Validation Study of Genes with Hypermethylated Promoter Regions Associated with Prostate Cancer Recurrence

Marni Stott-Miller; Shanshan Zhao; Jonathan L. Wright; Suzanne Kolb; Marina Bibikova; Brandy Klotzle; Elaine A. Ostrander; Jian-Bing Fan; Ziding Feng; Janet L. Stanford

doi:10.1158/1055-9965.EPI-13-1000

DOI: 10.1158/1055-9965.EPI-13-1000 Published July 2014

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Figure 1.
Kaplan–Meier plots for time to prostate cancer recurrence according to the average promoter region methylation in ABHD9 and HOXD3.
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Figure 2.
Box plots of each significantly hypermethylated CpG site in the ABHD9 promoter region in prostate tumor tissue versus adjacent benign prostate tissue.
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Figure 3.
Box plots of each significantly hypermethylated CpG site in the HOXD3 promoter region in prostate tumor tissue versus adjacent benign prostate tissue.

Table 1.

Distributions for selected characteristics of the prostate cancer patient cohort by recurrence status

	Recurrence (n = 104)	No recurrence (n = 303)
Characteristic	n (%)	n (%)
Age at diagnosis, y
35–54	33 (31.7)	92 (30.4)
55–59	18 (17.3)	77 (25.4)
60–64	34 (32.7)	86 (28.4)
65–69	12 (11.5)	28 (9.2)
70–74	7 (6.7)	20 (6.6)
Mean age (± SE)	58.5 (7.18)	58.2 (7.10)
PSA at diagnosis, ng/mL
0–3.9	13 (12.5)	54 (17.8)
4–9.9	45 (43.3)	189 (62.4)
10–19.9	24 (23.1)	28 (9.2)
20 +	15 (14.4)	14 (4.6)
Missing	7 (6.7)	18 (5.9)
Pathologic stage
Local	47 (45.2)	235 (77.6)
Regional	57 (54.8)	68 (22.4)
Gleason score
2–5	8 (7.7)	37 (12.2)
6	16 (15.4)	136 (44.9)
7 (3 + 4)	45 (43.3)	101 (33.3)
7 (4 + 3)	17 (16.4)	16 (5.3)
8–10	18 (17.3)	13 (4.3)

Table 2.

Number of CpG sites by location in 14 candidate genes evaluated for differential methylation profiles in prostate cancer patients with recurrence versus no recurrence

Candidate gene	Total No. CpG sites evaluated^a	No. CpG sites evaluated in promoter region^b	No. CpG sites evaluated in 5′ region^b	No. CpG sites evaluated in gene body region^b
ABHD9	20	12 (7)	11 (8)	6 (1)
APC	39	26 (0)	22 (0)	2 (0)
ASC	19	12 (2)	3 (0)	4 (0)
CD44	32	5 (0)	6 (0)	21 (0)
CDH13	61	10 (0)	4 (1)	47 (0)
GPR7	13	10 (4)	3 (1)	0 (0)
GSTP1	19	11 (0)	2 (0)	6 (0)
HOXD3	28	11 (9)	10 (9)	7 (1)
MDR1	31	7 (0)	25 (3)	11 (0)
PITX2	69	17 (1)	13 (0)	51 (0)
PTGS2	17	9 (3)	2 (0)	6 (0)
RARβ	29	11 (0)	11 (0)	12 (0)
RASSF1A	56	40 (0)	30 (0)	46 (0)
RUNX3	91	40 (0)	5 (0)	74 (0)

↵^aThe total number of CpG sites evaluated is greater than the sum of the CpG sites in each region for some genes because certain CpGs have multiple annotations due to alternative transcription start sites. The total number of CpG sites evaluated in each gene was used to determine statistical significance based on t tests of differential methylation between patients with recurrence versus no recurrence, with Bonferroni correction for multiple testing within each gene.
↵^bShown in parentheses is the number of CpG sites with significantly higher methylation (i.e., hypermethylated) in patients with prostate cancer recurrence compared with those with no evidence of recurrence.

Table 3.

Genes with over 50% of the promoter region CpG sites having higher methylation in tumor tissue from prostate cancer patients with recurrence versus no recurrence

Candidate gene	Illumina CpG site	Avg. β value: recurrence	Avg. β value: no recurrence	P^a	CpG location	Third IQR
ABHD9	cg18366919	0.581264	0.522453	0.000295	Island	0.649
ABHD9	cg26010734	0.507842	0.442909	0.000385	N_Shore	0.565
ABHD9	cg17476026	0.429670	0.357897	0.000540	N_Shore	0.525
ABHD9	cg17399362	0.512639	0.432249	0.000838	N_Shore	0.614
ABHD9	cg08457898	0.569149	0.518281	0.001475	N_Shore	0.643
ABHD9	cg16184495	0.589635	0.530117	0.001796	N_Shore	0.672
ABHD9	cg15826897	0.483290	0.434591	0.002572	N_Shore	0.535
HOXD3	cg13316854	0.438143	0.364137	9.46E-06	Island	0.476
HOXD3	cg24704177	0.453488	0.377573	2.93E-05	Island	0.498
HOXD3	cg06145336	0.671013	0.594325	3.33E-05	Island	0.742
HOXD3	cg01014615	0.646853	0.575173	9.87E-05	Island	0.719
HOXD3	cg12634591	0.488850	0.419412	0.000159	N_Shore	0.538
HOXD3	cg09387749	0.646414	0.581765	0.000419	N_Shore	0.715
HOXD3	cg25938806	0.456353	0.391304	0.000618	N_Shore	0.502
HOXD3	cg02773086	0.623405	0.569983	0.001001	N_Shore	0.691
HOXD3	cg18702197	0.426674	0.362498	0.001237	Island	0.472

Abbreviation: IQR, interquartile range.

↵^aP values with Bonferroni correction for multiple testing within each gene.

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Supplementary Data

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Supplementary Table 1 - PDF file - 57K, Genes with over 50% of the 5' region CpG sites having higher methylation in tumor tissue from prostate cancer patients with recurrence versus no recurrence.