Title Increase Risk of Squamous Cell Carcinoma in the ... · Alcohol Consumption and Multiple Dysplastic Lesions Increase Risk of Squamous Cell Carcinoma in the Esophagus, Q2 Head,

TitleAlcohol Consumption and Multiple Dysplastic LesionsIncrease Risk of Squamous Cell Carcinoma in the Esophagus,Head, and Neck.

Author(s)

Katada, Chikatoshi; Yokoyama, Tetsuji; Yano, Tomonori;Kaneko, Kazuhiro; Oda, Ichiro; Shimizu, Yuichi; Doyama,Hisashi; Koike, Tomoyuki; Takizawa, Kohei; Hirao, Motohiro;Okada, Hiroyuki; Yoshii, Takako; Konishi, Kazuo;Yamanouchi, Takenori; Tsuda, Takashi; Omori, Tai;Kobayashi, Nozomu; Shimoda, Tadakazu; Ochiai, Atsushi;Amanuma, Yusuke; Ohashi, Shinya; Matsuda, Tomonari;Ishikawa, Hideki; Yokoyama, Akira; Muto, Manabu

Citation Gastroenterology (2016)

Issue Date 2016-08-01

URL http://hdl.handle.net/2433/216918

Right© 2016 by the AGA Institute. Published by Elsevier Inc. Thisis an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Type Journal Article

Textversion publisher

Kyoto University

Alcohol Consumption and Multiple Dysplastic LesionsIncrease Risk of Squamous Cell Carcinoma in the Esophagus,Head, and NeckQ2

Q23 Chikatoshi Katada,1 Tetsuji Yokoyama,2 Tomonori Yano,3 Kazuhiro Kaneko,3 Ichiro Oda,4

Yuichi Shimizu,5 Hisashi Doyama,6 Tomoyuki Koike,7 Kohei Takizawa,8 Motohiro Hirao,9

Hiroyuki Okada,10 Takako Yoshii,11 Kazuo Konishi,12 Takenori Yamanouchi,13

Takashi Tsuda,14 Tai Omori,15 Nozomu Kobayashi,16 Tadakazu Shimoda,17 Atsushi Ochiai,18

Yusuke Amanuma,19 Shinya Ohashi,19 Tomonari Matsuda,20 Hideki Ishikawa,21

Akira Yokoyama,22 and Manabu Muto19

1Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara, Japan; 2Department of HealthPromotion, National Institute of Public Health, Wako, Japan; 3Department of Gastroenterology, Endoscopy Division, NationalCancer Center Hospital East, Kashiwa, Japan; 4Endoscopy Division, National Cancer Center Hospital, Tokyo, Japan;5Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 6Department ofGastroenterology, Ishikawa Prefectural Central Hospital, Kanazawa, Japan; 7Division of Gastroenterology, Tohoku UniversityGraduate School of Medicine, Sendai, Japan; 8Division of Endoscopy, 17Division of Diagnostic Pathology, Shizuoka CancerCenter, Shizuoka, Japan; 9Department of Surgery, National Hospital Organization, Osaka National Hospital, Osaka, Japan;10Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry andPharmaceutical Sciences, Okayama, Japan; 11Department of Gastroenterology, Kanagawa Cancer Center, Yokohama, Japan;12Division of Gastroenterology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan; 13Department ofGastroenterology, Kumamoto Regional Medical Center, Kumamoto, Japan; 14Department of Clinical Oncology, St. MariannaUniversity School of Medicine, Kawasaki, Japan; 15Department of Endoscopy Center, Kawasaki Municipal Kawasaki Hospital,Kawasaki, Japan; 16Department of Diagnostic Imaging, Tochigi Cancer Center, Utsunomiya, Japan; 18Division of Pathology,National Cancer Center Hospital and Hospital East, Tokyo, Japan; 19Department of Therapeutic Oncology, Kyoto UniversityGraduate School of Medicine, Kyoto, Japan; 20Research Center for Environmental Quality Management, Kyoto University,Otsu, Japan; 21Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kyoto, Japan;22Clinical Research Unit, National Hospital Organization Kurihama Medical and Addiction Center, Yokosuka, Japan

BACKGROUND & AIMS: Some patients develop multiplesquamous cell carcinomas (SCCs) in the upper aerodigestivetract, attributed to field cancerization; alcohol consumption hasbeen associated with this process. We examined the associationbetween multiple areas of dysplastic squamous epithelium withthe development of SCC of the esophagus or head and neckcancer, as well as alcohol consumption and smoking.METHODS: We examined 331 patients with early stageesophageal SCC using Lugol chromoendoscopy to evaluate thedysplastic squamous epithelium in the esophagus. Patients thenwere assigned to 3 groups, based on the number of Lugol-voiding lesions: A, no lesion; B, 1–9 lesions; or C, 10 or morelesions. Participants completed lifestyle surveys on their his-tory of drinking, smoking, and diet. All participants wereevaluated by laryngopharyngoscopy before registration; onlythose without head and neck cancer were included, except forpatients with superficial SCC limited to the subepithelial layer.Lesions detected in the esophagus and head and neck by sur-veillance were considered to be metachronous. The study endpoint was the cumulative incidence of metachronous SCCs inthe esophagus and head and neck after endoscopic resection ofesophageal SCC, according to the grade of Lugol-voiding lesions.At study entry, all patients were instructed to abstain fromalcohol and smoking. RESULTS: Over the 2-year study period,metachronous SCCs of the esophagus were detected in 4% ofpatients in group A, in 9.4% of patients in group B, and in24.7% of patients in group C (P < .0001 for patients in group Avs B or B vs C). Head and neck SCCs were detected in none of

the patients in group A, in 1.7% of the patients in group B, andin 8.6% of the patients in group C (P ¼ .016 for patients ingroup A vs C and P ¼ .008 for patients in group B vs C). SCC ofthe esophagus or head and neck developed in 4.0% of patientsin group A, in 10.0% of patients in group B, and in 31.4% ofpatients in group C (P < .0001 for group A vs B or A vs C).Alcohol abstinence decreased the risk of multiple SCCs of theesophagus (adjusted hazard ratio, 0.47, 95% confidence inter-val, 0.25–0.91; P ¼ .025), whereas smoking abstinence did not.CONCLUSIONS: Multiple dysplastic lesions in the esophagusincrease the risk of multiple SCCs. Alcohol abstinence reducesthe risk of metachronous SCCs. Clinical Trials registry:UMIN000001676 and UMIN000005466.

Keywords: Drinking Alcohol; Carcinogenesis; Risk Factor; Ge-netics Methods.

