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New insights into the genetic contribution of ALDH2 rs671 in pancreatic carcinogenesis: Evaluation by mediation analysis

Abstract A functional variant on ALDH2 rs671 (G>A) confers a protective effect against alcohol‐induced carcinogenesis through an indirect pathway mediated by decreased alcohol consumption. Conversely, this variant also contributes to the accumulation of carcinogenic agents, resulting in a direct carcinogenic effect. This study aimed to separately quantify these two opposing effects of the rs671 A allele on pancreatic cancer risk and explore the impact of the rs671 A allele and alcohol consumption on pancreatic carcinogenesis. We included 426 cases and 1456 age‐ and sex‐matched controls. Odds ratio (OR) and 95% confidence interval (CI) for alcohol consumption were estimated using a conditional logistic regression model. By defining rs671 A allele and alcohol consumption as exposure and mediator, respectively, we used mediation analysis to decompose the total‐effect OR of the rs671 A allele into direct‐ and indirect‐effect ORs. Alcohol consumption (10 g/d) was associated with pancreatic cancer risk (OR, 1.05; 95% CI, 1.01‐1.10), but tests for interaction between the rs671 A allele and alcohol consumption were nonsignificant, indicating that the effect of alcohol consumption did not vary by genotype. Mediation analysis showed that the nonsignificant total effect (OR, 1.15; 95% CI, 0.92‐1.44) can be decomposed into the carcinogenic direct (OR, 1.34; 95% CI, 1.04‐1.72) and protective indirect effect (OR, 0.86; 95% CI, 0.77‐0.95). This study supports the association between alcohol consumption and pancreatic cancer risk and indicates the potential contribution of the rs671 A allele to pancreatic carcinogenesis through impaired metabolism of known or unknown ALDH2 substrates.


| INTRODUC TI ON
Pancreatic cancer remains a fatal type of cancer, with a 5-year survival rate in Japan of only 8.5%. 1,2 Investigation of modifiable risk factors is therefore of critical importance. One candidate factor is alcohol consumption. Although alcohol consumption has been categorized as a risk factor with limited suggestive evidence, several epidemiological studies have shown a significantly increased pancreatic cancer risk with heavy alcohol consumption. 3 Nevertheless, the biological mechanism underlying an association between alcohol consumption and pancreatic cancer has not been fully elucidated.
One important hypothesis among several possible mechanistic hypotheses is the involvement of ethanol metabolites, in particular acetaldehyde, which is considered one of the most likely carcinogens in alcohol. 4 Studies on the East Asian-specific functional SNP of ALDH2 rs671 (c.1510G>A [p. Glu504Lys]) have revealed that acetaldehyde is a plausible carcinogen in some alcohol-related cancers. 5 Ingested alcohol is metabolized predominantly to acetaldehyde through an oxidative pathway in the liver, and acetaldehyde is further oxidized to acetate by ALDH enzymes. 6 Aldehyde dehydrogenase 2 has the highest affinity for acetaldehyde (Km < 1 µM) among ALDH isoforms and is predominantly responsible for the oxidation of acetaldehyde. 6,7 The enzymatic activity of ALDH2 is dramatically reduced by the ALDH2 rs671 A allele, and individuals with this variant experience rapid accumulation of blood acetaldehyde after alcohol ingestion. 6 The rs671 variant-drinking interaction observed in studies of alcohol-related cancers (eg, head and neck and esophageal cancers) 8 is accordingly evidence for the possible involvement of accumulated ethanol-derived acetaldehyde in alcohol-induced carcinogenesis. 5 However, with regard to pancreatic cancer, although a few epidemiological studies 9,10 have reported a possible association of rs671 with pancreatic cancer risk, no clear evidence for the rs671 variant-drinking interaction is yet available.
In intriguing contrast to its carcinogenic effect, the rs671 A allele also contributes to decreasing alcohol consumption. This effect is due to the occurrence of acetaldehyde-related adverse effects (eg, flushing, headache, palpitation, and nausea), which induce the individual to decrease their alcohol intake 11,12 and thereby protect against alcohol-induced carcinogenesis. 13 Due to these mutually opposing effects, the impact of the carcinogenic direct effect of the rs671 A allele appears attenuated by its indirect protective effect, mediated by reduced drinking intensity. Nevertheless, the conventional approach to risk using multivariate analysis focuses on simple adjustment for covariates, and cannot fully examine the mediation effects between rs671 and drinking intensity on the risk of pancreatic cancer.
Here, we undertook a pancreatic cancer case-control study using mediation analysis 14 to decompose the total effect of the rs671 A allele on pancreatic cancer risk into direct and indirect effects. We previously used mediation analysis to successfully decompose the total effect of the rs671 A allele on digestive tract cancer risk into direct and indirect effects and determined the site-specific impact of the carcinogenic direct effect as well as the site-agnostic impact of the protective indirect effect. 13 In the present study, by quantifying the direct and indirect effects of the rs671 A allele on the risk of pancreatic cancer separately, we aimed to explore the impact of the ALDH2 rs671 variant in conjunction with alcohol consumption on pancreatic carcinogenesis.

