Sorafenib is an oral drug that suppresses tumor growth by inhibiting the serine-threonine kinases C-Raf, wild-type B-Raf, and mutant (V600E) B-Raf, all of which are components of the Raf/MEK/ERK pathway (mitogen-activated proteins kinase pathway). This pathway acts downstream of the vascular endothelial growth factor receptor (VEGFR), the platelet-derived growth factor receptor (PDGFR), and the epidermal growth factor receptor. It also exerts anti-tumor effects by suppressing neovascularization. It achieves tumor neovascularization by inhibiting the tyrosine kinases VEGFR1, VEGFR2, VEGFR3, PDGFRβ, RET, and fms-related tyrosine kinase 3 (FLT-3). Two large-scale pivotal trials (the SHARP and Asia-Pacific trials) of sorafenib reported significant prolongation of overall survival (OS) compared with placebo[31,32]; indeed, sorafenib is now the standard therapeutic agent for advanced HCC. However, its ability to shrink tumors is weak and its systemic toxicity is relatively high. Therefore, novel molecular targeted agents with more potency or similar effects, but less toxicity, have been unmet need.

Although eight clinical trials with various agents/modalities comparing with sorafenib conducted in the last decade has shown negative outcomes, the results of the REFLECT trial with use of lenvatinib met its primary endpoint of non-inferiority of prolonging OS compared with sorafenib. Lenvatinib is an oral kinase inhibitor that selectively inhibits receptor tyrosine kinases involved in neovascularization and progression to high malignancy grade tumors and a poor prognosis; targeted kinases include VEGFR1, VEGFR2, VEGFR3, fibroblast growth factor receptor (FGFR) 1, FGFR2, FGFR3, FGFR4, PDGFRα, KIT, and RET. In particular, strong inhibition of FGFR4 is considered important for preventing aggressive growth or progression to a higher malignancy grade of HCC. The drug also suppresses invasion and metastasis. A single-arm phase II study of lenvatinib as a treatment for advanced HCC reported a time to progression (TTP) of 7.4 mo and an OS of 18.7 mo, which are very favorable[33]. Subsequently, a phase III study comparing sorafenib with lenvatinib, the REFLECT trial, was conducted[34].

The REFLECT trial was a global phase III study to show the non-inferiority of lenvatinib to sorafenib, in which patients with unresectable HCC, not previously treated with systemic chemotherapy, were allocated randomly to the lenvatinib or sorafenib arms at a 1:1 ratio. Stratification factors were Asian/non-Asian, vascular invasion and/or extrahepatic spread (presence or absence), Eastern Cooperative Oncology Group performance status 0 or 1, and body weight < 60 kg or ≥ 60 kg. Administration was continued until disease progression or development of a non-tolerable adverse event. The primary endpoint was verification of non-inferiority in terms of OS; the non-inferiority margin of OS was set at 1.08, which is very strict. PFS, TTP, objective response rate (ORR), and safety were evaluated as secondary endpoints.

Of the 954 patients registered, 478 and 476 were allocated to the lenvatinib and sorafenib groups, respectively. Overall, 67% of patients in the lenvatinib group were from the Asia-Pacific region and 33% were from western countries. Of these, 32% weighed less than 60 kg and 68% weighed 60 kg or more in lenvatinib arm. Vascular invasion and/or extrahepatic spread were noted in 69%, and BCLC stage C HCCs accounted for 78% in the lenvatinib arm. Regarding the cause of disease, HCC due to hepatitis C accounted for 19% in the lenvatinib arm and 27% in the sorafenib arm, suggesting an advantageous imbalance toward sorafenib[34]. In contrast, HCC due to hepatitis B accounted for 53% of cases in the lenvatinib arm and for 48% of cases in the sorafenib arm. In the lenvatinib arm, 46% of patients had an alpha fetoprotein (AFP) level exceeding 200 ng/mL vs 39% in the sorafenib group, also indicating an advantageous imbalance toward sorafenib.

