| 1 Background and ObjectiveIn China,esophageal cancer(EC)is a common malignant tumor,and its morbidity and mortality ranks in the forefront of malignant tumors nationwide,which threatens the health of the people seriously.Despite the growing awareness of public on esophageal cancer and the continuous advancement of medical technology,the mortality rate of patients has not decreased significantly.In particular,some patients with postoperative pathological diagnosis of early cancer die soon in one-year time after surgery.For the Surgeons and physicians,it is still a problem for them to make suitable plan to cure the patients.Currently,the effect of lymph node metastasis(LNM)has been confirmed that it has a severe influence on the survival of esophageal cancer.Patients with LNM are prone to suffer from recurrence and metastasis after surgery,which worsen the prognosis of them.Due to the unique anatomy of the esophagus,the submucosal lymphatic vessels are rich,crisscrossed,and have the characteristics of jumping and bidirectional.A wide range of LNM can occur in the early stage.However,there are still a considerable number of patients without LNM even their tumors had invaded the surrounded tissues of the esophagus.Therefore,it is worthy to explore that what are the characteristics of LNM of esophageal cancer,and which clinicopathological factors would affect LNM.In recent years,studies on the relationship between gene mutations and tumorigenesis have been carried out in full swing.Which genes are involved in the development of esophageal cancer,and which genes are involved in lymph node metastasis are also needed to be resolved urgently.There are mountain clinical studies on LNM worldwide,and some consensus has been reached.However,there are still arguing on some aspect.Beside,the samples enrolled in those studies are small and there is lack of demonstration of large data analysis.There are also little papers on the molecular level to find the mechanism of LNM.Here,we enrolled 69,039 esophageal squamous cell carcinoma(ESCC)with clinicopathological information.To our knowledge,it is the largest number of studies on esophageal cancer LNM,and we attempts to analyze the relationship between LNM and clinicopathological features,and their impact on postoperative survival.We want to use our results to compare the former study.In addition,in this study,112 pairs of ESCC samples were subjected to whole exon sequencing,and 411 pairs of samples received target Region Sequencing.Then,the significant high frequency genes were enrolled to under Immunohistochemical test for us to explore the expression of the protein 5,806 ESCC patients,it would help us to find out the genes and proteins which may play roles on the LNM of EC2 Materials and methods2.1 Clinicopathological features and effect on survival of esophageal cancer lymphnode metastasis2.1.1 PopulationThere were 44,408 males with an average age of 59.6±8.45 years and 24,631 females with an average age of 60.4±8.37 years,And 41,860 cases(60.4%)with negative lymph node metastasis.2.1.2 MethodsAccording to the hospital where the patient is treated,the patient's clinical pathological information is verified by searching the patient's name and the case number.The patients with esophageal cancer who were included in the study were followed up by telephone or face-to-face.The follow-up information was verified and entered into the computer.The data was analyzed by SPSS 21.0,and the results were analyzed using a chi-square test and a T-test.The Kaplan-Meier method was used to analyze the patient's survival.Multivariate analysis was performed using Logistic regression and COX models.A statistically significant criterion was set for ?=0.05.Stochastic Configuration Networks was initially used to evaluate the influence of different factors on LNM.2.2 whole exome sequencing,targeted sequencing and related protein expression in lymph node metastasis of esophageal cancer2.2.1 Patients112 ESCC patients were enrolled in the Whole genome exon sequence,including 90 males,with an average age of 62.5±7.71 years and 22 females with an average age of 63.5±6.09 years.Target region gene sequencing verification 411,including 320 males,with an average age of 62.2±7.64 years and 91 females with an average age of 61.0±9.80 years.Tissue microarray immunohistochemistry were did on a total of 5806 cases of esophageal squamous cell carcinoma,including 3806 males,with an average age of 60.5±8.40 years and 2,000 females with an average age of 61.5±8.15 years.2.2.2 MethodsDNA of the cancer tissues and adjacent normal epithelial tissues of ESCC patients were extracted.After quality control,the library was constructed.After quality inspection,the samples were suquenced.To evaluate the quality of sequencing,standardized process was used to compare the data,filter the genes that meet the target requirements,and analyze the high frequency mutant gene which may has influence on LNM.Then,tissue microarray immunohistochemistry was performed on the target gene to analyze the relationship between protein expression and LNM and survival.Kasplan-Meier method was used to analyze the relationship between target gene mutation and LNM and the expression of protein in different ESCC patients with different grade of LNM.Test level:?=0.05.3 Results3.1 Clinicopathological features and effect on survival of esophageal cancer lymphnode metastasis3.1.1 Characteristics of lymph node metastasis of esophageal cancerOf the patients enrolled in the study,27,179(39.4%)were positive for LNM and 41,860(60.6%)had no LNM.The survival of patients with positive LNM was significantly worse than that of patients with negative LNM(p<0.001),The 5-year survival rate of patients was significantly decreased along with the increasing grade of N by numbers of 0,1-2,3-6,?7 LNM numbers.(p<0.001);as well as the other method to divided the LNM numbers according to 1,2-3,?4.the survival period gradually decreased with the increase metastatic region numbers(p<0.001),but in patients received three-field lymph node resection,there was no statistical difference between the number of region between 2 and 3(p=0.049,?