Relationship between chronic hepatitis B virus infection and tumor progression in Chinese patients with advanced prostate cancer following combined androgen blockade therapy: a retrospective cohort study

2.1 Inclusion criteria

The inclusion criteria were as follows: (1) Patients diagnosed with advanced prostatic adenocarcinoma (locally advanced and/or distant metastases) confirmed by prostate needle biopsy, magnetic resonance imaging (MRI) of the pelvis, and computed tomography (CT) scans of the abdomen and chest, and radionuclide bone scan at the First People’s Hospital of Neijiang (Neijiang, China) between 01 January 2010, and 31 December 2023; (2) Patients with PCa received ADT using one of the following regimens: subcutaneous injection of leuprorelin (3.75 mg) every 4 weeks, or subcutaneous injection of goserelin (3.6 mg) every 4 weeks, or bilateral orchiectomy, or subcutaneous injection of goserelin (10.8 mg) every 12 weeks, and all regimens were administered in combination with oral bicalutamide (50 mg daily); and (3) Patients who underwent regular follow-up: after initiating CAB therapy, prostate-specific antigen (PSA) levels were measured monthly during the first three months. Subsequently, the follow-up interval for PSA testing was adjusted based on PSA trends: testing was performed every 3 months if levels demonstrated a stable decline, or every 2–4 weeks if an upward trend was detected. Imaging studies were scheduled according to PSA levels and the presence of symptoms (e.g., new-onset bone pain or recurrent urinary obstruction). Serum testosterone levels were measured every 3–6 months.

2.2 Exclusion criteria

The following were defined as exclusion criteria: (1) Patients lacking complete personal information; (2) Patients diagnosed with other forms of malignant tumors; (3) Patients who underwent prostatectomy after CAB treatment; (4) Patients who received other treatments (e.g., chemotherapy, radiotherapy, immunotherapy, or targeted therapy) prior to progression to CRPC; (5) Patients with viral hepatitis other than hepatitis B.

2.3 Data collection

The follow-up period was set to conclude on 01 March 2025. Based on previous literature reports [22, 23] and clinical experience, we collected the following baseline characteristics and clinical data: age; hemoglobin (Hb); initial PSA; body mass index (BMI); alanine aminotransferase (ALT); albumin; tumor-node-metastasis (TNM) clinical stage; Gleason score; bone metastasis status; smoking and drinking history; diabetes mellitus (DM); hypertension; HBsAg status; PSA nadir (nPSA) and time to PSA nadir (TTN). Additionally, the results of the hepatitis B serological panel test conducted after at least six months of CAB treatment were also collected for patients who were positive for HBsAg at baseline. For patients ultimately included in the analysis, stratification was performed based on their baseline HBsAg status, dividing them into the HBsAg-negative group and the HBsAg-positive group. HBV infection was defined by a positive HBsAg test. Patients were categorized as drinkers or smokers if they had a history of alcohol consumption or tobacco use within one year prior to their PCa diagnosis.

2.4 Endpoint selection rationale

The primary endpoint of this study was the diagnosis of CRPC, as defined by the 2016 European Association of Urology (EAU) guideline [24]. CRPC was selected as the endpoint, rather than overall survival (OS), for two reasons: (1) prior studies have demonstrated that the time from diagnosis to CRPC is a significant prognosticator of OS and thus serves as a valid surrogate endpoint [25]; (2) additionally, due to the high cost of novel ARAT and concerns about chemotherapy-related toxicities, many patients in this region did not receive effective treatment after progression to CRPC, leading to a significant increase in patients lost to follow-up.

