Prognostic Value of Drinking Status and Aldehyde Dehydrogenase 2 Polymorphism in Patients With Head and Neck Squamous Cell Carcinoma

Background The association between alcohol drinking, aldehyde dehydrogenase 2 (ALDH2) polymorphism, and survival in patients with head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods We performed a retrospective cohort study of 267 HNSCC patients at Aichi Cancer Center. Of these, 65 patients (24%) were non-drinkers, 104 (39%) were light drinkers (ethanol <46 g or <5 days/week), 46 (17%) were moderate drinkers (ethanol intake 46–68 g/day and ≥5 days/week), and 52 (20%) were heavy drinkers (ethanol intake ≥69 g and ≥5 days/week). The prognostic value of pre-treatment drinking status and ALDH2 polymorphism was investigated using multivariate proportional hazard models. Results Drinking status was associated with disease-free survival (DFS) in HNSCC patients, with marginal statistical significance (5-year DFS: 67.9% [95% confidence interval {CI}, 53.8–78.4%] for non-drinkers, 57.6% [95% CI, 47.4–66.6%] for light drinkers, 46.1% [95% CI, 30.8–60.1%] for moderate drinkers, and 43.5% [95% CI, 29.3–56.9%] for heavy drinkers; P = 0.088). However, this association lost significance when multivariate analyses were adjusted for established prognostic factors. ALDH2 genotype was not significantly associated with DFS in HNSCC patients (5-year DFS: 85.7% [95% CI, 53.9–96.2%] for Lys/Lys, 56.2% [95% CI, 47.4–64.1%] for Glu/Lys, and 50.5% [95% CI, 40.3–59.7%] for Glu/Glu; P = 0.154). After stratification by ALDH2 genotype, we observed a significant positive dose-response relationship between drinking status and DFS in HNSCC patients with ALDH2 Glu/Glu (P trend = 0.029). Conclusions In this study, we identified a significant positive dose-response relationship between pre-treatment drinking status and DFS in HNSCC patients with ALDH2 Glu/Glu. To confirm this association, further study is warranted.


INTRODUCTION
Worldwide, almost 600 000 new cases of head and neck cancer are reported each year. 1 Alcohol drinking is an established risk factor for head and neck squamous cell carcinoma (HNSCC). 2,3In general, ethanol is oxidized by alcohol dehydrogenase (ADH) enzymes to acetaldehyde, which is then further oxidized to acetate by aldehyde-dehydrogenase (ALDH) enzymes.This latter oxidation is largely dependent on the ALDH2 enzyme. 4The metabolism of ethanol leads to accumulation of acetaldehyde, which is toxic and established as a strong carcinogen, and differences in ethanol metabolism that result from polymorphisms in the genes that code for these enzyme affect cancer etiology among drinkers. 5][8][9][10][11][12][13] Light drinkers with ALDH2 Lys/Lys and Glu/Lys have 18 times and 5 times higher average peaks of acetaldehyde concentrations in blood, respectively, than moderate drinkers with ALDH2 Glu/Glu. 14][17][18][19][20][21] Previously, we clarified the association between lifestyle factors and prognosis in HNSCC patients. 224][25] This suggests that alcohol drinking might affect the survival of HNSCC patients and that ALDH2 polymorphism might interact with this association.
Here, we conducted a retrospective cohort study to clarify the potential association of these factors among 267 patients treated for HNSCC at Aichi Cancer Center (ACC).

Patients
We selected patients from the database of the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), based at ACC in Nagoya, Japan.The HERPACC framework has been detailed elsewhere. 26,27riefly, 23 408 HERPACC-enrolled, first-visit outpatients treated between January 2001 and November 2005 at ACC were asked to provide blood samples and information on lifestyle factors.Among those who participated, 22 727 patients (97.1%) completed a self-administered questionnaire including lifestyle factors, which was checked by a trained interviewer, and approximately 60% provided blood samples.The HERPACC study was approved by the Institutional Ethics Review Board of ACC, and all participants provided written informed consent.
HERPACC-enrolled patients diagnosed as having primary head and neck cancer who met the following criteria were included: (a) no prior history of cancer; (b) no malignant neoplasms of the salivary glands, nasopharynx, nasal, or paranasal sinuses (these cancers were excluded, as they have a distinct etiology); (c) histological diagnosis of squamous cell carcinoma; (d) Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 to 2 28 ; and (e) availability of information about drinking status and ALDH2 genotype.Ultimately, 267 patients were eligible for this study.

