© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60026-6

 

 

Contributors: TAM and DT contributed equally to the paper. DT and PI made the study concept and design. DS and GR acquired the data. TAM, DT and PP made the interpretation of data. TAM and DT made the analysis and drafted the manuscript. All authors made the critical review of the manuscript for important intellectual content. PI is the guarantor.

Funding: None.

Ethical approval: This study was approved by the Local Ethics Committee.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: The global risk of hepatocellular carcinoma (HCC) is largely due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. In recent years, however, an increased prevalence of non-viral HCC has been noted. The clinical impact of the presence/absence of viral infections in HCC remains controversial. The present study aimed to assess the effect of hepatitis viruses on demographics, clinical and pathological features and long-term outcome in a large cohort of Romanian patients who underwent surgery for HCC.

 

(Hepatobiliary Pancreat Dis Int 2014;13:162-172)

 

KEY WORDS: hepatitis B virus; hepatitis C virus; hepatocellular carcinoma; recurrence; prognosis

Hepatocellular carcinoma (HCC) is the seventh most common cancer and the third leading cause of cancer-related death worldwide.^[1] Thus, HCC is not only common but also very deadly, with a 5-year survival rate of less than 10%.^[2] According to the age-adjusted HCC incidence per 100 000 habitants, different areas are classified as low (<5), intermediate (5-15) and high (>15).^[3] Romania is considered to be at an intermediate incidence (8 HCC/100 000 habitants).^[4]

 

For the past century, the global risk of HCC was largely due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, along with aflatoxin intoxication, excessive alcohol consumption, obesity and diabetes.^{[5, 6]} Furthermore, diabetes mellitus has a negative impact on both overall and disease-free survival after curative-intent treatments for HCC.^[7] Chronic hepatitis B and C, mostly in the cirrhotic stage, are responsible for the great majority of cases of HCC worldwide,^[8] as appears to be the case in Romania.^{[4, 9]} Co-infection with both viruses is often noticed in HCC patients due to their similar ways of transmission; this situation has significantly impacted the overall burden of chronic liver disease.^[10] HBV and HCV co-infection is associated with more severe forms of liver disease compared with chronic hepatitis caused by a single virus.^[11] At the molecular level, distinct chromosomal abnormality patterns and transcriptomic signatures were observed in HCC without viral infection.^{[12, 13]} Therefore, there should be some clinico-pathological differences between HCC in patients without viral infection and those associated with hepatitis viruses.

 

With regards to its natural history and its clinical presentation, HCC is a heterogeneous disease. The incidence of HCC varies across the world due to geographic differences in the prevalence of risk factors.^[14] Furthermore, in addition to different etiology rates, other significant differences among countries are commonly noticed regarding patient age, gender ratio, tumor size or degree of underlying liver damage.^[15] Nevertheless, biological characteristics appear to affect the natural history of HCC.^[16]

 

Curative-intent treatment options for HCC include liver resection, liver transplantation and ablative therapy, depending on the tumor stage and functional status of the liver.^[17-20] However, despite the above-mentioned heterogeneity, no differences regarding long-term survival were observed between Western and Eastern centers when adjusted for clinico-pathological factors.^[15] Early detection and curative resection are considered the most important factors for long-term outcome.^[21] The effect of HBV and HCV infection on clinico-pathological features and long-term outcome in HCC-treated patients has been previously studied; however, most of these studies are from Eastern Asia.^[22] In Eastern Europe there is a conspicuous paucity of reports regarding the epidemiology^[23-25] and surgical management^{[17, 26]} of HCC. Furthermore, none of the works published thus far have addressed the effect of hepatitis on long-term prognosis. We assessed the potential differences determined by the presence of HBV or HCV in a large cohort of HCC patients from a single surgical center of an Eastern European country. Only patients who were surgically treated with liver resection, liver transplantation or radiofrequency ablation were considered.

