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.

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.

Leukemic Stem Cell Quantification Is Of Prognostic Value In Newly Diagnosed Patients In Chronic Phase Chronic Myeloid Leukemia (CML-CP) Receiving Nilotinib Therapy: Results From The ENEST1st Stem Cell Substudy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 649-649
Author(s):  
Noortje Thielen ◽  
Johan Richter ◽  
Gisela Barbany ◽  
Thoas Fioretos ◽  
Frank Giles ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Research Funding ◽  
Prognostic Value ◽  
Spleen Size ◽  
Newly Diagnosed ◽  
First Line ◽  
Cell Compartment ◽  
Stem Cell Compartment ◽  
Sokal Score

Abstract Background Leukemic stem cells (LSCs) are considered to be very important therapeutic targets in CML, as it has been shown both in vitro and in vivo that tyrosine kinase inhibitor (TKI) therapy is not able to eradicate these entirely. We previously reported that LSC burden at diagnosis has significant prognostic impact on therapy outcome in imatinib and dasatinib treated CML patients. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in patients in CML-CP. This translational, multinational, multicenter ENEST1st substudy addresses the predictive value of the stem cell profile in these patients. Patients and methods Bone marrow (BM) and/or peripheral blood (PB) samples were collected from newly diagnosed CML-CP patients before and after 1 and 3 months of nilotinib treatment. The LSC burden was analyzed by FISH and flow cytometry (FC). With the FISH method, CD34+ cells were pre-selected with paramagnetic beads after which they were fractionated into CD38+ (upper 80%, “progenitor”) and CD38- (lowest 5%, “stem cell”) pools using flow sorting. The proportion of Ph+ cells was assayed by counting 1,000 cells with interphase FISH using specific BCR-ABL1 probes. In the FC method, the stem cell compartment was defined as the lowest 1% of CD34+CD38- cells. LSCs were distinguished from normal hematopoietic stem cells (nHSCs) by either higher light scatter properties, and/or aberrant expression of CD7, CD11b and CD56, and/or higher CD45 and CD90 expression. Patient samples were assigned to as “residual nHSCs present” or “no residual nHSC present” at diagnosis and as “residual LSCs present” or “no residual LSCs present” at 1 and 3 months. Altogether, 48 patients from 6 European countries were investigated. Results By FISH analysis at diagnosis, the proportion of BCR-ABL+ cells in the stem cell compartment varied markedly in individual patients (1%-100%) and was lower (85%) than in progenitor (96%) or whole BM fractions (96%, p<0.05). This LSC burden correlated significantly with high WBC count (r=0.44, p=0.008), hemoglobin (r=-0.51, p=0.002), spleen size (r=0.51, p=0.002) and PB (r=0.57, p=0.0002) and BM (r=0.55, p=0.0005) blast percentage. Weak correlation was also found with Sokal score (r=0.37, p=0.03). By FC method, similar to FISH, the range of LSCs was 0%-100% with a median of 73%. Forty percent of patients had detectable residual nHSC at diagnosis. These patients had significantly lower Sokal score (p=0.001), smaller spleen size (p=0.006), lower platelet count (p=0.04) and lower PB blast percentage (p=0.001). With both methods no prognostic relationship existed between LSC burden and eosinophil/basophil counts. Interestingly, by FISH method, the LSC burden at diagnosis correlated also with BCR-ABL transcript levels according to the international scale at 3 (r=0.39, p=0.03), 9 (r=0.41, p=0.01) and 18 months (r=0.42, p=0.02). Borderline significant associations were also found at 6 (p=0.06) and 12 (p=0.07) months. All patients lacking major molecular remission at 12 or 18 months, had >80% of BCR-ABL+ cells in the stem cell fraction at diagnosis. When LSCs were analyzed by the FC method, patients with residual nHSCs at diagnosis had significantly lower BCR-ABL levels at 3 (0.27 vs 1.74, p=0.025) and 6 (0.13 vs 1.21, p=0.03) months, but this was not reflected in lower BCR-ABL levels at 12 or 18 months. During nilotinib therapy, the proportion of BCR-ABL+ cells by FISH decreased rapidly both the in progenitor and stem cell compartments. At 3 months the median LSC percentage was 0.28%, and it did not differ significantly from the values detected either in the progenitor fraction (0.34%) or whole BM (0.29%). Similarly, only a few patients had detectable LSCs left during follow-up time-points with the FC method. Conclusions LSC burden at diagnosis reflects the biology of the disease in newly diagnosed CML-CP patients. It also carries a prognostic value in first-line treated nilotinib patients, and a substantial number of patients not meeting the optimal response criteria at the 12-month time-point have high LSC burden at diagnosis. Furthermore, the presence of residual nHSCs at diagnosis is predictive for molecular response at 3 and 6 months. Nilotinib therapy markedly decreases LSC pool during the first 3 months of treatment. Noteworthy, the reduction is as potent in the LSC compartment as it is in more mature progenitor cell or whole BM fractions. Disclosures: Richter: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Fioretos:Novartis: unrestricted research grant Other. Giles:Novartis: Consultancy, Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Ossenkoppele:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Porkka:Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Wolf:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Meyers Squibb: Honoraria. Janssen:Novartis: Consultancy, Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

