DNA methylation and breast tumor clinicopathological features: The Western New York Exposures and Breast Cancer (WEB) study

Figures & data

Table 1. Selected characteristics of WEB study cases by inclusion in methylation analyses^a.

	Premenopausal		Postmenopausal
	Included (n = 226)	Not Included(n = 100)	Included(n = 494)	Not Included(n = 350)
	mean (SD)
Age (years)	45 (5)	45 (5)	63 (9)	63 (8)
BMI (kg/m^2)	27 (7)	27 (6)	29 (6)	29 (6)
Education (years)	14 (2)	14 (2)	13 (3)	13 (3)
Age at menarche (years)	13 (2)	12 (1)	13 (1)	13 (2)
	n (%)
Race
Caucasian	206 (91)	93 (93)	463 (94)	315 (90)^*
Other	20 (9)	7 (7)	31 (6)	35 (10)
ER negative	62 (27)	28 (28)	82 (17)	54 (15)^*
PR negative	72 (32)	35 (35)	155 (31)	103 (29)^*
Metastatic disease	76 (37)	22 (22)^*	109 (22)	55 (16)^*
Tumor size >2 cm	73 (32)	26 (26)^*	107 (22)	57 (16)^*
Nuclear grade
I	25 (11)	13 (13)	104 (21)	64 (18)^*
II	72 (22)	28 (28)	166 (34)	104 (30)
III	107 (47)	45 (45)	167 (34)	100 (29)
TNM Stage
0	30 (13)	13 (13)^*	53 (11)	52 (15)
I	72 (32)	36 (36)	227 (46)	150 (43)
IIA/IIB	85 (38)	24 (24)	136 (28)	68 (19)
III/IV	22 (10)	7 (7)	20 (4)	13 (4)

^a Numbers for some analyses are less than total for entire group because of some variables missing information

Abbreviations: BMI, body mass index; cm, centimeters; ER, estrogen receptor; PR, progesterone receptor; TNM, tumor-node-metastasis

* P < 0.05, P-values estimated with t-tests or chi-square tests as appropriate.

Figure 1. Distribution of average percent DNA methylation in breast tumors, selected genes, WEB study. The distance between the 25^th and 75^th percentiles, the inter-quartile range, is represented by the length of the box. The vertical lines represent 1.5 times the 25^th and 75^th percentile. The symbols outside the vertical lines represent values outside 1.5 times the inter-quartile range. The mean is represented by the symbol within the box and the median by the horizontal line within the box.

Table 2. Association of methylation above the median for selected genes with selected clinicopathological factors for breast cancer, premenopausal women WEB study 1996–2001.

