Telomere Parameters Research Articles

High-throughput single telomere analysis using DNA microarray and fluorescent in situ hybridization.

The human telomere system is highly dynamic. Both short and long leucocyte average telomere lengths (aTL) are associated with an increased risk of cancer and early death, illustrating the complex relationship between TL and human health and the importance of assessing TL distributions with single TL analysis. A DNA microarray and telomere fluorescent in situ hybridization (DNA-array-FISH) approach was developed to measure the base-pair (bp) lengths of single telomeres. On average 32000 telomeres were measured per DNA sample with one microarray chip assaying 96 test DNA samples. Various telomere parameters, i.e. aTL and the frequency of short/long telomeres, were computed to delineate TL distribution. The intra-assay and inter-assay coefficient of variations of aTL ranged from 1.37% to 3.98%. The correlation coefficient (r) of aTL in repeated measurements ranged from 0.91 to 1.00, demonstrating high measurement precision. aTLs measured by DNA-array-FISH predicted aTLs measured by terminal restriction fragment (TRF) analysis with r ranging 0.87-0.99. A new accurate and high-throughput method has been developed to measure the bp lengths of single telomeres. The large number of single TL data provides an opportunity for an in-depth analysis of telomere dynamics and the complex relationship between telomere and age-related diseases.

Analysis by TeloView® Technology Predicts the Response of Hodgkin's Lymphoma to First-Line ABVD Therapy.

Classic Hodgkin's lymphoma (cHL) is a curable cancer with a disease-free survival rate of over 10 years. Over 80% of diagnosed patients respond favorably to first-line chemotherapy, but few biomarkers exist that can predict the 15-20% of patients who experience refractory or early relapsed disease. To date, the identification of patients who will not respond to first-line therapy based on disease staging and traditional clinical risk factor analysis is still not possible. Three-dimensional (3D) telomere analysis using the TeloView® software platform has been shown to be a reliable tool to quantify genomic instability and to inform on disease progression and patients' response to therapy in several cancers. It also demonstrated telomere dysfunction in cHL elucidating biological mechanisms related to disease progression. Here, we report 3D telomere analysis on a multicenter cohort of 156 cHL patients. We used the cohort data as a training data set and identified significant 3D telomere parameters suitable to predict individual patient outcomes at the point of diagnosis. Multivariate analysis using logistic regression procedures allowed for developing a predictive scoring model using four 3D telomere parameters as predictors, including the proportion of t-stumps (very short telomeres), which has been a prominent predictor for cHL patient outcome in a previously published study using TeloView® analysis. The percentage of t-stumps was by far the most prominent predictor to identify refractory/relapsing (RR) cHL prior to initiation of adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) therapy. The model characteristics include an AUC of 0.83 in ROC analysis and a sensitivity and specificity of 0.82 and 0.78 respectively.

Telomere Status of Advanced Non-Small-Cell Lung Cancer Offers a Novel Promising Prognostic and Predictive Biomarker

Telomere length appears to correlate with survival in early non-small-cell lung cancer (NSCLC), but the prognostic impact of telomere status in advanced NSCLC remains undetermined. Our purpose was to evaluate telomere parameters as prognostic and predictive biomarkers in advanced NSCLC. In 79 biopsies obtained before treatment, we analyzed the telomere length and expression of TERT and shelterin complex genes (TRF1, TRF2, POT1, TPP1, RAP1, and TIN2), using quantitative PCR. Non-responders to first-line chemotherapy were characterized by shorter telomeres and low RAP1 expression (p = 0.0035 and p = 0.0069), and tended to show higher TERT levels (p = 0.058). In multivariate analysis, short telomeres were associated with reduced event-free (EFS, p = 0.0023) and overall survival (OS, p = 0.00041). TERT and TRF2 overexpression correlated with poor EFS (p = 0.0069 and p = 0.00041) and OS (p = 0.0051 and p = 0.007). Low RAP1 and TIN2 expression-levels were linked to reduced EFS (p = 0.00032 and p = 0.0069) and OS (p = 0.000051 and p = 0.02). Short telomeres were also associated with decreased survival after nivolumab therapy (p = 0.097). Evaluation of telomere status in advanced NSCLC emerges as a useful biomarker that allows for the selection of patient groups with different clinical evolutions, to establish personalized treatment.

