Comparison of hepatitis B virus reactivation in hepatocellular carcinoma patients who received tyrosine kinase inhibitor alone or together with programmed cell death protein-1 inhibitorsComparison of hepatitis B virus reactivation in hepatocellular carcinoma patients who received tyrosine kinase inhibitor alone or together with programmed cell death protein-1 inhibitors

Abstract

Background & Aims:  Programmed cell death protein-1 (PD-1) inhibitors plus tyrosine kinase inhibitor (TKI) has dramatically improved survival of patients with advanced hepatocellular carcinoma (HCC). However, the risk of hepatitis B virus (HBV) reactivation from these antitumor medications remains unclear.

Methods: Patients receiving TIK monotherapy (TKI group) or TKI combined with PD-1 inhibitors (combination group) were included. The primary endpoint was HBV reactivation as defined by an increase in HBV DNA titer by at least 1 log(10-fold) from baseline. The secondary endpoints included tumor progression, overall survival.

Results: A total of 499 patients met the inclusion criteria, including 296 patients in the TKI group and 203 patients in the combination group. The 3-month, 6-month and 12-month cumulative incidence rates of HBV reactivation in the TKI group vs combination group were 7.8%, 12.8% and 21.3% vs 9.9%,19.2% and 30.0%, respectively (P=0.02). The Cox proportional hazard model indicated that combination therapy (HR 1.41, 95% CI 1.00-1.99, P=0.05), ALT > 40 U/L (HR 1.50, 95% CI, 1.05-2.16, P=0.03), and tumor size > 5 cm (HR 1.58, 95% CI, 1.10-2.28, P=0.01) were independent risk factors for HBV reactivation. Compared with the HBV reactivation group, the progression-free survival and overall survival of patients in the HBV non-reactivation group were significantly prolonged (P<0.001 and P=0.001).

Conclusions: Patients who received TKI combined with PD-1 inhibitors had a greater risk for HBV reactivation, and those with HBV reactivation had a higher rate of tumor progression and shorter survival time, than those receiving TKI alone.

Introduction

Primary liver cancer ranks sixth among the most common cancers in the world(1). Approximately 54% of HCC cases are related to HBV(2). HBV reactivation is common in patients on immunosuppressive medications and on conventional cytotoxic chemotherapy, and it leads to complications including hepatitis as well as interruption of antitumor treatment(3). Although relevant guidelines indicate that patients with HBV-related HCC receiving systemic anticancer treatments should also receive antiviral prophylactic therapy(4), the existing antiviral drugs cannot completely eliminate covalently closed circular (ccc) DNA in chronic HBV patients (5), and HBV reactivation may still occur (6). Even patients who have HBsAg seroclearance may have HBV reactivation during the use of immunosuppressant medications(7).

In recent years, various anti-programmed cell death 1 (PD-1) and anti-programmed cell death ligand 1 (PD-L1) reagents have been used to treat patients with HCC. However, reactivation of HBV has continued to be a concern for clinicians during the use of antitumor therapy. Many studies have shown that HBV reactivation occurs in some cancer patients (3.5%~6.4%) after PD-1 inhibitors therapy (8–10). In addition, the definition of HBV reactivation used in above studies according to the American Association for the Study of Liver Diseases (AASLD) 2018 hepatitis B guidelines was as follows: (i) a ≥ 2 log (100-fold) increase in HBV DNA compared with the baseline level and (ii) HBV DNA ≥ 3 log (1000) IU/mL in a patient with a previously undetectable level (since HBV DNA levels fluctuate)(11). Whereas, before 2018, the definition of reactivation used in most studies was based on an increase in HBV DNA ≥ 1 log or ≥ 2 log⁽¹²⁾, but relevant research data using these definitions are not reported for HCC patients receiving PD-1 inhibitors.

