In recent years, immunotherapy for advanced G/GEJ cancer has attracted widespread attention. Prospective clinical trials, such as CheckMate 649[7], Orient 16[9], and RATIONALE 305[27] have demonstrated an effective clinical response in patients with advanced HER2-negative gastric cancer. Our results, consistent with the results of the ORIENT16 study, further demonstrate the efficacy and safety of adding anti-PD-1 sintilimab to platinum-based chemotherapy, which resulted in a statistically significant and clinically meaningful improvement in both progression-free survival and overall survival compared to chemotherapy alone, not only in the overall population but also in patients with strong PD-L1 expression.
However, the efficacy of anti-PD-1 treatment in tumors with low PD-L1 expression remains unclear. The results of patients with a CPS less than 5 have been shown to have a non-significant survival benefit in the ORIENT16 study. The level of PD-L1 expression was reclassified and two PD-L1 subgroups (CPS < 10 and CPS ≥ 10) were identified in our study, finding that for patients with CPS less than 10, no significant difference was observed between the anti-PD-1 immunotherapy plus chemotherapy group and placebo plus chemotherapy group (P = 0.761). This finding is further supported by Zhao et al.'s study[28], which demonstrated no significant differences in overall survival and progression-free survival between ICI-chemotherapy combinations and chemotherapy alone in the CheckMate-649 PD-L1 CPS 1–4 and KEYNOTE-062 PD-L1 CPS 1–9 subgroups (P ≥ 0.05). Interestingly, our landmark analysis demonstrated a survival advantage in patients with low PD-L1–expressing tumors approximately 300 days after treatment offered by the addition of anti-PD-1 blockade to conventional chemotherapy. This long-term survival discrepancy is likely attributable to the tailing effect of anti-PD-L1 treatment. Compared to traditional chemotherapy, which kills tumor cells directly, immunotherapy can be targeted to initiate immune-tumor interactions and amplify the immune effect. In addition, sustained antigen release shapes memory T cells and is responsible for long-term immunity. Gauci et al.[29] conducted a long-term analysis of solid tumor patients responding to anti-PD-1/PD-L1 therapy and found that 48/76 patients developed an objective response at 3 months regardless of PD-L1 expression. Similarly, the tailing effect, which depends on the immune status of an individual and tumor heterogeneity, was also embodied in long-term follow-up data of some clinical trials such as CheckMate153 of NSCLC[30] and RATIONALE-305[31] of advanced GC, especially striking in patients with high PD-L1 expression. This conclusion has also been validated in patients with CPS < 10 in our study and a time point of 300 days was proposed. Moreover, consolidation chemotherapy was initiated after six cycles of combination therapy in both groups and continued for up to a maximum of 2 years. Therefore, we proposed an alternative hypothesis that applications of immunotherapy might increase the sensitivity of the tumor to subsequent chemotherapy. Exposure to immune checkpoint inhibitors may elicit the antitumor activity of chemotherapy, which can lead to a high response rate[32]. A retrospective CLARITY study[33] also supported that for NSCLC patients who progressed to previous ICIs administered as first- or second-line therapy, salvage chemotherapy could yield a survival-favorable response. However, the underlying mechanisms and impact factors need to be investigated. Taken together, these findings could provide recommendations for clinicians in selecting first-line treatments for patients with CPS < 10, based on their clinical benefits. The combination therapeutic effect was restricted for early treatment owing to the involvement of immunotherapy in patients with a limited life expectancy. For most patients, immunotherapy should be initiated as early as possible in the treatment course and could be continued as maintenance therapy after 4–6 cycles of complete routine first-line therapy, and disease control (CR/PR/SD) was reached in order to increase the survival benefit.
In addition, we found that only a subset of patients could benefit from anti-PD-1 immunotherapy or platinum-based chemotherapy due to the high heterogeneity of gastric cancer (GC). For patients receiving anti-PD-1 immunotherapy, high PD-L1 expression was correlated with long PFS and OS which has been widely recognized in clinical trials such as KEYNOTE-061[34] and KEYNOTE-062[35]. This notion was further supported by PD-L1 staining of mIF analysis. While our study suggests that the expression of PD-L1 could predict outcomes in advanced G/GEJ cancer, PD-L1 expression could not be a single indicator for predicting the efficacy of immunotherapy.
