Large-Scale Plasma Proteome Epitome Profiling is an Efficient Tool for the Discovery of Cancer Biomarkers.

Current proteomic technologies focus on the quantification of protein levels, while little effort is dedicated to the development of system approaches to simultaneously monitor proteome variability and abundance. Protein variants may display different immunogenic epitopes detectable by monoclonal antibodies. Epitope variability results from alternative splicing, posttranslational modifications, processing, degradation, and complex formation and possesses dynamically changing availability of interacting surface structures that frequently serve as reachable epitopes and often carry different functions. Thus, it is highly likely that the presence of some of the accessible epitopes correlates with function under physiological and pathological conditions. To enable the exploration of the impact of protein variation on the immunogenic epitome first, here, we present a robust and analytically validated PEP technology for characterizing immunogenic epitopes of the plasma. To this end, we prepared mAb libraries directed against the normalized human plasma proteome as a complex natural immunogen. Antibody producing hybridomas were selected and cloned. Monoclonal antibodies react with single epitopes, thus profiling with the libraries is expected to profile many epitopes which we define by the mimotopes, as we present here. Screening blood plasma samples from control subjects (n = 558) and cancer patients (n = 598) for merely 69 native epitopes displayed by 20 abundant plasma proteins resulted in distinct cancer-specific epitope panels that showed high accuracy (AUC 0.826-0.966) and specificity for lung, breast, and colon cancer. Deeper profiling (≈290 epitopes of approximately 100 proteins) showed unexpected granularity of the epitope-level expression data and detected neutral and lung cancer-associated epitopes of individual proteins. Biomarker epitope panels selected from a pool of 21 epitopes of 12 proteins were validated in independent clinical cohorts. The results demonstrate the value of PEP as a rich and thus far unexplored source of protein biomarkers with diagnostic potential.

Benefit of an association of an antioxidative substrate and a traditional chinese medicine on telomere elongation.

Telomere shortening is involved in age-related disorders, such as cancer and cardiovascular diseases. Recently, telomerase re-activation strategies have been proposed to counteract telomere shortening and its consequences. Here, we investigated the benefit of dietary supplementation with a mix of S-adenosyl-methionine (SAMe) and a polysaccharide extract of Astragalus (APS) on telomere length of circulating lymphocytes of healthy volunteers. Blood lymphocytes of a cohort of 26 healthy volunteers who were administrated the mix of SAMe and APS in a food supplement for one year were collected. In vitro treatment of blood lymphocytes of healthy volunteers with the mix was also performed. A cohort of 150 healthy volunteers was used as a control. Telomere length was measured by Q-FISH. The micronucleus assay was performed to detect genotoxicity of the mix. The telomeres of circulating lymphocytes of the cohort of 26 donors supplemented with the mix were significantly longer than those of matched controls (p < 10-4). This elongation was essentially observed in the lymphocytes of older donors. Similarly, in vitro treatment of circulating lymphocytes with the mix significantly increased telomere length and decrease the proportion of cells with short telomeres. Here, we observed an increase in telomere length after in vivo and in vitro administration of a mix with SAMe and APS.  The benefit of dietary supplementation with this mix opens a new horizon for the battle against aging and could be used in the treatment of chronic age-related disorders.

Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries.

A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.

Is Response to Genotoxic Stress Similar in Populations of African and European Ancestry? A Study of Dose-Response After in vitro Irradiation.

Background: Exposure to genotoxic stress such as radiation is an important public health issue affecting a large population. The necessity of analyzing cytogenetic effects of such exposure is related to the need to estimate the associated risk. Cytogenetic biological dosimetry is based on the relationship between the absorbed dose and the frequency of scored chromosomal aberrations. The influence of confounding factors on radiation response is a topical issue. The role of ethnicity is unclear. Here, we compared the dose-response curves obtained after irradiation of circulating lymphocytes from healthy donors of African and European ancestry. Materials and Methods: Blood samples from six Africans living in Africa, five Africans living in Europe, and five Caucasians living in Europe were exposed to various doses (0-4 Gy) of X-rays at a dose-rate of 0.1 Gy/min using an X-RAD320 irradiator. A validated cohort composed of 14 healthy Africans living in three African countries was included and blood samples were irradiated using the same protocols. Blood lymphocytes were cultured for 48 h and chromosomal aberrations scored during the first mitosis by telomere and centromere staining. The distribution of dicentric chromosomes was determined and the Kruskal-Wallis test was used to compare the dose-response curves of the two populations. Results: No spontaneous dicentric chromosomes were detected in African donors, thus establishing a very low background of unstable chromosomal aberrations relative to the European population. There was a significant difference in the dose response curves between native African and European donors. At 4 Gy, African donors showed a significantly lower frequency of dicentric chromosomes (p = 8.65 10-17), centric rings (p = 4.0310-14), and resulting double-strand-breaks (DSB) (p = 1.32 10-18) than European donors. In addition, a significant difference was found between African donors living in Europe and Africans living in Africa. Conclusion: This is the first study to demonstrate the important role of ethnic and environmental factors that may epigenetically influence the response to irradiation. It will be necessary to establish country-of-origen-specific dose response curves to practice precise and adequate biological dosimetry. This work opens new perspective for the comparison of treatments based on genotoxic agents, such as irradiation.

