Abstract
Apolipoprotein E (apoE) mediates lipid metabolism both in peripheral and in the brain. The human APOE gene has three polymorphic alleles that influence the risk for various types of cancer and neurodegenerative diseases. A potential association between APOE allele and the risk for gastric cancer has been implicated, but the specific allele involved and potential associations with the subtype and the grade of cancer malignancy need further clarification. We screened the APOE genotype in 550 gastric cancer patients and 550 non-cancer control individuals and found that the presence of the APOE ε2 and lower serum total cholesterol are associated with an increased risk for gastric cancer (all P ≤ 0.0005). Interestingly, APOE ε2 is also correlated with increased risk for both intestinal and diffuse histotypes but not with TN classification or stage in gastric cancer patients, suggesting that APOE polymorphic alleles are associated with the risk of development but unlikely the progression of gastric cancer. Since ε2 carriers have lower levels of serum total cholesterol than non-ε2 carriers, our findings suggest that the increased risk for gastric cancer by APOE ε2 allele might be mediated through lowered serum total cholesterol levels.
Gastric cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide1,2. The high mortality associated with this disease might be attributed to the limited understanding on the genetic and environmental risk factors for early diagnosis, prevention and targeted therapy. The alterations of gene expression, such as the improper over-expression of oncogenes or the under-expression or disabling of tumor suppressor genes, have been associated with tumorigenesis3. In the study of differential gene expression between gastric cancer and the corresponding normal mucosa, apoE was found to be highly expressed in gastric cancer4. In particular, high apoE expression was correlated with deeper tumor invasion or more positive lymph node metastasis, contributing to shorter survival4. Therefore, the expression level of apoE may be a potential biomarker for predicting the malignancy of gastric cancer.
ApoE is a 299-amino acid glycoprotein that plays a key role in lipid transport and lipoprotein metabolism by binding to members of the low-density lipoprotein receptor family5,6,7. In addition, apoE has been shown to be involved in several biological events including nerve regeneration, antioxidant activities, immune response, as well as the modulation of tumor cell growth, metastasis induction and angiogenesis8,9,10,11. The APOE gene consists of four exons and three introns, and is polymorphic on two single nucleotides resulting in three different alleles (ε2, ε3 and ε4) and six APOE genotypes (ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4 and ε4/ε4)12. The ε2, ε3 and ε4 alleles exhibit different affinity for lipids and receptors; and have a world-wide frequency of 8.4%, 77.9% and 13.7%, respectively13,14. In addition, the APOE genotypes were shown to determine its protein levels in the brain, cerebrospinal fluid, and serum, with ε2/ε2 > ε3/ε3 > ε4/ε415.
APOE gene polymorphism leads to an alteration in lipid and lipoprotein metabolism16,17. In general, compared to the individuals with the ε3 allele, serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol) and apolipoprotein B (apoB) tend to be lower for those with the ε2 allele and higher for ε4 carriers16,18,19,20. Correlation between APOE gene polymorphism and high-density lipoprotein cholesterol (HDL-cholesterol) level was noted in some studies16,21, but not in others20,22. So far, a large number of cross-sectional and prospective studies have reported that low serum cholesterol levels are associated with higher risk for various cancers including gastric cancer23,24,25,26.
In our current study, we also observed a strong inverse association between serum total cholesterol levels and the risk for gastric cancer. In addition, we observed significant lower levels of total cholesterol, LDL-cholesterol and apoB in the ε2 carriers which are consistent with previous reports. However, we only observed a trend of higher levels of total cholesterol, LDL-cholesterol in the ε4 carriers. Intriguingly, the presence of the APOE ε2 allele is also associated with higher risk for both intestinal and diffuse types of gastric cancer. Since ε2 carriers have lower levels of serum total cholesterol than non-ε2 carriers, our findings suggested that the increased risk for gastric cancer by APOE ε2 allele might be mediated through lowered serum total cholesterol levels.
