Expression of CD 34 in cirrhotic liver – reliance to dedifferentiation Ekspresija CD 34 u ciroznoj jetri − zavisnost od dediferencijacije

Background/Aim. The vascular supply of dysplastic nodules (DN) is altered compared with surrounding cirrhotic nodules. Dysplastic nodules contain unpaired arteries which are isolated arteries unaccompained by bille ducts. In adition, capillarization or neovascularization is evident on CD34 and CD31 staining. The investigation of angiogenic profile of regenerative, dysplastic and nodules of hepatocellular carcinoma aimed at assessing whether vascular profile is in reliance to the process of dedifferentiation of hepatocytes during the course of cirrhosis. Methods. Thirty four liver nodules from surgical biopsies of 12 patients previously undiagnosed to have cirrhosis, were classified as regenerative, dysplastic and small hepatocellular carcinomas (HCC). The investigation included 8 large regenerative nodules (LRN), 11 low grade dysplastic nodules (LGDN), 12 high grade dysplastic nodules (HGDN) and 3 early HCC. Serial sections of the nodules and surrounding cirrhotic liver tissue were immunostained against CD34. The vascular counting method was performed. The results were analysed using SPSS computer statistical program. Results. The number of capillary unites showed significant differences among nodular types, with the largest number of capillaries in hepatocellular carcinoma as well as strong reliance to dedifferentiation. Conclusion. There is a significant correlation of sinusoidal capillarization to dediferentiation of the liver tissue during the course of cirrhosis. From diagnostic view, capillary counting may be helpfull to distinguish dysplastic from nondysplastic nodules. The appearance of dysplastic nodules in nonselected surgical biopsies is frequent enough to challenge caution during the follow-up of cirrhotic patients.

lesions that differ from surrounding hepatic parenchyma in terms of size, color, texture or degree of bulging at the cut surface 2 .Dysplastic nodules are characterized by a number of cytoarchitectural and angioarchitectural abnormalities [3][4][5][6] .Still, there are a lot of uncertainties in imaging diagnostic procedures that are not precise enough to qualify DN [7][8][9] .Morphological differentiation between an early stage of welldifferentiated hepatocellular carcinomas (HCC) and DN is often difficult and diagnostic confusion concerning those lesions is a controversal issue.Based on clinical and pathological details of early HCC, the pathway for human hepatocarcinogenesis has been well-established during the last decade 6 .It is evident that many HCC develop through a progressive pathway from premalignant lesions to HCC in cirrhotic liver.As HCC show tendency to increasing incidence, the pathologists are, and will be, frequently faced with nodular lesions and small HCC.Diagnostic uncertainity between well-differentiated HCC in the early stage and DN, in particular HGDN, do exist.
We analysed nodular lesions for CD34 expression in surgical biopsies of liver cirrhosis to: 1) compare capillarization of the hyperplastic and dysplastic nodules; 2) investigate the incidence of these changes in nonselected surgical liver biopsies and 3) demonstrate if vascular count can make the distinction between premalignant and nonmalignant lesions.

Methods
Thirty four liver nodules from surgical biopsies of 12 patients, 7 women and 5 men, mean age 52.33 years, were analysed.The biopsies were taken during the laparoscopic surgery for: acute cholecystitis (7 patients), obstructive jaundice (2 cases), acute hemorrhagic gastric ulcer (1 case), splenectomy after trauma (1 case) and liver carcinoma (1case).None of patients was previously diagnosed as cirrhotic.
All of examined lesions were detected grossly as expansive growths in surrounding nodular background and measured 0.2-1.2cm.Microscopically, they were classified as LRNs (those without architectural differences in comparison to adjacent cirrhotic nodules), as LGDN (showing normal architecture and large cell changes) or as HGDN (containing uneven foci of architectural abnormalities, nuclear crowding and small cell changes).
Serial sections of each nodule and surrounding cirrhotic liver and associated HCC -three casses, were immunostained with monoclonal antibody against CD34, a specific and sensitive marker to detect capillary unites.
The assessment of capillary units was performed in all dysplastic, large regenerative, and malignant nodules according to the method proposed for vascular counting 15 as follows: 3 mostly vascularized areas were identified by low magnification (×40) and those "hot spots" were marked by coloured pen to avoid topografical confusion.Vessel counting was performed under the high magnification (×200) within every "hot spot" area; this was performed to avoid topografical bias owing to a random evaluation 14 .Mean values of CD34 positive units were calculated for each single lesion and after that, mean value (± SD) was calculated for each nodule type.The results were statistically analysed using SPSS computer statistical program.