| Study population
The study population was selected from participants in HERPACC, which is described in detail elsewhere. 15 Controls were first-visit outpatients confirmed not to have cancer or a history of neoplasm. The most common reason for the visit to the Aichi Cancer Center Hospital among the control subjects was secondary screening after primary screening (31%), followed by patient discretion (25%), and referral from another clinic (24%). We obtained written informed consent and collected a self-administered questionnaire and a peripheral blood sample from each participant. The study was approved by the Institutional Ethics Committee of Aichi Cancer Center and carried out in accordance with the Declaration of Helsinki.

| Genotyping
DNA of each participant was extracted from the buffy coat fraction with a DNA Blood mini kit (Qiagen). Single nucleotide polymorphism and pancreatic cancer risk and indicates the potential contribution of the rs671 A allele to pancreatic carcinogenesis through impaired metabolism of known or unknown ALDH2 substrates.

K E Y W O R D S
alcohol consumption, aldehyde dehydrogenase 2, case-control study, mediation analysis, pancreatic cancer genotyping was carried out using TaqMan Assays on the 7500 Real-Time PCR System (Applied Biosystems). Genotype distributions in the controls were assessed for the Hardy-Weinberg equilibrium using the χ 2 test.

| Environmental factors
Information on environmental factors was obtained using a selfadministered questionnaire that questioned participants on their exposure status before the development of the symptoms for which they first visited the hospital. Responses were checked by trained interviewers.
To measure drinking intensity, we adopted daily alcohol intake (g/d). Conversion from the information in the questionnaire to the total amount of daily alcohol consumption has been described in detail elsewhere. 13 In brief, the participants were first asked about their drinking status (never, including "almost never", former, or current drinkers). We then required former and current drinkers to describe the frequency of alcohol drinking, type of beverage, and average consumption during each drinking session based on their average drinking behavior before stopping drinking (former drinkers) and during the year before the development of the symptoms or diagnosis (current drinkers). Each category of drinking frequency was assigned a score, as follows: 0 for never drinkers, 0.5 for <1 d/wk, 1.5 for 1-2 d/wk, 3.5 for 3-4 d/wk, and 6 for ≥5 d/wk in HERPACC-2; and 0 for never drinkers, 0.5 for 1-3 d/mo, 1.5 for 1-2 d/wk, 3.5 for 3-4 d/wk, 5.5 for 5-6 d/wk, and 7 for every day in HERPACC-3.
Using the information on type of beverage and average consumption during each drinking session, we estimated the total amount of pure alcohol consumed per single drinking session. Based on the concentration of alcohol in each beverage, the HERPACC Study assumed that 23 g alcohol was contained in 180 mL (one "go") of Japanese sake, 633 mL (one large bottle) of beer, 90 mL (half a "go") of shochu (distilled spirit), 60 mL (double shot) of whiskey, and 200 mL (two and a half glasses) of wine. Finally, daily alcohol intake was estimated by multiplying the total amount of pure alcohol consumed per single drinking session by the frequency score/7. As a measure of cumulative smoking exposure, we used pack-years, calculated by multiplying the number of packs consumed per day by the number of years of smoking. Body mass index (BMI) was estimated as the self-reported weight (kg) divided by the square of the self-reported height (m).
Frequencies of meat (beef or pork) and processed meat intake were categorized into the three categories of <1 time/wk, 1-4 times/wk, and ≥5 times/wk. A family history of pancreatic cancer in parents and siblings and history of diabetes were classified as "yes" or "no".

