HCV nonstructural protein 4 is associated with aggressiveness features of breast cancer.

BACKGROUND: Hepatitis C virus (HCV) has the lymphotropic feature that is supposed to be the reason of related extrahepatic manifestation. HCV viral oncoproteins may participate in the regulation of some gene expression that has been implicated in tumorigenesis. Our aim is to evaluate the HCV-NS4 circulating levels in breast cancer (BC) and to investigate its relation with BC tumor aggressiveness. METHODS: This study was performed among 158 Egyptian women (120 with BC and 38 with benign breast diseases). ELISA was used for detection of anti-HCV antibodies, HCV-NS4, fibronectin, and CA 15-3. RESULTS: No association between HCV detection in this group of BC patients (27.5% in BC vs. 23.7% in breast benign diseases, P = 0.687). Among HCV-infected patients, the mean HCV-NS4 serum level in BC was significantly higher than benign group (61.7 µg/mL vs. 33.9 µg/mL, P = 0.0005). Fibronectin levels were higher (P = 0.014) in patients infected with HCV than noninfected BC patients. Elevated HCV-NS4 levels were associated with tumor severity features like large size, late stages, high grades, and infiltrated lymph nodes. The elevated levels of HCV-NS4 (> 40 µg/mL) yielded an estimated odds ratio (95% confidence intervals) of 2.5 (0.98-6.36), 1.2 (0.44-3.33), 1.9 (0.53-7.00), and 2.5 (0.87-7.33) for developing large size, late stages, high grades, and infiltrated lymph nodes, respectively. Interestingly, HCV-NS4 levels significantly correlated with other BC tumor marker like CA15-3 (r = 0.535; P = 0.0009) and fibronectin (r = 0.432; P < 0.0001). CONCLUSIONS: HCV-NS4 appears to be associated with BC progression features. Oncologists treating such BC patients should consider HCV screening to enable the early identification and to prevent progression of the disease.

Simplified HCC-ART score for highly sensitive detection of small-sized and early-stage hepatocellular carcinoma in the widely used Okuda, CLIP, and BCLC staging systems.

BACKGROUND: Small-sized HCC can be effectively cured by surgery with good clinical outcomes. A highly sensitive HCC α-fetoprotein routine test (HCC-ART) for HCC diagnosis as well as a simplied form of the HCC-ART were reported in the British Journal of Cancer. Here, we verified and studied the applicability of the HCC-ART to the detection of early-stage HCC. METHODS: 341 cirrhotic patients and 318 HCC patients were included in this study. For each, the HCC-ART score was calculated, and then the sensitivity, specificity, and results of an ROC curve analysis were compared between the HCC-ART and AFP when these biomarkers were used to detect small-sized HCC. RESULTS: Different HCC-ART cutoffs were set for the detection of different tumor sizes. The HCC-ART (AUC = 0.871, 70% sensitivity, 97% specificity) and the simplified HCC-ART (AUC = 0.934, 82% sensitivity, 100% specificity) were found to have high predictive power when attempting to separate cirrhotic patients from those with small-sized HCC. The simplified HCC-ART score was superior to AFP for determining stages according to the early Okuda (0.950 AUC, 84% sensitivity, 99% specificity), CLIP (0.945 AUC, 84% sensitivity, 99% specificity), and BCLC (1.000 AUC, 100% sensitivity, 99% specificity) staging systems. The simplified HCC-ART score was more strongly correlated than AFP and other staging systems with HCC tumor size (P < 0.0001; r = 0.8). CONCLUSION: The HCC-ART is superior to AFP for diagnosing early-stage HCC. Due to its advantages of minimal variability and a wide continuous scale for assessing HCC severity, the simplified HCC-ART has the potential to be more widely used than the original HCC-ART.

Levels of Schistosoma mansoni Circulating Antigen in Chronic Hepatitis C Patients with Different Stages of Liver Fibrosis.

The goal of this study was to determine the levels of S. mansoni antigen in different liver fibrosis stages with chronic hepatitis C (CHC) Egyptian patients. A total of 174 CHC patients showing HCV-NS4 antigen and HCV- RNA in their sera were included. S. mansoni antigen was detected in serum using Western blot and ELISA. The levels of interferon-γ (IFN- γ) were determined using ELISA. The 50 kDa S. mansoni antigen discriminated patients infected with S. mansoni from healthy individuals with 0.93 area under curve (AUC), 92% sensitivity, and 97% specificity. The level of S. mansoni antigen (µg/ml) was significantly (P < 0.0001) increased with the progression of liver fibrosis stages (26.9 ± 17.5 in F1, 42.1 ± 25.2 in F2, 49.8 ± 30.3 in F3 and 62.2 ± 26.3 µg/mL in F4 liver cirrhosis), 26.9 ± 17.59 in significant fibrosis (F2-F4); 51.2 ± 27.9 in advanced fibrosis (F3-F4). A significant correlation (r = 0.506; P < 0.0001) was shown between the levels of the S. mansoni antigen and the HCV-NS4 antigen. In conclusion, the presence of S. mansoni antigen in different liver fibrosis stages of CHC patients confirming that concomitant schistosome infection aggravates liver disease.

