Training of prescreening cytotechnicians. A report on 10-year experience.

Institutional training of prescreening cytotechnicians started in 1972 in the Research Institute of Oncopathology, Budapest. Up to 1983 ten classes, i.e. 230 students have been trained. Authors acknowledge the good achievements of their students and state that the diagnostic reliability of the qualified cytotechnicians has been recognized by professionals. Their activity resulted in a considerably increased number of screenings and recognized cervical cancers. In 1964 merely 25 600 examinations, whereas in 1982 altogether 1 030 000 examinations were performed. Cervical cancers announced in 1978 were 597, but in 1982 this number rose to 1022, being 661 (66%) in stage 0. A so-called Cervix Program based on the activity of cytotechnicians has been initiated by the Hungarian Ministry of Health. In view of the authors' ten-year experience the execution of this enormous enterprise seems possible.

Derivatives of benzo(c)fluorene. X. Inhibitory effect of Benfluron on cellular immunity.

The effect of a cytostatic drug Benfluron-- 5(2-N,N-dimethyl-amino-ethoxy)-7-oxo-7H-benzo(c)fluorene hydrochloride was tested in mice on skin graft survival, graft-versus-host reaction (GVHR) and for mitogenic stimulation of human lymphocytes. Application of Benfluron resulted in a prolonged skin graft survival. The regional and systemic GVHR was potentially inhibited by p. o. administration of Benfluron at a dose of 100 mg/kg of body weight. Allogeneic spleen cells incubated with Benfluron at concentration of 5-20 micrograms/ml showed reduced capability to induce GVHR. PHA or PWM stimulation of lymphocytes was completely inhibited in the presence of 2 micrograms/ml Benfluron. Benfluron as a potent immunosuppressive agent can be considered useful for the prevention of GVHR.

Peripheral blood leukocyte adherence inhibition (LAI) reactivity before and during progressive growth of B77 tumor in rats.

Sequential changes in B77-rat fibrosarcoma (B77-RF) extract-induced leukocyte adherence inhibition (LAI) reactivity of peripheral blood leukocytes (PBL) were investigated in rats from the time of tumor inoculation through progressive growth and/or surgical excision of tumor. The time-course profiles of LAI reactivity of PBL in tumor bearing rats correlated with the stage of tumor growth. LAI reactivity of PBL in tumor bearing rats increased during 5-15 days after cell inoculation. Simultaneously, using in vitro 3H-uridine incorporation method for detection of T-cell population, the significantly increased number of immunocompetent lymphocytes was found. During progressive tumor growth (16 days and more after tumor cell inoculation) the LAI reactivity decreased. In this period moderate depletion of immunocompetent population of lymphocytes was observed. A significant decline in LAI reactivity was noted 10 days after a thorough excision of the tumors as compared to that prior to the surgical intervention. The kinetics of the observed time-course appearance of LAI reactivity and the demonstration of a rapid response following tumor challenge indicate further that LAI is a reliable in vitro measure of in vivo tumor immunity.

In vivo and in vitro effectivity of some platinum complexes.

The following platinum complexes have been tested: cis-DDP--cis-diamminedichloroplatinum(II), Platinex--1,2-diaminocyclohexaneplatinum(II)citrate, Platuran--1,2-diaminocyclohexaneplatinum(II)-glucarate , TMA--1,2-diaminocyclohexaneplatinum(II)-4-carboxyphthalate,o xo-PT--cis-diamminedichloro-trans-dihydroxyplatinum(IV), CHIP--cis-dichloro-bis-(isopropylamine)-trans-dihydroxyplatinum(IV ), CBDCA--cis-diammine-cyclobutane-1,1-dicarboxylatoplatinum(II ). The activity of all tested complexes againt L1210 cells was higher in soft agar colony assay when compared with suspension culture of the same target cells. Using various doses and schedules oxo-Pt, CBDCA and cis-DDP exhibited the highest in vivo activity against P388 leukemia.

Chromosomal aberrations, sister chromatid exchange (SCEs) and micronuclei induction with three platinum compounds (cis-DDP, CHIP, CBDCA) in V79 cells in vitro.

Three platinum compounds (cis-DDP, CHIP, CBDCA) were tested or induction of chromosome aberrations and micronuclei as well as for SCEs in V79 cells in vitro. There was a clear relationship between the degree of inhibitory activity of the tested drugs and the increase of studied markers of cytogenetic damage. Statistical significance was proved in increase of SCEs values in cis-DDP and CHIP-treated cells when compared with untreated controls.

