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Introduction

Thymic epithelial neoplasms are uncommon thymic neoplasms that arise from epithelial cells of the thymus; they are the most frequent type of anterior mediastinal tumors in adults (1). Thymic epithelial neoplasms are divided into thymomas, thymic carcinomas and thymic neuroendocrine tumors (2). In clinical practice, different classifications have been proposed and used. The latest histological classification released in 2015 by the World Health Organization (WHO) suggested two main thymoma types; types A and B. More specifically, thymomas can be classified into five histological types (A, AB, B1, B2 and B3) based on the morphology of epithelial cells and the lymphocyte to epithelial cell ratio (3). Traditionally, the most commonly used classifications are the Masaoka and Masaoka-Koga staging systems (4). Thymomas are known to be associated with a variety of immunological diseases (5). Myasthenia gravis (MG) is the most frequent syndrome accompanying thymomas and occurs in 15–20% thymoma patients (6,7).

The etiology and molecular pathogenesis of thymoma has not yet been elucidated. There are various mechanisms by which the pathogenesis of thymoma occurs, including epigenetic alterations, which are a hallmark of cancer due to their role in carcinogenesis initiation (8,9). Recent evidence has indicated that miR-145-5p is an important epigenetic regulation factor that may be involved in tumor progression and treatment response in thymic epithelial tumors (10). A set of prognostic and subtype-specific potential miRNAs have been identified in thymoma (11). A large miRNA cluster on chr19q13.42 was revealed as a transcriptional hallmark of type A and AB thymomas (12). Previous research has also provided evidence that DNA hypermethylation in promoter regions and global DNA hypomethylation serve an important role in the tumorigenesis of thymic epithelial tumors (13,14). Specific DNA methylation aberrations, which are associated with different thymic epithelial tumor histotypes or thymomas accompanied by MG, have previously been identified (15,16). However, the function of aberrant DNA methylation in thymomas is less clear. The specific DNA methylation aberrations in thymoma vs. control, type A vs. type B thymomas and MG- vs. non-MG-thymomas remains largely unknown.

Therefore, an array-based approach was used to uncover genome-wide DNA methylation profiles in fresh frozen thymoma and adjacent normal tissues in the present study. Following differential methylation analysis, a set of differentially methylated CpGs (DMCs) was identified. Furthermore, functional annotation analysis was performed on the corresponding differentially methylated genes. The present study may provide valuable insights into the epigenetic regulation of DNA methylation in thymoma and different thymoma subtypes.

Materials and methods

Study participants

For the genome-wide methylation analysis, eight patients with thymoma or atypical thymic carcinoid undergoing sternotomy were recruited at Peking Union Medical College Hospital (Beijing, China) between October 2014 and July 2015. The patients' age range was 26–80 years (mean age, 49 years; 1:1 male:female). WHO histological subtypes were recorded as follows: Atypical type A (n=1), type A (n=1), type AB (n=1), type B1 (n=1), type B2 (n=1), type B3 (n=2) and atypical thymic carcinoid (n=1). Atypical thymic carcinoid is an extremely rare thymic neuroendocrine tumor derived from the neuroendocrine system (17). In total, 16 paired surgically resected tumor and adjacent normal tissue samples were collected and stored at −80°C until DNA extraction. Written informed consent was provided by all participants. The present study was approved by the Ethics Committee of Peking Union Medical College Hospital (Beijing, China) and was performed in compliance with the Declaration of Helsinki. Patient demographics and clinical characteristics are presented in Table I.

Table I. Clinicopathological variables of thymoma patients used for methylation analysis.

Table I.

Clinicopathological variables of thymoma patients used for methylation analysis.

ID	Sex	Age	Myasthenia gravis	WHO histological classification	Masaoka stage	Adjuvant treatment
1	Male	36	No	Atypical type A	I	No
2	Female	52	Yes	Type A	I	No
3	Male	48	No	Type AB	2B	No
4	Female	47	No	Type B1	I	No
5	Male	33	Yes	Type B2	I	No
6	Male	30	Yes	Type B3	−	No
7	Male	80	No	Type B3	G3	Yes
8	Female	26	No	Atypical thymic carcinoid	G3	No

[i] T, tumor; N, adjacent normal tissues.

DNA isolation and bisulfite treatment

Genomic DNA was obtained from both the surgically resected tumor and adjacent normal tissue using the TIANamp Genomic DNA kit (Tiangen Biotech, Co., Ltd., Beijing, China) according to the manufacturer's instructions. The concentration of extracted DNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The quality of extracted DNA was checked in 0.8% agarose gel electrophoresis. Only samples with a purity of 1.8–2.05 were selected for the microarray study. In total, four DNA samples were excluded from further analysis due to poor quality. Finally, DNA samples from six tumors (1T, 2T, 4T, 6T, 7T and 8T) and six normal tissues (2N, 3N, 4N, 5N, 6N and 7N) were maintained for downstream analysis. Genomic DNA (200–500 ng) from each sample was chemically modified and bisulfite-converted using the EZ DNA Methylation kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer's instructions, which converts unmethylated cytosines into uracil. Methylated cytosines remained unchanged during treatment.

