Genetic polymorphisms in inflammatory response genes and their associations with breast cancer risk.

AIM: To explore the association of NFKB1 c.-798_-795delATTG (rs28362491), NFKBIA c.-949C>T (rs2233406), IL-8 c.-352A>T (rs4073), IL-10 c.-854T>C (rs1800871), TNF c.-418G>A (rs361525), and TNF c.-488G>A (rs1800629) polymorphisms with breast cancer risk in an East Chinese population.
METHODS: We conducted a case-control study including 975 study participants (474 breast cancer patients and 501 female controls without cancer) and genotyped the polymorphisms employing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression was used to assess the association of the polymorphisms with breast cancer risk.
RESULTS: We found that the ins/del and del/del genotypes of NFKB1 polymorphism and TT genotype of IL-10 polymorphism significantly increased breast cancer risk (NFKB1 ins/del odds ratio [OR] 1.69, 95% [CI] 1.23-2.33, P=0.001; NFKB1 del/del OR 2.42, 95% CI 1.72-3.42, P<0.001; IL-10 TT OR 2.36, 95% CI 1.58-3.52, P<0.001). On the other hand, the TT genotype of IL-8 polymorphism, GA and AA genotypes of TNF c.-418G>A polymorphism, and GA genotype of TNF c.-488G>A polymorphism significantly reduced breast cancer risk (IL-8 TT OR 0.48, 95% CI 0.33-0.72, P<0.001; TNF c.-418 GA OR 0.58, 95% CI 0.41-0.80, P=0.001; TNF c.-418 AA OR 0.38, 95% CI 0.14-0.98, P=0.044; TNF c.-488 GA OR 0.68, 95% CI 0.48-0.96, P=0.029). When stratified by menopausal status, the CT genotype of NFKBIA polymorphism significantly reduced the risk among pre-menopausal women (OR 0.63, 95% CI 0.40-0.99, P=,043), but not among post-menopausal women.
CONCLUSIONS: NFKB1, NFKBIA, IL-8, IL-10, and TNF polymorphisms could serve as useful predictive biomarkers for breast cancer risk among women in East China.

Breast cancer is the most frequent form of cancer and leading cause of cancer-related deaths among women around the world (1). The cancer accounts for almost one quarter of new cancer cases annually (2), and the incidence continues to increase rapidly, both in China and worldwide (3). Although it has been well-established that breast carcinogenesis is a result of the complex interactions between multiple environmental and genetic factors, the mechanisms of the oncogenesis at the molecular level remain poorly understood. Genetic factors can serve as a susceptibility variable for breast cancer development, and their identification can help to reduce the incidence of breast cancer (4). However, several breast cancer susceptibility genes identified so far, such as BRCA1 and BRCA2, account for only less than 5% of the total breast cancer incidence (5).

Single nucleotide polymorphisms (SNPs) have been extensively investigated for their associations with the risk of various cancers (6-11). As inflammation is caused by a molecular network underlying breast carcinogenesis (12), we propose that SNPs within inflammatory response genes could modify breast cancer predisposition risk. The associations of various inflammatory response gene polymorphisms with breast cancer risk in the Chinese population, especially the East Chinese population, have been understudied. In the current study, we investigated the associations of NFKB1 c.-798_-795delATTG (rs28362491), NFKBIA c.-949C>T (rs2233406), IL-8 c.-352A>T (rs4073), IL-10 c.-854T>C (rs1800871), TNF c.-418G>A (rs361525), and TNF c.-488G>A (rs1800629) polymorphisms with breast cancer risk in East China. Since all these polymorphisms are located in the promoter region, they could affect the transcriptional activity of the gene, resulting in enhanced or reduced cDNA, and eventually protein levels, among their carriers (6,7,13). In addition, despite the relatively well established associations of the polymorphisms with cancer risks in other populations (6-9), little is known about their association with breast cancer risk in East China population, which further motivated us to undertake this research.

