Single Telomere Length Analysis Research Articles

T cell memory revisited using single telomere length analysis.

The fundamental basis of T cell memory remains elusive. It is established that antigen stimulation drives clonal proliferation and differentiation, but the relationship between cellular phenotype, replicative history, and longevity, which is likely essential for durable memory, has proven difficult to elucidate. To address these issues, we used conventional markers of differentiation to identify and isolate various subsets of CD8+ memory T cells and measured telomere lengths in these phenotypically defined populations using the most sensitive technique developed to date, namely single telomere length analysis (STELA). Naive cells were excluded on the basis of dual expression of CCR7 and CD45RA. Memory subsets were sorted as CD27+CD45RA+, CD27intCD45RA+, CD27-CD45RA+, CD27+CD45RAint, CD27-CD45RAint, CD27+CD45RA-, and CD27-CD45RA- at >98% purity. The shortest median telomere lengths were detected among subsets that lacked expression of CD45RA, and the longest median telomere lengths were detected among subsets that expressed CD45RA. Longer median telomere lengths were also a feature of subsets that expressed CD27 in compartments defined by the absence or presence of CD45RA. Collectively, these data suggested a disconnect between replicative history and CD8+ memory T cell differentiation, which is classically thought to be a linear process that culminates with revertant expression of CD45RA.

The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

O019 Studying telomere biology and genomic instability in gliomas

Abstract Introduction Glioblastoma (GBM) is the most aggressive primary brain cancer. Identifying novel markers which stratify patients and predict response to targeted therapies is a priority. Telomeres are caps on chromosome ends protecting them from aberrant DNA damage response. Telomere length (TL) alterations and fusions are key markers for genomic instability and high-resolution telomere analysis provides powerful prognostic and predictive information in several tumour types. We therefore studied telomeric profiles and fusions in GBM patients and related this to clinical outcome. Methods Tumours from 151 adults with isocitrate dehydrogenase-wildtype GBMs were analysed for TL using the precision High-Throughput Single Telomere Length Analysis technique. 67 patients underwent further multiplex long-range polymerase chain reaction to identify fusions via Southern hybridisation using telomere-adjacent 33P radioisotope-labelled probes. Telomere fusions from 10 tumours that exhibited the highest frequency of fusions were characterised with long-read Nanopore sequencing. Results GBMs display short telomeres with a median length of 4.0kb. Shorter TLs in patients with unmethylated O6-methylguanine DNA methyltransferase status (commonest current biomarker) conferred worse survival (HR 2.13, p = 0.002). 66/67 (98.5%) patients analysed revealed telomeric fusions. Nanopore sequencing validated these events, while confirming the presence of microhomology and telomeric deletion. This mutational spectrum is consistent with utilisation of specific DNA repair pathways in mediating telomere fusion. Conclusion This study provides the first mechanistic evidence of dysfunctional telomeres in adult GBMs and suggests TL analysis may provide independent prognostic information. The presence of telomere fusions indicates significant genomic instability and may favour targeted therapies for patients exhibiting this signature.

Symptoms of Prenatal Depression Associated with Shorter Telomeres in Female Placenta.

Background. Depression is a common mood disorder during pregnancy impacting one in every seven women. Children exposed to prenatal depression are more likely to be born at a low birth weight and develop chronic diseases later in life. A proposed hypothesis for this relationship between early exposure to adversity and poor outcomes is accelerated aging. Telomere length has been used as a biomarker of cellular aging. We used high-resolution telomere length analysis to examine the relationship between placental telomere length distributions and maternal mood symptoms in pregnancy. Methods. This study utilised samples from the longitudinal Grown in Wales (GiW) study. Women participating in this study were recruited at their presurgical appointment prior to a term elective caesarean section (ELCS). Women completed the Edinburgh Postnatal Depression Scale (EPDS) and trait subscale of the State-Trait Anxiety Inventory (STAI). Telomere length distributions were generated using single telomere length analysis (STELA) in 109 term placenta (37–42 weeks). Multiple linear regression was performed to examine the relationship between maternally reported symptoms of depression and anxiety at term and mean placental telomere length. Results: Prenatal depression symptoms were significantly negatively associated with XpYp telomere length in female placenta (B = −0.098, p = 0.026, 95% CI −0.184, −0.012). There was no association between maternal depression symptoms and telomere length in male placenta (B = 0.022, p = 0.586, 95% CI −0.059, 0.103). There was no association with anxiety symptoms and telomere length for either sex. Conclusion: Maternal prenatal depression is associated with sex-specific differences in term placental telomeres. Telomere shortening in female placenta may indicate accelerated placental aging.

