Electronic Library of Scientific Literature


Volume 50 / No. 5 / 2003

Artemis, a novel guardian of the genome.

Dudasova Z, Chovanec M.

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

B and T lymphocytes recognize foreign antigen through specialized receptors: the immunoglobulins and the T cell receptors, respectively. The highly polymorphic antigen-recognition regions of these receptors are composed of variable (V), diversity (D), and joining (J) gene segments that undergo somatic rearrangement prior to their expression by the V(D)J recombination process. Proper joining of the V, D, and J segments requires the participation of the Rag proteins as well as the non-homologous end-joining (NHEJ) factors. Recently, a novel V(D)J recombination/NHEJ factor, Artemis, has been identified. Mutations in the ARTEMIS gene cause human severe combined immunodeficiency with increased radiosensitivity (RS-SCID), an autosomal recessive disease characterized by the absence of the T and B lymphocytes and by a defect in the V(D)J recombination. This minireview compiles all mutations in the ARTEMIS gene identified so far. Furthermore, phenotypes of RS-SCID patients and links to the particular mutations are described. Biochemical and structural properties of the Artemis proteins are reviewed and integrated into the processes of V(D)J recombination and NHEJ. A genomic caretaker function is assigned to Artemis.

Neoplasma. 2003; 50(5): 311-318.

Relationship between K-ras mutation and the expression of p21WAF1/CIP1 and p53 in chronic pancreatitis and pancreatic adenocarcinoma.

Hermanova M, Lukas Z, Kroupova I, Kleibl Z, Novotny J, Nenutil R, Pazourkova M, Brazdil J, Kren L, Dite P.

Department of Pathology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno, Brno, Czech Republic. marherman@post.cz

Overexpression of p21WAF1/CIP1 was recently described as an early event in the development of pancreatic intraepithelial neoplasia. Since activating K-ras mutations are described in more than 80% of pancreatic cancers and are known to increase intracellular levels of p21WAF1/CIP1 in experimental models, the possible role of activating K-ras mutations in an induction of the p21WAF1/CIP1 expression was investigated in our study. We examined 71 surgical specimens, 29 of chronic pancreatitis and 42 of invasive ductal adenocarcinoma both having a large spectrum of PanIN (pancreatic intraepithelial neoplasia) lesions. Expression of p53 and p21WAF1/CIP1 was examined immunohistochemically and codon 12 K-ras mutational analysis was performed using the very sensitive mutant-enriched PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis. Our study demonstrated the overexpression of p21WAF1/CIP1 as an early event in the development of pancreatic intraepithelial neoplasia in the group of chronic pancreatitis and invasive adenocarcinoma as well. Overexpression of p21WAF1/CIP1 increased progressively from normal ducts through the spectrum of PanIN lesions to invasive carcinomas. The p53 overexpression increased again progressively according to the severity of the lesion and seems to be a later event in the development of pancreatic intraepithelial neoplasia if compared to p21WAF1/CIP1 expression. Our results confirmed also the possible p53 independent p21WAF1/CIP1 expression in some PanIN2, PanIN3 lesions and invasive carcinomas. K-ras mutations were not revealed in samples with only low grade PanIN lesions (PanIN1a and PanIN1b). K-ras mutations were detected in 69,4% adenocarcinomas and in only one case of chronic pancreatitis. Two codon 12 K-ras positive pancreatic carcinomas showed K-ras mutations in the surrounding normal pancreatic tissue. In adenocarcinomas, no statistically significant correlation was found between K-ras mutational status and p21WAF1/CIP1 and p53 expression, respectively. The possible role of activating K-ras mutations in an induction of p21WAF1/CIP1 expression was not confirmed in this study.

Neoplasma. 2003; 50(5): 319-325.

Comparison of differential PCR and immunohistochemistry for the evaluation of c-erbB-2 in breast carcinoma and relationship to other prognostic factors.