Patients and methods

Study participants and ethical considerations

A total of 1032 female study participants – 514 breast cancer patients and 518 controls without cancer were identified at the Jiujiang First People’s Hospital. 474 breast cancer patients and 501 female controls without cancer agreed to participate in the study. The participants were interviewed by trained professionals and data related to smoking, oral contraceptive use, and menopausal status were collected. The patients’ histopathological types and cancer grading were retrieved from their medical records. All the participants were Han Chinese. The study received approval from the Ethics of Human Research Board of Jiujiang First People’s Hospital. Informed consent was obtained from the participants before inclusion in the study.

Genotyping

Polymorphisms were genotyped on the DNA isolated from the peripheral blood samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and the genotypes were verified by direct sequencing of PCR products. For NFKB1 c.-798_-795delATTG (rs28362491), the PCR primers used were 5′-TGG GCA CAA GTC GTT TAT GA-3′ and 5′-CTG GAG CCG GTA GGG AAG-’3 (6) and the annealing temperature was 63.5°C. The PCR product 281 bp (deletion allele) or 285 bp (insertion allele) was digested with PflMI (Van91I) restriction enzyme. The insertion genotype was identified as 2 bands on agarose gel, at 240 bp and 45 bp.

For NFKBIA c.-949C>T (rs2233406) polymorphism, the forward primer was 5′-GGT CCT TAA GGT CCA ATC G-3′ and the reverse primer was 5′-GTT GTG GAT ACC TTG CAC TA-3′ (7). The annealing temperature was also 63.5°C; the 200 bp product was digested with BfaI restriction enzyme; and the CC genotype was identified as 180 + 20 bp bands.

For IL-8 c.-352A>T (rs4073) polymorphism, the forward primer was 5′-CCA TCA TGA TAG CAT CTG T-3′ and the reverse primer was 5′-CCA CAA TTT GGT GAA TTA TTA A-3′ (8). The annealing temperature was 57°C; the 173 bp PCR product was digested with AseI restriction enzyme; and the AA genotype was identified as 152 + 21 bp bands.

For IL-10 c.-854T>C (rs1800871) polymorphism, the forward primer was 5′-TGA GCA AAC TGA GGC ACA GAA AT-3′ and the reverse primer was 5′-GAC AAC ACT ACT AAG GCT CCT TTG GGA-3′ (14). The annealing temperature was 59°C; the 315 bp PCR product was digested with SspI restriction enzyme; and the TT genotype was identified as 291 + 24 bp bands.

For TNF c.-418G>A (rs361525) polymorphism, the primers used were 5′-AAA CAG ACC ACA GAC CTG GTC-3′ and 5′-CTC ACA CTC CCC ATC CTC CCG GAT C-3′ (15). Annealing temperature was 59°C; the 150 bp PCR product was digested with BamHI restriction enzyme; and the GG genotype was identified as 130 + 20 bp bands.

For TNF c.-488G>A (rs1800629) polymorphism, the primers used were 5′-GAG GCA ATA GGT TTT GAG GGC CAT-3′ and 5′-GGG ACA CAC AAG CAT CAA G-3′ (15). The annealing temperature was 61°C; the 107 bp product was digested with NcoI restriction enzyme; and the GG genotype was identified as 87 + 20 bp bands.

Statistical analysis

Statistical analysis was done by using SPSS, version 17.0 (SPSS Inc., Chicago, IL, USA) The differences in age, smoking habit, oral contraceptive use, menopausal status, and genotypic distribution between cases and controls were assessed using a χ2 test. Risk association between the polymorphisms and breast cancer was evaluated using logistic regression analysis. P values of <0.05 were considered significant.

Results

There were no significant differences in mean age, smoking, oral contraceptives use, and menopausal status between patients and controls (Table 1).

