Evidence of Polyclonal Hematopoiesis in a Large Proportion of Patients with Low-Risk Myelodysplastic Syndrome.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2364-2364
Author(s):  
Luca Malcovati ◽  
Matteo G. Della Porta ◽  
Anna Galli ◽  
Sabrina Boggi ◽  
Lucia Malabarba ◽  
...  
Keyword(s):  
Telomere Length ◽  
Peripheral Blood ◽  
Low Risk ◽  
Refractory Anemia ◽  
Monocytic Differentiation ◽  
Telomere Shortening ◽  
Clonal Population ◽  
Clonal Nature ◽  
Methylation Patterns

Abstract Myelodysplastic syndromes (MDS) are usually defined as clonal proliferations of pluripotent stem cells, which retain their capacity to differentiate but do so in an inefficient manner, so that mature cells in the peripheral blood are variably altered and reduced. Various approaches can be used to prove the existence of a clonal population of cells. Analysis of X inactivation-dependent methylation patterns in double heterozygous females has been used in MDS, and this approach showed clonal hematopoietic progenitors capable of granulocytic-monocytic differentiation in most cases. However, in a recent study (Blood2003;102:1211–6) clonality, defined as a clonal population accounting for 35% or more of total granulocytes, was confirmed in only one third of patients with refractory anemia. We studied X-chromosome inactivation patterns (XCIP) in 50 consecutive female patients with MDS. These included 18 RA, 6 RARS, 12 RCMD, 4 RCMD-RS, 2 MDS with del(5q), 4 RAEB-1 and 4 RAEB-2. The XCIP was established by analysis of the IDS gene expression and of DNA methylation at the HUMARA and PGK loci. In order to assess the clonality status of single hematopoietic lineages, the relative expression of HUMARA alleles in reticulocytes, granulocytes and platelets was evaluated by RT-PCR. Finally, telomere length, as a major determinant of the replicative lifespan of hematopoietic cells, was analyzed on peripheral blood granulocytes by flow-FISH (Dako Telomere PNA kit/FITC). XCIP and telomere length analyses were performed at the diagnosis (21 cases) or during the follow-up (29 cases, median time from diagnosis 10 months, range 1–230). XCIP were informative in 43 patients: 20 displayed clonal or ambiguous XCIP (46.5%), while 23 showed polyclonal XCIP (53.5%). Polyclonal XCIP were found in 20 of 36 informative MDS patients without excess blasts (55.5%), and 3 of 7 (42.9%) with excess blasts. Among MDS patients with polyclonal hematopoiesis, clonal cytogenetic abnormalities were found in 9 out of 17 informative cases, all of them showing a chimeric karyotype with normal and abnormal metaphases. Telomere length was significantly shorter in patients with monoclonal patterns than in patients with polyclonal hematopoiesis (P=.01). A preliminary study of XCIP by expression of HUMARA alleles showed that 6 patients with polyclonal XCIP on PMN had clonal XCIP on both reticulocytes and platelets. In brief, these data provide evidence that more than 50% of patients with low risk MDS had some polyclonal hematopoiesis. However, detection of karyotypic abnormalities in these patients confirmed the clonal nature of the disease, the myelodysplastic clones coexisting with residual normal hematopoiesis. Preliminary data indicate that the myelodysplastic clone may originate in a common erythroid-megakaryocytic progenitor. Finally, the telomere data suggest that monoclonal hematopoiesis is associated with telomere shortening.

