The altered expression of α1 and ß3 subunits of the gamma-aminobutyric acid A receptor is related to the hepatitis C virus infection.

The modulation of the gamma-aminobutyric acid type A (GABA A) receptors activity was observed in several chronic hepatitis failures, including hepatitis C. The expression of GABA A receptor subunits α1 and ß3 was detected in peripheral blood mononuclear cells (PBMCs) originated from healthy donors. The aim of the study was to evaluate if GABA A α1 and ß3 expression can also be observed in PBMCs from chronic hepatitis C (CHC) patients and to evaluate a possible association between their expression and the course of hepatitis C virus (HCV) infection. GABA A α1- and ß3-specific mRNAs presence and a protein expression in PBMCs from healthy donors and CHC patients were screened by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. In patients, HCV RNA was determined in sera and PBMCs. It was shown that GABA A α1 and ß3 expression was significantly different in PBMCs from CHC patients and healthy donors. In comparison to healthy donors, CHC patients were found to present an increase in the expression of GABA A α1 subunit and a decrease in the expression of ß3 subunit in their PBMCs. The modulation of α1 and ß3 GABA A receptors subunits expression in PBMCs may be associated with ongoing or past HCV infection.

Expression of microRNA-155 precursor in peripheral blood mononuclear cells from Hepatitis C patients after antiviral treatment.

Chronic hepatitis caused by Hepatitis C virus (HCV) is the main source of liver cirrhosis, hepatocellular carcinoma, and extra-hepatic diseases. After treatment-induced resolution of hepatitis C, the persistence of HCV RNA in serum and peripheral blood mononuclear cells (PBMCs) is often observed. An expression of the precursor of microRNA-155 (miR-155) called BIC can be the factor responsible for a course of HCV infection. Therefore, we assessed the relationship between BIC expression and HCV RNA status in sera and PBMCs samples of 64 hepatitis C patients treated with interferon alpha(IFN-alpha)+ribavirin. High expression of BIC in PBMCs was determined in 100% of patients that harbored HCV RNA in serum and PBMCs. Further, we found that 83% of PBMCs samples were BIC-positive in a group of patients that eliminated HCV RNA only from serum. The lowest expression of BIC was found in patients that eliminated HCV RNA from both serum and PBMCs.

Various dietary fats differentially change the gene expression of neuropeptides involved in body weight regulation in rats.

Various high-fat diets are obesogenic but not to the same extent. The aim of the present study was to investigate the effects of saturated fat n-6 and n-3 polyunsaturated fatty acids (PUFAs) on the central neuropeptidergic system in adult rats. Using reverse transcriptase-polymerase chain reaction and in situ hybridisation, we evaluated the net effect of feeding in these fats, comparing the effects of a high- to low-fat diet, and the diversity of the effects of these fats in the same amount within the diet. We also determined plasma lipids, glucose, insulin and leptin concentrations. Six-week feeding with high-saturated fat evoked hyperpahagia and the largest weight gain compared to both high-PUFA diets. Rats fed high-saturated fat were found to have decreased neuropeptide Y (NPY) mRNA expression in the arcuate nucleus (ARC) and the compact zone of the dorsomedial nucleus (DMHc), unchanged pro-opiomelanocortin (POMC), galanin-like peptide (GALP) mRNA expression in the ARC, as well as melanin-concentrating hormone (MCH) and prepro-orexin (preORX) mRNA expression in the lateral hypothalamus, compared to low-saturated fed rats. By contrast, feeding with both high-PUFA diets increased POMC and GALP mRNA expression in the ARC compared to the corresponding low-fat diet and the high-saturated fat diet. Furthermore, feeding with both low-PUFA diets reduced NPY mRNA expression compared to the low-saturated fat diet exclusively in the DMHc. Uniquely, the high n-3 PUFA feeding halved MCH and preORX mRNA expression in the lateral hypothalamus compared to the other high-fat and low n-3 PUFA diets. In rats fed three high-fat diets, plasma insulin and leptin concentrations were significantly increased and the type of fat had no effect on these hormone levels. Rats fed high-saturated fat had both hyperglycaemia and hypertriacylglycerolemia and rats fed high n-3 PUFA only had hyperglycaemia. The present study demonstrates that various forms of dietary fat differentially change the expression of neuropeptide genes involved in energy homeostasis.