The primary endpoint OS in the lenvatinib and sorafenib arms was 13.6 and 12.3 mo, respectively, and the hazard ratio (HR) was 0.92 (0.79-1.06); this was lower than the non-inferiority margin with a specified upper limit of a 95% confidence interval (CI) of 1.08. These data suggest that lenvatinib was not inferior to sorafenib in terms of OS[34]. In addition, the PFS reported by institutional investigators according to the modified Response Evaluation Criteria in Solid Tumors (modified RECIST) criteria was 7.3 and 3.6 mo for the lenvatinib and sorafenib arms, respectively. TTP was 7.4 and 3.7 mo, respectively, and the ORR was 40.6% and 12.6% according to the results of Masked Independent Review Committee, respectively. Thus, lenvatinib had significantly more favorable anti-tumor effects than sorafenib (Table 3)[34]. Evaluation by Masked Independent Review Committee based on RECIST1.1 also confirmed that lenvatinib had favorable anti-tumor effects with regard to PFS, TTP, and ORR as well. Furthermore, when analysis was confined to intermediate stage HCC, the PFS in the lenvatinib arm was 9.1 mo, which is fairly long, and the ORR of BCLC B-stage HCC in the lenvatinib arm for the Japanese population was 61.3%, which is extremely high[35].

Because AFP was not included as a stratification factor, more patients with an AFP level exceeding 200 ng/mL were enrolled in the lenvatinib group. When this was corrected by analysis of covariance, the lenvatinib arm showed a superior OS (HR = 0.856; 95%CI = 0.736-0.995; nominal P-value = 0.0342)[34]. Based on these findings, if AFP was included as a stratified factor, the study would likely have confirmed the superiority of lenvatinib over sorafenib[36,37]. The probability that lenvatinib was superior to sorafenib after adjustment for AFP is as high as 0.743[38].

Sub-analysis of OS revealed that the OS-prolonging effects of lenvatinib were superior to those of sorafenib in almost all subsets. It should be noted that specifically that in the group with body weight < 60 kg, the OS benefits of lenvatinib were superior to those of sorafenib at a dose of 8 mg, and the HR was more favorable than that in the group weighing ≥ 60 kg or higher at a dose of 12 mg (HR, 0.85 vs 0.95, respectively). This suggests that weight-based dosing is successful[37,39]. In addition, the HR in the group with a baseline AFP of ≥ 200 ng/mL was 0.78 (95%CI, 0.63-0.98), confirming that lenvatinib has favorable OS benefits even in a group with a high AFP level and a poor prognosis. The treatment durations for the lenvatinib and sorafenib arms were 5.7 and 3.7 mo, respectively; therefore, lenvatinib can be given orally for a longer duration and the frequency of subjective AEs such as hand foot skin reaction and diarrhea is lower, indicating superior tolerability.

Based on the above findings, lenvatinib is not inferior to sorafenib in terms of OS; indeed, lenvatinib showed statistically significant and clinically meaningful improvement in all secondary endpoints (PFS, TTP, and ORR), confirming its efficacy as a first-line agent for patients with unresectable HCC. Regarding the AEs, hypertension, proteinuria and hypothyroidism were more frequently observed in lenvatinib arm although hand-foot skin reaction, diarrhea and alopecia were less frequently observed in lenvatinib arm as compared with sorafenib arm[34]. This AE profile suggests that AEs in lenvatinib arm are more favorable and tolerable than sorafenib since most of AEs observed in lenvatinib arm are asymptomatic and can be manageable by medication (hypertension, hypothyroidism) or by dose reduction (proteinuria). Based on these results, lenvatinib has been approved to treat HCC on March 23, 2018 in Japan. It was also approved in HCC by the Food and Drug Administration on August 16, 2018, in Europe on August 23, 2018, in South Korea on September 4, 2018, in China on August 29, 2018 and in Taiwan on November 28, 2018.

Regorafenib is an oral kinase inhibitor. It targets protein kinases such as VEGFR1, VEGFR2, VEGFR3, TIE2, PDGFRβ, FGFR, KIT, RET, RAF-1, and BRAF[40]. Because it is synthesized simply by binding fluorine to sorafenib, it has almost the same molecular structure as sorafenib and a similar, but stronger toxicity profile. Therefore, unlike other drugs, a placebo-controlled phase III study (RESORCE trial) was performed that included only patients who progressed under sorafenib treatment and those intolerant to sorafenib patients were excluded. The primary endpoint, OS, in the regorafenib arm was 10.6 mo and that in the placebo arm was 7.8 mo, which is a significant improvement[41]. PFS and TTP were also significantly more favorable (Table 4). This is the first drug for which efficacy was proved as a second-line systemic agent who progressed on sorafenib.

Regarding the AE profiles of regorafenib is as follows; grade 3/4 drug-related treatment-emergent AEs include hand-foot skin reaction in 13%, fatigue in 6%, hypertension in 13% and diarrhea in 2%[41]. However, most these AEs were manageable by medication or dose reduction/interruption. Based on these results, its use as a second-line agent for HCC in Japan was approved in May 2017. However, the drug is unsuitable for patients with sorafenib intolerance; this means that there is an unmet need for a second-line treatment in this group. However, this was soon solved by successful trials of cabozantinib and ramucirumab in 2018 as mentioned later.

