The authors have declared that no competing interests exist.
Conceived and designed the experiments: CTY. Performed the experiments: MLC TCC TSY WCL. Analyzed the data: MLC CTY. Contributed reagents/materials/analysis tools: YJL CJC CY. Wrote the paper: MLC CTY.
The roles of chronic hepatitis B virus (HBV) co-infection (CI) in carcinogenesis of hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) remained controversial. To gain new insights into this issue, we investigated the postoperative prognostic value of HBVCI in HCV-associated HCC.
A study cohort of 115 liver tissues obtained from the noncancerous parts of surgically removed HCV-associated HCCs were subjected to virological analysis in a tertiary care setting. Assayed factors included clinicopathological variables, tissue amounts of viral genomes, genotypic characterization of viruses, as well as the presence of overt (serum HBsAg positive) or occult (serum HBsAg negative but tissue HBV-DNA positive) HBVCI. Cox proportional hazard model was used to estimate postoperative survivals.
Of the 115 patients, overt and occult HBVCIs were detected in 35 and 16 patients, respectively. Multivariate analysis revealed that tumor size >3 cm (adjusted hazard ratio (AHR), 2.079 [95% confidence interval, 1.149∼3.761]), alpha-fetoprotein >8 ng/mL (AHR, 5.976 [2.007∼17.794]) albumin <4 g/dL(AHR, 2.539 [1.399∼4.606]), ALT >50 U/L (AHR,1.086 [1.006∼1.172]), presence of occult HBVCI (AHR, 2.708 [1.317∼5.566]), and absence of overt HBVCI (AHR, 2.216 [1.15∼4.269]) were independently associated with unfavorable disease-free survival. Patients with occult HBVCI had a shorter disease-free (
Occult and overt HBVCI served as independent predictors for postoperative survival in HCV-associated HCC.
Hepatocellular carcinoma (HCC) accounts for >90% of primary liver malignancies and ranks the fifth most common solid cancer and the third leading cause of cancer-related death worldwide
HCV infection is the most important risk factor for HCC in Hispanics and African Americans, whereas HBV infection is the dominant risk in Asians
Co-infection of HCV and HBV could result in more severe liver damage and thus accelerate the deterioration of liver function as well as the oncogenic process
Despite the accumulated reports on virological factors associated with the development of HCV-related HCC
This study was conducted under the approval of the institutional review board of Chang Gung Medical Center (CGMC). To conduct this study, the clinical records of 342 HCC patients receiving total removal of liver tumors from July 1998 to Aug 2001 in CGMC, Taiwan were reviewed. The liver tissues of these patients were stored in the tissue bank of CGMC after informed consents were obtained. Of them, 115 HCC patients who were positive for antibody against HCV (anti-HCV) and negative for antibody against hepatitis delta virus were included. All samples were frozen to –70°C, immediately after surgical resection until use. The following clinicopathological data were reviewed: gender, age, presence of liver cirrhosis, alcohol usage, Edmondson's histologic grade, microvascular invasion, macrovascular invasion, presence of tumor capsule, number of tumors, largest tumor size, presence of ascites upon surgery, alpha-fetoprotein (AFP), albumin, bilirubin, prothrombin time, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), date of surgical resection, date of tumor recurrence, and date of last follow-up or HCC related death. In our medical center, patients with main portal vein thrombosis were excluded from surgical management. Minor portal vein invasion discovered during or after surgery was categorized as macrovascular invasion.
Similarly, the Cox proportional hazard model was used to examine the association between clinicopathological and virological factors and overall survival after surgical resection of HCV-associated HCC. No factor associated with overall survival was found in univariate analysis. However, patients with occult HBVCI had a significantly shorter overall survival compared with those with overt HBVCI (67.39±11.47 vs. 115.14±4.6 months,
(A) Patients stratified by the risk score calculated from the 6 independent predictors. Grey line, risk score ≦2; Purple line, risk score 3 or 4; Red line, risk score ≥5. (B) Patients classified according to viral infection status. Black line, HCV infection with overt HBVCI; Blue line, HCV infection only; Green line, HCV infection with occult HBVCI. (C) Comparison of the overall survival between HCV-associated HCC patients with occult HBVCI (green line) and those with overt HBVCI (black line).