The potential immune microenvironment[36] on the prognostic impact of ICIs on gastric cancer, such as interferon (IFN)[37], neutrophil-to-lymphocyte ratio (NLR)[38], and specific genes like ARID1A[39], POLE or POLD1[40], and TP53[41], have attracted significant attention in recent year. Interestingly, our mIF analysis for the immune TME showed that CD8 + T cells, which act as the key effector of anti-tumor immunity, were more significantly enriched in well responders compared with poor responders in patients with advanced gastric cancer. However, the role of CD8 + T cells in the immunotherapy response remains unclear, which may be due to the functional heterogeneity of CD8 + T cell subtypes on one hand. Tc1 cells, which possess typical CD8 + T cell-related cytotoxic signatures, have been shown to promote the production of perforin, granzymes B and interferon(IFN)-γ[42] in order to kill tumor cells. Tc2[43] and Tc22[44] cells expressing granzyme B are highly cytolytic and demonstrate robust cytotoxic abilities. A special subtype of CD8 + tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, was also been found to positively correlated with patient survival time[45]. Conversely, the CD8 + Tc9 subtype[46] has been shown to produce relatively little IFN-γ and granzyme B, and is thought to promote disease progression, as well as the Tc17 subtype[47–49]. In our mIF analysis, the prognosis of patients with high CD8 + T expression was significantly better than that of patients with low CD8 + T expression, suggesting that CD8 Tc subsets with strong cytotoxic functions such as Tc1s, Tc2s, or Tc22s account for the majority of the TME in our gastric cancer samples. Regrettably, no trials included in this analysis provided CD8 + T cell subset analysis, and the underlying predictive mechanism remains unclear and requires further investigation.
On the other hand, some researchers have uncovered that a high degree of CD8 + T cell infiltration could increase the expression of immune checkpoint receptors such as PD-1 and CTLA-4 and sensitize tumors to ICI, which may be another reason for the good immunotherapy response of CD8 + T cells. Chiaravalli et al.[50] demonstrated that a high number of CD8 + TILs was associated with a high level of PD-L1 mRNA and represented a favourable prognostic factor. Therefore, immune treatment based on CD8 + T cells could be a promising strategy to potentiate the efficacy of anti-PD‐1 immunotherapy. The immune checkpoint receptor CTLA-4 is expressed on CD8 + effector T cells, and CTLA-4 blockade enhances the response of CD8 + T cells in the TME[51]. Currently, the combination of anti-PD-1 and anti-CTLA-4 mAb (such as nivolumab plus ipilimumab) has been approved for the treatment of melanoma[52], non-small cell lung cancer[53], hepatocellular carcinoma[54], mismatch repair deficient colorectal cancer[55], while lack of sufficient evidence on advanced gastric cancer. The COMPASSION-04 clinical trial[56], reported by the 2024 American Association for Cancer Research (AACR), showed promising clinical activity and manageable safety of a bispecific antibody targeting PD-1 and CTLA-4 as first-line treatment in patients with G/GEJ adenocarcinoma, regardless of PD-L1 expression. Furthermore, several clinical trials of anti-CTLA immunotherapy for gastric cancer are still underway. Taken together, our results demonstrate that CD8 + T cells play a pivotal role in predicting ICI treatment responses. A combination of PD-1 and CTLA-4 blockade could have synergistic effects by potentiating the antitumor activity and provide more therapeutic options for patients with low PD-L1 expression, and, hopefully, to be a new first-line treatment option for gastric or GEJ adenocarcinoma.
