JAK2V617F Allele Burden In Patients Research Articles

Relationship between TET2 Gene SNP rs3733609 C/T and JAK2V617F Allele Burden in Patients with Myeloproliferative Neoplasms

To investigate the relationship between the polymorphism of TET2 gene SNP rs3733609 and JAK2V617F allele burden in patients with myeloproliferative neoplasms (MPN). The exon 9 of TET2 gene was amplified by RT-PCR, and the nucleotide sequence of SNP rs3733609 site was analyzed by gene sequencing. The MGB Taqman probe PCR method was used to detect the JAK2V617F allele burden. The correlation of TET2 gene SNP rs3733609 C/T with the JAK2V617F allele burden and clinical parameters was analyzed. TET2 gene rs3733609 C/T heterozygosity (normal T/T) could be detected in 19 cases of 85 cases of JAK2V617F positive MPN (22.4%) patients, while the TET2 gene rs3733609 C/T heterozygosity could be detected only in 9 of the 106 healthy volunteers, and the incidence was only 8.5% (9/106). Compared with the negative group (TET2 rs3733609 T/T), there was no significant difference in the median age, hemoglobin level and platelet count in the patients with TET2 gene SNP rs3733609 (CT/TC) positive, but the WBC count of peripheral blood and JAK2V617F allele burden significantly increased. In JAK2V617F high allele burden group, TET2 gene SNP rs3733609 was positive in 7 cases (36.8%, 7/19), the ratio was higher than that in the low allele burden group(18.2%, 12/66). TET2 SNP rs3733609 C/T may be a new susceptible allelee, which affects the clinical characteristics and clonal evolution of MPN patients.

A novel quantitative JAK2V617F detection kit: prospective clinical performance study comparing MPN patients and healthy subjects

The JAK2V617F mutation is the commonest major genetic mutation of myeloproliferative neoplasms (MPNs) and has been defined in the WHO diagnostic criteria for MPNs. However, there is still no approved in vitro diagnostic test kit available in Japan. We evaluated a JAK2V617F allele quantification kit (test method) in a prospective, multicenter clinical performance study involving patients with MPNs who were diagnosed with polycythemia vera, essential thrombocythemia, and primary myelofibrosis; healthy volunteers were also included in the analysis. Good correlation was observed between the allele burden determined using the test method vs. that determined using next-generation sequencing (NGS) in the patient group (r=0.998, y=1.071x-0.069; n=156). Furthermore, all allele burdens in the healthy group (n=54) were below the lower limit of the measurement range of the test method (0.042%). Our results confirmed that the test method could quantitatively measure the JAK2V617F allele burden in patients with MPN. Thus, the novel JAK2V617F allele quantification kit can be considered useful for the diagnosis of MPNs.

Mechanisms of thrombogenesis in polycythemia vera

Thrombotic and cardiovascular events are among the leading causes of death for patients with polycythemia vera (PV), and thrombosis history is a key criterion for patient risk stratification and treatment strategy. Little is known, however, about mechanisms of thrombogenesis in patients with PV. This report provides an overview of thrombogenesis pathophysiology in patients with PV and elucidates the roles of conventional and nonconventional thrombotic risk factors. In addition to several conventional risk factors for thrombosis, clinical data have implicated increased hematocrit and red blood cell adhesiveness, activated platelets, leukocytosis, and elevated JAK2V617F allele burden in patients with PV. Furthermore, PV-related inflammation may exacerbate thrombogenesis through varied mechanisms, including endothelial damage, inhibition of natural anticoagulant pathways, and secretion of procoagulant factors. These findings suggest a direct link between myeloproliferation and thrombogenesis in PV, which is likely to provide new opportunities for targeted antithrombotic interventions aimed at decreasing PV-related morbidity and mortality.