Illumina 850K methylation microarray

Following bisulfite treatment, the DNA methylation status of case and control subjects was assayed using the recently developed Infinium MethylationEPIC BeadChip microarray from Illumina, Inc. (San Diego, CA, USA) according to the manufacturer's instructions, which measured the methylation status of 853,307 CpG sites distributed over the whole genome. The image intensities were extracted using the Illumina iScan system (Illumina, Inc.) and quality-controlled using RnBeads (version 3.5) (18) in R (www.r-project.org/).

Microarray data preprocessing

The Illumina iScan system was used for image and data analysis of the BeadChips. The raw (.idat) files obtained from the methylation microarray were then transferred to the RnBeads software and a quality check of the raw data of each probe analysis was performed, including background correction, adjustment of probe type differences and probe exclusion. The single nucleotide polymorphism-associated probes were filtered, while those corresponding to the sex chromosomes were not. To avoid batch effect, all samples were processed together. Following these intra-sample normalization procedures, DNA methylation was scored as a β value, ranging from 0 (no methylation) to 1 (100% methylation). Unsupervised hierarchical clustering was then performed with Euclidian distance and complete linkage.

Searching for DMCs

To identify DMCs, the average β value was compared between the groups of interest (thymoma tumor tissues vs. adjacent normal tissue; type A vs. type B thymoma; MG- vs. non-MG-thymoma). Briefly, the CpGs were considered DMCs at an average DNA methylation differences (Δβ) between two groups of >0.2. The DMCs for each comparison were determined, following which DMCs were annotated with respect to defined CpG islands (CGIs), shores, shelves and relative to RefSeq genes 3′ untranslated region (UTR), gene body, exon 1, 5′UTR, transcription start site (TSS)1500, TSS200 and intergenic, according to the Infinium MethylationEPIC Microarray annotation file (www.illumina.com).

Expression data of thymomas

Gene expression data of thymomas (GSE29695) (19) were downloaded from the public data repository of Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/gds) (20). These expression data were obtained from 36 patients with thymomas, divided into two main groups: Type A (1 type A and 9 type AB) and B thymomas (20 type B1-B2 and 6 type B3). Genes with a poor signal quality across a maximal number of arrays were removed. As a result, 6,486 genes were found to have signals significantly above background. The raw data were quantile-normalized and log2-transformed prior to statistical analysis. Differential expression analysis was performed between type A and B thymomas. Statistical analysis was performed using Wilcoxon rank-sum tests. P-values were further corrected for false discovery by applying the Benjamini-Hochberg procedure. The genes were defined as differentially expressed genes (DEGs) at P<0.05.

Functional annotation analysis of differentially methylated genes

Once DMCs were annotated to the genes, those that showed significant differences in DNA methylation between different groups underwent functional annotation analysis, using the online GeneCodis3 tool (genecodis.cnb.csic.es/analysis) (21). Gene Ontology (GO) enrichment analysis was performed to classify the differentially methylated genes into categories of cellular component, biological process and molecular function (22). In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to detect the potential pathways of the differentially methylated genes (23).

Receiver operating characteristic (ROC) analysis

In order to assess the diagnostic value of candidate DNA methylation markers, ROC analysis was performed using the pROC package (24) in R. The area under the curve (AUC) was then calculated to assess the performance of each DNA methylation marker.

Results

Illumina 850K methylation microarray of subjects

In total, eight paired tumor samples and corresponding adjacent normal tissues were evaluated. The qualified DNA of six tumor (1T, 2T, 4T, 6T, 7T and 8T) and six normal (2N, 3N, 4N, 5N, 6N and 7N) tissues was used for genome-wide DNA methylation profiling using the Illumina 850K methylation microarray. A Manhattan plot was produced to display P-values that were generated by the-log10 (P-value) function for each CpG site (Fig. 1A). In addition, a volcano plot of CpG sites was constructed using Δβ and P-value, representing the methylation difference between tumors and controls by magnitude of change and statistical significance (Fig. 1B).

Figure 1. Epigenome-wide results for six thymoma and six control samples. (A) Manhattan plot of CpG site. Each point represents the observed -log10 P-value at a CpG site. (B) Volcano plot of CpG site. The plot was produced by-log10 (P-value) against ∆β, representing the methylation difference between tumor and control samples.

