Treatment with camrelizumab plus tyrosine kinase inhibitors with or without TACE for intermediate-advanced hepatocellular carcinoma: a clinical e ﬃ cacy and safety study

Objectives: This research compared the curative e ﬀ ect and safety of camrelizumab plus tyrosine kinase inhibitors (TKIs) combined with transcatheter arterial chemoembolization (TACE) to those of camrelizumab plus TKIs for the treatment of intermediate-advanced hepatocellular carcinoma (HCC). Methods: From January 2019 to July 2021, 47 patients with intermediate-advanced HCC were included in this retrospective study. After screening, 44 eligible patients were split into two arms: the camrelizumab + TKI + TACE arm (n=28) and the camrelizumab + TKI arm (n=16). The primary endpoints were overall survival (OS) and progression-free survival (PFS), while tumor response and adverse events (AEs) served as secondary endpoints. Results: The median OS was 12.60 months for the 44 patients. The median PFS (p=0.0248, 7.20 vs. 3 months), objective response rate (ORR) (21.43 vs. 6.25%) and disease control rate (DCR) (57.14 vs. 18.75%) were better for patients in the camrelizumab + TKI + TACE arm than in the camrelizumab + TKI arm. After correcting for the e ﬀ ects of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (TBil) levels, the treatment e ﬀ ect of the camrelizumab + TKI + TACE arm (HR=0.330, 95% CI=0.130 – 0.880, p=0.026) was still superior. The common AEs in the two groups included reactive cutaneous capillary hyperplasia (n=17), myelosuppression (n=19) and liver dysfunction (n=12). However, AEs of grade ≥ 3 were equal between the groups. Conclusions: Camrelizumab + TKI + TACE therapy was more e ﬀ ective than camrelizumab + TKI therapy for intermediate-advanced HCC, and toxicity was manageable.


Introduction
Primary liver cancer (PLC), which includes hepatocellular carcinoma (HCC) (75-85%), intrahepatic cholangiocarcinoma (10-15%), and other rare types, was the sixth most common cancer and the third main cause of cancer-related deaths worldwide in 2020 [1][2][3]. HCC is the predominant type of PLC; therefore, in this study, we used HCC to represent PLC. In western countries, HCC mainly develops secondary to hepatitis C virus (HCV) infection [4][5][6][7]. In China, 76 and 11% of HCC cases are caused by hepatitis B virus (HBV) and HCV, respectively, with approximately 466,000 new HCC cases and 422,000 deaths occurring each year [8,9]. Because of the latent and asymptomatic progression of HCC, the majority of patients are not diagnosed until the disease has progressed to an intermediate or advanced stage [10]. Therefore, many patients with liver cancer miss the opportunity for surgical treatment [11]. Currently, the therapeutic selections for patients with middleto late-stage HCC are limited; nonsurgical local treatment and systemic treatment are the main treatment options, which can prolong the patient's life [12].
Considerable advances in nonsurgical therapy for HCC have been reported in recent years. Medication-based therapy is a nonsurgical treatment approach. In particular, immune checkpoint inhibitors (programmed death receptor-1 [PD-1] inhibitors), including pembrolizumab, camrelizumab, and nivolumab, have emerged as promising systemic therapies for intermediate-advanced HCC [13,14]. Camrelizumab is a selective humanized high-affinity anti-PD-1 antibody that blocks the PD-1 pathway [15], and it has shown good antitumor efficacy and safety in patients with intermediateadvanced HCC in both clinical trials and real-world settings [16]. However, in a multicenter phase II clinical study, only 32 of 217 patients with HCC who were treated with camrelizumab achieved an objective response (14.70%), and the 6-month overall survival (OS) rate for all patients in the trial was 74.40% [17].
Regarding targeted drug therapy for HCC, tyrosine kinase inhibitors (TKIs), such as lenvatinib, regorafenib, cabozantinib and ramucirumab, have been further developed over the past few years and have been added to firstor second-line systemic treatment regimens [1,[18][19][20]. TKIs increase the survival rate of patients with advanced HCC, but the effect is not fully satisfactory. Studies have shown that the combination of immune checkpoint inhibitors (ICIs) with vascular endothelial growth factor (VEGF)-targeted drugs has advantages over monotherapy against many solid tumors [21]. However, the median progression-free survival (mPFS) of patients treated with ICIs plus TKIs as firstand second-line therapy is reported to be 5.70 and 5.50 months, respectively [22].
At present, nonsurgical local therapies for intermediateadvanced HCC mainly include transcatheter arterial chemoembolization (TACE), radiofrequency ablation, microwave ablation, cryoablation, and percutaneous ethanol injection. TACE is the first choice for the treatment of unresectable medium-term HCC according to the Barcelona Clinic Liver Cancer (BCLC) staging classification [23,24]. In Asian countries, TACE is widely recommended for patients with HCC presenting various clinical conditions [25] Patients with a history of targeted treatment, biotherapy, radiotherapy, or chemotherapy; 3. Patients who underwent previous surgery or local treatment; 4. Patients with incomplete clinical data; and 5. Patients who stopped treatment halfway through the protocol or who experienced a serious adverse reaction. The design and execution of this research complied with the Declaration of Helsinki, and this study was authorized by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (2022ER181-1). The requirement for informed consent was waived because the study was retrospective.

