Persistence Of Aberrations Research Articles

A Pilot Study on COVID-19 Positive Subjects: An Excerpt of Post-Infection-Pro-Diabetic Disposition & Related Consequences in Correlation to Hepato-Pancreatic Bio-Markers, Pro-Inflammatory Cytokines and Other Risk Factors.

COVID-19, a global pandemic that led to increased morbidity and mortality worldwide since its outcome at the end of the year 2019. A newly discovered variant of severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) was the arbitrator for spreading the syndrome by droplet transmission causing multi-organ failure in many occasions. A post-infection-pro-diabetic disposition was found evident in this study with the persistence of hepato-pancreatic aberrations in respect of reference range of tissue specific bio-markers in hospital admitted COVID-19 cases. The results of this study show that hyperglycemia is a risk factor in precipitating disease oriented complications to the patients with COVID-19 disease. A post-infection follow- up on glycemic-index and related complexities is a vital need to the COVID-19 infected convalescent subjects. Implementation of guidelines on social measure and awareness of anti-viral interventions may be the only way to prevent COVID-19 transmission.

Persistence of radiation-induced aberrations in patients after radiotherapy with C-ions and IMRT

Background and purposeChromosomal aberrations in peripheral blood lymphocytes are a biomarker for radiation exposure and are associated with an increased risk for malignancies. To determine the long-term cytogenetic effect of radiotherapy, we analyzed the persistence of different aberration types up to 2.5 years after the treatment. Materials and methodsCytogenetic damage was analyzed in lymphocytes from 14 patients that had undergone C-ion boost + IMRT treatment for prostate cancer. Samples were taken immediately, 1 year and 2.5 years after therapy. Aberrations were scored using the multiplex fluorescence in situ hybridization technique and grouped according to their transmissibility to daughter cells. ResultsDicentric chromosomes (non-transmissible) and translocations (transmissible) were induced with equal frequencies. In the follow-up period, the translocation yield remained unchanged while the yield of dicentrics decreased to ≈40% of the initial value (p = 0.011 and p = 0.001 for 1 and 2.5 years after compared to end of therapy). In 2 patients clonal aberrations were observed; however they were also found in samples taken before therapy and thus were not radiotherapy induced. ConclusionThe shift in the aberrations spectrum towards a higher fraction of translocations indicates the exposure of hematopoietic stem and progenitor cells underlining the importance of a careful sparing of bone marrow during radiotherapy to minimize the risk for secondary cancers.

Persistence of chromosomal aberrations in peripheral blood lymphocytes of Hodgkin's lymphoma patients after ABVD chemotherapy

Persistence of chromosomal aberrations in peripheral blood lymphocytes of Hodgkin's lymphoma patients after ABVD chemotherapy

Hypoxia Increases Repopulating Ability of Myelodysplastic Syndrome Bone Marrow Cells

