BACKGROUND:No neoadjuvant treatment has been considered to be standard therapy for patients with resectable intrahepatic cholangiocarcinoma with high-risk factors for recurrence. The GOLP regimen (gemcitabine-oxaliplatin, lenvatinib, and an anti-programmed death 1 antibody) has shown promising efficacy with a manageable safety profile in advanced intrahepatic cholangiocarcinoma and biliary tract cancer. METHODS:In a phase 2-3 trial, we randomly assigned, in a 1:1 ratio, patients with resectable high-risk intrahepatic cholangiocarcinoma to the neoadjuvant group (intravenous gemcitabine-oxaliplatin plus toripalimab every 3 weeks for three cycles and oral lenvatinib once daily for 9 weeks, followed by curative resection) or the control group (curative resection and no neoadjuvant treatment). All patients received adjuvant capecitabine for eight cycles after surgery. The primary end point was event-free survival. Secondary end points included overall survival and safety. RESULTS:A total of 178 patients underwent randomization (88 patients to the neoadjuvant group and 90 to the control group). At the interim analysis at a median follow-up of 16.9 months, the median event-free survival was significantly longer in the neoadjuvant group (18.0 months; 95% confidence interval [CI], 13.8 to 27.6) than in the control group (8.7 months; 95% CI, 7.2 to 12.4) (P<0.001). Overall survival at 24 months was 79% (95% CI, 70 to 90) in the neoadjuvant group and 61% (95% CI, 50 to 75) in the control group (hazard ratio for death, 0.43; 95% CI, 0.23 to 0.79; P = 0.005, which did not meet the significance criterion [two-sided alpha, 0.0019]). Across all treatment phases, adverse events occurred in 97% of the patients in the neoadjuvant group and in 70% of those in the control group. During the neoadjuvant phase, adverse events of grade 3 or higher occurred in 28% of the patients, and treatment-related adverse events of grade 3 or higher in 26%. No treatment-related adverse event led to death. CONCLUSIONS:Neoadjuvant GOLP led to significantly longer event-free survival than control therapy, with mainly low-grade adverse events, among patients with resectable high-risk intrahepatic cholangiocarcinoma. (Funded by the Clinical Research Plan of Shanghai Hospital Development Center and others; ZSAB-neoGOLP ClinicalTrials.gov number, NCT04669496.).

BACKGROUND:Preoperative hypoalbuminemia is a risk factor for complications after hepatectomy for hepatocellular carcinoma (HCC). The impacts of preopeRative sErum Albumin Levels on postoperative outcomes in Chinese HCC Patients treated with surgical operation (REAL) studyaimed to evaluate the impact of preoperative human albumin administration on postoperative outcomes in patients with HCC and hypoalbuminemia undergoing hepatectomy. METHODS:This retrospective study analyzed patients with HCC and preoperative hypoalbuminemia (serum albumin < 36 g/L) who underwent liver resection at Zhongshan Hospital, Fudan University. Patients were categorized into those who received albumin supplementation and those who did not. Surgical outcomes included durations of hospital and intensive care unit (ICU) stay, incidence and grade of posthepatectomy liver failure (PHLF), 30-day complication rates, and hospital mortality. RESULTS:Patients with hypoalbuminemia who did not receive supplementation had significantly higher rates of PHLF grades A/B and 30-day complications compared with the normal albumin group, along with a shorter overall survival. However, patients with hypoalbuminemia who received supplementation achieved outcomes comparable to those of the normal albumin group, with no significant differences in hospital stay, ICU stay, PHLF grade distribution, or 30-day complication rates. Among patients with hypoalbuminemia, those who received albumin supplementation had a significantly lower PHLF rate. No in-hospital mortality occurred in any group. CONCLUSION:Preoperative albumin supplementation in patients with hypoalbuminemiais associated with significantly improved postoperative outcomes in this retrospective cohort. These real-world findings provide preliminary evidence supporting targeted albumin supplementation to reduce surgical risk among such vulnerable patients. However, large prospective randomized controlled trials are still required to validate our conclusions before widespread clinical application.