Abbreviations used in this paper: ALDH2, aldehyde dehydrogenase-2; CI,confidence interval; EMR, endoscopic mucosal resection; ESD, endo-scopic submucosal dissection; LVL, Lugol voiding lesion; RR, relative risk;SCC, squamous cell carcinoma.

© 2016 by the AGA Institute. Published by Elsevier Inc. This is an openaccess article under the CC BY-NC-ND license (http://creativecommons.

org/licenses/by-nc-nd/4.0/).0016-5085

http://dx.doi.org/10.1053/j.gastro.2016.07.040

FLA 5.4.0 DTD � YGAST60613_proof � 29 September 2016 � 11:53 am � ce

Gastroenterology 2016;-:1–10

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Q6 Q7Q8 Synchronous and metachronous development of

squamous cell carcinoma (SCC) in the upper aero-digestive tract including the esophagus and the headand neck region has been referred to as the phenomenonof “field cancerization”1 and adversely affects patient sur-vival.2 Recent endoscopic imaging technology such asnarrow-band imaging can detect early SCC, and minimallyinvasive endoscopic treatment can achieve cure andorgan preservation.3–6 Conversely, cancer survivors aftercurative treatment are at risk for the development ofmetachronous multiple SCCs in the preserved organ.However, little is known about the interval from the firstSCC to the metachronous SCC and the risk of metachronousSCC.

Squamous dysplasia has been believed to be a preneo-plastic lesion of SCC, and it is identified easily as Lugol-voiding lesions (LVL) on Lugol chromoendoscopy.7,8 Somepatients with esophageal SCCs have multiple LVLs in thebackground esophageal mucosa.9–13 However, prospectivedata on the association between the grade of LVL and therisk of metachronous SCC are lacking. In addition, mutationof the TP53 gene in the background esophageal mucosa wasshown as an early event of esophageal carcinogenesis.Moreover, TP53 gene mutations were detected even in themicroscopically normal epithelium.14 However, data on theassociation between the grade of LVL and TP53 status alsoare lacking. Follow-up data on LVL might provide importantinformation about the risk of metachronous SCC.

Although field cancerization is associated closely withalcohol consumption and insufficient alcohol meta-bolism,15,16 the benefit of alcohol abstinence on the fieldcancerization phenomenon remains unclear. In this study,we evaluated the cumulative incidence of metachronousmultiple SCCs in the esophagus and the head and neckregion according to the grade of LVL, as well as the riskfactors of metachronous multiple SCCs and the effect ofalcohol and smoking abstinence on field cancerization.

MethodsStudy Design

From September 2005 through May 2010, we prospectivelyrecruited patients from 16 hospitals throughout Japan. Thiscohort study was approved by the institutional review boardat each hospital, and we obtained written informed consentfrom all patients (UMINQ9 Clinical Trials Registry ID:UMIN000001676). The genetic analysis also was approvedby the Institutional Review Board of Kyoto University Hospital,and all participants provided written informed consent (UMINClinical Trials Registry ID: UMIN000005466).

Study PopulationInclusion criteria of this cohort study were as follows: (1)

newly diagnosed early SCC of the esophagus; (2) completeendoscopic resection by endoscopic mucosal resection (EMR)or endoscopic submucosal dissection (ESD); (3) tumor-negativevertical margins of resected specimens; (4) tumor invasionlimited to the mucosa on histopathologic examination ofresected specimens; (5) no additional treatment (surgical

resection, radiotherapy, chemotherapy, and so forth) immedi-ately after EMR or ESD; (6) no active head and neck cancer; (7)patients were evaluated by their attending physicians to be ingood general condition, allowing at least 2 years of follow-upevaluation; and (8) written informed consent obtained fromthe patient. Exclusion criteria were as follows: (1) a history ofchemotherapy for any other cancer or a history of surgicaltreatment or radiotherapy for head and neck cancer; (2) ahistory of iodine allergy; and (3) patients whom the investi-gator considered unsuitable as subjects.

We also prospectively examined TP53 mutations in thebackground esophageal mucosa in other patients with earlyesophageal SCC who were treated at Kyoto University Hospital.

Grading of LVLLVL was graded according to the number of LVLs per

endoscopic view (A, no lesions; B, 1–9 lesions; C, �10 lesions)(Figure 1). Endoscopic images obtained from eligible patients atstudy entry were reviewed centrally in a blinded fashion by 3endoscopists to determine the grade of LVL.

Survey of Lifestyle and FlushingReaction After Drinking

At study entry, lifestyle surveys were conducted using aself-administered questionnaire (Supplementary Figure 1). Thehistories of drinking, smoking, and consumption of high-temperature foods, green-yellow vegetables, and fruit weredocumented carefully. The same questionnaire was used toobtain detailed information on each subject. Patient reports offacial flushing after drinking alcohol were presumed to bemarkers of insufficient alcohol metabolism owing to inactivealdehyde dehydrogenase-2 (ALDH2) genotype (present Q10andpast flushing and never flushing were indicative of activeALDH2).17

Definition of Synchronous andMetachronous Cancer

All candidates were surveyed by laryngoscopy by anotolaryngologist to confirm whether they had synchronousmultiple cancers in the head and neck region before registra-tion. The surveillance interval was within 6 months. Only pa-tients without active head and neck cancer were eligible, exceptfor patients with superficial SCCs limited to within the sub-epithelial layer. Lesions detected by surveillance before regis-tration and lesions including the esophagus were defined assynchronous lesions. After registration, all of the patients werefollowed up by a planned schedule according to the protocol. Ifa new lesion was detected at the first follow-up evaluation (3months after registration) or thereafter, such a lesion wasdefined as a metachronous lesion.

Follow-Up ExaminationsEndoscopic examinations of the head and neck, esophagus,

and stomach, as well as Lugol chromoendoscopy, were per-formed at 3-month intervals for up to 6 months after EMR orESD. Subsequently, these examinations were repeated every 6months. The head and neck region was examined by laryn-goscopy by an otolaryngologist at the time of EMR or ESD andat 1-year intervals thereafter.18 In addition to otolaryngologists,

2 Katada et al Gastroenterology Vol. -, No. -


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gastrointestinal endoscopists also examined the oropharynxand hypopharynx during surveillance endoscopy.

Alcohol and Smoking AbstinenceAt study entry, the physicians in charge handed a document

describing the importance of alcohol and smoking abstinence toall patients and verbally instructed them to stop drinking andsmoking. At each examination, we surveyed smoking status(nonsmoker or smoker; for smokers, the number of cigarettessmoked per day) and drinking status (nondrinker or drinker;for drinkers, the drinking frequency and amount of alcoholconsumed) by self-reporting and instructed patients not todrink or smoke.

