Histological Presence of HBsAg in Liver Pathologies in Southeast Mexican Patients

AFP: Alpha-Fe-toprotein; HCV: Abstract To investigate the presence of Hepatitis B Virus (HBV) in hepatic lesions and Hepatocellular Carcinoma (HCC) in southeast Mexican patients, liver biopsies and resections specimens from HCC and hepatic lesions (cholestasis and steatosis, hepatitis and necrosis, fibrosis and cirrhosis) were collected and immunohistochemically examined. The expression of HBsAg and a liver tumor marker: Alpha-Fetoprotein (AFP) was quantified in liver specimens by quanti tative and semiquantitative methods. The correlation between expression level of AFP and HBsAg and degree of liver injury was analyzed statistically. Expression of AFP was found in all hepatic lesions and HCC samples in a different intensity level. There was no significant dif ference of AFP positive area in each group analyzed. The HBsAg expression was detectable as a weak stain in samples of all groups. The age of patients with hepatic lesions in contrast to HCC did show a significant difference. The age mean was significant higher in HCC (55.9) with respect to hepatic lesions (38.2). In the patients without HCC, the proportion of HBsAg positive area was higher than in HCC group, and this difference was statistically significant. A negative correlation of HBsAg in hepatic lesions and hepatocellular carcinoma was found. AFP was detected in hepatic lesions and HCC; hence additional diagnosis techniques should be used in Mexican southeast patients to have an accurate diagnose and a clinical treatment. There was a negative correlation between HBsAg presence and degree of liver injury. However, supplementary molecular techniques in search of occult HBV infection in HCC patients must be developed.

serious consequences and terminate in untreatable liver cancer [19,20]. The oncogenic capacity of HBV to enhance malignant transformation is due to interplay mechanisms of cell host defense against viral replication [21]. Moreover, the virus proteins them self-promote alterations of signal transduction pathways related to hepatocyte proliferation [22][23][24]. The immune-mediated liver damage contributes to hepatic pathogenesis [25]. The chronic liver inflammation (hepatitis) generated by HBV infection, stimulates continuous cycles of lowlevel liver cell destruction and regeneration that, over long periods of time, lead to hepatic lesions like steatosis, fibrosis, cirrhosis, and HCC [26]. The Hepatitis B Surface Antigen (HBsAg) and Alpha-Fetoprotein (AFP) are considered serum markers to HBV infection and HCC respectively. The HBsAg in a blood test indicates current HBV infection (acute or chronic) and the person can transmit the infection to others [27,28]. And the serum levels of AFP are correlated with the size and volume of the tumor at the time of diagnosis [29,30]. In Mexico, the impact of chronic HBV infection as etiologic factor on HCC is still unknown. Data about general chronic liver diseases prevalence underestimates the real outlook [31]. Although it is assumed that the majority of HCC reported in Mexico develops from alcohol-related cirrhosis, the incidence of viral hepatitis increased [32][33][34]. Alcohol consumption is the principal cause usually explored in a cirrhotic patient, and the assessment of a hepatic viral infection is not taken in consideration in all cases [31]. In accordance with Mexican Health Ministry and National Institute of Statistics and Geography reports, Veracruz is a southeast state that refers to record the highest rates of liver cancer mortality. Therefore, it is necessary to explore the presence of HBsAg and AFP levels in the hepatic lesions and HCC in southeast Mexican population. As a negative control (CN), a cervix tissue sample was used.

Anatomic-Histological Analysis
All specimens were sliced to hematoxylin-eosin staining for routine histological diagnosis. The histopathological examination was carried out and hepatic specimens were grouped according to

Tissue Microarray Design and Construction
In order to obtain the best magnification area to immunological

Immunohistochemical Staining and Quantification
Formalin-fixed paraffin liver sections (4 micron) were blocked it is based on grade staining: 0 (negative), 1(weak), 2 (moderate), and 3 (strong) as described by Zhang [35] and Chadha [36]. The final score was the total sum of the product of the staining intensity and its corresponding area percentage. For example, if a tumor showed 50% moderate staining and 50% strong staining, the final score would be (50X2)+(50X3)=250. A final score of at least 100 was considered positive expression [35].

Statistical Analysis
Statistical analyses were carried out using PASW Statistics v.

Staining Levels of AFP in Hepatic Lesions and Hepatocellular Carcinoma
The utility of AFP in serum as a tumor marker of HCC has been established. However, the specificity and sensibility of the test remains contradictory [37].

Relatively Low HBsAg Intensity Level in Hepatic Lesions and HCC
As mentioned above, one of the main risk factors associated to

Equivalent Intensity Protein Levels of Manual and Automatic Quantification
The presence of HBsAg and AFP proteins, was quantified through two methods described before: a semiquantitative visual score [35] or a quantitation by image analysis software [38]. Due to significant coefficient correlation by Spearman test, between visual and automated procedure, we decided to use the dataset deployed by automated software for statistical analysis.