Accelerated Telomere Shortening Identifies a Subgroup of Patients with Myelodysplastic Syndrome and Isolated 5q Minus Deletion with a Higher Probability of Response to Lenalidomide Treatment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3809-3809
Author(s):  
Fabian Beier ◽  
Ralph P Schneider ◽  
Guntram Buesche ◽  
Jens Panse ◽  
Ulrich Germing ◽  
...  
Keyword(s):  
Stem Cell ◽  
Telomere Length ◽  
Peripheral Blood ◽  
Preliminary Analysis ◽  
Research Funding ◽  
Disease Duration ◽  
Treatment Initiation ◽  
Telomere Shortening ◽  
Length Analysis

Abstract Abstract 3809 Introduction: Myelodysplastic syndromes (MDS) are heterogeneous clonal stem cell disorders characterized by ineffective hematopoiesis and an increased risk for leukemic transformation. Lenalidomide (LEN) was found to be an effective treatment particularly in a subset of MDS patients with isolated 5q minus deletion (del5q). A high proportion of these patients show erythroid response with transfusion independence and even complete cytogenetic response (CCR). However, particularly in patients not responding to LEN, disease progression to acute leukemia is observed. Accelerated telomere length shortening is regularly observed in hematopoietic stem cell disorders with increased stem cell turnover and/or altered telomere maintenance. Dysfunctional telomeres have been found to play an important role in the development of chromosomal instability and malignant transformation. The aim of this study was to investigate telomere length as a potential predictive biomarker in MDS del5q patients treated with LEN with regard to disease progression and treatment response. Methods and Patients: Telomere length (TL) was determined using confocal Q-FISH on paraffin-embedded BM biopsies of 54 MDS patients enrolled in the LEMON5 study (NCT01081431). Criteria for study inclusion were isolated del5q, transfusion dependence of at least one unit per 8 weeks and IPSS low risk and intermediate-1. TL was analyzed in a blinded fashion on specimen obtained before treatment initiation with LEN, control biopsies of 11 patients with newly diagnosed Morbus Hodgkin without BM affection were used for age-adaption of TL. At the time of this preliminary analysis, the study is ongoing, initial clinical data were available for 94% (51/54) and detailed follow up data for 63% (34/54) of the patients with a median follow up of 22 months. Mean age of the MDS patients was 68.6 years (range 40–87) and average disease duration before enrolment was 2.9 years. Results: We found that TL of the 54 MDS patients was significantly shorter compared to the age-adjusted TL (−0.57 kb, p=0.02, n=54). Interestingly, analysis according to the respective IPSS showed significant shorter telomeres in the low risk group (−0.91 kb, p=0.04, n=27) than in the intermediate-1 group (−0.55 kb, p=0.24, n=19). Focusing on the peripheral blood counts, cut-off values were set according to the distribution pattern representing the approximate median value. Patients with ANC counts <2000/μl (−0.98 kb, p=0.03, n=27), haemoglobin values <9g/dl (−0.89 kb, p=0.02, n=26) and platelets counts <300/nl (−0.87 kb, p=0.01, n=27) had significantly shortened telomeres compared to the age-adjusted controls. In contrast, patients with ANC counts >2000/μl (0.06 kb, p=0.9, n=20), haemoglobin >9g/dl (−0.23 kb, p=0.23, n=25) and platelet counts >300/nl (−0.07 kb, p=0.58, n=24) did not differ from the age-adjusted TL. Furthermore, patients with a history of more than 2 years of MDS had significantly shortened age-adjusted telomere length (−0.94 kb, p=0.02, n=26), but that was not the case in patients with a short disease duration (<2 years; −0.32 kb, p=0.36, n=28). Interestingly, with regards to response to LEN, patients later achieving a CCR under LEN had significantly shortened TL at treatment initiation (−1.47 kb, n=14, p=0.005) whereas this was not the case in patients with no response, relapse or progressive disease during follow-up (−0.23 kb, n=20, p=0.62). Furthermore, correlation with treatment duration showed that patients receiving more than 12 cycles of LEN (in which 93%, i.e. 13/14 patients were responding) had significantly shorter telomeres before start of LEN (−1.41 kb, n=17, p=0.02) compared to the group of patients with less than 12 cycles (0.22 kb, n=14) in which 41%, i.e. 7/17 patients were responding. Conclusions: Patients with MDS and isolated del5q undergo significant telomere shortening. Using telomere length analysis on paraffin-embedded BM biopsies using confocal Q-FISH, we were able to identify a subgroup of patients with lower peripheral blood counts and accelerated TL shortening that seemed to preferentially profit from LEN treatment. In summary and pending further confirmation with longer follow up of this preliminary analysis within the ongoing LeMon5 study, we conclude that telomere length analysis may identify a distinct biological subentity of MDS del5q patients more likely to benefit from treatment with LEN. Disclosures: Germing: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brümmendorf:Celgene: Research Funding.

