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ObjectiveRevealing the single-cell immune ecosystems in true versus de novo hepatocellular carcinoma (HCC) recurrences could help the optimal development of immunotherapies.DesignWe performed 5’and VDJ single-cell RNA-sequencing on 34 samples from 20 recurrent HCC patients. Bulk RNA-sequencing, flow cytometry, multiplexed immunofluorescence, and in vitro functional analyses were performed on samples from two validation cohorts.ResultsAnalyses of mutational profiles and evolutionary trajectories in paired primary and recurrent HCC samples using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The tumour immune microenvironment (TIME) of truly recurrent HCCs was characterised by an increased abundance in KLRB1+CD8+T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8+T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed elevated GDF15 expression on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited antitumour immunity in truly recurrent lesions. In contrast, myeloid cells’ cross talk with T cells-mediated T cell exhaustion and immunosuppression in the TIME ofde novorecurrent HCCs. Consistent with these findings, a phase 2 trial of neoadjuvant anti-PD-1 immunotherapy showed more responses in de novo recurrent HCC patients.ConclusionTrue and de novo HCC recurrences occur early, have distinct TIME and may require different immunotherapy strategies. Our study provides a source for genomic diagnosis and immune profiling for guiding immunotherapy based on the type of HCC recurrence and the specific TIME.

Mutations of the RAS oncogene are found in around 30% of all human cancers yet direct targeting of RAS is still considered clinically impractical except for the KRASG12C mutant. Here we report that RAS-ON (RASON), a novel protein encoded by the long intergenic non-protein coding RNA 00673 (LINC00673), is a positive regulator of oncogenic RAS signaling. RASON is aberrantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) patients, and it promotes proliferation of human PDAC cell lines in vitro and tumor growth in vivo. CRISPR/Cas9-mediated knockout of Rason in mouse embryonic fibroblasts inhibits KRAS-mediated tumor transformation. Genetic deletion of Rason abolishes oncogenic KRAS-driven pancreatic and lung cancer tumorigenesis in LSL-KrasG12D; Trp53R172H/+ mice. Mechanistically, RASON directly binds to KRASG12D/V and inhibits both intrinsic and GTPase activating protein (GAP)-mediated GTP hydrolysis, thus sustaining KRASG12D/V in the GTP-bound hyperactive state. Therapeutically, deprivation of RASON sensitizes KRAS mutant pancreatic cancer cells and patient-derived organoids to EGFR inhibitors. Our findings identify RASON as a critical regulator of oncogenic KRAS signaling and a promising therapeutic target for KRAS mutant cancers.

Supplementary Figure from Combination Neoantigen-Based Dendritic Cell Vaccination and Adoptive T-Cell Transfer Induces Antitumor Responses Against Recurrence of Hepatocellular Carcinoma

A high rate of recurrence after curative therapy is a major challenge for the management of hepatocellular carcinoma (HCC). Currently, no effective adjuvant therapy is available to prevent HCC recurrence. We designed a personalized neoantigen-loaded dendritic cell vaccine and neoantigen-activated T-cell therapy, and used it as adjuvant therapy to treat 10 patients with HCC who had undergone curative resection or radiofrequency ablation in the first stage of a phase II trial (NCT03067493). The primary outcomes were safety and neoantigen-specific immune response. Disease-free survival (DFS) was also evaluated. The immunotherapy was successfully administered to all the patients without unexpected delay and demonstrated a reasonable safety profile with no grade ≥3 treatment-related side effects reported. Seventy percent of patients generated de novo circulating multiclonal neoantigen-specific T-cell responses. Induced neoantigen-specific immunity was maintained over time, and epitope spreading was observed. Patients who generated immune responses to treatment exhibited prolonged DFS compared with nonresponders (P = 0.012), with 71.4% experiencing no relapse for 2 years after curative treatment. High expression of an immune stimulatory signature, enhanced immune-cell infiltration (i.e., CD8+ T cells), and upregulated expression of T-cell inflammatory gene profiles were found in the primary tumors of the responders. In addition, neoantigen depletion (immunoediting) was present in the recurrent tumors compared with the primary tumors (7/9 vs. 1/17, P = 0.014), suggesting that immune evasion occurred under the pressure of immunotherapy. Our study indicates that neoantigen-based combination immunotherapy is feasible, safe, and has the potential to reduce HCC recurrence after curative treatment.