Targeting the acute myeloid leukemic stem cell compartment by enhancing tumor cell-based vaccines

Immunotherapy ◽  
2013 ◽  
Vol 5 (8) ◽  
pp. 859-868 ◽  
Author(s):  
Jurjen M Ruben ◽  
Lindy L Visser ◽  
Hetty J Bontkes ◽  
Theresia M Westers ◽  
Gert J Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tumor Cell ◽  
Leukemic Stem Cell ◽  
Cell Compartment ◽  
Stem Cell Compartment ◽  
Acute Myeloid

Targeting MUC1 as a Marker for Myeloid Leukemia Stem Cells by DC/AML Fusions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1794-1794
Author(s):  
Jacalyn Rosenblatt ◽  
Zekui Wu ◽  
Corrine Lenahan ◽  
Adam Bissonnette ◽  
Baldev Vasir ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Fold Increase ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Cell Compartment ◽  
Stem Cell Compartment

Abstract The epithelial mucin antigen (MUC1) is aberrantly expressed in many epithelial tumors and hematologic malignancies and promotes oncogenesis and tumor progression. MUC1 is recognized by the T cell repertoire and has served as a target for cellular immunotherapy. In the present study, we examined MUC1 as a marker for myeloid leukemia cells and their progenitors and its capacity to serve as a target for leukemia stem cells. Myeloid leukemia cells were isolated from bone marrow aspirates or peripheral blood in patients with high levels of circulating disease. MUC1 was not expressed on unselected leukemia samples (mean expression 3%, n=12). Similarly, low levels of MUC1 expression were seen in leukemic blasts with monocytoid differentiation (mean expression 2.7%, n=5). A subset of leukemia specimens underwent CD34 selection by magnetic bead separation. In contrast to unselected cells, 38% of CD34+ leukemia cells expressed MUC1 (n=5). The leukemia stem cell compartment was isolated by separating CD34+/CD38−/ lineage- fractions by flow cytometric sorting. Leukemia stem cells demonstrated strong expression of MUC-1 by immunohistochemical staining and FACS analysis. Similarly, we examined MUC1 expression on progenitor cells derived from chronic phase chronic myeloid leukemia and following blast transformation. MUC1 was seen in only 4% of CD34+ cells obtained from chronic phase CML samples (n=4) while uniform expression was observed in samples derived from patients with accelerated/blastic phase disease. These data suggest that MUC1 serves as a marker for early leukemia progenitors and is associated with blastic transformation. We assessed the capacity of a cancer vaccine consisting of dendritic cell (DC)/myeloid leukemia fusions to stimulate immune responses that target MUC1 and other antigens expressed by the stem cell compartment. DCs were generated from adherent mononuclear cells that were cultured with GM-CSF and IL-4 and matured with TNFa. DCs were fused with patient derived myeloid leukemia cells using polyethylene glycol as previously described. Fusion cells were quantified by determining the percentage of cells that expressed unique DC and leukemia antigens. DC/AML fusions induced the expansion of MUC1 specific T cells. Stimulation of autologous T cells with DC/AML fusions resulted in a mean 3 fold increase in CD8+ cells binding the MUC-1 tetramer (N=4). DC/AML fusions stimulated anti-tumor immune responses that targeted leukemia stem cells. Fusion stimulated T cells demonstrated increased expression of IFNγ following exposure to lysate generated from unselected leukemia cells (29 fold) and leukemia stem cells (28 fold). In contrast, exposure to renal carcinoma lysate generated only a 5 fold increase in IFNγ. In summary, these findings suggest that leukemic progenitors in AML and accelerated/blast phase CML express MUC-1. DC/tumor fusion vaccines target the MUC-1 protein and the stem cell compartment, and may be a potent immunotherapeutic strategy to eliminate the malignant stem cell clone in AML.

