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.

Telomere Length Shortening in the CML Leukemic Stem Cell Compartment Correlates with the Respective Clone Size: Evidence for Early Vs. Late Chronic Phase

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4243-4243
Author(s):  
Anne-Sophie Bouillon ◽  
Monica S. Ferreira ◽  
Shady Adnan Awad ◽  
Johan Richter ◽  
Henrik Hjorth-Hansen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Telomere Length ◽  
Research Funding ◽  
Chronic Phase ◽  
Disease Stage ◽  
Leukemic Stem Cell ◽  
Cell Compartment ◽  
Clone Size ◽  
Telomere Shortening ◽  
Stem Cell Compartment

Abstract Introduction: Chronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the bcr-abl translocation. Recent data provides evidence that CML chronic phase (CP) can be classified into early and late CP depending on the degree of expansion of the leukemic stem cell (LSC) clone. Patients in late CP have a higher LSC burden going along with an inferior response to TKI therapy. Telomeres shorten with each cell division and telomere length (TL) reflects the replicative history of a cell. We postulate that the LSC burden correlates with accelerated telomere shortening due to clonal replicative expansion of bcr-abl positive cells. Previous studies evaluating TL in peripheral blood cells of CML patients already revealed a correlation of age-adapted TL with disease stage, response to treatment and duration of CP. However, the high intra-individual, mostly genetic inter-individual variability in TL limits the predictive value of TL measurements when no patient specific bcr-abl negative cells were available for comparison. The aim of our study was to analyze TL in the LSC and non-clonal HSC compartment of patients with CP using a modified Q-FISH technique allowing the TL analysis in bcr-abl positive and negative cells. Methods and Patients: 11 patients diagnosed with CML in CP of the NCT00852566 study (Nordic CML Group) were included into our retrospective analysis. Mean age of the patients was 58.9 years. Bone marrow samples from initial diagnosis were sorted for CD34+/CD38- cells reflecting the leukemic stem cell compartment. Samples were analyzed with the FISH method using dual fusion dual color BCR-ABL1 probe following standard procedures. After capturing the bcr-abl staining using confocal microscopy, samples were re-processed for TL analysis by Q-FISH using established protocols. Optimized microscopy techniques allowed for TL to be assessed in all previously captured cells allowing the identification of bcr-abl positive and negative cells within the same sample. Analysis and quantification of bcr-abl FISH staining and TL measurement by Q-FISH were performed in single-blinded fashion. Results: Bcr-abl negative cells represent the non-clonal hematopoiesis and were used to correct TL. We observed significant shortened TL in the bcr-abl positive cells compared to bcr-abl negative cells (-2.18 ± 2.08 kilobases (kb), p=0.01) in line with previously published results. Next, we correlated the clone size (i.e. the proportion of bcr-abl positive cells) with the degree of telomere shortening in the leukemic stem cell compartment. Mean clone size of the patients was 67.4 ± 21.7 % S.D. Despite of the relatively small sample size studied, we found a significant negative correlation (R²=0.45, p=0.04) between TL and clone size strongly supporting the notion that increased expansion of the bcr-abl positive LSC pool leads to accelerated telomere shortening. Conclusions: In this study, we provide evidence for accelerated telomere shortening in bcr-abl positive LSC as compared to their normal CD34+/CD38- counterpart in CP CML samples at diagnosis. Furthermore, the degree of TL shortening correlates with the clone size in the HSC compartment, i.e. a parameter that reflects duration of CP. Thus, this allows the discrimination of early vs. late CP and might as such be used as a prognostic (and potentially predictive) biomarker in CML. Disclosures Richter: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Fioretos:Cantargia: Equity Ownership. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Brümmendorf:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties.

