Genome-wide DNA methylation profiling of recurrent and non-recurrent chordomas

Chordomas are an aggressive rare type of malignant bone tumors arising from the remnant of the notochord. Chordomas occur mainly in vertebral bones and account for 1–4% of malignant bone tumors. Management and treatment of chordomas are difficult as they are resistant to conventional chemotherapy; therefore, they are mainly treated with surgery and radiation therapy. In this study, we performed DNA methylation profiling of 26 chordomas and normal nucleus pulposus samples plus UCH-1 chordoma cell line using the Illumina Infinium HumanMethylation450 BeadChips. Combined bisulfite restriction analysis and bisulfite sequencing was used to confirm the methylation data. Gene expression was analyzed using RT-PCR before and after 5-aza-2’-deoxycytidine (5-azaDC) treatment of chordoma cell lines. Analysis of the HumanMethylation450 BeadChip data led to the identification of 8,819 loci (2.9%) that were significantly differentially methylated (&gt;0.2 average β-value difference) between chordomas and nucleus pulposus samples (adjusted <i>P &lt;</i> 0.05). Among these, 5,868 probes (66.5%) were hypomethylated, compared to 2,951 (33.5%) loci that were hypermethylated in chordomas compared to controls. From the 2,951 differentially hypermethylated probes, 33.3% were localized in the promoter region (982 probes) and, among these, 104 probes showed cancer-specific hypermethylation. Ingenuity Pathway Analysis indicates that the cancer-specific differentially methylated loci are involved in various networks including cancer disease, nervous system development and function, cell death and survival, cellular growth, cellular development, and proliferation. Furthermore, we identified a subset of probes that were differentially methylated between recurrent and non-recurrent chordomas. BeadChip methylation data was confirmed for these genes and gene expression was shown to be upregulated in methylated chordoma cell lines after treatment with 5-azaDC. Understanding epigenetic changes in chordomas may provide insights into chordoma tumorigenesis and development of epigenetic biomarkers.

脊索瘤（Chordomas）是一类侵袭性罕见恶性骨肿瘤，起源于脊索残留组织。脊索瘤主要发生于椎骨，占恶性骨肿瘤的1%~4%。由于脊索瘤对常规化疗存在耐药性，其诊疗难度较大，临床主要采用手术与放射治疗手段。本研究利用Illumina Infinium HumanMethylation450 BeadChips芯片，对26例脊索瘤组织、正常髓核（nucleus pulposus）样本以及UCH-1脊索瘤细胞系进行了DNA甲基化谱（DNA methylation profiling）分析。本研究采用联合亚硫酸氢盐限制性分析（Combined bisulfite restriction analysis）与亚硫酸氢盐测序（bisulfite sequencing）对甲基化数据进行了验证。针对脊索瘤细胞系，本研究在施加5-aza-2’-脱氧胞苷（5-azaDC）处理前后，利用逆转录聚合酶链式反应（RT-PCR）分析了基因表达水平。对HumanMethylation450 BeadChips芯片数据的分析显示，脊索瘤与正常髓核样本间存在8819个显著差异甲基化基因座（占比2.9%），其平均β值差异大于0.2，校正后P值<0.05。其中5868个探针（66.5%）呈低甲基化状态，剩余2951个基因座（33.5%）在脊索瘤中呈高甲基化状态。在这2951个差异高甲基化探针中，33.3%（共982个探针）定位于启动子区域，其中104个探针呈现肿瘤特异性高甲基化特征。Ingenuity Pathway Analysis（IPA）通路分析结果显示，肿瘤特异性差异甲基化基因座参与了多个调控网络，包括肿瘤疾病、神经系统发育与功能、细胞死亡与存活、细胞生长、细胞发育及增殖等过程。此外，本研究还鉴定出一组在复发与未复发脊索瘤间存在差异甲基化的探针。针对上述基因的芯片甲基化数据得到了验证，且在施加5-azaDC处理后，甲基化脊索瘤细胞系中的基因表达呈上调趋势。阐明脊索瘤的表观遗传改变，可为揭示脊索瘤的肿瘤发生机制以及开发表观遗传生物标志物提供理论依据。