Chromosomal breakage tests in the differential diagnosis of Fanconi anemia and aplastic anemia.

BACKGROUND: FA patients are hypersensitive to preconditioning of bone marrow transplantation. OBJECTIVE: Assessment of the power of mitomycin C (MMC) test to assign FA patients. METHODS: We analysed 195 patients with hematological disorders using spontaneous and two types of chromosomal breakage tests (MMC and bleomycin). In case of presumed Ataxia telangiectasia (AT), patients' blood was irradiated in vitro to determine the radiosensitivity of the patients. RESULTS: Seven patients were diagnosed as having FA. The number of spontaneous chromosomal aberrations was significantly higher in FA patients than in aplastic anemia (AA) patients including chromatid breaks, exchanges, total aberrations, aberrant cells. MMC-induced ≥10 break/cell was 83.9 ± 11.4% in FA patients and 1.94 ± 0.41% in AA patients (p < .0001). The difference in bleomycin-induced breaks/cell was also significant: 2.01 ± 0.25 (FA) versus 1.30 ± 0.10 (AA) (p = .019). Seven patients showed increased radiation sensitivity. Both dicentric + ring, and total aberrations were significantly higher at 3 and 6 Gy compared to controls. CONCLUSIONS: MMC and Bleomycin tests together proved to be more informative than MMC test alone for the diagnostic classification of AA patients, while in vitro irradiation tests could help detect radiosensitive-as such, individuals with AT.

Prostate Cancer Survivors Present Long-Term, Residual Systemic Immune Alterations.

BACKGROUND: The development of cancer and anti-tumor therapies can lead to systemic immune alterations but little is known about how long immune dysfunction persists in cancer survivors. METHODS: We followed changes in the cellular immune parameters of prostate cancer patients with good prognostic criteria treated with low dose rate brachytherapy before and up to 3 years after the initiation of therapy. RESULTS: Patients before therapy had a reduced CD4+ T cell pool and increased regulatory T cell fraction and these alterations persisted or got amplified during the 36-month follow-up. A significant decrease in the total NK cell number and a redistribution of the circulating NK cells in favor of a less functional anergic subpopulation was seen in patients before therapy but tumor regression led to the regeneration of the NK cell pool and functional integrity. The fraction of lymphoid DCs was increased in patients both before therapy and throughout the whole follow-up. Increased PDGF-AA, BB, CCL5 and CXCL5 levels were measured in patients before treatment but protein levels rapidly normalized. CONCLUSIONS: while NK cell dysfunction recovered, long-term, residual alterations persisted in the adaptive and partly in the innate immune system.

Spontaneous Chromosomal Aberrations in Lymphocytes and Development of Tumor in Hospital Workers.

BACKGROUND: Cytogenetic analysis of chromosomes in blood lymphocytes can be used to reveal biomarkers of tumor risk. The frequency of chromosomal aberrations (CAs) appears to correlate with the later incidence of cancer. PATIENTS AND METHODS: In our work, a total of 515 healthy Hungarian medical workers and 725 controls were enrolled in our investigation. The CAs in peripheral blood lymphocytes were analyzed. RESULTS: The frequency of CAs was significantly higher in the groups working with ionizing radiation and with cytostatic agents compared to unexposed controls and in male smokers rather than non-smokers. The frequency of dicentric chromosomes, however, was not significantly different between control and exposed groups. Among 82 cancer cases (6.6%), the most frequent types were cancer of the breast (20.5%), colon (12.8%), lung and thyroid gland (9-9%). Our analysis showed 8.1% cancer cases in smokers compared to 5.7% in non-smokers. CONCLUSION: The potential exposure to carcinogens did not modify the effect of CAs on cancer risk but tobacco smoking did increase risk.

Relationship between biodosimetric parameters and treatment volumes in three types of prostate radiotherapy.

