Telomerase Complex Genes Research Articles

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers.

BackgroundComprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer.Methods and ResultsIn this study, we extensively characterize telomere‐related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom‐designed RNA‐seq panel, we identified five telomerase holoenzyme‐complex genes upregulated in clinically aggressive tumours compared to tumours from long‐term disease‐free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re‐expression revealed that promoter mutations, methylation and/or copy gains exclusively co‐occurred in clinically aggressive tumours. Quantitative‐FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki‐67 immunohistochemistry. RNA‐seq data analysis indicated that short‐telomere tumours exhibit an increased transcriptional activity in the 5‐Mb‐subtelomeric regions, site of several telomerase‐complex genes. Gene upregulation enrichment was significant for specific chromosome‐ends such as the 5p, where TERT is located. Co‐FISH analysis of 5p‐end and TERT loci showed a more relaxed chromatin configuration in short telomere‐length tumours compared to normal telomere‐length tumours.ConclusionsOverall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT‐locus.

Lactate is a potential promoter of tamoxifen resistance in MCF7 cells

BackgroundTamoxifen is a widely used estrogen receptor inhibitor, whose clinical success is limited by the development of acquired resistance. This compound was also found to inhibit mitochondrial function, causing increased glycolysis and lactate production. Lactate has been widely recognized as a signaling molecule, showing the potential of modifying gene expression. These metabolic effects of tamoxifen can by hypothesized to contribute in driving drug resistance. MethodsTo test this hypothesis, we used MCF7 cells together with a tamoxifen resistant cell line (MCF7-TAM). Experiments were aimed at verifying whether enhanced lactate exposure can affect the phenotype of MCF7 cells, conferring them features mirroring those observed in the tamoxifen resistant culture. ResultsThe obtained results suggested that enhanced lactate in MCF7 cells medium can increase the expression of tafazzin (TAZ) and telomerase complex (TERC, TERT) genes, reducing the cells' attitude to undergo senescence. In long term lactate-exposed cells, signs of EGFR activation, a pathway related to acquired tamoxifen resistance, was also observed. ConclusionsThe obtained results suggested lactate as a potential promoter of tamoxifen resistance. The off-target effects of this compound could play a role in hindering its therapeutic efficacy. General significanceThe features of acquired tamoxifen resistance have been widely characterized at the molecular level; in spite of their heterogeneity, poorly responsive cells were often found to display upregulated glycolysis. Our results suggest that this metabolic asset is not simply a result of neoplastic progression, but can play an active part in driving this process.

Utility of Telomerase Gene Mutation Testing in Patients with Idiopathic Pulmonary Fibrosis in Routine Practice.

Recent studies have suggested that causative variants in telomerase complex genes (TCGs) are present in around 10% of individuals with idiopathic pulmonary fibrosis (IPF) regardless of family history of the disease. However, the studies used a case-control rare variant enrichment study design which is not directly translatable to routine practice. To validate the prevalence results and to establish the individual level, routine clinical practice, and utility of those results we performed next generation sequencing of TCGs on a cohort of well-characterized consecutive individuals with IPF (diagnosis established according to ATS/ERS/JRS/ALAT guidelines). Of 27 IPF patients, three had a family history of idiopathic interstitial pneumonia (familial IPF) and 24 did not (sporadic IPF). Pathogenic/likely-pathogenic variants (according to American College of Medical Genetics criteria) in TCG were found in three individuals (11.1%) of the whole cohort; specifically, they were present in 2 out of 24 (8.3%) of the sporadic and in 1 out of 3 (33.3%) of the patients with familial IPF. Our results, which were established on an individual-patient level study design and in routine clinical practice (as opposed to the case-control study design), are roughly in line with the around 10% prevalence of causative TCG variants in patients with IPF.

Hematopoiesis under telomere attrition at the single-cell resolution

The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells’ self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells’ skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance.

