Utilization of hepatitis B virus surface antigen positive grafts in liver transplantation: A matched study based on a national registry cohort

Donor shortage has become worldwide limitation in liver transplantation (LT). Use of hepatitis B virus surface antigen positive (HBsAg+) donors could be an alternative source of donor organs. This study aims to investigate the safety and efficacy of LT using HBsAg+ liver grafts and associated long‐term outcome.


Introduction
Currently, liver transplantation (LT) remains the only curative therapeutic option for patients with end-stage liver diseases. The number of LT performed worldwide has been increasing progressively over the past decades owing to the remarkable advances in this field. 1 While mortality rates on the waiting list are increasing due to the relative scarce of organ donation. The disparity between the demand of organ transplantation and the limited supply of donor organs has become a global concern. 2 Living donors 3 and split grafts 4 have been widely accepted to expand liver grafts, but the underling ethical issues and technical complexities make these two approaches less ideal to reduce the donor shortage and waitlist mortality. To relieve organ shortage, current efforts on expanding organ pool have resulted in higher utilization of extended criteria donors (ECDs), as well as pushing the limits of what is acceptable for LT. 5 Although there is no precise definition for ECD liver, it includes liver grafts from donors with signs of past or present hepatitis B virus (HBV) infection. The most concern of such grafts is the increased risk of HBV transmission from donor to recipient. HBV infection is a global disease with an estimated prevalence of 4.9% or 364 million infections. 6 A nationwide survey of HBV in the general Chinese population showed that approximately 7.2% were HBV carriers. 7 Therefore, using grafts from such population may have significant potential to expand organ pool. Discarding these grafts would be not practical, especially in highly endemic areas such as Asia and Africa. Now, utilizing liver grafts from HBV core antibody positive (HBcAb+) donors has been proven safe and feasible without affecting post-transplant outcomes while requiring lifelong antiviral prophylaxis. [8][9][10][11][12][13] In contrast, using HBV surface antigen positive (HBsAg+) liver grafts is far more controversial due to the paucity of clinical experience. Recently, there has been increasing interest in using HBsAg+ liver grafts and early results from most studies were promising. [14][15][16][17][18][19][20][21][22][23] We have also reported our single-center data and demonstrated that HBsAg+ liver grafts can be safely used without increasing postoperative mortality and morbidity under post-transplant oral antiviral therapy. 24 However, all the previous studies were either case reports or small size cohort retrospective studies that were not sufficient to draw a firm conclusion.
In the present study, we analyzed the China Liver Transplant Registry (CLTR) database from January 2015 and September 2018 to investigate the safety and feasibility of HBsAg+ liver grafts in LT. To our knowledge, this is the largest case-controlled study from a national wide cohort so far, thus aiming to provide strong evidence on the use of HBsAg+ liver graft in clinical practice.

Patients and methods
Patients and data collection. This study was a retrospective analysis of a national database from the CLTR. The academic board of the CLTR approved the study protocol. In brief, patients who underwent orthotropic LT between January 2015 and September 2018 were collected and divided into two groups according to their corresponding donor HBsAg status (HBsAg+ group vs HBsAgÀ group). Clinical data recorded in the CLTR including both recipients and donors were screened for the analyses. The deadline of data record for the analysis was May 7, 2020. The relevant recipients' data included age, sex, blood type, body mass index, type of liver disease, model for end-stage liver disease (MELD) score, serology of HBV and hepatitis C virus (HCV), main complications, and the outcome and follow-up results. The donors' data included age, sex, blood type, body mass index, donor type, and graft cold ischemia time. The exclusion criteria were (i) younger than 14 years; (ii) living donor; (iii) split donor; (iv) reduced-size donor; (v) dual graft donor; (vi) re-transplantation; (vii) multi-organ transplantation; (viii) HCV donor; (ix) incomplete HBV serology record; and (x) lost in follow-up. All patients signed a written consent form and the extra possible risk was informed if receiving HBsAg+ grafts. No donor organs were obtained from executed prisoners or other institutionalized persons.
