The most intriguing and unexpected finding of the current study was that occult rather than overt HBVCI led to shorter postoperative disease-free survival in patients with HCV-associated HCC. Overt HBVCI not only did not accelerate the recurrence of HCC but it suppressed its occurrence, resulting in longer disease-free survival. To decipher this puzzle, the clinicopathological and virological factors were compared between the two groups. As expected, patients with overt HBV infection indeed had a higher HBV DNA level and a higher rate of tissue viral antigen expression. However, several supposedly important oncogenic factors, such as HCV RNA level, HCV genotype, HBV genotype, HBV BCP mutation, and HBV integration rates, did not differ between the two groups. Despite these inconvenient findings, it was found that the patients with occult HBVCI had a higher bilirubin level and a higher prevalence of precore stop codon G1896A mutation.
Taiwan is a hyperendemic area of HBV infection with 15–20% of the general population chronically infected with HBV 
. Majority of Taiwanese HBV carriers were infected with HBV perinatally before 3 years of age. Infection occurring after 3 years of age was less frequent and rarely developed into a chronic status 
. Hence, most cases enrolled in our study with dual HBV and HCV infection were likely chronic HBV carriers superinfected by HCV 
. A reasonable assumption is that HBV serves as an initiator and HCV serves as a promoter for hepatocarcinogenesis in such patients. Therefore, a higher HCV RNA level should have associated with a higher HCC recurrence rate. In the present study, however, no significant difference was found for the intrahepatic HCV RNA level between these two HBVCI groups, despite the presence of significant survival difference. In fact, the intrahepatic HCV RNA level was not a predictor for postoperative survivals in any of our patient groups (). On the other hand, it was known that HBV genomic DNA can integrate into host chromosome, independently of the amount of free HBV DNA 
. The integration potentially disrupts cellular genes and enhances chromosomal instability, leading to an increased risk of HCC development. However, in this study, the prevalence of HBV DNA integration was not significantly different between the occult and overt HBVCI patients.
It was not surprising that cases with overt HBVCI had higher intrahepatic HBV DNA levels than those with occult HBVCI. However, in 5 cases with occult HBVCI, high intrahepatic HBV DNA levels (up to 384286 IU/g, ) were found, regardless of the negativity of serum HBsAg. The possibility that these patients harbored vaccine escape like mutations was excluded by sequence analysis of the whole S gene 
. It is therefore curious that in these patients, the presence of a high intrahepatic viral load of HBV did not result in a detectable amount of serum HBsAg. The fact that in these occult HBVCI patients, hepatic HBsAg and HBcAg were still detectable by immunohistochemistry suggested that there was a secretory defect for HBsAg (). Intrahepatic retention of viral proteins could result in hepatocytopathy and subsequent necroinflammation, leading to an increased risk of postoperative recurrence. In the remaining 11 occult HBVCI patients, however, the intrahepatic HBV DNA was low and viral protein expression was undetectable. A possible explanation for the shorter disease-free survival in these patients is that they might be in a later stage of HCV and HBV co-infection. Presumably, in these patients, HCV and HBV might have taken turn to elicit numerous episodes of necroinflammation prior to suppression of HBV infection into the occult status. The poorer hepatic reserve in occult HBVCI patients (high bilirubin levels and higher AST/ALT ratios) might represent a liver suffered from more rounds of damage and thus carried a higher risk for postoperative HCC recurrence.
Whether a higher prevalence of the precore G1896A mutant in occult HBVCI patients had any effect on the increased risk of HCC recurrence remained to be determined. Reports regarding association between precore G1896A mutant and development of HBV-associated HCC were very conflicting so far 
. However, development of the precore G1896A mutation prevents the production of HBeAg, which provides a possible means to evade immune clearance and contributes to the persistence of occult HBVCI. Furthermore, precore G1896A mutation usually developed in patients suffering from repeated hepatitis flares during a long course of HBV infection. The increased prevalence of this mutation in occult HBVCI was consistence with the view that occult HBVCI was a later stage of HBV and HCV co-infection. In addition to the presence of occult HBVCI and absence of overt HBVCI, tumor size >3 cm, alpha-fetoprotein >8 ng/mL, albumin <4 g/dL, ALT >50 U/L, were also found to be independent predictors for the disease-free survival in the HCV-associated HCC patients. These results indicated that the degree of inflammation (ALT), hepatic functional reserve (albumin) and the tumor burden (AFP and tumor size) were three important factors associated with postoperative prognosis. Presumably, chronic hepatic necroinflammation with its subsequent generation of reactive oxygen species can induce chromosomal mutations and eventually lead to malignant transformation of proliferating hepatocytes. Likewise, poor liver function reserve, suggested by hypoalbuminemia, was found to be significantly associated with HCC occurrence in other studies 
. By combining the 6 independent predictors, three groups with significantly different postoperative prognosis were distinguishable. Therefore, in an area hyperendemic for HBV infection, recognition of the status of HBVCI becomes mandatory for a better assessment of the postoperative prognosis. Finally, whether anti-HBV therapy should be given after operation for those who suffered from occult HBVCI becomes a viable question. Although microvascular invasion is usually recognized as important predictors in HCC patients undergone tumor resection 
, it was not present as the prognostic factor here. In our study, almost half (51/115) of the cases were co-infected with HBV. Necroinflammation elicited by host immune response to dual-infection with HBV and HCV 
may mask the predicting role of microvascular invasion in determining disease-free survival.
The major limitation of the current study is that the number of patients with HCV-associated HCC and occult HBVCI was relatively small. Nonetheless, this is the largest prospective study with 115 liver tissues obtained from the noncancerous parts of surgically removed HCV-associated HCCs worldwide so far (even in terms of the case number of occult HBVCI) 
. Most studies trying to address the impacts of occult HBVCI on HCV-associated HCC were restricted by either inadequate HBV DNA sampling (from serum rather than liver) or small total cases number (cases number: 11~85) 
. Furthermore, to our knowledge, there is still no study focusing on the role of occult HBVCI in postoperative prognosis of HCV-associated HCC patients. In Taiwan, HBsAg prevailed in 12% of anti-HCV-positive patients 
. In our series, the prevalence of occult HBV CI was 20%. In view of this high prevalence, the roles of occult HBVCI in HCV-associated HCC cannot be overlooked in an area hyperendemic for HBV.
In conclusion, occult and overt HBVCIs were independent prognostic predictors for postoperative survival in HCV-associated HCC. In an area hyperendemic for HBV infection, recognition of the status of HBVCI is mandatory for a comprehensive assessment of the postoperative prognosis.