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

  • Author Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    ZhiCheng Lai
    Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Author Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    MinKe He
    Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Author Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    XiaoYun Bu
    Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Author Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    YuJie Xu
    Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • YeXing Huang
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • DongSheng Wen
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • QiJiong Li
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Li Xu
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • YaoJun Zhang
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Wei Wei
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • MinShan Chen
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Anna Kan
    Correspondence
    Corresponding author: Department of Hepatobiliary Oncology, Cancer Centre, Sun Yat-sen University, Guangzhou, 510060, China. Fax: +86 020 87343115.
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Ming Shi
    Correspondence
    Corresponding author: Department of Hepatobiliary Oncology, Cancer Centre, Sun Yat-sen University, Guangzhou, 510060, China. Fax: +86 020 87343585.
    Affiliations
    Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou, China
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  • Author Footnotes
    1 The first four authors (ZCL, MKH, XYB, YJX) contributed equally to this work.
Open AccessPublished:August 15, 2022DOI:https://doi.org/10.1016/j.ejca.2022.07.005

      Highlights

      • High-risk advanced HCC is associated with poor outcomes of systemic therapy.
      • Combination FOLFOX-HAIC with lenvatinib and toripalimab was tolerable and effective.
      • CCL28 and BTC levels before therapy might be the predictive biomarkers of efficacy.

      Abstract

      Introduction

      The combination of lenvatinib, toripalimab and hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) suggested encouraging antitumour activity in our retrospective study. We hereby prospectively establish the efficacy, safety and predictive biomarkers of the combination therapy as a first-line treatment in patients with high-risk advanced hepatocellular carcinoma (HCC).

      Materials and methods

      This phase II, single-centre, single-arm trial enrolled advanced HCC participants with high-risk. Of 51 screened participants, 36 received lenvatinib, toripalimab plus FOLFOX-HAIC. Participants received 21-day treatment cycles of lenvatinib, toripalimab, and FOLFOX-HAIC. The primary end-point was the progression-free survival (PFS) rate per RECIST at six months.

      Results

      Thirty-six participants (86.1% with high-risk features) were enrolled in our study. The primary end-point was met with a PFS rate of 80.6% (95% CI, 64.0%–91.8%) at six months. The median PFS was 10.4 months (95% CI, 5.8–15.0), and the median OS was not reached at the prespecified final analysis and was 17.9 months (95% CI, 14.5–21.3) after follow-up was extended. The ORR per RECIST was 63.9%, and per mRECIST was 66.7%. The median duration of response was 14.4 months (95% CI, 8.9–19.9). The most common adverse events were thrombocytopenia, elevated aspartate aminotransferase, and hypertension, and no treatment-related death was reported. Participants with low levels of both CCL28 and BTC had unsatisfactory prognosis.

      Conclusions

      Lenvatinib, toripalimab and FOLFOX-HAIC showed safe and encouraging antitumour activity for advanced HCC with high-risk features. The levels of CCL28 and BTC might be the predictive biomarkers for the triple combination therapy.

      Keywords

      Abbreviations:

      BTC (Betacellulin), CCL28 (C-C motif chemokine ligand 28), FOLFOX (Oxaliplatin, leucovorin, and 5-fluorouracil), HAIC (Hepatic arterial infusion chemotherapy), HCC (Hepatocellular caricinoma), PD-1 (Programmed death receptor-1)