Figure S1. Protein sequence alignment of HPyV6, HPyV7 and MCPyV small T antigens. Figure S2. Expression of HPyV sT induces p53-dependent senescence in human fibroblasts. Figure S3. Proliferation of BJ fibroblasts with expression of HPyV sT. Figure S4. Analysis of RNA-Seq from HPyV6, HPyV7, and MCPyV sT expressing BJ fibroblasts. Figure S5. Time course and extended analysis of additional SASP gene expression in BJ cells after sT expression. Figure S6. MCPyV sT shRNA confirms role of sT in SASP gene expression. Figure S7. Impact of LSD motif of MCPyV sT on gene expression and proliferation. Figure S8. MCPyV st, but not HPyV6/7 sT, stabilizes and activates c-Myc in an LSD motif dependent manner. Figure S9. Chromatin remodeling signatures induced by MCPyV sT. Figure S10. Mechanism of ncNF-κB and SASP induction by MCPyV sT. Figure S11. ncNF-κB signaling is required for SASP and EZH2 expression and affects cell proliferation. Figure S12. PyV sT conditioned media is not sufficient for growth in low serum. Figure S13. ST expression in VP-MCC lines and tumors. Figure S14. Impact of ncNF-κB inhibition on VP-MCC and VN-MCC. Figure S15. Summary Figure.

Primers used for cloning and qRT-pCR

Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB signaling prevented VP-MCC cell growth in vitro and in xenografts. We identify MCPyV sT–induced ncNF-κB signaling as an essential tumorigenic pathway in MCC.Implications:This work is the first to identify the activation of ncNF-κB signaling by any polyomavirus and its critical role in MCC tumorigenesis.

Table S5 shows detailed information of structure variations in the six HCC patients with WGS analysis.

Fig.S1 shows flow chart of enrollment for multifocal HCC patients with anti-PD-1 therapy. Fig.S2 shows CT images of the six HCC patients with two tumors in the liver. Fig.S3 shows purity of tumor content of the six HCC patients with two tumors in the liver. Fig.S4 shows HBV integration sites in the six HCC patients with two tumors in the liver. Fig.S5 shows expression level of TERT in the six HCC patients with two tumors in the liver. Fig.S6 shows landscape of inter-chromosomal translocations and copy number alterations in small and large tumors of four cases. Fig.S7 shows number and types of chromosomal rearrangement in the six HCC patients with two tumors in the liver. Fig.S8 shows copy number aberrations in the six HCC patients with two tumors in the liver. Fig.S9 shows mutational landscape of the six HCC patients with two tumors in the liver. Fig.S10 shows non-silent mutations in the six HCC patients with two tumors in the liver. Fig.S11 shows profile of mutational cancer cell fractions of small and large tumors in the six HCC patients. Fig.S12 shows unsupervised clustering of RNA expression in tumors and adjacent normal liver tissues of 12 multifocal HCC patients. Fig.S13 shows expression level of immune functional genes in small and large tumors of 12 multifocal HCC patients. Fig.S14 shows expression level of immune stimulators in small and large tumors of 12 multifocal HCC patients. Fig.S15 shows expression level of immune inhibitors in small and large tumors of 12 multifocal HCC patients. Fig.S16 shows mutations of immune-related genes in the six HCC patients with two tumor nodules in the liver. Fig.S17 shows images of the small and large tumors before and after anti-PD-1 therapy.

