Molecular Markers Of DNA Copy Number Aberration Related To Extrohepatic Metastasis Of Postoperative Hepatocellular Carcinoma

Objective Hepatocellular carcinoma(HCC)is one of the most malignant tumors in China.Surgery is still the main radical treatment of HCC,but postoperative extrahepatic metastasis is an important factor contributing to poor prognosis.However,little is known about the molecular features that are prone to extrahepatic metastasis of HCC.Therefore,investigation of the molecular markers related to extrohepatic metastasis of postoperative hepatocellular carcinoma has an important significance.Previous studies have shown that copy number aberrations(CNAs)is associated with tumor progression,intrahepatic recurrence and prognosis in postoperative HCC,but the relationship between CNAs and extrohepatic metastasis of postoperative HCC remain unclear.The purpose of this study was to investigate the molecular markers of CNAs related to extrohepatic metastasis of postoperative HCC.Methods Sixty-six cases of postoperative HCC were followed up,and non-extrahepatic-metastasis survival time is defined as the interval from surgery to extrahepatic metastasis or last follow-up time.High-resolution array-based comparative genomic hybridization(a CGH)of Agilent 244 K platform was used to detect the genomic CNAs in postoperative HCC samples.Agilent G2565 BA DNA microarray scanner was used to scan the hybrid chip,and the data were extracted and analyzed using Feature Extraction v9.5.The association between HCC CNAs and extrahepatic metastasis was analyzed by Cox proportional hazards model and Log-rank test,and the survival curve was drawn by Kaplan-Meier.Two-side P < 0.05 were defined as statistically significant.All statistical analyses were performed with Stata 13.0(Stata Corporation,College Station,TX)Results 1.All the 66 HCC patients were followed up,and the median follow-up time was 39.9 months(range,1.6-90.5 months)with 2633.4 months of total analysis time.Extrahepatic metastasis-free survival was expressed as the number of months from the date of surgery to the date of diagnosis of extrahepatic metastasis.Totally 25 patients(37.9%)occured extrahepatic metastasis during the follow-up period.2.Univariate Cox analysis showed that 5 clinicopathological factors were associtated with the postoperative extrahepatic metastasis of HCC(all P < 0.05),including the risk factors of history of hypertension,absence of complete tumor capsule and high TNM stage(stage III),and the protective factors of low platelet counts(<100×109/L)and prolonged prothrombin time(>12 seconds).3.Univariate Cox analysis of whole genome CNAs with extrahepatic metastasis showed that 5 CNAs related to extrohepatic metastasis of postoperative HCC(all P < 0.05): 6p21.32 gain,15q11.2 gain and 20q12-13.13 gain were risk factors,while 4q12 loss and 4q28.1-35.2 loss were protective factors.4.The multiple stepwise Cox regression analysis showed that 6p21.32 gain,4q28.1-35.2 loss,high TNM stage(stage III)and the history of hypertension were independent prognostic factors for extrahepatic metastasis of postoperative HCC.Among them,6p21.32 gain,high TNM stage(stage III)and the history of hypertension were risk factors,while 4q28.1-4q35.2 loss was the protective factor.5.The association analysis between CNAs combinations,based on the status of 6p21.32 gain and 4q28.1-35.2 loss,and the extrahepatic metastasis of postoperative HCC showed that all the patients can be categorized into 3 groups: 6p21.32 gain,6p21.32 non-gain/4q28.1-35.2 loss and 6p21.32 non-gain/4q28.1-35 non-loss,which presented significant difference in extrahepatic metastasis-free survival(Log-rank test,P < 0.01).Compared to 6p21.32 non-gain/4q28.1-35.2 non-loss group,the hazard ratio(HR)of extrahepatic metastasis of HCCs with 6p21.32 non-gain/4q28.1-35.2 loss was reduced to 0.18-fold(95% CI = 0.06~0.57,P < 0.01),and the HR of HCCs with 6p21.32 gain was significantly increased to 2.91-fold(95% CI = 1.13~7.46,P < 0.05).Conclusions 1.6p21.32 gain,15q11.2 gain and 20q12-13.13 gain are risk factors for extrahepatic metastasis of postoperative HCC,while 4q12 loss and 4q28.1-35.2 loss are protective factors.2.6p21.32 gain and 4q28.1-35.2 loss are independent prognostic factors for extrahepatic metastasis of postoperative HCC.3.The optimization combinations based on the status of 6p21.32 gain and 4q28.1-35.2 lost could predict the risk of developing extrahepatic metastasis for patients with postoperative HCC,which would provide valuable information for clinical treatment decision.