Treatment and follow-up
We considered surgery and chemoradiotherapy (CRT) or radiotherapy (RT) with or without induction chemotherapy (ICT) as treatment modalities for HNSCC.Attending surgeons determined treatment of each individual patient based on clinical disease stage, primary tumor site, and PS.Patients were followed up with a history and physical examination, complete blood cell count, and imaging examination every 3 to 6 months for 5 years after their treatment.We confirmed vital and disease status by checking medical records at the date of last follow-up visit.Vital status in patients lost to follow-up was confirmed by a census registration conducted annually.

Genotyping of ALDH2
We extracted DNA from the buffy coat fraction with a DNA Blood mini kit (Qiagen, Tokyo, Japan) or BioRobot EZ1 and EZ1 DNA Blood 350 mL Kit (Qiagen).We genotyped ALDH2 Glu504Lys polymorphism (rs671) by the TaqMan method (Applied Biosystems, Foster City, CA, USA).The quality control of genotyping was assessed statistically by using the Hardy-Weinberg test and by retyping of a random sampling of 5% of subjects.
Assessment of drinking and smoking status Details of the assessment of drinking and smoking status have been described elsewhere. 22Our questionnaire consisted of items related to smoking and drinking habits in the period preceding the development of the present symptoms or reason for visit to ACC.
Levels of alcohol consumption were divided into four groups: non-, light, moderate, and heavy drinkers.Moderate drinkers were defined as individuals who consumed alcoholic beverages in a daily amount of ≥46 g ethanol (equivalent to two Japanese drinks) but <69 g ethanol for ≥5 days per week.Heavy drinkers were defined as individuals who consumed alcohol beverages in a daily amount of ≥69 g ethanol for ≥5 days per week.Light drinkers were defined as individuals who consumed alcoholic beverages in a daily amount of <46 g ethanol for <5 days per week.The remaining patients were categorized as non-or light drinkers using data from the HERPACC study for head and neck cancers. 29umulative exposure to cigarette smoking was quantified as pack-years of smoking (PY), the product of the number of packs consumed per day and number of years of cigarette smoking.We divided patients into four groups based upon PY: non-, light (PY < 20), moderate (PY 20-39), and heavy smokers (PY ≥ 40).

Statistical methods
The primary endpoint of this study was disease-free survival (DFS), which was defined as the number of days from the beginning of treatment to the date of relapse.The associations between drinking status (0: non, 1: light, 2: moderate, and 3: heavy), ALDH2 genotypes (0: Glu/Lys, 1: Glu/Glu, and 2: Lys/ Lys), and DFS were evaluated by the Kaplan-Meier productlimit method and uni-and multivariate Cox proportional hazards models.Confounders considered in the multivariate analyses were age (continuous), sex (male or female), ECOG PS (0-2), smoking status (non-vs light vs moderate vs heavy), Union for International Cancer Control (UICC) stage (1-4), and treatment method (surgery or CRT/RT).
In addition, the interaction between drinking status and ALDH2 genotype was examined by adding an interaction term between the two items in multivariate models.Distribution of patient characteristics was assessed by the χ 2 test or Fisher's exact test, as appropriate.All statistical analyses were performed using the software STATA ver. 10 (Stata Corp, College Station, TX, USA).All tests were two-sided, and P-values of <0.05 were considered statistically significant.

Patient characteristics and survival
Table 1 summarizes subject characteristics in the study.Median age was 61 (range, 21-78) years, and median follow-up time was 5.0 years (range, 0.7 months-9.1 years).Drinking was more prevalent among males than females.Similarly, drinking was more prevalent among smokers, but less prevalent among oral cavity cancer patients than patients without oral cavity cancer.Five-year DFS among all patients was 55.5% (95% confidence interval [CI], 49.1-61.4%).
Table 2 shows the results of uni-and multivariate analysis for DFS.In univariate analysis, drinking status showed a significant trend in decreasing DFS (P trend = 0.013).However, neither drinking status nor ALDH2 genotype showed a significant association with DFS in multivariate analysis.

Interaction between drinking status and ALDH2
genotype on DFS Kaplan-Meier survival curves of DFS for drinking status according to ALDH2 genotype are shown in Figure 3. Drinking status was significantly associated with DFS in ALDH2 Glu/Glu patients (59.4% [95% CI, 30.9-79.4%] for  In multivariate analysis, drinkers with ALDH2 Glu/Glu showed a significant trend toward decreased DFS (P trend = 0.029; Table 3).In contrast, among patients with ALDH2 Glu/ Lys, drinking status showed no significant association with DFS.In addition, we observed suggestive heterogeneity between drinking status and ALDH2 genotype on DFS (P for heterogeneity = 0.100).

