scholarly journals Mutational Landscape in Myeloproliferative Neoplasms: Implications on Prognosis and Clinical Management

2021 ◽  
Vol 19 (11.5) ◽  
pp. 1362-1364
Author(s):  
Aaron T. Gerds
Keyword(s):  
Disease Progression ◽  
Clinical Management ◽  
Myeloproliferative Neoplasms ◽  
Diagnostic Classification ◽  
Mutational Landscape

Mutations are a critical piece in understanding how myeloproliferative neoplasms (MPNs) occur, specifically the pathobiology of JAK/STAT activation. Mutations play such an important role, in fact, that they are a key part of the diagnostic classification for these diseases. Furthermore, the mutational landscape of MPNs affects both the prognosis and the biology of disease progression. Current research in the field is focused on understanding how and why these mutations occur, as well as how to attack them to address disease at the time of progression or even before disease progression has occurred.

Outcomes of Patients with Myelodysplastic Syndromes/Myeloproliferative Neoplasms with Emphasis On MDS/MPN-Unclassified

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5025-5025
Author(s):  
Charikleia Kelaidi ◽  
Varnavas Constantinou ◽  
George Papaioannou ◽  
Niki Stavroyianni ◽  
Chrysanthi Vadikoliou ◽  
...  
Keyword(s):  
Disease Progression ◽  
Myelodysplastic Syndromes ◽  
Response Rate ◽  
Conflicts Of Interest ◽  
Systemic Mastocytosis ◽  
Myeloproliferative Neoplasms ◽  
Univariate Analysis ◽  
Jak2 V617f ◽  
Female Ratio ◽  
Wbc Count

Abstract Abstract 5025 Background: Data on outcomes of patients (pts) with myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN), especially MDS/MPN-unclassified (MDS/MPN-U), are scarce. Patients/methods: We retrospectively studied pts followed in our center, with MDS/MPN according to WHO 2008 criteria. Because of overlap characteristics of MPN and MDS, pts with systemic mastocytosis associated with MDS (SM/MDS) were also included. Pts with previous MDS or MPN were excluded. Response and disease progression were defined according to IWG 2006 criteria. Results: Twenty-five pts with MDS/MPN were included. Median age was 70 y (range 19–79). Male/female ratio was 1.77/1. Diagnosis was CMML-1 N=7, CMML-2 N=7, JMML N=1, MDS/MPN-U N=8, systemic mastocytosis (SM)/MDS N=2, with one additional pt with CMML subsequently developing SM. At diagnosis, median WBC count was 18.8 G/L (range 3–120), ANC 15.5 G/L (0.6–70), monocytes 1.9 G/L (0.1–16), left shift 16% (0–28), Hb 11.2 g/dL (6–17), platelets 99 G/L (10–680), peripheral and bone marrow (BM) blasts 5% (0–17) and 7% (2–19), respectively (resp.). 25% of pts had platelets count ≥400 G/L. Splenomegaly, B-symptoms and BM fibrosis were present in 23%, 57% and 27% of pts, resp. Karyotype was fav, int and unfav in 55%, 36% and 9% of pts, with −7, +8, del(12)(p11), del(12)(q14;q21), +10, +21, and previously unreported t(9;12)(q13;q13) in 3, 6, and 1 pt each, resp., while +21 and i(17)(q10) appeared during disease progression other than AML transformation. IPSS was low/int-1 and int-2/high in 50% and 50% of pts, resp. JAK2 V617F and CKIT D816V mutations were detected in 2/6 pts and 2/2 SM/MDS pts, resp. 70% and 29% of pts were transfused at diagnosis with PRBC and platelets, resp. Treatment included erythropoiesis stimulating agents (ESAs), low dose chemotherapy, intensive chemotherapy (IC) and azacitidine (AZA) in 40%, 36%, 16% and 48% of pts resp. Response rate to ESAs, IC and AZA was 60%, 14% and 14% resp. Response rate to AZA in CMML-1 pts was 33%. Dasatinib yielded no response in 1 SM/MDS pt with CKIT D816V. 3-year cumulative incidence of AML and median overall survival (OS) in pts with CMML-1, CMML-2 and MDS/MPN-U were 20%, 40% and 0 (P=0.059) and 39, 8, and 20 mo (P=0.50), resp. The pt with JMML died from AML transformation 3 months after diagnosis. 2/3 pts with SM/MDS died from disease progression w/o AML at a median of 10 mo after diagnosis. Median survival after disease progression other than AML transformation was 35, 15 and 14 mo in pts with CMML-1, CMML-2 and MDS/MPN-U, resp. (P=0.88). Cause of death was disease progression other than AML, AML transformation and unrelated to disease in 50%, 50%, and 0 and 80%, 0 and 20% of cases in CMML and MDS/MPN-U, resp. (P=0.10). Percentage of circulating blasts ≥5% was the only independent factor affecting risk of AML transformation in the overall population (P=0.0004). Diagnosis other than CMML-1, WBC ≥30 G/L, % of circulating blasts ≥5% and IPSS high/int-2 were associated with worse survival in univariate analysis (P=0.06, 0.03, 0.04 and 0.08, resp.). No predictive factor of OS was found in multivariate analysis. Conclusion: MDS/MPN are heterogeneous disorders with respect to disease progression and AML transformation. MDS/MPN-U tended to differ from CMML-1 by shorter survival after disease progression other than AML, and from CMML-2 by lower risk of AML transformation. Mortality of pts with MDS/MPN-U was mainly attributed to disease progression without AML transformation. Alternatively to hypomethylating agents, therapeutic options in pts with MDS/MPN-U could include JAK2 inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 482-482
Author(s):  
Koki Ueda ◽  
Kazuhiko Ikeda ◽  
Kazuei Ogawa ◽  
Akiko Shichishima-Nakamura ◽  
Kotaro Shide ◽  
...  
Keyword(s):  
Disease Progression ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Epigenetic Modifiers ◽  
One Year