      1. Introduction

      Hepatocellular carcinoma (HCC) has been the fourth leading cause of cancer [
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • Siegel R.L.
      • Torre L.A.
      • Jemal A.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      ], and approximately half of the patients are diagnosed with advanced HCC [
      • Kim B.H.
      • Lim Y.S.
      • Kim E.Y.
      • Kong H.J.
      • Won Y.J.
      • Han S.
      • et al.
      Temporal improvement in survival of patients with hepatocellular carcinoma in a hepatitis B virus-endemic population.
      ]. Atezolizumab plus bevacizumab was recommended as the preferred first-line treatment for advanced HCC by the National Comprehensive Cancer Network (NCCN) Guidelines [
      • Finn R.S.
      • Qin S.
      • Ikeda M.
      • Galle P.R.
      • Ducreux M.
      • Kim T.Y.
      • et al.
      Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma.
      ,
      • Benson A.B.
      • D'Angelica M.I.
      • Abbott D.E.
      • Anaya D.A.
      • Anders R.
      • Are C.
      • et al.
      Hepatobiliary cancers, version 2.2021, NCCN clinical practice guidelines in oncology.
      ]. In other clinical trials such as KEYNOTE-524 trial and RESCUE study, systemic treatments combining tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) also displayed promising antitumour activity [
      • Finn R.S.
      • Ikeda M.
      • Zhu A.X.
      • Sung M.W.
      • Baron A.D.
      • Kudo M.
      • et al.
      Phase Ib study of lenvatinib plus pembrolizumab in patients with unresectable hepatocellular carcinoma.
      ,
      • Xu J.
      • Shen J.
      • Gu S.
      • Zhang Y.
      • Wu L.
      • Wu J.
      • et al.
      Camrelizumab in combination with apatinib in patients with advanced hepatocellular carcinoma (RESCUE): a nonrandomized, open-label, phase II trial.
      ].
      However, a high tumour burden was usually associated with a poor outcome of systemic therapy for advanced HCC [
      • Chen S.-C.
      • Chao Y.
      • Chen M.H.
      Lenvatinib for the treatment of HCC: a single institute experience.
      ,
      • Tsuchiya K.
      • Kurosaki M.
      • Marusawa H.
      • Abe T.
      • Ogawa C.
      • Sohda T.
      • et al.
      The efficacy and safety of lenvatinib in patients who did not meet the inclusion criteria of the phase 3 trial (REFLECT trial) in real-world practice in Japan: a nationwide multicenter study in Japan.
      ,
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ,
      • Breder V.V.
      • Vogel A.
      • Merle P.
      • Finn R.S.
      • Galle P.R.
      • Zhu A.X.
      • et al.
      IMbrave150: Exploratory efficacy and safety results of hepatocellular carcinoma (HCC) patients (pts) with main trunk and/or contralateral portal vein invasion (Vp4) treated with atezolizumab (atezo) + bevacizumab (bev) versus sorafenib (sor) in a global Ph III study.
      ]. There is still an unmet need for patients with high tumour burden, especially for patients with high-risk features (Vp4, and/or bile duct invasion and/or tumour occupancy of ≥50% of the liver). The outcome data about high-risk patients were limited, because they had been usually excluded from previous trials [
      • Finn R.S.
      • Ikeda M.
      • Zhu A.X.
      • Sung M.W.
      • Baron A.D.
      • Kudo M.
      • et al.
      Phase Ib study of lenvatinib plus pembrolizumab in patients with unresectable hepatocellular carcinoma.
      ,
      • Xu J.
      • Shen J.
      • Gu S.
      • Zhang Y.
      • Wu L.
      • Wu J.
      • et al.
      Camrelizumab in combination with apatinib in patients with advanced hepatocellular carcinoma (RESCUE): a nonrandomized, open-label, phase II trial.
      ]. Recently, the updated IMbrave150 showed that atezolizumab plus bevacizumab has limited benefit in these patients with a median overall survival (OS) of 7.6 months [
      • Breder V.V.
      • Vogel A.
      • Merle P.
      • Finn R.S.
      • Galle P.R.
      • Zhu A.X.
      • et al.
      IMbrave150: Exploratory efficacy and safety results of hepatocellular carcinoma (HCC) patients (pts) with main trunk and/or contralateral portal vein invasion (Vp4) treated with atezolizumab (atezo) + bevacizumab (bev) versus sorafenib (sor) in a global Ph III study.
      ].
      To improve the survival of patients with high-risk advanced HCC, combination of locoregional therapies with systemic therapies were suggested [
      • Zhou J.
      • Sun H.
      • Wang Z.
      • Cong W.
      • Wang J.
      • Zeng M.
      • et al.
      Guidelines for the diagnosis and treatment of hepatocellular carcinoma (2019 edition).
      ,
      • Park J.W.
      • Chen M.
      • Colombo M.
      • Roberts L.R.
      • Schwartz M.
      • Chen P.J.
      • et al.
      Global patterns of hepatocellular carcinoma management from diagnosis to death: the BRIDGE Study.
      ]. Locoregional therapies can effectively reduce the tumour burden and expose tumour antigens [
      • Zhou J.
      • Sun H.
      • Wang Z.
      • Cong W.
      • Wang J.
      • Zeng M.
      • et al.
      Guidelines for the diagnosis and treatment of hepatocellular carcinoma (2019 edition).
      ,
      • Park J.W.
      • Chen M.
      • Colombo M.
      • Roberts L.R.
      • Schwartz M.
      • Chen P.J.
      • et al.
      Global patterns of hepatocellular carcinoma management from diagnosis to death: the BRIDGE Study.
      ,
      • Galluzzi L.
      • Buque A.
      • Kepp O.
      • Zitvogel L.
      • Kroemer G.
      Immunogenic cell death in cancer and infectious disease.
      ]. Furthermore, chemotherapeutic agents have been shown to exert a synergistic anticancer effect with TKIs and ICIs in preclinical studies [
      • Lesterhuis W.J.
      • Punt C.J.
      • Hato S.V.
      • Eleveld-Trancikova D.
      • Jansen B.J.
      • Nierkens S.
      • et al.
      Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice.
      ,
      • Mathew M.
      • Enzler T.
      • Shu C.A.
      • Rizvi N.A.
      Combining chemotherapy with PD-1 blockade in NSCLC.
      ,
      • Liu W.
      • Fowler D.
      • Smith P.
      • Dalgleish A.
      Pre-treatment with chemotherapy can enhance the antigenicity and immunogenicity of tumours by promoting adaptive immune responses.
      ].
      We had demonstrated that patients with advanced HCC and high tumour burden receiving sorafenib plus hepatic arterial infusion chemotherapy with oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX-HAIC) had a significantly longer median OS (13.37 versus 7.13 months) and acceptable safety profiles compared with patients receiving sorafenib alone [
      • He M.
      • Li Q.
      • Zou R.
      • Shen J.
      • Fang W.
      • Tan G.
      • et al.
      Sorafenib plus hepatic arterial infusion of oxaliplatin, fluorouracil, and leucovorin vs sorafenib alone for hepatocellular carcinoma with portal vein invasion: a randomized clinical trial.
      ]. In addition, one of our retrospective study suggested that compared with lenvatinib alone, the combination therapy of lenvatinib, toripalimab (a recombinant, humanised programmed death receptor-1 (PD-1) monoclonal antibody), and FOLFOX-HAIC had acceptable toxic effects and improved progression-free survival (PFS) and the response rate in advanced HCC [
      • He M.-K.
      • Liang R.-B.
      • Zhao Y.
      • Xu Y.-J.
      • Chen H.-W.
      • Zhou Y.-M.
      • et al.
      Lenvatinib, toripalimab, plus hepatic arterial infusion chemotherapy versus lenvatinib alone for advanced hepatocellular carcinoma.
      ].
      Herein, we aimed to prospectively explore the efficacy, safety and predictive biomarkers of lenvatinib, toripalimab, plus FOLFOX-HAIC as the first-line treatment in advanced HCC with high-risk.

