A Phase 1b Study of Lenvatinib plus Nivolumab in Patients with Unresectable Hepatocellular Carcinoma.

Introduction
In 2022, liver cancer was estimated to be the sixth most prevalent cancer and the third leading cause of cancer-related deaths globally [1]. Approximately 75%–85% of the cases of liver cancer are hepatocellular carcinoma (HCC) [1], and most patients with HCC present with unresectable disease [2–4].
Given the highly vascularized nature of HCC, multi-kinase inhibitor monotherapy (i.e., sorafenib and lenvatinib) for the first-line treatment of patients with unresectable HCC (uHCC) [2, 5] has shown success. Lenvatinib, a multi-kinase inhibitor targeting vascular endothelial growth factor (VEGF) receptors 1–3, fibroblast growth factor (FGF) receptors 1–4, platelet-derived growth factor receptor-α, RET, and KIT, is a standard-of-care first-line treatment for patients with uHCC who are not candidates for treatment with immune checkpoint inhibitors (ICIs) [6–12].
Notably, anti-programmed death ligand 1 antibodies have been successfully used and approved for the first-line treatment of patients with advanced HCC in combination with anti-VEGF (atezolizumab plus bevacizumab) [3, 6] and an anti-cytotoxic T-lymphocyte-associated antigen 4 antibody (durvalumab plus tremelimumab) [4, 7]. The combination of nivolumab, an anti-programmed death receptor-1 antibody, and ipilimumab, an anti-cytotoxic T-lymphocyte-associated antigen 4 antibody, has also shown efficacy as a first-line treatment for patients with uHCC [13]. Despite continued advances in treatments, the median overall survival (OS) seen across immune-based combination treatments for advanced HCC in the first-line setting is 16–24 months, and the objective response rate (ORR) is between 20% and 36% [3, 4, 13, 14]. Given the need to prolong OS in this patient population and the high percentage of patients who do not respond to these therapies [3, 4], additional investigation is warranted to evaluate potentially more potent combination therapies.
Multi-kinase inhibitors, in combination with ICIs, have shown clinically meaningful efficacy for the treatment of patients with other types of cancer [15]. Mechanistically, targeting multiple signaling pathways has the potential to benefit patients with advanced HCC, given the heterogeneity observed in the disease. Combinations of multi-kinase inhibitors and ICIs have been evaluated as first-line treatment for patients with uHCC [14]; however, these combinations have not yet been approved in this setting [6, 7, 16]. In the LEAP-002 trial [14], the combination of lenvatinib with pembrolizumab achieved a median OS of 21.2 months versus 19.0 months for lenvatinib plus placebo in the first-line treatment of advanced HCC. While this represents a numerical increase in OS, these results did not reach prespecified statistical significance. Nivolumab, in combination with ipilimumab, has recently been approved in the USA, Europe, and Japan as a first-line treatment for patients with HCC [7, 17–21]. Additionally, both lenvatinib (in combination with pembrolizumab) and nivolumab (in combination with cabozantinib) are used as multi-kinase inhibitors with ICI combination therapies for the first-line treatment of patients with advanced renal cell carcinoma and are considered the standard-of-care treatment for these patients [22–25]. Herein, we present the results of the first prospective phase 1b study of the combination of lenvatinib and nivolumab, study 117 (NCT03418922), evaluating the tolerability, safety, and antitumor activity in patients with advanced/uHCC.