Abstract Myeloproliferative neoplasms (MPN) are characterized by chronic proliferation of myeloid cells, extramedullary hematopoiesis and occasional leukemic transformation. Mutations in JAK2, CALR and MPL have been established as drivers of myeloproliferative phenotype, but their roles in disease progression with clonal expansion remain unclear. In addition, studies have shown mutations in epigenetic modifiers including TET2, DNMT3A, ASXL1 and EZH2, and aberrant expressions of microRNAs in MPN, but downstream of these changes is also largely unknown. Recently, we showed high expression of HMGA2 mRNA partly correlated with reduced microRNA let-7 in granulocytes of patients with MPN, including 100% patients with primary myelofibrosis (MF) and 20% polycythemia vera and essential thrombocythemia (Harada-Shirado et al, Brit J Haematol, 2015). In mice, loss of epigenetic modifiers such as BMI1 and EZH2, along with the Arf/Ink4a knockout (Oguro et al, J Exp Med, 2012) or the JAK2 V617F (Sashida et al, ASH, 2013), leads to overexpression of HMGA2 with accelerating MPN. We have generated transgenic (Tg) mice of Hmga2 cDNA with truncated 3'UTR (ΔHmga2) lacking binding sites of let-7 thatrepresses expression of HMGA2 (Ikeda et al, Blood, 2011). Δ Hmga2 mice overexpress HMGA2 and develop MPN-like disease, and represent a clonal advantage in competitive repopulations with serial bone marrow (BM) transplants (BMT). Here, to clarify if HMGA2 affect JAK2 V617F+ hematopoiesis, we crossed Δ Hmga2+/- mice with JAK2 V617F+/- Tg mice (Shide et al, Leukemia, 2008). Δ Hmga2-/-JAK2 V617F-/- wild type (WT), Δ Hmga2+/-JAK2 V617F-/- (Δ Hmga2 -Tg), Δ Hmga2-/-JAK2 V617F+/- (JAK2 V617F-Tg) and Δ Hmga2+/-JAK2 V617F+/- (double-Tg) mice were born at expected Mendelian ratios and we could analyze 5 - 6 of each. At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly were most severe in double-Tg compared with JAK2 V617F-Tg or Δ Hmga2 -Tg mice. Relative to WT, peripheral leukocyte and platelet counts were nearly 16- and 4-fold higher in double-Tg, while 3- and 2-fold higher in JAK2 V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, Δ Hmga2 -Tg, JAK2 V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 107/femur, respectively. However, JAK2 V617F-Tg and double-Tg equally showed MF whereas no MF was detected in WT and DHmga2-Tg, suggesting that HMGA2 partly recovers cellularity in fibrotic BM. In the absence and presence of JAK2 V617F, HMGA2 augments lineage- Sca1+ Kit+ cells (WT: Δ Hmga2-Tg: JAK2 V617F-Tg: double-Tg= 0.17%: 0.19%: 0.17%: 0.27% in BM cells), endogenous erythroid colonies (1: 11: 13: 21 CFU-E/104 BM cells) and CD71+ Ter119+ erythroblasts (23%: 29%: 5.7%: 10% in BM and 2.0%: 4.4%: 7.9%: 16% in spleen cells), indicating HMGA2 contributes to expansion of hematopoietic stem/progenitor cells (HSPC) and erythroid commitment in JAK2 V617F+ hematopoiesis. Most Δ Hmga2-Tg and JAK2 V617F-Tg survived for over one year, but all double-Tg mice died within 4 months after birth due to severe splenomegaly and MF with no acute leukemia. To study the effect of HMGA2 on JAK2 V617F+ HSPC activity, we performed BMT with 0.25 x 106 Ly5.2+Δ Hmga2-Tg, JAK2 V617F-Tg or double-Tg cells with 0.75 x 106 Ly5.1+ competitor WT cells to lethally irradiated Ly5.1+ WT mice. Proportions of Ly5.2+ cells were higher in recipients of Δ Hmga2 -Tg than double-Tg cells, while JAK2 V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2 V617F+ hematopoiesis, we performed another BMT with 1 x 104 KIT+ cells of JAK2 V617F-Tg mice transduced with retroviral vector of Hmga2 with each let-7 -site-mutated full-length 3'UTR (Hmga2-m7) to sublethally irradiated WT mice. Recipients of JAK2 V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2 V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2 V617F by expanding HSPC, leading to disease progression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mutational landscape of the transcriptome offers putative targets for immunotherapy of myeloproliferative neoplasms