Preoperative diagnosis of HCC was made by one of the following methods: echo-guided liver biopsy, fine needle aspiration cytology, high AFP level (>200 ng/mL) plus at least one dynamic imaging study (dynamic computed tomography or magnetic resonance imaging), or one dynamic imaging study plus angiography (if AFP <200 ng/mL). Tumors were completely resected, with a safety-margin of >1 cm. Post-operative follow-up was performed by ultrasonography, chest X-ray, AFP, and blood biochemistry every 1 to 3 months in the first year and every 3 to 6 months thereafter. Abnormal findings were verified by computed tomography or magnetic resonance imaging. Intrahepatic recurrence was established by use of the aforementioned criteria. Extrahepatic recurrence was confirmed by biopsy, aspiration cytology, computed tomography or magnetic resonance imaging, with the choice of study dependent upon the location of the lesions as well as the condition of the patient.
HBsAg levels were measured by radioimmunoassay (Ausria-II, HBsAg-RIA; Abbott Laboratories, North Chicago, IL). Anti-HCV antibody levels were measured by a third-generation enzyme immunoassay (HCV EIA III; Abbott Laboratories). Antibodies against hepatitis delta antigen were detected by enzyme immunoassay (anti-HD; Formosa Biomedical Technology Corporation, Taiwan).
Hepatic and serum HCV RNA was measured by COBAS TagMan HCV test (Roche Diagnostics, Tokyo, Japan). Serum levels were assayed according to the standard protocol provided by the manufacturer. For hepatic HCV-RNA assay, 20 mg of liver tissue was submitted for RNA extraction using the corresponding TRIzol reagents (Invitrogen, USA). The extracted samples were dissolved in pure water and submitted for the same standardized commercial assays. The final amounts of hepatic HCV RNA were calculated as IU per gram after corrected for the dilution factor, with a lower limit of detection of 1500 IU/g.
HCV genotype was determined by use of the InoLipa method (COBAS AmpliPrep/COBAS TaqMan HCV Test, Roche Diagnostics, Tokyo, Japan).
The hepatic and serum HBV-DNA concentration was quantified using Roche Tagman HBV Monitor (Roche Diagnostics, Basel, Switzerland). Serum levels were assayed according to the standard protocol provided by the manufacturer. For tissue HBV-DNA assay
Target regions | Stages of PCR | Primer sequences | Expected Sizes of PCR products |
Pre-S | |||
First | PSFo: 5′-ttgcgggtcaccttattcttg-3′ | ||
PSRo: 5′- agcaggggtcctaggagtc-3 | 598 bp | ||
Second | PSFi: 5′-ggaacaagagctacagc-3′ | ||
PSRi: 5′- ctgatgcgatgttctccatgtt-3′ | 558 bp | ||
Precore/X | |||
First | PCFo: 5′- gccttctcatctgccggtcc-3′ | ||
PCRo: 5′- gtatggtgaggtgaacaatg-3′ | 505 bp | ||
Second | PCFi: 5′- cataagaggactcttggactcc-3′ | ||
PCRi: 5′- aaagaattcagaaggcaaaaaaga-3′ | 320 bp | ||
Virus-Host junction (Alu-PCR) | |||
First | Alu3: 5′- aUUaacccUcacUaaagccUcgaUagaUYRYRccaYUgcac-3′ | ||
Alu5: 5′ – cagUgccaagUgUUUgcUgacgccaaagUgcUgggaUUa-3 | |||
Second | Tag3: 5′- attaaccctcactaaagcctcg-3′ | ||
Tag5: 5′- caagtgtttgctgacgccaaag-3′ |
Y = c+t; R = a+g, U = dUTP; In Alu-PCR, four sets of PCR were performed using Alu3/PCFo, Alu3/PCRo, Alu5/PCFo, and Alu5/PCRo as the first stage PCR primers. Subsequently, uracil DNA glycosylase was added to break the DNA strands at apurinic dUTP sites. Finally, Tag3/PCFi, Tag3/PCRi, Tag5/PCFi, and Tag5/PCRi were used respectively for the second stage PCR.
