| Background and objective:As one of the pathological nature characters,heterogeneity exists even in the early stage of gastric cancer.Even in early gastric cancer,mixed cancer with more than two histological types can be commonly detected.Based on the guidelines of the Japanese Gastric Cancer Society and the Nakamura classification of gastric cancer,early gastric cancer with a dominant proportion of differentiated cancer is classified as differentiated gastric cancer,while early gastric cancer with a dominant proportion of undifferentiated cancer is classified as undifferentiated gastric cancer.However,there is a lack of descriptive research based on the degree of mixing between differentiated and undifferentiated gastric cancer.Recent studies have shown that mixed histological type early gastric cancer has higher malignant potential than non-mixed early gastric cancer.At the same time,it is reported that the lymph node metastasis rate of mixed early gastric cancer is not higher than that of non-mixed early gastric cancer.There are contradictions in the existing research on mixed gastric cancer,and there is no continuous and complete research on early gastric cancer with different mixed degrees.Therefore,we conducted this study to reveal the clinicopathological characteristics of early gastric cancer with different mixed degrees and the role of this variable in clinical prediction of lymph node metastasis of early gastric cancer.Methods:In the first part of the study,all cases diagnosed as gastric cancer in our clinical center from 2016 to 2019 and pathologically showed mixed early gastric cancer were screened continuously.According to the retrospective review of the film by the pathologist,each case belongs to a mixed type of cancer with differentiated predominance or undifferentiated predominance.The representative wax samples corresponding to each case were collected from the pathological database,and the mucus phenotype of mixed gastric cancer(gastric type/intestinal type/gastrointestinal mixed type/empty line)was determined by immunohistochemical staining,and then the difference of mucus phenotype between differentiated dominant mixed type cancer and undifferentiated dominant mixed type cancer was compared.The mucus phenotype was determined by immunohistochemistry staining.The criteria for determining the mucus phenotype in this study are as follows:1)Gastric mucus phenotype:MUC6 and/or MUC5AC showed positive staining,while MUC2 and CD 10 staining were both negative;2)Intestinal mucus phenotype:positive staining for MUC2 and/or CD 10,while negative staining for MUC6 and MUC5AC;3)Gastrointestinal mixed mucus phenotype:MUC6 and/or MUC5AC staining positive while MUC2 and/or CD 10 staining positive;4)Empty type:If all four markers(MUC6,MUC5 AC,MUC2,and CD 10)stain negative,the mucinous phenotype is classified as empty type.The difference of lymph node metastasis rate between differentiated dominant mixed type cancer and undifferentiated dominant mixed type cancer was analyzed based on the lymph node metastasis obtained from the retrospective pathological results after operation.For the following parts of the study,through the medical record system of the clinical base,the clinical medical records of patients who had undergone radical surgery including lymph node dissection procedure in the First Affiliated Hospital of Dalian Medical University from January 2014 to January 2022 were retrospectively collected and analyzed.According to the clinical and postoperative pathological results,a total of 655 cases diagnosed as early gastric cancer after surgery were continuously selected.After further screening by exclusion criteria,a total of 626 cases with a total of 626 lesions were finally included in the study.The exclusion criteria are as follows:1)Special pathological types(neuroendocrine tumors,lymphostromal carcinoma,hepatoid carcinoma)that do not meet the criteria of this study were included(a total of 6 cases were excluded);2)Cases of multiple concurrent cancers(excluding a total of 12 cases);3)Experienced endoscopic treatment before surgery(excluding 2 cases);4)Local recurrence cases(excluding 2 cases);5)Cases who underwent preoperative radiotherapy or chemotherapy(0 cases were excluded);6)Cases with missing preoperative endoscopic examination results or images(a total of 7 cases were excluded).Finally,626 cases(including 626 early gastric cancer lesions)were included in this study.All clinical pathological data of the included cases were collected or evaluated according to certain methods and used for subsequent study.The proportion of undifferentiated components(PUC)of the 626 early gastric cancer lesions was obtained through the overall evaluation of all pathological sections of all cases after operation.Cases with a single pathological type of differentiated