BACKGROUND AND AIMS:Pathological complete response (pCR) following conversion therapy for initially unresectable hepatocellular carcinoma (uHCC) remains challenging to predict preoperatively. This study developed and validated a model integrating clinicopathological and radiomic features of the tumor to predict pCR. METHODS:In this multicenter retrospective study, temporal radiomics features were extracted from baseline, post-treatment, and delta (change) MRIs. Serum AFP response was calculated as log₁₀(preoperative AFP)/log₁₀(baseline AFP). Univariate analysis, collinearity assessment, LASSO, and random forest were employed to perform feature selection. Fourteen machine learning models were benchmarked, with performance evaluated by using comprehensive metrics AUC, NPV, PPV, sensitivity, specificity, calibration, and decision curve analysis. RESULTS:The model was developed and validated in a training (n=78), an internal test (n=32), and an independent validation cohort (n=44). The delta radiomic model significantly outperformed both baseline (test AUC: 0.835 vs. 0.483, p <0.05; validation AUC: 0.783 vs. 0.434, p <0.05) and preoperative models (test AUC: 0.685, p <0.05; validation AUC: 0.506, p <0.05), demonstrating superior predictive performance and generalization capability in predicting lesion-level pCR. Notably, when predicting patient-level pCR, the radiomic model also showed robust discrimination, with AUCs of 0.819 in the test set and 0.781 in the validation set. The combined radiomics-AFP model achieved even higher AUCs of 0.920 (test) and 0.857 (validation) in predicting lesion-level pCR. CONCLUSIONS:Dynamic radiomic changes effectively predict pCR in uHCC after conversion therapy. Combining delta radiomics with AFP response significantly improves predictive performance, offering a non-invasive method for assessing pCR and potentially guiding personalized treatment decisions.

Background:The criteria for identifying patients with potentially resectable hepatocellular carcinoma (HCC) at the baseline stage have helped identify patients who are more likely to achieve successful conversion. However, the suitable timing and selection of candidates for conversion surgery after systemic therapy remained underexplored. Methods:Based on real-world evidence, we analyzed a cohort of 222 patients with potentially resectable HCC treated with immune-based therapy between January 2019 and May 2024. The treatment response was assessed using the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) and modified RECIST (mRECIST). Depth of response (DpR) was defined as the maximum tumor shrinkage from baseline, based on the sum of the longest diameters, as assessed by an independent review facility per RECIST v1.1. Conversely, stable disease (SD) with a DpR < 0%/> 0% was categorized as SD_shrink/SD_non-shrink. Survival outcomes were compared between patients who underwent surgery and those who did not, with different DpRs, and assessed using Kaplan-Meier methods. Results:A total of 85 patients with partial response (PR), 86 with SD_shrink, and 51 with SD_non-shrink were included in this study. Generally, patients who underwent surgery had a better prognosis than those who did not (p < 0.001). Patients with PR and SD_shrink significantly benefited from the surgery in both overall survival and event-free survival. However, patients with SD_non-shrink failed to obtain the same benefits. Additionally, surgical patients with a DpR <0% had a better prognosis than those with a DpR >0% (p < 0.001 and p = 0.004, respectively), whereas the survival of patients who did not undergo surgery did not differ. Conclusion:DpR is an important factor in choosing the right candidates and the timing of conversion therapy. Patients only at a DpR <0% receiving various immune-based treatments who met the "potentially resectable" criteria can benefit from surgical resection. These findings can help increase the success rate of conversion therapy in patients with advanced HCC.