      2. Material and methods

      2.1 Study design and participants

      We performed this phase II, single-arm trial at the Sun Yet-sen University Cancer Centre. The trial protocol is available in Supplement 1. The trial was approved by the Research Ethics Board of Sun Yet-sen University Cancer Centre and was performed in accordance with the Declaration of Helsinki. All participants provided written informed consent. This study is registered with Clinicaltrials.gov, NCT04044313.
      The key inclusion criteria were 18 years or older, HCC with the diagnosis based on the American Association for the Study of Liver Diseases practice guidelines [
      • Heimbach J.K.
      • Kulik L.M.
      • Finn R.S.
      • Sirlin C.B.
      • Abecassis M.M.
      • Roberts L.R.
      • et al.
      AASLD guidelines for the treatment of hepatocellular carcinoma.
      ], no previous treatment, vascular invasion or extrahepatic metastasis and had measurable disease according to Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1 [
      • Eisenhauer E.A.
      • Therasse P.
      • Bogaerts J.
      • Schwartz L.H.
      • Sargent D.
      • Ford R.
      • et al.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      ]. The detailed inclusion and exclusion criteria were shown in the eMethods in Supplement 2.

      2.2 Procedures

      Participants initiated lenvatinib 3–7 days prior to initial HAIC to confirm tolerability and then underwent 21-day treatment cycles of lenvatinib, toripalimab, and FOLFOX-HAIC. A catheter will be superselectively placed into the feeding arteries of the tumour and the tumour thrombus. And the patients were transferred to inpatient ward for drug infusion via the hepatic artery. After HAIC was completed, the catheter and sheath were removed. The detailed procedures were written in eMethods in Supplement 2. The criteria for dose reduction, interruption and discontinuation of therapy, and assessment of tumour response and toxic effects are shown in the eMethods in Supplement 2.

      2.3 Outcomes

      The primary end-point was the PFS rate at 6 months, which was defined as the proportion of participants alive, assessable, and free from progression at 6 months. The secondary end-points were OS, PFS, objective response rate, and adverse events (AE). The definitions of outcomes were written in the eMethods in Supplement 2. Safety was evaluated according to NCI CTCAE Version 4.03. The immune-related adverse events (irAEs) were evaluated according to the consensus recommendations from the Society for Immunotherapy of Cancer (SITC) Toxicity Management Working Group [
      • Puzanov I.
      • Diab A.
      • Abdallah K.
      • Bingham 3rd, C.O.
      • Brogdon C.
      • Dadu R.
      • et al.
      Managing toxicities associated with immune checkpoint inhibitors: consensus recommendations from the Society for Immunotherapy of Cancer (SITC) Toxicity Management Working Group.
      ]. Tumour assessments were evaluated by two independent radiologists and the best responses were documented. All responses were confirmed by a second assessment after 4 weeks. All observations pertinent to the safety of the study medication were recorded on the case report form and included in the final report.

      2.4 Statistical analysis

      We used Simon's two-stage design with a one-sided α error of 5% and a power of 80% [
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      ], and the sample size was 36. The detail was written in the eMethods in Supplement 2. Three populations were used to analyse the data: the intention-to-treat population, the per-protocol population, and the safety population. The definitions of three populations were written in the eMethods in Supplement 2.
      We used SPSS (version 25.0) for all analyses. We calculated the PFS rate at 6 months, and the ORR with the 95% CIs was calculated by using the Clopper-Pearson method. The OS and PFS with associated 95% CIs were analysed by the Kaplan–Meier method.