Table 1

Demographic characteristics of cases with breast cancer and control participants

Variable	Cases	Controls	P
Mean age, mean ± standard deviation	59.1 ± 7.9	59.4 ± 8.0	0.567
Smoking, n
Yes	138	137	0.540
No	336	364
Oral contraceptive, n
Use	135	159	0.268
No	339	342
Menopausal status, n
Pre	179	213	0.130
Post	295	288
Histopathological type, n *
IDC	346	-	-
DCIS	71	-
ILC	57	-
Grade, n†
1	42	-	-
2	228	-
3	204	-

*IDC – invasive ductal carcinoma; DCIS – ductal carcinoma in situ; ILC – invasive lobular carcinoma.

†Grade 1 – well differentiated; Grade 2 – moderately differentiated; Grade 3 – poorly differentiated.

Genotype distribution

Significant differences between cases and controls were observed for NFKB1 ins/del and del/del genotypes, IL-8 TT genotype, IL-10 CC and TT genotypes, and TNF c.-418 and c.-488 GG and GA genotypes (Table 2). The two TNF polymorphisms were in strong linkage disequilibrium (R2 = 0.819). All the genotypic distributions followed Hardy-Weinberg equilibrium.

Table 2

Genotype distribution of the polymorphisms in cases with breast cancer and control participants

Gene	Genotype	Case, n/%	Controls, n/%	P
NFKB1	ins/ins	93/19.6	162/32.2	<0.001
	ins/del	210/44.3	216/43.1	0.708
	del/del	171/36.1	123/24.6	<0.001
NFKBIA	CC	288/60.8	297/59.3	0.637
	CT	147/31.0	162/32.3	0.657
	TT	39/8.2	42/8.4	0.930
IL-8	AA	192/40.5	186/37.1	0.281
	AT	231/48.7	213/42.5	0.052
	TT	51/10.8	102/20.4	<0.001
IL-10	CC	186/39.2	234/46.7	0.018
	CT	198/41.8	219/43.7	0.054
	TT	90/19.0	48/9.6	<0.001
TNF c.-418	GG	399/84.2	374/74.7	0.774
	GA	69/14.6	112/22.4	0.002
	AA	6/1.3	15/3.0	0.071
TNF c.-488	GG	404/85.2	397/79.2	0.015
	GA	66/13.9	95/19.0	0.034
	AA	4/0.8	9/1.8	0.206

Association between the polymorphisms and breast cancer risk

Significant associations were observed for at least one genotype of all the polymorphisms, with the exception of NFKBIA polymorphism. NFKB1 c.-798_-795delATTG ins/del and del/del genotypes, and IL-10 c.-854 TT genotype were associated with increased breast cancer risk, while IL8 c.-352 TT genotype, TNF c.-418 GA and AA genotypes, and c.-488 GA genotype were significantly associated with a reduced risk (Table 3).

Table 3

Association between the polymorphisms and breast cancer risk in cases with breast cancer and control participants

Gene		Genotype	Cases, n/%	Controls, n/%	Odds ratio (95% confidence interval)	P
NFKB1		ins/ins	93/19.6	162/32.2	-	-
		ins/del	210/44.3	216/43.1	1.69 (1.23-2.33)	0.001
		del/del	171/36.1	123/24.6	2.42 (1.72-3.42)	<0.001
NFKBIA		CC	288/60.8	297/59.3	-	-
		CT	147/31.0	162/32.3	0.94 (0.71-1.23)	0.637
		TT	39/8.2	42/8.4	0.96 (0.60-1.52)	0.855
IL-8		AA	192/40.5	186/37.1	-	-
		AT	231/48.7	213/42.5	1.05 (0.80-1.38)	0.724
		TT	51/10.8	102/20.4	0.48 (0.33-0.72)	<0.001
IL-10		CC	186/39.2	234/46.7	-	-
		CT	198/41.8	219/43.7	1.14 (0.87-1.50)	0.354
		TT	90/19.0	48/9.6	2.36 (1.58-3.52)	<0.001
TNF c.-418		GG	399/84.2	374/74.7	-	-
		GA	69/14.6	112/22.4	0.58 (0.41-0.80)	0.001
		AA	6/1.3	15/3.0	0.38 (0.14-0.98)	0.044
TNF c.-488		GG	404/85.2	397/79.2	-	-
		GA	66/13.9	95/19.0	0.68 (0.48-0.96)	0.029
		AA	4/0.8	9/1.8	0.44 (0.13-1.43)	0.171