Background The efficacy of immune checkpoint inhibitors (ICIs) for hepatocellular carcinoma (HCC) is limited by heterogeneity in individual responses to therapy. The heterogeneous phenotypes and crucial roles of cancer-associated fibroblasts (CAFs) in immunotherapy resistance remain largely unclear.Methods A specific CAF subset was identified by integrating comprehensive single-cell RNA sequencing, spatial transcriptomics and transcriptome profiling of patients with HCC with different responses to antiprogrammed cell death protein 1 (anti-PD-1) therapy. Mouse orthotopic HCC models and a coculture system were constructed, and cytometry by time-of-flight analysis was performed to investigate the functions and mechanisms of specific CAFs in the immune context of HCC.Results We identified a distinct flavin-containing monooxygenase 2 (FMO2)+ CAF subset associated with a favorable response to anti-PD-1 therapy and better clinical outcomes. FMO2+ CAFs increase anti-PD-1 treatment efficacy by promoting tertiary lymphoid structure formation and increasing the infiltration of CD8+ T cells and M1-like macrophages through the C-C motif chemokine ligand 19 (CCL19)-C-C motif chemokine receptor 7 axis. Mechanistically, FMO2 promotes nuclear factor kappa B/p65-mediated CCL19 expression by competitively binding to glycogen synthase 1 (GYS1) with praja ring finger ubiquitin ligase 1 (PJA1), thereby suppressing the PJA1-mediated proteasomal degradation of GYS1. CCL19 treatment potentiated the therapeutic efficacy of anti-PD-1 therapy in mouse orthotopic HCC models. A favorable immunotherapy response was observed in patients with HCC with high serum levels of CCL19.Conclusions We identified a novel FMO2+ CAF subset that serves as a critical regulator of microenvironmental immune properties and a predictive biomarker of the immunotherapy response in patients with HCC. CCL19 in combination with anti-PD-1 therapy may constitute a novel therapeutic strategy for HCC.

BackgroundImmune checkpoint blockade, particularly targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), shows promise in treating hepatocellular carcinoma (HCC). However, acquired resistance, especially in patients with 'hot tumours', limits sustained benefits. Lysine-specific demethylase 1 (LSD1) plays a role in converting 'cold tumours' to 'hot tumours', but its involvement in PD-1 inhibitor resistance in HCC is unclear.MethodsLSD1 and PD-L1 expression, along with CD8+ T cell infiltration, were assessed using immunohistochemistry in HCC tissues, correlating these markers with patient prognosis. The impact of LSD1 deletion on tumour cell proliferation and CD8+ T cell interactions was examined in vitro. Mouse models were used to study the combined effects of LSD1 inhibition and anti-PD-1 therapy on tumour growth and the tumour microenvironment (TME). The clinical relevance of LSD1, CD74 and effector CD8+ T cells was validated in advanced HCC patients treated with PD-1 blockade.ResultsLSD1 overexpression in HCC patients correlated with reduced PD-L1 expression, less CD8+ T cell infiltration and poorer prognosis. LSD1 deletion increased PD-L1 expression, boosted effector CD8+ T cells in vitro and inhibited tumour growth in vivo. While anti-PD-1 monotherapy initially suppressed tumour growth, it led to relapse upon antibody withdrawal. In contrast, combining LSD1 inhibition with anti-PD-1 therapy effectively halted tumour growth and prevented relapse, likely through TME remodelling, enhanced CD8+ T cell activity and improved CD74-mediated antigen presentation. Clinically, low LSD1 expression was associated with better response to anti-PD-1 therapy.ConclusionLSD1 deletion reshapes the TME, enhances CD8+ T cell function and prevents acquired resistance to anti-PD-1 therapy in HCC. The combination of LSD1 inhibitors and PD-1 blockade offers a promising strategy for overcoming resistance in advanced HCC.Key points Uncovering the synthetic lethality resulting from LSD1 deletion and PD1 inhibitor co-administration, evaluating their combined effects on tumour growth and TME remodelling. Elucidating the mechanism underlying the combined therapy of LSD1 deletion with PD1 inhibition for HCC. Exploring the implications of LSD1, CD74 and effector CD8+ T cell expression levels in advanced HCC patients undergoing anti-PD1 treatment.