Methods

Study Design and Participants
This open-label, single-arm study evaluated the tolerability, safety, and preliminary antitumor activity of treatment with lenvatinib in combination with nivolumab in patients with HCC. The study was conducted at 6 sites in Japan between January 16, 2018, and December 28, 2022. Patients were assigned to receive monotherapy-approved doses of either 8 mg (body weight <60 kg) or 12 mg (body weight ≥60 kg) oral lenvatinib once daily plus 240 mg intravenous nivolumab every 2 weeks (days 1 and 15) in 4-week cycles.
The study was conducted in 2 parts. Part 1 planned to enroll 6 patients with HCC to evaluate the tolerability of lenvatinib in combination with nivolumab by assessing dose-limiting toxicities (DLTs) over 1 treatment cycle. At least 3 patients treated with 12 mg lenvatinib were enrolled for the DLT evaluation. After confirming the tolerability of study treatment in part 1, additional patients were enrolled in part 2. Part 2 evaluated the safety and preliminary antitumor activity of lenvatinib in combination with nivolumab. Treatment continued until disease progression, development of unacceptable toxicity, or withdrawal of consent.
Adult patients (≥20 years old at the time of informed consent) with histologically or cytologically confirmed diagnosis of HCC (excluding fibrolamellar, sarcomatoid, or mixed cholangio-HCC tumors) or clinically confirmed diagnosis of HCC according to American Association for the Study of Liver Diseases criteria (including cirrhosis of any etiology and/or chronic hepatitis B or C virus infection) with measurable lesions by modified (m) Response Evaluation Criteria in Solid Tumors (RECIST) were eligible to participate in the study. Part 1 enrolled patients with HCC for whom no other appropriate therapy was available, and part 2 enrolled previously untreated patients with advanced/uHCC. Eligible patients had disease categorized as stage B or stage C based on the Barcelona Clinic Liver Cancer (BCLC) staging system, Child-Pugh class A, an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0 or 1, and adequate organ function. Exclusion criteria for part 1 included imaging findings of HCC corresponding to any of the following: HCC with ≥50% liver occupation, clear invasion into the bile duct, or portal vein invasion with Vp4. Full inclusion and exclusion criteria are presented in the online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000549681).

Endpoints and Clinical Assessments
In part 1, the primary endpoints evaluated were tolerability (including DLTs) and safety of lenvatinib in combination with nivolumab. Part 2 further evaluated the safety and preliminary efficacy of the combination therapy. Secondary endpoints were to evaluate ORR (the proportion of patients that experienced the best overall response of complete response or partial response) and the pharmacokinetic (PK) profile of lenvatinib and nivolumab. Exploratory endpoints included evaluation of disease control rate (the proportion of patients that experienced a complete response, partial response, or stable disease with a duration of ≥7 weeks from cycle 1 day 1), clinical benefit rate (defined as the proportion of patients who experienced a complete response, partial response, or durable stable disease with a duration of ≥23 weeks), pharmacodynamic (PD) biomarkers and pharmacogenomics (PGx) of lenvatinib and nivolumab, and, in part 2 only, duration of response, time to response, progression-free survival (PFS), and OS.
Adverse events (AEs) were monitored throughout the study and graded according to Common Terminology Criteria for Adverse Events version 4.03. Blood samples for PK assessment of lenvatinib in plasma and nivolumab in serum were collected as presented in online supplementary Figure 1. Tumor imaging assessments were performed at screening, every 8 weeks from cycle 1, day 1, or more frequently if clinically indicated, and at treatment discontinuation if ≥4 weeks had passed since the preceding assessment. Efficacy was assessed by mRECIST per investigator review, or additionally, in part 2, by mRECIST or RECIST version (v) 1.1 per independent review (i.e., post hoc independent imaging review was conducted in part 2) [26, 27]. Patients in part 1 were followed until the last observation visit. In part 2, survival was assessed every 12 weeks (±2 weeks) from the date of last observation except if a patient withdrew consent or the sponsor chose to halt survival follow-up. Survival follow-up continued until study discontinuation of the last patient or up to 2 years after the last patient began study treatment, whichever occurred later. Given that immunotherapies can induce antidrug antibodies (ADAs), which can potentially cause infusion-related reactions, the ADA status of patients was summarized (i.e., percentage of ADA-positive/-negative patients). Blood samples for ADA and biomarker analyses were collected as presented in online supplementary Figure 1. ADA analyses were performed similarly to analyses by Agrawal S et al., J Clin Pharmacol 2017 [28].

Biomarker Analyses
Serum biomarkers were assayed using a custom multi-analyte profile immunoassay panel (VEGF, ANG2, CXCL9, CXCL10, and CXCL11), ultrasensitive single molecule array immunoassays (interferon gamma [IFNγ]), or by enzyme-linked immunosorbent assays (FGF19 and FGF23) from Myriad RBM (Austin, TX, USA). For circulating tumor DNA (ctDNA) assays, the baseline mutational status of genes (wild-type or mutant) in ctDNA was measured at a sponsor laboratory by a PGDx elio plasma complete panel, which assessed 521 genes for single-nucleotide variants and insertions/deletions. Detected mutations were annotated using OncoKB (OncoKB v4.24).

