| Background and Purpose:Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide and is characterized by multiple nodules or early spread in the liver.Local treatment,including radiofrequency ablation(RFA)and microwave ablation(MWA),was recommended for those who were not eligible for surgical resection or liver transplantation in the early stage of HCC.However,tumor recurrence and metastasis caused by insufficient ablation have become the main factors affecting the long-term efficacy of thermal ablation.Although there have been many clinical reports on the prognosis of HCC ablation,little attention has been paid to the risk and prognostic factors of insufficient ablation after HCC ablation.In addition,the fact that local ablation had a high recurrence rate and induced a specific anti-tumor immune response through the release of tumor associated antigen(TAA)promoted the development of studies on combined ablation and immunotherapy.Previous studies have focused on the effect of insufficient ablation on the in-situ focal immune microenvironment,while there have been no relevant reports on the effect of insufficient ablation on non-target tumors and whether there is a synergistic effect of combined immunotherapy.Therefore,the present study was divided into two parts.Study 1 was designed to explore whether insufficient thermal ablation was associated with a higher risk of tumor progression in HCC patients,and to compare the efficacy of repeated ablation or transhepatic arterial chemotherapty and embolization(TACE)for residual tumor after insufficient ablation.Study 2 was designed to evaluate the effect and immunological effect of insufficient ablation of unilateral tumor in bilateral tumor-bearing mice on distant non-ablation tumors,and to explore the effectiveness of combined immunotherapy after incomplete ablation and the potential immune regulatory mechanism,so as to provide a new therapeutic idea for the intervention of HCC relapse after ablation.Materials and methods:Study 1:This study retrospectively analyzed the clinical data of284 HCC patients who underwent thermal ablation from June 2014 to September 2020.They were divided into the sufficient ablation group(n=236)and the insufficient ablation group(n=48)based on their responses after the first ablation.In order to reduce the impact of potential confounding factors on selection bias,a logical regression analysis was used to conduct a propensity score matching(PSM)between the two groups.Firstly,the progression free survival(PFS),overall survival(OS)and prognostic factors of HCC patients with sufficient and insufficient ablation after the first thermal ablation were compared and analyzed.Then,the difference in clinical outcomes between re-ablation and TACE treatment of residual tumors after insufficient ablation was compared.Study 2:Bilateral hepatocellular carcinoma model was established in BALB/c mouse,when the tumor in one side reached 0.8-1cm,mice were randomly divided into insufficient microwave ablation(i MWA)and false ablation group,unilateral larger tumors were treated with i MWA and sham ablation.The growth of distant tumor on the non-ablated side was dynamically monitored after surgery,and the immune status of distant tumor infiltration lymphocytes(TILs)was compared and analyzed by flow cytometry,immunohistochemistry,immunofluorescence and RNA-seq.In addition,the effect and immunological effect of i MWA combined with anti-PD-1 treatment on distant tumors were evaluated.Tumor bearing mice were randomly divided into four groups:control group,i MWA group alone,anti-PD-1 group alone and i MWA+anti-PD-1 treatment group,and the growth and immune status of distant tumors were compared in each group.Results:Study 1:After PSM of 284 patients,a total of 46 patients were matched,and PFS were significantly higher in the sufficient ablation group than in the insufficient ablation group(P<0.001),while OS was not different between the two groups(P=0.296).For patients in the insufficient ablation group,29 and 19 patients underwent re-ablation and TACE,respectively,and there were no significant differences in PFS(P=0.437)and OS(P=0.178)between the two groups.In multivariate Cox regression analysis,insufficient ablation response after first ablation(P<0.001)and Child-Pugh grade B(P=0.017)were independent risk factors for tumor progression.High AFP level(P=0.011)and Child-Pugh grade B(P=0.026)were independent risk factors for poor OS.Study 2:Compared with the control group,the distant unablated tumors in i MWA group grew significantly faster.The contralateral tumors were cut and analyzed by flow cytometry.The results showed that i MWA could up-regulate the expression of programmed cell death protein-1(PD-1),lymphocyte activation gene 3(LAG3)in CD8~+T cells,while down-regulate the expression of Perforin and GZMB.Further analysis of infiltrating immune cells in distant tumors showed that the proportion of myeloid-derived suppressor cells(MDSCs)increased significantly in distant tumors.Histopathological results showed that the number of Ki67and programmed cell death 1 ligand 1(PD-L1)positive cells in distant tumors in i MWA group was significantly increased compared with the control group(P<0.01),while the infiltrating CD8~+lymphocytes in distant tumors in i MWA group was significantly decreased(P<0.01).RNA-seq analysis showed that immunosuppressive genes such as IDO and CD36were significantly up-regulated,and differential genes were significantly enriched in the negative regulation of immune system and fatty acid metabolism.In addition,compared with the control group,anti-PD-1treatment alone could significantly inhibit the growth of distant tumors(P<0.01).Although the tumor inhibition effect of the combined treatment group was not as significant as that of the anti-PD-1 treatment group alone,the proportion of MDSCs infiltrating distant tumors in the combined treatment group was significantly reduced compared with the i MWA group(P<0.05).Conclusion:Study 1:Although HCC patients in the insufficient ablation group were more likely to develop tumor progression compared to those in the sufficient ablation group,their OS was not affected by insufficient ablation.In addition,acitve treatment strategies targeting residual or recurrent tumors after insufficient ablation can improve long-term survival in patients with HCC.Study 2:In this study,we revealed transcriptome and immune function changes in distant non-ablative tumors after unilateral insufficient ablation,and found that T cell depletion induced by MDSC aggregation may be the main cause of distant tumor progression after insufficient ablation.In addition,the application of immunocheckpoint inhibitors following insufficient ablation may have the potential to overcome T cell depletion and temporarily increase the activity of invasive cytotoxic T cells in distant tumors,thereby killing the tumor and promoting the effect of ablative therapy. |
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