Brachytherapy (BT) and external beam radiotherapy (EBRT) apply different dose rates, overall treatment times, energies and fractionation. However, the overall impact of these variables on the biological dose of blood is neglected. As the size of the irradiated volume influences the biological effect as well, we studied chromosome aberrations (CAs) as biodosimetric parameters, and explored the relationship of isodose surface volumes (ISVs: V1%, V1Gy, V10%, V10Gy, V100%, V150%) and CAs of both irradiation modalities. We performed extended dicentrics assay of lymphocytes from 102 prostate radiotherapy patients three-monthly for a year. Aberration frequency was the highest after EBRT treatment. It increased after the therapy and did not decrease significantly during the first follow-up year. We showed that various types of CAs 9 months after LDR BT, 3 months after HDR BT and in a long time-range (even up to 1 year) after EBRT positively correlated with ISVs. Regression analysis confirmed these relationships in the case of HDR BT and EBRT. The observed differences in the time points and aberration types are discussed. The ISVs irradiated by EBRT showed stronger correlation and regression relationships with CAs than the ISVs of brachytherapy.

Smoking, chromosomal aberrations, and cancer incidence in healthy subjects.

Chromosomal aberrations (CAs) in peripheral blood lymphocytes can be used as biomarkers of cancer risk. Cytogenetic tests were conducted on 2396 healthy Hungarian individuals and cancer incidence was followed up from 1989 to 2018. Venous blood samples were obtained from the subjects and metaphases from lymphocyte cultures were prepared. We compared the CA frequencies of the various smoking (1-5; 6-10; 11-19; or 20-40 cigarettes/day) and exposure (irradiation; chemical industry; chemical research laboratory) groups. Chromatid break (p = 0.0002), total aberration (p = 0.002), and aberrant cell (p = 0.001) frequencies were higher in smokers than in non-smokers. For very heavy smokers, total CAs were significantly higher than for non-smokers (<0.001) or less intensive smokers (p = 0.003-0.0006). Intensity of smoking was a predictor of chromosomal aberrations, while duration was not. During follow-up, 177 (7.3 %) cancer cases were found. A Cox-regression model showed that subjects with cell values ≥2 CAs developed cancer more frequently (hazard ratio = 1.39; 95 % CI, 1.02-1.90). The relative risks of cancer were 1.06 (95 % CI 0.53-2.06) for light smokers and 1.74 (95 % CI 1.08-2.77) for very heavy smokers. The distributions of cancer sites showed differences between smoker and non-smoker groups: in male smokers, lung cancer, in non-smokers, prostate, and in females (both groups) breast cancer were most common. Cancer incidence correlated with chromosome aberrations; smoking was not a confounder in this relationship.

[And what about doctors? Associations between spontaneous chromosomal aberrations and cancer morbidity in healthcare workers]. / És mi van az orvosokkal? Egészségügyi dolgozók spontán kromoszómaaberrációi és rákmorbiditása közti kapcsolat.

Healthcare workers may be occupationally exposed to low dose rate radiation or different chemicals during their work. There are strong associations between the increased frequency of spontaneous chromosomal aberrations in blood lymphocytes and the risk of cancer. Cytogenetic tests were conducted on 1240 healthy medical workers and cancer incidence was followed up between 1997-2018. Both structural and numerical chromosome aberrations were evaluated and the results were compared taking into account gender, age, and smoking. The frequency of aberrant cells was significantly higher in smoker males than in non-smokers (p=0.009). Within the same study period, there was no significant difference in chromosome aberrations between the potentially exposed group of workers and the control group. Among 82 cancer cases (6.6%) the most common tumors were breast (16), colon (12), lung (7) and thyroid gland cancers (7). Our analysis showed 7.3% cancer occurrence among smokers compared to 6.2% among non-smokers. These results suggest that in our cases cytogenetic effects of smoking are more deleterious than occupational exposures.

[Biological dose estimation for photons of different qualities in radiation therapy]. / Biológiai dózisbecslés a sugárterápiában alkalmazott különbözo sugárminoségu fotonnyaláboknál.

Flattening filter free mode (FFF) has been introduced in radiotherapy during the past decades, however, not much has been reported on its radiobiological effect. The purpose of our study was to compare the radiobiological effects of flattening filter and flattening filter free photon beams on chromosomal aberrations in peripheral blood lymphocytes. In our study the blood of the same healthy donor was irradiated with linear accelerator using both conventional flattening filter (FF) and FFF photon beams at dose rate of 3.57-23.08 Gy/min, using 6 or 10 MV. The dose-response calibration curves for dicentric + ring chromosomes induced by irradiation were fitted with linear-quadratic model. CABAS (Chromosomal Aberration Calculation Software) was used to prepare the curves. The coefficients and equations of the curves were calculated and compared with the results of other authors. We found significant differences in the number of aberrations at different irradiation parameters. Based on our results, FFF mode has a 10-20% higher biological effect than FF mode. These results can be used during radiotherapy or to estimate the biological doses in case of an accidental exposure to radiation.