Statistical Analyses
Part 1 of the study aimed to enroll 6 patients, which was deemed appropriate to evaluate the dose tolerability and perform a preliminary safety assessment. Part 2 aimed to enroll 20 patients based on the associated estimated probabilities of 87.8%, 96.1%, or 98.8% to detect the development of ≥1 intolerable treatment-related AEs (TRAEs) with a true incidence of 10%, 15%, or 20%, respectively.
DLTs were assessed in all part 1 patients who completed cycle 1 without a major protocol deviation, had at least 75% lenvatinib compliance and received at least two doses of nivolumab, or who experienced any DLT during cycle 1. Safety was assessed in all patients who received ≥1 dose of lenvatinib or nivolumab. PK was assessed in all patients who received ≥1 dose of lenvatinib and nivolumab and who had evaluable concentration data of lenvatinib (for lenvatinib PK analysis) or nivolumab (for nivolumab PK analysis). Efficacy was assessed in all patients who received ≥1 dose of lenvatinib and nivolumab. PD and PGx were assessed in all patients who received ≥1 dose of lenvatinib and nivolumab and had ≥1 post-dose PD or PGx dataset.
Baseline characteristics and safety data were summarized using descriptive statistics. AE-preferred terms were defined according to the Medical Dictionary for Regulatory Activities terminology Version 25.1. Plasma concentrations for lenvatinib and serum concentrations for nivolumab were summarized by dose level using summary statistics, and PK parameters of lenvatinib in part 1 were derived by non-compartmental analysis. The best overall response was summarized, and ORR, disease control rate, and clinical benefit rate with corresponding exact 2-sided 95% confidence intervals (CIs) were calculated. The Kaplan-Meier method was used to estimate the median duration of response, PFS, and OS; the corresponding 95% CIs were calculated. Summary statistics were reported for time to response. The effect of lenvatinib in combination with nivolumab on PD biomarker levels was summarized using descriptive statistics and was compared between day 1 of cycles 2–4 and baseline using the Wilcoxon signed rank test. Fisher’s exact test was used to test the association between mutation status (mutation-negative or mutation-positive) and responder class (responder or nonresponder).

Results

Patients
The study enrolled 39 patients in part 1 and part 2. In total, 30 patients (part 1, n = 6; part 2, n = 24) received study treatment. In part 1, all 6 patients completed DLT evaluation in cycle 1. As of the data cutoff date in part 2, the median follow-up time was 45.32 months (95% CI, 42.97–not estimable), and all patients discontinued study treatment. The reasons for treatment discontinuation were disease progression (part 1, n = 4; part 2, n = 18), AEs (part 1, n = 2; part 2, n = 2), patient choice (part 2, n = 1), and other reasons (part 2, n = 3 [decision of investigator and patient, physician judgment, and death]). Baseline patient characteristics are presented in Table 1. The majority (80%) of patients were male with a median age of 70 years (range, 36–81). Overall, 28 (93.3%) patients had an ECOG PS of 0, 17 (56.7%) had BCLC stage B and 23 (76.7%) had a Child-Pugh score of 5. No patients had macroscopic vascular invasion (portal vein involvement was counted separately) and 25 (83.3%) patients had a serum alpha-fetoprotein level of <200 ng/mL.

