Germline mutation of the RET proto-oncogene in members of Slovak families with multiple endocrine neoplasia 2.

Detection of mutations in RET proto-oncogene in Slovak families from different localities and of different ethnic origin with MEN 2 syndrome is reported. Despite the fact that the same mutation of RET oncogene was found in different family members, the latency period of tumor appearance and their pathogenicity differed substantially. In addition, also different phenotypes of the disease were expressed in various family members having the same RET gene mutation. The data indicate that the mechanism of MEN2 syndrome is not only due to the RET gene mutation, and strongly support the conclusion that additional genetic events are involved in the disease formation.

Mutation screening of the BRCA1 gene in Slovak patients.

Breast cancer is the most commonly observed malignancy in women of the western world. The family history is the strongest risk factor for the disease. Two major genes, BRCA1 and BRCA2 that are involved in the familial breast and ovarian cancer have been described. Germ-line mutations of the BRCA1 gene have been linked to 85% of all hereditary breast and ovarian cancers. We performed a mutation screening ofthe entire codingregion of the BRCA1 gene in 29 Slovak families suspected of having inherited predisposition to breast cancer. For the analysis we used a combination of a single strand conformation polymorphism (SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing. Genetic alterations were consistently indicated by SSCP and DHPLC and consequently confirmed by DNA sequencing as previously described pathogenic mutations. The patients with inherited BRCA1 mutations will undergo genetic counseling and cancer prevention health care program.

Effect of retinoic acid on the actin cytoskeleton in HL-60 cells.

The cytoskeleton, in addition to its structural and kinetic functions, is also involved in modulating signal transfer in cell proliferation, differentiation and death. In some myeloid leukemic cell lines, the process of cell differentiation accompanied by apoptosis, can be induced by all-trans retinoic acid (ATRA). In this report, we describe the morphological changes in actin cytoskeleton, taking place during apoptosis in cells of the human leukemic HL-60 cell line. By using fluorescent microscopy, the morphology of microfilaments and the proportion of apoptotic cells in the cell populations untreated or treated with 10(-6) M ATRA were detected. Interphase HL-60 cells showed aggregations of short, thick microfilament bundles in the region between the plasma membrane and the nucleus. In comparison with both interphase and mitotic cells, the cells with apoptotic nuclear fragmentation showed a different organisation of the actin cytoskeleton. The following types of F-actin structures were observed: (i) Cells with a high number of large dots/patches of F-actin under the plasma membrane. These dots might be localised only in the part of the cell or occurred under the whole plasma membrane. This arrangement was often associated with a diffuse signal for F-actin. (ii) Cells with 3D-network of F-actin fibres through the cytoplasm between remnants of the cell nucleus. This 3D-structure probably played an important active role in the process of apoptotic bodies formation. (iii) Cells without any detectable signal for F-actin or cells with only a very low F-actin signal. Both of these showed typical apoptotic collapse of chromatin. It is concluded that the actin cytoskeleton is a dynamic structure actively involved in the executive phase of the process of apoptosis. It is suggested that the rearrangement of the microfilament network and its subsequent degradation are necessary for the main morphological changes of apoptotic cells, i.e., plasma membrane blebbing and apoptotic bodies formation.

Plasminogen activator inhibitor type 2: potential prognostic factor for endometrial carcinomas.

The clinical determination of proteases which are involved in carcinogenesis, invasion and metastasis may contribute to the detection of the early stage of disease, and to the prognostic assessment of patients with the cancer. The aim of the present study was to determine the level of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1) and plasminogen activator inhibitor type 2 (PAI-2) in normal and malignant tissues of corpus uteri and to evaluate the possible correlation with clinical and histopathological prognostic factors. UPA, PA-I and PAI-2 were determined by the ELISA assay in tissue cytosol of matched pair samples from 27 patients with endometrial carcinoma. Results show that significantly higher levels of these proteins were found in malignant than in normal tissue samples (uPA: 1.266 versus 0.633 ng/mg protein, PAI-1:4.468 versus 1.958 ng/mg protein, and PAI-2:3.428 versus 0.483 ng/ml protein). The levels of uPA and PAI-1 did not correlate with clinical staging or pathohistological grading. However, in tumor tissues with clinical stages II and III, myometrial invasion > 50%, and lymphovascular invasion, increased levels of PAI-2 were determined. Our results indicate that components of the plasminogen activation cascade are up-regulated in endometrial cancer and suggest the role of PAI-2 in determining invasive potential of endometrial carcinomas.

Identification of a pepsin-sensitive type III-like collagen in breast cancer MCF-7 cells.