carcinoma(PUC=0%)were included in the PD(pure differentiated,PD)group.Cases with undifferentiated carcinoma(PUC=100%)were included in the pure undifferentiated group(PUD).The other cases that contain both differentiated and undifferentiated cancer components were classified as mixed cancer and grouped proportionally according to their mixing degree.The mixed type cancers were classified into G1 group,G2 group,G3 group,and G4 group according to the proportion of undifferentiated cancer.LNM rates of G1 and G2 groups were compared with the PD group under the indication framework of ESD treatment for differentiated early gastric cancer.We compared the LNM rates of G3,G4,and PUD groups under the indication framework of ESD treatment for undifferentiated early gastric cancer.To explore the differences in lymph node metastasis rates of gastric early cancer with different degrees of mixing based on ESD(endoscopic submucosal section,ESD)indications.Absolute indication:According to the relevant guidelines for the treatment of early gastric cancer using the new version of ESD,the absolute indication criteria for ESD are as follows:1)Pathological properties are determined to be differentiated cancer,with lesions limited to the mucosal layer,without ulcers,and unlimited tumor diameter.2)Pathologically,it is classified as differentiated cancer,with lesions limited to the mucosal layer and accompanied by ulcers.The tumor diameter is less than or equal to 3cm.3)The pathological nature is determined to be undifferentiated cancer,with lesions limited to the mucosal layer and no ulcers.The tumor diameter is less than or equal to 2cm.Relative indication:Based on a combination of four dimensions:pathological classification,lesion diameter,presence of ulcer formation,and depth of lesion infiltration,early gastric cancer lesions that exceed the absolute indication range are classified as gastric relative indications.Based on the ESD indications for the treatment of early gastric cancer,we calculated the lymph node metastasis rates of early gastric cancer with different mixed degrees under the absolute and relative indications of ESD to determine their applicability,respectively.According to the guidelines proposed by the Japanese Gastric Cancer Society,when evaluating the ESD indications and postoperative cure degree of early gastric cancer,gastric cancer lesions with the main histological types of high and medium differentiated tubular adenocarcinoma and papillary adenocarcinoma were uniformly evaluated according to the differentiated cancer standard,while gastric cancer lesions with the main histological types of low and ring cell carcinoma were uniformly evaluated according to the undifferentiated cancer standardIn the third part of the study,mixed type cancers were further subdivided into M1(0%<PUC≤20%),M2(20%<PUC≤0%),M3(40%<PUC≤60%),M4(60%<PUC≤80%),and M5(80%<PUC<100%)based on PUC levels.By further grouping the cases with undifferentiated cancer in different proportions,we explored the differences in clinicopathological data between early gastric cancer with different proportions of mixed degree.In this part,the patient’s gender,age,tumor size,tumor location,general morphology,presence or absence of ulcers,depth of tumor infiltration,presence or absence of vascular invasion,presence or absence of nerve invasion,degree of intraoperative lymph node clearance,and number of intraoperative lymph node dissection were collected or evaluated.The overall average age of patients in the reference study was 63.6(± 8.8)years,and the cutoff value for the age group of patients was set at 60 years old.Given that the overall arithmetic mean of tumor size is(2.4 ± 1.3cm),we divided the lesion size into two groups:less than or equal to 2cm and greater than 2cm,with a cutoff value of 2cm.We divide the tumor into three groups based on its actual location in the stomach:upper 1/3,middle 1/3,and lower 1/3.According to the Paris classification,the general morphology of early gastric cancer can be divided into five subtypes:Type 0-1(protrusion type),Type 0-Ⅱa(superficial protrusion type),Type 0-Ⅱb(flat type),Type 0-Ⅱc(superficial depression type),Type 0-Ⅲ(depression type)Ulcer judgment:UL(+)if ulcer formation or ulcer scar is observed,UL(-)if no ulcer formation or ulcer scar is observedIn this study,the depth of tumor invasion of early gastric cancer is divided into three levels:1.The tumor is limited to M(mucosal),including the mucosal epithelium,mucosal lamina propria and muscularis mucosae.2.The tumor is limited to SM1(sub mucosal layer 1,shallow layer of submucosa),which means the depth of the tumor breaks through the muscularis mucosae and the deepest infiltration is less than 500 microns.3.The tumor reaches SM2(sub mucosal layer 2,deep submucosa),which means the deepest tumor invasion reaches or exceeds 500 