In 2020, the incidence of Hepatocellular carcinoma (HCC) in the world ranked sixth among malignant tumors, and the mortality rate was the third in malignant tumors (1).Most patients are already at the advanced stage at the time of diagnosis and are thus not candidates for surgical treatment due to its insidious onset (2),systemic therapy is a only potentially treatment option for patients with advanced HCC (3, 4).The SHARP trial and Asia-Pacific trial (5, 6)was shown that sorafenib can prolong overall survival over placebo and has become the standard therapy for advanced HCC. Due to its low response rate and relatively high toxicity, many patients eventually develop progressive disease or intolerance to drug. Systemic therapy for advanced HCC has changed drastically in recent years. In the phase 3 trial on regorafenib, the median PFS was 3.1 months in the sorafenib group and 1.5 months in the placebo group (P < 0.001) and the median OS was 26.0 months (7). REFLECT (8)study demonstrated that lenvatinib was non-inferior to sorafenib in overall survival in advanced HCC (median survival time: 13.6 months for lenvatinib vs. 19.6 months for sorafenib). According to the National Comprehensive Cancer Network (NCCN) Guidelines for Hepatobiliary Cancers, second-line treatment with lenvatinib or regorafenib is a valid option for advanced HCC after sorafenib failure. The emergence of immunotherapy has dramatically changed the landscape of HCC treatment. Based on the CheckMate 040 study (9) and KEYNOTE-224 study (10)show that Nivolumab and Pembrolizumab have definite curative effect on liver cancer after sorafenib treatment. With the publication of IMbrave150 (11) and KEYNOTE-524 (12)research results, immune therapy combined targeted therapy has gradually become a new treatment option. At the same time, it also brings new methods and new ideas to the second-line treatment of advanced HCC.

There are currently few data on immune checkpoint inhibitors (ICIs) combined with molecular targeted agent (MTA) in the second-line treatment of advanced HCC, therefore, we analyzed the efficacy and safety of the patients who received PD-1 inhibitors combined with lenvatinib (PL) versus those who received PD-1 inhibitors plus regorafenib (PR) after sorafenib resistance.

At present, sorafenib is still the first-line treatment of advanced HCC (15). However, the disadvantages of poor tumor control effect and relatively more toxicity are the most common cause of failure and mortality. Systemic therapy for second-line treatment of HCC has changed significantly (16), since the official approval of the molecular targeted agent (Regorafenib, Cabozantinib, Ramucirumab) and immune checkpoint inhibitors (Nivolumab and Pembrolizumab) (17, 18). But relevant studies show that their potential survival benefit is still very limited (ORR 9.2%~24.1%, PFS 3.1m~5.2m OS 8.5m~15m) (19, 20).According to the latesresearch results, ICIs combined with MTA can further improve the ORR、PFS and OS. After patients with advanced hepatocellular carcinoma were treated with atezolizumab plus bevacizumab, pembrolizumab combined with lenvatinib, pembrolizumab combined with regorafenib, nivolumab combined with lenvatinib and camrelizumab combined with apatinib, the ORR reached 33.2% ~ 54.2%,PFS 5.4m~8.6m and OS 20.3m~21.5m (21–24). Phase 1b study of lenvatinib/regorafenib plus pembrolizumab as first-line treatment for advanced HCC, which results showed that they were expected to produce better results (12, 22).And there are few data on ICIs combined with MTA in the second-line treatment of advanced HCC. Therefore, the purpose of this retrospective study was to investigate the efficacy and safety of PD-1 inhibitors plus lenvatinib and PD-1 inhibitors plus regorafenib in the treatment of advanced HCC patients who experienced disease progression during sorafenib treatment.

The clinical benefit found in the study may be attributed to the synergistic antitumor effect of PD-1 inhibitors and MTA, which can not only reshape the immune microenvironment (25, 26), but also promote the normalization of immune-competent cell functions (27–29).Lenvatinib, an inhibitor of VEGF receptors 1-3, FGF receptors 1-4, PDGF receptor α, RET, and KIT, which can normalize tumor blood vessels and destroy the hypoxic microenvironment of tumors, so as to enhance PD-1 checkpoint blocking of HCC (30, 31).Keynote-524 study (12)was conducted to explore pembrolizumab combined with lenvatinib as a first-line treatment for patients with advanced HCC. Combination treatment demonstrated that ORR of 36% and DCR of 88%, and mPFS of 9.7 months. Identically, studies also have shown that the combination of regorafenib and PD-1 inhibitors has synergistic effect, which can normalize tumor vasculature and induce sustained M1 polarization (32, 33). It plays immunosuppressive role of the tumor microenvironment (driven by regulatory T cells which may be targeted with anti-PD1) (34, 35). Safety signals and encouraging antitumor activity were also confirmed by clinical studies. The Phase Ib Trial (REGONIVO) demonstrated that the combination of regorafenib plus nivolumab had a manageable safety profile and encouraging antitumor activity in patients with gastric and colorectal cancer (36).The updated data (abstract No: 4078) published in the 2021 ASCO, Phase Ib study of regorafinib combined with Pembrolizumab in the first-line treatment of advanced HCC (NCT03347292) showed that ORR was 31% and DCR was 89%(7 had PR and 14 had SD), showing good antitumor activity and overall safe tolerance (22).