Purpose:Targeted therapy and immunotherapy are transforming the treatment approach for intrahepatic cholangiocarcinoma (ICC). However, little is known about the intertumor heterogeneity (ITH) of multifocal ICC and its impacts on patient response to these treatments. We aimed to characterize the immunogenomic and epigenomic heterogeneity of multifocal ICC to guide treatment decision making.Experimental Design:We obtained 66 tumor samples from 16 patients with multifocal ICC and characterized the tumor and immune heterogeneity using whole-exome sequencing, bulk and single-cell RNA sequencing, methylation microarray, and multiplex immunostaining. Patients were divided into high- or low-ITH groups according to the median ITH index. Two independent cohorts were used to validate findings. Responses to anti-PD-1 therapy were assessed.Results:Multifocal ICC presented considerable intertumor genomic, transcriptional, and epigenomic heterogeneity within a patient in high ITH group. The immune profile among multiple tumors within a patient was relatively less heterogeneous in high- or low-ITH group, and consistent responses of multiple tumors to anti-PD-1 immunotherapy were observed. Unsupervised clustering of immune markers identified one low and one high immune subtype, with higher immune cell infiltration, closer tumor–immune cell interactions, and upregulated IFN-signature expression in high-immune subtype. Determining expression levels of CD8B and ICOS facilitated this immune classification and prediction of patient prognosis. Finally, promoter DNA methylation contributed to different immune profiles of two subtypes by regulating immune-gene expression.Conclusions:There is comprehensive heterogeneity in the genome, transcriptome, and epigenome of multifocal ICC. On the basis of the less heterogeneous immune profile of ICC, we suggest an immune classification that stratifies patients' prognosis and may support personalized immunotherapy.

Supplementary Data from Multiomic Analysis Reveals Comprehensive Tumor Heterogeneity and Distinct Immune Subtypes in Multifocal Intrahepatic Cholangiocarcinoma

Table S1 shows clinicopathologic information of 12 multifocal HCC patients. Table S2 shows PCR primers used for validation of HBV integration sites. Table S4 shows HBV integration analysis in HCC patients with WGS data. Table S8 shows P-value of pathways enrichment analysis in the small and large tumors of 12 multifocal HCC patients. Table S10 shows correlation between the number of rearrangements and immune activity. Table S11 shows detailed clinical data of eight patients with anti-PD-1 therapy.

Purpose:Immune checkpoint inhibitor therapy is emerging as the promising option for patients with advanced hepatocellular carcinoma. We aimed to investigate the heterogeneity of different tumor nodules of the same patient with multifocal hepatocellular carcinomas in response to immunotherapy and its molecular mechanisms.Experimental Design:We attained 45 surgical tumor samples including 33 small and 12 large nodules from 12 patients with multifocal hepatocellular carcinoma and evaluated genomic and immune heterogeneity among tumors through whole-genome sequencing and RNA sequencing. IHC was performed to validate the expression of immune markers. The responses to anti–programmed cell death protein-1 (PD-1) therapy in patients with multifocal hepatocellular carcinoma were evaluated.Results:The small and large tumors within the same patient presented with similar genomic characteristics, indicating their same genomic origin. We further found the small tumors had higher immune cell infiltration including more CD8+ T cells, M1 macrophages, and monocytes as compared with large tumors. Besides, the expression of interferon signature predictive of response to anti–PD-1 therapy was significantly upregulated in the small tumors. Moreover, the immune pathways were more vigorous along with less active proliferation pathways in the small tumors. In keeping with this, we found that small nodules were more sensitive to anti–PD-1 therapy than large nodules in patients with multifocal hepatocellular carcinoma.Conclusions:The small tumors in patients with multifocal hepatocellular carcinoma had higher immune cell infiltration and upregulation of immune pathways as compared with the large tumors, which can partially explain the different responses of small and large tumors in the same case to anti–PD-1 therapy.