Cell sensitivity to irradiation and DNA repair processes. II. The cell sensitivity to ionizing radiation of different LETs.

A new model of DNA single-strand break (SSB) and double-strand break (DSB) induction by radiations of different LET has been developed. Utilizing quadratic dependence of the dose that delta-electrons depart in the track of heavy particles the fraction of heavy particle energy deposited in the target of DNA dimensions has been calculated. SSBs arise from energy depositions in one strand of DNA, direct DSBs arise from two SSBs on opposite strands of DNA in the track of one particle. It is concluded that DSBs induced by gamma-radiation are mostly of enzymatic origin, meanwhile DSBs induced by high-LET radiation are direct DSBs. The dependence of the radiosensitivity D0-1 on LET (L) for isogenic mutants of E. coli with different sensitivity to gamma-radiation has been determined on the bases of the model and considering microscopic energy fluctuations. The shape of D0-1 (L) function is formed both by physical characteristics of radiation and by the ability of cells to repair some types of DNA damage. The model provides a basis for further investigation.

Experimental and clinical results of application of antitumor vaccines obtained by means of bacterial metabolism.

By means of metabolic products of the AB-56 strain of Bac. mesentericus it was possible to produce specific antitumor vaccines which both in experimental and clinical conditions proved to have prophylactic and therapeutic effects. 60-100% of the animals prophylactically immunized with these vaccines became resistant to a subsequent injection of living virulent cells, the percentage depending on the number of immunizations. The employment of similar autovaccines in the treatment of relapses and metastases in humans resulted in a 56% five-year survival of patients operated on for gastric cancer, in contrast to a 28% five-year survival of those without any vaccination.

Alkaline phosphatase from human uterine myoma. I. Purification and some properties.

Alkaline phosphatase from human uterine myoma was purified to homogeneity by butanol extraction, acetone precipitation, column chromatography on DEAE-cellulose, QAE-Sephadex and Con-A-Sepharose, gel filtration on Sephadex G-200 and preparative electrophoresis. The relative molecular mass (145 000), subunit mass (35 000), neutral sugars (7 micrograms/mg) and sialic acid (1 microgram/mg) content were estimated. pH optimum of the enzyme activity was 10.0-10.4, and Km for p-nitrophenyl phosphate was 0.23 mM. The enzyme was inhibited by Zn2+, phosphate, fluoride, EDTA and by treatment with neuraminidase. Mg2+ activated alkaline phosphatase and showed protective effect towards inhibition by EDTA and Zn2+. The uterine muscle alkaline phosphatase was also purified. It showed lower specific activity than myoma phosphatase, higher molecular mass (160 000) and subunit mass (76 400), higher neutral sugar and sialic acid content.

Monoclonal cryoglobulins and pyroglobulins.

In a series of 1182 monoclonal Ig, the authors demonstrated 10 cases with monoclonal cryoglobulin (0.8%) and 5 cases with pyroglobulin (0.42%) in the serum; in two of these cases both thermoproteins were present simultaneously. One observation revealed mixed cryoglobulin consisting of monoclonal IgM-kappa and polyclonal IgG. In 70 Waldenström's macroglobulinemias, monoclonal cryoglobulin occurred in 10%, pyroglobulin in 5.71%. In a subgroup of 744 monoclonal IgG, monoclonal cryoglobulin was demonstrated in 3 cases (0.4%), pyroglobulin in one case (0.13%).

Dose response effect of retinyl acetate on DMBA induced carcinogenesis in the hamster cheek pouch.

The role of vitamin A in the induction and growth of chemically induced tumors is still controversial. While in some studies vitamin A influenced inhibition of chemically induced tumors was observed, other studies report on an enhancing influence of this substance on tumor induction. The cheek pouch epithelium of sixty five Syrian golden hamsters was exposed to DMBA and different doses of retinyl acetate, singly and in combination with DMBA. The findings suggest that 1) a significant delay occurs in tumor induction in animals treated with DMBA in combination with a higher concentration (2.0%) of retinyl acetate, 2) retinyl acetate at lower concentration (0.5 and 1.0%) does not inhibit or delay tumor induction.

Effect of tissue selection on melanization of MI hamster melanoma.

Selection of the most melanized tissue of the slightly melanotic MI melanoma in hamster resulted in an origin of a new highly melanotic tumor line. It differed from the parental MI melanoma in the 5-fold higher melanin content, but its tyrosinase activity, growth rate and histological structure remained unchanged. Selection of the least melanotic tissue of the MI melanoma did not give positive results.