Blood ◽  
2019 ◽  
Vol 134 (2) ◽  
pp. 199-210 ◽  
Author(s):  
Fiorella Schischlik ◽  
Roland Jäger ◽  
Felix Rosebrock ◽  
Eva Hug ◽  
Michael Schuster ◽  
...  
Keyword(s):  
Mhc Class I ◽  
Myeloproliferative Neoplasms ◽  
Class I ◽  
Detailed Knowledge ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Mutational Landscape ◽  
Cell Based Therapy ◽  
Hematological Cancers

Abstract Ph-negative myeloproliferative neoplasms (MPNs) are hematological cancers that can be subdivided into entities with distinct clinical features. Somatic mutations in JAK2, CALR, and MPL have been described as drivers of the disease, together with a variable landscape of nondriver mutations. Despite detailed knowledge of disease mechanisms, targeted therapies effective enough to eliminate MPN cells are still missing. In this study of 113 MPN patients, we aimed to comprehensively characterize the mutational landscape of the granulocyte transcriptome using RNA sequencing data and subsequently examine the applicability of immunotherapeutic strategies for MPN patients. Following implementation of customized workflows and data filtering, we identified a total of 13 (12/13 novel) gene fusions, 231 nonsynonymous single nucleotide variants, and 21 insertions and deletions in 106 of 113 patients. We found a high frequency of SF3B1-mutated primary myelofibrosis patients (14%) with distinct 3′ splicing patterns, many of these with a protein-altering potential. Finally, from all mutations detected, we generated a virtual peptide library and used NetMHC to predict 149 unique neoantigens in 62% of MPN patients. Peptides from CALR and MPL mutations provide a rich source of neoantigens as a result of their unique ability to bind many common MHC class I molecules. Finally, we propose that mutations derived from splicing defects present in SF3B1-mutated patients may offer an unexplored neoantigen repertoire in MPNs. We validated 35 predicted peptides to be strong MHC class I binders through direct binding of predicted peptides to MHC proteins in vitro. Our results may serve as a resource for personalized vaccine or adoptive cell–based therapy development.