Abstract Background Predicting the efficacy of immune-based therapy in patients with unresectable hepatocellular carcinoma (HCC) remains a clinical challenge. This study aims to evaluate the prognostic value of the systemic immune-inflammation index (SII) in forecasting treatment response and survival outcomes for HCC patients undergoing immune-based therapy. Methods We analyzed a cohort of 268 HCC patients treated with immune-based therapy from January 2019 to March 2023. A training cohort of 93 patients received atezolizumab plus bevacizumab (T + A), while a validation cohort of 175 patients underwent treatment with tyrosine kinase inhibitors (TKIs) combined with anti-PD-(L)1 therapy. The SII cutoff value, determined using X-tile analysis based on overall survival (OS) in the training cohort, divided patients into high (> 752*109) and low (≤ 752*109) SII groups. Prognostic factors were identified through univariate and multivariate logistic and Cox regression analyses, and survival outcomes were assessed using Kaplan–Meier methods. The predictive accuracy of SII was evaluated using receiver operating characteristic (ROC) curves. Results An optimal SII cutoff of 752*109 stratified patients into high and low SII groups. Univariate and multivariate logistic regression indicated that SII was a significant predictor of the objective response rate (ORR), which was markedly different between the low and high SII subgroups (34.72% vs. 9.52%, P = 0.019). This finding was consistent in the validation cohort (34.09% vs. 16.28%, P = 0.026). SII also demonstrated prognostic value in Cox regression and Kaplan–Meier analyses. ROC curves confirmed that SII had superior predictive accuracy compared to common clinical indicators, with predictive relevance even in AFP-negative patients. Furthermore, a lower SII was associated with a higher T cell ratio and an increased number of CD8+ T cells and Granzyme B+ CD8+ T cells in peripheral blood. Conclusion SII is a promising predictor of both therapeutic efficacy and prognosis in HCC patients undergoing immune-based treatments. Its application may enhance clinical decision-making, thereby improving patient outcomes from immune-based therapy.

Adding a PD-1/PD-L1 inhibitor to gemcitabine plus cisplatin (GemCis) has shown survival benefits in advanced biliary tract cancer (BTC). Dual inhibition of PD-1/PD-L1 and TIGIT may act synergistically, and further enhance antitumor effects. ZSAB-TOP was a single-arm, multicenter, phase 2 study (NCT05023109) evaluating efficacy and safety of first-line tislelizumab (a PD-1 inhibitor) plus ociperlimab (a TIGIT inhibitor) and GemCis in advanced BTC. Eligible patients received tislelizumab (200 mg) and ociperlimab (900 mg) on day 1 until unacceptable toxicity or disease progression, in combination with cisplatin (25 mg/m²) and gemcitabine (1000 mg/m²) on days 1 and 8 of a 21-day cycle for a maximum eight cycles. The primary endpoint was confirmed objective response rate (ORR) evaluated by the investigator, which was compared with a historical ORR of 25% with GemCis, with a statistical superiority setting at p ≤ 0.05. From March 8, 2022, to January 18, 2023, 45 patients were enrolled. Among the 41 patients in the efficacy analysis set, the confirmed ORR was 51.2% (95% CI 35.1–67.1), achieving the statistical superiority criteria (p = 0.0003). Patients who had TIGIT+/PD-L1+ (n = 16) tended to have a numerically greater confirmed ORR (75.0% [95% CI 47.6–92.7]). After a median follow-up of 14.6 months, median progression-free survival was 7.7 months (95% CI 6.0–9.4), with a median overall survival of 17.4 months (95% CI 11.7-not reached). Treatment-related adverse events of grade ≥3 occurred in 60.0% of patients; immune-mediated adverse events of any grade was observed in 42.2%, with the majority being grade 1 or 2. In conclusion, first-line tislelizumab and ociperlimab plus GemCis yielded clinically promising tumor response and survival outcomes in advanced BTC and were generally well tolerated without new safety signals.

