Autophagy, a process for degrading intracellular substances to maintain basal metabolic turnover, is known to be perturbed in gastric cancer. Programmed cell death-1 (PD-1) with its ligand (PD-L1) are important immune checkpoint proteins and their regulation by autophagy has been reported in mouse melanoma and human ovarian cancer. Here, we explored the interplay between autophagy and the PD1/PD-L1 axis in gastric cancer. The expression of PD-L1 in gastric cancer cells was detected by Western blot and flow cytometry analysis. The effect of autophagy inhibition on PD-L1 expression was examined in vitro and in vivo. The molecular mechanisms of the regulation of PD-L1 by autophagy were evaluated in gastric cancer cell lines. The clinical relevance of autophagy-related markers p62/SQSTM1 and LC3 with PD-L1 was evaluated in 137 patients with gastric cancer. We found that inhibition of autophagy by pharmacological inhibitors or small interfering RNAs increased the levels of PD-L1 in cultured gastric cancer cells and in xenografts. Interferon (IFN)-γ also promoted PD-L1 gene transcription, whose action was enhanced by autophagy inhibition. Mechanistically, autophagy inhibition led to the accumulation of p62/SQSTM1 and activation of nuclear factor (NF)-κB, in which NF-κB inhibition or p62/SQSTM1 knockdown attenuated PD-L1 induction by autophagy inhibition. Immunohistochemical staining of primary tumor tissues of 137 patients with gastric cancer showed that LC3 and p62/SQSTM1 protein levels were positively correlated with PD-L1 (LC3, p < 0.001; p62/SQSTM1, p < 0.05). The expression of PD-L1 was also positively correlated with tumor lymphocyte infiltration (p < 0.001). We discovered that autophagy regulates PD-L1 expression in gastric cancer through the p62/SQSTM1-NF-κB pathway. Pharmacological modulation of autophagy may thus influence the therapeutic efficacy of PD-L1 blockade in gastric cancer.

Background: Epstein-Barr virus (EBV)-associated gastric cancer (GC) (EBVaGC) is a distinct molecular subtype of GC with a favorable prognosis. However, the exact effects and potential mechanisms of EBV infection on immune checkpoint blockade (ICB) efficacy in GC remain to be clarified. Additionally, EBV-encoded RNA (EBER) in situ hybridization (ISH), the traditional method to detect EBV, could cause false-positive/false-negative results and not allow for characterizing other molecular biomarkers recommended by standard treatment guidelines for GC. Herein, we sought to investigate the efficacy and potential biomarkers of ICB in EBVaGC identified by next-generation sequencing (NGS). Design: An NGS-based algorithm for detecting EBV was established and validated using two independent GC cohorts (124 in the training cohort and 76 in the validation cohort). The value of EBV infection for predicting ICB efficacy was evaluated among 95 patients with advanced or metastatic GC receiving ICB. The molecular predictive biomarkers for ICB efficacy were identified to improve the prediction accuracy of ICB efficacy in 22 patients with EBVaGC. Results: Compared with orthogonal assay (EBER-ISH) results, the NGS-based algorithm achieved high performance with a sensitivity of 95.7% (22/23) and a specificity of 100% (53/53). EBV status was identified as an independent predictive factor for overall survival and progression-free survival in patients with DNA mismatch repair proficient (pMMR) GC following ICB. Moreover, the patients with EBV+/pMMR and EBV−/MMR deficient (dMMR) had comparable and favorable survival following ICB. Twenty-two patients with EBV+/pMMR achieved an objective response rate of 54.5% (12/22) on immunotherapy. Patients with EBVaGC with a high cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) level were less responsive to anti-programmed death-1/ligand 1 (PD-1/L1) monotherapy, and the combination of anti-CTLA-4 plus anti-PD-1/L1 checkpoint blockade benefited patients with EBVaGC more than anti-PD-1/L1 monotherapy with a trend close to significance (p=0.074). There were nearly significant differences in tumor mutational burden (TMB) level and SMARCA4 mutation frequency between the ICB response and non-response group. Conclusions: We developed an efficient NGS-based EBV detection strategy, and this strategy-identified EBV infection was as effective as dMMR in predicting ICB efficacy in GC. Additionally, we identified CTLA-4, TMB, and SMARCA4 mutation as potential predictive biomarkers of ICB efficacy in EBVaGC, which might better inform ICB treatment for EBVaGC.

Immunotherapy has exhibited promising but controversial results in gastric cancer; determining criteria for choosing the appropriate target population is still problematic. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) exhibits distinctive genomic aberrations and clinicopathologic features, the positive status of EBV is a potential biomarker. We prospectively recruited 9 patients who were diagnosed with stage-IV EBVaGC, and all of the patients were treated by immune-checkpoint inhibitors. The median age of the patients was 62 years old. The clinicopathologic characteristics demonstrated a male predominance and poor differentiation status of EBVaGC. Lymph nodes were demonstrated to represent the most common metastatic site. Immunochemistry and polymerase chain reaction analysis revealed that all of the patients were proficient mismatch repair, and microsatellite instability-stable and programmed cell death-ligand 1 were detected in 7 patients. Three patients with positive programmed cell death-ligand 1 showed partial response, 5 patients showed stable disease, 1 patient without measurable lesion showed decreasing ascites and tumor marker level after immunotherapy. The longest duration of response was 18 months by the time of the last follow-up. EBVaGC exhibits distinctive clinicopathologic characteristics, and EBV-positive status may be a potential biomarker for gastric cancer immunotherapy.

