3D Nuclear Organization Of Telomeres Research Articles

3D Telomere Structure Analysis to DetectGenomic Instability and Cytogenetic Evolutionin Myelodysplastic Syndromes.

The disease course of myelodysplastic syndromes (MDS) features chromosome instability and clonal evolution, leading to the sequential acquisition of novel cytogenetic aberrations and the accumulation of these abnormalities in the bone marrow. Although clonal cytogenetic abnormalities can be detected by conventional cytogenetics in 50% of patients with MDS, such distinguishing patterns are lacking in the other 50%. Despite the increase in the prognostic value of some biomarkers, none of them is specific and able to discriminate between stable and unstable patients that subsequently progress to acute myeloid leukemia. This pilot study aimed to investigate the potential use of the 3D telomere profiling to detect genomic instability in MDS patients with or without clonal cytogenetic evolution. The comparison between different time points in patients with cytogenetic changes showed that in the CD34+ MDS cells, there was a significant decrease in the total number of telomeric signals, the average intensity of signals and the total intensity of telomeres. By contrast, the number of aggregates increased during cytogenetic evolution (p < 0.001). This pattern was observed only for MDS patients with cytogenetic evolution but was absent in patients without cytogenetic changes. In conclusion, we demonstrated that the 3D nuclear telomere organization was significantly altered during the MDS disease course, and may have contributed to cytogenetic clonal evolution.

Abstract 367: Methods to enhance the optical resolution for 3D-SIM for the study of Hodgkin's lymphoma nuclei

Abstract In spite of a great deal of knowledge already available, the precise higher order organization of chromatin is still a subject of an debate. Moreover, cancerous cells have an altered chromatin organization in comparison to their healthy counterparts. This is also the case for Hodgkin's lymphoma (HL) - a lymphoid malignancy of B cell origin. HL comprises two cell types, the Hodgkin cells and the diagnostic bi- or multinucleated Reed-Sternberg (RS) cells readily distinguishable from healthy lymphocytes. Patient stratitifcation has been proposed through the quantitative analysis of 3D nuclear telomere organization. With the advent of super resolution microscopy (SRM), further insights into the spatial organization of the cancer genome become feasible. This includes the study of chromatin arrangement with SRM. However, its application to HL remains limited due to technical difficulties: HL cells display thickness of &amp;gt;10µm resulting in light scattering and decreased signal-to-noise ratio. Structured Illumination Microscopy (SIM, a SRM type) in particular has advanced studies of the sub-cellular structures. It relies on projection of excitation pattern on to a fluorescently labeled sample. Sample's information is then shifted to a detectable band doubling the 3D resolution. Sample's refractive index homogeneity and counteracting photo-bleaching are both crucial for maintenance of the sharp illumination pattern and high-quality SIM, particularly in thick samples. Nevertheless, sample mounting media for SIM have not undergone a significant evolution since almost a decade. By identification and systematic comparison of non-hazardous components of mounting media we demonstrate increased sample's transparency and consequently improved SIM-quality. Using these novel sample clearing protocols we demonstrate high-resolution chromatin organization at ~120nm level in large RS cells of HL cell lines and in patient samples compared to lymphocytes. This work is of relevance of identification of new diagnostic structural biomarkers for HL. Citation Format: Aleksander T. Szczurek, Fabio Contu, Roberta Vanni, Hans Knecht, Nathalie Johnson, Tina Haliotis, Sabine Mai. Methods to enhance the optical resolution for 3D-SIM for the study of Hodgkin's lymphoma nuclei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 367.

XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711–2719, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

Effect of XPO1 inhibition in cancer cells on the 3D nuclear organization of telomeres: Quantitative analysis of short-term effects.

e22148 Background: The three-dimensional (3D) organization of the nucleus, especially the nuclear pore complex (NPC), is key to its normal function. Cancer cells have a disrupted nuclear organization that can be quantified by using 3D nuclear telomere imaging. Perturbation of NPCs can alter cell proliferation and signal transduction pathways and can lead to cancer cell death. We hypothesize that the 3D nuclear architecture of cancer cells is affected when NPCs are compromised through XPO1 inhibition by Selective Inhibitors of Nuclear Export (SINE). Methods: We used three SINE compounds; KPT-330, KPT-185, and KPT- 8602. They were individually administered at 1 μM concentration for 4 hours to proliferating cancer cell lines. The cancer cells investigated included lymphoid (Raji and Jurkat) and breast cancer cell lines (T47D and HCC1937). After four hours of incubation, the cells were 3D fixed, processed for 3D telomere hybridizations, and 3D imaged. The 3D nuclear telomere organization was measured and quan...

Abstract B19: Three-dimensional nuclear telomere organization and clinical significance in non-small cell lung cancer patients.

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death worldwide. Survival of NSCLC patients remains unfavorable prompting a better understanding of its molecular biology and causality, along with advances in diagnostic methods and multidisciplinary therapeutic approaches. Aberrant three-dimensional (3D) nuclear telomere organization can identify patient subgroups in various cancers and its correlation with tumor and patient characteristics may play a major role as a predictive and prognostic factor in NSCLC of different subgroups. This study aimed to explore the clinical application of 3D nuclear telomeric organization in NSCLC patients of different EGFR mutational status and smoking backgrounds. Paraffin-embedded NSCLC tissue specimens from non-smoking (N=6) and smoking (N=4) patients who underwent surgical resections at the British Columbia Cancer Research Centre of Canada were examined using the 3D fluorescent in situ hybridization (FISH). 3D nuclear-telomeric architecture analyses were performed using the TeloView program (Vermolen et al., 2005). The 3D nuclear telomere organization of 100 interphase nuclei per specimen was quantified by telomere numbers, telomere signal intensities, and frequencies of telomeric aggregates. Comparisons of 3D nuclear telomere organization between patient subgroups are shown below. In the smoking group, more telomeres, higher signal intensities, and greater numbers of aggregates were observed, compared to the non-smokers. The same pattern of 3D nuclear telomere organization was observed in the EGFR-negative/unknown group, compared to the EGFR-positive patients. This study provides a better understanding of 3D telomeric organization in NSCLC patients with different smoking backgrounds and EGFR mutational status, leading to a rationale of further exploration of this molecular profile in this cancer. 3D Telomeric parameters Average number of telomere per cell : Non-smokers 77.71, Smokers 80.37 Average number of aggregates per cell : Non-smokers 21.36, Smokers 27.59 Percentage of cells with aggregates : Non-smokers 2.16, Smokers 3.03 Average number of telomere per cell : Pos. EGFR 73.69, Neg./Unknown EGFR 80.95 Average number of aggregates per cell : Pos. EGFR 19.97, Neg./Unknown EGFR 25.52 Percentage of cells with aggregates : Pos. EGFR 1.96, Neg./Unknown EGFR 2.75 Citation Format: Patrapim Sunpaweravong, Chirawadee Sathitruangsak, Kelsie Thu, Wan L. Lam, Sabine Mai. Three-dimensional nuclear telomere organization and clinical significance in non-small cell lung cancer patients. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B19.

Genomic Instability: The Driving Force behind Refractory/Relapsing Hodgkin’s Lymphoma

