Prediction Model Of Recurrent Laryngeal Nerve Lymph Node Metatasis In Esophageal Carcinoma And Application Of Related Molecular Markers

Background and significanceAccording to the epidemiological data of the He jie in 2020,the annual number of new cases of esophageal cancer in our country is 246,000(43%of total world),and the annual death number is 188,000.Its morbidity and mortality rank 6th and 4th among all malignant tumors in our country.At present,the staging of esophageal cancer is the main basis for determining the treatment strategy.We mainly treat mucosal layer esophageal cancer tumors by ESD,EMR or endoscopic energy therapy,such as photodynamic therapy,Laser treatment,radiofrequency ablation and other methods.For early-stage esophageal cancer that invades the submucosa,the main choice is the comprehensive treatment based on surgery.Surgery includes radical dissection of the lesion and lymph node.Before 2000,the surgical methods for esophageal cancer in our country were:1.The sweet operation with left thoracotomy,which can complete the lymph node dissection(LND)of the lower mediastinum and upper abdomen.2.The Ivor-Lewis procedure through the right chest and upper abdomen approach can complete LND of two fields.The recurrence rate of lymph nodes after esophageal cancer is about 20%-40%,and the above mediastinum and neck are the main area.The multi-center retrospective study reported by the Japanese scholar Isono in 1991 suggested that the McKeown operation of threeincision esophageal cancer resection with radical LND in the neck,chest and upper abdomen reduced postoperative recurrence.Then,the 5-year survival rate of Japanese patients with esophageal cancer has increased to 34.3%compared with 26.7%of the two-field dissection.Therefore,many Japanese scholars have begun to take McKeown method for ESCC.However,this procedure has significant shortcomings such as complicated operation,huge trauma,and many postoperative complications,especially when the lymph nodes adjacent to the recurrent laryngeal nerve are dissected,it can cause complications such as recurrent laryngeal nerve injury and secondary lung infection.Studies have shown that not all patients can benefit from the removal of lymph nodes adjacent to the recurrent laryngeal nerve.For example,a Japanese scholar Taniyama's study found that for lower thoracic and abdominal esophageal cancer,whether or not to perform lymph node removal at this site has no statistical difference for 5-year survival rate.Accurately assessing the metastasis status of lymph nodes adjacent to the recurrent laryngeal nerve before treatment has important guiding significance for determining the surgical plan and the extent of lymph node dissection during the operation.However,there is no high-accuracy detection method for determining the metastasis status of the lymph nodes adjacent to the bilateral recurrent laryngeal nerve.At present,spiral CT is still the most commonly used method for judging the extent of tumor invasion and lymph node metastasis.Imaging experts usually judge lymph node metastasis with a short diameter greater than 1 cm as the boundary,but its sensitivity and specificity are less than 80%.It is obviously improper for lymph nodes of different parts to share the same criteria.The CT diagnostic criteria of lymph nodes adjacent to the recurrent laryngeal nerve of esophageal cancer was discussed in Part I.There are many factors that predict and affect the metastasis status of lymph nodes adjacent to the recurrent laryngeal nerve.The accuracy in judging the metastasis status of lymph nodes by CT or some other inspection method alone is low.Mathematical prediction model is a mathematical formula obtained by quantifying and weighting multiple risk factors in the clinic.Logistic multivariate regression analysis,as a special probability prediction mathematical model,is more and more widely used in clinical practice.In 2003,Professor Van Zee of the Memorial Sloan-Kettering Cancer Center in New York,USA conducted a multiple regression analysis on the clinicopathological data of 702 breast cancer patients who found sentinel lymph node metastasis and then scanned the axillary lymph nodes.He screened out 8 risk factors related to lymph nodes and established an MSKCC nomogram model to predict ymph node metastasis in breast cancer.The model was published on the website of Memorial Sloan-Kettering Cancer Center and was widely verified all over the world,the obtained AUC(area under the curve)value is 0.58-0.86,which has high accuracy.In the second part of this study,a multi-factor analysis of factors that predict and affect the metastasis status of the recurrent laryngeal nerve lymph nodes in patients with esophageal cancer was carried out to establish a mathematical model.This provides a more effective predictive method,which provides a reference basis for