Dynamic of Telomeric Parameters in Relapsing or Refractory (R/R) Chronic Lymphocytic Leukemia (CLL), an Analysis of the Filo ICLL001 Bomp Trial

Dynamic of Telomeric Parameters in Relapsing or Refractory (R/R) Chronic Lymphocytic Leukemia (CLL), an Analysis of the Filo ICLL001 Bomp Trial

Telomere Elongation and Clinical Improvement in Telomeropathy Patients: A Prospective Clinical Trial of Nandrolone in Telomeropathies

Telomeres are a complex of hexameric repetitive DNA sequences at the end of linear chromosomes. Telomeres erode during mitosis, and this process is accelerated in patients carrying germline pathogenic mutations in telomere-biology genes (Calado and Young, NEJM 2009). In vitro, the exposition to androgens stimulates TERT transcription and telomerase activity of lymphocytes and bone marrow CD34+ cells (Calado et al. Blood 2009). In a phase 1-2 prospective trial, the use of danazol led to telomere elongation in patients with telomeropathies (Townsley et al. NEJM 2016). The hematological response was observed in 79% of evaluable patients. Here, we describe a phase 1-2 single-center prospective study assessing the safety and the effect of the male hormone nandrolone decanoate on telomere erosion in patients with telomere diseases (ClinicalTrials.gov Identifier: NCT02055456). Entry criteria comprised patients older than two-year-old with age-adjusted mean telomere length below the 1st percentile ± identified mutations in telomerase complex genes, associated with at least one cytopenia and/or diagnosis of idiopathic pulmonary fibrosis (IPF). Patients were treated with nandrolone decanoate intramuscular at a dose of 5 mg/kg every 14 days for 24 months. The primary efficacy end point was a 20% decrease in the annual rate of telomere erosion, and the incidence of toxic effects was the primary safety end point. Secondary endpoints were hematologic response and pulmonary response. Flow-FISH was used to determine the telomere length of peripheral blood leukocytes. From May 2014 to October 2017, 20 consecutive patients were eligible for participation, and 17 were enrolled. The median age was 36 years (range, 4 - 59 years), and five patients were female. In all but one patient germline pathogenic mutations were identified (TERT, 7; RTEL1, 4; TERC, 2; WRAP53, 1; TINF2, 1; and SAMD9, 1). Eleven patients were diagnosed with bone marrow failure (9 with moderate and one with severe AA, and one with myelodysplasia). Four patients were diagnosed with IPF, and 2 patients had both moderate AA and IPF. One patient diagnosed with IPF had received an allogeneic bone marrow transplant before enrollment. Four patients had additional hepatic involvement, and 5 displayed cutaneous features of dyskeratosis congenita. The most common adverse events associated with drug were elevations in liver enzyme levels in 88%, acne in 59%, and virilization in 59%. Severe adverse events possibly related to drug occurred in six instances. Dose reductions were necessary in 5 patients due to severe or moderate adverse events. Seven patients withdrew from the study before 2 years: two patients discontinued therapy due to grade 3 adverse events (depression and acne); one patient sought alternative therapies; and four patients died during the study period, two due progressive IPF and two due intracranial hemorrhage. Out the 17 patients enrolled, a total of 15 reached 12 months of treatment and 13 were evaluable (one patient was transplanted and telomere length not evaluated; and for one patient the telomere sample was missing). Ten patients met the primary efficacy end point, showing telomere elongation. As of July 2019, nine patients reached 24 months of treatment and telomere length measured for seven patients at this time-point; all of them met end point criteria and showed telomere elongation at two years. The average increase in telomere length at 12 months was 1,119 bp (95% CI, 180-2,059 bp; P<0.04), and 1,257 bp (95% CI, 675-1,839 bp; P<0.02) at 24 months. The hematologic response rate was 60% at 3 months and 80% at 6 months. Only one patient relapsed during nandrolone use. Five of 8 transfusion-dependent patients became transfusion-independent, and one patient showed a reduction in transfusion requirements >50%. The pulmonary response rate was observed in 2 of 6 patients who had a pulmonary disease at baseline, and one patient eventually became oxygen-independent. In our study, nandrolone decanoate treatment was safe and led to telomere elongation in patients with telomeropathies. Nandrolone also improved hematologic function in patients with marrow failure. Pulmonary function remained stable or ameliorated in two-thirds of patients with pulmonary involvement. Known nandrolone side effects were common, especially liver toxicity and virilization. OffLabel Disclosure: We want to determine if treatment with male hormone nandrolone decante is safe and improves hematologic response as first-line treatment in patients with telomere disease.

Aplastic anemia: Etiology, molecular pathogenesis, and emerging concepts.

