Characteristic of expression levels of HepPar-1, alpha-fetoprotein, cytokeratin 7 and 20 by the cells of cholangiocellular cancer in trephine biopsy of the liver

Aim. Expression level of immunohistochemical markers such as HepPar-1, AFP, CK7, CK20 and the area of immunopositive cells in cholangiocellular liver cancer, and their differences from hepatocellular carcinoma vere investigated. Methods and results. Histopathological, histochemical and immunohistochemical research of trephine was determined in the liver in 90 patients with biopsy. Among them 53 patients had hepatocellular, 36 – сholangiocellular liver cancer, 1 patient had mixed hepato-cholangiocellular carcinoma. Level of expression of immunohistochemical markers of tumor cells and the area of immunopositive tumor cells in the tumor was determined by photo-digital morphometry. It was established that expression of α-fetoprotein is determined in 47.22% of patients with cholangiocellular liver carcinoma in tumor cells, when AFP-immunopositive cells represent 17,25 ± 9,67% of the total area of tumor cells. Positive expression of HepPar-1 cells in cholangiocellular liver cancer wasn’t detected (unlike hepatocellular carcinoma, when cytoplasmic expression of HepPar-1 by tumor hepatocytes is determined in 92.45% of cases). Expression of СK7 by cholangiocellular carcinoma cells was observed in 97.22% of patients, and the expression of CK20 – in 45.29% patients, immunopositive cells represent 43,55 ± 9,93% and 50,28 ± 16,35% of the tumor area, respectively. Medium strength correlation was determined between the level of AFP and CK7 expression by tumor cells in cholangiocellular carcinoma. Direct strong bond was determined between level of AFP and CK20 expression. Negative weak correlation was determined between the level of CK7 and CK20.

Immunohistochemical (IHC) researches with trephine biopsy puncture of the liver acquired special differential diagnostic importance, because of the containing of a limited amount of diagnostic material. Without them, it is almost impossible to identify hepatoor cholangiocellular phenotype of solidcellular and low-grade cancer, differential diagnosis is impossible when a trephine biopsy doesn't contain the characteristic patterns of hepatocellular carcinoma or tubular pattern of cholangiocellular cancer. Contradictory results of extended researches of last years, outlined in modern manuals [2,3,5], have shown a great variability of expression levels of HepPar-1, AFP, CK7, 8,18,19 and 20 by tumor cells of hepato-and cholangiocellular cancer, so this problem needs to be further developed.

Aim of the study
To determine level of expression of immunohistochemical markers HepPar-1, AFP, CK7, CK20 and area of immunopositive cells in cholangiocellular carcinoma of the liver and their differences from hepatocellular carcinoma.
Columns of trephine liver bioptats from patients with HCC and CCK were fi xed in 10% buffered formalin and embedded in paraffi n. With help of rotary microtome HM-3600 (MICROM Laborgerate GmbH -Germany) serial 3-4 micron thick sections were made for staining with hematoxylin and eosin, Van Gison and Masson-tricolor and for IHC studies.
Liver tissue in paraffi n sections undergone IHC study in accordance with standardized protocols after the temperature antigen unmasking and suppression of endogenous peroxidase activity. Primary antibodies and visualization system DAKO EnVision + System («DAKO», Denmark) with diaminobenzidine (DAB)were used. Monoclonal antibodies CK7 were used (Ks20.8 clone) to determine the cytokeratin profi le of HCC and CCK cells. Polyclonal antibodies CK20, cells of CCK and HCC were marked with help of monoclonal antibodies Hepatocyte Specifi c Antigen (HepPar-1 clone OCH1E5) and polyclonal antibodies against α-fetoprotein (AFP) (all reagents are «DAKO», Denmark).
The Program Image J was used for morphometric measurements of the area, which is occupied by Hep Par-1, AFP-, CK7-and CK 20-immunopositive cells in the digital images of immunohistochemical hepato-and cholangiocellular liver cancer. Total area of the expression of each listed marker was determined, which was represented as a percentage ratio of immunopositive pixels number of corresponding marker to the total number of pixels in the image, expressed in %.
Statistical processing of the results was performed on a personal computer using program «STATISTICA® for Windows 6.0» (StatSoft Inc., License № AXXR712D833214FAN5). The average value (M), standard deviation (σ), the standard error of the representativeness of the mean value (m) were calculated, also the 95% confi dence interval of the mean was calculated as well. Correlation was identifi ed by calculating of the Pearson's coeffi cient (for nonparametric data). Results were considered as signifi cant at p <0.05.