scholarly journals Rapid Engraftment Without Significant Graft-Versus-Host Disease After Allogeneic Transplantation of CD34+ Selected Cells From Peripheral Blood

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3967-3973 ◽  
Author(s):  
Alvaro Urbano-Ispizua ◽  
Ciril Rozman ◽  
Carmen Martı́nez ◽  
Pedro Marı́n ◽  
Javier Briones ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Graft Versus Host Disease ◽  
Peripheral Blood ◽  
Allogeneic Transplantation ◽  
Median Number ◽  
Refractory Anemia ◽  
Host Disease ◽  
Graft Versus Host ◽  
Cd34 Cells

Abstract We have prospectively evaluated the feasibility and results of the biotin-avidin immunoadsorption method (Ceprate SC system) for a phase I/II study of T-cell depletion of granulocyte colony-stimulating factor (G-CSF ) mobilized peripheral blood progenitor cells (PBPC) for allogeneic transplantation. Twenty consecutive patients, median age, 40 years (21 to 54) and diagnoses of chronic myeloid leukemia in chronic phase (n = 5), acute myeloblastic leukemia (n = 7), acute lymphoblastic leukemia (n = 2), chronic myelomonocytic leukemia (n = 1), refractory anemia with excess of blasts in transformation (n = 3), histiocytosis X (n = 1), and chronic lymphocytic leukemia (n = 1), were conditioned with cyclophosphamide (120 mg/kg) and total body irradiation (13 Gy; 4 fractions). HLA identical sibling donors received G-CSF at 10 μg/kg/d subcutaneously (SC); on days 5 and 6 (19 cases) and days 5 to 8 (1 case) donors underwent 10 L leukapheresis. PBPC were purified by positive selection of CD34+ cells using immunoadsorption biotin-avidin method (Ceprate SC) and were infused in the patients as the sole source of progenitor cells. No growth factors were administered posttransplant. The median recovery of CD34+ cells after the procedure was of 65%. The median number of CD34+ cells infused in the patients was 2.9 (range, 1.5 to 8.6) × 106/kg. The median number of CD3+ cells administered was 0.42 × 106/kg (range, 0.1 to 2). All patients engrafted. Neutrophil counts <500 and <1,000/μL were achieved at a median of 14 days (range, 10 to 18) and 15 days (range, 11 to 27), respectively. Likewise, platelet counts <20,000 and <50,000/μL were observed at a median of 10 days (range, 6 to 23) and 17 days (range, 12 to 130), respectively. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine plus methylprednisolone. No patient developed either grade II to IV acute or extensive chronic GVHD. After a median follow-up of 7.5 months (range, 2 to 22) three patients have relapsed, and one of them is again in hematologic and cytogenetic remission after infusion of the donor lymphocytes. Two patients died in remission: one on day +109 of pulmonary aspergillosis and the other on day +251 of metastasic relapse of a previous breast cancer. Sixteen of the 20 patients are alive in remission after a median follow-up of 7.5 months (range, 2 to 22). In conclusion, despite the small number of patients and limited follow-up, it appears that this method allows a high CD34+ cell recovery from G-CSF mobilized PBPC and is associated with rapid engraftment without significant GVHD, and with low transplant related mortality.

