HCC-Mark: a simple non-invasive model based on routine parameters for predicting hepatitis C virus related hepatocellular carcinoma.

BACKGROUND: Early detection of hepatocellular carcinoma (HCC) is crucial in providing more effective therapies. As routine laboratory variables are readily accessible, this study aimed to develop a simple non-invasive model for predicting hepatocellular cancer. METHODS: Two groups of patients were recruited: an estimation group (n = 300) and a validation group (n = 625). Each comprised two categories: hepatocellular cancer and liver cirrhosis. Logistic regression analyses and receiver operating characteristic (ROC) curves were used to develop and validate the HCC-Mark model comprising AFP, high-sensitivity C-reactive protein, albumin and platelet count. This model was tested in cancer patients classified by the Barcelona Clinic Liver Cancer (BCLC), Cancer of Liver Italian Program (CLIP) and Okuda systems, and was compared with other non-invasive models for predicting hepatocellular cancer. RESULTS: HCC-Mark produced a ROC AUC of 0.89 (95% CI 0.85-0.90) for discriminating hepatocellular carcinoma from liver cirrhosis in the estimation group and 0.90 (0.86-0.90) in the validation group (both p < 0.0001). This AUC exceeded all other models, that had AUCs from 0.41 to 0.81. AUCs of HCC-Mark for discriminating patients with a single focal lesion, absent macrovascular invasion, tumour size <2 cm, BCLC (0-A), CLIP (0-1) and Okuda (stage Ι) from cirrhotic patients were 0.88 (0.85-0.90), 0.87 (0.85-0.89), 0.89 (0.85-0.93), 0.87 (0.84-0.89), 0.85 (0.82-0.87) and 0.86 (0.83-0.89), respectively (all p < 0.0001). CONCLUSION: HCC-Mark is an accurate and validated model for the detection of hepatocellular cancer and certain of its clinical features.

Immunoreactivity evaluation of mutant p53 gene product with DNA ploidy pattern in colorectal carcinoma.

BACKGROUND/AIMS: Studying p53 protein expression in tumor cells is one of the effective methods for detecting p53 gene mutations. This study attempted simultaneous monitoring of p53 overexpression in colon cancer using immunohistochemical and immunoblotting techniques and also to compare abnormalities of p53 with DNA ploidy and clinicopathological variables. METHODOLOGY: The occurrence of p53 protein expression was analyzed in forty-nine fresh colorectal cancer specimens by immunohistochemical and p53 protein expression also demonstrated by Western immunoblotting technique in 28 colorectal cancer specimens, using an anti-human p53 monoclonal antibody (Do-7), and 25 normal colon mucosa as a negative control. DNA ploidy in 36 specimens of colon cancer tissues was determined by Flow cytometry. RESULTS: Overexpression of p53 protein was detected immunohistochemically in 53.1% (26 of 49) of the tumor specimens. DNA ploidy was performed in 36 cases, 55.6% (20 of 36) of colon cancer specimens were DNA aneuploidy, p53 immunostaining was positive in 60% of cases with DNA aneuploidy compared to 31.3% in diploid tumors (p<0.001). There was no significant association between p53 immunostaining and clinicopathological variables. Overexpression of p53 protein was demonstrated in nuclear protein extract by immunoblotting in 75% (21 of 28) of colorectal carcinoma. Aneuploidy carcinomas were more frequently p53 positive by immunoblotting than DNA diploidy carcinomas; 76.5% (13 of 17) vs. 72.7% (8 of 11) (p<0.2). P53 expression by immunoblotting was more frequently found in good lymphocytic infiltration than moderate and poor lymphocytic infiltration (p<0.001). Also, p53 expression in right colon was significant with rectum (p<0.009). The incidence of p53 expression in Duke's stage B was significant if compared with Duke's stage C (p<0.005). Immuno-reactivity of p53 expression was detected by immunostaining and immunoblotting in 89.3% (25 of 28) of colorectal cancer. P53 immunoreactivity by immunostaining and immunoblotting were closely related to the clinicopathological variables such as pathological type (p<0.01), lymphocytic infiltration (p<0.0001), tumor grade, and tumor site (p<0.001). DNA aneuploidy was more frequently p53 positive than DNA diploid tumor by immunostaining and immunoblotting (p<0.001). CONCLUSIONS: Immunohistochemistry confirmed by immunoblotting assay is a sensitive method for detecting the trace amount of p53 protein and provides valuable information for the understanding of colorectal cancer biology.

Dysregulation of blood lymphocyte subsets and natural killer cells in schistosomal liver cirrhosis and hepatocellular carcinoma.