A high rate of recurrence after curative therapy is a major challenge for the management of hepatocellular carcinoma (HCC). Currently, no effective adjuvant therapy is available to prevent HCC recurrence. We designed a personalized neoantigen-loaded dendritic cell vaccine and neoantigen-activated T-cell therapy, and used it as adjuvant therapy to treat 10 patients with HCC who had undergone curative resection or radiofrequency ablation in the first stage of a phase II trial (NCT03067493). The primary outcomes were safety and neoantigen-specific immune response. Disease-free survival (DFS) was also evaluated. The immunotherapy was successfully administered to all the patients without unexpected delay and demonstrated a reasonable safety profile with no grade ≥3 treatment-related side effects reported. Seventy percent of patients generated de novo circulating multiclonal neoantigen-specific T-cell responses. Induced neoantigen-specific immunity was maintained over time, and epitope spreading was observed. Patients who generated immune responses to treatment exhibited prolonged DFS compared with nonresponders (P = 0.012), with 71.4% experiencing no relapse for 2 years after curative treatment. High expression of an immune stimulatory signature, enhanced immune-cell infiltration (i.e., CD8+ T cells), and upregulated expression of T-cell inflammatory gene profiles were found in the primary tumors of the responders. In addition, neoantigen depletion (immunoediting) was present in the recurrent tumors compared with the primary tumors (7/9 vs. 1/17, P = 0.014), suggesting that immune evasion occurred under the pressure of immunotherapy. Our study indicates that neoantigen-based combination immunotherapy is feasible, safe, and has the potential to reduce HCC recurrence after curative treatment.

Imaging traits including nonsmooth tumor margins, internal arteries, peritumoral enhancement, and absence of hypodense halos can reflect tumor aggressiveness preoperatively and may affect treatment selection. This study aimed to explore the role of these four imaging traits in treatment selection between surgical resection (SR) and radiofrequency ablation (RFA) for patients with single ≤ 5 cm hepatocellular carcinoma (HCC).Three hundred eight-one patients with single ≤ 5 cm HCC who underwent SR (n = 202) or RFA (n = 179) in the First Affiliated Hospital of Sun Yat-sen University from April 2010 to December 2019 were retrospectively enrolled. The efficacy of SR and RFA in patients with the imaging traits that significantly influenced recurrence-free survival (RFS) or overall survival (OS) was compared and analyzed.Multivariable Cox regression analysis identified that having internal arteries (P = 0.001) was an independent influencing factor for RFS, while internal arteries (P = 0.005) and peritumoral enhancement (P = 0.001) were independent influencing factors for OS. In patients with internal arteries, subgroup analysis based on tumor size demonstrated that both RFS and OS of SR were superior to those of RFA in patients with 3-5 cm HCC (RFS, P = 0.023; OS, P = 0.015). In patients with peritumoral enhancement, both RFS and OS of SR were superior to those of RFA (RFS, P = 0.019; OS, P = 0.042).SR may be associated with improved survival compared with RFA in patients with single 3-5 cm HCC having internal arteries and patients with single ≤ 5 cm HCC having peritumoral enhancement.

Background and Purpose The clinical role of postoperative adjuvant therapy in hepatocellular carcinoma (HCC) is still unclear. The purpose of our study was to explore the clinical value of postoperative adjuvant anti-programed cell death 1 antibody (PA-PD-1) on the prognosis of HCC patients with high relapse risks after surgery. Patients and Methods Consecutive HCC patients with high recurrence risks treated with liver resection at our center during January 2019 and March 2021 were prospectively enrolled. Baseline differences were balanced between HCC patients with (PA-PD-1 group) or without PA-PD-1 (non-PD-1 group) after hepatectomy by propensity-score matching (PSM). Between these two groups, we compared overall survival (OS) and recurrence-free survival (RFS). Independent prognostic risk factors for OS and RFS were confirmed by Cox regression analysis and subgroup analysis was also performed. Results 47 pairs of patients with or without PD-1 treatment after hepatectomy were matched. After PSM, the 1-year and 2-year RFS were 58.4% and 44.1% in the PA-PD-1 group, and 34.0% and 21.3% in the non-PD-1 group (p = 0.008). The OS at 1-year and 2-year were 91.2% and 91.2% in the PA-PD-1 group, compared with 85.1% and 61.7% in the non-PD-1 group (p= 0.024). Multivariable analyses demonstrated that PA-PD-1 was an independent protective predictor associated with RFS and OS. Through subgroup analysis, we concluded that HCC patients with portal venous tumor thrombus (PVTT) or tumor size ≥ 5 cm significantly benefited from PA-PD-1 therapy in RFS and OS. Conclusions Adjuvant anti-PD-1 antibody can effectively improve the survival outcomes of HCC patients with high relapse risks after hepatectomy.