In classical Hodgkin’s lymphoma (HL) the malignant mononuclear Hodgkin (H) and multinuclear, diagnostic Reed-Sternberg (RS) cells are rare and generally make up <3% of the total cellular mass of the affected lymph nodes. During recent years, the introduction of laser micro-dissection techniques at the single cell level has substantially improved our understanding of the molecular pathogenesis of HL. Gene expression profiling, comparative genomic hybridization analysis, micro-RNA expression profiling and viral oncogene sequencing have deepened our knowledge of numerous facets of H- and RS-cell gene expression deregulation. The question remains whether disturbed signaling pathways and deregulated transcription factors are at the origin of refractory/relapsing Hodgkin’s lymphoma or whether these hallmarks are at least partially related to another major factor. We recently showed that the 3D nuclear organization of telomeres and chromosomes marked the transition from H- to RS-cells in HL cell lines. This transition is associated with progression of telomere dysfunction, shelterin disruption and progression of complex chromosomal rearrangements. We reported analogous findings in refractory/relapsing HL and identified the shelterin proteins TRF1, TRF2 and POT1 as targets of the LMP1 oncogene in post-germinal center B-cells. Here we summarize our findings, including data not previously published, and propose a model in which progressive disruption of nuclear integrity, a form of genomic instability, is the key-player in refractory/relapsing HL. Therapeutic approaches should take these findings into account.

3D nuclear organization and genomic instability in cancer.

To understand the genetic changes that occur at tumor initiation and during tumor progression, we focus on changes in nuclear architecture that promote the onset of genomic instability [1,2]. To determine changes in nuclear organization, we measure the 3D nuclear organization of telomeres, the ends of chromosomes. The measurements of telomeres allow one to trace the positions of chromosomes in interphase nuclei and, by using a fluorescent telomere-specific probe, all telomeres can be visualized in a single image. During the past years, we have defined the organization of telomeres in nuclei of normal, immortalized and tumor cells. We have developed quantitative software that enables us to measure the three-dimensional (3D) organization of telomeres [3,4]. The parameters we measure include: number of telomeres, sizes of telomeres, nuclear distribution of telomeres, and the presence of telomeric aggregates. The latter are clusters of telomeres that are absent from normal cells. More recently, we started to automate the 3D image acquisition and analysis and we are able to scan 15 000 cells per hour [5]. We observed changes in the nuclear organization of telomeres as a result of conditional c-Myc deregulation dependent on a functional myc box II [6,7]. We found Epstein-Barr Virus-induced changes in 3D telomere organization and resulting genomic instability [8]. Encouraged by these findings, we focused on tumor initiation and progression in patient samples and observed cancer-associated 3D nuclear telomere changes in lymphoid and solid tumors [9-12]. Moreover, the use of 3D nuclear telomere profiling permitted, for the first time, the identification of patient (and tumor) subpopulations that were not detectable up to that point. For example, we blindly defined three distinct subpopulations that correlated with short-term, intermediate and long-term survival in glioblastoma [11]. Using the same 3D imaging approach, we defined subpopulations in myelodysplastic syndromes and acute myeloid leukemias [12]. Additional studies are currently ongoing. We provided evidence that genomic instability is a result of these nuclear changes. Dynamic nuclear alterations directly result from 3D telomere aberrations [1,2,6,13-15]. These genomic changes include aneuploidy, Robertsonian fusions, breakage-bridge fusion (BBF) cycles with resulting terminal deletions and unbalanced translocations and continued rounds of BBF cycles. While these changes can be followed during cancer progression in patients, their true origin can only be examined in conditional expression studies or longitudinally in mouse models. Using conditional c-Myc deregulation, we demonstrated that changes in 3D telomere profiles precede the onset and propagation of genomic instability [6,13]. In Hodgkin’s lymphoma, in collaboration with Dr. Hans Knecht, we showed that mono-nucleated H cells become multinucleated Reed Sternberg (RS) cells through telomere dysfunction as measured by 3D nuclear profiles [10,16]. These changes coincide with localized shelterin dysfunction, aberrant centrosome duplication as well as spindle formation and significantly elevated levels of DNA damage foci [10,16]. Spectral karyotyping (SKY) and super resolution 3D imaging confirmed the dynamics and complexity of these genetic changes in which RS cells are the end-stage cells generated through multiple defects traced back to the H cells and propagated from there on [15]. Most recently, we have reported that Hodgkin’s patients with recurrent/non-recurrent disease display distinct 3D telomeric profiles [17]. Ongoing studies focus on a variety of cancers where clinicians currently lack the ability to define the risk of an individual patient to progress, the ability of early detection or of monitoring of disease progression. In conclusion, 3D telomere profiling provides a platform technology able to determine normal and aberrant nuclear organization that is a measure of genomic instability and cancer.