 

 

The data from 404 patients who had been treated from January 2001 to November 2010 with a final pathological diagnosis of HCC were retrospectively reviewed from a prospective-gathered electronic database established at the Center of General Surgery and Liver Transplant from the Fundeni Clinical Institute (Bucharest, Romania). Only patients who underwent surgical treatment (i.e., liver resection, liver transplantation or radiofrequency ablation) were considered. Informed consent was obtained from each patient, and the study was approved by the Local Ethics Committee.

 

The preoperative work-up included a serologic examination for HBV (hepatitis B surface antigen) and HCV (hepatitis C antibody) for all patients. The serum tumor marker alpha-fetoprotein (AFP) was preoperatively assessed in 207 patients (51%); the cut-off value was <32 ng/dL. Computed tomography (CT) and/or magnetic resonance imaging (MRI) examinations were performed to evaluate the extent of the disease, tumor location and characteristics. For patients who underwent ablation, the size and number of tumors were noted with imaging examinations and the tumor type was established through biopsy (patients without a positive biopsy for HCC were excluded). The choice of treatment modality was performed according to the patient status [Child-Pugh score or Barcelona Clinic Liver Cancer (BCLC) classification] and tumor features (localization, size, number and stage).

 

The patients with HCC were divided into four groups: 85 patients with only hepatitis B virus infection (HBV group), 164 patients with only hepatitis C virus infection (HCV group), 39 patients with hepatitis B and C virus infection (HBCV group), and 116 patients with no viral infection (non-BC group). The comparative analyses included demographics (age, gender and residence), serum AFP level, Child-Pugh score, staging systems such as Okuda, BCLC, the Cancer of the Liver Italian Program (CLIP), Milan criteria, type of treatment, pathology data (number and size of the tumors, underlying liver cirrhosis, Edmonson-Steiner grading system), disease-free and overall survival rates.

 

The follow-up protocol included clinical examination, serum AFP level and ultrasound and/or CT and/or MRI every 3 months in the first year and every 6 months thereafter. Data for AFP were available for 48 patients in the HBV group (56%), 90 patients in the HCV group (55%), 20 patients in the HBCV group (51%) and 49 patients in the non-BC group (42%). The last follow-up was April 1, 2011.

 

All statistical analyses were performed using SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA). The data were expressed as the number (percentage) and median (range), except for age, which was expressed by the mean±SD. Fisher's exact test (two-tailed) was used to compare the categorical variables, and the Mann-Whitney U test (two-tailed) was used for continuous variables. The median disease-free and overall survival rates were estimated using the Kaplan-Meier method, and the median follow-up time was estimated using reversed Kaplan-Meier curves. Comparisons were made using the log-rank test (for univariate analysis). Multivariate analysis used a forward stepwise Cox proportional hazards model and variables from the univariate analysis with P values less than 0.10 were included. A P value less than 0.05 was considered statistically significant.

 

 

Of the 404 patients with HCC, 288 (71%) had viral infection. Among them, 124 patients (31%) had HBV infection and 203 patients (50%) had HCV infection (Table 1).

 

Both patients in the HBV (56.0±11.3 years) and HBCV groups (56.0±9.9 years) were significantly younger than those in the HCV (61.0±8.5 years, P=0.001 and P=0.002) or non-BC groups (61.0±13.0 years, both P=0.001). No differences regarding patient age were observed between the HBV and HBCV groups (P=0.970) or between the HCV and non-BC groups (P=0.061) (Fig. 1A). Patients were predominantly male in all groups. Significant differences were observed between the HBV and HCV groups (P<0.001), the HCV and HBCV groups (P=0.010) and the HBV and non-BC groups (P=0.002), respectively. No differences were noted between the HCV and non-BC groups (P=0.362), the HBV and HBCV groups (P=0.763) or the non-BC and HBCV groups (P=0.054) (Fig. 1B). Regarding the origins, patients in the non-BC group were more frequently from rural areas than those in the HBV (P=0.005) and HCV groups (P=0.056). Overall, the non-BC patients were more frequently from a rural area than from an urban neighborhood in comparison with all viral etiologies (P=0.009) (Fig. 1C). In addition, patients from outside Bucharest were more prevalent in the non-BC group than in the HBCV group (P=0.042). The city-countryside distribution was not different among the groups of virus-infected patients (Fig. 1C, D).

