DNA methylation-based reclassification of olfactory neuroblastoma
Original Paper
First Online:
Abstract
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.
Notes
Acknowledgements
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.
Compliance with ethical standards
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.
Supplementary material
401_2018_1854_MOESM1_ESM.pdf (1.6 mb)
Supplementary Figure 1. Analysis of possible sub-clusters within “Core ONB”. A. Unsupervised hierarchical cluster analysis of DNA methylation data from “Core ONB” group samples (n = 42) of the initial methylation cluster analysis revealed further subdivision of the “Core ONB” DNA methylation group into two putative clusters: “Core ONB A” (n = 24) and “Core ONB B” (n = 18). B. The methylation subgroups “Core ONB A” and “Core ONB B” were compared on the level of histomorphology (H&E staining; B-C) and for expression patterns of selected immunohistochemical markers S100 (E–F), chromogranin A (H-I) and cytokeratin (K-L). The images illustrate representative staining results for each marker in both groups. Scale bar is 100 µm (only visualized in B). Bar graphs display comparisons of distributions of values for variables “Hyams grade” (D) as well as “S100” (G), “chromogranin A” (J) and “cytokeratin” (M) between tumor groups. The distributions of staining patterns between “Core ONB A” and “Core ONB B” tumors did not differ significantly for any variable tested (D, G, J and M). (PDF 1587 kb)
401_2018_1854_MOESM2_ESM.pdf (493 kb)
Supplementary Figure 2. Comparative copy number variation (CNV) profiling of DNA methylation subgroups “Core ONB A” and “Core ONB B”. A. CNV profiles of the n = 42 “Core ONB” tumor samples are displayed according to the clustering order of Supplementary Figure 1 with highlighted subdivision into putative methylation subgroups “Core ONB A” (n = 24) and “Core ONB B” (n = 18). B. The comparison of cumulated CNV profiles between “Core ONB A” and “Core ONB B” showed identical complex cytogenetic aberrations with recurrent losses of chromosomes 1, 2, 3, 4, 8, 9, 10, and 12 in both groups, further justifying their merge to an “Core ONB” DNA methylation group. (PDF 492 kb)
401_2018_1854_MOESM3_ESM.pdf (440 kb)
Supplementary Figure 3. Assessment of overall CpG methylation and CpG island methylation levels. A. The distribution of mean beta-values for all CpG probes of the methylation array was displayed as box plots for the sinonasal tumor groups “Core ONB”, Sinonasal IDH2 carcinoma”, “Sinonasal tumors, high meth.”, Other sinonasal tumors” and two additional brain tumor groups with established CIMP status (IDH-mutated astrocytoma and oligodendroglioma) to allow for comparison of “overall CpG methylation” between tumor groups. “Sinonasal IDH2 carcinoma” and “Sinonasal tumors, high meth.” demonstrated relatively high overall CpG methylation. B. The same analysis for CpG probes of the methylation array mapped to CpG islands revealed similar high levels of CpG island methylation between CIMP reference brain tumor groups and “Sinonasal IDH2 carcinoma”, prompting the assumption of CIMP status for this group in accordance with detected recurrent IDH2 mutations. C. Density plot analysis of the distribution of beta-values (“methylation ratio”) of all probes mapped to CpG islands for individual tumor samples confirmed relative hypermethylation of CpG islands for tumor samples of the “Sinonasal IDH2 carcinoma” methylation group in comparison to “Core ONB” and “Other sinonasal tumors”. An individual case from the “Sinonasal tumors, high methylation” group showed similar pronounced CpG island methylation, but no IDH1/2-mutation. (PDF 439 kb)
401_2018_1854_MOESM4_ESM.pdf (134 kb)
Supplementary Figure 4. Baseline characteristics and survival analysis of tumor groups. A. Baseline characteristics of tumor groups: The clinical attributes “Median age” and “Sex” were operationalized and compared by Kruskal–Wallis and Dunn’s post testing for “Median age” and by Chi-square testing for “Sex” between tumor groups. “Median age” was significantly different between “Core ONB” and “Sinonasal tumors, high methylation”. B. Kaplan–Meier survival analysis for tumor groups: A meaningful calculation of overall survival (OS) and group comparison was not possible due to limited clinical follow-up data. (PDF 134 kb)
401_2018_1854_MOESM5_ESM.xlsx (13 kb)
Supplementary Table 1. Overview of the institutionally diagnosed (histopathologically defined) olfactory neuroblastoma cohort. (XLSX 13 kb)
401_2018_1854_MOESM6_ESM.xlsx (22 kb)
Supplementary Table 2. List of genes included in the OncoPanel version 3 exome sequencing panel. (XLSX 22 kb)
401_2018_1854_MOESM7_ESM.xlsx (12 kb)
Supplementary Table 3. List of likely somatic non-synonymous variants with possible pathogenic and biological impact in “Core ONB” and “Sinonasal tumors with IDH2 mutation”. (XLSX 12 kb)
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