Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

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A Heterogeneous Response Pattern to Interferon-alpha2 with Induction of a Significant Decrease in the Calreticulin Mutant Allele Burden in a Subset of Patients with Essential Thrombocythemia and Primary Myelofibrosis

Blood ◽

10.1182/blood.v126.23.4057.4057 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4057-4057

Author(s):

Sabrina Cordua ◽

Lasse Kjaer ◽

Morten Orebo Holmström ◽

Niels Pallisgaard ◽

Vibe Skov ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Allele Burden ◽

Travel Grant ◽

Ifn Treatment

Abstract Introduction The discovery of mutations in the calreticulin (CALR) gene in the majority of JAK2 -V617F negative patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) (Klampfl et al., 2013; Nangalia et al., 2013) has improved the diagnostic accuracy considerably, and most recently distinct clinical and hematological characteristics according to mutational status have been described (Park et al., 2015). The perspective is to personalize and optimize treatment according to the molecular and clinical landscape. This may be achieved by obtaining more information on responses in myeloproliferative neoplasms (MPN) to existing treatment strategies as assessed by the allele burden. Mutations in the CALR gene have proven to play a major role in oncogenic and immunologic processes (Lu, Weng, & Lee, 2015). In this context, it is highly relevant to explore the effectiveness of interferon-alpha2 (IFN) in reducing the CALR -mutated clone. Until now, only one paper has reported a decrease in allele burden in two patients during IFN treatment (Cassinat, Verger, & Kiladijan, 2014). The objective of this report is to expand current knowledge on this important topic by describing the mutant CALR allele burden over time in a larger group of IFN-treated patients. Method Clinical data were collected retrospectively from a single institution on all IFN-treated CALR positive MPN patients with sequential determinations of the mutant allele burden. Type 1 and type 2 mutations were initially identified by a previously published fragment analysis (Klampfl et al 2013). We have developed a Taqman qPCR assay for precise determination of the mutant allele burden of type 1 and type 2 mutations. Stored DNA was subsequently analysed to increase follow-up time. Results Twenty-one patients were included. Fifteen patients had a diagnosis of PMF; 7 of these were diagnosed with prefibrotic myelofibrosis. Six patients had ET. The type 1 and 2 mutations were found in 15 and 6 patients, respectively. Median age was 60 years (range 42-79) and the sex ratio (M/F) was 8/13. Fifteen patients (71%) were in ongoing treatment with IFN, whereas treatment was discontinued in 6 (29%) because of side effects. Median time of IFN treatment was 756 days (range 42-3927). The IFN prescribed was either subcutaneous injection of Pegasys® (median: 45 microgram (ug) per week), PegIntron® 25-50 ug per week, or Multiferon® 3 x 3 million IU per week. Median follow up time since the first CALR measurement was 756 days (range 294-2108). Fourteen patients (67%) maintained an unchanged allele burden during follow up; 1 patient (5%) presented a temporary decrease (from 39% to 27% in allele burden) but increased to the initial level within months while still on IFN treatment (presumably due to low compliance); 1 patient (5%) displayed an increase in allele burden during transformation to acute myelogenous leukemia (Figure 1); and 5 patients (24%) exhibited a marked decrease in allele burden (median decrease: 32%, range 18-45) during treatment with IFN (Figure 2). All 5 patients with decreasing allele burden (Table 1) normalized their platelet counts within a median time of 5 weeks (range 4-20) after initiating treatment with IFN. Conclusion Using a novel sensitive assay for the CALR mutant allele burden, we have demonstrated and substantiated the effectiveness of IFN to reduce the allele burden in a larger series of CALR positive patients with PMF and ET. Importantly, we report for the first time on highly heterogeneous response patterns. Our observation of one fourth of the CALR positive patients responding to treatment with IFN strongly suggests that IFN significantly influences the CALR mutational load. Further clinical and molecular studies are urgently needed to explore the mechanisms behind the heterogeneous response patterns and the clinical implications in regard to clonal evolution and disease progression in non-responding patients. We are currently analysing these issues to assess the definite role of IFN in future treatment strategies in CALR positive MPN patients. Table 1. Patients responding to interferon-alpha2 Characteristics Number/median (range) Patients 5 Age, years 53 (42-62) Sex (M/F) 1/4 Diagnosis- Essential thrombocythemia- Primary myelofibrosis- Prefibrotic myelofibrosis 221 Calreticulin mutation type- type 1- type 2 50 Duration of interferon-alpha2 treatment, days 960 (177-2790) Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Cordua: Janssen-Cilag: Other: travel grant. Off Label Use: interferon alpha2 for myeloproliferative neoplasms. Holmström:La Roche Ltd: Other: travel grant. Pallisgaard:Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Novartis: Other: travel grant, Research Funding, Speakers Bureau; Roche: Other: travel grant. Hasselbalch:Novartis: Research Funding.