1071P - 3d Telomere Signatures of Hodgkin-Cells at Diagnosis Identify Patients with Poor Response to Conventional Chemotherapy

ABSTRACT In classical Hodgkin's lymphoma (HL) the malignant mononuclear Hodgkin (H) and multinuclear Reed-Sternberg (RS) cells are characterized by a distinct 3D nuclear telomere organization with shortening of the telomere length and the formation of telomeric aggregates. We asked if the severity of these telomere changes correlates with clinical behaviour of the disease. We evaluated (mainly prospectively) the 3D telomere organization by quantitative fluorescent in situ hybridization (Q-FISH) on diagnostic biopsies from 20 patients who were good responders and compared them with 20 diagnostic biopsies of 11 patients with refractory or relapsing HL (11 initial biopsies, five confirming progressions and four confirming relapses). The H-cells from patients with refractory/relapsing disease contained a significantly higher percentage of very small telomeres (p Disclosure All authors have declared no conflicts of interest.

Abstract 4725: The 3D nuclear telomere organization of Hodgkin-cells is different between patients entering rapid remission and patients with refractory or relapsing disease

Abstract Our recent study has utilized innovative 3D Q- FISH and three-dimensional (3D) imaging to define the 3D nuclear telomere organization in mono-nucleated Hodgkin (H) and multi-nucleated Reed-Sternberg (RS) cells of Hodgkin's lymphoma (HL) derived cell lines and diagnostic patient biopsies (Leukemia. 2009). These characteristics were found in both, classical EBV negative and EBV-associated, LMP1 expressing HL (Lab Invest. 2010). However, it is still unknown whether the 3D telomere profile at diagnostic biopsy is different in patients entering rapid remission after initiation of standard chemotherapy (ABVD) compared to patients with refractory or relapsing disease. In this study, we investigated by 3D telomere Q-FISH diagnostic biopsies of HL patients entering rapid complete remission and diagnostic biopsies from patients with refractory or relapsing disease. 8 diagnostic biopsies of 8 patients entering rapid remission (after 1-4 cycles of ABVD) and 8 diagnostic biopsies of 5 patients including 2 with primary refractory disease (progressing after 4-8 cycles of ABVD) and 3 cases relapsing 1-3 years after late remission (post 6 -8 cycles of ABVD) were analyzed the 3D telomeric structure by 3D Q-FISH and TeloView software (Cytometry A. 2005). We found that RS-cells of all patients from both groups showed significant increase in very short telomeres when compared to the mononuclear precursor H-cells (p&amp;lt;0.01). Additionally, the number of telomere aggregates was significantly higher in RS cells than in H cells in both, the rapid remission group (p&amp;lt;0.001) and the refractory or relapse group (p&amp;lt;0.01). Most importantly, all diagnostic biopsies of the relapse group contained a very high percentage of very small telomeres in both, H-cells (72.4 ± 0.1%) and RS-cells (84.6 ± 9.9%). These differences were highly significant compared the percentage of very small telomeres of the rapid remission group for both, H-cells (40.0± 0.2%) (p=0.002) and RS-cells (59.9 ± 0.2%) (p=0.008). Remarkably, the percentage of very short telomeres was even higher in H-cells of the relapse group than in RS-cells of the rapid remission group. The average number of telomere aggregates per H-cell was significantly higher in the relapse group (3.8 ± 1.9) compared to that one in the rapid remission group (1.3 ± 0.6) (p=0.0003). RS-cells in the relapse contained still more telomere aggregates compared to RS cells of the remission group (5.8 ± 2.8 versus 3.1 ± 1.1) (p=0.03). However, H-cells of the relapse group contained already more aggregates than RS cells of the remission group (p: no significant). These results indicate that the 3D nuclear telomere organization of H and RS cells in refractory or relapsing patients is different from that of patients in rapid remission. In particular, the 3D nuclear telomere profile of H-cells allows identifying aggressive disease already at diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4725. doi:10.1158/1538-7445.AM2011-4725