scholarly journals Factors Influencing Long-Term Hematopoietic Function Following Autologous Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2186-2186
Author(s):  
Alissa Visram ◽  
Natasha Kekre ◽  
Christopher N. Bredeson ◽  
Jason Tay ◽  
Lothar B. Huebsch ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Infection Rates ◽  
Advisory Committees ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background/Objective: Mobilized peripheral blood hematopoietic progenitor cells are the most common stem cell source for autologous hematopoietic stem cell transplantation (auto-HSCT). Successful short-term stem cell engraftment requires collection of at least 2x106 CD34+ cells/kg. The American Society of Bone Marrow Transplantation (ASBMT) recommends a stem cell infusion target of 3-5 x106 cells/kg (Giralt et al. 2014). However, the number of CD34+ cells to reinfuse to ensure long-term engraftment has not been established. Plerixafor, a reversible CXCR4 antagonist, increases CD34+ cell yield at collection even in patients who are predicted poor mobilizers (PPM). Although plerixafor could be used universally for all collections, this may not be the most cost-effective strategy (Veltri et al. 2012). This study sought to determine the minimum number of CD34+ cells/kg required for adequate long-term hematopoiesis, identify factors associated with poor long-term hematopoiesis, and determine if plerixafor mobilization improved long-term peripheral blood counts. Methods: A retrospective chart review was conducted on patients who underwent auto-HSCT between January 2004 and September 2013 at The Ottawa Hospital, for management of hematological malignancies. Peripheral blood cell counts were collected from 1 to 5 years after auto-HSCT, or until disease relapse. Poor long-term hematopoiesis was defined as an ANC <1 x109/L, hemoglobin <100 g/L, or platelets <100 x109/L. Patients were stratified into groups based on the infused CD34+ concentration (in cells/kg), and the proportion of patients with poor long-term hematopoiesis at 1, 2, 3, 4, and 5 years post auto-HSCT was compared with chi square tests. Long-term clinical outcomes (platelet and packed red blood cell transfusions, and post auto-HSCT infection rates) were compared between plerixafor-mobilized patients and PPM (defined as patients with pre-collection CD34+ <2 x 106 cells/kg) with standard mobilization regimens. Results: This study included 560 patients who underwent auto-HSCT, 210 with multiple myeloma and 350 with lymphoma. At 1 and 5 years post auto-HSCT 377 and 104 patients were included, respectively. A dose dependent improvement 1 year after auto-HSCT was seen in patients who received 0-2.99 x 106 CD34+ cells/kg (24.4%, n= 41) compared to patients who received 5-9.99 x 106 CD34+ cells/kg (11%, n=154, p=0.051) and ³10 x 106 CD34+ cells/kg (4.5%, n=66, p=0.006). Though there was a trend towards lower CD34+ infusions and poorer hematopoietic function (see table 1), there was no statistically significant difference in hematopoietic function based on CD34+ infusion concentrations after 1 year post auto-HSCT. 10 patients received <2 x106 CD34+ cells/kg, of whom the rate of inadequate hematopoiesis was 33% at 1 year (n=6) and 0% (n=1) at 5 years post auto-HSCT. Factors that increased the risk of poor hematopoiesis over the course of study follow up, based on a univariate analysis, included advanced age (OR 1.189, p=0.05), multiple prior collections (OR 2.978, p=0.035), and prior treatment with more than two chemotherapy lines (OR 2.571, p=0.02). Plerixafor-mobilized patients (n=25), compared to PPM (n=197), had a significantly higher median CD34+ cell collection (4.048 x109/L and 2.996 x109/L cells/kg, respectively, p=0.005). There was no significant difference in overall cytopenias, transfusion requirements, or infection rates between plerixafor-mobilized and PPM patients over the course of the study follow up. Conclusion: Low pre-collection CD34+ counts, advanced age, multiple prior collections, and more than two prior chemotherapy treatments adversely affected long-term hematopoiesis post auto-HSCT. We support the transfusion target of 3-5 x 106 cells/kg, as proposed by the ASBMT, given that at 5 years post auto-HSCT there was no statistical or clinically significant difference in hematopoietic function with higher CD34+ infusion targets. While mobilization with plerixafor significantly increased overall CD34+ cell collection when compared with PPM, long-term hematopoietic function and clinical outcomes were not different. This finding supports the practise of limiting plerixafor use only to patients who are PPM, thereby facilitating adequate stem cell collection and early engraftment, as opposed to universal plerixafor mobilization. Disclosures Sabloff: Lundbeck: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Canada: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Alexion: Honoraria.