Study Drug Exposure, Safety, and Immunogenicity
In all treated patients (n = 30), the median number of treatment cycles with lenvatinib and nivolumab was 11 (range, 1–39). The median duration of treatment with lenvatinib was 10.1 months (range, 0.5–52.0 months). The median duration of treatment with nivolumab was 10.1 months (range, 0.0–35.4 months). The duration of treatment for individual patients is presented in online supplementary Figure 2.
Patients were administered the approved doses of the study drug, 8 mg (if body weight was <60 kg) or 12 mg (if body weight was ≥60 kg) of oral lenvatinib once daily plus 240 mg intravenous nivolumab every 2 weeks (days 1 and 15) in 4-week cycles; no DLTs were observed in the patients enrolled in part 1 (n = 6). Treatment-emergent AEs (TEAEs) of any grade were observed in all treated patients in part 1 and part 2 (total, n = 30). Grade 3 or higher TEAEs were observed in 22/30 (73.3%) patients. Overall, 15/30 (50.0%) patients experienced serious AEs. AEs leading to death (bronchopulmonary aspergillosis, aortic dissection, and hepatic failure) occurred in 3/30 (10.0%) patients; none of these events were considered to be treatment-related. TRAEs of any grade were observed in all treated patients in part 1 and part 2 (total, n = 30). Grade 3 or higher TRAEs were observed in 18/30 (60.0%) patients. The most common TRAEs (occurring in ≥30% of patients overall) were palmar-plantar erythrodysesthesia syndrome (18/30, 60.0%), dysphonia (16/30, 53.3%), decreased appetite (15/30, 50.0%), diarrhea and proteinuria (each 14/30, 46.7%), hypertension (11/30, 36.7%), and fatigue, hypothyroidism, malaise, and stomatitis (each 9/30, 30.0%) (Table 2). The incidence of hepatic encephalopathy was 13.3% (4/30) overall. TRAEs led to a dose reduction of lenvatinib in 22/30 (73.3%) patients. TRAEs led to a lenvatinib dose interruption in 20/30 (66.7%) patients and a nivolumab dose interruption in 8/30 (26.7%) patients. Overall, 4/30 (13.3%) discontinued lenvatinib, and 4/30 (13.3%) discontinued nivolumab due to TRAEs. Albumin-bilirubin scores remained consistent from baseline to the end of treatment (online supplementary Fig. 3).
Results of serum ADA analysis against nivolumab are summarized in online supplementary Table 1. Overall, 3 (10.0%) patients were ADA-positive at baseline, and 7 (23.3%) patients were ADA-positive after study treatment. No patients were persistent positive, defined as ADA-positive at 2 or more consecutive time points, where the first and last ADA-positive were at least 15 weeks apart. Of the 7 patients who were ADA-positive, neutralizing antibody was detected in 1 (14.3%) patient.

PK Analysis
The PK profile of lenvatinib at cycle 1, day 1 and cycle 1, day 15 in part 1 is presented in online supplementary Table 2. On cycle 1, day 1 and cycle 1, day 15, approximate dose-proportional increases in mean maximum observed concentration (Cmax) and area under the concentration (AUC)-time curve from zero time to time of last quantifiable concentration (AUC(0-t)) of lenvatinib were observed in both the 8 mg once daily and 12 mg once daily dose groups. However, there was an overlap in the distribution of individual values between both dose groups. The mean accumulation ratios based on Cmax and AUC(0-t), Rac (Cmax) and Rac (AUC), were 1.49 and 1.60 in the 8 mg once daily dose group, respectively, and 1.41 and 1.47 in the 12 mg once daily dose group. The serum concentration of nivolumab throughout study treatment is presented in online supplementary Figure 4. The mean (standard deviation) serum concentrations of nivolumab just before completion of nivolumab administration of cycle 1, day 1 and cycle 5, day 1 were 67.3 (9.54) μg/mL and 149 (21.9) μg/mL, respectively, in part 1 and 57.7 (16.5) μg/mL and 112 (30.2) μg/mL in part 2. The mean (standard deviation) serum trough concentrations of nivolumab at 4-, 8-, 12-, and 16-week post-dose were 39.5 (8.56) μg/mL, 56.5 (19.1) μg/mL, 60.5 (19.0) μg/mL, and 77.6 (4.89) μg/mL, respectively, in part 1 and 36.5 (8.79) μg/mL, 49.5 (13.4) μg/mL, 58.3 (15.8) μg/mL, and 61.2 (20.0) μg/mL in part 2. Serum trough concentrations of nivolumab generally remained constant after 12 weeks of administration, and a steady state was reached by 12 weeks in most patients.

Efficacy
Tumor response data are presented in Table 3 and Figure 1. ORR by mRECIST per investigator review was 66.7% (n = 4/6; 95% CI, 22.3–95.7) in part 1 and 79.2% (n = 19/24; 95% CI, 57.8–92.9) in part 2. A complete response as best overall response by mRECIST per investigator review was not observed in part 1 and was observed in 4/24 (16.7%) patients in part 2. Tumor response rates in part 2 by mRECIST and by RECIST v1.1 per independent review are presented in Table 3. In part 2, among patients with an objective response (n = 19), the median (95% CI) duration of response was 6.57 (2.07–9.23) months, and the median time to response was 1.87 (range, 1.6–5.6) months by mRECIST per investigator review. In part 2 (n = 24), the median PFS (95% CI) by mRECIST per investigator review was 9.07 (3.71–11.07) months (Fig. 2a); the median OS (95% CI) was 26.94 (16.03–38.80) months (Fig. 2b).
Anticancer medications and procedures during survival follow-up are summarized in online supplementary Table 3. Of the 24 patients treated in part 2, 20 (83.3%) received any anticancer treatments; 13 (54.2%) received anticancer medications; and 18 (75.0%) received anticancer procedures.

