Alcohol and Dietary Folate Intake and Promoter CpG Island Methylation in Clear-Cell Renal Cell Cancer

Figures & data

Figure 1. Flowchart of available subcohort members and renal cell cancer cases after 20.3 yr of follow-up, Netherlands Cohort Study on Diet and Cancer, 1986–2006. Notes: ccRCC, clear-cell renal cell cancer; FFQ, food-frequency questionnaire; NCR, Netherlands Cancer Registry; PALGA, Netherlands Pathology Registry; RCC, renal cell cancer.

Table 1. Descriptive data on methylation in clear-cell renal cell cancer (ccRCC) cases, Netherlands cohort study on diet and cancer, 1986–2006.

			Methylation status of selected gene promoters: n (%)
	Total genotyped successfully ^a		Total		Low^b		Intermediate		High
	n	%	n	%	n	%	n	%	n	%
Total	297		297		112		81		104
FZD10	277	93.3	146	52.7	20	20.4	50	66.7	76	73.1
FST	290	97.6	36	12.4	0	0.0	4	5.1	32	31.1
SFRP1	290	97.6	55	19.0	0	0.0	4	5.1	51	49.0
SFN	284	95.6	274	96.5	90	90.0	81	100.0	103	100.0
CDO1	279	93.9	114	40.9	2	2.0	27	35.1	85	84.2
NEURL	295	99.3	121	41.0	10	9.1	26	32.1	85	81.7
GRE1	285	96.0	114	40.0	4	3.9	26	32.9	84	81.6
NEFH	270	90.9	76	28.2	2	2.0	13	18.6	61	60.4
LAD1	288	97.0	87	30.2	5	4.8	18	22.5	64	61.5
RASGFR2	269	90.6	63	23.4	0	0.0	4	5.4	59	57.8
VHL	292	98.3	23	7.9	1	0.9	3	3.8	19	18.3

CDO1, cysteine dioxygenase type 1; FST, Follistatin; FZD10, frizzled class receptor 10; GREM1, Gremlin 1, DAN family BMP antagonist; LAD1, Ladinin 1; NEFH, neurofilament, heavy polypeptide; NEURL, neuralized E3 ubiquitin protein ligase 1; RASGFR2, Ras-specific guanine nucleotide-releasing factor 2; SFN, Stratifin; SFRP1, secreted frizzled-related protein 1; and VHL, Von Hippel-Lindau gene.

^a Only ccRCC cases with at least four genes genotyped successfully.

^b Low = 0–19%, intermediate = 20–39%, and high = 40% or more of the selected gene promoter regions methylated.

Table 2. Baseline characteristics of subcohort members and renal cell cancer cases according to the methylation status of selected genes, Netherlands cohort study on diet and cancer, 1986–2006.

