benefits for preventing and treating MSI tumors. Therefore, we speculate that combination therapy of the FSP vaccine and immune checkpoint inhibitors based on anti-PD-1/PD-L1 antibodies may be able to achieve greater benefits than the single use of immune checkpoint inhibitors for MSI/PD-L1 + tumor patients.
Tumor stage is the most important prognostic factor in SBA (Zaaimi et al. 2016), and we found that the survival of patients gradually worsened with increasing tumor stage. It is well known that advanced cancer patients have a worse prognosis, as reported by many studies (Aparicio et al. 2013; Huffman et al. 2019; Ocasio Quinones and Woolf 2021; Raghav and Overman 2013; Zaaimi et al. 2016).
Many previous studies have been carried out to evaluate the prognostic value of PD-L1 expression in tumor cells and immune cells in different cancers (Fu et al. 2020; Giuffrida et al. 2020; Klose et al. 2020; Liu et al. 2020; Morihiro et al. 2019; Zhou et al. 2020), though the association between PD-L1 expression and prognosis is controversial. Klose et al (Klose et al. 2020), Giuffrida et al (Giuffrida et al. 2020), and Liu et al (Liu et al. 2020) reported that patients with PD-L1-positive expression have better survival outcomes than PD-L1-negative patients. At the same time, PD-L1 positive expression has been related to worse OS, as reported by Morihiro et al (Morihiro et al. 2019), Zhou et al (Zhou et al. 2020), Dai et al (Dai et al. 2018), and Fu et al (Fu et al. 2020). In our current study, no significant correlation was observed between PD-L1 expression and OS, which is consistent with a recent report (Bonanno et al. 2018). This discrepancy may be due to the different therapeutic methods provided to patients, such as chemotherapy and immunotherapy. For those with positive expression of PD-L1, immune checkpoint inhibitors, such as antibodies against PD-1/PD-L1, may improve survival.
Our research has some limitations. First, NADA is a rare disease, and the sample size included in this study was quite small. Second, due to the lack of sufficient tissue, we were unable to detect the density of tumor-infiltrating lymphocytes by IHC. Recently, it has been reported that PD-L1 expression with MSI/CD8 + lymphocyte infiltration is considered an important and useful indicator to predict the effectiveness of immune checkpoint inhibitors (Noh et al. 2020).
In conclusion, the MSI phenotype and an early tumor stage were significantly associated with better survival than MSS and a late tumor stage, and both were independent better prognostic factors. However, there was no significant correlation between the MSI phenotype and PD-L1 expression. Moreover, expression of PD-L1 in tumor cells/immune cells was not related to prognosis. A high proportion of MSI phenotypes and PD-L1 expression may be helpful when considering immune checkpoint inhibitors as a therapeutic strategy. In addition, a comprehensive assessment of predictors of immune checkpoint inhibitors, including the tumor mutational burden, density of tumor-infiltrating lymphocytes and expression of PD-1 and other molecular checkpoints, is needed in addition to PD-L1 expression and microsatellite status evaluations.