Endogenous Erythroid Colonies Research Articles

Childhood and adolescent essential thrombocythemia and prefibrotic primary myelofibrosis: insights into diagnosis, outcomes, and treatment from a large Chinese cohort.

The paucity of essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (pre-PMF) in individuals younger than 18 years highlights several unresolved issues in diagnosis, clinical outcomes, and treatment strategies. To address these knowledge gaps, we analyzed a large bidirectional cohort consisting of childhood and adolescent ET (CAA-ET, n = 156) and pre-PMF (CAA-preMF, n = 13), as well as adult ET (n = 349). We introduced immunophenotypic abnormalities as novel clonal markers in CAA-ET and CAA-preMF, establishing a comprehensive method for clonal marker detection that integrated driver and non-driver mutations, positive endogenous erythroid colony formation, immunophenotypic abnormalities, and chromosomal aberrations. Next-generation sequencing revealed distinct mutational profiles between CAA-ET and adult ET, along with different age-related trends in the distribution of driver mutations. Venous thrombosis was more prevalent in CAA-ET, with JAK2 V617F emerging as a potential risk factor (P = 0.018). Immunophenotypic abnormalities were identified as risk factors for disease progression (P = 0.027). Significant differences between expected and actual treatment practices were identified. Compared to CAA-ET, CAA-preMF demonstrated poorer progression-free survival (P < 0.001) and faster disease progression (P = 0.019). This study provides a critical foundation for refining diagnostic, prognostic, and therapeutic approaches for CAA-ET and CAA-preMF.

Determination of the JAK2 V617F mutation in thrombosis patients

Background: Janus kinase 2 (JAK2) gene mutation causes uncontrolled myeloproliferation independent of cytokines and abnormal formation of the endogenous erythroid colony. JAK2 mutations were frequently observed in myeloproliferative neoplasms (MPNs), especially in polycythemia vera (PV) and essential thrombocythemia (ET). MPNs represent a risk factor for the development of thrombosis that is a significant cause of morbidity and mortality in patients. Materials and methods: We aimed to study the correlation between JAK2 mutation and thrombosis. Thirty-nine patients with a clinical diagnosis of thrombocytosis and Budd-Chiari syndrome were collected to determine the JAK2 V617F mutation. Genomic DNA from all specimens was amplified and detected the presence of JAK2 V617F mutation by AS-PCR. Results: We demonstrated JAK2 V617F mutation in both patients with a clinical diagnosis of thrombocytosis and Budd-Chiari syndrome. We found that 11 of 37 (29.7%) thrombocytosis patients had a JAK2 V617F mutation. Moreover, one of two patients who represented as Budd-Chiari syndrome was positive for JAK2 V617F mutation. In addition, JAK2 V617F mutation was associated with thrombosis. However, further study in large series is needed to support this finding. Conclusion: Determination of the JAK2 V617F mutation may be helpful for screening latent or occult MPNs patients who have an occurrence of thrombosis to adjust the appropriate treatment for good patient outcomes.

Novel mutations in EPO-R and oxygen-dependent degradation (ODD) domain of EPAS1 genes-a causative reason for Congenital Erythrocytosis

Congenital Erythrocytosis (CE) can be primary or secondary due to the mutations in genes involved in the erythropoietin receptor and oxygen sensing pathway. In this study, 42 patients with 38 unrelated patients and one family (4 patients) who were JAK-2 mutation (both exon 12 and exon 14) negative with high haematocrit values were investigated. The Endogenous Erythroid colony (EEC) assay was performed in all patients, interestingly EEC colonies were high in EPAS1 and EPOR mutated patients compared to non-mutated patients. The sequence analysis of EPAS1 (exon 12), EPO-R (exon-8), VHL (exon-3), and EGLN1 (exon-1) genes in all these patients showed 19% of patients (8/42) had mutations, in exon12 of EPAS1 and exon 8 of EPO-R genes. Two novel missense mutations MW_600850:c.1183G>C, MW_600851:c.1028A>C in EPO-R gene were observed in the study group. One new MW_600849:c.1969C>T nonsense mutation and five MW_619914:c.1715A>G, MW_619915:c.1694G>T, MW_619916:c.1634T>C, MW_600852:c.1771C>G, MW_600848:c.1859G>A novel missense mutations were observed in the EPAS1 gene. Among them, 4 mutations p. (Gln572Arg), p. (Ser565Ile), p. (Ile545Thr), p. (Gln591Glu) in the ODD (Oxygen-dependent degradation) domain of HIF2α, all these variations contributed to the formation of non-functional HIF2α. No mutations were observed in VHL and EGLN1 genes. Using in silico analysis we observed that these mutations contributed to major conformational changes in the HIF2α protein making it non-functional. The mutations in the EPAS1 gene were heterozygous and show autosomal dominant inheritance patterns and we observed in one family. These novel mutations in the EPAS1 (75% (6/8)) and 25% (2/8) EPO-R genes correlating with EEC positivity were observed for the first time in India in CE patients.

