Tumor immune microenvironment and human high grade serous ovarian carcinoma: immunophenotyping of tumor-infiltrating B cells

BACKGROUND Ovarian cancer (OC) still represents the leading cause of death among gynecological malignancies in developed countries. Over the past years, advances in molecular and cellular biology have demonstrated the pivotal role of the tumor microenvironment (TME) in shaping OC outcomes. Among the components of the TME, immune infiltrates have gained increasing attention due to their multifunctional roles in influencing tumor growth, metastasis, and therapeutic responses. In this context, the role of B cells has been less investigated but is emerging as an important player in the TME. The objectives of this study are to develop a specific panel to analyze B cells in peritoneal biopsies of high grade serous ovarian carcinoma (HGSOC) patients by cytofluorimetry and to study TME, specifically assessing the role of B cells. MATERIALS AND METHODS This prospective observational study was conducted at University Hospital of Udine. Patients with advanced HGSOC and women with germline BRCA1-2 mutation submitted to risk reducing salpingo-oophorectomy (RRSO) were recruited. Peritoneal biopsies were collected at the time of surgery before any other treatment and fresh tissue samples were sent to Department of Medicine. In case of neoadjuvant chemotherapy (NACT), during interval debulking surgery (IDS), a new peritoneal biopsy was collected. Fresh tumor tissue was submitted to mechanical and enzymatic dissociation. Cells were then stained using the fluorochrome-conjugated monoclonal/polyclonal antibodies. Events were acquired by the Flow Cytometer. It allowed to verify the presence of B cell but especially to characterize the subpopulations of B cells. RESULTS A total of 15 patients were enrolled between January 2023 and September 2023. Thirteen patients underwent surgery for HGSOC, while two women underwent RRSO and were enrolled as healthy controls. The first aim of our study was to design a specific panel to analyze B cells in peritoneal biopsies of HGSOC patients by cytofluorimetry. Interrogating the samples, we evaluated the presence of immune cells (CD45+) and of B cell (CD19+). Then, we developed a panel to analyze the subpopulations of B cells: active and resting cell, antibody secreting cells, memory switched and unswitched cells and naïve cells. Cytofluorimetry analysis identified B cells in all our samples confirming a significant heterogeneity in the proportion of B cells (0.70-28.6%). Memory B cells were highly represented in our samples (20.2-51.4%). We discovered for the first time in HGSOC biopsies the presence of double negative cells (CD27-/IgD-). After refining the panel, we collected and tested the peritoneal sample of a patient submitted to IDS after NACT to compare the immune infiltrate before and after chemotherapy. The immune infiltrate increased from 24.6% to 59.8% while the B cell decreased (6.67% to 3.74%) with an increase of activated B cell (from 55% to 85.1%) and memory switched B cells (26.9% to 42.5%). CONCLUSIONS This is the first study to develop an extended panel to analyze B-cell subpopulations in peritoneal biopsies of HGSOC patients by cytofluorimetry. Improved knowledge of the TME, integrated with genetic and molecular characteristics, could help to better understand OC behavior.