Aplastic anemia (AA) is rare disorder of bone marrow failure which if severe and not appropriately treated is highly fatal. AA is characterized by morphologic marrow features, namely hypocellularity, and resultant peripheral cytopenias. The molecular pathogenesis of AA is not fully understood, and a uniform process may not be the culprit across all cases. An antigen-driven and likely autoimmune dysregulated T-cell homeostasis is implicated in the hematopoietic stem cell injury which ultimately founds the pathologic features of the disease. Defective telomerase function and repair may also play a role in some cases as evidenced by recurring mutations in related telomerase complex genes such as TERT and TERC. In addition, recurring mutations in BCOR/BCORL, PIGA, DNMT3A, and ASXL1 as well as cytogenetic abnormalities, namely monosomy 7, trisomy 8, and uniparental disomy of the 6p arm seem to be intimately related to AA pathogenesis. The increased incidence of late clonal disease has also provided clues to accurately describe plausible predispositions to the development of AA. The emergence of newer genomic sequencing and other techniques is incrementally improving the understanding of the pathogenic mechanisms of AA, the detection of the disease, and ultimately offers the potential to improve patient outcomes. In this comprehensive review, we discuss the current understanding of the immunobiology, molecular pathogenesis, and future directions of such for AA.

Abstract 3687: Dot1l inhibition selectively impairs androgen receptor dependent prostate cancer growth through loss of telomere integrity

Abstract The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and this histone mark is associated with activation of gene expression. DOT1L has a well-known role in MLL fusion leukemogenesis, however, its role in prostate cancer is yet to be elucidated. Here we show that DOT1L is overexpressed in many solid tumors including prostate cancer and is associated with a poor outcome. Genetic and chemical targeting of DOT1L showed a preferential loss in cell viability and colony formation in androgen receptor (AR)-signaling competent prostate cancer cells, including castration-resistant and enzalutamide-resistant cells. DOT1L inhibition reduced AR levels and upregulated enzymes involved in testosterone catabolism but only affected the expression of a subset of AR target genes. More importantly, loss of DOT1L selectively impaired telomere integrity by promoting telomere uncapping and shortening in AR-signaling competent cells. DOT1L inhibition also reduced the expression of telomerase and Shelterin complex genes including TRF2 and POT1. Recruitment of AR to telomeres and K79 methylation at sub-telomeric regions was also diminished. Consequently, loss of DOT1L leads to induction of Telomere dysfunction induced foci (TIF) and activates a cellular senescence program in these cells. Hence, these results provide compelling evidence for DOT1L inhibition as a novel therapeutic approach in advanced prostate cancers. Citation Format: Rajita Vatapalli, Yara Rodriguez, Changsheng Zhao, Vinay Sagar, Jonathan Anker, Sahithi Pamarthi, Kenji Unno, Benedito Carneiro, Debabrata Chakravarti, Sarki Abdulkadir. Dot1l inhibition selectively impairs androgen receptor dependent prostate cancer growth through loss of telomere integrity [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 3687.

Fanconi Anemia germline variants as susceptibility factors in aplastic anemia, MDS and AML.

Using next generation sequencing we have systematically analyzed a large cohort of 489 patients with bone marrow failure (BMF), including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), aplastic anemia (AA), and related conditions for the presence of germline (GL) alterations in Fanconi Anemia (FA) and telomerase genes. We have detected an increased frequency of heterozygous FA gene mutations in MDS and to lesser degree in AML suggesting that the presence of one normal allele may not be completely protective and indeed heterozygous FA lesions may have a long latency period before hematologic manifestation. In contrast, GL telomerase gene mutations were not associated with increased disease risk. When compared to large control cohorts, we have not detected an increased frequency of damaging variants among telomerase complex genes, including those previously believed to be involved in the pathogenesis of AA. Our results may suggest that while low penetrance and delayed disease onset can confound identification of genetic predisposition factors, GL FA alterations can be also associated with MDS.

Normal and Tumour Tissue mRNA Expressions of Telomerase Complex Genes in Several Types of Cancer.