Clonality in Granulocytes Detected by an Improved HUMARA Assay: A Good Prognostic Marker in Bone Marrow Failure Patients Exhibiting Chromosomal Abnormalities of Indefinite Significance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3702-3702
Author(s):  
Ken Ishiyama ◽  
Chiharu Sugimori ◽  
Hirohito Yamazaki ◽  
Akiyoshi Takami ◽  
Shinji Nakao
Keyword(s):  
Bone Marrow ◽  
Chromosomal Abnormalities ◽  
Bone Marrow Failure ◽  
Bone Marrow Examination ◽  
Refractory Anemia ◽  
Clonal Population ◽  
Dividing Cells ◽  
Humara Assay

Abstract Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p<0.01). In two AA patients who respectively exhibited add(5)(q13) in 10% and +8 in 38% dividing cells, clonality was not detected and these abnormal clones became undetectable at the time of subsequent bone marrow examination. Clonality was detected in the other 2 AA patients respectively exhibiting 9q–9q+ in 40% and del(7)(q14q22) in 25% dividing cells, and in all 5 RA patients respectively exhibiting +8 in 10%, del(5)(q13q31), dup(1)(q32q12) in 90%, del(5)(q13), add(11)(q23), inv(9) in 65% and X,-X in 100% of dividing cells. None of the 50 AA patients including 2 patients with clonality and chromosomal abnormalities underwent evolution to AML during 2-year follow up while one of 28 RA patients who exhibited del(5)(q13q31) progressed to AML. The proportion of clonal granulocytes in total granulocytes estimated by the HUMARA assay remained unchanged in most patients with clonality except for the transformed one. These data indicate that the chromosomal abnormality in bone marrow dividing cells is not necessarily associated with presence of clonal granulocyte population in peripheral blood and that detection of clonality in granulcytes in bone marrow failure patients with chromosomal abnormalities of indefinite significance is useful in predicting prognosis of these patients.

Maintenance of Telomere Length in Peripheral Blood CD4+CD25+ Regulatory T-Cells of Cancer Patients Despite Active Proliferation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3309-3309
Author(s):  
Dominik Wolf ◽  
Holger Rumpold ◽  
Christian Koppelstaetter ◽  
Guenther Gastl ◽  
Eberhard Gunsilius ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients ◽  
Peripheral Blood ◽  
Telomerase Activity ◽  
Telomere Shortening ◽  
Active Proliferation

Abstract CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the up-regulation of telomerase activity, whose regulation also remains unknown for Treg. We therefore isolated Treg and the respective CD4+CD25− control T-cell population from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17). Analysis of their content of T-cell receptor excision circles (TREC) revealed that the observed increase of Treg frequencies in peripheral blood is due to active cycling rather than to redistribution from other compartments (i.e. secondary lymphoid organs or bone-marrow), as Treg from cancer patients are characterized by a significant decrease of TREC content when compared to TREC content of Treg isolated from healthy age-matched controls. Surprisingly, despite their proven in vivo proliferation, telomere length is not further shortened in Treg from peripheral blood of cancer patients as shown by Flow-Fish, Real-Time PCR and Southern Blotting. Accodingly, telomerase activity of Treg was readily inducible in vitro by OKT3 together with IL-2. Notably, sorting of in vitro proliferating Treg using dilution of CFSE revealed a significant telomere shortening in Treg with high proliferative capacity (i.e. CFSElow fraction) under conditions of strong in vitro stimulatory growth conditions despite a high telomerase activity. Thus, under conditions of strong in vitro stimulation induction of telomerase seems to be insufficient to avoid progressive telomere shortening. In contrast, in actively proliferating peripheral blood Treg from patients with epithelial malignancies induction of telomerase activity is likely to compensate for further telomere erosion.

TERT Mutations in Patients with Squamous Cell Carcinoma of the Tongue and Refractory Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3096-3096
Author(s):  
Geraldine Aubert ◽  
Mark Hills ◽  
Carol Cremin ◽  
Irma Vulto ◽  
Barbara McGillivray ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Pulmonary Fibrosis ◽  
Cell Carcinoma ◽  
Telomere Length ◽  
Squamous Cell ◽  
Peripheral Blood ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Refractory Anemia ◽  
Normal Cells