SummaryRevealing differential tumor immune microenvironment (TIME) characteristics between true versus de novo hepatocellular carcinoma (HCC) recurrence could help optimal development and use of immunotherapies. Here, we studied the TIME of recurrent HBV-related HCCs by 5’and VDJ single-cell and bulk RNA-sequencing, flow cytometry, and multiplexed immunofluorescence. Analyses of mutational profiles, evolutionary trajectories, and clonal architecture using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The TIME of truly recurrent HCCs was characterized by an increased abundance in KLRB1+CD8+ T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8+ T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed an upregulated GDF15 expression level on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited anti-tumor immunity in the TIME of truly recurrent lesions. In contrast, we found that myeloid cells’ crosstalk with T cells mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCC. In conclusion, our results support genomic diagnosis and immune profiling for guiding immunotherapy implementation based on the type of HCC recurrence and TIME.HighlightsTruly recurrent lesions are seeded before primary tumor diagnosis, and that de novo cancer can occur earlier than the clinically used 2-year limit.ScRNA-seq unravels distinct immune ecosystems in true versus de novo HCC recurrences, highlighting the need for different immunotherapy strategies for two types of HCC recurrence.CD8+ T cells in de novo recurrence displayed cytotoxic and exhausted phenotypes while those in truly recurrent lesions showed a memory phenotype with weak cytotoxicity.HCC cells expressing the inhibitory molecule GDF15 were in the proximity of DCs only in truly recurrent lesions.High GDF15 expression level was associated with truly recurrent HCC and worse prognosis.Graphical abstract

Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB signaling prevented VP-MCC cell growth in vitro and in xenografts. We identify MCPyV sT–induced ncNF-κB signaling as an essential tumorigenic pathway in MCC. Implications: This work is the first to identify the activation of ncNF-κB signaling by any polyomavirus and its critical role in MCC tumorigenesis.

Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy.Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo.Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with NOur data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

Background The aim of the study is to compare the safety and efficacy of modified ALPPS (laparoscopic microwave ablation and portal vein ligation for staged hepatectomy, LAPS) and classical associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in the treatment of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Methods Clinical data of patients with HBV-related HCC who underwent LAPS or ALPPS surgery in our institute from April 2013 to October 2020 were retrospectively analyzed. Results 31 patients with HBV-related HCC were retrospectively collected in this study (LAPS = 8, ALPPS = 23). 7 patients with LAPS and 19 patients with ALPPS proceeded to resection (resection rate: 87.5% vs. 82.6%, P > 0.05). The hypertrophy rate of future liver remnant (FLR) caused by ALPPS was higher than that of LAPS (24.3 vs. 11.7 mL/d, P = 0.024). Compared with the ALPPS, LAPS was associated with less blood loss (300ml vs. 40ml, P Conclusions Compared with ALPPS, LAPS can reduce the occurrence of complications in patients with HBV-related HCC and improve patients’ prognoses.

Background Gut microbiota and microbe-derived metabolites are involved in the development of HCC. Bile acids (BAs) are the most important gut microbiota-modulated endogenous signaling molecules. Methods We tested serum bile acid levels and gut microbiome compositions in patients with HCC, chemical-induced HCC mouse models (DEN-HCC mice) and mouse orthotopic implanted liver tumor models with vancomycin treatment (vancomycin-treated mice). Then, we screened an important kind of HCC-related BAs, and verified its effect on the growth of HCC in vivo and in vitro. Results We found that the remarkably decreasing percentages of serum secondary BAs in the total bile acids of patients and DEN-HCC mice, especially, conjugated deoxycholic acids (DCA). The relative abundance of the bile salt hydrolase (BSH)-rich bacteria (Bifidobacteriales, Lactobacillales, Bacteroidales, and Clostridiales) was decreased in the feces of patients and DEN-HCC mice. Then, in vancomycin-treated mice, vancomycin treatment induced a reduction in the BSH-rich bacteria and promoted the growth of liver tumors. Similarly, the percentage of conjugated DCA after vancomycin treatment was significantly declined. We used a kind of conjugated DCA, Glyco-deoxycholic acid (GDCA), and found that GDCA remarkably inhibited the growth of HCC in vivo and in vitro. Conclusions We conclude that the remarkably decreasing percentages of serum conjugated DCA may be closely associated with HCC, which may be induced by the reducing gut BSH-rich bacteria. The mechanisms may be correlated with conjugated DCA directly inhibiting the growth and migration of HCC cells.

