Toll-like receptor 7 mRNA is reduced in hepatitis C-based liver cirrhosis and hepatocellular carcinoma, out-performs alpha-fetoprotein levels, and with age and serum aspartate aminotransferase is a new diagnostic index.

BACKGROUND: Hepatitis B and C viruses are leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Toll-like receptor 7 (TLR-7) has been implicated in the pathogenesis of HCC linked to hepatitis B. We hypothesised a role of leukocyte TLR-7 mRNA in hepatitis C related liver cirrhosis and HCC, using alpha-fetoprotein (AFP) and liver function tests as comparators. METHODS: We recruited 102 patients with HCV-related HCC, 97 with HCV-related liver cirrhosis and 60 healthy controls. Quantification of TLR-7 mRNA was performed using real-time PCR, AFP and routine LFTs by standard techniques. RESULTS: TLR-7 mRNA levels were significantly lower in HCC patients compared to cirrhotic patients and lower again in healthy controls (p < 0.001 for trend). In multivariate analysis, age, aspartate transaminase (AST), AFP, and TLR-7 mRNA were significant predictors of HCC. The ROCC/AUC for age, AST and TLR-7 mRNA were all between 0.64 and 0.78 (all P < 0.01), but for AFP was 0.57 (95% CI 0.48-0.65, P = 0.09). We derived an index score using age, AST and TLR-7 mRNA for the diagnosis of HCC. The ROCC/AUC for the index was superior to all three root indices in the prediction of HCC. The index linked significantly with the Tokyo and Vienna liver cancer staging systems, but not with those of the CLIP and Okuda systems, in distinguishing HCC from liver cirrhosis. CONCLUSION: The combination of TLR-7 mRNA levels with age and AST improves the performance of TLR-7 in HCC diagnosis, out-performs alpha-fetoprotein and predicts early HCC.

A panel of a mitogenic (PDGF), biochemical (albumin) and demographic (age) parameters for the non-invasive assessment of hepatic fibrosis.

Background: Several studies have investigated certain fibrosis markers that incorporate liver function tests, fragments of liver-matrix components and/or degraded products generated by hepatic stellate cells for determining the degree of hepatic fibrosis. However, the role of these molecules in the development of hepatic fibrosis is unclear. This work aimed (a) to determine whether platelet-derived growth factor (PDGF) is linked to different stages of hepatic fibrosis and (b) investigate its diagnostic performance alongside other laboratory and demographic factors in assessing liver fibrosis in chronic hepatitis C infection. Methods: Liver-fibrosis was staged according to Fibroscan, PDGF quantified using ELISA, and liver function tests and other analytes determined by standard techniques in 239 patients with chronic hepatitis C virus infection. Results: Patients with significant (F2-F4), advanced fibrosis (F3-F4) and cirrhotic liver disease (F4) showed significantly (P<0.0001) higher PDGF levels increase respectively compared to stage F0/1. We used this to construct the PARA-Index (PDGF/albumin ratio, age), which performed well in assessing hepatic-fibrosis stages with AUCs of 0.91, 0.87 and 0.86 for identifying F2-F4, F3-F4 and F4, respectively. Additionally, the PARA-Index correlated strongly (r=0.65, P<0.0001) with the severity of the fibrosis. An elevated PARA-index provided odds ratios of 21.0, 20.7 and 10.3 for developing F2-F4, F3-F4 and F4, respectively. Conclusion: A panel of mitogenic (PDGF), biochemical (albumin) and demographical (age) parameters may improve liver-fibrosis staging with a high degree of accuracy in those with a hepatitis C virus infection.

Clinical value of a diagnostic score for colon cancer based on serum CEA, CA19-9, cytokeratin-1 and mucin-1.

BACKGROUND: Although established markers such as CEA and CA19-9 are important for diagnosing early stages of colon cancer, they are not ideal. Developing promising markers include cytokeratin 1 (CK1) and mucin-1 (MUC1), but the combined value of each of these markers is unclear. We therefore evaluated the value of a combined laboratory-based score of these four markers in the diagnosis of colon cancer. METHODS: Two hundred patients who had undergone colonoscopic examination (150 colon cancer, 50 benign growths) were recruited. The study was controlled by 35 healthy subjects. CEA, CA19-9, CK1 and MUC1 were measured by ELISA and evaluated for cancer diagnosis using area under the receiver operating characteristic curve (AUC). RESULTS: Serum levels of all four markers were increased in the order colon cancer > benign disease > healthy controls (p < 0.001). In multivariate analysis, CA19.9 (p = 0.025), CK1 (p < 0.001) and MUC1 (p = 0.009) were significant independent predictors of colon cancer. A score that gave the greatest power of discrimination for colon cancer was defined as 1.06 + [0.001 × CA19.9 result] + [0.003 × CEA result] + [0.03 × CK1 result] + [0.05 × MUC1 result]. The colon score provided superior discrimination, AUC, and sensitivity and specificity for colon cancer versus benign growth than each of the individual markers. Similarly, the colon score provided superior AUC, and sensitivity and specificity that each individual marker for tumour stage, lymph node invasion and distant organ metastases than each individual marker. CONCLUSION: A colon score derived from serum CEA, CA19-9, CK1 and MUC1 is a potential valuable non-invasive index that could be used for detection and screening early stage colon cancer patients.