Background and Aims: Many patients with HCC present inadequate responses to lenvatinib therapy. Therefore, it is important to elucidate the underlying mechanisms of resistance and to formulate effective reversal strategies. Approach and Results: We conducted transcriptome and proteome sequencing analyses of lenvatinib-resistant cell lines and patient-derived tissues, identifying microtubule-associated serine/threonine kinase-like (MASTL) as a critical factor associated with lenvatinib resistance in HCC. Then, we utilized subcutaneous mouse models, half maximal inhibitory concentration (IC 50 ) measurements, and colony formation assays to determine the biological function of MASTL in promoting tumor growth and mediating resistance to lenvatinib. To further elucidate the underlying mechanisms, we performed co-immunoprecipitation and mass spectrometry analyses, revealing that MASTL facilitates the phosphorylation of Y-box binding protein-1 (YBX1). Using chromatin immunoprecipitation assays, we subsequently confirmed that phosphorylated YBX1 transcriptionally activates PAK4, identifying PAK4 as a downstream effector of the MASTL pathway. Moreover, mass spectrometry and phosphorylation analysis indicated that serine/threonine protein kinase 24 (STK24), a stress-responsive kinase, can activate MASTL in HCC under lenvatinib exposure. Notably, disruption of the MASTL/YBX1/PAK4 signaling axis restored HCC sensitivity to lenvatinib. Conclusions: We propose that the MASTL/YBX1/PAK4 axis, which is activated by stress-induced STK24, plays a crucial role in lenvatinib resistance. Inhibiting this axis by targeting MASTL effectively overcomes lenvatinib resistance in HCC.

BACKGROUND:The optimal conversion regimen that allows more patients with unresectable biliary tract cancer to access surgery remains unclear; there is currently no standard conversion therapy for biliary tract cancer in China, with commonly used regimens including immunotherapy-based combinations and local therapy. The ZSAB-TransGOLP study aimed to assess the efficacy and safety of tislelizumab plus lenvatinib and GEMOX (gemcitabine plus oxaliplatin) chemotherapy (GOLP) in patients with this disease. METHODS:This single-arm, phase 2 study was conducted at two centres in China. Eligible patients aged 18-70 years with previously untreated locally advanced unresectable biliary tract cancer (intrahepatic cholangiocarcinoma, perihilar bile duct cancer, and gallbladder cancer), and an Eastern Cooperative Oncology Group performance status of 0 or 1, Child-Pugh score of A, and at least 3 months' life expectancy were enrolled. Patients received 200 mg intravenous tislelizumab on day 1 and intravenous GEMOX (0·5 h of 1000 mg/m2 gemcitabine on days 1 and 8; and 2 h of 85 mg/m2 oxaliplatin on day 1) in a 21-day cycle for three cycles, and 8 mg oral lenvatinib once daily. Tumour resectability was determined by the multidisciplinary team every 3 cycles of conversion therapy; patients who were ineligible for R0 resection and did not require surgery after six cycles received maintenance therapy with tislelizumab plus lenvatinib at the same dose as used in conversion therapy until completing 1 year of treatment, disease progression, intolerable toxicity, death, consent withdrawal, or investigators' decisions. The primary endpoint was the R0 resection rate. All treated patients were evaluable for safety and primary endpoint. The trial is registered with ClinicalTrials.gov (NCT05156788) and is ongoing but closed for recruitment. FINDINGS:Between Dec 27, 2021 and July 3, 2023, 52 patients were screened, 11 were excluded for ineligiblity, and 41 patients were enrolled and received the GOLP regimen. All patients were Chinese, with median age of 58 years (IQR 54-65); 21 patients (51%) were male and 20 patients (49%) were female. Median duration of GOLP treatment was 3 cycles (IQR 3-6). 28 (68%) of 41 patients underwent surgery. At a median follow-up of 19·5 months (IQR 14·6-25·0) by data cutoff on Jan 20, 2025, the R0 resection rate was 63% (26 of 41 [95% CI 47-78]). All patients had at least one any-grade treatment-related adverse event (TRAE); grade 3-4 TRAEs occurred in 20 (49%) of 41 patients, with neutropenia (14 [34%] of 41) being most common. Serious TRAEs occurred in 4 (10%) of patients and included neutropenia (three [7%]) and decreased platelet count (one [2%]). No TRAE-related deaths occurred. INTERPRETATION:With promising efficacy and manageable safety, GOLP represents a potentially feasible and high-efficiency conversion regimen for unresectable locally advanced biliary tract cancer. FUNDING:Program of Shanghai Academic Research Leader, the Key Disease Joint Research Program of Xuhui District, Shanghai Health Commission Clinical Research Special Project, Fellowship from the China Postdoctoral Science Foundation, National Science and Technology Major Project of China, the Outstanding Resident Clinical Postdoctoral Program of Zhongshan Hospital Affiliated to Fudan University, National Natural Science Foundation of China, and the Shanghai Sailing Program.

