Somatic copy number alterations associating with alcohol drinking and smoking in head and neck squamous cell carcinoma. Homo sapiens

Background: The most important risk factors for head and neck squamous carcinoma (HNSCC) are tobacco smoking and alcohol consumption, while subgroups are caused by infection with human papillomaviruses (HPV) or Epstein-Barr virus (EBV). Here, we studied alterations of somatic copy-number in whole genome, p16 protein and TP53 mutations by alcohol drinking, smoking and viral infections. Methods: We conducted a prospective cohort study. DNA obtained from tumors and margin samples as well as peripheral blood was assayed by array comparative genomic hybridization using Agilent Whole Human Genome 180K. Mutations of p53 gene by direct sequencing, detection of HPV by polymerase-chain-reaction (PCR), quantification of EBV by reverse transcription-PCR and p16 immunohistochemical (IHC) staining were also performed. Overall design: prospective cohort study: 225 tumor samples were analyzed by Agilent-022060 SurePrint G3 Human CGH miroarray 4x180K. Log2 Ratio (tumor sample DNA/normal DNA) were compared among none alcohol drinkers, moderate alcohol drinkers, heavy alcohol drinkers, none smokers, moderate smokers, heavy smokers and both. Sample characteristics weres scored as following; Human Papilloma Virus Infection (gene detected by PCR in tumor sample): positive=1; negative=0 p16 protein immunohistochemistry: positive=1; negative=0 tumor grade: matured=0; moderate=1; immature=2 Radiotherapy with or without chemotherapy after operation: performed =1; not performed =0 Recurrence/relapse: rec =1; not rec =0 suvival status:death=1; survive=0 smoking status (Pack year of smoking): none smoker=0; pack-year <20 = 1 (moderate smoker); pack-year <=20 = 2 (heavy smoker) alcohol drinking: Alcohol drinkers were divided into non-drinker or less than one drink per day =0: one and more but less than two drinks per day = moderate drinker 1: two and more drinks per day=heavy drinker 2: during the 20 years preceding their treatment for HNSCC. One drink was defined as containing approximately 10g of alcohol, which is considered equal to 30 ml of hard liquor, 100 ml of wine containing 12% alcohol, or 360 ml of beer.

背景：头颈部鳞状细胞癌（head and neck squamous carcinoma, HNSCC）最主要的危险因素为烟草吸烟与酒精摄入，而部分亚型由人乳头瘤病毒（human papillomaviruses, HPV）或爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）感染引发。本研究针对饮酒、吸烟及病毒感染相关的全基因组体细胞拷贝数变异、p16蛋白及TP53突变展开分析。
方法：本研究采用前瞻性队列研究设计。通过安捷伦（Agilent）全人类基因组180K芯片的阵列比较基因组杂交（array comparative genomic hybridization）技术，对肿瘤组织、切缘样本及外周血提取的DNA进行检测。此外还开展了p53基因直接测序、人乳头瘤病毒（HPV）聚合酶链反应（polymerase-chain-reaction, PCR）检测、爱泼斯坦-巴尔病毒（EBV）逆转录聚合酶链反应（reverse transcription-PCR）定量分析，以及p16免疫组化（immunohistochemical, IHC）染色检测。
整体设计：本研究为前瞻性队列研究，共纳入225份肿瘤样本，采用安捷伦-022060 SurePrint G3人类CGH微阵列4x180K（Agilent-022060 SurePrint G3 Human CGH microarray 4x180K）进行分析。将非饮酒者、适量饮酒者、大量饮酒者，非吸烟者、适量吸烟者、大量吸烟者以及兼具吸烟与饮酒暴露者的Log2比值（肿瘤样本DNA/正常对照DNA）进行组间比较。
样本特征评分标准如下：
1. 人乳头瘤病毒感染（通过PCR在肿瘤样本中检测到病毒基因）：阳性=1，阴性=0；
2. p16蛋白免疫组化检测：阳性=1，阴性=0；
3. 肿瘤分级：成熟型=0，中分化型=1，未成熟型=2；
4. 术后辅助放疗联合或不联合化疗：实施=1，未实施=0；
5. 复发/复发转移：复发=1，未复发=0；
6. 生存状态：死亡=1，存活=0；
7. 吸烟状态（吸烟包年数）：非吸烟者=0；包年<20=1（适量吸烟者）；包年≤20=2（大量吸烟者）；
8. 饮酒状态：在头颈部鳞状细胞癌（HNSCC）治疗前20年内，饮酒者分为：非饮酒者或每日饮酒<1杯=0；每日饮酒1杯及以上但<2杯=适量饮酒者=1；每日饮酒≥2杯=大量饮酒者=2。标准1杯酒定义为含约10g酒精，相当于30ml烈酒、100ml酒精度12%的葡萄酒或360ml啤酒。