Purposes To evaluate the postsurgical prognostic implication of contrast-enhanced ultrasound (CEUS) for combined hepatocellular-cholangiocarcinoma (CHC). To build a CEUS-based early recurrence prediction classifier for CHC, in comparison with tumor-node-metastasis (TNM) staging. Methods The CEUS features and clinicopathological findings of each case were analyzed, and the Liver Imaging Reporting and Data System categories were assigned. The recurrence-free survival associated factors were evaluated by Cox proportional hazard model. Incorporating the independent factors, nomograms were built to estimate the possibilities of 3-month, 6-month, and 1-year recurrence and whose prognostic value was determined by time-dependent receiver operating characteristics, calibration curves, and hazard layering efficiency validation, comparing with TNM staging system. Results In the multivariable analysis, the levels of carbohydrate antigen 19-9, prothrombin time and total bilirubin, and tumor shape, the Liver Imaging Reporting and Data System category were independent factors for recurrence-free survival. The LR-M category showed longer recurrence-free survival than did the LR-4/5 category. The 3-month, 6-month, and 1-year area under the curves of the CEUS-clinical nomogram, clinical nomogram, and TNM staging system were 0.518, 0.552, and 0.843 versus 0.354, 0.240, and 0.624 (P = .048, .049, and .471) vs. 0.562, 0.545, and 0.843 (P = .630, .564, and .007), respectively. The calibration curves of the CEUS-clinical model at different prediction time pionts were all close to the ideal line. The CEUS-clinical model effectively stratified patients into groups of high and low risk of recurrence in both training and validation set, while the TNM staging system only works on the training set. Conclusions Our CEUS-clinical nomogram is a reliable early recurrence prediction tool for hepatocellular-cholangiocarcinoma and helps postoperative risk stratification.

The dynamic regulation of β-cell abundance is poorly understood. Since chromatin remodeling plays critical roles in liver regeneration, these mechanisms could be generally important for regeneration in other tissues. Here, we show that the ARID1A mammalian SWI/SNF complex subunit is a critical regulator of β-cell regeneration. Arid1a is highly expressed in quiescent β-cells but is physiologically suppressed when β-cells proliferate during pregnancy or after pancreas resection. Whole-body Arid1a knockout mice are protected against streptozotocin-induced diabetes. Cell-type and temporally specific genetic dissection show that β-cell-specific Arid1a deletion can potentiate β-cell regeneration in multiple contexts. Transcriptomic and epigenomic profiling of mutant islets reveal increased neuregulin-ERBB-NR4A signaling. Chemical inhibition of ERBB or NR4A1 blocks increased regeneration associated with Arid1a loss. Mammalian SWI/SNF (mSWI/SNF) complex activity is a barrier to β-cell regeneration in physiologic and disease states.

Objectives To construct a preoperative model for survival prediction in intrahepatic cholangiocarcinoma (ICC) patients using ultrasound (US) based radiographic-radiomics signatures. Methods Between April 2010 and September 2015, 170 patients with ICC who underwent curative resection were retrospectively recruited. Overall survival (OS)-related radiographic signatures and radiomics signatures based on preoperative US were built and assessed through a time-dependent receiver operating characteristic curve analysis. A nomogram was developed based on the selected predictors from the radiographic-radiomics signatures and clinical and laboratory results of the training cohort (n = 127), validated in an independent testing cohort (n = 43) by the concordance index (C-index), and compared with the Tumor Node Metastasis (TNM) cancer staging system as well as the radiographic and radiomics nomograms. Results The median areas under the curve of the radiomics signature and radiographic signature were higher than that of the TNM staging system in the testing cohort, although the values were not significantly different (0.76-0.82 versus 0.62, P = .485 and .264). The preoperative nomogram with CA 19-9, sex, ascites, radiomics signature, and radiographic signature had C-indexes of 0.72 and 0.75 in the training and testing cohorts, respectively, and it had significantly higher predictive performance than the 8th TNM staging system in the testing cohort (C-index: 0.75 versus 0.67, P = .004) and a higher C-index than the radiomics nomograms (0.75 versus 0.68, P = .044). Conclusions The preoperative nomogram integrated with the radiographic-radiomics signature demonstrated good predictive performance for OS in ICC and was superior to the 8th TNM staging system.