Telomere aberrations, including telomere loss, doublets, and extreme shortening, are increased in patients with infertility.

OBJECTIVE: To test the hypothesis that telomere shortening and/or loss are risk factors for infertility. DESIGN: Retrospective analysis of the telomere status in patients with infertility using conventional cytogenetic data collected prospectively. SETTING: Academic centers. PATIENT(S): Cytogenetic slides with cultured peripheral lymphocytes from 50 patients undergoing fertility treatment and 150 healthy donors, including 100 donors matched for age. INTERVENTION(S): Cytogenetic slides were used to detect chromosomal and telomere aberrations. MAIN OUTCOME MEASURE(S): Telomere length and telomere aberrations were analyzed after telomere and centromere staining. RESULT(S): The mean telomere length of patients consulting for infertility was significantly less than that of healthy donors of similar age. Moreover, patients with infertility showed significantly more extreme telomere loss and telomere doublet formation than healthy controls. Telomere shortening and/or telomere aberrations were more pronounced in patients with structural chromosomal aberrations. Dicentric chromosomes were identified in 6/13 patients, with constitutional chromosomal aberrations leading to chromosomal instability that correlated with chromosomal end-to-end fusions. CONCLUSION(S): Our findings demonstrate the feasibility of analyzing telomere aberrations in addition to chromosomal aberrations, using cytogenetic slides. Telomere attrition and/or dysfunction represent the main common cytogenetic characteristic of patients with infertility, leading to potential implications for fertility assessment. Pending further studies, these techniques that correlate the outcome of assisted reproduction and telomere integrity status may represent a novel and useful diagnostic and/or prognostic tool for medical care in this field.

A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining.

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.

Telomere and Centromere Staining Followed by M-FISH Improves Diagnosis of Chromosomal Instability and Its Clinical Utility.

Dicentric chromosomes are a relevant marker of chromosomal instability. Their appearance is associated with telomere dysfunction, leading to cancer progression and a poor clinical outcome. Here, we present Telomere and Centromere staining followed by M-FISH (TC+M-FISH) for improved detection of telomere dysfunction and the identification of dicentric chromosomes in cancer patients and various genetic syndromes. Significant telomere length shortening and significantly higher frequencies of telomere loss and deletion were found in the peripheral lymphocytes of patients with cancer and genetic syndromes relative to similar age-matched healthy donors. We assessed our technique against conventional cytogenetics for the detection of dicentric chromosomes by subjecting metaphase preparations to both approaches. We identified dicentric chromosomes in 28/50 cancer patients and 21/44 genetic syndrome patients using our approach, but only 7/50 and 12/44, respectively, using standard cytogenetics. We ascribe this discrepancy to the identification of the unique configuration of dicentric chromosomes. We observed significantly higher frequencies of telomere loss and deletion in patients with dicentric chromosomes (p < 10-4). TC+M-FISH analysis is superior to classical cytogenetics for the detection of chromosomal instability. Our approach is a relatively simple but useful tool for documenting telomere dysfunction and chromosomal instability with the potential to become a standard additional diagnostic tool in medical genetics and the clinic.

Lipid metabolite levels of prostaglandin D2 and eicosapentaenoic acid recovered from bronchoalveolar lavage fluid correlate with lung function of chronic obstructive pulmonary disease patients and controls.

One-step global profiling of analyte (mRNA, protein, metabolite) biomarkers may soon replace conventional blood and histological/biopsy diagnostics technologies. It is important to establish whether the numerous blood and other body fluid-derived potential novel diagnostics will be sufficiently efficacious and precise to replace, for example, imaging and functional diagnostic tests. Currently, imaging technologies and spirometry are indispensable for the diagnosis and management of chronic obstructive pulmonary disease (COPD). To validate the concept of using body fluid biomarkers in COPD and to address the question of whether biomarker levels correlate with lung function, we measured the level of a number of biologically relevant lipids and metabolites in the bronchoalveolar lavage (BAL) fluid of COPD and control subjects and examined whether these correlate with numeric parameters of lung function. Both the diagnosis and management of COPD rely on costly and labor intensive lung function tests. Thus, there is an imminent need to replace the current diagnostic approaches with simpler clinical assays. As a first step, we demonstrate proof of principle; the correlation of lipid biomarkers as measured by LC-MS with lung function. In the apparently BAL-accessible fluid compartment, the total recovered lipid metabolite amount, particularly prostaglandin D(2) and eicosapentaenoic acid show a remarkable linear correlation with lung function (R(2)>0.7). The study outcome is encouraging for the continuation of the work toward the measurement of lipid metabolite levels in more easily obtainable biological fluids such as sputum, exhaled air condensate, urine and plasma.

