Mammary carcinogenesis induced in Wistar:han rats by the combination of ionizing radiation and dimethylbenz(a)anthracene: prevention with melatonin.

The primary cancer chemoprevention is an important topic of experimental oncology. We have analyzed the possible oncostatic properties ofmelatonin in a combined model of radiation plus chemocarcinogen-induced mammary carcinogenesis. Virgin female rats of Wistar:Han strain were continuously irradiated with daily dose 96 mGy of gamma rays up to 15 days. At the end of irradiation, between 52-60 postnatal days, 7,12-dimethylbenz(a)anthracene was administered by gavage, in three 10 mg/rat consecutive doses. A part of animals drank melatonin in a concentration 100 microg/ml of tap water, continuously from the beginning of irradiation and 26 weeks after its end. The aim of the experiment was to investigate the preventive effect of melatonin on mammary tumor patterns. Relatively low incidence of mammary tumors in the noninfluenced group was probably connected with generally very low sensitivity of Wistar:Han female rats to single dose of chemocarcinogen in mammary carcinogenesis induction. In our trial melatonin decreased markedly the volume of mammary tumors, but did not influence any other tumor characteristics. The chemopreventive effect of melatonin, derived from in vivo realized mammary carcinogenesis study in female Wistar:Han rats was limited. The cancer preventive properties of melatonin should be investigated in the future especially from the standpoint of susceptible strain, effective doses, and mode plus sufficient length of application.

Repeated administration of carcinogen in critical developmental periods increases susceptibility of female Wistar: han rats to mammary carcinogenesis induction.

Analysis and knowledge of individual strain susceptibility of experimental animals to induction of carcinogenesis is important especially in regard to possibility of transfer of these facts to human pathology, first of all to chemopreventive projects. Our group (AHLERS et al. [1]) reported very low sensitivity of female Wistar:Han rats to induction of mammary carcinogenesis by 7,12-dimethylbenz(a)anthracene (DMBA) and by N-methyl-N-nitrosourea (NMU). The aim of this paper was to increase the sensitivity of females of this strain to mammary carcinogenesis induction by repeated administration of NMU in a dose 50 mg/kg of b.w. in critical periods: on 3-4 postnatal days, on 21 day (critical period for development of ductal parts of mammary gland) and between 50-55 days (maximal proliferation of whole gland). In comparison with 38% incidence of mammary tumors after the single dose and 65% incidence after 3 subsequent doses between 50-60 days, the combination of administration (only) on 21 day and between 50-55 postanatal days resulted in 88% incidence the sensitivity of animals reached the level of highly susceptible rat strains. The latency period was significantly increased in groups with NMU given on 3-4, 21 days and between 45-55 days respectively, on 21 day and between 45-55 days in comparison with control group (one dose of NMU). The tumor frequency per group and per animal in all groups with repeated NMU administration was significantly higher than that of control group. The volume of tumors was not influenced either by repeated carcinogen application or by time of its administration. These results expand the possibilities of analysis of carcinogen effects in individual periods of rat postnatal development.

Chromosomal aberrations, sister chromatid exchanges and high frequency cells in young patients with neurofibromatosis 1 (NF1).

Chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and high frequency cells (HFCs) have been assessed in peripheral blood lymphocytes of 10 neurofibromatosis (NF1) patients and 10 healthy controls. In both groups, the spontaneous rates and the induced (bleomycin for CA and MMC for SCE) frequencies were analyzed. No differences between cells from NF1 patients and controls were observed with respect to spontaneous or bleomycin induced CA. Spontaneous or MMC induced SCE frequencies were also similar in NF1 patients and controls. HFCs, on the contrary, were statistically lower in NF1 patients.

Sequential intermediate high-dose therapy with etoposide, ifosfamide and cisplatin for patients with germ cell tumors.

Intermediate high dose VIP (etoposide, ifosfamide, cisplatin) achieved comparable efficacy and improved tolerance in comparison with high-dose chemotherapy plus PBSC in poor risk germ cell tumors. The aim of this study was to confirm the effectivity and tolerance of this regimen in clinical practice. Twenty-five consecutive patients, 9 previously untreated with poor prognosis and 16 relapsed, were treated with 1.6 VIP or 1.9 VIP+PBSC. A relative dose intensity of 1.6 VIP was used in 14 patients and 11 patients received the intensity of 1.9 VIP. Clinical response was achieved in 56% of patients. Fifty-eight percent of patients have survived more than 1 year and 44% more than 2 years. No significant difference was noted between previously treated and untreated patients, as well as between the patients on 1.6 VIP and 1.9 VIP, with the exception of improved 1-year survival of patients on 1.9 VIP. One of four cisplatin-refractory patients achieved durable partial remission with a normal level of tumor markers. Serious non-hematological toxicity was rare. Myelotoxicity of 1.9 VIP was less serious in comparison with 1.6 VIP regimen, but the difference was not significant. Sequential intermediate high-dose therapy is an effective and tolerable regimen for patients with poor risk germ cell tumor as well as for relapsed patients.