Fibro-Mark: a panel of laboratory parameters for predicting significant fibrosis in chronic hepatitis C patients.

BACKGROUND: Fibrosis markers are useful for the prediction of cirrhosis but clinical scores such as King's score, AST-Platelet ratio index (APRI), Biotechnology research center (BRC), Fibrosis routine test (FRT), Fibro-α score and Fibro-quotient (FibroQ) have limited accuracy for diagnosing significant fibrosis. We hypothesised that new markers (reflecting the balance between hepatic fibrogenesis and fibrolysis) together with other indirect fibrosis markers would together construct a more sensitive and specific score capable of identifying fibrosis than existing scores. METHODS: Collagen IV, hyaluronic acid, platelet-derived growth factor (PDGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and AST, ALT, platelet count, albumin, total bilirubin, INR and AFP by routine methods in 148 patients with hepatitis C induced liver disease. Stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs) were used to create a predictive score and compare it to others. RESULTS: Patients with significant fibrosis (n = 100, F2-F4) showed 2.08, 2.14, 1.80 and 1.90-fold increase in collagen IV, hyaluronic acid, PDGF and TIMP-1, respectively, over patients with no or mild fibrosis (n = 48, F0/F1)(all p < 0.01). Significant independent predictors of F2-F4 were AFP (AUC 0.79), age (0.76), PDGF (0.74), collagen IV (0.78) and TIMP (0.75), which together formed a five-marker score 'Fibro-Mark' for predicting F2-F4. In comparison with other scores, AUC for Fibro-Mark was 0.89, BRC was 0.83, followed by FRT and King's score (both 0.82), APRI (0.80), Fibro-α (0.70) and finally Fibro Q (0.63). CONCLUSIONS: The Fibro-Mark score provides better discrimination in hepatic-fibrosis staging in chronic hepatitis C patients than existing scores.

Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients.

BACKGROUND: Hepatocellular carcinoma (HCC) is a multistage process resulting from various genetic changes. We aimed to determine nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 expression and to evaluate their importance in HCC diagnosis. METHODS: One hundred and twenty chronic hepatitis C (CHC) patients (60 non-HCC CHC patients and 60 HCC patients who had a single small (<5 cm) tumour) were recruited. The gene products of c-Myc and p53 were identified in liver tissues and serum samples using immunostaining, western blot and ELISA. RESULTS: Immunohistochemical detection of c-Myc and p53 with monospecific antibodies revealed intense and diffuse cytoplasmic staining patterns. Accumulated mutant proteins, released from tumour cells into the extracellular serum, were detected at 62 KDa, for c-Myc, and 53 KDa, for p53, using western blotting. In contrast to alpha feto-protein, there was a significant increase (p < 0.0001) in the positivity rate of c-Myc (86.7% vs. 6.7%) and p53 (78.3% vs. 8.3%) in the malignant vs. non-malignant patients. The parallel combination of c-Myc and p53 reach the absolute sensitivity (100%), for more accurate and reliable HCC detection (specificity was 87%). CONCLUSION: c-Myc and p53 are potential HCC diagnostic biomarkers, and convenient combinations of them could improve diagnostic accuracy of HCC.

Development and evaluation of a novel score for prediction of large oesophageal varices in patients with hepatitis c virus-induced liver cirrhosis.

OBJECTIVE: Variceal bleeding is one of the most common life-threatening complications of liver cirrhosis. This study aimed to develop and evaluate a predictive score, named Platelet count, Alpha fetoprotein (AFP) and Prothrombin-INR (PAP) for the prediction of large oesophageal varices and to compare PAP score with eight common liver fibrosis scores (AAR, APRI, GUCI, BRC score, Fibro-Alfa, FIB4, Lok and Fibro-Q) in patients with hepatitis C virus (HCV) induced liver cirrhosis. METHODS: A total of 277 patients with HCV-induced liver cirrhosis were evaluated by upper gastrointestinal endoscopy for presence of varices. Liver biochemical profile, complete blood count, prothrombin time and AFP were estimated. Stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs) were used to create a predictive score (PAP score) comprising platelet count, AFP and prothrombin-INR. RESULTS: PAP score predicts large oesophageal varices in patients with HCV-induced liver cirrhosis with AUC of 0.85. The optimum cut-off for predicting large oesophageal varices using ROC curve analysis was 0.27. At this point the PAP score had 77% sensitivity, 86% specificity, 94% negative predictive value and 84% efficiency. The diagnostic performances (AUC) of eight common liver fibrosis scores were 0.58 for the AAR score, 0.63 for APRI, 0.66 for GUCI, 0.68 for BRC, 0.72 for Fibro-Alfa, 0.70 for FIB4, 0.72 for Lok and 0.77 for Fibro-Q. CONCLUSION: PAP scores a non-invasive, inexpensive and simple score that could predict the presence of large oesophageal varices reducing the need of endoscopy. The PAP score has a superior AUC score than other scores, suggesting improved clinical value.

