الفهرس 
Introduction
Hypothesis and specific aim of the workOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma is one of the most common cancers in the world and is the third leading cause of cancer-related death. In Egypt, HCC is the second most common cancer in men and the 6th most common cancers in women. Nearly 80% of the Egyptian patients with HCC have HCV infection (in which genotype 4 predominates). Hepatocarcinogenesis is a multi-step process that involves many genetic alterations with an endpoint malignant transformation of hepatocytes. chronic liver diseases are widely considered to be pre-neoplastic conditions for HCC. However, the molecular alterations underlying pre-neoplastic conditions that predispose to HCC remain poorly understood.
In addition, accurate diagnosis of HCC is critically important to appropriate clinical management of the patients and assessment of the prognosis. Although histopathologic diagnosis of HCC may be straight forward in many cases, challenges are sometimes faced by pathologists in the distinction from non neoplastic hepatocellular lesions. Currently, various approaches are used to select the most sensitive and specific markers for diagnosis and many immunomarkers are used but all have limitations with respect to the differential diagnosis of HCC.
The present study explored the immunohistochemical expression of GPC3, HSP70 and CHC in HCV positive specimens among Egyptian patients. Then, we correlate the findings with the clinicopathological features to investigate the role of these markers in the development of HCC, followed by assessment the diagnostic value of these markers in differentiating HCC from non neoplastic hepatic lesions.
Formalin fixed paraffin embedded blocks of sixty eight liver specimens were studied. These specimens were divided into 3 groups:
1- Neoplastic group: include 30 specimens of surgically resected HCC in patients with positive HCV infection. Histological examination of the adjacent tissue was done (chronic hepatitis and cirrhosis present in 3 and 27 specimens respectively) of which 18 adjacent cirrhotic liver tissues were available in the same slide of HCC.
2- Non- neoplastic group: include 30 needle liver biopsies with chronic hepatitis C.
3- Control group: composed of eight morphologically normal liver tissues.
Glypican3 was expressed in 93.3% of HCC specimens which was higher than its frequency in non-neoplastic group. Within the non-neoplastic group; GPC3 expressed in 36.7 % of the chronic hepatitis specimens (all of them had grade 3 necroinflammatory grade). In contrast, GPC3 was undetectable in all of the 18 cirrhotic livers adjacent to HCC and the 8 morphologically normal liver specimens. Regarding GPC3 staining in different grades of HCC, it was found that all poorly differentiated HCC specimens were positive. However, well differentiated and moderately differentiated HCC specimens are less frequently expressed GPC3 being 83.3% and 92.9% respectively.
With respect to the relationship between GPC3 expression and the clinicopathological features of the studied specimens. A significantly higher mean of GPC3 IRS was detected in high grade tumors (p=0.010), in HCC more than 2 cm (p=0.044), in non capsulated tumors than capsulated one (p=0.010) and in HCC with vascular invasion (P=0.004). In addition, It was found that the mean of GPC3 immunoreactivity score was significantly higher in grade 3 chronic hepatitis than grade 1 and 2 necroinflammation (p= 0.000). Also, it was observed that the mean of GPC3 immunoreactivity score was significantly higher in stage 4 (active cirrhosis) than the other lower stages (p= 0.000). HCC specimens had a significantly higher GPC3 IRS score than the non-neoplastic group (p=0.000). The mean of GPC3 IRS was higher in chronic hepatitis than that in cirrhotic liver (p=0.004).
Heat shock protein 70 was expressed in 90% of HCC specimens which was higher than its frequency in non-neoplastic group. Within the non-neoplastic group; 40% of the chronic hepatitis specimens and 44.4% of the cirrhotic liver adjacent to HCC specimens showed HSP70 expression. In contrast, all of the morphologically normal liver specimens were negative. Regarding HSP70 staining in different grades of HCC, it was found that all (6/6) well differentiated HCC specimens were positive for HSP70. However, HSP70 expression was detected 92.9% and 80% specimens of moderately differentiated and poorly differentiated HCC respectively.
Regarding the relationship between HSP70 expression and the clinicopathological features of the studied specimens, a significantly higher mean of HSP70 IRS was detected in HCC with vascular invasion than those without (p=0.015). However, no significant difference in mean of HSP70 expression and the other clinicopathological features was detected. In addition, HCC specimens had a significantly higher HSP70 IRS score than the non-neoplastic group (p=0.000). However, no statistically significant difference was detected between the mean of HSP70 expression in cirrhotic liver adjacent to HCC and chronic hepatitis (p=0.774).
Clathrin heavy chain was expressed in 43.3% of HCC specimens which was higher than its frequency in non-neoplastic group (4.16%).Within the non-neoplastic group; 11.1% of the cirrhotic liver tissues adjacent to HCC specimens showed expression of CHC. In contrast, all chronic hepatitis and morphologically normal liver specimens were not expressed CHC. With respect to the association between CHC expression and the clinicopathological features of the studied cases, a statistically significant association was detected only between CHC expression and HCC grade (p=0.01), it was found that the highest CHC expression was found in moderately differentiated HCC (71.4%) followed by poorly differentiated (20%) then well differentiated HCC (16.7%). HCC specimens had a significantly higher CHC expression than the non- neoplastic group (p=0.000).
With respect to the performance of staining with different panels in differentiating HCC versus non neoplastic groups, it was found that the best sensitivity obtained by using a single marker immunostain with GPC3 alone while the best specificity obtained by using two markers panel with combination of GPC3 and CHC or by using three markers panel. The combined use of GPC3 and HSP70 had the best accuracy.
With respect to the performance of staining with different panels in differentiating well differentiated HCC from adjacent cirrhosis, it was found that GPC3 alone or in combination with HSP70 or CHC or the use of three markers panel showed the best specificity. HSP70 alone showed a sensitivity of 100% which was superior to the sensitivity of two or three markers panel and a single marker immunostain (GPC3 or CHC). The best accuracy for diagnosis of well differentiated HCC obtained by using GPC3 alone or in combination with HSP70.
Conclusions:
• The findings of the present study confirm a high frequency of expression of GPC3, HSP70 and CHC in HCC. The lower frequency of the expression of these markers in non-neoplastic liver tissues, suggest their role as early markers of hepatocarcinogenesis.
• The expression of the three markers was significantly associated with poor prognostic factors of HCC specimens. Glypican3 and clathrin heavy chain expression was correlated to high histologic grade. GPC3 and HSP70 expression was correlated to the presence of vascular invasion. Also, GPC3 expression was correlated to large tumor size. Targeting these genes might be beneficial in treatment of HCC.
• Glypican3 alone or in combination with HSP70 is useful in differentiating HCC from non-neoplastic lesions (chronic hepatitis and cirrhosis). However, CHC has a very low sensitivity. Also, GPC3 alone or in combination with HSP70 is useful in differentiating well differentiated HCC from adjacent cirrhotic liver. However, CHC has a very low sensitivity and accuracy. Also, we should emphasize that all the three markers may be negative in HCC, so negative staining does not exclude HCC diagnosis.