Electrophoretic analysis of [3H]proline-labeled culture medium proteins of MCF-7 cells revealed the presence of disulfide-bonded, bacterial collagenase-sensitive component which comigrated with pro(alpha)1 chains of type III and type I collagens. However, it was pepsin- and trypsin-sensitive. Within 1 min of pepsin-digestion, a component with a size of alpha1 chain of type I or III collagen was produced which degraded after 5 min of digestion. Similarly, the pepsin-sensitive band was completely degraded by trypsin at 30 degrees C within 5 min. We examined CNBr peptides of the collagenous band and demonstrated that it was alpha1 chain of type III collagen. When MCF-7 cells were cultured in the presence of 2 nM estradiol, a marked increase in the level of collagen secreted into medium was found. The identified proteinase-sensitive type III-like collagen as major protein of extracellular matrix, would be expected to be more susceptible to degradation which might contribute to tumor progression.

Significance of P-glycoprotein expression in childhood malignant tumors.

Resistance to chemotherapy significantly affects the treatment results in various cancers. Multidrug resistance caused by P-glycoprotein expression is now widely studied in human malignancies. We present the results of P-glycoprotein expression examination in 91 tumor tissue samples obtained from children treated for different malignant tumors in the Dept. of Pediatric Oncology, Prague. The correlation between the level of P-glycoprotein expression and tumor histology, clinical outcome, use of therapy, relapse rate and metastatic disease was made. P-glycoprotein expression was found significantly more frequent in soft tissue sarcomas, neuroblastomas, and hepatoblastomas, and generally in disseminated disease. On the contrary, a high expression of P-glycoprotein was not found in malignant brain tumors and nephroblastomas. The data strongly support the possibility that the percentage of P-glycoprotein expressing cells in selected tumors (soft tissue sarcomas, neuroblastomas), may have a clinical importance.

Establishment, morphological, growth and cytoskeletal properties of 135-BCA carcinoma cell line derived from lung brain metastasis.

Many cell lines have been established from lung cancer but carcinoma cell lines derived from brain metastases occur rarely. The carcinoma cells growth relatively slowly in comparison with brain cells which often overgrow the tumor cells in early passages. The origin of these rapidly dividing brain cells in carcinoma cultures is discussed with respect to the previous studies on adult human brain tissue cultures. It was found that the majority of cells in adult human brain cultures derived from brain biopsies of patients with non-cancer diseases do not express glial markers. Based on the previous studies we suggest that they are glial precursor cells. The high proliferative capacity and non-glial phenotype of these brain cells may lead to the suggestion that they are of cancer origin. In this study the establishment and characterization of a new carcinoma cell line 135-BCA is described. The tissue cultures were derived from brain metastasis of lung large cell carcinoma. The cell line is specific by the epithelial cell morphology and evident cytokeratins expression during the whole subcultivation. All tumor cells were strongly immunoreactive for vimentin and negative stained for glial fibrillary acidic protein (GFAP). The new cell line may prove of value in biological and therapeutic studies of lung cancer. In addition, the further comparative analysis may reveal the environmental influence of brain tissue on carcinoma cells.

Expression of apoptosis-regulating proteins p53, Bcl-2, and Bax in primary resected esophageal squamous cell carcinoma.

Apoptosis plays a key role in the pathogenesis, aggressiveness, and therapy responsiveness of cancer. Proteins of the Bcl-2 family as well as p53 are important regulators of apoptosis. The present study retrospectively examines the expression of apoptosis-regulating proteins in primary resected esophageal squamous cell carcinoma (ESCC) and the correlation between the outcome of patients' treatment and the expression of the proteins. We used antibodies specific for the human p53, Bcl-2 and Bax proteins to examine the expression of these apoptosis-regulating proteins in 40 archival specimens of patients with primary resected esophageal squamous cell carcinoma. The overall expression of p53, Bcl-2, and Bax was 73%, 18%, and 100%, respectively. No significant correlations were found between the expression of p53, Bcl-2, and Bax. The expression of Bcl-2 had a negative influence on survival in this population of primary resected ESCC patients (p=0.03). But no differences in survival were observed in relation to the expression of p53 or Bax. In conclusion, Bcl-2 expression may provide additional and prognostic information for the clinical course of the disease and therefore to be developed as a prognostic indicator for primary resected esophageal squamous cell carcinoma.

Expression of p53 and bcl-2 proteins in acute leukemias: an immunocytochemical study.