Nested PCRs targeted to the Pre-S and precore regions were performed by use of the intrahepatic HBV DNA as a template. The primer sequences and product sizes were listed in the
The methods to detect HBV basal core promoter (BCP) A1762T/G1764A mutations, precore stop codon G1896A mutation and pre-S deletion were performed as previously described
A PCR based technique (Alu-PCR) was employed using specific primers directed to human Alu sequences and to HBV sequences to amplify virus-host junctions, according to previous publications
Immunohistochemistry for HBV antigens was performed on sections from paraffin-embedded liver biopsies by use of the Avidin Biotin Complex method. The primary antibodies used were the mouse monoclonal antibodies against HBsAg (Chemicon, Temecula, CA) and HBc Ag (Dako, Carpinteria, CA), respectively. Visualization was achieved through reaction of the substrate 3,3′-Diaminobenzidine. The samples from patients with negative serum HBsAg and negative tissue HBV DNA was taken as the negative controls while tissue samples from chronic hepatitis B patients as the positive controls. Intensity of staining was judged semiquantitatively as absent, minimal, moderate, or strong and staining pattern was reported as nuclear, cytoplasmic, sub-membranous, or absent, as described previously
Overall survival was calculated from the date of diagnosis to the date of death or last follow-up. Disease-free survival was measured from the date of diagnosis to the date of recurrence, metastasis, death or last follow-up. The Cox proportional hazard model was used to identify clinicopathological and virological factors associated with postoperative survivals. To determine the cutoffs of a factor with parametric data for Cox proportional hazard analysis, experimental univariate analysis was performed to evaluate the association between the factor and disease-free survival using a series of increasing values as the cutoffs. The experimental cutoffs were determined by using R version 2.13 (The R Foundation for Statistical Computing) which based on searching the highest coefficient after partition the variants from the classification and regression tree. Verifications of the experimental cutoffs were performed by subsequent stepwise Cox proportional hazard models, which were also used to predict independent predictors associated with disease-free survival. The Kaplan-Meier method was used to estimate the survival probability, and the log-rank test was used to compare the survival curves between groups. Statistical analysis to compare the variables among the patients with various virological statuses was performed by a one-way analysis of variance (ANOVA), and its significance was assessed by Sheffe's post-hoc test. Statistical analysis was conducted by the use of Statistical Product and Service Solutions (SPSS, version 17.0).
The demographics and clinical features of the patients with HCV-associated HCC were listed in
Clinical parameters | Gender | P | |
Female (n = 37) | Male (n = 78) | ||
Age (years) | 64.3±9.4 | 62.2±11.7 | 0.333 |
Cirrhosis | 27 (73.0%) | 50 (64.1%) | 0.464 |
Tumor number | |||
1 | 23 (62.2%) | 45 (57.7%) | 0.800a |
2 | 6 | 20 | |
3 | 7 | 9 | |
4 | 1 | 4 | |
Size (Diameter, cm) | 4.0±3.0 | 4.6±3.1 | 0.333 |
Ascites | 3 (8.1%) | 5 (6.4%) | 0.711 |
Alpha-fetoprotein (ng/mL)b | 50.3 (3∼9688) | 33.0 (3∼16057) | 0.165c |
Albumin (g/dL) | 3.8±0.6 | 3.9±0.6 | 0.775 |
Bilirubin (mg/dL) | 1.0±0.5 | 1.4±0.8 | 0.202 |
Prothrombin time (sec) | 12.2±1.1 | 12.2±1.3 | 0.940 |
Creatinine (mg/dL) | 1.2±1.1 | 1.2±0.8 | 0.849 |
AST (U/L) | 72.1±56.8 | 88.7±128.8 | 0.456 |
ALT (U/L) | 73.9±57.5 | 106.4±185.4 | 0.300 |
Alcoholism | 1 (2.7%) | 16 (20.5%) | 0.026 |
Detectable serum HBsAg | 14 (37.8%) | 21(26.9%) | 0.389 |
Detectable Hepatic HBV DNA but undetectable serum HBsAg | 4(10.8%) | 12(15.3%) | 0.578 |
a Comparison between patients with tumor number = 1 and those with tumor number >1.
b Median (range).
c Mann-Whitney test.