Chromosomal Aberrations in Blood Lymphocytes as Predictors of Respiratory Function After Stereotactic Lung Irradiation.

Due to the profound difference in radiosensitivity of patients and various side effects caused by this phenomenon, a radiosensitivity marker is needed. Prediction by a marker may help personalise the treatment. In this study, we tested chromosomal aberrations (CA) of in vitro irradiated blood as predictor of pulmonary function decrease of nonsmall cell lung cancer (NSCLC) patients and also compared it with the CAs in the blood of irradiated patients. Peripheral blood samples were taken from 45 lung cancer patients before stereotactic radiotherapy (SBRT) and immediately after the last fraction and 3, 6, 9, 12, 15, 18, 21, and 24 months later. Respiratory function measurements were performed at the same time. Diffusing capacity of lung for carbon monoxide (DLCO), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1s), and FEV1s/FVC (FEV1%) were monitored. Metaphase preparations of lymphocytes were made with standard procedures, and chromosome aberrations were analysed. In our cohort, the 36-month local relapse-free survival was 97.4%, and the distant metastasis-free survival was 71.5% at 36 months. There was no change in the mean of the pulmonary function tests (PFTs) after the therapy. However, there was a considerable variability between the patients. Therefore, we subtracted the baseline and normalised the PFT values. There were significant decreases at 12-24 months in relative FEV1s and relative FEV1%. The tendentious decrease of the PFTs could be predicted by the in vitro chromosome aberration data. We also found connections between the in vitro and in vivo CA values (i.e., dicentrics plus rings after 3 Gy irradiation predicts dicentric-plus-ring value directly after the radiotherapy/V54 Gy (p = 0.001 24.2%)). We found that-after further validation-chromosome aberrations resulted from in vitro irradiation before radiotherapy can be a predictive marker of pulmonary function decrease after lung irradiation.

Quantifying biological effects of radiation from high-energy linear accelerators on lymphocytes.

The aim of this study was to investigate the radiobiological effects of flattening filter (FF) and flattening filter-free (FFF) modes of linear electron accelerators and to understand whether there is any difference between the effects of these modes. We evaluated the number of chromosome aberrations following irradiation of lymphocytes from healthy volunteers with X-ray photons at two energy levels, 6 and 10 MV; the dose rate ranged between 5.50 and 23.08 Gy/min and absorbed doses ranged between 0.5 and 8 Gy. A 60Co curve was employed for comparison. Metaphases from the lymphocyte cultures were prepared using standard cytogenetic techniques and chromosome analysis was performed. Our results allow the performance of biodosimetry at higher energies and doses than the currently used reference dosimetry. We observed significant differences in aberration frequencies when different irradiation techniques were used. FFF mode has a higher radiobiological effect than the FF mode. Linear-quadratic dose response calibration curves were constructed and relative biological effectiveness (RBE) values were calculated. Average RBE values using 6 MV (5.50 Gy/min) as a reference radiation were 1.28 for 60Co γ irradiation, 1.11 for 6 FFF and 0.79-0.92 for 10 FFF. Since there are compelling differences between radiation modalities in cases of hypofractionation, these results may be even more important in a therapeutic situation. In case of an accidental overdose of a patient, use of the appropriate calibration curves for biodosimetry are also essential for quantifying the overdose.

Progressive breast fibrosis caused by extreme radiosensitivity: Oncocytogenetic diagnosis and treatment by reconstructive flap surgery.

BACKGROUND: Fibrosis, a proliferative response of fibrocytes after tissue injury, is a common sequela of external radiotherapy and can vary greatly among patients even in the absence of DNA repair syndromes, due to their different intrinsic radiosensitivity. Fibrosis is also a serious cosmetic problem for patients, and in some cases it can also imply pain. CASE: Here, we report a case of a severe fibrosis 2 years after breast conserving surgery and postoperative 3D conformal breast irradiation. Furthermore, our patient had the suspicion of tumour recurrence. Our examinations were aimed at diagnosing recurrence or the lack of recurrence and investigating whether the symptoms occurred due to overdosing or extreme intrinsic radiosensitivity. Therefore, examining the patients' radiosensitivity, a cytogenetic test was performed, which revealed the patient's increased susceptibility to ionizing radiation, and therefore we rejected the prospect of overdosage. As a solution for the fibrosis, mastectomy was effectuated, and a latissimus dorsi musculocutaneous flap was used for reconstruction. CONCLUSIONS: We suggest a multi-disciplinary approach to manage fibrosis and propose cytogenetic markers to be used as predictors to identify patients who most likely benefit from a certain therapeutic regimen in terms of reduction of therapy-related side effects.