Carboplatin and cyclophosphamide in the treatment of metastatic seminoma.

Cisplatin-based chemotherapy is highly effective in advanced seminoma, but at the cost of a considerable toxicity. The response rate of carboplatin is comparable with cisplatin combinations but the relapse rate is higher. Our study assesses the efficacy and the toxicity of the combination of carboplatin and cyclophosphamide in patients with advanced seminoma. Nineteen consecutive patients received 6 cycles of intravenous cyclophosphamide 750 mg/m2 and carboplatin 350 mg/m2, repeated every 21 days. The overall objective response rate was 100%, 11 patients (58%) achieved a complete response and 8 patients (42%) showed a partial response. At median follow up of 4.2 years 3 patients (15%) relapsed. The 2-year disease-free survival and the overall survival are 72 and 94%, respectively. This outpatient treatment was well tolerated and the toxicity was mild. One patient had granulocytopenic fever and one patient had grade 3 cystitis. The combination therapy with carboplatin and cyclophosphamide is an effective and tolerable regimen in advanced seminoma.

Double modulation of 5-fluorouracil by leucovorin and low-dose methotrexate in advanced colorectal cancer.

A phase II study was carried out to evaluate the efficacy and toxicity of a double biochemical modulation of 5-fluorouracil (5-FU) by methotrexate (MTX) and leucovorin (LV) in patients with advanced unresectable colorectal cancer. Forty-two patients with measurable metastases of colorectal cancer were treated with 5-FU in daily doses of 600 mg/m2 given in a 6-hour intravenous (i.v.) infusion on days 1-5, LV 50 mg/m2 i.v. on days 2,3 and 5, and MTX 40 mg/m2 i.v. on days 1 and 4, every 4 weeks. Twenty-eight patients had a single metastatic site, eleven double, whereas three had more than two metastatic sites. Objective response (one complete response) was observed in 12 of 40 patients (30%) (95% confidence interval 16-48), stable disease in 19 patients (47%) and progression in 9 patients (23%). Overall median survival was 12 months. Median time to progression was 6 months. Treatment was generally well tolerated. The most frequent adverse reactions were stomatitis (38%), nausea and vomiting (35%), diarrhea (31%), leukopenia (18%), and plantar-palmar erythroderma (15%). The combination of 5-FU, LV and MTX seems to be an active regimen in advanced colorectal cancer.

Long-term (15 years) results of extended field radiotherapy in Hodgkin's disease.

From 1975 to 1990, 214 patients with the pathological Stage IA, IB, IIA, IIB and IIIA of Hodgkin's disease were treated by supradiaphragmatic and/or infradiaphragmatic mantle technique. Complete remission was achieved in 70 patients (8%) by means of radiotherapy only. Partial remission was achieved in 9 patients (2%). The survival at 10 years was 86% and 15 years it was 66%. The most frequent late complications were hypothyreosis, Lhermitte's syndrome and radiation pneumonitis.

Clinical stages of cutaneous malignant melanoma in Bulgaria.

The aim of the study was to investigate the distribution of the newly diagnosed cases with cutaneous malignant melanoma by clinical stages in Bulgaria over the period 1993-1995 as a reason for improving both mclanoma prevention and control. Over the period 827 new cases with cutaneous malignant melanoma are registered in the country. A representative sample of 671 cases has been taken. The cases with a localized melanoma (Stage I and II) were prevalent - 509 (75.8% of all studied cases). The thick melanomas (Stage IIB) were most frequently encountered among the primary lesions. They were 207 cases (30.8%). The thin melanomas (Stage IA) were only 41 (6.1%). The proportion of the cases with nodal and in-transit metastases (Stage III) -122 (18.2%) and the proportion of the cases with distant metastases (Stage IV) -40 (6.0%) were quite high. The analysis of the results shows that the cases with cutaneous malignant melanoma in Bulgaria are detected quite late. The cases with early-diagnosed melanoma are prevalent among women, young persons and urban population, and the cases with advanced melanoma are more frequent among men, persons older than 50 and rural population.

Trends in the incidence of non-melanoma skin cancer in Slovakia, 1978-1995.