Treatment protocols
Camrelizumab was administered at a dose of 200 mg via intravenous injection every 3 or 4 weeks. A TKI (sorafenib at a single dose of 200 mg or 400 mg twice daily, lenvatinib at a single dose of 4-12 mg once daily, or apatinib at a single dose of 250 mg once daily) was administered for more than 1 month but was discontinued during TACE treatment. In the combination arm, the cycles and doses of the camrelizumab regimen were the same as those in the camrelizumab plus TKI monotherapy arm, and TACE treatment either preceded or followed camrelizumab plus TKI treatment. After the first treatment cycle, patients who tolerated the regimens well continued treatment, except for those who experienced unacceptable side effects, rapid disease progression, unexpected death or interruption of treatment for another reason.

Information collected
Clinical data, laboratory data, and imaging reports of the enrolled patients were collected prior to camrelizumab plus TKI or camrelizumab plus TKI treatment in combination with TACE. The basic clinical data included age, sex, ethnicity, BCLC stage, presence or absence of HBV, body mass index (BMI), liver function (Child-Pugh classification) and ECOG score. The laboratory detection results included the white blood cell (WBC) count, alanine aminotransferase (ALT) level, platelet count (PLT), plasma prothrombin time (PT), total bilirubin (TBil) level, alphafetoprotein (AFP) level, aspartate aminotransferase (AST) level and albumin (ALB) level. Imaging reports included the size of the tumor, number of tumors, blood vessel invasion and extrahepatic metastasis.

Endpoints and assessments
The time from the first use of camrelizumab plus TKI or camrelizumab plus TKI plus TACE to the date of last follow-up or death was defined as OS. The time from the start of camrelizumab plus TKI or camrelizumab plus TKI plus TACE treatment to the day of the first imaging diagnosis confirming tumor progression was defined as PFS. The tumor response was assessed when patients were readmitted after a treatment cycle. In accordance with the Modified Response Evaluation Criteria in Solid Tumors (mRECIST), the response of the tumor was categorized as a complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). The sum of CR and PR (CR + PR) was defined as the objective response rate (ORR). The sum of CR, PR and SD was defined as the disease control rate (DCR) (CR + PR + SD).
During treatment, adverse reactions were monitored and recorded. This study used the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE version 5.0) to assess adverse events (AEs).