scholarly journals Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53

2018 ◽  
Vol 2 (24) ◽  
pp. 3581-3589 ◽  
Author(s):  
Bridget K. Marcellino ◽  
Ronald Hoffman ◽  
Joseph Tripodi ◽  
Min Lu ◽  
Heidi Kosiorek ◽  
...  
Keyword(s):  
Disease Progression ◽  
Chromosomal Abnormalities ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Primary Myelofibrosis ◽  
Chromosome 17 ◽  
Transcript Levels ◽  
History Of ◽  
Tumor Protein P53

Abstract The Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and the prefibrotic form of primary myelofibrosis (PMF), frequently progress to more overt forms of MF and a type of acute leukemia termed MPN-accelerated phase/blast phase (MPN-AP/BP). Recent evidence indicates that dysregulation of the tumor suppressor tumor protein p53 (TP53) commonly occurs in the MPNs. The proteins MDM2 and MDM4 alter the cellular levels of TP53. We investigated in 1,294 patients whether abnormalities involving chromosomes 1 and 12, which harbor the genes for MDM4 and MDM2, respectively, and chromosome 17, where the gene for TP53 is located, are associated with MPN disease progression. Gain of 1q occurred not only in individuals with MPN-BP but also in patients with PV and ET, who, with further follow-up, eventually evolve to either MF and/or MPN-BP. These gains of 1q were most prevalent in patients with a history of PV and those who possessed the JAK2V617F driver mutation. The gains of 1q were accompanied by increased transcript levels of MDM4. In contrast, 12q chromosomal abnormalities were exclusively detected in patients who presented with MF or MPN-BP, but were not accompanied by further increases in MDM2/MDM4 transcript levels. Furthermore, all patients with a loss of 17p13, which leads to a deletion of TP53, had either MF or MPN-AP/BP. These findings suggest that gain of 1q, as well as deletions of 17p, are associated with perturbations of the TP53 pathway, which contribute to MPN disease progression.

Transforming Growth Factor β (TGF-β)- and P-Selectin-Dependent Fibroblast Peripolesis of Megakaryocytes Contributes to Development of Myelofibrosis in Gata1low mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1741-1741
Author(s):  
Fabrizio Martelli ◽  
Maria Zingariello ◽  
Barbara Ghinassi ◽  
Fiorella Ciaffoni ◽  
Rosa Alba Rana ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Disease Progression ◽  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Lysyl Oxidase ◽  
Myeloproliferative Neoplasms ◽  
Extramedullary Hematopoiesis ◽  
Collagen Fibers ◽  
Hematopoietic Stem ◽  
Gold Particles

