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IMPORTANCE: Whether patients with Child-Pugh class B (CP-B) cancer with unresectable hepatocellular carcinoma (uHCC) benefit from active anticancer treatment vs best supportive care (BSC) is debated. OBJECTIVE: To evaluate the association of immune checkpoint inhibitor (ICI)–based therapies vs BSC with overall survival (OS) of patients with uHCC and CP-B liver dysfunction. DESIGN, SETTING, AND PARTICIPANTS: This retrospective, multicenter, international clinical case series examined data of patients with CP-B with uHCC who were receiving first-line ICI-based regimens from September 2017 to December 2022 whose data were extracted from an international consortium and compared with a cohort of patients with CP-B receiving BSC. Patients were treated in tertiary care centers across Europe, US, and Asia in routine clinical practice. After applying the inclusion criteria, 187 and 156 patients were left in the ICI and BSC groups, respectively. The propensity score was calculated for the following variables: age, alpha-fetoprotein levels, Child-Pugh score, extrahepatic spread, portal vein tumor thrombosis, cirrhosis, ascites, and baseline Eastern Cooperative Oncology Group performance status. EXPOSURES: Patients in the ICI group received first-line systemic therapy with either atezolizumab plus bevacizumab (A+B) (n = 141) or nivolumab (n = 46). MAIN OUTCOMES AND MEASURES: OS in the inverse probability of treatment weighting (IPTW) populations was the main outcome, and it was estimated with Kaplan-Meier method; univariable Cox regression test was used to make comparisons between the 2 groups. RESULTS: The median age was 66 (IQR, 61-72) and 73 (IQR, 66-81) years in the ICI (33 women [18%]) and BSC groups (41 women [26%]), respectively. In the IPTW populations, median OS was significantly longer in the ICI group (7.50 months; 95% CI, 5.62-11.15) compared with BSC (4.04 months; 95% CI, 3.03-5.03; hazard ratio, 0.59; 95% CI, 0.43-0.80; P < .001). Multivariable analysis confirmed that ICI exposure was associated with a reduction of approximately 50% in the risk of death (hazard ratio, 0.55; 95% CI, 0.35-0.86; P < .001), and the presence of portal vein tumor thrombosis, an Eastern Cooperative Oncology Group performance score of greater than 1, and alpha-fetoprotein levels of 400 ng/mL or greater were associated with increased risk of death. CONCLUSIONS AND RELEVANCE: The results of this case series provide comparative evidence of improved survival in association with ICI treatment compared with BSC in patients with uHCC with CP-B liver dysfunction.

There is an unmet need for effective therapies in pretreated advanced biliary tract cancer. We aimed to evaluate the efficacy of nanoliposomal irinotecan and fluorouracil plus leucovorin compared with fluorouracil plus leucovorin as second-line treatment for biliary tract cancer.

Autoimmune liver diseases (AILD) are rare causes hepatocellular carcinoma (HCC), and data on the efficacy and tolerability of anti-tumor therapies are scarce. This pan-European study aimed to assess outcomes in AILD-HCC patients treated with tyrosine kinase inhibitors (TKIs) or transarterial chemoembolization (TACE) compared with patients with more common HCC etiologies, including viral, alcoholic or non-alcoholic fatty liver disease.