Background:Bevacizumab, immune checkpoint inhibitors (ICIs), interventional therapy, either alone or in combination, have demonstrated promising anti-tumor activities in unresectable hepatocellular carcinoma (HCC). However, the optimal dosing strategy for bevacizumab within combination regimens remains undetermined. This study aimed to compare the efficacy and safety of different bevacizumab doses in triple therapy combining bevacizumab, ICIs, and interventional therapy for unresectable HCC. Methods:A retrospective study included patients with unresectable HCC treated with interventional therapy combined with bevacizumab (full dose or half dose) and ICIs between December 2020 and July 2023 was conducted. Propensity score matching (PSM, 1:1) was applied to minimize confounding effects. The progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and disease control rate (DCR) were compared and evaluated. Treatment-related adverse events (AEs) were analyzed to assess safety. Results:Among 66 enrolled patients, 36 received full-dose bevacizumab, and 30 received half-dose bevacizumab. The full-dose group exhibited significantly longer PFS compared to the half-dose group (median PFS: not attained vs. 8.0 months, P<0.001). Median OS was not reached in either group, with 1-year OS rate of 86% in the full-dose group and 83% in the half-dose group. Although full-dose group showed numerically higher ORR (55.6% vs. 36.7%, P=0.13) and DCR (88.9% vs. 83.3%, P=0.51), these differences did not reach statistical significance. Half-dose group experienced comparable AEs with full-dose group. Post-PSM analyses corroborated these findings. Conclusions:Although full-dose bevacizumab in triple therapy showed prolonged PFS compared to half-dose group, no statistically significant differences were observed in long-term survival outcomes or treatment-related AEs between the two groups. For unresectable HCC patients with severe liver cirrhosis, half-dose bevacizumab may serve as a safer alternative without compromising survival benefits.

The GOLP regimen (Gemcitabine, Oxaliplatin, Lenvatinib and anti-PD1 antibody) has been a promising first-line treatment for advanced intrahepatic cholangiocarcinoma (iCCA). A noninvasive tool to predict the response to GOLP regimen is needed for clinical practice. In this study, 188 cell-free DNA samples were collected before each cycle and after the third cycle (P0 to P3) from 47 iCCA patients receiving GOLP regimen. Genome-wide 5-hydroxymethylcytosine (5hmC) profiles of samples were generated by 5hmC-Seal. Tumor response was assessed per Response Evaluation Criteria in Solid Tumors 1.1. Differential and functional analyses revealed that cell proliferation and malignancies associated pathways exhibited higher 5hmC level in poor responders at P1. Immune response related pathways presented higher 5hmC level in good responders at P2. Patients were split into training and validation cohorts by simple randomization at a ratio of 2:1 and a 5-features weighted predictive (wp-) model showing an area under the curve of 0.967 in the validation samples was constructed by machine learning. The model-derived wp-scores showed an opposite trend between good responders and poor responders from P0 to P3. In conclusion, our study identified novel epigenetic modifications and pathways across treatment process to reflect response to the GOLP regimen for iCCA patients. We developed a highly sensitive and specific 5hmC-based 5-features predictive model for GOLP treatment, holding the promise as a noninvasive tool for precision care of iCCA patients.