Introduction: Myelodysplastic Syndromes (MDS) are clonal neoplasms. Whether the transforming event occurs in a myeloid committed cell or in earlier hematopoietic progenitor/stem cell it is still not ascertained. We evaluated the repopulating ability and stem cell potential of hypoxia maintained primary bone marrow (BM) progenitors derived from Myelodysplastic Syndromes (MDS) patients, and their capacity of engraftment in sublethally irradiated NOD-SCID mice.Methods: Thirty eight BM samples were obtained at diagnosis from MDS patients (WHO: 9 RA, 12 RCMD, 7 RAEB, 8 AML/post MDS, 2 Del(5)q). Mononuclear BM cells were isolated and cultured with TPO, FLT3-L, SCF, IL-3 in severe hypoxic conditions (0,3%O2, 5%CO2, 95%N2), for 10-13 days (LC1). The stem cell potential of these cultures was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, and according to the Culture-Repopulating Ability (CRA) assay methodology, cell proliferation was evaluated daily. Expression of CD34, CD38, CD117, CD133 was determined before and after hypoxia cultures, and when present, the persistence of chromosomal aberrations was analyzed by FISH at various stages of cell culture. In parallel, MDS mononuclear BM cells were intravenously injected in sublethally irradiated NOD-SCID mice before hypoxic culture and after LC1. Mice were sacrificed at day 56 and 90 after graft. The presence of CD45+ human cells in the peripheral blood of injected mice was evaluated by flow cytometry analysis every two weeks, and quantitated to evaluate the repopulating ability and the different engraftment capacity of cells before hypoxic culture and after hypoxic incubation. Mice marrow trephines as well as spleens were also evaluated morphologically to test the engraftment of human MDS cells.Results: In all 38 MDS cases studied, cultured cell number decreased of one log or more after 10-13 days of culture in hypoxic conditions. In 12/38 MDS cases we observed a significant repopulating ability at day 17 of LC2, all classified as Low/INT-1 IPSS risk category. In IPSS high and INT-2 risk cases, repopulating ability according to CRA method was absent; CD34+ cells were always decreased after hypoxia and did not coexpress CD38, confirming the selection of earlier hematopoietic progenitor cells. We demonstrated the persistence of chromosomal aberrations in CD34pos/CD38neg cells after hypoxic culture in 5 MDS cases (del5q, +8, del20q, -Y, complex karyotype). Sublethally irradiated NOD-SCID mice transplanted with hypoxia cultured cells showed in peripheral blood an higher percentage of CD45+ human cell than mice transplanted with non hypoxia selected cells. In particular, mice transplanted with hypoxia cultured BM mononuclear cells derived from a 5q- MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with non-hypoxia selected cells at day 28 after transplant (5,5% vs 0,56 %); mice transplanted with hypoxia cultured BM mononuclear cells derived from a CRDM MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with cells before hypoxia at day 56 (13,3% vs 1,4%), and at day 71 (43,05% vs 12,7%). Mice bone marrow cytometry analysis confirmed higher percentage of CD45+ cells in bone marrow derived from mice transplanted with hypoxia cultured cells than non-hypoxia cultured (1,9 % vs 0,1%).Conclusions: Severe hypoxic culture conditions are maintaining a sub-population of MDS cells endowed with increased repopulating capacity in vitro and in vivo. Most importantly, in our cohort of cases studied, repopulating ability was observed exclusively in IPSS lower risk MDS cases. DisclosuresSantini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding.

Eight-year follow-up study of three individuals accidentally exposed to 60Co radiation: Chromosome aberration and micronucleus analysis

We assessed dose levels and the persistence of chromosomal aberrations and micronuclei in three individuals in the 8 year following accidental 60Co radiation exposure. Venous blood samples were collected and used for analyses: traditional chromosome aberration (CA) measurement, G-banding, and the cytokinesis-block micronucleus (CBMN) assay. For CA analysis, we scored dicentric chromosomes (dic) and rings (r) in peripheral blood lymphocytes. The radiation doses (Gy) suffered by the individuals were estimated as: 1.79–2.43 (A), 2.36–2.86 (B), 1.58–1.82 (C), based on CA analysis; and 1.8–2.34 (A), 2.52–2.98 (B), 1.53–1.77 (C), based on CBMN frequencies. G-banding analysis was used to record translocations (t), inversions (inv), and deletions (del). Following the accident, unstable CAs reduced gradually, but stable aberrations persisted. Unstable CAs and CBMN may be valuable biomarkers for dose estimation shortly after high-dose radiation accidents, while stable aberrations may be more useful for assessing long-term effects.

Severe Hypoxia Promotes Selection of Repopulating Progenitor CELLS in Primary MDS BONE Marrow CELL Culture