Telomere-Associated Changes in Nuclear Architecture of Cancer-Associated Macrophage-like Cells in Liquid Biopsies from Melanoma Patients.

During phagocytosis, tumor-associated macrophages (TAMs) can incorporate genetic material from tumor cells. The incorporation of extra genetic material may be responsible for advanced malignant behavior observed in some TAMs, making TAMs potentially important players in cancer progression. More recently, similar cells were described in the blood as cancer-associated macrophage-like cells (CAMLs). CAMLs may be equivalent to TAMs cells in the blood, and they express macrophage markers. However, their origin is still unclear. In a previous study, we showed for the first time the distinct telomere 3D structure of circulating tumor cells (CTCs) in melanoma and other cancers. In the present pilot study, we investigated, comparatively, the 3D telomere structure of CAMLs, CTCs and leucocytes from nine melanoma patients with metastatic cutaneous melanoma stage IV. CTC capture was performed by size-based filtration followed by cytological and immunocytological evaluation. Three-dimensional Quantitative Fluorescent in situ Hybridization was performed to measure differences in five 3D telomere parameters. Telomere parameters, such as number, length, telomere aggregates, nuclear volume, and a/c ratio, were compared among different cellular types (CTCs, CAMLs, and normal leucocytes). Three telomere parameters were significantly different between CAMLs and leucocytes. The combination of two telomere parameters (telomere length against the number of telomeres) resulted in the identification of two CAMLs subpopulations with different levels of genomic instability. Those populations were classified as profile 1 and 2. Profile 2, characterized by a high number of short telomeres, was observed in four of the nine melanoma patients. To our knowledge, this is the first pilot study to investigate 3D telomere parameters as hallmarks of nuclear architecture in CAMLs’ population in comparison to leucocytes from the same patient. Further studies involving a larger patient sample size are necessary to validate these findings and explore their potential prognostic value.

Discriminating association of a common telomerase reverse transcriptase promoter polymorphism with telomere parameters in non-small cell lung cancer with or without epidermal growth factor receptor mutation

BackgroundThe role of epidermal growth factor receptor (EGFR) pathways in regulating telomerase is increasingly being recognised. We analysed the impact of rs2853669 single nucleotide polymorphism (SNP) on telomere parameters and its prognostic value for non-small cell lung cancer (NSCLC) with or without EGFR mutation. MethodsThe association of rs2853669 with telomerase reverse transcriptase (TERT) mRNA level and relative telomere length (RTL) was analysed using resected tumour samples from 250 NSCLC patients. We also investigated the patients’ clinical outcomes with a median follow-up of 57 months (2–99 months). ResultsThe rs2853669 T/C allele was significantly associated with lower TERT mRNA expression (versus C/C and versus T/T; p < 0.001 for both) and shorter RTL (versus C/C and versus T/T; p = 0.039 and 0.023) in patients without EGFR mutation. Such difference was not observed in their counterparts harbouring EGFR mutation. When considering the cohort as a whole, T/C allele was significantly associated with shortest overall survival compared with T/T or C/C allele (mean: 61.8, 80.9 and 88.7 months, plog-rank < 0.001) and disease-free survival (mean: 78.3, 87.9 and 91.5 months, plog-rank = 0.019). Stratification analyses showed that the negative prognostic effect of T/C on OS was constrained in patients without EGFR mutation. ConclusionOur study revealed significant associations of a common SNP within TERT promoter region on telomere parameters and survival in NSCLC patients without EGFR mutation. The result may help providing instruction for therapeutic interventions targeting telomerase and evidence for investigation of TERT-EGFR interacting mechanism in telomere biology.

Long-Term Dynamics of Three Dimensional Telomere Profiles in Circulating Tumor Cells in High-Risk Prostate Cancer Patients Undergoing Androgen-Deprivation and Radiation Therapy.