e16225 Background: In recent years, immune checkpoint inhibitors (ICIs) combined with targeted therapies have become the preferred first-line standard treatment for advanced hepatocellular carcinoma (HCC). However, the optimal second-line treatment for HCC following ICIs failure remains undefined. Benmelstobart, a novel PD-L1 inhibitor, has shown promising antitumor activity when combined with anlotinib, an antiangiogenic agent, in the treatment of second-line advanced HCC (NCT03825705). Consequently, FAITH study aimed to evaluate the efficacy and safety of anlotinib plus benmelstobart in patients with previously ICIs treated intermediate-to-advanced HCC clinically. Methods: This is a multi-center, single-arm, prospective phase II study. Intermediate-to-advanced HCC patients who failed the previous ICIs treatment were recruited. Eligible patients received anlotinib (10mg, po, d1-14, q3w) and benmelstobart (1200mg, iv, d1, q3w) until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) (RECIST v1.1). Secondary endpoints included ORR (mRECIST), progression free survival (PFS), disease control rate (DCR), overall survival (OS) and safety. Results: From December 2023 to January 2025, a total of 18 patients were enrolled. The median age was 54 years (range: 36-70 years), and 15 patients (83.3%) were male. Seven patients (38.9%) had an ECOG performance status of 1, and 15 (83.3%) patients had Child-Pugh A liver function. Fifteen patients (83.3%) had BCLC (Barcelona Clinic Liver Cancer) stage of C and three (16.7%) of B. Previous treatments included vascular endothelial growth factor inhibitors plus ICIs treatment (9 patients, 50.0%), tyrosine kinase inhibitors combined with ICIs (5 patients, 27.8%), cytotoxic T lymphocyte antigen 4 inhibitors plus ICIs (3 patients, 16.7%), and ICIs alone (1 patient, 5.6%). Fourteen patients (77.8%) were HBV-positive. Among the 14 evaluable patients, 1 (7.1%) achieved partial response, 11 (78.6%) had stable disease, and 2 (14.3%) experienced progressive disease. The ORR was7.1%, and the DCR was 85.7%. Among all 18 patients, 11 (61.1%) experienced treatment-emergent adverse events (TEAEs), with 4 (22.2%) reporting grade ≥3 TEAEs, included neutropenia (5.6%), thrombocytopenia (5.6%), bleeding (5.6%), and elevated bilirubin (5.6%). Conclusions: Anlotinib combined with benmelstobart demonstrated potential efficacy and acceptable safety profile in patients with intermediate-to-advanced HCC who failed prior ICI therapies, which is worthy of further exploration with continuous recruitment of the study subsequently. Clinical trial information: NCT06031480 .

The chemokine CXCL6 is identified as a pivotal regulator of biological processes across multiple malignancies. However, its function in cholangiocarcinoma (CCA) is underexplored. Tumor profiling for CXCL6 is performed using a public database. Both in vitro and in vivo experiments are utilized to evaluate the oncogenic effects of CXCL6 on CCA. Additionally, RNA-Seq is employed to detect transcriptomic changes related to CXCL6 expression in CCA cells and neutrophils. Molecular docking, fluorescence colocalization, and Co-IP are used to elucidate a direct interaction between JAKs and CXCR1/2. Additionally, LC-MS lipidomics and explored the impact of CXCL6 on immunotherapy in vivo. CXCL6 is upregulated in CCA tissues and promoted the proliferation and metastasis of CCA. Mechanistically, CXCL6 regulated the CXCR1/2-JAK-STAT/PI3K axis in CCA via autocrine signaling, leading to lipid metabolic reprogramming, and promoted neutrophil extracellular traps (NETs) formation by activating the RAS/MAPK pathway in neutrophils. Eventually, NETs formation induced immunotherapy resistance in CCA by blocking CD8+T cell infiltration. CXCL6 modulates CCA progression through the CXCR1/2-JAK-STAT/PI3K axis and reshaping its lipid metabolism. CXCL6 also mediates immunotherapy resistance through NETs, which may be a potential therapeutic target and biomarker for CCA.

