Electronic Library of Scientific Literature


Volume 50 / No. 6 / 2003

Involvement of the nucleotide excision repair proteins in the removal of oxidative DNA base damage in mammalian cells.

Rybanska I, Pirsel M.

Laboratory of Molecular Genetics; Cancer Research Institute, Slovak Academy of Sciences, Bratislava, 833 91 Slovak Republic. ivana.rybanska@savba.sk

Oxidative DNA base damage produced primarily by reactive oxygen species is assumed to be the most important endogenous damage. Lack of its repair may contribute to mutagenesis, carcinogenesis and aging. It is supposed that most oxidative DNA base damage is removed by the base excision repair pathway; although it was shown recently that other DNA repair pathways could be involved. This review is focused on the role of nucleotide excision repair (NER) and transcription-coupled repair (TCR) in the removal of oxidative DNA base damage in mammalian cells.

Neoplasma. 2003; 50(6): 389-395.

Paget's disease of the nipple: a copy number of the genes ERBB2 and CCND1 versus expression of the proteins ERBB-2 and cyclin D1.

Mrhalova M, Kodet R.

Department of Pathology and Molecular Medicine; 2nd School of Medicine, Charles University in Prague, Prague, 150 06 Czech Republic. marcela.mrhalova@lfmotol.cuni.cz

Paget's disease of the nipple (PDN) is characterized by Paget's cells infiltrating from an underlying breast carcinoma into the epidermis of the nipple. The tumor cells were reported to overexpress ERBB-2 protein. There is no evidence, whether the overexpression is caused by amplification of the ERBB2 gene (as is the case of invasive duct carcinomas) or by other causes. Because the Paget's cells proliferate vividly we were interested also in cyclin D1 expression and in its relation to the number of CCND1 gene copies. To address these issues, we performed a study using fluorescence in situ hybridization on interphasic nuclei (I-FISH) on formalin fixed / paraffin embedded tissue sections from twelve women with PDN. We compared immunohistochemical (IHC) expression of the ERBB-2 protein and cyclin D1 with the copy number of the gene ERBB2 and CCND1, respectively (I-FISH). In all cases of our series we found overexpression of the ERBB-2 protein (3+), and in all of them there was a strong amplification of the ERBB2gene (more than 10 signals per a nucleus, usually about 20). Keratinocytes of the epidermis had two signals of the gene. IHC and I-FISH revealed comparable findings in successive biopsies in two patients. Cyclin D1 was positive in seven cases. We found a moderate amplification of the CCND1 gene (up to 10 signals per a nucleus) in three patients only. Any correlation between the cyclin D1 overexpression anda copy number of the CCND1 gene was observed. Combined numerical changes of chromosome 17 and of chromosome 11 were found in seven women. It is concluded that in PDN, the ERBB-2 protein overexpression is caused by amplification of the ERBB2 gene. A specific immuno-therapy with trastuzumab (Herceptin) used in patients with invasive duct carcinoma may be also effective in patients with PDN.

Neoplasma. 2003; 50(6): 396-402.

The novel exon 11 mutation of BRCA1 gene in a high-risk family.

Ciernikova S, Tomka M, Sedlakova O, Reinerova M, Stevurkova V, Kovac M, Cente M, Ilencikova D, Bella V, Zajac V.

Cancer Research Institute; Slovak Academy of Sciences, Bratislava, 833 91 Slovak Republic. exonvzaj@savba.sk

Germline mutations in the BRCA1 and BRCA2 genes are required for the initiation of the development of hereditary forms of breast and ovarian cancer, which represent 10-15% of all cases. The course of the disease varies from case to case that can be due even to the possibility of multiple genetic changes including inactivation of other tumor suppressor genes - TP53 and APC genes or activation of oncogenes, especially K-ras oncogene. The combination of these changes results in an early expression of the broad variety of malignancies. The analyzed proband (II-5) comes from a high-risk family, in which various types of cancer were observed. The novel BRCA1 mutation in exon 11 (2057delCAGTGAAGAG) was detected by SSCP, HDA techniques and confirmed by automatic sequencing. Thesame deletion was observed in DNA sample of her first daughter (III-1), but DNA of her second one was without any mutational changes (III-2). Due to the occurrence of different types of cancer in this family, the incidental mutations in the APC; resp. TP53 tumor supressor genes and K-ras oncogene were searched as well. Any mutation was found after sequencing of SSCP interesting exons of these genes. The reasons for such strong malignant manifestation in this high risk family are discussed.