2.5 Statistical analysis

Statistical analysis was conducted using R version 4.2.1 (http://www.Rproject.org; The R Foundation, Vienna, Austria) and the Free Statistics software (version 2.0; Beijing Free Clinical Medical Technology Co., Ltd, Beijing, China). In this study, quantitative data were analyzed using either the mean ± standard deviation (SD) or the median and interquartile range (IQR), depending on the data type. Qualitative variables were expressed as proportions (%). For quantitative data exhibiting a normal distribution and homogeneous variance, the Student’s t-test was applied. The Mann-Whitney U test was utilized for quantitative data that did not conform to a normal distribution. Chi-square tests were employed to analyze categorical data.

Time to CRPC development in the two groups was estimated using the Kaplan-Meier survival curves, and the differences were determined using the log-rank test. The association between HBV infection and PCa progression after CAB was analyzed using Cox regression models. Both non-adjusted and multivariate adjusted models were used. Subgroup analyses were conducted to investigate the stability of the association between HBV infection and progression of advanced PCa after CAB treatment. Statistical significance was assessed by comparing adjusted hazard ratios (HRs) with 1.0 and describing 95% confidence intervals (CIs). In all tests, a p-value less than 0.05 was considered statistically significant.

3.1 Patient enrollment and HBV status characterization

The detailed exclusion process is shown in Fig. 1. Ultimately, a total of 385 patients were included in this study, including 28 HBsAg-positive patients (7.27%). All 28 HBsAg-positive patients tested positive for hepatitis B core antibody (HBcAb) and negative for hepatitis B surface antibody (HBsAb). The study follow-up concluded on 01 March 2025, with a median follow-up duration of 36.3 months. Prior to the diagnosis of PCa, 75% (21/28) of patients were unaware of their HBV infection, and none of them had received oral antiviral therapy for hepatitis B. Among these 28 patients, 26 were inactive HBsAg carriers (characterized by ALT levels below 30 U/L and HBV-DNA levels not exceeding 2000 IU/mL); the remaining 2 had active chronic hepatitis B (characterized by ALT levels exceeding 30 U/L and HBV-DNA levels greater than 2000 IU/mL). Notably, color doppler ultrasound examination revealed that none of the patients had liver cirrhosis.

Fig. 1.Flow diagram of this study. PCa: prostate cancer; ADT: androgen deprivation therapy; ARAT: androgen receptor axis-targeting; CRPC: castration-resistant prostate cancer; HBsAg: hepatitis B virus surface antigen.

3.2 HBV management and follow-up outcomes

After the diagnosis of PCa, the two patients with chronic hepatitis B in the active phase received entecavir treatment; both achieved undetectable HBV-DNA levels within 6 months. None of the patients with positive HBsAg at baseline underwent liver biopsy owing a lack of clinical indications. All patients in Group 2 received regular follow-up examinations for hepatitis B serological markers. During the follow-up period, conducted every six months to one year, none of the patients experienced HBsAg seroconversion to negative.

3.3 Comparative demographics and treatment responses between groups

The demographic details of the two groups are presented in Table 1. The mean age in the HBsAg-positive group was significantly lower than that in the HBsAg-negative group (66.6 ± 5.3 years vs. 75.9 ± 7.0 years, p < 0.001). After CAB treatment, patients in the HBsAg-positive group exhibited higher nadir PSA levels (p = 0.021) and a shorter time to nadir PSA (10.5 months vs. 17.0 months, p = 0.001).

3.4 CRPC progression and PFI comparison by HBsAg status

As of the follow-up cutoff date, a total of 168 (43.6%) patients who underwent CAB had demonstrated progression to CRPC. The Kaplan-Meier survival curves of the two groups are plotted in Fig. 2. The estimated median time to CRPC, development based on the Kaplan-Meier survival analysis was 40 (95% CI, 38.0–41.9) months in the HBsAg-negative group and 19 (95% CI, 4.6–33.4) months in the HBsAg-positive group. Comparison by log-rank test showed that the progression-free interval (PFI) was significantly shorter in the HBsAg-positive group than in the HBsAg-negative group (χ² = 15.895; p < 0.001).