Both HCV-RNA and HBV-DNA were extracted from noncancerous liver tissue for virological analysis. Of all patients, 58.2% (67/115) had HCV RNA levels greater than the lower detection limit for quantitative assessment (1500 IU/g) and 23.47% (27/115) had HCV RNA levels >13×106 IU/g (the cutoff used for survival analysis;
Parameter | No. of patients | Mean disease-free survival (95% CI) | HR (95% CI) | Adjusted HR (95% CI) |
Age (years) | ||||
≦47 | 11 | 91.7(62.5∼120.91) | ||
>47 | 104 | 42.58(34.14∼49.97) | 3.28(1.024∼10.508) |
2.018(0.067∼6.599) |
Gender | ||||
Female | 37 | 48.0 (34.5∼61.5) | ||
Male | 78 | 51.8 (38.9∼64.6) | 1.099 (0.631∼1.917) | |
Cirrhosis | ||||
No | 38 | 56.7 (37.6∼75.8) | ||
Yes | 77 | 45.3 (36.2∼54.4) | 1.064 (0.615∼1.841) | |
Alcoholism | ||||
No | 98 | 58.2 (46.1∼70.3) | ||
Yes | 17 | 39.9 (24.6∼55.2) | 1.393 (0.752∼2.581) | |
Tumor characteristics | ||||
Microvascular invasion | ||||
No | 76 | 53.4 (40.8∼66.0) | ||
Yes | 38 | 45.2 (31.0∼59.3) | 1.132 (0.654∼1.962) | |
Edmondson's grading | ||||
I-II | 40 | 55.9 (38.4∼73.4) | ||
III-IV | 75 | 46.2 (36.6∼55.8) | 1.018 (0.779∼1.331) | |
Encapsulation | ||||
No | 26 | 48.1 (31.1∼65.1) | ||
Yes | 88 | 48.5 (37.3∼59.7) | 1.238 (0.653∼2.349) | |
Tumor number | ||||
1 | 68 | 53.5 (39.9∼67.1) | ||
>1 | 47 | 40.5 (28.3∼52.6) | 1.313 (0.782∼2.206) | |
Largest tumor size (diameter, cm) | ||||
≦3 | 42 | 71.2 (53.8∼88.6) | ||
>3 | 73 | 37.5 (28.5∼46.5) | 2.167 (1.227∼3.827) |
2.079 (1.149∼3.761) |
Second branch macrovascular invasion | ||||
No | 101 | 50.0 (38.9∼61.1) | ||
Yes | 14 | 53.8 (29.2∼78.4) | 0.735 (0.311∼1.737) | |
AFP (ng/mL) | ||||
≦8 | 19 | 70.9 (54.9∼86.9) | ||
>8 | 96 | 46.2 (35.2∼57.3) | 3.259 (1.298∼8.183) |
5.976 (2.007∼17.794) |
Ascites | ||||
No | 107 | 51.5 (41.0∼62.0) | ||
Yes | 8 | 50.8 (30.4∼71.3) | 0.370 (0.051∼2.673) | |
Albumin (g/dL) | ||||
≦4.0 | 68 | 34.0 (24.1∼44.0) | 2.263(1.312∼3.904) |
2.539(1.399∼4.606) |
>4.0 | 47 | 68.4 (53.3∼83.5) | ||
Bilirubin (mg/dL) | ||||
≦1.0 | 48 | 62.6 (45.5∼79.8) | ||
>1.0 | 67 | 40.8 (31.4∼50.3) | 1.423 (0.833∼2.431) | |
Prothrombin time (sec) | ||||
≦12 | 67 | 58.3 (44.0∼72.6) | ||
>12 | 48 | 39.9 (28.5∼51.4) | 1.081 (0.951∼1.229) | |
Creatinine (mg/dL) | ||||
≦1.2 | 89 | 47.9 (37.0∼58.8) | ||
