Development of liver microtissues with functional biliary ductular network.

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

Immunohistochemical Study of Ezrin Expression in Colorectal Carcinoma: A Comparative Study between Objective Method and Digital Quantitative Assessment.

BACKGROUND: Colorectal cancer is one of the leading causes of cancer death in both developed and developing nations. It is the third most common type of cancer and the fourth leading cause of cancer-related deaths worldwide. Ezrin is involved in maintaining cell structure and cell motility. Expression levels of the ezrin gene correlate with numerous human malignancies. MATERIAL AND METHODS: Ezrin expression was evaluated in fifty one cases of colorectal carcinoma by using two methods; objective and quantitative method to determine the statistical relation between ezrin objective analysis score and clinicopathological parameters and to do a comparative study between both methods of analysis. RESULTS: Ezrin was expressed in 92.2% of cases, and it showed a statistical significant relation with tumor grade. A statistically significant relation was found between ezrin objective analysis score and ezrin quantitative analysis score (P-value <0.05). A strong positive Pearson correlation exists between both methods of analysis (R=0.868). CONCLUSION: Ezrin has a role in colorectal cancer progression and it might provide clinically valuable information in predicting the behavior of colorectal cancer. Digital pathology offers the potential for improvements in quality, efficacy and safety. It will be necessary to carry out similar studies on a larger sample size in order to elucidate the possible prognostic significance of ezrin in colorectal carcinoma and ensure the ability of digital pathology to transform the practice of diagnostic pathology. 
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Carbamoyl phosphate synthetase 1 (CPS1) as a prognostic marker in chronic hepatitis C infection.

This study aims to assess the value of carbamoyl phosphate synthetase 1 (CPS1), as a non-invasive serum marker, for the evolution of chronic HCV infection and hepatic fibrosis. Seventy-two patients with HCV positive serum RNA and 15 health volunteers were enrolled in this study. Out of 72 patients, 10 patients had decompensated liver with ascites. Quantitative analysis of CPS1 was performed in the harvested sera and corresponding liver biopsies using ELISA and immunohistochemistry techniques respectively. Also, mitochondrial count using electron microscopy, urea analysis and conventional liver tests were done. Patients were grouped into (F1 + F2) and (F3 + F4) representing stages of moderate and severe fibrosis respectively. Tissue and serum CPS1 (s.CPS1) correlated significantly in moderate and severe fibrosis. Patients with severe fibrosis showed significantly higher levels of s.CPS1 (p-value ≤ 0.05) and significantly lower mitochondrial counts (p-value = 0.0065) than those with moderate fibrosis. S.urea positively correlated with s.CPS1 only in the decompensated group, at which s.urea reached maximal levels. In conclusion, s.CPS1 is a potential non-invasive marker for the assessment of severity and progression of HCV in relation to mitochondrial dysfunction. Also, increased s.urea with the progression of the disease is mainly due to a concurrent renal malfunction, which needs further investigation.

Detection of Human Cytomegalovirus in Malignant and Benign Breast Tumors in Egyptian Women.

INTRODUCTION: Previous studies have reported a role for human cytomegalovirus (HCMV) in breast carcinogenesis. We sought to assess the role of HCMV infection in the development and/or progression of breast cancer (BC) among Egyptian patients. PATIENTS AND METHODS: The study included 61 patients with BC cases. Of these 61 patients, 40 had been assessed for HCMV in the blood, BC tissue samples, and adjacent non-neoplastic tissue samples, and 21 had been assessed for HCMV in the tissue only. Tissue samples from 20 patients with fibroadenoma (FA) were also included. As a control group, 41 blood samples obtained from healthy women with no history of cancer were used as a blood control group. HCMV was assessed using enzyme-linked immunosorbent assay, real-time polymerase chain reaction (PCR), and immunohistochemistry (IHC). RESULTS: A significant difference was found in the index value for the anti-CMV IgG antibodies between the BC patients and the control group (P = .001). Using real-time PCR, HCMV DNA was detected in 11 of 61 BC tissues (18%) compared with 1 of 20 FA tissues (5%). HCMV DNA was present in 8 of the 40 plasma samples (20%). Regarding the viral proteins, 21 of 61 samples (34.4%) were positive for early/immediate early (E/IE) and 49 (80.3%) were positive for PP65 expression by IHC. The concordance between the results obtained by the different assays was low. CMVPP65 expression was significantly associated with E/IE protein expression in the malignant and FA groups (P < .001). CONCLUSION: The presence of CMV proteins and DNA in BC tissues suggests a role for this virus. However, the basic criteria to support a causal association of HCMV with BC were not fulfilled.