		SCGB3A1				RARB				SYK
		n > 65.26%	n <65.26%	OR^a	95% CI	n > 4.09%	n <4.09%	OR^a	95% CI	n >2.50%	n < 2.50%	OR^a	95% CI
ER status	negative	18	25	1.0	referent	28	15	1.0	referent	55	27	1.0	referent
	positive	58	22	5.0	2.1–12.1	47	42	0.7	0.3–1.5	28	46	1.3	0.7–2.7
PR status	negative	22	30	1.0	referent	34	19	1.0	referent	35	29	1.0	referent
	positive	54	18	4.7	2.1–10.5	41	39	0.6	0.3–1.2	48	45	1.0	0.5–2.0
TNM Stage	0	8	10	1.0	referent	3	13	1.0	referent	13	10	1.0	referent
	I	27	19	2.3	0.7–7.4	29	25	4.7	1.1–19.0	35	30	0.8	0.3–2.2
	IIA/IIB	40	26	2.4	0.8–7.2	42	19	9.7	2.4–39.9	39	34	0.8	0.3–2.1
	III/IV	9	6	2.4	0.6–10.2	9	7	5.6	1.1–29.4	10	9	0.7	0.2–2.4
Tumor size	<2 cm	54	40	1.0	referent	50	47	1.0	referent	61	61	1.0	referent
	>2 cm	32	24	1.0	0.5–2.0	37	19	1.8	0.9–3.5	39	26	1.3	0.71–2.53
Vascular/lymph invasion	No	50	42	1.0	referent	50	46	1.0	referent	63	56	1.0	referent
	Yes	34	21	1.4	0.7–2.8	35	20	1.7	0.9–3.6	37	30	1.0	0.51–1.84
Nuclear Grade	I	12	5	1.0	referent	9	9	1.0	referent	14	9	1.0	referent
	II	32	21	0.7	0.2–2.2	29	25	1.2	0.4–3.6	29	33	0.4	0.2–1.2
	III	38	33	0.5	0.1–1.5	45	25	2.3	0.7–6.8	51	38	0.6	0.2–1.6
		FHIT				CCND2				BRCA1
		n >6.65%	n <6.65%	OR^a	95% CI	n > 9.55%	n < 9.55%	OR^a	95% CI	n > 4.24%	n < 4.24%	OR^a	95% CI
ER status	negative	9	17	1.0	referent	10	17	1.0	referent	23	27	1.0	referent
	positive	20	22	2.1	0.7–6.6	27	27	1.9	0.6–5.6	48	55	1.3	0.6–2.6
PR status	negative	10	19	1.0	referent	15	17	1.0	referent	30	31	1.0	referent
	positive	19	20	2.2	0.7–6.7	22	28	0.7	0.3–1.9	42	51	0.9	0.5–1.9
TNM Stage	0	9	4	1.0	referent	3	9	1.0	referent	13	5	1.0	referent
	I	8	17	0.2	0.1–0.9	12	14	2.8	0.5–15.0	33	32	0.3	0.10–1.1
	IIA/IIB	11	22	0.2	0.1–0.8	17	24	2.5	0.5–11.8	31	42	0.2	0.1–0.8
	III/IV	6	4	0.6	0.1–3.5	8	5	8.9	1.29;61.4	8	11	0.2	0.1–0.9
Tumor size	<2 cm	25	30	1.0	referent	21	34	1.0	referent	60	57	1.0	referent
	>2 cm	14	17	1.0	0.4–2.4	19	22	1.7	0.7–4.2	31	35	0.8	0.4–1.6
Vascular/lymph invasion	No	21	30	1.0	referent	25	36	1.0	referent	58	58	1.0	referent
	Yes	15	19	1.0	0.4–2.7	16	18	1.4	0.6–3.5	28	36	0.8	0.4–1.6
Nuclear Grade	I	7	6	1.0	referent	4	7	1.0	referent	10	11	1.0	referent
	II	11	14	0.7	0.2–2.7	13	17	1.9	0.4–9.0	31	27	1.1	0.4–3.0
	III	17	26	0.4	0.1–1.6	22	26	2.3	0.5–10.8	45	44	0.9	0.3–2.5

^a Odds ratios (OR) for methylation above the median value and 95% confidence intervals (CI) were estimated with unconditional logistic regression model adjusted for age in years, age at menarche, race, smoking status, and body mass index. Abbreviations: SCGB3A1, secretoglobin, family 3A, member 1; RARB, retinoic acid receptor, β; SYK, spleen tyrosine kinase; FHIT, fragile histidine triad ; CCND2, cyclin D2;BRCA1, breast cancer gene 1; ER, estrogen receptor; PR, progesterone receptor; TNM, tumor-node-metastasis. Statistically significant results (P < 0.05) are bolded.

Table 3. Association of methylation above the median for selected genes with selected clinicopathological factors for breast cancer, postmenopausal women WEB study 1996–2001.