JAK2 V617F Mutation Negative Polycythemia Diagnosed By IGF-1R, EEC Formation , and IRS2 over Expressions

We previously reported that by flow cytometry study of mononuclear cells (peripheral blood) of IGF-1 R (insulin growth factor -1 receptor) can help in differentiating polycythemia vera (PV) from secondary erythrocytosis (PLoS ONE 11(11): e016529), in patients negative for JAK2V617F mutation who requires frequent phlebotomy. EEC (Endogenous erythroid colony formation) formation which was shown to be related to IGF-1 signaling was employed as a minor criteria of PV prior to 2016 WHO criteria for the diagnosis of PV. IRS-2, an IGF-1 activation downstream signaling protein was found to be a docking protein, specific for erythropoietin-induced erythropoiesis. Current studies were designed to correlation between IGF-1R over-expression to EEC (Endogenous erythroid colony formation) formation and IRS-2 over expression in those patients who required frequent phlebotomy with elevated IGF-1R and JAK2 mutation negative patients. Materials and Methods: Patients: 8 patients withsecondary polycythemia (JAK2V617F mutation negative), and 12 MPN patients (6 ET, 3 PV and 3 MF) were studied. Of the 12 MPN patients, 11 were JAK2 mutation positive and 1 was CALR mutation positive. All 20 patients had elevated IGF-1 R. Methods: 1) IGF-1R expression: 106 cells blood mononuclear cells were incubated with 10 μL of PE-conjugated control or anti-IGF1R IgG (R&amp;D Systems) for 30 minutes. Fluorescent intensity was detected by a flow cytometry. 2) EEC formation: Erythroid colony cultures using Methyl cellulose based medium without (MethoCult H4230) or with pre-inclusion of cytokines (MethoCult H4230, including erythropoitin) from StemCell Technologies Inc. were used. On day 10-12, colonies were scored for Burst forming unit-erythroid (BFU-e), Colony forming unit-erythroid (CFU-e), and Colony forming unit cells (CFU-c) according to the manufacturer's graphic guide. 3) IRS-2 Expression: RT-PCR Predesigned primers for IRS-2 and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR blood mononuclear cells was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of IRS-2 was compared with each internal control and the final patient to normal control ratio was the average of the three. Results: 7 out of 8 patients with secondary PV, were found to have EEC formation. 12 MPN patients had increased expression of IRS-2 (Fig 1). 2 patients with secondary PV, so far studied also were found to have increased expression of IRS-2. Conclusion: 1) In patients with secondary PV (JAK2 mutation negative and increased IGF-1 and requiring frequent phlebotomy0, 7 out of 8 (87%) were found to have EEC formation. This suggests that IGF-1 by Flow cytometry can replace EEC as a supplement to the diagnosis of PV. 2) In JAK2 or CALR mutation positive patients , IGF-1R over-expression were found to have to have IRS-2 over-expression , 2 patients with secondary PV so far studied were also found to have elevated IRS-2 signaling. This suggests that the etiology of erythrocytosis in these secondary PV is likely through IRS-2 pathway. Further studies on this is still in progress. Disclosures No relevant conflicts of interest to declare.