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Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms

Blood ◽

10.1182/blood-2013-02-486050 ◽

2013 ◽

Vol 121 (21) ◽

pp. 4388-4395 ◽

Cited By ~ 65

Author(s):

Elisa Rumi ◽

Daniela Pietra ◽

Paola Guglielmelli ◽

Roberta Bordoni ◽

Ilaria Casetti ◽

...

Keyword(s):

Loss Of Heterozygosity ◽

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Neutral Loss ◽

Molecular Event ◽

Allele Burden ◽

Chromosome 1P ◽

Key Points ◽

Exon 10 ◽

Marrow Fibrosis

Key Points In MPL exon 10–mutated myeloproliferative neoplasms, the MPL-mutant allele burden varies considerably from about 1% to almost 100%. High mutation burdens originate from acquired copy-neutral loss of heterozygosity of chromosome 1p and are associated with marrow fibrosis.

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Lysine-Specific Histone Demethylase, LSD1, (KDM1A) As a Novel Therapeutic Target in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.601.601 ◽

2015 ◽

Vol 126 (23) ◽

pp. 601-601 ◽

Cited By ~ 2

Author(s):

Maria Kleppe ◽

Kaitlyn Shank ◽

Papalexi Efthymia ◽

Hugh Riehnhoff ◽

Ross L. Levine

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Serum Levels ◽

Advisory Committees ◽

Allele Burden ◽

Circulating Cells ◽

Cell Counts ◽

White Blood Cell Counts ◽

The Impact

Abstract Among BCR-ABL-negative myeloproliferative neoplasms (MPN), primary myelofibrosis (PMF) and post PV/ET myelofibrosis (MF) are associated with the highest degree of morbidity and mortality, including progressive bone marrow (BM) fibrosis and resultant BM failure. Although the JAK inhibitor ruxolitinib is now approved for the treatment of MF-associated splenomegaly and systemic symptoms, JAK inhibitor therapy does not reduce the proportion of JAK2-mutant cells in MPN patients. The limited ability of JAK inhibition to induce molecular or clinicopathologic responses in the majority of MPN patients underscores the need for the development of more effective therapies for JAK kinase-dependent malignancies. Recent studies have shown that the lysine-specific histone demethylase, LSD1 (KDM1A), participates in the balance between proliferation and differentiation in vivo by influencing state-specific gene expression patterns. In physiologic hematopoiesis, LSD1 is essential for normal myeloid differentiation affecting the erythroid, megakaryocytic and granulocytic lineages. Small molecule inhibitors of LSD1 have shown promising results in preclinical models of acute myeloid leukemia (AML) and solid cancers and have recently entered clinical trials in AML. However, the role and requirement for LSD1 in the pathogenesis of MPNs and the therapeutic targeting of LSD1 in MPN has not been investigated. In this study, we first tested the effects of IMG-98, a potent, selective LSD1 inhibitor, in the MPLW515L-driven ET/MF mouse model. After disease was established, mice were treated with IMG-98 or vehicle for 28 days. LSD1 inhibition in mice markedly suppressed myeloproliferation reducing granulocyte counts and spleen weights compared to mice treated with vehicle thus establishing therapeutic efficacy (Fig. 1a). Pathologic analysis of BM and spleen confirmed a marked reduction in myeloproliferation as well as a reversal of extramedullary hematopoiesis (EMH). Most notably, we observed a marked reduction in reticulin fibrosis with IMG-98 treatment (Fig. 1b). We next investigated the impact of IMG-98 therapy on inflammatory cytokine signaling; in contrast to the broad anti-cytokine effects of JAK1/2 inhibition, we observed a more specific anti-cytokine effect of IMG-98, a significant reduction in the secretion of the inflammatory cytokine Cxcl5 (Fig. 1c), a key participant in pathologic inflammatory states. We then investigated the in vivo impact of IMG-98 therapy on mutant disease burden. IMG-98 therapy reduced mutant allele burden to a degree not seen with JAK1/2 inhibitor therapy: whereas 74.6% of circulating cells in mice treated with vehicle were GFP-positive cells, only 43.2% of circulating cells were GFP-positive in IMG-98-treated mice (Fig. 1d). Flow cytometry analysis of spleen and BM revealed reduced numbers of CD11b/Gr1-positive myeloid cells and CD41-positive megakaryocytes. The numbers of mutant GFP-positive myeloid cells and megakaryocytes in these tissues were also significantly reduced by IMG-98 treatment. Studies of the impact of LSD1 inhibition on MPN stem cell function and on epigenetic regulation in MPN cells will be presented in detail. In summary, the LSD1 inhibitor IMG-98 had a highly significant therapeutic effect in an established preclinical model of ET/MF. LSD1 inhibition in diseased mice reduced JAK-STAT-driven myeloproliferation, markedly reversed EMH and BM fibrosis, and reduced the mutant clone burden. These data suggest LSD1 is a valid target in MPN and that clinical studies of LSD1 inhibitor IMG-98 alone and in combination with JAK inhibitors are warranted. Figure 1. a, b) LSD1 inhibition results in reduced white blood cell counts (WBC) and platelet counts (PLT). (a), and in near-complete elimination of BM fibrosis (b). c) Profound reduction of Cxcl5 serum levels in IMG-98 treated mice compared to vehicle treated mice. d) Significantly lower mutant allele burden in the peripheral blood of IMG-98 treated mice. * P<0.05, n =5. Figure 1. a, b). LSD1 inhibition results in reduced white blood cell counts (WBC) and platelet counts (PLT). (a), and in near-complete elimination of BM fibrosis (b). c) Profound reduction of Cxcl5 serum levels in IMG-98 treated mice compared to vehicle treated mice. d) Significantly lower mutant allele burden in the peripheral blood of IMG-98 treated mice. * P<0.05, n =5. Disclosures Riehnhoff: Imago: Employment, Equity Ownership. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