AbstractAbstract 4747 Introduction:Myelodysplastic Syndromes (MDS) are clonal neoplasms. Whether the transforming event occurs in a myeloid committed cell or in earlier hematopoietic progenitor/stem cell it is still not ascertained. In this study, we evaluated the repopulating ability of hypoxia selected cells obtained from primary MDS bone marrow cultures. We then characterized the “stemness” of MDS maintaining cells in different subtypes of this disease. Methods:We studied 31 samples obtained at diagnosis from patients with different WHO subtypes of MDS (6 RA, 12 RCMD, 7 RAEB, 6 AL/post MDS). Mononuclear bone marrow cells were isolated after density gradient centrifugation and cultured in RPMI 1640 medium supplemented with 20 % FBS and a cocktail of cytokines (TPO, FLT3-L, SCF, IL-3). Cells were incubated and selected in Ruskin Concept 400 anaerobic incubator, in severe hypoxia conditions by flushing with a performed gas mixture (0,3 % O2,5%CO2,95% N2), for 10–13 days (LC1) and daily counted (by Trypan blue dye). The stem and progenitor cell potential of these cultures at different times of incubation was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, according to the Culture-Repopulating Ability assay methodology (Cipolleschi et al, Leukemia 2000). At the same time, we evaluated clonal potential of selected cells by semisolid culture in methylcellulose, in the presence of the same cocktail of cytokines. The phenotype of hypoxia selected cells was evaluated by determining the expression of CD34, CD38, CD117, CD133, as well as beta-catenin expression and localization at fluoresence microscopy. When present, we analyzed by FISH the persistence of chromosomal aberrations in MDS cells, before and after hypoxic selection. Results:The hypoxic culture system allowed selection of a minute cell population: in 31/31 cases viable cell number decreased of one log after 10–13 days of culture in extreme hypoxic conditions. Only 11/31 MDS cases showed a significant repopulating ability at day 17 of LC2 and in the same cases we observed parallel formation of colonies ( CFU-GM and CFU-GEMM) in semisolid cultures. Ten of these eleven cases that showed repopulating ability belonged to the Low/INT1 IPSS risk category. In the other 20, all IPSS high and INT-2 risk cases, repopulating ability was absent. In particular, MDS cases presenting > 10% blasts in the bone marrow did not show repopulating cells after selective hypoxic conditions. In 12 cases, after hypoxia selection we observed a reduction in the number of CD34+ cells. Within this shrinked CD34+ population, we could isolate highly repopulating progenitors CD34+ /CD38−, that confirmed the selection of earlier hematopoietic progenitor cells. We analyzed by interphase FISH MDS cases presenting chromosomal aberration ( +8, −Y, complex karyotype and del7q). In all cases, the marker chromosomal aberrations were maintained after hypoxic culture. In particular, in one of these cases with marker chromosomal aberration, the percentage of cells carrying –Y at interphase FISH was 66% at baseline, and 82% after hypoxic selection, while 66% of the repopulating cells, after 8 days of normoxic LC2, still had −Y. Conclusions:We demonstrate here that it is possible to select by severe hypoxic conditions primary MDS progenitor cells with repopulating ability. MDS cases in earlier phases of the disease still show near-normal repopulating abilityafter hypoxic selection, whereas more progressive dysplastic disease is accompanied by a loss of repopulating ability, as measured by CRA assay and colony formation. Functional characterization of the neoplastic isolated repopulating cells is warranted. Disclosures:Santini:Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

Chronic myelogenous leukemia stem and progenitor cells demonstrate chromosomal instability related to repeated breakage-fusion-bridge cycles mediated by increased nonhomologous end joining

Chromosomal aberrations are an important consequence of genotoxic exposure and contribute to pathogenesis and progression of several malignancies. We investigated the susceptibility to chromosomal aberrations in chronic myelogenous leukemia (CML) progenitors after exposure to ionizing radiation. In normal progenitors, ionizing radiation induced both stable and unstable chromosomal lesions, but only stable aberrations persisted after multiple divisions. In contrast, radiation of chronic phase CML progenitors resulted in enhanced generation of unstable lesions that persisted after multiple divisions. CML progenitors demonstrated active cell cycle checkpoints and increased nonhomologous end joining DNA repair, suggesting that persistence of unstable aberrations was the result of continued generation of these lesions. CML progenitors demonstrated enhanced susceptibility to repeated cycles of chromosome damage, repair, and damage through a breakage-fusion-bridge mechanism. Perpetuation of breakage-fusion-bridge cycles in CML progenitors was mediated by classic nonhomologous end joining repair. These studies reveal a previously unrecognized mechanism of chromosomal instability in leukemia progenitors because of continued generation of unstable chromosomal lesions through repeated cycles of breakage and repair of such lesions.