Circulating levels and clinical implications of epithelial membrane antigen and cytokeratin-1 in women with breast cancer: can their ratio improve the results?

Immunohistochemical studies proved that the presence of breast cancer (BrCa) is accompanied by elevated levels of epithelial membrane antigen (EMA) and decreased levels of cytokeratin-1 (CK1). We, therefore, hypothesize that the serum EMA/CK1 ratio may serve as a promising biomarker for early diagnosis of breast cancer. The circulating levels of EMA and CK1 were determined by Western blot and enzyme-linked immunosorbent assay (ELISA) in sera from 102 women with BrCa and 90 women as controls (40 with benign breast disease and 50 healthy). EMA at 130 kDa and CK1 at 67 kDa were identified, purified, and quantified in sera of BrCa patients using ELISA. EMA/CK1 ratio values were found to discriminate BrCa patients from controls (P < 0.0001) with high diagnostic ability (area under the curve [AUC] = 0.901, sensitivity = 82, specificity = 76). The sensitivity and specificity for early-stage (≤ T2) BrCa were 72 and 76%, respectively. The ratio values of patients with late-stage (>T2) tumors were significantly higher than those of patients with early-stage (≤ T2) tumors. Moreover, higher grades (grades 2-3) were associated with higher values than grade 1 tumors. AUC values in different BrCa patients who had early stage, low grade, or size ≤ 2 cm were 0.855, 0.762, and 0.839, respectively. AUC values of patients with positive lymph node or positive distant metastasis were 0.907 and 0.913, respectively. We show for the first time the impact of serum EMA and CK1 ratio in BrCa detection. Differential EMA/CK1 values may serve as a diagnostic marker in early-stage breast cancer patients.

Noninvasive diagnosis of liver fibrosis and cirrhosis in chronic hepatitis C patients.

BACKGROUND: We aimed to derive a simple noninvasive test for liver-fibrosis staging and then estimate its performance against four simple noninvasive tests in chronic hepatitis C (CHC) patients. METHODS: CHC patients were divided into two cohorts: an estimation set (n = 324) and a validation set (n = 524). Liver fibrosis was staged according to the METAVIR scoring system. Statistical analysis was done using stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs). RESULTS: Biotechnology Research Center (BRC) score was constructed combining several blood markers that proved useful to stage liver fibrosis. Aspartate aminotransferase /alanine aminotransferase ratio (AAR), aspartate to platelet ratio index (APRI), Fibro-α, King, and BRC scores correlated with the histological fibrosis stages with correlation coefficient 0.26, 0.36, 0.58, 0.45, and 0.73, respectively. BRC score produced AUCs 0.87, 0.83, and 0.89 for significant (F2-F4), advanced fibrosis (F3-F4), and cirrhosis (F4), respectively. These results were reproduced in the validation study with no significant difference yielding AUCs 0.85 for F2-F4, 0.82 for F3-F4, and 0.88 for F4. CONCLUSION: BRC score, a novel noninvasive test, is a useful and easy tool to evaluate liver fibrosis in CHC patients and seems more efficient than AAR, APRI, Fibro-α score, and King's score in this group of Egyptian patients.

Development of a novel score for liver fibrosis staging and comparison with eight simple laboratory scores in large numbers of HCV-monoinfected patients.

BACKGROUND: This study aimed to develop and evaluate a predictive score named Fibrosis Routine Test (FRT) for liver fibrosis staging and to compare FRT with APRI, Lok, GUCI, FI, FibroQ, FCI, FIB-4 and 4RLB scores in large numbers of untreated HCV-monoinfected patients. METHODS: Large numbers of estimation (N=2045) and validation patients (N=3212) were included in this study. Stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs) were used to create a predictive score comprising age, AFP, APRI and albumin. RESULTS: In the estimation study, FRT produced AUCs 0.84, 0.85 and 0.86 for significant (F2-F4), advanced fibrosis (F3-F4) and cirrhosis (F4), respectively. FRT > 4 was 83% specific and 73% sensitive for F2-F4, FRT > 5 was 83% specific and 71% sensitive for F3-F4 and FRT > 5.5 was 81% specific and 73% sensitive for F4. In the validation study, FRT produced AUCs 0.81, 0.89 and 0.95 for F2-F4, F3-F4 and F4, respectively. The above eight scores demonstrated lower AUCs than FRT. CONCLUSION: While liver biopsy is invasive, costly and associated with complications, Fibrosis Routine Test (FRT) is a non-invasive, inexpensive, simple and may reduce the need of liver biopsy.