Fig. 2.K-M survival curves of the two groups after receiving CAB therapy. The endpoint of this study was the diagnosis of CRPC; HBsAg = 1: HBsAg-negative group; HBsAg = 2: HBsAg-positive group. HBsAg: hepatitis B virus surface antigen.

3.5 Univariate analysis

Based on univariate Cox regression analysis (Table 2), Hb, bone metastasis, age, Gleason score, albumin, initial PSA, nPSA, TTN, and HBV infection were all significantly associated with progression to CRPC (all p < 0.05). The collinearity assessment revealed that no collinearity existed among the specified independent variables.

3.6 Multivariate analysis

The findings from the multivariate Cox regression analysis are shown in Table 3. In each of the models (model I to Model IV), HBV infection demonstrated an association with an elevated risk of progression to CRPC. The corresponding HRs and 95% CIs were 2.75 (1.64–4.63), 2.93 (1.66–5.15), 2.1 (1.14–3.85), and 2.15 (1.15–4.04), respectively.

3.7 Subgroup analysis

In the subgroup analysis stratified by age, TNM stage, Gleason score, bone metastasis status, diabetes mellitus, and hypertension, the association between HBV infection and CRPC progression risk was explored in Fig. 3. No significant interactions were detected among the subgroups (All interaction p-values exceeded 0.05).

Fig. 3.Subgroup analysis of HBV-CRPC risk association. Each stratification was adjusted for all factors except the stratification factor itself. HR: hazard ratio; CI: confidence interval; TNM: tumor-node-metastasis.

3.8 Time-dependent ROC

As illustrated in Fig. 4, a time-dependent receiver operating characteristic (ROC) curve was generated. A thorough examination of this curve demonstrated that during the initial stage after the commencement of CAB therapy (for example, at t = 8 months), the HBV infection status demonstrated good discriminatory ability in predicting the progression of CRPC, as evidenced by an area under the curve (AUC) value of 0.71. However, as time progresses (such as at t = 28 months), the predictive efficacy of HBV infection status for CRPC progression declined substantially, with the AUC value decreasing to 0.55.

Fig. 4.Time-dependent ROC: HBV’s predictive efficacy change on CRPC. AUC: area under the curve.

4.1 The AR-dependent signaling pathway

The AR-dependent signaling involves the following mechanisms: amplification and overexpression of the AR gene, AR gene mutations, alterations in androgen biosynthesis, as well as the involvement of AR co-activators and expression of AR splice variants (AR-Vs). Zhu et al. [27] found that HBx was correlated with elevated AR expression in hepatocellular carcinoma (HCC) and induced AR expression by stimulating its transcription in liver cell lines. Based on this finding, we hypothesize that HBx in PCa tissues may influence the stability and activity of AR in PCa cells through the following signaling pathways:

1. It is plausible that HBx enhances AR stability and activity directly. This effect may occur by increased AR phosphorylation mediated by HBx-induced activation of the cellular Src (c-Src) kinase signaling pathway. Notably, HBx does not physically associate with ligand-bound AR in the nucleus [28].

2. It has been reported that augmented mitogen-activated protein kinase (MAPK) signaling, which is required for both ligand-dependent and ligand-independent activation of AR, is correlated with castration-resistance and metastatic progression [29]. HBx can transactivate the MAPK signaling pathway in association with the mobilization of cytosolic Ca²⁺, and the activated MAPK further activates AR [30].

3. In human PCa cells, AR can be activated by interleukin-6 (IL-6) even in the absence of androgens [31]. It has been found that serum levels of IL-6 are significantly higher in patients with acute and chronic hepatitis B compared to those in healthy individuals [32]. Elevated IL-6 concentrations may promote the occurrence of CRPC through multiple pathways: activation of the MAPK signaling pathway leading to AR activation [31]; overexpression of IL-6 increases PCa cell resistance to bicalutamide via transcriptional intermediary factor 2 (TIF2) [33]; and it can also directly activate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway to enhance AR stability and activity [34].