Combinations of polymorphisms and their associations with breast cancer risk

When NFKB1 and NFKBIA polymorphic genotypes were combined, positive ORs were observed for all the combinations. However, 5 out of 8 combinations showed significant association with breast cancer risk (Table 4) and only three combinations of IL-8 and IL-10 polymorphisms showed significant association with breast cancer risk (Table 4). Only four combinations of TNF c.-418 and c.-488 were analyzed due to the absence of other combinations in the study participants and two of them showed a significant association with breast cancer risk (Table 4).

Table 4

Combination of polymorphisms and their associations with breast cancer risk in cases with breast cancer and control participants

Genotype combination		Cases	Controls	Odds ratio (95% confidence interval)	P
NFKB1 ins/ins	NFKBIA CC	50	92	-	-
NFKB1 ins/del	NFKBIA CC	127	127	1.84 (1.21-2.81)	0.004
NFKB1 del/del	NFKBIA CC	111	78	2.62 (1.67-4.11)	<0.001
NFKB1 ins/ins	NFKBIA CT	33	57	1.07 (0.61-1.85)	0.822
NFKB1 ins/del	NFKBIA CT	66	68	1.79 (1.10-2.89)	0.019
NFKB1 del/del	NFKBIA CT	46	37	2.29 (1.32-3.98)	0.003
NFKB1 ins/ins	NFKBIA TT	9	13	1.27 (0.51-3.19)	0.604
NFKB1 ins/del	NFKBIA TT	16	21	1.40 (0.67-2.93)	0.369
NFKB1 del/del	NFKBIA TT	14	8	3.22 (1.26-8.20)	0.014
IL-8 AA	IL-10 CC	76	93	-	-
IL-8 AT	IL-10 CC	78	93	1.03 (0.67-1.57)	0.905
IL-8 TT	IL-10 CC	32	48	0.82 (0.48-1.40)	0.460
IL-8 AA	IL-10 CT	82	75	1.34 (0.87-2.07)	0.191
IL-8 AT	IL-10 CT	101	101	1.22 (0.81-1.84)	0.334
IL-8 TT	IL-10 CT	13	43	0.37 (0.19-0.74)	0.005
IL-8 AA	IL-10 TT	33	18	2.24 (1.17-4.29)	0.014
IL-8 AT	IL-10 TT	48	19	3.09 (1.68-5.70)	<0.001
IL-8 TT	IL-10 TT	6	11	0.67 (0.24-1.89)	0.446
TNF c.-418 GG	TNF c.-488 GG	399	374	-	-
TNF c.-418 GA	TNF c.-488 GG	3	17	0.17 (0.05-0.57)	0.004
TNF c.-418 AA	TNF c.-488 GG	2	6	0.31 (0.62-1.56)	0.156
TNF c.-418 GG	TNF c.-488 GA	0	0	N/A	N/A
TNF c.-418 GA	TNF c.-488 GA	66	95	0.65 (0.46-0.92)	0.014
TNF c.-418 AA	TNF c.-488 GA	0	0	N/A	N/A
TNF c.-418 GG	TNF c.-488 AA	0	0	N/A	N/A
TNF c.-418 GA	TNF c.-488 AA	0	0	N/A	N/A
TNF c.-418 AA	TNF c.-488 AA	4	9	0.42 (0.13-1.36)	0.148

Stratification of breast cancer risk association according to menopausal status

For pre-menopausal women, significant associations with breast cancer risk were observed for NFKB1 ins/del and del/del genotypes, NFKBIA CT genotype, IL-8 TT genotype, IL-10 TT genotype, and TNF c.-418 GA and AA genotypes. For post-menopausal women, significant associations with breast cancer risk were observed for NFKB1 ins/del and del/del genotypes, IL-8 TT genotype, IL-10 TT genotype, TNF c.-418 GA and AA genotypes, and TNF c.-488 GA genotype (Table 5).