A rapid, low-cost quantitative diagnostic method for hepatitis C virus infection using capillary zone electrophoresis.

Hepatitis C virus (HCV)-RNA amplification is a costly procedure in terms of time and reagents. Consequently, the search for more a cost-effective specific HCV diagnostic method is of great interest. Capillary zone electrophoresis (CZE) methods that detect HCV in serum, plasma, whole blood, and ascites without the need for sample pretreatment are not currently available. Here, a CZE method was developed that detects a larger specific peak in serum and other body fluids of HCV-infected patients than that found in healthy or hepatitis B virus (HBV)-infected individuals. The nature of the HCV peak was investigated using biochemical treatments, including RNase, DNase, and chymotrypsin enzymes. Electroeluted HCV peak was applied to transmission electron microscopy; electron micrographs showed that the HCV peak was attributed to virus-like particles with diameter and morphological properties similar to non-enveloped HCV nucleocapsids. The determination of CZE-HCV and HCV-RNA levels using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in 258 subjects revealed that these two tests were highly correlated (r = 0.92, p < 0.0001). One important issue of HCV testing is the storage conditions of serum to obtain reliable results. Serum samples at -20 °C showed the best preservation of the HCV peak up to one year. In conclusion, we detected HCV using CZE in a microliters volume from different body fluids. Besides the stability of samples in maintaining their peak height, the HCV-CZE test is rapid (<15 min) and a well-suited and low-cost technique. Thus, a major improvement in the quantitative diagnosis of HCV infection was established.

HCC-ART score, a simple, highly sensitive and specific test for early diagnosis of hepatocellular carcinoma: a large-scale, multicentre study.

BACKGROUND: A simple scoring system is needed to discriminate HCC from patients with chronic liver diseases (CLD). The simplest score would be one that requires only variables that can be documented simply from routine laboratory tests without the need for sophisticated tests. METHODS: Data from the estimation group (1351 patients) and the validation group (2208 patients) were retrospectively analysed. Liver fibrosis-negative control and liver cirrhosis were compared with HCC. Area under ROC curve (AUC) were used to develop HCC-α-fetoprotein-routine test (HCC-ART). RESULTS: Hepatocellular carcinoma-AFP-routine test showed diagnostic accuracy for liver cirrhosis vs HCC with ROC curves of 0.99%, sensitivity of 97%, and specificity of 96% in the estimation, and 0.95%, 90%, and 83%, respectively, in the validation. Sensitivity (97%) and specificity (100%) were obtained to discriminate HCC from liver fibrosis. Area under curve for AFP at 400 U l(-1) was 0.70, sensitivity was 41%, and specificity was 99% in the estimation, and 0.77%, 54%, and 99%, respectively, in the validation. The AUC for HCC-ART in HCC with single tumour, absent vascular invasion, size <2 cm and CLIP score (0-1) were 0.95, 0.93, 0.86, 0.87, respectively, compared with 0.72, 0.71, 0.71, 0.50, respectively, for AFP. CONCLUSION: Hepatocellular carcinoma-AFP-routine test could increase the accuracy of HCC screening and surveillances and could be used worldwide without extra efforts.

Evaluation of serum procollagen aminoterminal propeptide III, laminin, and hydroxyproline as predictors of severe fibrosis in patients with chronic hepatitis C.

In an attempt to identify biochemical analytes that could enhance the discrimination between the patients with severe liver fibrosis (F3-F4) and mild fibrosis (F1-F2) based on absolute values of biochemical markers, we measured 12 analytes, including procollagen III aminoterminal propeptide (PIIINP), laminin, proline, hydroxylproline, glycine, AST, ALT, alkaline phosphatase, albumin, total bilirubin, total protein, and prothrombin time in 252 individuals with chronic hepatitis C infection (CHC). PIIINP and laminin were determined by radio-immunoassay; the degraded amino acids were determined using high performance liquid chromatography. Statistical analyses were performed by logistic regression, and receiver operating characteristic (ROC) curves. The best linear combination of blood markers was selected by multivariate discriminant analysis (MDA) for construction of the fibrosis discriminant score (FDS). FDS, an index of five markers (PIIINP, laminin, hydroxyproline, prothrombin activity, and AST/ALT) correctly classified 82% of the patients with severe liver fibrosis at a discriminant cut-off score=-0.5 (i.e., less than -0.5 indicated severe liver fibrosis and greater than -0.5 indicated mild liver fibrosis with sensitivity (76%) and specificity (89%). This result was reproduced in a validation study with no significant difference. In conclusion, FDS is useful for identifying severe liver fibrosis in patients with CHC.