Enzymes of purine metabolism and membrane phenotype in malignant cells of some leukemia patients.

Lymphocytes from patients with chronic lymphatic leukemia, acute lymphoblastic leukemia and acute myeloid leukemia have been characterized by the activity of two enzymes involved in purine metabolism--adenosine deaminase and purine nucleoside phosphorylase and by the analysis of surface marker expression. The data obtained suggest that the activity of enzymes of purine metabolism can be used as a complementary diagnostic marker to conventional cell surface characteristics for the classification of lymphoproliferative diseases, especially in the case of T-acute lymphoblastic leukemia. The changes of the purine pathway enzymes may prove to be useful also as therapeutic determinants in hemopoietic neoplasia, particularly in the lymphoid malignancies.

Study of destructive mechanisms of the immune response to sarcoma I allograft in mice.

In B10 mice (H-2b) the Sarcoma I allograft (H-2a) was after a period of temporary progression definitely rejected by the allo-transplantation reaction. After a treatment of B10 mice with xenogeneic antithymocyte serum (ATS) the primary growth of the Sa I allograft was enhanced, later the allograft grew either permanently or, after a short regression, exhibited secondary growth, or permanently regressed. The destructive activity of spleen cells from untreated recipients, as measured according to the Winn neutralization test, increased at the time of tumor rejection and remained elevated. In ATS-treated recipients the destructive activity was markedly suppressed during primary tumor growth, at the time of temporary regression significantly increased and in permanent regressors remained elevated. In progressors there was again a decrease of this activity. The destructive activity in the spleen of regressors was found mainly in the cell fraction which did not adhere to nylon wool; these cells significantly accelerated the development and enhanced the destructive activity of normal spleen cells. The decrease of destructive activity in recipients with progressively growing tumors was detected both in cells adhering and in those which did not adhere to nylon wool. Besides this defect, the defect of proliferative activity of spleen cells was detected by the local graft-versus-host reaction test in non-treated as well as ATS-treated recipients. It could not be directly proven that the suppression of the alloimmune reactivity was caused by suppressor cells--in spleens of progressors there were found no cells capable to suppress the activity of effector cells nor the evolution of the destructive activity of normal spleen cells.

Derivatives of benzo(c)fluorene. XI. Effect of Benfluron on hemopoietic stem cells.

The effect of Benfluron-- 5-(2-N,N-dimethylamino-ethoxy)-7-oxo-7H-benzo(c)fluorene hydrochloride--on hemopoietic stem cells was tested by the production of spleen cell colonies (CFU-S) in irradiated mice following application of bone marrow cells and by the production of hemopoietic colonies (CFU-C) in semisolid agar. The reduced numbers of CFU-S were found in mice applied Benfluron or Benfluron-treated bone marrow cells. The increased numbers of CFU-S, however, were found in mice receiving bone marrow cells from Benfluron-treated donors. The production of CFU-C was decreased after 2 h incubation of bone marrow cells with 0.5-5 micrograms/ml of Benfluron, and fully inhibited at higher concentrations.

Immunosurgical approach to the treatment of malignant melanoma using 2,4-dinitrochlorobenzene (DNCB).

Non-specific local immunotherapy using 2,4-dinitrochlorobenzene (DNCB) was combined with additional excision of the primary melanoma. The treatment was completed by elective nodal dissection in cases of high risk invasive lesions. DNCB was also given in cases with advanced disease for the control of cutaneous lesions and disseminated metastases, respectively. Histological changes of regressed skin nodules and ways of DNCB administration are described. Comparison between surgery alone and the immunosurgical treatment was considered inappropriate at the present time due to limited number of cases.

The growth of lymphocyte colonies from peripheral blood of patients with malignant melanoma.

Lymphocytes from the peripheral blood of patients with malignant melanoma were cultivated using a modification of the two-layered cultivation method in a semisolid medium. The ability of the cultivated lymphocytes to form colonies and clusters in agar after stimulation with phytohemagglutinin was compared with the actual clinical state of patients. In patients with malignant melanoma, who show clinical signs of progress, significantly less colonies and clusters are formed than in controls. The number of colonies appears to be a more sensitive sign than the number of clusters. For orientation in clinical practice the number of large colonies is of an importance.

Immunological monitoring of melanoma patients on BCG immunotherapy. I. Enzyme-linked immunosorbent assay of human sera after BCG-treatment.

Purified lipopolysaccharide (LPS), isolated from BCG and purified protein derivative of tuberculin (PPD) were used as antigens in an enzyme-linked immunosorbent assay (ELISA) of sera from patients with malignant melanoma undergoing immunotherapy with BCG. It was found that LPS and ELISA are useful tools in the monitoring of humoral immune response against BCG.