      2.5 Quantitative measurement of human cytokines, lymphocyte enumeration and ELISA

      Peripheral blood samples were collected before and after two cycles of combination therapy. Peripheral blood mononuclear cells (PBMCs) were then isolated and used for flow cytometry. The quantitative measurement of cytokines using the The Quantibody Human Chemokine Array and the ELISA Kit were then performed according to the manufacturer's instructions. The details are written in the eMethods in Supplement 2.

      3. Results

      Between 1st August 2019 and 21st May 2020, 51 participants were screened. Of these, 36 participants were enrolled and received study treatment (Fig. 1). Two participants were excluded from the per-protocol population because they lacked post-baseline efficacy assessment data. The data cutoff point was February 1, 2021. The baseline characteristics are shown in Table 1. 31 participants (86.1%) were with high-risk features. All participants had macrovascular invasion or extrahepatic spread, including 86.1% of participants with portal vein invasion and 27.8% with extrahepatic metastasis. Participants infected with hepatitis B virus (88.9%) were given preemptive antiviral therapy.
      Table 1Baseline characteristics of intention-to-treat population (n = 36).
      CharacteristicsPatients
      Gender
       Male33 (91.7%)
       Female3 (8.3%)
      Age
       Median (IQR)49 (39–57)
       Mean (SD)48.5 (11.6)
      ECOG score
       012 (33.3%)
       120 (55.6%)
       24 (11.1%)
      Child-Pugh
       525 (69.4%)
       611 (30.1%)
      Hepatitis B infection
       Yes32 (88.9%)
       No4 (11.1%)
      Tumour size, cm
       Median (IQR)11.2 (8.4–12.9)
       Mean (SD)10.9 (4.7)
      ≤1012 (33%)
      >1024 (66.7%)
      Tumour number
       ≦35 (13.9%)
       >331 (86.1%)
      Degree of PVTT
       Vp 05 (13.9%)
       Vp 1-27 (19.4%)
       Vp 311 (30.6%)
       Vp 413 (36.1%)
      Extrahepatic spread
       No26 (72.2%)
       Yes10 (27.8%)
      AFP, ng/ml
       ≤40017 (47.2%)
       >40019 (52.8%)
      Four patients without HBV infection were excluded.
      HBV-DNA level, median (IQR), IU/ml
      1850 (166–13,600)
      ALT, median (IQR), U/L43 (29.6–60.2)
      AST, median (IQR), U/L72.3 (41.9–125.2)
      ALB, median (IQR), g/L39 (35.4–41.8)
      TBil, median (IQR), μmol/L14.9 (12.7–22)
      High-risk patients
      High-risk patients were defined as those who had tumour invasion of the main trunk of the portal vein and/or the portal vein branch contralateral to the primarily involved lobe (Vp4), and/or bile duct invasion and/or tumour occupancy of ≥50% of liver.
       Yes31 (86.1%)
       No5 (13.9%)
      a Four patients without HBV infection were excluded.
      b High-risk patients were defined as those who had tumour invasion of the main trunk of the portal vein and/or the portal vein branch contralateral to the primarily involved lobe (Vp4), and/or bile duct invasion and/or tumour occupancy of ≥50% of liver.
      Treatment administration is shown in eTable 1 in Supplement 2. The median study treatment duration for participants was 8.2 months. Eight participants achieved to be converted to resectable disease (eTable 2 in Supplement 2).

      3.1 Efficacy

      In the first ten participants enrolled, the PFS of seven participants was longer than 6 months which allowed the trial continued to stage two. Among 36 participants, the PFS of 29 participants (80.6%, 95% CI, 64.0%–91.8%) was longer than 6 months, and the primary end-point was met, with the median PFS of 10.4 months (95% CI, 5.8–15.0, Fig. 2B). The median OS was not reached at the prespecified final analysis and was 17.9 months (95% CI, 14.5–21.3, Fig. 2A) after follow-up was extended to 28th February 2022. Additionally, the OS was 17.4 months (95% CI, 12.2–22.6) and PFS was 10.4 months (95% CI, 6.4–14.4) in patients with high-risk features.
      Fig. 2
      Fig. 2Kaplan–Meier curves of progression-free survival (A, intention-to-treat population, n = 36), overall survival (B, intention-to-treat population, n = 36), and duration of response (C, patients with partial response, n = 23).
      Tumour response is shown in Table 2. In the intention-to-treat population (n = 36), a confirmed objective response was achieved in 23 (63.9%) participants. The median time to achieve response was 1.97 months (range, 1.8–4.03 months), and the reductions were enormous and durable (Fig. 3A and B). The median duration of response (DOR) was 14.4 month (95% CI, 8.9–19.9, Fig. 2C). In addition, 24 (66.7%) participants achieved a confirmed objective response based on the mRECIST, including five who achieved a complete response (CR). The tumour response of the per-protocol population was written in eResults in Supplement 2. Imaging scans of 12 representative participants are shown in Supplement 3.
      Table 2Tumour responses.
      Intention-to-treat population (n = 36)Per-protocol population (n = 34)
      Two patients without post-baseline efficacy assessments were excluded.
      RECISTmRECISTRECISTmRECIST
      Complete response05 (13.9%)05 (14.7%)
      Partial response23 (63.9%)19 (52.8%)23 (67.6%)19 (55.9%)
      Stable disease9 (25%)8 (22.2%)9 (26.5%)8 (23.5%)
      Progressive disease2 (5.6%)2 (5.6%)2 (5.9%)2 (5.9%)
      Not assessment2 (5.6%)2 (5.6%)
      Objective response rate23 (63.9%)24 (66.7%)23 (67.6%)24 (70.6%)
      Disease control rate32 (88.9%)32 (88.9%)32 (94.1%)32 (94.1%)
      Data are n (%) and n (%; 95% CI).
      a Two patients without post-baseline efficacy assessments were excluded.
      Fig. 3
      Fig. 3Antitumour activity. Patients in the efficacy-evaluable population were included (n = 34). (A) Best percentage change from baseline in intrahepatic target lesion per RECIST 1.1. The dashed line at −30% change represents the partial response. (B) Best percentage change from baseline in intrahepatic target lesion per mRECIST. The dashed line at −30% change represents the partial response. (C) Duration of treatment and response assessments by RECIST 1.1. The length of each bar represents the duration of treatment for each patient.

