Purpose

Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival.

Methods

FFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976–1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as “Early metastasis” (metastases and death within 2–5 years after enucleation, n = 8), “Late metastasis” (metastases and death within 9–21 years after enucleation, n = 7) and “No metastasis” (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR).

Results

DNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups (“Early”, “Late”, “No”) nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in “Subset Early metastasis” vs “Subset No metastasis” and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in “Subset Early metastasis vs “Subset No metastasis” and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM.

Conclusions

DNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.

中文翻译：

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福尔马林固定石蜡包埋的葡萄膜黑色素瘤标本的整合差异DNA甲基化和基因表达可鉴定与早期转移和不良预后相关的基因

目的

葡萄膜黑色素瘤（UM）是一种侵袭性恶性肿瘤，其中近50％的患者死于转移性疾病。DNA甲基化异常被认为是致癌作用中的重要表观基因组事件。福尔马林固定石蜡包埋（FFPE）样品代表了肿瘤组织的重要来源，并且最新技术已使这些样品能够用于全基因组DNA甲基化分析中。我们的目的是研究与组织病理学分类和生存数据有关的差异DNA甲基化。此外，我们试图鉴定可能与转移性疾病和不良生存有关的基因的异常DNA甲基化。

方法

包括从1976年至1989年进行眼摘除术的UM患者（n = 23）的FFPE样本。DNA甲基化使用Illumina Infinium HumanMethylation450阵列进行评估并结合组织病理学数据，挪威癌症登记处（已注册的UM转移）和挪威死亡原因登记处（时间和死亡原因）数据。差异DNA甲基化模式对比组织学分类，生存数据和聚类特性进行了调查。生存组定义为“早期转移”（摘除后2-5年内转移和死亡，n = 8），“晚期转移”（摘除后9-21年内转移和死亡，n = 7）和“无转移” ”（去核后≥18年未检测到转移，n = 8）。根据初步的多维标度（MDS）图，组织病理学分类，3号染色体状态，生存状态和聚类特性，选择了一部分样本。“子集早期转移”（n = 4）与“子集无转移”（n = 4）。在R统计软件中进行了生物信息学分析。评估了各种比较中的差异甲基化位置（DMP）和差异甲基化区域（DMR）。通过微阵列分析和定量逆转录聚合酶链反应（qRT-PCR）确定相关亚组的基因表达。

结果

DNA甲基化分析确定了2个簇，这些簇根据3号染色体的状态将样品分开。聚类1由具有3号染色体二体性（D3）的样本（n = 5）组成，聚类2由具有3号染色体二体性（M3）的样本（n = 15）组成。在M3 vs D3中鉴定出了1212个DMR和9386个DMP。根据我们预定的生存组（“早期”，“晚期”，“否”）或组织病理学分类（上皮样，混合，纺锤体），没有形成清晰的簇。我们确定了两个样本子集（n = 8）之间DNA甲基化的显着变化（βFC≥0.2，调整后的p <0.05）。“亚群早期转移”（n = 4）和“亚群无转移”（n = 4）确定了348个DMP和36个DMR，并且通过微阵列的差异基因表达显示14个DMP和2个DMR对应于基因表达的变化（FC） ≥1.5，p <0.05）。RNF13，Z NF217和HYAL1在“亚组早期转移”与“亚组无转移”中被高度甲基化并下调，可能是潜在的肿瘤抑制因子。TMEM200C，RGS10，ADAM12和PAM在“亚组早期转移vs”亚组无转移中被低甲基化和上调，并且可能是潜在的癌基因，因此是UM早期转移和不良预后的标志。

结论

DNA甲基化谱分析显示样品根据3号染色体状态的差异聚类：聚类1（D3）和聚类2（M3）。综合差异DNA甲基化和两个样本子集的基因表达确定了与早期转移和不良预后相关的基因。RNF13，ZNF217和HYAL1是甲基化程度高的候选肿瘤抑制剂，而TMEM200C，RGS10，ADAM12和PAM是甲基化程度低的候选肿瘤基因，与早期转移有关。UM FFPE样品代表了甲基化组研究的宝贵资源，可以进行长期随访。