On p. 3374, a section of the microarray analysis text should read as follows: Analysis was performed as described (Roepman et al., 2005). Data were analyzed using ANOVA (R version 2.2.1/MAANOVA version 0.98-7) (Wu et al., 2003). Genes with P<0.05 after false discovery rate correction were considered to be significantly changed. Mouse Whole Genome Gene Expression Microarrays V1 (Agilent Technologies, Belgium) containing mouse 60-mer probes were used and data have been deposited in ArrayExpress under accession number E-TABM-1104.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin which is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we used single-cell mRNA-sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche revealed evidence of an immunosuppressive microenvironment. We also identified an interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a putative cause of tumour-induced T-cell dysfunction. In malignant RMS cells we defined transcriptional programs reflective of normal myogenic differentiation. Furthermore, we showed that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the more clinically homogenous fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Congenital portosystemic shunts are developmental anomalies of the splanchnic vascular system that cause portal blood to bypass the liver. Large-breed dogs are predisposed for intrahepatic portosystemic shunts (IHPSS) and small-breed dogs for extrahepatic portosystemic shunts (EHPSS). While the phenotype resulting from portal bypass of the liver of the two types of shunt is identical, the genotype and molecular pathways involved are probably different. The aim of this study was to gain insight into the pathways involved in the different types of portosystemic shunting. Microarray analysis of mRNA expression in liver tissue from dogs with EHPSS and IHPSS revealed that the expression of 26 genes was altered in either IHPSS or EHPSS samples compared with that in liver samples from control dogs. Quantitative real-time PCR of these genes in 14 IHPSS, 17 EHPSS, and 8 control liver samples revealed a significant differential expression of ACBP, CCBL1, GPC3, HAMP, PALLD, VCAM1, and WEE1. Immunohistochemistry and Western blotting confirmed an increased expression of VCAM1 in IHPSS but its absence in EHPSS, an increased WEE1 expression in IHPSS but not in EHPSS, and a decreased expression of CCBL1 in both shunt types. Regarding their physiologic functions, these findings may indicate a causative role for VCAM1 in IHPSS and WEE1 for IHPSS. CCBL1 could be an interesting candidate to study not yet elucidated aspects in the pathophysiology of hepatic encephalopathy.

<p>Legends for Supplemental Figures 1-8</p>