Assessment of matrix metalloproteinase-1 for marking liver cirrhosis in chronic hepatitis C patients.

Our objective in the present study was to observe the change in the serum MMP-1 concentration using ELISA in 129 chronic hepatitis C (CHC) (78 non-cirrhotic and 51 with cirrhotic liver) and 50 healthy controls. The values of MMP-1 concentration increased in patients with CHC according to the stage of liver fibrosis. An area under the ROC curves (AUC) of the MMP-1 was 0.98 for discriminating patients with cirrhotic liver from healthy individuals and was 0.78 for discriminating patients with cirrhotic liver from non cirrhotic patients. The diagnostic potential of MMP-1 for discriminating cirrhosis from healthy individuals was very high with 98% sensitivity and 97% efficiency. MMP-1 detected cirrhosis in CHC with 71% sensitivity and 73% efficiency. In Conclusion, measurement of serum MMP-1 is useful for diagnosing liver cirrhosis in CHC patients.

Efficacy of passive immunization with IgY antibodies to a 58-kDa H. pylori antigen on severe gastritis in BALB/c mouse model.

Consecutive triple doses of 1 x 10(8) CFU/mL of a pathogenic H. pylori strain isolated from stomach of Egyptian patients with severe gastritis were used to establish infection in BALB/c mice model. White Leghorn hens were immunized with H. pylori whole cell lysate (HpLysate) antigen and with a highly reactive 58-kDa H. pylori (Hp58) antigen. Two months later, IgY antibodies (IgY-HpLysate & IgY-Hp58) were purified from egg yolk and its efficacy was evaluated in the adopted model. Microbiological culture and immunohistochemical staining revealed that H. pylori infection was inhibited 1 week after oral passive immunization in 70% of infected BALB/c mice with a significant decrease (p < 0.05) in the degrees of gastritis. In conclusion, we have adapted BALB/c mice model for human H. pylori pathogenic strain and oral passive immunization with specific IgY antibodies to the 58-kDa antigen inhibited active H. pylori infection and decreased gastritis.

AgNORs count and DNA ploidy in liver biopsies from patients with schistosomal liver cirrhosis and hepatocellular carcinoma.

OBJECTIVES: Argyrophilic nucleolar organizer regions (AgNOR) proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells, whereas their expression is very low in nonproliferating cells. The present study aimed to investigate the potential of DNA flow cytometry (FCM) and AgNORs count in the assessment of cellular kinetics of liver cirrhosis and hepatocellular carcinoma. DESIGN AND METHODS: Small-needle liver biopsies (217) were included and were taken from 84 patients with hepatocellular carcinoma (HCC) (one biopsy from tumor lesion and the other from residual nontumor) liver tissues. Only one biopsy was taken from 49 patients with liver cirrhosis. One part of biopsy was subjected to flow cytometry, and the other, to histopathology and AgNORs counting. RESULTS: An aneuploidy was shown in 44.5% of liver cirrhosis and in 78.6% of tumor sites. Aneuploid HCC cases showed high AgNORs count compared with diploid cases (3.407+/-1.18 vs. 1.74+/-0.9). An extremely significant increase in AgNORs count in tumor lesion (P<0.001) was found compared with residual liver tissues, liver cirrhosis and normal liver (3.89+/-0.827, 1.49+/-0.52, 1.62+/-0.29, and 1.3+/-0.17, respectively). In liver cirrhosis, dysplasia showed a significant relationship with ploidy (P<0.001) and AgNORs count (P<0.05). CONCLUSION: AgNORs count and DNA ploidy analysis of core biopsy specimens are useful in the assessment of cellular kinetics of liver cirrhosis and hepatocellular carcinoma.

Expression of p53 protein in liver and sera of patients with liver fibrosis, liver cirrhosis or hepatocellular carcinoma associated with chronic HCV infection.