Results
Subject description
A summary of the characteristics for the 550 gastric cancer patients and the 550 cancer-free controls is displayed in Table 1. Median age was 63 years (Range: 18–87 years) in gastric cancer patients and 59 years (Range: 22–84 years) in controls. Male gender was most common in both gastric cancer patients (71.5%) and controls (69.1%). Median BMI was 21.5 (Range: 14.5–32.2) in gastric cancer patients and 24.4 (Range: 17.5–59.7) in controls. In gastric cancer patients, T classification was most commonly either T3 (48.8%) or T4 (33.9%); N classification was fairly evenly distributed but most commonly N3 (40.0%); and stage was most commonly III (61.3%). There are 286 patients (52.0%) and 264 patients (48.0%) for intestinal and diffuse gastric cancer subtype, respectively. Serum lipid parameters and the prevalence of APOE genotypes for both patients and controls are also summarized in Table 1.
Table 1. Characteristics of the study population.
| Variable | Gastric cancer patients (N = 550) | Controls (N = 550) |
|---|---|---|
| Age (years) | 63 (18, 55, 69, 87) | 59 (22, 54, 64, 84) |
| Gender | ||
| Male | 393 (71.5%) | 380 (69.1%) |
| Female | 157 (28.5%) | 170 (30.9%) |
| BMI | 21.5 (14.5, 19.6, 23.4, 32.2) | 24.4 (17.5, 22.6, 26.3, 59.7) |
| T classification | ||
| T1 | 20 (3.6%) | N/A |
| T2 | 75 (13.7%) | N/A |
| T3 | 268 (48.8%) | N/A |
| T4 | 186 (33.9%) | N/A |
| N classification | ||
| N0 | 120 (21.9%) | N/A |
| N1 | 82 (15.0%) | N/A |
| N2 | 127 (23.2%) | N/A |
| N3 | 219 (40.0%) | N/A |
| Stage | ||
| I | 41 (8.0%) | N/A |
| II | 134 (26.1%) | N/A |
| III | 315 (61.3%) | N/A |
| IV | 24 (4.7%) | N/A |
| Gastric cancer subtype | ||
| Intestinal | 286 (52.0%) | N/A |
| Diffuse | 264 (48.0%) | N/A |
| APOE genotype | ||
| ε2/ε2 | 3 (0.5%) | 1 (0.2%) |
| ε2/ε3 | 81 (14.7%) | 60 (10.9%) |
| ε2/ε4 | 7 (1.3%) | 5 (0.9%) |
| ε3/ε3 | 371 (67.5%) | 396 (72.0%) |
| ε3/ε4 | 80 (14.5%) | 85 (15.5%) |
| ε4/ε4 | 8 (1.5%) | 3 (0.5%) |
| Triglycerides (mmol/L) | 1.01 (0.39, 0.79, 1.32, 8.69) | 1.18 (0.27, 0.84, 1.63, 6.14) |
| Total cholesterol (mmol/L) | 4.67 (1.80, 3.97, 5.45, 8.85) | 5.33 (0.91, 4.63, 6.03, 11.08) |
| HDL-cholesterol (mmol/L) | 1.24 (0.47, 1.00, 1.52, 2.78) | 1.41 (0.56, 1.16, 1.74, 3.18) |
| LDL-cholesterol (mmol/L) | 3.02 (0.73, 2.51, 3.76, 7.70) | 3.52 (0.36, 2.96, 4.14, 9.14) |
| ApoA1 (g/L) | 1.06 (0.35, 0.82, 1.35, 2.29) | 1.34 (0.77, 1.17, 1.48, 4.50) |
| ApoB (g/L) | 0.91 (0.29, 0.75, 1.08, 2.10) | 0.97 (0.08, 0.83, 1.11, 1.79) |
| ApoA1/apoB ratio | 1.20 (0.25, 0.90, 1.53, 8.07) | 1.37 (0.50, 1.12, 1.76, 5.93) |
The sample median (minimum, 25th percentile, 75th percentile, maximum) is given for continuous variables. Information was unavailable regarding T classification (1 gastric cancer patient), N classification (2 gastric cancer patients), stage (36 gastric cancer patients), ApoA1 (1 control), ApoB (1 control), and ApoA1/apoB ratio (1 control).