Patient-specific assessment, disease monitoring, and the development of an accurate early surrogate of the therapeutic efficacy of locally advanced prostate cancer still remain a clinical challenge. Contrary to prostate biopsies, circulating tumor cell (CTC) collection from blood is a less-invasive method and has potential as a real-time liquid biopsy and as a surrogate marker for treatment efficacy. In this study, we used size-based filtration to isolate CTCs from the blood of 100 prostate cancer patients with high-risk localized disease. CTCs from five time points: +0, +2, +6, +12 and +24 months were analyzed. Consenting treatment-naïve patients with cT3, Gleason 8-10, or prostate-specific antigen > 20 ng/mL and non-metastatic prostate cancer were included. For all time points, we performed 3D telomere-specific quantitative fluorescence in situ hybridization on a minimum of thirty isolated CTCs. The patients were divided into five groups based on the changes of number of telomeres vs. telomere lengths over time and into three clusters based on all telomere parameters found on diagnosis. Group 2 was classified as non-respondent to treatment and the Cluster 3 presented more aggressive phenotype. Additionally, we compared our telomere results with the PSA levels for each patient at 6 months of ADT, at 6 months of completed RT, and at 36 months post-initial therapy. CTCs of patients with PSA levels above or equal to 0.1 ng/mL presented significant increases of nuclear volume, number of telomeres, and telomere aggregates. The 3D telomere analysis of CTCs identified disease heterogeneity among a clinically homogeneous group of patients, which suggests differences in therapeutic responses. Our finding suggests a new opportunity for better treatment monitoring of patients with localized high-risk prostate cancer.

Identification of Neuroblastoma Subgroups Based on Three-Dimensional Telomere Organization

Using 3D telomere quantitative fluorescence in situ hybridization, we determined the 3D telomere organization of 74 neuroblastoma tissue samples. Hierarchical cluster analysis of the measured telomere parameters identified three subgroups from our patient cohort. These subgroups have unique telomere profiles based on telomere length and nuclear architecture. Subgroups with higher levels of telomere dysfunction were comprised of tumors with greater numbers of telomeres, telomeric aggregates, and short telomeres (P<.0001). Tumors with greater telomere dysfunction were associated with unfavorable tumor characteristics (greater age at diagnosis, unfavorable histology, higher stage of disease, MYCN amplification, and higher MYCN expression) and poor prognostic risk (P<.001). Subgroups with greater telomere dysfunction also had higher intratumor heterogeneity. MYCN overexpression in two neuroblastoma cell lines with constitutively low MYCN expression induced changes in their telomere profile that were consistent with increased telomere dysfunction; this illustrates a functional relationship between MYCN and 3D telomere organization. This study demonstrates the ability to classify neuroblastomas based on the level of telomere dysfunction, which is a novel approach for this cancer.

Telomere status in chronic lymphocytic leukemia with TP53 disruption.

In chronic lymphocytic leukemia (CLL), telomere dysfunction is associated with poor outcomes. TP53 is involved in cellular responses to dysfunctional telomeres, and its inactivation is the strongest adverse prognostic factor for CLL. Given the biological relationship between TP53 and telomeres, and their prognostic value, it is important to improve our understanding of the impact of TP53 alterations on telomeres. We performed a comprehensive study of the deletions and mutations of the TP53 gene and telomere parameters, including hTERT and the shelterin complex, in 115 CLL patients. We found that any type of TP53 alteration was associated with very short telomeres and high hTERT expression, independently of other biological CLL features. Patients with disrupted TP53 showed telomere deletions and chromosomal end-to-end fusions in cells with complex karyotypes. TP53 disruption was characterized by downregulation of shelterin genes. Interestingly, low expression of POT1, TPP1 and TIN2 was also found in some patients with wild-type TP53 and had an adverse impact on progression-free survival after standard genotoxic therapy. In conclusion, we have demonstrated that patients with disrupted TP53 have severe telomere dysfunction and high genomic instability. Thus, the telomeric profile could be tested as a biomarker in CLL patients treated with new therapeutic agents.

ERCC1 and telomere status in breast tumours treated with neoadjuvant chemotherapy and their association with patient prognosis.