Non-melanoma skin cancer (NMSC) incidence in Slovakia in the period 1978-1995 was analyzed. A total of 38,629 microscopically confirmed NMSC cases (19,600 in males and 19,029 in females) were registered by the National Cancer Registry: 31,714 (82.1%) were basal cell carcinomas (BCC), 6,396 (16.6%) squamous cell carcinomas (SCC) and only 519 (1.3%) other NMSC. Age standardized rates of NMSC increased in the given period by 59.1% in males and 58.5% in females. The greatest increase was observed for BCC, 70.4% and 65.0% in males and females respectively, the smaller for SCC, 13.5 and 18.8%. In the period 1993-1995 age-standardized incidence rates per 100,000 were 38.0 for BCC, 6.7 for SCC and 45.5 for all NMSC in males and 29.2, 3.8 and 33.6, respectively in females. The observed marked increase of incidence with age was particularly pronounced for SCC. In both sexes, head and neck was the most common localization of BCC and SCC (84.2 to 74.7%), followed by trunk for BCC (17.0% in males and 10.8% in females) and by upper limbs for SCC (with 11.6% in males and 12.5% in females). Very fast increase of BCC incidence over time, its slower increase with age as compared to SCC incidence and body-site distribution suggest that BCC etiology is much more similar to melanoma etiology than SCC one. Registration of NMSC in relation to changes in possible risk factors (i.e. sun exposure/protection, ozone layer decrease) is important to study the mechanism of disease occurrence and to support public health interventions.

Expression of Bcl-2 in dysplastic and neoplastic cervical lesions in relation to cell proliferation and HPV infection.

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death in a variety of tumors. The bcl-2 proto-oncogene is involved in the development of human follicular lymphomas and also in a number of solid tumors such as carcinomas of prostate, breast, lung and GIT. The present study was designed to analyze the role of Bcl-2 expression in cervical intraepithelial squamous neoplasias (CIN) and cervical invasive carcinomas. Special attention was given to the association of Bcl-2 expression with the grade of the lesion, proliferative activity (expression of nuclear antigen of proliferative cells - PCNA) and human papillomavirus (HPV) DNA positivity. We examined tissue samples obtained from 86 women with varying degrees of cervical disease. Bcl-2 and PCNA were investigated using immunohistochemical staining and detection of HPV DNA was performed by hybridization in situ. Increased Bcl-2 expression was observed in advanced degrees of dysplasia and in carcinomas. We found a strong association between the presence of Bcl-2 in pathological epithelium with both the degree of dysplasia and the proliferative activity. We also observed a significant correlation between the amount of Bcl-2 positive lymphocytes infiltrating the lesions and the degree of disease. We, therefore, suggest that Bcl-2 expression in these lymphocytes may influence the antiviral or antitumor immune response. On the other hand we did not detect any significant correlation between the Bcl-2 oncoprotein and the presence of HPV. These results indicate that Bcl-2 may play an important role in the development of cervical cancer.

Loss of heterozygosity on chromosome 5 in vicinity of the telomere in gamma-radiation-induced thymic lymphomas in mice.

Analysis of the loss of heterozygosity at the D5Mit143 locus was done for thymic lymphlomas induced by gamma-irradiation of mice from two reciprocal backcrosses (BALB/c x CcS-13)F1 x BALB/c and (BALB/c x CcS-13)F1 x CcS-13. BALB/c mice are susceptible to gamma-ray induction of lymphomas. The CcS-13 strain is one of 20 CcS/Dcm (CcS) series of recombinant congenic strains, and the CcS-13 mice are resistant to gamma-radiation-induced lymphomas [1, 8]. Our preliminary tests show 50% (6/12) frequency of allelic loss at the D5Mit143 locus in thymic lymphomas induced by gamma-irradiation of the mice from (BALB/c x CcS-13)F1 x BALB/c backcross. Yet, in gamma-radiation-induced lymphomas from the backcross made in opposite direction, namely, (BALB/c x CcS-13)F1 x CcS-13, the analysis with the DSMit143 marker revealed low incidence of the loss of heterozygosity, 6.7% (15). The D5Mit143 locus resides in the distal part of chromosome 5, close to the telomere. Allelic loss of heterozygosity at the D5Mit143 locus showed strain specificity. In each case, the lost allele derived from the CcS-13 resistant strain. Our current results and previously done) linkage analysis [8] let us to suspect existence of a putative tumor suppressor gene for gamma-radiation-induced lymphoma at the region of murine chromosome 5.

The expression of apoptosis-related proteins and the apoptotic rate in glial tumors of the brain.