Comparisons of demographic variables and serum lipid parameters according to the presence or absence of APOE ε4 and ε2 in all subjects
Comparisons of demographic variables and serum lipid profile according to the presence or absence of the APOE ε4 and ε2 alleles in all subjects are displayed in Table 2. After adjustment for multiple testing (P ≤ 0.005 considered significant), there were differences between subjects with and without a copy of ε4 regarding apoB (Median: 0.99 vs. 0.93, P = 0.0006) and apoA1/apoB ratio (Median: 1.22 vs. 1.31, P = 0.0001). There were significant differences between subjects with and without a copy of ε2 in the overall sample regarding total cholesterol (Median: 4.72 vs. 5.09, P = 0.0006), LDL-cholesterol (Median: 2.94 vs. 3.35, P < 0.0001), apoB (Median: 0.87 vs. 0.96, P < 0.0001), and apoA1/apoB ratio (Median: 1.47 vs. 1.27, P < 0.0001).
Table 2. Comparison of demographic variables and serum lipid parameters according to the presence or absence of the APOE ε4 and ε2 alleles in all subjects.
| Variable | Comparisons according to presence of ε4 |
Comparisons according to presence of ε2 |
||||
|---|---|---|---|---|---|---|
| ε4 allele present (N = 188) | ε4 allele not present (N = 912) | P-value | ε2 allele present (N = 157) | ε2 allele not present (N = 943) | P-value | |
| Age (years) | 60 (25, 84) | 60 (18, 87) | 0.37 | 61 (25, 81) | 60 (18, 87) | 0.046 |
| Gender | 1.00 | 0.35 | ||||
| Male | 132 (70.2%) | 641 (70.3%) | 105 (66.9%) | 668 (70.8%) | ||
| Female | 56 (29.8%) | 271 (29.7%) | 52 (33.1%) | 275 (29.2%) | ||
| BMI | 23.3 (14.7, 38.3) | 22.9 (14.5, 59.7) | 0.40 | 23.1 (15.2, 32.4) | 22.9 (14.5, 59.7) | 0.94 |
| Triglycerides (mmol/L) | 1.19 (0.46, 5.10) | 1.06 (0.27, 8.69) | 0.006 | 1.11 (0.45, 4.64) | 1.08 (0.27, 8.69) | 0.62 |
| Total cholesterol (mmol/L) | 5.10 (2.29, 10.24) | 5.03 (0.91, 11.08) | 0.16 | 4.72 (1.80, 8.22) | 5.09 (0.91, 11.08) | 0.0006 |
| HDL-cholesterol (mmol/L) | 1.27 (0.50, 3.18) | 1.34 (0.47, 3.02) | 0.034 | 1.39 (0.47, 2.78) | 1.32 (0.48, 3.18) | 0.035 |
| LDL-cholesterol (mmol/L) | 3.46 (1.24, 7.71) | 3.25 (0.36, 9.14) | 0.016 | 2.94 (0.73, 7.70) | 3.35 (0.36, 9.14) | < 0.0001 |
| ApoA1 (g/L) | 1.18 (0.44, 3.69) | 1.24 (0.35, 4.50) | 0.020 | 1.28 (0.35, 2.91) | 1.23 (0.40, 4.50) | 0.11 |
| ApoB (g/L) | 0.99 (0.37, 2.03) | 0.93 (0.08, 2.10) | 0.0006 | 0.87 (0.29, 1.97) | 0.96 (0.08, 2.10) | < 0.0001 |
| ApoA1/apoB ratio | 1.22 (0.39, 3.73) | 1.31 (0.25, 8.07) | 0.0001 | 1.47 (0.44, 8.07) | 1.27 (0.25, 5.93) | < 0.0001 |
The sample median (minimum, maximum) is given for continuous variables. P-values result from a Wilcoxon rank sum test or Fisher’s exact test. P-values of 0.0050 or lower were considered as statistically significant after applying a Bonferroni adjustment for multiple testing.