Neoplasma. 2003; 50(6): 403-407.

Argyrophilic nucleolar organizer regions (AgNORs) in relation to p53 and bcl-2 protein expression in leukemia patients.

Klobusicka M, Kusenda J, Babusikova O.

Cancer Research Institute; Slovak Academy of Sciences, Bratislava, 833 91 Slovak Republic. exonklo@savba.sk

The aim of this study was to assess the possible relationship between the silver stained nucleolar organizer regions (AgNOR) and immunocytochemically detected p53 and bcl-2 proteins in ALL, AML, B-CLL and CML patients (adults and children) at the initial presentation. AgNORs are loops of DNA, correlated with proliferative potential of cells. Alteration in p53 and bcl-2 proteins expression may characterize the malignant potential of leukemic cells. The patients were subdivided according to the p53 positivity and negativity. The frequency of p53-positive patients was relatively low in T-ALL (29%) and B-CLL (16%). B-ALL, AML and CML patients revealed higher frequency of p53 protein (46%, 47% and 88%, respectively). The overall frequency of positive cytoplasmic staining for bcl-2 protein was demonstrated in the majority of patients. No significant differences in the percentage of p53-positive cells among leukemia subtypes were seen. The proportion of bcl-2 protein positive cells did not differ significantly among various leukemia subtypes, except for significant differences between p53-positive and p53-negative peripheral blood (p=0.0073) and bone marrow (p=0.0175) cells of B-CLL patients. The samples from healthy subjects used as controls exhibited relatively low numbers of AgNOR dots in both, peripheral blood and bone marrow cells. Highly significant differences in AgNOR quantities between healthy donors and p53 protein positive peripheral blood as well as bone marrow cells of distinct leukemia subtypes (except for bone marrow cells in B-CLL patients, p=0.1727) were observed. Significant differences in AgNOR count between p53 protein positive and p53 protein negative samples of peripheral blood cells of B-ALL (p=0.0099) as well as B-CLL (p=0.0117) cases were found. No significant differences (except for B-CLL, p=0.0558) were encountered in bone marrow cells. P53 protein positivity or negativity did not influence the amount of AgNOR proteins in cells of our T-ALL and AML cases. Mutual comparing the number of AgNOR dots among different leukemias showed that for both peripheral blood and bone marrow cells the differences between ALL and AML (p=0.0383 and p=0.0033, respectively) as well as for peripheral blood of AML and CML (p=0.0302) were statistically significant. The bcl-2 protein positivity did not affect significantly the AgNOR distribution either in p53 protein positive or p53-negative cases of our leukemia patients. However, an association between the lowest AgNOR quantity and highest bcl-2 protein expression in p53-negative B-CLL patients was seen for both peripheral blood and bone marrow cells. The correlation between relatively high AgNOR numbers and relatively increased percentage of bcl-2 protein in the p53-positive cases of CML patients was found in some cases. Regarding the age and sex, the AgNOR distribution in p53-positive and p53-negative leukemia cases in children and adults showed neither relationship nor dependence. The WBC count differed evidently among distinct leukemia subtypes, with enormous heterogeneity in range as well. Larger studies are needed in order to consolidate these preliminary results and characterize the possible prognostic value of AgNOR in association with p53 and bcl-2 proteins expression.

Neoplasma. 2003; 50(6): 408-415.

Multiparametric flow cytometry in detection of minimal residual disease in acute lymphoblastic leukemia of early B-cell phenotype.

Mlcakova A, Babusikova O.