JAK2V617F allele burden in patients with myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are clonal malignant diseases that represent a group of conditions including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). The JAK2-V617F mutation is prevalent in almost all patients with MPNs and has become a valuable biomarker for diagnosis of MPNs. A different allele burden in these entities has long been noticed. The aim of our study was to assess the JAK2 allele burden in our JAK2V617F positive cases and its association with phenotype if any and to select a simple, sensitive assay for use in our clinical molecular diagnostic laboratory. Methodologies reported in this literature include amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR). We analyzed 174 cases by RQ-PCR for the quantification of JAK2V617F were initially screened by ARMS-PCR. We found that V617F allele burden in the entire population of patients was 73% ranging from 0.97 to 95%. The median V617F allele burden in PV patients was 40%, MF was 95%, and ET was 25%. ARMS-PCR and RQ-PCR were proven to be sensitive since ARMS-PCR is a qualitative method; it can be used to screen JAK2V617F mutation and RQ-PCR was used to quantify the V617F cells. Our study suggests that JAK2V617F positivity is associated with MPNs, and its allele burden is an excellent diagnostic marker for disease subtypes, prognosis, disease phenotype and complication, and evolution. The data indicates that ARMS-PCR is simple and can be easily performed for the primary screening of JAK2V617F mutation, and RQ-PCR is sensitive enough to detect low mutant allele levels (>10%), specific enough not to produce false positive results, and can be performed for the JAK2V617F allele burden quantification.

Abstract LB-294: Determination of JAK2V617F allele burden in a phase II study of patients with myelofibrosis treated with SAR302503 using a sensitive and robust allele-specific qPCR assay.

Abstract Janus kinase-2 (JAK2)/STAT pathway activation is commonly observed in myeloproliferative neoplasms and plays an important role in disease development and progression. The V617F activating mutation in the JAK2 pseudokinase domain is found in 95% of patients with polycythemia vera (PV) and in 50-70% of patients with primary myelofibrosis (MF) or essential thrombocythemia (ET). Studies have shown the clinical efficacy of JAK2 inhibitors in treating MF in terms of a reduction in splenomegaly and relief of constitutional symptoms. A reduction in the JAK2V617F allele burden after treatment with a JAK2-targeted agent could be a surrogate for a disease-modifying effect of this therapy. To demonstrate this, a robust method that can accurately measure the JAK2V617F allele burden is required. Here, we report the development of a sensitive quantitative PCR (qPCR) assay to determine the JAK2V617F allele burden in patients treated with SAR302503, the validation of the assay in a Phase 0 clinical study and implementation in a phase II study using samples from patients with MF. The assay demonstrated robust performance, with high sensitivity (LLOD=0.05% and LLOQ=0.5%) and accuracy. A phase 0 study in MF, PV, and ET patients showed that (1) there was minimal intra-subject sampling variability in JAK2V617F allele burden, and (2) whole-blood samples are stable for at least 48 hours at ambient conditions for DNA preparation without a significant impact on JAK2V617F allele burden measurement.Of the 31 patients with intermediate-2 or high-risk MF enrolled in a phase II clinical study (NCT01420770), baseline samples were available for 29 patients and of these, 26 were JAK2V617F-positive using this assay. Of the 19/26 patients for whom samples were available at all 3 time points, the median allele burden was 93% at baseline, 87% at the end of Cycle 3, and 78% at the end of Cycle 6. Among the 24 patients who were JAK2V617F-positive and for whom spleen measurements were available at the end of Cycle 6, a total of 15 (63%) had a spleen response (≥ 35% reduction in spleen volume by MRI versus baseline). In contrast, two JAK2V617F-negative patients did not have a spleen response. Patients with baseline allele burden levels of 0 to <25%, 25% to 75%, and ≥75% showed spleen response rates of 50%, 44%, and 69%, respectively, at the end of Cycle 6. JAK2V617F allele burden will continue to be assessed every 3 cycles. Longer follow up is needed to determine the clinical effect of allele burden reduction. Citation Format: Feng Liu, Moshe Talpaz, Animesh Pardanani, Catriona Jamieson, Nashat Gabrail, Ayalew Tefferi, Tianlei Lei, Rita Greco, Francisco Adrian, Nikki Daskalakis, Claudia Lebedinsky, Pamela Cohen, Donald Bergstrom. Determination of JAK2V617F allele burden in a phase II study of patients with myelofibrosis treated with SAR302503 using a sensitive and robust allele-specific qPCR assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-294. doi:10.1158/1538-7445.AM2013-LB-294

SAR302503: Interim Safety, Efficacy and Long-Term Impact on JAK2 V617F Allele Burden in a Phase I/II Study in Patients with Myelofibrosis,