Aims:To investigate the changes in mRNA expression levels of telomerase-related significant proteins in several types of cancer.Methods:Human telomerase reverse transcriptase, pontin, reptin and dyskerin expressions were measured in normal and tumour tissues obtained from 26 patients with colorectal, breast and gastric cancers, using the real-time reverse transcriptase-polymerase chain reaction method.Results:For all patients, no significant difference was found in mRNA expressions of human telomerase reverse transcriptase and dyskerin (p>0.05), although their levels in tumour tissues were found to be higher than in normal tissues. However, pontin and reptin mRNA expressions were significantly higher in tumour tissues than in normal tissues (p<0.01). While human telomerase reverse transcriptase showed a high correlation with only pontin (p<0.001) in normal tissues, high positive correlations were observed between human telomerase reverse transcriptase with pontin (p<0.005), reptin (p<0.01) and dyskerin (p<0.01) in tumour tissues.Conclusion:The increased mRNA expressions of all four genes in tumour tissues may suggest a role in cancer development. Correlations of pontin, reptin and dyskerin with human telomerase reverse transcriptase support the hypotheses describing their roles in telomerase complexes.

Brief Report: Differential Effects of Tenofovir, Abacavir, Emtricitabine, and Darunavir on Telomerase Activity In Vitro.

In vitro, tenofovir and abacavir induced a significant dose-dependent inhibition of telomerase activity at therapeutic concentrations in peripheral blood mononuclear cells of healthy subjects. Median inhibition of telomerase activity by tenofovir at 0.5 and 1 μM was 29% [Interquartile range (IQR) 29%-34%, P = 0.042] and 28% (IQR 28%-41%, P = 0.042), respectively. Abacavir inhibition was 12% (IQR 9%-13%, P = 0.043) at 3 μM and 14% (IQR 10%-29%, P = 0.043) at 10 μM. Tenofovir and abacavir did not change human telomerase reverse transcriptase (hTERT) levels or mRNA levels of other telomerase complex genes. Exposure to emtricitabine or darunavir did not affect telomerase activity, hTERT protein levels, or mRNA levels of telomerase/shelterin genes.

Deleterious Germline Mutations in Telomere Maintenance Genes Identified in a Subset of Patients with Myelodysplastic Syndrome and Idiopathic Pulmonary Fibrosis

The myelodysplastic syndromes (MDS) are the most common cause of bone marrow failure in adults, with an incidence of 40,000 cases per year. Next-generation sequencing of candidate genes has led to major advances in the description of the genetic landscape of MDS, identifying recurrently mutated genes and cellular pathways involved in disease pathogenesis. However, use of targeted panels indicates more comprehensive, unbiased sequencing techniques may yet identify additional recurrently mutated genes or cellular pathways important in MDS. Diseases caused by defects in telomere maintenance (telomeropathies) are variable in clinical and genetic presentation but often involve bone marrow failure. We hypothesized that acquired mutations in telomerase maintenance genes may be a recurrent event in MDS. This is significant as identification of recurrent somatic mutations in telomerase maintenance genes would provide further insight into MDS pathogenesis and identify a potential druggable pathway for MDS patients, as novel agents targeting the telomerase pathway are currently in clinical development. First, we identified three adults who presented in middle age with MDS and idiopathic pulmonary fibrosis (IPF), two of which also had a family history of IPF. All three patients had shortened telomeres (<1st centile), but no mutations detected in the telomerase pathway genes TERC, TERT, TINF2, or DKC2. In order to identify inherited and/or acquired genetic mutations affecting telomere biology, we performed exome sequencing on paired whole bone marrow (tumor) and skin (comparator/germline) samples from each patient. In patient 1 (see Table), we identified a heterozygous, inherited frameshift variant in RTEL1, (R132fs, not previously reported), a helicase required for telomere maintenance that has not been reported in MDS. Compound heterozygous mutations in RTEL1 have been implicated in Hoyeral-Hreiderson syndrome, and recent work showed ~5% of patients with familial IPF have heterozygous germline RTEL1 mutations (Cogan J.D. et al., Am J Resp Crit Care Med, 2015 and Stuart B.D. et al., Nat Genet, 2015). In patient 2, we identified a heterozygous inherited missense variant in ACD (V484I), a component of the telomerase shelterin complex also implicated in Hoyeral-Hreiderson syndrome that is not a common population variant (absent from 1000 genomes, the Exome Sequencing Project, and dbSNP). Analysis of constitutional alterations using the exome data from patient 3, did not identify an obvious causative variant; thus, whole genome sequencing is ongoing. These data show that inherited telomerase maintenance gene variants are found in patients with MDS and IPF. Next, to determine if telomerase complex genes were recurrently mutated in an unselected, de novo MDS population, we performed targeted next-generation sequencing of telomerase maintenance genes (15 genes) combined with sequencing of 335 myeloid malignancy-associated genes in 96 paired tumor/skin samples from MDS patients. Somatic variants were identified in the tumor compared to the paired skin using the Genome Modeling System, as previously reported (Griffith M. et al., PLoS Comput Biol. 2015). As expected, somatic analysis identified mutations in recurrently mutated MDS genes such as U2AF1, ASXL1, TET2, TP53 and others. A single, likely deleterious somatic inversion involving exon 9 of RTEL1 was identified; however, somatic analysis for telomere maintenance gene mutations was otherwise unremarkable. Evaluation of these genes for germline variants identified rare (ExAC allele frequency < 0.0007), apparently heterozygous, in-frame and frame-shift deletion variants affecting CTC1 (n=2), NOP10 (n=1) and TERT (n=1) in 4 individuals. The two CTC1 frame shift variants have been previously described in multiple patients with pediatric telomeropathies resulting from compound heterozygous mutations in CTC1. Additional heterozygous missense mutations previously associated with aplastic anemia were identified in 2 other individuals but are of uncertain significance. Collectively, these results indicate that germline mutations in telomere maintenance genes, other than TERT and TERC, underlie genetic predisposition for MDS and IPF in a subset of patients, which has not been previously appreciated, but are rare events in de novo MDS. [Display omitted] DisclosuresJacoby:Sunesis: Research Funding; Quintiles: Consultancy; Celgene: Speakers Bureau.

