Molecular Noninvasive Diagnosis of Hepatocellular Carcinoma Using Microsatellite Instability.

OBJECTIVE: Hepatocellular carcinoma (HCC) accounts for more than 80% of primary liver cancers. Moreover, in the next 10 years, more than one million patients are expected to die from liver cancer as estimated by the World Health Organization. The aim of the present study is to define the microsatellite phenotype in the blood, tumor and nontumor tissue samples from hepatocellular carcinoma cases to develop a simple non-invasive method for diagnosis and detection of the disease. METHODS: A total of 100 patients with histologically-proven HCC were enrolled in this study, blood samples and tissue specimens from tumor and nontumor tissue were obtained from each patient. DNA was extracted and microsatellite instability MSI status was determined by polymerase chain reaction (PCR) using 5 mononucleotide and 5 dinucleotide repeats. RESULTS: Among the 100 HCC tumors analyzed, (8%) considered as displaying a typical MSI-H phenotype as defined by instability in at least 3 of the 10 repeats analyzed, (61%) tumors displayed MSI-L and (31%) displayed MSS while in plasma the instability was (40%) for MSI-H, (44%) for MSI-L and (16%) for MSS. CONCLUSION: our findings could point to the achievement that HCC patients could be diagnosed by MSI analysis using blood sample as non-invasive way and this conclusion achieved our aim as the study shows impressive and promising results.

Evaluation of Osteopontin as a Biomarker in Hepatocellular Carcinomas in Egyptian Patients with Chronic HCV Cirrhosis

Background: Hepatocellular carcinoma (HCC) is a high incidence disease in Egypt with a poor prognosis and survival. Biomarkers are important for diagnosis of HCC at an early stage. Osteopontin (OPN), a glycoprotein secreted by macrophages, osteoblasts, and T cells, is also highly expressed in a variety of tumors, such as examples in the breast, colon, and stomach. The present study aimed to correlate the serum level of OPN in HCV-positive hepatocellular carcinoma patients, with OPN expression in tumor and non-tumor liver tissues in order to identify its efficacy as a biomarker for diagnosis. Material and Methods: Out of total of 146 patients, 80 were selected for inclusion in the study. Blood samples as well as specimens of tumor and non-tumor liver tissue were collected. In addition, blood samples from 20 healthy volunteers were obtained as controls. Serum OPN and alpha-fetoprotein (AFP) were evaluated by ELISA for HCC and control groups. OPN and AFP gene expression were examined by real-time PCR, after homogenization and DNA extraction from serum samples and liver tissues. Results: It was found that serum OPN levels were significantly higher in the HCC group compared to normal group (P=0.009), with a strong positive correlation with AFP expression. However, there was no significant difference between OPN expression in tumor and non-tumor liver tissue. Conclusion: Serum OPN is highly suggested to be a professional candidate for HCC early diagnosis, with a diagnostic ability and accuracy equal or higher than for AFP.

Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

Objective: To investigate the expression of TTF-1, RAGE, GLUT1 and SOX2 in HCV-associated HCCs and in surrounding non-tumorous liver tissue. Material and Methods: Tissue material from partial hepatectomy cases for HCC along with corresponding serum samples and 30 control serum samples from healthy volunteers were studied. Biopsies were classified into: non-tumor hepatic tissue (36 sections); HCC (33 sections) and liver cell dysplasia (LCD) (15 sections). All cases were positive for HCV. Immunohistochemistry (IHC), gene extraction and quantitative real-time reverse-transcription assays (qRT-PCR) were applied. Results: By IHC, LCD and HCC showed significantly high percentages of positive cases with all markers. SOX2 showed significant increase with higher HCC grades, while RAGE demonstrated an inverse relation and GLUT-1 and TTF-1 lacked any correlation. In nontumorous-HCV tissue, we found significantly high TTF-1, low RAGE and negative SOX2 expression. RAGE, GLUT-1 and SOX2 show non-significant elevation positivity in high grade HCV compared to low grade lesions. TTF-1, RAGE and SOX2 exhibited low expression in cirrhosis compared to fibrosis. Biochemical studies on serum and tissue extracts revealed significant down-regulation of RAGE, GLUT-1 and SOX2 genes, as well as significant up-regulation of the TTF-1 gene in HCC cases compared to controls. All studied genes show significant correlation with HCC grade. In non-tumor tissue, only TTF-1 gene expression had a significant correlation with the fibrosis score. Conclusion: Higher expression of TTF-1, RAGE, GLUT-1 and SOX2 in HCC and dysplasia compared to non-tumor tissues indicates up-regulation of these markers as early events during the development of HCV-associated HCC.

