Expression of Cytokeratin 7 as a Histological Marker of Cholestasis and Stages of Primary Biliary Cirrhosis

Summary. The aim of this study was to estimate cytokeratin 7 (CK-7) expression in biopsy specimens of patients with different stages of primary biliary cirrhosis and clinicopathological patterns (cholestatic and hepatitic) and its correlation with some biochemical and pathological parameters and to examine a diagnostic value of CK-7 expression. Material and Methods. A total of 82 biopsy specimens of patients with primary biliary cirrhosis were analyzed. CK-7 expression was graded by 4 grades depending on the extent into parenchymal areas and bile duct epithelium. The correlations of CK-7 expression grade with copper deposition, bile duct/portal tract ratio, bilirubin concentration, and activity of alkaline phosphatase and gamma-glutamyl transpeptidase were studied. CK-7 expression was evaluated as a marker of cholestasis (cholestatic pattern) and inflammation (hepatitic pattern). Results. A positive correlation of CK-7 expression grade with copper-binding protein grade (r=0.698, P<0.0001; OR=6.199, P<0.0001), serum bilirubin level (r=0.375, P=0.001), and alkaline phosphatase activity (r=0.276, P=0.014) was found. CK-7 expression grades correlated positively with histological stages of primary biliary cirrhosis (r=0.639, P<0.000) and negatively with granulomas (r=–0.432, P<0.0001; OR=0.173, P=0.0011). Conclusions. CK-7 expression is a sensitive marker of bile duct injury, which correlated well with histological stages of primary biliary cirrhosis, copper deposits, and biochemical markers of cholestasis: serum bilirubin level and alkaline phosphatase activity. Evaluation of CK-7 expression may improve the diagnosis of this serious and progressive disease. It is recommended to evaluate copper staining together with cytokeratin 7 expression in liver biopsy specimens for more precise diagnostic evaluation of asymptomatic primary biliary cirrhosis.

Although fl orid bile duct lesions and bile duct loss are known as important diagnostic features of primary biliary cirrhosis, their diagnostic usefulness is still controversial (12). Bile duct injury re-sembling that observed in primary biliary cirrhosis (PBC) to variable degree can be encountered in other diseases (15). For this reason, special staining techniques that highlight specifi c antigens and structures are very useful for better visualization of ductular reaction (11,14,16,17,(20)(21)(22)(23).
Cholestatic and infl ammatory patterns of lesions are the main tissue alterations underlying the clinical features and histopathological markers of PBC and determining direction and different mechanisms of disease progression.
The progression of liver damage in PBC is caused not only by cholestasis but also by portal and periportal infl ammation and fi brosis. These two types of lesions may be responsible for two types or two modes of PBC, which variably overlap even in the same biopsy (12).
The recent literature presented data on cytokeratin 7 (CK-7) mostly as a marker of bile duct damage and cholestasis, but there is a lack of studies on associations between CK-7 expression and morphological infl ammatory parameters (hepatitic pattern) as well as biochemical indices of liver damage. Therefore, it is reasonable to evaluate CK-7 expression as a marker of PBC hepatitic pattern more precisely. Cholestatic changes (cholestatic pattern) comprise bilirubinostasis, cholate stasis, cholestatic liver cell rosettes, feathery degeneration, accumulation of foamy lipid-laden macrophages, ductular reaction, and periductular and septal fi brosis. With the exception of bilirubinostasis, all these alterations develop progressively. However, in chronic cholestatic diseases characterized by progressive destruction of segments of the intrahepatic bile ducts, of which PBC is a paramount example, these morphological alterations are not always seen simultaneously. Cholate stasis in PBC has been demonstrated by the deposition of copper or copper-binding protein in hepatocytes (1). The aberrant expression of bile duct-type cytokeratins (CK-7 and CK- 19) in parenchymal cells has been demonstrated in various chronic cholestatic diseases, but CK-7 expression has been shown to be a more sensitive indicator than that of CK-19 (23). The discrepancy in CK-7 expression between cholestatic and infl ammatory patterns suggests that cholestasis and infl ammatory activity in the portal tract are independent events (16,17).
Several scoring systems for histological staging of the disease in PBC patients have been reported (24)(25)(26). Staging is important in assessing disease progression and therapeutic effi cacy, in spite of frequent discrepancy between histological staging and clinical and biochemical data (7). Evaluation of CK-7 expression extent may provide additional information for precise histological staging of primary biliary cirrhosis and could be used as a routine staining method (16,17).
The aim of this study was to evaluate CK-7 expression in bile duct reaction and cytoplasm of hepatocytes in biopsy specimens of PBC patients and to determine whether CK-7 expression could be related to some well-known PBC morphological lesions, such as epithelioid granuloma formation, changes in portal tract/bile duct ratio and grade of copper-binding protein expression in periportal hepatocytes. To evaluate a diagnostic value of CK-7 expression grade, we also estimated the correlation of CK-7 expression with some biochemical indices and histological stages of PBC.