The RESORCE trial was successful for the following reasons: (1) For second-line treatment with regorafenib, patients that discontinued sorafenib due to AEs were excluded, in other words, only patients with progressive disease (PD) under sorafenib treatment were included; (2) vascular invasion and extrahepatic spread were set as independent stratification factors to prevent imbalanced distribution between the two arms (active drug and placebo arms); (3) AFP, a strong poor prognostic factor, was also included as a stratification factor; and (4) only patients with sufficient sorafenib tolerance were selected (limited to patients who were able to tolerate sorafenib doses of 400 mg or higher for 20 d or longer during the 28-d period before PD). This study design prevented dropout due to AEs caused by regorafenib since molecular structure of regorafenib is very similar to sorafenib, and minimized the influence of post-treatment effects following PD after regorafenib treatment[41]. According to the RESORCE trial, the median survival time for patients treated with regorafenib was 10.6 mo (placebo: 7.8 mo; HR = 0.63; P < 0.0001). Sub-analysis of OS revealed that it was significantly more favorable for patients when sorafenib was first used at a Child-Pugh score of 5 than for patients introduced to sorafenib at a score of 6; this suggests that early switching from TACE to sorafenib for patients who become refractory to TACE at a Child-Pugh score of 5, and early switching to regorafenib for patients who become refractory to sorafenib, are important for extending survival. Furthermore, the median duration of pre-treatment with sorafenib before the RESORCE trial was 7.8 mo, which is relatively long. Many patients with long SD that responded to sorafenib well were enrolled in this trial. Thus, the effects on patients with rapid PD have been unclear. However, a recent report of sub-analysis of the RESORCE trial showed that the HR for OS in patients with rapid PD on sorafenib (TTP: 2.3 mo) was 0.66, thereby clarifying that regorafenib has OS benefit, even in patients showing rapid PD on sorafenib[42].

Based on the results of the RESORCE trial, sorafenib-regorafenib sequential therapy extended OS from the time of sorafenib treatment initiation to 26 mo (placebo group: 19.2 mo), thereby providing a favorable outcome[42,43]. This is a very important message to clinical practice. This outcome (OS = 26 mo) is almost comparable with that for intermediate stage HCC patients treated with conventional TACE[27]. At present, the BRISK-TA trial, a prospective phase III study involving the largest number of patients in the world, is the world largest TACE combination trial. Therefore, the outcome of the placebo arm of this trial is considered to be the world standard outcome of TACE treatment with no selection bias. In addition, the target groups of this trial comprised patients with BCLC B, BCLC A, and BCLC C (59%, 23%, and 17% of patients, respectively). Thus, 82% of all patients had early/intermediate stage disease and only 17% had advanced stage disease. In contrast, patients with advanced stage BCLC C accounted for 86% of patients enrolled in the RESORCE trial. When these two cohorts were compared, the OS of TACE-treated patients were 26.1 mo and that of patients who received sorafenib-regorafenib sequential therapy was 26 mo. Although it may be inappropriate to compare the results of the different randomized controlled trials (RCTs), it should be justified because there was no selection bias in the placebo group from the well-designed RCT. Considering that sorafenib-regorafenib sequential therapy targets far more advanced cases of HCC, the finding of a comparable OS result for TACE- and sorafenib-regorafenib-treated patients implicates very important issue. Although it was a highly selected patient population, the survival benefit of sorafenib-regorafenib sequential therapy for advanced stage HCC were comparable with those of TACE for intermediate stage HCC[27,42-44]. Therefore, sorafenib-regorafenib sequential therapy is expected to achieve a favorable outcome in the real-world clinical practice as well, suggesting that the timing of sorafenib introduction should be re-considered. Previously, a patient was switched from TACE to systemic therapy when they became refractory to TACE. However, it may be more important to identify a subgroup likely to become refractory to TACE and then introduce systemic therapy at an earlier time when the liver function reserve is maintained at a Child-Pugh score of 5 before becoming refractory to TACE[43,44].