Histologic AnalysisAlthough the diagnosis of invasive SCC did not usually differ

among the pathologists, the diagnosis of high-grade intra-epithelial neoplasia sometimes was discordant. To control thesedifferences, lesions with a diagnosis of high-grade intra-epithelial neoplasia were reviewed centrally in a blindedfashion by 3 pathologists. Lesions present at enrollment as wellas new lesions were examined. Lesions not diagnosed to behigh-grade intraepithelial neoplasia or SCCs on central reviewwere excluded from this analysis.

TP53 Mutation in BackgroundEsophageal Mucosa

Biopsy specimens were taken from noncancerous back-ground esophageal mucosa. Genomic DNA was extracted usingthe Gentra Puregene Tissue Kit protocol (Qiagen, Inc, Hilden,Germany) with several modifications, and a low frequency ofTP53 mutations was detected by deep sequencing of theamplicons by using the TruSeq Amplicon Cancer Panel (Illu-mina, Inc, San Diego, CA) to sequence mutational hotspots. Thiscancer panel includes hotspots of TP53 mutation, but not allTP53 exons. The amplicon libraries were subjected to massivelyparallel sequencing using MiSeq (Illumina, Inc) with 170-bppaired-end reads. The sequencing data were imported to theCLC Genomics Workbench softwareQ11 (version 6.0.1), and

low-quality data were trimmed by a quality limit of 0.001. Thecleaned-up reads were mapped to the human genome(GRCh37_dbSNP135), and the somatic mutations were detectedby the Quality-based Variant Detection command.

Statistical AnalysisWe examined whether the grade of LVL was associated with

the development of metachronous multiple SCCs in theesophagus and head and neck region. The number of patientsrequired to detect a difference between grades A and B with astatistical power of 80% and a significance level of 5% wasestimated assuming that the cumulative rate of metachronousSCCs in grades A, B, and C, would be 1%, 10%, and 30%,respectively, with a patient ratio of 4:4:2 and a drop-out rate of10%. The total number of patients required was estimated tobe 292. Under these conditions, the statistical power to detect adifference between grades A and C would be 99% or greater(significance level, 5%). The planned number of enrolledpatients was set at 330, taking into account drop-outs andineligible patients.

The Kaplan–Meier method and the log-rank test wereperformed for the analysis of development of a second pri-mary SCC in the esophagus and the head and neck region. Wedefined the time to development from the day of endoscopictreatment to the day of endoscopic diagnosis of a secondprimary SCC. The person-year method was used to calculatethe total number of multiple cancers arising in the esophagusand head and neck per 100 person-years (ie, a patient couldhave multiple events); the Poisson regression analysis wasused to estimate the sex- and age-adjusted Q12relative risk (RR)of all arising events. A Cox proportional-hazards model wasused to estimate the sex- and age-adjusted hazard ratio (RR)and 95% confidence interval (CI) of the primary event. Theanalysis of end points was conducted after the completion ofat least 2 years of follow-up observation for all patients fromthe time of enrollment. Data on patients in whom follow-upexaminations were not completed were censored at the lastobservation. All data were analyzed with SAS Q13(version 9). Allauthors had access to the study data and reviewed andapproved the final manuscript.

print&

web4C=FPO

Figure 1.Grading of LVLs. The number of LVLs per endoscopic view was counted, and the grading was divided into thefollowing 3 categories: (A) group A, no lesions; (B) group B, 1–9 lesions; and (C) group C, 10 or more lesions.

Q1- 2016 Alcohol, Dysplastic Lesion, and Field Cancerization 3


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ResultsStudy Population

A total of 332 patients with early esophageal SCC wereenrolled. One patient refused to participate in this cohortstudy. The other 331 patients were registered and under-went the preplanned follow-up examinations. One patientwas excluded because the lesion was not diagnosed defini-tively as high-grade intraepithelial neoplasia or invasive SCCon central review of the pathologic assessment (Figure 2).Table 1 summarizes the baseline characteristics of the 330patients according to the grade of LVL. The median follow-up period was 49.4 months (range, 1.3–81.2 mo). The pro-portion of men was higher in the more severe LVL grades(62.0%, 85.1%, and 93.4%, respectively; P < .0001 for A orB vs C). The largest subgroup according to age was 60 to 69years in grade C and 70 years or older in grades A and B.After adjusting for sex and age, the LVL grade was associ-ated with progressively higher proportions of heavydrinkers (27.7%, 26.2%, and 52.8%, respectively; P < .0001for trend, vs others), heavy smokers (41.1%, 65.9%, and71.0%, respectively; P < .0001 for trend), not eating green-yellow vegetables almost every day (52.0%, 54.9%, and71.3%, respectively; P ¼ .021 for trend), and patients with alow body mass index (23.4%, 32.0%, and 37.4%, respec-tively; P ¼ .027 for trend).

Primary and Secondary OutcomesLVL grade was associated with progressive increases in

the 2-year cumulative incidence of metachronous multipleSCCs (esophagus: 4.0%, 9.4%, and 24.7%, respectively;P < .0001 for A or B vs C; head and neck region: 0.0%, 1.7%,and 8.6%, respectively; P ¼ .016 for A vs C and P ¼ .008 forB vs C; both: 4.0%, 10.0%, and 31.4%, respectively; P <.0001 for A or B vs C) (Figure 3).

LVL grade was associated with progressive increases inthe total number of metachronous multiple SCCs per 100person-years (esophagus: RR, 1.91 and 7.41 for grades B andC, respectively, vs grade A; P < .0001 for trend; head andneck region: RR, 0.00 and 5.74 for grades A and C, respec-tively, vs grade B; P < .0001 for trend; both: RR, 2.19 and9.05 for grades B and C, respectively, vs grade A; P < .0001for trend) (Table 2). The frequency of events was notablyhigh in grade C (18.9, 5.8, and 24.7 per 100 person-years inesophagus, head and neck region, and both, respectively).

Grade C LVL was the most significant predictor ofmetachronous multiple SCCs on analysis with a univariateor multivariate Cox proportional-hazards model (esoph-agus: multivariate RR, 8.76; 95% CI, 3.02–25.5; for C vs. A;head and neck region: multivariate RR, 3.51; 95% CI,1.34–9.20; for C vs B; both: multivariate RR, 9.78; 95% CI,3.41–28.0; for C vs A) (Table 3). On multivariate analysis, analcohol flushing reaction after drinking was also a predictor

Figure 2. Enrollment andsubjects for analysis.