Biomarker Analysis
The effect of treatment with lenvatinib in combination with nivolumab on the level of PD serum biomarkers related to angiogenesis signaling, FGF signaling, and IFNγ signaling was evaluated. The median percentage change from baseline in biomarker levels through cycle 4, day 1 is presented in Figure 3. Statistically significant changes (p < 0.05) relative to baseline were observed for most evaluated biomarkers at cycle 2, day 1, cycle 3, day 1, and cycle 4, day 1. In relation to angiogenic signaling, VEGF levels increased and ANG2 levels decreased from baseline to cycle 4, day 1. In relation to FGF signaling, FGF19 and FGF23 increased from baseline to cycle 4, day 1. Downstream of IFNγ signaling, the levels of cytokines CXCL9, CXCL10, and CXCL11 increased from baseline to cycle 4, day 1.
The frequencies of mutations in the most common driver genes in HCC (TERT, TP53, and CTNNB1) in the ctDNA of all patients at baseline were evaluated. TERT promotor mutations were observed in 18/30 (60.0%) patients. TP53 mutations were observed in 13/30 (43.3%) patients. CTNNB1 mutations were observed in 12/30 (40.0%) patients (online supplementary Fig. 5).
The PGx of lenvatinib in combination with nivolumab was assessed by comparing antitumor activity in patients whose tumors were positive or negative for TERT, TP53, or CTNNB1 mutations. There were no statistically significant differences in tumor response rates based on the presence or absence of these mutations (Table 4).