A low-electrophoretic-mobility H1 histone subfraction from Kirkman-Robbins hamster hepatoma.

A protein showing lower electrophoretic mobility in acidic urea polyacrylamide gels than did the usual histone H1 subfractions has been detected among the H1 histones extracted from chromatin of a transplantable hamster hepatoma, originally induced by Kirkman and Robbins. It was proved to be a true H1 histone subfraction. It differs from the remaining ones by the total chain length, amino acid composition, and isoelectric point value. It is not a phosphorylated or phosphoribosylated metabolic form of another subfraction. Its proteolytic degradation products (obtained by thrombin and trypsin digestion) closely resembled those obtained from other H1 subfractions. The investigated hepatoma seems to provide an interesting model of neoplastic cells showing a distinct difference in histone composition from the homologous normal tissue.

Occurrence of the low-mobility H1 histones subfraction in embryonic, differentiated, and neoplastic tissues of the Syrian hamster.

Electrophoretically slow H1 histone subfractions with mobilities identical to that of the subfraction found in the Kirkman-Robbins hamster hepatoma chromatin have been shown to be present in 12-day hamster embryos and in a sarcoma-type hamster tumor induced by SV40. No subfractions of such mobility were found in hamster liver, regenerating liver, thymus, spleen, and a fast-growing transplantable amelanotic hamster melanoma. A suggestion is made that some defective mechanisms of differentiation may affect the regulation of expression of the genes coding for the H1 histone subfractions. The same mechanisms may possibly but not necessarily be connected with the molecular events leading to neoplastic growth.

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity--a staphylokinase variant. I. In vitro study.

We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent - hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity. The plasminogen activation rate (determined by K(M) and K(cat)) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.

Abnormalities of the P53, MDM2, BCL2 and BAX genes in acute leukemias.

Abnormalities of the P53 network have been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The purpose of this study was to define P53 gene mutations, to detect MDM2 gene amplification and to estimate mRNA expression of P53, MDM2, BCL2 and BAX genes in patients with ALL and AML. Twenty-five patients with ALL and 65 patients with AML, both recently diagnosed, were included into this study. Exons 5-8 of the P53 gene with flanking intronic sequence were amplified by the polymerase chain reaction (PCR) method and subjected to mutation screening by single-strand conformation polymorphism analysis (SSCP). Mutation of the P53 gene was found in one patient of the 25 with ALL and in five patients of the 65 with AML. Sequence analysis was subsequently performed. One mutation in intronic sequence in ALL and four missense mutations and one silent nucleotide substitution in AML were identified. Amplification of MDM2 gene was detected by multiplex-PCR analysis in only one sample from patient with ALL, but was not observed in any case of AML. To gain further insight into the role of P53 network in the evolution of acute leukemias, the P53, MDM2, BCL2 and BAX mRNAexpressions in portion samples from patients with ALL and AML were analyzed using multiplex RT-PCR. Although a low frequency of molecular disturbances of the P53 and the MDM2 genes was detected in this study, there was a high percentage of cases with increased mRNA level of P53 and MDM2. A high frequency of BCL2 mRNA overexpression and a relatively low frequency of BAX mRNA overexpression detected in both analyzed leukemias in this study, indicate that altered transcription of these genes may be involved in leukemogenesis.

A comparative study of P53/MDM2 genes alterations and P53/MDM2 proteins immunoreactivity in liver cirrhosis and hepatocellular carcinoma.