Cancer Research Institute; Slovak Academy of Sciences, Bratislava, 833 91 Slovak Republic. andrea.tomova@savba.sk

Leukemic cells can be distinguished from normal hematopoietic cells on the basis of chromosomal or molecular abnormalities, antigen receptor gene rearrangements and immunophenotype. Set of 3-, 4-combination of monoclonal antibodies was used for exact definition of immunophenotypic characteristics of B-cells populations from healthy donors and aberrant, asynchronous, over/under-expressed phenotypes and detection changes in intensity expression of markers that characterized pathological leukemic B-cells at diagnosis. These differences in normal and abnormal cell patterns were very important and could be utilized for analysis of minimal residual disease. On the basis of these findings we were able to clearly distinguish residual leukemic cells from hematogones (healthy B-lymphocyte precursors) too. We also verified that in some cases the CD58 marker is overexpressed on CD10+, CD34+ blast cells at diagnosis and can be feasible used for detection of minimal residual disease (MRD).

Neoplasma. 2003; 50(6): 416-421.

Leukemic cells and aberrant phenotypes in acute leukemia patients: a flow cytometry analysis.

Voskova D, Valekova L, Fedorova J, Hudecek J, Kubisz P.

Clinic of Hematology and Transfusiology; Faculty Hospital of Martin and Jessenius Medical Faculty, Comenius University, Martin, Slovak Republic. Peter.Kubisz@jfmed.uniba.sk

The aim of this study was to evaluate the heterogeneity of immunophenotype features in acute leukemia patients and to detect the presence of leukemia-associated immunophenotypes. We prospectively investigated the phenotype of blast cells from 44 adult acute leukemia patients using a large panel of monoclonal antibodies by multiparametric flow cytometry. Thirty-three patients were classified as AML according to the FAB classification. Eleven patients were diagnosed as ALL (10 cases B-ALL, 1 case T-ALL) according to both FAB and immunnophenotyping. We found leukemia-associated phenotypes in 28 of 33 AML patients (84.8%) and in 8 of 11 ALL patients (72.7%). In 61.1% of patients more than one aberrant phenotype was observed. Linear infidelity was the most frequent aberrancy in both AML (64.3%) and ALL (37.5%) subgroups. The present study shows that MFC is a helpful method for sufficient identification of leukemic cells and for determina- tion of blast cells immunophenotype heterogeneity. The double stain flow cytometry in our study revealed aberrant phenotypes in up to 81.8% patients.

Neoplasma. 2003; 50(6): 422-427.

NMU-induced mammary carcinogenesis in female rats is influenced by repeated psychoemotional stress.

Adamekova E, Markova M, Kubatka P, Bojkova B, Ahlers I, Ahlersova E.

Department of Animal Physiology, Institute of Biological and Ecological Sciences; Faculty of Science, P.J.Safarik University, Kosice, 041 67 Slovak Republic. iahlers@kosice.upjs.sk

Epidemiological and experimental studies indicate psychoemotional stress as an important factor in carcinogenesis. The aim of this study was to evaluate the effect of restraint stress on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis. Female Sprague-Dawley rats were injected with two intraperitoneal NMU doses each per 50 mg/kg b.w. between 39-49 postnatal days. Three experimental groups were created: 1. NMU (without restraint - control group, 12 animals), 2. NMU+1IMS (group with single restraint, IMS - immobilization stress, 12 animals), 3. NMU+7IMS (group restrained 7 times during aweek, 12 animals). Animals were immobilized daily in special boxes for 120 minutes or 7x120 minutes, respectively from third day after carcinogen administration. The observation lasted for 20 weeks. The incidence, frequency, latency and volume of mammary tumors were evaluated. In repeatedly immobilized group NMU+7IMS increase in tumor incidence by 57% (p<0.05), marked increase in frequency per group by 153% (p<0.01), increase in frequency per animal by 61% and shortened latency period by 7 days were recorded. The effect of single restraint was not seen. In this experiment repeated immobilization carried out early after carcinogen administration had a remarkable stimulatory effect on chemically-induced mammary carcinogenesis in female rats.