In this study, these analyses showed that using PD-1 inhibitors plus lenvatinib had better ORR and DCR than using PD-1 inhibitors plus regorafenib (ORR,12.5%vs. 10.3%, respectively, p = 0.557; DCR,71.9%vs. 58.6%, respectively; p < 0.207), but there was no statistical difference. However, median PFS and OS were not significantly different between the two groups in Kaplan-Meier survival analysis (PFS: 5.3 months vs 4.0 months, p = 0.512; OS: 14.1 months vs 13.7 months, p = 0.764 for the PL group vs PR group). Our finding is far worse than those in the RESCUE trial (37).

The study updated at the ASCO meeting in 2021, showed that camrelizumab combined with apatinib (selective VEGF 2 inhibitors) for second-line treatment of advanced HCC, with a median follow-up time of 29.1 months and an ORR of 22.5% (RECIST v1.1 standard), mOS was 21.8 months (17.3-26.8), and the 2-year OS rate was 44.6%. The patients included in RESCUE were only Child A, ECOG 0-1 score and macrovascular invasion (24.2%). However, the patients included in our study were relatively more late-staged with the majority of the patients in the PL/PR group [Child B (18.8%/10.3%), BCLC C (71.9%/79.3%), Extrahepatic spread (62.5%/79.3%)], reflecting real‐world data.

The author’s team believes that two combination regimes as the second-line therapy for patients with advanced HCC who failed sorafenib treatment can prolong survival time, compared with regorafenib or ICIs alone. The PL group seemed to have better tumor response and PFS compared with the PR group; however, their survival benefit of PL patients is not sustainable. In addition, patients in PR group have poor tolerance to regorafenib in clinical practice, which may be the latent reason for this result. In our study, it was found that the combination of locoregional treatment in the second-line treatment may not obtain survival benefit. These durations were different from the median PFS (7.4 months) and OS (14.3 months) of patients with HCC treated with regorafenib combined TACE for unresectable HCC with previous systematic treatment (38).The differences in the median PFS (4.2m vs 7.4m) outcomes between our study and previous study were likely due to differences in baseline patient characteristics. TACE combined with regorafenib included small proportions of patients with BCLC C (52.6%) or few patients with extrahepatic metastasis (34.2%), whereas our study included a larger proportion of patients with BCLC C (79.3%). Given the similar OS (14.9m vs 14.3m) associated with these combination therapies, patients with previous systematic treatment may not be ideal candidates for TACE plus systematic treatment as second line treatment. Although our outcome is currently negative, new advances in immunotherapy and locoregional therapy of HCC are paving the way for more powerful treatment strategies (39).

Treatment options using PD-1 inhibitors plus lenvatinib or regorafenib was not related to overall survival. Our study found that ECOG PS (0-1 vs 2) was an independent factor associated with mortality for patients with advanced HCC after sorafenib failure in multivariate analysis. The analyses showed that OS in the ECOG PS (0-1) group were significantly improved when compared with ECOG PS (2) group. (20.7m vs 6.8m, p<0.001). The author’s team believes that the basic physical state of patients is an important factor affecting the treatment benefits of patients in the practice of clinical practices. They have better tolerance to drugs and have the opportunity to receive locoregional treatment.

Overall, the adverse reactions incidence of two groups was similar to previous studies (12, 22).The study found that the PR group had significantly higher proportions of TRAEs than the PL group (79.3% vs 62.5%, p<0.001), with the leading four adverse events being HFSR (51.72%), hypertension (27.59%), fatigue (24.14%) and hypothyroidism (24.14%). Moreover, eight patients (10.2%) had severe TRAEs requiring permanent cessation of treatment, including five with HFSR, two with thrombocytopenia and one with RCEEP. Compared with PR group, the PL group had lower incidence of TRAEs during treatment, with hypertension (37. 5%), HFSR (28.13%) and hypothyroidism (18.75%). Only five patients (15.6%) had severe TRAE over grade 3, and presented with hypertension (n = 2,6.25%), proteinuria (n = 2,6.25%), and HFSR (3.13%). Although using PD-1 inhibitors plus regorafenib has more TRAE and poorer life quality, the long-term survival benefits appear equivalent to PR and PL in medical practices.

This article has several limitations. First of all, this is a retrospective study and the patients come from one medical center and may be affected by specific treatment practices. Secondly, the baselines of different ICIs groups are not balanced, and unable to perform subgroup analysis of immune checkpoint inhibitors used by patients. In the follow-up study, we will further explore the relationship between different types of immune checkpoint inhibitors and patient survival outcomes. Thirdly, about half of the enrolled patients using TACE treatment, and there was a certain bias in the evaluation of curative effect. Finally, the analysis of PFS or OS show no notable statistical significance due to the small sample size of the patients.

In conclusion, our results show that the combination of PD-1 inhibitors plus lenvantinib or regorafenib in the treatment of advanced HCC has acceptable survival benefits and controllable side effects, however, their PFS and OS did not differ significantly. Further research is needed on whether advanced HCC patients are combined with local treatment as second-line treatment. Multi-center, prospective, randomized controlled trials and large samples studies are needed to explore more economical and effective treatment regime in the future.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by The ethics committee of the Second Affiliated Hospital of Nanchang University. The patients/participants provided their written informed consent to participate in this study.

YX, KS and YM conceived the study. YX and SF conducted the work. YX, KS and JZ obtaining and analyzed the data. YX wrote the manuscript. YM reviewed the manuscript. All the authors listed have read and approved the manuscript. All authors contributed to the article and approved the submitted version.

This project was financially supported by the grants from the Natural Science Foundation of China (82160602).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.