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.

scholarly journals Genetic Dissection of Dyskeratosis Congenita-Causing Mutations in TINF2 Using Human Pluripotent and Hematopoietic Stem Cell Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2178-2178
Author(s):  
Seunga Choo ◽  
Franziska K. Lorbeer ◽  
Samuel G. Regalado ◽  
Sarah B. Short ◽  
Shannon Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Telomere Length ◽  
Hematopoietic Stem Cell ◽  
Research Funding ◽  
Dyskeratosis Congenita ◽  
Model Systems ◽  
Telomere Shortening ◽  
Hematopoietic Stem

Abstract The length of telomeres, which cap the ends of linear chromosomes and provide genomic stability, is tightly regulated in adult stem cells. The telomere reserve in the stem cell population sets the replicative potential of its differentiated progeny. For this reason, abnormally short telomeres in stem cells restrict the number of cell divisions that their differentiated progenies can undergo, eventually resulting in stem cell depletion and tissue failure syndromes. Telomere biology disorders (TBDs) display a broad range of clinical features, age of onset, and severity, which are all correlated with the extent of abnormal telomere shortening. One such early-onset TBD is dyskeratosis congenita (DC), which is a bone marrow predisposition syndrome characterized by a mucocutaneous triad (oral leukoplakia, nail dystrophy, and abnormal skin pigmentation) as well as other conditions driven by premature tissue aging. The leading cause of death in DC patients is bone marrow failure and hematopoietic stem cell transplantation is the only definite intervention to restore hematopoiesis. TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. TIN2 is a member of the shelterin protein complex bridging the double-strand binding shelterin proteins TRF1/TRF2, and the TPP1/POT1 heterodimer. Such interactions implicate a complex role of TIN2 in telomere length regulation: First, TIN2 stabilizes TRF1, which is a negative regulator of telomere length. Secondly, TPP1, which recruits telomerase, strictly requires TIN2 for telomere elongation and maintenance. TINF2-DC mutations are uniformly heterozygous and localize to a 30 amino acid coding stretch in exon 6 called the 'DC cluster'. TINF2-DC mutations usually arise de novo and result in an earlier disease onset, shorter telomeres, and a more severe manifestation compared to other heterozygous DC-causing mutations in genes such as TR and TERT, which encode the components of the telomerase enzyme. How TINF2-DC mutations cause telomere shortening is unknown. Specifically, whether telomere shortening is caused by reduced telomerase action at telomeres or by degradation of telomeric DNA remains unresolved as studies using different model systems report contrasting results. The discrepancy could be attributed to differences in the model systems used in the studies, highlighting the need for a genetically trackable, primary preclinical human model system. Here, we report the development of two novel endogenous, isogenic model systems to study TINF2-DC mutations. First, we generated human embryonic stem cells (hESCs) engineered to express the TINF2-DC T284R mutation from the endogenous locus, which recapitulated the short telomere phenotype observed in DC patients. Using this model, we identified a gene editing strategy that elongates telomeres in the mutant stem cells and eventually restores replicative potential of the differentiated cells. Next, we used a xenotransplantation model of donor-derived human hematopoietic stem cells (hHSCs) to test the effects of target gene modifications on telomere length and proliferative capacity in vivo. We demonstrate that our models robustly complement each other and offer direct insights into the disease mechanism as well as avenues to potential therapeutic approaches. Figure 1 Figure 1. Disclosures Bertuch: Elixirgen Therapeutics: Consultancy; ImmunityBio: Current equity holder in publicly-traded company; NIH/NCI,: Research Funding; DOD: Research Funding; Hyundai Hope on Wheels: Research Funding.

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.

Allogeneic Stem Cell Transplantation for Accelerated/Blastic Phase Philadelphia-Chromosome Negative MPN

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4532-4532
Author(s):  
Anjum Bashir Khan ◽  
Donal McLornan ◽  
Yogesh Jethava ◽  
Kavita Raj ◽  
Victoria T Potter ◽  
...  
Keyword(s):  
Stem Cell ◽  
Median Time ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloproliferative Neoplasm ◽  
Chronic Phase ◽  
Haematopoietic Stem Cell ◽  
Cell Transplant ◽  
Blastic Phase