Neoplasma. 2003; 50(6): 428-432.

Fludarabine combined with cyclophosphamid is highly effective in the treatment of chronic lymphocytic leukemia.

Tothova E, Kafkova A, Fricova M, Guman T, Stecova N.

Department of Hematology; UPJS and Faculty Hospital, Kosice, Slovak Republic. etothova@post.sk

Combined treatment of fludarabine (FLU) with cyclophosphamide (CY) may increase the complete remission (CR) rate, decreased minimal residual disease (MRD) and, possibly, prolong survival in B-chronic lymphocytic leukemia patient's (B-CLL). The aim of study was to evaluate the activity and toxicity of FLU in combination with CY, the FLU-CY schedule, in patients with previously untreated B-CLL. From May 1999 to December 2002, 57 patients with advanced or progressive B-CLL received treatment with FLU at a dose of 30 mg/m(2) for three consecutive days and CY at a dose of 300mg/m(2) for three days. The cycles were repeated at four week intervals or longer if severe myelosupression occurred. Guidelines for the evalution of response and toxicity were those developed by the National Cancer Institute Sponsored Working Group. Minimal residual disease (MRD) was detected by immunophenotyping only in patients with CR by standard criteria. In the analyzed group an overall response (OR) rate (CR+PR) of 89.5% (95% CI 80.6-94.7%) was achieved, including complete response in 29.8%. At the time of analysis 15 of 17 patients with CR are still in remission. Median duration of follow up in these is 12 (range 4-29.2) months. MRD was detected only in five out of 17(29.4%) patients with CR. Grade III/IV thrombocytopenia was seen in 3 (5.2%) patients and grade III/IV neutropenia in 6 (10.5%). Severe infections were noted in 14 (24%) patients. Two (3.5%) patients died, one due to sepsis, one as a result of disease progression. The FLU-CY regimen is highly effective combination in previously untreated CLL patients with acceptable toxicity. The efficacy of the regimen seems to be higher than that observed earlier after treatment with FLU alone.

Neoplasma. 2003; 50(6): 433-437.

The clinical characteristics and the role of surgery and imatinib treatment in patients with liver metastases from c-Kit positive gastrointestinal stromal tumors (GIST).

Rutkowski P, Nyckowski P, Grzesiakowska U, Nowecki ZI, Nasierowska-Guttmejer A, Pienkowski A, Dudek K, Krawczyk M, Ruka W.

Department of Soft Tissue/Bone Sarcoma; M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, 02-781 Poland. rutkowskip@coi.waw.pl

The purpose of this study was to analyze the clinical features of the group of c-KIT positive GIST patients with liver metastases evaluated and treated in two referral institutions as well as to attempt to define the role of surgery in the management of GIST given the emergence to imatinib as an important part of treatment strategy in GIST patients. Between August 2001 and December 2002, 90 patients with c-KIT positive GIST were referred to our institutions. In 50 patients metastatic disease were disclosed. Of these, 35 patients (35/50; 70%) were rendered to have liver metastases and therefore offered imatinib or surgical therapy depend on CT assessment. The median follow-up of these 35 patients calculated from the time of first operation was 23 months (range 3-246 months). Male patients comprised the majority of patients (70%) with liver metastases. In 14 patients (40%) the metastases were confined only to the liver, in the others 21 patients (60%) the liver metastases were accompanied by intraperitoneal dissemination (17; 48.6%) or local recurrences (4; 11.4%). The period of time between the diagnosis of primary lesion and occurring liver metastases ranged from 0 to 164 months (median time of liver metastases presentation was 16 months for patients undergone primary curative surgery). The liver metastases were estimated as resectable in 3 cases (8.6%) and hepatic resection of all gross lesions was possible. Group of 32 patients with unresectable liver involvement was considered to treatment with imatinib. The median time of imatinib treatment for survivors is 7.5 months (range: 3.5-18.5 months). Twelve patients (37.5%) demonstrated partial response (PR) and 16 patients (50%) stable disease (SD) according to RECIST criteria. We did not observe any complete response (CR). At median follow-up 7 months, 32 of 35 patients (91.4%) were alive, 3 patients (8.6%)remained free of disease and 28 patients (87.5%) remained on imatinib treatment and have maintained disease although with partial response or stabilization only. Radical surgical resection remains the only possibility of cure for GIST patients because the complete response after imatinib therapy is restricted to a few patients only. However, despite the advanced metastatic disease, approximately 90% of patients are alive and continue imatinib treatment with median follow-up time more than 7 months. Surgery in combination with adjuvant imatinib treatment may result in improved survival with patients with advanced GIST.