			Cases
					ccRCC, methylation status of selected genes
Baseline characteristics	Sex	Subcohort (n = 3980)	RCC total (n = 498)	ccRCCall (n = 306)	Low (n = 112) ^a	Intermediate (n = 81)	High (n = 104)
Intake of alcohol, n nonusers (%)	All	947 (23.8%)	111 (22.3%)	73 (23.9%)	24 (21.4%)	22 (27.2%)	26 (25.0%)
	Males	292 (14.9%)	48 (14.6%)	29 (14.9%)	8 (12.5%)	9 (17.3%)	12 (16.2%)
	Females	655 (32.3%)	63 (37.3%)	44 (39.6%)	16 (33.3%)	13 (44.8%)	14 (46.7%)
Intake of alcohol, mean (SD)^b	All	13.5 (15.1)	14.9 (15.0)	15.1 (15.0)	16.2 (18.4)	13.0 (12.4)	15.8 (12.4)
	Males	17.5 (17.0)	16.8 (15.6)	17.5 (15.8)	20.4 (20.0)	15.6 (13.1)	16.2 (12.8)
	Females	8.7(10.5)	10.0 (12.1)	9.1 (10.8)	8.8 (12.1)	6.0 (6.4)	14.1 (11.2)
Intake of folic acid (µg), mean (SD)	All	211.8 (72.5)	218.6 (76.0)	216.8 (79.8)	212.9 (86.6)	224.8 (85.7)	215.3 (67.3)
	Males	225.2 (75.0)	227.1 (77.9)	227.3 (80.3)	228.6 (104.5)	225.0 (66.0)	226.3 (64.9)
	Females	198.9 (67.7)	202.1 (69.6)	198.4 (75.8)	192.0 (48.0)	224.6 (114.1)	188.0 (66.4)
Sex, n males (%)		1954 (49.1%)	329 (66.1%)	195 (63.7%)	64 (57.1%)	52 (64.2%)	74 (71.2%)
Age at baseline, mean (SD)	All	61.3 (4.2)	60.9 (3.9)	60.7 (3.9)	60.5 (3.9)	61.0 (3.7)	60.8 (3.9)
	Males	61.2 (4.2)	60.8 (3.8)	60.5 (3.7)	60.6 (3.8)	60.5 (3.4)	60.4 (3.8)
	Females	61.4 (4.3)	60.9 (4.1)	60.9 (4.1)	60.3 (4.0)	61.9 (4.0)	61.5 (4.2)
Body mass index (kg/m^2), mean (SD)	All	25.0 (3.1)	25.4 (3.0)	25.5 (3.0)	25.0 (2.8)	25.8 (3.4)	25.7 (2.8)
	Males	24.9 (2.6)	25.3 (2.7)	25.4 (2.8)	24.6 (2.5)	26.0 (3.6)	25.7 (2.2)
	Females	25.0 (3.5)	25.8 (3.5)	25.6 (3.3)	25.5 (3.1)	25.4 (3.1)	25.9 (4.0)
Cigarette smoking, n current smokers (%)	All	1086 (27.3%)	153 (30.7%)	90 (29.4%)	33 (29.5%)	24 (29.6%)	32 (30.8%)
	Males	665 (34.0%)	120 (36.5%)	67 (34.4%)	22 (34.3%)	19 (36.5%)	25 (33.8%)
	Females	421 (20.8%)	33 (19.5%)	23 (20.7%)	11 (22.9%)	5 (17.2%)	7 (23.3%)
Cigarette smoking, cigarettes/day, mean (SD)^b	All	15.3 (10.3)	17.0 (12.2)	16.6 (11.6)	15.4 (10.3)	17.7 (10.5)	17.0 (13.6)
	Males	17.1 (10.6)	18.4 (12.5)	17.9 (12.0)	17.9 (10.6)	18.5 (10.6)	17.7 (14.2)
	Females	11.6 (8.4)	11.3 (8.5)	11.8 (8.4)	9.4 (6.5)	14.8 (9.9)	13.6 (9.4)
Cigarette smoking, years of smoking, mean (SD)^b	All	31.6 (12.2)	31.9 (12.0)	31.6 (11.9)	30.1 (13.2)	32.1 (11.7)	32.8 (10.8)
	Males	33.5 (11.7)	33.4 (11.4)	33.0 (11.2)	32.9 (12.3)	33.1 (11.2)	33.4 (10.4)
	Females	27.8 (12.4)	25.8 (12.4)	26.3 (13.0)	23.6 (13.3)	28.1 (12.9)	29.6 (12.4)
Hypertension, n diagnosed (%)	All	1059 (26.6%)	157 (31.5%)	95 (31.1%)	39 (34.8%)	27 (33.3%)	26 (25.0%)
	Males	472 (24.2%)	85 (25.8%)	45 (23.1%)	17 (26.6%)	11 (21.2%)	15 (20.3%)
	Females	587 (29.0%)	72 (42.6%)	50 (45.1%)	22 (45.8%)	16 (55.2%)	11 (36.7%)
Hypertension, n users of medication (%)	All	829 (20.8%)	119 (23.9%)	75 (24.5%)	31 (27.7%)	23 (28.4%)	19 (18.3%)
	Males	381 (19.5%)	66 (20.1%)	39 (20.0%)	16 (25.0%)	11 (21.2%)	10 (13.5%)
	Females	448 (22.1%)	53 (31.4%)	36 (32.4%)	15 (31.3%)	12 (41.4%)	9 (30.0%)
Diabetes mellitus, n diagnosed (%)	All	134 (3.4%)	22 (4.4%)	12 (3.9%)	4 (3.6%)	2 (2.5%)	6 (5.8%)
	Males	65 (3.3%)	11 (3.3%)	7 (3.6%)	1 (1.6%)	1 (1.9%)	5 (6.8%)
	Females	69 (3.4%)	11 (6.5%)	5 (4.5%)	3 (6.3%)	1 (3.5%)	1 (3.3%)
Family history of kidney cancer, n (%)	All	42 (1.1%)	5 (1.0%)	3 (1.0%)	—	2 (2.5%)	1 (1.0%)
	Males	14 (0.7%)	3 (0.9%)	1 (0.5%)	—	—	1 (1.4%)
	Females	28 (1.4%)	2 (1.2%)	2 (1.8%)	—	2 (6.9%)	—
Energy intake (kcal), mean (SD)	All	1926 (515)	2005 (514)	1987 (507)	2009 (533)	2016 (528)	1949 (465)
	Males	2171 (510)	2163 (498)	2164 (480)	2227 (496)	2209 (509)	2078 (444)
	Females	1689 (395)	1698 (391)	1676 (392)	1719 (435)	1671 (364)	1631 (355)
Sodium intake (g), mean (SD) ^c	All	2.3 (0.6)	2.5 (0.7)	2.5 (0.6)	2.3 (0.6)	2.6 (0.7)	2.5 (0.6)
	Males	2.6 (0.6)	2.7 (0.7)	2.7 (0.7)	2.4 (0.6)	2.8 (0.8)	2.7 (0.6)
	Females	2.1 (0.5)	2.1 (0.5)	2.1 (0.5)	2.0 (0.5)	2.3 (0.5)	2.1 (0.4)