Discussion
Unmet needs remain in the treatment of uHCC, with early phase studies needed to support the design of future phase 3 trials that can effectively assess the combination of treatments in different HCC subpopulations. We report the results of a phase 1b study evaluating the tolerability, safety, and preliminary antitumor activity of lenvatinib in combination with nivolumab for treating patients with HCC. Part 1 evaluated tolerability by assessing DLTs in patients with HCC for whom no other appropriate therapy was available. Part 2 additionally enrolled previously untreated patients with advanced HCC or uHCC to evaluate safety and antitumor activity. Treatment with the monotherapy-approved doses of lenvatinib in HCC, i.e., 8 mg (body weight <60 kg) or 12 mg (body weight ≥60 kg) oral once daily, plus 240 mg intravenous nivolumab every 2 weeks showed a manageable safety profile and promising efficacy in the 30 enrolled patients. High-risk patients with ≥50% liver occupation, clear invasion into the bile duct, or portal vein invasion with Vp4 were excluded. By creating a more homogenous participant group, we aimed to ensure the results were not skewed by cases where the disease is too aggressive for any treatment to be effective or well tolerated, thus allowing the potential benefits of this regimen in a population more likely to respond. The design of this current regimen was based on a synergistic mechanistic hypothesis, wherein lenvatinib’s immunomodulatory effects on the tumor microenvironment are thought to enhance the antitumor activity of nivolumab.
No DLTs were observed in the 6 patients with HCC who received lenvatinib in combination with nivolumab in part 1. The AEs observed in part 1 and part 2 were generally similar to those previously reported and established with either study treatment as monotherapy [5, 29–31]. Common AEs observed with lenvatinib monotherapy or with lenvatinib in combination with ICIs, including in this study, included palmar-plantar erythrodysesthesia syndrome, decreased appetite, diarrhea, proteinuria, hypertension, fatigue, hypothyroidism, weight decreased, nausea, and rash [5, 14]. Although hepatic encephalopathy occurred at a slightly higher rate in this study (TRAE: 13.3%) versus lenvatinib monotherapy in REFLECT (TEAE: 8%) [30], such differences may be accounted for by variations in study populations. In general, the overall safety profile of the combination treatment was manageable. Similar to rates observed in the phase 3 REFLECT trial of lenvatinib monotherapy in previously untreated patients with uHCC (serious AEs, 43%; discontinuation due to TEAEs, 14%) [5], 50.0% of patients in the current study experienced serious AEs, and 13.3% of patients discontinued lenvatinib due to TRAEs. Three fatal AEs occurred in this study, but these were not considered to be treatment-related. In the PK analysis, the distributions of lenvatinib AUC(0-t) were similar to those observed for lenvatinib in HCC previously [32]. They were within the distributions of AUC(0-τ) observed with lenvatinib monotherapy in the REFLECT trial [5], which suggests that nivolumab is unlikely to affect the PK of lenvatinib [5].
The ORR by mRECIST per investigator review was 66.7% in part 1 and 79.2% in part 2. In part 2, the median PFS was 9.07 months by mRECIST per investigator review, and the median OS was 26.94 months. Although caution should be used when comparing studies, ORR and PFS in this study were notable compared to a recent phase 3 study of lenvatinib plus pembrolizumab in the first-line treatment of uHCC (tumor assessments by independent review per mRECIST: ORR, 40.8%; median PFS, 8.4 months) [14]. In part 2, among the 19 patients who had an objective response by mRECIST per investigator review, the median duration of response was 6.57 months (95% CI: 2.07–9.23 months). Given the small number of patients in this group, the wide 95% CI suggests considerable variability in the duration of response. Considering this variability and that the duration of response analysis was limited to only 19 patients, it is difficult to conclude why the duration of response was relatively short despite a favorable ORR. Furthermore, given the median OS of 26.94 months, relatively short duration of response, and 10.1-month median duration of treatment, it is likely subsequent anticancer therapy may be attributed to longer survival. Higher rates of patients with baseline BCLC-B disease compared with other studies [3, 4, 14] may be partly attributable to better treatment outcomes in our analyses. Given the relatively high proportion of patients with BCLC-B disease and considering the results of the LEAP-012 trial [33], which evaluated the combination of lenvatinib and pembrolizumab with transarterial chemoembolization, future studies of lenvatinib-ICI combinations with loco-regional therapies in this patient population may be of interest.
The current study demonstrated promising antitumor signals that are favorable compared to several major Phase 3 trials for uHCC. The ORR (per RECIST v1.1) in part 2 of this trial (58%) was high compared to the ORR observed with atezolizumab plus bevacizumab in the IMbrave150 trial (30%) or the ORR with lenvatinib plus pembrolizumab in LEAP-002 (26.1%) [14, 34]. Furthermore, the median OS was 26.94 months compared to 19.2 months from IMbrave150 and 23.7 months from CheckMate 9DW [4, 35], while the median PFS of 9.07 months was also highly competitive. These results, however, should be interpreted with caution, due to the inherent limitations of a small, non-randomized phase 1b study compared to the large, randomized phase 3 trials. Overall, promising antitumor activity together with the combination’s manageable safety profile (which was consistent with previously reported monotherapy data [30, 31]) suggests that lenvatinib plus nivolumab may be of interest for further investigation.
Previous preclinical studies showed that lenvatinib inhibits VEGF- and FGF-driven angiogenesis and tumor growth by targeting VEGF and FGF receptors [36, 37]. Here, treatment with lenvatinib in combination with nivolumab resulted in changes in serum levels of PD biomarkers consistent with inhibition of VEGF and FGF receptor activity, increased VEGF, FGF19, and FGF23 levels, and decreased ANG2 levels. This is consistent with a previous report of changes in serum PD biomarker levels after treatment with lenvatinib monotherapy and with lenvatinib in combination with pembrolizumab [38, 39]. Expression of genes related to IFNγ signaling has previously been shown to be positively associated with favorable responses to anti-programmed death receptor-1 therapy in patients with advanced HCC [40]. In this study, treatment with lenvatinib in combination with nivolumab resulted in increased serum levels of factors downstream of IFNγ signaling.
The rates of genetic mutation observed in the ctDNA of patients in this study were consistent with previous reports, including the genes TERT, TP53, and CTNNB1, which are commonly mutated in patients with HCC [41–45]. No statistically significant differences in ORR were observed in patients whose ctDNA was positive for TERT, TP53, or CTNNB1 mutation compared with those whose ctDNA was negative for these mutations. In a ctDNA biomarker analysis of the phase 3 REFLECT study, lenvatinib monotherapy also showed similar ORR across these three mutations [46]. Thus, nivolumab may enhance the antitumor activity of lenvatinib regardless of the mutation status of these three key HCC-related driver genes. However, the analysis of ctDNA in this study was exploratory; therefore, any associations between common HCC mutations and treatment response should be interpreted with caution.
The interpretation of data in this study is limited due to the small size of the enrolled patient population; however, the combination of lenvatinib with nivolumab was well tolerated and showed encouraging antitumor activity in patients with HCC. AEs were manageable, and no unexpected safety signals were observed. Based on these results, evaluation of lenvatinib in combination with nivolumab in a larger, global patient population is warranted.