We have analyzed by immunocytochemistry the p53 and Bcl-2 proteins expression in 49 patients with B-ALL, T-ALL and AML at the time of initial diagnosis. The diagnosis was based on morphologic and cytochemical criteria and on immunophenotyping. To demonstrate the p53 protein expression, p53 specific mouse antihuman immunoreagent clone DO-1 that recognizes both wild and mutated p53 protein was used. To detect Bcl-2 a monoclonal antibody that recognizes the 26-kD Bcl-2 protein was applied. For evaluation of both proteins a sensitive Immunotech detection kit based on peroxidase labeled streptavidin biotin reagent was utilized. The patients were divided according to the presence or absence of both, nuclear p53 and cytoplasmic Bcl-2 proteins. A relative low frequency of p53 protein expression in B- and T-lineage acute lymphoblastic leukemia has been shown at diagnosis. In AML cases, the frequency of p53 expression was higher than that in ALL. Bcl-2 protein immunoreactivity has been found in the majority of acute leukemia patients. The marked heterogeneity in the percentage of p53 and Bcl-2 positive cells in individual patients was observed. Comparative analysis of the distinct acute leukemia subtypes according to the percentage of p53 and Bcl-2 positive cells showed no significant differences except for p53 protein positivity in relation between T-ALL and AML cases. The samples from healthy subjects used as a control exhibited very low proportion of positively stained cells and significantly differed from p53 as well as Bcl-2 positive cases. p53 and Bcl-2 positivity have not been significantly affected neither by age, sex nor WB C counts. Association between myeloid cells maturation and proportion of p53 and Bcl-2 positive cells was observed. Noteworthy was the inverse relation between the higher proportion of p53 positive cells and low Bcl-2 positivity in some cases of acute leukemia. Although our preliminary results need to be confirmed in a larger group of patients, immunocytochemical analysis of p53 and Bcl-2 proteins, indicators of cell alterations, may help to identify risk patients requiring intensive therapy.

A study on nitric oxide secretion by transplantable melanoma cell lines with regard to their spontaneous apoptosis.

In the study we investigated if there exists any correlation in the nitric oxide (NO) secretion by two types of melanoma cells of the same origin but differing in their biological properties and ability to undergo spontaneous apoptosis. Our results suggest that there exists an inverse correlation between the dynamics of NO secretion by cells of two melanoma lines and their ability to undergo spontaneous apoptosis.

Polymerase chain reaction detection of cells carrying t(14;18) in bone marrow of patients with follicular and diffuse large B-cell lymphoma: the importance of analysis at diagnosis and significance of long-term follow-up.

The t(14;18) is the most frequent chromosomal aberration observed in follicular lymphoma (FL), and is less frequent in diffuse large cell lymphoma (DLCL). The bcl-2/IgH rearrangement constitutes good target for polymerase chain reaction (PCR) detection that allows to find out one tumor cell in 100,000 normal cells. The PCR assay was used to detect bcl-2-rearranged cells in blood and bone marrow (BM) in 63 previously untreated patients with DLCL and in 53 patients with FL. Twenty five FL patients (47%) and 9 DLCL patients (14%) had PCR-detectable lymphoma cells in BM and peripheral blood. Minimal residual disease (MRD) was evaluated in 17 FL and 5 DLCL patients undergoing first-line chemotherapy. Three DLCL patients (60%) but only 1 FL (6%) patient achieved molecular response (PCR-negative status in BM). Two PCR bcl-2/IgH positive patients with FL were treated with rituximab (anti-CD20 antibody) and had no PCR-detectable lymphoma cells in BM after the therapy. Peripheral blood stem cells (PBSC) were harvested in 5 FL (1 PCR-negative) and in 2 DLCL (1 PCR-negative) patients. PCR-positive lymphoma cells contamined PBSC in all patients with BM PCR-positivity before harvesting. Five FL patients underwent autologous transplantation (AT). No bcl-2/IgH positive cells were detected in 4 patients (80%) at any point after AT. One patient achieved molecular response after rituximab treatment. All the patients are in CR 6, 22, 30, 31 and 42 months respectively, after AT. On the other hand, 4 FL patients in clinical complete remission, but with persistent PCR positivity in BM relapsed with median of 21 months (interval, 14-28 months) from the end of a first-line chemotherapy. Thus, the results show that PCR detection of the bcl-2/IgH rearrangement is a very useful method in evaluating the BM infiltration by lymphoma cells especially in the situation of MRD. Conventional chemotherapy did not eradicate bcl-2 positive cells in BM in most of lymphoma patients, but autologous transplantation or rituximab immunotherapy can induce molecular response in a significant proportion of them. Our results support the previous observations of the molecular response importance in view of better disease free and probably also overall survival.

The role of palliative radiotherapy in locally advanced non-small cell lung cancer.