Table 5

Association between the polymorphisms and breast cancer risk among pre- and post-menopausal women with and without breast cancer

Menopause	Genotype	Cases	Controls	Odds ratio (95% confidence interval)	P
Pre	NFKB1 ins/ins	34	67	-	-
Pre	NFKB1 ins/del	84	90	1.84 (1.11-3.06)	0.019
Pre	NFKB1 del/del	61	56	2.15 (1.24-3.72)	0.006
Post	NFKB1 ins/ins	59	95	-	-
Post	NFKB1 ins/del	126	126	1.61 (1.07-2.42)	0.022
Post	NFKB1 del/del	110	67	2.64 (1.69-4.12)	<0.001
Pre	NFKBIA CC	119	124	-	-
Pre	NFKBIA CT	44	73	0.63 (0.40-0.99)	0.043
Pre	NFKBIA TT	16	16	1.04 (0.50-2.18)	0.913
Post	NFKBIA CC	169	173	-	-
Post	NFKBIA CT	103	89	1.18 (0.83-1.69)	0.348
Post	NFKBIA TT	23	26	0.91 (0.50-1.65)	0.746
Pre	IL-8 AA	72	79	-	-
Pre	IL-8 AT	85	86	1.08 (0.70-1.68)	0.717
Pre	IL-8 TT	22	48	0.50 (0.28-0.91)	0.024
Post	IL-8 AA	120	107	-	-
Post	IL-8 AT	140	127	0.98 (0.69-1.40)	0.924
Post	IL-8 TT	29	54	0.48 (0.28-0.81)	0.006
Pre	IL-10 CC	73	104	-	-
Pre	IL-10 CT	72	92	1.11 (0.73-1.71)	0.620
Pre	IL-10 TT	28	17	2.35 (1.20-4.60)	0.013
Post	IL-10 CC	113	130	-	-
Post	IL-10 CT	120	127	1.09 (0.76-1.55)	0.644
Post	IL-10 TT	62	31	2.30 (1.40-3.79)	0.011
Pre	TNF c.-418 GG	150	162	-	-
Pre	TNF c.-418 GA	26	45	0.62 (0.37-1.06)	0.082
Pre	TNF c.-418 AA	3	6	0.54 (0.13-2.20)	0.389
Post	TNF c.-418 GG	249	212	-	-
Post	TNF c.-418 GA	43	67	0.55 (0.36-0.84)	0.005
Post	TNF c.-418 AA	3	9	0.28 (0.08-1.06)	0.061
Pre	TNF c.-488 GG	154	173	-	-
Pre	TNF c.-488 GA	24	37	0.73 (0.42-1.27)	0.266
Pre	TNF c.-488 AA	1	3	0.37 (0.04-3.64)	0.397
Post	TNF c.-488 GG	252	224	-	-
Post	TNF c.-488 GA	41	58	0.63 (0.41-0.97)	0.038
Post	TNF c.-488 AA	2	6	0.30 (0.06-1.48)	0.139

Risk association according to patient histopathological types

NFKB1 heterozygous and variant genotypes were associated with breast cancer risk in invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS), but not in invasive lobular carcinoma (ILC). IL10 variant genotype was associated with increased breast cancer risk in all three types of breast cancers. On the other hand, IL8 variant genotype and heterozygous genotypes of both TNF polymorphisms were associated with decreased risk of IDC but not of other types of breast cancer (Table 6).