Telomere Length in Cystic Fibrosis Patients – Are Patients with CF Ageing Too Quickly?

Life expectancy for patients living with Cystic Fibrosis (CF) is increasing year on year and there is growing interest in the ageing process in CF. Telomeres are repetitive sequences of DNA that cap the ends of eukaryotic chromosomes and shorten with ongoing cell division, thus providing a marker of replicative history and biological ageing. We aimed to investigate whether telomere length as a function of age differs between patients with CF and healthy individuals and whether telomere length is associated with severity of the patient’s CF condition. Peripheral blood samples and demographic data were collected from 47 consenting patients (age 1 to 57 years) with CF attending their routine annual review appointment at the All Wales Adult CF Centre and Noah’s Ark Children’s’ Hospital in Cardiff, UK. Telomere length profiles were assessed from peripheral blood samples, using the high resolution single telomere length analysis technique (STELA) and compared to healthy control telomere length data. Patients with CF had significantly shorter telomere lengths than healthy individuals, when adjusting for age (p<0.001). Telomere length is decreasing 70% more quickly in the CF cohort than healthy controls. Telomere length does not appear to correlate with markers of disease severity. Telomere lengths are significantly shorter in individuals with CF than in the age-adjusted healthy population. This is suggestive of premature biological ageing of peripheral blood leukocytes in CF patients.

Human Endometrial Carcinogenesis Is Associated with Significant Reduction in Long Non-Coding RNA, TERRA.

Telomeres are transcribed as long non-coding RNAs called TERRAs (Telomeric repeat containing RNA) that participate in a variety of cellular regulatory functions. High telomerase activity (TA) is associated with endometrial cancer (EC). This study aimed to examine the levels of three TERRAs, transcribed at chromosomes 1q-2q-4q-10q-13q-22q, 16p and 20q in healthy (n = 23) and pathological (n = 24) human endometrium and to examine their association with cellular proliferation, TA and telomere lengths. EC samples demonstrated significantly reduced levels of TERRAs for Chromosome 16p (Ch-16p) (p < 0.002) and Chromosome 20q (Ch-20q) (p = 0.0006), when compared with the postmenopausal samples. No significant correlation was found between TERRA levels and TA but both Ch-16p and Ch-20q TERRA levels negatively correlated with the proliferative marker Ki67 (r = −0.35, p = 0.03 and r = −0.42, p = 0.01 respectively). Evaluation of single telomere length analysis (STELA) at XpYp telomeres demonstrated a significant shortening in EC samples when compared with healthy tissues (p = 0.002). We detected TERRAs in healthy human endometrium and observed altered individual TERRA-specific levels in malignant endometrium. The negative correlation of TERRAs with cellular proliferation along with their significant reduction in EC may suggest a role for TERRAs in carcinogenesis and thus future research should explore TERRAs as potential therapeutic targets in EC.

Tissue-Specific Ultra-Short Telomeres in Chronic Obstructive Pulmonary Disease.

