The Value Of Circulating Tumor DNA High Frequency Gene Sequencing In Monitoring Tumor Recurrence After Liver Transplantation For HCC

BackgroundFor patients suffering hepatocellular carcinoma,liver transplantation can completely remove the tumor as well as the residual cirrhotic liver tissue,which is one effective clinical treatment to liver cancer.Among the HCC patients perfromed liver transplantation,more than one-third died of tumor recurrence or metastasis.Hence,the tumor recurrence and metastasis severely impact the prognosis of HCC patients post liver transplantation.Facing current organ shortage,selecting liver transplant candidates of HCC patients is the key to reduce the tumor recurrence after liver transplantation.So far,Milan Criteria is one only accepted selection criteria of liver transplantation for HCC patients globally.However,in China,most liver cancer patients are approaching advanced stage of tumor during their first hospitalization.If only and strictly applying the Milan Criteria,less than 5%of the liver tumor patients will be enrolled in the transplant waiting list.Fortunately,growing evidence has demonstrated that some patients exceeding Milan Criteria might also achieve good prognosis through liver transplantation.The Milan Criteria refers mainly to the number and size of the liver tumors,but its biological behavior and carcinogenesis seem more closely related to the tumor recurrence and metastasis as well as the survival after liver transplantation.Therefore,the Milan Criteria has certain limitations.Recent studies have found that circulating tumor DNA(ctDNA)can manifestate the tumor burden,malignancy,metastatic ability,and genetic mutations.While,detecting ctDNA before surgery can help to grasp the biological characteristics of the tumor and predict the risk of recurrence after liver transplantation.At present,the diagnosis and treatment of liver cancer is mainly based on histopathological classification and tumor staging.However,for patients with the same clinical stage and pathological grade of tumor,the outcome is quite different under similar therapeutical strategy.Therefore,the current accurate diagnosis and treatment to individual molecular typing has become a researching hotspot in the field of liver cancer.In this study,we intend to investigate the molecular classification of liver cancer from patients beyond Milan criteria by detecting ctDNA before and after liver transplantation.We also intend to demonstrate the value of monitoring ctDNA to the evaluation of tumor recurrence and patient prognosis after liver transplantation.Object iveTo evaluate whether the detection of ctDNA-related high-frequency mutated genes based on high-throughput sequencing technology can analyze the molecular typing of patients who exceeding Milan criteria.Monitoring the changing level of ctDNA after transplantation of patients who exceeding Milan criteria,in order to predict and monitor the patient’s postoperative recrudescence and metastasis.MethodsThis study was divided into three parts.The first part collected preoperative and postoperative serum samples from 30 liver cancer patients exceeding Milan criteria,as well as intraoperative tumor specimens.Using high-throughput sequencing technology,16 high-frequency mutant genes in serum ctDNA and tumor tissue DNA were collected and sequenced,then compared the serum ctDNA and tissue DNA sequencing results with clinicopathological data,analyzed the correlation between high frequency mutant genes and clinicopathological features of patients.In the second part,30 patients were enrolled to monitor ctDNA before and 1st,3rd,6th,9th and 12th month post liver transplantation.Abdominal contract-enhanced CT and thoracic CT scan was performed during 3rd,6th and 12th months post transplantation.Tumor recurrence and metastasis,as well as patient survival were collected and analyzed.The perioperative,post-transplant ctDNA titer,the tumor recurrence as well as patient tumor-free-survival were recorded.The patient overall survival was also compared and analyzed.In the third part,9 patients with tumor recurrence or metastasis within 1 year after operation were recorded and analyzed.Peripheral blood sample of each subject was extracted during 1st,3rd,6th,9th and 12th month after liver transplantation.The ctDNA was detected,and pathological examination of tumor tissue from these 9 patients was obtained.The value of ctDNA in monitoring the tumor recurrence and patient prognosis post liver transplantation was investigated.ResultsThe top 5 high frequency mutations in liver cancer tumor tissue sequencing results were TERT,TP53,CTNNB1,ALB and PIK3CA,which were consistent with the results of the TCGA database and COSMIC database.A comprehensive comparison between the sequencing results and the patient’s clinical and pathological data revealed a significant correlation between the "maximum tumor diameter" and the ctDNA mutation frequency(p=0.02).Comparing the results of ctDNA detection with the tissue DNA detection,it was found that ctDNA detection results of each patient was more consistent to the database than that of tissue DNA.There was no correlation between ctDNA level before liver transplantation and one month post operation.TERT,TP53 and TSC2 were selected and gathered in one prognosis panel.According to the panel results,the patients were divided into positive or negative group.Refering to the tumor-free-survival,the result indicated that positive group was significantly worse than the negative one.However,there presented no significant difference between these two groups when comparing 1-year overall survival(p>0.05).Continuous monitoring of the AFP and PIVKAII in patients with postoperative recurrence showed there had a significant reduction to almost normal levels.The"3T" gene in plasma ctDNA had a positive result at the corresponding time point,combining a 1-2 cm recurrent lesion within the graft liver found by abdominal contract-enhanced CT scan.Traditional AFP and PIVKAII serological monitoring methods are not adequate for monitoring tumor recurrence.Meanwhile,the study had also demonstrated that sirolimus,as a immunosuppressive agent,can effectively target the TSC2 deletion mutant liver cancer cells,resulting significant reduction of the invasiveness of TSC2 mutant cancer cells(p<0.05).Conclusions(1)In this study,the result of ctDNA grasping and detecting are similar to that of COSMIC database.The results of the study are reliable.The ctDNA detection can effectively overcome the impact of tumor heterogeneity on the detection results.(2)Circulating tumor DNA can objectively demonstrate the tumor genomic composing information of liver cancer cells,demonstrating the important clinical value of "liquid biopsy".(3)Preoperative ctDNA,such as three-gene detecting panel(TERT,TP53,TSC2),have the potential value of predicting the early prognosis of HCC patients exceeding Milan criteria post liver transplantation.