Use of Ppmx-T003 As a Potent Inhibitor of Erythrocytosis in Polycythemia Vera

Iron incorporation through transferrin (Tf) and its receptor, TfR1, plays a critical role in erythropoiesis. We hypothesized that blockade of TfR1 may be a promising strategy to suppress erythrocytosis observed in polycythemia vera (PV). To examine this possibility, we developed PPMX-T003, a monoclonal antibody against human TfR1, through phage display screening. PPMX-T003 was observed to bind to TfR1 with a high affinity(Kd= 3.56×10-10)and presented strong ability to suppress the growth of PV model cell lines such as HEL (EC50= 0.07 nM). PPMX-T003 induced TfR1 internalization but blocked the recycling of the receptor presumably because it was strongly bound, even in the endosome, which switched the fate of TfR1 from recycling to degradation. Consequently, the PPMX-T003 treatment greatly reduced the intracellular iron concentration, along withcell cycle arrest in the G2/M phase in HEL cells. In contrast, PPMX-T003 failed to block cell proliferation in human umbilical vein endothelial cells that expressed a subtle level of TfR1. These findings suggest that PPMX-T003 is a potent inhibitor for cells whose growth is dependent on iron incorporation by TfR1. This led us to examine whether PPMX-T003 blocked the endogenous erythroid colony (EEC) formation in erythroid progenitor cells in PV. We observed that PPMX-T003, at a concentration of 40 ng/mL, exhibited complete blockade of EEC formation inJAK2 V617F-positive PV patients' cells. Although hydroxycarbamide (HU) and phlebotomy are well-established treatment strategies for the management of hematocrit in PV patients, there is a risk of secondary leukemia associated with HU treatment and side effects such as fatigue and restless leg syndrome due to phlebotomy-induced iron deficiency. Therefore, we suggest PPMX-T003, which may preferentially suppress erythrocytosis without changing the iron level in peripheral blood, as an alternative treatment for PV patients. Disclosures Ohira: PPMX: Current Employment, Current equity holder in private company. Nomura:PPMX: Current Employment. Matsuura:PPMX: Current Employment. Komatsu:Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents &amp; Royalties: PCT/JP2020/008434, Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding.

Secondary Erythrocytosis Is Phenotypically Distinct from Polycythemia Vera but Associated with Comparable Rates of Thrombosis at Diagnosis