micronsDegree of intraoperative lymph node clearance was according to the Japanese guidelines for the treatment of gastric cancer.According to different surgical procedures,the criteria for determining the range of lymph node clearance in the treatment of early gastric cancer are as follows:1.Total gastrectomy:DO:The range of lymph node dissection is less than D1.D1:No.1-7.D1+:D1+No.8a,9,11p.D2:D1+No.8a,9,11p,11d,12a.2.Distal gastrectomy DO:The lymph node dissection range is less than D1.D1:No.1,3,4sb,4d,5,6,7.D1+:D1+No.8a,9.D2:D1+No.8a,9,11p,12a.3.Proximal gastrectomy:D0:D0:The lymph node dissection range is less than D1.D1:No.1,3,4sb,4d,5,6,7.D1+:D1+No.8a,9.In this study,the median value of the number of lymph node biopsy was 22.3(±9.6),and 22 was finally set as the grouping cutoff value of the number of lymph node biopsy.Including PUC level as one of the parameters,the independent risk factors for lymph node metastasis of early gastric cancer were determined by univariate analysis and multivariate logistic regression analysis.Finally,based on the results of logistic regression analysis,the nomograph prediction model of lymph node metastasis probability of early gastric cancer was further constructed by using R language statistical analysis software.The consistency between the probability of predicted outcomes and the probability of actual observed outcomes in this model is evaluated through a calibration curve,represented by the consistency index(C-index).Under the guidance of the Bootstrap method,a sample queue with the same sample size is repeatedly selected as the model training set,while the corresponding un-sampled queue set is used as the validation set to evaluate the performance of the model.This verification was repeated 1000 times to obtain the final calibration curve.Results:1.Differences in mucus phenotype and lymph node metastasis rate between differentiated and undifferentiated dominant early gastric cancer:In mixed early gastric cancer,there was no significant difference in mucus phenotype distribution between differentiated and undifferentiated dominant types(p=0.534).There was no statistically significant difference in lymph node metastasis rates among different mucus phenotypes(p=0.787).The lymph node metastasis rate of undifferentiated dominant mixed gastric early cancer was significantly higher than that of differentiated dominant gastric early cancer(31.3%vs 10.3%,p=0.039).2.Study on the adaptability of ESD indications to early gastric cancers of different mixed degrees.In this part of the study,the grouping results of mixed type cancer are as follows:312 cases classified into PD group(PUC=0%),34 cases in G1 group(0%<PUC≤25%),39 cases in G2 group(25%<PUC<50%),64 cases in G3 group(50%≤PU≤75%),55 cases in G4 group(75%<PUC<100%),and 122 cases in PUD group(PUC=100%).Group G1,G2 and PD were compared under the ESD indications for differentiated early gastric cancer,and group G3,G4 and PUD were compared under the ESD indications for undifferentiated early gastric cancer.There was no statistical difference in the rate of lymph node metastasis among early gastric cancer patients who met the absolute indication of ESD treatment for early gastric cancer.Among the mixed cancers that met the relative indication of ESD for the treatment of undifferentiated early gastric cancer,the lymph node metastasis rate of G4(75%<PUC<100%)in the mixed cancer group was significantly higher than that of other groups(G4 vs PU:44.9%vs 10.8%,p<0.001;G4 vs G3:44.9%vs 18.0%,p=0.002).3.In exploring the differences in clinicopathological data of early gastric cancer with different undifferentiated proportions,we divided the mixed type cancer into more five groups:M1(0%<PUC≤20%)26 cases,M2(20%<PUC≤40%)34 cases,M3(40%<PUC≤60%)30 cases,M4(60%<PUC≤80%)41 cases,M5(80%<PUC<100%)61 cases.There was no statistical difference in the difference of clinicopathologic factors,age,sex,tumor location,general shape of tumor,whether there were ulcers on the tumor surface,scope of lymph node dissection and dissected lymph node number during operation among the 7 groups(p>0.05 after Bonferroni correction).The number of lesions with nerve invasion in pure undifferentiated carcinoma group(PUD)was more than that in pure differentiated carcinoma group(PD)(p=0.025 after Bonferroni correction)Compared with the pure differentiated group(PD),the cases of mixed type cancer M4 group(60%<PUC≤80%,p<0.001)and mixed type cancer M5 group(80%<PUC<100%,p=0.035)showed more lymphatic or vascular invasion(LVI).At the same time,compared with PD group,the tumor diameter of M4 group was larger(p=0.025),and the tumor of M5 group showed more SM2 layer infiltration(p<0.05)(All p value were corrected under Bonferroni method).With regard to the comparison of lymph node metastasis rates among the groups,we found that the lymph