For unresectable biliary tract cancer (BTC), chemotherapy (CT) alone is associated with poor survival and severe toxicity. Immunotherapy (IO) is potentially effective for BTC, and radiation therapy (RT) may synergize with IO. We investigated CT versus combined RT and anti-programmed cell death-1 (PD-1) IO for unresectable BTC.We prospectively observed 117 participants with unresectable BTC either at initial diagnosis or at first recurrence at a single center who chose 1 of 2 treatment options between August 2018 and October 2020. The options were (1) external beam RT combined with anti-PD-1 IO (RT/IO) or (2) CT alone. In the RT/IO group, camrelizumab (200 mg intravenously every 3 weeks) was initiated within 7 days after the completion of RT and continued until progression or intolerable side effects were noted. The median dose per fraction was 55 Gy/25 fractions (range, 50-60 Gy/20-25 fractions). In the CT group, cisplatin and gemcitabine were delivered intravenously every 3 weeks for 8 cycles. We analyzed the adverse events (AEs), overall survival (OS), and disease-free survival (DFS), and performed subgroup analysis according to tumor mutational burden (TMB) and microsatellite status in the combination group.Thirty-nine and 78 participants received RT/IO and CT, respectively. The crude rate of severe AEs (grade ≥3 AEs) was higher in the CT group (79.4% vs 7.7%, P.001). The OS and DFS after RT/IO were longer than that after CT (median OS: 17.0 vs 11.5 months, P = .01; median DFS: 12.5 vs 7.9 months, P = .008). Participants with low TMB or microsatellite stability had a shorter median OS (13.6 vs 25.7 months, P = .03) and median DFS (9.8 vs 19.3 months, P = .012) than participants with high TMB or microsatellite instability.RT combined with anti-PD-1 IO may be well tolerated and associated with an improved response rate, DFS, and OS compared with CT alone in patients with unresectable BTC.

Background Epithelial-mesenchymal transition (EMT) is critical for cancer cell metastasis. Recently, EMT was reported to be associated with the inflammatory tumour microenvironment and, therefore, might be a predictive biomarker for immune checkpoint blockade agents. However, the underlying mechanism is still unclear. Methods Patient survival data for our HCC cohort, TCGA and GEO datasets were determined by Kaplan-Meier analysis. The functional roles of ELMO1 in HCC were demonstrated by a series of in vitro and in vivo experiments. Gene microarray analysis was used to demonstrate potential mechanisms of ELMO1. Data retrieved from the TCGA datasets were used to determine the relationships of ELMO1, EMT and TMB. Findings Here, we report an indispensable role for ELMO1 in linking EMT with tumour mutation burden (TMB), which is a promising biomarker for the immune checkpoint blockade agent response. Upregulated ELMO1 expression is associated with a poor prognosis in hepatocellular carcinoma (HCC), as well as increased cell growth, invasion, migration, angiogenesis and EMT in vitro and in vivo. Mechanistically, we provide evidence that ELMO1 regulates SOX10 expression and induces EMT through PI3K/Akt signalling. Moreover, ELMO1 is negatively associated with TMB, indicating a negative relationship between EMT and TMB. Interpretation ELMO1 serves as a link between EMT and TMB, providing a mechanistic basis for the further development of ELMO1 as a therapeutic target against HCC and potentially a promising biomarker of the immune checkpoint blockade agent response. Fund National Natural Science Foundation of China; Natural Science Foundation of Guangdong Province; Young Teacher Training Program of Sun Yat-sen University; Science and Technology Plan of Guangdong Province; Special Support Program of Guangdong Province, Science and Technology Innovation Youth Talent Support Program; the Pearl River Science and Technology New Talent of Guangzhou City; Medical Scientific Research Foundation of Guangdong Province.

