Study On The Relationship Between The Number Of Negative Lymph Nodes And Prognosis Of Patients With Esophageal Cancer And Immunotherapy For Esophageal Cancer

ENGLISH ABSTRASTS SECTION 1ANALYSIS OF THE RELATIONSHIP BETWEEN THE NUMBER OF NEGATIVE LYMPH NODES AND PROGNOSIS OF PATIENTS WITH THORACIC ESOPHAGEAL SQUAMOUS CELL CARCINOMA AFTER RADICAL RESECTIONBackgroundEsophageal cancer(EC)is one of the most common malignant tumors,with the seventh and sixth highest morbidity and mortality in the world.High incidence in Asia,Eastern and Southern African countries.In 2015,there were 477,900 newly diagnosed patients with EC and 375,000 EC-related deaths in China.The main pathological types of EC are adenocarcinoma and squamous cell carcinoma,mainly squamous cell carcinoma.Upwards of 90%of patients with EC in China have the squamous cell carcinoma subtype.Despite advancements in multidisciplinary treatment in recent years,radical resection is still the only treatment that can cure EC.Studies have confirmed that the number of metastatic lymph nodes(LNs)is an independent prognostic factor in patients with EC.The accurate number of metastatic LNs mainly depends on the degree of LN dissection,which reflects the accuracy of postoperative pathological staging to a certain extent.Although the 7th American Joint Committee on Cancer(AJCC)staging system recommends that a total dissection of at least 15 LNs are necessary to accurately stage patients undergoing esophagectomy without induction chemoradiation,the optimal extent of LN dissection during radical lymphadenectomy for EC are still controversial.In recent years,the number of NLNs has been confirmed to be an important factor related to patient survival in breast cancer,gastric cancer and colorectal cancer,proving to be more accurate than the number of metastatic positive LNs in predicting patients’ prognosis.Several researchers have analyzed the impact of NLNs on patient survival for EC.Greenstein et al.found that an increasing NLNs count was related to with long-term patient survival in those with LN-negative esophageal adenocarcinoma but not in those with the squamous cell subtype.Another study demonstrated that NLNs count was an advantageous prognostic indicator in LN-positive EC patients but not in those who were LN-negative.Wu et al.draw the conclusion that the number of NLNs was an independent prognostic factor for LN-negative EC through a retrospective review of 429 EC patients who underwent modern two-field lymphadenectomy.These results are inconsistent,and the conclusions are controversial and need to be further investigated.Therefore,in this study,we explore the correlation between the number of NLNs and prognosis of EC patients,through the retrospective analysis of patients with thoracic esophageal squamous cell carcinoma(ESCC)after radical resection,and to explore the clinical role of NLNs in EC.MethodsThis study retrospectively analyzed 579 patients with thoracic ESCC who underwent radical esophagectomy at the Department of thoracic surgery,Qilu hospital,Shandong University,China,between January 2009 and December 2012.The inclusion criteria were as follows:Patient ages ranging between 40 to 70 years;a diagnosis of thoracic ESCC(confirmed via histopathology);had not received preoperative chemotherapy and/or radiotherapy;no second primary tumor history;comprehensive preoperative examination including upper gastrointestinal imaging,chest and abdomen enhanced CT,neck color doppler ultrasonography,gastroscope and brain MRI;complete resection(RO);complete clinical data and at least 3 years of follow-up or death;survival time more than three months were all inclusion factors for this study.The exclusion criteria were as follows:patients with non-cancer related death;no RO resection;cervical esophageal cancer or those lost to follow-up.According to the number of NLNs,the patients were divided into 4 groups:0～9,10～14,15～19 and ≥20,respectively.Log-rank tests were used to carry out univariate analysis while Cox regression models were employed for multivariate analysis.Univariate analysis included age,gender,tumor length,tumor location,tumor differentiation,T stage,N stage and NLNs number.Multivariate analysis included tumor length,T stage,N stage and NLNs number.Patient survival was defined as the surgery date to the last point of follow-up or death and was determined using the Kaplan-Meier method.A P-value<0.05 was taken to indicate statistical significance.All calculations were carried out employing the SPSS program version 23.0(SPSS,IBM,Chicago,USA).Results1.Survival outcome of patientsAll 579 patients were followed up for a median of 36 months(total range between 3 to 80 months).Overall,patients had an MST of 36 months and 50.8%of them had a 3-year OS rate.The 3-year OS rates of patients with stage Ⅰ,stage Ⅱ and stage Ⅲ were 86.1%,53.7%and 31.6%respectively(χ2=71.864,P=0.000).When the numbers of NLNs were 9 or less,10 to 14,15 to 19 and 20 or more in 579 patients,the MST was 13 months,27 months,39 months and 47 months respectively,and the 3-year OS rates of patients were 21.7%,40.0%,61.2%and 77.5%respectively(χ2=120.475,P=0.000).There were 344 node-negative and 235 node-positive patients.When the numbers of NLNs were 9 or less,10 to 14,15 to 19 and 20 or more in node-negative patients,the MST was 18 months,36 months,42.5 months and 48 months and the 3-year OS rates of patients were 34.9%,50.9%,65.6%and 89.0%respectively(χ2=35.284,P=0.000).When the numbers of NLNs were 9 or less,10 to 14,15 to 19 and 20 or more in node-positive patients,the MST was 12 months,22 months,36 months and 45 months and the 3-year OS rates of patients were 14.3%,19.3%,51.8%and 68.9%respectively(χ2=67.604,P=0.000).2.Analysis of prognostic factors of patientsUnivariate analysis indicated that significant factors that influence the 3-year OS after esophagectomy were gender(P=0.023),tumor length(P=0.000),tumor differentiation(P=0.011),T stage(P=0.000),N