Multiomics Reveals Tumor Microenvironment Remodeling in Locally Advanced Gastric and Gastroesophageal Junction Cancer Following Neoadjuvant Immunotherapy and Chemotherapy

Background: Perioperative chemotherapy is the standard of care for patients with locally advanced gastric and gastroesophageal junction cancer. Recent evidence demonstrated the addition of programmed cell death protein 1 (PD-1) inhibitors enhanced therapeutic efficacy. However, the mechanisms of response and resistance remain largely undefined. A detailed multiomic investigation is essential to elucidate these mechanisms.

Methods: We performed whole-exome sequencing, whole-transcriptome sequencing, multiplex immunofluorescence and single-cell RNA sequencing on matched pretreatment and post-treatment samples from 30 patients enrolled in an investigator-initiated Phase 2 clinical trial (NCT04908566). All patients received neoadjuvant PD-1 inhibitors in combination with chemotherapy. A major pathologic response (MPR) was defined as the presence of no more than 10% residual viable tumor cells following treatment.

Results: Before treatment, the positive ratio of CD3+T cells in both the tumor parenchyma and stroma was significantly higher in the non-MPR group compared with the MPR group (p=0.042 and p=0.013, respectively). Least absolute shrinkage and selection operator regression was employed for feature gene selection and 13 genes were ultimately used to construct a predictive model for identifying MPR after surgery. The model exhibited a perfect area under curve (AUC) of 1.000 (95% CI: 1.000 to 1.000, p<0.001). Post-treatment analysis revealed a significant increase in CD3+T cells, CD8+T cells and NK cells in the tumor stroma of MPR patients. In the tumor parenchyma, aside from a marked increase in CD8+T cells and NK cells, a notable reduction in macrophage was also observed (all p<0.05). Importantly, forkheadbox protein 3 (FOXP3), the principal marker for regulatory T cells (Treg) cells, showed a significant decrease during treatment in MPR patients. FOXP3 expression in the non-MPR group was significantly higher than in the MPR group (p=0.0056) after treatment. Furthermore, single-cell RNA sequencing analysis confirmed that nearly all Treg cells were derived from the non-MPR group.

Conclusions: Our study highlights the critical role of dynamic changes within the tumor immune microenvironment in predicting the efficacy of neoadjuvant combined immunochemotherapy. We examined the disparities between MPR/non-MPR groups, shedding light on potential mechanisms of immune response and suppression. In addition to bolstering cytotoxic immune responses, specifically targeting Treg cells may be crucial for enhancing treatment outcomes.