H3 K27M-altered diffuse midline gliomas (DMGs) are highly malignant tumours that arise in the midline structures of the CNS. Most DMGs carry an H3 K27M-mutation in one of the genes encoding for histone H3. Recent studies suggested that epigenetic subgroups of DMGs can be distinguished based on alterations in the MAPK-signalling pathway, tumour localisation, mutant H3-gene, or overall survival (OS). However, as these parameters were studied individually, it is unclear how they collectively influence survival. Hence, we analysed dependencies between different parameters, to define novel epigenetic, clinically meaningful subgroups of DMGs. We collected a multifaceted cohort of 149 H3 K27M-mutant DMGs, also incorporating data of published cases. DMGs were included in the study if they could be clearly allocated to the spinal cord (n = 31; one patient with an additional sellar tumour), medulla (n = 20), pons (n = 64) or thalamus (n = 33), irrespective of further known characteristics. We then performed global genome-wide DNA methylation profiling and, for a subset, DNA sequencing and survival analyses. Unsupervised hierarchical clustering of DNA methylation data indicated two clusters of DMGs, i.e. subtypes DMG-A and DMG-B. These subtypes differed in mutational spectrum, tumour localisation, age at diagnosis and overall survival. DMG-A was enriched for DMGs with MAPK-mutations, medullary localisation and adult age. 13% of DMG-A had a methylated MGMT promoter. Contrarily, DMG-B was enriched for cases with TP53-mutations, PDGFRA-amplifications, pontine localisation and paediatric patients. In univariate analyses, the features enriched in DMG-B were associated with a poorer survival. However, all significant parameters tested were dependent on the cluster attribution, which had the largest effect on survival: DMG-A had a significantly better survival compared to DMG-B (p < 0.001). Hence, the subtype attribution based on two methylation clusters can be used to predict survival as it integrates different molecular and clinical parameters.

H3 K27M 改变的弥漫性中线神经胶质瘤 (DMG) 是产生于中枢神经系统中线结构的高度恶性肿瘤。大多数 DMG 在编码组蛋白 H3 的基因之一中携带 H3 K27M 突变。最近的研究表明，DMG 的表观遗传亚组可以根据 MA​​PK 信号通路、肿瘤定位、突变 H3 基因或总生存 (OS) 的变化来区分。然而，由于这些参数是单独研究的，目前尚不清楚它们如何共同影响生存。因此，我们分析了不同参数之间的依赖性，以定义新的表观遗传、有临床意义的 DMG 亚组。我们收集了 149 个 H3 K27M 突变 DMG 的多方面队列，还纳入了已发表病例的数据。如果 DMG 可以明确分配到脊髓（ n = 31；一名患有额外鞍区肿瘤的患者）、延髓（ n = 20）、脑桥（ n = 64）或丘脑（ n = 33），则将其纳入研究中，无论进一步已知的特征如何。然后，我们进行了全球全基因组 DNA 甲基化分析，并针对子集进行了 DNA 测序和生存分析。 DNA 甲基化数据的无监督分层聚类表明有两个 DMG 簇，即亚型 DMG-A 和 DMG-B。这些亚型在突变谱、肿瘤定位、诊断年龄和总体生存率方面存在差异。 DMG-A 富含具有 MAPK 突变髓质定位和成年年龄的 DMG。 13% 的 DMG-A 具有甲基化MGMT启动子。相反，DMG-B 对于TP53突变、 PDGFRA扩增、脑桥定位和儿科患者的病例有所丰富。在单变量分析中，DMG-B 丰富的特征与较差的生存率相关。 然而，测试的所有重要参数均取决于聚类归因，这对生存影响最大：与 DMG-B 相比，DMG-A 的生存率明显更高 ( p < 0.001)。因此，基于两个甲基化簇的亚型归因可用于预测生存，因为它整合了不同的分子和临床参数。