In the present study, the expression of P53 and MDM2 proteins were examined in specimens from a group of 20 patients (9 with primary hepatocellular carcinoma HCC and 11 with liver cirrhosis LC, linked to HBV infections as a major aetiologic factor) by immunohistochemistry. The immunostaining findings were correlated with P53 mutation analysis using PCR-SSCP, PCR-HDF and direct sequencing, and MDM2 amplification studies by differential PCR. P53 immunopositivity was found in 9 out of the 20 (45.0%) cases. Mutations of the P53 gene were detected in 5 (55%) tumors and 3 (27%) LC samples; 7 of these cases revealed P53 immunoreactivity. The mutations were base transitions at codons 175, 245 and 273; no changes were observed at codon 249, characteristic for aflatoxins action. MDM2 immunopositivity was revealed in 9 out of 20 (45.0%) specimens. MDM2 amplification occurred in 4 (44.4%) and 1 (9.1%) cases, HCC and LC specimens respectively; only in 2 tumors (10.0%), which exhibited MDM2 immunoreactivity. Overall, MDM2 positivity was not associated with MDM2 amplification in 7 out of the 20 studied samples (35.0%). Two HCC patients were found to have both gene abnormalities. Either the mutation rate of the P53 gene as well as the amplification level of the MDM2 gene was higher in HCC than in precancerous liver tissue stages. These results support the notion that besides P53 alterations, MDM2 gene deregulation seems to be an important event in hepatocarcinogenesis. Additionally, the mechanism of MDM2-mediated degradation of P53 protein, without involving stabilization and inactivation of P53 gene, should be considered for the understanding of all features of tumor progression processes.

Atypical chronic myelogenous leukemia following immunosuppressive therapy for severe aplastic anemia.

Late clonal complications of aplastic anemia (AA) such as acute leukemia, myelodysplastic syndromes or paroxysmal nocturnal hemoglobinuria have been recognized for a long time. To our knowledge, chronic myelogenous leukemia (CML) as a late complication of severe aplastic anemia has as yet not been reported. We report here a case of AA treated successfully with antilymphocytic globulin and cyclosporin in whom Ph1 negative, BCR/ABL negative CML developed 8 years after diagnosis of AA. This case of atypical, secondary CML was refractory to treatment with interferon alpha and hydroxyurea.

Richter's syndrome following cladribine therapy for chronic lymphocytic leukemia first manifested as pathologic fracture of the femur.

Richter's syndrome (RS) refers to the development of aggressive non-Hodgkin's lymphoma (NHL) during the course of chronic lymphocytic leukaemia (CCL). It occurs in approximately 3% of patients with CLL. The isolated form of this complication in bone is extremely rare and, so far, has not been described in a patient treated with cladribine (2-CdA). We report a case of CLL treated successfully with 2-CdA, where isolated diffuse large B-cell lymphoma (LBCL) developed 2 years after the diagnosis of CLL Rai II and one year after the completion of 2-CdA treatment. RS was first manifested as a pathologic fracture of the left femur. The LBCL was clonally distinct from the original CLL cells. The patient was successfully treated with CHOP and radiotherapy and obtained complete response of the LBCL.

Low-grade non-Hodgkin's lymphoma in a patient with systemic lupus erythematosus.

Coexistence of systemic lupus erythematosus (SLE) with low-grade non-Hodgkin's lymphoma (LGNHL) has been described occasionally in the literature with the potential pathogenetic role of monoclonal B CD5+/CD19+ cells. We report a case of LGNHL which developed 18 months after diagnosis of SLE. The monoclonal population of lymphocytes in the peripheral blood and bone marrow was CD5/CD19 negative but CD19/CD22 positive. The SLE responded well to treatment with prednisone and the course of the LGNHL was stable and cytotoxic treatment was not required.

16q heterozygosity loss in Wilms' tumour in children and its clinical importance.