Abstract 3838▪▪This icon denotes a clinically relevant abstract Background:SAR302503 (SAR503, formerly TG101348), a potent, oral JAK2-selective inhibitor was studied in a Phase I/II trial for the treatment of patients with high- or intermediate-risk primary, post-polycythemia vera (PV) and post-essential thrombocythemia (ET) myelofibrosis (MF). SAR503 was administered orally once daily in 28-day cycles. Eligibility criteria included platelet count of ≥50 × 109/L. Interim safety and efficacy data from this study up to April 2010 have been previously published (JCO 2011, 29(7):789–796). The aim of this presentation is to report updated safety and efficacy of ongoing patients as well as an analysis of the JAK2V617F allele burden in this cohort. Results:Overall, 59 subjects (median age 64 years) were treated. Forty four patients had PMF, 12 post-PV MF and 3 post-ET MF; 86% were JAK2 V617F-positive. Median palpable spleen size was 18 cm at study enrollment. Twenty eight patients were treated in the dose-escalation cohort (30–800 mg administered as a single daily dose); thirty one patients were treated at the MTD (680 mg) in the dose confirmation cohort. 43/59 patients (73%) completed 6 cycles of treatment and continued treatment on the extension study. Currently, 22 patients (37%) remain on treatment with a median number of 28.5 cycles (24–41 range) and a median of last dose of 440 mg/day. Safety:Treatment-emergent toxicities in cycle 1–6 have been previously reported; toxicities were dose-dependent and generally alleviated with dose-reduction. Five patients discontinued treatment beyond cycle 6 for treatment-related adverse events: thrombocytopenia, depression, mental status changes, creatinine elevation and subdural hematoma. For the subgroup of patients with a baseline platelet count between 50–100 × 109/L (n =13; median 73, range 51–94); the platelet count at defined times points during follow up was: cycle 3; median 50, range 21–138 (p=0.09) and cycle 6; median 47, range 13–85 (p=0.01). Despite 7 of the 13 patients being treated at ≥680 mg/day, only 2 instances of Grade 4 thrombocytopenia were noted in this group Spleen response:As previously reported, spleen responses were seen early, usually within first 3 cycles, with half or more patients in each dose level ≥240 mg/day showing a durable ≥50% decrease in palpable spleen size. Spleen size (mean, median, range, and proportion with ≥50% reduction) at the following time points was: Baseline (n=58; 18.33cm, 18cm, 4–38cm, NA) ; 6 months (n=57; 9.05cm, 9cm, 0–30cm, 54.4%;) 12 months (n=42; 8.55cm, 9cm, 0–28cm, 66.7%) 18 months (n=36; 8.03cm, 8.5cm, 0–33cm, 52.8%); 24 months (n=31; 8.10cm, 8cm, 0–30cm, 54.8%,) 30 months (n=18; 6cm, 7.5cm, 0–16cm, 61.1%,and) 36 months (n=9; 5.89cm, 3cm, 0–16cm, 66.7%).JAK2V617F allele burden: We previously reported a significant decrease in JAK2V617F allele burden at the end of cycles 6 and 12. A durable decrease was also demonstrable after 24 cycles of treatment (n =21; median 9%, range 0–100%) relative to baseline (n =51; median 20%, range 3–100%) (p=0.03). Similarly, for patients with JAK2 V617F allele burden >20% at baseline; there was a significant decrease after cycle 24 (n =12; median 21%, range 6–100%) relative to baseline (n =23; median 60%, range 23–100%) (p=0.03). Conclusions:SAR503 is safe and efficacious treatment with long term effect on spleen size and JAK2V617F allele burden in patients with high- and intermediate-risk myelofibrosis. Additional follow up information will be updated at the time of meeting. Disclosures:Jamieson:Wintherix: Equity Ownership; Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria. Gao:Sanofi-Aventis: Employment. Zhang:Sanofi-Aventis: Employment. Neumann:Sanofi-Aventis: Employment.

Influence of JAK2 46/1 haplotype in the natural evolution of JAK2V617F allele burden in patients with myeloproliferative neoplasms

JAK2V617F allele burden was prospectively measured in untreated patients with polycythaemia vera (PV, n=26) or essential thrombocythaemia (ET, n=36) and compared according to JAK2 46/1 haplotype status. The mean increase in JAK2V617F allele burden per year was 1%, 0.8% and 6% for PV patients with the JAK2 46/1 haplotype in negative, heterozygous and homozygous status, respectively (p<0.001). The JAK2 46/1 haplotype had no influence in JAK2V617 allele burden in ET. In conclusion, untreated PV patients homozygous for the JAK2 46/1 haplotype show a progressive increase in the JAK2V617F allele burden during the evolution of the disease.

