Clinical and biomarker analyses of hepatic arterial infusion chemotherapy plus lenvatinib and PD-1 inhibitor for patients with advanced intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with a dismal prognosis and few effective therapeutic approaches. This study aimed to investigate the efficacy, safety, and predictive biomarkers of hepatic arterial infusion chemotherapy (FOLFOX-HAIC) in combination with lenvatinib and PD-1 inhibitor for patients with advanced iCCA. Methods: Locally advanced or metastatic iCCA patients receiving the triple combination therapy of lenvatinib, PD-1 inhibitor, and FOLFOX-HAIC were included in this retrospective study. Primary endpoint was the progression-free survival, evaluated using the RECIST criterion. The secondary endpoints included overall survival, objective response rate, and safety. Whole exome and RNA sequencing of tumor biopsy tissues were performed for biomarker exploration. Results: Between May, 2019 and December 2022, a total of 46 patients were included in this study. The primary endpoint showed a median progression-free survival of 9.40 months (95% CI: 5.28-13.52), with a 6-month progression-free survival rate of 76.1%. The median overall survival was 16.77 months (95% CI, 14.20-19.33), with an objective response rate of 47.8% and disease control rate of 91.3% per RECIST. In addition, 4.3% and 8.7% of patients achieved complete response of all lesions and intrahepatic target lesions per mRECIST, respectively. The most common treatment-related adverse events were neutropenia, thrombocytopenia, elevated aspartate aminotransferase and alanine aminotransferase level. Furthermore, integrated analysis of genetic, transcriptomic, and immunohistochemistry data revealed that pre-existing immunity (high expression level of immune-related signatures and intra-tumoral CD8+ T cell density) in baseline tumor tissues was associated with superior clinical benefits. However, the evaluation of tumor mutation burden did not show potential predictive value in this triple combination. Conclusion: FOLFOX-HAIC in combination with lenvatinib and PD-1 inhibitor demonstrated a promising antitumor activity with manageable safety profiles in patients with advanced iCCA. Moreover, our study also revealed new perspectives on potential biomarkers for clinical efficacy.

MZF1 promotes tumour progression and resistance to anti-PD-L1 antibody treatment in hepatocellular carcinoma.

Background & Aims: The mechanism underlying resistance to immunotherapy involves engagement of immune checkpoint pathways. The transcriptional and epigenetic processes of checkpoint molecules, however, have not been well investigated. We thus studied whether the transcription factor myeloid zinc finger 1 (MZF1) may promote resistance to immunotherapy in hepatocellular carcinoma (HCC). Methods: Single-cell RNA-sequencing was performed to study the correlation between MZF1 and tumour microenvironment features in six patients with HCC. Combined immunohistochemistry and multi-immunofluorescence analyses were performed for verification. Ectopic expression of MZF1 was used in both orthotopic and genetically engineered hydrodynamic mouse HCC models for in vivo experiments. Proteome analysis, including protein degradation assays, ubiquitination assays, and co-immunoprecipitation assays, revealed the function of MZF1 in immune checkpoint pathways. Results: Single-cell RNA-sequencing suggested an immunosuppressive environment and a strong correlation with the immune checkpoint programmed death ligand 1 (PD-L1) in MZF1-overexpressing tumours. Analyses of 163 HCC samples demonstrated that MZF1 expression in HCC cells is associated with decreased T-cell infiltration. In vivo experiments showed that ectopic MZF1 expression in HCC cells impairs T-cell recruitment, resulting in resistance to immune checkpoint blockade. Mechanistically, MZF1 accelerated PD-L1 ubiquitination by binding to the cyclin-dependent kinase 4 (CDK4) activation site, while a direct bond between CDK4 and MZF1 led to increased MZF1 expression. Conclusions: MZF1 promotes PD-L1 ubiquitination via CDK4 and possibly MZF1. Inhibition of CDK4 can therefore restore PD-L1 expression and may be a potential strategy for combination with anti-PD-L1 antibodies. Impact and implications: Resistance to immune checkpoint blockade with anti-programmed death ligand 1 (PD-L1) antibody therapy is attributed to oncogenic alterations of tumour cells, however, effective countermeasures are yet to be established. Here, we report that the transcription factor myeloid zinc finger 1 (MZF1) can bind to the cyclin-dependent kinase 4 (CDK4) activation site and accelerate PD-L1 ubiquitination. A CDK4 inhibitor therefore enhances anti-PD-L1 antibody efficacy by blocking MZF1 signalling. This indicates a potential benefit of combining CDK4 inhibitors and anti-PD-L1 antibodies for the treatment of advanced HCC.