      3.2 Safety

      We analysed the safety data of all 36 participants (Table 3). Thirty-five participants (97.2%) in the safety population experienced at least one treatment-related adverse event. The most common treatment-related adverse events of any grade were anaemia (50%), thrombocytopenia (52.8%), elevated alanine aminotransferase (63.2%), elevated aspartate aminotransferase (66.7%), and hypoalbuminaemia (69.4%). Treatment-related grade 3 or 4 adverse events occurred in 26 (72.2%) participants, and the most common adverse events were thrombocytopenia (13.9%), elevated aspartate amin-otransferase (13.9%), and hypertension (11.1%). Serious treatment-related adverse events were observed in 14 (38.9%) participants (eTable 3 in Supplement 2). No treatment-related deaths occurred.
      Table 3Treatment related adverse events.
      Listed are adverse events, as defined by the National Cancer Institute Common Terminology Criteria (version 4.03), that occurred in at least 5% of patients.
      Adverse eventLeToHAIC group (n = 36)
      Any grade (%)Grade 1–2Grade 3 (%)Grade 4
      Neutropenia15 (41.7%)12 (33.3%)3 (8.3%)0
      Anaemia18 (50%)17 (47.2%)1 (2.8%)0
      Thrombocytopenia19 (52.8%)14 (38.9%)4 (11.1%)1 (2.8%)
      Infection2 (5.6%)02 (5.6%)0
      Fatigue17 (47.2%)16 (44.4%)1 (2.8%)0
      Hypertension14 (38.9%)10 (27.8%)4 (11.1%)0
      Weight loss12 (33.3%)12 (33.3%)00
      Hypothyroidism10 (27.8%)6 (16.7%)00
      Hand–foot skin reaction10 (27.8%)9 (25%)1 (2.8%)
      Rash8 (22.2%)6 (16.7%)2 (5.6%)
      Nausea14 (38.9%)14 (38.9%)0
      Vomiting10 (27.8%)9 (25%)1 (2.8%)
      Diarrhoea6 (16.7%)6 (16.7%)0
      Abdominal pain14 (38.9%)13 (36.1%)1 (2.8%)
      Sensory neuropathy11 (30.6%)9 (25%)0
      Proteinuria11 (30.6%)10 (27.8%)1 (2.8%)
      Ascites4 (11.1%)2 (5.6%)2 (5.6%)0
      Elevated ALT21 (58.3%)18 (50%)3 (8.3%)
      Elevated AST24 (66.7%)19 (52.8%)5 (13.9%)
      Hyperbilirubinaemia17 (47.2%)15 (41.7%)1 (2.8%)1 (2.8%)
      Hypoalbuminaemia25 (69.4%)25 (69.4%)00
      Immune-related adverse event15 (41.7%)
       Immune-related hepatitis01 (2.8%)0
       Immune-related pneumonitis01 (2.8%)0
       Immune-related dermatitis4 (11.1%)2 (5.6%)0
       Immune-related hypothyroidism8 (22.2%)00
      Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase.
      a Listed are adverse events, as defined by the National Cancer Institute Common Terminology Criteria (version 4.03), that occurred in at least 5% of patients.
      Immune-related adverse events of any grade were observed in 15 (41.7%) participants. The most common immune-related adverse events were dermatitis (22.2%) and hypothyroidism (13.9%). Treatment-related grade 3 or 4 immune-related adverse events occurred in 4 (11.1%) participants. All immune-related adverse events disappeared after participants stopped toripalimab and received hormone therapy.
      The dose modifications are shown in eResults in Supplement 2.

