Turning cold into hot: combination of pembrolizumab with bevacizumab and oral metronomic cyclophosphamide increases immune cell migration into the tumor microenvironment in responding patients with recurrent ovarian cancer (090)

Abstract

<b>Objectives:</b> Our single institute phase II clinical trial (NCT02853318) with pembrolizumab combined with bevacizumab and oral cyclophosphamide showed an objective response rate (ORR) of 47.5% with a median progression-free survival (PFS) of ten months (mo) with minimal toxicity and improved quality of life (QOL) in our recurrent ovarian cancer patients. In this clinical trial, 30% of the patients achieved durable clinical response (>12 mo), which is a marked improvement compared to the efficacy of single-agent pembrolizumab (median PFS 2.9 mo) in a similar cohort of patients. Our objective was to understand the changes induced by the triple combination in peripheral circulation and in the tumor microenvironment (TME), particularly in patients with a durable clinical response. <b>Methods:</b> Patients on NCT02853318 underwent baseline and on-treatment tumor biopsy and peripheral blood collection after three cycles of treatment. Out of the 40 patients, 18 early progressors (median PFS < 10 mo) and 16 late progressors (median PFS > 10 mo) had good quality paired RNA samples for analysis. RNASeq data analysis was performed in our Genomic and Bioinformatic cores. Blood samples at matching time points were analyzed using mass cytometry (CyTOF). To confirm our observations made from gene expression analyses with protein level expression, we selected seven patients with PFS < 10 mo and seven patients with PFS > 10 mo and applied NanoString Digital Spatial Profiling to visualize and further characterize the immune cells within the tumor and stroma compartments. <b>Results:</b> Pathway analysis of expression profiles from baseline tumor samples of late and early progressors revealed that the TME of late progressors was immunologically "hot" and enriched for B cell proliferation and B cell signaling/activating pathways, demonstrating increased CD40 activity, increased T cell differentiation, migration, T cell receptor signaling, antigen processing/presenting, INF-γ production and NFkB activation. Statistical analysis of protein expression levels between baseline and on-treatment biopsies showed a significant increase of T cells and B cells in tumors in patients with PFS of > 10 mo (Fig. 1), but not in patients with PFS of < 10 mo. CyTOF analysis demonstrated that the treatment decreased peripheral B cells, memory B cells, CD8 and CD4 T cells, as well as total lymphocytes, suggesting that the combination was able to drive both B and T cells to TME with larger magnitude in responding patients. CyTOF analysis also showed that responding patients had significantly higher CD4 and CD4 effector memory T cells in the circulation, suggesting that this could serve as a pretreatment biomarker to predict response to immune checkpoint blockade. <b>Conclusions:</b> Pembrolizumab combined with bevacizumab and oral cyclophosphamide significantly increased both T and B cell influx to tumor from stroma and increased CD4 effector memory T cells in circulation in patients with durable clinical response.Fig. 1