Identification of DMCs between tumor and control samples

In order to analyze DNA methylation differences between tumor and control samples, the average β values between the groups were examined. A total of 19,118 probes were found to be significantly differentially methylated (Δβ>0.2 and adjusted P<0.05), including 119 hypermethylated and 18,999 hypomethylated DMCs. Overall, there was a general decrease in tumor methylation, compared with the control. An unsupervised hierarchical clustering was performed and a heat map of the top 1,000 hypomethylated DMCs and all 119 hypermethylated DMCs was produced (Fig. 2). The heat map showed two robust DNA methylation clusters: One encompassing all tumors and another containing all controls. This indicated that tumors and controls had different DNA methylation characters and patterns.

Figure 2. Unsupervised hierarchical clustering dendrogram of the top 1,000 hypermethylated DMCs and all 119 hypomethylated DMCs between tumor and control samples. DNA methylation values were represented as colors, with red representing hypermethylated DMCs and green representing hypomethylated DMCs. DMCs, differentially methylated CpGs; T, tumor; N, normal control.

Genomic features of DMCs between tumors and controls

The methylation categories of DMCs were analyzed in relation to genomic locations. Significant differences were observed between the hypo- and hypermethylated DMCs according to the functional genomic distribution, as well as the CpG content and neighborhood context (Fig. 3).

Figure 3. Genomic features of DMCs between tumor and control samples. (A) Graph showing percentages of hypermethylated DMCs according to their functional genomic distribution and CpG content/neighborhood context. (B) Graph showing percentages of hypomethylated DMCs according to their functional genomic distribution and CpG content/neighborhood context. DMCs, differentially methylated CpGs; UTR, untranslated region; TSS, transcription start site.

Of the 119 significantly hypermethylated DMCs, 9% were located in the 3′UTR region, 56% in the gene body, 8% in exon 1, 14% in the 5′UTR, 3% in TSS1500, 9% in TSS200 and none in intergenic region (Fig. 3A). Of the annotated significantly hypermethylated DMCs, 16% belonged to the CGI, 24% to the shore area, 3% to the shelf area, and the remaining 57% to the open sea area (Fig. 3A).

In contrast to hypermethylation, 3% of the 18,999 significantly hypomethylated DMCs were located in the 3′UTR region, 67% in the gene body, 1% in exon 1, 15% in the 5′UTR, 9% in the TSS1500, 3% in the TSS200, and the remaining 1% in intergenic region (Fig. 3B). With regard to CpG content and neighborhood context, 84% belonged to the open sea area of the genome, 9% to the shore area, 6% to the shelf area and 1% to the CGI (Fig. 3B).

Functional annotation of differentially methylated genes between tumor and controls

Of the 119 significantly hypermethylated DMCs, 81 DMCs represented 72 genes. Functional annotation of the 72 genes indicated that the most significantly enriched pathway was ‘natural killer cell mediated cytotoxicity’, as shown in Table II.

Table II. KEGG pathway enrichment analysis for the 72 hypermethylated genes and 6,202 hypomethylated genes between tumor and control.

Table II.

KEGG pathway enrichment analysis for the 72 hypermethylated genes and 6,202 hypomethylated genes between tumor and control.

A, KEGG pathway for hypermethylated genes
ID	Items	FDR
hsa04650	Natural killer cell mediated cytotoxicity	0.006166
B, KEGG pathway for hypomethylated genes
ID	Items	FDR
hsa05200	Pathways in cancer	1.03×10−23
hsa04510	Focal adhesion	1.80×10−23
hsa04810	Regulation of actin cytoskeleton	1.07×10−22
hsa04360	Axon guidance	1.12×10−16
hsa04020	Calcium signaling pathway	1.18×10−16
hsa04514	Cell adhesion molecules (CAMs)	4.80×10−16
hsa04724	Glutamatergic synapse	6.56×10−15
hsa05412	Arrhythmogenic right ventricular cardiomyopathy (ARVC)	6.29×10−13
hsa05146	Amoebiasis	1.92×10−12
hsa04070	Phosphatidylinositol signaling system	2.85×10−12
hsa04530	Tight junction	4.58×10−12
hsa04520	Adherens junction	5.38×10−12
hsa04971	Gastric acid secretion	5.19×10−11
hsa04060	Cytokine-cytokine receptor interaction	5.45×10−11
hsa04080	Neuroactive ligand-receptor interaction	1.40×10−10
hsa05410	Hypertrophic cardiomyopathy (HCM)	2.06×10−10
hsa04730	Long-term depression	2.68×10−10
hsa04512	ECM-receptor interaction	4.27×10−10
hsa05222	Small cell lung cancer	4.27×10−10
hsa04970	Salivary secretion	6.28×10−10
hsa04144	Endocytosis	6.91×10−10
hsa05414	Dilated cardiomyopathy	7.09×10−10
hsa04270	Vascular smooth muscle contraction	1.15×10−9
hsa04380	Osteoclast differentiation	1.68×10−9
hsa05215	Prostate cancer	1.76×10−9
hsa04010	MAPK signaling pathway	2.65×10−9
hsa04910	Insulin signaling pathway	3.76×10−9
hsa04972	Pancreatic secretion	4.18×10−9
hsa04666	Fc gamma R-mediated phagocytosis	7.21×10−9
hsa04662	B cell receptor signaling pathway	1.26×10−8

[i] FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes.