Background: Since lowering the hemoglobin (Hb) and hematocrit (Hct) diagnostic thresholds for polycythemia vera (PV) to 165 g/L and 49% in men and 160 g/L and 48% in women respectively, by the World Health Organization (WHO) in 2016 (Blood. 2016;127:2391), hematologists have witnessed an increase in referrals for erythrocytosis, a significant proportion of which are found to have secondary erythrocytosis (SE). This has yielded novel challenges as comprehensive data on SE and reports comparing SE vs PV are scarce. Further, data regarding thrombosis in SE have been inconsistent (Clin Appl Thromb Hemost. 2013;19:363; J Clin Anesth. 1991;3:99; Ann Hematol. 2016;95:233). Methods: This multicentric retrospective study included PV patients enrolled in a pan-provincial registry of the chronic myeloid leukemia and myeloproliferative neoplasms Quebec Research Group (GQR LMC-NMP). SE patients were recruited from the Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada. PV diagnosis was per WHO 2016 criteria (Blood. 2016;127:2391). SE was defined as sustained elevation in Hb and/or Hct above WHO thresholds for PV with negative JAK2V617F testing and non subnormal serum erythropoietin (Epo) levels. Epo levels were drawn in therapy-naïve patients within 3 months of diagnosis using a standard immune-enzymatic assay (reference 3-30 mIU/mL). Endogenous erythroid colony assays used standard methods. JAK2 mutation screen and risk-stratification for PV were according to convention. Standard statistical methods were used to assess variables across i) SE and PV groups, and ii) subjects having experienced a thrombotic event vs no thrombosis. JMP® Pro 13.0.0 software was used for all analyses (SAS Institute, Cary, NC, USA). Results: Among 102 informative cases of erythrocytosis, 36 (35%) were SE and 66 (65%) were PV. Median age was significantly lower in the SE (57 years old; range 19-76) vs PV group (63.5 years old; range 20-89) (p=0.005), with a preponderance of males (75% in SE vs 45% PV; p=0.004). Median serum Epo levels were normal in the SE cohort (10.3 mIU/mL; range &amp;lt;1-148) vs subnormal in PV patients (2.3 mIU/mL; range &amp;lt;1-14.1) (p&amp;lt;0.0001). At diagnosis, SE vs PV patients had significantly lower baseline platelets (191 vs 417 x 109/L; range 125-476 vs 120-995), leukocyte counts (7.2 vs 10 x 109/L, range 4.1-15.1 vs 4.5-20.5) and lactate dehydrogenase (LDH) (182 vs 247 U/L, range 126-316 vs 157-874) (p&amp;lt;0.0001). Further, SE vs PV cohorts displayed less frequent palpable splenomegaly (6% vs 23%; p=0.02), higher median BMI (33.4 vs 26; p=0.02), and an excess of active smokers (31% vs 12%; p=0.03) (Table 1). The most prevalent etiology of erythrocytosis in the SE cohort was idiopathic, reported in 50% of cases (n=18), followed by smoking/chronic obstructive pulmonary disease in 22.2% (n=8), smoking plus additional factors in 8.3% (n=3), sleep apnea, polycystic kidney disease, or a combination of the latter two in 5.6% each (n=2 each) and post kidney transplant in 2.7% (n=1) (Table 1). Nearly as many SE (n=11, 31%) as PV patients (n=26, 39%) were exposed to bone marrow sampling (p=0.37) and SE patients were frequently managed with phlebotomy (42% vs 76% PV, p=0.0007) and aspirin therapy (56% vs 93% PV, p&amp;lt;0.0001) (Table 1). SE and PV populations had similar rates of thrombosis at/prior to diagnosis including events in 9 (25%) SE and 19 (29%) PV patients (p=0.68), both predominantly arterial (n=7 and n=16 respectively; p=0.58) (Table 2). History of thrombosis at any time in both groups clustered significantly with older age (p=0.001), hypertension (p&amp;lt;0.0001), diabetes (p=0.01), obesity (p=0.01) and hyperlipidemia (p&amp;lt;0.0001) (Table 3). Conclusions: SE patients are significantly younger, more likely to be male, active smokers, and obese, with normal-high Epo, normal LDH, and exceedingly rare leukocytosis and thrombocytosis, discriminating them phenotypically from PV patients. Importantly, rates of thromboembolic events prior to/at diagnosis are comparable in SE vs PV, suggesting that SE may not be benign from a thrombosis standpoint. Classic cardiovascular risk factors significantly cluster with thrombosis risk, emphasizing the importance of controlling these variables in both cohorts. Finally, a significant portion of SE patients are subject to bone marrow sampling and, though controversial, treatment with phlebotomy and aspirin, highlighting the need for formal studies to guide management in this population. Disclosures Szuber: Novartis: Honoraria. Busque:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Assouline:Takeda: Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding. Olney:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Quantification of IGF-1 Receptor Is Useful in the Differential Diagnosis of Essential Thrombocytosis from Reactive Thrombocytosis

To make a definite diagnosis of essential thrombocytosis (ET) from reactive thrombocytosis (RT), the most reliable criteria are the presence of driver mutations, namely JAK2, CALR, or MPL gene mutations. In the absence of these driver mutations, so-called triple-negative ET, the differential diagnosis could be difficult; bone marrow biopsy could be helpful, but in some cases it may be difficult, to do gene sequence analysis to find other clonal marker gene mutations than the driver mutations, only very few were found. We studied IGF-1R quantification by flow cytometry in mononuclear cells (MNC) from peripheral blood, a substitute for performing endogenous erythroid colony formation (EEC), in 33 patients with ET (untreated or off treatment with hydroxyurea), 28 patients with RT, and 16 normal volunteer controls. We found IGF-1R levels significantly elevated in ET patients compared to RT patients or controls. A cutoff value of 253 was chosen from the logistic regression to predict each patient's group, a value ≥253 meant that a patient belonged to the ET group (sensitivity of 68.6% and specificity 96.4.%). Therefore, we suggest adding quantification of IGF-1R in blood MNC by flow cytometry is useful in differentiating ET from RT , we suggest this maybe added to the minor criteria for diagnosing ET. Funding Statement: JCW received grants from Celgen Corp. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The author declares no conflicting interests with the commercial funder relating to using products, consultancy, patents, product development, or market products. The other authors declare no competing financial interests. Ethics Approval Statement: Peripheral blood was obtained from patients with written informed consent, the protocol for which was approved by the IRB of Brookdale University Hospital.