 

Liver cirrhosis was present in 289 patients (72%) and was less frequent in the non-BC group (52/116, 47%) than in the HBV (61/85, 72%; P=0.002), HCV (141/164, 86%; P=0.001) or HBCV groups (35/39, 90%; P=0.001). Liver cirrhosis was also less frequent in the HBV group than in the HCV (P=0.009) and HBCV groups (P=0.036). No differences in the prevalence of liver cirrhosis (P=0.792) were observed between the HCV and HBCV groups, nor differences in Child-Pugh scores (P≥0.199) among the groups (Table 1).

 

In these groups, there was no significant difference in the number of patients with multicentric tumors, the size of the tumor, or the Edmonson-Steiner grading system (Table 2).

 

The median serum AFP level was 30.2 ng/dL (range 2.0-60490.0) in the HBV group, 83.7 ng/dL (range 2.1- 5741.0) in the HCV group, 47.1 ng/dL (range 1.4-991.0) in the HBCV group, and 12.0 ng/dL (range 1.1-35500.0) in the non-BC group. But no statistical differences were observed among all the groups (P≥0.476).

 

At the time of initial diagnosis, more patients in the non-BC group (83/116, 72%) were assessed beyond the Milan criteria than those in the HBV (44/85, 52%; P=0.004), HCV (65/164, 40%; P=0.001) or HBCV groups (14/39, 36%; P=0.001). But there were no differences in this regard between the HBV and HCV groups (P=0.080), the HBV and HBCV groups (P=0.122) or between the HCV and HBCV groups (P=0.717), respectively.

 

With regard to clinical staging, no differences were observed in the Okuda or CLIP staging systems among the groups (Table 1). As expected, however, patients in the non-BC group had more advanced BCLC stages (62 patients with C/D stages, 53%) than those in the HCV (63 with C/D stages, 38%; P=0.020) or HBCV groups (12 with C/D stages, 31%; P=0.017). But there were no differences between the HBV (38 with C/D stages, 45%) and HCV (P=0.344), HBCV (P=0.169) and non-BC groups (P=0.257), nor differences between the HCV and HBCV groups (P=0.461).

 

The types of treatment given to each group are shown in Table 3. Liver resection was more frequently performed in the non-BC group than in the HCV (P=0.001), HBV or HBCV groups (P=0.005). But no differences in this regard were observed between the HBV and HCV groups (P=0.665), the HCV and HBCV groups (P=0.847) or the HBV and HBCV groups (P=1.000), respectively.

 

The median time of follow-up was 44 months (range 4-123 months). A total of 103 patients were excluded from the survival analysis because of postoperative deaths or loss from follow-up: 19 (22%) in the HBV group, 42 (26%) in the HCV group, 5 (13%) in the HBCV group and 37 (32%) in the non-BC group. Postoperative mortality was 4.5% (18 patients), and there were no differences between the groups (data not shown).

 

No differences were observed in the disease-free or overall survival rates of the groups (Fig. 2). Furthermore, survival analysis was made after adjustment of age and the presence or absence of liver cirrhosis. The median (60 years) and the first quartile (53 years) were used as cut-off points of age. No differences in the disease-free (P≥0.642) or overall survival rates (P≥0.654) were observed among the groups when the presence/absence of cirrhosis was adjusted (data not shown). Finally, no differences were observed among the groups after adjustment of age (disease-free survival P≥0.547; overall survival P≥0.702) (data not shown).