At present, the effect of PD-1 inhibitors on HBV reactivation in HCC patients is unclear, as previous studies with PD-1 inhibitors used only one definition of HBV reactivation (AASLD), did not include a control group, or evaluated quantitative changes in HBsAg. In view of this problem, we sought to investigate the HBV reactivation rate (by different definitions of HBV reactivation) and the HBsAg seroclearance rate in HCC patients treated with TKI alone or TKI combined with PD-1 inhibitors. We also attempted to evaluate the possible effects of tumor progression and overall survival (OS) on HBV reactivation.

Methods

Study design and participants

This was a retrospective study conducted at the Fifth Medical Center of the General Hospital of the Chinese People's Liberation Army in China. This study screened all HCC patients who visited our hospital from March 2017 to March 2021. The inclusion criteria were as follows: (1) HCC diagnosed by pathology or by two radiologic imaging techniques confirming HCC; (2) patients who received PD-1 inhibitors combined with TKI or TKI alone for at least one month; (3) patients seropositive for HBsAg; and (4) continued use of anti-HBV medications during antitumor therapy. The exclusion criteria were as follows: (1) the presence of other positive viral markers, including IgM antibody to hepatitis A virus (HAV), antibody to HCV, IgG antibody to hepatitis D virus (HDV), IgM antibody to hepatitis E virus (HEV), or antibody to HIV; (2) lack of HBV DNA and liver function tests before and during the first HCC treatment; and (3) patients with a follow-up data interval of more than 3 months.

In addition, we detected HBV-related indicators in plasma samples of 59 patients receiving TKI or TKI plus PD-1 inhibitors, including HBsAg quantification and HBV pregenomic RNA (pg RNA). Samples were taken twice for each patient at the beginning of systematic treatment and approximately 3 months after treatment. These samples came from plasma samples taken from previous major national special research and were stored at -80 ℃. All patients signed informed consent forms and received ethical support. 

Related definitions and study endpoints

The primary endpoint in this study was HBV reactivation, which is defined as one of the following: an increase in HBV DNA by at least 1 log(10-fold) compared with baseline or HBV DNA becoming detectable in a patient with previously undetectable levels (Definition A of reactivation)(13). The secondary endpoint (Definition B of reactivation) was HBV DNA increasing by ≥ 2 log compared with baseline, and Definition C of reactivation was based on the AASLD standard(11). Another secondary endpoint was tumor progression: during the follow-up period, the tumor status at the beginning of TKI or TKI combined with PD-1 inhibitors treatment was taken as the baseline, and tumor progression was defined by RECIST version 1.1 evaluation. Tumor progression and HBV reactivation occurring simultaneously: HBV reactivation and tumor progression (compared with initial imaging findings) were found during the same hospitalization. HBsAg seroclearance is defined as the loss of HBsAg detectability at one or more instances in chronic hepatitis B (CHB) patients(14). HBsAg activation and pg RNA activation were defined as the level of HBsAg and pg RNA increasing by ≥ 10-fold compared with baseline or the absolute value increasing by ≥ 200 units.

Data collection

Baseline date was defined as the date of initiation of TKI or TKI combined with PD-1 inhibitors treatment. Baseline data included sex, age, alcohol history, smoking history and other population characteristics, serological markers, radiologic imaging results, and antiviral therapy regimens (ETV, TDF, adefovirdipivoxil (ADV), tenofovir alafenamide fumarate (TAF) and a combination of two antiviral drugs). Thereafter, HBV DNA, HBsAg and abdominal imaging results were collected during follow-up.

Sample detection

Plasma samples were detected twice in 59 patients: at the beginning of treatment and approximately 3 months after treatment. HBV DNA was measured by real-time polymerase chain reaction (Roche Cobas instrument of American Roche company) with a low detection limit of 20 IU/mL. HBsAg quantification was performed by an AU5800-03 instrument from Beckman Coulter, Inc., and the low limit of detection was 0.05 IU/mL. pg RNA was detected by the AutoSAT instrument of Shanghai Rendu Biotechnology Co., Ltd., and it can be detected from 50 copies/ml to 100 copies/ml and quantified from 100 copies/ml to 108 copies/ml.