In addition, 10,953 of the 18,999 significantly hypomethylated DMCs represented 6,202 genes. To obtain further insight into pathways targeted by the hypomethylated DMCs, further functional annotation was performed. ‘Pathways in cancer’, ‘focal adhesion’, ‘regulation of actin cytoskeleton’, ‘axon guidance’, ‘calcium signaling pathway’ and ‘cell adhesion molecules (CAMs)’ were the most enriched KEGG pathways (Table II).

Identification of DMCs between type A and B thymomas

The DNA methylation profiling of two type A (1T and 2T) and three type B (4T, 6T and 7T) thymomas was obtained using the Illumina 850K methylation microarray. In total, 10,014 CpGs were differentially methylated at Δβ>0.2 and P<0.001 between type A and B thymoma subjects, which consisted of 3,998 hypermethylated and 6,016 hypomethylated DMCs. The top 1,000 DMCs were used for unsupervised hierarchical clustering. The results indicated that DNA methylation profiling of type A was significantly distinguished from that of type B thymoma (Fig. 4).

Figure 4. Unsupervised hierarchical clustering dendrogram of the top 1,000 DMCs between type A and type B thymomas. DNA methylation values were represented as colors, with red representing hypermethylated DMCs and green representing hypomethylated DMCs. DMCs, differentially methylated CpGs; T, tumor; N, normal control.

Genomic features of DMCs between type A and B thymomas

The functional genomic distribution, CpG content and neighborhood context for the hypo- sand hypermethylated DMCs are shown in Fig. 5. Out of the 3,998 hypermethylated DMCs, 1% were located in the 3′UTR region, 69% in the gene body, 2% in exon 1, 16% in the 5′UTR, 8% in the TSS1500, 3% in the TSS200 and 1% in the intergenic region (Fig. 5A). In addition, 1% of the hypermethylated DMCs belonged to the CGI, 6% to the shore area, 4% to the shelf area, and the remaining 88% to open sea area (Fig. 5A).

Figure 5. Genomic features of DMCs between type A and type B thymomas. (A) Graph showing percentages of hypermethylated DMCs according to their functional genomic distribution and CpG content/neighborhood context. (B) Graph showing percentages of hypomethylated DMCs according to their functional genomic distribution and CpG content/neighborhood context. DMCs, differentially methylated CpGs; UTR, untranslated region; TSS, transcription start site.

As shown in Fig. 5B, 3% of the 6,016 hypomethylated DMCs were located in the 3′UTR region, 70% in the gene body, 1% in exon 1, 12% in the 5′UTR, 8% in the TSS1500, 3% in the TSS200, and the remaining 2% intergenic region. With regard to CpG content and neighborhood context, 72% of the hypomethylated DMCs belonged to the open sea area of the genome, 14% to the shore area, 7% to the shelf area, and 7% to the CGI (Fig. 5B).

Integrated analysis of methylation and expression data of type A and B thymomas

Considering that aberrant DNA methylation may cause gene expression alterations in thymomas (25), methylation and expression data of type A and B thymomas from the GEO database were analyzed. Differential methylation analysis showed that a total of 377 hypermethylated DMCs between type A and B thymomas were located in proximal promoters (TSS1500 and TSS200), which were associated with 319 genes. In addition, a total of 658 hypomethylated DMCs between type A and B thymomas were located in proximal promoters, which were associated with 530 genes.

The expression data compared 10 type A and 26 type B thymomas, and 1,562 DEGs were identified between the two types. In total, 55 common genes were found between the methylation and expression data of type A and B thymomas (Table III). Among them, 36 genes showed an inverse correlation between DNA methylation and expression alterations, in which seven genes were hypermethylated with low expression (ICAM3, APBB1IP, IFI16, PARVG, CCM2, INPP5D, SP110) and 29 were hypomethylated with high expression (GALC, ALS2CR4, IQCC, RPL22, FEZ2, EPS15, KIF25, PACSIN2, PRKAR1A, PTPRE, ATP2A2, PNPLA8, SERPINB5, SGK3, CBLB, KLF11, C5orf45, SLC2A10, AUH, CPE, FBXO8, EEF1E1, STARD13, RAPGEF4, FSTL1, ZNF396, FRAS1, NAV2 and LCA5).