OBJECTIVES: Hepatitis C virus (HCV) is a major aetiological agent of chronic hepatitis and it leads to the development of liver cirrhosis and hepatocellular carcinoma (HCC). The significances of p53 protein and anti-p53 antibodies levels in HCV genotype IV infected patients with different liver pathology were evaluated. DESIGN AND METHODS: Immunostaining and western blot based on monospecific anti-p53 antibody were used for the identification of p53 protein in liver tissues and serum samples. The serum levels of p53 protein and anti-p53 IgG antibodies were evaluated using enzyme linked immunosorbent assay (ELISA). RESULTS: Mild and diffuse p53 cytoplasmic immunostaining was found in liver tissues of patients with liver fibrosis [F1-F3] and liver cirrhosis [F4] in comparison with strong and diffuse p53 cytoplasmic immunostaining in patients with HCC. The target p53 protein was identified in sera of patients with liver fibrosis, liver cirrhosis and HCC at 53-kDa. The detection rate of serum p53 protein increases significantly (p<0.05) with the progression of the liver pathology. However, a significant difference (p<0.05) was only shown between serum p53 protein level of HCC patients and those of other liver pathology. In contrast, anti-p53 IgG antibodies positive rates showed only a significant decrease (p<0.05) in HCC in comparison with liver cirrhosis. CONCLUSIONS: The serum and cytoplasmic p53 protein expressions were more pronounced in patients with HCC more than liver cirrhosis, and in liver cirrhosis more than liver fibrosis. These results suggest that HCV genotype IV and p53 protein levels may have a role in the development of HCC among Egyptian patients.

Immunochemical identification and partial characterization of a native hepatitis C viral non-structural 4 antigen in sera of HCV infected patients.

BACKGROUND: The identification of native HCV antigens may prove very useful in the diagnosis and early treatment of HCV infection. Here, we aimed to identify and partially characterize a native HCV-NS4 antigen. METHODS: The western blot, ELISA and immunohistochemical staining were used to identify the native antigen in sera and liver biopsies of HCV serotype 4 infected patients. RESULTS: The native NS4 antigen was identified in serum at 27-kDa molecular weight, in addition to a lower approximately 21-kDa antigen. The purified HCV antigen showed a polypeptide band at 27-kDa when analyzed by silver stained SDS-PAGE and a single peak at 7.6 min by capillary zone electrophoresis. The immunostaining pattern of hepatocytes was cytoplasmic with mainly coarse granular and diffuse pattern based on specific rabbit antisera to the native HCV antigen. A highly significant correlation (r=0.797, p<0.0001) was shown between serum concentrations of the HCV-NS4 antigen and HCV-RNA. Also, antigen detection rates were increased (p<0.05) with the progression of liver disease. CONCLUSION: A native HCV-NS4 antigen was identified and partially characterized as 27-kDa protein and the NS4 antigenemia based ELISA test can serve as a useful addition to HCV diagnostic methods, especially under field condition.

Immunochemical identification and detection of serum fibronectin in liver fibrosis patients with chronic hepatitis C.

Serum tests measuring the dynamic processes of fibrogenesis and fibrolysis may reflect the severity of liver disease. Fibronectin plays a role in liver fibrosis. The aim of this study was to assess the diagnostic value of fibronectin in chronic HCV infection among Egyptian patients. Fibronectin was identified using specific monoclonal antibody and Western blot at 90-kDa in sera of HCV infected patients with liver fibrosis. The purified serum fibronectin showed one peak at 8 min when analyzed by capillary zone electrophoresis. Fibronectin was quantified in serum using ELISA. The mean (+/-SD) serum level of fibronectin (mg/L) in liver fibrosis patients were 450.9 (+/-170.3) and 230.5 (+/-90.3) in control individuals, respectively. There was a significant correlation between METAVIR score and serum fibronectin (r=0.401; P<0.0001). The area under the receiver operating characteristic (ROC) curve of fibronectin for discriminating patients with liver fibrosis from those with no fibrosis livers and its p value were 0.78 and P<0.0001. The efficiency of fibronectin for discriminating patients with liver fibrosis from those with non fibrosis livers was 75%. In conclusion, serum fibronectin can differentiate HCV infected patients with liver fibrosis from patients with non fibrosis.

Immunodetection of collagen types I, II, III, and IV for differentiation of liver fibrosis stages in patients with chronic HCV.