node metastasis rate of early gastric cancer lesions in the mixed cancer group M4(60%<PUC≤80%)was higher(M4 vs PD:p<0.001,M4 vs M1:p=0.010,M4 vs M2:p=0.004,M4 vs M3:p=0.038,M4 vs M5:p=0.040,M4 vs PUD:p<0.001,all p value corrected by Bonferroni method).In this study,the lymph node metastasis rate of M5(80%<PUC<100%,p=0.035)in mixed cancer group was higher than that of PD group(p=0.009 after Bonferroni correction).The overall lymph node metastasis rate in M5 group was higher than that in pure undifferentiated cancer group PUD group(19.7%vs 9.0%),but there was no statistical difference in this study.In addition,there was no statistical difference in the rate of lymph node metastasis between any groups(p>0.05 after Bonferroni correction).4.The results of univariate analysis related to lymph node metastasis of early gastric cancer showed that the diameter of the tumor was greater than 2 cm(OR 4.015;95%CI 2.245-7.179;P<0.001),there was ulcer on the tumor surface(OR 2.038;95%CI 1.212-3.424;P=0.007),the depth of tumor infiltration reached the SM2 layer of submucosa(OR 7.809;95%CI 3.952-15.431;P<0.001),and there was vascular infiltration(OR 18.082;95%CI 10.201-32.052;P<0.001),The presence of nerve invasion(OR 2.973;95%CI 1.261-6.843;P=0.009),the proportion of undifferentiated cancer components in the lesions[(M4:OR 14.687;95%CI 6.812-31.666),(M5:OR 3.777;95%CI 1.725-8.267),p<0.001]were all related to lymph node metastasis of early gastric cancer(Table 3).Multivariate logistic regression analysis showed that the tumor size was more than 2 cm(OR 3.157;95%CI 1.581-6.303;P=0.001),and the depth of tumor infiltration reached the SM2 layer of submucosa(OR 2.869;95%CI 1.262-6.523;P=0.012),The presence of vascular invasion(OR 12.648;95%CI 6.246-25.611;P<0.001)and the proportion of undifferentiated cancer at M4 level(60%<PUC≤80%)(OR 12.205;95%CI 4.791-31.088;P<0.001)significantly predicted a higher risk of lymph node metastasis in early gastric cancer5.Establishment of probability prediction model for lymph node metastasis of early gastric cancer and its internal validation structure:According to the results of multivariate logistic regression analysis related to lymph node metastasis of early gastric cancer,a prediction model for lymph node metastasis of early gastric cancer was built by using R language software.The depth of tumor invasion to the SM2 layer of the submucosa,the size of the tumor is more than 2cm,the proportion of vascular invasion and undifferentiated cancer is the level of M4 group,and the scores in the nomograph prediction model are 39 points,42.5 points,38 points and 93 points respectively.According to the score of each clinicopathologic factor,the probability of lymph node metastasis was evaluated after the total score was added.The ROC curve was drawn to evaluate the predictive ability of the nomograph model for lymph node metastasis.The results showed that the area under the curve of the nomograph prediction model was 0.899(range 0.724-0.915)(95%CI 0.724-0.915,P<0.05).Bootstrap self-sampling internally verifies the prediction efficiency of the nomograph prediction model,and the C-index value is 0.899.The correction curve shows that the prediction probability of the nomograph model is consistent with the actual lymph node metastasis probability(Figure 4).Hosmer-Lemeshow tests that the fitting effect of the calculation model is good(χ=7.187,P>0.05).Conclusion:1.There is no significant statistical difference in lymph node metastasis rate between different mucus phenotypes in mixed early gastric cancer,and there is no significant difference between the mucus phenotypic expression profile of differentiated dominant mixed cancer and undifferentiated dominant cancer.In mixed early gastric cancer,there is a difference in lymph node metastasis rate between differentiated dominant early gastric cancer and undifferentiated dominant early gastric cancer.2.The absolute indication standard for ESD treatment of early gastric cancer is relatively applicable to the treatment of mixed early gastric cancer.The probability of lymph node metastasis is higher for the mixed type cancer with undifferentiated cancer that does not meet the absolute indication of ESD.3.The clinicopathological characteristics of early gastric cancer with different proportion of undifferentiated cancer are different.4.The tumor size is more than 2cm,the depth of tumor invasion reaches the SM2 layer of submucosa,the presence of vascular invasion and the proportion of undifferentiated cancer at the M4 level are independent risk factors for lymph node metastasis of early gastric cancer.5.The nomograph model based on the risk factors of lymph node metastasis of early gastric cancer can predict the probability of lymph node metastasis of early gastric cancer. |
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