Abstract Abstract 4532 Myeloproliferative Neoplasm (MPN)-Leukaemic Transformation (LT) uniformly carries a dismal prognosis. Effective therapy for such patients are currently lacking with no established evidence base to guide Allogeneic Haematopoietic Stem Cell Transplant (AHSCT) regimen. We report the outcome of a cohort of patients undergoing AHSCT at our 2 institutions over a 6-year period (2006–2012). 24 patients underwent AHSCT following diagnosis of MPN transformed to an accelerated phase (5–9% blasts on bone marrow (BM), n=9 & 10–19% BM blasts, n=2) or blastic phase (>20% blasts on peripheral blood or BM, n=13). Disease subtypes were: Polcythaemia Vera (PV, n=4), Essential Thrombocythaemia (ET, n=6), Primary Myelofibrosis (PMF, n=8), Myelodysplastic/Myeloproliferative neoplasm-Unclassified (MDS/MPN-U, n=6). Median age at diagnosis was 50 years (range 29–67) and median time to transformation was 50 months (range 0–271). Cytogenetics were abnormal at transformation in 11 patients (46%), with 6 (25%) demonstrating abnormalities of chromosomes 5, 7 or complex karyotype, and 5 displaying trisomy 8, whilst 1 had isolated chromosome 17p deletion. 13 patients harboured the JAK2V617F mutation. Patients received a median of 3 (range, 1–5) lines of therapy for chronic and acute phase prior to AHSCT, of which 20 patients received intensive AML-type induction therapy. Disease status at time of AHSCT was complete remission (CR) in 13 cases, partial response (PR) in 7, and 4 patients had persisting AML. Conditioning regimes were Reduced Intensity with T-depletion (Alemtuzumab or Anti-Thymocyte globulin) in 23/24 cases (Fludarabine/Busulfan-based n=12, FLAMSA (Fludarabine, Ara-C and Amsacrine, followed by TBI/Cyclophosphamide or Busulfan) n=9, Fludarabine Cyclophosphamide TBI haploidentical protocol n=1, Fludarabine/Melphalan n=1). Median CD34 dose was 6.48 × 10∧6 cells/kg (range 1.17 {BMH} −10.71). Stem cell source was Peripheral Blood in all but one case, from unrelated (n=17) or related (n=7) donors. Median time to both neutrophil and platelet engraftment was 13 days (range 9–25 and 7–68 respectively); 2 patients including the haploidentical transplant had Primary Graft Failure (8%). The incidence of severe (grade 3&4) acute GVHD was 3/24 (12.5%) and 10 patients developed NIH-defined chronic GVHD (8 moderate, 1 severe). Day 100 Non-relapse mortality was 12.5%. Patients underwent sequential chimerism monitoring. Median OS for the entire cohort was only 10 months with a median progression free survival (PFS) of only 6.5 months. 5 patients received therapeutic Donor Lymphocyte Infusion (tDLI) for relapse at a median dose of 1×10∧6 CD3+/kg. 2 patients received DLI alone for chronic phase relapse, of whom 1 achieved remission. 3 patients received chemotherapy + DLI and 1 achieved 2ndCR. At last follow-up, 11/24 patients were alive with median surviving patient follow-up of 25 months. The percentage of BM blasts at progression from chronic phase had a highly significant impact upon outcome post AHSCT, median OS 23 vs. 10 months for 5–9% BM blasts compared to 310% BM blasts (p=0.011) & PFS 11 vs 6 months respectively (p=0.033, Fig 1). This effect was replicated when considering disease response immediately prior to AHSCT, with a median OS of 28 months for those in CR, compared to 10 months for those with excess blasts (p=0.017) and median PFS 11 vs 6 months, p=0.019 (Fig 2). Disease duration, subtype, Jak2 status and age at allograft did not significantly affect survival. Of note for the 3 surviving patients with follow-up over 6 months, all received FLAMSA-RIC conditioning (n=9). 5 patients who received FLAMSA TBI; 2 died of treatment-related complications, and 2 with residual disease at time of AHSCT relapsed early. Of 4 patients who have received a hybrid FLAMSA-Busulfan regimen, 2 remain alive in CR and 2 achieved a relapse free period of 12 months. Interestingly, PFS for FLAMSA-Bu patients appears significantly improved compared to conventional RIC regimens (median PFS 12 vs. 6 months, p=0.035) on univariate analysis, although conclusions are limited by cohort size. Further work into optimising transplantation regimens for accelerated and blastic phase MPN is warranted. Early use of FLAMSA-Busulfan hybrid protocol, before transformation to overt blastic phase, in conjunction with early weaning of immunosuppressive therapy and prophylactic DLI may improve the proportion of long-term survivors. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: Harrison: Sanofi Aventis: Honoraria; Shire: Honoraria, Research Funding; YM Bioscience: Consultancy, Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