Relationship between spontaneous frequency of aneuploidy and cancer risk in 2145 healthy Hungarian subjects.

Numerical and structural chromosomal abnormalities are the hallmarks of cancer. Whereas the structural chromosome aberrations got more substantial attention for cancer risk assessment in a healthy population, the role of aneuploidy is much less understood in this respect. We analysed the frequency of numerical (and structural) aberrations in peripheral blood lymphocytes of 2145 healthy individuals between 1989 and 2010, taking into account different biological- and exposure-conditions. We also studied to what extent chromosome gains or losses may predict the probability of cancer. The average frequency of all aneuploid cells was 1.78±0.06% in the entire study population, which increased linearly with age. Gender and smoking did not influence the values, however, occupational exposures did. The highest frequency of aneuploidy was found in chemical industry-workers (1.89±0.05%) compared with the lowest value of medical radiation workers (1.44±0.10%), respectively. No correlation was found between numerical and structural chromosomal aberrations. Cancer incidence followed for 1-23 years after the chromosome analysis showed a 1.26-fold relative risk (confidence interval: 1.02-1.58; P = 0.04) for those with higher frequency of aneuploid cells (1.82% vs. 1.44% in controls). Hypodiploidy had higher impact on the cancer risk than hyperdiploidy (1.72% vs. 0.10%). Our findings on the frequency of numerical aberrations in a healthy cohort represent the largest cytogenetic database from one laboratory with an unchanged mechanistic scoring method during a 30-year period, and provide basic information not only for genotoxicological studies but also confirm the association between numerical aberrations and cancer risk.

[Frequency of spontaneous aneuploidy in the healthy Hungarian population]. / A spontán aneuploidia gyakorisága egészséges magyar populációban.

Aneuploidy plays very important role in tumor development as the consequence of either congenital or acquired mutations. In order to evaluate the adverse effects of various aneugens, the knowledge of the spontaneous frequency of numerical chromosome abnormalities in healthy population is fundamental. In our study we analyzed the spontaneous rate of numerical and structural chromosome aberrations in peripheral blood lymphocytes of 2145 healthy individuals, with special attention to the influence of biological (gender, age) and life-style factors (smoking, different occupational exposure). Correlation between aneuploidy and risk of cancer development were investigated according to National Cancer Registry data followed for 1-23 years. In the whole population the average frequency of aneuploid cells was 1.77±0.06%. This value increased by age linearly (r2=0.81) regardless of occupational exposures. Gender (biological factor) or smoking (life style factor) did not influence the values, however, the occupation of individuals modified the frequency of numerical aberrations. Individuals who worked at workplaces with radiation hazard had the lowest (1.44±0.10%), and those working in the chemical industry had the highest (1.89±0.05%) values of aneuploidy, respectively. We could not find any correlation between numerical and structural chromosome aberrations. In our population studied 97 individuals developed cancer and only those who had ≤2% aneuploidy survived more than 12 years in good health conditions. To our knowledge, this study has the highest case number investigated up to now. Our results support that aneuploidy, similarly to structural chromosomal aberrations, might be an additional cytogenetic biomarker of the genetic instability.

No short-term cytogenetic consequences of Hungarian red mud catastrophe.

