Optimization Of Drug Metabolism Capacity Of Human Primary Hepatocytes In Vitro And Feasibility Study Of Combined Immunotherapy And Targeted Downstaging For Advanced Hepatocellular Carcinoma Followed By Reoperatio

Objective:To explore the efficient isolation of primary human hepatocytes(PHHs)from discarded human liver tissues(DHLTs)which were obtained from resected human liver and to optimize the drug metabolic function of PHHs in vitro.Methods:DHLTs that were not necessary for pathological examination were obtained from resected human liver with a minimum warm ischemia time in the process of hepatic resection.PHHs were isolated from DHLTs by the perfusion of digestion solution.PHHs were cultured in a two-dimension system supplemented with five chemicals.Components from human plasma that promoted the morphological maintenance and drug metabolic function of PHHs in vitro were analysed.The morphology of PHHs in vitro was observed and analysed.The gene expression in PHHs of phase I drug metabolization enzymes,phase II drug metabolization enzymes,phase III drug transporters and nuclear receptor that regulated drug metabolization was analysed by q RT-PCR.The global gene expression profiles of PHHs were analysed by RNA-sequence.The drug metabolic function of PHHs was analysed by high performance liquid chromatography-mass spectrometry.Differences were statistically significant when P<0.05.Results:The success rate for isolation of PHHs from DHLTs was 96.4%(27/28).PHHs were successfully isolated from 27 cases of DHLTs,including 12 males and 15 females with a median age of 54.0 years.The diagnosis of patients included liver benign tumor(11/27),liver malignant tumor(9/11)and hepatolithiasis(7/27).The median warm ischemia time of DHLTs was 7.0 minutes.A median number of 2.9×10~9PHHs was isolated from a median mass of 64.7g DHLTs,with a median production of 5.7×10~6 PHHs/g.Isolation of PHHs was more efficient from DHLTs obtained from patients with liver benign or malignant tumor than patients with hepatolithiasis.The human plasma and human fibrin gel promoted the morphological maintenance of PHHs in vtro.The human fibrin gel and human thrombin promoted the up-regulation for gene expression of PHHs in vtro of phase I drug metabolization enzymes,phase II drug metabolization enzymes,phase III drug transporters and nuclear receptor,leading to a more similar global gene expression of drug metabolization to freshly isolated PHHs.The drug metabolic function of PHHs cultured by human thrombin in vitro was promoted to a similar level to freshly isolated PHHs.Conclusion:PHHs can be efficiently isolated from DHLTs which are obtained from resected human liver,with a reasonable selection of DHLTs and a control of liver warm ischemia injury.The human thrombin can promote drug metabolic function of PHHs in vitro.The two-dimension PHHs culture system supplemented with human thrombin could be applied as a tool for drug metabolism research in vitro.Objective: To explore the feasibility,safety and survival benefits of radical surgery after immune checkpoint inhibitors(ICIs)combined tyrosine kinase inhibitors(TKIs)downstaging therapy for advanced hepatocellular carcinoma(a HCC).Methods: A cohort of consecutive patients who were diagnosed with HCC at our single institute was retrospectively assigned to the Barcelona Clinic Liver Cancer stage 0 or A(BCLC-0/A)group or the BCLC-C group.BCLC-A patients underwent a radical surgery.BCLC-C patients underwent a radical surgery after the ICIs combined TKIs downstaging therapy.The primary outcome was overall survival(OS).Secondary outcomes were relapse-free survival(RFS),relapse,deaths,intraoperative information(including ASA grade,hepatectomy term,operation time,blood loss,blood transfusion and total hilar clamping time),postoperative information(including mechanical ventilation time,fasting time,abdominal drainage time,complications and hospital stay)and pathologic diagnosis.Before and after downstaging therapies,contrastenhanced or diffusion-weighted imaging was performed for BCLC-C patients to assess complete intrahepatic macrovascular tumor thrombus necrosis(CIMTTN).Image qualitative features including disappearance of arterial enhancement(DOAE)and no diffusion restriction(NDR)were analysed.The percentage of intrahepatic macrovascular tumor thrombus enhancement(IMTTE%)and apparent diffusion coefficient(ADC)values were calculated.Receiver operating characteristic analysis was performed for IMTTE% and ADC values.The CIMTTN was confirmed by pathologic analysis.Differences were statistically significant when P < 0.05.Results: Of 101 evaluable BCLC-C patients who received ICIs combined TKIs therapy,29 underwent a following surgery.18 patients with 36 imaging examinations were included in diagnostic test of imaging features.DOAE and NDR yielded 88.9% / 77.8% sensitivity,83.3% / 80.0% specificity,84.2% / 82.4% positive predictive value,88.2% /75.0% negative predictive value,and 86.1% / 78.8% diagnostic accuracy for diagnosing CIMTTN,respectively.The area under curve(AUC)for IMTTE% values in the diagnosis of viable IMTT was 0.94,with an optimal cut-off value of 145.5% resulting in a sensitivity/specificity of 88.9% / 88.9%.The AUC for ADC values in the diagnosis of CIMTTN was 0.79,with an optimal cut-off value of 1.3×10-3 mm2/s resulting in a sensitivity/specificity of 88.9% / 80.0%.There were 46 patients assigned to the BCLC-0/A(n=28)or the BCLC-C group(n=18).The median follow-up time was 11.1 months.The differences between BCLC-0/A and BCLC-C group in median OS(7.7 vs.12.2 months,P = 0.208),median RFS(6.3 vs.8.4 months,P = 0.919)and relapse rate(10.7% vs.11.1%,P = 0.966)were not statistically significant.In BCLC-0/A group,there was no death.In BCLC-C group,there was a case of death due to small cell lung cancer.There were a more complicated hepatectomy term,a longer operation time,a more blood loss,a more blood transfusion,a longer postoperative mechanical ventilation time,a longer postoperative abdominal drainage time and a longer postoperative hospital stay in BCLC-C group than in BCLC-0/A group(P < 0.05).The differences between two groups in ASA grade,total hilar clamping time,postoperative fasting time and postoperative complications were not statistically significant(P > 0.05).All BCLC-C patients were downstaged to BCLC-A(3/18)or BCLC-0(15/18)confirmed by pathologic analysis.In the BCLC-C group,a complete necrosis of tumor after downstaging therapy was confirmed in six patients by pathologic analysis,and a partial necrosis of tumor with the remaining viable cell median ratios of 5.0% in 12 patients.The microvascular invasion was confirmed in three(10.7%)BCLC-0/A patients by pathologic analysis,but none in BCLC-C patients.Conclusion: ICIs combined TKIs therapy bears the potential to downstage a HCC to early stage.Contrast-enhanced and diffusion-weighted imaging features show good diagnostic performance in the differentiation of CIMTTN after ICIs combined TKIs therapy.A radical surgery after ICIs combined TKIs downstaging therapy is safe and feasible for a HCC,featuring its pathologic benefits and comparable survival to the BCLC-0/A patients.