scholarly journals Autograft-Absolute Lymphocyte Count Infusion Predicts Survival in Double/Triple Hit Lymphomas Post-Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1836-1836
Author(s):  
Luis F. Porrata ◽  
David J. Inwards ◽  
Stephen M. Ansell ◽  
Ivana N. Micallef ◽  
Patrick B. Johnston ◽  
...  
Keyword(s):  
Stem Cell ◽  
Prognostic Factor ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Peripheral Blood ◽  
Lymphocyte Count ◽  
Research Funding ◽  
Absolute Lymphocyte Count ◽  
Hematopoietic Stem

Abstract The autograft absolute lymphocyte count (A-ALC) ≥ 0.5 x 10 9 cells/kg is a survival prognostic factor for lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). However, the A-ALC ≥ 0.5 x 10 9 cells/kg has not been tested as prognostic factor against double hit/triple hit lymphomas (DHL/THL). Thus, we set up to investigate if A-ALC ≥ 0.5 x 10 9 cells/kg is a prognostic factor for overall survival (OS) and progression-free survival (PFS) for DHL/THL post-APBHSCT. From January 2012 until December 2020, we identified 77 DHL/THL patients treated with APBHSCT. All patients required to have the diagnosis of DHL/THL by FISH for rearrangements of MYC, BCL2, and BCL6. Of the 77 patients, 62 patients were classified as DHL and 15 patients as THL. Of the DHL, 82 % (52/68) had the MYC/BCL2 and 16% (10/62) the MYC/BCL6 rearrangements. Dividing the cohort into two groups (A-ALC ≥ 0.5 x 10 9 cells/kg and A-ALC &lt; 0.5 x 10 9 cells/kg), both groups were balanced in regard to de novo versus transformed histology (p = 0.3), cell of origin (p = 0.6), DHL and THL (p = 0.6), MYC/BCL2; MYC/BCL6; and MYC/BCL2/BCL6 rearrangements (p = 0.6), first line chemotherapy (p = 0.2), salvage chemotherapy (p = 0.5), disease status prior to APBHSCT (CR1, CR, and PR) (p = 0.07), stem cell mobilization (Plerixafor versus not) (p = 0.6), and infusion of CD34 count (p = 0.2). With a median follow-up of 20.4 months (range, 0.4-94.5 months) for the entire cohort and with a median follow-up of 33.5 months (range, 3.5-94.5 months) for the patients that remained alive, DHL/THL patients infused with A-ALC ≥ 0.5 x 10 9 cells/kg experienced superior OS (HR = 0.251, 95%CI 0.117-0.539, P &lt; 0.0004) and PFS (HR = 0.347, 95%CI 0.160-0.753, P &lt; 0.007). The 5-year OS rates for the A-ALC ≥ 0.5 x10 9 cells/kg group was 73% (95% confidence interval [CI], 52%-87%) and for the A-ALC &lt; 0.5 x10 9 cells/kg group was 18% (95% CI, 7%-39%) (Figure 1A). The 5-year PFS rates for the A-ALC ≥ 0.5 x10 9 cells/kg group was 73% (95% CI, 59%-85%) and for the A-ALC &lt; 0.5 x10 9 cells/kg group was 13% (95% CI, 5%-33%) (Figure 1B). Multivariate analysis showed that A-ALC was an independent predictor for OS (HR =0.178, 95%CI 0.052-0.614, p &lt;0.005) and PFS (HR = 0.400, 95%CI 0.189-0.850, p &lt;0.02). Our study showed that A-ALC is a prognostic factor for survival in DHL/THL patients undergoing APBHSCT. Our current practice for all lymphoma patients is not only to collect enough stem cell for hematologic engraftment, but also A-ALC ≥ 0.5 x 10 9 cells/kg to improve clinical outcomes post-APBHSCT. Figure 1 Figure 1. Disclosures Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Paludo: Karyopharm: Research Funding.