Background Gut microbiota and the tumor microenvironment are thought to be critical factors that modulate the processes of liver diseases, including hepatocellular carcinoma (HCC). Interleukin-25 (IL-25) promotes type 2 immunity via alternative activation of macrophages, and is closely associated with inflammation-related diseases, even malignancies. However, it is not clear which role IL-25 plays in the development of HCC, and whether gut microbiota are involved. Methods IL-25 was detected by ELISA, Western blotting (WB), and immunohistochemistry. Chemokines were measured by RT-qPCR and WB. After co-culture with IL-25-stimulated macrophages, the cell growth, migration, invasion and EMT marker of HCC cell lines (MHCC97L and HepG2) were evaluated by Brdu proliferation, Transwell assays and WB. An antibody neutralization assay of chemokine CXCL10 was performed to confirm its role in HCC development. Furthermore, the effects of IL-25 in HCC were investigated in vivo. Dysbiosis of gut microflora was induced by antibiotics (vancomycin, cefoperazone or combination of ampicillin, neomycin, metronidazole, and vancomycin). We used feces suspension to treat colonic epithelial NCM460 cells, and detected IL-25 and tuft cell marker DCLK1 using WB and immunofluorescence staining. Results We found that the level of IL-25 was significantly elevated in HCC patients, and was negatively correlated with survival rate after hepatectomy. However, IL-25 did not directly promote the development of HCC cells. Then, we observed the significant positive correlation between IL-25 level and M2 percentage (CD206/CD68) in HCC tumors. In vitro and in vivo, IL-25 induced alternative activation of macrophages promoted HCC cell migration, invasion and tumorigenesis, increased the expression of vimentin, Snail and phospho-ERK, and decreased the expression of E-cadherin in HCC cells. After IL-25 treatment, chemokine CXCL10 was increased in macrophages. Neutralizing CXCL10 in macrophage-conditioned medium reversed the IL-25-mediated effect on HCC cells. Vancomycin-induced dysbiosis promoted the growth of orthotopic HCC homograft. Surprisedly, we found the hyperplasia of colonic epithelial tuft cells, from which more IL-25 was secreted . Conclusions IL-25 promotes the progression of HCC through inducing alternative activation and CXCL10 secretion of macrophages in tumor microenvironment, and IL-25 secretion may partly result from hyperplastic epithelial tuft cells in colon, induced by gut microbiota dysbiosis. Electronic supplementary material The online version of this article (10.1186/s13046-019-1271-3) contains supplementary material, which is available to authorized users.

FoxM1 is an oncoprotein that is significantly overexpressed in many malignancies including hepatocellular carcinoma, but its role in intrahepatic cholangiocarcinoma (ICC) remains unclear. This study explores the expression of FoxM1 in human ICC, its relationships with clinical outcomes, and its role in the proliferation, migration, and invasion of ICC in vitro and in vivo. The results show that FoxM1 was markedly elevated in tumor tissues versus the paired peritumoral tissues. Overexpression of FoxM1 was correlated with multiple tumor nodules, tumor size > 5 cm, positive lymph node metastasis and advanced TNM stage. Cox analysis revealed that overexpression of FoxM1 is an independent prognostic indicator for both the overall survival and disease-free survival of ICC patients after hepatectomy. Furthermore, up/downregulation of FoxM1 markedly promoted/inhibited ICC cell proliferation, migration, and invasion in vitro and in vivo. Bioinformatic analysis indicated that overexpression of FoxM1 resulted in the dysregulation of multiple signaling pathways in ICC, and selected components of some key signaling pathways such as c-Myc signaling were confirmed in vitro. In addition, overexpression of FoxM1 enhanced MMP-9 and MMP-2 protein expression in ICC cells. In conclusion, FoxM1 promotes ICC progression and is a reliable predictor of poor prognosis in ICC.

ObjectiveAltered metabolites are important for the tumourigenicity of hepatocellular carcinoma (HCC). We performed integrative metabolomics analysis of the metabolites changes in portal venous blood and in comparison with the metabolites changes in liver tissues and stool samples of HCC patients and healthy liver donors.DesignSerum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines.ResultsDetailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (pConclusionsThe integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development.