There were an estimated 905,700 new cases of liver cancer, and approximately 830,200 associated deaths in 2020 worldwide (1). Globally, liver cancer has being the second most common causation of cancer-related deaths (1).

Sorafenib, a multikinase inhibitor that targets raf, platelet-derived growth factor, vascular endothelial growth factor, and tyrosine kinases (2, 3) was made offical by the Food and Drug Administration in November 2007 for the treatment of advanced liver cancer and it remained the standard of care for over a decade, prolonging the median survival of patients in clinical trials (4). In August 2018, data from the Phase III REFLECT trial (NCT01761266) have enabled the same agency to approve another small-molecule tyrosine kinase inhibitor as first-line medication of advanced liver cancer, Lenvatinib, which inhibits the activity of the vascular endothelial growth factor receptor, platelet-derived growth factor receptor α, stem cell factor receptor, fibroblast growth factor receptor, and rearrangement during transfection (5, 6). However, the median survival time of patients receiving Lenvatinib monotherapy was 13.6 months in a clinical trial (7).

ICIs are monoclonal antibodies that can block the interaction between immune checkpoint proteins and their ligands, thereby enhancing the anti-tumor immune response by preventing T cell inactivation and restoring immune recognition and immune attack. At present, it mainly includes anti-PD-1, anti-PD-L1 and anti-CTLA-4 (8). Anti-PD-1 can bind to its ligands PD-L1 or PD-L2 which expressed in various tumors, including HCC (9). It was found that pembrolizumab treatment shown promising clinical effects in patients with advanced hepatocellular carcinoma after sorafenib treatment fails in KEYNOTE-224 clinical trial. The mPFS is 4.9 months and the median overall survival is 12.9 months in this trial (10). In 2017, the PD-1 inhibitor nivolumab received accelerated approval in the United States for the second-line treatment of patients with advanced HCC after sorafenib treatment (11).

Combination of immunotherapy strategies are being developed to enhance liver tumor response to immune checkpoint inhibitors (12). The combination of tyrosine kinase inhibitors or vascular endothelial growth factor inhibitors and immune checkpoint inhibitors may enhance dendritic cell and cytotoxic T lymphocyte activity and inhibit tumor-associated macrophage, regulatory T-cell, and myeloid-derived suppressor cell regulation of the immune microenvironment, thereby creating an inflammatory microenvironment associated with relatively effective and long-lasting responses to checkpoint inhibitors (13). In a phase Ib trial (KEYNOTE-524), the mPFS and mOS of HCC patients treated with Lenvatinib and anti-PD-1 were 8.6 and 22 months, respectively (14).

However, clinical trials may not fully reflect real-world treatment efficacy, while risk factors for liver cancer may differ among populations (15). In China, the dominating causation of HCC is chronic hepatitis B virus infection. While the major risk factor for HCC in developed countries is nonalcoholic fatty liver disease (16). Furthermore, clinical trials tend to exclude patients with clear invasion into the bile duct or main portal vein. Patients who had received systemic treatment such as chemotherapy or sorafenib tend to be also excluded (7). Therefore, this retrospective study comprised 48 patients with HCC or ICC treated with Lenvatinib alone or combined with anti-PD-1, aiming to appraise the efficacy and safety of these treatments in clinical practice in China.

We included 56 cases with unresectable liver cancer treated with Lenvatinib from December 2018 to March 2021 at the Comprehensive Cancer Center of Drum Tower Hospital of Nanjing University. There were 48 patients analyzable and well-documented. A total of 21 HCC patients were managed with Lenvatinib only, while a total of 18 and 9 patients with HCC and ICC, respectively, were treated with combination therapy. The patients’ average age was 59.7 years. Eight patients were female; 6 cases of them were HCC and other 2 cases were ICC. Twenty-one (43.75%) patients had progressive disease after receiving first-line treatment; meanwhile, 27 (56.25%) patients received no systemic treatment. According to the Child-Pugh score, 4 patients had grade B liver function, of which 3 were HCC; the remaining patients had grade A liver function. Forty patients had cirrhosis at baseline, including all ICC cases and 31 HCC cases. There were 4 ICC patients and 35 HCC patients had hepatitis B virus infection, respectively. Most patients had tumor node metastasis (TNM, American Joint Commission on Cancer 8th edition) stage IV; only 11 patients had stage III disease, and all of them were HCC cases. In addition, in HCC patients, 39 cases all presented with microvascular invasion; 33 cases had tumor invasion into the macroscopic portal vein or extrahepatic spread, or both; 12 cases had lung metastases. Only one HCC patient had no liver lesions. Six HCC patients were classified as Barcelona Clinic Liver Cancer stage B; the remaining patients were classified as stage C. The specific characteristics of HCC patients are presented in Supplementary Table 1 .