Matching procedures. Given the big differences in the population between two groups that may cause potential selection bias and confounders, propensity score matching analysis was used in this study to balance out such nonuniform effect. 25 Patients who received HBsAg+ donors were set as the study group (HBsAg+ donor group), whereas propensity score matched patients who received HBsAgÀ donors were selected as the control group (HBsAgÀ donor group). Propensity scores were estimated using a non-parsimonious multivariable logistic regression model according to the collected pre-transplant clinical data of both donor and recipient. A nearest-neighbor 1:1 matching scheme with a caliper width of 0.1 was set for propensity score matching. Statistical inference in the matched cohort was performed with a Cox proportional-hazards regression model stratified on the matched pair to preserve the benefit of matching. For further subgroup analysis on the basis of recipient HBsAg status or malignant diagnosis at transplantation, propensity score matching was re-conducted, respectively. Graft and patient survival and main complications after LT between two groups from each matched cohort were compared.
Statistical analysis. Continuous variables were presented as the mean value and standard deviation, while categorical values were expressed using relative frequencies and proportions. Statistical analyses were carried out using the chi-square test for non-continuous variables, Student's t-test for continuous variables with normal distribution, and the Mann-Whitney U-test for continuous variables with nonparametric distribution. The Kaplan-Meier method with log-rank test was used for cumulative survival analysis. For all analyses, the reported P values were all two-sided and a P value of < 0.05 was considered statistically significant. All statistical analyses were performed using SAS software package (version 9.4).

Results
A total of 15 623 LTs were registered in the CLTR between January 2015 and September 2018. A total of 4719 patients were excluded according to the inclusive criteria. Among them, 522 patients had incomplete HBV serological status or lost in followup. Finally, 10 904 adult LT patients were enrolled in the present study, of which 601 received HBsAg positive grafts and 10 303 received HBsAg negative grafts. After propensity score matching, 98 patients in the HBsAg+ donor group were not matched with the HBsAgÀ donor group and were excluded for further analyses. Finally, 503-paired patients from both HBsAg+ and HBsAgÀ donor groups were generated. The main clinical characteristics of both donors and recipients in the matched two groups were well balanced (shown in Table 1).

Outcomes of liver transplantation in relationship to the donor hepatitis B virus surface antigen status.
Our results showed that there were no differences in patient and graft survival between the HBsAg+ and HBsAgÀ donor groups. The 1-, 3-, and 5-year patient survival rates in the HBsAg+ donor group were 81.52%, 72.04%, and 66.65%, which were not significantly different compared with those in the HBsAgÀ donor group, respectively (83.93%, 77.27%, and 65.73%, P = 0.222) (Fig. 1a). Similarly, the 1-, 3-, and 5-year graft survival rates were also comparable between the two groups (P = 0.243, Fig. 1b).
The most main complications after LT were comparable between the two groups. The rates of graft-related complications such as primary non-function, delayed graft function, and acute rejection were 1.1%, 4.7%, and 2.0% in the HBsAg+ donor group and 1.6%, 3.5%, and 0.8% in the HBsAgÀ donor group. No significant differences were found between the two groups (P values were 0.73, 0.48, and 0.11, respectively). Other complications were also noted to be comparable between the two groups such as bile duct complication (6.4% vs 7.2%, P = 0.61), vascular complication (0.8% vs 1.0%, P = 1.0), postoperative infection (37.6% vs 33.7%, P = 0.36), and renal failure (6.8% vs 7.4%, P = 0.78). The rate of tumor recurrence was 17.4% in the HBsAg+ donor group, which was slightly higher than 13.1% in the HBsAgÀ donor group. But the difference was not statistically significant (P = 0.18).
Notably, HBV reinfection was very low in the HBsAgÀ donor group with an HBsAg positivity rate of 1.4%. Whereas in the HBsAg+ donor group, all the patients (100%) remained HBsAg positive indicating HBV infection retained in this population ( Table 2).

Outcomes of liver transplantation in recipients with different recipient hepatitis B virus surface antigen status.
