A multiplexed immunohistochemical consecutive staining on single slide (MICSSS) analysis of the immune microenvironment of bile duct cancers (BDC) pre and post neoadjuvantchemotherapy (NACT).

e16151 Background: NACT is increasingly being used in the management of locally advanced BTC. Emerging evidence suggests a potential key contributing role of tumor infiltrating immune cells in the prognosis & response to therapy. We set out to characterize immune modulation of tumor immune microenvironment composition in BTC following NACT. Methods: Patients (pts) with locally advanced BTC who underwent a diagnostic biopsy, then NACT followed by resection between 2014 & 2018 were identified & consented after IRB approval. MICSSS, a sample-sparing chromogenic consecutive multiplex tissue staining method, was performed with a series of immune markers (Table), to characterize T cell subsets, B cells, macrophages, mature dendritic cells (DCs), and immune checkpoints on pre & post NACT formalin-fixed paraffin-embedded tumor tissue sections. Density was calculated for each marker (+ve cells/mm2) following annotation of tissues by tumor, fibrosis, necrosis, stromal & tumor infiltrating lymphocyte-enriched areas. Results: Nine pts were enrolled. Final analysis included 5 pts with adequate tissue. Median age = 48 (41-56), with 4 female, 4 intrahepatic cholangiocarcinomas & 1 gallbladder. All pts received Gemcitabine/Cisplatin as NACT with a median of 5 (4-7) cycles. Median time from diagnosis to surgery was 4.3 (1.4-7.8) months & last cycle to surgery was 0.9 (0.6-1.5) month. All pts were MMR proficient, 1 Her2+ & 2 with FGFR2 amplification. NACT on average produced a depletion of all immune markers (Table). Given the small N, each pt was considered their own control & changes in mean cell densities post NACT were calculated. Pt2 with a 40-fold increase in PDL1 expression & 5-fold decrease in CD8:FOXP3 ratio notably had the shortest disease-free interval (DFI). Pt3 with the longest DFI had the largest increase in CD8:FOXP3 by about 8-fold combined with a decrease in PDL1. Conclusions: Preliminary results suggest NACT may modulate immune microenvironment despite overall immune cell depletion. Future studies should focus on strategies to expand immune modulation of the tumor microenvironment in BTC by NACT, including immune oncology agent priming prior to or after NACT.[Table: see text]