Table 6

Association between the polymorphisms and breast cancer risk according to histopathological type of patients

Histo-pathological type*	Genotype	Cases	Controls	Odds ratio (95% confidence interval)	P
IDC	NFKB1 ins/ins	64	162	-	-
IDC	NFKB1 ins/del	152	216	1.78 (1.25-2.54)	0.002
IDC	NFKB1 del/del	130	123	2.68 (1.83-3.91)	<0.001
DCIS	NFKB1 ins/ins	12	162	-	-
DCIS	NFKB1 ins/del	33	216	2.06 (1.03-4.12)	0.040
DCIS	NFKB1 del/del	26	123	2.85 (1.38-5.88)	0.005
ILC	NFKB1 ins/ins	17	162	-	-
ILC	NFKB1 ins/del	25	216	1.10 (0.58-2.11)	0.767
ILC	NFKB1 del/del	15	123	1.16 (0.56-2.42)	0.688
IDC	NFKBIA CC	212	297	-	-
IDC	NFKBIA CT	102	162	0.88 (0.65-1.19)	0.419
IDC	NFKBIA TT	32	42	1.07 (0.65-1.75)	0.795
DCIS	NFKBIA CC	46	297	-	-
DCIS	NFKBIA CT	25	162	0.99 (0.59-1.68)	0.989
DCIS	NFKBIA TT	0	42	N/A	N/A
ILC	NFKBIA CC	30	297	-	-
ILC	NFKBIA CT	20	162	1.22 (0.67-2.22)	0.510
ILC	NFKBIA TT	7	42	1.65 (0.68-3.99)	0.266
	IL-8 AA	137	186	-	-
IDC	IL-8 AT	174	213	1.10 (0.82-1.49)	0.496
IDC	IL-8 TT	35	102	0.46 (0.29-0.72)	0.001
DCIS	IL-8 AA	29	186	-	-
DCIS	IL-8 AT	33	213	0.99 (0.58-1.69)	0.981
DCIS	IL-8 TT	9	102	0.56 (0.25-1.24)	0.156
ILC	IL-8 AA	26	186	-	-
ILC	IL-8 AT	24	213	0.80 (0.44-1.45)	0.473
ILC	IL-8 TT	7	102	0.49 (0.20-1.17)	0.108
IDC	IL-10 CC	140	234	-	-
IDC	IL-10 CT	147	219	1.12 (0.83-1.50)	0.446
IDC	IL-10 TT	59	48	2.05 (1.33-3.17)	0.001
DCIS	IL-10 CC	29	234	-	-
DCIS	IL-10 CT	29	219	1.06 (0.61-1.84)	0.812
DCIS	IL-10 TT	13	48	2.18 (1.05-4.50)	0.034
ILC	IL-10 CC	17	234	-	-
ILC	IL-10 CT	22	219	1.38 (0.71-2.67)	0.335
ILC	IL-10 TT	18	48	5.16 (2.48-10.73)	<0.001
IDC	TNF c.-418 GG	298	374	-	-
IDC	TNF c.-418 GA	43	112	0.48 (0.32-0.71)	0.001
IDC	TNF c.-418 AA	5	15	0.41 (0.15-1.16)	0.095
DCIS	TNF c.-418 GG	61	374	-	-
DCIS	TNF c.-418 GA	10	112	0.54 (0.27-1.10)	0.092
DCIS	TNF c.-418 AA	0	15	N/A	N/A
ILC	TNF c.-418 GG	40	374	-	-
ILC	TNF c.-418 GA	16	112	1.33 (0.72-2.47)	0.358
ILC	TNF c.-418 AA	1	15	0.62 (0.08-4.84)	0.651
IDC	TNF c.-488 GG	302	397	-	-
IDC	TNF c.-488 GA	41	95	0.56 (0.38-0.84)	0.005
IDC	TNF c.-488 AA	3	9	0.43 (0.11-1.63)	0.219
DCIS	TNF c.-488 GG	61	397	-	-
DCIS	TNF c.-488 GA	10	95	0.68 (0.33-1.386)	0.293
DCIS	TNF c.-488 AA	0	9	N/A	N/A
ILC	TNF c.-488 GG	41	397	-	-
ILC	TNF c.-488 GA	15	95	1.52 (0.81-2.87)	0.188
ILC	TNF c.-488 AA	1	9	1.07 (0.13-8.70)	0.945

*IDC – invasive ductal carcinoma; DCIS – ductal carcinoma in situ; ILC – invasive lobular carcinoma.