Demographic Characteristics of Patients with Hepatic Lesions and Hepatocellular Carcinoma
The patient demographic characteristics with hepatic lesions (non-cancer samples: cholestasis and steatosis, hepatitis and necrosis, fibrosis and cirrhosis) and HCC are summarized in two great groups (Table 1). There was a prevalence of male gender in both groups, although this difference was not significant. On the other hand, the age of patients with or without HCC did show a difference. The age mean was higher in HCC (55.9 ± 13.6) with respect to hepatic lesions (38.2 ± 24.4). In the non-cancer samples, the median of HBsAg positive area was higher than in HCC group.
Finally, there was not difference of the AFP expression between HCC and non-cancer specimens.

Min-max value: Minimum and maximum value
Proportions were compared using Chi square test.
Means were compared using Student's test.
Medians were compared using Mann-Whitney U test.

Correlation Between Age, Degree of Liver Injury and HBsAg-Alpha fetoprotein Score
In Table 2 were not correlated significantly.

Discussion
The sensitivity and specificity of serological AFP level as hepatic tumor marker have displayed a controversial setting, ranging from characteristics of the diagnostic test used [39], the racial differences in AFP effectiveness [40] even the virological status affects the efficiency of AFP in patients with HCC and chronic liver diseases [41]. Our data indicated a non-exclusive expression of AFP in HCC and a widespread minor or major presence in each group tested. In accordance with our results, in a previous report the AFP levels rise further as the grade of liver steatosis increases [42]. The increased serum AFP level in patients with severe fatty liver was attributed to hepatic inflammation and/or fibrosis as underlying cause. Other chronic liver diseases (viral hepatitis, hepatic fibrosis, and cirrhosis), are connected with augmented AFP level [43]. It has even been reported in other malignancies such as gastric cancer [44][45][46]. The AFP expression in non-cancerous liver pathologies and non-liver tissues, converge in a permanent inflammatory condition regardless of its etiology. The previous studies and our results support that using only the measurement of AFP is insufficient for HCC diagnosis. In fact, nowadays the evaluation of fucosylated AFP is being used along with the standard imaging studies to improve accurate diagnosis [47,48].
Mexico is a Latin-American country considered part of low endemic HBV infection area. This situation may be due to diverse factors such as recent addition of the HBsAg detection from 1980 [49], to commercially available detection methods with a low sensibility and specificity used in healthcare institutions [50], to a new unsystematized epidemiological national surveillance [51] and occult HBV infection [34]. Nevertheless, although the national immunization program started since 1999, the population of sexually active adolescents and adults not covered by the program have a risk of becoming infected with HBV. The incidence and prevalence of liver diseases in Mexico have increased in the last two decades [32] and it will increase further [52]. Veracruz is one of the southeast Mexican states with major increase of liver cancer mortality [32]. However, there are not enough regional studies in which the etiology has been analysed. Maybe this could be explained due to do not exist a national specific program for hepatocellular carcinoma epidemiological surveillance.
The HBsAg, since its discovery in Australia in 1967 [53] is employed in both Mexico and the entire world, as a qualitative diagnostic marker for acute or chronic HBV infection. All three forms of HBsAg antigen (Dane particle, filamentous particle and spherical particle) can be detected in serum with commercial assays for diagnosis test in clinical practice [54]. in the sera at low replicative and transcriptional levels allowing inflammation, facilitating progression of disease [57,58].
This spontaneous sorcerous was associated to older age but could be implicated the HBV genotypes and geographic areas of high endemicity [59]. Moreover, HBsAg mutations described above [60], hamper the diagnostic performance and limit HBV detection with a determinant mutation [61]. The failure to detect HBsAg due to mutations justifies the HBsAg clearance and explains an Occult HBV Infection (OBI). OBI is defined as serologically or tissular undetectable HBsAg, despite HBV DNA circulating [62].
The discovery of OBI was made in order to elucidate the HBV transmission, even by blood components negative for HBsAg of donor's transfusions. Although we haven´t analyzed the HBV DNA to consider an occult HBV infection as a possible explanation results, there are several investigations describing an OBI among native Mexicans and southeast citizens [34,63], where the genotype H was the main circulating HBV strain with mutations in core region [64]. In conclusion, AFP was detected in hepatic lesions and HCC; hence additional diagnosis techniques should be used in Mexican southeast patients to accurate diagnostic and clinical treatment.
There was a positive significant correlation between age and degree of liver injury. Also found a negative correlation between HBsAg presence and degree of liver injury. However, supplementary molecular techniques to test presence of occult HBV infection in HCC patients must be developed.