Statistical analysis

Qualitative variables were reported as the frequency, and continuous variables were expressed as the mean ± SD or median (interquartile range [IQR]) values. Pearson χ² or Fisher’s exact test was used to detect the relationship between the two groups at baseline. The Kaplan–Meier method was used to estimate the cumulative probabilities of primary and secondary endpoints; log rank tests were used to compare the cumulative probabilities. We used Cox proportional hazards regression analyses to estimate adjusted hazard ratios (HRs). A 2-tailed P value of ≤ 0.05 defined statistical significance. All statistical analyses were performed using SPSS version 26.0 (IBM, Corp.).

Results

Patient characteristics

From March 2017 to March 2021, 2329 HCC patients were screened. A total of 499 patients met the inclusion criteria, including 296 patients in the TKI group and 203 patients in the combination group (Fig. 1). The baseline characteristics of all patients are shown in Table 1. The patients were mostly male (n = 439,78.9%), and the median age was 55 years old (IQR, 48-60). The median follow-up time in the TKI group was 6 months (IQR, 3-11), and the median follow-up time of the combination group was 5 months (IQR, 2-9). The antitumor drugs in the TKI group were mainly lenvatinib (n = 145, 48.9%) and sorafenib (n = 145, 48.9%). The combined group was dominated by lenvatinib plus sintilimab (n = 106, 52.2%) and sorafenib plus sintilimab (n = 30, 14.7%). Anti-HBV drugs were administered to all patients, mainly entecavir (n = 425, 85.1%) and tenofovir (n = 41, 8.2%). There were more local regional treatments in the TKI group than in the combination group (82.4% vs. 53.7%, P<0.001). 

HBV reactivation

Among the 499 HCC patients, 138 (27.7%) had HBV reactivation according to Definition A, 70 (14%) according to Definition B and 13 (2.6%) according to Definition C. The 3-month, 6-month and 12-month cumulative incidence rates of HBV reactivation (according to Definition A) in the TKI group and combination group were 7.8%, 12.8%, 21.3%, and 9.9%, 19.2% 30.0%, respectively (Fig. 2).

HBsAg seroclearance

During the follow-up of 499 HCC patients, only 5 patients (1%) developed HBsAg seroclearance, 2 patients received TKI, and 3 patients received TKI combined with PD-1 inhibitors (Table 2). After HBsAg seroclearance, 3 patients continued to use antiviral drugs, but all 3 had HBV reactivation, which occurred with a median onset of 20 weeks (range, 15-110 weeks). At reactivation, all patients had HBV DNA >2 log increase with the baseline undetectable even though the HBsAg remained negative.

HBV pgRNA and HBsAg quantitative analysis

Sixty-three eligible patients were selected from above cohorts with plasma samples collected before and after treatment (TKI group with 19 patients, TKI plus PD-1 inhibitors group with 44 patients). There was no significant difference in baseline characteristics between the two groups (suppl. Table 1). The incidence of HBV reactivation, pg RNA and HBsAg increasing rate in the TKI combined with PD-1 inhibitors group were higher than those in the TKI group (10.5% vs. 25%, 5.3% vs. 25%, 5.1% vs. 11.4%, respectively) (suppl. Table 2). 

Factors associated with HBV reactivation

Table 3 and suppl. Table 3 analyzed factors associated with HBV reactivation. The levels of HBV reactivation in the combination group were significantly higher than those in the TKI group no matter according to Definition A, Definition B or Definition C (P=0.02, P=0.01, P=0.04). The baseline levels of AFP (P=0.04) and ALT (P=0.01) showed significant differences only when Definition A was used. Additionally, HBsAb negativity was only significant when Definition B was used for reactivation (P=0.04). There was no significant difference in the HBV reactivation rate between patients with or without local treatment during TKI group and the combination group (suppl. Table 4).