Evaluation of Mir-224, Mir-215 and Mir-143 as Serum Biomarkers for HCV Associated Hepatocellular Carcinoma

HCV induced hepatitis and hepatocellular carcinoma as its sequel are major health problems world-wide and especially in Egypt. For diagnosis and during treatment of liver diseases, liver functions are monitored through determination of serum levels of liver enzymes and α-fetoprotein although the obtained information is generally not sufficient for either early detection of hepatic insult or effective follow up of therapeutic effects. More sensitive biomarkers may help to achieve these goals. MiRNAs are small non-coding RNAs that have an important role in gene expression and regulation. Many, such as miR-224, miR-215, miR-143 are correlated with tumor appearance and with the degree of fibrosis in lung, breast and colon cancer. This study was performed to estimate the level of these miRNAs in serum of patients with HCV-associated hepatitis and HCC in relation to grade of hepatitis, stage of fibrosis and differentiation of tumor tissue. In addition, correlations between serological and tissue levels were assessed. A total of 80 patients were examined, out of which 50 were included in the study. Blood samples and tissue specimens from malignant tumor and corresponding non-tumor tissue of HCV hepatitis patients were collected. Blood samples from 20 healthy volunteers were also obtained as controls. It was found that miRNAs profiles differed in HCC patients compared to controls and HCV-associated hepatitis cases. Distinction of tumor grade and fibrosis stage of patients as well as between different grades of tumor differentiation proved possible, making miRNAs promising biomarkers for diagnosis and assessment of treatment response of HCC patients.

Differential Expression of Glypican-3 and Insulin­Like Growth Factor-II mRNAs and Alpha-Fetoprotein and Ki-67 Markers in HCV Related Hepatocellular Carcinomas In Egyptian Patients

Background: Increasing evidence indicates that in hepatocellular carcinomas (HCCs) abnormal gene expression, for example of glypican-3 (GPC-3) and insulin-like growth factor-II (IGF-II), are associated with the occurrence and progression of HCC. The objective of this study was to evaluate the differential expression of GPC-3 and IGF-II mRNAs in HCC tissues with a background of chronic hepatitis C virus (HCV) genotype 4 cirrhosis, in relation to Ki-67 and alpha-feto protein (AFP) tissue markers. Methods: One hundred and five patients with HCCs who had undergone hepatectomy, were included, after obtaining informed consent. Total RNA was extracted from malignant and corresponding peri-malignant liver tissues, and GPC-3 and IGF-II mRNAs in addition to beta-actin mRNA as an internal control, were evaluated in all samples by reverse transcriptase-polymerase chain reactions (RT-PCR). Routine histopathological diagnosis as well as immunohistochemical (IHC) staining using monoclonal antibodies for Ki-67 and AFP were also performed. Result: Expression of GPC-3 mRNA was positive in all HCC malignant tissue, with overexpression in 86/105 (81.9%); in respect to the grade of the tumor (1-3 grades), while in peri-malignant tissue it was over expressed only in 20/105 (19%). The IGF-II mRNA was over expressed in only 10/105 (9.5%) malignant and peri-malignant samples. AFP was expressed in 33.3% of malignant samples but absent in peri-malignant tissues. Ki-67 expression was significantly increased in malignant compared to peri-malignant tissue. Conclusion: GPC-3 and IGF II mRNAs may be good molecular markers for HCC, especially with a background of cirrhosis due to chronic HCV infection. Significant correlations were noted with the pattern of AFP and Ki-67 expression.

Outcome of sublay mesh repair in non-complicated umbilical hernia with liver cirrhosis and ascites.

BACKGROUND: Umbilical hernia repair is often accompanied by complications in patients with liver cirrhosis and ascites. It appears that the early elective repair of umbilical hernias in these patients is safer and can be considered for selected patients. The objective of this study is to evaluate the feasibility, safety, complications and technical aspects of sublay mesh repair of umbilical hernia in cirrhotic patients with ascites. METHODS: Between October 2010 and April 2013, 70 patients with non-complicated umbilical hernia, liver cirrhosis and ascites were enrolled in this study. All patients underwent sublay mesh repair. Demographic data, preoperative variables, peri-operative course, and postoperative complications were recorded and analyzed. RESULTS: A total of 38 women and 32 men underwent operation at an average age 51.24 years. The patients mean MELD score was 18 (range 12-25). The mean operative time was 67.45 min and the average hospital stay was 3.8 days. 2 patients had wound infection, 3 patients developed seroma and 1 patient had an ascitic fistula. Recurrence occurred in 1 (1.4%) patient and no mortality related to the procedure. CONCLUSION: elective sublay umbilical hernia mesh repair is a safe approach and feasible technique in selected non-complicated cirrhotic patients with ascites.

Perioperative acute phase response and outcome of hepatic resection, an experimental study.

Identification of patients at risk of postoperative complications could have an impact on the indications for a procedure as well as permitting modifications of treatment to reduce the surgical risk. This experimental study evaluated the correlation between peri-operative acute phase response and outcome of hepatic resection. The study was conducted on sixty healthy golden hamsters, which underwent partial hepatectomy. They were arranged in 3 groups (20 per each). One day prior to resection, fracture of the left hind leg was done in group I (G I) & wound infection had been created in group II (GII); while nothing done in group III (GIII) that acted as a control. Blood samples to estimate SGPT and serum albumin (as basic investigations for hamsters liver function) and serum IL-6 and CRP (as acute phase reactants) were taken preoperatively, immediately after resection and for the consecutive 3 days post operatively. The mean serum level of both acute phase reactants increased in GI and GII preoperatively and continues to rise immediately after resection. Post-operatively; among the three groups, the mean serum level of both reactants was higher in GI than in GII that was in turn higher than in GIII except when the postoperative complications were more severe than the other group, then this relation changed.