DNA methylation-activated full-length EMX1 facilitates metastasis through EMX1-EGFR-ERK axis in hepatocellular carcinoma.

Altered DNA methylation is a crucial epigenetic event in hepatocellular carcinoma (HCC) development and progression. Through methylation-transcriptomic analysis, we identified a set of sixty potential DNA methylation-based epidriver genes. In this set of genes, we focused on the hypermethylation of EMX1, which is frequently observed in hepatobiliary tumors. Despite of its frequent occurrence, the function of EMX1 remains largely unknown. By utilizing bisulfite-next-generation sequencing, we have detected EMX1 DNA hypermethylation on the gene body, which is positively correlated with EMX1 mRNA expression. Further analysis revealed that EMX1 mRNA terminal exon splicing in HCC generated two protein isoforms: EMX1 full length (EMX1-FL) and alternative terminal exon splicing isoform (EMX1-X1). Cellular functional assays demonstrated that gain-of-function EMX1-FL, but not EMX1-X1, induced HCC cells migration and invasion while silencing EMX1-FL inhibited HCC cells motility. This result was further validated by in vivo tumor metastasis models. Mechanistically, EMX1-FL bound to EGFR promoter, promoting EGFR transcription and activating EGFR-ERK signaling to trigger tumor metastasis. Therefore, EGFR may be a potential therapeutic target for EMX1-high expression HCC. Our work illuminated the crucial role of gene body hypermethylation-activated EMX1-FL in promoting tumorigenesis and metastasis in HCC. These findings pave the way for targeting the EMX1-EGFR axis in HCC tumorigenicity and metastasis.

Lenvatinib, Toripalimab plus FOLFOX Chemotherapy in Hepatocellular Carcinoma Patients with Extrahepatic Metastasis: A Biomolecular Exploratory, Phase II Trial (LTSC).

PURPOSE: To investigate the efficacy, safety, and biomarkers of systemic chemotherapy with oxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) in combination with lenvatinib and toripalimab as the first-line treatment for advanced hepatocellular carcinoma (HCC) with extrahepatic metastasis. PATIENTS AND METHODS: In this biomolecular exploratory, phase II trial, eligible patients underwent the triple combination therapy of lenvatinib, toripalimab, plus FOLFOX chemotherapy. Primary endpoint was progression-free survival (PFS) rate at 6 months by RECIST v1.1. Single-nucleus RNA sequencing (snRNA-seq) of tumor biopsy samples was performed for exploratory biomarker analyses. RESULTS: Between November 19, 2019, and July 4, 2021, 30 patients were enrolled. The primary endpoint was a 6-month PFS rate of 66.7%, with a median PFS of 9.73 months [95% confidence interval (CI), 2.89-16.58]. The median overall survival (OS) was 14.63 months (95% CI, 11.77-17.50), with an objective response rate of 43.3%. Twenty-four (80.0%) patients exhibited high-risk features, among whom the median OS and PFS were 13.7 months (95% CI, 9.24-18.16) and 8.3 months (95% CI, 3.02-13.58), respectively. The most common adverse events were neutropenia, and increased aspartate aminotransferase and alanine aminotransferase levels. Exploratory analyses of snRNA-seq profiles suggested that patients with higher abundance of tumor-infiltrating immune cells were more likely to benefit from this combination. In addition, two subtypes of hepatocytes (AKR1C2+ and CFHR4+ malignant hepatocytes) were associated with reduced clinical benefits. CONCLUSIONS: FOLFOX chemotherapy in combination with lenvatinib and toripalimab showed promising antitumor activity with manageable toxicities in advanced HCC with extrahepatic metastasis. AKR1C2+ and CFHR4+ hepatocyte subtypes may be predictive biomarkers of resistance to the combination therapy.

Hepatic Arterial Infusion of Oxaliplatin, Fluorouracil, and Leucovorin Versus Sorafenib for Hepatocellular Carcinoma Refractory to Transarterial Chemoembolization: Retrospective Subgroup Analysis of 2 Prospective Trials.