PurposeTelomere biology, especially tissue-specific ultra-short telomeres, might provide a strong contribution to our current knowledge in COPD development as well as a predictive marker for prognosis. To test this hypothesis, we investigated telomere lengths in lung tissue and leukocytes in patients diagnosed with COPD.Patients and MethodsThirty-two patients were included in the current study. All patients showed a post-bronchodilator ratio of less than 70% post-bronchodilator predicted value of forced expiratory volume in second (FEV1%), mean 56%; range [19% to 86%]. To be able to investigate ultra-short telomeres, universal single telomere length analysis (U-STELA) was used.ResultsOur results showed a higher level of the ultra-short telomere presence in bronchoalveolar lavage (BAL) cells when compared to leukocytes with statistical significance t(62)=5.771, p<0.00001. The FEV1% was lower in subjects with ultra-short telomeres in BAL (50.6% vs 81.6%: p<0.001) and in ultra-short telomeres in blood leukocytes (37.3% vs 58.5%: p=0.051) when compared to subjects without ultra-short telomeres in leukocytes. Furthermore, the patients who had ultra-short telomeres in BAL samples were significantly older (p=0.014) than patients who did not have ultra-short telomeres. Ultra-short telomeres in BAL (p=0.05) but not in leukocytes (p=0.33) were associated with FEV1% in a regressions model adjusting for age (p<0.0001), ever smoking (p<0.0001) and sex (p=0.71). The patients with ultra-short telomeres were graded higher in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification (p=0.006).ConclusionThis study emphasizes the need to investigate the correct tissue to get a representative evaluation of the stage or advancedness of COPD. To our knowledge, this is the first study to show that there is a correlation between the presence of ultra-short telomeres in lung tissue and COPD severity. Our results suggest that ultra-short telomeres are involved in the molecular pathogenesis of COPD and might be used as a tissue-specific predictive biomarker.

Impaired reproductive function and fertility preservation in a woman with a dyskeratosis congenita.

To determine the impact of accelerated telomere shortening on the fertility parameters and treatment outcomes of a woman with dyskeratosis congenita (DKC). A case study of the clinical data, blood, discarded oocytes, and arrested embryos of a woman with DKC and donated cryopreserved embryos from unaffected patients. Mean telomere length in blood cells was analyzed by flow cytometry-fluorescence in situ hybridization (flow-FISH) and qPCR. The load of short telomeres in blood cells was measured by universal single telomere length analysis (Universal STELA). The mean telomere length in embryos was analyzed by single-cell amplification of telomere repeats (SCATR) PCR. Comparison of clinical parameters revealed that the DKC patient had reduced anti-Mullerian hormone (0.3 vs 4.1 ± 5.7ng/ML), reduced oocytes retrieved (7 vs 18.5 ± 9.5), reduced fertilization rate, and reduced euploidy rate relative to unaffected patients. Additionally, mean telomere length in DKC embryos were shorter than unaffected embryos. However, hormone treatment led to increased leukocyte telomere length, while the load of short telomeres was also shown to decrease during the course of treatment. We demonstrate for the first time the direct detrimental impacts of short telomeres on female fertility. We further demonstrate positive effects of hormone treatments for people with telomere disorders.

Measurement of Short Telomere Load in Individual Cells

Increasing evidence demonstrates that shortest more than mean telomere length predicts telomere dysfunction and genomic instability in association with a number of conditions, including cell senescence, aging and tumorigenesis. We developed Universal Single Cell Single Telomere Length Analysis (USC-STELA), based on a PCR-amplification and southern blotting, to measure short telomeres in individual cells. The mean short telomere length measured in individual cells by USC-STELA correlates with that from bulk cells, measured by Universal STELA (U-STELA). The validation and reproducibility of USC-STELA was confirmed using different cell types with known telomere lengths, as well as by using paired sister-cells from human embryos and cultured cells. Interestingly, individual cells known to elongate telomeres via alternative lengthening of telomeres (ALT) have more short telomeres, yet longer mean telomere length than control cells. Moreover, individual senescent fibroblasts carry more short telomeres compared to human embryonic stem cells (hESCs), consistent with the notion that short telomeres contribute to cellular senescence. Additionally, we found a greater load of short telomeres in polar bodies than in matching oocytes, providing further insights into the accelerated polar body DNA degradation following extrusion from the oocyte. USC-STELA provides a new method to study telomere dysfunction in individual cells, with potential to improve our understanding of the role of telomere dynamics in cancer, developmental biology and reproductive medicine.

Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA)

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.

Salivary Gland Stem Cells Age Prematurely in Primary Sjögren's Syndrome.

ObjectiveA major characteristic of the autoimmune disease primary Sjögren's syndrome (SS) is salivary gland (SG) hypofunction. The inability of resident SG stem cells (SGSCs) to maintain homeostasis and saliva production has never been explained and limits our comprehension of mechanisms underlying primary SS. The present study was undertaken to investigate the role of salivary gland stem cells in hyposalivation in primary SS.Methods SGSCs were isolated from parotid biopsy samples from controls and patients classified as having primary SS or incomplete primary SS, according to the American College of Rheumatology/European League Against Rheumatism criteria. Self‐renewal and differentiation assays were used to determine SGSC regenerative potential, RNA was extracted for sequencing analysis, single telomere length analysis was conducted to determine telomere length, and frozen tissue samples were used for immunohistochemical analysis.Results SGSCs isolated from primary SS parotid gland biopsy samples were regeneratively inferior to healthy control specimens. We demonstrated that SGSCs from samples from patients with primary SS are not only lower in number and less able to differentiate, but are likely to be senescent, as revealed by telomere length analysis, RNA sequencing, and immunostaining. We further found that SGSCs exposed to primary SS–associated proinflammatory cytokines we induced to proliferate, express senescence‐associated genes, and subsequently differentiate into intercalated duct cells. We also localized p16+ senescent cells to the intercalated ducts in primary SS SG tissue, suggesting a block in SGSC differentiation into acinar cells.ConclusionThis study represents the first characterization of SGSCs in primary SS, and also the first demonstration of a linkage between an autoimmune disease and a parenchymal premature‐aging phenotype. The knowledge garnered in this study indicates that disease‐modifying antirheumatic drugs used to treat primary SS are not likely to restore saliva production, and should be supplemented with fresh SGSCs to recover saliva production.

Metformin and insulin treatment prevent placental telomere attrition in boys exposed to maternal diabetes.

Shortened leukocyte and placental telomeres associated with gestational diabetes mellitus (GDM) suggest this exposure triggers telomere attrition contributing to adverse outcomes. We applied high resolution Single Telomere Length Analysis (STELA) to placenta from GDM pregnancies with different treatment pathways to determine their effectiveness at preventing telomere attrition. Differences in telomere length between control (N = 69), GDM lifestyle intervention (n = 14) and GDM treated with metformin and/or insulin (n = 17) was tested by Analysis of Covariance (ANCOVA) followed by group comparisons using Fisher’s least significant difference. For male placenta only, there were differences in mean telomere length (F(2,54) = 4.98, P = 0.01) and percentage of telomeres under 5 kb (F(2,54) = 4.65, P = 0.01). Telomeres were shorter in the GDM lifestyle intervention group compared to both controls (P = 0.02) and medically treated pregnancies (P = 0.003). There were more telomeres under 5 kb in the GDM lifestyle intervention group compared to the other two groups (P = 0.03 and P = 0.004). Although further work is necessary, we suggest that early adoption of targeted medical treatment of GDM pregnancies where the fetus is known to be male may be an effective strategy for ameliorating adverse outcomes for children.

