209 Preclinical mechanistic and clinical evaluation of the corticosteroid dexamethasone’s detrimental effects on immune checkpoint blockade in glioblastoma cancer

Abstract

BackgroundIncreasing data indicate that corticosteroids can exert a detrimental effect on immunotherapy for oncology patients. Dexamethasone, a uniquely potent corticosteroid, is frequently administered to brain tumor patients to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in glioblastoma patients – particularly in the context of immunotherapy.MethodsWe evaluated the dose-dependent effects of dexamethasone when administered with PD-1 blockade and/or radiotherapy on survival and tumor response in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A glioblastoma tumors. The immune microenvironment was comprehensively profiled using flow cytometry analysis. Clinically, the effect of dexamethasone on survival was evaluated in 181 IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors using multivariable Cox regression.ResultsDespite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner (figure 1). Concurrent dexamethasone also abrogated survival following anti-PD-1 with or without radiotherapy in immunoresistant CT-2A models (figure 2). Dexamethasone decreased T lymphocyte numbers (figure 3) by increasing apoptosis (figure 4), in addition to decreasing lymphocyte functional capacity (figure 3C/D). Myeloid and NK cell populations were also generally reduced by dexamethasone (figure 3). Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment by relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival, regardless of dose (referent no dexamethasone; <2 mg HR 2.16, 95%CI: 1.30–3.68, p=0.003; ≥2 mg HR 1.97, 95%CI: 1.23–3.16, p=0.005; table 1 and figure 5).Abstract 209 Figure 1Concurrent dexamethasone reduces the survival benefit of anti-PD-1 therapy in GL261-luc2 glioblastoma mouse models in a dose-dependent manner. (A) Experimental schema. Anti-PD-1 (αPD1, red arrows) was administered in a dose-intensive schedule, i.e. IP beginning on day 6 (500 µg) followed by 7 additional doses (250 µg/dose) at 3-day intervals, with dexamethasone delivered IP daily from days 6–27. (B) Kaplan-Meier survival estimates of anti-PD-1 therapy without dexamethasone (n=42) and anti-PD-1 therapy with concurrent dexamethasone at 1 mg/kg (n=34), 2.5 mg/kg (n=16), or 10 mg/kg (n=34, data derived from 4 experiments, with 8–10 mice each), compared to IgG (n=42) and 10 mg/kg dexamethasone-only (n=8) controls; with comparison by Cox regression. (C) The corresponding longitudinal bioluminescence imaging, displayed as change from baseline (day 6 after implantation, dotted gray line), for mice treated with anti-PD-1 alone (n=42) or anti-PD-1 with concurrent 1 mg/kg (n=34), 2.5 mg/kg (n=16), or 10 mg/kg dexamethasone (n=34), as compared to IgG control (n=16) and dexamethasone 10 mg/kg only control (n=8). Tumor response visualized in red and lack of response in blue. (D) Representative longitudinal MRI findings demonstrating increased tumor growth when low (1 mg/kg) or high (10 mg/kg) doses of dexamethasone were co-administered during PD-1 therapy, compared to anti-PD-1 without dexamethasone. Dotted red line outlines the tumor on coronal MRI plane ns, not significant, p≥0.05; *p<0.05; **p<0.01; ***p<0.001; Dex, dexamethasone; BLI, bioluminescence imaging; OS, overall survival; 95CI, 95% confidence interval; NR, not reached.Abstract 209 Figure 2Concurrent dexamethasone decreases the OS benefit of anti-PD-1 plus RT in syngeneic GL261-luc2 and CT-2A-luc glioblastoma mouse models. Kaplan-Meier OS estimates are depicted, with comparison by logrank test and Cox regression. (A) To assess concurrent dexamethasone’s effect on a dose-intensive schedule of anti-PD-1 with or without RT in GL261-luc2 mice (n=8/group), anti-PD-1 was administered IP via a loading dose (500 µg) followed by 5 additional doses (250 µg/dose) at 3-day intervals. RT was administered in 2 Gy fractions/day for 5 days beginning on day 6. Dexamethasone was delivered IP daily from days 6–27 at 10 mg/kg. (B) For GL261-luc2 mice (n=8/group), anti-PD-1 (αPD1) was administered IP via an abbreviated dosing schedule every 3 days beginning on day 6 for a total of 4 doses (250 µg/dose). (C) For CT-2A-luc mice (n=8–16/group), anti-PD-1 was administered IP via a loading dose (500 µg) followed by 7 additional doses (250 µg/dose) at 3-day intervals. RT was administered in 2 Gy fractions/day for 5 days beginning on day 6. Dexamethasone was delivered IP daily from days 6–27 at 10 