Erdokan S, Ergin M, Tuncer I.

Department of Pathology, School of Medicine; Cukurova University, 1330 Adana, Turkey. seydaer@yahoo.com

The c-erbB-2 gene codes for a membrane receptor protein that is homologous to the epidermal growth factor receptor. Differential polymerase chain reaction (PCR) is an alternative semi-quantitative method for evaluating gene amplification and can be performed in formalin-fixed paraffin-embedded specimens. We aimed to compare differential PCR and IHC (immunohistochemistry) in the determination of c-erbB-2 status of breast cancers. Correlation between the prognostic impact of c-erbB-2 gene amplification and protein overexpression with conventional prognostic factors were also evaluated. Differential PCR and IHC for c-erbB-2 were performed on formalin-fixed paraffin sections of 60 invasive breast cancers. Results and the relation with the other prognostic parameters were compared. A highly significant degree of concordance between differential PCR and IHC in the evaluation of c-erbB-2 status of breast carcinoma was detected. Amplification and overexpression were significantly related to the number of metastatic lymph nodes, histologic grade, and lymphatic invasion but not age, histologic type, tumor size and estrogen status. We demonstrated and confirmed the importance of c-erbB-2 overexpression and amplification as a single and combined prognostic parameter together with conventional factors and confirmed that it can be detected by both immunohistochemistry and differential PCR techniques in breast carcinoma. This semi-quantitative technique provides reliable results and can be used routinely.

Neoplasma. 2003;50(5): 326-330.

Binding analysis of HLA-G specific antibodies to hematopoietic cells isolated from leukemia patients.

Polakova K, Bandzuchova E, Hofmeister V, Weiss EH, Hutter H, Russ G.

Cancer Research Institute; Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic.

Expression of HLA-G on the surface of malignant hematopoietic cells isolated from leukemia patients was analyzed by flow cytometry using monoclonal antibodies (mAbs) recognizing both, intact HLA-G complex (87G, 01G and MEM-G9) as well as HLA-G free heavy chain (4H84, MEM-G/1 and MEM-G/2). Prerequisite of HLA-G detection by mAbs specific to free heavy chain was mild acid treatment, which dissociates intact HLA-G complex. All mAbs, with the exception of 4H84 mAb, did not indicate the presence of HLA-G antigen in leukemia cells. Positive staining with 4H84 mAb was detected in acid-treated cells isolated from 16 out of 30 patients. Intensity of staining increased after IFN-g pre-incubation in most cases. Immunoblot analyses and RT-PCR, however, failed to detect HLA-G antigen or HLA-G transcripts in cells that bind 4H84 mAb after acid-treatment. The binding of 4H84 mAb can be explained by the acid-induced cross-reactivity of this HLA-G specific mAb with classical HLA class I molecules [15]. The results described here further demonstrate that the HLA-G molecule is not expressed in freshly isolated human leukemia cells.

Neoplasma. 2003; 50(5): 331-338.

The rate of apoptosis and expression of Bcl-2 and Bax in leukocytes of acute myeloblastic leukemia patients.

Dabrowska M, Pietruczuk M, Kostecka I, Suchowierska M, Kloczko J, Nasilowska B, Bany-Laszewicz U, Marianska B.

Department of Hematological Diagnostics; Medical University of Bialystok, 15-274 Bialystok, Poland. mdabrows@amb.edu.pl