Limb-salvage angioplasty in poor surgical chronic liver disease and diabetic patients.

Critical limb ischemia (CLI) in high surgical risk patients with chronic liver diseases has a grave prognosis with a one-year mortality rate of 20% and a one-year amputation rate of 25% after the initial diagnosis. According to Trans-Atlantic Inter-Society Consensus (TASC)-II Guidelines, revascularization (surgical & endovascular) is the treatment of choice for patients with critical limb ischemia (CLI). The primary goal of revascularization is to relieve ischemic rest pain, heal ulcers, prevent amputation, improve patient's quality of life (limb salvage) and secondary goal was the periprocedural complications. Endovascular techniques include balloon angioplasty, stents, stent-grafts, and plaque debulking procedures. Surgical options, identification of patients at risk of postoperative complications could have an impact on the indications for a procedure as well as permitting modifications of treatment to reduce the surgical risk This study evaluated the treatment out comas after limb salvage angioplasty for patients who otherwise would be candidates for primary amputation due to poor co-morbid conditions as chronic liver disease and diabetes. The clinical evaluation, laboratory investigations and abdominal ultrasonography were performed to all patients to evaluate their liver status. Patients were classified according to Child-pugh classification into child A, B & C. All patients were subjected to either detailed arterial duplex or C.T. angiography to assess their arterial lesions from January 2008- January 2010. 95 patients with critical limb ischemia (Rutherford categories 4, 5, 6) were treated by primary percutaneous transluminal angioplasty (PTA). No patient was excluded on the basis of the extent of arterial occlusive disease. The primary end points were immediate technical success, clinical improvement and limb salvages rates. Secondary end points were periprocedural complications and mortality. Most of the patients were male (54.7%) with mean age 62 (48-70 years). Underlying cirrhosis due to HCV was (82.2%), HBV (5.4%), while mixed viral infections was (12.4%). 54% were categorized as Child B, 32% as child A and 14% as child C. Associated diabetes mellitus was present in 96% of the cases, hypertension in 64.2%, ischemic heart disease in 74% and hyperlipedemia in 32%. Rest pain, tissue loss, or both, were the presenting symptoms in 83% while infection and ulcer were present in the other 17% of patients. The total numbers of interventions were 154; the treated lesions were 89 in the tibial arteries, 12 in the popliteal artery, 44 in the superficial femoral artery, 3 in the common femoral artery and 6 in the iliac arteries with initial technical success rate of 93.6% and periprocedural complications of 12.6%. All patients were in Rutherford clinical category 4, 5, 6 none of these patients had a previous bypass operation. Mean follow-up was 15 months. The limb-salvage rate was 87.4%. Eighty patients (84.2%) of toe amputation sites healed primarily. three patients with rest pain had resolution of their symptoms after angioplasty. All technical failures were due to inability to cross the lesions. Of the 6 technical failures, 4 required amputation, and 2 refused any further therapy.

Effect of pancreatic biliary reflux as a cofactor in cholecystitis.

This study assessed the effect of pancreatico-biliary reflux (PBR) as co-factor in the process of chronic cholecystitis by measurement of the levels of active pancreatic enzyme amylase in gallbladder bile and serum of patients undergoing cholecystectomy. Pancreatic Amylase levels in bile from the gallbladder and serum were measured during surgery in 68 patients with chronic calcular cholecystitis subjected to elective open or laparoscopic cholecystectomy in the National Hepatology and Tropical Medicine Research Institution and Theodore Bilharz Research Institute. Bile amylase was detected in 64 patients (94.1%) indicating pancreatico-biliary reflux. Biliary amylase level ranged from 20-50 IU/L in 42 patients (61.76%), below 20 IU/l in 14 patients (20.59%), over 50 IU/L in 8 patients (11.76%) and undetectable in two patients. According to gallbladder bile amylase, the incidence of Occult PBR in patients operated upon for chronic calcular cholecystitis was 94.1%. The reason should be clarified by further research and wider scale study. Routinely investigating biliary amylase in every patient having cholecystitis can be a method for early detection of precancerous lesions.

Histopathological findings in appendectomy specimens: a retrospective clinicopathological analysis.

The prevalence and clinical features of the various obstructive lesions in appendectomy specimens studied pathologically with special emphasis on unusualetiological findings were evaluated. The clinic-pathological data of 251 patients who underwent appendectomies for presumed acute appendicitis from January; 2008 to December; 2010; were reviewed retrospectively. Among all appendectomies performed, eleven (4.38%) specimens revealed incidental abnormal pathological diagnoses: Four cases (1.59%) of Enterobiusvermicularis, Ascaris lumbricoides two cases (0.79 %), schistosomiasis eggs in three cases (1.19%), and parasitic amebiasis in two cases (0.79%). None suffered tumor in any appendectomies specimens. Mean age was lower, perforated and gangrenous appendicitis was absent and negative appendectomy rate was higher in patients with unusual appendix pathology.