Biological Dosimetry Network in Africa: Establishment of a Dose-Response Curve Using Telomere and Centromere Staining.

PURPOSE: Biological dosimetry, based on the relationship between the absorbed dose after exposure to ionizing radiation and the frequency of scored aberrations, has been and continues to be an important tool for estimating the dose after exposure. Dicentric chromosomes are considered to be the most specific and sensitive aberration related to radiation exposure. Here, we established the dose-response curve following in vitro irradiation of circulating lymphocytes from healthy donors from three African countries after scoring unstable chromosomal aberrations. MATERIALS AND METHODS: Blood samples from 16 African donors were exposed to various doses (0 to 4 Gy) using an X-RAD320 x-ray system with a maximum photon energy of 250 kV at a dose rate of 0.1 Gy min. Blood lymphocytes were cultured for 48 h, and chromosomal aberrations were scored during the first mitosis by telomere and centromere staining. The distribution of dicentric chromosomes was determined. RESULTS: No dicentric chromosomes were found after the analysis of 2,669 first-division metaphases before in vitro exposure. We established a linear-quadratic dose-response curve based on the frequency of dicentric and ring chromosomes and calculated double-strand breaks, taking into account all scored aberrations. CONCLUSION: The generation of a specific dose-response curve for African donors will allow the practice of precise biological dosimetry in these countries. This work is the first step towards realizing an African biodosimetry network and the establishment of a biological dosimetry laboratory, which could play a major role in the application of radioprotection norms.

Erratum: Cuceu, C., et al. Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability. Cancers 2018, 10, 233.

The authors wish to make the following corrections to this paper [...].

Chromosomal Instability in Hodgkin Lymphoma: An In-Depth Review and Perspectives.

The study of Hodgkin lymphoma (HL), with its unique microenvironment and long-term follow-up, has provided exceptional insights into several areas of tumor biology. Findings in HL have not only improved our understanding of human carcinogenesis, but have also pioneered its translation into the clinics. HL is a successful paradigm of modern treatment strategies. Nonetheless, approximately 15-20% of patients with advanced stage HL still die following relapse or progressive disease and a similar proportion of patients are over-treated, leading to treatment-related late sequelae, including solid tumors and organ dysfunction. The malignant cells in HL are characterized by a highly altered genomic landscape with a wide spectrum of genomic alterations, including somatic mutations, copy number alterations, complex chromosomal rearrangements, and aneuploidy. Here, we review the chromosomal instability mechanisms in HL, starting with the cellular origin of neoplastic cells and the mechanisms supporting HL pathogenesis, focusing particularly on the role of the microenvironment, including the influence of viruses and macrophages on the induction of chromosomal instability in HL. We discuss the emerging possibilities to exploit these aberrations as prognostic biomarkers and guides for personalized patient management.

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability †.

Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

Background: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10-3), event-free survival (p < 10-4), and freedom from progression (p < 10-3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.

Transmission of Induced Chromosomal Aberrations through Successive Mitotic Divisions in Human Lymphocytes after In Vitro and In Vivo Radiation.

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

Replication Timing of Human Telomeres is Conserved during Immortalization and Influenced by Respective Subtelomeres.

Telomeres are specific structures that protect chromosome ends and act as a biological clock, preventing normal cells from replicating indefinitely. Mammalian telomeres are replicated throughout S-phase in a predetermined order. However, the mechanism of this regulation is still unknown. We wished to investigate this phenomenon under physiological conditions in a changing environment, such as the immortalization process to better understand the mechanism for its control. We thus examined the timing of human telomere replication in normal and SV40 immortalized cells, which are cytogenetically very similar to cancer cells. We found that the timing of telomere replication was globally conserved under different conditions during the immortalization process. The timing of telomere replication was conserved despite changes in telomere length due to endogenous telomerase reactivation, in duplicated homologous chromosomes, and in rearranged chromosomes. Importantly, translocated telomeres, possessing their initial subtelomere, retained the replication timing of their homolog, independently of the proportion of the translocated arm, even when the remaining flanking DNA is restricted to its subtelomere, the closest chromosome-specific sequences (inferior to 500 kb). Our observations support the notion that subtelomere regions strongly influence the replication timing of the associated telomere.