Results and discussion
Immunohistochemical studies results showed that cytoplasmic and nuclear expression of α-fetoprotein was determined in malignant cells in 81.13% of patients with HCC and 47.22% of patients with CC liver cancer. Alpha-fetoprotein is carcinoembryonic protein and is produced in the liver and internal organs of the yolk sac endoderm, as well as in the cells of malignant tumors of the liver [6]. It was found that the level of AFP expres-№5 (86) 2014 ISSN 2306-4145 sion by tumor cells and area of the AFP-immunopositive cells in the CC is lower than in HCC of the liver. During analyzing of the expression of AFP by CC cells it was found that 27.78% of patients had moderate expression level of this glycoprotein and was 68,51 ± 15,09 points, in 19.44% of patients it was low (34,62 ± 10,51 points), 52.78% of patients had negative level of the AFP expression by CC cells (expression level was 6,51 ± 2,87 points).
With help of digital photo morphometry we found that the area occupied by AFP-immunopositive cells was 17,25±9,67% of the total area of cholangiocellular cancer cells. According to S.A. Geller, L.M. Petrovic еt al. [2] data no more than 10% of CC liver are AFP-immunopositive; most part of the pathologists notice that AFP is marker of hepatocellular liver differentiation of malignant cells, however, the sensitivity of this marker in the FCC, according to different authors, vary from 15% to 70% [2,7].
HepPar-1 expression (Hepatocytes paraffin-1) normally reacts with hepatocytes mitochondrial enzymes, renal tubular epithelium and intestinal epithelium [8]. Positive expression of HepPar-1 by cells in cholangiocellular liver cancer wasn't detected. Positive cytoplasmic expression of HepPar-1 by tumor cells was detected in 92.45% of patients with hepatocellular carcinoma [9], as well as in the tubular component of mixed hepato-cholangiocellular carcinoma of the liver. Our results are consistent with results of other researchers. According to S.A. Geller, L.M. Petrovic еt al. [2] and A. Lugli еt al. [10] all cholangiocarcinomas are HepPar-1-immunonegative, while HepPar-1 expression is detected in 80-90% of hepatocellular carcinomas [1,2], which indicates that expression of HepPar-1 is represented in some CC.
At the basis of using antibodies against cytokeratins for differential immunohistochemical diagnosis of primary liver cancer in adults is the idea that the most likely source of hepatocellular, cholangiocellular and mixed hepatocellular-cholangiocellular carcinoma are hepatic progenitor cells localized in the tubules Goering, which normally differentiate into hepatocytes and cholangiocytes [4,11]. Consequently, tumor hepatocytes (as normal) retain expression of low molecular cytokeratins 8 and 18 and cholangiocellular cancer cells (like cholangiocytes as as well biliary ducts as cholangioles) characterized by expression of cytokeratins 7, 19 and 20 [4].
Study of the expression of CK7 and CK20 by CC cells showed the following. Positive cytoplasmic expression of CK7 by tumor cells was detected in 97.22% of cholangiocellular carcinomas. At the same time in 54.72% of the cases of CC, the high level of CK7 expression was determined (114,03±11,53 points), in 31.17% of the patients moderate level of expression was found (71,06±14,68 points), in 8.55% weak level of CK7 expression by tumor cells was found (43,32±6,71 points), and in 2.78% of patients CK7 expression in the tumor was negative (detection limit was 10,99±3,71 points). Cytokeratin-7-positive cells were unevenly distributed in malignant cholangiocellular tumors, the average size of CK7-immunopositive cells in CCK was 43,55±9,93%. In accordance with our previously published results CK7 expression was also detected in 37.74% of HCC cases.
According to information published by David J. Dabbs in manual of diagnostic immunohistochemistry, the fetal hepatocytes contain cytokeratins CK8, 18 and 19; then after 10-th week of gestation, they lose CK19, so mature hepatocytes express only CK8 and CK18. Cholangiocytes of intrahepatic bile ducts in the adult patients liver express CK7, 8,18,19, and often are CK20-immunonegative [3]. Expression of CK7 by CC tumor cells is detected in the majority of CC and it is usually weakly positive or negative in HCC [12], that is why CK7 considered as the most appropriate marker of CC.
Normally, CK20-positive cells were localized in the gastric and intestinal epithelium, urothelium and Merkel cells of the skin appendages. Despite the fact that CK20 seemed like an intestinal epithelium marker, it is found in 20% of malignant liver tumors [3]. According to A.D. Burt еt al. data [1] presence of CK20-immunopositive cells in liver malignant tumors is used for the differential diagnosis between primary hepato, cholangiocellular carcinoma of the liver and liver metastasis in colorectal cancer, stomach or other organs.
Expression levels of AFP, CK7, CK20, HepPar-1 and correlations between them in CCK are shown in table 1.
Correlation analysis showed negative, weak force correlation (the Pearson coeffi cient r = -0,1) between expression level of CK7 and CK20 in the tumor cells in patients with CC. There was direct correlation of medium strength (the Pearson coeffi cient r = +0,5) between level of AFP and CK7 expression. And direct strong bond (the Pearson coeffi cient r = +1) between level of AFP and CK20 expression.

1.
Expression of α-fetoprotein by tumor cells was determined in 47.22% of patients with cholangiocellular liver carcinoma, AFP-immunopositive cells constituted 17,25 ± 9,67% of the total area of tumor cells.
5. Weak force correlation (the Pearson coeffi cient r = -0,1) was founded between expression level of CK7 and CK20 in the tumor cells in patients with CC. There was direct correlation of medium strength (the Pearson coeffi cient r = +0,5) between level of AFP and CK7 expression. And direct strong bond (the Pearson coeffi cient r = +1) between level of AFP and CK20 expression.

Conclusion
The main immunohistochemical difference between cholangiocellular carcinoma and hepatocellular carcinoma is the lack of HepPar-1 expression by its cells, and also more than 2-fold lower level of expression of α-fetoprotein. Variable level of expression of cytokeratins in cholangiocellular and hepatocellular carcinoma is likely due to the presence of various impurities clones with abnormal cholangiosimilar and hepatosimilar cellular differentiation. The resulting comparative data should be considered in the differential diagnosis of immunohistochemical cholangiocellular and hepatocellular carcinoma in trephine biopsy of the liver with limited diagnostic material.