Immunological factors are important in the pathogenesis of a wide spectrum of hepatobiliary diseases. Using flow cytometry, we determined the changes in lymphocyte subsets and natural killer cells in 123 individuals (81 patients with liver disease and 42 healthy volunteers). The liver diseases included periportal fibrosis (PPF, 10 patients), liver cirrhosis (LC, 31 patients), and hepatocellular carcinoma (HCC, 40 patients). Schistosomiasis and viral hepatitis B and C were the putative etiological agents of liver diseases. Immunophenotyping by indirect immunofluorescence was conducted using monoclonal antibodies to CD3 (T-lymphocytes), CD4 (helper/inducer T-cells), CD8 (suppressor/cytotoxic T-cells), and CD57 (natural killer cells) cell surface markers. Immunophenotyping of PPF patients showed no significant changes in all markers compared with the healthy controls. However, there was a significant decrease ( P<0.01) in CD3 and CD4 T-cells, and a highly significant increase ( P<0.001) in CD57 T-cells in patients with LC or HCC. In addition, LC and HCC patients showed no significant change in CD8 T-cells compared with controls. In conclusion, the progression of liver diseases is associated with a dysregulation of cellular immune responses. T-lymphocytes and natural killer cells may play a role in the immunopathogenesis of liver cirrhosis and HCC.

Identification and characterization of a 26- to 28-kDa circulating antigen of Fasciola gigantica.

As a disease of domestic ruminants, fascioliasis is of considerable economic importance. Although serological tests are available for the diagnosis of the disease, they are of generally low specificity because of cross-reactivity with antigens from other parasites. There is a need to identify other Fasciola antigens on which more specific tests could be based. In the present study, a specific rabbit anti-serum and western-blot analyses were used to demonstrate the presence of a highly reactive antigen of 26-28 kDa not only in an extract of adult F. gigantica but also in the excretory/secretory products of the worms and in the bile secretions and sera of cattle that were naturally infected with this parasite. The 26- to 28-kDa antigen was isolated from preparative polyacrylamide gels, by electro-elution. The purified antigen showed a single peak at 5.8 min when analysed by capillary zone electrophoresis. It was characterized as protein containing 47.5% hydrophilic and 29.3% hydrophobic amino acids. Immunostaining demonstrated that the target epitope was located in the gut and tegument of adult F. gigantica and within the bile ducts, the portal tracts of the livers and the mucosa and muscularis of the gallbladders of infected cattle. A simple and rapid dot-ELISA technique based on the specific rabbit anti-serum was 100% specific when tested on the sera from nine cattle infected with F. gigantea and 27 uninfected cattle. In conclusion, the 26- to 28-kDa Fasciola antigen may be a promising candidate for the immunodiagnosis of fascioliasis.

Immunochemical characterization and diagnostic potential of a 63-kilodalton Schistosoma antigen.

Schistosoma circulating antigens were used for the detection of active infection. Anti-S. mansoni IgG2a monoclonal antibody (MAb) designated C5C4 was generated. The target epitope of this MAb was detected in adult worms, eggs, and cercariae antigenic extracts of S. mansoni and S. haematobium, had a molecular size of 63 kD, and was not detected in Fasciola hepatica and Ascaris. In addition, a 50-kD degradation product was identified only in the urine of infected individuals. Analysis by high-performance liquid chromatography of the purified antigen demonstrated only one peak. The 63-kD antigen was characterized as a protein containing 40.4% hydrophobic, 7.5% acidic, and 8.8% basic amino acids. The C5C4 MAb was used in a Fast Dot-ELISA for rapid and simple diagnosis of human schistosomiasis. The 63-kD circulating antigen was detected in 92% of urine samples from 330 S. mansoni-infected individuals, with 16% false-positive results among 130 noninfected individuals.

Vaccination against Schistosoma mansoni infection using 74 kDa Schistosoma protein antigen.

An IgG2a anti-Schistosoma mansoni mouse monoclonal antibody was shown to passively protect Swiss mice. The 74 kDa target antigen was isolated from antigenic extracts of S. mansoni adult worms. Swiss and C57 BL/6J mice were immunized with 30, 50, 100 and 200 microg antigen/mouse doses with and without Freund's adjuvant. Sera of immunized mice showed high reactivity against 74 kDa antigen. The highest protection level (76.6% in Swiss mice and 50.1% in C57 BL/6J mice) was obtained using the 50 microg antigen dose with and without Freund's adjuvant. A marked reduction in granuloma number and intensity of collagen and reticular granuloma fibers was observed. The 74 kDa antigen has the ability to protect mice of different strains and to modulate the host immune system.

DNA ploidy of liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma: a flow cytometric analysis.

Flow cytometric DNA analysis was used to assess cellular kinetics of needle liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma (HCC). An abnormal DNA content was shown in 44.5% of liver cirrhosis cases and in 78.6% of tumor sites. The number of proliferating cells (S + G2M%) was significantly increased in cirrhotic liver (P < 0.05). Dysplasia was found in 66% of cirrhotic specimens. All negative dysplasia specimens showed a diploid pattern while 69% of positive dysplastic specimens were aneuploid (P < 0.001). In conclusion, cell proliferation, aneuploidy and liver cell dysplasia are important indicators in liver cirrhosis for the development of HCC.

Rapid detection of a Schistosoma mansoni circulating antigen excreted in urine of infected individuals by using a monoclonal antibody.