Purpose:Sorafenib is recommended for patients with hepatocellular carcinoma refractory to transarterial chemoembolization but with unsatisfactory overall survival and tumor response rate. Previously published studies showed hepatic arterial infusion chemotherapy of oxaliplatin, fluorouracil, and leucovorin was an effective and safe treatment. The aims of this study were to compare the clinical efficacy and safety of oxaliplatin, fluorouracil, and leucovorin-based hepatic arterial infusion chemotherapy with sorafenib in patients with hepatocellular carcinoma refractory to transarterial chemoembolization. Methods: This was a retrospective subgroup analysis of 2 prospective clinical trials, including 114 patients with hepatocellular carcinoma who were confirmed to be transarterial chemoembolization refractoriness. Of these, 55 patients received hepatic arterial infusion chemotherapy of fluorouracil, and leucovorin (FOLFOX-HAIC group, oxaliplatin 85 or 130 mg/m2, leucovorin 400 mg/m2, fluorouracil bolus 400 mg/m2, and 2400 mg/m2 for 23 or 46 h, every 3 weeks), and 59 patients were treated with sorafenib (sorafenib group, 400 mg sorafenib twice daily). Overall survival, progression-free survival, objective response rate, and treatment-related adverse events were compared between the 2 groups. Results: The FOLFOX-HAIC group showed a longer overall survival (17.1 months [95% confidence interval 13.4-20.8] vs 9.1 months [95% confidence interval 7.5-10.6]; hazard ratio 0.35 [95% confidence interval 0.23-0.53]; P < .001), a higher objective response rate (RECIST: 18 [32.7%] vs 1 [1.7%], P < .001), and a longer progression-free survival (7.6 months [95% confidence interval 5.6-9.6] vs 3.9 months [95% confidence interval 2.3-5.4]; hazard ratio 0.49 [95% confidence interval 0.33-0.72]; P < .001) than the sorafenib group. The safety results suggested that both oxaliplatin, fluorouracil, and leucovorin-based hepatic arterial infusion chemotherapy and sorafenib had acceptable treatment-related toxic effects. No significant difference was observed in the overall occurrence of any grade, grade 3/4, or serious adverse events between the 2 groups. Conclusions: Oxaliplatin, fluorouracil, and leucovorin-based hepatic arterial infusion chemotherapy might be a better choice than sorafenib for patients with hepatocellular carcinoma refractory to transarterial chemoembolization.

One day versus two days of hepatic arterial infusion with oxaliplatin and fluorouracil for patients with unresectable hepatocellular carcinoma.

BACKGROUND: Hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin and 5-fluorouracil was effective in unresectable hepatocellular carcinoma (HCC). The program of FOLFOX-HAIC in HCC was performed for 1 day (HAIC 1d) or 2 days (HAIC 2d). We hereby retrospectively compared the efficacy and safety between these two treatment regimens and explored the predictive power of thymidylate synthase (TYMS), an enzyme involved in the DNA synthesis process and metabolism of fluorouracil. METHODS: This study included patients with a primary diagnosis of unresectable HCC. These patients received HAIC for 1 day or 2 days. The overall survival (OS), progression-free survival (PFS), tumor response, and adverse events were compared. The propensity score matching (PSM) was used to reduce bias. Peripheral blood samples before the treatments were collected and used to measure the concentration of TYMS through enzyme-linked immunosorbent assay (ELISA). ELISA was performed according to the manufacturers' guidelines. RESULTS: We included 368 patients for this study: 248 in the HAIC 1d group and 120 in the HAIC 2d group. There was no significant difference of OS between the two groups (14.5 for HAIC 1d vs 15.3 months for HAIC 2d, p=0.46). Compared with the HAIC 1d group, the HAIC 2d group did not prolong the PFS (7.3 vs 7.5 months, p=0.91) or elevate the tumor response (42.5% vs 39.1%, p=0.53) per RECIST 1.1. In the PSM cohort, the efficacy between the two groups was similar. The total frequencies of grade 3-4 events were higher with the HAIC 2d group than with the HAIC 1d group, especially in the PSM cohort (p=0.043). Additionally, patients with TYMS low level might benefit longer OS from the HAIC 2d group (18.7 vs 13.6 months, p=0.014). CONCLUSIONS: There was not much of a difference in efficacy between the two groups, but the HAIC for 1 day might be safer, which needed further research. The level of TYMS might be the predictive biomarkers.