In the multivariate analysis using Definition A for reactivation, combination therapy (HR 1.41, 95% CI 1.00-1.99, P=0.05), ALT>40 U/L (HR 1.50, 95% CI 1.05-2.16, P=0.03) and tumor diameter greater than 5 cm (HR 1.58, 95% CI 1.10-2.28, P=0.01) were independent risk factors for HBV reactivation. When Definition B was used, combined treatment (HR 1.78, 95% CI 1.09-2.92, P=0.02) and HBsAb negativity (HR 2.57, 95% CI 1.16-5.67, P=0.02) were independent factors. However, the combination of PD-1 inhibitors with TKI was the only significant risk factor for HBV reactivation when Definition C was used (HR 3.47, 95% CI 1.08-11.14, P=0.04).

HBV reactivation and prognosis

The constituent ratio of simultaneous tumor progression in patients with HBV reactivation is shown in Supplementary Table 5. Among 138 patients with HBV reactivation using Definition A, 79 (57.2%) had HBV reactivation and tumor progression at the same hospitalization, but the rates were higher in both the Definition B and Definition C groups (65.7% and 76.9%, respectively). The cumulative tumor progression rate and OS in patients with and without HBV reactivation were compared by log rank tests, and the risk curves are shown in Figure 3. The rate of tumor progression in both the Definition A and Definition B reactivation groups was significantly higher than that in the non-HBV reactivation group (P <0.001, P =0.008). Compared with the HBV reactivation group, the non-HBV reactivation group had longer median OS according to Definition A and Definition B, 20 months vs. 14 months (P=0.001), and 19 months versus 15 months (P=0.016). Due to insufficient cases, the survival curve of patients with reactivation according to Definition C (n = 13) was not drawn.

Discussion

To the best of our knowledge, this is the first study to compare HBV reactivation in HCC patients receiving TKI alone vs. TKI combined with PD-1 inhibitors. Using different definitions of HBV reactivation, we showed that TKI combined with PD-1 inhibitors was an independent risk factor. The 3-month, 6-month and 12-month cumulative incidence rates of HBV reactivation (according to Definition A) in the TKI group and combination group were 7.8%, 12.8% and 21.3% vs 9.9%, 19.2% and 30.0%, respectively. The test results of plasma samples further showed that the rate of HBsAg and pg RNA increasing were higher in the combined group.

This study shows that HBV reactivation according to Definition C was rare (2.6%), but HBV reactivation using Definition A (27.7%) was approximately 10 times higher than that of Definition C. At present, due to the availability of high-efficiency and low drug resistance antiviral drugs, HBV reactivation events have been significantly reduced. Relevant research shows that when rituximab is used in patients with HBsAg positivity with lymphoma, the rate of HBV reactivation may be reduced from 59.1–22.9% (P < 0.001) in the group receiving prophylactic antiviral therapy(15). Our results are consistent with previous studies showing that the rate of HBV reactivation in patients assessed using Definition C is very low (3.5%)(6). Therefore, it is very difficult to further analyze the risk factors associated with HBV reactivation using Definition C. We found that high levels of ALT and tumor size greater than 5 cm were independent risk factors for HBV reactivation (according to Definition A), which were not significant according to Definition C. In addition, the correlation between HBV reactivation and tumor progression and survival was also evaluated well by Definition A, and it could not be evaluated by Definition C. Therefore, HBV reactivation (Definition A) should be recommended for better management of HBV related HCC patients treated with PD-1 inhibitors.

Our study found that only high levels of ALT, negative HBsAb, tumor diameter greater than 5 cm, and combined treatment were independent risk factors for HBV reactivation. Previous studies have also shown that HBsAb negative patients are more likely to have HBV reactivation(16), which may be related to the role of HBsAb in neutralizing HBV infectivity. Other HBV reactivation relevant factors, such as male sex, older age, liver cirrhosis, high baseline HBV-DNA level and HBeAg positive, local treatment reported by previous studies were not found in our results (17).