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Table 1.Clinical Characteristics of Patients According to the LVL Grade

LVL grade

A (n ¼ 50) B (n ¼ 174) C (n ¼ 106) P

SexMale, % 62.0% 85.1% 93.4% <.0001Age, y

40–59 22.0% 17.2% 19.8%60–69 36.0% 36.8% 55.7%�70 42.0% 46.0% 24.5% .005 a

Means ± SD 66.4 ± 9.9 67.7 ± 8.6 64.8 ± 6.5 .018 a

Sex- and age-adjusted % or mean

Drinking alcoholb,c

Never/rare 25.9% 7.9% 4.7%Light 13.8% 21.4% 8.9%Moderate 27.8% 30.9% 25.5%Heavyd 27.7% 26.2% 52.8%Ex-drinker 4.7% 13.6% 8.2% <.0001 a

Means ± SEe 10.4 ± 1.8 13.1 ± 0.9 19.9 ± 1.2 <.0001 f

Strong alcoholic beveragesb

Frequently 6.7% 11.8% 12.2%Sometimes 15.1% 20.1% 23.2%Never 78.2% 68.1% 64.6% .17 f

Smoking, pack-yearsb

Never, 0 22.7% 14.8% 12.4%Light, <30 36.2% 19.3% 16.6%Heavy, �30 41.1% 65.9% 71.0% <.0001 f

High-temperature foodb

Likes very much 18.4% 20.8% 20.6%Likes somewhat 24.5% 25.1% 24.0%Neither likes nor dislikes 45.8% 41.8% 36.8%Dislikes somewhat 9.3% 9.9% 15.9%Dislikes very much 2.0% 2.3% 2.7% .81 f

Green-yellow vegetablesb

Seldom 0.0% 3.8% 2.1%1–2 d/mo 3.8% 5.7% 5.5%1–2 d/wk 20.5% 16.5% 35.3%3–4 d/wk 27.7% 29.0% 28.4%Almost every day 48.0% 45.1% 28.7% .021 f

Fruitb

Seldom 11.1% 9.6% 12.8%1–2 d/mo 5.2% 15.2% 12.6%1–2 d/wk 33.4% 22.9% 33.1%3–4 d/wk 26.2% 20.2% 17.0%Almost every day 24.1% 32.0% 24.5% .31 f

Body mass index, kg/m2g

<21.2 23.4% 32.0% 37.4%21.2–23.6 18.8% 37.1% 33.9%�23.7 57.8% 30.9% 28.7% .003 f

Means ± SE 23.5 ± 0.44 22.3 ± 0.24 22.1 ± 0.31 .027 f

Alcohol flushingb

Never flushing 35.2% 31.5% 40.6%Flushingh 64.8% 68.5% 59.4% .12 a

aP for trend across the 3 groups.bPercentage values were adjusted for sex and age by direct method (using all patients as the standard population) andstatistically tested by the Cochran–Mantel–Haenszel test. Mean values were adjusted for sex and age and were statisticallytested by analysis of covariance.cNever/rare, <1 U/wk; light, 1–8.9 U/wk; moderate, 9–17.9 U/wk; heavy, �18 U/wk (1 U ¼ 22 g ethanol).dP < .0001 for trend, vs others.eExcluding ex-drinkers.fP for homogeneity among the 3 groups.gCategorized by the overall tertile. Missing data for 1 patient.hPresent or past flushing.

- 2016 Alcohol, Dysplastic Lesion, and Field Cancerization 5


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of metachronous esophageal SCC (RR, 1.07; 95% CI,1.02–2.82) and esophageal and head and neck SCC (RR,1.60; 95% CI, 1.00–2.57). Small or large body mass indexalso was associated with metachronous esophageal SCC (RR,2.09; 95% CI, 1.14–3.83; and RR, 1.86; 95% CI, 1.00–3.47,respectively) and esophageal and head and neck SCC (RR,1.73; 95% CI, 0.99–3.02; and RR, 1.55; 95% CI, 0.88–2.73,respectively).

TP53 Gene Alteration in BackgroundEsophageal Mucosa

To examine whether mutations of genes such as TP53occurred in the noncancerous esophageal mucosa withmultiple LVLs and whether the mutation frequency wasassociated with the severity of LVLs, we performed deepsequencing of the TP53 gene amplicon using biopsy speci-mens of noncancerous esophageal mucosa in patients withor without multiple LVLs. These samples were obtainedfrom 38 early esophageal SCCQ14 cases independent from ourpresent cohort study. The clinical characteristics are sum-marized in Supplementary Tables 1 and 2Q15 , and the detectedlow frequency of TP53 mutations (�2% of total reads) arelisted in Supplementary Table 3. Our study showed that thenumber of cases that had a TP53 mutation was 3 of 7(42.9%) in grade A, 5 of 9 (55.6%) in grade B, and 17 of 22(77.3%) in grade C (Supplementary Figure 2). Of note,several mutations (2–6 mutations) of the TP53 gene werefound simultaneously in biopsy specimens of noncancerousesophageal mucosa in patients with grade C LVL(Supplementary Figure 2). The median mutation frequencyof TP53 genes in biopsy specimens was 0%, 2.0%, and10.1% in grades A, B, and C, respectively. The median ingrade C was significantly higher than that in grade A(P ¼ .04) (Supplementary Figure 3).

Effect of Alcohol and Smoking Abstinence on theDevelopment of Metachronous SCC

As shown in Supplementary Figure 4, 69 (30.9%) of 223patients who drank 1 U/day (22 g of ethanol) or more atbaseline abstained from drinking alcohol, and 69 (53.5%) of129 patients who had smoked at baseline abstained fromsmoking. The cumulative incidence of metachronous

multiple SCCs in the esophagus was shown according to thestatus of drinking alcohol and smoking (Figure 4). Hazardratios were adjusted for sex, age, LVL grade, alcohol intakeat baseline, and alcohol flushing.

Alcohol abstinence decreased the risk of developingmetachronous multiple SCCs of the esophagus (adjustedhazard ratio, 0.47; 95% CI, 0.25–0.91; P ¼ .025) (Figure 4A).The risk reduction was especially large in grade C LVL(adjusted hazard ratio, 0.23; 95% CI, 0.09–0.60; P ¼ .003)(Figure 4B). In contrast, smoking abstinence did notdecrease the risk of developing metachronous multiple SCCsof the esophagus (all: adjusted hazard ratio, 0.59; 95% CI,0.29–1.18; P ¼ .13; grade C LVL: adjusted hazard ratio, 0.73;95% CI, 0.32–1.67; P ¼ .45) (Figure 4C and D, respectively).