The aim of this study was to evaluate retrospectively the treatment results of non-small cell lung cancer (NSCLC) patients treated with palliative intent in Dokuz Eylul University Hospital, Radiation Oncology Department. One hundred and fifteen inoperable, non-metastatic and symptomatic NSCLC patients were treated with palliative radiotherapy (PRT) between July 1991 and May 2000. PRT was used in patients with low performance status, weight loss more than 10% within last 6 months, secondary malignancies, co-morbid diseases and socio-economic problems. Parallelly opposed isocentric antero-posterior fields including both the parenchymal and mediastinal masses were used. 10-55 Gy total doses were delivered in 1-23 fractions with a median of 30 Gy. Nineteen patients received systemic chemotherapy before PRT. Survival analysis was made from the treatment beginning date, and subjective palliation rates were assessed according to clinical improvements in symptomatology evaluated 1-6 weeks after PRT. The median follow-up time was 28 weeks (1-234 weeks). Totally, 245 disease-related symptoms were detected in 115 patients. Overall "improvement" in symptomatology was found to be 90% (221/245) with a "near-total response" rate of 46% (113/245). Hemoptysis was the best palliated symptom. Median survival time was 30 weeks. Karnofsky performance status (KPS) (p=0.015), weight loss (p=0.0015), histologic tumor type (p=0.0024) and tumor size (p=0.02) were found to effect overall survival rates significantly in uni-variant analysis. Multi-variant analysis revealed statistically significant effect with histological tumor type and weight loss status. Only 16% of patients (3/19) showed partial and 5% (1/19) complete response to systemic treatment. Median survival time was 46 weeks in this group. In conclusion, this retrospective study of patients with poor prognostic factors confirms that PRT is an effective treatment modality in symptomatic locally advanced NSCLC patients resulting in 90% symptomatic improvement rate and a median survival of 30 weeks.

Genetic susceptibility to malignant diseases--ethical issues. Minireview.

Ethical problems connected with genetic testing with the intention of the measurement of the susceptibility or predisposition to malignant tumors are presented (respect for autonomy, beneficence, nonmaleficence, confidentiality, privacy, veracity and truth-telling, informed consent, right to know, right not to know, informational self-determination, etc.). Various aspects dealing with ethics of screening and research projects involving human subjects are discussed as well.

Activity of some lysosomal enzymes in serum and in tumors of patients with squamous cell lung carcinoma.

The aim of the study was an assessment of some lysosomal enzymes activity in serum and in tumors of patients with lung cancer histopathologically confirmed as squamous cell lung carcinoma. The first group constisted of 10 patients with stage II of the disease and the second group consisted of 11 patients with stage III of the disease. Lysosomal enzymes activities were assayed in serum before surgery and on the 10th day after surgery in serum and in tumors. Arylsuphatase, cathepsin D and acid phosphatase activities were higher in the patients serum than in that of the control group. The decrease of arylsulphatase and cathepsin D activities after surgery was statistically significant in both groups of patients, but the cathepsin D activity was still 3 times higher in patients than in those from the control group. The decrease of acid phosphatase activity after surgery was about 50% in both groups of patients and this decrease was statistically significant. The arylsulphatase and acid phosphatase activity in tumors was nearly 3 times higher in stage III patients than it was in stage II patients, but the cathepsin D activity was nearly the same in both patient groups. Higher lysosomal enzyme activity may be a useful factor in diagnosing and monitoring of lung cancer. However, further investigations are needed.

The immunohistochemical and serological determination of p53 protein in patients with malignant lymphomas.

The product of mutated p53 gene is a protein with abnormal conformation, impaired DNA binding, and a prolonged half life, the latter of which results in immunohistochemically detectable levels within nuclei of malignant cells. The present study was aimed at the immunohistochemical determination of p53 overexpression in patients with various histological types of nonHodgkin's lymphomas (NHL), with a particular interest in gastric lymphomas. In these patients, as well as in controls, also serological determinations of p53 protein were performed using an ELISA method. Immunohistochemical overexpression of p53 protein was found in 21% of NHL patients, with the highest incidence of p53 immunoreactivity in cases of Burkitt's lymphoma, follicle center lymphoma grade III, and diffuse large B-cell lymphoma. In gastric lymphomas the overall incidence of p53 immunoreactivity was as high as 46%. Serological ELISA determinations of p53 protein in NHL patients and in controls remained below the lowest detection limit of the method in all 128 cases. Considering that p53 mutations are associated with poor response to therapy, and consequently with poor prognosis, it is of great importance to determine the subset of patients that are particularly at risk for an unfavorable outcome and should be treated more aggressively. Immunohistochemical determinations of p53 overexpression represent a rapid and simple, yet somewhat imperfect technique for an estimation of the frequency of mutational events. On the other hand, serological determinations of p53 protein are completely inadequate for the evaluation of p53 status.

Switching (overtargeting) of estrogen effects and its potential role in hormonal carcinogenesis.