To compare the outcomes in HBsAg+ patients who received either HBsAg+ or HBsAgÀ liver grafts, these patients were further stratified by recipient HBsAg status: HBsAg + and HBsAgÀ. After propensity score matching, 445-paired HBsAg+ and HBsAgÀ recipients were generated for the analyses. The main clinical characteristics of the paired two groups were well balanced (Table 3). Results showed that the 1-, 3-, and 5-year patient survival rates were 82.32%, 72.51%, and 66.73% in the HBsAg+ donor group, which were lower than 87.06%, 79.85%, and 77.07% in the HBsAgÀ donor group, respectively (P = 0.027). However, after excluding 30 ABO-incompatible patients in both groups, the difference became narrowed and reached no significance (P = 0.055 and P = 0.057, respectively) (Fig. 2). The main complications were similar between the two groups. The rate of tumor recurrence was 17% in the HBsAg+ donor group, which was slightly higher than that in the HBsAg+ donor group (11.7%). The difference was not statistically significant but approaching significance (P = 0.1) (Table 3).
Similarly, the outcomes in HBsAgÀ patients who received either HBsAg+ or HBsAgÀ grafts were also compared. Between 55-paired HBsAgÀ patients, those who received HBsAg+ grafts showed comparable patient (P = 0.598) and graft (P = 0.646) survival than those who received HBsAgÀ grafts (Fig. 3). However, in both groups, the survival rates were lower than those in HBsAg + patients. This might be explained by that those HBsAgÀ patients with HBsAg+ grafts seemed severer because the MELD scores in these two groups were higher (Table S1).
Outcomes of patients with hepatocellular carcinoma in relationship to donor hepatitis B virus surface antigen positivity. To define whether carrying an HBV-infected graft under immune suppression exposed the recipients of an increased risk of hepatocellular carcinoma (HCC) recurrence, HCC patients at transplantation were further stratified for analysis. After propensity score matching, 188-paired HCC patients who received either HBsAg+ or HBsAgÀ grafts were matched for further analyses (Table S2). The 1-, 3-, and 5-year patient survival rates in HCC patients who received HBsAg+ grafts  were 84.79%, 68.85%, and 57.17%, which were comparable with 87.41%, 70.13%, and 70.13% in those received HBsAgÀ grafts (P = 0.340) (Fig. 4a). In similar, the 1-, 3-, and 5-year graft survival rates were also comparable (84.79% vs 87.41%, 67.66% vs 69.89%, and 57.04% vs 69.89%, respectively) between the two groups (P = 0.347) (Fig. 4b). A total of 31 patients with HBsAg + grafts and 29 with HBsAgÀ grafts developed tumor recurrence during the follow-up. The 1-, 3-, and 5-year cumulative recurrence  rates were comparable between the two groups (P = 0.775), thus indicating the use of HBsAg+ grafts did not increase early tumor recurrence in HCC recipients (Fig. 4c).

Discussion
The big gap between scarce availability and high demand of organs continue to be the main limitation of LT nowadays. Efforts have been made to relieve the organ shortage, including the implement of ECDs. 2,3,5 There is no precise definition for ECDs but can be divided into two categories: grafts with risk of impaired function due to graft injury and risk of disease transmission between donor and recipient. The latter includes donors with serological evidence of HBV infection either past or present. The most concern of such grafts is the risk of transmitting HBV infection after LT. The existence of intrahepatic covalently closed circular DNA in HBcAb+ liver grafts is the main likely reason for de novo HBV infection in recipients after transplant. 26 However, in the era of highly effective antiviral therapy, many studies have demonstrated that HBcAb+ liver grafts have very low risk of de novo HBV and non-inferior survival with proper antiviral prophylaxis after LT. 9,10 The use of HBcAb+ liver grafts has now been widely accepted with consensus of using nucleotide analogues after transplantation. However, using liver grafts from HBsAg+ donors is more controversial as such grafts definitely carry and transmit HBV to recipients. Even in present clinical practices, HBsAg+ donors may still be one principal cause for rejection of liver graft by some transplant centers with a term so-called risk avoidance attitude. 27 Although evidence of using HBsAg+ liver grafts is quite limited, several studies have been recently reported regarding the feasibility and safety of transplanting those grafts. At the early beginning, only single-center case reports and small case series were sporadically reported since the first successful liver transplant of HBsAg+ graft in the USA in 1994. Thereafter, more studies reported encouraging data obtained in larger sample sizes that are from not only single centers but also nationwide databases. A retrospective study on the clinical outcome of 23 HBV-infected patients underwent deceased-donor LT using HBsAg+ liver grafts in China suggested the safety and effectiveness of this procedure. 