Ropeginterferon alpha-2b targets JAK2V617F-positive polycythemia vera cells in vitro and in vivo

Polycythemia vera is characterized by the acquisition of the JAK2V617F mutation. Recommended treatments include hydroxyurea and interferon-alpha. Several groups have reported a reduction in the JAK2 mutant allele burden in interferon-treated patients, but significance of this observation is questioned. We characterized the activity of ropeginterferon alpha-2b, a novel form of interferon-alpha recently shown to be safe and efficacious in polycythemia vera. Ropeginterferon was able to inhibit the proliferation of the HEL, UKE-1, and UT-7 JAK2-mutant cell lines while sparing JAK2-wild-type UT-7 and normal CD34+ cells growth. In vitro treatment of erythroid progenitors derived from PV patients showed that ropeginterferon could considerably inhibit the growth of endogenous erythroid colonies, a hallmark of polycythemia vera. Finally, we could study in sequential samples the clonal architecture of erythroid progenitors derived from patients included in a randomized study comparing hydroxyurea to ropeginterferon. After 1 year of treatment with ropeginterferon, the ratio of JAK2-mutated to wild-type colonies grown from bone marrow progenitors was reduced by 64%, compared to 25% in patients receiving hydroxyurea. This study shows that ropeginterferon has a potent targeted activity against JAK2-mutant cells and is able to drastically reduce the proportion of malignant progenitors in patients treated with this drug.

The WHO diagnostic criteria for polycythemia vera—role of red cell mass versus hemoglobin/hematocrit level and morphology

Regarding diagnosis of polycythemia vera (PV), discussion persists about hemoglobin (Hb) and/or hematocrit (Hct) threshold values as surrogate markers for red cell mass (RCM) and the diagnostic impact of bone marrow (BM) morphology. We performed a retrospective study on 290 patients with PV (151 males, 139 females; median age 65years) presenting with characteristic BM features (initial biopsies, centralized evaluation) and endogenous erythroid colony (EEC) formations. This cohort included (1) a group of 229 patients when following the 2008 versus 256 patients diagnosed according to the 2016 World Health Organization (WHO) guidelines, all presented with increased RCM; (2) masked PV patients with low Hb (n = 143)/Hct (n = 45) recruited from the 2008 WHO cohort; (3) a cohort of 17 PV patients with elevated diagnostic Hb/Hct levels but low RCM; and (4) nine PV patients with increased RCM, opposing low Hb/Hct values. All patients were treated according to current PV guidelines (phlebotomies 87%, hydroxyurea 79%, and acetylsalicylic acid 87%). Applying the 2016 WHO criteria significantly increased concordance between RCM and Hb values compared with the 2008 WHO criteria (90 vs. 43% in males and 83 vs. 64% in females). Further analysis of the WHO 2016 PV cohort revealed that increased RCM is associated with increased Hb/Hct (93.8/94.6%). Our study supports and extends the diagnostic impact of the 2016 revised WHO classification for PV by highlighting the importance of characteristic BM findings and implies that Hb/Hct threshold values may be used as surrogate markers for RCM measurements.

