Examination of the tumor immune microenvironment (TIME) with multispectral immunofluorescence (m-IF): Association of markers with prognosis and bevacizumab (bev) benefit in NRG Oncology/NSABP C-08.
Authors: Pogue-Geile Katherine L. , Joy Marion , Wang Ying , Song Nan , Kim Rim S. , Yothers Greg , Allegra Carmen Joseph , Srinivasan Ashok , Finnigan Melanie , Jacobs Samuel A. , Paik Soonmyung , Suga Jennifer Marie Marie , Hopkins Judith O. , DiBella Nicholas J. , Saito Naoyuki G. , Lucas Peter C. , Wolmark Norman 
Published: June 2021

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Background: The purpose of this study was to quantify different molecules of TIME including T cells, macrophages, and immune checkpoint proteins (ICPs), and determine their association with clinical outcomes and treatment benefit in pts enrolled in C-08, which tested the efficacy of adding bev to 5-fluoruracil+leucovorin+oxaliplatin. Our pre-specified, NCTN-CCSC approved primary objective hypothesized that pts with more CD8 cells would have a better prognosis and receive benefit from bev. Methods: Tissue microarrays were used to assess TIME of 1,509 C-08 pts using m-IF and the Vectra Pathology System. Three m-IF panels were used to quantitatively assess T cells (CD3, CD8, CD45RO, FOXP3), macrophages (CD68, CD163), and ICPs (PD-1, PD-L1, CTLA4, TIM3, LAG3, OX40) in stromal and tumor (panCK) regions. The primary objective was to determine the association between overall survival (OS) and high (top 3rd) v low CD8 expression in both stromal and tumor regions. All markers were tested for associations with OS and recurrence-free interval (RFI) and with bev prediction using Cox models and median cut points. Results: Based on our pre-specified analysis, pts with high CD8 cells had better OS, HR=0.66 (95%CI: 0.49-0.88), p=0.005 but pts with high CD8 cells did not receive bev benefit. All T cells and double stained CD8/PD-1 were associated with better RFI. CD3, CD8, CD68, PD-1, PD-L1, and LAG3 cells were associated with better OS. PD-1 and CD8/PD-1 were associated with RFI in pts with deficient mismatch repair (dMMR) and proficient (p)MMR but TIM3, CD3/CD45RO and CD163 were only associated with RFI in dMMR. Association of CD8 cells with bev benefit (RFI) was seen in dMMR pts, HR 0.27 (95% CI: 0.1-0.73), p=.01 and OS, HR=0.27, (95% CI: 0.12-0.64), p=0.0028 but there was no significant interaction. Single staining CD8, PD-1, and double staining CD8/PD-1 cells were associated with bev benefit in dMMR pts but with bev harm in pMMR pts. However, pts with tumors having >1% of PD-1 and PD-L1 cells (n=197 including 76 dMMR, 100 pMMR, and 21 unknown), received significant bev benefit (int p=.0056). Conclusions: CD8 cells were associated with better OS but were not associated with bev benefit. All T cells and PD-1, PD-L1, and LAG3 cells, were associated with better prognosis in the entire cohort but when pts were stratified for MMR status differences in their association with prognosis and bev benefit emerged. PD-1, CD8, and CD8/PD-1 cells were associated with bev harm in pMMR but bev benefit in dMMR. A significant interaction for the association of high % PD-1 and PD-L1 with bev benefit regardless of MMR status may be a chance finding. However, VEGF has immunosuppressive effects and bev may block these effects in tumors with high PD-L1 and PD-1, regardless of MMR status. NCT: 00096278 PA DOH, U10CA-180868, -180822, -196067, Genentech, Sanofi; NSABP. Clinical trial information: 00096278.