Evaluation of risk factors for gastric cancer
An evaluation of risk factors for gastric cancer is provided in Table 3. In single variable analysis without adjusting for potential confounding variables, there was strong evidence of an association with increased risk of gastric cancer for older age (P < 0.0001) and decreased BMI (P < 0.0001), but no association was evident for gender (P = 0.39). Additionally, risk of gastric cancer was significantly higher for individuals with lower levels of triglycerides (P < 0.0001), total cholesterol (P < 0.0001), HDL-cholesterol (P < 0.0001), LDL-cholesterol (P < 0.0001), apoA1 (P < 0.0001), and apoB (P < 0.0001). There was a trend toward an increased risk of gastric cancer for subjects with a copy of the APOE ε2 allele; however, this did not survive correction for multiple testing (P = 0.032). There was no statistically significant evidence of an association with risk of gastric cancer for APOE ε4 (P = 0.87) or apoA1/B ratio (P = 0.087).
Table 3. Evaluation of risk factors for gastric cancer.
| Variable | Single variable analysis |
Multivariable analysis |
||
|---|---|---|---|---|
| OR (95% CI) | P-value | OR (95% CI) | P-value | |
| Age1 | Overall test of difference: P < 0.0001 | Overall test of difference: P < 0.0001 | ||
| ≤55 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 55.01–60 | 0.56 (0.40, 0.79) | 0.001 | 0.60 (0.38, 0.94) | 0.024 |
| 60.01–65 | 1.08 (0.76, 1.53) | 0.66 | 0.88 (0.56, 1.39) | 0.58 |
| >65 | 2.28 (1.66, 3.13) | <0.0001 | 2.12 (1.40, 3.22) | 0.0004 |
| Gender1 | ||||
| Female | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| Male | 1.12 (0.86, 1.45) | 0.39 | 1.00 (0.70, 1.42) | >0.99 |
| BMI1 | Overall test of difference: P < 0.0001 | Overall test of difference: P < 0.0001 | ||
| >25 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 23.01–25 | 2.69 (1.84, 3.93) | <0.0001 | 2.96 (1.87, 4.70) | 0.0004 |
| 21.01–23 | 4.82 (3.30, 7.03) | <0.0001 | 6.69 (4.17, 10.75) | <0.0001 |
| ≤21 | 17.02 (11.26, 25.73) | <0.0001 | 27.23 (15.96, 46.45) | <0.0001 |
| APOE ε4 allele2 | ||||
| Not present | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| Present | 1.03 (0.75, 1.40) | 0.87 | 1.25 (0.82, 1.92) | 0.30 |
| APOE ε2 allele1 | ||||
| Not present | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| Present | 1.45 (1.03, 2.05) | 0.032 | 2.34 (1.46, 3.76) | 0.0004 |
| Triglycerides3 | Overall test of difference: P < 0.0001 | Overall test of difference: P = 0.061 | ||
| >1.50 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 1.091–1.50 | 1.64 (1.17, 2.31) | 0.004 | 1.74 (1.09, 2.77) | 0.020 |
| 0.811–1.09 | 2.02 (1.44, 2.85) | <0.0001 | 1.65 (1.01, 2.72) | 0.046 |
| ≤0.81 | 2.08 (1.48, 2.92) | <0.0001 | 1.24 (0.71, 2.15) | 0.45 |
| Total cholesterol4 | Overall test of difference: P < 0.0001 | Overall test of difference: P < 0.0001 | ||
| >5.75 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 5.031–5.75 | 1.78 (1.26, 2.53) | 0.001 | 1.75 (1.16, 2.65) | 0.008 |
| 4.291–5.03 | 2.22 (1.57, 3.14) | <0.0001 | 1.91 (1.25, 2.92) | 0.003 |
| ≤4.291 | 5.97 (4.13, 8.63) | <0.0001 | 6.28 (3.98, 9.89) | <0.0001 |
| HDL-cholesterol5 | Overall test of difference: P < 0.0001 | Overall test of difference: P < 0.0001 | ||