3D nuclear organization of telomeres in the Hodgkin cell lines U-HO1 and U-HO1-PTPN1: PTPN1 expression prevents the formation of very short telomeres including "t-stumps"

BackgroundIn cancer cells the three-dimensional (3D) telomere organization of interphase nuclei into a telomeric disk is heavily distorted and aggregates are found. In Hodgkin's lymphoma quantitative FISH (3D Q-FISH) reveals a major impact of nuclear telomere dynamics during the transition form mononuclear Hodgkin (H) to diagnostic multinuclear Reed-Sternberg (RS) cells. In vitro and in vivo formation of RS-cells is associated with the increase of very short telomeres including "t-stumps", telomere loss, telomeric aggregate formation and the generation of "ghost nuclei".ResultsHere we analyze the 3D telomere dynamics by Q-FISH in the novel Hodgkin cell line U-HO1 and its non-receptor protein-tyrosine phosphatase N1 (PTPN1) stable transfectant U-HO1-PTPN1, derived from a primary refractory Hodgkin's lymphoma. Both cell lines show equally high telomerase activity but U-HO1-PTPN differs from U-HO1 by a three times longer doubling time, low STAT5A expression, accumulation of RS-cells (p < 0.0001) and a fourfold increased number of apoptotic cells.As expected, multinuclear U-HO1-RS-cells and multinuclear U-HO1-PTPN1-RS-cells differ from their mononuclear H-precursors by their nuclear volume (p < 0.0001), the number of telomeres (p < 0.0001) and the increase in telomere aggregates (p < 0.003). Surprisingly, U-HO1-RS cells differ from U-HO1-PTPN1-RS-cells by a highly significant increase of very short telomeres including "t-stumps" (p < 0.0001).ConclusionAbundant RS-cells without additional very short telomeres including "t-stumps", high rate of apoptosis, but low STAT5A expression, are hallmarks of the U-HO1-PTPN1 cell line. These characteristics are independent of telomerase activity. Thus, PTPN1 induced dephosphorylation of STAT5 with consecutive lack of Akt/PKB activation and cellular arrest in G2, promoting induction of apoptosis, appears as a possible pathogenetic mechanism deserving further experimental investigation.

The Epstein-Barr Virus (EBV) Encoded Oncoprotein LMP1 Mediates Down Regulation of Shelterin Proteins, Formation of Telomere Aggregates and Multinuclearity.