RCC, renal cell carcinoma; ccRCC, clear-cell RCC; SD, standard deviation.

^a Low = 0–19%, intermediate = 20–39%, and high = 40% or more of the selected gene promoter regions methylated.

^b Among users/consumers only.

^c Residuals.

Table 3. Multivariable hazard ratios for the association between alcohol and folate intakes and the risk of renal cell cancer, Netherlands Cohort Study on Diet and Cancer, 1986–2006

			Renal cell cancer			Clear-cell renal cell cancer
	Median intake	Subcohortperson years	No. of cases	HR^a^,^b	95% CI	No. of cases	HR	95% CI
Alcohol intake (g/day)
0	0	15 882	111	1	(ref)	73	1	(ref)
0.1–4.9	1.8	19 946	132	0.91	(0.69–1.20)	76	0.79	(0.56–1.11)
5–14	9.3	15 026	102	0.77	(0.57–1.03)	62	0.72	(0.50–1.03)
15–29	21.8	10 544	100	0.92	(0.67–1.25)	63	0.92	(0.63–1.34)
30+	39.9	5849	53	0.79	(0.54–1.16)	32	0.78	(0.48–1.24)
P for trend			0.27			0.46
Continuous per 10 g		67 247	498	0.96	(0.90–1.03)	306	0.97	(0.89–1.06)
Dietary folate intake (µg/day)
Q1	160/139 ^c	13 336	100	1	(ref)	66	1	(ref)
Q2	190/166	13 522	87	0.85	(0.62–1.17)	52	0.76	(0.51–1.12)
Q3	212/188	13 500	101	1.01	(0.74–1.39)	59	0.89	(0.60–1.32)
Q4	241/213	13 560	96	0.94	(0.68–1.30)	58	0.86	(0.58–1.28)
Q5	298/267	13 329	114	1.13	(0.80–1.61)	71	1.07	(0.70–1.64)
P for trend			0.36			0.59
Continuous per 100 µg		67 247	498	1.08	(0.89–1.30)	306	1.08	(0.85–1.39)
Interaction of alcohol and dietary folate intake
Low alcohol, high folate^d		25 380	174	1	(ref)	103	1	(ref)
Low alcohol, low folate		25 474	171	0.99	(0.77–1.26)	108	1.06	(0.78–1.44)
High alcohol, high folate		8136	88	1.14	(0.85–1.53)	56	1.29	(0.89–1.87)
High alcohol, low folate		8257	65	0.83	(0.59–1.17)	39	0.87	(0.56–1.35)
P for interaction			0.15			0.08