Expression of ERG Protein and TMRPSS2-ERG Fusion in Prostatic Carcinoma in Egyptian Patients.

AIM: Prostate cancer (PCa) is the second most common cancers in men worldwide. Its incidence can be influenced by several risk factors including genetic susceptibility. Therefore the search for the expression of a certain gene (ERG) and its rearrangement could give us clues for proper identification of PCa. And the study of ERG expression and its comparison to FISH in Egyptian patients can show whether ERG immunophenotype could be used instead of FISH, as it is cheaper. MATERIALS AND METHODS: This study was performed on 85 cases of PCa, showing 30 cases with HGPIN and 30 cases of prostatic hyperplasia. All were immunohistochemistry stained using ERG monoclonal rabbit antihuman antibody was used (clone: EP111). FISH analysis was performed in 38 biopsies of PCa cases to detect TMRPSS2-ERG rearrangement using the FISH ZytoLight TriCheck Probe (SPEC TMRPSS2-ERG). RESULTS: ERG expression was found in 26% of PCa cases and 20% of HGPIN cases. FISH analysis showed fusion of 21 cases of PCa (out of 22 cases showing ERG immunoexpression). CONCLUSION: Our findings emphasise that only malignant and pre-malignant cells and not benign cells from the prostate stain positive. ERG expression may offer a simpler, accurate and less costly alternative for evaluation of ERG fusion status in PCa.

Expression of FGFR3 Protein and Gene Amplification in Urinary Bladder Lesions in Relation to Schistosomiasis.

BACKGROUND: Bladder cancer represents the fifth most common malignancy worldwide and a major cause of cancer-related morbidity and death. Incidence and mortality rates have remained relatively constant over the past four decades. Urothelial bladder cancers have identified multiple risk factors. AIM: We aimed at evaluating the expression of the FGFR3 protein and gene amplification in the urothelial cells of neoplastic and non-neoplastic urothelial lesions of the urinary bladder, and correlation with tumour grade, stage and associated bilharziasis. MATERIAL AND METHODS: One hundred and five different urinary bladder lesions were studied, including 15 cystitis cases (9 bilharzial and 6 non-bilharzial cystitides), 75 urothelial carcinoma cases (18 bilharzial associated and 57 non-bilharzial associated) and 15 squamous cell carcinoma associated with bilharziasis, beside 5 control cases. Data concerning age, sex, tumour grade, stage, and associated bilharziasis were obtained. Each case was studied for FGFR3 expression, and FISH technique was applied on forty malignant cases that show high protein expression. RESULTS: The highest incidence of cystitis was in the fourth decade while of bladder cancer was in the seventh decade. Tumour grade was correlated significantly with tumour stage. FGFR3 correlates significantly with tumour grade, stage and with a bilharzial infestation. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. CONCLUSIONS: FGFR3 overexpression in malignant cases was significantly higher than in chronic cystitis. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. FGFR3 may be further studied as a subject for target therapy of bladder cancer.

Expression of Epidermal Growth Factor Receptor and Transforming Growth Factor Alpha in Cancer Bladder: Schistosomal and Non-Schistosomal.

INTRODUCTION: Overexpression of epidermal growth factor receptor (EGFR) has been described in several solid tumors including bladder cancer. Transforming growth factor alpha (TGFα) is frequently deregulated in neoplastic cells and plays a role in the development of bladder cancer. TGFα-EGFR ligand-receptor combination constitutes an important event in multistep tumorigenesis. METHODS: This study was done on 30 bladder biopsies from patients with urothelial carcinoma, 15 with squamous cell carcinoma, 10 with cystitis and 5 normal control bladder specimens. All were immuohistochemically stained with EGFR and TGFα antibodies. RESULTS: EGFR and TGFα were over-expressed in higher grades and late stages of bladder cancer. Moreover, they show higher expression in squamous cell carcinoma compared to urothelial carcinoma and in schistosomal associated lesions than in non-schistosomal associated lesions. CONCLUSION: EGFR and TGFα could be used as prognostic predictors in early stage and grade of bladder cancer cases, especially those with schistosomal association. In addition they can help in selecting patients who can get benefit from anti-EGFR molecular targeted therapy.

Her2/neu Protein Expression and Oncogene Amplification in Gastric Carcinoma with Clinico-Pathological Correlation in Egyptian Patients.