Dysfunctional telomeres and DNA damage repair (DDR) play important roles in cancer progression. Studies have reported correlations between these factors and tumour aggressiveness and clinical outcome in breast cancer. We studied the characteristics of telomeres and expression of ERCC1, a protein involved in a number of DNA repair pathways and in telomere homeostasis, to assess their prognostic value, alone or in combination, in 90 residual breast tumours after treatment with neoadjuvant chemotherapy (NCT). ERCC1 status was investigated at different molecular levels (protein and gene expression and gene copy‐number variations) by immunohistochemistry, qRT‐PCR and quantitative multiplex fluorescent‐PCR (QMF‐PCR). A comprehensive analysis of telomere characteristics was performed using qPCR for telomere length and qRT‐PCR for telomerase (hTERT), tankyrase 1 (TNKS) and shelterin complex (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2) gene expression. Short telomeres, high hTERT and TNKS expression and low ERCC1 protein expression were independently associated with worse survival outcome. Interestingly, ERCC1 gains and losses correlated with worse disease‐free (p = 0.026) and overall (p = 0.043) survival as compared to survival of patients with normal gene copy‐numbers. Unsupervised hierarchical clustering of all ERCC1 and telomere parameters identified four subgroups with distinct prognosis. In particular, a cluster combining low ERCC1, ERCC1 gene alterations, dysfunctional telomeres and high hTERT and a cluster with high TNKS and shelterin expression correlated with poor disease‐free (HR= 5.41, p= 0.0044) and overall survival (HR= 6.01, p= 0.0023) irrespective of tumour stage and grade. This comprehensive study demonstrates that telomere dysfunction and DDR can contribute synergistically to tumour progression and chemoresistance. These parameters are predictors of clinical outcome in breast cancer patients treated with NCT and could be useful clinically as prognostic biomarkers to tailor adjuvant chemotherapy post‐NCT.

Short Telomere Load, Telomere Length, and Subclinical Atherosclerosis: The PESA Study

BackgroundLeucocyte telomere length (LTL) shortening is associated with cardiovascular ischemic events and mortality in humans, but data on its association with subclinical atherosclerosis are scarce. Whether the incidence and severity of subclinical atherosclerosis are associated with the abundance of critically short telomeres, a major trigger of cellular senescence, remains unknown. ObjectivesThe authors conducted a cross-sectional exploration of the association between subclinical atherosclerosis burden and both average LTL and the abundance of short telomeres (%LTL<3 kb). MethodsTelomere length was assessed by high-throughput quantitative fluorescence in situ hybridization in circulating leukocytes from 1,459 volunteers without established cardiovascular disease (58% men, 40 to 54 years of age) from the PESA (Progression of Early Subclinical Atherosclerosis) study. Subclinical atherosclerosis was evaluated by coronary artery calcium scan and 2-dimensional/3-dimensional ultrasound in different aortic territories. Statistical significance of differences among multiple covariates was assessed with linear regression models. Independent associations of telomere parameters with plaque presence were evaluated using general linear models. ResultsIn men and women, age was inversely associated with LTL (Pearson’s r = −0.127, p < 0.001) and directly with %LTL<3 kb (Pearson’s r = 0.085; p = 0.001). Short LTL reached statistical significance as a determinant of total and femoral plaque in men, but not in women. However, this association was not sustained after adjustment for age or additional adjustment for cardiovascular risk factors. No significant independent association was found between %LTL<3 kb and plaque burden. Serum-oxidized low-density lipoprotein levels were directly associated with %LTL<3 kb in men (p = 0.008) and women (p < 0.001). ConclusionsIn a cross-sectional study of a middle-aged population, average LTL and short telomere load are not significant independent determinants of subclinical atherosclerosis. Longitudinal follow-up of PESA participants will assess long-term associations between telomere length and progression of subclinical atherosclerosis.

Absolute qPCR for measuring telomere length in bone marrow samples of plasma cell disorders.

Telomere length (TL) is currently used as an emerging biomarker in understanding the development/progression of hematological malignancies. The absolute quantitative PCR (qPCR) methodology has allowed the study of TL from a variety of mammalian tissues, but it has not been tested for bone marrow (BM) samples. In this study, we have examined the relationship between TL data generated by absolute qPCR versus those obtained by terminal restriction fragments (TRF) in 102 BM samples from patients with plasma cell disorders. A significant linear correlation between both methodologies was observed (p < 0.0001; r (2) = 0.70). Results were also analyzed in relation to clinical characteristics and significant associations between telomere shortening and parameters of adverse prognosis were observed. Furthermore, another set of 47 BM samples from patients with low quantity of DNA for TRF assay were suitably analyzed by qPCR, indicating the usefulness of the absolute qPCR methodology for the inclusion of patients with scarce material to the study. Taken together, these findings are of interest considering the importance of telomere dysfunction in the pathogenesis of cancer and give a new alternative to measure TL in hematologic disorders with substantial time and cost savings.