AbstractAbstract 3625 Introduction:Tumour cells in classical Hodgkin's lymphoma (HL) and Burkitt's lymphoma (BL) are derived from germinal center B-cells and may or may not harbour the EBV genome. In both, EBV-negative and EBV-associated HL, the 3D nuclear organization of telomeres marks the transition from mononuclear Hodgkin (H-cell) to diagnostic multinuclear Reed-Sternberg (RS-cell) cells. Moreover, transient expression of the EBV-encoded oncoprotein LMP1 in the HL cell lines L-428 and HD-MyZ significantly increases the percentage of RS cells. However, the molecular mechanisms through which this selective LMP1 oncoprotein expression promotes the transition from H- to RS-cells is still unknown. In order to answer this question we analyzed the 3D telomere dynamics and the expression of key shelterin proteins at the transcriptional, translational and topographical localization level in the EBV-negative Burkitt cell line BJAB, stably transfected with an inducible LMP1 system (tetracycline on/off system). To mimic the permanent LMP1 expression as observed in tumour cells of EBV-associated Hodgkin's lymphoma, we extended the period of LMP1 expression up to 21 days and examined key proteins of the shelterin complex, 3D telomere dynamics and formation of multinuclear cells in interphase nuclei of BJAB Burkitt's lymphoma cells at day 1, 3, 7, 10, 14 and 21. Statistical analysis was performed using nested or two-way analysis of variance. Results:Already at day 3 LMP1 induced substantial down regulation of the shelterin key-components TRF1, TRF2 and POT1 at the transcriptional and protein level. This down regulation was reversible after LMP1 suppression; in particular, suppression of LMP1 induction at day 7 returned shelterin-key components to the initial level of expression at day 14. Massive LMP1 mediated down regulation of shelterin proteins was also confirmed by immunofluorescence. At day 7, stable LMP1 expression led to a significant increase of 3D telomere aggregates, nuclear volume and polyploid cells, most of them multinucleated, when compared to non-LMP1 expressing BJAB controls (p < 0.02). These changes progressed over time and at day 21 LMP1 expressing BJAB cells were characterized by highly significant increase of 3D telomere aggregates (p = 0.0005) and increased nuclear volume (p < 0.0001), compared to the LMP1-suppressed control. Discussion: In an experimental setting mimicking the in vivo conditions of EBV-associated HL, i.e. a permanent LMP1 expression in the H-cells, the LMP1 oncoprotein deregulates shelterin-expression, leads to telomere aggregates and mediates multinuclearity. Our findings point to a shelterin/telomere related key function of LMP1 in Reed-Sternberg cell formation in EBV-associated Hodgkin's lymphoma and may explain the four fold increased risk to develop HL within a median incubation time of four years after symptomatic EBV infection. The LMP1-dependent mechanisms involved are probably unprotected telomeres (shelterin down regulation) favouring chromosomal rearrangements as a result of telomeric aggregate formation and ongoing bridge-fusion- bridge cycles which lead to disturbed cytokinesis and finally multinuclearity. Disclosures:No relevant conflicts of interest to declare.

3D Telomere Dynamics In Hodgkin's Lymphoma

AbstractAbstract 745 Introduction:Innovative 3D telomere q-FISH allows a mechanistic understanding of the transition from the mononuclear Hodgkin (H) to the multinuclear Reed-Sternberg (RS) cell in in Hodgkin's lymphoma (HL) derived cell lines and diagnostic patient biopsies (Leukemia. 2009; 23:565-573). In RS-cells the telomere protecting shelterin complex appears to be disrupted and deregulation of DNA repair mechanisms is observed. These changes occur in both, classical EBV negative and EBV-associated, LMP1 expressing HL (Lab Invest. 2010; 90:611-619). However, it is not known whether the 3D telomere profile at diagnostic biopsy is different in patients entering rapid remission after initiation of standard chemotherapy (ABVD) compared to that-one of patients with relapsing or refractory disease. In order to answer this question we analyzed by 3D telomere q-FISH diagnostic biopsies of HL patients entering rapid complete remission and compared them to diagnostic biopsies of patients with relapsing or refractory disease. Patients and methods:Rapid remission group (after 1–4 cycles of ABVD): 7 diagnostic biopsies of 7 patients, 19–57 years old, 5 male, 3 LMP1 expressing, 4 nodular sclerosis subtype, 3 mixed cellularity subtype, stages IA, IIA, IIIA × 2, IIIB x2, IVA. Relapse group: 7 diagnostic biopsies of 4 patients, 40–77 years old, 2 male, 1 LMP1 expressing, 3 nodular sclerosis subtype, 1 mixed cellularity subtype, stages IIA bulky, IIIB × 2, IVB; first remission after 6–8 cycles of ABVD in 3 patients, 1 patient died from progressive disease after 11 months. 3D telomere q-FISH was performed as described (Lab Invest. 2010; 90:611-619) and statistical analysis was performed using nested or two-way analysis of variance. Results:Bi- or multinuclear RS-cells of all patients from both groups showed a significant increase of very short telomeres and telomere aggregates when compared to the mononuclear precursor H-cells. However, most importantly, all diagnostic biopsies of the relapse group contained a very high percentage of very small telomeres, including so-called “t-stumps”, in both, H-cells (76,8 ± 11,8%) and RS-cells (87,9 ± 7,3%). Compared to the percentage of very small telomeres identified in both, H-cells (33,7 ± 9,4%) and RS-cells (54,6 ± 15,0%) of the rapid remission group, this increase is highly significant (p <0.001). Moreover, analogous findings are observed for the number of telomere aggregates. In the relapse group the average numbers of telomere aggregates per cell were 4,3 ± 2,4 aggregates per H-cell and 5,4 ± 3.0 aggregates per RS-cell, compared to 1,2 ± 0,7 aggregates per H-cell and 3,3 ± 1,1 aggregates per RS-cell in the rapid remission group. Discussion:The 3D nuclear telomere organization of H- and RS- cells in diagnostic biopsies of relapsing or refractory HL is characterized by both, H- and RS-cells with abundant “t-stumps” and numerous telomere aggregates. Very short telomeres, including t-stumps, and telomere aggregates, both, are characteristics of aggressiveness in cancer biology (Mol Cell. 2007;28:315-327; J Cell Biochem. 2010; 109:1095-1102). Thus, H- and RS-cells of refractory or relapsing HL show significant differences in the 3D telomere dynamics already at first, diagnostic biopsy when compared to H- and RS-cells of HL entering rapid remissions. Disclosures:No relevant conflicts of interest to declare.