Telomere Length Predicts for Outcome to FCR Chemoimmunotherapy in CLL

Telomeres are structures that cap the ends of chromosomes and play a critical role in maintaining genomic integrity. Telomere length is a key determinant of telomere function, with short telomeres being subjected to aberrant DNA repair activity that leads to telomere fusion and large scale genomic rearrangements. Single telomere length analysis (STELA) is a high-resolution approach to determine telomere length and is capable of detecting telomeres within the length range at which fusions can occur. Using STELA, we have previously shown a link between short telomeres, telomere fusion and genomic instability in CLL. We went on to define the telomere length threshold at which the chromosome end-capping function is lost resulting in telomere fusion events and genomic instability, the so-called ‘fusogenic’ range. We have shown that dividing patients with CLL into those with telomeres inside the fusogenic range (IFR) and outside the fusogenic range (OFR) is powerful prognostic tool. Consequently, we developed a high-throughput version of the assay (HT-STELA) to allow for rapid evaluation of large numbers of clinical samples.In this study, we used HT-STELA to evaluate whether telomere length could predict for outcome following fludarabine, cyclophosphamide, rituximab (FCR)-based treatment using samples collected from two concurrent phase II studies, ARCTIC and ADMIRE (n = 275). Patients with a mean telomere length IFR had significantly shorter progression-free survival (P<0.0001; HR=2.17) and shorter overall survival (P=0.0002; HR=2.44) when compared to patients with a mean telomere length OFR. In contrast, CD38 expression and β2-microglobulin were not informative and IGHV mutation status was only predictive of progression-free survival (P=0.0016; HR=1.9). Bifurcation of the IGHV-mutated and unmutated subsets according to telomere length revealed that patients with short, dysfunctional telomeres in each subset had shorter progression-free survival (HR = 4.35 and HR = 1.48 respectively) and shorter overall survival (HR = 3.81 and HR = 2.18 respectively). Although the mean telomere length of the IGHV-unmutated group was significantly shorter than the IGHV-mutated group (P<0.0001), patients with long (OFR) telomeres in both subsets has a significantly better overall survival (P = 0.025; 79% at 5 years and P = 0.006; 83% at 5 years respectively). Indeed, the overall survival of the OFR and IFR subsets were not significantly different regardless of IGHVmutation status (P = 0.61; HR = 1.24 and P = 0.41; HR = 1.47 respectively). In multivariate modelling using Cox proportional hazards with forward selection, telomere length was the dominant co-variable for progression-free survival (P=0.0002; HR=1.85) and overall survival (P=0.05; HR=1.61); when telomere length was added into the model no other parameter retained independent significance in the presence of telomere length. Taken together our data suggest that telomere length is a powerful predictor of outcome to FCR-based treatment and could be used to inform the design of future risk-adapted clinical trials.Figure 1. Stratification of patients by telomere length predicts for progression-free and overall survival following FCR-based treatment. Bifurcation of the patient cohort according to the previously defined telomere length threshold for telomere dysfunction was predictive of (A)PFSand (B) OS. Patients whose telomere length were ≤ the mean of the fusogenic range (IFR) showed shorter PFS and OS than those patients with mean telomere length outside of the fusogenic range (OFR). (C) In terms of OS, the short telomere subsets in both (C)IGHV-mutated and (D)IGHV-unmutated groups showed significantly reduced survival. It is of particular interest that the OS of patients with (E)long telomeres (OFR) and (F) short telomeres (IFR) were not significantly different in IGHV-mutated and IGHV-unmutated groups. [Display omitted] DisclosuresNorris:Cardiff University: Patents & Royalties: Telomere measurement patent. Hillmen:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Novartis: Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Gilead Sciences, Inc.: Honoraria, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Hills:Daiichi Sankyo: Consultancy, Honoraria. Baird:Cardiff University: Patents & Royalties: Telomere measurement patents. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents. Fegan:Janssen: Honoraria; Gilead Sciences, Inc.: Honoraria; Abbvie: Honoraria; Napp: Honoraria; Roche: Honoraria.