Hepatocellular carcinoma (HCC) exhibits significant plasticity, enabling phenotypic switching that promotes a drug-tolerant state and circumvents drug-induced cytotoxicity. In this study, we identify the hepatic-to-biliary lineage transition (HBT), associated with Claudin 4 (CLDN4), a tight junction protein, as a potential target for mitigating lenvatinib resistance in HCC. CLDN4 expression is more prevalent in lenvatinib-resistant patients. Palmitoylation of CLDN4 at cysteine residues C104 and C107 regulates ubiquitination at lysine residue K103, inhibits clathrin-mediated endocytosis, and sustains CLDN4 anchoring within lipid rafts. Anchored CLDN4 facilitates the phenotypic transition of HCC cells, resulting in increased resistance to lenvatinib by driving the mobilization of contactin-1 to lipid rafts and activating the Notch signaling pathway. Salvianolic acid B, an inhibitor of CLDN4, is demonstrated to reduce both HBT and lenvatinib resistance in HCC. Additionally, combination chemotherapy appears to be an effective therapeutic strategy for HCC patients undergoing HBT.

(A-B) Flow cytometry assays of tumor infiltration proportions of CD11b+ F4/80+ MHC-II+ macrophages and CD3+ CD8+ PD-1+ T cells in orthotopic Hepa1-6 tumors with Slamf7 overexpression and control groups. (C) Heatmap showing the immunological activity ssGSEA scores in tumor samples from TCGA-LIHC cohort. The upper horizontal bars represent the expression level of SLAMF7. (D) Correlation matrix showing the relationships between SLAMF7 level and various immune activity scores in tumors from TCGA-LIHC cohort. (E) Violin diagrams showing the percentages of 22 immune cell types calculated using the CIBERSORT method in HCC tumors with SLAMF7 high and low expression groups. (F) The standardized risk score of a T cell exhaustion (TEX) signature between tumors with low and high SLAMF7 expression in the TCGA-LIHC cohort. (G) Correlation of the risk score of a M2-like macrophage-related gene signature with mRNA level of SLAMF7 in GSE14520 cohort. (H) Immunologic signature gene sets enriched in HCC with high SLAMF7 expression from TCGA-LIHC cohort. Student’s t test.

Representative IHC staining images of p53 protein in murine liver tumor tissues in the indicated groups. Scale bar: 50 μm.

(A) Immunoblot assay of SHB protein level in the indicated HCC cells. (B-C) Immunoblot assays of phosphorylated and non-phosphorylated SHIP1, and K63-linked ubiquitinated TRAF6 level in the indicated HCC cells with and without SHB silence.

Objective This phase Ib trial aimed to assess the safety and efficacy of sintilimab plus bevacizumab (sintilimab/bev), followed by resection in patients with potentially resectable intermediate-stage hepatocellular carcinoma (HCC) and explore the clinical implications of circulating tumour DNA (ctDNA) and T cell receptor (TCR) repertoire.Methods and analysis Eligible patients with intermediate-stage HCC received sintilimab/bev treatment. Patients with partial response or stable disease for at least two consecutive evaluations and technically resectable received hepatectomy. Postoperatively patients continued to receive sintilimab/bev until tumour recurrence or intolerable toxicities for up to 12 months. The primary endpoints were treatment safety and event-free survival (EFS). Plasma ctDNA measurements and TCR repertoire were analysed.Results 30 patients were enrolled. 17 (56.7%) patients received liver resection. Grade 3 treatment-related adverse events occurred in seven patients (23.3%). No grade 4/5 AE or postoperative mortality was observed. The median EFS of the 30 patients was 16.3 months (95% CI 13.4 to 19.2). The 12-month and 24-month survival rates were 93.2% and 82.0%, respectively. Of the 17 patients who received hepatectomy, the median recurrence-free survival was 14.1 months (95% CI 8.9 to 19.4). A lower ctDNA measurement and higher TCR repertoire were associated with better tumour response or patients’ survival.Conclusions The study suggested systemic therapy with sintilimab/bev was safe and effective in patients with intermediate-stage HCC, and resection in selected patients was associated with improved survival. ctDNA measurement and TCR repertoire may help identify patients who may benefit from sintilimab/bev treatment and patients with a higher risk of tumour recurrence.Trial registration number NCT04843943.