INTRODUCTION: The loss of heterozygosity (LOH) of 16q is a structural change detected in about 20-30% of Wilms' tumour cases. Aberrations which result in deletion of 16q are also found in breast cancer, prostate cancer and liver cancer, where they are connected with a worse prognosis. The hypothesis of a bad prognosis in nephroblastomas with LOH 16q was first formulated by scientists from NWTS (National Wilms Tumor Study) on the basis of 232 cases of Wilms' tumour. However, SIOP studies (International Society of Paediatric Oncology) which included 28 cases of Wilms' tumour, did not show any clinico-pathological correlations with LOH 16q. Therefore, we aimed to evaluate the importance of LOH 16q in relation to clinico-pathological factors in a group of children, treated according to the SIOP criteria. AIMS: The aim of this work was to evaluate the frequency of LOH 16q in sporadic unilateral Wilms' tumour and to study the relationship between LOH 16q and selected patho-clinical parameters. The study comprised 66 children (31 girls and 35 boys) aged from 2 days to 13 years. METHODS: LOH 16q was studied by the examination of polymorphism of marker sequences in the region 16q24. DNA was isolated from paraffin sections of tissue for routine microscopic examination by the microdissection method. The method of study involved the amplification of polymorphic sequences from the 16q24 region by polymerase chain reaction (PCR) and separation of the products of amplification by polyacrylamide gel electrophoresis. The results were the subject of statistical analysis in relation to gender, age of child at first diagnosis, stage of clinical advancement and histological type of tumour. The connection between LOH 16q and recurrences, metastases and death, and failure free survival and absolute survival of children followed-up for over 24 months after nephrectomy were studied. RESULTS: The study revealed a lack of correlation between LOH 16q and gender, however LOH 16q was more frequent in children with Wilms' tumour aged >24 months, P<0.05. Also, LOH 16q was more frequent in tumours classified as clinical stage (CS) II or III than in CS I, P<0.05, but there were no differences in the occurrence of LOH 16q in tumours classified as CS II and CS III. We have found no correlation between LOH 16q and the histological type of tumour. However, LOH 16q has been found three times as frequently in tumours from children who died than in tumours of children who survived, P<0.0024.

Loss of heterozygosity of WT1 gene in the prognosis of sporadic Wilms' tumour in children.

The aim of this report was to evaluate the prognostic value of allele loss of the WT1 gene in children with sporadic Wilms' tumour. Allele loss of the WT1 gene was evaluated using microsatellite polymorphisms in the 3' untranslated region of WT1 in a radioactive PCR assay. The study comprised 66 children (30 girls and 36 boys), aged from 2 days to 13 years, treated for Wilms' tumour according to the SIOP-09 and PGGL scheme. We have used DNA isolated from the neoplastic versus normal kidney tissue from the paraffin embedded sections using microdissection procedure. Loss of heterozygosity (LOH) of the WT1 gene was found in 12 children (19.6%), 5 cases were non-informative. No significant correlation could be found between the LOH of WT1 gene and sex and age. Significantly more frequent occurrence of LOH in tumor in low stage of advancement and low degree of malignancy was found. However, no significant effect of LOH of WT1 gene was observed on frequency of recurrences, metastasis and deaths. Study of allele loss of the WT1 gene may be recommended in difficult cases as an additional factor useful for the diagnosis and in the assignment of the tumour to the appropriate risk group.

Microsatellite instability and expression of DNA mismatch repair genes in malignant astrocytic tumors from adult and pediatric patients.

Microsatellite instability (MSI) is used as a molecular marker for defective DNA mismatch repair (MMR) genes. We report here alterations of MSI in 15 malignant astrocytomas (WHO grade III) and glioblastomas (GBM; WHO grade IV) of pediatric patients (2 - 21 years) and 12 GBM from adults (44 - 68 years) by comparative analysis of BAT25/BAT26 loci and 10 other microsatellite markers. High-level microsatellite instability (MSI-H) occurred in 4 of the 15 pediatric cases (26.7%) and in 1 of the 12 adult GBM cases (8.3%). Low-level microsatellite instability (MSI-L) was observed in 6 pediatric cases (40%) and 8 adult GBM (66.7%). Unstable BAT-25 locus was found in 1 of the MSI-H pediatric cases. Thus, 2 unstable cases showed no instability of this marker. For BAT-26, such a discordance was even more profound: in 1 of MSI-H cases, we obtained no PCR product and the remaining 3 showed no alterations of this marker. MSH2 (Human MutS, Homologue2) protein was detected in all but 3 pediatric cases (1 highly unstable and 2 low-level unstable) and in all adult cases. MLH1 (Human MutL, Homologue 1) protein was detected in all but 2 pediatric cases (1 highly unstable and 1 low-level unstable). Thus, 2 highly unstable pediatric cases showed no detectable MLH1/MSH2 proteins. Our data support earlier observations that MSI occurs predominantly in malignant astrocytic tumors of young patients, which lends support to the hypothesis of different molecular mechanisms of pediatric brain tumors. Surprisingly, we found no significant correlation between the status of 10 microsatellite markers and that of either BAT25 or BAT26 loci or with the expression of MMR genes.

