{
  "abstract": "Background Immune checkpoint inhibitors (ICIs) have significantly improved overall survival in metastatic urothelial carcinoma (mUC). However, predictive biomarkers for therapeutic response remain insufficiently defined. Although some genomic alterations have been implicated in modulating tumor immunogenicity and ICI sensitivity in other cancers, evidence in mUC remains limited and warrants further investigation.Methods We retrospectively analyzed 67 patients with mUC treated with ICIs and performed targeted next-generation sequencing using a 440-gene cancer panel. Tumor mutational burden (TMB), genomic alterations, and clinical outcomes were evaluated to identify biomarkers associated with ICI response. Functional studies were conducted using urothelial carcinoma cell lines co-cultured with peripheral blood mononuclear cells (PBMCs) with or without anti-programmed death-ligand 1 (PD-L1) treatment to evaluate tumor viability, cytokine responses, and antigen presentation. Gene knockdown and overexpression experiments, quantitative PCR, immunofluorescence, and western blotting were used to assess cytokine production, antigen presentation machinery components, and major histocompatibility complex (MHC) class I expression. In vivo validation was performed using CRISPR/Cas9-mediated knockout syngeneic murine models treated with anti-PD-L1 to assess tumor growth, immune infiltration, and therapeutic response through immunohistochemistry and flow cytometry.Results Responders to ICI monotherapy exhibited significantly higher TMB and enriched mutations in PIK3CA, ADAMTSL1, NSD1, and PRKDC. Hotspot PIK3CA mutations correlated with elevated TMB across multiple TCGA pan-cancer, breast and colorectal cohorts. Functionally, PIK3CA-mutant tumor cells demonstrated enhanced cytotoxicity when co-cultured with PBMCs and anti-PD-L1, whereas PIK3CA knockdown abolished this effect. Silencing PIK3CA reduced expression of pro-inflammatory cytokines (interferon-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-6) and CD8+ effector molecules (granzyme B, perforin A), while PIK3CA E545K overexpression restored these immune mediators. Mechanistically, PIK3CA mutations enhanced antigen presentation via the IRF1-NLRC5-MHC-I axis, increasing HLA-A and B2M expression. In vivo, PIK3CA-deficient tumors displayed diminished response to anti-PD-L1 therapy, reduced CD8+ T-cell infiltration, and attenuated MHC-I expression.Conclusions This study is the first to identify PIK3CA mutation as an immune-modulating driver that enhances tumor immunogenicity through activating IRF1-NLRC5-MHC-I pathway in urothelial carcinoma.",
  "authors": [
    {
      "affiliations": [
        "Doctoral Program of Clinical and Experimental Medicine, National Sun Yat-sen University College of Medicine, Kaohsiung, Taiwan",
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Yu-Li Su"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Shih-Yu Huang"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Chung-Wen Kuo"
    },
    {
      "affiliations": [
        "Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan"
      ],
      "name": "Ling-Yi Xiao"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Chang-Ting Lin"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Yi-Hua Chen"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Li-Chung Chang"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Ming-Chun Kuo"
    },
    {
      "affiliations": [
        "Division of Hematology Oncology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Chia-Che Wu"
    },
    {
      "affiliations": [
        "Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Jei-Ming Peng"
    },
    {
      "affiliations": [
        "ACT Genomics Co., Ltd, Taipei, Taiwan"
      ],
      "name": "Chia-Ling Wu"
    },
    {
      "affiliations": [
        "Department of Anatomic Pathology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan",
        "College of Medicine, Chang Gung University, Taoyuan, Taiwan"
      ],
      "name": "Hsuan-Ying Huang"
    },
    {
      "affiliations": [
        "Doctoral Program of Clinical and Experimental Medicine, National Sun Yat-sen University College of Medicine, Kaohsiung, Taiwan",
        "Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan"
      ],
      "name": "Chih-Yen Chien"
    }
  ],
  "title": "Oncogenic PIK3CA mutation enhances tumor immunogenicity through the IRF1–NLRC5–MHC-I axis in urothelial carcinoma",
  "uid": "58b2de5f-745a-5037-92e9-1a5307f4406b"
}