Bone Marrow Histology Characteristics in MPL515 Mutated Thrombocythemia with Various Degrees of Myelofibrosis: A Cross Sectional Follow-up Study in Eight Cases

MPLW515l/K mutated Essential Thrombocythemia (ET) usually present with increased platelet counts around 1000 x 109/L as the only abnormal laboratory finding with normal values for hemoglobin and leukocytes and minor splenomegaly on palpation only in a few. Early stage MPL515 mutated ET show the presence of clustered small and giant megakaryocytes with pronounced deeply lobulated nuclei, which are not seen in JAK2V617F positive ET, prodromal PV, and classical PV. Indicating that MPL515 mutated ET has no clinical, laboratory and bone marrow features of prodromal PV. Clustering of large mature large to giant megakaryocytes with pronounced hyper lobulated nuclei in a normocellular bone marrow is the hallmark of JAK2-wild type MPL515 mutated thrombocythemia. Bone marrow histology in MPL515 mutant patients revealed isolated megakaryocytic proliferation in a normocellular bone marrow at diagnosis with a reduction of erythropoiesis. MPL515 Thrombocythemia do not have PV features at diagnosis, do not evolve into PV during follow-up, have normal LAP score, serum EPO and ferritin levels. In contrast to JAK2V617F positive ET, no spontaneous endogenous erythroid colonies (EEC) was found in any of evaluated MPLW515L cases. Spontaneous megakaryocyte growth in culture with an overall normal response to thrombopoietin (TPO) has been demonstrated in two MPL515 mutated cases.

The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis

AimsWe investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis...

Generation, Characterization and Genetic Modification of Human iPSCs Containing Calr, MPL and JAK2 Mutations Found in MPN Patients

Introduction: Myeloproliferative neoplasms (MPNs) that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are associated with multiple somatic and myeloid-specific mutations. A major challenge to the study of these diseases has been the lack of model systems that can accurately represent their complexity; the clonal heterogeneity within the hematopoietic stem/progenitor cells (HSPC) population is confounding and it is technically challenging to isolate and expand patient HSPC clones in vitro. To overcome this hurdle, we have utilized induced pluripotent stem cell (iPSC) technology to generate disease and clone-specific iPSC lines that preserve the genetic identity of HSPC clones with the JAK2-V617F mutation (Ye et al., 2009 in Blood). Recent advances in MPN genetics have identified CALR and MPL mutations in the majority of JAK2-V617F negative MPN. We therefore created additional panels of iPSCs from patients carrying CALR and MPL mutations in addition to our JAK2-mutated iPSCs. In addition, we used CRISPR/Cas9 technology to study genetic lesions and their contribution the phenotypes by using iPSC in vitro hematopoietic differentiation assays (Ye, et al., 2015 in Molecular Therapy).Methods: We generated iPSC lines from MPN patients with JAK2V617F, CALR or MPL mutations using peripheral blood CD34+ cells by transient expression of reprogramming factors in plasmid vectors. We characterized the pluripotency and karyotype of the derived iPSC lines, and determined if they contained the same somatic mutation as found in blood samples of each MPN patient. Then the derived iPSC cell lines were differentiated to CD34+ HSPCs, erythroid or megakaryocytic cell lineages using defined in vitro culture systems. Erythroid differentiation was determined by flow cytometric analysis of CD235a (Glycophrin A)-positive and CD45-negative cells. Megakaryocytic differentiation was determined by the levels of CD41+CD42+ cells.Results: In vitro differentiation assays have shown increased erythroid differentiation of HSPCs derived from patient-specific iPSCs that carry the JAK2V617F mutation, recapitulating a major PV disease feature. Editing a PV-derived JAK2V617F/V617F iPSC line to JAK2V617F/WT abrogated endogenous erythroid colony (EEC) formation, erythroid expansion and altered erythropoietin dose-response curve characteristic of the parent line. Conversely, gene editing a JAK2V617F/WT to a JAK2V617F/V617F iPSC line from a different PV patient conferred EEC formation and expanded erythropoiesis. In addition, data generated from these isogenic iPSCs suggested the contribution of additional genetic lesions to MPN disease phenotypes in JAK2-V617F positive patients. We also generated iPSC lines from MPN patients with the CALR type II (+5 bp) mutation and the rare MPL-V501L mutation. We found that the iPSC line containing a heterozygous MPL-V501L mutation (derived from an ET patient) appeared normal with iPSC typical morphology and phenotypes (expressing OCT4 and TRA-1-60 pluripotency markers) as the wildtype and JAK2-V617F iPSCs. We also generated four iPSC lines from a MF patient: all the four clones contained the homozygous CALR type II (+5 bp) mutation as found in the blood cells of the MF patient. Two iPSC clones we examined had a normal karyoptype. However, the iPSC lines showed signs of increased spontaneous differentiation when being cultured under the standard culture condition, based on cell morphology and reduction of OCT4 & TRA-1-60 expression. We are currently testing the potential of these iPSC lines containing the CALR and MPLmutations in hematopoietic differentiation, focusing on erythrocytic and megakaryocytic differentiation activities.Conclusions: we believe that by comparative analysis of isogenic iPSCs containing the common and rare MPN driver mutations, both common and specific pathways essential for MPNs can be determined. Precise genome editing or surgery of these mutations by engineered targeting may also provide a novel approach to eliminate the malignant MPN clones.This project is supported in part by the MPN Research Foundation and LLS. DisclosuresNo relevant conflicts of interest to declare.