individualized treatment of patients and guides clinical treatment.In order to further improve the accuracy of our prediction of lymph node metastasis of thoracic ESCC,we hope to improve our prediction of lymph node metastasis of it by adding molecular diagnostic indicators.In addition,the related molecular indicators that affect the occurrence and development of tumors are usually closely related to the degree of disease,recurrence and metastasis,and prognosis.Indepth research on them has important practical clinical value for judging the nature of the disease,the stage of the disease,and the prognosis.Proliferating cell nuclear antigen(Ki-67)is a potential proliferation marker of tumor cells widely used at present,and has certain reference value for predicting the malignant transformation of tumors.Studies have shown that its expression rate gradually increases in different stages of the evolution of normal esophagus to dysplasia and esophageal cancer,which plays an important role in distinguishing early esophageal cancer and precancerous lesions.Minichromosome Maintenance Proteins 7(MCM7)is a new proliferation indicator.It plays a key role in maintaining the activity of the helicase of the MCM complex and controlling the initiation of DNA replication.MCM7 is expressed,but it is not affected by DNA repair and has better specificity.Some scholars suggest that it be used as a reliable biomarker to monitor the process of cell proliferation.Previous research found that MCM7 overexpression exists in many tumor cells,such as NSCLC,hepatocellular carcinoma,neuroblastoma and other tumors.Podoplanin(PDPN)is closely related to the malignant progression of tumors,such as epithelial-mesenchymal transition and distant metastasis and invasion.It promotes the progression and metastasis of a variety of tumors including oral squamous cell carcinoma,bladder tumors,brain tumors,etc.,but its relationship with the occurrence and development of esophageal squamous cell carcinoma is still unclear.The third part of this study explored the correlation between the expression of MCM7,Ki-67,and PDPN with the clinicopathological characteristics of ESCC and its clinical prognosis prediction value.In addition,we observed the influence of Ki-67 and Podoplanin on the biological behavior of esophageal cancer cells from the cytological level,which provides a theoretical basis for accurately predicting the condition of esophageal cancer and selecting tumor treatment targets.Part ? The criteria for diagnosis of recurrent laryngeal nerve lymph node metastasis by multiplanar reconstruction of spiral CT inthoracic oesophageal squamous cell carcinomaObjectiveTo study the diagnostic value of multiplanar reconstruction(MPR)of spiral CT in recurrent laryngeal nerve lymph node metastasis and work out the best criteria for it.MethodsBased on the of with thoracic esophageal cancer who underwent radical in our hospital During December 2016 and June 2019,138 patients were treated with resection of esophageal cancer,the clinical data was collected.Multi-plane reconstruction technology was used for image post-processing to the previous multi-slice spiral CT scan data.Measure the short diameter and long diameter of the lymph nodes adjacent to the bilateral recurrent laryngeal nerve,calculate the short diameter and length diameter ratio(SLR),compare the above three parameters with the postoperative pathological results,and draw the ROC Curve of the above three indicators for predicting the corresponding lymph node metastasis status to compare the sensitivity and specificity of different diagnostic criteria used in clinical practice.ResultsIn the 138 patients,291 of left RLN LNs were cleared,with an average of 2.11 ± 0.41(0-14)in each case,the metastasis rate is16.70%;436 of the right RLN LNs were cleared,with an average of 3.16 ± 0.45(0-17)in each case,the metastasis rate is 21.00%,with a total metastasis rate of 29.7%.The area under ROC curve was 0.808,0.779 and 0.621 respectively when we use the short diameter,long diameter,and short length diameter ratio of the left RLN LNs as diagnostic criteria.When we use 3.10 mm as the the cut-off value of short diameter of the left RLN LNs.The sensitivity was 91.3%and the specificity was 41.7%.The area under ROC curve was 0.865,0.807 and 0.637 respectively when we use the short diameter,long diameter,and short length diameter ratio of the right RLN LNs as diagnostic criteria.When we use 5.68 mm as the the cut-off value of short diameter of the right RLN LNs,The sensitivity and specificity of the diagnosis were 96.6%and 67.9%,respectively.Conclusion1.The short diameter of lymph node has higher diagnostic efficiency than the long diameter of lymph node and SLR of lymph node.2.It has high clinical clinical significance when we set the cutoff value of the short diameter of the left recurrent laryngeal nerve lymph