HBV DNA load is associated with tumor recurrence and OS (18, 19). Our results further demonstrate that patients with HBV reactivation have significantly shorter median progression-free survival and OS. This relationship is considered to be the result of the joint action of tumor and HBV. On the one hand, the immunosuppressive environment of tumor progression may facilitate HBV reactivation. First, tumor progression can increase the expression of vascular endothelial growth factor (VEGF)(20), which inhibits T-cell function and dendritic cell maturation, increases the enrichment of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) that inhibit immune function, and increases the recruitment of tumor-associated macrophages and mast cells that secrete immunosuppressive factors(21). Meanwhile, tumor progression can also cause an increase in transforming growth factor-β (TGF-β) and PDL-1, which can inhibit the effects of effector T cells, natural killer cells (NK) and dendritic cells (22, 23). On the other hand, HBV reactivation (a sudden increase in HBV DNA load) may cause an increase in the inhibitory receptors Tim-3, PD-1 and exocrine signaling, which may lead to low immune function and eventually tumor progression. HBV reactivation results in an increase in HBV X protein (HBx) production(24), and HB_X may promote the development of HCC (25). Additionally, the immunosuppressive receptors mucin domain 3 (Tim-3) and PD-1 expressed by CD8 + T cells in patients with a high HBV DNA load were significantly increased (27). In addition, HBV-secreted exosomes may damage the function, proliferation and survival of NK cells(28). Tumor progression and HBV reactivation have a damaging effect on immune function, and the increase in Tregs inhibits HBV antigen specificity and the HCC tumor antigen-specific immune response(29).

The mechanism of HBV reactivation in HCC patients receiving TKI or TKI combined with PD-1 inhibitors is unclear. To evaluate the effect of TKIs on the anti-HBV effect of antiviral drugs, we performed in vitro experiments of TKIs cotreated with ETV on HepG2-NTCP cells infected by HBV virus inoculum, the results showed that there were no significant changes in HBV DNA level, intracellular HBsAg level or HBeAg level (suppl. Figure 1).

We speculate that this high HBV reactivation rate may be caused by disorder of the immune environment caused by PD-1 inhibitors. PD-1 inhibitors may cause more unbound PD-1 and PDL-1 molecules in the body and may become more severe in patients with HBV-related HCC(26). When PD-1 inhibitors cannot resist the binding of PD-1 molecules to PDL-1 molecules, the PD-1/PD-L1 axis may be reactivated, and HBV-specific T cells with high expression of PD-1 molecules may be the first to be consumed. The significant increase in the HBV reactivation rate in HCC patients treated with TKI combined with PD-1 inhibitors also may be the result of the combination of the two regimens, but it still needs to be confirmed by multi-mechanistic studies.

In 2020, the American Society of Clinical Oncology (ASCO) published a Provisional Clinical Opinion stating that patients with CHB receiving any systemic anticancer therapy should receive antiviral prophylactic therapy for the duration of anticancer therapy, but it was not recommended for patients with HBsAg seroclearance when receiving PD-1 inhibitors (4). Our results showed that HBV reactivation occurred in 3 of 5 patients with HBsAg seroclearance. This suggests that even if HBsAg seroclearance occurs in HCC patients during treatment with PD-1 inhibitors, the withdrawal of antiviral drugs should be cautious.

In conclusion, HCC patients treated with TKI combined with PD-1 inhibitors had a higher HBV reactivation rate than those treated with TKI alone. In the era of highly effective antiviral HBV drugs, the use of a low standard reactivation definition (Definition A) may be more sensitive to the analysis of risk factors for HBV reactivation and benefit for HCC prognostic management. The tumor progression rate and OS are significantly associated with HBV reactivation. Such results suggest that if HBV reactivation is found during the treatment of HCC with PD-1 inhibitors, the progression of the tumor should be assessed through imaging to adjust the therapeutic regimen in a timely fashion.

