Epigenetic studies in rhabdomyosarcoma

为了扩展我们在RMS肿瘤中DNA甲基化的研究，我们使用HM450阵列评估了21个FP和17个FN肿瘤的全基因组DNA甲基化模式。无监督分析证实了我们之前的发现，DNA甲基化模式和融合状态密切相关。除了FP和FN类别之间的DNA甲基化差异外，我们的DNA甲基化分析还揭示了FP集群中的两个主要子集，以及FN集群中的两个主要子集。我们的评估显示，PAX3-FOXO1和PAX7-FOXO1融合体分别在FP类别的两个亚群中富集。同样，RAS突变型和RAS野生型病例在FN类别的两个亚群中均富集其中一个。我们在另外一组21例FP和27例FN肿瘤中验证了这些发现。在发现队列中发现，有两个主要的FP病例群和两个主要的FN病例群。比较FP病例中PAX3-FOXO1和PAX7-FOXO1融合状态证实了该验证队列中基因融合亚型与DNA甲基化模式之间的显著关联。此外，比较FN病例的RAS突变状态也证实了RAS突变状态与DNA甲基化模式之间具有统计学意义的相关性。为了进一步探索PAX3-FOXO1和PAX7-FOXO1亚型之间的DNA甲基化差异，我们将发现和验证队列结合起来。通过监督生物信息学方法比较这两个融合亚群显示，与pax7 - foxo1阳性肿瘤相比，PAX3-FOXO1-中高甲基化探针比低甲基化探针多17倍。与pax7 - fox01阳性肿瘤相比，pax3 - fox01 -中启动子超甲基化显著增加的一个值得注意的基因是CDKN1C，这是一种推定的肿瘤抑制基因，定位于RMS和其他肿瘤中11p15.5等位基因缺失的区域。RNA表达研究表明，与pax7 - foxo1阳性肿瘤相比，PAX3-FOXO1-中CDKN1C mRNA的表达明显减少，随后的免疫组织化学研究证实，PAX3-FOXO1-和pax7 - foxo1阳性肿瘤中CDKN1C蛋白的表达存在显著差异。为了进一步研究FN类别内的DNA甲基化差异，我们结合了来自发现和验证队列的FN RMS肿瘤。对这些FN病例DNA甲基化的监督分析显示，与RAS野生型FN RMS肿瘤相比，RAS突变体的CpG高甲基化位点比低甲基化位点多1.5倍。利用这些差异甲基化的CpG位点，随后对FN组合队列和正常骨骼肌样本进行聚类分析，再次确定了两个主要亚群。正常肌肉样本和大多数RAS野生型病例聚集在一个亚组中，所有RAS突变病例以及额外的RAS野生型病例聚集在第二个亚组中。对后一个RAS突变富集亚组的DNA甲基化模式的检查显示了两个较小的簇，一个簇由大多数RAS突变肿瘤组成，没有RAS野生型肿瘤，另一个簇由RAS突变和RAS野生型肿瘤混合组成。进一步的分析表明，在RAS野生型肿瘤中，另外两个RAS通路基因（NF1和SOS1）的突变频率很高，从而支持了两个主要FN亚群之间的DNA甲基化差异与涉及RAS信号通路的更大突变集相关的假设。基于这一前提，一项比较具有突变RAS通路基因的FN肿瘤与野生型RAS通路基因的监督方法发现，与野生型RAS通路相比，突变RAS通路的FN RMS肿瘤中低甲基化的CpG位点是高甲基化的16倍。在突变型RAS通路肿瘤中，启动子低甲基化显著增加的一个值得注意的基因是ALDH1A3，该基因先前被报道为胚胎性RMS中癌症干细胞的潜在标记物。RNA表达研究表明，与野生型肿瘤相比，RAS通路突变体中ALDH1A3 mRNA的表达明显增加。为了确定是否有RMS模型系统再现了人类原发RMS肿瘤中发现的DNA甲基化模式，我们比较了原发RMS肿瘤与长期RMS细胞系、来自这些长期RMS细胞系的异种移植物（CDXs）和患者来源的异种移植物（PDXs）的全基因组甲基化模式。虽然分层聚类再次显示了FP簇和FN簇，但这些主要簇中的每一个都被细分为两个较小的簇，一个包含几乎所有的细胞系和cdx，另一个包含几乎所有的pdx和原发肿瘤。此外，热图显示，绝大多数分析的CpG位点在细胞系和cdx中都是高甲基化的，而在原发肿瘤和pdx中发现的低甲基化和高甲基化分布更广泛。两个pdx与细胞系和cdx聚集的发现表明pdx可能在某些未知的情况下失去天然甲基化模式。进一步的研究表明，pdx与原发肿瘤之间的总体甲基化水平无统计学差异，而细胞系和cdx的总体甲基化水平明显较高，这证实了pdx是研究RMS肿瘤中DNA甲基化模式生物学意义的最佳实验模型。为了扩展这一结论，我们评估了PDX肿瘤短期培养（1-2个月）时DNA甲基化模式的稳定性。一项比较RMS PDX肿瘤和短期培养与一组长期RMS细胞系的无监督分析表明，所有FN短期培养和大多数FP短期培养在短期培养期间保持了整体DNA甲基化模式。因此，这些发现提供了在将这些细胞返回体内环境之前使用短期培养对pdx进行实验操作的可能性。

To extend our studies of DNA methylation in RMS tumors, we used the HM450 array to assess genome-wide DNA methylation patterns in a discovery cohort of 21 FP and 17 FN tumors. Unsupervised analyses confirmed our previous finding of a close association of DNA methylation pattern and fusion status. In addition to the DNA methylation differences between the FP and FN categories, our DNA methylation analysis also revealed two major subsets within the FP cluster, and two major subsets within the FN cluster. Our evaluation revealed that the PAX3-FOXO1 and PAX7-FOXO1 fusions were each enriched in one of the two subsets in the FP category. Similarly, RAS mutant and RAS wild-type cases were each enriched in one of the two subsets in the FN category. We validated these findings in an additional cohort of 21 FP and 27 FN tumors. As was found in the discovery cohort, there were two major clusters of FP cases and two major clusters of FN cases. Comparison of the PAX3-FOXO1 and PAX7-FOXO1 fusion status in the FP cases confirmed a significant association between the gene fusion subtype and DNA methylation pattern in this validation cohort. In addition, comparison of the RAS mutation status in the FN cases also confirmed a statistically significant association between RAS mutation status and DNA methylation pattern. To further explore DNA methylation differences between the PAX3-FOXO1 and PAX7-FOXO1 subtypes, we combined the discovery and validation cohorts. Comparison of these two fusion subsets by a supervised bioinformatic approach revealed greater than 17-fold more hypermethylated than hypomethylated probes in PAX3-FOXO1- compared with PAX7-FOXO1-positive tumors. One notable gene with significantly more promoter hypermethylation in PAX3-FOXO1- than PAX7-FOXO1-positive tumors is CDKN1C, a putative tumor suppressor gene that is localized within a region of 11p15.5 allelic loss in RMS and other tumors. RNA expression studies demonstrated significantly less expression of CDKN1C mRNA in PAX3-FOXO1- compared