HR, Hazard ratio; 95% CI: 95% confidence interval; Q1–Q5: Quintile 1–5; ref: Reference.

^a Cox Proportional Hazards Model with age as timescale.

^b Adjusted for: sex, year of birth (5-yr categories), BMI (kg/m²), cigarette-smoking status (current, not current), years of cigarette smoking, number of cigarettes/day, hypertension (yes/no; including use of antihypertensive treatment), energy intake (kcal/day), intake of alcohol (categories), methionine (continuous), vitamin B2, and vitamin B6.

^c Median intake for men/women.

^d Cut-offs:

Alcohol intake: low = 0–14 g/day; high = 15 + g/day.

Dietary intake of folate: high = above median intake in subcohort; low = below median intake in subcohort median intake: 212 µg/day in males and 188 µg/day in females.

Table 4. Multivariable hazard ratios for the association between alcohol and folate intake and the risk of clear-cell renal cell cancer according to molecular subtypes, Netherlands Cohort Study on Diet and Cancer, 1986–2006

			ccRCC, by methylation status of selected genes
			Low methylation ^a			Intermediate methylation			High methylation
	Median intake	Subcohortperson years	No. of cases	HR^b	95% CI	No. of cases	HR	95% CI	No. of cases	HR	95% CI	P for hetero-geneity
Alcohol intake (g/day)
0	0	15 882	24	1^c	(ref)	22	1	(ref)	26	1	(ref)
0.1–4.9	1.8	19 946	33	1.06	(0.62–1.81)	19	0.66	(0.35–1.23)	20	0.58	(0.32–1.05)
5–14	9.3	15 026	23	0.87	(0.48–1.56)	17	0.66	(0.34–1.26)	19	0.56	(0.30–1.04)
15–29	21.8	10 544	16	0.79	(0.40–1.55)	17	0.82	(0.41–1.63)	30	1.08	(0.60–1.94)
30+	39.9	5849	16	1.29	(0.65–2.58)	6	0.46	(0.17–1.25)	9	0.55	(0.24–1.27)	0.23^d
P for trend			0.97			0.25			0.76
Continuous per 10 g		67 247	112	1.05	(0.93–1.19)	81	0.86	(0.72–1.03)	104	0.96	(0.84–1.10)	0.19
Dietary folate intake (µg/day)
Q1	160/139^e	13 336	26	1^f	(ref)	16	1	(ref)	23	1	(ref)
Q2	190/166	13 522	21	0.85	(0.47–1.53)	16	0.95	(0.44–2.03)	12	0.46	(0.22–0.95)
Q3	212/188	13 500	22	0.95	(0.52–1.76)	13	0.80	(0.36–1.77)	22	0.84	(0.44–1.63)
Q4	241/213	13 560	20	0.90	(0.49–1.65)	16	0.94	(0.44–2.04)	21	0.75	(0.38–1.48)
Q5	298/267	13 329	23	1.09	(0.56–2.13)	20	1.15	(0.52–2.57)	26	0.93	(0.45–1.93)	0.84
P for trend			0.77			0.74			0.72
Continuous per 100 µg		67 247	112	1.03	(0.65–1.62)	81	1.22	(0.77–1.93)	104	1.01	(0.71–1.44)	0.38
Interaction of alcohol and dietary folate intake
Low alcohol, high folate^g		25 380	38	1^f	(ref)	28	1	(ref)	32	1	(ref)
Low alcohol, low folate		25 474	42	0.95	(0.58–1.57)	30	1.17	(0.69–1.98)	33	1.16	(0.67–2.00)
High alcohol, high folate		8136	18	1.17	(0.65–2.10)	13	1.11	(0.53–2.29)	25	1.74	(0.96–3.17)
High alcohol, low folate		8257	14	0.73	(0.36–1.46)	10	0.89	(0.39–2.04)	14	1.09	(0.51–2.33)	0.78
P for interaction			0.32			0.47			0.16