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Prevalence of the JAK2 V617F Mutation in Patients with Peripheral Arterial Disease

Blood ◽

10.1182/blood.v124.21.3165.3165 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3165-3165

Author(s):

Elena Kinz ◽

Klaus Gasser ◽

Axel Muendlein ◽

Andreas Leiherer ◽

Michael Steurer ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Arterial Disease ◽

Jak2 V617f Mutation ◽

Allele Burden ◽

Cell Counts ◽

V617f Mutation ◽

Artery Disease ◽

Peripheral Arterial

Abstract Introduction: The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms and increases thrombotic risk. We previously showed that JAK2 V617F is also found in healthy subjects as well as in patients with coronary artery disease (0.6% and 1.3%, respectively). Peripheral arterial disease (PAD) is an important manifestation of diffuse atherosclerosis and PAD patients are at exceptionally high risk for cardiovascular events, showing a worse prognosis than that of patients with coronary artery disease Due to the close relation of the JAK2 V617F mutation to thrombotic events we hypothesized that this mutation may play an important role in the risk management of PAD patients. However, prevalence of JAK2 V617F or of occult myeloproliferative neoplasms is unknown in PAD patients. Methods: In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD. JAK2 mutational status from 997 age-matched healthy people was available from a previous study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with allele-specific quantitative real-time PCR. Results: From a total of 287 PAD patients samples, 9 (3.1%) were tested positive for JAK2 V617F mutation corresponding to a 5-fold, highly significant increase compared with healthy people (p<0.001). Mutant allele burden of JAK2 V617F positive samples was ranging between 0.2% and 96.2% (median=0.75%). Generally, our study showed no significant association of the JAK2 V617F mutation with abnormal blood cell counts. However, the patient with the highest mutant allele burden showed elevated hemoglobin values (> 18.5 g/dL) indicating polycythemia vera (PV). Conclusion: We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. For this reason mutation analysis should be considered in PAD patients with abnormal blood cell counts to identify occult myeloproliferative neoplasms and to adjust therapeutic treatment, possibly reducing the risk of future vascular complications. Disclosures No relevant conflicts of interest to declare.