AIM: Amplification of the Her2/neu gene and overexpression of the Her2/neu protein in gastric carcinoma (GC) is a golden criterion for target therapy with trastuzumab (Herceptin). We aim to evaluate the immunohistochemical protein expression and amplification of the oncogene Her2/neu by FISH technique in the epithelial gastric carcinoma and to compare their association with different clinicopathologic parameters aiming at identifying positive cases that may benefit from targeted therapy. MATERIALS AND METHODS: This study was done on eighty-five tumour tissue samples from patients with GC as well as thirty non-malignant lesions (Gastritis, intestinal metaplasia, adenoma with low-grade dysplasia, adenoma with high-grade dysplasia). All were immunohistochemically stained with Her2/neu antibody. RESULTS: All equivocal and some selected GC cases were submitted for FISH technique to detect Her2/neu gene amplification. By immunohistochemistry twenty-three cases (27%) were defined as positive for Her2/neu gene amplification and/or protein overexpression. The levels of Her2/neu positive (3+), Her2/neu equivocal (2+) and Her2/neu negative (1+/0) were measurable in 14.2%, 32.9% and 52.9% of the samples, respectively. FISH showed that Her2/neu gene was amplified in 22 cases, 10 Her2/neu positive (3+), 11 (39.3%) Her2/neu equivocal (2+) and 1 Her2/neu negative (1+) cases with IHC staining those who can benefit from anti Her2/neu target therapy. Her2/neu was overexpressed positivity (3+) more in intestinal type and mixed carcinoma, and moderately differentiated tumours. None of gastritis, intestinal metaplasia or adenoma with low-grade dysplasia cases showed positivity for Her2/neu (3+). The Her2/neu positivity (3+) was associated with both adenocarcinoma cases and high-grade dysplasia (P = 0.002). CONCLUSIONS: The results highlight the necessity of FISH test for further categorization when gastric cancer cases are equivocal (2+) by IHC to determine eligibility for the targeted therapy. Stepwise increase in the expression of Her2/neu was seen in low-grade dysplasia, high-grade dysplasia and carcinoma cases implying its role in cancer evolution. Overexpression of Her 2/neu in GC patients can be promising in selecting those who can get benefit from anti-Her2/neu target therapy.

Fibrosis in Chronic Hepatitis C: Correlation between Immunohistochemically-Assessed Virus Load with Steatosis and Cellular Iron Content.

AIM: We aimed study impact of hepatocytic viral load, steatosis, and iron load on fibrosis in chronic hepatitis C and role of VEGF and VEGFR overexpression in cirrhotic cases in evolving HCC. MATERIAL AND METHODS: Total of 120 cases were included from TBRI and Beaujon Hospital as chronic hepatitis C (CHC), post-hepatitis C cirrhosis, and HCC. Cases of CHC were stained for Sirius red, Prussian blue and immunohistochemically (IHC) for HCV-NS3/NS4. HCC were stained IHC for VEGF and by FISH. RESULTS: Stage of fibrosis was significantly correlated with inflammation in CHC (P < 0.01). Noticed iron load did not correlate with fibrosis. Steatosis was associated with higher inflammation and fibrosis. The cellular viral load did not correlate with inflammation, steatosis or fibrosis. VEGF by IHC was significantly higher in cases of HCC when compared to cirrhotic group (P < 0.001). Amplification of VEGFR2 was confirmed in 40% of cases of HCC. Scoring of VEGF by IHC was the good indicator of VEGFR2 amplification by FISH (P < 0.005). CONCLUSION: Grade of inflammation is the factor affecting fibrosis in CHC. The degree of liver damage is not related to cellular viral load or iron load. Steatosis is associated with higher inflammation and fibrosis. VEGF by IHC is correlated with overexpression of VEGFR2 by FISH.

The clinical significance of HER2 protein amplification/expression in urinary bladder lesion.

OBJECTIVE: To evaluate HER2 oncoprotein expression by both immunohistochemical (IHC) staining and fluorescence in situ hybridisation (FISH) in different benign and malignant bladder lesions, and the effect of bilharzial infestation on this expression. PATIENTS AND METHODS: In a prospective controlled study, 72 patients were classified into a control group, and groups with cystitis, urothelial carcinoma, and squamous cell carcinoma (SCC). HER2 was detected using standard IHC staining and FISH in all groups. The correlation of HER2 expression with tumour type, stage and grade in relation to normal urothelium and cystitis was assessed. The effect of schistosomal infestation was evaluated. RESULTS: HER2 expression was statistically significantly higher in patients with malignant lesions than in the other groups, and in high-stage and -grade tumours than in low-stage and -grade tumours. The use of FISH increased the detection of HER2-positive tumours. Schistosomal infestation did not affect HER2 expression in patients with transitional cell carcinoma. CONCLUSION: High-stage and -grade bladder malignancies expressed HER2 much more than did benign lesions. FISH is more sensitive for detecting HER2 expression. The treatment of HER2-positive tumours might benefit from novel targeted-treatment protocols.