Many studies have reported potential biomarkers of GC for ICI therapy. Whether immune cells in the TME are associated with traditional platinum-based chemotherapy remains to be elucidated. Generally, chemotherapeutic drugs can specifically induce a cellular immune response, which results in tumor cell death or stimulates immune effector molecules to relieve the suppressed immune response. Therefore, tumor immune status can be used to evaluate the effects of chemotherapy. Interestingly, we observed that those with a CPS of 10 or more were more insensitive to chemotherapy and had significantly shorter PFS than patients with a CPS of less than 10. To further validate the association between PD-L1 expression and chemotherapy efficacy, we also analyzed the microenvironmental components using multiple immunohistochemical analyses and found that high PD-L1 expression may be more resistant to chemotherapy. Lou et al.[57] used intracellular PD-L1 as an RNA-binding protein that enhances the efficacy of chemotherapy by inhibiting DNA damage response and repair. Another study by Frank Sinicrope[58] examined the potential role of tumor cell-intrinsic PD-L1 in regulating chemosensitivity in human CRC cells and found that the deletion of PD-L1 could suppress BH3-only BIM and BIK proteins, causing chemoresistance and survival reduction. In contrast to the ability of PD-L1 mentioned above, knockdown of PD-L1 has been shown to sensitize breast cancer[59], small-cell lung cancer[60], and lymphoma cells[61] to chemotherapy-induced apoptosis. These reported differences may be attributed to the functional diversity of PD-L1 and tumor cell type specificity. In GC, the team of Shin K[62] evaluated the correlation among serum-derived exoPD-L1, plasma sPD-L1, immune-related markers, and circulating immune cells. The low sPD-L1 group showed significantly better overall survival (OS) and progression-free survival (PFS) than the high sPD-L1 group, considering pretreatment plasma-derived sPD-L1 level as a prognostic marker for patients receiving cytotoxic chemotherapy. In the tumor microenvironment, low PD-L1 expression also predicted sensitivity in our study. However, few studies in the prior literature have reported the role of PD-L1 in TME against traditional chemotherapy and the underlying mechanisms remain to be further explored.
In addition, we found significantly higher expression of CD4 + T cells in the poor-responder group than in the well-responder group, supporting that the relatively higher expression of CD4 + T cells in tumor tissues limits patients from benefiting from chemotherapy. CD4 + T cells could be used as promising markers for predicting the efficacy of chemotherapy. Immunohistochemistry (IHC) of the T-cell marker CD4 + was performed on GC patients who received XELOX therapy to evaluate tumor-infiltrating lymphocytes (TILs) at Fudan University[63]. As a result, from patients in the XELOX non-sensitive group(XNSG), we observed that the percentage of CD4 positive cells was significantly higher than that in the XELOX sensitive group(XSG)(54.2% and 2.9%, respectively) (P < 0.05). Similarly, another study on esophageal cancer (EC)[64] showed that regulatory T cells (Tregs), a subset of CD4 + T cells, play a pivotal role in the prognosis of non-operative chemoradiotherapy. Patients with a high proportion of regulatory T cells before chemoradiotherapy had a significantly worse OS than patients with a low proportion of regulatory T cells, which may be partially related to the expression of inhibitory receptors (such as IL-10) and depletion of T cells. Consequently, platinum-based chemotherapy may also be a surprising option for patients with high CD4 + proportion. Despite the close association between CD4 + T cell density and chemotherapy response, our study had notable limitations. Further validation in a larger sample is needed, and the mechanism of chemoresistance in CD4 + Ts is far from fully understood.
LIMITATIONS
Despite its strengths and implications for future research, our study has some limitations. First and most importantly, combined with results from the ORIENT16 clinical trial, the subgroup analyses regarding PD-L1 expression should be stratified based on CPS 0–5, 5–10 or CPS ≥ 10 for more precision treatment. There were a limited number of data and also a limited number of patients to allow meaningful analyses of this subgroup. Additionally, although we observed some significant tendency toward treatment response in patients with different components of the tumor microenvironment, the trend is still require further evaluation in larger patient populations. Finally, we only performed a quantitative analysis of each biomarker separately. Further studies, including colocalization analyses and spatial transcriptomics, are essential, with the potential to provide additional biological insights into the TME in GC and present an opportunity to improve patient selection for novel diagnostics and therapeutics.