Twenty patients died at the termination of the follow-up duration. The overall mOS was 22.43 months (95% confidence interval (CI) was not reached, range: 4.20-28.30 months, Figure 1 ). The 6-month, 1-year, and 18-month OS rates were 95.74% (95% CI, 84.01-98.92), 73.08% (95% CI, 57.45-83.74), and 63.22% (95% CI, 46.99-75.70), respectively. The overall mPFS was 8.93 months. (95% CI, 6.76-11.10, range: 3.20-28.30 months, Figure 1 ). The mOS and mPFS of HCC patients treated with monotherapy were 22.43 months (95% CI was not reached, Figure 2 ) and 11.60 months (95% CI, 7.46-15.74, Figure 2 ), respectively. In the HCC combination treatment group, the corresponding were 21.77 months (95% CI, 3.68-39.86, Figure 3 ) and 7.10 months (95% CI, 2.8-11.4, Figure 3 ), respectively. However, patients with ICC in the combination treatment group did not reach mOS ( Figure 4 ); meanwhile, the mPFS was 8.63 months (95% CI, 0-17.67, Figure 4 ).

Lenvatinib was approved in 2018 as a first-line treatment for advanced HCC (6). This study used real-world data to assess the clinical efficacy of Lenvatinib used with or without anti-PD-1. The present ORR of patients with HCC conducted with Lenvatinib monotherapy was lower than that reported by the REFLECT trial; however, both mOS (22.43 vs. 13.6 months) and mPFS (11.60 vs. 7.3 months) in the present research were greater than those reported by the REFLECT trial (7). However, the present mOS of combined therapy were similar to those reported by the KEYNOTE-524 trial (14), which used Lenvatinib plus pembrolizumab (an anti-PD-1 antibody) in the treatment of unresectable liver cancer (21.77 vs. 22 months). The present ORR were comparable to those reported by the KEYNOTE-524 study (50.00% vs. 36%) (14). The mPFS (7.10 vs. 8.6 months) was slightly shorter than that previously reported, which may be attributed to the later disease condition of patients treated with combination therapy than that of those treated with monotherapy.

In the present study, nine patients with ICC did not reach mOS, and their mPFS was slightly greater than that previously reported by Zhao et al. (8.63 vs. 4.9 months); the present ORR was also higher than that previously reported by these authors (44.44% vs. 25%) (16). These findings suggest that PD-1 combined with Lenvatinib may benefit patients with ICC. However, the number of ICC patients included in this study was small and the follow-up time was short. In order to make our results more credible, we will enroll more patients for analysis and extend the follow-up time of patients.

The quality of life is becoming the focus of oncology research. The incidence of serious adverse events in clinical trials of sorafenib was 52% (4); the rates associated with Lenvatinib were comparable to those associated with sorafenib (7). In phase III clinical REFLECT trial, a 12-mg therapeutic dose was used for patients weighing >60 kg; in contrast, patients weighing ≤60 kg were given a dose of 8 mg (7). Reducing the dose may delay the onset of treatment-related adverse events; however, it may affect efficacy (13). In the present study, given disease stage, large tumor burden, and the overall poor liver function of the included patients, the dose of 8 mg was used. At the end of the follow-up period, patients with body weight of > 60 kg had slightly poorer mOS and mPFS than did those with body weight of ≤60 kg; however, this difference was not statistically significant. In the present study, the 8 mg dose did not affect treatment efficacy especially in the combined therapy group. However, further studies are required to validate the present findings and elucidate the relationship between drug dosage and treatment efficacy and safety.

HCC is a complex disease with multiple pathogenic mechanisms and associated with multiple risk factors; the use of a single biomarker is insufficient for prognostication (17). AFP is the most widely used and recognized serum marker in this context. Zhang found that AFP- and ultrasound-based screening every 2 years may reduce HCC mortality by 37% (18). However, this approach remains controversial. In the present study, baseline AFP levels correlated with survival, including the mOS of HCC patients; this findings is consistent with that of the REFLECT trial. However, the associated mechanism remains unclear and further research is needed. In addition, CA19-9 is a biomarker for the diagnosis of cholangiocarcinoma (19, 20). We found that CA19-9 may have a certain relationship with the prognosis of ICC. However, due to the low incidence of cholangiocarcinoma, our center currently collects very little data, and we would expand the sample size to further support our conclusion.

At present, there are many clinical studies on lenvatinib and anti-PD-1 in the treatment of advanced liver cancer malignancies, such as NCT02579616 (21), NCT03006926 (14), NCT04044313 (22), NCT03895970 (16). There are also some retrospective analysis of the efficacy of lenvatinib in the real world (23–26). In our central study, based to the limited data available so far, it was found that Lenvatinib alone or in combination with anti-PD-1 could be an effective treatment for unresectable HCC and ICC. Moreover, baseline AFP and CA19-9 levels may contribute to predict the prognostication. However, the relationships among body weight and Lenvatinib dose and toxicity require further studies. Of course, in the future work, we will expand the sample size and extend the follow-up time to collect more data to make our conclusion more credible.

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 Ethics committee of Comprehensive Cancer Center of Drum Tower Hospital of Nanjing University. The patients/participants provided their written informed consent to participate in this study.

SZ, JShe, and BL conceived and designed the experiments. SZ, CL, YD, JShe, and JSha performed the experiments and analyzed the samples. SZ, CL, YD, and JSha analyzed the data. SZ, JShe, and BL wrote the manuscript. All authors interpreted the data, critically revised the manuscript for important intellectual contents and approved the final version.

This study was supported by National Natural Science Foundation of China (No. 81902914); Jiangsu Provincial Medical Youth Talent (No. QNRC2016043); and the Key Medical Science and Technology Development Project of Nanjing (No. ZKX16032).

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.

The handling editor HL declared a shared parent affiliation with the authors at the time of the review.

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