Abstract Abstract 1741 The fibrosis observed in patients with primary myelofibrosis (PMF), the most severe of the Ph-chromosome negative myeloproliferative neoplasms, and in mouse models (TPOhigh or Gata1low) of the disease is thought to be caused by fibroblasts activated by TGF-β released by megakaryocytes (MK) engaged in a pathological process of emperipolesis with the neutrophils. Gata1low mice have a life span > 2 years and develop myelofibrosis in precise sequential stages which closely recapitulate the human disease. Mice are born thrombocytopenic and anemic and remain thrombocytopenic all their life but recover from anemia at one month by developing extramedullary hematopoiesis in spleen. These mice develop fibrosis and increased angiogenesis by 6–8 months and increased hematopoietic stem/progenitor cell trafficking and extramedullary hematopoiesis in liver at 10-months (Martelli et al Blood 2005; 106:4102). Eliades et al (J Biol Chem 2011; 286:27630) recently reported that MK from Gata1low mice express high levels of lysyl oxidase, a matrix cross-linking protein responsible for collagen polymerization, identifying a an additional mechanism of fibrosis induction by Gata1low MK. By electron microscopy, we observed that 10–20% of MK in the spleen of both PMF patients and Gata1low mice contain numerous collagen fibers embedded within their cytoplasm. This observation suggested to us that lysyl oxidase-dependent collagen polymerization may occur within the cytoplasm of the MK raising the question of the mechanism that allowed collagen to enter within these cells. To clarify this issue, extensive optical and electron microscopy studies of the spleen of Gata1low mice during disease progression were performed. With disease progression, the numbers of fibroblasts in the spleen of Gata1low mice greatly increased [fibroblasts/mm2= 165±28, 245±24 and 312±60 vs 80±20 in 3-, 6- and 15-month old Gata1low mice vs 15-month-old wild type (WT) littermates, respectively p<0.05-0.01 Gata1low vs WT and 3-month vs 6- and 15-month Gata1low mice]. Furthermore Gata1low fibroblasts had an activated morphology that included long protrusions containing numerous fibronectin- and collagen-gold particles (67±10 vs 11±1 fibronectin- and 45±14 vs 13±2 collagen-gold-particles/mm2 in Gata1low and WT fibroblast protrusions, p<0.05-0.01). In numerous cases, the protrusions surrounded MK penetrating their cytoplasm in a process of peripolesis that could involve either close (see Figure) or distal cell/cell interactions. The numbers of MK engaged in fibroblast peripolesis was 23±22, 80±34 and 186±12/section in 3-, 6- and 15-month Gata1low mice vs below detection in 15-month WT littermates. Once in the cytoplasm, the fibroblast protrusions fused their membranes with those of the MK releasing fibronectin and collagen into the MK cytoplasm. MK engaged in fibroblast peripolesis presented morphological nuclear changes in which the condensed chromatin of cells was similar to that observed in para-apoptosis, a Tunel-negative process of cell death. Based on the nuclear condensation state, peripolesis-engaged MK were classified in 4 stages, the most advanced of which was characterized by the presence of collagen fibers in the cytoplasm. These collagen fibers contained great numbers of TGF-β and myeloperoxidase immunogold-particles embedded in the heavy electron dense pocket of the polymer twists (see Figure). Nude para-apoptotic MK nuclei were found surrounded by cytoplasmic MK ghosts rich in TGF-β-collagen fibers which also contained clusters of 2–6 myofibroblasts (undetected in spleen from WT mice). Fibroblast/MK peripolesis was not detectable in spleen from double Gata1lowP-selectinnull mice or in Gata1low mice treated with an inhibitor of TGF-β signaling. These mice also did not express fibrosis and neo-angiogenesis. These results indicate that fibrosis and TGF-β accumulation in the microenvironment of Gata1low mice is not a random process but occurs in “hot-spots” localized at sites of MK and fibroblasts interaction. We suggest that this previously undescribed TGF-β (to activate fibroblast)- P-selectin (to promote fibroblast/MK interaction)-dependent MK/fibroblast interaction may facilitate scaring and vessel formation not only in PMF but in other diseases as well. Disclosures: No relevant conflicts of interest to declare.

Next-Generation Deep-Sequencing Enables a Quantitative Monitoring of RUNX1 Mutations in 534 Patients with Myelodysplastic/Myeloproliferative Neoplasms and Myelodysplastic Syndromes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2792-2792
Author(s):  
Alexander Kohlmann ◽  
Vera Grossmann ◽  
Stefan Harbich ◽  
Frank Dicker ◽  
Niroshan Nadarajah ◽  
...  
Keyword(s):  
Disease Progression ◽  
Deep Sequencing ◽  
Myelodysplastic Syndromes ◽  
Myeloproliferative Neoplasms ◽  
Refractory Anemia ◽  
Patient Specific ◽  
Next Generation ◽  
Employment Equity ◽  
Clone Size ◽  
Equity Ownership