Background Lenvatinib is approved as first-line treatment for patients with advanced hepatocellular carcinoma (HCC). The efficacy of lenvatinib in Caucasian real-world patients is insufficiently defined. The purpose of this study was to evaluate the efficacy of lenvatinib in a multi-center cohort (ELEVATOR) from Germany and Austria. Methods A retrospective data analysis of 205 patients treated with first-line systemic lenvatinib at 14 different sites was conducted. Overall survival, progression free survival, overall response rate and adverse event rates were assessed and analyzed. Results Patients receiving lenvatinib in the real-world setting reached a median overall survival of 12.8 months, which was comparable to the results reported from the REFLECT study. Median overall survival (mOS) and progression free survival (mPFS) was superior in those patients who met the inclusion criteria of the REFLECT study compared to patients who failed to meet the inclusion criteria (mOS 15.6 vs 10.2 months, HR 0.55, 95% CI 0.38-0.81, p=0.002; mPFS 8.1 vs 4.8 months HR 0.65, 95% CI 0.46-0.91, p=0.0015). For patients with an impaired liver function according to the Albumin-Bilirubin (ALBI) grade, or reduced ECOG performance status ≥2, survival was significantly shorter compared to patients with sustained liver function (ALBI grade 1) and good performance status (ECOG performance status 0), respectively (HR 1.69, 95% CI 1.07-2.66, p=0.023; HR 2.25, 95% CI 1.19-4.23, p=0.012). Additionally, macrovascular invasion (HR 1.55, 95% CI 1.02-2.37, p=0.041) and an AFP ≥200 ng/mL (HR 1.56, 95% CI 1.03-2.34, p=0.034) were confirmed as independent negative prognostic factors in our cohort of patients with advanced HCC. Conclusion Overall, our data confirm the efficacy of lenvatinib as first-line treatment and did not reveal new or unexpected side effects in a large retrospective Caucasian real-world cohort, supporting the use of lenvatinib as meaningful alternative for patients that cannot be treated with IO-based combinations in first-line HCC.

Background and Aims:The multikinase inhibitor cabozantinib has been approved for hepatocellular carcinoma (HCC) previously treated with sorafenib. We report safety and efficacy data of an international, multicenter, real-life cohort of patients with advanced HCC treated with cabozantinib. Methods:Patients with HCC who were treated with cabozantinib were retrospectively identified across 11 centers in Austria, Switzerland, and Germany. Patients’ characteristics, adverse events, duration of treatment and overall survival (OS) data were analyzed until April 1, 2020. Results:Eighty-eight patients from 11 centers were included. The predominant underlying liver diseases were NAFLD/NASH in 26 (30%) and hepatitis C infection in 21 (24%) patients. Seventy-eight patients (89%) were classified as Barcelona clinic liver cancer (BCLC) stage C. Sixty patients (68%) were Child-Pugh A, whereas 22 (25%) were Child-Pugh B, respectively. Cabozantinib was used as systemic second- and third-line or later treatment in 41 (47%) and 46 (52%) patients, respectively. The following best responses under cabozantinib were documented: partial response in 6 (7%), stable disease in 28 (32%), and progressive disease in 28 (32%) patients, respectively. Fifty-two patients (59%) died during follow-up. The median OS from start of cabozantinib treatment was 7.0 months in the entire cohort and 9.7 months in Child-Pugh A patients, while Child-Pugh B patients had a median OS of 3.4 months, respectively. Thirty-seven (42%) patients fulfilled the CELESTIAL inclusion and exclusion criteria, showing a median OS of 11.1 months. Most common adverse events were fatigue (15.6%) and diarrhea (15.6%). Conclusion:Cabozantinib treatment was effective, safe, and feasible in patients with advanced HCC in patients with compensated cirrhosis. Patients in the real-life setting had more advanced liver disease – in which 25% of patients were Child-Pugh B. However, OS in patients with Child-Pugh A cirrhosis was similar to that reported in the phase 3 trial (CELESTIAL).

