Hepatitis B virus (HBV) accounts for over 50% of cases of hepatocellular carcinoma (HCC) worldwide. Although nucleos(t)ide analog (NA) therapy reduces risk for HCC, several studies have shown persistent risk for HCC despite antiviral therapy. The clinical outcomes of patients reaching breakthrough HCC has not been observed. Longitudinal observations up to a period of 24 years at our center have shown poorer survival outcomes in patients successfully treated with antiviral therapy, as opposed to those naïve to antiviral therapy. Proposed theories for this disparity include HBV integration with host DNA, the role of androgens, and altered host immune responses.

Hepatitis B virus (HBV) infection is a public health problem, accounting for more than 257 million cases of chronic infection and a major cause of hepatocellular carcinoma (HCC) worldwide [1]. With the vaccination and the advent of nucleos(t)ide analogues (NAs) as antiviral therapy, chronic HBV infection currently accounts for approximately 50% of HCC cases worldwide, a significant decrease from >80% in the 1980’s [2-5]. The reduced incidence of HBV-related HCC (HBV-HCC) with NAs with lamivudine, entecavir, and tenofovir disoproxil fumarate is well documented [6-9]. On the other hand, several investigations, including large multicenter studies, have demonstrated that despite successful viral suppression, there remains a persistent risk for HCC while anti- HBV therapy [10-16].

However, although these studies demonstrated that a persistent risk for HBV-HCC remains despite successful antiHBV therapy, there have been no longitudinal observations showing the clinical outcome and survival of these patients who develop breakthrough HCC despite viral suppression. In effort to address this knowledge gap, the case series described here observed survival outcomes up period of 24 years between patients who developed HBV-HCC despite successful treatment with NA therapy (antiviral-treated) and patients who developed HBV-HCC without prior NA therapy (antiviral-naïve)

Study population

The study was a retrospective case series at a single tertiary medical center. Patients included in this study were observed for a period up to 24 years, with the earliest starting in 1996. Inclusion criteria were patients diagnosed with chronic hepatitis B (CHB) who developed HBV-HCC with active follow-up at our center upon chart review. Patients who were identified as antiviral-treated patients have had successful viral suppression with undetectable HBV DNA in serum. After diagnosis of their initial HCC, all patients received either surgical resection or loco-regional therapies, such as percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), microwave ablation (MWA), or radioembolization. Furthermore, all patients were treated with NA therapy after HCC diagnosis. The primary endpoint observed in this study was death.

Statistical analysis

Statistics were performed using SPSS 27. The categorical data was analyzed with descriptive statistics. This was expressed with percentages along with ranges and median values. The Kaplan-Meier method was used to estimate overall survival curve, and the log rank test was adopted to evaluate the difference between groups.

This case series included a total of 26 patients diagnosed with HBV-HCC. Thirteen patients were on NA therapy at their first diagnosis of HCC (Table 1 – antiviral-treated). Conversely, the other thirteen were naïve to antiviral therapy at the time of their first HCC diagnosis (Table 2 – antiviral-naïve). Antiviral experience patients had no detectable HBV DNA in serum at time of HCC diagnosis, whereas antiviral-naïve patients had varying levels of HBV DNA. Antiviral therapy used for antiviral treated patients Table 1 prior to HCC diagnosis included lamivudine (LAM) + tenofovir disoproxil fumarate (TDF) (patient #1-4, 8, 10, 12), TDF (patient #5-6, 9, 13), entecavir (ETV) (patient #11), and ETV + TDF (patient #7). The NA therapy was continued after diagnosis of HCC while treatment for the newly diagnosed HCC was initiated.

F: Female; M: Male; Lt : Left; Rt: Right; FHx: Family History; Dx: Diagnosis; UD: Undetectable; yrs: Years; LAM: Lamivudine (150mg daily); TDF: Tenofovir Disoproxil Fumarate (300mg daily); ETV: Entecavir (1mg daily); TACE: Transarterial Chemoembolization; MWA: Microwave Ablation; TARE: Transarterial Chemoembolization; OSH Ablation: Outside Hospital Ablation; Lap-MWA: Laparoscopic Microwave Ablation

*For list of HCC recurrences please refer to Figure 2.

Table 1: Development of HCC during anti-HBV Treatment.