DISCUSSION
In this study, we demonstrated that high pre-treatment alcohol consumption worsens DFS in HNSCC patients.This effect was evident only among the ALDH2 Glu/Glu patients, suggesting that it might differ by ALDH2 genotype.To our knowledge, this is the first report to evaluate the impact of alcohol drinking combined with ALDH2 genotype on clinical outcome in HNSCC patients.Although the mechanism behind this association between drinking status and survival of HNSCC patients remains unclear, several explanations appear plausible based on existing evidence.First, alcohol drinking may induce activation of NF-κB, a transcription factor that has been linked with the transformation of cells and survival of cancer stem cells. 30Additionally, NF-κB regulates the expression of genes associated with the apoptosis, proliferation, invasion,   angiogenesis, and metastasis of cancer.Second, alcohol consumption may induce TP53 mutation, which is associated with HNSCC survival. 23,31Third, alcohol drinking may modify the DNA methylation profile in HNSCC cells.DNA methylation modifications may affect the prognosis of HNSCC. 32Fourth, alcohol drinking damages normal mucosa in the head and neck region.Long-term continuation of this mucosal damage, called "field cancerization", may be associated with a predisposition to relapse or development of a second primary tumor (SPT). 25owever, the incidence of SPT was not associated with drinking status in this study (data not shown).
In addition, our findings suggest that the association between alcohol drinking and prognosis of HNSCC may differ by ALDH2 genotype.Although this mechanism is also unclear, we speculate that this association might be affected by alcohol dependence. 33Individuals who are heterozygous or homozygous for the Lys allele of ALDH2 Glu504Lys polymorphism (rs671) have greatly reduced ability to metabolize acetaldehyde, which greatly decreases their risk for alcohol dependence. 34After definitive treatment, ALDH2 Glu/Glu patients might maintain higher levels of alcohol consumption than ALDH2 Glu/Lys patients.However, this point should be assessed in other studies evaluating post-treatment or undertreatment drinking behavior according to ALDH2 genotype.
Our study has several methodological strengths.First, because clinicians involved in the care of study patients were not aware of the exposure status examined in this study, information bias was less likely to have been introduced.Second, because the analyses were adjusted for established prognostic factors, including clinical disease stage and PS, the observed associations were theoretically independent.However, the exclusion of residual confounding by unevaluated factors, such as human papilloma virus (HPV) infection, cannot be completely ruled out.
Several methodological limitations also warrant mention.First, our information on drinking habits reflected pretreatment drinking status only, and we were therefore unable to evaluate the impact of changes in drinking behavior during the study.Second, the prognostic value of drinking-related comorbidities, including coronary artery disease, cerebral infarction, peripheral vascular disease, and chronic liver disease, might bias this study.If death induced from these comorbidities were not uncommon in the heavy drinkers, our study would have overestimated the prognostic value of heavy drinking in HNSCC patients.Actually, while the ratio of patients who died from HNSCC alone was 58.8%, information on the cause of death was unavailable for 29.5%.Third, although we tried to minimize the effect of bias by considering potential confounders in multivariate analysis, the impact of residual confounding, including that due to HPV infection, cannot be fully excluded.Finally, the moderate sample size may have limited the ability of the study to detect differences between the groups.We concluded that high pre-treatment alcohol drinking worsened DFS in patients with HNSCC.This effect might be clearer among patients with ALDH2 Glu/Glu.Our results suggest a possible gene-environmental interaction in the clinical outcome of HNSCC patients.Replication in a larger study is warranted.

ONLINE ONLY MATERIAL
Abstract in Japanese.

Table 1 .
Characteristics of head and neck squamous cell carcinoma patients according to drinking status and ALDH2 genotype

Table 2 .
Impact of drinking status and ALDH2 genotype on disease-free survival in patients with head and neck squamous cell carcinoma CI, confidence interval; HR, hazard ratio.Drinking status was classified as follows: non, light (<46 g ethanol or <5 days/week), moderate (46-68 g ethanol for ≥5 days/week), and heavy (≥69 g ethanol for ≥5 days/week).Adjusted for age, sex, performance status, clinical disease stage, primary tumor site, smoking, and definitive treatment.

Table 3 .
Impact of drinking status according to ALDH2 genotype on disease-free survival in patients with head and neck squamous cell carcinoma ALDH2, aldehyde dehydrogenase 2; HR, hazard ratio; CI, confidence interval.Drinking status was classified as follows: non, light (<46 g ethanol or <5 days/week), moderate (46-68 g ethanol for ≥5 days/week), and heavy (≥69 g ethanol for ≥5 days/week).Adjusted for age, sex, performance status, clinical disease stage, primary tumor site, smoking, and definitive treatment.