ccRCC, clear-cell renal cell carcinoma; HR, hazard ratio, 95% CI: 95% confidence interval; Q1–Q5, Quintile 1–5; ref, Reference.

^a Low = 0–19%, intermediate = 20–39%, and high = 40% or more of the selected gene promoter regions methylated.

^b Cox Proportional Hazards Model with age as timescale.

^c Adjusted for: sex, year of birth (5-yr categories), BMI (kg/m²), cigarette-smoking status (current, not current), years of cigarette smoking, number of cigarettes/day, hypertension (yes/no; including use of antihypertensive treatment), energy intake (kcal/day).

^d Test for heterogeneity of alcohol intake and ccRCC according to methylation status, for the five categories of alcohol intake combined.

^e Median intake for men/women.

^f Adjusted for: sex, year of birth (5-yr categories), BMI (kg/m²), cigarette-smoking status (current, not current), years of cigarette smoking, number of cigarettes/day, hypertension (yes/no; including use of antihypertensive treatment), energy intake (kcal/day), intake of alcohol (categories), methionine (continuous), vitamin B2, and vitamin B6.

^g Cut-offs:

Alcohol intake: low = 0–14 g/day; high = 15+ g/day

Dietary intake of folate: high = above median intake in subcohort; low = below median intake in subcohort median intake: 212 µg/day in males and 188 µg/day in females.

Table 5. Multivariable hazard ratios for the association between alcohol and folate intake and the risk of (clear-cell) renal cell cancer according to the duration of follow-up, Netherlands Cohort Study on Diet and Cancer, 1986–2006

			Renal cell cancer			Clear-cell renal cell cancer
	Median intake	Subcohort person years	No. of cases	HR^a^,^b	95% CI	No. of cases	HR	95% CI
Duration of follow-up and alcohol intake (g/day)
Follow-up 0–20.3 yr
Alcohol intake (g/day)
0	0	15 882	111	1	(ref)	73	1	(ref)
0.1–14	4.1	34 972	234	0.85	(0.66–1.08)	138	0.76	(0.57–1.03)
15+	25.4	16 393	153	0.88	(0.66–1.18)	95	0.88	(0.62–1.25)
P for trend			0.47			0.56
Follow-up 0–11.3 yr
Alcohol intake (g/day)
0	0	15 882	75	1	(ref)	56	1	(ref)
0.1–14	4.1	34 972	133	0.72	(0.54–0.97)	83	0.61	(0.43–0.86)
15+	25.4	16 393	90	0.74	(0.53–1.05)	52	0.59	(0.39–0.91)
P for trend			0.12			0.02
Follow-up 11.3–20.3 yr
Alcohol intake (g/day)
0	0	15 882	36	1	(ref)	17	1	(ref)
0.1–14	4.1	34 972	101	1.11	(0.74–1.66)	55	1.28	(0.73–2.23)
15+	25.4	16 393	63	1.21	(0.76–1.94)	43	1.91	(1.02–3.57)
P for trend			0.43			0.04

HR, hazard ratio; 95% CI: 95% confidence interval; Q1–Q5, Quintile 1–5; ref, Reference.

^a Cox Proportional Hazards Model with age as timescale.

^b Adjusted for: sex, year of birth (5-yr categories), BMI (kg/m²), cigarette-smoking status (current, not current), years of cigarette smoking, number of cigarettes/day, hypertension (yes/no; including use of antihypertensive treatment), energy intake (kcal/day), intake of alcohol (categories), methionine (continuous), vitamin B2, and vitamin B6.