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.

Mutational analysis of telomere complex genes in Indian population with acquired aplastic anemia

BackgroundAcquired aplastic anemia (AAA) is rare disorders caused due to the profound or almost complete bone marrow failure. It is a life threatening hematopoietic stem cells disorder, which is characterized by pancytopenia or complete loss of blood-forming cells. The aim of the present study is to screen for the mutations in telomerase complex genes, and to establish a molecular and hematological profile of Indian sub population. MethodologyWe have conducted a case control study of total 70 participants; 50 patients, who fulfilled the blood count and bone marrow criteria of the International agranulocytosis & AAA, and 20 healthy controls. These samples were selected from hematology clinics at Jaipur, India, during the period of two years (January 2012–December 2013). We screened four telomere complex genes; TERT, DKC1, NOP10 and NHP2 of mutations at single base pair in sampled blood and bone marrows. We have predicated the consequences of mutations on protein structure using 3D multilevel modeling protein structure software Phyre2, PolyPhen2 and YASARA. ResultsThe hematological and molecular basis of acquired aplastic anemia was investigated in 50 anemia patients and 20 healthy controls. AAA patients showed hematologic abnormalities (macrocytic anemia, thrombocytopenia, & granulocytopenia) in peripheral blood and severe hypoplastic bone marrows. Screening of telomere complex genes TERT, DKC1, NOP10 and NHP2 in AAA patients and controls revealed; novel and reported mutations in TERT and DKC1, whereas, no pathogenic mutations were observed in NOP10 and NHP2 genes. In TERT gene, one non-synonymous mutation (Chr5: 1287,825 C→T; Arg979Trp) was identified in exon 12 and two heterozygous non-synonymous mutations (Chr X: 153,994,542 T→K; Val105Gly & Chr X: 153,994,591 T→K; Ser121Arg) were found in exon 5 of DKC1 gene. To determine and visualize the possible effect of TERT and DKC1 mutations on protein structure YASARA with FoldX functionality has been used and many structural consequences were found that might destabilize the protein. Predicated structural consequences may destabilize the TERT and DKC1 proteins ultimately causing blood disorders.. ConclusionThe present study indicates the mutation spectrum in the genes implicated in AAA, i.e. TERT, DKC1, NOP10 and NHP2 on small case-control group in an Indian sub population.

Telomere Elongation and Hematologic Improvement in Humans Treated with Androgens: A Prospective Clinical Trial of Danazol in Telomeropathies