Introduction:The number of efficacious systemic agents for advanced hepatocellular carcinoma (HCC) has rapidly increased over the past 3 years. However, guidance for optimal sequential systemic treatment in patients with advanced disease and experience with outcome and safety profiles are lacking. Objective:We aimed to assess efficacy and tolerability of sequential systemic therapy of advanced HCC. Methods:Our single-center study prospectively followed 14 patients who received multiple, sequential systemic therapies after progression or intolerance to sorafenib. Endpoints were overall and progression-free survival (OS, PFS), objective response rate (ORR), and treatment-emergent adverse events (TEAE). Results:Patients had well-compensated liver function and good performance status at start of each systemic therapy. Agents included sorafenib (n= 14), regorafenib (n= 10), immunotherapy with nivolumab or pembrolizumab (n= 10), lenvatinib (n= 3), ramucirumab (n= 2), and others, with a median of 3 lines of systemic therapy per patient. Median OS was 37.4 months from initiation of first-line therapy with sorafenib. PFS and ORR for sorafenib, regorafenib, and immunotherapy were 6.6, 5.3, and 6.6 months, and 15.4, 11.1, and 22.2%, respectively. TEAE were frequent (46–80%), but mostly manageable during tyrosine kinase inhibitor therapy and without the need for termination in most patients. However, TEAE due to immunotherapy (60%) led to cessation of treatment in 40% of the patients. Conclusions:Sequential systemic therapy is able to prolong median OS in selected patients with advanced HCC to more than 3 years. TEAE are frequent, but manageable, and the quality of adverse events depends on the respective agent. Further investigation of potential predictive biomarkers for treatment allocation is needed.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+T cells or TNF neutralization, suggesting that CD8+T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Background:Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and the second most lethal malignancy worldwide. In the Western world, HCC predominantly develops in patients with liver cirrhosis. Therefore, application of locoregional interventions and systemic agents should be based on an interdisciplinary evaluation, most importantly, taking the functional liver reserve into account. This review summarizes current treatment lines and novel strategies in the management of HCC. For the most part, randomized controlled trials and large meta-analyses are reported, with an emphasis on systemic therapies. Summary:In patients with limited hepatic disease and sufficient liver function, resection and local ablation are the most frequently employed curative locoregional therapies. Due to recurrence rates of up to 70% within 5 years and in patients with compromised liver function not amenable to these local modalities, liver transplantation remains superior in terms of tumor control and long-term survival. However, its applicability is limited because of the increasing gap between available donor organs and patients on the waiting list. Transarterial chemoembolization is commonly employed to bridge patients to transplantation and also serves as standard of care for patients not suitable for other local therapies. Recently, various phase 3 trials have reported a clinical benefit for the tyrosine kinase inhibitors lenvatinib, regorafenib, and cabozantinib in HCC. In addition, ramucirumab, an angiostatic antibody, also improves survival in second-line systemic therapy. This opens new avenues in the sequential application of treatment lines, and thus early response assessment is necessary to fully utilize the clinical impact of locoregional therapies and systemic therapies and to shift patients to further treatment lines before hepatic deterioration. Key Messages:Clinical decision-making in hepatocellular carcinoma is based on an interdisciplinary evaluation. Liver transplantation should always be considered as long-term curative treatment option, especially in T2 patients. In palliative treatment, early response assessment is required to advance patients to the next treatment line before decompensation.

Cytotoxic chemotherapy is generally ineffective in patients with hepatocellular carcinoma. We assessed the intravenous perfusion of doxorubicin-loaded nanoparticles in patients with hepatocellular carcinoma in whom previous sorafenib therapy had failed.

Background Sorafenib is the recommended treatment for advanced hepatocellular carcinoma (HCC), but transarterial chemoembolization (TACE) is performed in individual cases with limited extrahepatic spread. The aim of this study was to compare the outcome of patients with HCC and extrahepatic disease (EHD) treated with sorafenib and TACE.Methods A total of 172 patients with HCC and EHD treated with sorafenib (n= 98) or TACE (n= 74) at three German referral centers (Hannover, Mainz and Hamburg) were included in this study. In order to reduce selection bias, patients were matched for significant demographic differences using a propensity score analysis.Results Patients with liver cirrhosis, higher extrahepatic tumor burden and/or infiltration of adjacent organs/structures were significantly more often treated with sorafenib. Median overall survival (OS) was similar for sorafenib- and TACE-treated patients (7 versus 8 months, p= 0.312). In a propensity score analysis matched for demographic differences, median OS remained similar with 4 versus 8 months for sorafenib versus TACE (p= 0.613).Conclusion Treatment with TACE is not inferior to treatment with sorafenib in patients with limited EHD of HCC. TACE represents an effective therapeutic option in selected patients with EHD.

Sorafenib is the recommended treatment for advanced hepatocellular carcinoma (HCC), but transarterial chemoembolization (TACE) is performed in individual cases with limited extrahepatic spread. The aim of this study was to compare the outcome of patients with HCC and extrahepatic disease (EHD) treated with sorafenib and TACE. A total of 172 patients with HCC and EHD treated with sorafenib (n?=?98) or TACE (n?=?74) at three German referral centers (Hannover, Mainz and Hamburg) were included in this study. In order to reduce selection bias, patients were matched for significant demographic differences using a propensity score analysis. Patients with liver cirrhosis, higher extrahepatic tumor burden and/or infiltration of adjacent organs/structures were significantly more often treated with sorafenib. Median overall survival (OS) was similar for sorafenib- and TACE-treated patients (7 versus 8 months, p?=?0.312). In a propensity score analysis matched for demographic differences, median OS remained similar with 4 versus 8 months for sorafenib versus TACE (p?=?0.613). Treatment with TACE is not inferior to treatment with sorafenib in patients with limited EHD of HCC. TACE represents an effective therapeutic option in selected patients with EHD.