>1.2 | 26 | 59.6 (42.1∼77.2) | 0.751 (0.517∼1.091) | |
AST (U/L) | ||||
≦42 | 49 | 71.9 (55.2∼88.6) | ||
>42 | 66 | 34.4 (25.5∼43.4) | 1.253 (1.088∼1.443) |
1.050 (0.870∼1.266) |
ALT (U/L) | ||||
≦50 | 49 | 67.5 (50.9∼84.1) | ||
>50 | 66 | 38.4 (28.7∼48.1) | 1.071 (1.008∼1.137) |
1.086 (1.006∼1.172) |
AST/ALT | ||||
≦0.89 | 55 | 56.27(45.70∼66.83) | ||
>0.89 | 60 | 40.7(26.95∼54.45) | 2.072(1.23∼3.491) |
1.621(0.944∼2.784) |
Fat metamorphosis | ||||
No | 76 | 46.68(34.55∼58.80) | ||
Yes | 39 | 52.97(40.01∼65.94) | 0.68(0.39∼1.18) | |
Virological factors | ||||
HCV-RNA (all patients) | ||||
≦13×106 IU/g | 88 | 43.1 (34.6∼51.6) | ||
>13×106 IU/g | 27 | 66.4 (43.1∼89.7) | 0.636 (0.322∼1.256) | |
HCV genotype (all patients) | ||||
Non-1 | 22 | 41.9 (28.3∼55.5) | ||
1 | 45 | 59.5 (41.5∼77.6) | 0.776 (0.386∼1.561) | |
HCV-RNA (non-HBVCI patients only) | ||||
≦13×106 IU/g | 50 | 36.9 (25.7∼48.1) | ||
>13×106 IU/g | 14 | 54.0 (37.9∼70.1) | 0.355 (0.108∼1.168) | |
HCV genotype (non-HBVCI patients only) | ||||
Non-1 | 14 | 28.6 (17.0∼40.3) | ||
1 | 25 | 53.3 (34.6∼72.0) | 0.531 (0.202∼1.397) | |
Intrahepatic HBV-DNA(By nested PCR detection) | ||||
Negative | 64 | 41.6 (31.1∼52.1) | ||
Positive | 51 | 57.4 (42.5∼72.2) | 0.771 (0.461∼1.291) | |
Occult HBV superinfection | ||||
No | 99 | 56.9 (45.3∼68.5) | ||
Yes | 16 | 29.2 (12.7∼41.8) | 2.028 (1.069∼3.849) |
2.708 (1.317∼5.566) |
Overt HBV superinfection | ||||
No | 80 | 38.5 (29.6∼47.5) | 2.062(1.14∼3.73) |
2.216(1.15∼4.269) |
Yes | 35 | 70.4 (52.3∼88.6) |
P<0.05.
HR, hazard ratio; CI, confidence interval.
Among the 35 patients with positive serum HBsAg (i.e. overt HBVCI), it was found that the HBV DNA level was lower than the detection limit of our quantitative test (120 IU/gram of liver tissue) in 3 patients. All the 3 patients were positive for hepatic HBV DNA on nested PCR assay. Of the 16 patients with occult HBVCI, 9 had HBV-DNA levels >120 IU/g, whereas in the remaining 7 patients, hepatic HBV DNA was positive only when nested PCR assays were performed. The intrahepatic HBV DNA levels varied greatly regardless of the status of serum HBsAg. For those with occult HBVCI, the intrahepatic HBV DNA levels ranged from undetectable to 3.4×105 IU/g; for those with overt HBVCI, the levels ranged from undetectable to 2.0×107 IU/g.