Cabozantinib is an oral multi-kinase inhibitor that inhibits the activity of VEGF, c-MET, RET, AXL, TIE2, and FLT3. The survival-prolonging effects of cabozantinib as a second-line agent for patients with HCC refractory/intolerant to sorafenib treatment compared with placebo control were presented at the American Society of Clinical Oncology Gastrointestinal Cancer Symposium (ASCO-GI), held in January 2018. In total, 707 patients with unresectable HCC were allocated to the cabozantinib and placebo groups (2:1 ratio). The median OS for the cabozantinib group (n = 470) was 10.2 mo (95%CI, 9.1-12.0), demonstrating significant survival benefit when compared with the placebo group [8.0 mo (95%CI, 6.8-9.4)] (Table 5)[45,46]. Systemic chemotherapy with a drug other than sorafenib was allowed during this trial, with the drug positioned as the second- or third-line therapy.

Regarding the AE profiles, grade 3/4 AEs include diarrhea (10%), decreased appetite (6%), hand-foot skin reaction (17%), fatigue (10%) and hypertension (16%)[45]. However, most of AEs were manageable by medication or dose reduction/interruption. As this trial was not performed in Japan, a bridging phase 2 study is now underway; therefore, its approval as a second-line agent is expected in the near future in Japan as well.

Ramucirumab is a recombinant monoclonal human immunoglobulin IgG1 antibody specific for VEGFR-2. It is injected intravenously and inhibits VEGFR-2 activity by blocking its binding to VEGF-A, VEGF-C, and VEGF-D. Thus, it exerts anti-tumor effects by inhibiting endothelial cell proliferation, migration, and survival, thereby preventing tumor neovascularization.

The previous phase of the REACH trial examined the survival benefit of ramucirumab compared with placebo in patients with unresectable advanced HCC that was refractory/intolerant to sorafenib treatment. No survival benefit was proven in this trial[15]. However, sub-analysis limited to patients with an AFP level ≥ 400 ng/mL revealed improved survival; this result was reproducible regardless of the geographic region[47,48]. Thus, the REACH-2 trial was planned and conducted. The study design was not markedly different from that of the original REACH trial except that the subjects were limited to patients with an AFP level of ≥ 400 ng/mL, and macroscopic vascular invasion was included as a stratification factor. The results were presented at ASCO in June 2018. The study results were positive in terms of the primary endpoint: improvement of OS[49,50]. The median OS in the ramucirumab group was 8.5 mo and that in the placebo group was 7.3 mo, confirming a significant survival benefit over placebo (HR = 0.710; P = 0.0199) (Table 6).

Regarding the AE profiles, grade 3/4 AEs include hypertension (12%), thrombocytopenia (5%), hepatic encephalopathy (3%) and neutropenia (3%). These AEs were manageable by medication and/or dose reduction/interruption. Most importantly, relative dose intensity was as high as 98.5%, which suggests tolerability of this drug is fairly high[49]. Based on these results, ramucirumab is expected to be approved for patients refractory or intolerant to sorafenib treatment and with an AFP level ≥ 400 ng/mL.

Nivolumab is the first recombinant monoclonal human immunoglobulin IgG4 antibody specific for human PD-1. A phase I/II study of advanced HCC, the Checkmate-040 trial, reported a response rate of 20%, which included three complete responders. The disease control rate was 64%, which is very promising. The drug also showed a durable and long-lasting response effect in responders[58]. AE profiles are fairly mild; grade 3 AE was only observed in 1 % (fatigue). Grade 1/2 AEs include rash (19%), pruritus (10%), diarrhea (10%), decreased appetite (10%) and fatigue (8%)[58]. Subsequently, a trial involving an increased number of patients was performed and the updated results were reported at ASCO in 2017. The median survival of patients treated with nivolumab as a first-line therapy was 28.6 mo whereas that of patients treated with nivolumab as a second-line was 15 mo; again, very promising. Based on the results of the phase I/II study described above, the United States marked nivolumab for priority review as a second-line agent after sorafenib; it was approved by the Food and Drug Administration in September 2017. The head-to-head phase III study (CheckMate-459) of nivolumab vs sorafenib as a first-line agent is now ongoing and the results are eagerly awaited. If this study is positive, 1^st option among 1^st line agents will undoubtedly become nivolumab because of its durable long-lasting response in responders.

Similar to nivolumab pembrolizumab is a recombinant monoclonal human immunoglobulin IgG4 antibody specific for human PD-1. A phase II study of HCC patients reported a response rate of 17%, indicating that the effects are comparable with those of nivolumab[59]. AE profiles are also mild and comparable with those of nivolumab: grade 3 AEs were observed only in 4% (fatigue) and 1% (decreased appetite). Grade 1/2 AEs include fatigue (17%), pruritus (12%), diarrhea (11%) and rash (10%)[59]. A placebo-controlled phase III study of pembrolizumab as a second-line treatment for patients refractory/intolerant to sorafenib was conducted[60], however, the information was press released on Feb 19, 2019 that this trial did not meet its primary endpoints of prolonging PFS nor OS.

