下咽頭癌患者の予後に対するアルコール・アルデヒド脱水素酵素の遺伝子多型の影響

概要

Introduction:
Hypopharyngeal squamous cell cancer (HPSSC) has the worst prognosis among the head and neck cancers, with an overall 5-year disease-specific survival rate about 30% to 35%. Epidemiological risks for Hypopharyngeal Cancer include alcohol consumption. However, not all consumers develop HPSCC, indicating a role of host susceptibility factors in cancer development. Some research has recommended that genetic polymorphisms might explain individual variations in susceptibility to HPSCC. Even though the mechanism through which alcohol influences HPSCC risk remains not fully understood, there is sufficient evidence for the carcinogenicity of acetaldehyde, the carcinogenic metabolite of ethanol. In general, two enzymes are essentially involved in alcohol metabolism. Alcohol is initially oxidized to acetaldehyde by alcohol dehydrogenase 1B (ADH1B), then to acetic acid by aldehyde dehydrogenase 2 (ALDH2). The genes encoding both enzymes exhibit polymorphism and ethnic variations. Amino acid coding single nucleotide polymorphisms (SNPs) of ADH1B (G/A, rs1229984) and ALDH2 (G/A, rs671) are well known, and the ADH1B *1/*1 and ALDH2 *2 alleles of those SNPs have markedly reduced enzymatic activities, resulting accumulation of toxic acetaldehyde. Therefore, these SNPs are strongly connected with alcohol related cancers within the upper aerodigestive tract. Recently, the role of genes within the alcohol metabolic process pathways is of specific interest. ADH and ALDH genes are highly studied, yet some SNPs have been identified to be related with cancer risk. However the association with survival is still not clear. The aim of this research was to investigate the association between ADH1B, ALDH2 polymorphism and clinical outcome of HPSCC patients in Japanese population.

Materials and Methods:
The participants in this study were consecutive Japanese males with primary HPSSC treated at the Dept. of Otorhinolaryngology, Head & Neck Surgery, Graduate School of Medicine, Kobe University, Kobe, JAPAN. All were diagnosed between January 2005- June 2013 and met the following inclusion criteria: 1) histologically diagnosed HPSSC, 2) complete resection. Patients with a history of previous cancer or any form of anti-cancer treatment were excluded. All cases were followed-up after surgical treatment every 3 to 6 months. The vital and disease status were verified by evaluating medical records at the date of last follow-up visit.The most recent follow-up data in this analysis were obtained in January 2017. The experimental procedures were approved by the Institutional Review Board for Human Studies at the Kobe University Graduate School of Medicine, and informed consent was obtained from each patient.

Lifestyle, demographic, clinical data and pathologic characteristics of all participants were collected from patient medical records. Alcohol consumption was basically defined in terms of the average number of beverages (beer, whiskey, wine, Japanese sake and shochu) per day. Details of smoking status were evaluated as pack- years (number of cigarettes/20 per day x number of years of smoking). HPSCC was confirmed in each patient by pathological examination. The tumors were staged based on the TNM classification system [9] and categorized as early clinical stage (T1 + T2) or advanced clinical stage (T3 + T4). The histologic grade was confirmed according to the classification system recommended by the World Health Organization.

Since, multiple lugol-voiding lesions (M-LVLs) have been related with a high risk of multiple cancers arising in the head and neck, we included multiple LVLs as a clinical risk factor in univariate and multivariate proportional hazard Cox regression analysis.

Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens using a QIAamp DNA FFPE Tissue Kit (QIAGEN, Germany) based on the manufacturer’s instructions, and stored at -20 °C until real-time polymerase chain reaction (PCR). The single-nucleotide polymorphisms were genotyped by utilising pre- designed TaqMan® allelic discrimination assays for the ADH1B Arg47His (rs1229984) and ALDH2 Glu504Lys polymorphism (rs671) under conditions recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). The obtained data were divided into two groups according to individuals’ ADH1B-ALDH2 genotypes; the high risk cancer group ADH1B *1/*1 and the low risk cancer group ADH1B *1/*2, ADH1B *2/*2; and high risk cancer group ALDH2 *1/*2, ALDH2 *2/*2 and the low risk cancer group ALDH2 *1/*1.

Result:
The study population consisted of 85 patients, with available clinicopathologic, epidemiologic (including smoking and alcohol consumption) and genetic analysis. Their median age was 69 years (range, 45 to 92 years). Mean and median follow-up times from the diagnosis date were 56.7 and 51 months, respectively. Among those patients, 41 (47.6%) had a large number of irregular shaped, well-defined unstained lesions throughout the esophageal mucosa after application of Lugol dye solution. Those patients were defined as having M-LVLs.

Of the patients, 21 (24.4%), 50 (58.1%) and 14 (16.2%) carried the *2/*2, *2/*1 and *1/*1 genotypes of ADH1B, and 8 (9.3%), 42 (48.8) and 35 (40.6) carried the *2/*2, *2/*1 and *1/*1 genotypes of ALDH2, respectively. ADH1B and ALDH2 genotype was not significantly associated with 5 year OS and DFS in HPSCC patients (p=0.974, p=0.946 and p=0.201, p=0.156, respectively). In subgroup analyses, heavy drinkers with ALDH2 *2 allele resulted in significantly worse OS (p=0.028) and DFS (p=0.029) compared to the other patients. The results remained significant when patients with stages I and II disease were excluded (p=0.009 and P= 0.007, respectively). Whereas, heavy drinkers with ALDH1B*1*1 genotype were not significantly associated with 5-year OS and DFS (p=0.155 and p=0.199, respectively). Moreover, heavy drinkers with ALDH2 *2 allele remained statistically significant in multivariate analysis for OS and DFS, indicating independent poor prognostic factor (HR=2.251, 95 % CI 1.018-4.975 and HR=2.261, 95 % CI 1.021-5.006, respectively). The results remained significant when patients with stages 1 and 2 disease were excluded.

Conclusion:
Up to date, prognosis in head and neck squamous cell carcinoma (HNSCC) is estimated by patient (age and medical status at diagnosis), tumor characteristics (site and size), and treatment response. However, outcome can vary remarkably between patients with tumors from the similar location and the same stage, histological grade, and nodal status. New reliable prognostic markers may serve a substantial role for more effective classification of patients for adjuvant therapy or close observation. To develop a more reliable prognostic factor, studying the impact of genes on susceptibility and outcome is another option. We conclude that patients carrying the *2 allele of ALDH2 were associated with worse survival only in those who were heavy drinkers, suggesting that this SNP exposes patients with HPSCC who are also heavy drinkers to worse clinical outcome. This association was also significant in advanced HPSCC (stages III-IV). These findings provide reliable evidence for a gene– environmental connection between the ALDH2 *2 allele and alcohol consumption. Prohibiting the use of alcohol and early detection of cancer are strongly recommended for such individuals.