{
  "abstract": "Background Cholangiocarcinoma (CCA) is a deadly and heterogeneous cancer originating from the biliary tract. Age-standardized incidence rates across the globe range from 0.3 to 85 per 100,000 and the 5-year survival is <20%. 1 Current treatments including surgery, radiation, chemotherapy, and immunotherapy provide limited efficacy.2 Mesothelin (MSLN) is a tumor-associated antigen with minimal expression on normal tissues and high expression on several solid tumors. Building on success in treating hematologic malignancies, chimeric antigen receptor (CAR) T cells targeting MSLN are now being explored in clinical trials for solid tumors,3 including ovarian carcinomas and mesotheliomas.4–6 Although well-documented in some tumors, the expression of MSLN in CCA has yet to be thoroughly evaluated. Here, we aimed to assess MSLN expression across a limited number of archival formalin-fixed, paraffin-embedded (FFPE) tumors from CCA patients.Methods FFPE tumors from 10 CCA patients were sourced from the Tumor Tissue and Biospecimen Bank at the University of Pennsylvania, and analyzed for MSLN expression using immunohistochemistry (IHC). Detection was performed using a mouse monoclonal antibody (5B2) that specifically recognizes the first 100 amino acids present in the N-terminal region of MSLN. Following optimization of MSLN detection in archival tissues, the differentiation of the cholangiocarcinoma was determined by standard histologic evaluation by an expert pathologist and the MSLN expression analyzed in the malignant cells by annotation and measuring DAB mean cell intensity using QuPath3.2 7 with the percentage of positive tumor cells and their mean expression intensity reported.Results MSLN was expressed in 4 out of the 10 CCA tumors analyzed with expression correlating with differentiation as 100% of well, 50% of moderate and 0% of poorly differentiated cases having expression. No MSLN expression was observed in adjacent normal tissue control areas or negative control samples, confirming antibody specificity. MSLN staining was mostly cytoplasmic with variable degrees of membranous expression, with one case displaying clear and strong membrane staining. Of the MSLN-positive cases, 52-93% of tumor cells had expression.Conclusions Our preliminary results show that IHC detection of MSLN expression in CCA is feasible, specific, and confirms MSLN expression in 4 of 10 randomly selected CCA tumors. These results support further development of CAR T cell therapy targeting MSLN in CCA with prospective tumor MSLN testing for potential correlates with outcomes. SynKIR-110 is a MSLN-targeted KIR-CAR T cell therapy in a Phase 1 clinical trial treating patients with CCA, ovarian cancer, and mesothelioma (# NCT05568680).Acknowledgements Verismo Therapeutics would like to thank all patients who contributed to this study and those currently enrolled in the Phase 1 trial. We are also very thankful to the staff members of Tumor Tissue and Biospecimen Bank (TTAB), Pathology Clinical Service Center (PCSC) at the University of Pennsylvania for their help with sourcing tissue samples and conducting staining.References Qurashi M, Vithayathil M, Khan SA. Eur J Surg Oncol. 2025;51(2):107064.Feng Q, Sun B, Xue T, et al. Front Immunol. 2022;13:1025608.Klampatsa A, Dimou V, Albelda SM. Expert Opin Biol Ther. 2021;21(4):473–486.Beatty GL, Haas AR, Maus MV, et al. Cancer Immunol Res. 2014;2(2):112–120.Haas AR, Tanyi JL, O’Hara MH, et al. Mol Ther. 2019;27(11):1919–1929.Hassan R, Butler M, O’Cearbhaill RE, et al. Nat Med. 2023;29(8):2099–2109.Bankhead P, Loughrey MB, Fernández JA, et al. QuPath: open source software for digital pathology image analysis. Sci Rep. 2017;7(1):16878.Ethics Approval This study was approved by the University of Pennsylvania’s Ethics Board and informed consent was obtained from all patients.",
  "authors": [
    {
      "affiliations": [
        "Verismo Therapeutics, Philadelphia, PA, USA"
      ],
      "name": "Adina Vultur"
    },
    {
      "affiliations": [
        "University of Pennsylvania, Philadelphia, PA, USA"
      ],
      "name": "Emma E Furth"
    },
    {
      "affiliations": [
        "Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"
      ],
      "name": "Michael C Milone"
    },
    {
      "affiliations": [
        "Verismo Therapeutics, Philadelphia, PA, USA",
        "University of Pennsylvania, Philadelphia, PA, USA",
        "Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"
      ],
      "name": "Susan K Howard"
    },
    {
      "affiliations": [
        "Verismo Therapeutics, Philadelphia, PA, USA"
      ],
      "name": "Emily A Winters"
    },
    {
      "affiliations": [
        "Verismo Therapeutics, Philadelphia, PA, USA"
      ],
      "name": "Andrea Campanile"
    },
    {
      "affiliations": [
        "Verismo Therapeutics, Philadelphia, PA, USA"
      ],
      "name": "Jun Xu"
    },
    {
      "affiliations": [
        "Verismo Therapeutics, Philadelphia, PA, USA"
      ],
      "name": "Laura A Johnson"
    }
  ],
  "title": "1238 Histopathologic analysis of mesothelin expression in cholangiocarcinoma supports inclusion in biomarker-targeted clinical trials",
  "uid": "266745b9-9d48-50af-a6f7-ac6d55bc709f"
}