The rate of apoptosis as well as expression of Bcl-2 and Bax was evaluated before and after induction therapy in leukocytes of 70 patients with acute myeloblastic leukemia (AML), retrospectively divided into group A (with longer survival) and group B (with shorter survival). We found, that leukocytes of untreated AML patients showed susceptibility to apoptosis similar to control cells. Marked increase in percentage of apoptotic leukocytes was observed after induction therapy exclusively in patients with longer survival, which was accompanied by better normalization of routine hematological parameters. In this group, the Bcl-2/Bax ratio was similar to the control and remained unchanged after treatment. In AML patients with shorter survival, a twofold increase in this ratio was observed both before and after the completion of induction therapy. In both groups of untreated patients, western blot analysis revealed the presence of prominent additional bands reacting with anti-Bcl-2 or anti-Bax antibody, which were undetectable in control leukocytes. After the therapy, these bands disappeared, especially in patients from group A. In conclusion, the lack of therapy-induced enhancement in leukocyte apoptosis, an increased ratio of Bcl-2/Bax as well as persistent presence of abnormal Bcl-2 and Bax protein bands after induction therapy in AML patients may be considered as factors associated with unfavorable clinical outcome.

Neoplasma. 2003; 50(5): 339-344.

Low antigen-dependent activity of T cells after repeated stimulation using dendritic cells and expansion with interleukin-2.

Buchler T, Hajek R, Kovarova L, Musilova R, Bourkova L, Cech Z, Vanova P, Tuzova E, Vidlakova P, Vorlicek J, Penka M.

Laboratory of Experimental Hematology and Immunotherapy, Department of Clinical Hematology, University Hospital Brno-Bohunice, Brno, Czech Republic. tbuchler@fnbrno.cz

Both CD8+ and CD4+ T cells with specific activity against tumor antigens are needed for an efficient antitumor immune response. Activation and proliferation of T cells require cellular interactions including adhesion, recognition of peptides presented by MHC molecules to the T cells receptor, and costimulation. In a series of experiments we attempted to generate and expand specific T cells by repeated stimulation using antigen-loaded autologous dendritic cells (DCs). DCs were obtained from peripheral blood mononuclear cells (PBMC) in the presence of IL-4 and GM-CSF. TNF-a was added to induce maturation. A conjugate of myeloma idiotypic protein with keyhole limpet hemocyanin was used as antigen. Nonadherent peripheral blood mononuclear cells were cultured in the presence of Il-2 and IL-7. Autologous DCs were added to the lymphocyte cultures on days 3, 10, and 17. The lymphocytes were stimulated by high concentration of IL-2 between days 21 and 27. Lymphocytes harvested on day 27 proliferated in response to antigen-loaded DC but failed to do so if less than 0.3 x 10(6) DCs were added for stimulation during culture. However, no cytotoxic activity against autologous DCs was detected and IFN-g production in the T cell cultures was low at the end of culture. In conclusion, the generation and expansion of T cells using repeated stimulation by autologous DCs is feasible but defective cytotoxic response of these cells occurs, possibly as a consequence of repeated frequent exposure to antigen.

Neoplasma. 2003; 50(5): 345-349.

Hairy cell leukemia: early immunophenotypical detection and quantitative analysis by flow cytometry.

Babusikova O, Tomova A.

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

The abnormal coexpression of the so-called 'HCL-restricted' markers (CD22+CD11c, CD25 and CD103) identified on monotypic, slightly large B-lymphocytes in the large cell-gate of dot-plots has previously been shown to be highly characteristic of hairy cell leukemia (HCL). The main aim of our present study was to determine if patterns with low levels of neoplastic cells in bone marrow (BM) or peripheral blood (PB) are of a value the early diagnosis and/or detection of minimal residual disease (MRD) in HCL. Next we wished to determine if quantitative immunophenotyping given by molecules of equivalent soluble fluoresceine (MESF) could help to distinguish pathologic B-lymphocytic pool from that of normal residual B-cells also in patients with low numbers of HCL cells. The abnormal immunophenotypes were studied in 174 specimens from 19 patients with suspect HCL or during follow-up of already treated patients. For evaluation of marker density fluorescent calibration microbeads were used. In 12 HCL patients (67%) permanent complete remission was observed after treatment. In 6 patients (33%) transient MRD+ phenotype was identified but the clinical manifestation of relapse was followed till now in only three patients. One patient was phenotyped just only at diagnosis. The pathological cells in low levels were found in 5 patients at diagnosis (in the range from 2 to 12%) and in patients with MRD+ phenotype they were recognized repeatedly in the range from 2 to 8%. Furthermore, we observed in hairy cells significantly higher values of molecule numbers of some B-cell markers, comparing to that of residual B-cells in nonleukemic lymphocyte gate of the same sample. We found profound and persistent CD4+ lymphopenia in all but one studied patients after CdA treatment. Conclusions: Flow cytometric immunophenotyping of PB and BM is highly sensitive and specific method and is capable to detect low levels of malignant cells in HCL. Quantitative analysis of MESF values of pathological B-cells comparing to normal residual B-cells seems to be another new marker of HCL in common, which is reliable detecting also small cell numbers in examined sample. A long-term decline of CD4+ T-cells correlated with the relatively low incidence of clinical progression of HCL.