scholarly journals TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment

Haematologica ◽  
2019 ◽  
Vol 105 (5) ◽  
pp. 1306-1316
Author(s):  
Barbara Depreter ◽  
Karin E. Weening ◽  
Karl Vandepoele ◽  
Magnus Essand ◽  
Barbara De Moerloose ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Leukemic Stem Cell ◽  
Cell Compartment ◽  
Stem Cell Compartment ◽  
Acute Myeloid

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.

Longterm Serial Transplantability of BCR-ABL+ Chronic Phase CML Cells Is Predicted by a 6-Week LTC-IC Assay.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2770-2770
Author(s):  
Ivan Sloma ◽  
Philip A Beer ◽  
Kyi Min Saw ◽  
Matthiew Chan ◽  
Karen Lambie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Chronic Phase ◽  
Cell Compartment ◽  
Qrt Pcr ◽  
Nsg Mice ◽  
Stem Cell Compartment ◽  
Cd34 Cells

Abstract Abstract 2770 Since the advent of tyrosine kinase inhibitors (TKIs) to treat CML patients, the development of methods to measure and characterize the CML stem cell compartment has stimulated increasing interest. Here we compared different methods for quantifying the relative frequency of very primitive Ph+/BCR-ABL+ in 33 chronic phase CML patients that had not been previously treated with TKIs. Materials and Methods Longterm culture-initiating cells (LTC-ICs) were assayed by coculturing immunomagnetically isolated CD34+ cells for 5-weeks in LTCs containing murine stromal cells (n=10) and/or for 6-weeks in LTCs containing murine stromal cells secreting human SCF, IL-3 and G-CSF (n=33). Genotyping of the LTC-ICs was based on genotyping individual colonies generated in methylcellulose assays of cells harvested at the end of the 5 or 6 weeks of coculture, either by karyotyping G-banded metaphases (n=16) or by qRT-PCR of extracted RNA (n=17). In vivo assays were performed on 2 samples by injecting 106 CD34+ cells (>97% and 44% Ph+ 6-week LTC-ICs) IV into primary sublethally irradiated NOD/SCID IL2Rγc−/− (NSG) mice and after 30–35 weeks further into secondary NSG mice. All mice were analyzed periodically for human hematopoietic cells by flow cytometry of marrow aspirates. CD34+CD38− cells isolated by FACS from primary CML samples (n=17) were spotted on slides and examined by FISH for the presence of the BCR-ABL gene. Results The proportion of 6-week LTC-ICs that was Ph+/BCR-ABL+ ranged from <5% to 100%. Although the CML LTC-ICs represented >80% of the LTC-ICs in 36% of the 33 cases studied, these represented <50% in 45% of these cases. Ph+/BCR-ABL+ LTC-ICs were more prevalent when measured with the 5-week LTC-IC assay (86±7% Ph+/BCR-ABL+) than with the 6-week LTC-IC assay (11±8% Ph+/BCR-ABL+, n=10). FISH analysis of the initial CD34+CD38- cells showed that >80% of these were BCR-ABL+ in 70% of the 17 cases studied. Notably, these latter values were not correlated with the proportion of leukemic 6-week LTC-ICs in the same samples (Spearman rank correlation r = 0.43, p = 0.08). Primary NSG mice transplanted with CD34+ cells from the patient with no detectable normal LTC-ICs regenerated almost exclusively differentiated human myeloid cells for up to 35 weeks and at increasing levels at the later time points (35% of total marrow cells after 35 weeks). Cells obtained 8 and 35 weeks post-transplant showed these contained readily detectable clonogenic cells which were exclusively BCR-ABL+ (84 genotyped colonies). Secondary recipients were again repopulated with exclusively BCR-ABL+ myeloid cells. Recipients of the second sample showed a transient early peak of myeloid cells followed by a peak of B lymphoid cells at 8 weeks, at which time only 20% of the human CFCs present were BCR-ABL+. At 35 weeks post-transplant, human cells were still detectable in these mice (5±3% of the marrow) and myeloid cells had again become the predominant lineage with BCR-ABL+ cells detectable by qRT-PCR but no CFCs were identified. Secondary recipients of these cells were reconstituted with myeloid cells but only transiently. Conclusion Functionally defined chronic phase CML stem cells represent a very minor subset of the CD34+CD38- compartment and assessment of these cells, like assessment of the most commonly used 5-week LTC-IC assay, overestimates the BCR-ABL+ stem cell compartment. Both of these endpoints also fail to provide a reliable indicator of the prevalence of BCR-ABL+ stem cells defined by more stringent functional assays. We also show for the first time that BCR-ABL+ CML stem cells are capable of serial transplantability spanning one and a half years in NSG mice and this may be anticipated by results from the 6-week LTC-IC assay. Disclosures: No relevant conflicts of interest to declare.

Telomere Shortening At Diagnosis Of Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Is Significantly More Pronounced In Patients With Less-Than-Optimal Versus Optimal Molecular Response (ELN criteria) After 12 Months Of Treatment With Nilotinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1471-1471
Author(s):  
Katrin Wenn ◽  
Lena Tomala ◽  
Fabian Beier ◽  
Stefan Wilop ◽  
Lucia Vankann ◽  
...  
Keyword(s):  
Telomere Length ◽  
Board Of Directors ◽  
Research Funding ◽  
Chronic Phase ◽  
Response To Treatment ◽  
First Line ◽  
Advisory Committees ◽  
Telomere Shortening ◽  
Tki Treatment ◽  
First Line Treatment