=0.05/6).3.1.2 The effect on survival and lymph node metastasis of clinicopathological factors on esophageal cancerThe lymph node metastasis rate of male patients was significantly higher than that of female patients(p<0.001),and the survival was also worse than that of females(p<0.001).people at 50-69 years old seem to be prone to suffered from esophageal cancer,and there are no difference among different age grade in the LNM rate(p=0.066),but in terms of prognosis,survival gradually worsened with age(p<0.001);smoking and drinking were harmful to patients,lymph node metastasis rate and survival in patients with a history of smoking or drinking are worse than patients with not them;patients with a family history of cancer or patients lived in high incidence of esophageal cancer have lower lymph node metastasis rate and their survival was better(p<0.001).Along with the depth of infiltration increased,the rate of metastasis increased(p<0.001),and survival continued to deteriorate(p<0.001).In patients with stage T1,the lymph node metastasis rate was 16.7%,and the lymph node metastasis rate of the mucosal muscle layer was lower than that of the submucosa(p<0.001).The 5-year survival rate of patients gradually decreased from early to advanced stage in pathological stage(p<0.001).With the decrease of differentiation degree,the lymph node metastasis rate gradually increased(p<0.001),and the survival gradually became worse(p<0.001).With the increase of tumor length,the lymph node metastasis rate gradually increased(p<0.001),and the survival gradually become worse.(p<0.001).In carcinoma in situ and superficial esophageal cancer,the rate of concealed lymph node metastasis is low and the prognosis is best.In patients with advanced esophageal cancer,the ones whose type were mushroom type would had lower lymph node metastasis rate and the better survival rate than other types;as the tumor position moved down,the lymph node metastasis rate also increased gradually(p<0.001).However,the difference can only be founded in the upper and mid-thoracic survival(p<0.001);there was a positive correlation between the total number of lymph node resection and the number of positive lymph node metastases(r=0.141,p<0.001).Patients with ?15 had a higher lymph node metastasis rate than patients with less than 15(p<0.001),but had worse survival(p<0.001).3.1.3 Effect of supraclavicular lymph node metastasis on survival of ESCC patientsIn patients with lymph node metastasis,supraclavicular lymph node metastasis was not an independent factor(HR:1.116;95%CI:0.940-1.324).Under the same number of metastatic lymph nodes,there was no difference in survival between patients having lymph node metastasis with or without supraclavicular lymph node metastasis.3.1.4 Stochastic Configuration Networks Analysis of Factors Affecting Lymph Node MetastasisAmong the univariate factors,the depth of invasion had the greatest influence on lymph node metastasis(accuracy=0.57792).In the univariate combination of two groups,the depth of invasion plus gender had the greatest influence on lymph node metastasis(accuracy=0.68940).3.2 whole exome sequencing,tareted sequencing and related roteinp expression in lymph node metastasis of esophageal cancerA total of 114 high frequency mutation genes in esophageal squamous cell carcinoma were screened by Whole genome exon sequence.In 411 cases of esophageal squamous cell carcinoma,13 high frequency mutation genes were detected through targeted sequencing.One of them is COL12A1.The total mutation rate of COL12A1 was different between patients with positive lymph node metastasis and patients with negative lymph node metastasis(p=0.014).The survival rate of patients with mutation was worse than that of patients without mutation(p=0.013).The expression of COL12A1 protein in 5,806 esophageal squamous cell carcinomas was verified by tissue microarray.Effective staining was performed in 5,777 cases.COL12A1 protein was mainly expressed in cytoplasm.The positive expression rate was 70.9%.There were differences in lymph node metastasis positive/negative patients(p=0.028)and infiltration depth(p=0.024).The infiltration depth and differentiation degree of COL12A1 patients with(+),(++)and(+++)were different.The survival time of COL12A1 positive patients was worse than that of COL12A1 negative patients(p<0.001),and it was an independent factor(HR:1.134;95%CI:1.062-1.210).The survival of patients with(+++)expression of COL12A1 was worse than that of the other two positive expression types.4 Conclusions1)Here,We present a total number of 69,039 ESCC patients,to our knowledge,it is the largest data to study the clinicopathological features and related effects of LNM of esophageal squamous cell carcinoma.LNM is closely related to the prognosis of patients with esophageal cancer.Along with the number of LNM increases or the number of metastatic region,the patient's survival deteriorates;2)gender,degree of invasion,degree of differentiation,location of tumor,number of lymph node resection,high and low incidence area are independent influencing factors of lymph node metastasis;gender,age,degree of infiltration,degree of differentiation,tumor location,high and low incidence area are affecting patients with esophageal cancer Independent factor of survival;3)History of smoking,history of drinking,family history of tumors,length of tumor,and general type can affect ly,ph node metastasis and patients survival;4)Patients with a number of lymph node dissections of ?15 survived<15 patients,but the metastatic rate was higher than those of less than 15 patients;5)The survival of patients with supraclavicular lymph node metastasis is not significantly worse than that of lymph node metastasis without supraclavicular lymph node metastasis,so it should not be transferred to distant metastasis;6)Preliminary attempts have shown that the introduction of Stochastic Configuration Networks to evaluate the extent of LNM related factors in esophageal cancer has shown a certain significance,and further research is needed in the future;5)The mutation of COL 12A1 has a significant difference in the survival of patients with esophageal cancer.There is also a difference between patients with lymph node metastasis and those without metastasis.The lymph node metastasis rate of patients with negative expression of COL12A1 protein is lower than that of positive patients,and the survival is better than that of patients with positive expression. |
PDF Full Text Request