 

The following potential risk factors for disease-free and overall survival rates were considered upon univariate analysis: age, area of residence (urban vs rural/Bucharest and suburbs vs outside), gender, cirrhosis, the Milan criteria, Okuda, CLIP and BCLC classifications, tumor diameter and number, Edmonson-Steiner grading, HBV, HCV, and HBV and HCV viral co-infections, and different types of treatment (partial liver resection, liver transplantation and radiofrequency ablation). For overall survival, the Milan criteria, Okuda and BCLC classifications, number of tumors, Edmonson-Steiner grading and radiofrequency ablation were identified as statistically significant prognostic factors by the univariate analysis. For disease-free survival, in addition to the above-mentioned factors, liver transplantation was identified as a prognostic factor in the univariate analysis. HBV infection, HCV infection, and a co-infection of HBV and HCV were not identified as prognostic factors after surgery for HCC in the univariate analyses for disease-free (P≥0.311) or overall survival (P≥0.128). The multivariate analyses for potential risk factors are shown in Tables 4 (for disease-free survival) and 5 (for overall survival).

 

 

A recently study^[22] has addressed the effect of viral hepatitis status on clinico-pathological features and patient survival after surgical treatment of HCC. Notably, only a single study included European patients.^[27] Moreover, few studies included a comparative analysis among all four groups of patients: HBV-only, HCV-only, HBV and HCV co-infection and non-viral HCC.^[22] It is well-known that both HBV and HCV carriages are highly prevalent in the Romanian population and therefore represent a serious public health problem.^[28-33] A similarly worrying situation is not encountered in Western Europe^[31] and can only be compared with what is observed in neighboring countries such as the Republic of Moldova^[34] and perhaps Bulgaria.^[35]

 

Most (71%) of the HCC cases in the present series are related to viral infections, primarily to HCV infection and secondarily to HBV infection. Similar features were previously reported in another smaller series of Romanian patients with HCC.^[9] The prevalence of viral infection in HCC cases worldwide is reported to range between 13%-90.5% for HBV and 4%-88% for HCV.^[36-38] Although we have previously shown that the molecular epidemiology of HCC in Romania appears to be slightly different from that observed in Western Europe,^[25] the association of HCC with HBV (31%) or HCV (50%) in the present study is similar to that reported in Western Europe^[39-42] but differs from the situation in Greece, where HBV continues to dominate HCC causes.^[43] HBV-associated HCC incidence is currently decreasing in the autochthonous European population because of systematic immunization policies implemented in the European Union. Thus, it is highly probable that Romania will follow this trend because there is a decrease in HBV prevalence because of immunizations.^[44] The HCV-related HCC incidence was predicted to rise until 2015-2020 and decrease thereafter because of preventive measures taken during the 1990s to control the spread of HCV.^[42] A similar situation will occur in Romania, in which the health care system has greatly improved in the past two decades.^{[45, 46]} Thus, the non-BC subset of HCC patients will be increasingly important in the future, as is the case in Northern Europe.^[43]

Patients in the present series without viral infections were older than those with HBV-only or co-infection of HBV and HCV. A similar trend was observed in the HCV-only-infected patients. An older age was shown in non-viral HCC patients compared with HBV- or HCV-only patients.^{[22, 47, 48]} However, a younger age was seen in patients without viral infection.^[15] In our study, a small number of non-BC patients were younger than the rest of the population. This finding suggests the presence of alleles predisposing to liver cancer in early-onset HCC patients or in 2%-3% of the Romanian population.^[49-51]

 

In the present study, patients with HCV-only were older than those with HBV-only or a co-infection of HBV and HCV. Previous studies^[52-54] have shown a significant older age in patients with HCV-only compared with those with HBV-only HCC. This situation could be explained by a dominating role of HBV over HCV, with HBV setting the pace of a fast liver disease progression. Alternatively, this situation could merely reflect an age difference at contamination, with HBV acquired earlier in a patient's life span and thereby triggering earlier complications of chronic infection. In Romania, the mode of HBV infection is known to be predominantly horizontal, thus occurring later in life than in the Far Eastern countries where HBV transmission was previously vertical/perinatal.^{[29, 55, 56]} This may explain why the mean age difference between those who are HBV- and HCV-infected is only 5 years, whereas the difference in Taiwan region is 12 years.^[57]

