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The aim of the study was to identify and quantify lymphocytes with asynchronous replication of the AURKA and TP53 genes in cancer patients versus controls and to assess the diagnostic capabilities of this approach. MATERIALS AND METHODS: The study was carried out with peripheral blood lymphocytes probed for the AURKA and TP53 genes using the interphase fluorescence in situ hybridization (FISH) method (Vysis, USA and Kreatech, The Netherlands). The control group included 70 people: clinically healthy donors and patients with non-oncological diseases such as gastritis, pancreatitis, chronic calculous cholecystitis, bronchial asthma, peptic ulcer disease, inguinal hernia, arthrosis, myoma, hepatitis, epilepsy, chronic prostatitis, chronic tonsillitis, and rectal adenoma. The group of cancer patients included 219 people with various oncological diseases: gastric cancer (n=68), colorectal cancer (n=30), chronic lymphocytic leukemia (n=52), Hodgkin lymphoma (n=33), and polyneoplasia (n=41). RESULTS: In the control group, the mean frequency of lymphocytes with asynchronous gene replication (AGR) was 22.0±3.4% for AURKA and 18.0±3.2% for TP53; in the group of cancer patients, that was 36.8±4.8 and 28.4±5.1%, respectively. The excessive presence of lymphocytes with the AGR in cancer patients was consistent and statistically significant (p<0.0001). For the AURKA gene, the AGR-based cancer detection showed a sensitivity of 98.6±0.7%, a specificity of 100%, and an accuracy of 98.3±0.8%, and for the TP53 gene - 78.6±3.1, 98.5±0.9, and 85.9±2.6%, respectively. CONCLUSION: This pilot study on lymphocytes with AGR of AURKA and TP53 genes in cancer patients can serve a basis for creating a new molecular cytogenetic technology for detecting malignant neoplasms in humans.

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Molecular-biochemical and cytogenetic analyses were made on blood cells of 17 radiation accident victims who, from 1.7 to 43.8 years previously, had suffered acute radiation sickness (ARS) ranging from severity grades I to IV. Molecular-biochemical data were obtained with patients' leukocytes and with mononuclear cells on their oxidative status by a) the level of an anion-radical O2*- in the 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide (MTT) test and b) the sum of reactive oxygen species in the 2,7-dichlorodihydrofluorescein diacetate (DCFDA) test together with a test for DNA strand unwinding in alkaline conditions by measurements of fluorescence intensity of ethidium bromide. Each parameter was measured in freshly sampled cells and during a 5-hour incubation as absolute means on 5 measurements and in % change from the initial values. Cytogenetic data were obtained from the standard metaphase preparations scored for routine unstable chromosomal aberrations (us-CA)--dicentrics; and stable aberrations (sCA)--translocations. The latter ones were assayed by the FISH method using whole chromosome 2, 4 and 12 fluorescent probes and scaled up to genome equivalence. For all patients reduced oxidative status of about 25-30% was obtained by the MTT-test (p < 0.005), and the DCFDA-test (p < 0.027). The yield of usCA depended on the time after irradiation with higher yields associated with the shorter postirradiation times and reducing almost to expected background frequencies. The yield of sCA was high for all patients, correlating with the severity of ARS whilst the molecular-biochemical parameters showed no relationship with ARS. A correlation was observed between parameters of oxidative status and % of cells with usCA: by the MTT-test r = from 0.50 up to 0.61 (p = from 0.06 up to 0.003), but by the DCFDA-test the strength of correlation was smaller: r = from 0.38 up to 0.48. An inverse correlation was found between initial oxidative state of mononuclear cells and the frequency of CA in lymphocytes. Similarly a marked inverse correlation between degree of DNA unwinding by the ethidium bromide assay on leukocytes and sCA in lymphocytes was also noted. The feasibility of radiation-induced delayed genomic instability in vivo for humans long time after irradiation is discussed.