Endoscopic resection techniques and ablative therapies for Barrett’s neoplasia

Esophageal adenocarcinoma is the most rapidly increasing cancer in western countries. High-grade dysplasia (HGD) arising from Barrett's esophagus (BE) is the most important risk factor for its development, and when it is present the reported incidence is up to 10% per patient-year. Adenocarcinoma in the setting of BE develops through a well known histological sequence, from non-dysplastic Barrett's to low grade dysplasia and then HGD and cancer. Endoscopic surveillance programs have been established to detect the presence of neoplasia at a potentially curative stage. Newly developed endoscopic treatments have dramatically changed the therapeutic approach of BE. When neoplasia is confined to the mucosal layer the risk for developing lymph node metastasis is negligible and can be successfully eradicated by an endoscopic approach, offering a curative intention treatment with minimal invasiveness. Endoscopic therapies include resection techniques, also known as tissue-acquiring modalities, and ablation therapies or non-tissue acquiring modalities. The aim of endoscopic treatment is to eradicate the whole Barrett's segment, since the risk of developing synchronous and metachronous lesions due to the persistence of molecular aberrations in the residual epithelium is well established.

LONG-TERM CELLULAR EFFECTS IN HUMANS CHRONICALLY EXPOSED TO IONIZING RADIATION

The aim of the study was to investigate hematopoietic injury and recovery in residents of the Techa riverside villages who had been chronically exposed to radiation as a result of the activities of the Mayak Nuclear Facility, and evaluate late effects from chronic, low-dose radiation exposures. Whole blood samples were drawn from 338 unexposed individuals resident in noncontaminated villages, and 692 individuals chronically exposed externally (to primarily gamma radiation) and internally from Sr since 1949 at decreasing dose-rates which have currently reached the background levels. The mean cumulative dose in the exposed cohort was 0.62 Gy over the years 1949 to 2008 using the Techa River Dosimetry System (TRDS) 2000. The frequency of chromosome aberrations and mutations in peripheral lymphocytes, and other aspects indicative of cellular and molecular repair of radiation damage, were measured. The subjects were divided into two study groups: (a) 171 individuals who during the early exposure period (where the highest dose-rates were prevalent) were noted to manifest leucopenia and/or were diagnosed with chronic radiation syndrome (CRS), and (b) 521 exposed individuals without cytopenia and CRS. The first group demonstrated an increased frequency of micronuclei, dicentric chromosomes, somatic mutations (CD3-CD4+cells) in lymphocytes, and mutations in the TP53 gene. In addition, they demonstrated a lower Cu/Zn-SOD concentration, a significantly increased concentration of nitric oxide, and a greater apoptotic frequency in peripheral blood lymphocytes compared to exposed individuals without leucopenia. Similar to the unexposed individuals, the second group demonstrated "background levels" of mutational frequencies several years after their exposures, but they did show an increased number of cells with delayed cell cycles based on Chk-2 concentrations compared to the unexposed population. The data are consistent with the idea that a chronic radiation exposure within a dose range from 0.01 Gy to 1.96 Gy results in more severe late hematopoietic effects in a select cohort of highly radiosensitive individuals, rather than an overall increase in late effects in cells of each exposed individual. The authors state that radiation-exposed subjects demonstrating CRS showed an activation of barrier anti-oxidative stress mechanisms at late periods after radiation exposure, apparently in response to a more severe radiation damage than subjects exposed to similar radiation doses but not demonstrating CRS. Finally, the persistence of chromosome aberrations and somatic mutations in the CRS cohort is indicative of an exhaustion of the anti-oxidative stress mechanisms responding for so many years after the exposure, leading to genomic instability.

FOLLOW-UP STUDY BY CHROMOSOME ABERRATION ANALYSIS AND MICRONUCLEUS ASSAYS IN VICTIMS ACCIDENTALLY EXPOSED TO 60Co RADIATION

The goal of this study was to assess the persistence of chromosomal aberrations and micronuclei of three victims 2 y after accidental radiation exposure to Co gamma rays. Traditional chromosome aberration analysis was performed by scoring the dicentric chromosomes (dic) and rings (r) in peripheral blood lymphocytes. Micronuclei were detected using the cytokinesis block micronucleus assay. G-banding and semi-automatic karyotype analysis was used to record translocations (t), inversions (inv) and deletions (del). The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at high levels 6 mo after the accident. Two years after exposure, the frequency was reduced to 4-11% in the three victims. However, stable chromosome aberrations, which were detected by G-banding and included t, inv, and del, remained at a high level and have an obvious dose-dependent relationship even 2 y post-exposure. The frequency of micronuclei decreased faster than that of chromosome aberrations, reaching almost a normal level two years after the accident, especially for the child victim. Unstable chromosome aberrations reduced gradually, but the stable aberration remained at a high level along with the time-lapse. The micronucleus assay was less valuable for assessing long-term effects after high dose irradiation.