Telomere Length Is Associated with Epigenetic Programming in CLL and Is a Superior Predictor of Clinical Outcome with the Ability to Bifurcate Patients with the Same CLL-IPI Score

The characteristic clinical heterogeneity observed in chronic lymphocytic leukemia (CLL) reflects its biological diversity and as a consequence, better understanding of the biology of this disease has led to the identification of a number of useful prognostic tools. Advances in massively parallel sequencing technologies have unearthed genetic and epigenetic heterogeneity and reinforced the concept that specific genetic lesions and the development of genomic complexity can modulate the pathology of CLL. In addition, DNA methylation profiling has shown that CLL patients can be grouped into at least three clinically relevant epigenetic subgroups with distinct outcomes. However, despite this explosion in knowledge, defining the prognosis of individual CLL patients remains a significant clinical challenge. Consequently, there is a need to identify prognostic tests that can provide reliable personalized risk assessments.We have recently shown that a subset of CLL patients manifest telomere dysfunction, which drives genomic instability and clonal evolution. We went on to show an association between short, dysfunctional telomeres and ‘high-risk’ genetic lesions1,2. Importantly, longitudinal analysis confirmed that tumor cell telomere length remained remarkably stable throughout the course of the disease suggesting that the telomere length is fixed at an early point in disease pathogenesis making it a reliable predictor of both clinical progression and overall survival (OS) regardless of when the measurement is made. In this study, we generated telomere length profiles in 224 newly diagnosed, early stage CLL patients seen at the Mayo clinic in Rochester using high-throughput single telomere length analysis (HT-STELA). We then tested the ability of telomere length to predict time to treatment (TTT) and OS and compared its predictive power with DNA methylation profiling and CLL-IPI scores.Patients with a mean telomere length inside the fusogenic range (IFR) had a significantly shorter time to treatment (P<0.0001; HR = 3.2) and OS (P = 0.0027; HR = 2.3) when compared to patients with a mean telomere length outside the fusogenic range (OFR) (Figure 1A and B). When considering epigenetic subgroups, we found a strong association between telomere length and ‘epitype’; high programmed (HP) CLL cells had significantly longer telomeres than intermediate programmed (IP), and low programmed (LP) CLL cells (P = 0.0002 and P<0.0001 respectively). Furthermore, bifurcation of the HP and IP groups into telomere length OFR and IFR subsets identified patients with distinctly different TTT (P = 0.0008; HR = 2.74) and OS (P = 0.004; HR = 2.4). 79/81 (97.5%) of the LP group had short telomere length IFR, but when we divided the LP group above and below the mean telomere length of the fusogenic range (≤/>2.17kb), patients with the shortest telomeres had a significantly worse OS (P = 0.034; HR = 2.83). Telomere length was also strongly associated with CLL-IPI score; the CLL-IPI low-risk group (CLL-IPI 1) had the longest telomeres and the CLL-IPI high-risk group (CLL-IPI 4) had the shortest telomeres (P<0.0001). More heterogeneity in telomere length was observed in the CLL-IPI 1 and 2 groups and patients with telomere length IFR in these ‘low-risk’ groups showed significantly shorter TTT (P<0.0001; HR = 4.24) and overall survival (P<0.0001; HR = 10.1) (Figure 1C and 1D). Finally, multivariate modelling, using Cox proportional hazards with forward selection, revealed that telomere length retained independent prognostic significance when CLL-IPI score was entered in the model for time to treatment (HR = 1.42; P = 0.023) and OS (HR = 1.68; P = 0.008). In conclusion, telomere length analysis provides more accurate prognostic information about the progression and outcome of CLL patients and can augment the prognostic power of the CLL-IPI for early stage CLL.ReferencesLin TT et al. Blood. 2010;116(11):1899-907.Britt-Compton et al. Leukemia. 2012; 26(4):826-30. [Display omitted] DisclosuresNorris:Cardiff University: Patents & Royalties: Telomere measurement patent. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Jansen: Research Funding. Kay:Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Fegan:Napp: Honoraria; Gilead Sciences, Inc.: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Baird:Cardiff University: Patents & Royalties: Telomere measurement patents. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents.

Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing

Telomeres play important roles in genome stability and cell proliferation. Telomere lengths are heterogeneous and because just a few abnormal telomeres are sufficient to trigger significant cellular response, it is informative to have accurate assays that reveal not only average telomere lengths, but also the distribution of the longest and shortest telomeres in a given sample. Herein we report for the first time, the development of single telomere length analysis (STELA) - a PCR-based assay that amplifies multiple, individual telomeres - for Ustilago maydis, a basidiomycete fungus. Compared to the standard telomere Southern technique, STELA revealed a broader distribution of telomere size as well as the existence of relatively short telomeres in wild type cells. When applied to blm∆, a mutant thought to be defective in telomere replication, STELA revealed preferential loss of long telomeres, whose maintenance may thus be especially dependent upon efficient replication. In comparison to blm∆, the trt1∆ (telomerase null) mutant exhibited greater erosion of short telomeres, consistent with a special role for telomerase in re-lengthening extra-short telomeres. We also used STELA to characterize the 5’ ends of telomere C-strand, and found that in U. maydis, they terminate preferentially at selected nucleotide positions within the telomere repeat. Deleting trt1 altered the 5’-end distributions, suggesting that telomerase may directly or indirectly modulate C-strand 5’ end formation. These findings illustrate the utility of STELA as well as the strengths of U. maydis as a model system for telomere research.

Telomere length heterogeneity in placenta revealed with high-resolution telomere length analysis

IntroductionTelomeres, are composed of tandem repeat sequences located at the ends of chromosomes and are required to maintain genomic stability. Telomeres can become shorter due to cell division and specific lifestyle factors. Critically shortened telomeres are linked to cellular dysfunction, senescence and aging. A number of studies have used low resolution techniques to assess telomere length in the placenta. In this study, we applied Single Telomere Length Analysis (STELA) which provides high-resolution chromosome specific telomere length profiles to ask whether we could obtain more detailed information on the length of individual telomeres in the placenta. MethodsTerm placentas (37–42 weeks) were collected from women delivering at University Hospital of Wales or Royal Gwent Hospital within 2 h of delivery. Multiple telomere-length distributions were determined using STELA. Intraplacental variation of telomere length was analysed (N = 5). Telomere length distributions were compared between labouring (N = 10) and non-labouring (N = 11) participants. Finally, telomere length was compared between female (N = 17) and male (N = 20) placenta. ResultsThere were no significant influences of sampling site, mode of delivery or foetal sex on the telomere-length distributions obtained. The mean telomere length was 7.7 kb ranging from 5.0 kb to 11.7 kb across all samples (N = 42) and longer compared with other human tissues at birth. STELA also revealed considerable telomere length heterogeneity within samples. ConclusionsWe have shown that STELA can be used to study telomere length homeostasis in the placenta regardless of sampling site, mode of delivery and foetal sex. Moreover, as each amplicon is derived from a single telomeric molecule, from a single cell, STELA can reveal the full detail of telomere-length distributions, including telomeres within the length ranges observed in senescent cells. STELA thus provides a new tool to interrogate the relationship between telomere length and pregnancy complications linked to placental dysfunction.

WS09.6 Telomere length in cystic fibrosis patients – are patients with CF ageing too quickly?

Life expectancy for patients living with Cystic Fibrosis (CF) is increasing year on year and there is growing interest in the ageing process in CF. Telomeres are repetitive sequences of DNA that cap the ends of eukaryotic chromosomes and shorten with ongoing cell division, thus providing a marker of replicative history and biological ageing. We aimed to investigate whether telomere length as a function of age differs between patients with CF and healthy individuals and whether telomere length is associated with severity of the patient’s CF condition. Peripheral blood samples and demographic data were collected from 47 consenting patients (age 1 to 57 years) with CF attending their routine annual review appointment at the All Wales Adult CF Centre and Noah’s Ark Children’s’ Hospital in Cardiff, UK. Telomere length profiles were assessed from peripheral blood samples, using the high resolution single telomere length analysis technique (STELA) and compared to healthy control telomere length data. Patients with CF had significantly shorter telomere lengths than healthy individuals, when adjusting for age (p<0.001). Telomere length is decreasing 70% more quickly in the CF cohort than healthy controls. Telomere length does not appear to correlate with markers of disease severity. Telomere lengths are significantly shorter in individuals with CF than in the age-adjusted healthy population. This is suggestive of premature biological ageing of peripheral blood leukocytes in CF patients.