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The results of comparative analysis of gene and structural mutations found in peripheral blood lymphocytes of inhabitants of Orel district areas contaminated with radionuclides as a result of Chernobyl accident are presented. The average level of 137Cs contamination in those areas ranged about 22-113 kBq/m2. In the study group was found the enhanced frequency of somatic cells with gene and structural mutations compared with laboratory control level by synchronous applying a T-cell receptor (TCR) loci mutation assay and cytogenetic analysis of unstable aberrations. The case-control comparison was carried out using the measured mutation frequencies and cases of various thyroid gland sickness recognized by ultrasonic examination. The cytogenetic assay did not show the statistical difference between healthy group and subjects with thyroid gland sickness. The average frequency of TCR loci mutation cells in the subjects with thyroid gland sickness was found to be statistically higher comparing with healthy persons. This finding was true for each study region and for Orel district in total. The subgroup of subject exposed in utero in 1986, soon after accident was analyzed. Both cytogenetic and TCR loci mutation assays shown enhancement of average mutation frequency in somatic cells in the subjects of this subgroup with thyroid gland sickness comparing with healthy persons.

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A study of frequency of unstable chromosome aberrations in 50 workers of nuclear chemical plants in remote period after beginning or finishing professional contact with ionizing radiation was carried out. 14 persons from this cohort were mainly whole-body exposed to external gamma-rays and 36 were exposed to combined external and internal radiation from incorporated Pu nuclides. In results of this irradiating practically every subject had a chronical radiation sickness. In the 1-st group the frequency of unstable aberrations varied from 0.2 to 3.6 per 100 cells and exceeded reliably control level in 5 persons. In the 2-nd group the frequency of unstable aberrations varied from 0 to 11.6 per 100 cells and exceeded reliably control level in 20 examined workers. The FISH study of frequency of stable aberrations was performed in 13 subjects who were exposed to combined external and internal radiation. Total frequency of complete and incomplete translocations varied from 0.6 to 18.5 aberrations per genome per 100 cells and reliable exceeded control level in 9 subjects. Non-random participation in exchange rearrangements (translocations) was revealed for used set of chromosomes (2, 3 and 8).

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Results of multiyear cytogenetic study of children and teenagers living in areas, radioactive by contaminated after Chernobyl accident, were adduced. Mean density of 137Cs contamination in two compared living areas were 111 and 200 kBq/m2 and mean external doses accumulated for 1986-2001 were 6.7 and 11.4 mGy correspondingly. Averaged thyroid doses receives by subjects of all age groups in the second area were approximately 1.5 times higher than in the first area; in the youngest group (0-1 year) the doses were 114.3 and 174.3 mGy. During 17 years cytogenetic investigation approximately from 30% to 60% of examined persons were observed the increased level of chromosome aberrations in lymphocytes of peripheral blood. Average frequency of unstable aberrations (acentrics, dicentrics and centric rings) constituted about 0.4 per 100 cells (0.22 per 100 cells in controls) during all period of observation. Level of marker aberrations (dicentrics and centric rings) was increased almost all times of study and varied within 0.04-0.19 per 100 cell (0.03 in control group). The parallel investigation of frequency of stable aberrations by FISH method showed up their level about 3 times exceeding observed dicentrics level. Comparably higher indexes of cytogenetic disturbances were revealed in group exposed in utero during period of accident.

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A follow-up of 10 highly irradiated men, mostly reactor crew, from the Chernobyl accident is described. Their pre-accident medical conditions and relevant medical status approximately 10-13 y later are listed. A comparison is made between estimates of their average whole-body penetrating radiation doses derived from several biological parameters. First estimates were based on their presenting severity of prodromal sickness, early changes in blood cell counts and dicentric chromosome aberrations in lymphocytes. In three cases ESR measurements on tooth enamel were also made. Retrospective dosimetry using FISH translocations was attempted 10-13 y later. This showed good agreement for those patients with the lower earlier dose estimates, up to about 3 Gy. For the others, extending up to about 12 Gy, the translocations indicated lower values, suggesting that in these cases translocations had somewhat declined. Repeated chromosomal examinations during the follow-up period showed an expected decline in dicentric frequencies. The pattern of decline was bi-phasic with a more rapid first phase, with a half-life of approximately 4 months followed by a slower decline with half-lives around 2-4 y. The rapid phase persisted for a longer time in those patients who had received the highest doses. 10-13 y later dicentric levels were still above normal background, but well below the translocation frequencies.