Spontaneous and radiation-induced chromosomal instability and persistence of chromosome aberrations after radiotherapy in lymphocytes from prostate cancer patients

The aim of the study was to compare the spontaneous and ex vivo radiation-induced chromosomal damage in lymphocytes of untreated prostate cancer patients and age-matched healthy donors, and to evaluate the chromosomal damage, induced by radiotherapy, and its persistence. Blood samples from 102 prostate cancer patients were obtained before radiotherapy to investigate the excess acentric fragments and dicentric chromosomes. In addition, in a subgroup of ten patients, simple exchanges in chromosomes 2 and 4 were evaluated by fluorescent in situ hybridization (FISH), before the onset of therapy, in the middle and at the end of therapy, and 1 year later. Data were compared to blood samples from ten age-matched healthy donors. We found that spontaneous yields of acentric chromosome fragments and simple exchanges were significantly increased in lymphocytes of patients before onset of therapy, indicating chromosomal instability in these patients. Ex vivo radiation-induced aberrations were not significantly increased, indicating proficient repair of radiation-induced DNA double-strand breaks in lymphocytes of these patients. As expected, the yields of dicentric and acentric chromosomes, and the partial yields of simple exchanges, were increased after the onset of therapy. Surprisingly, yields after 1 year were comparable to those directly after radiotherapy, indicating persistence of chromosomal instability over this time. Our results indicate that prostate cancer patients are characterized by increased spontaneous chromosomal instability. This instability seems to result from defects other than a deficient repair of radiation-induced DNA double-strand breaks. Radiotherapy-induced chromosomal damage persists 1 year after treatment.

Persistence of Radiation-Induced Chromosome Aberrations in a Long-Term Cell Culture

The aim of the present study was to evaluate the persistence of chromosome aberrations induced by X rays. FISH painting and mFISH techniques were applied to long-term cultures of irradiated cells. With painting, at 2 Gy the frequency of apparently simple translocations remained almost invariable during all the culture, whereas at 4 Gy a rapid decline was observed between the first and the second samples, followed by a slight decrease until the end of the culture. Apparently simple dicentrics and complex aberrations disappeared after the first sample at 2 and 4 Gy. By mFISH, at 2 Gy the frequency of complete plus one-way translocations remained invariable between the first and last sample, but at 4 Gy a 60% decline was observed. True incomplete simple translocations disappeared at 2 and 4 Gy, indicating that incompleteness could be a factor to consider when the persistence of translocations is analyzed. The analysis by mFISH showed that the frequency of complex aberrations and their complexity increased with dose and tended to disappear in the last sample. Our results indicate that the influence of dose on the decrease in the frequency of simple translocations with time postirradiation cannot be fully explained by the disappearance of true incomplete translocations and complex aberrations. The chromosome involvement was random for radiation-induced exchange aberrations and non-random for total aberrations. Chromosome 7 showed the highest deviations from expected, being less and more involved than expected in the first and last samples, respectively. Some preferential chromosome-chromosome associations were observed, including a coincidence with a cluster from radiogenic chromosome aberrations described in other studies.

Chromosome aberrations and telomere length modulation in bone marrow and spleen cells of melphalan‐treated p53+/− mice

The p53 gene regulates cell cycle and apoptotic pathways after induction of DNA damage. Telomeres, capping chromosome ends, are involved in maintaining chromosome stability; alterations of their length have been related to increased levels of chromosomal aberrations. To study a possible interaction between chromosome aberrations, telomere dysfunction, and p53, we investigated via painting analysis the induction and persistence of chromosome aberrations in bone marrow and spleen cells of p53+/- (and wild type) mice exposed for 4, 13, or 26 weeks to 2 mg/kg melphalan (MLP), a chemotherapeutic agent with carcinogenic potential. In addition, telomere length was evaluated in bone marrow cells by quantitative fluorescence in situ hybridization (Q-FISH). Chromosome aberrations were significantly increased in both tissues after MLP treatment. The p53 genotype did not influence the response of spleen cells, whereas a slight but significant increase of the aberration frequency was measured in the bone marrow of p53+/- mice exposed to MLP for 13 weeks with respect to the level detected in the matched wild-type group. The main finding of our still preliminary results on telomere length modulation was again a difference between the two genotypes. In bone marrow cells of wild-type mice, MLP treatment was associated with telomere shortening, while in p53+/- mice telomere elongation was the prevalent response to MLP exposure. In agreement with previous literature data, our in vivo study suggests that even the lack of a single functional copy of the p53 gene might have an impact on the quantity and quality of chromosome alterations induced in cycling cells by a clastogenic exposure.