		SCGB3A1				RARB				SYK
		n > 65.26%	n <65.26%	OR^a	95% CI	n > 4.09%	n <4.09%	OR^a	95% CI	n >2.50%	n < 2.50%	OR^a	95% CI
ER status	negative	16	45	1.0	referent	26	35	1.0	referent	34	31	1.0	referent
	positive	100	99	2.9	1.5;5.6	104	138	1.1	0.6–2.0	129	159	0.7	0.4–1.2
PR status	negative	34	70	1.0	referent	48	71	1.0	referent	60	74	1.0	referent
	positive	82	74	2.3	1.4–4.0	82	102	1.3	0.8–2.1	103	116	1.1	0.7–1.7
TNM Stage	0	10	13	1.0	referent	20	17	1.0	referent	23	25	1.0	referent
	I	66	69	1.1	0.4–2.7	75	90	0.7	0.3–1.5	83	107	0.8	0.4–1.5
	IIA/IIB	43	62	0.8	0.3–2.0	52	53	0.8	0.4–1.8	61	62	1.1	0.6–2.1
	III/IV	6	6	1.3	0.3–5.6	5	8	0.6	0.2–2.2	9	5	2.1	0.6–7.2
Tumor size	<2 cm	100	117	1.0	referent	129	139	1.0	referent	145	170	1.0	referent
	>2 cm	39	45	1.0	0.6–1.7	36	47	0.8	0.5–1.3	54	41	1.7	1.0–2.7
Vascular/lymph invasion	No	103	103	1.0	referent	112	138	1.0	referent	133	158	1.0	referent
	Yes	29	52	0.6	0.3–1.0	45	37	1.6	0.9–2.6	49	51	1.2	0.8–1.9
Nuclear Grade	I	23	30	1.0	referent	35	37	1.0	referent	35	54	1.0	referent
	II	51	51	1.3	0.6–2.5	49	76	0.7	0.4–1.3	66	71	1.5	0.9–2.6
	III	54	66	1.1	0.6–2.5	71	55	1.3	0.7–2.3	78	66	2.0	1.2–3.6
		FHIT				CCND2				BRCA1
		n >6.65%	n <6.65%	OR^a	95% CI	n > 9.55%	n < 9.55%	OR^a	95% CI	n > 4.24%	n < 4.24%	OR^a	95% CI
ER status	negative	17	13	1.0	referent	16	18	1.0	referent	39	31	1.0	referent
	positive	71	62	0.8	0.4–1.8	69	68	1.0	0.5–2.1	138	129	0.8	0.5–1.4
PR status	negative	28	28	1.0	referent	27	35	1.0	referent	74	56	1.0	referent
	positive	60	47	1.2	0.6–2.3	58	51	1.4	0.7–2.7	103	104	0.7	0.5–1.1
TNM Stage	0	10	12	1.0	referent	3	6	1.0	referent	21	22	1.0	referent
	I	47	50	1.0	0.4–2.6	42	53	1.6	0.3–7.5	102	85	1.2	0.6–2.4
	IIA/IIB	33	19	1.9	0.6–5.3	38	28	2.4	0.5–11.4	49	61	0.8	0.4–1.6
	III/IV	4	5	1.0	0.2–4.9	6	1	10.5	0.8–144.9	9	8	1.2	0.4–3.7
Tumor size	<2 cm	77	76	1.0	referent	66	77	1.0	referent	155	150	1.0	referent
	>2 cm	23	16	1.4	0.7–2.9	32	22	1.6	0.8–3.1	44	43	1.0	0.6–1.6
Vascular/lymph invasion	No	66	69	1.0	referent	63	76	1.0	referent	145	134	1.0	referent
	Yes	31	18	1.8	0.9–3.6	29	15	2.2	1.0–4.5	44	49	0.8	0.5–1.3
Nuclear Grade	I	27	20	1.0	referent	17	19	1.0	referent	39	42	1.0	referent
	II	23	30	0.6	0.3–1.3	40	35	1.2	0.5–2.8	66	71	1.0	0.6–1.7
	III	41	29	1.2	0.6–2.6	37	37	1.3	0.6–3.0	70	70	1.1	0.6–1.9

^a Odds ratios (OR) for methylation above the median value and 95% confidence intervals (CI) were estimated with unconditional logistic regression model adjusted for age in years, age at menarche, race, smoking status, and body mass index. Abbreviations: SCGB3A1, secretoglobin, family 3A, member 1; RARB, retinoic acid receptor, β; SYK, spleen tyrosine kinase; FHIT, fragile histidine triad ; CCND2, cyclin D2; BRCA1, breast cancer gene 1;ER, estrogen receptor; PR, progesterone receptor; TNM, tumor-node-metastasis. Statistically significant results (P < 0.05) are bolded.

Table 4. Hazard ratios (HR) and 95% confidence intervals (CI) for death from breast cancer by methylation of selected genes, WEB study premenopausal cases of breast cancer.

% methylation^a	n deceased	n alive	Unadjusted HR	95% CI	Adjusted^b HR	95% CI
SCGB3A1
<65.26%	12	43	Referent		Referent
>65.26%	9	71	0.47	0.20–1.11	0.41	(0.17–0.99)
GSTP1
<6.73%	9	63	Referent		Referent
>6.73%	12	65	1.24	0.52–2.93	1.18	0.46–3.02
RARB
<4.09%	6	49	Referent		Referent
>4.09%	15	72	1.62	0.63–4.18	1.60	0.61–4.17
SYK
<2.50%	11	68	Referent		Referent
>2.50%	14	77	1.14	0.52–2.52	0.84	0.36–1.94
FHIT
<6.65%	4	42	Referent		Referent
>6.65%	7	24	2.81	0.82–9.61	2.57	0.71–9.24
CDKN2A
<2.72%	10	52	Referent		Referent
>2.72%	9	59	0.81	0.33–1.98	0.80	0.32–2.00
CCND2
<9.55%	6	41	Referent		Referent
>9.55%	10	29	2.13	0.77–5.85	3.41	1.11–10.48
BRCA1
<4.24%	18	73	Referent		Referent
>4.24%	7	72	0.42	0.18–1.01	0.40	0.16–0.97
SFN
<54.64%	11	61	Referent		Referent
>54.64%	10	64	0.92	0.39–2.16	0.81	0.33–2.00