Statistical analysis
Continuous variables are reported as the means ± standard deviations or as the medians (interquartile ranges), and categorical variables are presented as frequencies (percentages). Student's t test or Mann-Whitney U tests were used to assess differences in continuous variables between the two groups. Chi-square tests or Fisher's exact test were used to assess differences in the categorical variables between groups. The Kaplan-Meier approach was used to calculate OS and PFS, while differences in survival were assessed using the log-rank sum test. The

Patients
Three patients were excluded based on the inclusion and exclusion criteria (two with survival of less than 3 months and one with previous local treatment with TACE), and 44 patients were ultimately enrolled and split into the camrelizumab + TKI + TACE arm (n=28) and the camrelizumab + TKI arm (n=16) (Figure 1). Baseline characteristics of all patients are listed in Table 1. The camrelizumab + TKI arm included 13 males (81.25%) and 3 females (18.75%), the median age was 62.5 years (36-76), and the median BMI was 22.50 kg/m 2 (18.40-27.20). The camrelizumab + TKI + TACE arm included 22 males (78.57%) and 6 females (21.43%); their median age was 54 years (32-77), and their median BMI was 21.4 (18.00-34.70). Nine patients (56.25%) and 16 patients (57.14%) had a history of HBV infection in the camrelizumab + TKI arm and the camrelizumab + TKI + TACE arm, respectively. According to the ECOG scoring standard, 9, 27, and 8 patients scored zero points, one point, and two points, respectively. Extrahepatic metastases were present in 26 patients, while 18 patients did not have metastasis. Ultimately, among all treated patients, only 3 patients in the camrelizumab + TKI + TACE arm achieved downstaging and successfully underwent radical resection of liver cancer (R0). The differences in the baseline characteristics between patients in the two arms were not statistically significant (all p>0.05, Table 1).

OS and PFS
The median OS was 12.60 months for the 44 patients enrolled. The OS rates at 6 and 12 months were 80.80% and 50.30% in the camrelizumab + TKI arm and 82.10% and 49.20% in the camrelizumab + TKI + TACE arm, respectively (p=0.4827) (Figure 2A). The mPFS for all enrolled patients was 6 months. Regarding mPFS in both groups, the camrelizumab + TKI + TACE arm had a longer mPFS than the camrelizumab + TKI dual therapy arm (p=0.0248, 7.20 vs. 3.00 months) ( Figure 2B). The 6-and 12-month PFS rates were 60.70 and 55.70% in the camrelizumab + TKI + TACE arm, respectively, compared with 26.80 and 13.40% in the camrelizumab + TKI arm, respectively. Figure 3 shows no significant differences in OS between the two subgroups. As shown in Figure 4, patients who were male, positive for HBsAg, had an ECOG-PS score of 0-1, a single

Tumor response after treatment
The solid tumor response to treatment was assessed using the mRECIST criteria. In the camrelizumab + TKI arm, PR and SD were observed in 1 patient and 2 patients, respectively, yet 13 of 16 patients had PD. In the camrelizumab + TKI + TACE arm, 2 patients achieved a CR, 4 patients attained a PR, 10 patients had SD, and 12 developed PD. As shown in Table 4, the ORR and DCR were higher in the camrelizumab + TKI + TACE arm than in the camrelizumab + TKI arm (ORR: 21.43 vs. 6.25%, DCR: 57.14 vs. 18.75%).

Adverse reactions after treatment
The incidence of adverse reactions observed among the 44 enrolled patients during treatment is presented in Table 5. The most common adverse reactions in the two groups were reactive cutaneous capillary endothelial proliferation (RCCEP) (n=17), myelosuppression (n=19), and abnormal liver function (n=12). Only patients in the camrelizumab + TKI + TACE treatment arm experienced adverse reactions such as hypertension, dizziness, vomiting, gastrointestinal bleeding and renal insufficiency. The number of grade ≥3 AEs reported by patients in the camrelizumab + TKI arm was the same as that for patients in the camrelizumab + TKI + TACE arm. However, one patient in the camrelizumab + TKI + TACE arm discontinued treatment due to severe RCCEP.