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A Rapid, Sensitive and Specific Method for Quantifying Calr Mutant Allele Burden in Persons with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.5210.5210 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5210-5210

Author(s):

Jiao Zhou ◽

Qiumei Yao ◽

Robert Peter Gale ◽

Jinlan Li ◽

Lingdi Li ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Mutation Screening ◽

Heterozygous Mutation ◽

Specific Method ◽

Wild Type ◽

Allele Burden ◽

Exon 9 ◽

Calr Mutations ◽

Calr Mutation

Abstract Background: CALR mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive and specific methods to detect and quantify these mutations are needed. Methods: We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. Results: We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. Conclusions: PCR amplification followed by fragment length analysis is a rapid, sensitive and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PV with JAK2V617F and for estimating mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Disclosures No relevant conflicts of interest to declare.

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Clonal Analysis of SF3B1, JAK2 and MPL in Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis

Blood ◽

10.1182/blood.v120.21.172.172 ◽

2012 ◽

Vol 120 (21) ◽

pp. 172-172

Author(s):

Ilaria Ambaglio ◽

Anna Gallì ◽

Daniela Pietra ◽

Matteo G Della Porta ◽

Marta Ubezio ◽

...

Keyword(s):

Mutant Allele ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Refractory Anemia ◽

Wild Type ◽

Mutation Status ◽

Allele Burden ◽

Ring Sideroblasts ◽

Exon 10

Abstract Abstract 172 Somatic mutations of the RNA splicing machinery have been recently identified in patients with myelodysplastic syndrome (MDS). In particular, a strong association has been found between SF3B1 mutation and the MDS subtype defined as refractory anemia with ring sideroblasts (RARS). Similarly, within myelodysplastic/myeloproliferative neoplasms (MDS/MPN) a high prevalence of SF3B1 mutations has been reported in the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). These findings strongly support a causal relationship between SF3B1 mutations and ring sideroblasts. Interestingly, a high proportion of RARS-T patients also harbor JAK2 and/or MPL mutations. The available evidence suggests that somatic mutations of SF3B1 might be an early pathogenetic event determining myelodysplastic features, and that subsequent occurrence of JAK2 and/or MPL mutations may cause the myeloproliferative phenotype. In this work, we studied the mutation status of SF3B1, JAK2 and MPL in circulating granulocytes and bone marrow cells from RARS-T patients. We also studied the in vitro growth of hematopoietic progenitors (BFU-E, CFU-GM), and genotyped individual colonies to examine the mutation status of the above genes. The coding exons of SF3B1 were screened using massively parallel pyrosequencing. A real time PCR-based allelic discrimination assay was used for the detection of JAK2 (V617F), while Sanger sequencing was employed for JAK2 exon 12 and MPL exon 10 mutation analysis. Twenty-eight patients affected with RARS-T were assessed for SF3B1, JAK2 and MPL exon 10 mutation status. Eighteen patients (64%) showed somatically acquired mutation of SF3B1. The median mutant allele burden was 43%, consistent with the presence in the majority of patients of clonal hematopoiesis characterized by a dominant clone carrying a heterozygous SF3B1 mutation. Fourteen patients carried the JAK2 (V617F) mutation (median allele burden 6.5%, range 0.4–29.5%), while one had a JAK2 exon 12 mutation. In 13 cases, the JAK2 mutation was detected at the time of diagnosis, whereas in 2 patients, who had a typical RARS phenotype and were negative for JAK2 mutations at clinical onset, JAK2 (V617F) was detected 18 and 32 months after diagnosis, respectively, and concomitantly with a progressive increase in platelet count. Four patients, two of whom were JAK2 (V617F)-positive, carried the MPL (W515L) mutation (median allele burden 27.5%, range 25–50%). Concomitant mutations of SF3B1 and JAK2 or MPL were observed in 8 cases. Seven patients carried an SF3B1 mutation and JAK2 (V617F), while one carried SF3B1 (K700E), JAK2 (V617F), and MPL (W515L). In all these cases, the SF3B1 mutant allele burden was higher than that of JAK2 or MPL, indicating the existence of an SF3B1-mutated dominant clone with minority JAK2- or MPL-mutated clones. We genotyped individual colonies from peripheral blood in 2 patients with concomitant mutations. In a patient with granulocyte SF3B1 and JAK2 mutant allele burdens equal to 45% and 8%, respectively, SF3B1 (H662Q) was detected in 9 of 11 colonies, three of which also carried JAK2 (V617F); the remaining two colonies had wild type SF3B1 and JAK2. These data are consistent with the existence of a dominant hematopoietic clone carrying the SF3B1 mutation and the subsequent emergence of a JAK2-mutated subclone. The other patient, who was initially SF3B1- mutated and JAK2 wild type, at the time of colony assay had a mutant allele burden equal to 50% and 1% for SF3B1 (K700E) and JAK2 (V617F), respectively. Forty-three of 45 colonies were heterozygous for SF3B1 (K700E) and wild type for JAK2. The opposite pattern was observed in the remaining 2 colonies, which carried just JAK2 (V617F). These data indicate the coexistence of two distinct clones, a dominant one carrying the SF3B1 mutation and a minority one carrying JAK2 (V617F). In summary, these observations suggest that the occurrence of an SF3B1 mutation represents an early event in patients with RARS-T, likely causing mitochondrial iron overload, ring sideroblasts, ineffective erythropoiesis and anemia, typical myelodysplastic features. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the emergence of minority clones and the acquisition of myeloproliferative features. JAK2- mutated clones may emerge as subclones of the dominant SF3B1-mutated clone or as independent clones. Disclosures: No relevant conflicts of interest to declare.