3D structural and functional characterization of the transition from Hodgkin to Reed-Sternberg cells

Recent research using an innovative 3D quantitative FISH approach of nuclear remodelling associated with the transition from mononuclear Hodgkin to diagnostic multinuclear Reed-Sternberg cells revealed profound changes in the 3D nuclear organization of telomeres. Analogous 3D telomere dynamics were identified in Hodgkin's lymphoma derived cell-lines and diagnostic patient biopsies. These changes were observed in both, EBV positive and EBV-negative Hodgkin's lymphoma and independent of the age of the patients at presentation. Compared to mononuclear Hodgkin cells, multinuclear Reed-Sternberg cells are characterized by a highly significant increase of telomere aggregates, often composed of very short telomeres, telomere shortening and loss. RS-cells with telomere free "ghost" nuclei are regularly observed. The telomere protecting shelterin complex appears to be disrupted and deregulation of DNA-repair mechanisms is observed. Our findings are consistent with the hypothesis that distinct 3D telomere changes and shelterin disruption represent a common pathogenetic denominator in the generation of Reed-Sternberg cells.

3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost' nuclei and very short telomeres

In Epstein–Barr virus (EBV) negative Hodgkin’s cell lines and classical EBV-negative Hodgkin’s lymphoma (HL), Reed–Sternberg cells (RS cells) represent end-stage tumor cells, in which further nuclear division becomes impossible because of sustained telomere loss, shortening and aggregation. However, the three-dimensional (3D) telomere organization in latent membrane protein 1 (LMP1)-expressing RS cells of EBV-associated HL is not known. We performed a 3D telomere analysis after quantitative fluorescent in situ hybridization on 5 μm tissue sections on two LMP1-expressing HL cases and showed highly significant telomere shortening (P<0.0001) and formation of telomere aggregates in RS cells (P<0.0001), when compared with the mononuclear precursor Hodgkin cells (H cells). Telomere-poor or telomere-free ‘ghost' nuclei were a regular finding in these RS cells. These nuclei and their telomere content strongly contrasted with the corona of surrounding lymphocytes showing numerous midsized telomere hybridization signals. Both H cells and RS cells of two EBV-negative HL cases analyzed in parallel showed 3D telomere patterns identical to those of LMP1-expressing cases. As a major advance, our 3D nuclear imaging approach allows the visualization of hitherto unknown profound changes in the 3D nuclear telomere organization associated with the transition from LMP1-positive H cells to LMP1-positive RS cells. We conclude that RS cells irrespective of LMP1 expression are end-stage tumor cells in which the extent of their inability to divide further is proportional to the increase of very short telomeres, telomere loss, aggregate formation and the generation of ‘ghost' nuclei.