Telomere length is an independent prognostic marker in MDS but not in de novo AML.

Telomere dysfunction is implicated in the generation of large-scale genomic rearrangements that drive progression to malignancy. In this study we used high-resolution single telomere length analysis (STELA) to examine the potential role of telomere dysfunction in 80 myelodysplastic syndrome (MDS) and 95 de novo acute myeloid leukaemia (AML) patients. Despite the MDS cohort being older, they had significantly longer telomeres than the AML cohort (P<0·0001) where telomere length was also significantly shorter in younger AML patients (age <60years) (P=0·02) and in FLT3 internal tandem duplication-mutated AML patients (P=0·03). Using a previously determined telomere length threshold for telomere dysfunction (3·81kb) did not provide prognostic resolution in AML [Hazard ratio (HR)=0·68, P=0·2]. In contrast, the same length threshold was highly prognostic for overall survival in the MDS cohort (HR=5·0, P<0·0001). Furthermore, this telomere length threshold was an independent parameter in multivariate analysis when adjusted for age, gender, cytogenetic risk group, number of cytopenias and International Prognostic Scoring System (IPSS) score (HR=2·27, P<0·0001). Therefore, telomere length should be assessed in a larger prospective study to confirm its prognostic role in MDS with a view to integrating this variable into a revised IPSS.

Telomere erosion in NF1 tumorigenesis.

Neurofibromatosis type 1 (NF1; MIM# 162200) is a familial cancer syndrome that affects 1 in 3,500 individuals worldwide and is inherited in an autosomal dominant fashion. Malignant Peripheral Nerve Sheath Tumors (MPNSTs) represent a significant cause of morbidity and mortality in NF1 and currently there is no treatment or definite prognostic biomarkers for these tumors. Telomere shortening has been documented in numerous tumor types. Short dysfunctional telomeres are capable of fusion and it is considered that the ensuing genomic instability may facilitate clonal evolution and the progression to malignancy. To evaluate the potential role of telomere dysfunction in NF1-associated tumors, we undertook a comparative analysis of telomere length in samples derived from 10 cutaneous and 10 diffused plexiform neurofibromas, and 19 MPNSTs. Telomere length was determined using high-resolution Single Telomere Length Analysis (STELA). The mean Xp/Yp telomere length detected in MPNSTs, at 3.282 kb, was significantly shorter than that observed in both plexiform neurofibromas (5.793 kb; [p = 0.0006]) and cutaneous neurofibromas (6.141 kb; [p = 0.0007]). The telomere length distributions of MPNSTs were within the length-ranges in which telomere fusion is detected and that confer a poor prognosis in other tumor types. These data indicate that telomere length may play a role in driving genomic instability and clonal progression in NF1-associated MPNSTs.

Telomere length profiles in primary human peritoneal mesothelial cells are consistent with senescence

Mesothelial cell (MC) senescence contributes to malignancy and tissue fibrosis. The role of telomere erosion in MC senescence remains controversial, with evidence for both telomere-dependent and telomere-independent mechanisms reported. Single telomere length analysis revealed considerable telomere length heterogeneity in freshly isolated human peritoneal MCs, reflecting a heterogeneous proliferative history and providing high-resolution evidence for telomere-dependent senescence. By contrast the attenuated replicative lifespan, lack of telomere erosion and induction of p16 expression in in vitro-aged cells was consistent with stress-induced senescence. Given the potential pathophysiological impact of senescence in mesothelial tissues, high-resolution MC telomere length analysis may provide clinically useful information.