Lenvatinib, toripalimab plus hepatic arterial infusion chemotherapy in patients with high-risk advanced hepatocellular carcinoma: A biomolecular exploratory, phase II trial.

INTRODUCTION: The combination of lenvatinib, toripalimab and hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) suggested encouraging antitumour activity in our retrospective study. We hereby prospectively establish the efficacy, safety and predictive biomarkers of the combination therapy as a first-line treatment in patients with high-risk advanced hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This phase II, single-centre, single-arm trial enrolled advanced HCC participants with high-risk. Of 51 screened participants, 36 received lenvatinib, toripalimab plus FOLFOX-HAIC. Participants received 21-day treatment cycles of lenvatinib, toripalimab, and FOLFOX-HAIC. The primary end-point was the progression-free survival (PFS) rate per RECIST at six months. RESULTS: Thirty-six participants (86.1% with high-risk features) were enrolled in our study. The primary end-point was met with a PFS rate of 80.6% (95% CI, 64.0%-91.8%) at six months. The median PFS was 10.4 months (95% CI, 5.8-15.0), and the median OS was not reached at the prespecified final analysis and was 17.9 months (95% CI, 14.5-21.3) after follow-up was extended. The ORR per RECIST was 63.9%, and per mRECIST was 66.7%. The median duration of response was 14.4 months (95% CI, 8.9-19.9). The most common adverse events were thrombocytopenia, elevated aspartate aminotransferase, and hypertension, and no treatment-related death was reported. Participants with low levels of both CCL28 and BTC had unsatisfactory prognosis. CONCLUSIONS: Lenvatinib, toripalimab and FOLFOX-HAIC showed safe and encouraging antitumour activity for advanced HCC with high-risk features. The levels of CCL28 and BTC might be the predictive biomarkers for the triple combination therapy.

Hepatic Arterial Infusion Chemotherapy of Oxaliplatin, Fluorouracil, and Leucovorin With or Without Sorafenib as Initial Treatment for Advanced Hepatocellular Carcinoma.

PURPOSE: Our previous study showed that hepatic arterial infusion chemotherapy (HAIC) using oxaliplatin, fluorouracil, and leucovorin (FOLFOX) plus sorafenib provided a significant survival benefit over sorafenib for advanced hepatocellular carcinoma. However, it is unclear whether the survival benefit should be attributed to the synergism between HAIC and sorafenib or just HAIC alone. We aim to compare HAIC using FOLFOX plus sorafenib with HAIC alone in patients with advanced hepatocellular carcinoma. MATERIALS AND METHODS: This was a retrospective study including 225 eligible patients treated with HAIC using FOLFOX (HAIC alone group, n=126, oxaliplatin 85 mg/m², leucovorin 400 mg/m², fluorouracil bolus 400 mg/m² and 2400 mg/m² for 46 hours, every 3 weeks) alone or HAIC plus sorafenib (soraHAIC group, n=99, sorafenib 400 mg twice daily). Survival curves were calculated by the Kaplan-Meier method, and propensity-score matching was used to reduce bias. RESULTS: The soraHAIC group showed a longer overall survival (12.9 [95% CI, 10.4-15.4] vs. 10.5 [95% CI, 9.5-11.5] months, HR=0.71 [95% CI, 0.53-0.96]; P=0.025), a better progression free survival (7.0 [95% CI, 5.3-8.8] vs. 5.3 [95% CI, 3.5-7.1] months, HR=0.76 [95% CI, 0.58-0.99]; P=0.046), and a higher disease control rate (RECIST 1.1: 74.8% vs. 61.1%, P=0.030) than the HAIC alone group. In multivariate analysis, soraHAIC was an independent favor factor for survival. In terms of the grade 3/4 adverse event, hand-foot skin reaction was more frequent in the soraHAIC group than the HAIC alone group. In the propensity-score matched cohorts (93 pairs), the overall survival, the progression free survival and disease control rates in the soraHAIC group were also better than those in the HAIC group (P<0.05). CONCLUSION: HAIC plus sorafenib may improve overall survival and progression free survival compared with HAIC alone as initial treatment for advanced hepatocellular carcinoma.