      3.3 The triple combination therapy increased CCL28 level and the number of CD8+ and CD4+ T cells in peripheral blood

      We performed a preliminary experiment to examine cytokines that were up-regulated in the peripheral serum of the participants.
      The results suggested that CCL14, CCL28, TNFSF14 and CCL26 significantly up-regulated after two cycles of treatment. AXL and BTC, on the other hand, had high expression before treatment, and their concentration decreased after two cycles of treatment. These cytokines were therefore considered as potential predictive markers (Fig. 4A). Then we performed ELISA to quantify the concentration of these candidate cytokines. The serum level of CCL28 was significantly elevated and BTC was significantly decreased after two cycles of combination treatments (Fig. 4B), while other cytokines had no significant difference (data not shown). Further, The Cancer Genome Atlas (TCGA) analysis indicated that the expression of PD-1 and lenvatinib targets were significantly higher in participants with high CCL28 expression (Fig. 4C).
      Fig. 4
      Fig. 4The levels of CCL28 or BTC before the treatment might be a predictive biomarker for the efficacy of combination therapy. (A) The heatmap of quantitative measurement of human cytokines in the pre-experiment. (B) The change of peripheral blood level of CCL28 (upper) and BTC (lower) before and after the treatment. (C) The expression of PD-1 and lenvatinib target between CCL28 high group and CCL28 low group from TCGA database. (D) Kaplan–Meier curves of overall survival between CCL28 high group and CCL28 low group (cut-off value, 5.9). (E) Kaplan–Meier curves of overall survival between BTC high group and BTC low group (cut-off value, 387.8). (F) Kaplan–Meier curves of overall survival between CCL28lowBTClow group and non-CCL28lowBTClow group. (G) The number of CD8+ (left) and CD4+ (right) T cells before and after the treatment.
      Furthermore, the participants were divided according to the concentration of CCL28 or BTC. The cut-off value for CCL28 was 5.9 ng/ml and 387.8 pg/ml for BTC. The results suggested that the participants in the CCL28 high group (19.5 versus 10.8 months, p = 0.01) (Fig. 4D) and BTC high group (20.3 versus 10.4 months, p < 0.001) had longer median OS (Fig. 4E). Moreover, participants with low level of both CCL28 and BTC had unsatisfactory median OS (10.2 versus 20.3 months, p < 0.001) (Fig. 4F).
      Additionally, we performed flow cytometry and the results suggested that the number of CD8+ and CD4+ T cells were significantly elevated after the combination therapy (Fig. 4G).

