No treatment-related deaths or drug-related grade 3 or 4 4 adverse reactions were observed. 18F-FDG PET/CT Five patients underwent 18F-FDG PET/CT at baseline and after 6?weeks. cut-off, 11 patients were deceased due to osteosarcoma. Median overall survival was 6.6?months (95% CI 3.8C9.3). No treatment-related deaths or drug-related grade 3 or 4 4 adverse events were observed. PD-L1 expression was positive in one of 11 evaluable tumor samples, and the Chrysophanol-8-O-beta-D-glucopyranoside positive sample was from a patient with a mixed treatment response. Conclusion In this phase 2 study in advanced osteosarcoma, pembrolizumab was well-tolerated but did not show clinically significant antitumor activity. Future trials with immunomodulatory agents in Chrysophanol-8-O-beta-D-glucopyranoside osteosarcoma should explore combination strategies in patients selected based on molecular profiles associated with response. Eastern Cooperative Oncology Group. Treatment and radiological response The median number of cycles of pembrolizumab administered was 2 (range 1C6). Four patients received only one cycle, all due to clinical progression prior to the second cycle. Ten patients underwent at least one radiological evaluation, while two patients with clinical progression were not evaluated radiologically. PD was observed in 9 of 10 patients, of whom six had PD at the first evaluation after 6?weeks and three at the second evaluation after 12?weeks. One patient had SD at first evaluation and stopped study treatment after cycle 3 because steroid therapy was initiated due to dyspnea and hemoptysis caused by a progressive lung metastasis, and was not subsequently evaluated. Thus, 0 of 12 patients reached the primary endpoint of clinical benefit defined as SD, PR, or CR at 18?weeks of treatment. Best overall response is shown in Fig.?1. Due to rapid disease progression, only four patients completed EORTC QLQ-C30 questionnaires during treatment, and quality of life results are thus not reported. Open in a separate window Fig. 1 Waterfall plot showing best RECIST response. Individual patients are represented by vertical bars and the change in tumor size according to RECIST v1.1 PECAM1 is depicted on the Y-axis. Two patients did not undergo radiological evaluation and are not included Survival outcome Nine patients (75%) had confirmed disease progression. Estimated median progression-free survival was 1.7?months (95% CI 1.2C2.2). At time of data cut-off, 11 patients were deceased, all from osteosarcoma, and estimated median overall survival was 6.6?months (95% CI 3.8C9.3). Adverse events Adverse events of grade 3 or higher occurred in 7 of 12 patients (58%). Anemia grade 3 was reported in two patients, and increased alkaline phosphatase (grade 3), medullary compression (grade 3), pneumothorax (grade 3), and tumor-related pain (grade 3) in one patient each. No treatment-related deaths or drug-related grade 3 or 4 4 adverse reactions were observed. 18F-FDG PET/CT Five patients underwent 18F-FDG PET/CT at baseline and after 6?weeks. The median SUVmax at baseline was 15.4 (range 4.1C20.5) and at first evaluation 11.4 (range 2.3C28.0). One patient had an increase in SUVmax from 20.5 to 28.0. SUVmax was reduced after 6?weeks in the other four patients, with an absolute reduction of 1.3C4.4 (12C44%) compared to baseline values. Three patients had progressive metabolic disease (PMD) at first evaluation using PERCIST v1.0. One patient with stable metabolic disease (SMD) at the first evaluation had PMD at the second PET/CT after 18?weeks, and one patient had SMD at both response evaluations. PD-L1 expression Pretreatment tumor samples from 11 patients were available for analysis of PD-L1 expression. From three patients tumor tissue was obtained by a study-specific biopsy before treatment and from 8 patients archival tumor material was used for the analyses. In one sample, there was a strong positive membranous expression of PD-L1 ( ?50% positive tumor cells), whereas the other ten samples were negative (Fig.?2). The PD-L1 positive specimen was a study biopsy of a soft tissue metastasis in the abdominal wall of a 41-year-old woman. She had PD and PMD after two treatment cycles, but with a mixed radiological and metabolic response. Uptake of 18F-FDG Chrysophanol-8-O-beta-D-glucopyranoside and tumor size were reduced in the lung and kidney metastases (Fig.?3, arrows), accompanied by an improved general condition and less tumor pain. There was, however, significant progression of abdominal and pelvic metastases (Fig.?3, arrowheads), and study treatment was discontinued. Open in a separate window Fig. 2 Photomicrographs of immunohistochemical staining with monoclonal anti-PD-L1 antibody (clone 22C3; Dako). a? ?50% positive membranous staining of tumor cells. Scale bar 50?m. b Negative staining. Scale bar 100?m Open in a separate window Fig. 3.