Peculiarities of the estrogens influence on target tissues is one of the crucial problems in understanding of the estrogen-induced carcinogenesis and anticarcinogenesis mechanisms. Conditions or factors enhancing the genotoxic component in total effect of estrogens (on the uterine tissue, in particular) are very important, since these factors may influence both the hormonal carcinogenesis type and biological properties of the developing hormone-dependent tumors. In this study female rats (3 months of age at the beginning of experiment) have been given plain water (group 1) or 5% ethanol solution over 4 months. Rats which received ethanol were further divided into 6 groups (groups 2-7). During last 2 months of the experiment N-acetylcysteine was given to rats in group 3, ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E)--to group 4, melatonin--to group 5, carnosine--to group 6; the rats in group 7 swam for 5 days a week according to the so called developing schedule. 2.5 weeks before the end of experiment all rats underwent bilateral ovariectomy, and over 11 days preceding the last day of the experiment they received injections of estradiol (2 microg intramuscularly daily). When the experiment was over, estradiol and cholesterol blood levels, progesterone receptors content, peroxidase activity, proliferation index, percent of cells in S and G2/M phases, thickness of endometrium and rate of DNA damage in uterine tissue (COMET assay) and estradiol 2-hydroxylase activity in liver tissue were measured. The conclusion was that administration of 5% ethanol combined with estrogen injections results in genotoxic (G) changes in the uterus, which may be prevented by giving N-acetylcysteine or melatonin. Combination of vitamins C and E enhances some features of hormonal (H) estrogen effects (uterine weight, induction of progesterone receptors), but attenuates the other (proliferation index). Consequently, the combination of N-acetylcysteine and optimal doses of ascorbic acid and alpha-tocopherol may be recommended for prevention of the phenomenon of switching of estrogen effects [PSEE] (e.g. enhancement of G-component and decrease of H-component), observed particularly in cases of the treatment with tobacco smoke or ethanol consumption in more than moderate (15%) concentrations, which lead to the increased risk of genotoxic type of hormonal carcinogenesis.

Cytotoxic T lymphocyte precursor frequency analysis in the selection of HLA matched unrelated donors for hematopoietic stem cell transplantation: the correlation of CTLp frequency with HLA class I genotyping and aGVHD development.

The selection of human leukocyte antigen (HLA) compatible unrelated donors for hematopoietic stem cell transplantation (HSCT) is based on the direct genotyping of HLA class I and class II alleles (HLA-A, -B, -C, -DRB1, -DQB1 loci). The cellular test estimating the frequency of cytotoxic T lymphocyte precursors (CTLp) has been included into the selection procedure of unrelated donors to detect the class I alloreactivity and to predict acute graft versus host disease (aGVHD) occurrence and severity. The relationship between HLA-A, -B, -C high/medium resolution genotyping and CTLp activation was analysed in the cohort of 78 unrelated donor/patient pairs indicated for HSCT. The high frequency of CTLp (> 1:100,000) correlated significantly (p < or = 0.0002) with the incompatibilities in alleles of HLA-A, -B, -C loci. Nevertheless, the results of HLA-A, -B, -C genotyping and CTLp assay are not fully alternative, suggesting that the CTLp test gives its specific information. The high CTLp frequency (CTLpf) in 14/35 pairs fully matched by HLA class-I alleles genotyping could reflect the influence of another factors upon the CTLp activation. On the contrary, the low CTLp frequency values (< or = 1:100,000) found in 8/43 pairs with existing HLA class-I alleles incompatibilities could indicate the immunological permissivity of these particular mismatches. The clinical relevance of the CTLp test for aGVHD prediction has been also analysed. The relationship between CTLp activation in vitro and the incidence and severity of aGVHD was evaluated in 37 patients who underwent allogeneic HSCT. The severe form of aGVHD (grade III-IV) developed in 9 of 18 cases (50%) with the high pretransplant CTLpf value. The patients with the low CTLpf (n = 19) suffered from the severe form of aGVHD in 2 cases (10%) only, the remaining 17 patients from this group were without aGVHD symptoms or developed only the mild form of aGVHD (I-II). The relationship between CTLp results and the incidence and severity of aGVHD was found statistically significant (p < or = 0.01).

Determination of optimal conditions for detection of acute myeloid leukemia t(8;21) and t(15;17) translocations using RT-PCR.

Prognosis of patients with acute myeloid leukemia (AML) is not satisfactory. Long time remission can be achieved only in 25-40% children with AML. The aim of the study is to improve diagnosis of AML in cases characterized by specific chromosomal translocations t(8;21) and t(15;17). These translocations can be efficiently detected using reverse transcriptase-polymerase chain reaction (RT-PCR). To determine optimal conditions for detection of the t(8;21) and t(15;17) translocations in human cells, we performed a detail study of experimental conditions for RT-PCR on human cell lines NB-4 and KASUMI-1. For detection of t(8;21) using Pfx polymerase, the optimal PCR conditions included magnesium ion concentration in the range of 0.6 to 1 mM, 0.4 microM primer concentration, 5 degrees C annealing temperature and 1 microl of template cDNA. For detection of t(15;17) using the Pfx polymerase, the optimal conditions included 1 mM magnesium ion concentration, 0.6 microM primer concentration, 63 degrees C-66 degrees C annealing temperature and 2 microl of template cDNA. The results proved to be valid for clinical diagnostics of these chromosomal aberrations in blood and/or bone marrow samples of AML patients.