Neoplasma. 2003; 50(6): 438-442.

Effects of flavonoids on glutathione and glutathione-related enzymes in cisplatin-treated L1210 leukemia cells.

Cipak L, Berczeliova E, Paulikova H.

Cancer Research Institute; Slovak Academy of Sciences, Bratislava, 833 91 Slovak Republic. exoncip@savba.sk

Connections between the ability of quercetin (Qu)and galangin (Ga) to differentially modulate cis-Pt-induced apoptosis and their effects on glutathione system of murine L1210 leukemia cells were studied. The results showed that total glutathione (GSHt) level is increased significantly (cca 123% of control level), both in cells treated with 10 microM Qu and in cells treated with 4 microM cis-Pt and 10 microM Qu in combination. 10 microM Ga had no effect on GSHt content. Activities of glutathione S-transferase (GST) and glutathione reductase (GR) were not changed significantly when 10 microM flavonoids were used. Significant inhibition of GR activity was observed when flavonoids were used in concentrations higher than 25 microM.The presented data indicate that Qu change the redox state of the cells that is implicated in regulation of apoptosis, due to its ability to increase the GSHt level, and thus may potentiate cis-Pt-induced apoptosis of L1210 cells.

Neoplasma. 2003; 50(6): 443-446.

Serum thrombopoietin levels in patients with reactive thrombocytosis due to lung cancer and in patients with essential thrombocythemia.

Werynska B, Ramlau R, Podolak-Dawidziak M, Jankowska R, Prajs I, Usnarska-Zubkiewicz L, Kuliczkowski K.

Department of Pulmonary Diseases; Medical University of Wroclaw, Wroclaw, Poland. 1111@hemat.am.wroc.pl

In patients with thrombocytosis normal to increased serum thrombopoietin (TPO) levels havebeen reported. Theaim of this study was to investigate the relationship between serum TPO concentration, platelet number and plasma levels of fibrinogen in patients with reactive thrombocytosis (RT) due to lung cancer and in patients with essential (primary) thrombocythemia (ET). A total of 70 newly diagnosed patients with RT or ET (platelet counts > 600 G/l) were studied: 45 with RT due to lung cancer (25 with non-small cell lung cancer, NSCLC and 20 with small cell lung cancer, SCLC), and 25 with ET. Twenty normal volunteers were used as controls. TPO was measured by immunoassay technique (ELISA). Mean serum TPO values in patients with RT due to lung cancer were statistically significantly higher than those in patients with ET or in controls. The highest platelet count was seen in patients with ET, and mean plasma fibrinogen levels were the highest in RT patients. In NSCLC patients mean serum TPO concentrations were higher (not statistically significant) than in SCLC, and a statistically significant relationship between TPO serum concentration and fibrinogen level was observed. No correlations between platelet counts and TPO serum concentrations were found. Our results indicate increased serum TPO levels in patients with thrombocytosis in lung cancer which may be related to the activity of neoplasms. In addition, it is postulated that the relatively low TPO values in patients with ET may result from a dysregulation of the feedback loop involved in platelet production.

Neoplasma. 2003; 50(6): 447-451.

Hemoglobin as a factor influencing the outcome in inoperable oropharyngeal carcinoma treated by concomitant radiochemotherapy.

Oblak I, Strojan P, Zakotnik B, Budihna M, Smid L.