Modern molecular biology methods allow a more precise analysis of the biological characteristics of tumors and, consequently, a more precise treatment plan. The determination of apoptotic rate and expression of apoptosis-related proteins belong among the important prognostic/diagnostic markers in many tumors. The validity of these factors had not yet been sufficiently analyzed in astroglial tumors. The aim of this work was therefore to study mutual relationships between apoptotic rate, expression of apoptosis-regulating proteins and some clinical and histopathological data. The TUNEL method was used for the determination of apoptosis in 44 astroglial tumor specimens. The percentage of TUNEL positive cells was expressed by the TUNEL index (TI). The TI data was compared with the immunohistochemically detected expression of proteins involved in apoptosis (BCL-2, FAS, FAS-L, and caspase 1), with grading, age, proliferative activity (assessed by PCNA expression analysis) and overall survival of patients. The statistical evaluation of results was done by two-way sample analysis of variance. We have demonstrated significantly higher values of both TI and expression of FAS-L and caspase 1 in low grade tumors, which were characterized by a longer survival, lower average age and a lower expression of PCNA. FAS-L expression correlated significantly with the expression of the caspase 1. No significant difference was found between the expression of BCL-2 and FAS. These results suggest that the determination of TI in astroglial tumors may be an important prognostic marker. The expression of FAS-L and caspase 1 in low grade astroglial tumors could indicate the increased readiness to apoptosis via the FAS/FAS-L cascade.

Detection of Epstein-Barr virus in Hodgkin's lymphoma (patients in the Czech Republic).

The frequency of EBV demonstrated in patients with Hodgkin's lymphoma (HL) shows geographical variability. In the present study, we investigated the frequency of EBV in HL patients in the Czech Republic. The presence of EBV was determined by immunohistochemistry (IHC) with anti LMP-1 antibody and by in situ hybridization (ISH) method for EBERs. We studied 142 cases with HL. The age of patients ranged from 4 to 82 years. The male to female ratio was 1.2 (males 55.6%). In the series of 142 patients 47 (33%) positive cases were found. The incidence of EBV-positive results was significantly, higher in males than in females (70.2 vs. 29.7%, p = 0.023). Five patients were found in the age group below 10 years. They were positive with LMP-1 antibody and for EBERs in ISH method. The same results were discovered in two patients above the age of eighty. The most frequent histologic types of HL were nodular sclerosis (64 cases) and mixed cellularity (62 cases), respectively. The former type contained 16 EBV-positive cases (25%) and the latter 24 (38%) positive cases. The lymphocyte depletion type 2 (67%); lymphocyte rich type 5 (38%). EBV-positivity examined by ISH and IHC methods determined not only diagnostic Hodgkin cells and Reed-Sternberg cells but also small lymphocytes. In IHC method were small lymphocytes positive in 11 cases, more sensitive ISH revealed 32 positive cases.

Application of cytogenetic endpoints and Comet assay on human lymphocytes treated with vincristine in vitro.

The genotoxic potential of vincristine is assessed on human peripheral blood lymphocytes following administration of the drug at a dose 0.0875 microg/ml by use of single cell gel electrophoresis - Comet assay (SCGE), analysis of structural chromosome aberrations (CA), micronucleus assay (MN) and sister chromatid exchange (SCE) analysis. In vitro treatment of human lymphocytes with vincristine was performed on cells in G0 phase, as well on lymphocyte cultures 24 hours after stimulation with mitogen phytohemagglutinine. For the Comet assay at 24, 48 and 72 h the treated cells were embedded in agarose on slides, lysed with alkaline lysis solution and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depends on the amount of DNA damage. For the analysis of structural CA cells were grown on F-10 medium for 48 hours, and for MN and SCE analysis for 72 hours. The results on SCGE showed an increase in tail length compared to control both in cells treated in G0 and in cells treated 24 h after mitogen stimulation. The amount of DNA damage was higher in cells treated with vincristine 24 h after mitogen stimulation. Administered concentration of drug caused total inhibition of lymphocytes growth in 72-h cultures for MN and SCE analysis indicating strong microtubule distruptive effects of vincristine. Analysis of structural CA reveals chromatid breaks and acentric fragments as the main aberration types both in cells treated in G0 and in cells treated 24 h after mitogen stimulation. Number of these aberrations was higher in cells treated in G0 phase. Results obtained in this study by use of different cytogenetic endpoints confirmed that vincristine exhibits both aneugenic and clastogenic effects on human lymphocytes.

Dexamethasone does not enhance antileukemic activity of cladribine in mice with leukemias L1210 and P388.

