Whole Exome Sequencing To Identify Novel Gene Mutations Associated With Postoperative Relapse Of Node Negative Thoracic Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC) is one of malignant digestive tumors with extremely aggressive nature and poor survival rate worldwide. Loco-regional lymph node recurrence is the main disease relapse pattern after esophagectomy with radical lymphadenectomy. About 30% to 50% ESCC patients without lymph node metastasis(pN0) after surgical resection will occur relapse in 5 years,and it was the main cause of the treatment failure and death for them.Identifying high risk factors of postoperative relapse and taking individual adjuvant therapy for pN0 ESCC patients are the keys for controlling the relapse and improving the cure rate after esophagectomy.Predicting the postoperative relapse and survival according to TNM stage systems is lack of sensibility and accuracy. Structural and functional abnormalities of many genes and proteins are involved in carcinogensis, development, invasion and metastasis of ESCC. Intensifying the research on the change of molecular genetics unique for ESCC and exploring the molecular biomarkers for predicting the lymph node relapse in pN0 ESCC patients have important clinical significance.For this reason, we first identify the somatic mutations closely related to lymph node metastasis of ESCC patients by whole-exome sequencing and validate them by Sanger sequencing. To explore the relationships between the somatic mutations and relapse and survival after surgical resection of pN0 ESCC, We then detect the somatic mutations selected in the first step by Sanger sequencing in 112 pT1bN0M0 ESCC. Our experiment includes two parts. PartⅠ: Study on the somatic mutations closely related to lymph node metastasisof ESCC patients by whole-exome sequencing.Object: To identify the somatic mutations closely related to lymph node metastasis of ESCC patients.Methods: We use whole-exome sequencing on an Illumina 2500 Platform to identify the somatic mutations in 2 ESCC patients with lymph node metastasis.Results: Through sequencing 6 samples of 2 ESCC patients’ primary carcinoma, metastatic lymph node and non-cancerous esophagus, and followed analyzing according to described method, we finally identified 11 frameshift mutations and 9 non-synonymous SNP mutations which located on exon. They were all co-existed in primary carcinoma and metastatic lymph node, but not in non-cancerous esophagus. They were all confirmed as high disease-causing. And then they were selected for further test by Sanger sequencing. The results suggest that mutation c.251C>T(p.A84V)on CXCR1 and mutation c.1672C>G(p.Q558E)on PABPC1 could be potential cancer gene mutations related to lymph node metastasis of ESCC patients.Conclusions: We identified CXCR1 and PABPC1 as new potential cancer genes related to lymph node metastasis of ESCC patients.Part Ⅱ: Study on the clinic-pathological variables and CXCR1、PABPC1 mutations affecting postoperative relapse and survival of pT1b-4aN0M0 ESCC.Object: To explore the clinic-pathological variables and CXCR1、PABPC1 mutations affecting postoperative relapse and survival of pT1b-4aN0M0 ESCC.Methods: 112 pT1b-4aN0M0 ESCC patients enrolled in this study. CXCR1 and PABPC1 mutations were detected by Sanger sequencing. Logistic regression analysis was employed to identify the factors affecting the relapse and Cox regression analysis was employed to identify the factors affecting survival after esophagectomy of pT1b-4aN0M0 ESCC patients.Results: CXCR1 mutation was detected in 17 patients. Of them, 11 were involved in postoperative relapse. PABPC1 mutation was detected in 8 patients. Of them, 4 were involved in postoperative relapse. In multivariate analysis based on Logistic regression, CXCR1 mutation, Tumor differentiation, Tumor length and Tumor location affected overall relapse significantly; CXCR1 mutation, Tumor differentiation also affected loco-regional relapse significantly; Pathological stage affected distant relapse significantly. Multivariate analysis based on Cox regression revealed that, CXCR1 mutation, T status, Tumor differentiation,Tumor location and age affected postoperative overall survival(OS) significantly; Tumor differentiation, Tumor length and Age affected postoperative disease-free survival(DFS) significantly. PABPC1 mutation has no impact on postoperative relapse or survival of pT1b-4aN0M0 ESCC patients.Conclusions:1) CXCR1 mutation was involved in postoperative relapse and survival of pT1b-4aN0M0 ESCC patients, CXCR1 could be a potential cancer gene related to lymph node relapse of pT1b-4aN0M0 ESCC patients.2) CXCR1 mutation, Tumor differentiation, Tumor length and Tumor location were the independent predictive factors for overall relapse.3) CXCR1 mutation, Tumor differentiation were the independent predictive factors for loco-regional relapse and Pathological stage was the independent predictive factors for distant relapse. We speculated that there were different variables affecting loco-regional relapse and distant relapse after surgical resection of pT1b-4aN0M0 ESCC patients, respectively.4) Tumor differentiation, Tumor length and Age were the independent prognostic factors for postoperative disease-free survival(DFS); T status, CXCR1 mutation, Tumor differentiation,Tumor location and age were the independent prognostic factors for postoperative overall survival(OS).