Background Immunotherapy has changed the therapeutic landscape in oncology. Advanced uterine leiomyosarcoma (ULMS) remains an incurable disease in most cases, and despite new drug approvals, improvements in overall survival have been modest at best. The goal of this study was to evaluate programmed-death 1 (PD-1) inhibition with nivolumab in this patient population Methods This single-center phase 2 trial completed enrollment between May and October 2015. Patients received 3 mg/kg of intravenous nivolumab on day 1 of each 2-week cycle until disease progression or unacceptable toxicity. The primary endpoint was objective response rate. We assessed PD-1, PD-ligand 1 (PD-L1), and PD-L2 expression in archival tumor samples and variations in immune-phenotyping of circulating immune cells during treatment Results Twelve patients were enrolled in the first stage of the 2-stage design. A median of 5 (range, 2-6) 2-week cycles of nivolumab were administered. Of the 12 patients, none responded to treatment. The overall median progression-free survival was 1.8 months (95% confidence interval, 0.8-unknown). The study did not open the second stage due to lack of benefit as defined by the statistical plan. Archival samples were available for 83% of patients. PD-1 (>3% of cells), PD-L1, and PD-L2 (>5% and >10% of tumor cells, respectively) expression were observed in 20%, 20%, and 90% of samples, respectively. No significant differences were observed between pre- and posttreatment cell phenotypes. Conclusion Single-agent nivolumab did not demonstrate a benefit in this cohort of previously treated advanced ULMS patients. Further biomarker-driven approaches and studies evaluating combined immune checkpoint-modulators should be considered.

Objective Extracellular traps-(ETs) consist of DNA-protein-complexes formed following tissue injury contributes to the inflammatory and thrombosis cascades thereby exacerbating injury. Exogenous DNase-1 has been suggested as a therapeutic strategy to limit injury in the brain and myocardium.. These studies were designed to evaluate the effects of exogenous DNase-I treatment on skeletal muscle injury following acute hind limb ischemia-reperfusion (IR) injury in mice, and to determine whether neutrophils were a major source of ETs in postischemic muscle tissue. Methods C57BL6 mice were subjected to 1.5 hrs tourniquet ischemia and 24 hrs reperfusion with and without human recombinant DNase-I treatment. A separate set of mice was subjected to neutrophil depletion, followed by the same intervals of IR. Laser Doppler imaging and tissue harvesting was done at 24 hrs for assessment of limb perfusion, muscle fiber injury, ATP, markers of inflammation, thrombosis, and ETs formation. Results DNase-I treatment significantly reduced ETs detection in post-ischemic muscle but did not alter skeletal muscle fiber injury, levels of pro-inflammatory molecules or ATP. DNase-I treatment did enhance postischemic hind limb perfusion, decreased infiltrating inflammatory cells and reduced the expression of Thrombin-Anti-Thrombin-III. Neutrophil depletion resulted in a significant yet small reduction in ETs in the post ischemic muscle. Neutrophil depletion did not alter skeletal muscle fiber injury, hind limb perfusion, or ATP levels. Conclusions These data suggest that neither DNase-I treatment nor Neutrophil depletion were protective against IR injury, even though both decreased ETs detection in skeletal muscle following IR. Neutrophils are not the only source of ETs following IR.

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Background: Squamous cell carcinoma of the anal canal (SCCA) is an uncommon malignancy with limited therapeutic options. Nivolumab and pembrolizumab show promising results in patients with SCCA. Human papillomavirus (HPV)-negative tumors are frequently TP53-mutated (TP53-MT) and often resistant to therapy. Methods:We present a large molecularly-profiled cohort of SCCA, exploring the underlying biology of SCCA, differences between TP53-wild type (TP53-WT) and TP53-MT tumors, and differences between local and metastatic tumors. SCCA specimens (n=311) underwent multiplatform testing with immunohistochemistry (IHC), in situ hybridization (ISH) and next-generation sequencing (NGS). Tumor mutational burden (TMB) was calculated using only somatic nonsynonymous missense mutations. Chisquare testing was used for comparative analyses.Results: The most frequently mutated genes included PIK3CA (28.1%), KMT2D (19.5%), FBXW7 (12%), TP53 (12%) and PTEN (10.8%). The expression of PD-1 was seen in 68.8% and PD-L1 in 40.5% of tumors. High TMB was present in 6.7% of specimens. HER2 IHC was positive in 0.9%, amplification by chromogenic in situ hybridization (CISH) was seen 1.3%, and mutations in ERBB2 were present in 1.8% of tumors. The latter mutation has not been previously described in SCCA. When compared with TP53-WT tumors, TP53-MT tumors had higher rates of CDKN2A, EWSR1, JAK1, FGFR1 and BRAF mutations.PD-1 and PD-L1 expression were similar, and high TMB did not correlate with PD-1 (P=0.50) or PD-L1 (P=0.52) expression.Conclusions: Molecular profiling differences between TP53-MT and TP53-WT SCCA indicate different carcinogenic pathways which may influence response to therapy. Low frequency mutations in several druggable genes may provide therapeutic opportunities for patients with SCCA.

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