 

The present study have shown that patients with HCC without viral infection are more frequently from rural areas compared with HBV-only patients; a similar trend was observed compared with HCV-only patients; however, statistical significance was not reached. These findings are surprising because previous studies have shown a higher prevalence of HBV and HCV in rural areas of Romania compared with urban areas.^{[28, 29]} However, data from the literature are controversial. A report from Poland indicated that urban dwellers were more likely to be carriers of hepatitis viruses than those who live in rural areas.^[58] However, symmetric studies showed no differences in Asian countries^[59] or even an increased prevalence of HBV in rural areas in Western Europe, China or Africa.^{[52, 60]} Alternatively, the cause of the larger non-BC proportion among rural dwellers could be linked to the presence of specific environmental or lifestyle HCC risk factors. In such settings, an excess of HCC cases should be observed in some regions of Romania. Several risk factors for non-viral HCC, such as excessive alcohol consumption and diabetes were found to prevail in rural areas of Romania.^{[61, 62]} Interestingly, Central European surveys have reported increased rates of cancers affecting rural populations.^{[63, 64]} Further epidemiological studies are warranted to confirm this hypothesis. Furthermore, non-BC HCC patients are more frequently from outside Bucharest areas compared with HBCV patients. HBV and HCV were recently found to have the highest prevalence in the south of Romania,^{[28, 29]} including the Bucharest area.^[32]

 

Liver cirrhosis was present in a large number of patients of the present series (72%). The prevalence of liver cirrhosis in HCC patients displays some geographical variability and is high everywhere (more than 80%), except in sub-Saharan Africa.^{[3, 65]} Although rare in Europe, HCC on a non-fibrotic liver may result from hepatitis B, alcohol abuse and infrequently from chronic hepatitis C.^[66] In the present series of patients and as shown previously, liver cirrhosis was significantly less frequent in HCC without viral infection compared with all other patient groups.^{[47, 67]} This situation suggests that some of the patients within the non-BC subset were submitted to a more directly tumorigenic process (either genetic or environmental). Liver cirrhosis prevalence was significantly lower in the HBV-only patients compared with HCV-only or HBCV patients. This finding confirms that HBV does not require cirrhosis to systematically induce carcinogenesis.^[66] The highest rate of liver cirrhosis (90%) was observed in the present study in patients with a co-infection of HBV and HCV. Co-infection is known to generate intense hepatic inflammation that induces the stimulation of fibrogenesis by cytokines, resulting in an accelerated pathophysiologic process.^{[15, 39, 57, 68, 69]} As a consequence, co-infection is more strongly associated with HCC than either single infection, suggesting a synergistic effect for fibrogenicity and carcinogenicity.^[70] HBV and HCV are non-redundant inducers of hepatocarcinogenesis: HBV is a DNA virus that is able to integrate into host genomic DNA and may generate genomic instability or encode proteins important in carcinogenesis, whereas HCV is a strictly cytoplasmatic RNA virus endowed with different tumor-promoting activities either indirectly through chronic liver inflammation or more directly through viral products interacting with onco-suppressive or oncogenic proteins.^[5]

 

Although no differences were observed between the groups of the present series regarding the Child-Pugh score, serum AFP level, Okuda or CLIP staging systems, uninfected HCC patients were more frequently assessed outside the Milan criteria compared with HBV- and HCV-only/HBCV patients. Furthermore, patients without viral hepatitis had significantly more advanced BCLC stages at diagnosis compared with HCV-only or HBCV patients. It was previously shown that more advanced CLIP and BCLC stages prevailed in patients with HBV-only compared with HCV-only HCC.^[52]

 