Prognostic significance of telomerase-associated parameters in glioblastoma: effect of patient age

Glioblastoma multiforme (GBM) is a heterogeneous, highly aggressive primary brain tumor with strongly variable patient survival. Because reliable prognostic biomarkers are lacking, we investigated the relation between telomerase-associated parameters and the disease course. Telomerase-associated parameters were determined in 100 GBM tissues and associated with clinical characteristics and overall survival. Expressions of telomere length, telomerase activity (TA), and human telomerase reverse transcriptase (hTERT) were analyzed by quantitative PCR, telomeric repeat amplification protocol assay, and reverse transcriptase-PCR, respectively. Mutation status of isocitrate dehydrogenase (IDH)1 was determined by direct sequencing, and O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation by methylation-specific PCR. Of 100 GBM tissues, 61 were positive for both hTERT mRNA and TA, with a highly significant correlation between both parameters (linear regression, P < .0001). Telomere length determination revealed a significant difference between the hTERT/TA-positive and -negative subgroups, with markedly longer telomeres in the hTERT/TA-negative cohort (unpaired Student's t-test, P = .0001). Accordingly, significantly shorter telomeres were detected in GBM tissues derived from older patients (>60 y at diagnosis, P < .0001). While no association of telomere parameters with MGMT promoter status was found, all tumors with IDH1 mutation (6/100) were negative for both hTERT expression and TA and harbored significantly longer telomeres. Patients with tumors lacking hTERT expression/TA showed a significant survival benefit (Kaplan-Meier test, both P < .01), which, however, was based exclusively on the younger patient subgroup (≤60 y, both P < .005; >60 y, both ns). Telomerase activation is not an independent prognostic parameter in GBM but predicts aggressive tumor behavior solely in a younger patient cohort.

Three-Dimensional Nuclear Telomeric Organization (3D) of Chronic Myeloid Leukemia Patients Predicts Accelerated Phase and Blast Crisis.

AbstractAbstract 2771One major cause of genomic instability and cellular apoptosis is telomere dysfunction. Telomere loss or dysfunction results in breakage–bridge –fusion cycles, aneuploidy, and ongoing chromosomal abnormalities. The three-dimensional (3D) nuclear organization of telomeres allows for a distinction between normal and tumor cells: nuclei of the latter tend to be disorganized and commonly contain telomeric aggregates. However, few studies have addressed the impact of telomeres dynamics in CML progression. The frequency of additional chromosomal abnormalities in CML is around 5% in chronic phase and increases to 50–80% in the advanced phases. Clonal evolution often precedes progression and is predictive for inferior therapeutic outcome. In order to better understand cellular and molecular mechanisms in CML progression, the objectives of this investigation were examine telomere dysfunction and alterations in the 3D nuclear telomere architecture. Eighteen CML patients, in total, 54 bone marrow samples (chronic phase, accelerated phase and blast crisis) were eligible for 3D nuclear telomeric investigation. The quantitative FISH (QFISH), cytologic diagnosis and the cytogenetic determination for additional chromosomal abnormalities were assessed according to standard protocols. 3D image analysis on 30 interphase nuclei per slide was obtained by using an Axio Imager A1 microscope (Carl Zeiss, USA). Sixty z-stacks were acquired at a sampling distance of x,y: 102 nm and z: 200 nm for each slice of the stack. AxioVision 4.8 software (Carl Zeiss, Canada) was used for 3D image acquisition, and deconvolution analysis. Three CML subgroups were defined on the basis of their 3D telomeric profiles. The telomeric parameters (number, length, telomere aggregates and nuclear volumes) were compared between these three subgroups. Distribution of telomere intensities in CML phases was compared between the patient’s subgroups. All patients of a same subgroup displayed similar 3D telomeric profiles. Comparison with clinical diagnosis after the classification according to telomere profile showed that all CML patients were classified in the three distinct subgroups. Statistical analyses showed significant differences between the CML subgroups (P<0.001). Each of the quantitative telomere parameters exhibited significant differences. Furthermore, statistical analyses combining all 3D telomere parameters revealed significant differences between all subgroups (P<0.05). According to our data these profiles are correlated to the disease evolution and increased telomere dysfunction in these subgroups. It seems that the evolution of CML progresses from low to high level of telomere dysfunction, that is, from early stage to more aggressive stage, followed by disease transformation. We concluded that telomere 3D organization is a highly accurate tool to distinguish CML stages. We propose that monitoring 3D telomere dysfunction might be a very powerful marker to measure this transformation. Furthermore, it may be a better indicator of therapeutic response because an optimal response will lead to a normal cellular biology, including elimination of abnormal telomeric aggregates and the increase of normal 3D telomeric profiles.Financial support: FAPESP (2011/01647-2). Disclosures:No relevant conflicts of interest to declare.