Abstract Dyskeratosis Congenita (DC) is a marrow failure syndrome characterized by skin and nail abnormalities, oral leukoplakia and very short telomeres in circulating leukocytes. Heritable defects in telomere maintenance have been directly implicated in DC by the discovery of mutations in genes encoding components of the telomerase complex: DKC1, TERT, and TERC as well as mutations in the gene encoding the telomere binding protein TINF2. Defective telomeres in DC result in impaired hematopoiesis and predispose to myeloproliferative disorders. Heritable mutations in TERT and TERC have also been implicated in patients presenting with aplastic anemia (AA) and idiopathic pulmonary fibrosis (IPF) without clinical signs of DC. Because short telomeres appear to be associated with increased risks for various human cancers, including head and neck cancer, we sequenced TERT and TERC in two patients with oral carcinoma and anemia. The first patient presented at age 47 with invasive squamous cell carcinoma (SCC) of the tongue. The patient had a male sibling said to be also suffering from SCC which was not available for analysis and his mother died at age 37 from lymphoma. The patient displayed mild macrocytic anemia and oral leukoplakia. The telomere lengths of peripheral blood cells from the patient, determined by flow-FISH, were found to be below the first percentile expected for his age. In contrast, the leukocyte telomere lengths for the patient’s father and a female sibling were within the normal range. Bi-directional sequence analysis of TERT and TERC was conducted on DNA isolated from whole blood for the three family members. A novel mutation in exon 9 of TERT, C842T, situated within the reverse transcriptase domain of the telomerase enzyme catalytic component was identified in the patient but not in the 2 unaffected relatives. This suggested inheritance of a TERT mutation from the mother. The function of TERT C842T was compared to wildtype (WT) TERT by transfecting WT and mutant TERT cDNA into clonal Jurkat T cells and measuring telomere elongation by flow-FISH following 4 weeks of culture. TERT C842T showed 30% of the elongation obtained with WT TERT (p=0.0034). The second patient is a 60 yr old male with SCC of the tongue and refractory anemia with ring sideroblasts. The leukocyte telomere length was around the 1st percentile expected for his age. TERT sequencing revealed a three nucleotide deletion resulting in loss of 441E while retaining frame that is expected to impair telomerase activity. Our data support the concept that mutations in TERT can cause defective telomere maintenance and thereby compromise the proliferation of hematopoietic as well as epithelial cells. The resulting loss of normal cells selects for cells with defective DNA damage checkpoints that are triggered by chromosome ends without telomere repeats. Such cells are at high risk of becoming malignant because their proliferation will be stimulated by the loss of normal cells and their genome is very unstable as telomere function is compromised. Together these factors facilitate and enable clonal evolution of abnormal cells by DNA repair defects and cycles of chromosome fusions/bridge/breakage. Hematological and pathological findings consistent with Dyskeratosis Congenita together with peripheral blood telomere length measurements appear useful parameters to screen for telomere defects in patients and facilitate the discovery of mutations in “telomere maintenance” genes. The TERT mutations in patients with oral carcinomas illustrate that disease manifestations of telomere dysfunction in humans can be very diverse and range from DC, to defective hematopoiesis, pulmonary fibrosis and cancer predisposition.

scholarly journals Telomere length dynamics over 10-years and related outcomes in patients with COPD

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
E. Córdoba-Lanús ◽  
S. Cazorla-Rivero ◽  
M. A. García-Bello ◽  
D. Mayato ◽  
F. Gonzalvo ◽  
...  
Keyword(s):  
Lung Function ◽  
Gas Exchange ◽  
Telomere Length ◽  
Telomere Shortening ◽  
Lung Hyperinflation ◽  
Risk Of Death ◽  
Copd Patients ◽  
Increased Risk ◽  
Over Time