Neoplasma. 2003; 50(5): 350-356.

Glutathione S-transferase M1 and P1 metabolic polymorphism and lung cancer predisposition.

Reszka E, Wasowicz W, Rydzynski K, Szeszenia-Dabrowska N, Szymczak W.

Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 90-950 Lodz, Poland.

Individual susceptibility to different environmental agents is expected to be associated with alterations in metabolism of xenobiotics. Thus, genetic polymorphism of glutathione S-transferase (GST) can be recognized as a potential risk modifier in lung cancer development. The distribution of GSTM1 and GSTP1 genotypes was studied in a group of 138 diagnosed lung cancer patients and in 165 controls living in central Poland and RFLP-PCR technique was applied. The frequency of GSTM1 null genotype and GSTP1 Val single and duplicated alleles was similar among patients and controls. GSTM1 homozygous deletion was most prevalent in small-cell carcinoma groups (adjusted odds ratio (OR): 2.32, 95% confidence interval (CI): 0.98-5.52). In patients and controls, GSTM1A genotype was most frequent (34.1% vs. 37.0%). The estimated lung cancer risk for GSTM1 null, GSTP1 Ile/Val and GSTP1 Val/Val combined genotype was 1.44 (95% CI: 0.73-2.83), suggesting the absence of modifying effect of defective GSTM1 and GSTP1 alleles on lung cancer predisposition.

Neoplasma. 2003; 50(5): 357-362.

A contribution to examination of propidium iodide and annexin V plasma cells indices in multiple myeloma.

Scudla V, Ordeltova M, Bacovsky J, Vytrasova M, Sumna E, Martinek A, Horak P.

3rd Department of Internal Medicine, University Hospital, Medical Faculty of Palacky University, Olomouc, 775 20 Czech Republic. vlastimil.scudla@fnol.cz