node as 3.10 mm and the right 5.68 mm.Part ?A nomogram prediction model for recurrent laryngeal nerve lymph node metastasis in thoracic oesophageal squamous cell carcinomaObjectiveTo explore the risk factors of RLN LNs metastasis in thoracic esophageal squamous cell carcinoma,and to establish a nomogram model to predict the status of them.MethodsCollect the information of 265 patients with thoracic esophageal squamous cell carcinoma from 2015-1-1 to 2018-6-1 in the 960 Hospital of the People's Liberation Army and the Second Hospital of Shandong University,such as the short diameter of the left and right lymph nodes adjacent to the recurrent laryngeal nerve(RLN LNs),the location of the lesion and postoperative pathology.The risk factors related to lymph node metastasis to the recurrent laryngeal nerve were preliminarily screened by univariate analysis,then we analyzed them by binary logistic regression and a model was established.Draw a Nomogram graph,then evaluate the discrimination and effect of the model by calibration curve and the area under the ROC curve(AUC).The performance of the model was internally verified through the Bootstrap method,and 114 consecutive patients collected from July 2018 to October 2020 were used as an independent verification data set for external verification.ResultsAmong the 265 patients,222 were males and 43 were females.A total of 6255 lymph nodes were dissected,with an average of 23.60±1.04(7-58)lymph nodes.2.34±0.33(0-16)for the left RLN LNs.2.94±0.31(0-17)for the right RLN LNs.The metastasis rate of RLN LNs was 28.3%.(1)We screened out 6 independent risk factors by multivariate analysis for RLN LN metastasis.The calibration curve shows that the predicted values in the training set and the validation set are in good agreement with the actual observations.The Bootstrap corrected AUC of the training set and external validation set were 0.809(95%CI:0.751?0.861)and 0.819(95%CI:0.733?0.895),respectively.(2)Short diameter>5 mm is an independent risk factor for left RLN LN metastasis.(3)We screened out 3 independent risk factors by multivariate analysis for right RLN LNs metastasis.AUC of the Nomogram model was 0.868(0.812-0.916).The calibration curve shows that the predicted values in the training set and the validation set are in good agreement with the actual observations.The Bootstrap correction AUC of its training set and external validation set are 0,868(0.812-0.916)and 0.893(0.828-0.946),respectively.ConclusionThe Nomogram model can predict the possibility of lymph node metastasis next to the recurrent laryngeal nerve,which has high clinical practical value.Part ? Application value of MCM7,Ki-67 and Podoplanin Expression in Esophageal Squamous Cell CarcinomaObjectiveTo observe the correlation between MCM7,Ki-67 and Podoplanin and clinicopathological factors and clinical prognosis of esophageal squamous cell carcinoma.Observe the effects of Ki-67 and Podoplanin on the biological behavior of esophageal cancer cell lines.MethodsWe collected the clinical information of 105 ESCC patients,including their degree of differentiation,LNM status,tumor stage,TNM stage,progression-free survival(PFS)and overall survival(OS).Compare its expression with the differentiation degree of thoracic esophageal squamous cell carcinoma,lymph node metastasis status,T staging,TNM staging and other clinicopathological factors,PFS,OS.In order to observe the influence of Ki-67 and Podoplanin on the biological behavior of ESCC cells,we knocked down the expression of Ki-67 and PDPN in the ESCC cell line KYSE150,and observed the proliferation and migration ability by a vareaty of tests.Results(1)Ki-67 is related to the differentiation degree of thoracic ESCC(p<0.001)and LNM(p=0.006),Podoplanin is related to T stage(p=0.008),and the difference is statistically significant significance.(2)The postoperative median PFS of patients with thoracic esophageal squamous cell carcinoma with low and high expression of MCM7 was 40 months and 24 months,respectively(P=0.042).(3)CCK8,EdU and clone formation experiments showed that the proliferation ability of KYSE150 cells in the Ki-67 and PDPN interference group decreased.Scratch experiment and transwell experiment showed that the migration and invasion ability of KYSE150 cells in the Ki67 and PDPN interference group decreased.Conclusion(1)Ki-67 and PDPN plays an important role in the proliferation,migration and invasion of ESCCcells.It is related to the depth of invasion and differentiation of esophageal cancer and lymph node metastasis.(2)Patients with thoracic esophageal squamous cell carcinoma with high MCM7 expression have a poor prognosis,which is more prone to early postoperative recurrence,and the overall survival time is shorter.(3)Ki-67,Podoplanin and MCM7 can be used as potential prognostic markers and tumor treatment targets for esophageal cancer.