Homeostasis of telomere length rather than telomere shortening after allogeneic peripheral blood stem cell transplantation

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 358-362 ◽  
Author(s):  
Helene Roelofs ◽  
Elmar S. D. de Pauw ◽  
Aeilko H. Zwinderman ◽  
Sonja M. Opdam ◽  
Roel Willemze ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Telomere Length ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Blood Stem Cell ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Blood Stem Cell Transplantation

Abstract Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell–depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.

scholarly journals Large Granular Lymphocytosis and Its Impact on Long Term Clinical Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation: 14-Year Follow-up Data

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2019-2019
Author(s):  
Guldane Cengiz Seval ◽  
Atilla Uslu ◽  
Ekin Kircali ◽  
Sinem Civriz Bozdag ◽  
Klara Dalva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Support Research

Introduction: Several studies have attempted to describe the characteristics associated with large granular lymphocytosis (LGL) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and its clinical significance. However the clinical features of LGL lymphocytosis in the allo-HSCT setting is still sparse. The current study represents a detailed review of 667 patients transplanted in a single center with the objective to define the incidence of LGL lymphocytosis, to identify associations with transplant-related clinical parameters and to assess the impact on transplant related outcomes. Patients and Methods: During a 14-year follow up period (2005-2017) in this unicentric cohort study, we identified 19 patients (2.8%) with a significant LGL lymphocytosis, among 667 consecutive adult patients who underwent allo-HSCT. LGL lymphocytosis was defined as the presence of at least two of the following criteria: (1) sustained lymphocytosis above 3.0x109/L observed in at least three consecutive determinations over a time frame of 2-3 months, (2) predominance (that is, >30%) of LGLs in the peripheral blood, (3) confirmation of clonality by T-cell receptor analysis using PCR. Flow cytometry analyses were performed using the flow cytometry system FACSCalibur (BD Biosciences, San Jose, CA). The immunophenotyping of the lymphocytes included the following antibody panel: CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD30, CD56, CD57, HLA-DR, TCRab, and TCRgd. T-LGL expansion was defined as an abnormal T cell population type CD31, CD81, or CD41, with expression of at least 1 of the NK markers (CD16, CD57, or CD56), and with presence of LGLs in peripheral blood films. Results: A total of 19 (Female/ male: 10 [52.6 %]/ 9 [47.4 %]) patients included into the study met the morphological criteria for LGL lymphocytosis. The median age of the patients was 46 years (range, 18- 62 years). The majority of the patients (64.7 %) had the diagnosis of acute myeloid leukemia. The stem cell source was peripheral blood stem cells (PBSC) in 15 patients (88.2 %) and most of the patients underwent an allo-HSCT with a MAC (n= 13) regimen at a median of 25.1 months from allo-HSCT. The median onset of LGL lymphocytosis was 11.5 (2.1- 55.7) months and median lymphocyte count at the time of diagnosis of LGL lymphocytosis was 5400/ mL (5170- 8700/ mL). None of the patient showed cytopenia, palpable splenomegaly, and none of them had typical signs or symptoms of an autoimmune disease. In addition; GvHD, viral infections, disease relapse and loss of donor chimerism were excluded during lymphocytosis. Samples from 19 patients were phenotyped by flow cytometry. These studies confirmed a T cell phenotype of LGLs in the majority of patients (n=12). Two patients presented with LGLs consistent with NK cells and seven showed properties of a mixed NK/T-cell lineage. A monoclonal LGL population of T-cell origin was identified in eight (42.1%) of these patients. With a median follow-up of 12.2 months none of the patients demonstrating increased LGL values has progressed to LGL leukemia or any other lymphoproliferative disorder. Four patients experienced cutaneous acute GVHD followed by a progressive chronic GVHD. Two patient developed a grade II acute cutaneous GVHD which rapidly responded to steroids in addition to cyclosporin A. Five patients had de novo chronic GVHD. In subgroup analysis, we compared the OS of monoclonal and oligoclonal LGL lymphocytosis and 1-year-OS was longer but non-significantly in monoclonal LGL lymphocytosis group; 75% ± 1.6% vs. 44.4% ± 2.2%, respectively (p= 0.21) (Figure). Median PFS was 28.8 months in oligoclonal LGL lymphocytosis group and 8.3 months in monoclonal LGL lymphocytosis group but the number of patients in this group does not provide enough statistical power to confirm whether the differences in PFS were statistically significant (p= 0.3). At the time of this report, three patients have died. The primary cause of death was relapse of the primary disease in one of the patients, whereas 2 patients died of TRM (10.5%). Discussion: In conclusion, we observed LGL lymphocytosis in 2.8 % of a large cohort of post allo-HSCT survivors. Our data indicate that, even if monoclonal, post-transplantation LGL expansion may be considered as an expression of chronic stimulation triggered by allo-HSCT rather than the result of a malignant transformation. Disclosures Özcan: Amgen: Honoraria, Other: Travel support; BMS: Other: Travel support; Jazz: Other: Travel support; Sanofi: Other: Travel support; Bayer: Research Funding; Novartis: Research Funding; Roche: Other: Travel support, Research Funding; Archigen: Research Funding; Takeda: Honoraria, Other: Travel support, Research Funding; Abdi Ibrahim: Other: Travel support; MSD: Research Funding; AbbVie: Other: Travel support, Research Funding; Janssen: Other: Travel support, Research Funding; Celgene Corporation: Research Funding, Travel support. Ilhan:Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Beksac:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 239: Stem Cell Therapy Improves Critical Limb Ischemia