The aim of this study was a contemporaneous measurement and a mutual comparison of plasma cells proliferative activity and grade of apoptosis in patients with monoclonal gammopathy of undetermined significance (MGUS) and various phases of MM i.e. smoldering (SMM), stable/plateau and active (progression/relapse) forms of this disease. The analyzed group of 197 patients consisted of 30 MGUS, 21 SMM, 82 patients examined at the time of MM diagnosis and 64 patients analyzed during various phases of the disease after previous chemotherapy. Plasma cell proliferative activity was measured by means of a propidium iodide index (PC-PI) examined by flow cytometry using a DNA/CD138 double staining technique. For detection of plasma cells entering apoptosis (PC-AI) flow cytometry method with annexin V FITC and MoAb CD138 was used. The individuals with MGUS, SMM and stable/plateau form of MM had overall low levels of PC-PI (M-1.8, 1.7% and 2.1%) and relatively high levels of PC-AI (M-9.1, 10.8 and 9.0%). The correlation between PC-PI and PC-AI was in all the groups mutually highly statistically significant (p=0.000). Analysis of plasma cells proliferative activity (PC-PI) was statistically significant in comparison of MGUS or SMM and versus: patients examined at the time of MM diagnosis (p=0.018 or 0.016); patients evaluated during various phases of MM after previous chemotherapy (p=0.021 or 0.019); stable/plateau MM phase in the cohort of all patients (p=0.017 or 0.040); in the plateau phase after chemotherapy (p=0.008 or 0.024) but insignificant in comparison of MGUS and SMM and with the stable group examined at the time of MM diagnosis. Analysis of the apoptotic process revealed significant differences when comparing PC-AI of SMM but not MGUS group versus all cohort of stable/plateau MM patients (p=0.045); there were also insignificant differences in comparison of MGUS and SMM groupsand versus the stable form of MM measured at the time of MM diagnosis or plateau phase after chemotherapy. There was observed a statistically significant difference in the PC-AI in comparison of SMM group versus group of all patients examined at the time of MM diagnosis (p=0.001) or in various phases of this disease (p=0.015) and the group of MGUS patients compared with patients evaluated at the time of MM diagnosis (p=0.03). Very significant statistical differences of plasma cell proliferative (PC-PI) and apoptotic (PC-AI) activity were found when comparing the levels of both the indices of MGUS, SMM and stable/plateau MM group versus the active (progression/relapse) form of MM marked by a higher level of PC-PI (3.2%, p=0.000) and PC-AI (4.8%, p=0.000) in the whole cohort of MM patients, but also in comparison with both the active forms at the time of MM diagnosis or active forms evaluated during various phases of the disease after chemotherapy. Highly significant inverse relationship between PC-PI versus PC-AI was also revealed in the group of patients in the active (progression/relapse) phase of MM (p=0.000). These results revealed importance of measurement not only of proliferative but also of apoptotic plasma cells indices for a complex evaluation of the cells kinetics of plasma cells compartments in patients with MGUS or MM. This study confirmed the initial hypothesis of a common 'inverse relationship between the proliferative (PC-PI) and the apoptosis activity (PC-AI) in plasma cells compartments in patients with MGUS, smoldering, stable/plateau and active (progression/ relapse) forms of MM'.

Neoplasma. 2003; 50(5): 363-371.

Evaluation of dose homogenization and radiation carcinogenesis risk in total body irradiation for bone marrow transplantation.

Oysul K, Dirican B, Beyzadeoglu M, Surenkok S, Arpaci F, Pak Y.

Department of Radiation Oncology, Gulhane Military Medical Academy, Ankara, Turkey. kaan@gata.edu.tr

The purpose of this study is to report on the dose homogeneity in total body irradiated patients undergoing Bone Marrow Transplantation (BMT), and carcinogenic risk in surviving patients. Between 1987 and 2001, 105 patients received hyperfractionated (6 fractions in 3 days) 12 Gy Total Body Irradiation (TBI) in our institution with lateral opposed fields. All the patients had measurements with thermoluminiscence dosimetry (TLD100) placed on seven bilateral body sites in vivo, controlled by the randophantom measurements to verify reasonable dose homogeneity achievement. The comorbid effects in the whole TBI conditioning group with at least three months post BMT follow-up were noted and surviving patients who had a minimum 5-year and maximum 14-year follow-up (median 7.8 years) have been evaluated for carcinogenic radiation risk on the basis of tissue weighting factors as defined by ICRP 60. Reasonable dose homogeneity by lateral opposed beam TBI has been obtained in all 105 patients in whom lateral TLD100 measurement means were within +5% of the planned doses. Calculated carcinogenesis risk factor was 11.34% for males and 12.40% for females, and no second-cancer has been detected whilst radiation-induced 5 cataracts and 10 interstitial pneumonia comorbidities were noted. Dose homogenization can be well achieved for hyperfractionated lateral-beam TBI with acceptable comorbidities and estimated second-cancer risk is significant but relatively low compared to the risk from the clinical indications for TBI.

Neoplasma. 2003; 50(5): 372-376.