Material and Methods
Eighty-two patients (5 males and 77 females; mean age, 58.4±10.7 years) with primary biliary cirrhosis confi rmed by clinical data and standard biochemical and immunological tests and without signs of PBC-autoimmune hepatitis (AIH) overlap or/and viral or metabolic hepatitis were studied. All the patients were tested for anti-HCV by a microparticle enzyme immunoassay (AxSYM hepatitis C virus version 3.0; ABBOTT, 65205 Wiesbaden, Germany) for HBsAg (AxSYM hepatitis B Ag [V2], ABBOTT) and underwent ultrasound examination by LOGIQ 500 PRO series (General Electric Company, 3135 Fairfi eld CT, USA).
Necroinfl ammatory activity was evaluated according to the histological activity index (HAI) by Ishak et al. (1995) (29), and histological staging of PBC was estimated by the Ludwig's scoring system (22). CK-7 expression in proliferated bile ducts was examined by immunohistochemical staining with monoclonal mouse anti-human CK-7 (clone OV-TL12/30; DAKO, DK-2600 Glostrup Denmark) and graded semiquantitavely according to Yabushita et al. as follows (16) Rhodanine staining was used for visualization of copper-associated protein in hepatocytes. Copper (Cu) deposition was graded as follows: grade 0, no deposits; grade I, Cu deposition only in the cytoplasm of some periportal hepatocytes; grade II, Cu deposition in most periportal hepatocytes; and grade III, Cu deposition in deeper intralobular hepatocytes in addition to periportal hepatocytes (30,31). The number of recognizable bile ducts per portal tract was expressed as bile duct/portal tract ratio. The presence of epithelioid granulomas around the damaged bile ducts was recognized as groups of epithelioid cells near damaged bile ducts characterized by epithelial atrophy, vacuolization, with increased number of intraepithelial lymphocytes inside the altered basement membrane.
Statistical Analysis. The comparison of serum bilirubin concentration and activity of ALP, γ-GTP, ALT, and AST in four groups of patients with different grades of CK-7 expression was made by the ANOVA test. Differences in the presence of epithelioid granulomas in biopsies of above-mentioned groups of patients were evaluated by the chi-square test. The Fisher exact test was employed for the evaluation of differences in copper-binding protein deposits and Ludwig's PBC stages in groups of patients with various grades of CK-7 expression.
Pearson's correlation coeffi cient was calculated for estimation of correlation between continuous variables; for ordinal data, Spearman's correlation coeffi cient was calculated. The estimation of the odds ratio with 95% confi dence interval for Ludwig's PBC stages, serum bilirubin level, activity of ALP, γ-GTP, ALT, and AST, grades of copperbinding protein deposits, and presence of granulomas adjusted for CK-7 expression grades was made via multivariate logistic regression analysis. A P value of ≤0.05 was considered statistically signifi cant.
Statistical analysis was carried out with SPSS (SPSS Inc., Chicago, Illinois 60606, USA) and SAS software (SAS Institute Inc., Cary, NC 27513 USA).

Results
According to the Ludwig's staging system, 82 PBC patients were classifi ed as follows: 11 cases had stage I PBC, 25 cases stage II PBC, 34 cases stage III PBC, and 12 cases stage IV PBC (Table 1). CK-7 was expressed in epithelial cells of interlobular bile ducts in all PBC biopsy specimens, whereas expression in cholangiocytes of ductules (in areas of ductular reaction) and cytoplasm of hepatocytes varied signifi cantly. The grade of CK-7 staining and grades of copper-binding protein deposits showed a strong positive correlation (Spearman's correlation coeffi cient r=0.698, P<0.0001). This clear tendency of the presence of more numerous copper-binding protein deposits in the biopsies of patients with higher CK-7 grade (Fig. 5) was confi rmed by multivariate logistic regression analysis (Table 2).
Meanwhile, the CK-7 expression grades showed a strong positive correlation with PBC histological stages (Spearman's correlation coeffi cient, r=0.639; P<0.0001) (Fig. 10). The certain discrepancy was observed in the specimens with grades 1 and 2 CK-7 expression where all 4 Ludwig's stages were presented (Fig. 10). Grade 3 and 4 CK-7 expression was mostly seen in the specimens of patients with stage III (70% and 60 %, respectively) and stage IV (20% and 40%, respectively) PBC.