A Novel Assay for Development of Chromosomal Lesions in Human CD34+ Cells Following Exposure to Ionizing Radiation Reveals Increased Susceptibility To Acquire Unstable Chromosomal Lesions in CML CD34+ Cells.

Acquisition of chromosomal lesions likely plays an important role in pathogenesis of primary and therapy-related leukemia, as well as in disease evolution in myeloproliferative and myelodysplastic disorders. However the process of acquisition of chromosomal lesions in hematopoietic stem cells (HSC) is not well understood. Development of persistent chromosomal abnormalities may involve several factors including acquisition of DNA lesions, repair of DNA damage, sensing of nonrepaired or misrepaired lesions and activation of cell cycle checkpoint and apoptotic pathways, and clonal growth advantage conferred by the lesions. In the current study we assessed the frequency, nature and kinetics of chromosomal lesions following exposure to genotoxic agents in normal human HSC and determined whether these were altered in CML, a prototypic HSC malignancy associated with genetic instability and acquisition of new chromosomal abnormalities during disease progression. CD34+ cells were selected from 4 normal donors and 4 newly diagnosed, untreated chronic phase CML patients. Cells were exposed to increasing doses of γ-radiation, cultured with growth factors and metaphase spreads assessed for development of chromosomal lesions. Chromosome painting was performed using chromosomes 1, 3, 5, 7, 11 and 21 probes, representing 32% of genomic DNA, with >100 metaphases scored per dose per time. Radiation exposure resulted in induction of chromosomal lesions in normal CD34+ cells in a dose-dependent manner. Chromosomal lesions were not seen in cells cultured without radiation exposure. The frequency of aberrant metaphases after 72 h culture (shown by cell cycle analysis to represent the first cell division for normal CD34+ cells) was 2.7% with 0.5 Gy, 4.8% with 1.0 Gy, 9.9% with 2.0 Gy and 23.5% with 4.0 Gy exposures. Chromosome aberrations observed at first division included both stable (translocations, insertions) and unstable (excess fragments, dicentrics) lesions. Subsequent results are shown for exposure to 2.0 Gy. The frequency of aberrant metaphases dropped to 5.3% at 144 h (3–4 cell divisions); only stable lesions persisted at this time. In contrast to normal CD34+ cells, first cell division in CML CD34+ cells was seen at 24 h, at which time 15.3% of the metaphases showed aberrations, whereas 11.2% of metaphases showed aberrations after 72 h (3–4 divisions) and 11.2% after 144 h (6–7 divisions). A striking difference between normal and CML cells was persistence of unstable aberrations after several cell divisions in the latter (42.9% unstable lesions present even after 6–7 divisions). These observations suggest impaired ability to sense and eliminate cells with chromosomal lesions or continued generation of such lesions after initial radiation exposure in CML cells. In conclusion we have developed a novel chromosome painting based assay for evaluation of acquisition of chromosomal lesions in primitive hematopoietic cells. We have demonstrated an inherent chromosomal instability that may contribute to clonal evolution and disease progression in CML CD34+ cells. This assay will provide a useful platform for: i) assessment of mechanisms underlying development of chromosomal lesions in response to DNA damage; and ii) assessing susceptibility to genotoxic agents, and allow improved understanding of pathogenesis and disease evolution in myeloid malignancies.

