Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer mortality in the United States, with a 5-year survival rate of 13%. For patients with locally advanced PDAC, neoadjuvant chemotherapy with FOLFIRONOX (FFX) or Gemcitabine is first-line treatment with the goal of increasing resectability. Recent studies have shown tertiary lymphoid structures (TLS), ectopic aggregations of lymphocytes within tumor and non-tumor tissue, are associated with improved clinical outcomes in PDAC. There is less understanding, however, of how the spatial and transcriptomic features of TLS correlate with neoadjuvant therapy response, particularly between two different first-line chemotherapies, FFX and Gemcitabine. Using QuPath, machine learning software for digital pathology image analysis, we analyzed multispectral stained tumor tissue samples from n=67 PDAC patients. Using markers for CD4, CD20, and CD21, we were able to assess the composition and state of TLS, with a particular focus on the presence of CD21+ follicular dendritic cells (fDC), which mark the formation of germinal centers (GCs). We assessed TLS activity by measuring Ki67 for proliferating lymphocytes and AID for B cells undergoing somatic hypermutation within the TLS. GC formation is often associated with more active TLS and improved survival but is not abundant in human tumors. If less active TLS are present, they can still contribute to B cell education through extrafollicular differentiation, an alternate route of B cell activation that doesn’t require GC education as B cells will differentiate to antibody secreting cells (ASC) depending on the inflammatory cues within patient tumors. Using AI training models and custom scripts, immune aggregates were identified, immunophenotyped at single-cell resolution, and classified as a lymphoid aggregate, TLS, or TLS with GC based on composition. Patient TLS profiles were analyzed by comparing structure count, proximity to tumor, and the density, activity, and proliferation of T and B cells. Clinical correlates included overall survival, change in tumor size and CA19-9 levels, and recurrence. Preliminary analysis reveals that FFX patients had a better response and harbored a greater number of total immune aggregates. Gemcitabine patients had a higher number of GCs but still demonstrated less AID activity than the FFX cohort. To investigate whether B cells in the extrafollicular pathway were ultimately associated with a better response in patients with low TLS activity, we isolated healthy donor naïve B cells and differentiated them in extrafollicular conditions in media collected from treatment naïve, FFX or Gemcitabine PDAC patient derived organoids. B cell differentiation to ASCs was assessed by spectral cytometry. Testing the regulatory dynamics of the extrafollicular response in vitro could uncover new immunotherapeutic targets that would augment TLS function in PDAC patients. Ultimately, this work will aid us in understanding how standard of care neoadjuvant chemotherapies modulate B cell education within and outside of TLS. Citation Format: Adam Tcharni, Charu Arora, Renee Anderko, Amer Zureikat, Tullia C. Bruno. FOLFIRONOX and Gemcitabine treatment alters formation and activity of tertiary lymphoid structures in pancreatic ductal adenocarcinoma patients abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₃): Abstract nr A087.
Tcharni et al. (Sun,) studied this question.