This study has several limitations. First, there is no uniform time interval for HBV DNA monitoring. Second, we did not use patients treated with a single PD-1 inhibitor as a control group, nor did we find HCC patients without any antitumor treatment for comparison. Third, our study did not include patients with HBsAg-negative advanced HCC because such patients were rarely encountered in our research center. In view of these limitations, we strongly encourage further studies to include enlargement of the patient sample size, grade the definition of HBV reactivation to explore risk factors, and optimize the monitoring, prevention and management of HBV reactivation in HBsAg-positive HCC patients receiving immunotherapy.

Declarations

Acknowledgments We thank the patients and their families.This work was supported by the Issued by the Science, Technology and Innovation Commission of Shenzhen Municipality (KCXFZ202002011006448).

Conflict of Interest Statement: The authors declare no conflicts of interest that pertain to this work

Author Contributions: JL and TY: study design, statistical analysis, and drafting of the manuscript. LZ, BC and JC: data interpretation and critical revision of the manuscript. XG, ZL and YL: study design, study supervision, and critical revision of the manuscript. SZand YL: study supervision, data interpretation, and critical revision of the manuscript. All authors approved the final submission.

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Tables

TKI, tyrosine kinase inhibitor; TKI plus PD-1 inhibitor, tyrosine kinase inhibitor plus programmed cell death protein-1 inhibitors; Tumor progression, referring to the events that occurred from the beginning of follow-up to the end of the study compared with baseline; AFP, alpha fetoprotein; ALT, alanine aminotransferase; Tumor size, maximum tumor diameter; ETV, Entecavir; TDF, Tenofovir disoproxil fumarate; TACE, Transarterial chemoembolization; BCLC, Barcelona Clinic Liver Cancer; a, Including tenofovir alafenamide fumarate(TAF, n=11), adefovirdipivoxil (ADV, n=5),ETV plus TDF(n=7),ETV plus ADV(n=8),ETV plus TAF(n=2); b, Including Two or more times of the same local treatment(n=68) or Two or more different local treatments（n=51); c, Including Lenvatinib(n=145),Sorafenib(n=145),Regorafenib（n=3),Apatinib (n=2), Anlotinib (n=1); d, Including Lenvatinib plus Sintilimab (n=106),Lenvatinib plus Pembrolizumab(n=11),Lenvatinib plus Camrelizumab (n=15),Lenvatinib plus Toripalimab (n=6),Lenvatinib plus Tislelizumab(n=2),Lenvatinib plus Atezolizumab(n=1), Lenvatinib plus Nivolumab(n=1), Sorafenib plus Sintilimab (n=30), Sorafenib plus Toripalimab (n=11), Sorafenib plus Camrelizumab (n=3), Regorafenib plus Camrelizumab (n=2), Regorafenib plus Atezolizumab(n=1), Regorafenib plus Toripalimab (n=1), Apatinib plus Toripalimab (n=1), Apatinib plus Sintilimab(n=1), Anlotinib plus Sintilimab (n=1).

M, male; ETV, Entecavir; TDF, Tenofovir; ADV, adefovirdipivoxil; HBsAg, hepatitis B surface antigen; AFP, Alpha fetoprotein; BCLC, Barcelona Clinic Liver Cancer; NA, not applicable; a, Tumor progression occurred 76 weeks after starting sorafenib treatment. After turning HBsAg seroclearance, antiviral therapy continued to be used, and the drug was stopped after 8 weeks and then during the follow-up of 80 weeks, the surface antigen remained negative without HBV reactivation; b, This patient continued to follow up for 4 weeks after turning HBsAg seroclearance, continued regular antiviral therapy, continued negative hepatitis B surface antigen and no HBV reactivation; c, Toripalimab plus Lenvatinib; d, Nivolumab plus Lenvatinib; e, Sintilimab plus Sorafenib

HR, hazard ratio; ALT, alanine aminotransferase.