Yun Gu

Background The epithelial-mesenchymal axis frames gastric cancer heterogeneity but mainly captures phenotypic extremes. CCNE1 gain, encompassing amplification or Cyclin E1 overexpression, represents one intermediate state linked to chromosomal instability and therapeutic resistance. However, its clinicopathologic identity and immune features in gastric cancer remain insufficiently characterised. Methods We analysed 1273 gastric cancer patients across six independent cohorts: ZSHS (n = 453), TCGA (n = 410), ACRG (n = 300), SMC (n = 43), MSKCC (n = 22), and ZSHS NGS cohort (n = 45). Tumours were classified into CCNE1 gain, epithelial, or mesenchymal subtypes using protein, copy-number, or transcript-level data. Clinicopathologic features, survival outcomes, treatment responses, and immune contexture were evaluated across subtypes. Results CCNE1 gain tumours retained E-cadherin positivity but showed increased proliferation, more nerve and venous invasion. They were associated with poor prognosis and reduced response to adjuvant chemotherapy, HER2-targeted therapy, anti-angiogenic agents, and PD-1 blockade independent of epithelial-mesenchymal classification. The immune contexture exhibited an immune-desert phenotype with reduced lymphocyte infiltration, impaired cytotoxicity, increased M2 macrophage polarisation, and elevated TGF-β. Conclusion CCNE1 gain delineates a clinic-ready, therapy-refractory subtype of gastric cancer beyond the epithelial-mesenchymal framework, representing over one in ten patients. These tumours preserve epithelial morphology yet exhibit aggressive proliferative and invasive behaviour, coupled with immune desert and myeloid-driven suppression.

CD8 ⁺ T cell infiltration is essential for antitumor immunity across cancers while its clinical significance in gastric cancer (GC) remains unclear. This reflects molecular heterogeneity of GC, as defined by The Cancer Genome Atlas (TCGA) into four subtypes: Epstein–Barr virus (EBV)‐positive, microsatellite instability (MSI), chromosomal instability (CIN), and genomically stable (GS), each with distinct immune features. We aimed to characterize distribution, clinical relevance, and immune associations of CD8 ⁺ T cell infiltration within this molecular framework. TCGA ( n = 336) and Zhongshan Hospital (ZSHS, n = 455) cohorts were analyzed. CD8 ⁺ T cell infiltration and immune features were compared across TCGA subtypes. Prognostic and predictive significance of CD8 ⁺ T cells was evaluated in ZSHS cohort. CD8 ⁺ T cell infiltration was elevated in the EBV‐positive and MSI subtypes (ZSHS: p = 0.026; TCGA: p < 0.001). In ZSHS cohort, high CD8 ⁺ T cell infiltration was associated with better overall survival ( p = 0.040), particularly in the EBV‐positive ( p = 0.036) and CIN ( p = 0.065) subtypes, but not in MSI ( p = 0.440) or GS ( p = 0.860). Notably, low CD8 ⁺ T infiltration predicted superior response to adjuvant chemotherapy in MSI patients (HR = 0.210, p = 0.022). Immune profiling revealed associations of CD8 ⁺ T cells with antigen presentation in EBV‐positive, tertiary lymphoid structure signatures in CIN, and podoplanin+ cells in GS tumors, instead of neoantigen burden in MSI or pan‐fibroblast TGFβ response signature in GS. CD8 ⁺ T cell infiltration demonstrates subtype‐specific prognostic and therapeutic significance in GC—beneficial in EBV‐positive and CIN tumors, and predictive of chemotherapy response in MSI with low infiltration, which accompanied by divergent immune features, reflecting heterogeneous immunological landscape of GC.

