Construction And Optimization Of A Long-term Survival Prediction Model For Hepatocellular Carcinoma Patients After Liver Transplantation

Objective According to the World Cancer Report published by WHO in 2014,liver cancer is the sixth most frequent and the second most deadly cancer in the world.Hepatocellular carcinoma(HCC)is the main pathological type of liver cancer,accouting for 80% of all cases.Liver transplantation(LT)is the best treatment of patients with unresectable early hepatocellular carcinoma,allowing disease-free survival rates of 60–80% at 5 years.Despite these good results,about 10%-20% of recipients experience a posttransplant HCC recurrence,even for those who meet the Milan criteria.Therefore,limits exist in the current system of liver transplantation criteria for HCC.In this study,we developed a criteria-specific long-term survival prediction model for HCC patients after LT(MHCAT)based on the Milan,UCSF,Fudan and Hangzhou criteria.Thereafter,MHCAT was validated in a large cohort of HCC patients after LT in China.And we tried to establish a novel three dimensional(3D)culture method for circulating tumor cells(CTCs)of HCC patients.This 3D culture system can be used as a powerful tool for determining biological invasion behaviours of HCC,and might equip the MHCAT to be a more useful model for prognostic evaluation and liver donor distribution.Methods Part 1.Construction an validation of MHCAT and role of intraoperational blood loss for postoperational HCC recurrence.MHCAT was generated using a single-center cohort of 223 HCC patients receiving liver transplantation,by Cox regression analysis.Another cohort of 1371 HCC patients recorded in China liver transplant registry(CLTR)was used for validation of MHCAT.Survival predictions for each patient were calculated using MHCAT scores and the Metroticket formula separately,and the prediction efficacy of MHCAT and Metroticket was compared using the area under ROC curve(c-statistic).The Kaplan-Meier method with the log-rank test was used to analyse long-term survival rates.We further analysed the role of intraoperational blood loss(IBL),an independent risk factor in MHCAT,for postoperational HCC recurrence.A total of 479 patients who underwent LT for HCC from January 2001 to December 2012 at our institution wereenrolled in this retrospective study.Kaplan–Meier and Cox regression methods were used to assess the recurrence rate,as well as its risk factors.Stratified analysis was performed to further examine the effect of IBL on HCC recurrence according to different characteristics of tumors.We also investigated the independent risk factors for excessive IBL using logistic regression analysis.Part 2.Construction of 3D culture system for HCC-CTCs and study on biological invasion behaviours of HCC-CTCs A novel 3D culture method for HCC-CTCs was originally developed in our lab.Ten milliliter peripheral blood were obtained for 79 HCC patients during liver resection or liver transplantation.The number of HCC-CTCs clones and cellular morphology were observed under the 3D culture system.The status of HCC-CTCs clones was analysed with reference to HCC oncological indicators.Different types of HCC-CTCs clones were isolated to injected subcutaneously into nude mice for tumorigenesis.Results Using single-center data,MHCAT was constructed with reference to the Milan,University of California San Francisco(UCSF),Fudan and Hangzhou criteria.Pre-operative alfa-fetoprotein(AFP)level,IBL and retransplantatio were common significant predictors of long-term survival in HCC patients with reference to the Milan,UCSF and Fudan criteria,whereas in MHCAT based on the Hangzhou criteria,total bilirubin,IBL and retransplantation were independent predictors.A total of 1371 patients were taken part into the validation analysis for MHCAT,with a median follow-up time of 22.2 months(IQR 6.1-72.4 months).The proportions meeting the Milan,UCSF,Fudan and Hangzhou criteria were 34.4%,39.7%,44.2% and 51.9%,respectively.The c-statistics for MHCAT predictions of 3-and 5-year survival rates of HCC recipients were 0.712–0.727 and 0.726–0.741,respectively.There were no significant difference for prediction efficacy among MHCAT with reference to four criteria(p > 0.05).Among these patients,1298 LTs for HCC were ultimately selected for the In comparison analysis for prediction efficacy of MHCAT and Metroticket.,The the c-statistic of MHCAT for predictions of 3-year survival with reference to the Milan,UCSF and Fudan criteria was significantly increased higher compared with that for Metroticket(p < 0.05),while there were no significant difference for 5-year survival predictions(p > 0.05).Kaplan–Meier analysis with the log-rank test according to IBL at per liter intervals showed that IBL > 4 L was significantly associated with a higher recurrence rate(P < 0.001).Multivariate analysis identified that IBL > 4 L(P < 0.001;hazard ratio [HR] = 2.32,95 % confidence interval [CI] = 1.60–3.36)was an independent risk factor for post-LT HCC recurrence,as well as age < 60 years,exceeding Milan criteria,AFP levels > 400 ng/m L,and micro-and macrovascular invasion.IBL > 4 L(P < 0.001;HR = 2.45,95 % CI = 1.64–3.66)was also independently associated with early(within 1 year)recurrence after LT.Furthermore,a significant correlation between IBL > 4 L and vascular invasion(P = 0.019)was found.IBL > 4 L was independently associated with HCC recurrence for patients with vascular invasion,but not for patients without vascular invasion.Finally,we found that the presence of ascites,model for end-stage liver disease score,and operation time were independent risk factors for IBL > 4 L.A novel 3D culture system was successfully developed.Using this culture method,,HCC-CTCs formed clones from 96.9% of HCC patients beyond the Milan criteria.The number of CTCs clones was statistically related with the tumor number,size,and vascular invasion(p < 0.01).We also noted there were two types of HCC-CTCs clones,Multi-cellar(MC)ones and Oligo-cellar(OC)ones.The preliminary experiment showed that the MC type had a more aggressive invasiveness.Conclusion MHCAT can effectively predict long-term survival for HCC recipients following LT.This helps clinicians estimate who of the candidates with HCC would gain better prognosis after liver transplantation.The number and cellular morphology of HCC-CTCs under the 3D culture systerm may be potential indicators for HCC biological invasion behaviours and prognosis.However,this need to be further validated in a large cohort of blood samples from HCC patients and a long-term follow up.Combined with HCC-CTCs status,MHCAT may be optimized and helps clinicians estimate who of the candidates with HCC would gain better prognosis after liver transplantation.