The results of a phase 1b study of combination therapy with an anti-PD-L1 antibody, atezolizumab, and another antibody to VEGF, bevacizumab, were reported at ASCO in 2018. Although there were only 23 patients, the response rate according to a RECIST 1.1-based blind review reached 65%, with combination therapy showing a synergistic effect[61]. Based on this result, FDA designated this combination trial as a breakthrough therapy in July 2018. However, updated results presented at ESMO 2018 on October 21, 2018 revealed ORR according to modified RECIST- based independent review assessment dropped to 34% as a result of increased numbers of patients to 73 (Table 8)[62]. Therefore, we have to be cautious on the results derived from small numbers of patients in phase 1/2 trial. A randomized controlled phase III study of combination therapy versus sorafenib is underway (the IMbrave150 trial) (Table 1)[60,63]. Other combination cancer immunotherapies in HCC are also ongoing (Table 8)[64,65].

VEGF released by cancer cells suppresses anti-tumor immune responses (Figure 3). VEGF activates the main players in an immunosuppressive tumor microenvironment; these include regulatory T cells (Tregs), tumor-associated macrophages, and myeloid-derived suppressor cells. Cytokines released by these cells inhibit dendritic cell maturation and activation and proliferation of NK cells and CD8-positive cells, thereby driving an immunosuppressive microenvironment (Figure 3)[66]. Accordingly, combination treatment with a molecular targeted drug plus an anti-VEGF agent, bevacizumab, is appropriate to induce synergistic effects.

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Figure 3 Tumor Immuno suppressive microenvironment caused by vascular endothelial growth factor produced by tumor[66]. VEGF-A up-regulates tumor-associated macrophage, regulatory T-cell, and myeloid-derived suppressor cell, which cause immune suppressive microenvironment through the down-regulation of the dendritic cell maturation, NK activation, T cell activation and T cell proliferation. VEGF-A: Vascular endothelial growth factor A; MDSC: Myeloid-derived suppressor cell; DC: Dendritic cell; TAM: Tumor-associated macrophage; Treg: Regulatory T cell; IL: Interleukin.

The results of a phase 1b study of combination treatment with lenvatinib plus pembrolizumab were reported at the same ASCO meeting in 2018. The results of this study were also promising. The effects were evaluated in 26 patients; although the mean duration of follow-up was less than 3 mo, the response rate was 42.3%, with no PD patients (Table 8)[67]. However, again we have to be cautious on the results derived from small numbers of patients in phase 1/2 trial similar to atezolizumab/bevacizumab combination trial. The synergistic effects of lenvatinib plus an anti-PD-1 antibody were also suggested in a mouse model (Figure 4)[68,69]. Thus, combination therapy with immune checkpoint inhibitor/molecular targeted drugs may play an important role in the HCC treatment paradigm in the very near future (Figure 5)[70,71]. Biomarker that predicts response to immunotherapy or combination immunotherapy is still an unmet need in immunotherapy of HCC and extensive effort to identify such biomarkers is warranted.

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Figure 4 Mechanism underlying the synergistic effect of combination therapy on hepatocellular carcinoma with lenvatinib and anti-PD-1/PD-L1 antibodies[68]. Lenvatinib inhibits tumor angiogenesis and growth through VEGFR1-R3 and FGFR1-R4 inhibition. Lenvatinib also inhibits VEGF-mediated tumor suppressive microenvironment, such as immunosuppressive cells (tumor associated macrophage, regulatory T cells and myeloid-derived suppressor T cells) or tumor suppressive cytokines (IL10 or TGF-β). Lenvatinib also suppress the co-inhibitory checkpoint inhibitor, TIM 3 and increase the co-stimulatory molecules, CD137, OX40 or ICOS. Finally, PD-1/PD-L1 Ab restores the exhausted T cell activity to kill the cancer cell. Therefore, synergistic effect is obtained by this combination.

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Figure 5 Improved Overall Survival after combination therapy with molecular targeted agents and immune checkpoint inhibitors[70]. Molecular targeted agents improve survival compared with chemotherapy by cytotoxic agents, but will become resistant sooner or later. Durable long-lasting response is obtained by immune checkpoint inhibitors, but only small portion of patients (15%-20%). Durable long-lasting response will be expected by a combination therapy with molecular targeted agents and immune checkpoint inhibitors in the majority of the patients (50%-70%) with advanced hepatocellular carcinoma.