Red mud is an industrial waste produced in the process of alumina extraction from bauxite with concentrated NaOH. When the red mud-containing reservoir collapsed in Ajka Alumina Plant Hungary in October 2010, the most serious immediate effects were caused by the high alkalinity (pH ≥ 13) of the flood. Many persons suffered burn-like damage to tissues and contact with caustic desiccated ultra-fine dust with traces of toxic metals also caused irritation of upper respiratory tract and eyes. This catastrophe was unique from the point of view of genotoxic effects as well. Therefore cytogenetic examinations were carried out on inhabitants, either with burns (17 persons) or on those inhaling desiccated caustic dust (42 persons). Chromosomal aberration (CA) analysis and bleomycin (BLM)-sensitivity assays, as possible markers of effects, were studied in peripheral blood lymphocytes of persons within 4-6 weeks following the catastrophe. Controls were matched for age, sex and smoking habits, and also places of residence with different constituents of air pollution either from rural (59 persons), or from urban environments (59 persons). Neither spontaneous rate of CAs (1.47% vs. 1.69%) nor BLM-induced in vitro chromosomal breakage (0.79 vs. 0.83 break/cell) showed elevated rates when cytogenetic biomarkers of genotoxicity were compared between controls and exposed persons. Time spent in cleaning did not affect cytogenetic changes either (R(2) = 0.04). BLM-induced mutagen sensitivity was similar in exposed and control persons (27.1% vs. 30.5%). It seems that the red mud exposure does not appear to pose an immediate genotoxic hazard on residents when measured with cytogenetic methods. We recommend, however, that those involved in clean-up activities should be followed closely not only for overall health, but also for further genotoxic risk assessment, because the long-term hazards of ultra-fine fugitive dust particles with alkalinity of residual NaOH in red mud are still unknown.

[Problems occurring in the application of cytogenetic biomarkers for alcoholics with and without malignant diseases]. / Biomarkerek alkalmazása során felmerülo problémák malignus és nem malignus betegségben szenvedo alkoholisták esetében.

Applicability of alcohol- and smoking-related cancer-risk biomarkers might be modified by several factors. Among those, reality of self-reports on alcohol consumption of alcoholic patients with different diseases and extreme high mutagen hypersensitivity of Hungarians, as well as the immunologic role of peripheral lymphocytes as experimental objects of cytogenetic biomarkers seem to be new viewpoints of interest. To clarify these problems, 432 head and neck cancer patients (HNCP), 62 alcoholics with alcoholic hepatitis (ALCL), and 101 disease-free chronic alcoholics (ALC) were examined. Despite clinically confirmed alcohol-related diagnoses (and GGT and MCV values) only about half of HNCPs and ALCLs reported about any alcohol consumption, in contrast to the realistic self-reports of ALCs. In cytogenetic case control investigations no difference between the spontaneous rate of chromosomal aberrations (CAs) of healthy controls and ALCs was found, however, genetic instability expressed as a 40-50% elevation rate of CAs in HNCPs and ALCLs might be associated with systemic inflammatory reaction of lymphocytes. Bleomycin sensitivity assay showed the highest break/cell (b/c) values not in HNCPs (1.06 b/c) as it was reported earlier, but in "healthy" ALCs (1.52 b/c). This phenomenon can be related to the local effect of genotoxins (alcohol, smoking, and in particular the diet), which probably reflects merely a reaction of mucosal immune system. Nearly 50% of mutagen-hypersensitive Hungarian controls, in contrary to the expected 10-20% ones, might also be explained by this. Similarly, HNCPs with oral cancer, where the local mutagen effect was the most intensive, had the highest b/c values. In conclusion, when cytogenetic biomarkers of alcoholism are examined, the subjective character of self-reports at epidemiologic level and immunologic role of lymphocyte subpopulations as genetic confounders must also be taken into consideration.

Radiosensitivity of subterranean bacteria in the Hungarian Upper Permian Siltstone Formation.

The main purpose of this work was to study the radioresistance of subterranean aerobic and anaerobic isolates from the Hungarian Upper Permian Siltstone (Aleurolite) Formation, in order to assess the safety of potential sites of future underground repositories for nuclear waste. A total of 93 isolates were studied. The radiosensitivities of these aerobic and anaerobic bacteria isolates were determined: the D10 values (decimal reducing doses) of the aerobic spore-formers lay in the range 0.80 -2.44 kGy, and those of the anaerobic spore-formers lay in the range 1.86 4.93 kGy. The D10 values of the aerobic and anaerobic vegetative isolates were much lower, in the ranges 0.11 0.57 and 0.22-0.40 kGy. respectively. The variability in bacterial radioresistance indicates the biodiversity at this potential disposal site. These results can affect the construction of a future underground repository, since knowledge of the most resistant microorganism may be of importance as concerns calculation of the time required to inactivate the bacteria surrounding the containers.