^a methylation values were dichotomized at the median

^b adjusted for age, smoking status, parity, race, and body mass index

Abbreviations: SCGB3A1, secretoglobin, family 3A, member 1; GSTP1, glutathione S-transferase pi 1; RARB, retinoic acid receptor, β; SYK, spleen tyrosine kinase; FHIT, fragile histidine triad; CDKN2A, cyclin-dependent kinase inhibitor 2A; CCND2, cyclin D2; BRCA1, breast cancer gene 1; SFN, stratifin

Table 5. Hazard ratios (HR) and 95% confidence intervals (CI) for death from breast cancer by methylation of selected genes, WEB study postmenopausal cases of invasive breast cancer.

% methylation^a	n deceased	n alive	Unadjusted HR	95% CI	Adjusted^b HR	95% CI
SCGB3A1
<65.26%	14	141	Referent		Referent
>65.26%	11	122	0.93	0.42–2.04	0.85	0.38–1.91
GSTP1
<6.73%	12	150	Referent		Referent
>6.73%	13	144	1.14	0.52–2.50	1.16	0.52–2.57
RARB
<4.09%	14	162	Referent		Referent
>4.09%	10	140	0.81	0.36–1.82	0.79	0.35–1.78
SYK
<2.50%	16	178	Referent		Referent
>2.50%	11	170	0.70	0.33–1.51	0.73	0.34–1.59
FHIT
<6.65%	3	78	Referent		Referent
>6.65%	9	84	2.81	0.76–10.38	2.09	0.54–8.19
CDKN2A
<2.72%	9	136	Referent		Referent
>2.72%	13	125	1.42	0.61–3.33	1.37	0.58–3.23
CCND2
<9.55%	7	88	Referent		Referent
>9.55%	9	88	1.38	0.51–3.70	1.37	0.49–3.84
BRCA1
<4.24%	15	164	Referent		Referent
>4.24%	11	173	0.71	0.32–1.54	0.71	0.32–1.55
SFN
<54.64%	14	132	Referent		Referent
>54.64%	9	133	0.63	0.27–1.44	0.58	0.25–1.36

^a methylation values were dichotomized at the median

^b adjusted for age, smoking status, parity, race, and body mass index

Abbreviations: SCGB3A1, secretoglobin, family 3A, member 1; GSTP1, glutathione S-transferase pi 1; RARB, retinoic acid receptor, β; SYK, spleen tyrosine kinase; FHIT, fragile histidine triad; CDKN2A, cyclin-dependent kinase inhibitor 2A; CCND2, cyclin D2; BRCA1, breast cancer gene 1; SFN, stratifin

Table 6. Summary of functions of genes included in analysis.

Gene	Function
Secretoglobin, family 3A, member 1 (SCGB3A1)	Also known as high in normal-1 (HIN-1) tumor suppressor gene that regulates epithelial cell differentiation.^37 Previous analyses of gene methylation and clinicopathological features have found methylation is associated with ER positive status.^9,19,24
Glutathione-s-transferase p 1 (GSTP1)	Involved in the detoxification of xenobiotics, including carcinogens. Hypermethylation and subsequent loss of expression have been observed in various types of carcinoma.^3,37
Retinoic acid receptor, β (RARB )	Steroid hormone receptor that mediates cellular signaling in embryonic morphogenesis and cell growth and differentiation.^37 Related to unfavorable lymph node metastasis, higher tumor grade, and higher proliferation rate in women with invasive ductal carcinoma.^13
Spleen tyrosine kinase (SYK)	Thought to restrain breast tumor invasion by controlling cell division.^55 Hypermethylation has been positively correlated with breast cancer invasiveness.^56
Fragile histidine triad (FHIT)	Tumor suppressor gene localized in an active fragile site.^32 Lost or reduced expression has been associated with more invasive and aggressive breast cancers.^31
Cyclin-dependent kinase inhibitor 2A (CDKN2A)	Cell cycle regulator that is frequently methylated in several types of cancer, including breast cancer.^4
Cyclin D2 (CCND2)	Regulates cyclin dependent kinases.^37 Previously found to be frequently methylated in breast cancer and ductal carcinoma in situ.^4
Breast cancer susceptibility gene 1(BRCA1)	Damage repair gene, if inactivated through methylation it could increase the frequency of sporadic mutations.^57
Stratifin (SFN)	Also referred to as 14-3-3σ .^58 Involved in a wide variety of cell response functions including DNA damage checkpoints and apoptosis; tumor suppressor gene.^7,59,60