Molecular diversity of gammadelta T cells in peripheral blood from patients with B-cell chronic lymphocytic leukaemia.

To characterize circulating gammadelta T cell subpopulations in B chronic lymphocytic leukaemia patients (n=30), TCR Vgamma and Vdelta gene-segment use was analyzed by RT-PCR using a panel of subfamily-specific oligonucleotide primers. All results were compared with those obtained with specimens from healthy donors (n=10). The cells expressing Vdelta1+ TCR displayed the highest relative increase in B-CLL patients (particularly observed in 60% of cases), but Vdelta3+ T lymphocytes also expanded in leukaemic peripheral blood (10% of studied cases). Both mentioned gammadelta T cell subsets were significantly more frequent in the most severe stages of disease--Rai III+IV. The analysis of Vgamma region usage in TCR formation revealed that gammadelta T cells from B-CLL patients predominantly expressed a Vgamma9 segment (26 of 30 cases), usually linked to Cgamma1 region. It should be noticed that the dominant TCR genes expression in a 50% of healthy donors was Vdelta2+/Vgamma9+, however, Vgamma4 and Vgamma8 transcripts were also observed (2 and 3 of 10 cases, respectively). The above results indirectly indicate that gammadelta T lymphocyte expansion was driven by the oligo- or polyclonal proliferation and can reflect specific response against the autologous tumor cells.

Expression of gene encoding P65 oncofetal protein in acute and chronic leukemias.

In this study we have established conditions for p65 gene expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR). On the basis of this technique we analyzed p65 gene expression in various types of leukemia: acute myeloblastic leukemia (AML) (n=26); acute lymphoblastic leukemia (ALL) (n=26) and chronic lymphocytic leukemia (CLL) (n=40). The highest frequency of p65 gene expression was found in the patients with CLL (66%). No relationship between the expression of p65 gene and clinical stage of leukemia was observed. The lower percentage of positivity (presence of gene transcript) was seen in patients with ALL (42%) and AML (46%).

Medullary thyroid carcinoma (MTC)--clinical and molecular aspects on the basis of own experience.

In our clinic 19615 patients were operated over 25 years on for goiter. Malignant thyroid neoplasms were found in 1049 (5.3%) patients including 875 (83.4%) women and 174 (16.6%) men. Sixty two adult patients (42 women and 20 men were operated on for medullary thyroid carcinoma (MTC). Thyroid cancer was diagnosed in this group pre or intraoperatively in 44 (71%) patients and postoperatively, on histologic examination, in 18 (29%) patients. These patients were reoperated. Radical operations (total thyroidectomy with regional lymph node removal) were conducted in 43 (69.3%) patients and palliative ones in 19 (30.7%) patients. After MTC surgery, MEN 2A (MTC and an adrenal tumor) were diagnosed by means of imaging techniques (USG, CT) in 6 (9.7%) patients. All adrenal tumors were unilateral. Five of these patients were operated, and pheochromocytoma was confirmed by histopathologic examination. Two years after the MTC operation, 1 women was lost to follow-up. After a year, she was admitted to hospital for severe hypertension and died of cerebral hemorrhagia. Pheochromocytoma was revealed by autopsy. All patients were treated complementarily after the MTC operation. Different combinations of teleradiotherapy, chemotherapy and substitutive doses of levothyroxine were used. Ten (23.2%) of 43 patients operated radically were reoperated 1-3 years after the first operation due to loco-regional tumor recurrence. Radical reoperations were performed in 4 patients, and palliative ones in 6. Over a 0.5-23-year follow-up period, 26 (41.9%) patients died, including 20 of cancer, and 6 of other reasons. Four out of 36 living patients have clinical or biochemical symptoms of neoplastic disease. The follow-up period of MEN 2 patients operated on ranged from 1 to 6 years. Up to now, no tumor in the second adrenal gland has been diagnosed in any of these patients. Genetic (molecular) tests performed in 31 out of 36 living patients revealed mutations of RET gene in 4 (12.9%).

p65 and c-erbB2 genes expression in breast tumors: comparison with some histological typing, grading and clinical staging.