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Alaa Marzouk
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Limb Ischemia ◽  
Control Group ◽  
Chronic Limb Ischemia

Introduction: The journey from single cell to complex being is attributable to stem cells role. Adult stem cells originate during ontogeny & persist in specialized niches within organs. Asymmetric division of each stem cell during differentiation produces : one daughter stem cell & one daughter transit amplifying/intermediate cell having migratory properties. Forced migration of hematopoietic stem/progenitor cells (HSPC) from bone marrow into peripheral blood is called mobilization. Accumulating evidence suggests that attenuation of the chemokine stromal derived factor-1(SDF-1)-CXCR4 axis that plays a pivotal role in retention of HSPC in bone marrow (BM) results in the release of these cells from the BM into peripheral blood. Recently, adult cells have been genetically reprogrammed to an embryonic stem cell like state. Induced pluripotent stem cells (IPSCs) were similar to human embryonic stem cells in morphology, proliferative capacity, expression of cell surface antigens, & gene expression. Treatment of ischemic vascular disease of lower limbs remains a significant challenge. Unfortunately, if medical & surgical salvage procedures fail, amputation is an unavoidable result for those patients. Aim of Work: (Hypothesis) To assess the application of implantation of autologous stem/progenitor cell in the treatment of chronic limb ischemia & to evaluate the safety, efficacy & feasibility of this novel therapeutic approach. Methods: A total of 24 patients with chronic limb ischemia not eligible for arterial reconstruction or endovascular procedures were enrolled & randomized (1:1) to either the implanted group or the control group. Control group: Conventional medical therapy in the form of anti platelet therapy & vasodilators. Implanted group: Subcutaneous injection of 300μ g/day of recombinant human granulocyte colony stimulating factor (G-CSF) for 5 days to mobilize stem/progenitor cells from BM. Total leucocytic count is measured daily to follow up successful mobilization of bone marrow mononuclear cells (BMMNCs). Stem cell Harvesting After 5 days peripheral blood mononuclear cells (PBMNCs) were harvested using a cell separator. Samples from apheresis products are subjected to TLC measurement & immunophenotypic characterization of CD34+ cells by flow cytometry. The collected PBMNCs were implanted by multiple intramuscular injections into ischemic limbs. Results: There was significant increase in pain free walking distance & ankle/brachial index (ABI) & significant decreased rest pain. Effectiveness was documented by : reduced number of amputation, increase ABI & improvement of the quality of life in therapeutic group compared to control group. Conclusion: The novel therapeutic approach of PBMNCs implantation in patients with chronic limb ischemia is safe, feasible & effective in decreasing co-morbidity & rate of amputation. Safety was manifested by absence of complications during G-CSF therapy or during harvesting & injection of the stem cells. Recommendations: 1- Future studies on larger number of patients & longer follow up. 2- Controlled studies using different methods & different cell population (PBMNCs, BMMNCs or MSCs) to compare the outcome of each. 3-Studing the role of endothelial progenitor cell dysfunction in different ischemic diseases to develop successful gene therapy.

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