Combination of corticosteroids with new purine analogs such as cladribine 2-chlorodeoxyadenosine, (2-CdA) and fludarabine (FAMP) is controversial. The possibility of potentiation of antineoplastic activity of 2-CdA or FAMP by corticosteroids has not been documented so far. On the other hand, such combination may increase immunosuppression and the risk of infections. The aim of our study was to evaluate the influence of 2-CdA and dexamethasone (DEX) on the survival time of mice bearing lymphoid leukemias L1210 and P388. CD2F1 strain mice (132 male) were used in the experiment. The animals were injected i.p. on day 0 with 10(6) leukemic cells. The drugs were given on days 1-5 i.p. in the following concentrations: 2-CdA - 20 mg/kg, DEX 1.25 mg/kg, DEX 2.5 mg/kg, DEX 5 mg/kg, DEX 10 mg/kg, alone and in combination. The animals were observed daily for survival for a minimum of 30 days. The efficacy of the therapy against leukemia (defined as increase in lifespan - ILS) was assessed as the percentage of the median survival time (MST) of the treated group(t) to that of the control group(c): ILS(%) = (MSTt/MSTc) 100. The survival time of mice bearing L1210 or P388 leukemia treated with both drugs simultaneously was not longer than that of mice treated with either of drugs alone. Combination of 2-CdA and DEX in doses 5 and 10 mg/kg resulted in decrease of survival time of animals bearing P388 leukemia as compared with the control group without any treatment. Our study revealed that combination of 2-CdA with DEX in both leukemias is not more effective than 2-CdA alone. These results may indicate that routine addition of corticosteroids to purine analogs in the treatment of lymphoid malignancies is not warranted.

Comparison of three in vitro assays at evaluation of IC50 of acetylsalicylic acid, ferrous sulfate, amitriptyline, methanol, isopropanol and ethylene glycol in human cancer cells HeLa.

Evaluation of the 50% inhibitory concentration (IC50) of acetylsalicylic acid, ferrous sulfate, amitriptyline, methanol, isopropanol and ethylene glycol was done on human cancer cells cultured in in vitro conditions. Three different in vitro assays were used in this study: the plating efficiency test, the microprotein test and the neutral red uptake test. Obtained results were evaluated by statistical methods. All used methods seem to be useful for screening a cytotoxic potential of the tested chemicals. The knowledge of cytotoxic effects of frequently used chemicals on mammalian cells is important not only for necessary in vitro genotoxicity and carcinogenicity studies but also for assessing the toxicity of chemicals to find out possible hazards to the human health. Results presented in this paper underline the usefulness of the wider methodological approach for the comparison of the different endpoints as well as a necessity for selection of a battery of in vitro cytotoxicity tests allowing to estimate the possible harmful effects of xenobiotics.

Adjuvant clodronate therapy in patients with locally advanced breast cancer--long term results of a double blind randomized trial. Slovak Clodronate Collaborative Group.

Between March 1990 and May 1993 seventy three patients with previously untreated breast cancer, Stage III or IV without osseal metastases were randomized to sodium clodronate 1600 mg daily p.o.(arm A = 37 patients) or placebo (arm B = 36 patients) over 2 years, additionally to standard therapy. Ten patients were not evaluable for response because of short duration of therapy (less than 2 months). Bone metastases developed in 30% of patients in arm A and 23% patients in arm B. Median time to appearance of bone metastases was 13 months in arm A and 28 months in arm B. Non-bone metastases appeared in 48% patients in arm A and in 48% patients in arm B. Time to development of non-bone metastases was 20 months in arm A and 16 months in arm B. Five-year survival was 41% in arm A and 39% in arm B. There were no significant differences between the treated and control arms.

The impact of radiotherapy on the incidence and time of occurrence of local recurrence in early-stage breast cancer after breast conserving therapy.

There is still little information on the delay of local recurrence after conservatively treated and irradiation breast cancer. To evaluate the impact of radiation therapy (RT) on the incidence and on the time of occurrence of ipsilateral breast tumor recurrence (IBTR), we reviewed the treatment results in 415 women with UICC Stage I or II unilateral breast cancer. All underwent breast conserving surgery (BCS) and full axillary dissection between 1983 and 1987. Out of them 309 patients were irradiated and 106 were not. The median dose of RT was 50 Gy in five weeks to the whole breast. Systemic therapy, when it was given, consisted of 6-cycles of CMF for node positive premenopausal women and 20 mg tamoxifen for three years for postmenopausal women. The median follow up time was 120 months in survivors. The 10-year actuarial IBTR rate was 36.6% for the nonirradiated and 9.1% for the irradiated women (p = 0.0000); 48.6% for patients treated with CMF and 4.2% for those treated with CMF plus RT (p = 0.0051); 29.0% for patients treated with tamoxifen and 7.9% for those treated with tamoxifen plus RT (p = 0.0318). The patient's age and the presence of an extensive intraductal component (EIC) were both highly associated with the likelihood of tumor recurrence in the treated breast. Patients under 41 years of age had an actuarial 10-year IBTR rate of 75% without RT and 17. 1% with RT (p = 0.0006). Women with an EIC positive tumor had an IBTR rate of 88.9% when RT was not given and 27.2% when RT was given (p = 0.0003). In invasive lobular cancer, irradiated patients had a IBTR rate of 2.3%, compared to 53.2% for nonirradiated patients (p = 0.0008). RT resulted in a significant delay in the appearance of IBTR (p = 0.0250) and the median time was increased by 20.0 months. We conclude that RT has the property of not only preventing but also delaying IBTR. In invasive lobular tumors the risk of IBTR is very high when RT is omitted, but BCS plus radiation therapy is effective treatment. Patients wih EIC positive tumor are at high risk of IBTR even when a median dose of 50 Gy is given to the whole breast.