Abstract Abstract 2792 Introduction: Somatic mutations of key candidate genes have gained interest as biomarkers predicting poor survival in myelodysplastic/myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX1 (runt-related transcription factor 1) deregulations constitute such a disease-defining molecular aberration and are usually tested applying a combination of denaturing high-performance liquid chromatography and direct Sanger sequencing. Patient-specific RUNX1 mutations were proposed to represent clinically useful molecular alterations to follow disease progression from MDS to s-AML. Study design: Using genomic DNA obtained from mononuclear cells a next-generation amplicon deep-sequencing (NGS) assay targeting the complete coding region of RUNX1 was developed on a longitudinal series of 116 retrospective samples obtained from 25 patients collected between 11/2005 and 6/2010 (454 Life Sciences, Branford, CT). Subsequently, this assay was applied to characterize an unselected prospectively collected MPN/MDS patient cohort during their course of disease. Results: Here, we present analyses on a cohort of 534 patients (females: 200; males 334). The median age was 72.0 years (25.2–95.7 years). The cohort included 149 chronic myelomonocytic leukemias (CMML), 11 cases with 5q- syndrome, 10 cases with refractory cytopenia with unilineage dysplasia (RCUD), 15 cases with refractory anemia with ring sideroblasts (RARS), 105 cases with refractory cytopenia with multilineage dysplasia (RCMD), 135 cases with refractory anemia with excess blasts-1 (RAEB-1), 87 cases with refractory anemia with excess blasts-2 (RAEB-2), and 22 cases with t-MDS, respectively. In total, 130 RUNX1 mutations were observed in 17.8% (95/534) of these patients. The mutational clone size ranged from 1.7% to 94% and amounted to a median of 31%. In comparison to our data from an AML cohort, i.e. 460 patients at diagnosis with 112 (24.3%) cases mutated, the median clone size was about 10% lower in MPN/MDS. In detail, 74.7% (71/95) of patients harbored one mutation, whereas 25.3% (24/95) of cases harbored two (17.9%; 17/95) or >=3 (7.4%; 7/95) mutations. The 130 RUNX1 mutations were characterized as follows: 29% frame-shift mutations, 42% missense, 14% nonsense, 13% exon-skipping, and 2% in-frame insertion/deletion alterations, respectively. The following codons were recurrently mutated: Arg174 (8/95 patients; 9.4%), Arg177 (6/95 patients; 7.0%), and Arg135 (5/95 patients; 5.3%). The mutations were predominantly located in the RHD domain (55%) and TAD domain (13%) and in cases with 2 or more alterations only 15% (4/24) harbored mutations outside of these regions. In all cases with 3 concomitant mutations both domains were affected (4/4 patients). Further, cases with >1 RUNX1 mutation were more frequently observed in CMML (33.3%; 8/24 mutated), RAEB-1 (17.2%; 5/29 mutated) and RAEB-2 (34.5%, 10/29 mutated) as compared to other disease groups, respectively. In subsequent serial analyses including 56 samples from 22 cases the amplicons carrying the respective known alteration were analyzed with increased coverage for disease status monitoring (in median 833 reads/amplicon were sequenced; leading to a sensitivity of ∼1:800). With a median time span of 2.5 months between the molecular analyses a total of 2 to 4 samples per patient were analyzed. In 5/22 patients, this assay then allowed to monitor the treatment success of allogeneic stem cell transplantation: in 3 cases the mutations known before transplantation became undetectable; in 2 cases the same mutated clones still remained detectable at a level of 0.2% and 23%, respectively. Further, in 17 patients quantitative assessment of mutated RUNX1 read counts was used to monitor stable disease (n=12) or allowed to follow an increasing clone size in 3 patients that progressed into s-AML (39% -> 53% increase; 31% -> 42% increase; 7% -> 37% increase). Summary: Unbiased techniques such as deep-sequencing provide the required diagnostic specificity and sensitivity to enable classification and individualized monitoring of disease progression. We here demonstrate that amplicon-based NGS is a suitable method to accurately detect and quantify the broad spectrum of molecular RUNX1 aberrations with high sensitivity. It is therefore suitable for therapy guidance. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Harbich:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Evaluation of hMICL As a Marker Associated with Disease Progression in BCR-ABL Negative Myeloproliferative Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1724-1724
Author(s):  
Line Nederby ◽  
Laura Laine Larsen ◽  
Peter Hokland ◽  
Anne Stidsholt Roug
Keyword(s):  
Disease Progression ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Stable Phase ◽  
Blast Transformation ◽  
Color Flow ◽  
Flow Cytometric ◽  
Stem And Progenitor Cells ◽  
Disease Stages