| >1.63 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 1.331–1.63 | 1.77 (1.25, 2.50) | 0.001 | 2.52 (1.65, 3.88) | <0.0001 |
| 1.081–1.33 | 1.81 (1.28, 2.55) | 0.0007 | 3.08 (1.97, 4.81) | <0.0001 |
| ≤1.08 | 3.34 (2.36, 4.73) | <0.0001 | 5.99 (3.67, 9.78) | <0.0001 |
| LDL-cholesterol6 | Overall test of difference: P < 0.0001 | Overall test of difference: P = 0.35 | ||
| >3.97 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 3.271–3.97 | 1.37 (0.97, 1.93) | 0.072 | 1.11 (0.71, 1.72) | 0.65 |
| 2.711–3.27 | 1.96 (1.39, 2.77) | 0.0001 | 1.15 (0.74, 1.81) | 0.53 |
| ≤2.71 | 4.16 (2.92, 5.94) | <0.0001 | 1.54 (0.94, 2.51) | 0.084 |
| ApoA17 | Overall test of difference: P < 0.0001 | Overall test of difference: P < 0.0001 | ||
| >1.45 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 1.231–1.45 | 0.87 (0.61, 1.24) | 0.43 | 1.03 (0.67, 1.56) | 0.90 |
| 0.991–1.23 | 1.44 (1.02, 2.03) | 0.037 | 1.73 (1.12, 2.62) | 0.013 |
| ≤0.99 | 10.82 (7.11, 16.47) | <0.0001 | 13.41 (7.93, 22.67) | <0.0001 |
| ApoB8 | Overall test of difference: P < 0.0001 | Overall test of difference: P = 0.48 | ||
| >1.09 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 0.951–1.09 | 0.83 (0.59, 1.16) | 0.27 | 0.85 (0.54, 1.33) | 0.48 |
| 0.791–0.95 | 1.10 (0.79, 1.54) | 0.57 | 0.74 (0.47, 1.16) | 0.19 |
| ≤0.79 | 1.83 (1.30, 2.56 | 0.0005 | 0.99 (0.61, 1.61) | 0.97 |
| ApoA1/apoB ratio9 | Overall test of difference: P = 0.087 | Overall test of difference: P < 0.0001 | ||
| >1.65 | 1.00 (reference) | N/A | 1.00 (reference) | N/A |
| 1.291–1.65 | 1.35 (0.96, 1.90) | 0.082 | 2.89 (1.87, 4.47) | <0.0001 |
| 1.041–1.29 | 1.49 (1.06, 2.10) | 0.023 | 4.60 (2.86, 7.42) | <0.0001 |
| ≤1.04 | 3.73 (2.62, 5.30) | <0.0001 | 11.84 (7.18, 19.52) | <0.0001 |
OR = odds ratio; CI = confidence interval. ORs, 95% CIs, and p-values result from logistic regression models. Multivariable models were adjusted for age and BMI, as well as other variables that were associated with risk of gastric cancer in single variable analysis with a p-value of 0.10 or lower. Because many of these other variables were very highly correlated with one another which results in high potential for collinearity, all variables satisfying this criteria could not always be adjusted for in all models, and therefore model adjustments in multivariable analysis were as follows:
1Adjusted for age, BMI, APOE ε2, triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, ApoA1, ApoB, and ApoA1/apoB ratio.
2Adjusted for age, BMI, APOE ε4, triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, ApoA1, ApoB, and ApoA1/apoB ratio.
3Adjusted for age, BMI, APOE ε2, triglycerides, HDL-cholesterol, LDL-cholesterol, ApoA1, ApoB, and ApoA1/apoB ratio.
4Adjusted for age, BMI, APOE ε2, total cholesterol, and ApoA1/apoB ratio.
5Adjusted for age, BMI, APOE ε2, triglycerides, HDL-cholesterol, LDL-cholesterol, and ApoB.
6Adjusted for age, BMI, APOE ε2, triglycerides, LDL-cholesterol, ApoA1, and ApoB.
7Adjusted for age, BMI, APOE ε2, triglycerides, HDL-cholesterol, LDL-cholesterol, and ApoA1.
8Adjusted for age, BMI, APOE ε2, triglycerides, HDL-cholesterol, ApoA1, and ApoB.
9Adjusted for age, BMI, APOE ε2, triglycerides, and total cholesterol. P-values of 0.0042 or lower were considered as statistically significant after applying a Bonferroni adjustment for multiple testing.
