HOXD12 defines an age-related aggressive subtype of oligodendroglioma

Abstract

Oligodendroglioma, IDH-mutant and 1p/19q-codeleted has highly variable outcomes that are strongly influenced by patient age. The distribution of oligodendroglioma age is non-Gaussian and reportedly bimodal, which motivated our investigation of age-associated molecular alterations that may drive poorer outcomes. We found that elevated HOXD12 expression was associated with both older patient age and shorter survival in the TCGA (FDR < 0.01, FDR = 1e-5) and the CGGA (p = 0.03, p < 1e-3). HOXD12 gene body hypermethylation was associated with older age, higher WHO grade, and shorter survival in the TCGA (p < 1e-6, p < 0.001, p < 1e-3) and with older age and higher WHO grade in Capper et al. (p < 0.002, p = 0.014). In the TCGA, HOXD12 gene body hypermethylation and elevated expression were independently prognostic of NOTCH1 and PIK3CA mutations, loss of 15q, MYC activation, and standard histopathological features. Single-nucleus RNA and ATAC sequencing data showed that HOXD12 activity was elevated in neoplastic tissue, particularly within cycling and OPC-like cells, and was associated with a stem-like phenotype. A pan-HOX DNA methylation analysis revealed an age and survival-associated HOX-high signature that was tightly associated with HOXD12 gene body methylation. Overall, HOXD12 expression and gene body hypermethylation were associated with an older, atypically aggressive subtype of oligodendroglioma.

Keywords: ATAC-seq; DNA methylation; HOX; HOXD12; Oligodendroglioma; RNA-Seq.

Fig. 1

Investigation of the non-Gaussian age distribution in oligodendroglioma identifies HOXD12 expression as age and survival associated. a The distribution of oligodendroglioma patient age was significantly and marginally significantly non-Gaussian in six independent oligodendroglioma cohorts. b Older age was associated with worse outcomes in cohorts with survival data. c Unbiased differential gene expression analysis between older and younger patients returned 16 significantly differentially expressed genes, including several genes in the HOX, GATA, and keratin gene families. d, e Of the four differentially expressed genes that also stratified TCGA oligodendroglioma survival, HOXD12 had the strongest association with poor outcomes. f In addition to the TCGA, older oligodendroglioma patients were also enriched for HOXD12-positive expression status in the CGGA. g HOXD12-positive expression status was prognostic in the CGGA, validating the results from the TCGA. h HOXD12-positive expression status was prognostic independent of patient age and CNS WHO grade in the TCGA and independent of CNS WHO grade in the CGGA. i Age distributions of TCGA and CGGA oligodendrogliomas when partitioned by HOXD12-positive and HOXD12-negative expression status

Fig. 2

HOXD12 Hypermethylation is Associated with Age and Predictive of Poor Survival Independent of HOXD12 Expression. a All HOXD12-associated methylation probes (N = 14) were significantly positively correlated with HOXD12 expression in the TCGA. b HOXD12-associated probes were tested for age, univariate survival, and multivariate survival associations in the TCGA using specified thresholds. Only the three probes located within HOXD12’s gene body passed all such tests. c HOXD12 hypermethylation was associated with age in the TCGA and the Capper et al. cohort. d HOXD12 hypermethylated oligodendrogliomas from Capper et al. and the TCGA were enriched for higher CNS WHO grade. e HOXD12 hypermethylation was prognostic in the TCGA. f Nomogram for predicting 5- and 10-year overall survival using patient age, HOXD12 methylation level, and CNS WHO grade. g Multivariate survival analyses showed HOXD12 hypermethylation was prognostic independent of HOXD12-positive expression status, age, and CNS WHO grade. the TCGA. h Oligodendroglioma TCGA patients harboring HOXD12-positive expression status and HOXD12 gene body hypermethylation had the worst outcomes (overall survival = 2.6 years)

Fig. 3

HOXD12-Positive Expression and Gene Body Hypermethylation are Independently Prognostic of Key Histopathologic, Genomic, and Radiographic Features. a List of key tested histopathologic, genomic, and radiographic features. b HOXD12 gene body hypermethylation was linked to high Ki-67 proliferative index and the presence of mitotic figures. HOXD12-positive expression status and hypermethylation were associated with the loss of chromosome arm 15q and MYC activation. HOXD12 hypermethylation was associated with the presence of T1-post-contrast enhancement. c Representative H&E-stained sections of a HOXD12-positive and gene body hypermethylated TCGA oligodendroglioma (TCGA-DU-7018) showed increased mitotic activity, necrosis, and MVP. d MRI of the same patient showed strong contrast enhancement (blue) and extensive peritumoral edema (red) on T1-post contrast and FLAIR MRI sequences, respectively. e, f HOXD12-positive expression status and hypermethylation were independently prognostic of all tested histopathologic and genomic variables. g HOXD12-positive expression status was independently prognostic of all tested radiographic features

Fig. 4

HOXD12 activity is associated with neoplastic nuclei, proliferating cells, and an increased stem-like phenotype. a UMAP embeddings of single-nuclei RNA-seq and single-nuclei ATAC-derived single-nuclei RNA-seq from the Blanco-Carmona et al. and Wang et al. cohorts, respectively, labeled by neoplastic cell state or non-neoplastic cell type. Neoplastic nuclei are labeled as Gradient, Astro-like, OPC-like, or Cycling. b In both the Blanco-Carmona et al. and Wang et al. cohorts, single-nucleus HOXD12 RNA reads and single-nucleus ATAC HOXD12 reads were more prevalent in neoplastic tissue compared to non-neoplastic tissue. c, d Among neoplastic nuclei, HOXD12 RNA and ATAC reads were most common in OPC-like and cycling cells, which are associated with stem-like and proliferative phenotypes. e Lineage and stemness scores for oligodendroglioma tumors nuclei in the Blanco-Carmona et al. and Wang et al. cohorts. f Nucleus stemness scores are higher in tumor nuclei that have HOXD12 RNA or ATAC reads than those without in both the Blanco-Carmona et al. and Wang et al.

Fig. 5

A Pan-HOX signature led by HOXD12 is associated with poor outcomes and proliferation. a A UMAP embedding of all HOX gene body associated DNA methylation probes for oligodendroglioma patients in the TCGA, Capper et al., and GLASS datasets (N = 365) formed two clear clusters termed HOX-high and HOX-low. b The mean value of HOX gene body probes is significantly higher in the HOX-high cluster than in the HOX-low cluster. c The mean value of 98.4% of HOX gene body probes is higher in the HOX-high cluster than in the HOX-low cluster. d The HOX-high cluster is associated with patient age and tumor WHO grade in the cohorts of the TCGA and Capper et al. Differences in TCGA and Capper et al. age distributions are likely attributable to the abundance of young, WHO grade 3 oligodendrogliomas in the Capper et al. cohort. e The TCGA patients in the HOX-high cluster harbor significantly worse outcomes than those in the HOX-low cluster. f TCGA tumors in the HOX-high cluster more commonly had mitotic figures and high Ki67 index; the presence of palisading necrosis and MVP were also elevated. g In a differential gene expression analysis between the two methylation classes, HOXD12 had the highest RNA expression log fold change. h TCGA patients in the HOX-high cluster were associated with the mitotic cell cycle pathway in a gene ontology analysis. i In a differential methylation analysis, gene body probes on the HOXD locus had higher log fold change on average compared to gene body probes from other HOX loci. j In this differential methylation analysis, gene body probes associated with HOXD12 had the highest average log fold change compared to other HOX genes