Abstract 2044: Three-dimensional nuclear telomeric architecture defines cytopenias of myelodysplastic syndrome and its transformation to acute myeloid leukemia

Abstract Background Myelodysplastic Syndromes (MDS) are a group of disorders characterized by cytopenias, with a propensity for evolution into Acute Myeloid Leukemias (AML). This transformation is governed by acquisition of additional mutations, which in turn is driven by genomic instability and epigenetic events. However, how genomic instability occurs in this disease remains unknown. Telomere dysfunction might be the generator of genomic instability leading to cytopenias and disease progression from MDS to AML. Methods We undertook a prospective pilot study of 95 patients to investigate the nuclear telomere organization in MDS (57 patients) and AML (38 patients). Three-dimensional (3D) quantitative fluorescent in situ hybridization was performed to label telomeres of bone marrow samples. Nuclear telomere organization was analyzed using TeloViewTM and TeloScanTM. Findings Our cohort was composed of 57 cases of MDS and 38 AML. MDS cohort was constituted of Refractory Anaemia with Multilineage Dysplasia (RCMD = 32 cases), Refractory Anaemia (RA = 12 cases), Refractory Anaemia with Excess of Blasts 1 and 2 (RAEB-1 = 8 cases; RAEB-2 =1 case), Refractory Anaemia with Ring Sideroblasts (RARS = 2 cases), MDS associated with isolated del(5q) (1 case), and MDS unclassified (MDS-U = 1 case). AML were composed of AML-M4 (12 cases), AML-M2 (10 cases), AML-M5 (5 cases), AML-M0 (5 cases), AML-M1 (2 cases), AML-M4eo (1 case), and AML with multidysplasia related changes (1 case). We analyzed the 3D nuclear telomeric architecture, and determined the telomere numbers, presence of telomere aggregates, telomere signal intensities, nuclear volume, and telomere distribution. From these parameters we constructed 3D telomeric profiles, and we were able to subdivide the MDS patients into 9 subgroups and the AML patients into 6 subgroups. Each of the quantitative telomere parameters showed significant difference between MDS and AML. Furthermore, statistical analyses combining all the telomere parameters displayed significant difference between all subgroups. These profiles were linked to the evolution of telomere dysfunction in these subgroups allowing us to define a model of progression of MDS to AML. Our 3D telomeres profiling reflect better genomic abnormalities in MDS and AML than the cytogenetic categorisations. Telomere profiles seem to be a potential biomarker, which will improve the clinical management of these patients combined to other indexes such as IPSS and WPSS in MDS, and the cytogenetic risk factor classifications of AML. Interpretation Our results define for the first time the chronological and evolutionary process of telomere dysfunction in MDS and AML. These results are consistent with the hypothesis that telomere dysfunction generates genomic instability, leading to worsening cytopenias and to the transformation of MDS to AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2044. doi:1538-7445.AM2012-2044