Discussion
Primary biliary cirrhosis histopathologically is characterized by chronic nonsuppurative destructive cholangitis leading to cholestasis and ductopenia of interlobular bile ducts. Nevertheless, sometimes it is diffi cult to prove bile duct injury and differentiate this serious progressive disease from bile duct damage because of chronic viral hepatitis, drug-induced and autoimmune hepatitis (7,9).
Staging and classifi cation of PBC is still controversial (13,17). This may be due to the striking variability in clinical and histopathological features of the disease, refl ecting the overlapping and discontinuous nature of the pathological processes.
We examined and compared the extent of immunohistochemically demonstrated CK-7 expression in bile duct cells and hepatocytes in PBC patients with different clinicopathological patterns (cholestatic and hepatitic) of this disease to prove the value of CK-7 immunostaining in the histopathological diagnosis and staging of PBC.
Different grade of CK-7 expression depended on histopathological pattern of liver injury, i.e., predominance of cholestatic or hepatitic patterns usually found but expressed differently in biopsy specimens of PBC patients. The relationship between CK-7 expression and copper-binding protein in hepatocytes, cholestatic biochemical data (bilirubin and ALP activity), and bile duct/portal tract ratio points to the cholestatic clinicomorphological pattern of injury (8,11). Our results obtained show that the most valuable marker of a cholestatic pattern could be the relationship between CK-7 expression and presence of copper-binding protein in the hepatocytes.
The grade of CK-7 expression and grade of copper-binding protein deposits showed a strong positive correlation. This clear tendency toward the presence of more numerous copper-binding protein   deposits in biopsies of patients with higher CK-7 grade (Fig. 5) was confi rmed by multivariate logistic regression analysis (Table 2). A higher level of Cu deposition was found in the specimens with grade 3 and 4 CK-7 expression suggesting that CK-7 expression was a more sensitive marker of chronic cholestatic condition than the deposition of copperbinding protein, because it was weakly expressed in the specimens with grade 1 and 2 CK-7 expression. A strong positive correlation between CK-7 grading and copper-binding protein suggests Cu deposition to be an independent morphological marker of cholestasis.
Nevertheless, CK-7 expression can be considered a more sensitive marker of cholestasis as emphasized by other authors (16). According to our data, serum bilirubin level and ALP but not γ-GT activity showed a weak positive correlation with the grades of CK-7 expression and proved CK-7 expression to be a marker of cholestatic pattern too, although a discrepancy between biochemical data and histological fi ndings exists that was confi rmed by other authors (7).
The precise assessment of PBC histological stage is important for the evaluation of prognosis often used in survival models as well as in the evaluation of treatment effi cacy in therapeutic trials on PBC (26). Scheuer's classifi cation includes chronic nonsuppurative destructive cholangitis as a characteristic histological feature of PBC and has been widely used for staging PBC (25).
Data that a part of patients with even advanced Ludwig's (III and IV) stages present with grade 1 and 2 CK-7 expression (Fig. 10) may suggest that cholestatic and infl ammatory patterns are independent events (1,7,19). Other reason for fi nding low CK-7 expression (grade 1 and grade 2) in Ludwig's stages II, III, and even IV could be due to topographic heterogeneity of the lesions causing considerable overlap between different PBC stages in the same biopsy as also noted by others (11). Nevertheless, in comparison between the CK-7 grading and histological staging, we consider that cholestasis progresses in more advanced stages of Ludwig's classifi cation and that it is less expressed in Ludwig's stages I and II where infl ammatory activity and bile duct lesions dominate over fi brosis and cholestasis. The fact that there is a positive correlation between the CK-7 grading and histological staging does not support the concept that histological staging is an independent marker of cholestasis as suggested by multivariate logistic regression analysis in the comparison with copper-binding protein expression. This could be due to the essence of the Ludwig's classifi cation, which is based only on parameters of infl ammation and fi brosis, analogous to the grading and staging of chronic viral hepatitis, and does not include the cholestatic pattern, which nevertheless is one of the main histological criteria of PBC.
The fi nding that epithelioid granulomas were observed in specimens with all grades of CK-7 expression emphasizes the heterogeneity and discontinuity of the cholestatic and infl ammatory patterns in PBC, even in the same liver biopsy, whereby early bile duct lesions can be seen in the livers of patients with advanced-stage disease (25). However, the fi nding of fewer granulomas in more advanced stages indicates that cholestatic lesions predominate in more advanced stages of PBC -similar to what was shown comparing CK-7 grading and histological Ludwig's stages -the more expressed fi brosis, the more severe cholestasis but fewer granulomas (the main pathological PBC features).
As infl ammation and cholestasis are of importance in the progression of PBC (11,15,16), their characteristic morphological patterns deserve to be included in histological staging especially when dealing with liver biopsies for the evaluation of therapeutic effi cacy.
For more precise and adequate evaluation of PBC histological stages, it is advisable to assess CK-7 expression and its grades that could be used as a diagnostic and prognostic marker in the management and follow-up of patients with PBC.

Conclusions
Our data confi rm the expression of cytokeratin 7 as a sensitive marker of bile duct injury and cholestasis in patients with primary biliary cirrhosis that correlated well with the stages of primary biliary cirrhosis and biochemical parameters of choles tasis -bilirubin concentration and alkaline phosphatase activity. The results support the inclusion of 4-grade cytokeratin 7 expression staging for more accurate biopsy-based staging of primary biliary cirrhosis and improvement of diagnosis and prognosis of patients with primary biliary cirrhosis. It is recommended to evaluate copper staining together with cytokeratin 7 expression in liver biopsy specimens as these both markers have been shown to have diagnostic and prognostic value while examining patients with primary biliary cirrhosis.