Department of Radiation Oncology; Institute of Oncology, Ljubljana, SI-1000 Slovenia. pstrojan@onko-i.si

The aim of the study was to analyze the prognostic significance of hemoglobin (Hb) concentration for loco-regional control and survival of patients with inoperable carcinoma of the oropharynx. Seventy patients with inoperable squamous cell carcinoma of the oropharynx were prospectively treated by concomitant regimen of conventional radiotherapy and chemotherapy with Mitomycin C and Bleomycin. The prognostic value of Hb concentration before the therapy (Hb-S) and at the end of the therapy (Hb-E), the difference between both (DHb), and the average Hb concentration (Hb-Av) were analyzed. Hb concentration was falling significantly (median values, from 139 g/L to p<0.0001) during the first three weeks of the therapy; after that, it reached a plateau. In the last week of therapy, a slight increase (p=0.08) in Hb concentration was recorded. Significant correlation (p<0.0001) was found between Hb-S and other Hb-related parameters. The median follow-up of the patients alive on close-out date was 5.7 years (range 4-10.5 years). Longer disease-free survival (DFS) and disease-specific survival (DSS) correlated with higher values of Hb-S (p=0.0005, p=0.008) and Hb-E (p=0.02, p=0.02), while the Hb-Av was predictive for DFS only (p=0.004). The most significant difference between low- and high-Hb groups was calculated at cut-off concentrations of 122 (Hb-S), 116 (Hb-E), and 120 (Hb-Av) g/L. Only Hb-S was tested in multi- variate model where its independent value for predicting both, DFS (p=0.002; RR 3.6) and DSS (p=0.01; RR 2.9), was confirmed. In our patients, Hb-Swas proved to bean independent prognostic factor in predicting DFS and DSS. We believe that the concentration of Hb >/=120 g/L should be maintained during radiotherapy course.

Neoplasma. 2003; 50(6): 452-458.

Lens-sparing external beam radiotherapy of intraocular metastases: our experiences with twenty four eyes.

Bajcsay A, Kontra G, Recsan Z, Toth J, Fodor J.

Department of Radiotherapy; National Institute of Oncology, Budapest, H-1122 Hungary. bajcsay@oncol.hu

Occurrence of uveal metastases is higher, than the number of clinically diagnosed cases, furthermore all cases are not amenable to therapy. Treatment of primary cancer is permanently improving, as a result life prospective is better, with an increasing number of late distant metastases in an unusual location, as e.g. intraocular metastasis. As surgical approach is not suitable, and chemo/hormonal therapy often has a limited effect on intraocular dissemination, other treatment modalities are needed for the maintenance of visual acuity, and prevention of further deterioration of the quality of life. The study was made to evaluate the efficacy of external beam radiotherapy (EBRT) with lens-sparing techniques in the management of patients developing intraocular metastases (IOM). Between March 1994 and March 2002, 24 eyes of 17 patients with tumors metastatic to the eye were treated by EBRT. The female:male ratio was 8.5:1, age ranged between 37 and 74 years (mean: 56 years). The site of the primary tumor was: breast (11), lung (4) and others (2). The visual acuity at the beginning of irradiation was between 0.1-0.7 (mean 0.5) and a mean KPS of 60% was detected. The mean time elapsed from the diagnosis of primary tumor and recognition of metastasis was 38.9 months in case of breast, and 6.7 months in lung cancer. Eyes were treated by 6 MV photon beams, using a modified technique of Schipper's lens-sparing retinoblastoma treatment method. The following parameters were studied: visual acuity changes, local response rate, survival times from irradiation and ocular complications. Mean follow-up time was 24 months. Mean visual acuity improved two lines on the Snellen chart. The mean survival time after treatment of IOM was 21 months in breast and 4.9 months in lung cancer patients. Local response rate was 78%. No radiation cataract was observed. Only one patient developed radiation retinopathy 32 months after the treatment. External beam radiotherapy is recommended for the treatment of intraocular metastases to improve quality of life. In selected cases - especially breast cancer patients - lens-sparing technique is the treatment of choice.

Neoplasma. 2003; 50(6): 459-464.

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