All patients previously treated with NA therapy were identified as Child-Pugh (C-P) class A cirrhosis, except for patient #3.

Among patients naïve to antiviral therapy Table 2 at the time of HCC diagnosis, all patients were C-P class A, with the exception of patient #6, 13 who were not cirrhotic. As it has been clearly established in prior studies with favorable outcomes, antiviral naïve patients diagnosed with HCC underwent locoregional therapy along with initiation of anti-HBV therapy [17-23]. Antiviral-naïve patients NA therapies were TDF (patient #1, 4-10, 13), LAM (patient #3), LAM+TDF (patient #2, 11), or ETV+TDF (patient #12). Additionally, all patients had an initial single tumor size ≤ 5cm, except for patient #3 in the antiviralnaïve group with a 9 cm tumor.

TDF: Tenofovir Disoproxil Fumarate; F: Female; M: Male; Lt: Left; RT: Right; Dx: Diagnosis; FHx: Family History; yrs: Years; UNK: Unknown; LAM: Lamivudine (150mg daily); TDF: Tenofovir Disoproxil Fumarate (300mg daily); ETV: Entecavir (1mg daily); RFA: Radiofrequency Ablation; OSH ablation: Outside Hospital Ablation; TACE: Transarterial Chemoembolization; MWA: Microwave Ablation; TARE: Transarterial Radioembolization; PCEI: Percutaneous Ethanol Injection; Lap-MWA: Laparaoscopic Microwave Ablation; Lap-RFA: Laparoscopic Radiofrequency Ablation.

*For list of HCC recurrences please refer to Figure 3.

Table 2: Anti-HBV treatment naïve HCC patients.

At the time of their first HCC diagnosis, patients in each group were of similar ages with a median of 61 and 57 in the antiviral-treated and antiviral-naïve groups respectively Table 3. Patients in the antiviral-treated group were predominantly male with only 1 female (92.3%), whereas 7 were male among the 13 (53.8%) in the antiviral-naïve group. Positive family history of HBV was present in 69.2% of antiviral-treated patients and was noted in 53.8% of treatment-naïve patients (although patient #11 had an unknown family history).

^aIQR =interquartile range
^bIndependent-Sample Median

Table 3: Summary of antiviral treated and antiviral naïve patients.

Figure 1 demonstrates the years of successful antiviral treatment (HBV DNA undetectable) of the 13 patients who developed HCC while on therapy. The median number of years of successful NA therapy prior to HCC diagnosis for these patients was 8 years, with patient #1 having the shortest duration of NA therapy of 3 years prior to HCC diagnosis, and patient #9 the longest years of successful NA therapy of 12 years.

Figure 2 is modified from Figure 1 to display the survival from the time of HCC diagnosis in antiviral-treated patients. As demonstrated in Figure 2, the survival of patients who developed HCC while on successful antiviral therapy carried poor survival. Six of the thirteen patients (46.2%) died during the observed period, with 5 of 6 patients dying less than 3 years after HCC diagnosis. Only three patients in this cohort reached liver transplantation and survived (patients #1, #3, and #10).

Figure 3 illustrates the length of survival of patients who were treatment naïve before the first diagnosis of HCC. These patients have lower mortality with only 2 of 13 Patients (15.4%) dying during the observation period.

It is clear that there is a stark survival disparity between the antiviral-treated and naïve groups. Apart from three posttransplantation patients (#1, #3, #10), most antiviral-treated patients, 46.2% (6 of 13) died early, especially 5 of the 6 within 3 years after HCC diagnosis. In contrast, patients naïve to antiviral therapy had lower mortality with 15.4% of patients (2 of 13) dying during the observation period. Of note, all patients were started or continued antiviral therapy after HCC diagnosis and remained with undetectable HBV DNA levels, except for patient #7 who had a brief three-year period of interrupted antiviral therapy in the setting of compliance. The rates of death were 46.2% and 15.4% in the antiviral-treated and antiviral-naïve (X2=2.89, p=0.089), respectively. The rates of overall survival were lower in patients previously treated with antiviral therapy at the time of their HCC diagnosis than those naïve to antiviral therapy (X2=7.057, p=0.008) Figure 4.