| Statistical analysis
Analyses were carried out for HERPACC-2 and HERPACC-3 separately. In order to obtain more accurate point estimates, a pooled analysis was further undertaken by combining the two studies. We used the t test or χ 2 test to evaluate differences in the distribution of covariates between cases and controls. Odds ratios and their two-sided 95% CIs for drinking intensity (0, 0< to <23, 23≤ to <46, and ≥46 g/d) were estimated using a conditional logistic regression model, with adjustment for age (continuous), pack-years (continuous), BMI (kg/m 2 ) (continuous), frequency of meat intake (<1, 1-4, ≥5 times/wk), frequency of processed meat intake (<1, 1-4, ≥5 times/ wk), family history of pancreatic cancer (yes/no), history of diabetes (yes/no), and ALDH2 rs671 genotype. Trend analysis for drinking intensity (unit OR per 10 g daily alcohol intake) was also carried out with the same covariate adjustment. The interaction term that corresponded to multiplying a one-allele change in rs671 by a 10-g change in daily alcohol intake was further added into the model. We assessed the variant-drinking interaction on both the additive and multiplicative scales using the relative excess risk due to interaction 17 and the Wald test of the coefficient of the interaction term, respectively.
A mediation analysis was carried out to decompose the totaleffect OR of ALDH2 rs671 A allele on pancreatic cancer risk into direct-and indirect-effect ORs with the paramed command of STATA. 18 In the mediation analysis, we assumed a hypothetical causal structure between rs671 A allele (exposure), drinking intensity (mediator), and pancreatic cancer risk (outcome) as depicted in the directed acyclic graph presented in Figure 1. Details of the methodology of this analysis are described elsewhere. 14,19,20 Briefly, we estimated direct-and indirect-effect ORs by combining two models, namely: (a) the linear regression model for the mediator (drinking intensity) conditioned on the exposure (rs671 A allele) and covariates; and (b) the logistic regression model for the outcome (pancreatic cancer risk) conditioned on the exposure, the mediator, and covariates. As covariates, the model included age, sex, pack-years, BMI, frequency of meat intake, frequency of processed meat intake, family history of pancreatic cancer, and history of diabetes. Daily alcohol intake (g/d) as a measure of drinking intensity was square-root transformed and entered in the model. In the pooled analysis, we additionally included HERPACC version (2/3) as a covariate. The direct effect represents F I G U R E 1 Directed acyclic graph illustrating assumed causal structure. A hypothetical causal structure between rs671 A allele (exposure), drinking intensity (mediator), and pancreatic cancer risk (outcome) is shown. We decomposed the total effect of rs671 A allele on pancreatic cancer risk into direct and indirect effects using mediation analysis. ALDH2, aldehyde dehydrogenase 2 the effect of the rs671 A allele on pancreatic cancer risk through pathways that are independent of drinking intensity, whereas the indirect effect is interpretable as the effect controlled by drinking intensity. The direct-effect OR multiplied by the indirect-effect OR is equivalent to the total-effect OR. 14 To preclude concerns about residual confounding by smoking, analyses restricted to never smokers were also carried out.
Some data for daily alcohol intake, pack-years, BMI, frequency of meat intake, frequency of processed meat intake, and history of diabetes were incomplete (Table 1). These missing values were handled by applying multiple imputation using chained equations. 21 The results from the 10 generated imputed datasets were combined using Rubin's rules. 22 All analyses were carried out using STATA version 15 (Stata Corporation). We interpreted two-sided P values of less than .05 as statistically significant.