The A3669G Polymorphism of GR Is a Host Genetic Modifier Associated with Polycythemia Vera and Primary Myelofibrosis

AbstractAbstract 3067The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) which are all associated with a gain-of-function mutation (JAK2V617F) of the JAK2 gene. There is ongoing discussion on the mechanism by which this single mutation may cause diseases with three different phenotypes. The consensus is that while the JAK2V617F may increase proliferation of hematopoietic cells in general, the lineage affected is determined by host genetic modifiers still to be identified. The glucocorticoid receptor (GR) is an important regulator of erythropoiesis in vivo and in vitro. It exerts its activity by synergistic transcriptional and membrane-associated mechanisms. Upon activation, GR forms a transcriptional complex with phosphorylated STAT-5 (P-STAT-5) which binds and alters the expression of a subset of the erythropoietin (EPO)- and stem cell factor (SCF)-target genes. Physical interaction between GR and the EPO receptor (EPO-R) on the cell membrane suppresses the ability of both receptors to phosphorylate STAT-5 when activated together. Insufficient GR/P-STAT-5 transcriptional activity has been suggested to result in delayed erythroid maturation and to induce eythrocytosis in patients exposed to excessive glucocorticoids. We had previously determined that erythroid cells (EBs) generated ex vivo from peripheral blood mononuclear cells of patients with PV and those obtained from normal donors (ND) stimulated with dexamethasone have similar biological (increased proliferation with delayed maturation) and biochemical (great levels of NFE-2, WT-1 and GATA2 and low levels of GATA1 and β-globin expression) properties (Varricchio et al,Blood 2009;114:3899a). This similarity is counterintuitive because STAT-5 phosphorylation was reduced in normal EBs exposed to dexamethasone and EPO while in EBs from PV patients it was constitutive due to the presence of the JAK2V617F mutation. In addition to the canonical GRα signalling isoform, cells may express the dominant negative GRβ isoform which binds poorly to glucocorticoids because its unique structure impairs ligand binding and determines nuclear retention. Expression of GRβ is regulated by the presence of the A3669G SNP in exon 9 which specifically stabilizes GRβ mRNA. The presence of this polymorphism has been associated with glucocorticoid resistance but its effects on erythropoiesis are unknown. To clarify the similarities between the biological properties of EBs expanded ex vivo from ND in the presence of dexamethasone and those obtained from PV patients, we determined the levels of expression (at the mRNA and protein level) of GRα and GRβ in EBs expanded ex vivo from 16 ND and 16 PV patients. While EBs from ND expressed GRα only, those expanded from PV patients expressed both GRα and GRβ. Therefore, in spite of constitutive STAT-5 phosphorylation, in EBs from PV patients formation of transcriptionally active GR/P-STAT-5 complexes is reduced by expression of GRβ. To clarify the mechanism underlying expression of GRβ in EBs obtained from PV patients, the frequency of the A3669G polymorphism was determined by specific PCR followed by sequencing of the amplified products using DNA obtained from the mononuclear cells of ND (7) and patients with PV (10). DNA from patients with PMF (10) and ET (14) was analysed for comparison. The polymorphism was detected in 60% of patients with PV but not in ND, indicating that GRβ mRNA is particularly stable in EBs from PV patients. In addition, the polymorphism was observed in 50% of patients with PMF but was not detected in patients with ET. A trend toward higher JAK2V617F allele burden was observed in A3669G positive PV patients (69.8±23.0) compared to A3669G negative PV patients (37.3±22.6) and ET patients (28.13±27.9) while the JAK2V617F allele burden in patients with PMF with or without the A3669G SNP was the same (51.2±37.5 vs 42.8±17.9). These data suggest that in addition to mutations of EPO-R and Von Hippel-Lindau gene, erythrocytosis may be induced by excess GR activation by glucocorticoids or, in association with the JAK2V617F mutation, by the presence of the A3669G SNP which favours GRβ expression. Moreover, the A3669G SNP may represent a host genetic modifier and a potential diagnostic tool to predict development of erythrocytosis in patients with MPN and GRβ may represent a potential therapeutic target for PV. Disclosures:No relevant conflicts of interest to declare.