Abstract Introduction Chronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the BCR-ABL translocation. Telomere length (TL) reflects the replicative history of eukaryotic cells and progressive telomere shortening is associated with genetic instability. Clinically, accelerated telomere shortening has been demonstrated in patients with CML and was found to correlate with disease progression and clinical risk score in the pre-tyrosine kinase inhibitor (TKI) era. The aim of the current study was to investigate whether telomere length (TL) at diagnosis might predict response to treatment in patients receiving nilotinib as first line treatment of chronic phase CML on the ENEST1st study (NCT01061177) Methods and Patients TL analysis of peripheral blood leukocytes was analyzed using monochrome multiplex quantitative PCR in blood samples from 93 newly diagnosed CML patients enrolled on study in Germany. One extreme outlier was excluded from the analysis. 89 healthy controls were used for age-adaption of TL. Median age of the analyzed CML patients was 49.6 years (range: 19-83), Sokal (32 low, 31 intermediate, 13 high risk) and Euro (34 low, 36 intermediate, 6 high risk) scores were available in 76 patients. Response to treatment according to standard criteria was available at 3 month (mo, n=79 patients), 6 mo (n=75), 12 mo (n=71) and 18 mo (n=55) after treatment start. Results Mean age-adjusted TL in CML patients was significantly shortened compared to normal individuals (ΔT/S ratio: -0.30 +/- 0.67, p=<0.001). Interestingly, whereas TL followed an expected linear decline over time in the control population of healthy individuals, this age-correlation was no longer detectable in CML patients pointing to a significantly more pronounced TL deficit in younger as opposed to older patients with CML. In univariate analysis, no significant correlation between age-adjusted TL at diagnosis and Euro or Sokal risk score nor with any standard individual prognostic parameters was detected with the exception of the peripheral blast count which was shown to be inversely correlated with age adjusted telomere (ΔT/S ratio) length by linear regression (p=0.01). When ΔT/S ratio measured at diagnosis was correlated with response to treatment at defined time points according to the ELN criteria 2013 (i.e. <10% BCR-ABL/ABL ratio at 3 mo, <1% at 6 mo, and <0.1% at 12 mo), we could demonstrate that while all cohorts had shortened telomere length compared to age-adjusted controls, less-than-optimal responders had more accelerated telomere shortening compared to optimal responders, i.e. ΔT/S -0.66 (n=4) vs. -0.25 (n=75) at 3 mo; ΔT/S -0.37 (n=10) vs. -0.28 (n=65) at 6 mo and ΔT/S -0.67 (n=15) vs. -0.21 (n=56) at 12 mo. While this pattern seems rather consistent, only the difference at the 12 mo time point reached statistical significance (p=0.028), potentially due to the imbalance of the groups induced by the low number of less-than-optimal responders to treatment according to ELN criteria observed under nilotinib first line treatment. Conclusions These data were generated on the first prospective study of the role of telomere length as a potential biomarker for TKI treatment in CML. We could confirm that TL is significantly shortened in CML patients at diagnosis. Even more so, telomere shortening seems to be significantly more accelerated in younger as opposed to older patients pointing to a threshold value of telomere length in CML similar to observations in patients with bone marrow failure syndromes. Furthermore, telomere shortening tends to be more pronounced in patients not meeting the criteria for optimal response according to ELN 2013. Further follow up will reveal whether TL has the potential to serve as a long-term predictive biomarker for frequency and durability of response as well as for the sustainability of treatment cessation in CML patients responding well to TKI treatment. Disclosures: Frank: Novartis: Employment. Walasek:Novartis: Employment. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Giles:Novartis: Consultancy, Research Funding. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bristol Myer Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy.

scholarly journals Stem Cell Biomarkers in Chronic Myeloid Leukemia

2008 ◽  
Vol 24 (4-5) ◽  
pp. 201-216 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Yun Zhao ◽  
Donna Forrest ◽  
Clayton Smith ◽  
Allen Eaves ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Myeloproliferative Disease ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Stem Cell Compartment ◽  
Treatment Responses

Chronic myeloid leukemia (CML) is a clonal multi-step myeloproliferative disease that is initially produced and ultimately sustained by a rare subpopulation of BCR-ABL+ cells with multi-lineage stem cell properties. These BCR-ABL+ CML stem cells are phenotypically similar to normal hematopoietic stem cells which are also maintained throughout the course of the disease at varying levels in different patients. Defining the unique properties of the leukemic stem cells that produce the chronic phase of CML has therefore had to rely heavily on access to samples from rare patients in which the stem cell compartment is dominated by leukemic elements. Here we review past and ongoing approaches using such samples to identify biologically and clinically relevant biomarkers of BCR-ABL+ stem cells that explain their unusual biology and that may help to design, or at least predict, improved treatment responses in CML patients. These studies are of particular interest in light of recent evidence that chronic phase CML stem cells are not only innately resistant to imatinib mesylate and other drugs that target the BCR-ABL oncoprotein, but are also genetically unstable.

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