DNA methylation markers for risk of metastasis in a cohort of men with localized prostate cancer

Figures & data

Table 1. Candidate genes selected for prognostic evaluation and results.

Functional categories	Genes selected for prognostic evaluation	Genes found significant
Cell cycle control	CCNA2[29], CCNB1[28,29], CCNB2[29], CCND1[25,26], CCND2[27], CDC2[29], CDC25[29], DDIT4[28], EPR1[29], MGMT[30], Ki-67[27,29], USP22[29], XIAP[29]	CCND1*, CDC2*, USP22
Metastasis suppressors and tumour suppressors	APAF1[40], APC[37,38] §, BAX[51], BCL2[49], BCL6[50], BRMS1[33], CD82[32], CDKN1A[36], CEBPD[42], FOXO1[45], FOXO3[45], FOXO4[45], FOXO6[45], HBEGF[34], IGFBP3[41], KISS1[31], LPL[46], MAPSK4[31], MDM2[48], NDRG1[31], NME1[31], OLFM4[47], PEBP1[35], PTEN[43], RAP1GAP[44], RARB[39], RASSF1A[39], TGFB1[31]	EGF, RAP1GAP, RARB*, RASSF1A, TGFB1
Cell signalling and transcription factors	CD44[55], CTGF[34], CXCR4[52], EREG[34], FOS[46], GPI[34], HSP90[58,59] §, HSPA1A[59], LTBP1[34], PITX2[56,57], PSCA[53], MTA1[54], SPP1[52]	CTGF, CXCR4, HSPA1A
Cell adhesion/motility/ invasion	ADAMTS1[34], AGRN[34], CAV1[53], CDH1[31], COL1A1[24], COL1A2[34], CTNNB1[31], CTNND2[31], EFEMP1[34], FBLN2[34], GPI[31], HGF[31], ICAM1[60], MATN2[34], MMP2[31], MMP9[31], MTDH[31], PECAM1[60], PLAU[31], RHOC[52], RPSA[62], SPARC[34], TMSB15A[53], TMSB15B[53], TNC[34], VCAM1[61]	ADAMTS1, CTNNB1, FBLN2, ICAM1, MATN2, RHOC, TMSB15B, VCAM1*
Angiogenesis	ADM[28], ANGPTL4[34], DDAH1[69], EGF[63], eNOS[65,69] §, EPHA2[68], FGFR2[67], GLI1[66], HIF1A[64], KDR[67], NRP2[70], PTN[71], VEGF[28]	ADM*, DDAH1, EPHA2, FGFR2, GLI1, PTN
Immune function/Inflammation	CCL2[72], CPM[34], CPN1[34], CSF1[52], CXCL1[34], IL13RA2[34], PTGS2[73,74]	CPN1, CSF1
Pluripotency genes[19]	KLF4, MYC, OCT4§, SOX2§
Genes over-expressed in human embryonic stem cells[75]	AEN, C14ORF115, CDC25A, CHEK2, CLDN6, CYP26A1, CXORF15, DBNDD1, DNMT3A, DSCC1, DTYMK, EPHA1, EPHX3, ERCC6L, ETV4, GDF3, GINS4, GJC1, GPR19, HELLS, HESX1, L1TD1, LIN28, LPAR4, MCM10, MYBL2, NANOG, NCAPH, NDUFAF7, ORC1L, ORC2L, POUSF1§, PRDM14, PWP2, RBM14, RRP9, SLC5A6, TDGF1, WSCD1, ZIC3	AEN*, DBNDD1, DNMT3A, EPHX3*, GINS4, L1TD1, MYBL2, NANOG, ORC2L, PRDM14, WSCD1

*Among those showing consistent statistical significance, ADM, AEN, CCND1, CDC2, EPHX3, ICAM1, RARB, and VCAM1 were found significant in both statistical methods.

^§These candidate genes (APC, HSP90, eNOS) and stem cell genes (OCT4, SOX2, POUSF1) were not found in our array and therefore omitted from our analyses.

Figure 1. Study population flow chart.

Table 2. Demographic and clinical characteristics of the study population.