Background: Common treatments for metastatic/unresectable HER2‐negative gastric cancer include chemotherapy, immune checkpoint inhibitor monotherapy and chemotherapy plus immune checkpoint inhibitor. However, significant drug resistance exists regardless of the treatment regimen.Methods: Patients with metastatic/unresectable HER2‐negative gastric/gastroesophageal junction adenocarcinoma were enrolled. All patients were divided into three groups according to the treatment regimen and were further divided into responders and non‐responders according to efficacy evaluation. Metagenomics sequencing were performed to analyze gut microbiome signature of patients receiving different treatments at baseline and throughout treatment.Results: One hundred seventeen patients with HER2‐negative advanced gastric or gastroesophageal junction adenocarcinoma receiving chemotherapy alone, anti PD‐1/PD‐L1 immunotherapy alone or combined regimen were included in this study. Microbiome signatures related to clinical response are distinct among the three treatment groups. Among which, 14, 8 and 13 species were significantly different between responders and non‐responders in immunotherapy, immunotherapy plus chemotherapy and chemotherapy group, respectively. Patients with higher relative abundance of Lactobacillus possessed higher microbiome diversity and significantly better response to anti‐PD‐1/PD‐L1 immunotherapy and had a trend to achieve better progression‐free survival. Another cohort of 101 patients has been used as an external validation set to confirm the stability and reliability of these findings.Conclusions: Gut microbiome affects response of treatments in HER2‐negative advanced gastric cancer in a treatment‐specific way, immunotherapy plus chemotherapy did not equal to a simple superposition of immunotherapy and chemotherapy. Lactobacillus is expected to become a novel choice as an adjuvant agent in promoting the efficacy of immunotherapy in gastric cancer.

Background: Immunotherapy dramatically changed the treatment landscape of gastric cancer in recent years. PD-L1 expression was proposed as a biomarker; however, the treatment strategy according to PD-L1 is still uncertain. Here, we aimed to find the appropriate cutoff value of PD-L1 expression for gastric cancer immunotherapy. Methods: We did a systematic electronic research of prospective clinical trials of gastric cancer immunotherapy across databases. Studies that provided subgroup analysis results stratified by PD-L1 expression were included. Objective response rate (ORR), disease control rate (DCR), hazard ratio (HR), and 95% confidential interval (CI) of progression-free survival (PFS) and overall survival (OS) at different PD-L1 cutoff values were extracted. Results: Twelve studies and 6,488 patients in total were finally included for pooled analysis. ORR in allover, PD-L1-negative, combined positive score (CPS) ≥1, CPS ≥5, and CPS ≥10 population was 10%, 3%, 13%, 20%, and 23%, respectively. Immune checkpoint inhibitor (ICI) monotherapy failed to show survival advantage in allover and PD-L1-negative patients. Single-agent ICI therapy prolonged OS (HR = 0.84, 95% CI: 0.74–0.96) but not PFS (HR = 1.38, 95% CI: 0.91–2.09) in PD-L1 CPS ≥1 patients. For combined immunotherapy, ORR in allover, PD-L1-negative, CPS ≥1, CPS ≥5, and CPS ≥10 population was 64%, 57%, 48%, 60%, and 58%, respectively. Allover population could gain survival benefit from combined immunotherapy based on the results from Checkmate-649. OS (HR = 0.81, 95% CI: 0.71–0.92) and PFS (HR = 0.77, 95% CI: 0.69–0.86) were significantly prolonged in PD-L1 CPS ≥1 patients receiving combined immunotherapy. Conclusion: Efficacy and survival advantages improved with PD-L1 CPS. CPS ≥1 was the cutoff value for ICI monotherapy to gain survival benefit. Combined immunotherapy prolonged PFS and OS in allover population but needs further study to confirm it.

Introduction: The wide application of immune checkpoint inhibitors has significantly improved the survival expectation of cancer patients. While immunotherapy brings benefits to patients, it also results in a series of immune-related adverse events (irAEs). Increasing evidence suggests that the gut microbiome is critical for immunotherapy response and the development of irAEs.Methods: In this prospective study, we recruited 95 patients with advanced/unresectable gastrointestinal cancers treated with immunotherapy and report a comprehensive analysis of the association of the gut microbiome with irAEs. Metagenome sequencing was used to analyze the differences in bacterial composition and metabolic pathways of baseline fecal samples.Results: In summary, we identified bacterial species and metabolic pathways that might be associated with the occurrence of irAEs in gastric, esophageal, and colon cancers. Ruminococcus callidus and Bacteroides xylanisolvens were enriched in patients without severe irAEs. Several microbial metabolic pathways involved in the urea cycle, including citrulline and arginine biosynthesis, were associated with irAEs. We also found that irAEs in different cancer types and toxicity in specific organs and the endocrine system were associated with different gut microbiota profiles. These findings provide the basis for future mechanistic exploration.