The liver has a remarkable capability to restore its functional capacity following liver injury. According to the current paradigm, differentiated and usually quiescent hepatocytes are the primary cell type responsible for liver repair. As reserve compartment, bipotent hepatic progenitor cells are activated, especially if extensive loss or damage of hepatocytes with impaired replication occurs, e.g. in cirrhotic liver tissue. Recently, animal studies have suggested that liver regeneration following partial hepatectomy is associated with telomerase activation. Telomerase, a ribonucleoprotein with reverse transcriptase activity, plays a pivotal role in maintaining telomere length and chromosomal stability in proliferating cells. In cells lacking telomerase activity, replication-associated telomere shortening limits the replicative lifespan. Therefore, in the context of liver regeneration, telomerase activation might be a cellular mechanism to confer an extended lifespan to replicating hepatocytes and hepatic progenitor cells. On the other hand, high levels of telomerase activity are a hallmark of cancer, including hepatocellular carcinoma. Moreover, recent data indicate that telomerase activation may be an early event in hepatocarcinogenesis. At present, it is unclear, whether telomerase activation preserves the non-malignant phenotype and replicative longevity of liver cells or constitutes an early alteration obligatory for an unlimited proliferation and malignant transformation.

Embryonic stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells. We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro. Various combinations of culture media, growth and differentiation factors, and substratum precoatings were evaluated, and it was determined that a combination of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone, and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage. Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver. The optimal culture condition also induced albumin gene expression in differentiated human ES cells 1% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocyto-chemistry. In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels 10-fold higher than with other hepatocyte differentiation media. In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage. This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.

Currently, different techniques to expand human hepatocytes in vitro are being investigated to generate enough cells for liver-directed cell therapies. However, based on observations in fibroblasts and other cell types, telomere attrition limits the proliferative capacity of normal somatic cells. Therefore, we explored whether telomere-dependent replicative aging restricts the in vitro proliferation of human hepatocytes. Subpopulations of cells isolated from a neonatal liver and characterized as hepatocyte derived by RT-PCR and flow cytometry started to proliferate 5–7 days after plating and were termed proliferating human hepatocytes (PHH). Following retroviral-mediated transduction of the catalytic telomerase subunit, telomerase reverse transcriptase (hTERT), telomerase activity increased from almost undetectable levels to levels as high as in HepG2 and other telomerase-positive cell lines. As expected, untransduced PHH progressively lost telomeric repeats and arrested after 30–35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerase-reconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.

Disease progression in CML is associated with accelerated telomere shortening caused by increased turnover of Bcr-Abl positive cells. We have recently proposed a model in which continuous telomere shortening is correlated with increasing genetic instability in Bcr-Abl-positive cells thus facilitating the acquisition of secondary genetic abnormalities and, as a consequence clonal succession and eventually clinical progression of the disease. Based on this hypothesis, telomerase upregulation in late stage disease is a prerequisite for prevention of replication-induced senescence in Bcr-Abl positive cells. As a consequence, treatment of CML with Telomerase inhibitors (TI) represents an attractive strategy aiming at the potential eradication of cycling CML stem cells. Therefore, we have exploited both pharmacological (small molecule inhibitor) and genetic strategies (dominant negative hTERT mutants, DN-hTERT) of telomerase inhibition in CML cells in vitro We first treated K562 cells with the pharmacological telomerase inhibitor BIBR1532 in vitro. After around 400 population doublings (PD), no differences in growth kinetics nor signs of senescence or apoptosis were observed in BIBR1532 treated cells despite of significant telomere shortening (22 base pairs (bp) per PD) compared to control cells. Furthermore, neither significant differences in mRNA expression of telomere/telomerase-associated proteins, nor accumulation of double strand breaks (DSBs) under irradiation was observed in treated cells with short telomeres as opposed to untreated control cells. The very slow shortening rate of 22bp/PD plus the lack of stigmata pointing to induction of senescence in K562 cells lead to the assumption that telomerase activity is not complete inhibited by the compound. In order to verify the potency of telomerase directed treatments in CML, we therefore expressed DN-hTERT in K562 cells. Integration of DN-hTERT led to a significant decrease in telomerase activity (measured by RQ-TRAP). Furthermore, DN-hTERT expressing cells underwent accelerated telomere shortening at a substantially higher rate (>100 bp/PD) from 15kb to around 4kb within 110 days of culture. In contrast to BIBR1532-treated cells, DN-hTERT expressing K562 cells slowed down growth kinetic in comparison to control cells after 80 days of culture. By using Annexin 5 staining, 25% of apoptotic cells could be detected in cells with critically short telomeres as compared to control cells (<3%). Finally, a significantly increased accumulation of double strand breaks (DSBs) detected by gammaH2AX foci after exposure to irradiation was observed in DN-hTERT K562 cells as compared to control cells pointing to an impaired DNA repair machinery in Bcr-Abl positive cells with disrupted telomere maintenance. In summary, the data suggest that pharmacological telomerase inhibition by BIBR1532 is insufficient to induce telomere-mediated senescence in Bcr-Abl-positive cells. However, accelerated telomere shortening, slowing down of growth kinetics, elevated apoptosis and increased radiosensitivity induced by expression of DN-hTERT indicate a therapeutic potential for telomerase-directed treatment strategies in CML.