Interferon-α targets JAK2V617F-positive hematopoietic progenitor cells and acts through the p38 MAPK pathway

Interferon-alpha (IFNalpha) therapy leads to hematological remissions and a reduction of the JAK2V617F allele burden in patients with polycythemia vera (PV). In this study, the cellular target by which IFNalpha affects hematopoiesis in PV patients was evaluated. CD34(+) cells were isolated from normal bone marrow and the peripheral blood of patients with PV and were treated in vitro with each of the three commercially available forms of IFNalpha: IFNalpha 2b, pegylated IFNalpha 2a (Peg-IFNalpha 2a), and pegylated IFNalpha 2b (Peg-IFNalpha 2b). Each form of IFNalpha was equally potent in suppressing hematopoietic colony formation by normal CD34(+) cells, but Peg-IFNalpha 2a and IFNalpha 2b were more effective than Peg-IFNalpha 2b in inhibiting burst-forming unit erythroid-derived colony formation by PV CD34(+) cells. In addition, exposure of PV CD34(+) cells to equal doses of Peg-IFNalpha 2a and IFNalpha 2b resulted in a 38% to 40% reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells (HPC), while equivalent doses of Peg-IFNalpha 2b did not reduce the number of malignant HPC. Further studies explored the mechanism by which IFNalpha induced PV HPC growth inhibition. Treatment of Peg-IFNalpha 2a increased the rate of apoptosis of PV CD34(+) cells and the phosphorylation/activation of p38 mitogen-activated protein kinase in PV CD34(+) cells, while the p38-specific inhibitor SB203580 reversed the growth inhibition and apoptosis induced by Peg-IFNalpha 2a. These data suggest that low doses of IFNalpha selectively and directly suppress PV JAK2V617F HPC and that these agents act through the p38 mitogen-activated protein kinase pathway.

Study of the Relation Between the Time Course of the JAK2V617F Allele Burden and the Clinical Evolution in Patients with Myeloproliferative Neoplasms (MPN); A Single Center Study

Introduction: The JAK2V617F mutation is frequent in MPN, however its clinical implication is still in debate. There are few publications that analyze changes in JAK2V617F allele burden.Methods: We performed a single centre study on 65 patients (19 polycythemia vera (PV), 42 essential thrombocythemia (ET) and 4 primary myelofibrosis (PMF)) all of them with at least two JAK2V617F determination separated by minimum 12 months. The JAK2 mutation was determinated in DNA from peripheral blood granulocytes (in 3 cases from bone marrow smear) by MutaScreenTMKit (IPSOGEN).Results: The mean follow-up period was 26 months (range 12–176). In 42/65 (64.6%) patients JAK2 mutation was positive and in 24/42 (57%) changes in JAK2V617F allele burden (>10%) were observed. Interestingly, in 7/18 (39%) patients who showed an increase of JAK2V617F allele burden progression was observed. In this group, one case of PMF with initial increase of JAK2V617F allele burden during transformation to acute myeloid leukemia (AML) and posterior decrease after transformation* and one case of ET with an increase during transformation to myelofibrosis (MF) and posterior conversion to JAK2V617F negativity after allo-HSCT** were observed (table1). 36/42 (85.7%) of JAK2V617F positive patients were under cytoreductive treatment: 30 with hydroxyurea (HDU), 1 anagrelide (ANA) and 5 mixed. There was no decrease of JAK2-V617F allele burden in patients without cytoreductive treatment. Data of patients with HDU treatment are presented in table 2. 3/5 of patients with mixed cytoreduction showed an increase of JAK2V617F allele burden: all of them presented bad clinical control but no data of progression were observed. None of JAK2V617F negative patients (n=23: 1 PV, 2 PMF, 20 ET) became positive for the mutation with the mean follow-up period of 19.37 months. There were no cases of transformation in this group of patients.Conclusions: According to our study, JAK2V617F allele burden can change during the follow-up. Increase (or stability) of JAK2V617F allele burden are more frequent in transformation from ET to PV or MF; in cases of progression to AML we can observe decrease, stability or initial increase with posterior decrease of JAK2V617F allele burden. Decrease of JAK2 allele burden can be related to cytoreductive treatment. Increase of JAK2V61F in patients with cytoreductive treatment can be related to transformation or bad clinical control. More prospective studies are needed to conclude whether changes in JAK2-V617F allele burden could be useful to predict transformation of MPN patients.Table1. Changes of JAK2V617F allele burden and clinical evolution in JAK2V617F positive patients.JAK2Clinical evolution (N=42)STABILITYPROGRESSIONDECREASE6/42 (14%)51 [1 PV-AML]STABILITY18/42 (33%)153 [1 PV-MF; 1 ET-MF; 1 ET-AML]INCREASE18/42 (43%)117 [3 ET-PV; 3 ET-MF (one case with posterior allo-HSCT**); 1 PMF-AML*]Table2. Changes of JAK2V617F allele burden in JAK2V617F positive patients with HDU and no cytoreductive treatment.TreatmentJAK2V617F: changes of allele burdenDECREASESTABILITYINCREASEUntreated-42 [ET-PV]HDU newly started25 [PV-MF and ET-AML after 14 and 7 years of treatment respectively]7 [2 ET-PV; PMF-AML*]HDU already treated3 [PV-AML]76 [3 ET-MF (one case with posterior allo-HSCT**)]