18 We previously introduced our experience on the safe use of HBsAg+ liver grafts by reporting so far the largest number of  patients with HBsAg+ liver grafts in a single transplantation center. 24 With effective antiviral prophylaxis, the post-transplant mortality and morbidity did not significantly differ between a group of 42 patients with HBsAg+ liver grafts and 327 consecutive recipients of HBsAgÀ grafts. More recently, another study from a group of Taiwan concerning the situation of living donor LT also provided a positive result in 14 consecutive patients. 20 Similar promising results had also been described in either multicenter or national database studies. Liver transplant patients from three Italian centers were retrospectively evaluated by Ballarin et al. showing that 2% of the population utilized grafts from HBsAg+ donors and confirmed a satisfactory long-term survival without affecting graft stability. 19 Based on the United Network for Organ Sharing (UNOS) database, both Saidi et al. 17 and Krishnamoorthi et al. 21 showed comparable graft and patient survival between patients who received HBsAg+ versus HBsAgÀ liver grafts. We previously selected 78 HBsAg+ liver graft patients and matched each of them with 4 recipients of HBsAgÀ grafts among the 92 157 patients in the same database. 22 Cox hazards regression analysis showed that the graft and patient survival were not reduced by HBsAg+ liver grafts. Furthermore, a latest paper regarding this specific theme involving a total of 259 matched cases collected from the CLTR database again revealed the post-transplant complications and long-term survival were not significantly different. 23 To provide more evidence-based practices, the present study enrolled so far the largest number of patients with HBsAg+ liver grafts from a national database. Propensity score matching analysis was performed to simulate a randomized controlled study. Our results showed that there was no significant difference in long-term survival of patients received HBsAg+ liver graft compared with those received HBsAgÀ liver graft. The main post-transplant complications were also comparable except for the recurrent/retain of HBV infection. HBsAg positivity rate in the HBsAgÀ donor group represents HBV recurrence after LT. Our result showed it was 1.4%, which was similar to literature reports. According to a literature review, the overall HBV recurrence rate of HBV patients receiving HBsAgÀ liver grafts varied from 0% to 24% under different regimen of prophylaxis in 16 different centers. 28 Whereas in the HBsAg+ donor group, all the patients (100%) remained HBsAg positive regardless the recipients were HBsAg positive or not before LT, indicating HBV infection retained in this population.
We did not analyze the strategies of antiviral prophylaxis after LT in patients received HBsAg+ liver graft because of the big variations of antiviral strategy in different centers. Consensus guidelines regarding a prophylactic regimen for HBV recurrence after LT have been introduced since 2015 in the USA 29 and 2016 in China 30 ; however, there has been no unified strategy recommendation for HBsAg+ patients or grafts to date. Each patient, either HBsAg+ or received HBsAg+ liver graft, took antiviral regimens based on different institutional protocols. It included one or two nucleotide/nucleoside analogues with or without different dosage of hepatitis B immunoglobulins (HBIG). Our rough result showed the HBV serological status was stable after LT with antiviral regimens and no graft loss was directly attributed to HBV infection. HBV infection was well controlled in patients received HBsAgÀ grafts with an HBsAg positivity rate of 1.4%. For patients with HBsAg+ grafts, it was not surprising that HBsAg retained positive even after antiviral therapy. This might be explained by hypothesis that an HBsAg+ liver graft has already established chronic HBV infection and may continuously produce large amount of HBsAg. This result was also in line with our previous single-center study that showed negative conversion of HBsAg could not be achieved by either an oral antiviral agent alone or in combination with HBIG. 24 To date, HBsAg+ liver grafts might have been preferentially allocated to HBsAg+ recipients due to the most concern of otherwise iatrogenic acquiring of HBV infection in naïve recipients. However, it remained unclear whether outcomes were different for HBV-infected or naïve patients after transplanting HBsAg+ versus HBsAgÀ liver grafts. Our