Disease progression in CML is associated with accelerated telomere shortening caused by increased turnover of Bcr-Abl positive cells. We have recently proposed a model in which continuous telomere shortening is correlated with increasing genetic instability in Bcr-Abl-positive cells thus facilitating the acquisition of secondary genetic abnormalities and, as a consequence clonal succession and eventually clinical progression of the disease. Based on this hypothesis, telomerase upregulation in late stage disease is a prerequisite for prevention of replication-induced senescence in Bcr-Abl positive cells. As a consequence, treatment of CML with Telomerase inhibitors (TI) represents an attractive strategy aiming at the potential eradication of cycling CML stem cells. Therefore, we have exploited both pharmacological (small molecule inhibitor) and genetic strategies (dominant negative hTERT mutants, DN-hTERT) of telomerase inhibition in CML cells in vitro We first treated K562 cells with the pharmacological telomerase inhibitor BIBR1532 in vitro. After around 400 population doublings (PD), no differences in growth kinetics nor signs of senescence or apoptosis were observed in BIBR1532 treated cells despite of significant telomere shortening (22 base pairs (bp) per PD) compared to control cells. Furthermore, neither significant differences in mRNA expression of telomere/telomerase-associated proteins, nor accumulation of double strand breaks (DSBs) under irradiation was observed in treated cells with short telomeres as opposed to untreated control cells. The very slow shortening rate of 22bp/PD plus the lack of stigmata pointing to induction of senescence in K562 cells lead to the assumption that telomerase activity is not complete inhibited by the compound. In order to verify the potency of telomerase directed treatments in CML, we therefore expressed DN-hTERT in K562 cells. Integration of DN-hTERT led to a significant decrease in telomerase activity (measured by RQ-TRAP). Furthermore, DN-hTERT expressing cells underwent accelerated telomere shortening at a substantially higher rate (>100 bp/PD) from 15kb to around 4kb within 110 days of culture. In contrast to BIBR1532-treated cells, DN-hTERT expressing K562 cells slowed down growth kinetic in comparison to control cells after 80 days of culture. By using Annexin 5 staining, 25% of apoptotic cells could be detected in cells with critically short telomeres as compared to control cells (<3%). Finally, a significantly increased accumulation of double strand breaks (DSBs) detected by gammaH2AX foci after exposure to irradiation was observed in DN-hTERT K562 cells as compared to control cells pointing to an impaired DNA repair machinery in Bcr-Abl positive cells with disrupted telomere maintenance. In summary, the data suggest that pharmacological telomerase inhibition by BIBR1532 is insufficient to induce telomere-mediated senescence in Bcr-Abl-positive cells. However, accelerated telomere shortening, slowing down of growth kinetics, elevated apoptosis and increased radiosensitivity induced by expression of DN-hTERT indicate a therapeutic potential for telomerase-directed treatment strategies in CML.