As reported previously, tissue HCV-RNA levels were higher than the detection limit in 94 patients but were lower than the detection limit in 21 patients. Of the 94 patients, 82 (87.2%) had serum samples available for quantitative measurement, which were all found to be positive for HCV-RNA. Regression analysis showed a positive correlation (beta = 0.012; 95% CI = 0.003 to 0.020; P = 0.007). Of the 21 patients with undetectable HCV-RNA in liver tissues, 17 (81.0%) had serum samples available for quantitative test. Of them, 5 were positive for HCV-RNA in the serum (125 to 2.8×105 IU/mL), whereas 12 were negative for HCV-RNA test. Interestingly, 10 of the 12 patients were positive for HBV-DNA in the liver tissues by PCR test, suggesting a suppression effect on HCV by HBV. Of these 10 patients, HBsAg was positive in 9 of them. Taken together, although 12 of our patients were negative for HCV-RNA in the tissue and serum samples, it was likely caused by viral interference in HBV+HCV coinfection. Therefore, in this study, we considered all anti-HCV positive patients as HCV-related HCC but with HBV superinfection as a variable for prognosis analysis.
In 51 patients who had positive tissue HBV-DNA by PCR assay (overt or occult HBV coinfection), 40 (78.4%) had serum samples available for quantitative analysis. Of them, 10 patients who had tissue HBV DNA <120 IU/g were also negative for serum HBV DNA. In the remaining 30 patients, the serum HBV-DNA was above detection limit. Regression analysis showed a positive correlation (beta = 0.170; 95% CI = 0.044 to 0.297; P = 0.010).
Cox proportional hazard model was used to examine the association between clinicopathological and virological factors, and disease-free survival after surgical resection of HCV-associated HCC (
Kaplan-Meier survival analysis was thus performed to verify the predictive values of these 6 independent factors identified by multivariate analysis (
Patients separated into two groups according to tumor size (A), alpha-fetoprotein (AFP) (B), albumin (C), ALT (D), overt HBVCI (E), and occult HBVCI (F) respectively were subjected for survival analysis. Green line, patients with assayed values greater than the cutoff (A to D) or patients with HBVCI (E and F); Blue line, patients with assayed values lower or equal to the cutoff (A to D) or patients with no HBVCI (E and F).
The clinicopathological factors were compared among patients with HCV infection only, occult HBVCI, and overt HBVCI (
HCV Ab (+) | ||||
Classification | HCV infection alone | HCV infection with occult HBVCI | HCV infection with overt HCVCI | P |
Case number | 64 | 16 | 35 | |
Sex (male) | 45 (71.4%) | 12 (75%) | 21 (60%) | 0.47 |
Age (years) | 64.5±9.44 | 63.0±11.8 | 59.4±12.8 | 0.073 |
Cirrhosis | 43 (66.6%) | 11 (68.75%) | 23 (65.7%) | 0.976 |
Fatty metamorphosis | 17 (26.9%) | 4 (25%) | 18 (51.4%) | 0.031a |
Tumor number | 1.6±0.9 | 1.6±0.7 | 1.8±0.9 | 0.299 |
Size (Diameter, cm) | 4.2±3.0 | 5.9±3.5 | 4.2±2.7 | 0.113 |
Histology grade | 2.61±0.60 | 2.87±0.8 | 2.8±0.71 | 0.1 |
Microvascular invasion | 23 (36.5%) | 6 (37.5%) | 10 (27.5%) | 0.523 |
Macrovascular invation | 8 (12.7%) | 1 (6.25%) | 5 (14.2%) | 0.717 |
Ascites | 5 (7.9%) | 1 (6.25%) | 2 (5.7%) | 0.921 |
Alpha-fetoprotein (ng/mL) | 809 (2∼16057) | 68.3 (0∼17141) | 48.2 (0∼>100000) | 0.9 |