	Cases (N = 215)	Controls (N = 404)	p-value
Age (mean (SD))	72.7 (8.8)	72.9 (8.4)	0.74
Race/ethnicity			0.16
White	122 (56.7%)	248 (61.4%)
Hispanic	27 (12.6%)	31 (7.7%)
Black	46 (21.4%)	77 (19.1%)
Asian	14 (6.5%)	26 (6.4%)
Others	6 (2.8%)	22 (5.4%)
Year of diagnosis			0.84
1997–1999	56 (26.1%)	117 (29.0%)
2000–2002	60 (27.9%)	111 (27.5%)
2003–2006	99 (46.0%)	176 (43.6%)
2007
Stage at diagnosis			0.12
I	208 (96.7%)	399 (98.8%)
II	7 (3.2%)	5 (1.2%)
Gleason score			<0.01
Range	3–10	4–10
≤6	56 (26.0%)	207 (51.2%)
7	90 (41.9%)	109 (26.8%)
≥8	69 (32.1%)	91 (22.5%)
PSA at diagnosis (mean (SD))	34.9 (86.15)	14.8 (25.09)	<0.01
PSA doubling time			0.01
Negative or stable	81 (37.7%)	191 (46.9%)
<3 years	51 (23.7%)	71 (17.4%)
≥3 years	27 (12.6%)	71 (17.4%)
Missing	56 (26%)	74 (18.2%)
Number of biopsy positive cores (mean (SD))	5.9 (3.38)	4.7 (3.02)	<0.01

*T-tests or chi-square tests were used to test for statistical significance and report the p-values above.

Figure 2. Volcano plots for the (a) CpG site-level analysis and (b) PC-based analysis.

Table 3. Twenty-four candidate genes found to be significant in the CpG site-level analysis.

Gene	Number of Significant Sites	CpG Site (Illumina ID)	Location of CpG Site	OR	Adjusted p-value (DFDR)
ADM	1	cg21039208	TSS1500|N_Shore	0.09	0.0749
AEN	3*	cg24687276	TSS1500|N_Shore	0.07	0.0885
CCND1	10*	cg24387864	TSS1500|Island	0.06	0.0096
CDC2	1	cg19147990	TSS1500|N_Shore	0.00	0.0885
CPN1	2*	cg23197945	1stExon	14.73	0.0623
DNMT3A	6*	cg11354105	Body|TSS1500|Island	8.91	0.0645
EPHA2	9*	cg05797770	Body	10.80	0.0797
EPHX3	7*	cg18366919	TSS1500|Island	7.22	0.0482
FBLN2	3*	cg09459808	Body|Island	0.10	0.0797
FGFR2	12*	cg18566515	5´UTR|TSS200|N_Shore	5.59	0.0817
GINS4	5*	cg23688868	TSS1500|N_Shore	0.06	0.0482
ICAM1	3*	cg20036207	TSS1500|3´UTR|N_Shore	35.74	0.0812
L1TD1	5*	cg23049458	5´UTR|Island	5.17	0.0623
MATN2	6*	cg13255750	Body	0.01	0.0099
ORC2L	3*	cg02786368	5´UTR|N_Shelf	0.00	0.0715
PRDM14	5*	cg13267264	TSS200|Island	4.91	0.0167
PTN	10*	cg03464514	5´UTR|1stExon	9.17	0.0952
RAP1GAP	3*	cg11285442	Body|5´UTR	0.01	0.0096
RARB	13*	cg10712623	TSS1500|TSS200	34.29	0.0749
RASSF1A	4*	cg19811994	Body|5´UTR|N_Shore	0.05	0.0099
TGFB1	2*	cg15160195	Body|N_Shore	0.04	0.0797
TMSB15B	3*	cg13933773	TSS1500|N_Shore	0.12	0.0715
VCAM1	3*	cg26265881	5´UTR|1stExon	8.45	0.0838
WSCD1	8*	cg21172077	Body	0.06	0.0045

*For the candidate genes with more than one significant marker, the CpG site, location, OR and p-value reported are that of the marker with the smallest DFDR adjusted p-value. See supplemental table for the complete list of sites that were significantly associated with metastasis.

Table 4. Candidate genes found to be significant in PC-based analysis.

Gene	Number of Significant PCs	OR for PC	Adjusted p-value (DFDR)
ADM	1	1.32	0.0082
AEN	1	1.86	8.5926 x 10^−5
CCND1	1	1.18	0.0677
CDC2	1	0.65	0.0677
CTNNB1	2*	2.18	0.0677
DDAH1	1	1.55	0.0677
EPHX3	1	1.15	0.0677
MYBL2	1	0.58	0.0891
RARB	3*	1.16	0.0063
VCAM1	1	0.75	0.0052

*For the candidate genes with more than one significant PC, the OR and DRDR-adjusted p-value reported are that of the PC with the smallest adjusted p-value.

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