Table III. Fifty-five common genes between the methylation and expression data of type A and B thymomas.

Table III.

Fifty-five common genes between the methylation and expression data of type A and B thymomas.

NCBI gene ID	Symbol	Mean.a	Mean.b	log2FC	P-value.wilox
51149	C5orf45	−2.102930058	−2.503322201	−0.251443123	0.021177724
9637	FEZ2	1.303466656	1.098450577	0.246883717	0.001037259
1363	CPE	1.504873609	1.021808511	0.558517465	0.000425847
549	AUH	0.361937179	−0.093619066	NA	0.009478003
89797	NAV2	0.357340345	−0.374725063	NA	0.001568131
11069	RAPGEF4	−0.556887935	−1.1271287	−1.017193317	0.001193108
7919	BAT1	0.383356739	1.017168635	−1.407799434	0.039674448
3834	KIF25	−1.524919975	−1.76772686	−0.21316184	0.04690174
26269	FBXO8	−0.359657144	−0.850239994	−1.241247864	0.0105367
11167	FSTL1	0.2920439	−0.375401601	NA	0.000672044
2581	GALC	1.009684914	0.913221129	0.144869007	0.021177724
23051	ZHX3	−1.431590388	−1.06661393	0.424580688	0.00430993
6146	RPL22	1.92145252	1.723732502	0.15666142	0.014340868
57176	VARS2	0.504252427	0.852942998	−0.758303208	0.043164899
5791	PTPRE	0.648380999	0.358704002	0.85404797	0.005445117
57404	CYP20A1	−1.075059329	−0.82894206	0.375073109	0.021177724
2060	EPS15	0.601014623	0.359845244	0.740023504	0.006104164
80144	FRAS1	1.224452937	0.541981128	1.175822802	5.26×10−5
3853	KRT6A	−0.968952762	−0.078244194	3.630370725	0.001568131
5268	SERPINB5	0.884731379	0.530946778	0.736672239	0.0105367
868	CBLB	0.621699125	0.254483425	1.288644852	1.51×10−5
5573	PRKAR1A	1.630734363	1.38168313	0.239095003	0.00232546
488	ATP2A2	0.613248964	0.293021363	1.065467044	0.000158687
50640	PNPLA8	−0.118260809	−0.44273736	−1.904479067	0.039674448
84283	TMEM79	−1.351520231	−0.790482225	0.77377818	0.008511842
9521	EEF1E1	−0.664623538	−1.15528475	−0.797639192	0.017478649
167691	LCA5	−1.152514072	−1.982068102	−0.782222167	7.71×10−5
10109	ARPC2	1.176142371	1.309877505	−0.155369195	0.019253419
51389	RWDD1	0.229588154	0.734803706	−1.678310694	0.04690174
90627	STARD13	−1.152950212	−1.659424944	−0.525353163	0.004848756
8462	KLF11	0.31155478	−0.083381262	NA	0.021177724
252884	ZNF396	−0.062746326	−0.739148969	−3.558262253	9.29×10−5
65062	ALS2CR4	1.134639505	0.971836256	0.22344884	0.033385967
55721	IQCC	−1.638095756	−1.819151371	−0.151245901	0.04690174
10978	CLP1	−1.334499175	−0.955373735	0.482161294	0.039674448
128387	TATDN3	−1.64574706	−1.425240499	0.207537237	0.023260885
11252	PACSIN2	1.408366082	1.165251567	0.273380936	0.002641037
23678	SGK3	−0.155180551	−0.520928994	−1.747138985	0.000899667
64098	PARVG	0.104295597	0.587763121	−2.494556581	0.019253419
160728	SLC5A8	−0.811122514	−0.389094826	1.059798045	0.033385967
81031	SLC2A10	1.032303784	0.622080161	0.730695183	0.009478003
2	A2M	0.50047177	−0.092486832	NA	0.000133173
3635	INPP5D	−1.025761944	−0.798974115	0.360475283	0.043164899
8452	CUL3	−0.41681927	−0.934666023	−1.165028973	0.002993599
3431	SP110	0.653415681	0.813894131	−0.316840067	0.021177724
3428	IFI16	−0.554778354	−0.001533575	8.498868544	0.019253419
152330	CNTN4	0.055184231	−0.701012597	NA	0.005445117
2690	GHR	0.828244257	0.288386478	1.522052773	0.012960556
3385	ICAM3	−0.828251179	−0.004174698	7.632252662	0.043164899
83605	CCM2	0.909215687	1.225045194	−0.430140494	0.021177724
54518	APBB1IP	−0.245298433	0.422097639	NA	0.017478649
81846	SBF2	0.52478967	0.114005332	2.20263802	4.32×10−5
27252	KLHL20	0.692117181	0.541111423	0.355090623	0.036418783
10068	IL18BP	0.367427467	0.117710084	1.642221566	0.043164899
185	AGTR1	−1.260285672	−1.835884547	−0.542724545	0.002993599