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A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms

Hematology ◽

10.1179/1607845415y.0000000009 ◽

2015 ◽

Vol 20 (9) ◽

pp. 517-522 ◽

Cited By ~ 6

Author(s):

Qiu-Mei Yao ◽

Jiao Zhou ◽

Robert Peter Gale ◽

Jin-Lan Li ◽

Ling-Di Li ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Accurate Method ◽

Allele Burden

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Characterization of CD34+ Hematopoietic Progenitor Cells in JAK2V617F and Calr-mutated Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v124.21.4572.4572 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4572-4572

Author(s):

Anna Angona ◽

Alberto Alvarez ◽

Raquel Longarón ◽

Laura Camacho ◽

Concepción Fernández ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Mutant Allele ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Differentiation Potential ◽

Allele Burden ◽

Cd34 Cells

Abstract Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells. Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN. Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results: CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p<0.001). The mutant allele burden in granulocytes and CD34+ cells was higher in patients with JAK2V617F-positive MF than in those with CALR-mutated MF (CD34+CD38-: 71% vs 47.2% p=0.05, CD34+CD38+: 68.4% vs 40.6% p=0.018, granulocytes: 76.9% vs 53.7% p=0.05). Finally, we could demonstrate that the mutant load was lower in CALR-mutated ET patients than in CALR-mutated MF at progenitor level and in granulocytes (CD34+CD38-: 30.6% vs 47.1% p=0.08, CD34+CD38+: 17.8% vs 40.6% p=0.03, granulocytes: 29.6% vs 53.7% p=0.004). Conclusion: CALR-mutated ET patients have a higher mutant load in CD34+CD38- than JAK2V617F-positive ET and PV patients, whereas the JAK2V617F-positive hematopoietic progenitor cells have more differentiation potential than those CALR-mutated. Moreover, in the MF phase of MPN, the expansion of the mutated clone at the progenitor level is greater in JAK2V617F-positive than in CALR-mutated patients. Disclosures No relevant conflicts of interest to declare.

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Effects of the Sympathicomimetic Agonist Mirabegron on Disease Course, Mutant Allele Burden, Marrow Fibrosis, and Nestin Positive Stem Cell Niche in Patients with JAK2-Mutated Myeloproliferative Neoplasms. a Prospective Multicenter Phase II Trial SAKK 33/14

Blood ◽

10.1182/blood.v128.22.3108.3108 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3108-3108 ◽

Cited By ~ 3

Author(s):

Beatrice Drexler ◽

Jakob Passweg ◽

Martin Bigler ◽

Alexandre PA Theocharides ◽

Nathan Cantoni ◽

...