In multivariable analysis we adjusted for age, BMI, and other variables (see Table 3 legend for details). In this multivariable analysis, there was still strong evidence of an association with increased risk of gastric cancer for age (P < 0.0001), BMI (P < 0.0001), total cholesterol (P < 0.0001), HDL-cholesterol (P < 0.0001), apoA1 (P < 0.0001), and apoA1/B ratio (P < 0.0001). Additionally, the association between APOE ε2 and increased risk of gastric cancer strengthened and became significant after adjustment for multiple testing (P = 0.0004). Associations with gastric cancer for triglycerides (P = 0.061), LDL cholesterol (P = 0.35) and apoB (P = 0.48) weakened in multivariable analysis and were no longer even nominally significant, while the lack of association of gender and APOE ε4 with risk of gastric cancer remained unchanged (P ≥ 0.30). Intriguingly, when examining the association between APOE ε2 and gastric cancer subtype, an increased risk of gastric cancer was noted in ε2 carriers for both intestinal gastric cancer (P = 0.017) and diffuse gastric cancer (P = 0.0006) (Table 4).
Table 4. Association between APOE ε2 and gastric cancer subtype.
| Gastric cancer subtype | Single variable analysis |
Multivariable analysis |
||
|---|---|---|---|---|
| OR (95% CI) | P-value | OR (95% CI) | P-value | |
| Intestinal | 1.41 (0.94, 2.11) | 0.10 | 2.01 (1.13, 3.56) | 0.017 |
| Diffuse | 1.51 (1.00, 2.27) | 0.051 | 2.86 (1.57, 5.21) | 0.0006 |
OR = odds ratio; CI = confidence interval. ORs, 95% CIs, and p-values result from logistic regression models. Multivariable models were adjusted for all variables that were associated with risk of overall gastric cancer in single variable analysis with a p-value of 0.10 or lower, which were age, BMI, triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, ApoA1, ApoB, and ApoA1/apoB ratio.
Analysis of the associations of APOE genotypes with TN classification and stage in gastric cancer patients
Associations of APOE ε4 and ε2 with T classification, N classification, and stage in gastric cancer patients are displayed in Table 5. There was no evidence of an association between ε4 and T classification (P = 0.38), N classification (P = 0.96), or stage (P = 0.59), and also no evidence of an association between ε2 and T classification (P = 0.63), N classification (P = 0.78), or stage (P = 0.23).
Table 5. Association of APOE ε4 and ε2 with TN classification and stage in gastric cancer patients.
| Variable | Comparison of TN classification and stage according to presence of the APOEε4 allele |
Comparison of TN classification and stage according to presence of the APOEε2 allele |
||||
|---|---|---|---|---|---|---|
| ε4 allele present (N = 95) | ε4 allele not present (N = 455) | P-value | ε2 allele present (N = 91) | ε2 allele not present (N = 459) | P-value | |
| T classification | ||||||
| T1 | 3 (3.2%) | 17 (3.7%) | 0.38 | 5 (5.6%) | 15 (3.3%) | 0.63 |
| T2 | 12 (12.6%) | 63 (13.9%) | 9 (10.0%) | 66 (14.4%) | ||
| T3 | 54 (56.8%) | 214 (47.1%) | 49 (54.4%) | 219 (47.7%) | ||
| T4 | 26 (27.4%) | 160 (35.2%) | 27 (30.0%) | 159 (34.6%) | ||
| N classification | ||||||
| N0 | 23 (24.2%) | 97 (21.4%) | 0.96 | 17 (18.9%) | 103 (22.5%) | 0.78 |
| N1 | 11 (11.6%) | 71 (15.7%) | 19 (21.1%) | 63 (13.8%) | ||
| N2 | 22 (23.2%) | 105 (23.2%) | 20 (22.2%) | 107 (23.4%) | ||
| N3 | 39 (41.1%) | 180 (39.7%) | 34 (37.8%) | 185 (40.4%) | ||
| Stage | ||||||
| I | 8 (9.0%) | 33 (7.8%) | 0.59 | 6 (7.3%) | 35 (8.1%) | 0.23 |
| II | 23 (25.8%) | 111 (26.1%) | 25 (30.5%) | 109 (25.2%) | ||
| III | 56 (62.9%) | 259 (60.9%) | 51 (62.2%) | 264 (61.1%) | ||
| IV | 2 (2.2%) | 22 (5.2%) | 0 (0.0%) | 24 (5.6%) | ||
P-values result from a Wilcoxon rank sum test. Information was unavailable regarding T classification (1 gastric cancer patient), N classification (2 gastric cancer patients), and stage (36 gastric cancer patients). P-values of 0.0167 or lower were considered as statistically significant after applying a Bonferroni adjustment for multiple testing.