Quantification of IGF-1 Receptor May Be Useful in Diagnosing Polycythemia Vera-Suggestion to Be Added to Be One of the Minor Criterion.

Endogenous erythroid colony (EEC) formation is one of the minor criteria for diagnosing polycythemia vera (PV) according to 2008 WHO diagnostic criteria. But EEC requires bone marrow aspiration and sophisticated laboratory procedures; therefore, practically it is rarely used to diagnose PV. Insulin-like growth factor 1 receptor (IGF-1R) was found to be constitutively phosphorylated and was responsible for the EEC formation in PV; therefore, we measured IGF-1R levels in the peripheral blood of 26 PV patients and compared them with those of 33 patients with secondary polycythemia and 29 normal controls. Among the PV patients, 16 were treated with only phlebotomy, 9 received hydroxyurea, and 1 was treated with ruxolinitinib. We found that PV patients treated with only phlebotomy had significantly higher IGF-1R levels than did those PV patients treated with hydroxyurea or ruxolinitinib. None of the secondary PV patients or normal controls had elevated IGR-1R levels, while 14 of 16 (87%) PV patients had significantly elevated IGF-1R levels. The new 2016 WHO has eliminated EEC as a minor criterion for diagnosing PV, but there are still some cases that cannot be definitively diagnosed by the current criteria. Therefore, we suggest that quantifying the IGF-1R level in peripheral blood by flow cytometry to replace EEC as the minor criterion for diagnosing PV.

47-Year-Old Man With Pruritus

47-year-old man with a medical history of headaches and seasonal allergies presented to the primary care clinic in late fall for evaluation of diffuse pruritus of 6 weeks’ duration that had occurred after initiating a new diet. The diet mostly restricted foods like dairy, soy, nuts, grains, and processed foods, but he stated that he had substantially increased his consumption of coconut and various spices including turmeric. The itching was present over most of his body but most prominent on the inner thighs and most notable after a hot shower. He provided picturesfromrecent episodes thatillustrated linear excoriations on the thighs. When he showered in cooler water, the diffuse pruritus was still present but minimally improved compared with a hot shower. He had been using the same body soap for years but tried a new soap without relief from the itching. In addition, he changed laundry detergent from one he had used for years with no relief. He had no occupational or habitual exposures that are suspected to contribute to pruritus. Overthe-counter hydrocortisone cream applied to his thighs provided minimal relief. His weight had been stable, and he reported no constitutional symptoms. On physical examination, the patient was a

Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

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. DisclosuresNo relevant conflicts of interest to declare.