| RE SULTS
The baseline characteristics of cases and controls are shown in Table 1. Genotype frequencies among controls did not deviate from the values predicted from the Hardy-Weinberg equilibrium. Table 2 shows ORs for drinking intensity, along with tests for variantdrinking interaction from the two studies and a pooled analysis. The pooled estimate showed a significantly increased pancreatic cancer risk among those with daily alcohol intake of 46 g or more (OR, 1.57; 95% CI, 1.00-2.46). A significant dose-dependent association between alcohol intake and pancreatic cancer risk (per 10-g change in daily alcohol intake: OR, 1.05; 95% CI, 1.01-1.10) was also shown.
Individual results from the two studies showed the same tendency.
Tests for interaction were not significant on either the additive or multiplicative risk scales, indicating that the effect of alcohol drinking on the risk of pancreatic cancer did not vary by rs671 genotype.
We therefore carried out the mediation analysis without considering exposure-mediator interaction.  (Table 3).
To explore the heterogeneous impact between sexes, we undertook a sex-stratified analysis (Table S1)

| DISCUSS ION
In this study, we undertook a mediation analysis with the aim of ex- (1.11-1.28) for heavy drinking compared with nondrinkers and occasional drinkers. 23 The protective indirect effect of the rs671 A allele also supported this association. Furthermore, we observed a lack of variant-drinking interaction, consistent with previous studies. 10,24 This indicates that the accumulation of ethanol-derived acetalde-  Abbreviations: ALDH2, aldehyde dehydrogenase 2; CI, confidence interval; HERPACC, Hospital-based Epidemiologic Research Program at Aichi Cancer Center; OR, odds ratio. a ORs were calculated by a conditional logistic regression model adjusted for age, pack-years, body mass index, meat intake, processed meat intake, family history of pancreatic cancer, history of diabetes, and ALDH2 rs671 genotype. b Multiplicative interaction was assessed using a Wald test of the coefficient of the interaction term corresponding to a one-allele change in the genetic variant multiplied by a 10-g change in daily alcohol intake; the test for additive interaction was carried out using the relative excess risk due to interaction.  -2 Study and the HERPACC-3   Study were carried out between 2001 and 2005 and between   2005 and 2013, respectively, there was no overlap between the two studies in the participants they each included, and these two studies can accordingly be regarded as independent studies. By analyzing HERPACC-2 and -3 separately, we successfully showed the consistency of the results between these two independent studies and ensured their robustness. Second, we had detailed information on potential confounding factors such as smoking, meat/processed meat intake, and BMI. (d) no mediator-outcome confounders that are affected by the exposure. 40 Because exposure was a genetic variant occurring in a single ethnic group, our study conformed with assumptions (a) and (c). Given the specific functions of rs671 in drinking intensity, assumption (d)

TA B L E 3 Direct and indirect effects of the ALDH2 rs671 A allele on pancreatic cancer
was also likely to hold. However, it is unclear whether assumption (b) was upheld. Nevertheless, given that we controlled for major potential confounders, we accordingly consider that assumption (b) is also likely upheld, to some degree at least. Finally, although we confirmed the consistency of the results between the two individual studies, validation of these observed associations requires replication in a larger sample size and in other populations.
In conclusion, we showed that alcohol consumption, especially heavy alcohol consumption, is a preventable exposure factor for pancreatic cancer and that the total effect of the ALDH2 rs671 A allele on pancreatic cancer can be decomposed into a carcinogenic direct effect and a protective indirect effect. This epidemiological study using mediation analysis highlighted the impact of alcohol consumption and the possible contribution of impaired metabolism of ALDH2 substrates on pancreatic cancer, and thereby provides additional evidence on the pathogenesis of pancreatic carcinogenesis. | 1449