Ovarian dysgerminoma. A clinico-pathologic study.

The authors present the clinical, pathologic and therapeutic report on 5 cases of ovarian dysgerminoma in patients between 15 and 30 years of age, classified as Stage Iai in 3 cases and Stage III in 2 cases. Treatment was surgical in 4 cases; in 1 case the surgery was followed by radiation and chemotherapy. All the patients survive free of disease from 4 to 11 years. One of these neoplasms showed a particular histologic pattern in some way similar to the "spermatocytic seminoma" of the testis. This should be the first time that such a type of ovarian dysgerminoma has been reported in the literature.

The epidemiological risk factors of the larynx cancer among the native and migrant male population.

In the present paper relative risk of larynx cancer has been evaluated in 272 cases in the stationary (native Upper Silesians) (65.4%) and migrant (34.6%) population of men. The corresponding control groups (total 739 men) consisted of 401 (54.3%) and 338 (45.7%) men, respectively, who did not suffer from neoplastic diseases. The important relationship between larynx cancer incidence and cigarette (with or without filter), pipe and cigar smoking has been proved. It has also been shown that the evaluation of larynx cancer risk according to the birth place of populations is more useful, because it enables to discover new epidemiological aspects of the larynx cancer. The higher larynx cancer risk has also been disclosed in men who are in manual work, and who are immediately exposed to the influence of various dusts and gases in their place of work. It has been discovered, that chronic inflammation of the upper respiratory tract, pneumonia, tuberculosis, emphysema or bronchitis also influence higher larynx cancer incidence risk in the native and migrant populations analysed.

Prospects of short-term tests in primary cancer prevention.

Prognostic significance of lymphocytic infiltration in radiation-treated uterine cervix carcinoma.

In a retrospective series of 45 patients with uterine cervix carcinoma Stage Ib and IIa who had been treated by radiation, biopsy material obtained prior to treatment was reevaluated for the presence of lymphocytic infiltration. The analysis based on 5 degrees of the intensity of stromal reaction has shown that the presence of lymphocytic infiltration, the intensity of which was significantly related to the proportion of blood vessels in stroma, was connected with a favorable outcome of treatment. The results suggest that vascular density being the determinant factor in oxygenation of the cancer tissue and its radiotherapeutic control may also play an important role in the immunological reaction against the tumor.

The fluctuation of circulating immune complexes level in patients with malignancy.

Serum levels of circulating immune complexes (CIC) in 13 patients with breast cancer, 12 patients with uterine cervix cancer, 4 patients with uterine corpus cancer, 11 patients with lung cancer and 15 healthy persons were estimated by means of the PEGIKEM test during one day at 6 a. m., 11 a. m. and 5 p. m. Maximal deviations from the highest daily levels were expressed in percentage of them. These deviations range on average between 18-26 percent and are not significantly different from those of healthy persons (29 percent). Fluctuation of CIC levels in various patients differs in an individual manner. There is not the same tendency of changes of CIC level within two consecutive days, either. To interpret right a single value of CIC level one must be aware of its possible fluctuation.

Serum lysozyme activity in patients with breast cancer treated by mastectomy and radiation.

In 37 patients with breast cancer, lysozyme activity in undiluted and diluted sera was determined. Blood was collected from peripheral vein before surgical intervention, during surgery from vein draining when radiotherapy was finished. In parallel experiments the lysozyme activity was tested in peripheral blood of 40 healthy persons. Increased lysozyme activity both in undiluted and diluted sera was observed and radiotherapy did not change the activity of the tested enzyme.

Assessment of lethal and cytostatic effects of alkylnitrosoureas on the cells of experimental tumors.

The action of a number of active anticancer agents belonging to the alkylnitrosoureas (MNU, DMNU, ADEKO, BCNU, CCMU, MeCCNU), as well as the effectiveness of a combined use of DMMU and ADEKO with cyclophosphamide was assessed on experimental tumors transplanted by inocula of different size. A mathematical model of the connection between survival of tumor-bearing animals with the kinetics of tumor growth was proposed to analyze the treatment effect. The contribution of lethal and cytostatic factors to the resultant effect of the drugs was evaluated. A combination of DMNU and cyclophosphamide was found to possess an additive effect. The synergism of the therapeutic action shown in a combined application of ADEKO and cyclophosphamide was stipulated by the potentiation of the lethal effect of these drugs on tumor cells.