Abstract Background Chronic obstructive pulmonary disease (COPD) has been proposed as a disease of accelerated aging. Several cross-sectional studies have related a shorter telomere length (TL), a marker of biological aging, with COPD outcomes. Whether accelerated telomere shortening over time relates to worse outcomes in COPD patients, is not known. Methods Relative telomere length (T/S) was determined by qPCR in DNA samples from peripheral blood in 263 patients at baseline and up to 10 years post enrolment. Yearly clinical and lung function data of 134 patients with at least two-time measures of T/S over this time were included in the analysis. Results At baseline, T/S inversely correlated with age (r = − 0.236; p < 0.001), but there was no relationship between T/S and clinical and lung function variables (p > 0.05). Over 10 years of observation, there was a median shortening of TL of 183 bp/year for COPD patients. After adjusting for age, gender, active smoking and mean T/S, patients that shortened their telomeres the most over time, had worse gas exchange, more lung hyperinflation and extrapulmonary affection during the follow-up, (PaO2 p < 0.0001; KCO p = 0.042; IC/TLC p < 0.0001; 6MWD p = 0.004 and BODE index p = 0.009). Patients in the lowest tertile of T/S through the follow-up period had an increased risk of death [HR = 5.48, (1.23–24.42) p = 0.026]. Conclusions This prospective study shows an association between accelerated telomere shortening and progressive worsening of pulmonary gas exchange, lung hyperinflation and extrapulmonary affection in COPD patients. Moreover, persistently shorter telomeres over this observation time increase the risk for all-cause mortality.

scholarly journals Androgen Derivatives Improve Blood Counts and Elongate Telomere Length in Patients with Dyskeratosis Congenita

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2585-2585 ◽  
Author(s):  
Martin Kirschner ◽  
Monica Sofia Ventura Ferreira ◽  
Anne-Sophie Bouillon ◽  
Marcin W. Wlodarski ◽  
Michaela Schwarz ◽  
...  
Keyword(s):  
Telomere Length ◽  
Peripheral Blood ◽  
Research Funding ◽  
Somatic Mutations ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Treatment Start ◽  
Increased Risk

Abstract Introduction: Classical Dyskeratosis Congenita (DKC) is a systemic disorder characterized mainly by mucocutaneous features and bone marrow failure. DKC is caused by mutations affecting proper telomere maintenance leading to premature telomere shortening. Clinically, assessment of telomere length (TL) is being used for screening and diagnosis of DKC. Previous studies showed that androgen derivatives (AD) such as danazol or oxymetholone can improve blood counts and reduce transfusion frequency in patients with DKC. Reports from in vitro studies suggest that AD can increase the expression of telomerase and elongate telomeres reversing at least partially the mutation-related haploinsufficiency of the telomerase complex. However, whether telomere elongation can be observed in vivo is still controversial. Patients with DKC have an increased risk of developing solid tumors and acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Malignant transformation occurs mostly by chromosomal instability mediated by critical short telomeres and not via clonal hematopoiesis (CHIP) and eventual selection for MDS-related somatic mutations. The question whether increased telomerase activity by AD increases the risk for additional MDS-related mutations is unclear. In our study, we aimed to investigate TL and MDS-related somatic mutations in DKC patients undergoing treatment with AD. Methods and Patients: 5 patients enrolled in the Aachen Telomeropathy Registry (ATR) that underwent AD treatment were included in the analysis. All patients had molecularly confirmed DKC (4 patients having mutations in TERC, 1 patient in TERT). TERC mutated patients received danazol treatment (mean dosage 625 mg per day) while the patient with TERT mutation was treated with low dose oxymetholone (0.22mg/kg) per day. Patients were at a median age of 43.1 (range from 21.7 to 53.8) years. Median duration of treatment with AD was 14 months (3 to 29 mo) and is actually ongoing in all patients treated with danazol. Follow-up for blood counts and TL length assessment was carried out after median 14 months after treatment start with AD. TL assessment and blood counts of the patient receiving oxymetholone was carried out at the end of AD treatment after 29 months. All patients underwent next-generation sequencing (NGS) analysis using custom NGS-panel including frequent genes implicated in MDS development. Quality parameters of the NGS analysis were satisfactory (Q30>85%) and 95% of the expected area was covered at minimum 300x. To minimize risk of detecting sequencing errors, a threshold of 10 (absolute) and 5% (relative) variant allele frequency (VAF) was chosen. TL assessment of peripheral blood granulocytes and lymphocytes was carried out by Flow-FISH and all results are given in kb. Results: Analysis of the peripheral blood counts revealed a significant increase in platelets counts from mean 56/nl ±50 S.D. before treatment to 88/nl ±49 (p=0.03) during treatment. Similar results were observed for leukocyte counts increasing significantly from 3.83/µl±1.86 to 4.70/µl±2.88 (p=0.04). Hemoglobin counts showed a non-significant increase from 8.9 g/dl ±2.6 to 10.2 g/dl ±2.9 (p=0.13, all student paired t-test). Focusing on TL, lymphocyte TL increased significantly from 4.32kb±0.47 to 5.13kb ±0.57 (p=0.001). TL in the granulocyte subpopulation increased from 4.73kb±0.33 before treatment start to 6.10kb±0.50 under treatment (p=0.026). Calculated median increase in TL per months for lymphocytes and granulocytes was 0.092 kb (0.019 to 0.223 kb) and 0.166 kb (0.019kb to 0.513kb). Finally, NGS analysis for possible MDS-related mutations did not reveal any mutations before and under AD treatment. Conclusions: Based on our data in this genetically homogenous cohort of 5 patients with mutations in the telomerease genes TERC and TERT and short TL, AD significantly improve blood counts and elongate telomeres in granulocytes and lymphocytes. No MDS-related somatic mutations were observed during telomerase activation with AD. Pending longer follow up, treatment with AD seems to represent an efficient and safe therapy for patients with TERT or TERC mutations. Whether AD persistently elongate telomeres in DKC patients and how much this is dependent on the underlying DKC-related mutation requires further investigation. Disclosures Kirschner: Basilea Pharmaceutica: Other: travel support; BMS: Consultancy; Bayer: Consultancy; Roche: Consultancy. Wilop:Medizinwelten-Services GmbH: Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria, Other: Travel grant; Bristol-Myers Squibb: Honoraria. Brümmendorf:Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Merck: Consultancy. Beier:Gilead: Other: travel support; Celgene: Other: travel support.