Telomere Shortening and Associated Chromosomal Instability in Peripheral Blood Lymphocytes of Patients With Hodgkin’s Lymphoma Prior to Any Treatment Are Predictive of Second Cancers

To investigate a potential link between telomere length, chromosomal instability, and the advent of a second cancer (SC) in patients with Hodgkin's lymphoma (HL), who are known to be at risk for SCs. This study was premised on the finding that telomere dysfunction and DNA repair pathways were related to many pathologic conditions. Three cohorts of patients with HL were studied: 73 who were prospectively followed >5 years after diagnosis (prospective HL cohort), 28 who developed a SC (SC HL cohort), and 18 long-term survivors with no evidence of disease or complication since their initial treatment (NED HL cohort). Telomere length was analyzed by a telomeric restriction fragment assay in peripheral blood lymphocytes. Thirty healthy donors and 70 patients with a newly diagnosed solid tumor were the control population. Compared with controls, patients from the prospective HL cohort, before any treatment, showed age-independent shorter telomeres (mean, 8.3 vs. 11.7 kb in healthy donors; <6 kb in 18% in HL patients), increased spontaneous chromosomal abnormalities, and increased in vitro radiation sensitivity (p < 10(-4) each). After treatment, telomere shortening was associated with cytogenetic profiles characterized by the persistence of complex chromosomal rearrangement and clonal aberrations. Moreover, the two cases of SC in the prospective HL patients had short telomeres and CCR initially. In addition, the SC HL cohort was characterized by markedly short telomeres (6.6 vs. 9.7 kb in the NED HL cohort), the presence of complex chromosome rearrangements, and increased in vitro radiation sensitivity. An intimate relationship between pre-treatment telomere shortening, chromosomal instability, radiation sensitivity and occurrence of SC was found in HL patients.

Persistence of chromosome aberrations following acute radiation: I, PAINT translocations, dicentrics, rings, fragments, and insertions

Chromosome translocations are used to estimate the doses of radiation received following occupational or accidental exposure. Biodosimetry relies on the assumption that translocations are not cell-lethal and persist with little or no loss over time. While translocations do exhibit substantially greater persistence than other aberration types (e.g., dicentrics), there is evidence that translocation frequencies also decline over time, at least following acute doses above 1 Gy. To the extent that translocation frequencies decline, the predicted absorbed doses will be underestimated. Yet unknown is whether translocations induced by ionizing radiation at doses below 1 Gy also show significant declines. Here we report on the persistence of translocations induced by 137Cs gamma-rays at acute doses ranging from 0.2 to 4 Gy using peripheral blood lymphocytes from two unrelated healthy male donors. Chromosome aberrations were evaluated by simultaneously painting chromosomes 1, 2, and 4 in red and 3, 5, and 6 in green in cells harvested 2-7 days following exposure and were scored using the PAINT system. Translocations were also enumerated using several other methods and these results are reported separately by us in this issue. For comparison, the persistence of dicentrics, rings, acentric fragments, and color junctions was also evaluated and showed rapid losses with time. The results from both donors provide evidence that translocation frequencies decline with time in a statistically significant manner at doses as low as 0.2-0.3 Gy. The frequency of translocations for all dose groups declined from day 2 to 7 by averages of 39% and 26% for donors 1 and 2, respectively. These data emphasize the importance of considering translocation loss in biological dosimetry long times after exposure.

Persistence of chromosome aberrations following acute radiation: II, Does it matter how translocations are scored?

Chromosome breaks and rearrangements resulting from ionizing radiation can be much more complicated than many investigators thought possible some years ago. The realization that not all translocations are reciprocal, that multiway exchanges occur, and that some double-strand breaks are not repaired prior to mitosis have all contributed to the difficulty of knowing how best to identify, record, evaluate, and report chromosome translocations. Here we describe the results of a series of experiments in which blood from two normal healthy subjects was obtained, irradiated with 137Cs gamma-rays in vitro at doses ranging from 0 (controls) to 4 Gy, and cultured. Cells from each dose group and donor were harvested at days 2, 2.5, 3, 4, 5, and 7 and evaluated for chromosome damage by simultaneously painting chromosomes 1, 2, and 4 in red and 3, 5, and 6 in green. The persistence of dicentrics, fragments, rings, insertions, and PAINT translocations are reported separately by us in this issue. In this article, we focus on translocations, characterizing the various types in detail and comparing and contrasting their persistence across all dose groups for both donors. The results indicate that the persistence of all translocation types was sufficient to be used for retrospective dosimetry, although nonreciprocal translocations exhibited diminished persistence compared to the other types. We also characterize the kinetics of the radiation dose responses of the two donors who exhibited significant differences in the induction as well as the persistence of translocations. Based on the evidence presented here, we hypothesize that these individuals differ in the recognition and repair of radiation-induced damage as well as in cell cycle checkpoint control. Despite these differences, the temporal frequency of translocation losses at both the high and low doses was similar for both subjects.