      4. Discussion

      The single-arm, phase II trial investigated the combination therapy of systemic therapy (lenvatinib and toripalimab) and locoregional therapy (FOLFOX-HAIC), and it met the primary end-point, with a progression-free survival rate of 80.6% at 6 months. The triple combination therapy as the first-line treatment in patients with high-risk advanced HCC achieved a manageable toxicity profile and promising antitumour activity, which might be due to the elevation of CCL28 to increase CD4+ and CD8+ T cells and decrease of BTC.
      The results in this prospective trial were consistent with those of our retrospective study [
      • He M.-K.
      • Liang R.-B.
      • Zhao Y.
      • Xu Y.-J.
      • Chen H.-W.
      • Zhou Y.-M.
      • et al.
      Lenvatinib, toripalimab, plus hepatic arterial infusion chemotherapy versus lenvatinib alone for advanced hepatocellular carcinoma.
      ]. Notably, there were 86.1% of participants with high-risk features, and most studies excluded these participants [
      • Finn R.S.
      • Ikeda M.
      • Zhu A.X.
      • Sung M.W.
      • Baron A.D.
      • Kudo M.
      • et al.
      Phase Ib study of lenvatinib plus pembrolizumab in patients with unresectable hepatocellular carcinoma.
      ,
      • Xu J.
      • Shen J.
      • Gu S.
      • Zhang Y.
      • Wu L.
      • Wu J.
      • et al.
      Camrelizumab in combination with apatinib in patients with advanced hepatocellular carcinoma (RESCUE): a nonrandomized, open-label, phase II trial.
      ,
      • Kudo M.
      • Finn R.S.
      • Qin S.
      • Han K.H.
      • Ikeda K.
      • Piscaglia F.
      • et al.
      Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial.
      ]. Even so, participants in this study achieve a great improvement in ORR, OS, PFS, and DOR. 15 participants (41.7%) achieved PR at the initial radiographic evaluation, and five participants received post-study curative treatment.
      The powerful antitumour activity may be mainly due to the synergistic antitumour effect of lenvatinib, toripalimab, and FOLFOX-HAIC. First, lenvatinib and chemotherapy may modulate cancer immunity of tumour microenvironment, restore immunosurveillance and increase antigenicity to improve the immune response when combined with PD-1 antibodies [
      • Lesterhuis W.J.
      • Punt C.J.
      • Hato S.V.
      • Eleveld-Trancikova D.
      • Jansen B.J.
      • Nierkens S.
      • et al.
      Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice.
      ,
      • Mathew M.
      • Enzler T.
      • Shu C.A.
      • Rizvi N.A.
      Combining chemotherapy with PD-1 blockade in NSCLC.
      ,
      • Liu W.
      • Fowler D.
      • Smith P.
      • Dalgleish A.
      Pre-treatment with chemotherapy can enhance the antigenicity and immunogenicity of tumours by promoting adaptive immune responses.
      ,
      • Kato Y.
      • Tabata K.
      • Kimura T.
      • Yachie-Kinoshita A.
      • Ozawa Y.
      • Yamada K.
      • et al.
      Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8+ T cells through reduction of tumor-associated macrophage and activation of the interferon pathway.
      ,
      • Fukumura D.
      • Kloepper J.
      • Amoozgar Z.
      • Duda D.G.
      • Jain R.K.
      Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges.
      ]. Our results suggested that the triple combination therapy significantly elevated the number of CD4+ and CD8+ T cells in peripheral blood to improve immune response through the increasing of CCL28, a chemokine had been proven to chemoattract CD4+ and CD8+ T cells [
      • Wang W.
      • Soto H.
      • Oldham E.R.
      • Buchanan M.E.
      • Homey B.
      • Catron D.
      • et al.
      Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2).
      ]. Furthermore, we found CD4+ and CD8+ T cells increased more in participants with CCL28 elevated (Supplement figure). Second, lenvatinib and PD-1 inhibitors can increase chemotherapeutic drug delivery by inhibiting angiogenesis and promoting vascular normalisation [
      • Tohyama O.
      • Matsui J.
      • Kodama K.
      • Hata-Sugi N.
      • Kimura T.
      • Okamoto K.
      • et al.
      Antitumor activity of lenvatinib (e7080): an angiogenesis inhibitor that targets multiple receptor tyrosine kinases in preclinical human thyroid cancer models.
      ,
      • Shigeta K.
      • Datta M.
      • Hato T.
      • Kitahara S.
      • Chen I.
      • Matsui A.
      • et al.
      Dual programmed death receptor-1 and vascular endothelial growth factor receptor-2 blockade promotes vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma.
      ]. Our results suggested that the combination treatment also significantly decreased the level of betacellulin (BTC) which was a member of the epidermal growth factor involved in the tumour angiogenesis and progression [
      • Moon W.S.
      • Park H.S.
      • Yu K.H.
      • Park M.Y.
      • Kim K.R.
      • Jang K.Y.
      • et al.
      Expression of betacellulin and epidermal growth factor receptor in hepatocellular carcinoma: implications for angiogenesis.
      ]. Finally, the results may also be because participants with Child-Pugh class A and adequate organ function were selected so that participants could tolerate treatments.
      Our results also suggested that participants might benefit longer OS from the CCL28 high group or BTC high group. TCGA analysis indicated patients with low CCL28 expression had significantly lower expression of PD-1 and lenvatinib targets, which might affect the efficacy of lenvatinib and toripalimab. Similarly, the low levels of BTC might indicate that tumour vascular were not abundant, which might affect the efficacy of lenvatinib and the chemotherapeutic drug delivery. Therefore, the levels of CCL28 or BTC before the treatment might be a predictive biomarker for patients undergoing the triple combination treatment.
      The safety profile showed no unexpected or new toxic effects relative to single-agent lenvatinib, toripalimab, or HAIC [
      • Kudo M.
      • Finn R.S.
      • Qin S.
      • Han K.H.
      • Ikeda K.
      • Piscaglia F.
      • et al.
      Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial.
      ,
      • He M.K.
      • Le Y.
      • Li Q.J.
      • Yu Z.S.
      • Li S.H.
      • Wei W.
      • et al.
      Hepatic artery infusion chemotherapy using mFOLFOX versus transarterial chemoembolization for massive unresectable hepatocellular carcinoma: a prospective non-randomized study.
      ,
      • Wang F.H.
      • Wei X.L.
      • Feng J.
      • Li Q.
      • Xu N.
      • Hu X.C.
      • et al.
      Efficacy, safety, and correlative biomarkers of toripalimab in previously treated recurrent or metastatic nasopharyngeal carcinoma: a phase II clinical trial (POLARIS-02).
      ]. Only grade 3–4 thrombocytopenia, elevated aspartate aminotransferase and hypertension occurred in more than 10% of participants. All these adverse events were not unexpected and were alleviated by treatment interruption or dose modification. Hypertension and hand-foot skin reaction were mainly att-ributed to lenvatinib, with incidences similar to those in the REFLECT trial [
      • Kudo M.
      • Finn R.S.
      • Qin S.
      • Han K.H.
      • Ikeda K.
      • Piscaglia F.
      • et al.
      Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial.
      ]. Bone marrow suppression, gastrointestinal reaction, and liver dysfunction were mainly attributed to HAIC, consistent with our retrospective studies [
      • He M.
      • Li Q.
      • Zou R.
      • Shen J.
      • Fang W.
      • Tan G.
      • et al.
      Sorafenib plus hepatic arterial infusion of oxaliplatin, fluorouracil, and leucovorin vs sorafenib alone for hepatocellular carcinoma with portal vein invasion: a randomized clinical trial.
      ,
      • He M.K.
      • Le Y.
      • Li Q.J.
      • Yu Z.S.
      • Li S.H.
      • Wei W.
      • et al.
      Hepatic artery infusion chemotherapy using mFOLFOX versus transarterial chemoembolization for massive unresectable hepatocellular carcinoma: a prospective non-randomized study.
      ]. In addition, there were potentially overlapping toxicities among lenvatinib, toripalimab, and HAIC, such as weight loss and hypothyroidism fatigue. Notably, the grade 3–4 toripalimab-related adverse events (11.1%) reported in this study were consistent with those reported in a previous study [
      • Wang F.H.
      • Wei X.L.
      • Feng J.
      • Li Q.
      • Xu N.
      • Hu X.C.
      • et al.
      Efficacy, safety, and correlative biomarkers of toripalimab in previously treated recurrent or metastatic nasopharyngeal carcinoma: a phase II clinical trial (POLARIS-02).
      ].
      Unlike previous studies in which participants with hepatitis B virus (HBV) infection must have a viral load less than 100 IU/ml at screening [
      • Zhu A.X.
      • Finn R.S.
      • Edeline J.
      • Cattan S.
      • Ogasawara S.
      • Palmer D.
      • et al.
      Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial.
      ,
      • El-Khoueiry A.B.
      • Sangro B.
      • Yau T.
      • Crocenzi T.S.
      • Kudo M.
      • Hsu C.
      • et al.
      Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial.
      ], our study did not limit the baseline HBV-DNA load. A total of 25 participants had a baseline HBV-DNA load greater than 100 IU/ml. Immune-related hepatitis occurred in one patient with a low baseline HBV-DNA load. The results of this study showed that the PD-1 antibody is safe for participants with a high viral load at the screening when participants receive antiviral treatments, consistent with our previous study [
      • He M.K.
      • Peng C.
      • Zhao Y.
      • Liang R.B.
      • Lai Z.C.
      • Kan A.
      • et al.
      Comparison of HBV reactivation between patients with high HBV-DNA and low HBV-DNA loads undergoing PD-1 inhibitor and concurrent antiviral prophylaxis.
      ].
      This trial had several limitations. First, this was a single-arm study with no control group for comparison, and thus, selection bias could not be ruled out. Second, the small sample size and single-centre reduced the certainty of effectiveness observed, and the cut-off value of CCL28 and BTC were needed further investigation in a larger population. Third, the primary end-point was not the OS. However, we think OS may be affected by post-study treatment, and may not reflect the effect of the combined therapy. Fourth, because it is difficult to obtain HCC tissue, the number of lymphocytes in the tumour microenvironment could not be evaluated, and potential efficacy predictors such as PD-L1 expression and tumour mutational burden were not measured in this study. Finally, major participants were infected with HBV, which might affect the generalisability of the results to the broader population.