Comparison of Individual Radiosensitivity to γ-Rays and Carbon Ions.

Carbon ions are an up-and-coming ion species, currently being used in charged particle radiotherapy. As it is well established that there are considerable interindividual differences in radiosensitivity in the general population that can significantly influence clinical outcomes of radiotherapy, we evaluate the degree of these differences in the context of carbon ion therapy compared with conventional radiotherapy. In this study, we evaluate individual radiosensitivity following exposure to carbon-13 ions or γ-rays in peripheral blood lymphocytes of healthy individuals based on the frequency of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) that was either misrepaired or left unrepaired to form chromosomal aberrations (CAs) (simply referred to here as DSBs for brevity). Levels of DSBs were estimated from the scoring of CAs visualized with telomere/centromere-fluorescence in situ hybridization (TC-FISH). We examine radiosensitivity at the dose of 2 Gy, a routinely administered dose during fractionated radiotherapy, and we determined that a wide range of DSBs were induced by the given dose among healthy individuals, with highly radiosensitive individuals harboring more IR-induced breaks in the genome than radioresistant individuals following exposure to the same dose. Furthermore, we determined the relative effectiveness of carbon irradiation in comparison to γ-irradiation in the induction of DSBs at each studied dose (isodose effect), a quality we term "relative dose effect" (RDE). This ratio is advantageous, as it allows for simple comparison of dose-response curves. At 2 Gy, carbon irradiation was three times more effective in inducing DSBs compared with γ-irradiation (RDE of 3); these results were confirmed using a second cytogenetic technique, multicolor-FISH. We also analyze radiosensitivity at other doses (0.2-15 Gy), to represent hypo- and hyperfractionation doses and determined that RDE is dose dependent: high ratios at low doses, and approaching 1 at high doses. These results could have clinical implications as IR-induced DNA damage and the ensuing CAs and genomic instability can have significant cellular consequences that could potentially have profound implications for long-term human health after IR exposure, such as the emergence of secondary cancers and other pathobiological conditions after radiotherapy.

Global quantification of γH2AX as a triage tool for the rapid estimation of received dose in the event of accidental radiation exposure.

The phosphorylation of the H2AX histone to form γH2AX foci has been shown to be an accurate biomarker of ionizing radiation exposure. It is well established that there is a one-to-one correlation between the number of γH2AX foci and radiation-induced double strand breaks in cellular DNA, which can be translated to the received dose. However, manual counting of foci is time-consuming, and cannot accommodate high throughput analysis required to obtain rapid results for medical triage purposes in the case of large-scale accidental exposure. Furthermore, the accuracy of γH2AX measurements could potentially be compromised by delays between the time of exposure and analysis of results, as well as inter-cellular and inter-individual variability of this biological response. To evaluate more rapid approaches of quantifying γH2AX for use in an emergency situation, and to determine the impact of inter-individual variability, we compared two methods of global γH2AX fluorescence quantification (low magnification immunofluorescence microscopy and flow cytometry) to the well-established γH2AX foci scoring method in human primary fibroblasts. All three approaches were well correlated, indicating that global γH2AX fluorescence measurements are suitable for dose estimation. For rapid triage in an emergency situation, we propose the use of flow cytometry, as it is more highly correlated with foci scoring and because of the speed and ease of the method. Dose response curves (0.25-6Gy) using flow cytometry measurements showed that inter-individual variability in global γH2AX fluorescence is statistically insignificant at 4h post-irradiation. Based on these data, we propose calibration curves that can be applied to populations exposed to moderate radiation doses to estimate individual received doses, independent of individual radiosensitivity, at this specific time point post-irradiation using human fibroblasts and lymphocytes. Furthermore, we define three triage categories that could facilitate immediate and follow-up care in the case of a radiological accident.

Detection and automated scoring of dicentric chromosomes in nonstimulated lymphocyte prematurely condensed chromosomes after telomere and centromere staining.

PURPOSE: To combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose-response curve and automation of the process. METHODS AND MATERIALS: Blood samples from healthy donors were exposed to (60)Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory. RESULTS: We successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose-response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings. CONCLUSION: The introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw.

Restriction endonuclease accessibility as a determinant of altered chromatin structure.

Active genes in eukaryotic genomes are typically found in open, nuclease-sensitive regions of chromatin. This chapter presents an overview of the techniques used to assay restriction endonuclease cleavage of chromosomal DNA as an approach to assess general accessibility at a genomic region of interest. We describe protocols to (1) prepare nuclei templates, (2) treat chromosomal DNA with a restriction enzyme(s), and (3) visualize and quantify chromosomal cleavage(s), with an emphasis on ligation-mediated (LM) PCR techniques.