Functional annotation of differentially methylated genes between type A and B thymomas

Based on the combination analysis result of the 36 genes, the GO and KEGG pathway enrichment analysis was performed with a threshold FDR value of <0.05. The most enriched biological processes for the seven genes that were hypermethylated with a low expression were ‘negative regulation of neutrophil differentiation’, ‘blood vessel endothelial cell differentiation’ and ‘negative regulation of monocyte differentiation’. The most enriched cellular components were ‘focal adhesion’, ‘cytoplasm’ and ‘cortical cytoskeleton’. The most enriched molecular functions were ‘phosphatidylinositol trisphosphate phosphatase activity’, ‘inositol-4, 5-bisphosphate 5-phosphatase activity’ and ‘PTB domain binding’. Furthermore, functional annotation showed that the seven genes that were hypermethylated with a lower expression were highly involved in five KEGG pathways: ‘Insulin signaling pathway’, ‘Fc gamma R-mediated phagocytosis’, ‘Fc epsilon RI singling pathway’, ‘CAMs’ and ‘focal adhesion’ (Table IV).

Table IV. Enrichment analysis of the 7 genes hypermethylated with lower expression between type A and type B thymoma.

Table IV.

Enrichment analysis of the 7 genes hypermethylated with lower expression between type A and type B thymoma.

Term	ID	Items	FDR
Biological processes	GO:0045659	Negative regulation of neutrophil differentiation	0.004604
	GO:0060837	Blood vessel endothelial cell differentiation	0.004604
	GO:0045656	Negative regulation of monocyte differentiation	0.005524
	GO:0061154	Endothelial tube morphogenesis	0.005524
	GO:0001885	Endothelial cell development	0.006138
	GO:0045409	Negative regulation of interleukin-6 biosynthetic process	0.006575
Cellular components	GO:0005925	Focal adhesion	0.003788
	GO:0005737	Cytoplasm	0.011388
	GO:0030863	Cortical cytoskeleton	0.015032
	GO:0030054	Cell junction	0.015433
	GO:0005886	Plasma membrane	0.017133
	GO:0005884	Actin filament	0.017957
Molecular functions	GO:0034594	Phosphatidylinositol trisphosphate phosphatase activity	0.003274
	GO:0030487	Inositol-4,5-bisphosphate 5-phosphatase activity	0.003274
	GO:0051425	PTB domain binding	0.004364
	GO:0004445	Inositol-polyphosphate 5-phosphatase activity	0.005727
	GO:0005515	Protein binding	0.023332
	GO:0005178	Integrin binding	0.0412
KEGG pathways	hsa04910	Insulin signaling pathway	0.031387
	hsa04666	Fc gamma R-mediated phagocytosis	0.032684
	hsa04664	Fc epsilon RI signaling pathway	0.036521
	hsa04514	Cell adhesion molecules (CAMs)	0.035423
	hsa04510	Focal adhesion	0.039626

[i] FDR, false discovery rate.

In addition, the 29 genes that were hypomethylated with high expression were predominantly involved in the following biological processes: ‘Morphogenesis of an epithelium’, ‘regulation of protein phosphorylation’ and ‘cell communication’. ‘Cytoplasm’, ‘cytosol’ and ‘cAMP-dependent protein kinase complex’ were the most enriched cellular components. ‘cAMP binding’, ‘cAMP-dependent protein kinase regulator activity’ and ‘galactosylceramidase activity’ were the most enriched molecular functions (Table V). No significantly enriched KEGG pathways were identified for the 29 genes that were hypomethylated with high expression at a threshold FDR value of <0.05.

Table V. Enrichment analysis of the 29 genes hypomethylated with higher expression between type A and type B thymoma.

Table V.

Enrichment analysis of the 29 genes hypomethylated with higher expression between type A and type B thymoma.