stage(P=0.000)as well as the number of NLNs(P=0.000).Further analysis using multivariate methods indicated that significantly factors affecting the 3-year OS after esophagectomy were tumor length(P=0.042),T stage(P=0.000),N stage(P=0.000)and the number of NLNs(P=0.000).ConclusionThe numbers of NLNs is a significant independent prognostic indicator for thoracic ESCC patients’survival after curative esophagectomy.It is suggested that under the premise of controlling the operative complications,the number of LNs dissection of EC should be increased in order to improve the survival of patients.ENGLISH ABSTRASTS SECTION 2EPCAM EXPRESSION IN ESOPHAGEAL CANCER AND ITS CORRELATION WITH IMMUNOTHERAPYBackgroundEsophageal cancer(EC)is one of the most common malignant tumors in the world.Radical resection is the main treatment for resectable EC,but local recurrence and distant metastasis are the main reasons for postoperative patients’ treatment failure.Nearly two-thirds of patients had locally advanced stage or distant metastasis at the time of diagnosis and lost the opportunity for surgery.Systemic chemotherapy is currently the main treatment for advanced EC.However,drug resistance and tumor progression occurred soon after systemic chemotherapy,and the overall 5-year overall survival(OS)rate of the patients remains at 15-25%.Studies have reported that EC stem cells are the main reason for radiotherapy and chemotherapy resistance and treatment failure.These cancer stem cells(CSCs)harbor unique properties,including self-renewal,high tumorigenicity,proliferation,migration,evasion,and therapy resistance which leads to treatment failure.Therefore,finding effective drugs to target these cells resistant to radiotherapy and chemotherapy is the key to the treatment of fatal EC.Epithelial cell adhesion molecule(EpCAM)is considered to be one of the important biomarkers of cancer and is expressed in epithelial tumors and overexpressed in some CSCs.It is also considered as a marker of some CSCs.However,the expression of EpCAM in EC stem cells is not clear.Solitomab is an EpCAM/CD3 bispecific single-chain antibody construct,which can bind to tumor cells expressing EpCAM and T cells to activate T cells to kill tumor cells.Studies have confirmed that Solitomab can suppress uterine sarcoma,colon cancer,pancreatic cancer cells proliferation.However,the effect of Solitomab on EC cells has not been reported.Therefore,in this study,EC Eca109 cell spheres cultured in serum-free medium were tested for cell proliferation,cloning,invasiveness and multidrug resistance in vitro,and were detected the expression of EpCAM,and the cytotoxicity of bispecific antibody Solitomab combined with γ δ T cells on EC cell spheres was observed,so as to explore effective immune drugs for EC.Methods1.Eca109 cell spheres were cultured in serum-free medium,and the morphological differences between wild-type Eca109 cells and Eca109 cell spheres were compared by microscope and flow cytometry.2.MTT method and colony formation assay were used to detect and compare the proliferation ability of wild-type Eca109 cells and Eca109 cell spheres in vitro.3.Using transwell migration assay to detect and compare the invasive ability of wild-type Eca109 cells and Eca109 cell spheres.4.Using different concentrations of Nab-PTX and cisplatin to treat wild-type Eca109 cells and Eca109 cell spheres and testing cell viability by MTT assay.5.The expression level of EpCAM on wild-type Eca109 cells and Eca109 cell spheres was detected by flow cytometry.6.The cytotoxicity of γδ T cells combined with bispecific antibody Solitomab on wild-type Eca109 cells and Eca109 cell spheres was detected by flow cytometry.Results1.Eca109 cells gradually formed suspension-growing cell spheres in serum-free medium for 3 days and were passaged after cultured for 12 days.Compared with the first generation of cell spheres,the second generation formed faster and were smaller in size and more regular in shape.Flow cytometry analysis showed that the forward scatter(FSC)and side scatter(SSC)values of Eca109 cell spheres were lower than those of wild-type Eca109 cells,suggesting that Eca109 cell spheres were smaller in size and had less cytoplasmic granules.2.MTT method and colony formation assay showed that the proliferation ability and clone formation rate of more than 50 cells of Eca109 cell spheres was significantly higher than wild-type Eca109 cells,the difference was statistically significant(P<0.05).3.The number of Eca109 cell spheres passed through Matrigel matrix glue to filter membrane was higher than that of wild-type Eca109 cells,and there was significant difference between the two groups(P<0.05).4.MTT assay showed that viability of Eca109 cell spheres treated with different concentrations of albumin-bound paclitaxel and cisplatin was significantly higher than that of wild-type Eca109 cells(P<0.05).5.Flow cytometry showed that high EpCAM expression level was observed in wild-type Eca109 cells and Eca109 cell spheres,but the expression level on Eca109 cell spheres was higher than that of wild-type Eca109 cells.6.Co-culture experiment showed that there was no significant difference in the cytotoxicity of y 8 T cells to wild-type Eca109 cells or Eca109 cell spheres without Solitomab.After adding Solitomab,y 8 T cells could significantly enhance the cytotoxicity of both cells.The cytotoxicity of y 8 T cells to Eca109 cell spheres was significantly higher than that of wild-type Eca109 cells(P<0.05).Conclusions1.EC Eca109 cell spheres have strong stem cell characteristics such as proliferation,invasion and multidrug resistance,and may contain a high proportion of EC stem cells.2.EC Eca109 cell spheres had a high expression level of EpCAM,and EpCAM may be one of the markers of EC stem cells.3.EpCAM may be a potential molecular target of immunotherapy for EC,and Solitomab may become an effective immunotherapeutic drug for EC resistant to chemotherapy.