الفهرس 
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Abstract
Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy of adults. It is a malignant neoplasm composed of cells that differentiate in some way in the manner of hepatocytes. HCC is the sixth most common cancer worldwide and is associated with increasing mortality. HCC is featured by the high recurrence rate as well as high tumor metastasis rate.
Great efforts have been made in the treatment of HCC in the past decade. However, recurrence after ablation or resection is a major drawback. The difficulty in eradicating tumors may be due to the fact that conventional treatments target the bulk of the tumor cells leaving behind the cancer stem cells, which like their normal counterparts, maintain the ability to initiate tumorigenicity.
Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that hepatocellular carcinoma CSC will identify a group of patients at high risk for progression.
Toll like receptor 4 is a protein that in humans is encoded by the TLR4 gene. TLR4 is a transmembrane protein, member of the Toll like receptor family, which belongs to the pattern recognition receptor (PRR) family. Its activation leads to an intracellular signaling pathway nuclear factor-kB (NF-κB ) and inflammatory cytokine production which is responsible for activating the innate immune system. It appears that TLR4 can use some mechanisms to induce survival of HCC cells. Moreover, TLR4 positive HCC cells have stem-like properties including migration, chemotherapy resistance and tumor invasion.
CD133 is the most frequently used cell surface antigen to detect and isolate CSCs from various solid tumors including brain, colon, pancreas, prostate, lung, and liver. Multiple cancer stem cell associated markers have been identified, among which CD133 has received considerable attention. Some contrasting evidence of the accuracy associated with using CD133 as a marker for CSC detection and/or isolation. Many researchers nowadays discuss the clinical relevance of CD133 in cancer and thoroughly describe the utility and limitations of using CD133 for CSC identification and therapeutic targeting.
CK19 expression in the liver is a marker of biliary differentiation and progenitor cell phenotype. CK19 is a novel HCC biomarker associated with poor prognostic factors in HCC patients due to high risk of microvascular invasion and distant metastasis, as well as worse treatment outcome. However, focal expression of CK19 in a considerable proportion of HCC has been shown by several studies. Typically, CK19 is detected in isolated or discrete clusters of small tumor cells in 10% to 27% of HCC.
The present study included 60 paraffin blocks of hepatocellular carcinoma specimens in Egyptian patients, collected from the archives of The National Cancer Institute in Cairo University during the time period from January 2016 to December 2018.
The mean age of the studied patients was 63.93 years. The tumor size was <5 cm in 16/60 (26.7%) cases and ≥5cm in 44/60 (73.3%) cases.
The H & E stained sections of 60 specimens of hepatocellular carcinoma were evaluated according to the WHO classification, into the following histological types: 40/60 (66.7%) cases were Trabeculae & acini pattern of HCC. 20/60 (33.3%) cases were solid pattern of HCC. HCC was graded into: 12/60 (20%) were grade I; well differentiated tumor. 15/60 (25%) were grade II; moderately differentiated tumor. 33/60(55%) were grade III; poorly differentiated tumor. TNM pathological classification and lymph node status were carried out according to the AJCC. Regarding tumor stages of HCC cases, in accordance of the size of the tumor there is: 23/60 (38.3%) were considered T1. 23/60 (38.3%) were considered T2. 14/60 (23.3%) were considered T3a.
Sections from the 60 cases were immunostained with TLR4, CD133 and CK19 to detect their expressions and they revealed that TLR4 expression was negative in 11/60 (18.3%) and positive in 49/60 (81.7%) among the positive cases TLR4 expression was strongly expressed in 30/60 (50%) of cases of HCC and TLR4 was moderately expressed in 19/60 (31.7%) of cases. TLR4 showed significant correlation with cirrhosis, tumor grade, stage, surgical resection margin and vascular invasion. However, TLR4 expression according to age, sex, tumor size, histological pattern showed no significant correlation.
CD133 expression was negative in 19/60 (31.7%) and positive in 41/60 (68.3%) among the positive cases, CD133 expression was strongly expressed in 10/41 (16.7%) of cases of HCC, CD133 was moderately expressed in 16/41 (26.7%) of cases and expression of CD133 was weak expression in15/41 (25%). CD133 showed significant correlation with tumor focality, cirrhosis, grade, stage (T), and vascular invasion, while statistical evaluation of CD133 expression according to age, sex, tumor size, tumor location, histological pattern and surgical margin showed no significant correlation.
Ck19 expression was negative in 43/60 (71.7%) and positive in 17/60 (28.3%) among the positive cases, Ck19 expression was strongly expressed in 2/17 (3.3%) of cases of HCC, Ck19 was moderately expressed in 5/17 (8.3%) of cases and expression of CK19 was weak expression in10/17 (16.7%). Ck19 expression showed significant correlation with cirrhosis, and with grade, while statistical evaluation of Ck19 expression according to age, sex, tumor size, tumor location, tumor focality, histological pattern, tumor stage (T), vascular invasion and surgical margin showed no significant correlation.
There was significant correlation between TLR4 and CD133 expression but no significant correlation was found between TLR4 and CK19 expression and between CD133 and CK19 expression.
Conclusion:
The present study indicates that:
• TLR4 expression is correlated with tumor aggressiveness and progression, as indicated by increasing grade, stage, surgical involved resection margin and vascular invasion of HCC and presence of liver cirrhosis.
• TLR4 as a potential CSC marker and may have a modulatory effect on malignant behaviors as it is correlated with poor prognostic parameters in HCC.
• CD133 expressions significantly corresponded to presence of liver cirrhosis, higher tumor grades, stages, and vascular invasion.
• CD133 expression as a CSCs marker can enhance our understanding of HCC initiation, progression, prognosis, metastasis and relapse, in addition to the help in the development of novel therapeutic agents targeting and/or preventing HCC.
• CK19-positive HCCs expressed in a minor number of the studied HCC and correlated with increasing grades of HCC and presence of cirrhosis.
• CK19 could be used as a prognostic marker in HCC.
• Our findings may lend themselves to a new strategy of individualized adjuvant therapy selection and post-treatment surveillance aimed at identifying patients with the highest disease recurrence or progression.
Recommendations:
Studying TLR4, CD133 and CK19 expression on a large number of cases and different varieties of HCC.
Our findings will help to open the possibility to design further studies on the impact of TLR4 and CD133 in the prognosis HCC prognosis and to provide a therapeutic target for this frequent tumor.
Prospective studies properly powered based on this study should do a follow up for the patients and to get enough information about distant metastasis to emphasize the correlation between TLR4 and CD133 expression and the patient survival and disease outcome.
Studying TLR4, CD133 and CK19 in correlation with other HCC-CSC markers; CD29, CD44, CD166, OV6 and CD90, to clarify their role in HCC tumorgenesis.