The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches.

The HER family of receptor tyrosine kinase couples binding of extracellular growth factor ligands to intracellular signal transduction pathways, contributing in this fashion to the ability of the cell to respond correctly to its environment. The HER family and its ligands are critically involved in the carcinogenesis of the mammary gland. Abnormal function of the members of HER family resulting in receptor hyper-activation (due to gene amplification, protein overexpression or abnormal transcriptional regulation) has been linked with breast cancer prognosis. It is also extensively studied as the predictive factor and target for therapy. There are clinical indications supporting the concept that none of the receptors: EGFR, HER2, HER3 and HER4 can be considered as the stand-alone receptor in breast cancer development and clinical course of the disease. There is a growing body of evidence that cooperation between them contributes to more aggressive tumor phenotype and influences the response to therapy. This underlines the importance of quantification of all HER family members and indicates the urgent need for implementation of methods that can efficiently and reliably examine four HER receptors as a whole panel in breast cancer patients.

An epidermal growth factor receptor intron 1 polymorphism in healthy women in Poland.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation.

Dynamics of hepatitis B virus quasispecies heterogeneity in association with nucleos(t)ide analogue treatment determined by MALDI-TOF MS.

Minor drug-resistant variants may preexist in every subject infected with hepatitis B virus (HBV). However, understanding the dynamic of genotypic evolution within the HBV population requires accurately following allele frequencies through time. We used MALDI-TOF MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) for localization and quantitative allele frequency detection to investigate preexisting HBV quasispecies and the genotypic evolution of drug-resistant variants during nucleos(t)ide analogue therapy. We found a significant difference between the genotypic evolution of drug-resistant variants depending on response to treatment.

Coexistence of HFE and rare UGT1A1 genes mutations in patients with iron overload related liver injury.

This report describes two patients hospitalised in Hepatology Unit, Infectious Diseases Department Medical University of Gdansk because of liver damage discovered in family doctor's practice. Hereditary hemochromatosis was diagnosed in both cases. Diagnosis was established basing on medical records review, and biochemical, molecular and liver specimen tests. The analysis of polymorphism of UGT1A1 gene was done in these cases because those patients were a part of the larger study on prevalence of UGT1A1 gene mutations in patients with hereditary hemochromatosis. We discovered rare variant forms of UGT1A1 gene coexisting with HFE gene mutations.

Hepatitis delta virus infection in chronically HBV-infected patients from northern Poland.

The objective of this study was to estimate the presence of hepatitis delta virus RNA in chronically HBV-infected patients from northern Poland. Three out of 63 studied samples (4.8%) were positive in a qualitative test for total antibodies to HDV antigen. Five samples (7.9%) turned out to be HDV-RNA-positive by RT-PCR, four of them were sequenced in the region of L-HDAg, and phylogenetic analysis was performed. All four examined samples belonged to genotype I. Two RNA-positive/anti-HD-negative samples possessed a few uncommon nucleotide substitution sites within the L-HDAg sequence, which could suggest unique variants in the Polish population of HDV-infected patients.

European multicenter evaluation of high-density DNA probe arrays for detection of hepatitis B virus resistance mutations and identification of genotypes.

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 10(3) genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

An epidermal growth factor receptor intron 1 polymorphism in healthy women in Poland.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation. (Int J Biol Markers 2005; 20: 184-8).

HCV-RNA detection in liver bioptates--a comparison of automatic and 'home-made' protocols combined with a new procedure of HCV-RNA extraction.

BACKGROUND: For HCV-RNA detection in liver tissue a generally accepted reference method has not been established yet. Therefore, we have developed a procedure of HCV-RNA extraction from liver tissue and compared two methods of HCV-RNA detection. MATERIALS AND METHODS: A set of 32 liver biopsy specimens, obtained from chronically HCV infected patients, has been examined. At the time of biopsy, serum HCV-RNA was detectable in 20 patients in RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 (Roche Molecular Systems, Inc, Pleasanton, CA, USA) 2 serum samples were negative for HCV-RNA and 10 patients has not been tested. Liver tissue has been homogenized in the presence of CRSR-Green (Fast RNA Kit-Green, Bio101, Inc, Vista, CA, USA) and a mixture of phenol/chloroform/isoamylic alcohol, using a FastPrep homogenizator (Bio101, Inc, Vista, CA, USA). Then RNA has been isolated from the material obtained using the Total RNA Prep Plus procedure (A&A Biotechnology, Gdansk, Poland). Presence of HCV-RNA was next tested by means either of 'home-made' nested RT-PCR procedure or the RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0. In case of an inhibition of PCR detected in the first run of automatic assay, both PCR procedures have been repeated. RESULTS: In 5 cases of automatic assay an inhibition of PCR reaction has been detected in the first run, the RT-PCR procedures has been then successfully repeated in the second run. The presence of HCV-RNA in the liver tissues was detected in a total of 22 cases (69%) by mean of automatic assay and in a total of 20 cases (63%) by means of 'home-made' RT-PCR procedure. Except for two cases of HCV-RNA positivity in a biopsy tissue, detected by means of automatic but not 'home-made' procedure, the results obtained employing the methods have been identical. In one serum HCV-RNA positive case, the presence of HCV RNA has not been detected in a liver tissue using both methods. CONCLUSIONS: Our procedure of RNA isolation combined either with automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 or 'home-made' RT-PCR procedure may be useful for establishing presence of HCV-RNA in liver tissue. In this combination, the automatic assay seems to be more sensitive than the 'home-made' procedure.