Influence of previous treatment on palliative effect of HDR brachytherapy in advanced esophageal cancer.

General effectiveness and influence of previous treatment on value of palliative HDR brachytherapy were assessed in 35 patients with advanced esophageal cancer treated from 1992 till 1997 with brachytherapy HDR (BT). Twelve of them were treated only with BT, II received previously chemoradiotherapy (CHTT), 12 teleradiotherapy (TT). BT appeared to be effective method of palliation. No significant differences in effectiveness of BT in analyzed groups were observed. Sever complications were observed in 9 cases (26%), and that in patients treated previously. Brachytherapy seems to be efficient after previous treatment, however, in this case, the risk of complications increases.

Male breast cancer. Does the prognosis differ compared to female?

Due to the low incidence of breast cancer in males there are not many reports in the literature. In this study we analyzed results of treatment in 65 breast cancer males, who had been treated in one institution. Radical surgery was performed in 45 patients. Lymph node metastases were found in 25 patients (55.5%), the tumor was usually moderately differentiated (21 pts - 46.7%). Median survival after radical surgery was 73 months compared to 38 months for nonsurgical patients (p < 0.0001). In the group of males after radical surgery the results of 5-, 10- and 15-year survival rates were 69.8, 59.7 and 31.3% respectively. Comparable analysis of two subgroups of patients with favorable (T1 or T2, N0, grade I or II) and unfavorable (T3 or N+ or grade III) prognostic factors was also performed. In the first subgroup the 5-, 10- and 15-year survival rates were 90, 77.4 and 62%, compared to 61.8, 23.1 and 23. 1% for the second subgroup. The multivariate analysis showed grading and node status as the strongest parameters influencing survival. Relative risk of death was over 3 times higher for nodal metastases and near 3 times higher for high grade carcinomas (p < 0.01), compared to patients without metastases and low grade of tumor. Similar analysis was performed when 45 males were compared to 500 selected women, with similar clinical parameters (age, node status, grading). Again, data indicated grading and lymph node status as the strongest prognostic factors. It was not unlikely, that gender had some influence on prognosis, when relative risk of death for males was over 1.5 times higher than for females, but this result was not clearly significant (p < 0.1 ). The question, whether male breast cancer prognosis is worse then in female remains open. Multiinstitutional prospective studies are needed in this area.

The current look at high-dose chemotherapy in breast cancer minireview.

High-dose chemotherapy (HDC) in high-risk breast cancer is one of the possible approaches how to improve therapeutic results, eventually, to overcome the incurability of the disease. In recent randomized studies superiority of HDC to conventional therapy has not been unambiguously established. Nevertheless, many oncologists, as well as, patients are so convinced of HDC benefits, that they are not willing to take part in randomized studies. At an ASCO Annual Meeting (American Society of Clinical Oncology) in May 1999 in Atlanta - preliminary results of five large randomized studies phase III were presented (2 studies on metastatic breast cancer and 3 studies on high-risk breast cancer with more than 10 positive lymph nodes). The ASCO was informed of an investigation into serious scientific misconduct in a clinical trial that was presented in a plenary session of its Annual Meeting. The results of Dr. Bezwoda's research were presented at ASCO's Meeting as one of four plenary papers on the investigational therapy and was the only one to clearly indicate a survival benefit in the high-dose regimen. Preliminary results presented there, however, did not confirm the original hypothesis of the high efficacy of HDC. It is necessary to wait for definite results (within two or three years, because enrollment of patients either has been finished or is being finished just now) and several parameters may change. In view of hitherto results, some investigators think that there is no need to continue in similar intensive studies. Still some believe that different modifications of therapeutic regimens or new, less toxic drugs should be tested which may lead to more effective and safer HDC.

Chronic myelogenous leukemia as gene activation model in oncology minireview.

Many unique features of chronic myelogenous leukemia (CML) make it as a model for studying the development of leukemia in humans. Chronic myeloid leukemia is a disease of the hematopoietic stem cell that progress in a multistep fashion. The biphasic or triphasic clinical course of the disease exemplies the multistep process of tumor progression from the indolent chronic phase to a more aggressive and terminal blast crisis. CML was the first neoplastic disease shown to be associated with consistent karyotypic abnormality now known as the Philadelphia (Ph) chromosome. The result of the Philadelphia chromosome translocation t(9;22)(q34:q11) is the transposition of the c-abl oncogene from chromosome 9 to chromosome 22, where it is fused with part of the her gene. The translocation generates a new hybrid bcr-abl gene which plays a crucial role in the pathogenesis of CML. Presently, CML is perhaps the best understood cancer in humans and the model of oncogenesis mediated by the Ph chromosome translocation is one of the best-characterized example of gene activation in leukemia.