Risk association according to patient cancer grading

Increased risk associations were observed for NFKB1 heterozygous genotype (in Grade 2 and 3 patients), NFKB1 variant genotype (in all patients), NFKBIA variant genotype (in Grade 1 patients), IL10 heterozygous genotype (in Grade 1 patients), IL10 variant genotype (in all patients), and TNF c.488 heterozygous genotype (in Grade 1 patients). Decreased risk associations were observed for IL8 heterozygous and variant genotypes, TNF c.418 heterozygous genotype, and TNF c.488 heterozygous genotype (all in Grade 2 and 3 patients) (Table 7).

Table 7

Association between the polymorphisms and breast cancer risk according to cancer grading of patients

Grade*	Genotype	Cases	Controls	Odds ratio (95% confidence interval)	P
1	NFKB1 ins/ins	10	162	-	-
1	NFKB1 ins/del	12	216	0.90 (0.37-2.13)	0.811
1	NFKB1 del/del	20	123	2.63 (1.19-5.83)	0.017
2	NFKB1 ins/ins	44	162	-	-
2	NFKB1 ins/del	101	216	1.72 (1.14-2.59)	0.009
2	NFKB1 del/del	83	123	2.48 (1.60-3.83)	<0.001
3	NFKB1 ins/ins	39	162	-	-
3	NFKB1 ins/del	97	216	1.86 (1.22-2.84)	0.004
3	NFKB1 del/del	68	123	2.29 (1.45-3.63)	<0.001
1	NFKBIA CC	14	297	-	-
1	NFKBIA CT	16	162	2.09 (0.99-4.40)	0.051
1	NFKBIA TT	12	42	6.06 (2.62-13.98)	<0.001
2	NFKBIA CC	144	297	-	-
2	NFKBIA CT	67	162	0.81 (0.57-1.15)	0.253
2	NFKBIA TT	17	42	0.83 (0.45-1.51)	0.554
3	NFKBIA CC	130	297	-	-
3	NFKBIA CT	64	162	0.90 (0.63-1.28)	0.571
3	NFKBIA TT	10	42	0.54 (0.26-1.11)	0.097
1	IL-8 AA	17	186	-	-
1	IL-8 AT	16	213	0.82 (0.40-1.67)	0.588
1	IL-8 TT	9	102	0.96 (0.41-2.24)	0.935
2	IL-8 AA	90	186	-	-
2	IL-8 AT	111	213	0.54 (0.33-0.89)	0.017
2	IL-8 TT	27	102	1.07 (0.76-1.51)	0.669
3	IL-8 AA	85	186	-	-
3	IL-8 AT	104	213	1.06 (0.75-1.51)	0.709
3	IL-8 TT	15	102	0.32 (0.17-0.58)	<0.001
1	IL-10 CC	5	234	-	-
1	IL-10 CT	28	219	5.98 (2.26-15.77)	<0.001
1	IL-10 TT	9	48	8.77 (2.81-27.34)	<0.001
2	IL-10 CC	91	234	-	-
2	IL-10 CT	91	219	1.06 (0.75-1.50)	0.706
2	IL-10 TT	46	48	2.46 (1.53-3.94)	<0.001
3	IL-10 CC	90	234	-	-
3	IL-10 CT	79	219	0.93 (0.65-1.33)	0.722
3	IL-10 TT	35	48	1.89 (1.15-3.12)	0.012
1	TNF c.-418 GG	30	374	-	-
1	TNF c.-418 GA	12	112	1.33 (0.66-2.69)	0.419
1	TNF c.-418 AA	0	15	N/A	N/A
2	TNF c.-418 GG	190	374	-	-
2	TNF c.-418 GA	33	112	0.58 (0.37-0.88)	0.012
2	TNF c.-418 AA	5	15	0.65 (0.23-1.83)	0.421
3	TNF c.-418 GG	179	374	-	-
3	TNF c.-418 GA	24	112	0.44 (0.27-0.72)	0.001
3	TNF c.-418 AA	1	15	0.13 (0.01-1.06)	0.057
1	TNF c.-488 GG	21	397	-	-
1	TNF c.-488 GA	21	95	4.17 (2.19-7.96)	<0.001
1	TNF c.-488 AA	0	9	N/A	N/A
2	TNF c.-488 GG	199	397	-	-
2	TNF c.-488 GA	27	95	0.56 (0.35-0.89)	0.016
2	TNF c.-488 AA	2	9	0.44 (0.09-2.07)	0.301
3	TNF c.-488 GG	184	397	-	-
3	TNF c.-488 GA	18	95	0.40 (0.23-0.69)	0.001
3	TNF c.-488 AA	2	9	0.48 (0.10-2.24)	0.350