mg/kg*p<0.05; **p<0.01; ***p<0.001; Dex, dexamethasone; 95CI, 95% confidence interval; NR, not reachedAbstract 209 Figure 3Concurrent dexamethasone negatively affects intratumoral and systemic adaptive and innate immune cell populations in the GL261-luc2 glioblastoma mouse model. (A) Experimental schema. Tissue was collected at day 16 of a dose-intensive regimen of anti-PD-1, in which anti-PD-1 (αPD1) was administered IP beginning on day 6 (500 µg loading dose) followed by 3 additional doses (250 µg) at 3-day intervals, with dexamethasone (10 mg/kg) administered IP on days 6–16. Tissue (n=4–8/group) was harvested on day 16 and analyzed by flow cytometry. Immune cell counts were evaluated by multiple linear regression, normalized to the corresponding IgG control group’s mean count (displayed as dashed gray line), and displayed as mean ± SE. (B) Differences in CD45+ leukocytes and CD45+ CD3+ lymphocytes, including CD4+ and CD8+ T cells between treatment groups. (C) Percentage of splenic IFNγ+ CD4+ and CD8+ lymphocytes by treatment group. (D) Change in the number of early activated CD69+ T cells by site for each treatment group. Additionally, differences between treatment groups in innate immune cells including (E) myeloid cells (CD45hi CD11bhi), macrophages (Ly6Clo-int Ly6G-), monocytes (Ly6Chi Ly6G-), and microglia (in the brain, CD45lo CD11bhi), (F) dendritic cells (DCs; CD45+ CD11c+) and NK cells (CD45+ CD3- NK1.1+); as well as (G) activated (CD80+ CD86+) myeloid cells and DCs, PD-L1+ myeloid cells, and Ki67+ NK cells were analyzed cLN, cervical lymph node; Dex, dexamethasone; ns, not significant, p≥0.05; *p<0.05; **p<0.01; ***p<0.001Abstract 209 Figure 4Concurrent dexamethasone increases apoptosis of CD4+ and CD8+ T cells in the GL261-luc2 glioblastoma mouse model. (A) Late apoptosis was evaluated by 7-AAD+ and annexin-V+ staining in non-tumor-bearing mouse spleens (n=3/group) either 1 hour after the first dexamethasone dose or 1 hour after the sixth daily dexamethasone dose. Apoptosis differences were tested by two-way ANOVA with post-test correction. Cell counts normalized to the corresponding IgG control group’s mean count (B) and percent (C) of proliferating CD4+ and CD8+ T cells were evaluated by Ki67 staining, using the same dosing schema and analyses as figure 3 (n=4–8/group) cLN, cervical lymph node; Dex, dexamethasone; hr, hour; ns, not significant, p≥0.05; *p<0.05; **p<0.01; ***p<0.001Abstract 209 Figure 5Baseline dexamethasone is associated with decreased OS among glioblastoma patients receiving anti-PD-(L)1 therapy, irrespective of dexamethasone dose. Kaplan-Meier OS estimates for 181 IDH-wildtype glioblastoma patients treated with anti-PD-(L)1 therapy, who were either on ≥2 mg (dashed gray line), <2 mg (dashed black line), or no (solid black line) baseline dexamethasone are depicted; including both (A) unadjusted analyses (n=181) and (B) analyses adjusted (by a Cox regression model; n=163) for relevant prognostic factors including disease setting (newly-diagnosed vs. recurrent), patient age, MGMT promoter methylation, KPS and tumor volume prior to anti-PD-(L)1 initiation, and extent of resection**p<0.01; ***p<0.001; Dex, dexamethasone ; mos, monthsAbstract 209 Table 1Multivariable Cox regression analysis of the effect of baseline dexamethasone on overall survival in glioblastoma patients treated with anti-PD-(L)1.ConclusionsWe demonstrate that concurrent dexamethasone administration, even at a low dose, limits the therapeutic benefit of anti-PD-1 therapy both in mouse glioblastoma models and in a retrospective cohort of 181 IDH-wildtype glioblastoma patients. Mechanistically, dexamethasone decreased intratumoral T cells and systemic levels of T cells, natural killer cells, and myeloid cells, while qualitatively impairing lymphocyte function. The mechanism of T cell depletion included induction of apoptosis. These findings indicate that dexamethasone hinders both adaptive and innate immune responses, intratumorally and systemically, and that its administration should be carefully assessed among glioblastoma patients undergoing second-generation immunotherapy clinical trials. Our findings also have ramifications for brain metastasis patients where immune checkpoint inhibitors are part of standard-of-care management.AcknowledgementsWe thank Min Wu for assistance in generating CT-2A luciferase-transduced cells, and Drs. Geoffrey Young, Lei Qin, Xin Chen, and Jing Li for assistance in evaluation of patients‘ radiographic imaging.Ethics ApprovalApproved under DFCI Institutional Review Board protocol 10-417.