Discussion
Our case-control study of 550 gastric cancer patients and 550 cancer-free controls evaluated for the first time the effects of APOE gene polymorphism and serum cholesterol levels on the risk of gastric cancer among a Chinese Han population. Our results show that the presence of APOE ε2 allele and low serum levels of total cholesterol, HDL-cholesterol, apoA1 and apoA1/B ratio are associated with an increased risk of gastric cancer. Interestingly, APOE ε2 is also correlated with increased risk for both intestinal and diffuse histotypes. However, APOE genotypes do not appear to influence the invasion and metastasis of gastric cancer as reflected by TN classification and stage.
In gastric cancer, serum cholesterol levels have been reported to inversely correlate with the risk of disease25,27,28. In our current study, we also observed a strong inverse association between serum total cholesterol levels and the risk of gastric cancer. To date, the underlying mechanism for such an association remains unclear. Previous reports have shown that the serum levels of total cholesterol, LDL-cholesterol and apoB levels are associated with APOE genotypes, with individuals carrying the ε2 allele having lower and those carrying the ε4 displaying higher levels compared to the more common ε3 allele16,18,19,20. In our studied population, we observed significant lower levels of total cholesterol, LDL-cholesterol and apoB in the ε2 carriers which are consistent with previous reports. Additionally, we observed a significant higher level of apoB in the ε4 carriers. However, we only observed a trend of higher levels of total cholesterol, LDL-cholesterol in the presence of ε4 allele (Table 2). Since ε2 carriers have lower levels of serum total cholesterol than non-ε2 carriers, our findings suggest that the increased risk effect for gastric cancer by APOE ε2 allele might be mediated through lowered total cholesterol level.
The effect of APOE genotypes on the risk of numerous cancers has been previously investigated, including breast, prostate, ovarian, colorectal, head and neck cancer29,30,31,32,33,34,35,36. However, the results have been conflicting which might be attributed to limited sample sizes or the inherent differences among different ethnic populations. Our current study in a Chinese Han population observed that the presence of ε2 allele is associated with an increased risk for gastric cancer with OR of 2.34 in multivariable analysis. In contrast, a previous study reported a protective effect of the APOE ε2 allele against gastric cancer in an Italian Caucasian population37. The discrepancy between these two studies might be attributed to the different ethnic groups that were studied, or potentially due to unmeasured confounding variables. Future extension of these findings is necessary to clarify and further understand the association between APOE ε2 and gastric cancer risk in both ethnic populations.
ApoE has recently been identified as a potential tumor-associated marker in gastric cancer from gene expression analysis4,38,39. Higher apoE expression was found in gastric cancer, particularly in advanced T and N grades. We therefore examined the association between APOE genotypes and T classification, N classification, and stage in gastric cancer patients. Our data showed no significant association between APOE genotypes and various classifications or stage of gastric cancer, suggesting that APOE polymorphic alleles are associated with the risk of development but unlikely the progression of gastric cancer.
In summary, our study confirmed the associations of lower levels of serum cholesterol with the incidence of gastric cancer in a Chinese Han population. Importantly, our study reports for the first time that APOE ε2 as a risk allele for both intestinal and diffuse types of gastric cancer in our studied population, which might be partly attributed to the lower serum total cholesterol of ε2 carriers compared to those with ε3 or ε4 alleles.
Methods
Approval for this study was obtained from the Ethics Committees of the Xiamen University and Fujian Medical University Union Hospital. All experiments were performed in accordance with the approved guidelines. All individuals who participated in this study gave written informed consent.
Study subjects
A total of 550 gastric cancer patients with histological confirmation and 550 cancer-free controls seen at the Fujian Medical University Union Hospital in China between 2011 and 2014 were included in this case–control study. Information was collected regarding age, gender, body mass index (BMI), and serum lipid parameters which include the levels of triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, apoA1, apoB, and apoA1/apoB ratio. None of the patients underwent pre-operative chemotherapy or radiation therapy. Histopathological evaluations were performed with reference to the Japanese Classification of Gastric Carcinoma, 3rd English edition40.