Telomeres, the terminal complex of hexameric repeats and shelterin protein of linear chromosomes, shorten with every mitosis. Telomere attrition is accelerated in patients with mutations in telomerase complex genes (Calado and Young, NEJM 2009) and with replicative stress, as in chronic bone marrow failure. Historically, male hormones were effective in some patients with aplastic anemia (AA), and case reports and retrospective observations have suggested hematologic improvement in patients with telomeropathies treated with male hormones. Exposure of normal lymphocytes and CD34+ cells to androgens increased telomerase activity in vitro, and in cells from individuals carrying loss-of-function TERT mutations to normal levels (Calado et al. Blood 2009). We have conducted a phase I/II single-center trial (www.clinicaltrials.gov NCT01441037) assessing the safety and the effect of male hormones on telomere attrition in patients with telomere disease. Entry criteria included age-adjusted mean telomere content ≤1%ile, ± identified mutations in telomerase complex genes, and low blood counts (hemoglobin <9.5g/dL, platelets <30,000/uL, or neutrophils <1,000/uL) and/or pulmonary fibrosis. Danazol, 800 mg/day, was administered for 2 years. Primary protocol objectives were safety and activity of danazol in slowing telomere attrition. Secondary endpoints were hematologic response at 3 and 6 months (increase in hemoglobin >1.5 g/dL or platelets >20,000/uL or neutrophils >500/uL). Twenty seven patients were enrolled, accrual commencing August 2011. Most patients had moderate (n=20) or severe (n=4) AA, one had myelodysplasia, and two pulmonary fibrosis. Median age was 41 years (range 17-66); 15 patients were females. There was only one severe adverse event possibly related to drug. Frequent reported symptoms were muscle cramping with dehydration and exacerbation of headaches. Changes in serum lipid profiles were observed in all patients, with increased serum LDL and decreased HDL. Severe elevation in liver enzymes was not observed. One death occurred on study, not treatment related (pneumonia in a pulmonary fibrosis case). Mean telomere content of leukocytes at enrollment was compared with mean telomere content at 6, 12, and 24 months on drug as well as available samples before starting danazol. Telomere attrition prior to protocol entry, determined by q-pcr, was estimated at loss of 227 bp/year (95% CI, 58-368bp; p=0.009). Androgen administration appeared to elongate telomeres: the average increase in telomere length at 6 months was 205 bp (95% CI, 82-329 bp; p=0.002), at 12 months 441 bp (95% CI, 263-620 bp; p=0.0001), and at 24 months 347 bp (95% CI, 87-607 bp; p=0.01). A similar trend of increase in mean telomere content with danazol was confirmed in flow-sorted lymphocytes. Hematologic response rate, as defined by protocol, was 67% at 3 months and 60% at 6 months. Nine of eleven patients who required RBCs became transfusion-independent; two of them also became platelet transfusion independent. Liver cirrhosis was present in 6 patients at enrollment; worsening of liver disease in one occurred with continued alcohol abuse. To date 8 patients have completed two years of danazol, all of them responders; 10 patients remain on danazol, and 9 patients stopped drug prior to 2 years. Blood counts in all patients have been stable with drug discontinuation, with median follow up of 258 days (range 31-335). In conclusion, male hormones are safe and efficacious in patients with inherited bone marrow failure associated with telomeropathies, producing clinically meaningful hematologic improvement. Increased mean telomere content in patients, suggests that in vitro demonstration of up-regulation by sex hormones of TERT and of telomerase enzymatic activity is the mechanism of action of androgens in vivo. To our knowledge, this is the first successful prospective effort to favorably modulate telomere length by pharmacologic intervention in humans. Sex hormones may be useful in other conditions of accelerated telomere attrition, as for example after chemotherapy, and other drugs and small molecules may be usefully screened for their effects on telomerase in vitro. DisclosuresOff Label Use: we want to determine if treatment with male hormone danazol is safe and improves hematologic response as first-line treatment in patients with AA and telomere disease(www.clinicaltrials.gov NCT01441037)..

Telomere Length Recovery Strongly Predicts Overall Survival in Acute Promyelocytic Leukemia