4. Seo et al. [35] reported that in the absence of androgens, the Wnt/β-catenin pathway can activate AR-mediated transcription by up-regulating the Hippo pathway effector Yes-associated protein (YAP). Furthermore, the Wnt/β-catenin pathway crosstalks with the androgen pathway, so β-catenin signaling therefore confers resistance to androgen inhibitors [36]. Many studies have shown that HBx can activate the Wnt/β-catenin signaling pathway and promote β-catenin accumulation through multiple pathways [37].

5. Glioma-associated oncogene homolog 2 (GLI2), a transcription factor in the Hedgehog (Hh) pathway, can potentially serve as a co-activator of AR [38]. Overexpression of GLI2 in lymph node carcinoma of the prostate (LNCaP) cells enhanced AR-specific gene expression in the absence of androgen [38]. Li et al. [39] also found that GLI2 may function by supporting the androgen signaling pathway in CRPC cells through the co-activation of full-length AR and its splice variants. HBx has been found to upregulate the level of GLI2 mRNA and increase the stability of GLI2 in liver cancer cells [40].

4.2 The AR-independent signaling pathway

AR-independent signaling pathways encompass several key mechanisms, including epithelial-mesenchymal transition (EMT), inactivation of the phosphatase and tensin homolog (PTEN) gene, activation of the PI3K/AKT signaling pathway, activation of the Wnt/β-catenin pathway, as well as the production and self-renewal of PCSCs [41].

1. EMT contributes to the development of CRPC through two distinct pathways: the Yes-associated protein 1 (YAP1) pathway, where YAP1, a crucial protein within the Hippo pathway, plays a significant role in promoting the transcriptional regulation of EMT and may induce the emergence of cancer stem cells (CSCs) [42], with studies reporting that overexpression of YAP1 can drive cell proliferation, castration-resistant growth, invasion, and enhance the metastatic potential of PCa cells [43], notably, HBx has been shown to up-regulate YAP1 expression in liver cancer cells to facilitate their growth [44], and moreover, Huang et al. [18] discovered that in HBsAg-positive NPC samples, HBx could upregulate YAP1 expression to further promote cell invasiveness and metastasis via EMT; and the signal transducer and activator of transcription 3 (STAT3) pathway, in which STAT3 can be activated through multiple signaling cascades, with sustained activation of STAT3 in PCa cells inducing EMT and thus increasing their metastatic and invasive capabilities [45], Liu et al. [46] demonstrated that persistent activation of STAT3 in PCa cells leads to the development of CRPC while downregulation of STAT3 expression restores the sensitivity of PCa cells to chemotherapy. In liver cells, HBx can activate STAT3 and subsequently promote EMT [47].

2. The PI3K/AKT pathway is negatively regulated by the PTEN tumor suppressor [48], and loss of PTEN or activation of the PI3K/AKT pathway results in enhanced cell proliferation, survival, migration, and castration-resistant growth in PCa [49]. HBx is thought to lead to PTEN inactivation and activation of the PI3K/AKT pathway through the following mechanisms: Firstly, HBx directly interacts with mitochondrial membrane proteins, which may then increase the levels of mitochondrial reactive oxygen species (ROS) and lipid peroxide production [50]; by promoting the oxidation of cysteine residues within PTEN, the ROS induced by HBx can inactivate PTEN and enhance the function of AKT [51]. Secondly, HBx may cause an increase in IL-6 concentration, and elevated IL-6 levels could further activate the PI3K/AKT pathway [34].