DiscussionWe found that patients with grade C LVL were at sig-

nificant risk for the development of metachronous multipleSCCs in the esophagus and the head and neck region.Furthermore, TP53 mutation in the background esophagealmucosa was associated with an increased grade of LVL.These results clearly supported the theory of field cancer-ization in which genetic alteration in the backgroundsquamous epithelium might accumulate and increase thepotential to develop metachronous multiple SCCs. In addi-tion, alcohol abstinence significantly decreased the devel-opment of metachronous SCCs, especially in the patientswith grade C LVL. This result indicates that alcohol absti-nence should be encouraged to reduce the risk of meta-chronous multiple SCCs after curative treatment.

Although the field cancerization phenomenon in theupper aerodigestive tract has been well known for morethan half a century, it has been difficult to detect earlycancer in this region. A randomized controlled trial foundthat narrow-band imaging effectively can detect early SCC inthe esophagus and the head and neck region.3 In patientswith early SCC, organs can be preserved by endoscopiccurative treatment, but the preserved organs are affectedstrongly by the field cancerization phenomenon. Therefore,an effective surveillance strategy after treatment andmethods to prevent metachronous multiple SCCs are neededin survivors of esophageal SCC.

Figure 3. Cumulative incidence of metachronous multiple SCCs according to the LVL grade: (A) esophagus, (B) head and neckregion, and (C) esophagus and head and neck region.



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Data on the interval from the first SCC to the develop-ment of a second primary metachronous SCC have beenlimited, largely because of dismal survival, completeesophageal resection on diagnosis, poor follow-up evalua-tion, and lack of effective surveillance programs. In thisstudy, we focused on patients with early esophageal SCCwho could achieve cure and organ preservation. The fre-quency of events was notably low in grade A. The 2-yearcumulative incidence of metachronous multiple SCCs andthe total number of metachronous multiple SCCs per 100person-years were 4.0%/2.8, 0.0%/0.0, and 4.0%/2.8 in theesophagus, head and neck region, and both, respectively. Onthe other hand, the frequency of events was notably high in

grade C. The 2-year cumulative incidence of metachronousmultiple SCCs and the total number of metachronous mul-tiple SCCs per 100 person-years were 24.7%/18.9, 8.6%/5.8, and 31.4%/24.7 in the esophagus, head and neckregion, and both, respectively. These results indicated thateffective surveillance programs according to the grade ofLVL or effective adjuvant strategies according to the gradeof LVL are required to detect metachronous SCC earlier.

There has been no effective biomarker for the risk of thefield cancerization phenomenon. In this study, we clearlyshowed that an increase in the grade of LVL is associatedclosely with the risk of metachronous SCC. In addition, gradeC LVL and a flushing reaction after drinking alcohol are

Table 2.Person-Years and Number of Primary and All Events According to the Grade of LVL

LVL grade

Total (n ¼ 330)A (n ¼ 50) B (n ¼ 174) C (n ¼ 106)

EsophagusPrimary eventsEvents, n 4 26 44 74Person-years 208.1 652.0 327.1 1187.2Per 100 person-years 1.9 4.0 13.5 6.2RRa 1 2.32 7.68 P < .000195% CI Referent 0.79–6.82 2.64–22.3 for trend

All eventsEvents, n 6 33 85 124Person-years 215.6 703.2 449.9 1368.6Per 100 person-years 2.8 4.7 18.9 9.1RRb 1 1.91 7.41 P < .000195% CI Referent 0.78–4.69 3.12–17.6 for trend

Head and neckPrimary eventsEvents, n 0 6 14 20Person-years 215.6 687.0 407.9 1310.4Per 100 person-years 0.0 0.9 4.1 1.5RRa 0.00 1 3.64 P ¼ .00295% CI Incalculable Referent 1.39–9.52 for trend

All eventsEvents, n 0 7 26 33Person-years 215.6 703.2 449.9 1368.6Per 100 person-years 0.0 1.0 5.8 2.4RRb 0.00 1 5.74 P < .000195% CI Incalculable Referent 0.08–0.40 for trend

Esophagus, head and neckPrimary eventsc

Events, n 4 29 50 83Person-years 208.1 640.0 306.0 1154.1Per 100 person-years 1.9 4.5 16.3 7.2RRa 1 2.48 8.52 P < .000195% CI Referent 0.85–7.19 2.97–24.4 for trend

All eventsd

Events, n 6 40 111 157Person-years 215.6 703.2 449.9 1368.6Per 100 person-years 2.8 5.7 24.7 11.5RRb 1 2.19 9.05 P < .000195% CI Referent 0.91–5.26 3.87–21.2 for trend

aSex- and age-adjusted hazard ratio by Cox proportional-hazards model.bSex- and age-adjusted relative risk by Poisson regression.cEsophagus or head and neck cancer.dEsophagus plus head and neck cancer(s).



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predictors of metachronous multiple SCCs. We previouslyreported that multiple LVLs in patients with SCC in theesophagus and the head and neck region was associatedwith ALDH2 inactive genotype and metachronous SCC in theesophagus and the head and neck region.13,19,20 These datamean that cancer survivors with flushing reaction afterdrinking alcohol or multiple LVLs in the background mu-cosa, or both, should be considered as having a higher riskof field cancerization.

A recent meta-analysis showed that the alcohol-relatedrisk of esophageal SCC is reversible after alcohol absti-nence, and 16 years are required until all increased riskdisappears.21 In our study, with a median follow-up periodof only 49.4 months (range, 1.3–81.2 mo), the risk ofmetachronous SCC significantly decreased after alcoholabstinence, especially among the patients with grade C LVL,in whom the hazard ratio decreased to 0.23. These data arevery important for reducing the risk of metachronous SCC incancer survivors who have received curative treatment.

The median follow-up time in our study was 49.4months, which might be too short to draw strong recom-mendations about the value of surveillance intervals.

However, even in this short period, the patients with gradeC LVL had a risk of metachronous multiple SCCs in theesophagus and the head and neck region. This means thatclose surveillance may be required for the patients withesophageal cancer who were treated successfully. Clinically,the grade of LVL on iodine staining of the backgroundmucosa should be evaluated initially. If the grade suggests arisk of cancer, narrow-band imaging should be performed assurveillance for the early detection of cancer.

We showed that the TP53 mutation was seen even innoncancerous lesions in patients with early esophageal SCC,and the frequency of the TP53 mutation was associated withthe severity of LVL grade. We believe these data help explainthe mechanisms of metachronous esophageal carcinogen-esis. In this study, we obtained biopsy samples from Lugol-stained lesions, in which glycogen granules are rich andstructural atypia is not seen. Such mucosa was assumed tobe “non Q16-cancerous” esophageal epithelium.22 However, wecould not exclude the possibility that the greater the num-ber of LVL lesions present in the patient, the more likely thepossibility that biopsy specimens from normal mucosa stillcontain cryptic dysplasia that would not have been detected

Table 3.Predictors of Metachronous Multiple Cancers by Cox Proportional-Hazards Model

Esophagus Head and neck Esophagus or head and neck

UnivariateRR (95% CI)a

MultivariateRR (95% CI)b

Univariate RR(95% CI)a


UnivariateRR (95% CI)a


Grade of LVLsA 1 (ref) 1 (ref) 0.00 (incalculable) 0.00 (incalculable) 1 (ref) 1 (ref)B 2.33 (0.79–6.85) 2.46 (0.84–7.24) 1 (ref) 1 (ref) 2.49 (0.86–7.23) 2.64 (0.91–7.68)C 7.66 (2.64–22.3) 8.76 (3.02–25.5) 3.61 (1.38–9.46) 3.51 (1.34–9.20) 8.51 (2.97–24.4) 9.78 (3.41–28.0)

Drinking alcohol, per þ1 U 1.12 (1.02–1.23) – 1.18 (1.01–1.38) – 1.11 (1.01–1.22) –

Alcohol flushingNever flushing 1 (ref) 1 (ref) 1 (ref) – 1 (ref) 1 (ref)Flushing 1.39 (0.84–2.29) 1.70 (1.02–2.82) 1.29 (0.49–3.37) – 1.30 (0.82–2.07) 1.60 (1.00–2.57)

Strong alcoholic beveragesSometimes/never 1 (ref) – 1 (ref–) – 1 (ref) –

Frequently 0.95 (0.45–1.99) – 1.17 (0.34–4.05) – 1.07 (0.55–2.08) –

Smoking, pack-yearsNever/light (<30) 1 (ref) – 1 (ref) – 1 (ref) –

Heavy (�30) 1.12 (0.66–1.92) – 1.80 (0.57–5.66) – 1.15 (0.69–1.91) –

High-temperature foodLikes very much 1.04 (0.57–1.90) – 0.93 (0.27–3.20) – 1.09 (0.62–1.92) –

Others 1 (ref) – 1 (ref) – 1 (ref) –

Green-yellow vegetablesAlmost every day 0.77 (0.46–1.26) – 0.59 (0.21–1.68) – 0.64 (0.39–1.03) –

Others 1 (ref) – 1 (ref) – 1 (ref) –

FruitAlmost every day 0.46 (0.24–0.87) 0.42 (0.22–0.82) 0.32 (0.07–1.45) 0.32 (0.07–1.43) 0.43 (0.23–0.81) 0.39 (0.21–0.74)Others 1 (ref) 1 (ref) 1 (ref) 1 (ref) 1 (ref) 1 (ref)

Body mass index, kg/m2

<21.2 2.06 (1.13–3.75) 2.09 (1.14–3.83) 1.29 (0.46–3.60) – 1.65 (0.95–2.85) 1.73 (0.99–3.02)21.2–23.6 1 (ref) 1 (ref) 1 (ref) – 1 (ref) 1 (ref)�23.7 1.59 (0.86–2.94) 1.86 (1.00–3.47) 0.67 (0.21–2.13) – 1.33 (0.76–2.33) 1.55 (0.88–2.73)

NOTE. One patient with missing data for body mass index was excluded (n ¼ 329).–, Not selected (P � .15). Sex and age also were adjusted but are not shown in the table.aSex- and age-adjusted RR for each variable.bVariables were selected from all the variables listed above Q18by the stepwise procedure with P < .15 for entry and removal, andthe relative risks were shown for the selected variables.



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by Lugol staining alone. In addition, we could not addressthe difference regarding TP53 mutations between the pri-mary lesion and the noncancerous background esophagealmucosa because we did not obtain DNA samples from pri-mary lesions. Moreover, we could not assess whether thedifferent TP53 mutations identified in the same case werederived from monoclonal or polyclonal origin. These limi-tations should be clarified further in future studies.

In conclusion, the grade of LVL is a useful predictor ofthe risk of metachronous multiple SCCs arising in theesophagus and the head and neck region in survivors ofesophageal cancer. Alcohol abstinence is required to reducethe risk of metachronous multiple SCCs in the esophagus.

Supplementary MaterialNote: To access the supplementary material accompanyingthis article, visit the online version of Gastroenterology atwww.gastrojournal.org, and at http://dx.doi.org/10.1053/j.gastro.2016.07.040.

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cerization” in oral stratified epithelium. Cancer 1953;6:963–968.

2. Matsubara T, Yamada K, Nakagawa A. Risk of secondprimary malignancy after esophagectomy for squamouscell carcinoma of the thoracic esophagus. J Clin Oncol2003;21:4336–4341.

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Figure 4. Cumulative inci-dence of metachronousmultiple esophageal SCCsaccording to the status ofdrinking alcohol andsmoking. (A) All patientsaccording to the status ofdrinking alcohol. (B) Pa-tients with grade C LVLaccording to the status ofdrinking alcohol. (C) Allpatients according to thestatus of smoking. (D) Pa-tients with grade C LVLaccording to the status ofsmoking.



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10. Shimizu Y, Tsukagoshi H, Fujita M, et al. Head and neckcancer arising after endoscopic mucosal resection forsquamous cell carcinoma of the esophagus. Endoscopy2003;35:322–326.

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13. Katada C, Muto M, Nakayama M, et al. Risk of superficialsquamous cell carcinoma developing in the head andneck region in patients with esophageal squamous cellcarcinoma. Laryngoscope 2012;122:1291–1296.

14. Mandard AM, Hainaut P, Hollstein M. Genetic steps inthe development of squamous cell carcinoma of theesophagus. Mutat Res 2000;462:335–342.

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18. Katada C, Muto M, Tanabe S, et al. Surveillance afterendoscopic mucosal resection or endoscopic submu-cosal dissection for esophageal squamous cell carci-noma. Dig Endosc 2013;25(Suppl 1):S39–S43.

19. Muto M, Takahashi M, Ohtsu A, et al. Risk of multiplesquamous cell carcinomas both in the esophagus andthe head and neck region. Carcinogenesis 2005;26:1008–1012.

20. Katada C, Muto M, Tanabe S, et al. Risk for the devel-opment of multiple Lugol-voiding lesions of the esoph-ageal mucosa in patients with esophageal squamous cellcarcinoma. Dis Esophagus 2014;27:457–462.

21. Jarl J, Gerdtham UG. Time pattern of reduction in risk ofoesophageal cancer following alcohol cessation – ameta-analysis. Addiction 2012;107:1234–1243.

22. Mori M, Adachi Y, Matsushima T, et al. Lugol stainingpattern and histology of esophageal lesions. Am J Gas-troenterol 1993;88:701–705.

Received December 7, 2015. Accepted July 18, 2016.

Reprint requestsAddress requests for reprints to: Manabu Muto, MD, PhD, Department ofTherapeutic Oncology, Kyoto University Graduate School of Medicine, 54Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. e-mail:[email protected]; fax: (81) 75-751-4594. Q3

AcknowledgmentsData were presented in part at Digestive Disease Week 2013 (abstract Su1486)in Orlando, FL. Q17

Chikatoshi Katada, Tetsuji Yokoyama, Hideki Ishikawa, Akira Yokoyama,Yusuke Amanuma, Shinya Ohashi, Tomonari Matsuda, and Manabu Mutowere responsible for the conception and design; Chikatoshi Katada, TetsujiYokoyama, Hideki Ishikawa, Yusuke Amanuma, Tomonari Matsuda, andManabu Muto were responsible for data analysis and interpretation; ManabuMuto and Kazuhiro Kaneko were responsible for financial support;Chikatoshi Katada and Manabu Muto provided administrative support; andall authors were responsible for the collection and assembly of data,manuscript writing, final approval of the manuscript, and provision of studymaterials or patients.

Conflicts of interestThe authors disclose no conflicts. Q4

FundingSupported by a grant from National Cancer Center Research and DevelopmentFund 36 by the Ministry of Health, Labour and Welfare of Japan. Q5



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mailto:[email protected]

Supplementary Figure 1. A self-administered questionnaire for lifestyle surveys.

web4C=FPO

Supplementary Figure 2. TP53 mutations in biopsy speci-mens of noncancerous background esophageal mucosa.Deep sequencing of the TP53 gene amplicon was performedto detect small frequencies of mutations (�2% of total reads).A rectangle shows a biopsy specimen of each case. A redplot shows a mutation of TP53, and the plot size conforms tothe mutation frequency. The 38 cases are divided accordingto their LVL grade (grade A, 7 cases; grade B, 9 cases; andgrade C, 22 cases). ALDH2*1/*1 (ALDH2 wild-type homozy-gotes), ALDH2*1/*2 (ALDH2 heterozygotes), and ALDH2*2/*2(ALDH2 mutant homozygotes) are shown with blue rectan-gles, red rectangles, and yellow rectangles, respectively.

- 2016 Alcohol, Dysplastic Lesion, and Field Cancerization 10.e1


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Supplementary Figure 3. Comparison of the TP53 mutationfrequency between LVL grades. A circle plot shows a sum-mation of TP53 mutation frequency in each case. The boxheight represents the interquartile range showing the lowerquartile (25th percentile values) and upper quartile (75thpercentile values) values. The line in the box represents themedian, and the whiskers represent the minimum andmaximum TP53 mutation frequencies. The P values werecalculated by the Wilcoxon signed-rank test.

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Supplementary Figure 4. Patient flow chart of alcohol and smoking abstinence according to the LVL grade. *P ¼ .90, †P ¼.61, and ‡P ¼ .98. (A) ¼ a, (B) ¼ j, (C) ¼ b, (D) ¼ k. Q19



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Supplementary Table 1.Clinical Characteristics of 38 Esophageal SCC Patients Analyzed With TP53 Deep Sequencing Q20

Sample ID LVL grade ALDH2 genotype Age, y Sex Alcohol drinkinga Smoking, pack-year

S1 A *1/*1 74 Female Never 35.25S2 A *1/*2 60 Female Never 0S3 A *1/*1 85 Female Light 0S4 A *1/*1 57 Male Light 40S5 A *1/*1 64 Male Light 40S6 A *1/*2 68 Male Moderate 30S7 A *1/*1 60 Male Moderate 40S8 B *1/*1 67 Male Heavy 129S9 B *1/*2 69 Male Moderate 60S10 B *1/*2 75 Male Moderate 45S11 B *1/*2 72 Male Moderate 2.5S12 B *1/*1 55 Female Heavy 12.5S13 B *1/*1 68 Male Moderate 45S14 B *1/*1 64 Female Heavy 40S15 B *1/*2 75 Male Moderate 87S16 B *1/*1 72 Male Moderate 45S17 C *1/*2 75 Male Heavy 5S18 C *1/*2 71 Male Never 38S19 C *1/*2 53 Male Heavy 70S20 C *1/*1 62 Female Moderate 20S21 C *1/*2 67 Male Heavy 70.5S22 C *1/*2 61 Male Moderate 40S23 C *1/*2 64 Male Moderate 34.5S24 C *1/*2 71 Male Moderate 75S25 C *2/*2 58 Male Heavy 40S26 C *1/*2 61 Male Moderate 20S27 C *1/*1 69 Male Heavy 21.5S28 C *1/*1 59 Female Moderate 40S29 C *1/*2 67 Male Moderate 100S30 C *1/*2 59 Male Moderate 20S31 C *1/*2 59 Male Heavy 0S32 C *1/*2 73 Male Heavy 30S33 C *1/*2 64 Male Heavy 39.6S34 C *1/*2 68 Male Heavy 50S35 C *1/*2 71 Male Heavy 75S36 C *1/*2 69 Male Heavy 51S37 C *1/*2 66 Male Heavy 86S38 C *1/*2 50 Male Heavy 270

aNever/rare, <1 U/wk; light, 1–8.9 U/wk; moderate, 9–17.9 U/wk; heavy, �18 U/wk (1 U ¼ 22 g ethanol).

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Supplementary Table 2.Clinical Characteristics of 38Esophageal SCC PatientsAccording to the LVL Grade

LVL grade

A (n ¼ 7) B (n ¼ 9) C (n ¼ 22)

SexMale, % 57.1% 77.8% 91.0%

Age, yMeans ± SD 67 ± 10 69 ± 6 64 ± 6

Drinking alcohola

Never/rare 28.6% 0.0% 4.5%Light 42.9% 0.0% 0.0%Moderate 28.6% 66.7% 36.4%Heavy 0.0% 33.3% 59.1%

Heavy smoker, �30 pack-years 71.4% 77.8% 72.7%ALDH2 genotype

*1/*1 71.4% 55.6% 13.6%*1/*2 28.6% 44.4% 81.8%*2/*2 0.0% 0.0% 4.5%

aNever/rare, <1 U/wk; light, 1–8.9 U/wk; moderate, 9–17.9U/wk; heavy, �18 U/wk (1 U ¼ 22 g ethanol).



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Supplementary Table 3.Summary of TP53 Mutations in 38 Esophageal SCC Patients

Sample ID Mutation type Coverage Frequency Start position End position Reference allele Mutated allele Amino acid change Cosmic occurrence

S5 Nonsynonymous SNV 891 2.5 7578446 7578446 T A I162F Q2110S6 Nonsynonymous SNV 1243 2.7 7578532 7578532 A C M133R 6S6 Frameshift deletion 1030 2.0 7577572 7577572 T – 2S6 Nonsynonymous SNV 649 2.3 7579358 7579358 C A R110L 25S7 Nonsynonymous SNV 1571 2.2 7578394 7578394 T C H179R 114S7 Nonsynonymous SNV 1280 3.6 7578203 7578203 C A V216L 7S7 Nonsynonymous SNV 1280 2.7 7578191 7578191 A G Y220H 12S7 Nonsynonymous SNV 944 2.9 7577524 7577524 T G T253P 3S12 Nonsynonymous SNV 197 2.0 7579362 7579362 A G F70L 2S13 Nonsynonymous SNV 522 2.1 7579358 7579358 C G R110P 9S14 Nonsynonymous SNV 967 5.5 7578190 7578190 T C Y220C 231S15 Splicing 1055 2.2 7574035 7574035 T G 0S15 Nonframeshift deletion 986 2.9 7577580 7577585 TAGTGG – 0S15 Nonsynonymous SNV 987 3.7 7577530 7577530 T A I251F 8S15 Nonsynonymous SNV 989 2.2 7577547 7577547 C T G254D 110S16 Nonsynonymous SNV 1445 13.2 7578522 7578522 T A Q136H 1S16 Nonsynonymous SNV 880 2.6 7578396 7578396 G T H178Q 4S16 Nonsynonymous SNV 646 2.8 7577139 7577139 G A R267W 26S22 Nonsynonymous SNV 869 2.2 7577539 7577539 G A R248W 495S23 Nonsynonymous SNV 810 2.5 7577139 7577139 G A R267W 26S24 Nonsynonymous SNV 345 2.6 7579358 7579358 C A R71L 25S25 Frameshift deletion 1844 3.0 7579879 7579879 G – 0S26 Nonsynonymous SNV 936 4.1 7578260 7578260 C T V197M 10S26 Stopgain SNV 825 3.5 7574003 7574003 G A R342* Q2274S27 Splicing 950 9.8 7577610 7577610 T A 10S28 Nonsynonymous SNV 485 2.5 7578448 7578448 G T A29D 9S28 Nonsynonymous SNV 800 2.9 7578190 7578190 T C Y220 231S28 Stopgain SNV 372 5.1 7577100 7577100 T A R148X 10S29 Nonsynonymous SNV 1504 2.3 7578266 7578266 T A I195F 21S29 Nonsynonymous SNV 748 3.6 7578235 7578235 T C Y205C 66S29 Nonsynonymous SNV 877 6.6 7577565 7577565 T C N239S 21S29 Nonsynonymous SNV 879 4.9 7577539 7577539 G A R248W 495S30 Nonsynonymous SNV 356 11.2 7577121 7577121 G A R141C 474S30 Stopgain SNV 1091 5.7 7574021 7574021 C A E336* 4S31 Nonsynonymous SNV 1225 5.7 7578526 7578526 C T C135Y 52S31 Frameshift deletion 694 3.7 7577599 7577599 C – 0S31 Nonsynonymous SNV 702 5.0 7577536 7577536 T A R249W 34S31 Nonsynonymous SNV 502 2.4 7577124 7577124 C A V272L 23S32 Nonsynonymous SNV 1079 2.5 7577559 7577559 G A S241F 73S32 Nonsynonymous SNV 1080 4.8 7577570 7577570 C T M237I 68S32 Nonsynonymous SNV 537 4.8 7577098 7577098 T A R280S 11S32 Nonsynonymous SNV 538 3.0 7577094 7577094 G A R282W 395S32 Nonsynonymous SNV 1638 2.6 7578538 7578538 T C N131S 4S33 Splicing 1416 4.5 7578370 7578370 C T 21

10.e6Katada

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Supplementary Table 3.Continued

Sample ID Mutation type Coverage Frequency Start position End position Reference allele Mutated allele Amino acid change Cosmic occurrence

S33 Nonsynonymous SNV 1393 4.5 7578406 7578406 C T R175H 818S33 Nonsynonymous SNV 339 5.0 7577124 7577124 C A V140L 23S33 Stopgain SNV 339 6.8 7577058 7577058 C A E162X 43S34 Nonsynonymous SNV 1394 13.8 7578190 7578190 T C Y220C 231S34 Nonsynonymous SNV 1346 9.2 7578536 7578536 T G K132Q 10S35 Nonsynonymous SNV 679 23.4 7577559 7577559 G A S241F 73S36 Nonsynonymous SNV 2181 2.5 7578395 7578395 G A H179Y 85S36 Nonsynonymous SNV 2181 4.4 7578393 7578393 A T H179Q 12S36 Nonsynonymous SNV 1147 4.3 7578271 7578271 T C H193R 81S36 Nonsynonymous SNV 674 9.5 7577538 7577538 C T R248Q 565S36 Nonsynonymous SNV 563 2.7 7577121 7577121 G A R273C 474S36 Frameshift deletion 583 2.2 7579391 7579391 G – 0S37 Nonsynonymous SNV 717 4.0 7578454 7578454 G A A159V 37S37 Nonsynonymous SNV 691 8.7 7577547 7577547 C A G245V 62S37 Nonsynonymous SNV 607 14.5 7577120 7577120 C T R273H 513S37 Nonsynonymous SNV 645 3.7 7579358 7579358 C A R110L 25S38 Splicing 896 6.5 7577609 7577609 C T 13S38 Frameshift insertion 899 6.3 7577530 7577530 – T I119fs 0S38 Nonsynonymous SNV 899 2.6 7577528 7577528 G C I251M 0S38 Nonsynonymous SNV 454 26.7 7577098 7577098 T G R148S 5

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Alcohol,DysplasticLesion,and

FieldCancerization

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Documents

Title Increase Risk of Squamous Cell Carcinoma in the ... · Alcohol Consumption and Multiple Dysplastic Lesions Increase Risk of Squamous Cell Carcinoma in the Esophagus, Q2 Head,