Telomeres are the ends of linear chromosomes composed of tandem hexameric nucleotide repeats, maintained by a telomerase enzyme complex, made of reverse transcriptase catalytic subunit (TERT) and an RNA template (TERC). Present in very specific cells, such as embryonic and adult stem cells, the telomerase complex functions to elongate and slow the attrition of telomeric repeats associated with cellular division during replicative cellular age, as well as conferring stability to the ends of the chromosomes. In humans, both telomere length (TL) and loss-of-function mutations in the telomerase complex genes have been linked to malignant transformation and alteration of normal hematopoiesis. Though very short telomeres and germline mutations in TERT and TERC are associated with acute myeloid leukemia (Calado et al. PNAS 2009), the association between TL or telomerase complex integrity and outcome in patients (pts) with acute promyelocytic leukemia (APL) has not been thoroughly analyzed.We present the largest analysis to date of mean telomere content, as a function of TL, and germline mutations in TERT and TERC in peripheral blood leukemic promyelocytes or total mononuclear cells (remission) from 164 APL pts at diagnosis, complete remission (CR) and relapse, enrolled in the intergroup trials, E2491 and C9710. The goal was to evaluate the correlation of TL in APL blasts at diagnosis and at relapse, in CR in normal total mononuclear cells, as well as delta TL (the difference between TL at diagnosis and CR) with clinical and laboratory parameters, including disease risk status by presenting white blood cell count (WBC ≤ or > 10,000/μl), early mortality, achievement of CR and overall survival (OS). Peripheral blood leukocytes from healthy subjects (ages 0-90 yrs) were collected and analyzed as controls. TL was measured by real-time quantitative PCR, and was dichotomized into short or long by the third quartile. Patient demographics and outcomes were compared using Fisher’s exact test, the Kruskal-Wallis test, and the Wilcoxon signed rank test. To evaluate the effect of TL on clinical outcomes, the Cochran-Mantel-Haenszel statistic was used in the association test on early mortality and CR. OS were compared using stratified log-rank tests. Hazard ratios were computed using stratified univariate and multivariate Cox models.Median TL at presentation was significantly shorter in the APL than in the control healthy volunteer group (5.1 vs. 7.8 kb, p<0.0001). At CR, the median increase in TL was 2.0 kb (p<0.0001 against 0). There was a statistically significant association between TL at diagnosis and the WBC risk group status (p=0.001). There was no statistically significant difference in pretreatment characteristics (age, sex, ECOG performance status (PS), and risk status) and early mortality (p=0.40) across studies/regimens. However, TL at presentation (p=0.004) and CR (p=0.0003) was shorter in study C9710 than study E2491, though there was no difference in delta TL (p=0.21). TL at diagnosis was not prognostic of achievement of CR (p=0.78) or early mortality (p=0.19). However, delta TL was identified as a powerful predictor for OS (p=0.006), which remained true after adjusting for WBC risk status (p=0.009). In patients who achieved a CR, results from Cox regression with the forward selection criterion demonstrated that delta TL was more potent in predicting OS than the WBC risk status, TL at presentation, age, sex, or ECOG PS. TL at CR (p=0.96) and delta TL (p=0.31) did not predict for likelihood of relapse. Relapsed pts had significantly shorter median OS (p<0.0001) compared to those who did not relapse (2.2 yrs vs. OS not reached). We did not find mutations in the TERT or TERC genes apart from codon 279 and 1062 polymorphisms, which were identified at a frequency similar to that found in normal healthy volunteers.In the largest study of APL pts analyzed to date, we demonstrate that APL blasts consistently harbor shorter TL at presentation compared to healthy people, and that TL at presentation correlates with higher risk disease. TL significantly recovers by the time of disease remission, and among those pts who achieve CR, delta TL is the most powerful predictor of OS. These findings warrant confirmation in a prospective fashion, and if corroborated, may offer TL measurement as a simple and inexpensive parameter in disease risk stratification and as meaningful predictor of OS in pts with APL. DisclosuresDumitriu:GSK: Research Funding. Stein:Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Larson:Novartis: Consultancy, Research Funding.

Abstract 550: Genomic instability and telomere characteristics as predictive biomarkers of therapeutic response in triple-negative breast cancer