Term	ID	Items	Hyp_c
Biological processes	GO:0002009	Morphogenesis of an epithelium	0.008491
	GO:0001932	Regulation of protein phosphorylation	0.009307
	GO:0007154	Cell communication	0.021245
	GO:0060512	Prostate gland morphogenesis	0.022083
	GO:0033003	Regulation of mast cell activation	0.022083
	GO:0023051	Regulation of signaling	0.022083
Cellular components	GO:0005737	Cytoplasm	0.000212
	GO:0005829	Cytosol	0.028305
	GO:0005952	cAMP-dependent protein kinase complex	0.000883
Molecular functions	GO:0030552	cAMP binding	0.004318
	GO:0008603	cAMP-dependent protein kinase regulator activity	0.006962
	GO:0004336	Galactosylceramidase activity	0.011623
	GO:0004490	Methylglutaconyl-CoA hydratase activity	0.011623
	GO:0031775	Lutropin-choriogonadotropic hormone receptor binding	0.011623
	GO:0005509	Calcium ion binding	0.023277

Evaluation of DNA methylation markers for type A and B thymomas

To assess the clinical functionality of DNA methylation markers as diagnostic biomarkers for type A and B thymomas, their sensitivity and specificity were determined using ROC curve analysis. For the seven genes that were hypermethylated with low expression, the AUC of ICAM3 (0.717), APBB1IP (0.748), IFI16 (0.745), PARVG (0.762), CCM2 (0.703), INPP5D (0.741) and SP110 (0.738) was >0.7, as shown in Fig. 6.

Figure 6. ROC curves for discrimination of type A and type B thymomas. ROC curves and AUC values were generated for (A) PARVG, (B) IFI16, (C) SP110, (D) INPP5D, (E) CCM2I, (F) APBB1IP and (G) ICAM3, to compare gene expression in 10 type A and 26 type B thymoma cases from GSE29695. ROC, receiver operating characteristic; AUC, area under the curve.

For the 29 genes that were hypomethylated with high expression, the AUC of FEZ2 (0.818), PTPRE (0.811), ATP2A2 (0.829), CBLB (0.853), C5orf45 (0.820), CPE (0.853), FSTL1 (0.815), ZNF396 (0.871), FRAS1 (0.885), NAV2 (0.801) and LCA5 (0.846) was > 0.8, as shown in Fig. 7A-K. Among these 11 genes, ZNF396 and FRAS1 had the largest AUC. For the diagnosis of type A and B thymomas, the sensitivity and specificity of ZNF396 was 84.6 and 90.9% (Fig. 7H), while that of FRAS1 was 100 and 72.7% (Fig. 7I), respectively. The combination analysis of the above 11 genes increased sensitivity to 96.2% (Fig. 7L). The AUC of all 29 genes was shown in Fig. 8.

Figure 7. ROC curves for the discrimination of type A and type B thymomas. ROC curves and AUC values were generated for (A) FEZ2, (B) PTPRE, (C) ATP2A2, (D) CBLB, (E) C5orf45, (F) CPE, (G) FSTL1, (H) ZNF396, (I) FRAS1, (J) NAV2 and (K) LCA5, to compare gene expression in 10 type A and 26 type B thymoma cases from GSE29695. (L) A ROC curve of the above 11 genes was also generated. ROC, receiver operating characteristic; AUC, area under the curve.

Figure 8. ROC curves for discrimination of type A and type B thymomas. ROC curves and AUC values were generated for 29 genes to compare gene expression in 10 type A and 26 type B thymoma cases from GSE29695. ROC, receiver operating characteristic; AUC, area under the curve.

Identification of DMCs between MG- and non-MG-thymomas

Methylation microarray analysis was also used to obtain the DNA methylation pattern of the 2 MG- and 4 non-MG-thymomas. Using Δβ>0.2 and P<0.001, 121 DMCs were identified between the MG- and non-MG-thymoma subjects, including 22 hypermethylated and 99 hypomethylated DMCs. The result of unsupervised hierarchical clustering showed that the DNA methylation profiling of MG-thymoma was significantly distinguished from that of non-MG-thymoma (Fig. 9).

Figure 9. Unsupervised hierarchical clustering dendrogram of the 121 DMCs between MG-thymoma and non-MG-thymoma. DNA methylation values were represented as colors, with red representing hypermethylated DMCs and green representing hypomethylated DMCs. MG, myasthenia gravis; DMCs, differentially methylated CpGs; T, tumor; N, normal control.

The 22 hypermethylated DMCs represented 20 genes, which were enriched in 4 KEGG pathways: ‘Mucin type O-Glycan biosynthesis’, ‘lysine degradation’, ‘p53 signaling pathway’ and ‘phototransduction pathway’. The 99 hypomethylated DMCs represented 73 genes, and no significantly enriched KEGG pathway was found.

Discussion

Epigenetic changes, particularly changes in DNA methylation, are important markers and widely studied in a variety of cancer types (26). The role of DNA methylation in oncogenesis is a topic of interest in the study of cancer biology. A previous report showed that epigenetic events have been implicated in thymomas (27). However, to the best of our knowledge, exhaustive analysis of genome-wide aberrant DNA methylation in the development and progression of thymomas has not been performed. In the current pilot study, the methylation profile of patients with thymoma was described. The landscape of methylation in thymomas was obtained using an Illumina 850K methylation microarray.