Acetaminophen modulations of chemotherapy efficacy in MDAH 2774 human endometrioid ovarian cancer cells in vitro.

Epidemiological data have correlated consumption of nonsteroidal antinflammatory drugs with lowered risk for many types of cancer, and some recent studies indicate a reverse correlation with acetaminophen consumption and ovarian malignancy. In this study we examined effects of acetaminophen on plating, S-phase and colony growth of MDAH 2774 human endometrioid ovarian carcinoma, as well as sensitivity of this cell line to carboplatin in all three tests, and paclitaxel to clonogenic assay. Acetaminophen significantly enhanced S-phase in first 72 hours and enhanced cell population in 96 hours of plating monitorization, but decreased one week colony growth by approximately 80%, which was in the range of cytotoxic drugs. Interestingly with low dose carboplatin in first 72 hours acetaminophen enhanced cell proliferation more profoundly, but only thereafter decreased cell growth synergistically with carboplatin. It did not effect paclitaxel colony growth inhibiting acitivity. MDAH-2774 cell line lack p-53 and MSH-2, which are both 'gatekeeper' apoptosis inducing genes against genome damaging insult. Thus, presence of lower doses of oxidizing drugs may help the induction of proliferative signals, but only their sustained presence may overcome such signals and ultimately bring to cell demise.

Chemoembolization for liver metastases from colorectal carcinoma: risk or a benefit.

Results from clinical trials do not allow definitive conclusions about the role of chemoembolization (ChE) in the treatment of colorectal cancer (CRC) liver metastases. The aim of present phase II study was to investigate toxicity and efficacy of ChE for patients, with unresectable colorectal liver metastases after failure of 5-FU based chemotherapy. Secondary endpoint was clinical benefit measurement. Eleven patients were enrolled in first stage (two-stage Simon design), 2 males/9 females, median age 60 (46-71). Performance status was I in 8 patients and II in 3 patients. All patients had radical surgery, 7 of them adjuvant chemotherapy and 4 systemic chemotherapy. The ChE regimen consisted of an injection of iodinated oil Lipiodol with mitomycin C (3 mg/ml). Repeated treatments were performed at 9- to 12-week intervals. We applied 17 ChE (median 1/pts.). Clinical benefit was a composite of measurements of pain, ECOG performance status, weight and tumor fever. Study was stopped after first stage because non of the patients (pts) achieved objective response (RECIST). Stable disease occurred in 5 pts (45%). Median time to progression was 3 months (range 3-9 months). Median survival was 9 months (range 4-16 months). A decrease of the baseline carcinoembryonic antigen level occurred in 0% of the cases. Clinical benefit was recorded in one patient. Common toxicity included a "postembolization syndrome," which consisted of fever, pain in the right upper quadrant, nausea, and vomiting. Grades 3-4 toxicity (NCI-CTC) followed transaminases 6/11, LDH 4/11. In addition, a drop in F V levels was noted in 5 pts, F VII in 9, F IX in 2 and F X in 10 pts. Decrease in At III levels occurred in 6 pts and FDP appeared in one. Thus, The ChE as performed in the present study did not appear to bring any benefit; furthermore, significant liver toxicity compromises the safety of such procedure.

Bilateral breast cancer.

The aim of this study was to evaluate the clinical and pathological features of breast cancer patients with bilateral breast cancer and to assess the impact of the second breast cancer on their prognosis. Thirty six breast cancer patients with bilateral (metasynchronous) cancer were treated in Greatpoland Cancer Center from 1983 to 1995. It constituted 4.1% of all breast cancer patients treated in those years. 5-year survival rates were compared with clinical data (age, clinical stage, histological diagnosis), methods of treatment and length of interval between occurring of both tumors. Five year survival of patients treated for second breast cancer was 55.6% (20/36), disease-free survived--15/36 (41.7%). 5-year survival rate was greater in group of patients with clinical stage T1 or T2 (69.6%, 16/23) than in group with T3 or T4 (30.8%, 4/13) (p=0.009). In group of 20 patients without nodal involvement (N0) 5 years survived 75.0% of patients (15/20), in group with N1--11.1% (1/9) (p=0.01). Length of interval between both breast tumors influenced 5-year survival rate. In group of patients with interval longer than 5 years, 5-year survival rate was 73.9% (17/23), in group of patients with interval shorter than 2 years--0% (0/6) (p=0.002). No correlations were observed between survival rate and age, histological diagnosis, methods of treatment.