      5. Conclusions

      This phase II study showed that lenvatinib, toripalimab, and FOLFOX-HAIC were tolerable, with a promising anti-tumour activity in participants with high-risk advanced HCC. The level of CCL28 and BTC before the treatment might be the predictive biomarkers for efficacy of the combination therapy. Although limited value of HAIC as an international standard of care for advanced HCC, this study indicated that this combination strategy might be suitable as a novel first-line treatment option for this patient population and needed to be confirmed in the phase III trial.

      Author contributions

      Ming Shi: Conceptualisation, Methodology, Writing - Review & Editing, Supervision. Anna Kan: Methodology, Resources, Data Curation. ZhiCheng Lai: Formal analysis, Investigation, Data Curation, Writing - Original Draft. MinKe He: Formal analysis, Investigation, Writing - Original Draft. XiaoYun Bu: Resources, Writing - Original Draft. YuJie Xu: Resources, Writing - Original Draft. YeXing Huang: Investigation. DongSheng Wen: Investigation. QiJiong Li: Investigation. Li Xu: Investigation. YaoJun Zhang: Investigation. Wei Wei: Investigation. MinShan Chen: Investigation.

      Funding

      This work was supported by National Natural Science Foundation of China (No. 82102985, No. 82072610), Development Planned Project in Key Areas of Guangdong Province (2019B110233002), and China Postdoctoral Science Foundation (No. 2021TQ0383).

      Conflict of interest statement

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgement

      We acknowledge Junshi Biosciences for providing toripalimab. Special thanks to Mrs. QiuHua Yu for collecting the clinical samples and Mr. YiLin Shi for maintaining research data and software code used in this study.

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:
      Supplement figure
      Supplement figureChanges in the number of CD8+ (left) and CD4+ (right) T cells between CCL28 elevated group and CCL28 not elevated group.

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