3D Telomeric Profiles of MGUS, MMN and Relapsed MM

Abstract 2899Multiple myeloma (MM) is recognized as the second most common cancer of the blood. It is a malignant disorder of plasma cells and commonly affects adults past the age of 50. Although risk factors have been established, it is currently not possible to assess the individual risk to cancer progression. Moreover, the causes of disease progression from its precursor condition, monoclonal gammopathy of undetermined significance (MGUS), to full-blown MM and its progression to relapsed MM remain elusive. We have performed previous studies on the three-dimensional (3D) nuclear organization of telomeres and found that normal and tumor cells display significant differences in their nuclear organization. These differences were objectively quantified with two software programs, TeloView and TeloScan developed by our group.We now report on a new double blinded preliminary study with 36 patients, including 20 MM, 12 MGUS and 4 relapsed MM. Using blood- and bone marrow-derived plasma cells from the respective patient groups, we have examined 3D nuclear telomeric profiles of the above patients. Plasma cells from MM, MGUS and relapsed MM exhibit specific 3D telomeric signatures. MM have the highest telomere numbers, followed by MGUS, while relapsed MM presents with the lowest numbers of telomeres and the shortest telomeres. Additional telomere parameters, such as cell cycle distribution profiles (a/c ratio), telomere aggregate numbers, distances from nuclear centre and also exhibited significance (p< 0.001).Within the MGUS and MM patient groups we studied are patients whose 3D telomeric profiles indicate the beginning of a new signature that resembles signatures of MM and relapsed MM respectively. Blood and bone marrow gave us comparable results opening the future opportunity to base diagnostics and monitoring on blood samples, sparing the patient form invasive bone marrow sampling with potentially adverse effects. Based on our current preliminary data, we propose the following 3D telomeric criteria that define individual signatures of MGUS and MM and progression within each of the groups: telomere numbers, telomere sizes, presence of telomeric aggregates, telomeres per nuclear volume, and a/c ratios. The clinical significance of these findings is the early identification of individuals with high risk of progression. This opens the possibility for better monitoring and early intervention with newer treatments with an acceptable efficacy and low toxicity ratio. Disclosures:No relevant conflicts of interest to declare.

The Epidemiology of Human Telomeres: Faults and Promises

There are neglected but growing problems in the epidemiological field of telomere biology. The focus of the field has been on leukocyte telomere dynamics, which ostensibly register the accruing burden of oxidative stress and inflammation. Important as they are, studies that have examined associations between leukocyte telomere length and indices of aging and diseases of aging also include many that are compromised by poor epidemiological and laboratory methodology. The shortcomings of these studies muddle findings, undermine conclusions, and compromise the ability of the field to attain its goals, which include a better understanding of human aging. Specific steps are delineated to resolve these problems. They include a call for an impartial evaluation of the two major methods (Southern blots and quantitative polymerase chain reaction) currently in use to measure telomere parameters and a proposal for a working model to test the potential connections of leukocyte telomere dynamics with human aging and longevity.

Telomere Length and Mortality: A Study of Leukocytes in Elderly Danish Twins

Leukocyte telomere length, representing the mean length of all telomeres in leukocytes, is ostensibly a bioindicator of human aging. The authors hypothesized that shorter telomeres might forecast imminent mortality in elderly people better than leukocyte telomere length. They performed mortality analysis in 548 same-sex Danish twins (274 pairs) aged 73-94 years, of whom 204 pairs experienced the death of one or both co-twins during 9-10 years of follow-up (1997-2007). From the terminal restriction fragment length (TRFL) distribution, the authors obtained the mean TRFL (mTRFL) and the mean values of the shorter 50% (mTRFL(50)) and shortest 25% (mTRFL(25)) of TRFLs in the distribution and computed the mode of TRFL (MTRFL). They analyzed the proportions of twin pairs in which the co-twin with the shorter telomeres died first. The proportions derived from the intrapair comparisons indicated that the shorter telomeres predicted the death of the first co-twin better than the mTRFL did (mTRFL: 0.56, 95% confidence interval (CI): 0.49, 0.63; mTRFL(50): 0.59, 95% CI: 0.52, 0.66; mTRFL(25): 0.59, 95% CI: 0.52, 0.66; MTRFL: 0.60, 95% CI: 0.53, 0.67). The telomere-mortality association was stronger in years 3-4 than in the rest of the follow-up period, and it grew stronger with increasing intrapair difference in all telomere parameters. Leukocyte telomere dynamics might help explain the boundaries of the human life span.