A cytochemical note on nucleoli of granulocytic precursors and granulocytes in patients suffering from the refractory anemia with excess blasts (RAEB) of the myelodysplastic syndrome (MDS).

Nucleoli were studied in the proliferation as well as maturation granulopoietic compartment in patients suffering from refractory anemia with excess blasts (RAEB) of the myelodysplastic syndrome (MDS) by means of simple cytochemical procedures for the demonstration of nucleolar RNA and silver stained proteins of nucleolus organizer regions. Regardless of the procedure used for the nucleolar visualization, early stages of the granulopoietic compartment and particularly myeloblasts of RAEB patients were characterized by reduction of the nucleolar number expressed by the nucleolar coefficient the values of which resembled those described previously in acute myeloid leukemias. The reduced values of the nucleolar coefficient of these cells in silver stained specimens of RAEB patients were accompanied by a decreased number of clusters of silver stained particles representing interphasic silver stained nucleolus organizer regions (AgNORs). The reduction of these clusters was also described previously in leukemic cells. In addition, the differences in the values of the nucleolar coefficient of granulocytic precursors between specimens stained for RNA and those stained with the silver reaction might reflect changing composition and proportions of nucleolar components in the course of the granulocytic development.

Characteristics of nasal T/NK-cell lymphoma among Brazilians.

Nasal T/NK-cell lymphomas are highly associated with Epstein-Barr virus (EBV). They are more frequent in Asia than in Western countries. In Central and South America there are few studies about nasal T/NK-cell lymphoma and they have shown a strong predominance of this phenotype in Native American descents, supporting the hypothesis of a racial predisposition for the disease. We studied the lymphomas involving midline facial region at a Brazilian institution. T/NK cell lymphomas (16/25) were more frequently found compared to B lymphomas (9 cases, all B large cell). T/NK cell lymphomas involved predominantly the nasal region. Histologically they showed angioinvasion and necrosis. All of them were positive for CD3 and CD56 and showed numerous tumor cells labeled by EBER-1. Although disease was localized in 61% at diagnosis, there was no tendency to cure. The racial distribution of patients with T/NK-cell phenotype was similar to that found in B-cell lymphomas. EBV was more frequently found in adenoids than in palatine tonsils. In inflammatory lesions of the nasal and palatal regions EBV was not found. In the present study the relative frequency of T/NK versus B cell sinonasal lymphomas was high and similar to that observed in other Latin American countries. However, there was not any racial association with T/NK-cell phenotype and the tumor showed an agressive behavior similar to that reported in Asia. The high frequency of EBV-positive lymphocytes in nasopharyngeal lymphoid tissue (adenoids) suggests that they could serve as a reservoir for the virus.

Tamoxifen therapy and hepatic steatosis.

Tamoxifen has been used for a long time as a hormonal treatment in breast cancer. Recent studies in pre and postmenopausal women have shown that tamoxifen exhibits favorable effects on the lipid profile. In this study we investigated the effects of tamoxifen on lipid profile and hepatic steatosis. Fifty two (31 postmenopausal and 21 premenopausal) women with breast cancer treated with tamoxifen at a dose of 20 mg daily were included in the study. Serum lipid parameters (total cholesterol, high and low density lipoprotein cholesterol and triglyceride) were measured baseline and at the 6th month of tamoxifen treatment. Upper abdominal ultrasonography was performed before and at the 6th month of therapy for assessment of liver steatosis. We obtained decreased levels of serum total cholesterol after 6 months of tamoxifen treatment (p < 0.05). However, we did not detect any changes in triglyceride and high-density lipoprotein cholesterol levels (p > 0.05). Increased liver steatosis was observed in 22 patients (42.3%) after tamoxifen treatment. We could not detect increase in lipid levels in these patients. There was no significant difference between the lipid levels in the patients with increased liver steatosis and stable or no liver steatosis. Whether hepatic steatosis is dependent on lipid changes in tamoxifen use should be further investigated.

Inhibition of carcinogenic and clastogenic effects of N-nitrosomorpholine in rats immunized with tularemia vaccine.