Human Leukocyte Antigen DR15 in Patients with Myelodysplastic Syndromes (MDS) – A Study in Chinese Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2463-2463
Author(s):  
Qiong Liao Master ◽  
Yan Zhang ◽  
Xiao Li ◽  
Zheng Zhang ◽  
Shaoxu Ying ◽  
...  
Keyword(s):  
Human Leukocyte Antigen ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Human Leukocyte ◽  
Low Risk ◽  
Chinese Patients ◽  
Refractory Anemia ◽  
Healthy Controls ◽  
Leukocyte Antigen ◽  
Significant Difference

Abstract We investigate the frequency of the human leukocyte antigen DR15 (HLA-DR15) allele in patients with myelodysplastic syndromes (MDS). We used polymerase chain reaction-sequence-specific primers (PCR-SSP) to detect HLA-DR15 in the peripheral blood of patients with MDS(n = 76) and healthy controls (n = 227). The frequency of HLA-DR15 in MDS patients (40.8%) was significantly higher than in controls (13.7%; P &lt; 0.01). The diagnoses of refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) accounted for 77.4% (24/31) and 62.2% (28/45) of the DR15-positive and the DR15-negative patients, respectively (difference not statistically significant). Although no statistically significant difference was observed, some trends were observed: IPSS low-risk MDS (IPSS score, ≤1) accounted for 80.6% of the DR15-positive patients compared to 64.4% among the DR15-negative patients. However, the difference between the numbers of DR15-positive and DR15-negative patients with chromosomal abnormalities was not statistically significant. Nevertheless, poor risk chromosome abnormalities (according to IPSS), were present in only 1 DR15-positive patient, while such abnormalities were present in 8 DR15-negative patients. In addition, the proportions of DR15-positive and DR15-negative patients with more than 5% blasts in marrow were 19.4% and 31.1%, respectively. Peripheral blood pancytopenia occurred in 51.6% of DR15-positive, and in 40.0% of DR15-negative patients. Although the HLA-DR15 allele appeared to be present more frequently in patients less than 60 years of age, this association was not significant. The frequency of HLA-DR15 was significantly higher in patients with MDS than in healthy controls suggesting the possibility that HLA-DR15 is associated with an enhanced susceptibility to develop MDS. The fact that HLA-DR15 was predominantly noted in patients with RA/RARS and low IPSS scores, suggested that HLA-DR15 might be associated more with bone marrow failure and less with leukemic transformation. clinical/experimental characteristics in HLA-DR15 positive or negative MDS patients Cohort HLA-DR15 positive(n=31) HLA-DR15 negative(n=45) P value RA/RARS(case/%) 24/31(77.4) 28/45(62.2) 0.161 Low risk (IPSS≤1) (case/%) 25/31(80.6) 29/45(64.4) 0.126 Karyotype abnormal(case/%) 13/31(41.9) 18/45(40.0) 0.866 Poor chromosome(case/%) 1/31(3.2) 8/45(17.8) 0.117 Blast&gt;5%(case/%) 6/31(19.4) 14/45(31.1) 0.253 Pancytopenia(case/%) 16/31(51.6) 18/45(40.0) 0.317 Male patients(case/%) 17/31(54.8) 25/45(55.6) 0.951 age(&lt;60 years) (case/%) 20/31(64.5) 20/45(44.4) 0.085 Figure Figure Figure Figure