Abstract Abstract 1724 Introduction: BCR-ABL negative myeloproliferative neoplasms (MPNs) (polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)) have preponderance to progress to hematopoietic insufficiency with fibrosis and/or blast transformation. This progression is often difficult to detect, but can be preceded by a stage of variable duration commonly termed acceleration, which is not homogeneously defined, but characterized by worsening cytopenias and constitutional symptoms. The monitoration of this process is clearly important, but at the present time biomarkers for this are lacking. We and others have described the human myeloid inhibitory C-type lectin-like receptor (hMICL) (also known as CLL-1) to be a very stable marker for the malignant cells in acute myeloid leukemia (AML), even in CD34- cases (van Rhenen et al. Leukemia 2007, Roug et al. Cytometry 2011). Given the need for new markers in MPN we hypothesized that determining the level of circulating hMICL expressing stem cells could be predictive of disease progression in MPN. Materials and Methods: Peripheral blood (PB) was obtained from 37 PV-, 19 ET-, and 8 PMF patients. A total of 57 (36 PV, 16 ET, and 5 PMF) patients were in stable phase while 7 (1 PV, 3 ET, and 3 PMF) were defined as being in an accelerated phase. Samples were prepared for five-color flow cytometry protocols using the antibodies anti-CD34, anti-CD38, anti-CD45, anti-CD14, and anti-hMICL. Acquisition was performed on a BD FACSCanto II and data analyses were carried out in FlowJo software. We focused on the following subsets: CD45lowSSClowCD14-CD34+hMICL+ and CD45lowSSClowCD14-CD34+CD38-hMICL+. Gates defining hMICL positivity were established on the basis of hMICL- lymphocytes as internal controls. One million cells were analyzed per sample and subsets of interest were calculated as percentage of these. Results: We found a percentage of CD34+hMICL+ (mean 1.176%, SD 0.908) and CD34+CD38-hMICL+ (mean 0.256%, SD 0.329) in PMF patients, which was statistically different from both PV- (mean 0.038%, SD 0.101, and mean 0.005%, SD 0.018, respectively) (p=0.0002 and p<0.0001, respectively) and ET patients (mean 0.116%, SD 0.252, and mean 0.009%, SD 0.024, respectively) (p=0.001 and p=0.0007, respectively). Somewhat surprisingly, compared to PV patients the CD34+hMICL+ compartment in ET patients was significantly higher (p=0.036). Importantly, the stem cell compartment in the accelerated phase patients was significantly higher than in the stable phase ones: CD34+hMICL+ (stable phase: mean 0.108%, SD 0.31, and accelerated phase: mean 0.983%, SD 0.917, respectively) (p=0.0002) and CD34+CD38-hMICL+ (stable phase: mean 0.017%, SD 0.081, and accelerated phase: mean 0.202%, SD 0.325, respectively)(p=0.0003). Conclusion: We have identified circulating CD34+hMICL+ and CD34+CD38-hMICL+ cells as an interesting biomarker for evaluating the disease state in MPN patients. The addition of hMICL discriminates between normal and neoplastic stem- and progenitor cells in MPN patients and reliably assigns disease stages in these patients superior to the mere evaluation of circulating CD34+ and CD34+CD38- cells. Importantly, hMICL+ cells, though rare, were dependably identified in PB. Thus, this study provides possible options for flow cytometric monitoring of MPN patients. Disclosures: No relevant conflicts of interest to declare.