The aim of the present work was to study whether immunization of rats with tularemia live vaccine (TLV) can influence carcinogenic and mutagenic action of N-nitrosomorpholine (NNM). The experiments were performed with male albino random-bred rats. The first group of rats was immunized with TLV 15 days before the start of experiment. These animals and the second group (positive control) were treated with NNM orally, (total dose was about 250 mg/rat). Rats including solvent (negative) control group were killed 12 months after the start of NNM treatment to study the carcinogenic effect. Experiments to study the influence of TLV on mutagenesis were performed with three groups of rats: the first (on 15th day after immunization with TLV) and the second group were injected intraperitoneally with NNM 100 mg/kg b.w. on 2 consecutive days, third group received only distilled water. The results of long-term experiment have shown that tumor incidence (both malignant and benign) in rats of positive control group was 74.2%. Immunized rats had significantly decreased incidence of tumors compared with the previous group--36.1%. Micronuclei level in bone marrow cells of non-immunized rats was statistically significantly higher than that in immunized rats. The inhibition of carcinogenic and clastogenic effects of NNM in rats immunized with TLV are probably due to a decrease in cytochrome P-450 activity. We suggest that immunization of rats with TLV can protect them against the cacinogenic and clastogenic actions of some chemicals.

Changes in apoptosis, mitosis, Her-2, p53 and Bcl2 expression in breast carcinomas after short-term tamoxifen treatment.

The short-term (7 days) effect of tamoxifen on apoptosis and mitosis index, p53, Bcl2 and Her-2/neu/c-erb2 expression in invasive ductal mammary carcinoma was studied histologically in the diagnostic biopsy and surgically removed tumor tissue of 10 patients. Following tamoxifen treatment expression of HER-2 and p53 decreased but Bcl2 remained unchanged. Mitotic activity decreased slightly, but not significantly. Apoptotic activity increased in six cases in the second sample compared to the values measured of the first biopsy. These changes may be attributed to the effect of antiestrogen therapy.

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation.

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.

Expression of c-erbB-2 in node negative breast cancer does not correlate with estrogen receptor status, predictors of hormone responsiveness, or PCNA expression.

The aim of this study was to analyse the relationships between the expression of c-erbB-2, estrogen receptor (ER), progesterone receptor (PR), Bcl-2 and PCNA in node negative breast cancer. Expression of these markers was determined by HercepTest, by immunohistochemistry and quantified by morphometry in the group of 125 selected breast carcinoma patients with broad spectrum of histological types and grades. Multivariate statistical analysis revealed only relationships between ER/PR, ER/Bcl-2, ER/grade and ER/age. There was not found any significant relationship between c-erbB-2 expression and any other immunohistochemical marker, apocrine metaplasia, histological type or patient characteristics. The same result was found in complete group of tumors as well as in individual groups divided according to histological type. These results indicate that in node negative breast tumors, c-erbB-2 expression does not correlate inversely with hormone receptor status and hormone responsiveness like previously reported metastasising breast cancer and that the prognostic significance of c-erbB-2 expression in these tumors is not clear.

Possible genotoxic activity of extracts of Bryonia alba roots on human lymphocytes and transformed cells.

Bryonia alba roots (BAR) are widely used as an adaptogenic and restorative drug with immunomodulatory and stress-protective properties that increase the non-specific resistance of an organism toward harmful stimuli. Potential genotoxic activity of aqueous and methanol extracts of BAR was studied on human normal (lymphocytes) and transformed (HeLa and Caco-2) cells using single cell gel electrophoresis (the comet assay). The results obtained did not show any evidence of genotoxic effects of BAR.

Concomitant radiotherapy and metronomic temozolomide in pediatric high-risk brain tumors.

Temozolomide, an oral alkylating agent has a significant activity in preclinical testing and in clinical trials in adults and children as well. Penetration across the blood brain barrier has been documented. In adult and pediatric phase I and II trials a five-day every 28 days schedule was first approved for clinical use. With respect to temozolomide proximal mechanism of resistance, and further to increase dose intensity, new schedules are proposed to use more prolonged drug exposure. Higher doses of metronomic temozolomide were piloted. Eight children with poor prognosis brain tumors were eligible. Treatment consisted from concomitant radiotherapy given 1x170 cGy, 5d/wk, for total dose 55/56 Gy, together with temozolomide 90 mg/m2/day for 42 days. No further dose escalation has been planned for this group of patients. Myelosuppression was the primary toxicity, occurring around day 21. Nonhematologic toxicities were infrequent and in no case dose limiting toxicity (DLT) occurred. The most common nonhematologic toxicity was vomiting, effectively managed with antiemetics. Six responses were documented. The best responses (CR) were seen in 2 patients with high-risk medulloblastomas, who have progressed early after neurosurgery. Furthermore, one more patient had CR and 3 patients PR at the end of temozolomide treatment. We have piloted novel dose schedule of temozolomide and evaluated clinical toxicities in a cohort of 8 children. This is the first study to report feasibility and tolerability of 90 mg/m2/day of temozolomide treatment in metronomic fashion. In addition, we have documented encouraging responses in children with medulloblastomas, progressing early after their initial surgery.