Purpose: Ewing sarcoma and osteosarcoma are primary bone sarcomas occurring most commonly in adolescents. Metastatic and relapsed disease are associated with dismal prognosis. Although effective for some soft tissue sarcomas, current immunotherapeutic approaches for the treatment of bone sarcomas have been largely ineffective, necessitating a deeper understanding of bone sarcoma immunobiology. Experimental Design: Multiplex immunofluorescence analysis of immune infiltration in relapsed versus primary disease was conducted. To better understand immune states and drivers of immune infiltration, especially during disease progression, we performed single-cell RNA sequencing (scRNAseq) of immune populations from paired blood and bone sarcoma tumor samples. Results: Our multiplex immunofluorescence analysis revealed increased immune infiltration in relapsed versus primary disease in both Ewing sarcoma and osteosarcoma. scRNAseq analyses revealed terminally exhausted CD8+ T cells expressing co-inhibitory receptors in osteosarcoma and an effector T-cell subpopulation in Ewing sarcoma. In addition, distinct subsets of CD14+CD16+ macrophages were present in Ewing sarcoma and osteosarcoma. To determine pathways driving tumor immune infiltration, we conducted intercellular communication analyses and uncovered shared mechanisms of immune infiltration driven by CD14+CD16+ macrophages and unique pathways of immune infiltration driven by CXCL10 and CXCL12 in osteosarcoma. Conclusions: Our study provides preclinical rationale for future investigation of specific immunotherapeutic targets upon relapse and provides an invaluable resource of immunologic data from bone sarcomas.

NRAS and BRAF mutations occur in congenital melanocytic nevi (CMN), but results are contradictory. Sixty-six prospectively collected CMN patients were analyzed for NRAS Q61 mutations using Sanger sequencing. Negative cases were evaluated for BRAF V600E mutation. NRAS Q61 mutations affected 51 patients (77.3%), and BRAF V600E was found in 5 (7.6%). NRAS Q61 mutation affected 29 (80.6%) of 36 giant, 16 (80.0%) of 20 large, and 5 (62.5%) of 8 medium-size CMN; BRAF mutation affected 1 (5%) of 20 large and 4 (11.4%) of 36 giant CMN. Compared to NRAS, BRAF-mutated nevi show scattered/extensive dermal and subcutaneous nodules (100% BRAF+ vs 34.8% NRAS+) ( P = 0.002). Neurocutaneous melanocytosis (NCM) affected 16 (24.2%) of 66 patients, with NRAS Q61 mutation in 12 (75.0%), and BRAF V600E in 2 (12.5%), P = 0.009. Two patients were negative for both mutations (12.5%). In conclusion, although NRAS Q61 mutations predominate, BRAF V600E mutation also affects patients with large/giant CMN (L/GCMN), and with NCM, a novel finding. BRAF V600E is also associated with increased dermal/subcutaneous nodules. These findings open the possibility of BRAF-targeted therapy in some L/GCMN and NCM cases.

Epidemiological studies have shown an increased prevalence of high blood pressure in pediatric patients. Today we know that risk factors can be detected during childhood and may help in preventing the disease. To evaluate risk and protection factors related to high blood pressure in childhood. We evaluated children aged 3 to 10 years, residing in the east and southwest sanitary districts of the City of Goiânia, Goiás, and obtained the following data: birth weight, breastfeeding, family history of high blood pressure and obesity, weight, height, body mass index (BMI), and blood pressure. We applied the Mann-Whitney U-test to these variables in order to compare pressure variation. In this sample, 519 children were evaluated, and 246 (47.4%) of them were male. The BMI assessment identified 109 (21%) overweight children, of which 53 (10.3%) were obese. Predominant and/or exclusive breastfeeding for less than 6 months was found in 242 (51.2%). The mean systolic pressure was significantly higher in children with exclusive and/or predominant breastfeeding for less than 6 months (p = 0.04), and in children with family history of high blood pressure (p = 0.05), and in overweight children (p <0.0001). These data were confirmed in multivariate analysis. In this sample, we observed that overweight and hereditary factors may be associated with elevated blood pressure, and that breastfeeding for more than 6 months seems to offer a protective effect.