Acknowledgments
The authors thank the patients, their families, the investigators, and the teams who participated in this trial. They also thank Aika Fueki for her contributions to the scientific and operational development associated with this manuscript.

Statement of Ethics
All patients provided written informed consent before participating in the study. The study protocol was approved by the appropriate Institutional Review Board or Independent Ethics Committee at participating institutions (National Cancer Center Hospital East, K0675; National Cancer Center Hospital; T4550; Toranomon Hospital, 18-34-B; Kindai University Hospital, 1884; Chiba University Hospital, 030012; Iizuka Hospital, 30-2). The study was conducted in accordance with the Good Clinical Practice guidelines and the principles of the Declaration of Helsinki.

Conflict of Interest Statement
Masafumi Ikeda: reports research funding paid to institution from AbbVie, AstraZeneca, Bayer, Bristol Myers Squibb, Chugai, Eisai, and MSD; consulting or advisory role for AbbVie, AstraZeneca, Bayer, Chugai, Eisai, Eli Lilly Japan, MSD, and Ono; honoraria from Abbott, AstraZeneca, Bristol Myers Squibb, Chugai, Eisai, Eli Lilly Japan, MSD, and Takeda. Masatoshi Kudo: reports research funding paid to institution from Chugai, Eisai, GE Healthcare Japan, Otsuka, and Taiho; consulting fees from AstraZeneca, Chugai, Eisai, F. Hoffmann-La Roche, and MSD; honoraria from AstraZeneca, Chugai, and Eisai. Kenta Motomura: reports honoraria from AstraZeneca, Chugai, Eisai, and Gilead; travel expenses from Eisai. Takuji Okusaka: report grants or contracts from AstraZeneca, Bristol Myers Squibb, Chiome Bioscience, Chugai, Eisai, Incyte, MSD, Syneos Health, and Sysmex Corporation; consulting fees from Amgen, AstraZeneca, Eisai, FUJIFILM Toyama Chemical, Jazz, Nihon Medi-Physics, and Nihon Servier; honoraria from AstraZeneca, Chugai, Daiichi Sankyo, Eisai, Incyte, Johnson & Johnson, Kyowa Kirin, Kanehara Shuppan, MSD, Myriad Genetics, Nihon Servier, Novartis, NHK, Ono, Taiho, Yakult Honsha, and Yodosha. Naoya Kato: reports grants or contracts from AstraZeneca, Chugai, Bayer Yakuhin, Ltd.; honoraria from AstraZeneca, Bristol Myers Squibb, Eisai, Takeda, Bayer Yakuhin Ltd., Chugai, and Eli Lilly. Takashi Hisai and Sari Shiba: employees of Eisai Co., Ltd. Hiroki Ikezawa and Taisuke Hoshi: employees of Eisai Co., Ltd.; stock or stock options from Eisai Co., Ltd. Masataka Kuroda: employee of Ono Pharmaceutical Co., Ltd. Hiromitsu Kumada: payment for lectures from AbbVie, Gilead Sciences, Eisai Co., Ltd., Chugai, and Sumitomo Pharma. Masahiro Kobayashi: nothing to disclose.

Funding Sources
This study was funded by Eisai Co., Ltd. The funder contributed to the study design, data collection, data analysis, and data interpretation in collaboration with the authors; all authors had full access to the data. Investigators and site personnel collected data. The corresponding author had full access to all the data and had final responsibility for the decision to submit the publication. Medical writing support was provided by Oxford PharmaGenesis, Newtown, PA, USA, and was funded by Eisai Inc., Nutley, NJ, USA.

Author Contributions
Conceptualization and methodology: M. Ikeda, M. Kobayashi, T. Hisai, H. Ikezawa, S. Shiba, T. Hoshi, and M. Kuroda. Data curation: T. Hisai, H. Ikezawa, S. Shiba, and T. Hoshi. Formal analysis: H. Ikezawa and S. Shiba. Investigation: M. Ikeda, M. Kudo, K. Motomura, T. Okusaka, N. Kato, H. Kumada, and M. Kobayashi. Validation: H. Ikezawa, T. Hisai, S. Shiba, T. Hoshi, and M. Kuroda. Visualization: M. Ikeda and M. Kobayashi. Writing – original draft and writing – review and editing: all authors.