Genetic and Epigenetic Alterations of Ph-Negative Myeloproliferative Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5549-5549
Author(s):  
Lyubov Polushkina ◽  
Irina Martynkevich ◽  
Vasily Shuvaev ◽  
Ekaterina Petrova ◽  
Lyudmila Martynenko ◽  
...  
Keyword(s):  
Disease Progression ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Disease Onset ◽  
Suppressor Gene ◽  
Homozygous Mutation ◽  
Epigenetic Alterations ◽  
Diagnosis And Prognosis ◽  
Ubiquitin Ligase Activity ◽  
Blastic Phase

Abstract Objectives and background: Genetic mutations result in abnormalities of myelopoietic proteins and lie in the basis of Ph-negative myeloproliferative neoplasms (MPNs) development and its subsequent progression. Several somatic mutations in JAK2, MPL, TET2, EZH2, ASXL1, CBL, IDH1, IDH2, IKZF1 genes were detected in chronic and blastic phase MPNs. Recent studies have revealed a number of epigenetic alterations that contribute to Ph-negative MPNs pathogenesis and determine the clinical outcome. Mutations involving the EZH2 gene are thought to result in loss of methyltransferase activity suggesting a potential role of tumor suppressor gene silencing as a mechanism in the disease progression. Decrease in ubiquitin ligase activity caused by mutations CBL gene leads to myeloid proliferation. EZH2, CBL mutations are thought to be of prognostic value in MPN’s at the time of transformation to the blastic phase but data are inconsistent and require the further verification.The goal of our research was to determine the significance of mutations genes EZH2, CBL in the diagnosis and prognosis of Ph-negative MPNs. Methods. We have examined 102 patients with Ph-negative MPNs (45 pts with PV, 30 pts with ET and 27 pts with PMF). For all patients the detection of V617F mutation of JAK2 gene was done. V617F-negative pts with PV and pts with ET or PMF underwent the analysis of mutations in 12-th exon of JAK2 and 515 codone of MPL gene respectively. For 80 pts (30 with PV, 28 with ET and 22 with PMF) cytogenetic analysis and EZH2 mutation status were performed. Identification of CBL mutations was performed in 24 patients with available RNA samples. Mutations in 8, 10, 17, 18, 19 exons of EZH2 and RING-domen of CBL were defined by sequence analysis. V617FJAK2 mutation was detected in 44/45 (97,8%) pts with PV, 16/30 (53,3%) pts with ET and 13/27 (48,1%) pts with PMF. 538-539del-insL in 12-th exon of JAK2 was found in 1/45 (2,22%) patient with PV. W515KMPL mutation was identified in 1/30 (3,33%) pt with ET and 1/27 (3,7%) pt with PMF. 2 mutations of EZH2 gene have been found in 2 individuals with PMF (2/22). Both mutations are located in the 19 exon. The Ile713Thr mutation was detected in the patient with a del(6)(q15) karyotype which is associated with an intermediate cytogenetics risk. This patient subsequently underwent transformation from PMF to myelodysplastic syndrome in 9 months after the disease onset. Another case of mutation harboring (Thr731Asp) was detected in a patient with PMF and poor prognosis karyotype (chromosome 7 monosomy). This patient had transformation PMF to acute myeloid leukemia and died after 20 months. Homozygous mutation Q420R in CBL gene was detected in 1/24 patient with complex karyotype. Disease progression was observed after 16 months from the diagnosis. Conclusion. Mutations in EZH2 and CBL genes could be assessed as additional prognostic markers of unfavourable prognosis in patients with BCR-ABL-negative MPNs with different chromosomal aberrations. The integration of cytogenetic and molecular analyses could be a valuable option for stratification of patients and optimising the treatment strategy. References: Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 2010; 24:1128–1138. Disclosures No relevant conflicts of interest to declare.

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