DNA methylation-based classification of sinonasal undifferentiated carcinoma

Abstract

Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignancy harboring IDH2 R172 mutations in >80% cases. We explored the potential of genome-wide DNA methylation profiling to elucidate tumor biology and improve the diagnosis of sinonasal undifferentiated carcinoma and its histologic mimics. Forty-two cases, including sinonasal undifferentiated, large cell neuroendocrine, small cell neuroendocrine, and SMARCB1-deficient carcinomas and olfactory neuroblastoma, were profiled by Illumina Infinium Methylation EPIC array interrogating >850,000 CpG sites. The data were analyzed using a custom bioinformatics pipeline. IDH2 mutation status was determined by the targeted exome sequencing (MSK-IMPACT^TM) in most cases. H3K27 methylation level was assessed by the immunohistochemistry-based H-score. DNA methylation-based semi-supervised hierarchical clustering analysis segregated IDH2 mutants, mostly sinonasal undifferentiated (n = 10) and large cell neuroendocrine carcinomas (n = 4), from other sinonasal tumors, and formed a single cluster irrespective of the histologic type. t-distributed stochastic neighbor embedding dimensionality reduction analysis showed no overlap between IDH2 mutants, SMARCB1-deficient carcinoma and olfactory neuroblastoma. IDH2 mutants demonstrated a global methylation phenotype and an increase in repressive trimethylation of H3K27 in comparison to IDH2 wild-type tumors (p < 0.001). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed no difference in pathway activation between IDH2-mutated sinonasal undifferentiated and large cell neuroendocrine carcinomas. In comparison to SMARCB1-deficient, IDH2-mutated carcinomas were associated with better disease-free survival (p = 0.034) and lower propensity for lung metastasis (p = 0.002). ARID1A mutations were common in small cell neuroendocrine carcinoma but not among IDH2 mutants (3/3 versus 0/18 and p < 0.001). IDH2 mutations in sinonasal carcinomas induce a hypermethylator phenotype and define a molecular subgroup of tumors arising in this location. IDH2-mutated sinonasal undifferentiated carcinoma and large cell neuroendocrine carcinoma likely represent a phenotypic spectrum of the same entity, which is distinct from small cell neuroendocrine and SMARCB1-deficient sinonasal carcinomas. DNA methylation-based analysis of the sinonasal tumors has potential to improve the diagnostic accuracy and classification of tumors arising in this location.

Figure 1.

DNA methylation-based classification of the sinonasal tumors. Semi-supervised hierarchical clustering analysis of a histologically diverse cohort of the sinonasal tumors (n=39) based on the top 10,000most variably methylated probes. IDH2 R172 mutant tumors formed a single cluster of IDH2-mutated carcinomas including sinonasal undifferentiated carcinoma and large cell neuroendocrine carcinoma. The other two distinct clusters are seen in olfactory neuroblastoma (n=4) and SMARCB1-deficient sinonasal carcinomas (n=5). Despite the small sample size, moderately-differentiated intestinal type adenocarcinoma (n=2) also showed a distinct methylation fingerprint and separation from other tumor categories. Dotted line separates carcinomas with neuroendocrine differentiation from poorly-differentiated carcinoma with focal glandular/acinar differentiation. A single case of a high-grade non-intestinal type adenocarcinoma (SN_18, red arrow) clustered with poorly-differentiated carcinoma with neuroendocrine and glandular differentiation (A). High-grade non-intestinal type adenocarcinoma (SN_18) displayed an entirely glandular growth pattern (H&E, top) and neuroendocrine differentiation was supported by positive chromogranin immunohistochemistry (200x magnification, B). t-SNE analysis of the sinonasal tumors was consistent with the DNA methylation-based clustering analysis as depicted in the heatmap in Figure 1A. IDH2 mutants are represented as the largest cluster in the lower right. Each tumor group is color-coded according to legend in Figure 1A (C). Abbreviations: PD non-ITAC= poorly-differentiated carcinoma non-intestinal type adenocarcinoma, SNUC=sinonasal undifferentiated carcinoma, LCNEC= large cell neuroendocrine carcinoma, ONB=olfactory neuroblastoma, MD ITAC=moderately-differentiated intestinal type adenocarcinoma, SMARCB1-def=SMARCB1-deficient sinonasal carcinoma, HG non-ITAC=high-grade non-intestinal type adenocarcinoma, PDCA-NE-GL=poorly-differentiated carcinoma with neuroendocrine and glandular differentiation, SCNEC-SQCC=combined small cell neuroendocrine carcinoma and squamous cell carcinoma, SCNEC=small cell neuroendocrine carcinoma, PDCA-GL=poorly-differentiated carcinoma with focal glandular/acinar differentiation.

Figure 2.

IDH2 R172 mutations in sinonasal carcinomas induce a hypermethylator phenotype and increase histone H3K27 trimethylation. Heatmap of semi-supervised hierarchical clustering analysis of the top 10,000 most variably methylated probes in IDH2 wild-type and IDH2 R172 mutated sinonasal tumors depicts a clear segregation of the two cancer methylomes and supports a global methylation in IDH2 R172 mutants (A). Scattered plots depict H-scores of H3K27me3 immunohistochemistry, which was significantly higher in IDH2 R172 mutants consistent with the induced histone H3K27 trimethylation (B). IDH2 wild-type SNUC (SN_07, H&E, left) was negative for mutant IDH2 (11C8B1) immunostain and showed patchy positive labeling for H3K27me3 (H-score 150). In contrast, IDH2 R172S mutant SNUC (SN_50, H&E, left) was mutant IDH2 immunopositive and showed an increased labeling for H3K27me3 (H-score 260, 200x magnification, C). Abbreviations: SNUC=sinonasal undifferentiated carcinoma, WT=wild-type.

Figure 3.

IDH2 mutant large cell neuroendocrine carcinoma is genetically similar to the IDH2 mutant SNUC and is distinct from small cell neuroendocrine carcinoma, which harbor recurrent ARID1A mutations. Oncoprint summarizes all oncogenic and select recurrent somatic alterations in large cell neuroendocrine carcinoma and other carcinomas with neuroendocrine features detected by MSK-IMPACT™. Oncogenic potential of the genetic alterations is defined by the OncoKB ([26], www.cBioPortal.org). SNUC cases previously published are not included (A). IDH2 R172S mutated large cell neuroendocrine carcinoma (SN_49, H&E, top) was immunopositive for IDH2 (11C8B1) and INSM1 (H-score 100, 200x magnification), (B). INSM1 immunoexpression defined by H-score was distinct among sinonasal carcinomas with neuroendocrine features showing the lowest expression in SNUC and the highest expression in small cell neuroendocrine carcinoma. Error bars represent standard deviation (C). Abbreviations: SNUC=sinonasal undifferentiated carcinoma, PDCA-NE-GL=poorly-differentiated carcinoma with neuroendocrine and glandular differentiation, LCNEC= large cell neuroendocrine carcinoma, SCNEC=small cell neuroendocrine carcinoma.

Figure 4.

Outcome of the sinonasal carcinomas based on IDH2/SMARCB1 mutation status. IDH2 mutated sinonasal carcinomas show non-significant trend in better disease-specific survival in comparison to all IDH2 wild-type carcinomas (A), IDH2/SMARCB1 wild-type carcinomas (B), and SMARCB1-deficient sinonasal carcinomas (C), but a significantly better disease-free survival than SMARCB1-deficient sinonasal carcinomas (D), (Log Rank test). Abbreviations: DSS=disease-specific survival, DFS=disease-free survival, WT=wild-type, SMARCB1-def= SMARCB1-deficient sinonasal carcinoma.