to PAX7-FOXO1-positive tumors, and subsequent immunohistochemistry studies on confirmed a significant difference in CDKN1C protein expression between PAX3-FOXO1- and PAX7-FOXO1-positive tumors. To further investigate DNA methylation differences within the FN category, we combined FN RMS tumors from the discovery and validation cohorts. A supervised analysis of DNA methylation in these FN cases revealed 1.5-fold more hypermethylated than hypomethylated CpG sites in RAS mutant compared to RAS wild-type FN RMS tumors. Using this collection of differentially methylated CpG sites, a subsequent clustering analysis of the combined FN cohorts and normal skeletal muscle samples again identified two main subgroups. The normal muscle samples and most RAS wild-type cases clustered in one subgroup and all RAS mutant cases along with additional RAS wild-type cases clustered in the second subgroup. Examination of the DNA methylation pattern within the latter RAS mutant-enriched subgroup revealed two smaller clusters, one cluster consisting of most RAS mutant tumors and no RAS wild-type tumors, and a second cluster consisting of a mixture of RAS mutant and RAS wild-type tumors. Further analysis demonstrated that mutations of two additional RAS pathway genes (NF1 and SOS1) were present at a high frequency in the RAS wild-type tumors in this latter subgroup, thus supporting the hypothesis the DNA methylation differences between the two main FN subsets are associated with a larger set of mutations involving the RAS signaling pathway. Based on this premise, a supervised approach comparing FN tumors with mutant RAS pathway genes versus wild-type RAS pathway genes identified 16-fold more hypomethylated than hypermethylated CpG sites in FN RMS tumors with a mutant RAS pathway compared to a wild-type RAS pathway. One notable gene with significantly more promoter hypomethylation in mutant RAS pathway tumors is ALDH1A3, which was previously reported as a potential marker for cancer stem cells in embryonal RMS. RNA expression studies demonstrated significantly more ALDH1A3 mRNA expression in RAS pathway mutant versus wild-type tumors. To determine which if any RMS model systems recapitulate the DNA methylation patterns found in human primary RMS tumors, we compared genome-wide methylation patterns in primary RMS tumors to long-term RMS cell lines, xenografts derived from these long-term cell lines (CDXs) and patient-derived xenografts (PDXs). Though hierarchical clustering again demonstrates a FP cluster and a FN cluster, each of these main clusters is subdivided into two smaller clusters, one containing nearly all cell lines and CDXs and one containing nearly all PDXs and primary tumors. Furthermore, the heat maps reveal that the vast majority of analyzed CpG sites are hypermethylated in cell lines and CDXs in contrast to the wider distribution of hypo- and hypermethylation found in primary tumors and PDXs. The finding of two PDXs clustering with cell lines and CDXs suggests that PDXs may lose the native methylation pattern under some unknown circumstances. Further studies showing no statistical differences in overall methylation levels between PDXs and primary tumors in contrast to the significantly higher overall methylation levels in cell lines and CDXs confirms that PDXs are the optimal experimental model to study the biological significance of DNA methylation patterns in RMS tumors. To extend this conclusion, we assessed the stability of the DNA methylation patterns when short-term cultures (1-2 months) were established from the PDX tumors. An unsupervised analysis comparing RMS PDX tumors and short-term cultures with a set of long-term RMS cell lines demonstrated that the overall DNA methylation pattern was maintained during short-term culture for all FN short term cultures and most FP short term cultures. These findings thus provide the possibility of using a short-term culture to perform experimental manipulations of PDXs before returning these cells to the in vivo environment.