LAUSR

Background. Two pre-operative radiotherapy (RT) regimens are in common use for patients (pts) with locally advanced rectal cancer (LARC): Conventionally fractionated, long course chemoradiation (LCCRT, 25x1.8Gy with concomitant chemotherapy) or hypofractionated short-course RT (5X5Gy) followed by systemic chemotherapy (SCRT-C). RT is thought to induce immune responses to cellular damage, but the understanding of their evolution and relation to fractionation is limited. We are conducting a serial biospecimen collection in such pts with baseline, 2, 6 and 12 week (wk) sampling. We report serial measurement of tumor and peripheral blood lymphocyte responses for each regimen. Method. Multiplex immunofluorescence quantified tumor infiltrating lymphocytes (CD8, FOXP3). Routine diagnostic flow cytometry quantified circulating lymphocytes. A multiplex ELISA quantified cytokines in blood plasma. Bulk RNA-sequencing (QuantSeq) quantified gene expression within the tumor. Result. We report results for 20pts who received RT for stage III/IV LARC (13 LCCRT, 7 SCRT-C). Biopsy results are available for 10pts, circulating lymphocytes for 20pts and peripheral blood cytokines for 16pts. In LCCRT patients (n=8), relative to baseline, tumor infiltrating cytotoxic (CD8+) T cells were uniformly decreased during (2wks, P<0.05) and directly after treatment (6wks, P<0.05) before returning to baseline at 12wks. T regulatory cells (FOXP3+) similarly significantly decreased at 2wks and 6wks but remained below baseline at 12wks (P<0.05). Circulating lymphocytes also fell at wk2 and wk6 after commencing LCCRT and had begun recovering by wk12(n=13; P<0.05). The concentrations of circulating interleukins secreted by (IL2, IL8) or which activate T-lymphocytes (IL2, IL15) were reduced in the circulation at wk2 (n= 9; P<0.05) and wk6 (NS). In SCRT-C patients, we noted an increase in CD8 T cells at 2wks in 2/2 pts, also reflected in gene expression data, and an increase in FOXP3 T cells in 1/2 pts. Circulating lymphocytes were similarly decreased at wk2 in both the SCRT-C (n=7) and LCCRT (n=13) pts, but this reduction was less marked within the SCRT-C cohort at wk6 (P<0.01) and wk12 (P<0.05) relative to LCCRT patients. IL2, IL8 and IL15 did not change during or after SCRT-C (n=7; NS). Conclusion. LCCRT caused a drop in T cells during treatment, whilst SCRT-C appears to induce intra-tumoral T cell responses from wk2 and abrogates systemic reactions to a lesser extent. These results require evaluation in a larger cohort but have implications for understanding how RT induces microenvironmental changes and impacts pelvic bone marrow. We show in vivo that SCRT may be more immunostimulatory in LARC, with implications for trials combining RT with immunotherapy. Citation Format: Lily V. Hillson, Ross K. McMahon, Kathryn A. Pennel, Jean A. Quinn, Leia Jones, Raheleh Amirkhah, Aula Ammar, Phimmada Hatthakarnkul, Annabelle Ferguson, Simon W. Milling, Alec McDonald, Philip D. Dunne, Joanne Edwards, Sean M. O'Cathail, Campbell S. Roxburgh. Temporal changes in intratumoral and systemic lymphocytes in response to short and long course radiotherapy regimens in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3231.