Background: The aberrant p16 ^INK4a expression has been identified in gastric cancer (GC). However, the staining pattern of p16 ^INK4a and its associations with clinical outcomes and immune contexture remains obscure. Methods: This study involved two patient cohorts, the Zhongshan Hospital (ZSHS, n = 443) and Samsung Medical Center (SMC, n = 43) cohort. Patients were divided into p16 ^INK4a loss, wild‐type (WT), and overexpression (OE) subgroups. The patient characteristics, overall survival (OS), response to adjuvant chemotherapy (ACT) and immune checkpoint inhibitor (ICI) treatment, as well as tumor immune contexture were investigated in each subgroup. Results: In ZSHS cohort, 90 of 443 (20.3%) patients with p16 ^INK4a OE gastric cancer, who were characterized by advanced pT stage ( p = .011), higher Ki‐67 ( p < .001), Rb loss ( p < .001), and CCNE1 OE incidence ( p < .001). Patients with p16 ^INK4a OE tumors presented with poor OS (ZSHS: OE vs. WT, p = .004; specific in MSI tumors, p = .019), inferior responsiveness to ACT (OE vs. WT, p = .011; specific in MSI tumors, p = .024) and ICI (OE vs. WT, p = .045; specific in programmed death‐ligand 1 CPS ≥1 and CIN tumors, p = .027 and p = .032, respectively), whereas patients with p16 ^INK4a loss and WT gastric cancer exhibited comparable clinical outcomes (loss vs. WT: OS, p = .424; ACT, p = .834; ICI, p = .223). Moreover, p16 ^INK4a OE gastric cancer was associated with lower antitumor M1, neutrophils, and CD8 ⁺ T cells infiltration and higher pro‐tumor TGF‐β, CD73, and IDO expression. Conclusion: This study identified a specific triple classification staining pattern of p16 ^INK4a expression in GC, and demonstrated that p16 ^INK4a OE was associated with poor clinical outcomes and evasive immune contexture.

Background SMAD4 mutation and homozygous deletion represent the most prevalent genomic events driving aggressive biological behavior in gastric cancer (GC). However, clinical outcome and therapeutic response in GC patients with Smad4-loss remains obscure. Methods This study included 990 GC patients from four independent clinical centers including the Zhongshan Hospital (ZSHS) cohort, the Cancer Genomic Atlas (TCGA) cohort, the Samsung Medical Center (SMC) cohort and the Memorial Sloan Kettering Cancer Center (MSKCC) cohort. Results In ZSHS cohort, 60/454 GC patients harbored Smad4-loss are characterized by lower pN stage, well histology differentiation, lower EBV infection, null p53 staining and lower tumor proliferation. Smad4-loss GC patients exhibit miserable overall survival across ZSHS cohort and TCGA cohort. Moreover, Smad4-loss GC patients yield no impact on adjuvant chemotherapy, poor outcome upon anti-PD-1 immunotherapy or anti-HER-2 therapy. Interestingly, Smad4-loss GC show more well and intermediate differentiation and lower Ki67 staining. Furthermore, Smad4-loss GC exhibit tumor immunosuppressive contexture characterized with enriched CXCL13⁺CD8⁺T cells, reduced IFN-γ⁺ cells and GZMB⁺ cells infiltration. Conclusions Smad4 loss yields poor clinical outcome, immunotherapy and anti-HER-2 treatment resistance and tumor immunosuppressive contexture in GC patients. Our findings provide clues for further detailed biological investigation and aggressive clinical management in Smad4-loss GC patients.

Objective: Immunotherapy has not yielded satisfactory therapeutic responses in gastric cancer (GC). However, targeting myeloid checkpoints holds promise for expanding the potential of immunotherapy. This study aims to evaluate the critical role of Siglec-10⁺ tumor-associated macrophages (TAMs) in regulating antitumor immunity and to explore the potential of the myeloid checkpoint Siglec-10 as an interventional target. Design: Siglec-10⁺ TAMs were assessed based on immunohistochemistry on tumor microarrays and RNA-sequencing data. Flow cytometry, RNA sequencing, and single-cell RNA-sequencing analysis were employed to characterize the phenotypic and transcriptional features of Siglec-10⁺ TAMs and their impact on CD8⁺ T cell-mediated antitumor immunity. The effectiveness of Siglec-10 blockade, either alone or in combination with anti-programmed cell death 1 (PD-1), was evaluated using an ex vivo GC tumor fragment platform based on fresh tumor tissues. Results: Siglec-10 was predominantly expressed on TAMs in GC, and associated with tumor progression. In Zhongshan Hospital cohort, Siglec-10⁺ TAMs predicted unfavorable prognosis (n=446, p+ TAMs demonstrated inferior response to pembrolizumab in GC (n=45, p=0.008). Furthermore, Siglec-10⁺ TAMs exhibited an immunosuppressive phenotype and hindered T cell-mediated antitumor immune response. Finally, blocking Siglec-10 reinvigorated the antitumor immune response and synergistically enhances anti-PD-1 immunotherapy in an ex vivo GC tumor fragment platform. Conclusions: In GC, the myeloid checkpoint Siglec-10 contributes to the regulation of immunosuppressive property of TAMs and promotes the depletion of CD8⁺ T cells, ultimately facilitating immune evasion. Targeting Siglec-10 represents a potential strategy for immunotherapy in GC.