Cerebrospinal fluid and serum carcinoembryonic antigen in brain tumors.

Batabyal SK, Ghosh B, Sengupta S, Ghosh SN, Chatterjee R.

Department of Biochemistry & Neurology, BR Singh Hospital & Center for Education & Research, Kolkata 700026, India.

Carcinoembryonic antigen (CEA) has been indicated to be a marker for brain tumors. In this study CEA was measured in serum and cerebrospinal fluid (CSF) of 14 patients with benign brain lesions, 16 with primary brain tumors and 8 with metastatic brain tumors by radioimmuno assay. Tumor cyst fluid CEA of 6 patients having intracranial tumors was also measured. The control group (n=20) had no neurological disease. The mean CEA levels in CSF for the control group, patients with benign tumors, primary tumors and metastatic tumors were 0.22 ng/ml, 0.31 ng/ml, 0.92 ng/ml, and 6.3 ng/ml respectively. Corresponding serum CEA levels were 2.5, 2.7, 3.0 and 5.2 ng/ml. Results showed that CEA level in CSF may play an important role in differential diagnosis of primary and metastatic brain tumors and consequently management of the treatment. To our knowledge this is the first such study on brain tumors from India.

Neoplasma. 2003; 50(5): 377-379.

Effect of smoking and aging on micronucleus frequencies in human exfoliated buccal cells.

Konopacka M.

Department of Experimental and Clinical Radiobiology; Institute of Oncology, 44-100 Gliwice, Poland. m_konopacka@pf.pl

In the present work the frequency of micronuclei (MN) in exfoliated buccal cells in 120 healthy individuals with relation to sex, age and smoking was investigated. Neither age nor sex showed any effect on the level of micronuclei. Smoking has shown a significant effect upon basal DNA damage. In the present study the calculated background frequency of micronuclei (per mille) in oral epithelial cells of 50 smokers and 70 non-smokers were 1.50 (+/-0.47) and 0.55 (+/-0.32), respectively.

Neoplasma. 2003; 50(5): 380-382.

Prognostic factors in the operative and palliative treatment of pancreatic cancer.

Talar-Wojnarowska R, Gasiorowska A, Strzelczyk J, Janiak A, Malecka-Panas E.

Department of Digestive Tract Diseases, Medical University of Lodz, 90-153 Lodz, Poland. r-wojnarowska@wp.pl

Recent studies have emphasized the importance of patient selection for the surgical resection of pancreatic adenocarcinoma based on reproducible prognostic factors. The aim of the study was to investigate the prognostic factors affecting long-term survival in patients with resectable and nonresectable pancreatic cancer and to evaluate their prognostic value. Forty six patients (25 women, 21 men, aged 44-80) with ductal adenocarcinoma of the pancreas were reviewed. Primary tumor size and regional enlargement of lymph nodes was assessed with enhanced CT scan. 13 patients were treated conservatively, 9 with standard Whipple procedure (pancreatoduodenectomy) and 24 - with palliative surgery. Survival probabilities were computed using univariate Kaplan-Meier analysis. Log-rank test was used to compare survival between groups. Overall median survival was 6 months with a 4 years survival of 2.2%. There was no difference in survival time (ST) between patients aged 65 years or younger and older (p=0.71). MeanST in patients after Whipple procedure was 10.3, after palliative surgery - 9.4 and after conservative treatment - 4.4 months (p<0.05). Thirty-day surgical mortality was 9.4%. ST was significantly longer in patients with tumors 3 cm or less of diameter compared with larger ones (p<0.05). Presenting signs and symptoms, like jaundice, diabetes, alkaline phosphatase, aspartate and alanine aminotransferase elevation and history of cholecystectomy did not have any significant impact on survival. The only significant independent factors improving survival were: operative treatment and tumor size smaller than 3 cm.

Neoplasma. 2003; 50(5): 383-387.

Electronic Library of Scientific Literature - Academic Electronic Press