*Grade 1 – well differentiated; Grade 2 – moderately differentiated; Grade 3 – poorly differentiated.

Discussion

This study established that the ins/del and del/del genotypes of NFKB1 polymorphism and TT genotype of IL-10 polymorphism significantly increased breast cancer risk, while the TT genotype of IL-8 polymorphism, GA and AA genotypes of TNF c.-418G>A polymorphism, and GA genotype of TNF c.-488G>A polymorphism significantly reduced breast cancer risk. Various lines of evidence have found that chronic inflammation was a risk factor for breast cancer development (16-18). Inflammation can cause DNA damage, and hence carcinogenesis, by inducing and activating oxidant-producing enzymes (19). Events that are linked to inflammation, such as postmenopausal status and obesity, have also been associated with an increased breast cancer risk (6). If inflammation represents an important pathway in carcinogenesis, polymorphisms in the inflammatory response genes could potentially modify cancer predisposition risk.

We analyzed not only the association of individual polymorphisms and breast cancer risk, but also the effects of combinations of functionally related polymorphisms (NFKB1 and NFKBIA; IL-8 and IL-10; and TNF c.-418 and c.-488), menopausal status, histopathological type, and cancer grading. To our knowledge, this is the first study investigating the association between NFKB1 polymorphism and breast cancer risk although there are a few reports on its association with several other cancers. Our findings are in agreement with a study from East China that found that del/del genotype increased the risk of bladder cancer (20). However, a study in Southern Chinese population (21) found that the ins/ins genotype increased the risk of colorectal cancer. Our report also presents the first evidence for the association of NFKBIA polymorphism with the risk of breast cancer in any Asian population. Thus far, only one study has examined this association but it was conducted in a Caucasian population (22). Similarly to our study, they found no association between NFKBIA polymorphism and breast cancer risk. For IL-8 polymorphism, one study conducted in East China showed no association with breast cancer risk (23). Our results are in disagreement with this study, whose genotype distribution deviate significantly from the Hardy-Weinberg equilibrium. However, our results are similar to an Iranian study, which also found an association between the variant genotype of the polymorphism and breast cancer risk (24). On the other hand, a study from East China showed no association between IL-10 polymorphism and breast cancer risk (25), which is different from our results. For TNF c.-418 and c.-488 polymorphisms, an Indian study (26), reported that the AA genotype resulted in an increased breast cancer risk, which is also different from our results. It should be noted, however, that this study had a small sample size with only 40 cases. Similar to our study, Park et al (27) reported a reduced risk of breast cancer among carriers of the A allele of the polymorphisms. However, this risk reduction was not statistically significant.

In conclusion, our study provided evidence for the association of various inflammatory response gene polymorphisms with the risk of breast cancer in East China. The strengths of the present study are the reasonably large sample size and the detailed combination and stratification analyses performed. The limitations of the study are the small number of polymorphisms studied within each gene and the small sample sizes obtained by stratification according to menopausal status, histopathological type, and cancer grading, which might have led to misleading interpretation. Therefore, further studies by independent research groups are needed to confirm our findings.