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Page 10 of 12 Fung et al. Hepatoma Res 2018;4:62 I http://dx.doi.org/10.20517/2394-5079.2018.92

patients may show detectable HBsAg using the hs-HBsAg assay, especially those who were negative for
[19]
anti-HBs . This would suggest that HBsAg seroclearance only implies undetectability below the standard
quantitative assay. For the first time, the current study demonstrated that quantitative HBsAg levels using
a highly sensitive assay could be used to predict higher chance of HCC recurrence after LT for HBV-related
HCC. By using a completely HBIG-free regimen for hepatitis B prophylaxis, the study population presented
a unique opportunity to study the characteristics of HBsAg both qualitatively and quantitatively after
transplantation, and its association with early HCC recurrence. The administration of HBIG will preclude
any useful determination of HBsAg, as these will largely become undetectable through binding to anti-HBs,
which is consistently kept at a high level through the use of regular injections. Oral nucleos(t)ide analogues,
although effective in suppressive HBV DNA to undetectable levels, has significantly less effect on the HBsAg
levels. Previous studies have demonstrated that even potent oral antiviral therapy may not reduce HBsAg levels
[20]
despite prolonged HBV DNA suppression to undetectable levels .
The current study showed a significantly higher rate of HCC recurrence in those who failed to achieve
HBsAg seroclearance after transplantation (50.0% vs. 23.4% respectively at 8 years post transplant, P = 0.024).
Furthermore, for patients who remained HBsAg positive after transplant, the recurrence of HCC was early
(within the first year of transplant). It is possible that micrometastasis present at the time of transplant may
be responsible for the persistence of HBsAg. In addition, a significantly higher HCC recurrence rate was
observed in those who had HBsAg seroreversion compared to those who remained negative for HBsAg (60.5%
vs. 9.4% respectively at 10 years post transplant, P < 0.001). The association of recurrence HCC and HBV have
[21]
also been described in a smaller cohort of patients previously . Since extra-hepatic tumour tissue stained
positive for HBsAg, it is likely that malignant tumour cells are able to support HBsAg production. Moreover,
there was correlation between the timing of HBsAg re-appearance and HCC recurrence (r = 0.551, P = 0.027),
raising the possibility that HBsAg may be useful as a marker of HCC recurrence. The temporal relationship
suggests that the HBsAg is unlikely to have a role in the pathogenesis of HCC recurrence, but rather HCC
may be responsible for the HBsAg reversion.

To date, the only tumour marker currently readily available for post transplant HCC recurrence is serum
AFP, which in the current study, showed low sensitivity (7% to 21%) as an early predictor after transplantation.
In addition, a significant proportion of HCC did not secrete AFP. The finding that hs-HBsAg levels above
the LLOD (≥ 0.0005 IU/mL) at 3 and 6 months can be associated with higher rates of early HCC recurrence
suggests that this may be used as an early tumour marker. In contrast, the less sensitive conventional HBsAg
measurement did not show a significant difference at 3 or 6 months post transplant with respect to a positive or
negative HBsAg status and subsequent HCC recurrence. This may be due to the fact that during the early post
transplant period, tumour load is likely to be extremely low for those with recurrence, and therefore the HBV
DNA may not be detectable and HBsAg may not be quantifiable by conventional assays.

One of the consistent observations with HBIG-free prophylaxis after transplantation is the development
of detectable transient levels of anti-HBs titers shortly after transplantation, despite the absence of HBIG
administration (including the current study) [22-24] . This is most likely due to passive transfer of antibodies from
the donor, and is largely non-sustainable. Importantly, this phenomenon may preclude the use of quantitative
HBsAg as a useful tool for predicting HCC recurrence in the very early phases (within 3 months) after
transplantation, and provide explanation as to why the HBsAg levels were not predictive at 1-month post
transplant period in the current study. Other intermediate replication markers such as the hepatitis B core-
related antigen may be more useful, and deserves further study.

There are several limitations to the current study. Firstly, quantitative hs-HBsAg was performed in available
stored samples only. A previous study looking at the change in HBsAg levels with the conventional assay in
[25]
stored sera found no significant changes over time . Secondly, this cohort consists of patients largely in the