Albumin (g/dL) | 3.8±0.5 | 3.7±0.8 | 4.1±0.4 | 0.057 |
Bilirubin (mg/dL) | 1.1±0.5 | 2.5±0.39 | 1.0±0.4 | 0.001b |
Prothrombin time (sec) | 12.1±1.2 | 11.9±1.0 | 12.4±1.6 | 0.442 |
Creatinine (mg/dL) | 1.2±1.0 | 1.4±1.3 | 1.4±0.3 | 0.409 |
AST (U/L) | 104.4±141.1 (24∼937) | 67.6±60.5 (20∼201) | 54.1±37.1 (13∼178) | 0.088 |
ALT (U/L) | 120.3±202.3 (18∼1462) | 59.8±52.5 (17∼199) | 70.3±61.7 (7∼286) | 0.205 |
AST/ALT | 1.06±0.71 | 1.21±0.47 | 0.93±0.40 | 0.292 |
Alcoholism | 7 (11.1%) | 5 (31.2%) | 5 (14.2%) | 0.124 |
a Sheffe's Post Hoc test revealed patients with HCV infection alone had significantly lower percentage of fat metamorphopsis than those with overt HBVCI (
b Sheffe's Post Hoc test revealed patients with occult HBVCI had the highest bilirubin level (occult HBVCI vs. HCV infection alone,
Since in the HCV-associated HCC patients, those with occult HBVCI had significant shorter disease-free and overall survivals than those with over HBVCI, we compared the immunohistochemical and virological parameters between these two groups of HBVCI patients (
(A) Tissue expression of HBcAg (200X). Upper panels from left to right: scattered distribution of nuclear staining (without counter stain), scattered distribution of nuclear membranous staining (without counter stain), and both nuclear and nuclear membranous staining (with counter stain). Lower panels, diffuse cytoplasmic staining. (B) Tissue expression of HBsAg (200X). Upper panels, scattered distribution of cytoplasmic staining. Lower panels, diffuse distribution of cytoplasmic staining. (C) Tissue expression of HBsAg (100X). Regional distribution of sub-membranous staining pattern.
Virological factor | HBVCI | P | |
Occult (n = 16) | Overt (n = 35) | ||
HBV-DNA (IU/g) | 478 (0∼342826)a | 2694 (0∼>2×107)a | 0.041b |
Tissue HBsAg expression | 5(31.25%) | 34(97.15%) | < 0.001 |
Tissue HBcAg expression | 3(18.75%) | 1(2.85%) | 0.051 |
Precore G1896A | 7 (43.8%) | 3 (8.6%) | 0.006 |
BCP mutations | 4 (25.0%) | 10 (28.6%) | 0.999 |
Pre-S deletion | 0 | 8 (22.9%) | 0.045 |
HBV genotype C | 3 (18.8%) | 5 (14.3%) | 0.694 |
HCV-RNA (× 106 IU/g) | 3.2 (0∼94.7)a | 0.4 (0∼49.2)a | 0.215b |
HCV genotype 1 | 8/10 (80%) | 12/18 (66.7%) | 0.669 |
HBV integration | 5(31/2%) | 14 (40%) | 0.454 |
Survival | |||
Recurrence-free survival (months) | 12.5 (5.42∼19.07)a | 77.64 (26.7∼128.58)a | 0.002c |
Overall survival (months) | 67.39±11.4d | 115.145±4.6d | 0.026c |
a Median (range); b Mann-Whitney test; c Kaplan-Meier analysis; d Estimation is limited to the largest survival time if it is censored.
After Bonferroni correction for the alpha error, P<0.006 was considered statisitical significant.
Lower detection limit of intrahepatic HBV-DNA, 120 IU/g; Lower detection limit of intrahepatic HCV-RNA, 1500 IU/g.
The genotypic characteristics of these 51 HBVCI patients were also assayed, including genotype, precore stop codon G1896A mutation, BCP A1762T/G1764A mutation, pre-S deletion mutation and sequence analysis of the whole S coding region (
Four sets of Alu-PCR were performed (See
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
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,
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
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)
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.