Using PCR technique we have analyzed p65 and c-erbB2 genes expression in 47 frozen tissue slides taken from patients diagnosed as ductal and lobular breast cancer, classified as G3, and in a limited panel of proliferative breast disease cases. Expression of p65 was generally connected with small tumor size and with absence of metastases in regional lymph nodes. We have found interdependence between p65 gene expression and negative states of lymph nodes. On the contrary, c-erbB2 expression was observed in patients with large tumors and with metastases to the regional lymph nodes. Between both genes (p65 and c-erbB2) opposite interdependence was found. No statistical dependence between estrogen/progesterone receptor levels and p65 or c-erbB2 expression were noticed. The presence of p65 expression appeared in the group of proliferating breast disease cases which were connected with higher risk of breast cancer. Lack of p65 expression accompanied cases which were classified as fibroadenoma.

A comparative study of P53/MDM2 genes alterations and P53/MDM2 proteins immunoreactivity in soft-tissue sarcomas.

In the present study, the expression of P53 and MDM2 proteins were examined in 94 soft-tissue sarcomas (35 malignant fibrohistiocytomas, 15 neurosarcomas, 14 liposarcomas, 13 leiomyosarcomas, 11 fibrosarcomas and 6 dermatofibrosarcomas) by immunohistochemistry. The immunohistochemical findings were correlated with P53 mutation analysis using PCR-SSCP, PCR-HDF and direct sequencing, and MDM2 amplification studies by differential PCR. P53 immunopositivity was found in 25 out of 94 (26.6%) cases. Alterations of the P53 gene were detected in 12 (12.8%) tumors; eight of these tumors revealed P53 immunoreactivity. A high number of P53 positive and P53 mutated tumors were histologically defined as poorly differentiated G3 (64.0% and 75.0%, respectively). MDM2 immunopositivity was revealed in 36 out of 94 (38.3%) cases. MDM2 amplification occurred in 17 tumors (18.1%); only nine of these tumors exhibited MDM2 immunoreactivity. Overall, MDM2 positivity was not associated with MDM2 amplification in 27 out of 94 tumors (28.7%). There was no significant correlation between MDM2 overexpression and histological grade. However, when the samples were stratified by immunophenotype, the majority of tumors (52.5%) with isolated MDM2 overexpression (dissociated from P53 positivity) were defined histologically as low grade (G1 + G2). These results support the notion that besides P53 alterations, MDM2 gene deregulation seems to be an important event in sarcomas evolution. Additionally, the mechanism of MDM2-mediated degradation of P53 protein, without involving stabilization and inactivation of P53 gene, should be considered for better understanding of all features of tumor progression processes.

A comparative study of epidermal growth factor receptor (EGFR) and MDM2 gene amplification and protein immunoreactivity in human glioblastomas.

The gene of epidermal growth factor receptor (EGFR) is often altered in human astrocytomas and its amplification, rearrangement and overexpression occur almost exclusively in high grade tumours (glioblastomas). MDM2 gene is amplified in a small proportion of glioblastomas, and MDM2 immunoreactivity has also been found in this group. However, the relation between gene amplification and protein overexpression depends on several factors. Thus, the study on mutual relationship between these events needs to be clarified. In a series of 28 glioblastomas, we analysed MDM2 and EGFR gene amplification by differential PCR and protein overexpression was evaluated by immunohistochemistry. Thirteen cases (45%) presented immunopositivity for EGFR. A significant amplification of EGFR gene (the EGFR/SOD ratio above the control value +/- 3 SD) was observed in 9 tumours among which, one revealed no EGFR-immunopositivity. Three tumours displayed the ratio +/- 2-3 SD but these tumours also presented immunoreactivity for EGFR. Two other glioblastomas, with weak EGFR-expression, showed no gene amplification. The immunohistochemical staining for MDM2 revealed strong positivity only in one case, and this tumour also presented MDM2 gene amplification. On the contrary, another tumour which showed MDM2 gene amplification showed no MDM2 immunopositivity. In conclusion, our results demonstrate that there is no strict correlation between gene amplification at the DNA level and protein overexpression.