DNA methylation-based reclassification of olfactory neuroblastoma
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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 four 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.
We are much obliged to the staff of the Center for Cancer Genome Discovery (CCGD) at the Dana-Farber Cancer Institute (Boston, US) for DNA sequencing and especially thank Paul van Hummelen and Aaron Thorner for assistance with interpretation of findings. We thank the Microarray unit of the Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ) for providing excellent DNA methylation services. We thank the DKFZ-Heidelberg Center for Personalized Oncology (DKFZ-HIPO) for technical support and funding through HIPO_036. In other parts, this work was supported by an Illumina Medical Research Grant. This work was further supported by the Deutsche Krebshilfe (Grant no. 111630) and the Fördergemeinschaft Kinderkrebs-Zentrum Hamburg e.V.
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Conflict of interest
Andreas von Deimling, David Jones, and David Capper share inventorship of a “DNA-methylation-based method for classifying tumor species of the brain”. A patent has been applied for this method (EP 3067432 A1). All terms are being managed by the German Cancer Research Center in accordance with its conflict of interest policies. G. Reifenberger has received research grants from Roche and Merck Serono, and honoraria for lectures or advisory boards from Amgen and Celldex. The other authors declare no conflicts of interest.
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