DNA methylation-based reclassification of olfactory neuroblastoma

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n=66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed 4 subgroups among institutionally diagnosed ONB. The largest group (n=42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n=7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10 and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB

Protein Phosphatase 1 Regulatory Subunit 1A in Ewing Sarcoma Tumorigenesis and Metastasis

Protein phosphatase inhibitors are often considered as tumor promoters. Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) is a potent protein phosphatase 1 (PP1) inhibitor; however, its role in tumor development is largely undefined. Here we characterize, for the first time, the functions of PPP1R1A in Ewing sarcoma (ES) pathogenesis. We found that PPP1R1A is one of the top ranked target genes of EWS/FLI, the master regulator of ES, and is upregulated by EWS/FLI via a GGAA microsatellite enhancer element. Depletion of PPP1R1A resulted in a significant decrease in oncogenic transformation and cell migration in vitro as well as xenograft tumor growth and metastasis in an orthotopic mouse model. RNA-sequencing and functional annotation analyses revealed that PPP1R1A regulates genes associated with various cellular functions including cell junction, adhesion and neurogenesis. Interestingly, we found a significant overlap of PPP1R1A-regulated gene set with that of ZEB2 and EWS, which regulates metastasis and neuronal differentiation in ES, respectively. Further studies for characterization of the molecular mechanisms revealed that activation of PPP1R1A by PKA phosphorylation at Thr35, and subsequent PP1 binding and inhibition, was required for PPP1R1A-mediated tumorigenesis and metastasis, likely by increasing the phosphorylation levels of various PP1 substrates. Furthermore, we found that a PKA inhibitor impaired ES cell proliferation, tumor growth and metastasis, which was rescued by the constitutively active PPP1R1A. Together, these results offered new insights into the role and mechanism of PPP1R1A in tumor development and identified an important kinase and phosphatase pathway, PKA/PPP1R1A/PP1, in ES pathogenesis. Our findings strongly suggest a potential therapeutic value of inhibition of the PKA/PPP1R1A/PP1 pathway in the treatment of primary and metastatic ES

Sarcoma classification by DNA methylation profiling

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications