| 1 Background and PurposeAccording to the World Health Organization(WHO),esophageal carcinoma(EC)is the eighth common malignant tumor all of the world and one of the six leading cancer-related lethal carcinoma.There are about 500,000 new cases of EC in the world each year,and more than half of them occur in China.By histopathological types,primary epithelial origin esophageal carcinoma are classified into esophageal squamous cell carcinoma(ESCC),esophageal adenocarcinoma(EAC),adenoid cystic carcinoma,mucoepidermoid carcinoma,adenosquamous carcinoma,undifferentiated carcinoma,neuroendocrine carcinoma carcinoma and mixed neuroendocrine-nonneuroendocrine tumors,among which ESCC and EAC are the two main types.There are significant differences between primary ESCC and primary EAC in terms of epidemiological characteristics,histological occurrence and risk factors.The main pathological type of esophageal cancer in China is ESCC.Patients with ESCC account for about 95%of EC patients,while primary EAC accounts for only about 2%.It has been reported that EACC has a high incidence in rural and mountainous areas with underdeveloped economic conditions;With the continuous improvement of the economy and living standards in recent years,the incidence of EAC among urban residents also shows an increasing trend year by year.ESCC mostly occurs in the middle and upper esophagus,while EAC mostly occurs in the lower esophagus.The occurrence pattern of ESCC in China is that the DNA damage in esophageal squamous epithelial cells cannot be repaired in time due to the lack of nitrosamine exposure,vitamins and other trace elements(especially riboflavin,vitamin A and folic acid,etc.),resulting in proliferation basal cells,atypical hyperplasia,carcinoma in situ,and eventually invasive ESCC.More and more studies have proved that there is another incidence mode of esophageal adenocarcinoma in China.When the esophageal proper glands normally located in the submucosa may appear benign hyperplasia,atypical hyperplasia and intestinal metaplasia may occur,and gradually destroy and replace the normal esophageal squamous cell epithelium,which eventually develops into esophageal adenocarcinoma.These phenomena all suggest that primary ESCC and primary EAC have different molecular basis,especially in China and in western countries may also be significantly different.In this study,based on the large database of clinical diagnosis,pathology and follow-up information of 500,000 patients from high and low incidence areas of esophageal cancer established by our team,the distribution characteristics and survival of patients with primary ESCC and primary EAC were systematically analyzed,and further explore the genomic changes of primary ESCC and primary EAC.This study aimed to explore the different genomic characteristics of primary ESCC and EAC to provide a theoretical basis for establishing molecular classification criteria suitable for ESCC and EAC,establishing a system and key technologies for early detection and early warning screening of high-risk groups of ESCC and EAC in China,achieving precise treatment and individualized prevention and treatment strategies for Chinese patients,and reducing esophageal cancer morbidity and mortality.2 Patients and methods2.1 Clinical phenotype,pathological characteristics and survival analysis of primary ESCC and EAC2.1.1 PatientsAll EC patients are from the large database of clinical diagnosis and treatment pathological information and biological sample information database of esophageal cancer patients established by State Key Laboratory of Esophageal Cancer Prevention and Treatment,postoperative pathological diagnosis was ESCC,EAC;no tumor of other organs occurred at the same time;and with complete clinical phenotypic information and postoperative pathological diagnosis.A total of 88,620 patients with primary ESCC and 6,308 patients with primary EAC were randomly screened.Among them,there were 56,016 male patients with primary ESCC,with an average age of 59.61±8.8 years,and 32,604 females,with an average age of 60.0±8.8 years;among the 6308 cases of primary EAC,there were 4,614 males with an average age of 62.4±9.8 years and 1,694 females with an average age of 62.4±9.6 years.2.1.2 MethodsThe clinical phenotype data and pathological data of all patients were standardized according to the standard,and the follow-up work of the patients was completed.The SPSS25.0 software was used to perform the chi-square test on the distribution characteristics of different clinical phenotypes of all patients,and Logistic regression was used to analyze the lymph node metastasis related data,Kaplan-Meier method and Cox regression were used to analyze the survival and mortality risk of patients.2.2 Genomics comparison of primary ESCC and primary EAC2.2.1 PatientsAll EC patients are from the large database of clinical diagnosis and treatment pathological information and biological sample information database of esophageal cancer patients established by State Key Laboratory of Esophageal Cancer Prevention and Treatment,postoperative pathological diagnosis was ESCC,EAC;without radiotherapy,chemotherapy or any other treatment before surgery;no tumor of other organs occurred at the same time;the postoperative pathological diagnosis of the patient was complete;the clinical phenotype and follow-up information were complete.A total of 405 patients finally met the above conditions,and a total of 382 patients were included in the analysis.Among the 303 cases of primary ESCC,206 cases were male,the patients have an average age of 62.47±8.68 years,and 97 cases were female,the patients have an average age of 64.91±8.57 years;among the 79 cases of primary EAC,54 cases were male,the patients have an average age of 61.87±7.58 years,and 25 cases were female,the average age of the patients was 63.92±8.57 years old.2.2.2 Methods2.2.2.1 Comparison of significantly mutated genes between ESCC and EACThe cancer tissues and adjacent normal tissues and peripheral venous blood of surgically resected gross specimens from patients with ESCC and EAC were collected.Libraries and captures,and whole-exome sequencing was performed with the Illumina HiSeqPE150 platform.By performing quality control on the sequencing raw data,high-quality sequencing sequences were obtained,which were compared and analyzed with the human reference genome sequence(human_B3 7)to obtain Bam files.Based on this data,Mutsig 2CV was used for somatic mutation analysis to map the high-frequency mutation gene spectrum of Chinese ESCC and EAC;Fisher’s exact test was used to compare and analyze the high-frequency mutation gene profiles of ESCC and EAC,and to further explore the similarities and differences of the high-frequency mutation gene profiles of ESCC and EAC.The software system is R language v4.0.5,and Kaplan-Meier method is mainly used for survival analysis.2.2.2.2 Construction of risk prediction and prognosis judgment models for ESCC and EACSPSS25.0 software and R language was used to univariate and multivariate Logistic regression and Cox regression analysis were performed on the differentially mutated genes in ESCC and EAC with different phenotypes.The genes and clinical phenotypes with significant statistical differences(p<0.05)were selected for risk prediction and prognostic judgment model construction.3 Results3.1 Clinical phenotype,pathological characteristics and survival analysis of primary ESCC and primary EAC3.1.1 Clinicopathological characteristics of patients with primary ESCC and primary EACIn this study,the distribution of 88,620 primary ESCC and 6,308 primary EAC with different clinical phenotypes,such as gender,age at diagnosis,high and low incidence area,urban/rural,different family history,smoking history,drinking history,etc.characteristics were analyzed(Table 3).The results showed that there were significant differences in the proportions of primary ESCC and primary EAC in gender,age at diagnosis,region,positive/negative family history,smoking history/no smoking history(p<0.05).The proportion of patients with positive lymph node metastasis in patients with primary EAC was significantly higher than that in patients with primary ESCC(48.29%vs 38.61%,p=0.000,OR:1.486,95%CI:1.399-1.575).3.1.2 Survival analysis of patients with primary ESCC and primary EACIn this study,survival analysis was performed based on the follow-up information of primary ESCC patients and primary EAC patients with different TNM stages,and Kaplan-Meier survival curves were drawn.The results showed that the survival of patients with ESCC or EAC in the early stage(stage 0-I),intermediate stage(stage Ⅱ)and advanced stage(stage Ⅲ-Ⅳ)was significantly different(p<0.05).3.2 Comparison of significantly mutated genes between ESCC and EAC3.2.1 Comparison of significantly mutated genes between primary ESCC and primary EACIn this study,15,146 genes and 260,096 mutation sites were detected in 303 Chinese patients with ESCC,of which 207,962 sites had single base substitutions,33,846 sites had deletions,and 18,288 sites had insertions.A total of 191 genes with mutation frequency≥2%were identified among 274 genes with q value<0.05.In 79 cases of Chinese ESCC,69,148 mutation sites were detected,of which 62,736 sites had a single base substitution,3,843 sites were deleted,and 2,569 sites were inserted,and 15 genes were identified.If the value is less than 0.05,the mutation frequency is 3.80%-78.48%.By comparison,it was found that TP53,TGFBR2,PPP1R12B,MYL1 and BRD7 genes were significantly different between ESCC and EAC(q<0.05).And the mutation frequencies of these five genes in ESCC and EAC were relatively consistent.A total of 186 genes with mutation frequency≥2%,including NOTCH1,KMT2D,FBXW7,PER3,CDKN2A,FAT1,and ABCF1 were significantly mutated only in ESCC(q<0.05).A total of 10 genes with mutation frequency≥2%,including CDC27,ZC3H11A,GART,PTPRZ1,SPDYE1 were significantly mutated only in EAC(q<0.05).3.2.2 Analysis of copy number variation in primary ESCC and primary EACThis study detected 39,224 and 9,356 copy number variations(CNVs)with qualified copy ratios in 303 primary ESCC and 79 primary EAC,respectively.The CNV comparison results and high-frequency mutation genes were further analyzed,and 42 genes including NOTCH1,ABCF1 and RSPH6A among the 186 specific high-frequency mutation genes in primary esophageal squamous cell carcinoma were identified with copy number variation.Copy number variation occurred in GART and GOLIM4 genes in specific 10 frequently mutated genes.3.2.3 Comparison of significantly mutated genes in patients with ESCC and EAC with different clinical phenotypes3.2.3.1 Comparison of significantly mutated genes in male patients with ESCC and EACA total of 76 genes were detected in male ESCC and male EAC gene mutation with significant statistical difference(p<0.05),of which 11 genes such as C1orf173,CDC27 and MUC12 mutation frequencies were all>3%.ABCF1,AJUBA and CDH10 genes were only mutated in male ESCC,and the mutation frequencies were 9.22%(19/206),9.22%(19/206)and 6.80%(14/206),respectively.ELF2 and ZP3 genes were only mutated in male EAC,with a mutation frequency of 9.26%(5/54).3.2.3.2 Comparison of significantly mutated genes in female patient with ESCC and EACThere were statistically significant differences in gene mutations of 10 genes between the female ESCC and the female EAC(p<0.05),among which the mutation frequencies of 8 genes including CDC27,PABPC3 and ZC3H11A were>3%.The ABCA13 gene was only mutated in female ESCC,and the mutation frequency was 15.46%(15/97),and the PPP4R2 gene was only mutated in female patients with EAC,and the mutation frequency was 24.00%(6/25).3.2.3.3 Comparison of significantly mutated genes between ESCC and EAC with aged<60 yearsThe gene mutations of CDC2,BRCA2,TG,NBPF20,NIN,ZNF850 and MGA genes were detected to be significantly different between ESCC with aged<60 years old and EAC with aged<60 years old(p<0.05),in which the mutation frequencies of 7 genes including CDC27,BRCA2 and TG were all>3%.No specific gene mutation was found in the groups diagnosed with ESCC under the age of 60 and EAC under the age of 60.3.2.3.4 Comparison of significantly mutated genes between ESCC and EAC with aged≥60 yearsA total of 43 genes were detected between the ESCC with aged≥60 years and EAC with aged≥60 years(p<0.05),among which ZC3H11A,C1orf173 and PER3 The mutation frequencies of 6 genes were all>3%.The RELN gene was only mutated in ESCC at diagnosis age≥60 years,and the mutation frequency was 9.85%(20/203).And no specific gene mutation was found in EAC at diagnosis age≥60 years.3.2.3.5 Comparison of significantly mutated genes between patients with ESCC and patients with EAC in high-incidence areasA total of 9 genes in the high-incidence area ESCC and the high-incidence area EAC were significantly different(p<0.05).Among them,7 genes including EPPK1,CDH23 and USP10 have higher mutation frequencies in EAC than in ESCC;the mutation frequencies of KMT2D and USH2A genes were higher in ESCC than in EAC(23.64%vs 7.41%,14.55%vs 3.70%);the AJUBA gene was only mutated in the high-incidence area of ESCC,and the mutation frequency was 7.27%(12/165);the UTP14C gene was only mutated in high-risk EAC,and the mutation frequency was 9.26%(5/54).3.2.3.6 Comparison of significantly mutated genes between ESCC and EAC with positive family historyThe mutation frequencies of CDC27,NBPF3,KMT2D,EPPK1 and FLG genes were all>3%.The KALRN and ZNF107 genes were only mutated in ESCC with positive family history,and the mutation frequencies were 11.93%(13/109)and 10.09%(11/109),respectively.The PPP4R2 gene was only mutated in EAC with positive family history,and the mutation frequency was 14.29%(6/42).3.2.3.7 Comparison of significantly mutated genes between ESCC and EAC with negative family historyA total of 43 genes between family history negative ESCC and family history negative EAC were found to be significantly different(p<0.05),and among them the mutation frequencies of 11 genes including MUC16,AHNAK and C1orf173 were all>3%.The GIMAP7 gene was only mutated in EAC with negative family history,and the mutation frequency was 13.51%(5/37).And no specific gene mutation was found in ESCC with negative family history.3.2.3.8 Comparison of significantly mutated genes in patients with positive smoking history between ESCC and EACA total of 39 genes was found to be significantly different in smoking history-positive ESCC and smoking history-positive EAC(p<0.05).Among them,10 genes including MUC12,C1orf173 and AHNAK mutation frequency of all genes were>3%.ABCF1 gene mutation only occurred in ESCC with positive smoking history,and the mutation frequency was 10.95%(15/137).K1AA1731 and SPDYE1 genes were only mutated in EAC with positive smoking history,and the mutation frequencies were both 14.63%(6/41).3.2.3.9 Comparison of significantly mutated genes in patients with no-smoking history between ESCC patients and EAC patientsA total of 14 genes were detected in the smoking history-negative ESCC and the smoking history-negative EAC,the frequency of gene mutation was significantly different(p<0.05),among which the mutation frequency of 7 genes including PABPC3,ABCC9 and RAPGEF6 were>3%.PKHD1 and PCDHA2 genes were only mutated in ESCC with negative smoking history,and the mutation frequencies were 10.84%(18/166)and 9.64%(16/166),respectively.The PPP4R2 gene was only mutated in EAC with negative smoking history,and the mutation frequency was 15.79%(6/38).3.2.3.10 Comparison of significantly mutated genes in patients with no-drinking history between ESCC and EACA total of 55 genes were detected in the frequency of gene mutation in ESCC with no-drinking history and EAC with no-drinking history(p<0.05),among which the mutation frequency of 10 genes including NBPF20,RNF145 and ZC3H11A were>3%.NAV3,RELN,SI,UNC13C and CCDC178 genes are only frequently mutated in ESCC with no-drinking history,and the mutation frequencies are 8.76%(17/194),8.25%(16/194),7.22%(14/194),7.22%(14/194)and 6.70%(13/194).PPP4R2,CLEC16A and ELF2 were only frequently mutated in EAC with no-drinking history,with mutation frequencies of 12.07%(7/58),8.62%(5/58)and 8.62%(5/58),respectively.3.2.3.11 Comparison of significantly mutated genes affecting lymph node metastasis in patients with ESCC and EAC(1)Significantly mutated genes associated with lymph node metastasis of ESCCA total of 10 genes were identified in the lymph node metastasis-positive group and the lymph node metastasis-negative group with significant statistical difference(p<0.01),among them the mutation frequencies of UNC13C,AGBL3,SACS and TECPR2 in lymph node metastasis-positive ESCC were significantly higher than those in the lymph node metastasis-negative group;CUX2,VWA8,AMPH,ANKZF1,EXD3 and MAGEC3 were only mutated in lymph node metastasis-positive ESCC;the mutation frequencies of CUX2 and VWA8 genes were 6.08%(9/148)and 5.41%(8/148),respectively;and the mutation frequencies of AMPH,ANKZF1,EXD3 and MAGEC3 genes were all 4.73%(7/148).(2)Significantly mutated genes associated with lymph node metastasis in ESCCIt was identified that the TAF1 gene was highly mutated only in lymph node metastasis-positive EAC,with a mutation frequency of 15%(6/40).The mutation frequency of FSIP2 gene in lymph node metastasis-positive EAC was significantly higher than that in lymph node metastasis-negative group(20.00%vs 2.56%,p=0.029).(3)Comparative analysis of significantly mutated genes related to lymph node metastasis in ESCC and EACThe mutation frequency of TAF1 gene associated with lymph node metastasis of EAC was 5.41%in the positive group of ESCC,and 3.23%in the negative group of ESCC,there are no significant statistical difference(p=0.405).There was also no statistically significant difference in the frequency of mutations in the FSIP2 gene between the two groups(9.46%vs 10.32%,p=0.849).The mutation frequencies of 10 mutated genes with significant differences between the lymph node metastasis-positive and lymph node metastasis-negative groups in ESCC were all<3%in EAC.3.2.3.12 Comparison of significantly mutated genes in patients with early stage between ESCC and EACZNF141,ZNF680 and CACNA2D4 genes were identified as specific high-frequency mutation genes in early stage ESCC.The mutation frequency of ZNF141 gene was 12.50%(5/40),and the mutation frequency of ZNF680 and CACNA2D4 gene was 10%(4/40).The MYO18B gene is a specific high-frequency mutation gene in patients with early stage EAC,with a mutation frequency of 75%(3/4).3.2.3.13 Comparative analysis of significantly mutated genes between ESCC and EAC with lower thoracic segmentA total of 5 statistically significant gene mutations were found.Among them,the NBPF8 gene was only frequently mutated in the lower thoracic EAC,CDKN2A and CDH10 were only frequently mutated in the lower thoracic ESCC.And both RGPD3 and USP10 genes were mutated in lower thoracic ESCC and lower thoracic EAC,but the mutation frequencies were significantly different(p<0.05).3.2.3.14 Comparative analysis of significantly mutated genes associated with the prognosis of ESCC patients and EAC patientsA total of 3,752 genes with a mutation rate of>1.5%were identified in 303 patients with ESCC,of which 126 genes were significantly associated with survival(p<0.05).A total of 2,846 genes with a mutation rate of>1.5%were identified from 79 patients with EAC,of which 223 genes were significantly associated with survival(p<0.05).A total of 8 genes mutations were identified that were associated with the prognosis of both ESCC and EAC,and 118 gene mutations only affected the survival of ESCC patients,and the prognosis of patients with ESCC with mutations in EPS8,HYDIN,MED23,OR7C1,OTUD4,GLTSCR1,GRIK3 and ZBTB20 genes was worse than that of wild-type patients.A total of 215 gene mutations only affected the survival of patients with EAC,and the prognosis of EAC patients with GOLGA3,LAMA2,ACAN,EIF4G3,GRIK1 and GIMAP7 gene mutations was worse than that of wild-type patients.3.2.4 Construction of risk prediction and prognosis judgment models for ESCC and EAC3.2.4.1 Construction of risk prediction models for ESCC and EACAccording to multivariate Logistic regression analysis,the family history and 4 frequently mutated genes(CDC27,GART,PTPRZ1 and SPDYE1)were used to construct the risk prediction model for ESCC and EAC.The areas under the ROC curve were 0.709 and 0.679,respectively,and the model was qualified.3.2.4.2 Establish of a prognostic model for ESCC and EACAccording to multivariate Cox regression analysis,seven factors(High-incidence area/Low-incidence area,lymph node metastasis(with/without),HYDIN(MUT/WT),MED23(MUT/WT),GLTSCR1(MUT/WT),OTUD4(MUT/WT)and ZBTB20(MUT/WT))were used to establish the risk prediction model for primary ESCC,five factors(EIF4G3(MUT/WT),GIMAP7(MUT/WT),GOLGA3(MUT/WT),GRIK1(MUT/WT)and RSPH6A(MUT/WT))were used to establish the risk prediction model for primary EAC.Through internal verified by Bootstrap method,the C indexes of these models were 0.647 and 0.631 respectively.4 Conclusions4.1 Clinical phenotype,pathological characteristics and survival analysis of primary ESCC and primary EAC(1)There were significant differences in the distribution characteristics of primary ESCC and primary EAC with different clinical phenotypes such as gender,age,high/low incidence area,rural/urban,family history,and smoking history;(2)The survival of primary ESCC was significantly poorer than that of EAC.4.2 Genomics comparison between primary ESCC and primary EAC(1)TP53,TGFBR2,and PPP1R12B genes are the significantly mutated genes of primary ESCC and EAC,NOTCH1,PER3,CDKN2A,FAT1,and ABCF1 genes are the unique significantly mutated genes of primary ESCC,CDC27,ZC3H11A,and GART genes are the unique significantly mutated genes of primary EAC.(2)Copy number variation occur in 42 genes of 186 unique significantly mutated genes of primary ESCC,copy number variation of GART and GOLIM4 occur in primary EAC.(3)The patients with the following characteristics are more probably with primary ESCC:age≥60 years old with PER3 gene mutation,female with PABPC3 gene mutation,high-incidence area with AASDH gene mutation,or negative family history and KCNMA1 gene mutation are more likely to have ESCC than EAC.(4)The patients with CDC27 gene mutation and with any of the four characteristics(younger than 60 years old,positive family history,smoking or alcohol consumption)have a higher probability of developing primary EAC than ESCC.The patients with ZC3H11A gene mutation and with any of the three characteristics(female,older than 60 years or without alcohol consumption)have a higher probability of developing primary EAC.The patients with GART gene mutation and without smoking have a higher probability of developing primary EAC.(5)ZNF141,ZNF680 and CACNA2D4 genes are significantly mutated genes in early stage of primary ESCC,MYO18B and PKHD1L1 genes are significantly mutated genes in early stage of primary EAC.(6)NBPF8,RGPD and USP10 genes may be significantly mutated genes of primary EAC located in the lower thoracic segment in China.(7)EPS8,GLTSCR1,OR7C1,OTUD4,HYDIN,ZBTB20,MED23 and GRIK3 gene mutations are associated with poor prognosis in patients with ESCC;LAMA2,EIF4G3,GRIK1,GOLGA3,GIMAP7 and ACAN gene mutations are associated with poor prognosis in EAC patients.(8)Successfully established risk prediction and prognosis judgment models for ESCC and EAC. |
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