Keyword(s):

Bone Marrow ◽

Phase Ii ◽

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Nerve Fibers ◽

Allele Burden ◽

Sympathetic Nerve Fibers ◽

Bone Marrow Histology ◽

Cell Niche

Abstract Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by aberrant proliferation of erythroid, megakaryocytic and myeloid lineages. They are associated with decreased survival, thromboembolic complications, hemorrhage and leukemic transformation. MPN can be subdivided into polycythemiavera(PV), essentialthrombocythemia(ET) and primary myelofibrosis (PMF). The JAK2-V617F mutation is present in 70-80% of all MPN patients. MPN is initiated and maintained by mutated hematopoietic stem and progenitor cells (HSPC). Bone marrow mesenchymal stem cells expressing the intermediate filament proteinnestin(nestin+ MSCs) that are innervated by sympathetic nerve fibers constitute an important component of the stem cell niche and regulate normal HSCs. Thesenestin+ MSCs are strongly reduced in bone marrow of JAK2-V617F positive MPN patients and in mice expressing JAK2-V617F due to damage of the sympathetic nerve fibers triggered by cytokines from the mutant cells. In a JAK2-V617F mouse model of MPN, treatment with a beta-3sympathicomimeticagonist corrected the damage inflicted by the MPN clones on their niches and ameliorated the MPN phenotype. To test the potentially beneficial effect on disease-control by modulating bone marrow niche cells in patients with MPN, we performed a phase II trial with the beta-3sympathicomimeticagonistmirabegron. Patients and Methods: The trial consisted ofmirabegrontreatment with 25 mg daily during the first week, followed by 50 mg daily for at least 24 weeks. Patients with acytohistologicallyconfirmed diagnosis of MPN and a JAK2-V617F allele burden >20% in granulocytes at study entry were eligible, if not treated with JAK2 inhibitors or interferon. Reduction of the JAK2-V617F mutant allele burden ³50% in granulocytes was defined as the primary end point. Secondary end points included changes in blood counts or MPN related symptoms. As a side study, bone marrow biopsies were quantified fornestin+ MSCs, fibrosis and CD34+ HSPCs. N=39 patients have been accrued in 10 institutions in Switzerland. Eight (21%) had ET, 22 (56%) PV, and 9 (23%) PMF. N=27 (69%) were male, the median age was 62 (Q1-Q3 53-72) years. Median mutated allele burden at study onset was 52% (Q1-Q3 33-73%). All patients had prior treatment, N=28 (72%) patients hadcytoreductivetreatment, the remaining patients hadantiaggregation, anticoagulation or phlebotomy. Results: No patient reached the primary endpoint of 50% reduction in allele burden, one patient achieved a 25% reduction by 24 weeks of treatment. Adverse events were mostly grade I or II on the CTCAE scale. Three patients had grade III events: two were considered to be at least possibly related to study medication. In the side study, 24 patients agreed to bone marrow biopsy prior to and at the end ofmirabegrontreatment and for 20 patients both measurements are available. In these patients an increase in thenestin+ MSCs cells from a median of 1.09 (Q1-Q3 0.38-3.27)/mm2 to 3.95 (Q1-Q3 1.98-8.79)/mm2 (p<0.0001, Wilcoxon signed-rank test) and a slight decrease of myelofibrosis from a median grade of 1.00 (Q1-Q3 0.50-3.00) to 0.75 (Q1-Q3 0.50-2.00) (p=0.02), were observed. The mean change in thenestin+ cells from baseline to week 24 was 3.52 (95% confidence interval 1.65-5.39)/mm2. Morphometric changes in thenestin+ MSCs were significant for PV (n=13, p=0.007) and PMF (n=5, p=0.04). Bone marrow CD34+ cells slightly increased from a median 2.50 (Q1-Q3 2.00-3.25) to 3.00 (Q1-Q3 2.00-3.75) (p=0.06). Conclusion: In this prospective phase II clinical trial treatment with the beta-3-sympathicomimetic agonistmirabegronfor 24 weeks failed to achieve the primary endpoint to reduce the JAK2-V617F mutant allele burden >50% in patients with MPN. However, an increase in thenestin+ MSCs in bone marrow and a slight decrease of myelofibrosis were found, which will be further investigated. Figure 1 Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Figure 1. Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Disclosures Theocharides: Novartis: Consultancy, Honoraria. Rüfer:Novartis: Consultancy, Speakers Bureau. Benz:Celgene: Consultancy. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau. Skoda:Novartis: Consultancy, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Consultancy, Speakers Bureau.

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