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Blood samples of ten Chernobyl and one non-Chernobyl victims were analysed both by conventional cytogenetics and by fluorescence in situ hybridization (FISH) using a cocktail of chromosomes 2, 3 and 8. The analysed group comprised men acutely irradiated mainly in 1986 and aged 26-47 years at the time of first blood sampling. All of them displayed acute radiation syndrome of varying severity. Chromosome analysis of the earliest blood samples was carried out by conventional scoring of unstable aberrations with the number of metaphases analysed per individual ranging from 35 to 300. Estimated individual doses ranged from 0.85 to 9.8 G y. After a 10 year delay, i.e. in 1996, blood samples were analysed both by conventional scoring of unstable aberrations and by FISH measurements of stable ones. Usually about 500 metaphases per individual were scored. Estimated by the FISH-method individual translocation (tc + ti) frequencies ranged from 2.2 to 116.8 per 100 cells full genome equivalent. Based on three different published dicentric dose response, in vitro curves individual doses were calculated from the earliest dicentric frequencies. A dose response curve for truly persisting translocations (tc + ti) was estimated over the range 1-10 Gy.

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Within the frames of activity of joint commission the cytogenetic studies of 98 persons from different areas of Semipalatinsk Province have been carried out in 1989. The studies revealed the higher level of chromosome aberrations in the settlements adjacent to firing ground. The possible connection of discovered cytogenetic lesions to the influence of radiation aftermath of ground and air nuclear weapon test is discussed.

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The paper presents the data on cytogenetic studies of 663 children inhabited radionuclide contaminated territories of Bryansk Province. Statistically significant difference with control was observed only for the territory with the highest radioactive contamination level of 137Cs (1000 kBq/m2). For two other territories with lesser contamination (100 and 700 kBq/m2) the rate of chromosome aberrations is nearly the same and does not differ significantly from control one. No difference in the level of cytogenetic damage was discovered for children affected by radiation pre- or postnatally.

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Comparative studies of the yield of stable and unstable chromosome aberrations have been carried out in human lymphocytes gamma irradiated in vitro within the doze range 0.1--1.0 Gy. It was observed that the yield of unstable chromosome aberrations (dicentrics and centric rings) is satisfactorily described for discovered dose range with linear-quadratic approximation. For stable chromosome aberrations in G-banded chromosome the dose response dependence was less expressed. The studies revealed non-random involvement of different chromosomes in translocations, and the most frequently involved are 1 and 13 chromosomes.

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It was shown by method of cytokinetic blocking that with neutron irradiation of human lymphocyte culture (mean energy of 0.85 MeV, doses of 0.05 to 2 Gy) the dose-response relationship, with respect to the share of binuclear cells with micronuclei and the frequency of micronuclei in binuclear cells, was of a multiphase nature with a more or less manifest plateau within the dose-range from 0.5 to 1.0 Gy. Both micronuclear tests may be used for indicating the degree of radiation injury to the organism caused by neutrons of the above-mentioned energy and doses of 0.05-0.5 Gy.

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The method of the cytokinetic blocking was used to determine, in 18 healthy male donors aged 21-35 years, the frequency of binuclear cells with micronuclei that amounted to 1.1 +/- 0.2% (0.3-2.4%) on the average. The dependence of this parameter upon radiation dose (60Co-gamma radiation, 0.05 to 2 Gy) was a linear quadratic function and was described by the following regression equation: Yx = 0.76 + 5.76 D +/- 2.61 D2. With a dose of 0.05 Gy the frequency of binuclear cells with micronuclei exceeded significantly the control level.

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