The Relationship Between Chromosomally Abnormal Hematopoiesis and the JAK2V617F Allele Burden in Patients (pts) with Ph-Negative Chronic Myeloproliferative Disorders (Ph-neg MPD)

The JAK2V617F mutation, present in >90% of pts with polycythemia vera (PV) and in approximately 50% of pts with primary myelofibrosis (PMF) and essential thrombocythemia (ET), has been identified in CD34+ cells from pts with PV and PMF, consistent with the notion that this mutation arises in an early hematopoietic progenitor cell (HPC). The relationship between the recurrent chromosomal abnormalities, occurring occurring in 8% of ET, 29% of PV and ~50% of PMF pts, to the JAK2V617F mutation is uncertain. In order to determine a relationship between the JAK2V617F allele burden and the frequency of HPC with chromosomal abnormalities, we identified 5 pts (3 PV, 1 PMF and 1 ET) who were JAK2V617F positive and had different cytogenetic abnormalities. At the time of the study Pt 1 (PMF) had +9, del(20)(q11q13) in 50% of cells and del(20)(q11q13),+der(9)t(1;9)(q21;q13) in 50% cells. Pt 2 (PV) had 85% of cells with +9. Pt 3 (PV) had 15% of cells with del(20)(q11q13). Pt 4 (ET) had 36% of cells with dup(1)(q21q31) and Pt 5 (PV) had +der(9)t(1;9)(q21;q13) in 55% cells. The JAK2V617F allele burden in granulocytes and CD34+ cells ranged from 55% to >95%. HPC (BFU-E, CFU-GM and CFU-Meg) were assayed from CD34+ cells and each individual colony was plucked, split in half, and tested for both the JAK2V617F mutation and the presence of the marker cytogenetic abnormality using FISH. Among the 289 colonies studied, 82% were JAKV617F homozygous, 8% were heterozygous for JAK2V617F and 10% contained only wild type (WT) JAK2. Cytogenetically abnormal colonies, containing the marker chromosome, as detected by FISH, were observed in 80% of the JAK2V617F homozygous, 64% of JAK2V617F heterozygous and 30% of the WT JAK2 colonies. Despite 100% of the colonies being homozygous for JAK2V617F in Pt 3 only 7% of colonies exhibited del(20q). In Pt 1, with two distinct cytogenetically abnormal populations, 2% of JAK2V617F homozygous colonies contained del(20q) alone, while 40% of colonies had both del(20q) and +9p and 52% had a combination of all three abnormalities. Further analysis of Pts 2 and 5 demonstrated that 87% and 47%, respectively, of JAK2 homozygous colonies contained gain of 9/9p. Duplication (1)(q21q31), a characteristic chromosomal abnormality of Pt 4, was identified in 18% of JAK2 WT colonies while its frequency in homozygous JAK2V617F HPC was 76%. These observations suggests that an increased JAK2V617F allele burden is associated with an increased frequency of cytogenetically abnormal HPC. A small proportion of homozygous JAK2 V617F HPC are involved by del(20q) (2%–7%) indicating their low proliferative capacity. Acquisition of trisomy 9 or trisomy 9p either in the presence of del(20q) or alone, involved a much larger population of JAK2V617F homozygous HPC (61%–93%) suggesting their higher proliferative capacity. In colonies containing only WT JAK2, 12% to 26% of cells within colonies had either +9 or dup(1q) suggesting that these chromosomal abnormalities might occur independently of JAK2V617F. These studies suggest a complex relationship between the JAK2V617F allele burden and occurrence of clonal chromosomal abnormalities in Ph-neg MPD HPC which might define important biological differences and clinical outcomes in MPD.