Discussion
To date, many different treatment approaches have been developed and explored for intermediate and advanced HCC, yet the clinical benefits have been limited. Continuous research has found that treatment with PD-1 inhibitors in combination with other therapies provides better results than individual treatments [26][27][28][29]. In particular, the combination of PD-1 inhibitors and anti-VEGF drugs has established the efficacy of immunotherapy in this field and has become a recommended first-line treatment [30]. Our study compared camrelizumab + TKIs and camrelizumab + TKIs + TACE for the treatment of intermediateadvanced HCC. The mPFS was longer for patients in the     [31]. PD-1 inhibitors bind to PD-1 receptors on the surface of T cells and block the interactions of the receptors with PD-L1 and PD-L2, leading to the activation of T lymphocytes [32]. TKIs mainly inhibit the growth and proliferation of tumor cells by inhibiting cellular signal transduction and promoting apoptosis, thereby reducing tumor angiogenesis. However, malignant liver tumors are highly heterogeneous and prone to vascular invasion in the early stage. TACE primarily embolizes the arteries supplying blood to tumors, causing tumor ischemia and hypoxia and thereby inhibiting tumor growth and promoting tumor cell necrosis and apoptosis. After PD-1 inhibitors are administered to activate T lymphocytes and TKIs are administered to suppress angiogenesis, local TACE treatment can be provided. TACE releases large amounts of tumor antigens, thereby improving the ability of T lymphocytes to fully eradicate the tumor cells. Thus, combination therapy with camrelizumab + TKIs + TACE may benefit patients with intermediate-advanced HCC.
Notably, previous studies have indicated that age, sex, HBsAg positivity, the BCLC stage, Child-Pugh classification, ECOG score, AFP levels, degree of tumor invasion into the portal vein and extrahepatic metastasis are related to a poor prognosis [28,[33][34][35]. The results from our study are consistent with those of previous reports. However, in the current study, the single-factor analysis indicated that the WBC count was independently associated with OS and that TBil, AST, and ALT levels and treatment methods significantly affected PFS. The multifactor analysis revealed that the WBC count and AFP levels were independent prognostic factors for a poor OS outcome. In addition, we also found that treatment may be an independent factor for prolonged PFS. However, TBil levels and tumor number were independent risk factors for PFS. The results from the aforementioned analysis imply that future treatment options for intermediate-advanced HCC may be based on multiple predictive factors to identify patients who will respond well to combination therapy and to improve antitumor efficacy.
Patients with intermediate-advanced HCC who receive camrelizumab (a PD-1 inhibitor) show good responses to treatment, but the accompanying immune-related AEs may affect the prognosis. Since PD-1 inhibitors activate T lymphocytes, the adverse reactions to this treatment are different from traditional chemotherapeutic toxicity because they are immune-mediated reactions, such as rash, hypertension, colitis, hepatitis, pneumonia, nephritis, pericarditis, reduced thyroid function, and fatigue [36,37]. Studies have shown that the most common adverse reaction to camrelizumab is RCCEP (66.8%) [38]. In the present study, RCCEP accounted for 38.64% of all AEs (13.64% in the camrelizumab + TKI arm and 25% in the camrelizumab + TKI + TACE arm), and 1 patient was unable to tolerate grade 3 RCCEP and stopped treatment. TKIs bind to VEGF, block the VEGFR-2 signaling pathway and inhibit RCCEP formation [39][40][41]. However, camrelizumab + TKI + TACE treatment elicited more adverse reactions than camrelizumab + TKI treatment, including myelosuppression, abnormal liver function and abnormal renal function (13 vs. 6, 7 vs. 5, 1 vs. 0). This increase in adverse reactions might reflect the induction of additional stress by TACE treatment in vivo. The results of this study corroborate the hypothesis that camrelizumab + TKI + TACE treatment is more effective than camrelizumab + TKI treatment, with manageable side effects.
A few limitations of our research should be noted. First, our study was a retrospective study, and thus subject selection bias was unavoidable. Second, the sample size was small, and the subjects were of a single ethnicity. Third, many types of TKIs were used; no fixed, single-drug treatment was administered. Fourth, the follow-up time was short, and thus the long-term curative effects require further study. Finally, the study was conducted at a single center; therefore, in the future, larger sample sizes and long-term multicenter controlled clinical studies are needed.

Conclusions
Camrelizumab plus TKIs combined with TACE is an effective treatment strategy for patients who suffer from intermediate-advanced HCC, and the incidences of toxicity and side effects are manageable. This triple combination therapy achieves better short-term efficacy than camrelizumab plus TKIs. Thus, the findings of this study provide a valuable clinical reference.