Dougan, Stephanie K.

Background Pancreatic ductal adenocarcinoma (PDAC) is a challenging target for immunotherapy because it has an immunosuppressive tumor microenvironment. Neoadjuvant chemoradiotherapy can increase tumor-infiltrating lymphocyte (TIL) density, which may predict overall survival (OS). We hypothesized that adding programmed cell death protein 1 (PD-1) blockade to chemoradiotherapy would be well tolerated and increase TILs among patients with localized PDAC. Methods Patients were randomized 2:1 to Arm A (receiving pembrolizumab plus chemoradiotherapy (capecitabine and external beam radiation)) or Arm B (receiving chemoradiotherapy alone) before anticipated pancreatectomy. Primary endpoints were (1) incidence and severity of adverse events during neoadjuvant therapy and (2) density of TILs in resected tumor specimens. TIL density was assessed using multiplexed immunofluorescence histologic examination. Results Thirty-seven patients were randomized to Arms A (n=24) and B (n=13). Grade ≥3 adverse events related to neoadjuvant treatment were experienced by 9 (38%) and 4 (31%) patients in Arms A and B, respectively, with one patient experiencing dose-limiting toxicity in Arm A. Seventeen (71%) and 7 (54%) patients in Arms A and B, respectively, underwent pancreatectomy. Median CD8+ T-cell densities in Arms A and B were 67.4 (IQR: 39.2–141.8) and 37.9 (IQR: 22.9–173.4) cells/mm2, respectively. Arms showed no noticeable differences in density of CD8+Ki67+, CD4+, or CD4+FOXP3+ regulatory T cells; M1-like and M2-like macrophages; or granulocytes. Median OS durations were 27.8 (95% CI: 17.1 to NR) and 24.3 (95% CI: 12.6 to NR) months for Arms A and B, respectively. Conclusions Adding pembrolizumab to neoadjuvant chemoradiotherapy was safe. However, no convincing effect on CD8+ TILs was observed.

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) trials have evaluated CTLA-4 and/or PD-(L)1 blockade in patients with advanced disease in which bulky tumor burden and limited time to develop antitumor T cells may have contributed to poor clinical efficacy. Here, we evaluated peripheral blood and tumor T cells from patients with PDAC receiving neoadjuvant chemoradiation plus anti–PD-1 (pembrolizumab) versus chemoradiation alone. We analyzed whether PD-1 blockade successfully reactivated T cells in the blood and/or tumor to determine whether lack of clinical benefit could be explained by lack of reactivated T cells versus other factors. Experimental Design: We used single-cell transcriptional profiling and TCR clonotype tracking to identify TCR clonotypes from blood that match clonotypes in the tumor. Results: PD-1 blockade increases the flux of TCR clonotypes entering cell cycle and induces an IFNγ signature like that seen in patients with other GI malignancies who respond to PD-1 blockade. However, these reactivated T cells have a robust signature of NF-κB signaling not seen in cases of PD-1 antibody response. Among paired samples between blood and tumor, several of the newly cycling clonotypes matched activated T-cell clonotypes observed in the tumor. Conclusions: Cytotoxic T cells in the blood of patients with PDAC remain sensitive to reinvigoration by PD-1 blockade, and some have tumor-recognizing potential. Although these T cells proliferate and have a signature of IFN exposure, they also upregulate NF-κB signaling, which potentially counteracts the beneficial effects of anti–PD-1 reinvigoration and marks these T cells as non-productive contributors to antitumor immunity. See related commentary by Lander and DeNardo, p. 474

4128 Background: Pancreatic cancer (PC) is a challenging target for immunotherapy due to suppressive immune-microenvironment. Neoadjuvant chemoradiation (CRT) can increase the presence of tumor-infiltrating lymphocytes (TILs). We hypothesized that the combination of CRT and pembrolizumab can lead to further increase in TILs and their activation. Methods: Patients with resectable or borderline resectable PC were randomized 2:1 to the investigational treatment (Arm A) of pembrolizumab 200mg IV every 3 weeks concurrently with CRT (capecitabine 825 mg/m2 orally twice daily and radiation 50.4 Gy in 28 fractions over 28 days) or CRT only (Arm B) prior to surgical resection. The primary endpoints were treatment safety and density of TILs with the objective to estimate differences in TILs density between the investigational and the control arms. Immune cell densities were assessed using multiplexed immunofluorescence on resected tumor specimens. Densities of CD8+TILs were measured in 2-10 representative regions containing residual cancer per case and then averaged to obtain overall densities. The study was amended after enrollment of 37 patients to allow FOLFIRINOX prior to CRT, given changes in standard of care. Results: 37 patients were enrolled (24 Arm A and 13 Arm B). Post-neoadjuvant therapy, 13 patients had unresectable disease (9 on A and 4 on B), and 24 patients underwent surgery and were evaluable for the TILs primary endpoint (17 arm A and 7 arm B). The mean difference (A-B) in CD8+ cell density was 36 cells/mm2 (95% CI -85 to 157, stdev 130) (p 0.48). Additional analysis did not show significant differences in CD8+Ki67+ (activated cytotoxic T-cells), CD4+, and CD4+FOXP3+ (regulatory T cells), M1- or M2-like polarized macrophages, or granulocytes. The median recurrence free survival (RFS) was 18.2 months on Arm A and 14.1 on Arm B (p 0.41) and Overall Survival was 27.8 months on Arm A and 24.3 on Arm B (p 0.68) with a median follow up of 2.2 years. The most common grade 3 treatment-related toxicities were lymphopenia reported in 29% on Arm A and 31% on Arm B followed by diarrhea in 8% on Arm A attributed to CRT. There was only 1 DLT of increased ALT attributed to the combination on Arm A that resolved after holding the treatment and receiving steroids. There were no major surgical complications reported within 30 days post-surgery. Conclusions: The combination of CRT and pembrolizumab is safe. Preliminary analysis shows that the addition of pembrolizumab to CRT has minimal effects on several immune cell populations including CD8+TILs in the PC microenvironment. The study is currently enrolling 25 more patients who receive FOLFIRINOX prior to randomization to CRT+/- Pembrolizumab, which will help to dissect the immune modulatory effect of chemotherapy followed by CRT. Clinical trial information: NCT02305186.