data showed that HBsAg+ recipients with HBsAg+ liver grafts seemed to have a lower graft and patient survival compared with those received HBsAgÀ grafts. However, after excluding the ABO-incompatible patients in both groups, the difference became narrowed and reached no significance. Only severe patients with poorly expected survival might be selected to transplant ABO-incompatible HBsAg+ liver grafts at urgent conditions. Excluding this population might eliminate partial selective bias. It was notable that the tumor recurrence rate was slightly higher in the HBsAg+ versus HBsAgÀ donor group (17% vs 11.7%, P = 0.1), approaching significant difference. This might potentially cause the apparently but not significantly poorer survival of patients with HBsAg+ liver grafts. However, whether the difference did exist and its true potential mechanisms need further studies. On the other hand, overall survival for HBsAgÀ patients who received either HBsAg+ or HBsAgÀ liver grafts were comparable. This suggested that transplanting HBsAg+ liver grafts did not result in worse outcomes even in HBV naïve patients. Although the survival rates in these groups were lower than those in HBsAg+ patents, it might be because the MELD scores were higher in these groups perhaps owning to higher acceptance of HBsAg+ liver grafts for severe patients. Therefore, our results suggested that it is safe and feasible to use HBsAg+ liver grafts regardless of the HBV status of the recipients themselves.
Worldwide, HBV infection is considered as the main cause of HCC. 31 It induces HCC through direct or indirect oncogenic mechanisms owing to HBV-DNA integration into the host genome. 32 However, it remains unclear whether carrying an occult HBV-infected graft under immune suppression exposes the recipients of an increased risk of HCC recurrence and de novo HCC after LT. Our result showed that the tumor recurrence rate seemed slightly increased in HCC patients who received HBsAg+ liver grafts. It should be noted that HBsAg+ liver grafts were usually at higher tendency to be allocated to HCC patients in advanced stage. To eliminate the selective bias by tumor staging, Milan criteria were added in propensity scoring for further matching analysis in subgroup of HCC patients. Results showed that the risk of tumor recurrence was not increased in HCC patients who received HBsAg+ versus HBsAgÀ liver grafts. The accumulative survival and tumor recurrence rates were comparable between the two groups. Furthermore, there was no de novo HCC being reported in any non-HCC patient of HBsAg+ liver graft. This could partially attribute to the antiviral prophylaxis therapy after LT. There has been accumulating evidence that effective antiviral therapy is associated with reduced but not eradicated risk of HCC in HBV-infected patients. 33 Consensus guidelines regarding antiviral prophylaxis of HBV infection have been widely implemented nowadays. Patients of HBV infection or those received HBV-related grafts were referred to at least one antiviral prophylactic regimen based on the protocols in different centers. However, the current setting only limited to a median follow-up time of 5 years. HCC carcinogenesis under HBV infection may take several years. Therefore, longer follow-up studies are still needed.
The strength of the present study includes a large cohort size from a national registry database and a propensity score match analysis, which simulates a randomized controlled multicenter study and makes the result more reliable. However, there are still some limitations of this study. First, this was a retrospective study. Second, data from a national registry cohort may subject to bias and confounding usually be generated by data heterogeneity, reporting bias, data entry errors or missing, and center-specific practices. Thirdly, detailed antiviral prophylaxis analysis was lacking in the present study because it was impossible as the big discrepancy of antiviral strategies among different transplant centers. Thus, more data such as well-designed multicenter studies are required before this practice is widely adopted.
In summary, our study showed that using HBsAg+ liver grafts had comparable outcomes as HBsAgÀ liver grafts. In the era of effective antiviral prophylaxis against HBV, transplanting HBsAg+ grafts were not associated with inferior outcomes irrespective of the HBV status of recipients themselves. Neither the transmission of the occult HBV infection from HBsAg+ donor to recipient could increase the risk of HCC recurrence and de novo HCC after LT. Therefore, we suggested the use of HBsAg+ liver grafts was safe and feasible and might play an important role in expanding donor organs especially in area of a high prevalence of HBV infection.