Schistosoma circulating antigens were used to indicate the infection intensity and to assess cure. An immunoglobulin G2a (IgG2a) mouse monoclonal antibody was used in a fast dot-enzyme-linked immunosorbent assay (ELISA; FDA) for rapid and simple diagnosis of schistosomiasis in the field. Seven hundred Egyptians were parasitologically examined for Schistosoma mansoni and other parasitic infections. A rectal biopsy was done as a "gold standard" for individuals showing no S. mansoni eggs in their feces. Egg counts were obtained by the Kato smear method for only 100 of 152 individuals with eggs in their feces. Specific anti-schistosome IgG antibodies were evaluated in sera by ELISA. Urine samples from the 700 individuals were tested by FDA for detection of the circulating antigen. The assay showed a sensitivity of 93% among 433 infected individuals and a specificity of 89% among 267 noninfected individuals. FDA showed the highest efficiency of antigen detection (91%) compared with the efficiency of antibody detection by ELISA (75%) and stool analysis (60%). In addition, FDA detected infected patients with 20 eggs/g of feces. Also, the sensitivity of FDA ranged from 90 to 94% among samples from patients with different clinical stages of schistosomiasis. All the assay steps can be completed within 30 min at room temperature for 96 urine samples. The monoclonal antibody identified a 74-kDa antigen in different antigenic extracts of S. mansoni and Schistosoma haematobium and in the urine of infected individuals. In addition, a 30-kDa degradation product was identified only in the urine samples. On the basis of these results, FDA should be used as a rapid tool for the sensitive and specific diagnosis of Schistosoma infection.

High prevalence of hepatitis B viral DNA in cirrhotic patients without surface antigen.

The diagnosis of liver diseases induced by hepatitis B virus (HBV) is supported by the detection of HBV surface antigen (HBsAg) in serum. The present study aimed to investigate the presence of HBV deoxyribonucleic acid (DNA) in patients with liver cirrhosis using a polymerase chain reaction (PCR) based on primers derived from the pre-S1 and pre-core regions. HBsAg was detected in 10 of 48 patients (21%), total anti-hepatitis B core antigen (HBc) antibodies in 54%, anti-hepatitis B e antigen (HBeAg) in 14.6%, anti-HBc immunoglobulin M in 8%, and anti-HBs in 26%; none had detectable HBeAg. HBV DNA was detected in 73% of the cirrhotic patients. All cirrhotic patients with HBsAg also had HBV DNA; HBV DNA was detected in 64.5% of those without HBsAg. We conclude that the clearance of HBsAg does not necessarily indicate termination of viraemia in patients with liver cirrhosis and the detection of HBV DNA using a PCR based on primers from the pre-S1 and pre-core regions should be included in the diagnosis of HBV infection.

Immunochemical purification and characterization of a 74.0-kDa Schistosoma mansoni antigen.

A polypeptide antigen of 74.0 kDa molecular weight was detected in the antigenic extracts of the 3 developmental stages of Schistosoma mansoni (eggs, cercariae, and adult worms) by western blotting using BRL4 monoclonal antibody (mAb) that significantly protected mice at the levels of 51.6%, 42%, and 53.8% against challenge S. mansoni infection in 3 separate experiments. This antigen was isolated and purified from crude soluble worm antigen preparation by immunoaffinity chromatography using CNBr-activated sepharose-4B beads coupled with the BRL4 mAb. The purified antigen showed a single peak when analyzed by both high-performance liquid chromatography and high-performance capillary electrophoresis. The 74-kDa antigen was characterized as a protein in nature with 56.9% hydrophilic amino acids and 43.1% hydrophobic amino acids. This antigen was detected in 93% of urine samples from infected cases with specificity of 89% among noninfected cases using an enzyme immunoassay-fast dot-enzyme-linked immunosorbent assay based on BRL4 mAb.

Detection of p53 protein overexpression and DNA ploidy analysis in colon cancer.

BACKGROUND/AIMS: This study was designed to demonstrate the accumulation of the mutant p53 protein in human neoplasms. The correlation of flow cytometric DNA ploidy pattern with p53 expression using the immunoblotting technique was also investigated. METHODOLOGY: In this study, the occurrence of p53 overexpression was analyzed in 34 cases of adenocarcinoma of the colon by western immunoblotting technique, using an anti-human p53 monoclonal antibody (Do-7). The nuclear protein extract from human colon tumor specimens was immunoblotted relative to protein standards of known molecular weight. Flow cytometric analysis was used to study the DNA ploidy pattern of the tumor cells. RESULTS: Monoclonal antibody p53-Do 7 detected a single band of 53 KDa in 70.5% (24 of 34) of the tumor specimens examined. Whereas, no bands were detected in the normal colon mucosa. The relation between p53 overexpression and the clinicopathological variable (Dukes' staging) was studied and no significant difference in p53 overexpression between Dukes' stages B and C was found. Flow cytometric analysis revealed a higher incidence of DNA aneuploidy in 75% (15 of 20) of p53 positive cases compared with 64.3% (9 of 14) in the diploid tumors. CONCLUSION: The immunoblotting technique can successfully detect the mutant p53 and is therefore expected to provide valuable information on the role of p53 in the process of carcinogenesis.