Abstract Background. Triple negative breast cancers (TNBC) represent about 15 % of invasive breast carcinomas. These tumors are identified in clinical practice according to their “triple negative” immunophenotype (estrogen receptor, progesterone receptor and HER2 negative). TNBC show aggressive behavior and an unusual sarcoma-like pattern of metastasis. Identification of predictive biomarkers is critical to optimal therapeutic management of these tumors. TNBC are characterized by increased genomic instability. These tumors present complex pattern of numerous low-amplitude genomic gains and losses as well as shorter telomeres. Aim. Identification of predictive biomarkers of response to neoadjuvant chemotherapy in TNBC by evaluating telomeric parameters and genomic instability. Methods. We studied 38 TNBC patients treated with neoadjuvant chemotherapy according to the TVA protocol (FEC 100, docetaxel, panitumumab). DNA and RNA were extracted from snap-frozen tumor samples obtained before neoadjuvant therapy. The pathological response to treatment was evaluated according to Chevallier's and Sataloff's classifications and correlated with aCGH profiles (8x60K, Agilent) and telomere characteristics, including telomere length (qPCR) and expression (qRT-PCR) of telomerase (hTERT) and shelterin complex genes (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2). Results. Telomere shortening and high hTERT expression were found in more aggressive tumors according to TNM classification (P&amp;lt; 0.04), and hTERT expression was also highly correlated with an increased tumor size (P= 0.0058). Short telomeres were strongly associated with incomplete histological response to neoadjuvant therapy according to Chevallier's (P= 0.0004) and Sataloff's classifications, (P= 0.0007). hTERT expression was significantly increased in cases with incomplete response (P&amp;lt; 0.04). Among shelterin complex genes, TRF2 and TPP1 overexpression was found in more aggressive TNBC (according to TNM classification and SBR grading, P&amp;lt; 0.05), but there was no correlation with pathological response. aCGH data indicated that genomic instability in non-responders was mainly represented by genomic gains. In particular, cases with incomplete therapeutic responses showed significantly larger size of gains than cases with complete responses. (P= 0.0089). Short telomeres were also associated with larger gains of genomic material (P= 0.0067). Of note, telomere shortening has been previously described as one of the mechanisms of genomic gains and amplifications. Conclusions. Telomere shortening and hTERT overexpression were observed in resistant TNBC. Genomic profile of non-responding tumors was characterized by larger genomic gains. Telomere length, hTERT expression and genomic instability profiles may represent predictive biomarkers of response to neoadjuvant chemotherapy in TNBC. Citation Format: Mathilde Gay-bellile, Nina Radosevic-Robin, Eleonore Eymard-Pierre, Fabrice Kwiatkowski, Marie-Mélanie Dauplat, Maud Privat, Catherine Abrial, Patricia Combes, Gwendoline Soler, Yves-Jean Bignon, Jean-Marc Nabholtz, Philippe Vago, Frédérique Penault-Llorca, Andreï Tchirkov. Genomic instability and telomere characteristics as predictive biomarkers of therapeutic response in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2014-550

Chapter Eight - Telomeres in Lung Diseases

Idiopathic pulmonary fibrosis is a progressive and fatal interstitial lung disease leading to respiratory failure. Mutations in telomerase complex genes (TERT or TERC) and short telomeres are genetic risk factors for the development of familial or sporadic idiopathic pulmonary fibrosis. Up to 15% of familial cases and approximately 5% of sporadic cases carry a heterozygous mutation in one of the genes, and patients' cells retain approximately 50% of telomerase activity. Pulmonary fibrosis also is a manifestation of dyskeratosis congenita, an inherited bone marrow failure syndrome caused by telomere dysfunction. Short telomeres even in the absence of telomerase mutations are a feature of most patients with idiopathic pulmonary fibrosis. Telomerase mutations also have been linked to pulmonary fibrosis and emphysema syndrome. Although short telomeres have been clearly linked to idiopathic pulmonary fibrosis, the mechanisms of disease are still unclear.

Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms

In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion.Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1).Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11).In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases. Disclosures:Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

Telomerase gene mutation screening and telomere overhang detection in Chinese patients with acute myeloid leukemia

Loss-of-function mutations in telomerase complex genes reduce telomerase activity, and can clinically manifest as bone marrow failure disease, which predisposes to acute myeloid leukemia (AML). Telomerase dysfunction also leads to short telomeric overhang, which is a crucial telomeric structural component, and potentially results in chromosome instability. We screened variants in telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) genes, and investigated the 3’-overhang length in bone marrow samples from 72 Chinese patients with AML (61 de novo, 11 secondary, excluding M3), aged 13–77. Cytogenetics, disease severity and short-term survival were evaluated. Three TERT mutations (n896G>A, n1079C>G and n1451G>C) were identified. Mutation carriers had short overhangs and a poor prognosis. We found that overhang lengths were much shorter in AML compared to normal controls (p < 0.001). Short overhangs were related to a high percentage of karyotype abnormalities and poor prognosis (73.8% in short overhang group vs. 30% in normal group, p=0.001). Multivariant analysis showed that overhang length, age and unfavorable chromosome abnormalities served as independent prognostic markers in AML (Cox regression, p=0.001). These data raise the possibility that short overhang length may predict poor prognosis in patients with AML. These findings would have to be confirmed in large, prospective studies.