Differential sensitivity of ovarian carcinoma cell lines to apoptosis induced by the IMPDH inhibitor benzamide riboside.

The differential sensitivity of examined human ovarian carcinoma cell lines (CH1, A-2780 and SKOV-3) to the IMPDH inhibitor, benzamide riboside (BR), was demonstrated with the aid of MTT assay. Present data show that all three examined ovarian carcinoma cell lines were sensitive to the cytotoxic effects of BR in the order of sensitivity CH1, SKOV-3, A-2780, (IC50 = 2.8, 4.0 and 7.4 microM, respectively). Although the IC50 of SKOV-3 cells was similar to that previously determined by others, more than 20% of SKOV-3 cells remained viable in a plateau up to 40 microM BR concentration. This relative resistance of SKOV-3 cells to BR corresponded to the absence ofBR-induced apoptosis in SKOV-3 cells, which together with clearly demonstrated sensitivity of CH1 cells to BR-induced apoptosis, established by flow cytometry (presence of nuclei with sub-G0 DNA content, Annexin V binding) and western blotting (poly-ADP-ribosyl-polymerase (PARP) cleavage), further stressed the role of drug-induced apoptosis in the over-all drug-induced cytotoxicity.

Nucleoli in cells of the granulopoietic proliferating compartment in patients suffering from the refractory anemia of the myelodysplastic syndrome.

Granulocytic precursors of the granulopoietic proliferating compartment (GPC) were investigated in patients suffering from refractory anemia (RA) of myelodysplastic syndrome (MDS) to provide an information on the number of nucleoli and incidence of main nucleolar types in these cells stained with the simple cytochemical procedure for the demonstration of RNA. The results demonstrated that the incidence of main nucleolar types in all stages of the granulopoietic proliferating compartment in RA patients of MDS generally did not differ in comparison with that of control patients without a disturbed granulopoiesis. In contrast, the number of nucleoli expressed by the values of the nucleolar coefficient in all stages of GPC in RA patients was significantly smaller than in control persons. In addition, the values of the nucleolar coefficient of myeloblasts in patients with RA of MDS were close to those in patients with acute myeloid leukemias.

Influence of ribavirin on the micronucleus formation and in vitro proliferation of human lymphocytes.

Cytotoxic effects of the antiviremic drug ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) were evaluated in vitro measuring micronucleus formation and cell proliferation kinetics in whole blood cultures employing cytokinesis block (CB) micronucleus test. The cells were exposed to ribavirin doses ranging from 0.05 0.65 micromol/ml at three different incubation times. The frequency of micronuclei in treated samples demonstrated relatively low ability of ribavirin to induce micronuclei. However, the lowest concentration ofribavirin markedly changed the frequency of mononucleated and multinucleated cells, particularly binucleated ones, which declined significantly. The decline in the frequency ofbinucleate cells was followed with accumulation of greater number of mononucleate cells. Decreased proliferation potential of lymphocytes treated with ribavirin indicates that cells are arrested prior to metaphase. The present investigation showed that ribavirin is capable to induce a delay in cellular proliferation at all the doses assayed. The study demonstrated that CB micronucleus assay is simple and rapid method that can be used to assess toxic effect of drugs in vitro.

The expression of proliferating cell nuclear antigen (PCNA) in leukemia cell lines HL-60 and K-562 at the light and electron microscope level.

PCNA antigen was localized at the light and electron microscopes level in two human leukemia cell lines HL-60 and K-562. PCNA expression was used to discriminate cycling from non-cycling cells. PCNA protein at the level of the light microscope was present in 70% of the cell in HL-60 cell line and in 65% of the cells in K-562 line. Streptavidin immunogold method was used for localization of PCNA expression at the ultrastructural level. Positive staining for this protein was seen as granular pattern in the nucleus and in the cytoplasm. In the nucleus the gold particles were seen to be associated with heterochromatin and euchromatin of the leukemia cells. In cytoplasm it was found on the endoplasmic reticulum and associated with ribosomes. Controls of the leukemia cells incubation with normal mouse serum showed no labelling at the light and electron microscope level.

