Anlotinib in the treatment of advanced hepatocellular carcinoma: an open-label phase II study (ALTER-0802 study)

Hepatocellular carcinoma (HCC) ranks the seventh most common and second most fatal malignant tumor worldwide [1]. Half of the prevalence and mortality occurred in China due to the high incidence of Hepatitis B Virus infection [2]. Angiogenesis-based targeted therapy has been standard management for advanced HCC since the approval of sorafenib in 2007. Though there was a limited survival benefit, especially in Chinese patients, with a median time to progression (TTP) of 2.8 months and overall survival (OS) of 6.5 months in a pivotal Oriental trials, sorafenib remained the unique targeted agent in first-line treatment until the approval of lenvatinib in 2019 [3]. Three other agents, regorafenib, carbozanitinib and ramucirumab, were approved for second-line treatment, and progression-free survival (PFS) varied from 2.8 to 5.2 months and OS from 8.5 to 10.6 months [4‐6]. It suggested the importance of continuous anti-angiogenic treatment for HCC.

Several issues on angiogenesis targeted therapy remain clinically relevant for HCC. First of all, resistance occurs inevitably. Though the exact mechanism of resistance remains unclear, it is believed the activation of the Fibroblast Growth Factor Receptor (FGFR) pathway, another angiogenic pathway, plays an important role [7]. Secondly, no predictive biomarkers have been clearly defined for angiogenesis-based therapy. A Japanese study found a relationship between the better efficacy of lenvatinib and a higher level of serum Fibroblast growth factor 19 (FGF19) or a lower level of Angiopoietin 2 (Ang-2) [8]. However, the markers have not been tested in large-scale studies.

The approval of Nivolumab in September 2017 unveiled a new era of immunotherapy for HCC. But it failed to show superiority to sorafenib as a first-line treatment. The embarrassment was terminated by IMbrave 150 study. The combination of Atezolizumab (Programmed Death Ligand-1 [PD-L1] inhibitor) and bevacizumab achieved significant improvements over sorafenib in median OS (19.2 vs. 13.4 months, p = 0.0009), PFS (6.9 vs. 4.3 months, p = 0.0001) and ORR (30% vs. 11%) [9]. While consistent activities are exhibited with other combinations of immune checkpoint inhibitors (ICIs) and different anti-angiogenic Tyrosine Kinase Inhibitors (TKIs), the safety profile has become more clinically concerning. Better tolerated TKIs are sought to be used in combination with PD-1/PD-L1 inhibitors. Overall, it is still vital to develop novel potent targeted agents with favorable safety profiles and abilities to overcome anti-angiogenesis resistance.

Anlotinib is a novel multi-targeting TKI, with substantial inhibitory activity against Vascular Endothelial Growth Factor Receptor (VEGFR) 1–3, Platelet-derived Growth Factor Receptor (PDGFR) α/β, FGF Receptor 1–4, and c-kit [10]. In vitro studies, anlotinib blocked VEGFR2 with an extremely low half-maximal inhibitory concentration (IC50) value of 0.2 nM [11]. Anlotinib downregulated phosphorylation of FGFR1 at an inhibition rate of 45.0% (p-FGFR1/FGFR1) at 1 μM, and showed an IC50 value of 25 nM in AN3Ca cells overexpressing mutant FGFR2 [11, 12]. Anlotinib has been approved for the treatment of non-small cell lung cancer, small cell lung cancer and soft tissue sarcoma in China. Due to its potent inhibitory activity and additional activity against FGFR2, anlotinib was believed to have favorable antitumor activity as first- or second-line treatment in HCC. In vitro studies, anlotinib significantly inhibited the proliferation of HCC cells and prompted apoptosis. Subsequent animal experiments also illustrated that anlotinib reduced HCC progression [13]. To clarify the clinical efficacy and safety of anlotinib in first- and second-line treatment for advanced HCC, we conducted this pilot phase II study (NCT02809534) since October 2016, when sorafenib stood as the unique available targeted agent worldwide. We also analyzed the plasma level of some concerned cytokines with an attempt to identify the predictive markers.

In this study, anlotinib showed promising antitumor properties against HCC as a first-line treatment. In Cohort 1, anlotinib achieved a median TTP of 5.9 months and a 12-week PFS rate of 80.8%. These results seemed numerically better than sorafenib in Chinese patients, that the median TTP was 2.8 and 3.7 months in the Oriental and REFLECT studies, respectively [3, 16]. So far, lenvatinib has shown the longest median TTP of 7.4 months in first-line TKI treatment [16]. In IMbrave 150 trial, the ORR of atezolizumab combined with bevacizumab was 30%, the median PFS and OS were 6.9 months and 19.2 months, respectively. In Chinese cohort, the updated median OS of combination therapy achieved 24 months [9]. Whether there was a numerical difference between monotherapy, such as anlotinib or lenvatinib, and combination therapy, the difference should be validated in the future.