A total of 19,118 DMCs were identified, 119 of which were hypermethylated and 18,999 hypomethylated. In general, a global decrease in methylation was observed in thymoma tissue, compared with the control. It is known that DNA methylation may be more dynamic outside the CGI (28); the results of the present study also indicated that the majority of DMCs were found within the gene body or open sea area. The DNA methylation of FHIT, MLH1, E-cadherin, MGMT, CDKN2A, HPP1 and DAP-K in thymomas has been previously described (13,14,29,30). The present study provided a more extensive list of candidate differentially methylated genes, which may be associated with thymomas. Further studies are required to evaluate the gene expression alterations in thymomas regulated by aberrant DNA methylation.

The latest histological classification recognizes two main thymoma types: A and B (1). The histological subtypes of thymomas seem to be of independent prognostic significance (31,32). Type A thymoma frequently follows a benign clinical course, whereas type B thymoma is considered a low to moderate malignant neoplasm (33). In the present study, the global methylation patterns of types A and B thymoma across the genome were studied. To avoid gender bias, all CpG probe and gene expression data were removed from chromosomes X and Y for the analysis. Differential methylation analysis identified 3,998 hypermethylated and 6,016 hypomethylated DMCs between type A and B thymoma subjects. Genomic features of DMCs also suggested that most DMCs were found within the gene body region or open sea area.

Furthermore, the methylation array data of type A and B thymomas were analyzed in relation to the gene expression array data from the GEO. According to the results, a set of 36 genes showed an inverse correlation between DNA methylation and expression alterations, which may have potential functional consequences, owing to aberrant promoter DNA methylation (TSS1500 and TSS200). Pathway enrichment analysis suggested that the seven genes that were hypermethylated with low expression (ICAM3, APBB1IP, IFI16, PARVG, CCM2, INPP5D and SP110) covered major pathways associated with Fc gamma R-mediated phagocytosis, Fc epsilon RI signaling pathway, cell adhesion molecules and focal adhesion, which serve an important role in tumor development and host-defense mechanisms. Therefore, the present results underlined the importance of aberrant DNA methylation in different subtypes of thymoma.

The methylation status of the 29 genes that were hypermethylated with a low expression were also evaluated using ROC curve analysis to distinguish type A from B thymomas. The results indicated that 11/29 genes (FEZ2, PTPRE, ATP2A2, CBLB, C5orf45, CPE, FSTL1, ZNF396, FRAS1, NAV2 and LCA5) may be potential biomarkers for the diagnosis of type A and B thymomas, with AUC>0.8. It has been reported that diagnostic information may be increased if the methylation of multiple genes is analyzed in combination (34). Herein, it was observed that combination analysis of the 11 genes increased sensitivity to 96.2%. The present results suggested that there are different epigenetic regulation mechanisms for type A and B thymomas. These 11 genes had potential functional consequences in type A and B thymomas, owing to aberrant promoter DNA methylation. Their roles in thymoma subtypes, as well as the utility of these biomarkers in a clinical setting, requires further study in a larger cohort of thymoma subjects.

In conclusion, the present study reported the dysregulated DNA methylation involved in thymoma using the Illumina 850K methylation microarray. Significant changes were observed in the DNA methylomes of thymoma tumor and normal samples, and between type A and B thymomas. To the best of our knowledge, the present study was the first global DNA methylation analysis of thymoma, which may set the foundation for understanding the mechanisms of tumorigenesis in thymoma, as well as for future investigation of epigenetic regulation in type A and B thymomas.

Acknowledgements

We thank Beijing Medintell Bioinformatic Technology Co., Ltd., for assistance in high-throughput sequencing and data analysis.

Funding

This study was funded by the Research Special Fund for the Public Welfare Industry of Health (grant no. 201402001), the National Natural Science Foundation of China (Youth Science Fund) (grant no. 81400068) and the CAMS Innovation Fund for Medical Sciences (grant no. 2016-I2M-1-002).

Availability of data and materials

The datasets used and analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

YB, YM, and YN designed the study. YB, YM, SL, HL and JH contributed to the materials and performed experiments. YN, YZ, NL and LL performed the data analyses. YZ, NL, XM and JY interpreted the data. LL, XM, JY, BL, ZL and ZW performed the microarray analysis of DNA methylation and contributed significantly to writing the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Written informed consent was provided by all participants. The present study was approved by the Ethics Committee of Peking Union Medical College Hospital (Beijing, China) and was performed in compliance with the Declaration of Helsinki.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References