Background: This study aimed to investigate the expression and clinical significance of Dendritic cell-associated C-type lectin-1 (Dectin-1) in gastric cancer (GC), and to explore the mechanism of Dectin-1 regulating tumour-associated macrophage (TAM)-mediated immune evasion in GC. Methods: The association of Dectin-1⁺ cells with clinical outcomes was inspected by immunohistochemistry on tumour microarrays. Flow cytometry and RNA sequencing were applied to detect characteristics of T cells, phenotypic and transcriptional features of Dectin-1⁺ TAMs. The effect of Dectin-1 blockade was evaluated using an in vitro intervention experiment based on fresh GC tissues. Results: High infiltration of intratumoral Dectin-1⁺ cells predicted poor prognosis in GC patients. Dectin-1⁺ cells were mainly composed of TAMs, and the accumulation of Dectin-1⁺ TAMs was associated with T-cell dysfunction. Notably, Dectin-1⁺ TAMs exhibited an immunosuppressive phenotype. Furthermore, blockade of Dectin-1 could reprogramme Dectin-1⁺ TAMs and reactivate anti-tumour effects of T cells, as well as enhanced PD-1 inhibitor-mediated cytotoxicity of CD8⁺ T cells against tumour cells. Conclusions: Dectin-1 could affect T-cell anti-tumour immune response by regulating the immunosuppressive function of TAMs, leading to poor prognosis and immune evasion in GC patients. Blockade of Dectin-1 can be used alone or in combination with current therapeutic strategies in GC.

In gastric cancer (GC), the therapeutic response of immune checkpoint blockade (ICB) remains suboptimal. Targeting myeloid cell checkpoints might be feasible as adjuvant to current ICB regimens. We sought to evaluate the crucial role of C5aR1⁺ TAMs in regulating antitumor immunity and the efficacy of combinatorial treatment with antiprogrammed cell death protein‐1 (PD‐1) and C5aR1 blockade. Here, we found that C5aR1 was predominantly expressed on macrophages and high level of C5aR1⁺ TAMs infiltration could predict poor prognosis and inferior chemotherapeutic response. The flow cytometry (FCM) and single‐cell RNA‐seq (scRNA‐seq) data revealed that C5aR1⁺ TAMs exhibited immunosuppressive property which might contribute to CD8⁺ T cell dysfunction. Blockade of C5aR1 could diminish the immunosuppressive function of TAMs and led to reinvigorated CD8⁺ T cells mediated antitumor immunity. Moreover, using in vitro intervention experiment based on fresh GC surgical specimens, we discovered that C5aR1 blockade exert a synergistic effect when combined with PD‐1 inhibitor for tumor clearance. Our study demonstrated that C5aR1 is a critical myeloid checkpoint and plays a crucial role in regulating the immunosuppressive property of TAMs and CD8⁺ T cell immune tolerance. C5aR1 blockade reprograms TAMs and reinvigorated the cytotoxicity of CD8⁺ T cells, thus improving the efficacy of anti‐PD‐1 therapy for tumor eradication in GC.

Original Article Development and validation of the artificial intelligence (AI)-based diagnostic model for bronchial lumen identification Ying Li, Xiaoxuan Zheng, Fangfang Xie, Lin Ye, Elena Bignami, Yasmeen K. Tandon, María Rodríguez, Yun Gu, Jiayuan Sun

Background: Although PD-1 has been reported to be a marker of T-cell exhaustion in several malignancies, the biological role of PD-1⁺CD8⁺ T cells in gastric cancer (GC) remains unclear. Herein, we aimed to investigate the role of PD-1⁺CD8⁺ T cells in the tumour microenvironment and its clinical significance in GC. Designs: This study included 441 tumour microarray specimens and 60 Flow cytometry specimens of GC patients from Zhongshan Hospital, and 250 GC patients from the Asian Cancer Research Group. Results: Here, we demonstrated that PD-1⁺CD8⁺ T cells functioned as an independent adverse prognosticator in GC. In addition, an abundance of intratumoral PD-1⁺CD8⁺ T cells indicated worse chemotherapeutic responsiveness to fluorouracil in Stage III GC patients. Mechanistically, PD-1⁺CD8⁺ T cell high infiltration indicated an exhausted phenotype of global CD8⁺ T cells in GC tissues, which was characterised by elevated immune checkpoint expression including CTLA-4 and TIM-3, whereas decreased expression of perforin. Furthermore, PD-1⁺CD8⁺ T cell high-infiltration patients with Stage III GC held elevated activity of several therapeutic signal pathways. Conclusions: Our study highlighted that PD-1⁺CD8⁺ T cell abundance predicts inferior prognosis in GC, and may serve as a novel predictive biomarker to guide therapeutic option.