Long-term persistence of chromosome aberrations in uranium miners.

Chromosome aberration analyses were performed on blood samples from 165 active underground uranium miners between 1981 and 1985. After decommissioning the mine in 1997 chromosome aberration analyses were also included in the medical laboratory investigations of health conditions of 141 subjects between 1998 and 2002 within the framework of a follow-up-study. The numerical data are presented as functions of the exposure categories expressed in working level month up to 600. In the active groups the dicentric level was 7 to 12 times higher than in the unexposed population, the acentrics also higher with more than an order of magnitude, the frequency of total aberrations--including dicentrics, acentrics, rings, deletions, minits and numerical aberrations, i.e. both chromatid and chromosome type of aberrations were also well above the control level. In the group of former uranium miners although there were slight decreases in the dicentrics after 8 to 25 yr, the values were not significantly different from the values of active miners. The frequency of deletions was also maintained in the post-mining period. The frequency of acentrics, however, decreased significantly, but even the lowest values remained 2-3 times higher than the values in the unexposed population.The possibility is suggested that for the long-term persistence of cytogenetic alterations the permanent production and presence of clastogenic factors might be responsible. The comparison of the two datasets suggest a long-term persistence of cytogenetic alterations above the population average values in a large fraction of persons investigated.

Persistence of chromosome aberrations in peripheral lymphocytes from Hodgkin's lymphoma remission patients

Purpose : To analyse spontaneous and in vitro bleomycin-induced chromosome aberrations in peripheral lymphocytes taken from Hodgkin's disease patients after prolonged (up to 31 years) remission periods, and to consider these data from the point of view of the carcinogenic potential of anticancer therapy. Materials and methods : Conventional analysis of chromosome preparations stained with azure-eosin. Results : The mean frequency and patterns of both spontaneous and induced aberrations in remission patients were significantly different from comparison groups (healthy donors and primary Hodgkin's disease patients). Individual values were characterized with high variation and did not show correlation with post-therapy time. New cancer cases diagnosed in remission patients were more frequent in subjects with high chromosome sensitivity to in vitro bleomycin challenge than in patients whose sensitivity to bleomycin was at a control level. Conclusions : The results are interpreted as suggesting that the tumorigenic potential of radiochemotherapy is mediated via induction of genetic instability in exposed cells. Long after the therapy, the instability may become an initiating event in the development of new malignancies in affected tissues, whereas the instability induced in haemopoietic stem cells may reveal itself in peripheral lymphocytes derived from formerly exposed precursors.

Reciprocal translocations in patients with testicular seminoma before and after radiotherapy

Purpose: The evaluation of radiation-induced chromosomal translocations in peripheral lymphocytes using fluorescent in situ hybridization is a promising method for retrospective dosimetry after a radiation accident. We evaluated the genomic frequency of chromosomal translocations in patients with testicular seminoma who received adjuvant radiotherapy to the retroperitoneal lymph nodes, to evaluate the time–effect relationship of radiation-induced stable aberrations after partial body irradiation. Methods: In 13 patients, peripheral lymphocytes could be evaluated before radiotherapy and at several time points after radiotherapy. In 17 additional patients, lymphocyte samples were obtained after radiotherapy. Thirteen healthy men served as age-matched controls for the aberration frequency before radiotherapy. Fluorescent in situ hybridization was performed using whole chromosome probes against chromosomes No. 4, No. 6, and No. 7. Results: Nearly all patients displayed an increased spontaneous rate of genomic translocations ( F G ) before radiotherapy compared to age-matched, healthy men. The difference was significant in the paired ranks test ( p < 0.0001). After adjuvant radiotherapy, the F G increased 2- to 8-fold in individual patients. Within 20 months after radiotherapy, the F G returned to pretherapeutic levels. Conclusions: The frequency of genomic translocations after partial body irradiation is time dependent. A persistence of chromosomal aberrations, which is to be expected after total body irradiation, could not be observed. It is likely that the dose and the volume of the irradiated bone marrow are playing a role in the persistence of stable chromosomal aberrations. Patients with testicular seminoma displayed an increased frequency of spontaneous genomic translocations before the initiation of radiotherapy. This chromosomal instability might be related to the known increased rate of secondary cancers in this patient group.