Screening of Telomere Length in Peripheral Blood Lymphocytes From Patients with Aplastic Anemia by Flow-Fluorescence in Situ Hybridization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3205-3205
Author(s):  
Nobuhiro Nishio ◽  
Yoshiyuki Takahashi ◽  
Hideki Muramatsu ◽  
Asahito Hama ◽  
Toshihito Nagata ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Telomere Length ◽  
Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Telomere Shortening ◽  
Blood Lymphocytes ◽  
Normal Individuals ◽  
Telomere Signal ◽  
Normal Controls

Abstract Abstract 3205 Poster Board III-142 Telomeres in peripheral blood cells from patients with aplastic anemia (AA) are shorter than those from normal individuals. One possible explanation is that telomere shortening reflects the extent and duration of damage to hematopoietic stem cells and the number of compensatory stem cell divisions. Patients with such stressed hematopoiesis probably do not respond to immunosuppressive therapy (IST). Cryptic forms of dyskeratosis congenita (DC) have been recognized in patients with AA who do not have physical abnormalities, but who harbor the gene mutation associated with telomere maintenance. To identify such patients is important because those who have these mutations might respond poorly to IST. The present study evaluates the usefulness of screening telomere length in peripheral blood lymphocytes from patients at the time of diagnosis with AA by flow-fluorescence in situ hybridization (flow-FISH) using Telomere PNA kits (Dako Cytomation, Glostrup, Denmark). After gating diploid cells based on propidium iodine staining, lymphocytes were isolated according to size and granularity. Relative telomere length (RTL) was calculated as the ratio between the telomere signal of each sample and the telomere signal of the control cell line. Because telomeres shorten with age, we obtained age-adjusted measurements for comparison. We used flow-FISH to measure the length of telomeres in peripheral blood lymphocytes from five and two patients with classical and cryptic DC who had the TERT mutation, respectively, to determine whether these two forms of DC can be screened. Significant telomere shortening was detected in all seven patients compared with age-matched normal individuals. To determine the association between telomere length and response to IST, we compared telomere lengths in peripheral blood lymphocytes from 61 normal controls and 27 patients with AA (very severe, severe and non-severe, n = 9 each) at diagnosis who received IST with antithymocyte globulin (ATG) and cyclosporine. The median age of the patients was 9.0 years (range, 1.5 to 14.8). The causes of AA were unknown etiology in 25 patients and hepatitis in 2. In total, 17 of the 27 patients (63%) responded to IST at 6 months after administration of ATG. Age-adjusted delta RTLs did not significantly differ between normal controls and responders (p = 0.181), but those in non-responders were significantly lower than those in normal individuals (p = 0.000). Our findings suggest that screening telomere length in patients with AA at diagnosis is useful for detecting cryptic DC and for predicting responses to IST. Disclosures No relevant conflicts of interest to declare.

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