Xu X, Gammon MD, Jefferson E, Zhang Y, Cho YH, Wetmur JG, Teitelbaum SL, Bradshaw PT, Terry MB, Garbowski G, et al. The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study. Epigenetics 2011; 6:1276-83; PMID:22048254; http://dx.doi.org/10.4161/epi.6.11.17744

 PubMed Web of Science ®Google Scholar

Saxena A, Dhillon VS, Shahid M, Khalil HS, Rani M, Prasad DT, Hedau S, Hussain A, Naqvi RA, Deo SV, et al. GSTP1 methylation and polymorphism increase the risk of breast cancer and the effects of diet and lifestyle in breast cancer patients. Exp Ther Med 2012; 4:1097-103; PMID:23226781; http://dx.doi.org/10.3892/etm.2012.710

 PubMed Web of Science ®Google Scholar

Marzese DM, Hoon DSB, Chong KK, Gago FE, Orozco JI, Tello OM, Vargas-Roig LM, Roqué M. DNA methylation index and methylation profile of invasive ductal breast tumors. J Mol Diagn 2012; 14:613-22; PMID:22925694; http://dx.doi.org/10.1016/j.jmoldx.2012.07.001

 PubMed Web of Science ®Google Scholar

Coopman PJP, Do MTH, Barth M, Bowden ET, Hayes AJ, Basyuk E, Blancato JK, Vezza PR, McLeskey SW, Mangeat PH, et al. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells. Nature 2000; 406:742-7; PMID:10963601; http://dx.doi.org/10.1038/35021086

 PubMed Web of Science ®Google Scholar

Yuan Y, Liu H, Sahin A, Dai JL. Reactivation of SYK expression by inhibition of DNA methylation suppresses breast cancer cell invasiveness. Int J Cancer 2005; 113:654-9; PMID:15455373; http://dx.doi.org/10.1002/ijc.20628

 PubMed Web of Science ®Google Scholar

Syeed N, Husain SA, Sameer AS, Chowdhri NA, Siddiqi MA. Mutational and promoter hypermethylation status of FHIT gene in breast cancer patients of Kashmir. Mutat Res 2011; 707:1-8; PMID:21095196; http://dx.doi.org/10.1016/j.mrfmmm.2010.11.001

 PubMed Web of Science ®Google Scholar

Zaki SM, Abdel-Azeez HA, El Nagar MR, Metwally KA, MM SA. Analysis of FHIT Gene Methylation in Egyptian Breast Cancer Women: Association with Clinicopathological Features. Asian Pac J Cancer Prev 2015; 16:1235-9; PMID:25735361; http://dx.doi.org/10.7314/APJCP.2015.16.3.1235

 PubMedGoogle Scholar

Jovanovic J, Ronneberg JA, Tost J, Kristensen V. The epigenetics of breast cancer. Mol Oncol 2010; 4:242-54; PMID:20627830; http://dx.doi.org/10.1016/j.molonc.2010.04.002

 PubMed Web of Science ®Google Scholar

Szyf M, Pakneshan P, Rabbani SA. DNA methylation and breast cancer. Biochem Pharmacol 2004; 68:1187-97; PMID:15313416; http://dx.doi.org/10.1016/j.bcp.2004.04.030

 PubMed Web of Science ®Google Scholar

Chavez-Munoz C, Hartwell R, Jalili RB, Jafarnejad SM, Lai A, Nabai L, Ghaffari A, Hojabrpour P, Kanaan N, Duronio V, et al. SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts. J Cell Biochem 2012; 113:2622-32; PMID:22422640; http://dx.doi.org/10.1002/jcb.24137

 PubMed Web of Science ®Google Scholar