LRN -large regenerative nodules; LGDN -low grade dysplastic nodules; HGDN -high-grade dysplastic nodules; HCC -hepatocellular carcinomas
Immunohistochemical expression of de novo formed capillary units is ilustrated in Figure 1.Sinusoidal capillarization and CD34 positive forms were at the periphery of the LRN and LGDN and were more centrally located in HGDN, while randomly in HCC (Figure 2).The number of capillaries was not significantly different among specific nodular types, but the greatest one was in HGDN (Table 2).The Tacmann's test for comparison of mean values showed statistical signifficant differences among tested groups (p < 0.001) and it confirmed the results of Anne's analysis (F = 184.75;p < 0.001).There is a statistically significant difference in vascular units number among the tested groups (p < 0.001) with the greatest number of CD34 positive units in HCC and the smallest ones in LRN (Table 2) .

Discussion
We investigated the sinusoidal capillarization in reliance to dedifferentiation in the cirrhotic liver.Nodules were classified according to standardized nomenclature 1 dividing them into hyperplastic (LRN) and dysplastic (low and high-grade).Biological and clinical significance of those nodules is not elucidated.Previous investigations had shown the progression from cirrhosis to HCC to be followed by a shift of vascular supply mainly from venous to arterial type [10][11][12][13][14] .It is obvious that HCC are higly vascularized tumors.The process of neovascularization runs in parallel to the process of dedifferentiation 6 .Putting these facts in connection to the appearance of morphologically different nodules in cirrhosis and the process of dedifferentiation, it was accepted that abnormal vascularization can be of diagnostic help to recognize those lesions with potential to neoplastic transformation 10,11,15,17 .A functional and biological background for these morphological entities is necessary as it was demonstrated that a number of entirely benign looking LGDN are monoclonal growths, as are some HGDN and HCC 18 .It is clear that clonality type together with morphological and biological characteristics are sufficient in concluding about the nature of nodules in cirrhosis.
Previous investigations of unpaired arteries and sinusoidal capillarization demonstrated an increased number of both structures in hyperplastic and dysplastic nodules.
In this study, we investigated the CD34 positive units in hyperplastic and dysplastic nodules of cirrhotic liver as well as in tree small, well-differentiated HCC.The analysis was performed on surgical liver biopsies taken during laparoscopic surgery in previously undiagnosed cirrhotic patients.The incidence of small HCC was 25% and morphologically specific nodules, other than cirrhotic, were found in 13.02% of all the analysed nodules.We found that incidence important as it reflected a native status in the moment of diagnosis, remaining on silent course of both cirrhosis and HCC, although the number of cases included in the study was small.
It was previously shown that there was no difference in angiogenic profile among cirrhotic nodules, LRN and LGDN 5,10,11,16 .The results of investigations of unpaired arteries and sinusoidal capillarisation by Rancolli et al. 16 suggest that the extent of capillarization but not the arterialization is increasingly upregulated in HGDN and fully malignant lesions.Contrary to other investigators 11,[16][17][18] we found significant differences in CD34 positive units among all tested types of nodules in cirrhotic liver, as well as in HCC.This discrepances could be the result of different selection of LGDN.We adopted very strict histomorphologic criteria and only nodules with a large cell change and expansive growth measuring 0.2-1.2cm were included as LGDN.We must be very careful in conclusion about diagnostic value of the results.It is necessery to undertake more investigations to establish a cut-off value of vascular units in premalignant and malignant LN.
Finally, we showed the differences between LRN and DN on the basis of vascular profile.As mentioned previously, some of them are in fact monoclonal.Undoubtedly, only the molecular caracterization of individual hepatocelular nodules will contribute to determine which nodules are dysplastic or neoplastic [19][20][21] .A correlation of basic biological analysis with morphophenotypic features is expected to provide a helpful information of clinical significance.
There is a huge attempt in following cirrhotic patients and tracing the processes of mild-to high-grade dysplastic, or carcinoma.Despite technological advances, imaging cir-  rhotic patients remain a challenging issue because nonmalignant DN mimic a small HCC.Through progression from regenerative nodules to LGDN, HGDN and HCC, it is possible to visualize new arterial vessels.It is neovascularity that allows HCC to be diagnosed and is a key for imaging cirrhotic patients 22,23 .The analysis of neovascularization in biopsies in combination to imaging results will help in following patients in risk for developing HCC and for early detection and treatment of carcinoma.

Conclusion
There is a strong correlation of sinusoidal capillarization with dedifferentiation of the liver tissue during the course of cirrhosis.From diagnostic view, capillary counting may be helpful to distinguish dysplastic from nondysplastic nodules.The appearance of dysplastic nodules in nonselected surgical biopsies is frequent enough to challenge caution during the follow-up of cirrhotic patients.