APOE genotyping and quality control
Genomic DNA was extracted from peripheral blood using a DNA extraction kit (Zeesan Biotech, Xiamen). Genotyping of the two APOE SNPs (rs429358:T/C; rs7412:T/C) was carried out using the APOE SNP genotyping kits (Memorigen Biotech, Xiamen, China) and the Applied Biosystems® 7500 real-time PCR Systems (Applied Biosystems, Foster City, CA). Data analysis was performed by measuring the allele-specific fluorescence. As a measure for quality control, three samples with known APOE genotypes were included in each assay. Additionally, the genotyping analysis was blinded to the subject’s case or control status. Finally, 10% of the total samples were randomly selected and retested with 100% concordance. There was no evidence of a departure from Hardy-Weinberg Equilibrium in study controls for either rs429358 (P = 0.79) or rs7412 (P = 0.71).
Statistical analysis
Continuous variables were summarized with the sample median, minimum, 25th percentile, 75th percentile, and maximum. Categorical variables were summarized with number and percentage of patients. Due to the small number of subjects with APOE ε4/ε4 and ε2/ε2 genotypes, we utilized a dominant model in analyzing both APOE ε4 (presence vs. absence of the ε4 allele) and ε2 (presence vs. absence of the ε2 allele) in all analysis. Comparisons of demographic variables and serum lipid parameters according to the presence of the APOE ε4 or ε2 allele were made using a Wilcoxon rank sum test or Fisher’s exact test. Risk factors for gastric cancer were evaluated using single variable (i.e. unadjusted) and multivariable logistic regression models. Multivariable models were adjusted for all variables that were associated with risk of gastric cancer in single variable analysis with a p-value of 0.10 or lower, although there were some exceptions to this due to the high degree of correlation between many of the variables of interest and the resulting potential for collinearity (see Table 3 legend for details). Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated. For easier interpretation of results, all continuous variables were categorized based on approximate sample quartiles for use in association analysis. The association between APOE ε2 and gastric cancer subtype was evaluated using single variable (i.e. unadjusted) and multivariable logistic regression models. Multivariable models were adjusted for all variables that were associated with risk of gastric cancer in single variable analysis with a p-value of 0.10 or lower (see Table 4 legend for details). Associations of APOE ε4 and ε2 with TN classification and stage in gastric cancer patients were examined using a Wilcoxon rank sum test.
In order to adjust for multiple testing, we utilized a Bonferroni correction separately for each group of similar tests. Specifically, p-values ≤ 0.0050 were considered as significant when comparing demographic variables and serum lipid parameters according to presence of ε4 and ε2; p-values ≤ 0.0042 were considered as significant when evaluating risk factors for gastric cancer; p-values ≤ 0.0167 were considered as significant when evaluating associations of ε4 and ε2 with TN classification and stage in gastric cancer patients; and p-values ≤ 0.025 were considered as significant when evaluating the association between APOE ε2 and gastric cancer subtype. All statistical analysis was performed using R Statistical Software (version 2.14.0; R Foundation for Statistical Computing, Vienna, Austria).
Additional Information
How to cite this article: Kang, R. et al. Apolipoprotein E epsilon 2 allele and low serum cholesterol as risk factors for gastric cancer in a Chinese Han population. Sci. Rep. 6, 19930; doi: 10.1038/srep19930 (2016).
Acknowledgments
The authors are grateful to all subjects for participation in our study. This study was supported by grants (81370459, 31400914) from the National Natural Science Foundation of China (to X.C.) and by the Fundamental Research Funds for the Central Universities (20720150051, to X.C.).
Footnotes
Author Contributions Conceived and designed the experiments: X.C., G.B., H.X. and Y.Z. Collected the samples and clinical data: P.L., C.H. and L.C. Performed APOE genotyping: R.K., T.W., X.L., Z.W., Z.Z. and L.Z. Analyzed the data: M.H., X.C., T.W. and R.K. Wrote the paper: X.C., M.H., G.B. and R.K.
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