As shown in Figure 3, among the antiviral-naïve patients, 7 of 13 patients developed the second new or recurrent HCC during their clinical course. With repeated local tumor ablation and continued antiviral therapy they survived. Patient #1 developed recurrence at the treated site (right lobe) 5 years after the first HCC, and 10 years later, a 1 cm new tumor appeared in the other lobe. On both occasions, the patient received local ablations and continued antiviral therapy. She survived over 20 years after the first HCC.

It has been well-established that although antiviral therapy improves survival in patients with HBV infection, there remains the persistent risk for HCC. Our case series provides an opportunity to demonstrate that patients who develop HCC while on successful antiviral therapy carry poorer prognosis compared to those naïve to antiviral therapy prior to the development of HCC. It is unclear why those with longterm successful antiviral therapy were observed with worse survival/prognosis upon HCC diagnosis.

One mechanism that can potentially contribute to worse survival in patients receiving long-term antiviral therapy is integration of HBV DNA into the host genome. Halting HBV replication at the reverse transcription phase of the viral life cycle for prolonged period may increase priming the HBVinfected hepatocytes for enhanced turnover of HBV DNA integration events which are known to cause insertional mutagenesis and genomic instability [24]. While HBV DNA can integrate randomly into the host genome, over the course of chronic HBV infection there is an increased potential for integration to occur at critical HCC driver genes giving rise to clonally-expanded aggressive malignant HBV-infected hepatocytes. Thus, despite successful suppression of viral replication with long-term anti-viral therapy, integration can still occur and potentially contribute to the survival disparity seen in this cohort. Additionally, HBV integration can produce chimeric HBV antigens potentially priming immune responses and exacerbating immune dysfunction [25]. With the continuous halting of HBV DNA transcription due to successful antiviral therapy, accumulation of unfinished viral products including several transcribed mRNAs may occur. This may increase integration between HBV DNA and host DNA. Upon HCC development in the presence of continuous suppression of viral replication, cancerous cells may be primed/evolved in a more vicious and malignant manner. It remains to be further explored the HBV integration sites between the two cohorts.

Furthermore, HBV DNA integration events in the TERT promoter have been shown to render TERT transcription responsive to sex hormones enhanced by the androgen receptor, a new mechanism proposed for the male dominance of HBV-HCCs [26]. Interestingly in this study, we predominantly see the male gender in patients previously treated with antiviral therapy compared to those naïve to antiviral therapy (92.3% vs. 53.8%). Male patients have been known to have a generally poorer prognosis than females in HCC [27]. This is likely in part to the role androgens have in promoting HBVHCC [28].

Another potential mechanism for our findings may include altered host immune responses to HBV-HCC in patients previously treated with antiviral therapy. Infection with CHB is known to exhaust host HBV-specific T-cells and increase expression of inhibitory checkpoint molecules such as PD-1 and CTLA-4 [29]. Luo et. al. demonstrated that in CHB patients, antiviral therapy restored these HBV-specific immune responses, and subsequently suppressed HBV replication [30]. However, it is unclear if this immune reconstitution may inadvertently contribute to the progression to HCC in certain individuals due to dysfunctional inflammatory responses causing DNA damage, genomic instability, etc.

Our longitudinal period of observation approaching 20 years allowed an opportunity to observe the difference in survival between antiviral-treated and antiviral-naïve patients who develop their first HBV-HCC.

These findings provide a unique opportunity for discussion and investigation into this disparity. As shown in prior studies of favorable outcomes with concomitant anti-HBV therapy, patients who are naïve to antiviral treatment and subsequently develop HCC may benefit from initial treatment with loco-regional therapy along with initiation of anti-HBV therapy. While the etiology for our findings remains unclear, these observations suggest that for patients who develop HCC despite successful long-term anti-HBV therapy, the best management would be early referral for liver transplantation. Our observation encourages further investigation of the possible causes and improvement of outcomes in patients who develop HCC despite successful viral suppression.

D Garrido contributed to the data analysis and writing of the manuscript. P Block contributed to the writing and editing of the manuscript. S Lin contributed to the interpretation of findings of the study. D Halegoua-DeMarzio contributed to the editing of the manuscript. HW Hann conceived the idea for the manuscript and assisted in writing and editing.

HW Hann: Serves the National Advisory Board of The Gilead Sciences. Receives grant from Gilead and Assembly Biosciences. All other authors declared that there are no conflicts of interest.