Changes in distribution of the phases of mitosis in human tumor xenografts following drug treatment.

Distribution of individual mitotic phases in different human xenografts on nude mice showed significant changes after treatment with a range of drugs when compared with untreated controls. The total amount of mitoses in prophase significantly decreased in all responsive tumors while those in all other phases increased but in a very variable degree. There was some correlation in between the range of the mitotic phase distribution changes and the degree of tumor response to treatment.

Relationship between melanogenesis, proliferative activity and response to chemotherapy of human melanoma xenografts.

Wide range of different drugs used in the cytotoxicity studies in an amelanotic human melanoma xenograft on nude mice showed a clear inverse relationship between melanogenesis and proliferation. Decreasing rates in proliferative activity estimated by mitotic answer expressed by phase distribution changes as well as by the tumor volume response to drug treatment were in direct relationship with increasing rates in the degree of melanogenesis expressed in percentage of melanin containing cells in histological specimens.

Chemical transformation of Swiss mouse embryo fibroblasts in primary culture.

Trypsinized secondary cultures of various cell systems have been frequently used by different investigators as models to study, in vitro chemical transformation. In the present study, non-trypsinized primary cultures of fibroblasts derived from fetuses of 14 days gestational age have been used to find out the timings of in vitro chemical transformation by 20-methylcholanthrene and has been designated as PMM-14 cells. These PMM-14 cells have been compared with trypsinized cultures of the same cell systems. The criteria for neoplastic transformation considered in the study involved the appearance of morphological changes, indefinite growth in tissue culture, acquisition of tumorigenic potential in vitro as evidenced by in vivo tests and in vivo latency period for palpable tumor formation. The time span of neoplastic transformation of non-trypsinized embryo fibroblasts in culture was remarkably reduced in comparison with trypsinized cultures of same cell systems. Such discrepancy seems to be due to repeated use of trypsin which may replace many of vital cell surface molecules causing a delay in the expression of carcinogen-induced malignancy. Similarly, repeated subculture before transformation may also have a role in the delayed expression of malignancy.

Characterization of cathepsin B-like proteinase from ascitic fluid of patients with primary liver cancer.

Ascitic fluid of patients with primary liver cancer was shown to contain a latent thiol proteinase which can be activated by pepsin treatment or by autolysis at acidic environment. This enzyme resembles cathepsin B (EC 3.4.22.1) in many physical-chemical properties including substrate specificity, requirement for thiol activators and inhibition both by thiol blocking reagents and by peptidyl diazomethyl ketones, but has a higher molecular size even after activation. Pepsin treatment and autolysis reduce its Mr from 41 800 to 33 400 and 27 700, respectively, but all these forms are larger than human liver cathepsin B. The latent enzyme may be, therefore, an enzyme--inhibitor complex or an inactive precursor of cathepsin B due to an altered processing in Golgi endoplasmatic reticulum-lysosome compartment.

The use of isoprenaline in experimental hyperthermia.

The paper presents the results of experiments in which the perfusion of Walker carcinoma 256 in rats was influenced by s.c. application of isoprenaline under conditions of 45 degrees C hyperthermia. Cumulation of heat energy in the tumor was selectively increased and higher speed of temperature rising was achieved. The changes in blood supply in tumor tissue and skin were quantitatively documented by a method of 86Rb uptake.

Malignant neoplasms of the pancreas. A study based on autopsy data from 1953 to 1982 in Bialystok, Poland. I. Frequency, age and sex distribution.

Among 18 751 autopsies in persons over 20 years of age 195 primary malignant neoplasms of pancreas (MNP) were stated (117 in males, 78 in females; the ratio being 1.5 : 1). In the period of the investigation (1953--1982) MNP comprised 1.0% of all autopsies, 4.1% of all malignant neoplasms (MN), and 11.0% of MN of alimentary system (MNAS). The frequency of MNP in all autopsies was from 0.5% during the years 1953--1957 to 1.2% in 1978--1982, in all MN from 2.7% to 4.5%, and in all MNAS from 5.7% to 12.7%, respectively. MNP were observed most frequently at the age of 61--70. The mean age was 63.6 and was higher in females than in males (66.3 and 61.8, respectively), and increased in the period under study from 54.6 to 65.6 (in males from 50.8 to 64.8, in females from 59.3 to 66.8). The significant increase in the incidence of MNP in the population of patients who died in all hospitals in Bialystok (the north-eastern Poland) between 1953--1982 and were autopsied in our Department correlates with data indicating the rise in the incidence of MNP in many countries of the world.