Offspring's leukocyte telomere length, paternal age, and telomere elongation in sperm.

Leukocyte telomere length (LTL) is a complex genetic trait. It shortens with age and is associated with a host of aging-related disorders. Recent studies have observed that offspring of older fathers have longer LTLs. We explored the relation between paternal age and offspring's LTLs in 4 different cohorts. Moreover, we examined the potential cause of the paternal age on offspring's LTL by delineating telomere parameters in sperm donors. We measured LTL by Southern blots in Caucasian men and women (n=3365), aged 18–94 years, from the Offspring of the Framingham Heart Study (Framingham Offspring), the NHLBI Family Heart Study (NHLBI-Heart), the Longitudinal Study of Aging Danish Twins (Danish Twins), and the UK Adult Twin Registry (UK Twins). Using Southern blots, Q-FISH, and flow-FISH, we also measured telomere parameters in sperm from 46 young (<30 years) and older (>50 years) donors. Paternal age had an independent effect, expressed by a longer LTL in males of the Framingham Offspring and Danish Twins, males and females of the NHLBI-Heart, and females of UK Twins. For every additional year of paternal age, LTL in offspring increased at a magnitude ranging from half to more than twice of the annual attrition in LTL with age. Moreover, sperm telomere length analyses were compatible with the emergence in older men of a subset of sperm with elongated telomeres. Paternal age exerts a considerable effect on the offspring's LTL, a phenomenon which might relate to telomere elongation in sperm from older men. The implications of this effect deserve detailed study.

High telomerase activity and long telomeres in advanced hepatocellular carcinomas with poor prognosis

Telomerase reactivation and telomere maintenance are crucial in carcinogenesis and tumor progression. In this study, the relationships between telomere parameters, chromosomal instability and clinicopathological features were evaluated in hepatocellular carcinomas (HCCs). Telomere length (TL), telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA levels were measured in 49 hepatitis B virus (HBV)-related HCCs and corresponding non-tumorous tissues. The results were compared with clinicopathological data, including differentiation, multipolar mitosis (MM), anaphase bridge, immunohistochemical stain results for cytokeratin 19 (CK19) and patient outcome. TL of HCCs ranged from 4.7 to 13.1 kb, and 44.4% of HCCs showed telomere lengthening. hTERT mRNA levels and TA were closely related (P=0.008), and were significantly higher in HCCs than non-tumorous tissues. TL was significantly higher in HCCs with strong TA (P=0.048), high hTERT mRNA levels (P=0.001) and poor differentiation (P=0.041). Frequent MM was associated with poor differentiation (P=0.007) and advanced stage (P<0.001). TA was positively correlated with MM, anaphase bridges and advanced stage (P=0.019, P=0.017 and P=0.029). Thirteen (28.3%) HCCs were CK19+ and demonstrated longer telomeres than CK19− HCCs (P=0.046). Overall survival was poor in HCCs with MM >0.4 per field (P=0.016), high TA (P=0.009) and high TL ratio (HCC/non-HCC) >0.8 (P=0.044). Our results show that long telomeres, high TA and high mitotic instability are poor prognostic markers for HBV-related HCCs and their close association suggests that telomere maintenance may be important for the progression of HCCs with high chromosomal instability to more aggressive ones.

Telomere Dynamics in Macaques and Humans

In humans, telomere length in proliferating tissues shortens with age--a process accelerated with age-related diseases. Thus, telomere length and attrition with age in the nonhuman primate may serve as a useful paradigm for understanding telomere biology in humans. We examined telomere parameters in tissues of young and old Macaca fascicularis and compared them with several tissues from humans. Macaque telomeres were variable in length and exhibited partial synchrony (equivalence) within animals. They were longer than humans, partially because of longer subtelomeric segments. As skeletal muscle telomere length was unchanged with age, we used it as an internal reference to offset interanimal variation in telomere length. We identified age-dependent telomere attrition in lung, pancreas, skin, and thyroid. Similar to humans, telomerase activity was detected in spleen, thymus, digestive tract, and gonads. We conclude that factors that modify telomere attrition and aging in humans may also operate in the macaque.