3. PCSCs, which exhibit unique characteristics of self-renewal and multilineage differentiation, have been documented to possess intrinsic resistance to various therapies, encompassing resistance to castration, chemotherapy, and radiotherapy [52]. It has been observed that PCSCs either do not express or exhibit extremely low levels of AR expression, and their proliferation is independent of AR [53]. ADT, along with the suppression of AR expression or activity, has been demonstrated to reprogram bulk PCa cells, leading to the generation of AR-/lo PCSCs [41, 54]. It has been established that numerous signaling pathways, including PI3K/AKT, Notch, hedgehog/GLI, Wnt/β-catenin, and STAT3, may positively regulate the properties of PCSCs [55]. Given that the PI3K/AKT signaling pathway has been previously discussed, the remaining signaling pathways will be examined in detail below, with a focus on causal interpretations and speculations: (1) Notch signaling pathway: In the normal prostate, Notch signaling is thought to govern the differentiation state and structural organization of the gland. Aberrant Notch expression within tissues may promote the development of PCa, with expression levels increasing alongside cancer grade [56]. It has been speculated that HBx within the body could activate the Notch pathway, triggering apoptosis and abnormal cell cycle alterations, ultimately culminating in liver cancer progression [57]. (2) Hedgehog/GLI Signaling Pathway: The Hedgehog signaling pathway is instrumental in maintaining stem cell populations, facilitating tissue repair, and promoting regeneration in normal adult tissues. Aberrant activation of the Hedgehog pathway has been noted in various cancers [58]. With respect to PCa, there is mounting evidence suggesting that Hedgehog signaling plays a pivotal role in the disease’s progression towards more aggressive and therapy-resistant states [59]. Glioma-associated oncogene homolog 1 (GLI1), a crucial transcriptional activator of the Hedgehog signaling pathway, when upregulated, fosters the growth and proliferation of these CSCs, such as esophageal squamous cell carcinoma (ESCC), lung squamous cell carcinoma (LSCC), ductal breast carcinoma (DBC), and PCa [60]. It has been established that in HBV-induced liver cancer, HBx activates the Hedgehog pathway through diverse mechanisms: Firstly, HBx can upregulate the expression of GLI1 and GLI2 mRNA and proteins, which serve as master regulators of Hedgehog signaling [40]. Secondly, HBx enhances the protein stability of GLI1 and GLI2 and induces the nuclear translocation of GLI1 through direct protein-protein interaction between HBx and GLI1 [40]. (3) Wnt/β-catenin signaling pathway: wingless-type MMTV integration site family (Wnt) proteins secreted by the tumor stroma contribute to therapy resistance [61]. Moreover, inhibition of androgen activity leads to upregulation of the Wnt/β-catenin pathway, which subsequently promotes androgen-independent growth of PCa cells [36]. In liver cancer cells, HBx activates the Wnt/β-catenin signal pathway through multiple routes, thereby facilitating the initiation, invasion, and metastasis of liver cancer [37]. (4) STAT3 signaling pathway: The loss of AR expression triggers STAT3 activation in PCa cells, which further leads to the development of PCSCs [54]. The activation of STAT3 induced by AR downregulation is mediated by a high concentration of IL-6 induced by HBx [47, 54].

In addition to the aforementioned factors, several other mechanisms may also contribute to the development of CRPC promoted by HBV infection. First, HBx suppresses the expression of tumor suppressor genes, such as p53, and inhibits the function of negative growth regulators [62]. Second, HBV-DNA integration into the host cell genome occurs at various stages of infection, including during the development of HCC [63], and HBV integration-targeted genes (ITGs) are significantly enriched in many cancer-related signaling pathways, such as the MAPK and Hedgehog pathways [63]. However, whether HBV-DNA integrates into normal prostate cells to promote the development of PCa or integrates into PCa cells to promote the development of CRPC remains unclear and requires further investigation.

There are several limitations to this study. Firstly, this was a retrospective analysis conducted at a single institution. Secondly, there was a small number of patients in the HBsAg-positive group. Thirdly, all patients are of Chinese Han ethnicity. Lastly, since this study is a preliminary one, the mechanisms by which HBV infection promotes the early progression of advanced patients to CRPC after CAB therapy remain speculative and are based solely on existing literature, without experimental validation or mechanistic investigation.