Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.

MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. In this study, using qRT-PCR, we examined the expression of six miRNAs (miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145) in tumors from 193 prospectively recruited patients with colorectal cancer, and associations with clinicopathological parameters and patient outcome were analyzed. The miRNAs were chosen based on previous studies for their biomarker potential and suggested biological relevance in colorectal cancer.

In colorectal cancer there is a need for molecular markers that can complement the histopathological staging in predicting the likelihood of disease recurrence following curatively intended surgery. B7‐H3 is an immunoregulatory protein shown to be overexpressed in several cancer forms, often associated with more advanced disease and poor prognosis. We wanted to examine whether B7‐H3 could be a potential prognostic marker in colorectal cancer. Paraffin‐embedded samples from 277 colorectal cancer patients were immunostained with anti‐B7‐H3 antibody. B7‐H3 was expressed in the tumor cell cytoplasm and cell membrane in 62% and 46% of the samples, respectively. Unexpectedly, B7‐H3 was expressed in the nucleus in 30% of the tumors. The nuclear localization was confirmed by Western immunoblotting of subcellular fractions. Importantly, in colon cancer, nuclear B7‐H3 expression was independently and significantly associated with reduced metastasis‐free, disease‐specific and overall survival. B7‐H3 expression in tumor‐associated vasculature and fibroblasts was observed in the majority of samples, and endothelial B7‐H3 expression was also significantly associated with poor outcome in colon cancer. In rectal cancer patients, the only significant association was between fibroblast B7‐H3 expression and shorter metastasis‐free survival. Few significant associations to clinicopathological parameters were seen. The results indicate that nuclear B7‐H3 might be involved in colon cancer progression and metastasis, and suggest that nuclear B7‐H3 could become a useful prognostic marker in colon cancer.

Aim: To evaluate the activity and safety of the PD-1 antibody pembrolizumab in adult patients with advanced osteosarcoma. Material and methods: The study was a single-arm, open-label, phase 2 trial in patients with unresectable, relapsed osteosarcoma. The primary endpoint was clinical benefit rate (CBR) at 18 weeks of treatment, defined as complete response, partial response, or stable disease using RECIST v1.1. The trial had a Simon´s two-stage design, and ≥ 3 of 12 patients with clinical benefit in stage 1 were required to proceed to stage 2. The trial is registered with ClinicalTrials.gov, number NCT03013127. NanoString analysis was performed to explore tumor gene expression signatures and pathways. Results: Twelve patients were enrolled and received study treatment. No patients had clinical benefit at 18 weeks of treatment, and patient enrollment was stopped after completion of stage 1. Estimated median progression-free survival was 1.7 months (95% CI 1.2–2.2). At time of data cut-off, 11 patients were deceased due to osteosarcoma. Median overall survival was 6.6 months (95% CI 3.8–9.3). No treatment-related deaths or drug-related grade 3 or 4 adverse events were observed. PD-L1 expression was positive in one of 11 evaluable tumor samples, and the 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 osteosarcoma should explore combination strategies in patients selected based on molecular profiles associated with response.

We have previously reported overexpression of the immunoregulatory protein B7-H3 in colorectal cancer and that nuclear expression predicted poor outcome in colon cancer patients. The present study was performed to examine the prognostic role of B7-H3 in an independent colorectal cancer cohort. Using tissue microarrays from 731 colorectal cancer patients, tumour B7-H3 expression was assessed by immunohistochemistry. Associations with clinicopathological parameters and patient outcome were investigated. Nuclear expression of B7-H3 in cancer cells was present in 27% of the samples in the total study cohort, while cytoplasmic/membrane and stromal expression was seen in 86% and 77% of the samples, respectively. Nuclear B7-H3 had no prognostic relevance in the complete outcome cohort, neither in colon cancer patients. However, nuclear B7-H3 was significantly associated with reduced recurrence-free survival in TNM stage I colorectal cancer patients. Overexpression of B7-H3 in colorectal cancer was confirmed, but in contrast to previous results, nuclear B7-H3 was not a strong prognostic biomarker in this cohort. The discrepancy might be related to the use of single-core tissue microarrays for detection of the heterogeneously expressed B7-H3, and the role of B7-H3 in colorectal cancer still needs further examination.