Regarding the pathology characteristics, we observed that in the present series, patients without viral infection tend to have larger tumors compared with all other groups (7 vs 4-5 cm), though these findings did not reach statistical significance. Larger^{[47, 69]} and less differentiated^{[69, 71]} tumors in patients with HCC in the absence of viral infection have been previously described. Because of their advanced ages and stages at diagnosis combined with their larger tumors, one may speculate that these HCC may have followed an indolent growth for years. Nevertheless, further studies are needed to demonstrate this feature. A more appropriate explanation for advanced ages, stages at diagnosis and larger tumors in the uninfected HCC patients of the present series could be due to the poor effectiveness of cancer surveillance in community practice, delayed presentation or low-quality medical care. These features must be corroborated with the fact that non-viral HCC patients are more frequently from rural areas where there is a lack of specialized medical centers and where people are more reluctant to seek medical help. Nevertheless, in the present study, the disease-free and overall survival rates do not appear to be impaired for patients from rural/outside Bucharest areas (Fig. 2). However, a non-significant trend towards a better prognosis in patients coming from the Bucharest area should be noted.

 

The impact of viral status on long-term outcome in patients with HCC is controversial. Thus, some studies have shown a significantly poorer disease-free^{[6, 27, 53, 72]} and overall survival rates in patients with HBV-/HCV- only or co-infection,^{[47, 72]} whereas other investigators did not identify differences regarding the recurrence^[52,69] or overall survival rates^{[15, 52, 53, 73]} between HCC patients with or without viral infection or between HBV and HCV patients^{[15, 47, 54]} as appears to be the case in the present series. Postoperative antiviral therapy may potentially reduce the recurrence rates^[74] and improve overall survival^{[38, 75]} after surgery for HCC with viral infection. Nevertheless, interferon-alpha appears to suppress tumor growth in HCC but promotes metastasis capacity.^{[76, 77]}

 

Although statistical significance was not reached, the data of the present study showed a trend for better disease-free and overall survival rates in HBV-only patients compared with all other groups of patients; a trend towards a worse long-term outcome was observed in the HBCV group of patients with HCC. This observation is at odds with a recent meta-analysis that showed a tendency for better overall survival rates in HCC patients without viral infection and an impaired disease-free survival for HBV or HCV.^[22] The differences between the reported meta-analysis and the outcome of the present study may be explained either by the fact that uninfected HCC patients had more advanced disease in Romania than in reported countries or because the risk factors and clinico-biological features of the tumors differ widely between different patient cohorts.

 

The lack of a significant difference in overall survival between groups may be explained by the fact that there were no significant differences in regard to the most important independent prognostic factors for overall survival identified in the present series (i.e., number of tumors and Edmonson-Steiner grading). Furthermore, based on the identified independent prognostic factors for disease-free survival in the present series, one could expect to have a worse survival in the non-BC group (because of the increased rates of advanced BCLC stages). However, this feature could be balanced by the significantly reduced rates of liver cirrhosis compared with all other groups. The trend for a worse disease-free survival in the HBCV group could be explained by the highest rate of liver cirrhosis (90%) in this group compared with all other groups. The presence of cirrhosis was identified as an independent risk factor for poor disease-free survival in the present study. The lack of statistical significance for disease-free survival rates in the HBCV group compared with all other groups could be explained by the highest rate of liver transplantations (31%); liver transplantation has been identified as an independent prognostic factor for better disease-free survival.

 

The present study is limited by its retrospective design and should be interpreted with caution. Thus, some data regarding clinics or pathology with a potential impact on long-term outcome after surgery for HCC were overlooked.

 

In conclusion, despite a lower incidence of liver cirrhosis, uninfected patients with HCC are diagnosed at advanced ages and stages, and these patients are likely to have larger tumors. This finding could be explained by the low effectiveness of cancer surveillance in community practice. A careful definition of liver cancer-prone individuals among the population free of viral hepatitis should be developed, and a surveillance program should be implemented either in community or tertiary care facilities. Furthermore, an improvement in the quality of medical care in rural areas, along with the implementation of medical educational programs for the general population, could contribute to an earlier diagnosis in patients with HCC without viral infection. The issue of the subset of young patients (<45 years) developing HCC without viral infections should be investigated in appropriate epidemiological studies.

 

 

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