Bovine seminal ribonuclease induces in vitro concentration dependent apoptosis in stimulated human lymphocytes and cells from human tumor cell lines.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease has been proven to have important biological properties as aspermatogenic, antitumor, embryotoxic and immunosuppressive activities. Recently we published preliminary results concerning the ability of bovine seminal ribonuclease (BS RNase) to induce time dependent apoptosis in Con-A stimulated human lymphocytes and in human tumor cells based on DNA content and cell cycle analysis. In this study we bring more confirmative data concerning the concentration dependent in vitro induction of apoptosis in stimulated human lymphocytes and tumor cells of three human cell lines using the most sensitive and specific cytometric method for at present apoptosis determination the indirect TUNEL. BS RNase 50 microg/ml was proven to induce 49.7, 54 and 68.1% apoptosis in the cells of the ML-2 myeloid cell line and two neuroblastoma cell lines: NB-1 and NB-2, respectively. In Con A-stimulated human lymphocytes, BS RNase also induced apoptosis, eventhough not so pronounced as in human tumor cell lines. In all cultures the induction of apoptosis was proportional to BS RNase concentration ranging from 2-50 microg/ml and correlated with proportional decrease in 3H-thymidine incorporation into the newly synthesized DNA. Side by side with the ability of BS RNase to suppress the growth of human tumors transplanted to nude mice, these biological properties determine this enzyme as a promising agent with potential clinical application.

Prognostic significance of genetic markers in chronic myelogenous leukemia patients after bone marrow transplantation.

Chronic myelogenous leukemia (CML) is a malignant disease of hematopoietic stem cell with a biphasic or triphasic clinical course and most often, with a fatal outcome. Significant progress in improving outcome for patients with CML has been achieved over past years. This can be attributed to marked improvement in therapeutic protocols and increased use of bone marrow transplantation (BMT) which remains the most effective option for long-term disease control of patient with CML. The residual leukemic activity in patients after BMT remains a central clinical question. To effectively monitor minimal residual disease leukemic activity after BMT, molecular genetic techniques are currently utilized in conjunction with cytogenetic assays. Because the clinical significance of detection minimal residual disease in CML remains to be determined, we performed cytogenetic analysis and PCR amplification technique in 37 Ph+ CML patients. All patients received transplants for CML in Bratislava between years 1992 and 1999. Our results suggest that PCR positivity after transplant is of limited prognostic significance for particular individuals and can be used to identified groups of individuals at elevated risk of relapse.

p53 single nucleotide polymorphisms and bladder cancer.

Two p53 germline polymorphisms, a BstUI in exon 4 and a MspI in intron 6 were studied using polymerase chain reaction (PCR) based methods in 50 patients with bladder cancer and 145 healthy controls. Increased frequencies of the BstUI and MspI A2 alleles were found to be associated with statistically non-significant (p = 0.2308 and p = 0.5959) but increased odd ratios for bladder cancer (OR 1.44, 95% CI 0.82-2.27 and OR 1.20, 95% CI 0.61-2.33). Statistically significant difference between patients with bladder cancer and controls was found in the distribution of MspI genotypes. There was a significantly lower proportion of the heterozygous A1A2 genotype in all patients but not in controls (p = 0.0354). The results of this study suggest that BstUI and Msp1 germ line polymorphisms of the tumor suppressor gene p53 marginally modify the risk of bladder cancer.

Bone marrow and peripheral blood leptin levels in lymphoproliferative diseases--relation to the bone marrow fat and infiltration.

Leptin is a nonglycosylated protein produced mostly by adipocytes. The role ofleptin in body weight regulation through its anorectic effect in hypothalamus is very well known. Less known are other leptin effects such as the stimulation of hematopoesis and some parts of immunity system. The role of leptin in the pathogenesis of some malignant tumors is discussed. Only a little is known about bone marrow adipocyte leptin production. We examined leptin concentrations in the sera from peripheral blood and bone marrow, the percentage of bone marrow fat, the degree of bone marrow infiltration, the body mass index (BMI) in 42 patients with lymphoproliferative diseases. We found that bone marrow has significantly lower leptin levels (6,6+/-10,9 ng/ml) than peripheral blood (9,1+/-11,5 ng/ml) (p < 0.0001). Bone marrow and peripheral blood leptin levels have also a significant thin correlation (r = +0.91, p < 0.0001). Bone marrow (r = +0.55, p < 0.0005) and peripheral blood (r = +0.52, p < 0.0005) leptin concentrations are significantly correlated to BMI. Blood serum leptin (r = +0.46, p < 0.003) and bone marrow leptin (r = +0.40, p < 0.01) are related to the bone marrow fat percentage. In addition we found a negative correlation of blood serum leptin (r = -0.59, p < 0.0001) and bone marrow leptin (r = -0.42, p < 0.005) to bone marrow malignant infiltration. When we divided the patients into groups with bone marrow infiltration more than 10% and without or less than 10% infiltration, the first group had significantly lower peripheral blood (p < 0.001) and bone marrow (p < 0.02) leptin. We also confirmed a relation of bone marrow fat and infiltration (r = +0.49, p < 0.001). Our results suggest a relationship among leptin levels in blood or bone marrow and bone marrow infiltration in lymphoproliferative diseases. This fact needs further investigation and an evaluation of its application in clinical practice.