The 3D nuclear organization of telomeres marks the transition from Hodgkin to Reed–Sternberg cells

To get an insight into the transition from mononuclear Hodgkin cells (H cells) to diagnostic multinuclear Reed-Sternberg cells (RS cells), we performed an analysis of the three-dimensional (3D) structure of the telomeres in the nuclei of the Hodgkin cell lines HDLM-2, L-428, L-1236 and lymph node biopsies of patients with Hodgkin's disease. Cellular localization of key proteins of the telomere-localized shelterin complex, the mitotic spindle and double-stranded DNA breaks was also analyzed. RS cells show significantly shorter and significantly fewer telomeres in relation to the total nuclear volume when compared with H cells; in particular, telomere-poor 'ghost' nuclei are often adjacent to one or two nuclei displaying huge telomeric aggregates. Shelterin proteins are mainly cytoplasmic in both H and RS cells, whereas double-stranded DNA breaks accumulate in the nuclei of RS cells. In RS cells, multipolar spindles prevent proper chromosome segregation. In conclusion, a process of nuclear disorganization seems to initiate in H cells and further progresses when the cells turn into RS cells and become end-stage tumor cells, unable to divide further because of telomere loss, shortening and aggregate formation, extensive DNA damage and aberrant mitotic spindles that may no longer sustain chromosome segregation. Our findings allow a mechanistic 3D understanding of the transition of H to RS cells.

3D Nuclear Organization of Telomeres in Hodgkin and Reed-Sternberg Cells.

Telomerase activity is high in Hodgkin (H) and Reed-Sternberg (RS) cells and abundant telomerase RNA template (hTR) is identified in both by in situ hybridization. However, nothing is known about the three-dimensional (3D) structure of the telomeres in H and RS and the molecular events associated with the transition from the mononuclear H to the multinuclear diagnostic RS are still unclear. We analyzed the 3D structure of telomeres in interphase nuclei in the Hodgkin cell lines HDLM-2, L-428 and L-1236, where about 90 to 99% are mononuclear H and only 1 to 10% are multinuclear RS. We also analyzed the 3D structure of telomeres within 5 mm thin sections of three lymph node biopsies diagnostic for Hodgkin's disease (HD). The stereometric (3D) organization of telomeres was investigated in 30 H and 30 RS of each cell line and in 30 H and 30 RS of each lymph node biopsy specimen as previously described (Proc Natl Acad Sci USA , 102: 9613). Cellular localization of key-proteins of the telomere localized shelterin-complex (TRF1 and TRF2), of the mitotic spindle (centrin and gamma-tubulin), and of double-stranded DNA breaks (gamma-H2AX), was also analyzed. In all three cell lines the multinuclear RS showed overall significantly shorter and also significantly less telomeres in relation to the total nuclear mass when compared to their mononuclear H precursors. Visualization of their 3D telomeric structure revealed that this difference was due to partial or nearly total loss of telomeres within single nuclei of multinuclear RS; in particular, one or two nearly telomere free nuclei were often adjacent to one or two nuclei displaying several huge telomeric aggregates. TRF1 and TRF2 were mainly cytoplasmic in H and RS whereas gamma-H2AX accumulated in the nuclei of RS but not H. Multiple pairs of centrosomes not correlating with the number of nuclei, as well as high numbers of multi-polar spindles and incomplete spindles were identified in RS. Importantly, results analogous to those described in the HD cell lines were found in the HD lymph node biopsy specimen. In conclusion, our results suggest that multinuclear RS represent end stage tumour cells, where further nuclear division gets impossible due to sustained 3D telomere aggregation, shortening or loss. In particular, the number of nuclei within RS correlates closely with the 3D organization of telomeres, thus allowing for the first time a mechanistic understanding of the transition from H to RS cells. This process is initiated in H and advances to end stage telomere free “ghost” nuclei as observed in many RS. The shelterin complex appears to be disrupted and the mitotic cycle is profoundly disturbed.