Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma.

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Extended lifespan and improved genome stability in HepaRG-derived cell lines through reprogramming by high-density stress.

The characteristics and fate of cancer cells partly depend on their environmental stiffness, i.e., the local mechanical cues they face. HepaRG progenitors are liver carcinoma cells exhibiting transdifferentiation properties; however, the underlying mechanisms remain unknown. To evaluate the impact of external physical forces mimicking the tumor microenvironment, we seeded them at very high density for 20 h, keeping the cells round and unanchored to the substrate. Applied without corticoids, spatial confinement due to very high density induced reprogramming of HepaRG cells into stable replicative stem-like cells after replating at normal density. Redifferentiation of these stem-like cells into cells very similar to the original HepaRG cells was then achieved using the same stress but in the presence of corticoids. This demonstrates that the cells retained the memory required to run the complete hepatic differentiation program, after bypassing the Hayflick limit twice. We show that physical stress improved chromosome quality and genomic stability, through greater efficiency of DNA repair and restoration of telomerase activity, thus enabling cells to escape progression to a more aggressive cancer state. We also show the primary importance of high-density seeding, possibly triggering compressive stress, in these processes, rather than that of cell roundness or intracellular tensional signals. The HepaRG-derived lines established here considerably extend the lifespan and availability of this surrogate cell system for mature human hepatocytes. External physical stress is a promising way to create a variety of cell lines, and it paves the way for the development of strategies to improve cancer prognosis.

Modeling Global Genomic Instability in Chronic Myeloid Leukemia (CML) Using Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

METHODS: We used a patient-specific induced pluripotent stem cell (iPSC) line treated with the mutagenic agent N-ethyl-N-nitrosourea (ENU). Genomic instability was validated using γ-H2AX and micronuclei assays and CGH array for genomic events. RESULTS: An increased number of progenitors (x5-Fold), which proliferated in liquid cultures with a blast cell morphology, was observed in the mutagenized condition as compared to the unmutagenized one. CGH array performed for both conditions in two different time points reveals several cancer genes in the ENU-treated condition, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Transcriptome GEO-dataset GSE4170 allowed us to associate 125 of 249 of the aberrations that we detected in CML-iPSC with the CML progression genes already described during progression from chronic and AP to BC. Among these candidates, eleven of them have been described in CML and related to tyrosine kinase inhibitor resistance and genomic instability. CONCLUSIONS: These results demonstrated that we have generated, for the first time to our knowledge, an in vitro genetic instability model, reproducing genomic events described in patients with BC.

High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview.

In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.

A Central Role of Telomere Dysfunction in the Formation of a Unique Translocation within the Sub-Telomere Region Resulting in Duplication and Partial Trisomy.

Telomeres play a major role in maintaining genome stability and integrity. Putative involvement of telomere dysfunction in the formation of various types of chromosomal aberrations is an area of active research. Here, we report a case of a six-month-old boy with a chromosomal gain encompassing the 11q22.3q25 region identified by SNP array analysis. The size of the duplication is 26.7 Mb and contains 170 genes (OMIM). The duplication results in partial trisomy of the region in question with clinical consequences, including bilateral renal dysplasia, delayed development, and a heart defect. Moreover, the karyotype determined by R-banding and chromosome painting as well as by hybridization with specific sub-telomere probes revealed the presence of an unbalanced t(9;11)(p24;q22.3) translocation with a unique breakpoint involving the sub-telomere region of the short arm of chromosome 9. The karyotypes of the parents were normal. Telomere integrity in circulating lymphocytes from the child and from his parents was assessed using an automated high-throughput method based on fluorescence in situ hybridization (FISH) with telomere- and centromere-specific PNA probes followed by M-FISH multicolor karyotyping. Very short telomeres, as well as an increased frequency of telomere loss and formation of telomere doublets, were detected in the child's cells. Interestingly, similar telomere profiles were found in the circulating lymphocytes of the father. Moreover, an assessment of clonal telomere aberrations identified chromosomes 9 and 11 with particularly high frequencies of such aberrations. These findings strongly suggest that telomere dysfunction plays a central role in the formation of this specific unbalanced chromosome rearrangement via chromosome end-to-end fusion and breakage-fusion-bridge cycles.

Patient-Derived iPSCs Reveal Evidence of Telomere Instability and DNA Repair Deficiency in Coats Plus Syndrome.

Coats plus (CP) syndrome is an inherited autosomal recessive condition that results from mutations in the conserved telomere maintenance component 1 gene (CTC1). The CTC1 protein functions as a part of the CST protein complex, a protein heterotrimer consisting of CTC1-STN1-TEN1 which promotes telomere DNA synthesis and inhibits telomerase-mediated telomere elongation. However, it is unclear how CTC1 mutations may have an effect on telomere structure and function. For that purpose, we established the very first induced pluripotent stem cell lines (iPSCs) from a compound heterozygous patient with CP carrying deleterious mutations in both alleles of CTC1. Telomere dysfunction and chromosomal instability were assessed in both circulating lymphocytes and iPSCs from the patient and from healthy controls of similar age. The circulating lymphocytes and iPSCs from the CP patient were characterized by their higher telomere length heterogeneity and telomere aberrations compared to those in control cells from healthy donors. Moreover, in contrast to iPSCs from healthy controls, the high levels of telomerase were associated with activation of the alternative lengthening of telomere (ALT) pathway in CP-iPSCs. This was accompanied by inappropriate activation of the DNA repair proteins γH2AX, 53BP1, and ATM, as well as with accumulation of DNA damage, micronuclei, and anaphase bridges. CP-iPSCs presented features of cellular senescence and increased radiation sensitivity. Clonal dicentric chromosomes were identified only in CP-iPSCs after exposure to radiation, thus mirroring the role of telomere dysfunction in their formation. These data demonstrate that iPSCs derived from CP patients can be used as a model system for molecular studies of the CP syndrome and underscores the complexity of telomere dysfunction associated with the defect of DNA repair machinery in the CP syndrome.

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.

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.

The Use of Natural Agents to Counteract Telomere Shortening: Effects of a Multi-Component Extract of Astragalus mongholicus Bunge and Danazol.

A link between telomere shortening and oxidative stress was found in aging people and patients with cancer or inflammatory diseases. Extracts of Astragalus spp. are known to stimulate telomerase activity, thereby compensating telomere shortening. We characterized a multi-component hydroethanolic root extract (HRE) of Astragalus mongholicus Bunge and assessed its effects on telomeres compared to those of danazol. Astragalosides I to IV, flavonoids, amino acids and sugars were detected in the HRE. Samples of peripheral blood lymphocytes with short telomeres from 18 healthy donors (mean age 63.5 years; range 3286 years) were exposed to a single dose of 1 µg/mL HRE or danazol for three days. Telomere length and telomerase expression were then measured. Significant elongation of telomeres associated to a less toxicity was observed in lymphocytes from 13/18 donors following HRE treatment (0.54 kb (0.15-2.06 kb)) and in those from 9/18 donors after danazol treatment (0.95 kb (0.06-2.06 kb)). The rate of cells with short telomeres (<3 kb) decreased in lymphocytes from all donors after exposure to either HRE or danazol, telomere elongation being telomerase-dependent. These findings suggest that the HRE could be used for the management of age-related diseases.

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 [...].

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.

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.

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.

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.

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.

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.