Porphyria cutanea tarda, hepatitis C virus and selected symptoms of cholestasis.

BACKGROUND: The influence of hepatitis C virus (HCV) infection on the development of porphyria cutanea tarda (PCT) was recently detected, but the status of trace elements in etiology of the diseases is relatively poorly recognized. Individually observed symptoms of cholestasis do not seem to be related to the above conditions. MATERIAL AND METHODS: A group of 22 PCT patients (37-73 years old, mean 54.9 years), including 10 cases (45.5%) of serum HCV-RNA positive, as evaluated by automatic Cobas AmplicorTM Hepatitis C ver. 2.0 assay (Roche Diagnostics), was assessed. None of the patients have recently abused alcohol. Serum levels of copper and bilirubin, activity of gammaglutamyltranspeptidase (GGTP) and alkaline phosphatase (ALP) were measured in all patients. RESULTS: We have found statistically significant differences between both mean serum activity of GGTP (145.9 U/l vs. 58.3 U/l, p = 0.004) and mean serum copper levels (131.9 mg/dl vs. 79 mg/dl, p = 0.003) in patients infected with HCV compared to the other PCT patients, respectively. Mean serum ALP activity was also higher, but the difference was not of statistical importance (106.9 U/l vs. 90.9 U/l, p = 0.24); mean serum bilirubin concentration was significantly lower in HCV-infected patients (0.55 mg/dl vs. 0.85 mg/dl, respectively, p = 0.035). CONCLUSION: In our group of PCT patients we have noticed a higher serum copper level and higher activity of both GGTP and alkaline phosphatase, but lower level of serum bilirubin in patients infected with HCV as compared with those not infected. This observation does not confirm suggestions concerning the role of HCV in promotion of cholestatic liver disease in PCT patients. The influence of HCV infection on the metabolism of copper and GGTP activity is connected rather with the inflammatory lesions in the liver of PCT patients infected with HCV. Bilirubin metabolism in PCT patients requires further studies for its elucidation.

Influence of hepatitis C virus (HCV) infection on porphyrin and iron metabolism in porphyria cutanea tarda (PCT) patients.

BACKGROUND: Porphyria Cutanea Tarda (PCT) is the most common form of porphyria. It is characterised by lowered activity of uroporphyrinogen decarboxylase. It seems possible that the hepatitis C virus (HCV) infection triggers the symptoms of PCT. MATERIAL AND METHODS: A group of 29 PCT patients (33-73 years old, mean 54 years) was evaluated. In these patients the serum HCV-RNA (by mean of automatic Cobas AmplicorTM Hepatitis C ver. 2.0 assay, Roche Diagnostics), activity of transaminases (ALT, AST), serum protein fractions, iron, total iron binding capacity (TIBC), ferritin, transferrin, transferrin saturation, and urinary porphyrins excretion were assessed. RESULTS: The presence of HCV-RNA was detected in 12 cases (41.4%). Statistically important differences between currently HCV infected patients and other PCT patients concerned the following parameters: mean body mass (68.5 vs. 78.0 kg, p = 0.02), mean body mass index (22.4 vs. 26.8 kg/m2, p = 0.007), mean serum activities of ALT (112.6 vs. 65.4 U/l, p = 0.02) and AST (85.0 vs. 56.5 U/l, p = 0.038), total serum protein concentration (78.0 vs. 71.6 g/l, p = 0.016) and serum alpha-2 protein concentration (7.2 vs. 5.5 g/l, p = 0.009), respectively. Some other not significant differences were also found: mean serum iron level 182 vs. 169 micrograms/dl, transferrin concentration 2.77 vs. 2.56 g/l, transferrin saturation 43 vs. 45%, serum TIBC 445 vs. 397 mg/dl and ferritin concentration 394 vs. 400 micrograms/l, respectively. The mean urinary uroporphyrin concentrations were 643 micrograms/l vs. 366 micrograms/l (p = 0.16) and those of coproporphyrins were 190 micrograms/l vs. 136 micrograms/l (p = 0.37), respectively. CONCLUSION: In the group of PCT patients investigated no significant relationship between current HCV infection and iron or porphyrin metabolism was noted. Nevertheless, HCV infection significantly influences the symptoms of liver damage and seems to influence other metabolic parameters in PCT patients.