As expected, anlotinib appeared to have similar activity in HCC refractory to anti-angiogenic TKIs, with a median TTP of 4.6 months and a PFS rate at 12 weeks of 72.5%. In previous studies, anti-angiogenic-based second-line treatments showed a median PFS of 2.8, 3.1 and 5.2 months, a median OS of 8.5, 10.6 and 10.2 months with ramucirumab, regorafenib and cabozantinib, respectively. ICIs in the second-line setting showed a PFS of 2.3 to 4.9 months. The median OS of 18.0 months in Cohort 2 was even more encouraging. The broad inhibition of FGFR1-4 might be one of the underlying mechanisms of being active in HCC refractory to anti-angiogenic TKIs [17]. The difference between with or without subsequent TKI treatment was enormous, which further indicated that continuous anti-angiogenic therapies could bring more survival benefit for patients with advanced HCC. Based on the rich blood perfusion and continuous inhibition of angiogenesis strategy of treatment in HCC, the anti-angiogenic tyrosine kinase inhibitors (TKIs) would be chosen as second-line regimens, even under the circumstance of first-line anti-angiogenic agents combined with ICIs therapy.

We found patients with lower baseline plasma CXCL1 levels achieved significantly longer TTP. Furthermore, the level of CXCL1 increased significantly as the disease progressed in Cohort 2. CXCL1 is involved in the induction of neutrophils migration, angiogenesis, arteriogenesis, inflammation, wound healing and tumorigenesis. It was demonstrated that CXCL1, secreted from tumor cells, induced migration of vascular endothelial cells and angiogenesis in vitro. In vivo studies indicated that the tumor angiogenesis could be reduced by CXCL1 pathway blocking [18]. CXCL1 pathway might be involved in sorafenib resistance. In sorafenib-treated osteosarcoma and ovarian cancer, the expression of CXCL1 increased significantly [19, 20]. Blockade of CXCR2, the receptor of CXCL1, elevated the efficacy of sorafenib and delayed resistance [20]. The microRNA 30A and microRNA 200A, could regulate the expression of CXCL1, was observed to be related to the efficacy of regorafenib. [21‐23]. Overall, we postulated that CXCL1 might be a critical angiogenic promotor and play an important role in both primary and secondary resistance to VEGF/VEGFR inhibitors in HCC. Those microRNAs related to CXCL1 expression might also be predictors for anlotinib, which could be verified in future studies.

Anti-angiogenesis of anlotinib could contribute to the regulation of tumor microenvironment and inflammation in tumors [24, 25]. In our study, the concentration of plasma VEGFA and CCL11 were significantly increased at PD, and level of PD-L2 and CCL5 were significantly decreased. The impact of these changes needs to be further illustrated.

Anlotinib has been investigated in a variety of malignant tumors [26‐29]. In this study, there were no unexpected AEs. The most common grade 3 or 4 AEs of anlotinib included hypertension (8%), diarrhea (8%) and hand-foot syndrome (6%). In the REFLECT study, the most common grade 3 or 4 AEs of lenvatinib were hypertension (23%), weight loss (8%), proteinuria (6%), thrombocytopenia (5%), and liver dysfunction (5%) [16]. In the RESORCE study of regorafenib, the most common grade 3 or 4 AEs were hypertension (15%), hand-foot syndrome (13%), elevated AST (10%), elevated bilirubin (10%), and fatigue (9%) [4]. Thus, the incidence of AEs in anlotinib seemed lower than other standard TKIs in hepatocellular carcinoma. All grade 3 or 4 AEs occurred in less than 10% of patients. The incidence of grade 3 or 4 hypertension in anlotinib seemed lower than that in lenvatinib and regorafenib. Besides, the safety profile was similar between anlotinib and other TKIs. In a randomized phase II trial of anlotinib in comparison with sunitinib as first-line treatment in renal cell carcinoma, grade 3 or 4 AEs in anlotinib were significantly less frequent than that in sunitinib (28.9% versus 55.8%, p < 0.01), especially in terms of thrombocytopenia and neutropenia [29].

Taken the safety profile together, anlotinib could be one of the potential partners for combination therapy with ICIs as first-line treatment in hepatocellular carcinoma. Exploratory trials of anlotinib with ICIs have been conducted already. A phase II trial (NCT04172571) aimed to evaluate the safety and efficacy of first-line anlotinib plus penpulimab, an IgG1 anti-PD-1 antibody, in which the preliminary ORR was 31% with acceptable toxicity [30]. We had begun another phase II trial (NCT03825705) to assess the safety and efficacy of anlotinib combined with TQB2450, an IgG1 anti-PD-L1 antibody, and had observed preliminary efficacy (unpublished).

This was an open-label phase II clinical study. The sample size of the two cohorts was limited. Only the preliminary efficacy and safety data of HCC treatment could be provided, which still needed to be further verified by expanded clinical research. More clinical data were needed to verify the predictive role of CXCL1. Some patients in both the cohorts received ICIs, which interfered with the interpretation of OS results.

Anlotinib showed promising and similar activities both in the first- and second-line treatment of advanced HCC. Anlotinib was well tolerated and is expected to be a favorable partner of ICIs. The role of anlotinib in advanced HCC warrants further validation in future clinical trials.