The current study is aimed at evaluating serum collagens and other serum biochemical markers as useful, non-invasive markers of hepatic fibrosis associated with chronic hepatitis C virus (HCV). Collagen types I, II, III, and IV were detected in serum using ELISA and Western blot techniques. The ELISA levels of collagen I, II, III, and IV increased significantly with the progression of fibrosis staging. Based on receiver-operating characteristic (ROC) curve analysis, the collagen type III (70 kDa) and type IV (200 kDa) were more useful than other serum bio-markers for differentiating severe fibrosis from mild fibrosis. Multivariate discriminant analysis (MDA) selected a fibrosis discriminant score (FDS) = [2.345 + Collagen III (microg/mL) x 1.923 + Collagen IV (microg/mL) x 1.544 + ALT (U/mL) x 0.005] - [albumin(g/L) x 0.046]. The FDS correctly classified 87% of the severe fibrosis patients at a cut-off score = 2.2 (i.e., more than 2.2 indicated severe fibrotic liver and less than 2.2 indicated mild fibrotic liver) with specificity of 97%. In a validation study, the FDS was applied to the second cohort of patients and the results were reproduced without significant difference. In conclusion, the developed four-parameter based FDS is useful for identifying severe liver fibrosis in patients with chronic HCV infection.

Rapid diagnosis and follow up of bladder cancer patients using urinary high molecular weight cytokeratins.

We have developed an office-based dot-EIA for the detection of a urinary high molecular weight cytokeratin (CK). Immunohistochemical staining and western blot based on CK1K10 monoclonal antibody were used to identify the CK. Urine of 192 patients with different types, grades, and stages of bladder tumor and 72 controls were evaluated using dot-EIA. An intense and diffuse cytoplasmic reaction was shown in bladder squamous cell carcinoma. The target epitope was identified in urine at 65, 56, and 40-kDa. The CK purified from urine showed single polypeptide at 65-kDa using SDS-PAGE and single peak at 7.4 min using capillary zone electrophoresis. The dot-EIA detected the CK with high sensitivity (97%) and specificity (94%). The CK was not detected in urine of bladder cancer patients showing response to radiotherapy. The sensitive and specific office-based detection of urinary cytokeratin would be helpful in rapid diagnosis and follow up of bladder carcinoma.

An office-based immunodiagnostic assay for detecting urinary nuclear matrix protein 52 in patients with bladder cancer.

OBJECTIVE: To report the rapid (5 min) and simple detection of a nuclear matrix protein (NMP) in the urine of patients with bladder cancer, using a newly developed office-based dot-enzyme-linked immunosorbent assay (ELISA). PATIENTS AND METHODS: Western blot and specific immunoglobulin-G antibody were used to identify the urinary NMP marker. Urine samples from 149 patients with bladder cancer and 72 controls were evaluated using the developed dot-ELISA. The initial responses of 43 patients treated by irradiation were followed using the assay. RESULTS: The NMP marker was identified in the urine of patients with bladder cancer at 52 kDa (NMP-52) by Western blot. The dot-ELISA detected the urinary NMP-52 marker in 92% of patients with squamous cell carcinoma, 98% with transitional cell carcinoma, and all six of those with adenocarcinoma of the bladder, with a specificity of 94%. The positive and negative predictive values (97% and 94%, respectively) and efficiency (96%) of the dot-ELISA were high. In addition, the NMP-52 tumour marker was not detected in the urine of patients who showed a response after radiotherapy. CONCLUSION: Detecting the urinary NMP-52 marker using dot-ELISA would be helpful in the rapid diagnosis and follow-up of patients with bladder cancer.

DNA ploidy and liver cell dysplasia in liver biopsies from patients with liver cirrhosis.

There is controversy among pathologists when assessing the presence or absence of liver cell dysplasia in liver biopsies taken from cirrhotic patients. The objective of the present study was to determine the DNA ploidy pattern of hepatocytes of patients with liver cirrhosis and its relationship to liver cell dysplasia. A total of 48 male patients diagnosed with liver cirrhosis based on clinical, laboratory and histopathological criteria were included in the study. A liver biopsy was taken from each patient; one part of the biopsy was subjected to histopathology, and the other to flow cytometry. The histopathological examination revealed liver cell dysplasia in 60% of patients with liver cirrhosis (62% of them had large cell dysplasia [LCD] and 38% had small cell dysplasia [SCD]). Abnormal DNA content (aneuploidy) was found in 81.5% of positive liver cell dysplasia specimens and found only in 11.1% of negative liver cell dysplasia specimens, with a statistically significant difference (P<0.001). Aneuploidy was found more commonly in LCD but without significant difference (P>0.05) in comparison with SCD. In conclusion, SCD (similar to LCD) is also associated with aneuploidy and elevated DNA index, and may carry the same risk for progression to hepatocellular carcinoma.

A novel antigen detection immunoassay for field diagnosis of hepatitis C virus infection.

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (approximately 5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.