Clinical Relevance of Molecular Markers in Taiwan Chinese Patients with Primary Myelofibrosis: Endogenous Erythroid Colony Growth Is the Most Important Predictor of Outcomes

Background and purpose: The clinical features and molecular markers of primary myelofibrosis (PMF) in Asian population have rarely been reported. We examined the clinical relevance of molecular markers in a large cohort of PMF patients in Taiwan.Methods: Bone marrow or blood samples at initial diagnosis from 145 patients consecutively diagnosed with PMF based on WHO criteria in Chang Gung Memorial Hospital-Linkou, Taiwan, were examined. EEC assay was performed in a serum free culture system. PRV-1 mRNA expression in granulocytes was measured by real-time RQ-PCR TaqMan assay. Pyrosequencing was used to detect JAK2 V617Fand its allele burden as well as 46/1 rs12343867 genotype in granulocytes. Mutational analysis of MPL (exon 10) was performed by PCR assay followed by direct sequencing. CALR (exon 9) mutations were screened by GeneScan analysis followed by sequencing for those with length changes. Ten of 20 patients progressed to secondary AML (sAML) had matched paired diagnosis and sAML samples available for comparative analysis.Results: Of the 145 patients with PMF, the median age was 64 years, 76 were male, IPSS low risk 25, Int I 23, Int II 41, and high risk 56 patients. In a median follow-up of 35.8 months (range 1.1 to 275.5 months), 20 patients progressed to sAML, 88 patients died with a median overall survival (OS) of 67.4 months. JAK2 V617F was detected in 52% (74/143) patients, CALR mutations in 30% (41/135) (type1 n=29; type 2 n=5; and others n=7), MPL mutations in 4% (5/141) (n=2/2/1 for W515L/K/A), and 11.0% of PMF patients were triple-negative. The incidence of 46/1 haplotype in 112 patients analyzed was TT 32 %, CT 36 %, and CC 32 %; C-allele frequency was significantly higher in PMF compared with 50 normal subjects (50% vs. 24%; P< 0.0001).EEC growth was detected in 48.9% (45/92) of patients examined. PRV-1 over-expression was present in 40% (28/70) of patients. Of the 10 matched paired PMF/sAML samples, 6 patients had CALR mutations with similar allele burden at both phases of disease whereas sAML evolved from a non-JAK2 V617F clone in one of the 3 patients carrying JAK2 V617F at diagnosis. Patients with EEC growth or PRV-1 over-expression were significantly associated with younger age, higher WBC and platelet counts. EEC-positive patients had higher Hb level and lower circulating blasts. JAK2 V617F was closely associated with higher WBC and platelet counts whereas patients with CALR mutations had lower WBC counts. None of these molecular markers had a correlation with constitutional symptom, IPSS, occurrence of thrombosis or risk of sAML transformation. EEC growth conferred a favorable leukemia-free survival (LFS) (P =0.019) and OS (P =0.013) compared with those without EEC. PRV-1 over-expression was associated with better OS (P =0.036). JAK2 V617F and MPL mutations did not influence LFS and OS. Allele burden of JAK2 V617F had no impact on outcomes. CALR mutations were associated with a favorable OS compared with mutation-negative patients (P =0.034). There were no difference in outcomes between type 1 and type 2 mutations of CALR. Patients with triple-negative mutations had a significantly inferior OS (P =0.020). CT genotype (46/1) was associated with shorter LFS (P =0.026). EEC growth was strongly associated with PRV-1 over-expression and JAK2 V617F mutation, whereas EEC formation and CALR mutations were mutually exclusive. In multivariate analysis, EEC growth was the most important predictor for LFS (HR 0.058; 95% CI: 0.005-0.676, P =0.023) and OS (HR 0.21; 95% CI 0.076-0.581, P =0.003) among the molecular markers; CALR mutations also held favorable OS (HR 0.245; 95% CI 0.085-0.709, P =0.009).Conclusions: Approximately 90% of PMF patients in Taiwan had JAK2 V617F, CALR, or MPL mutations, half were associated with C-allele genotype, 78% had EEC growth and /or PRV-1 over-expression. EEC growth was the most important independent factor for predicting better outcomes and CALR mutations also conferred a favorable OS. (Grant support: NSC96-2314-B-182-003, CMRPG330303, OMRPG3C0021, and MOHW103-TD-B-111-09) DisclosuresNo relevant conflicts of interest to declare.

Diagnostic and Treatment Guidelines for the Chronic Myeloproliferative Neoplasms: Current Challenges

ISSN 2045-1393 Int. J. Hematol. Oncol. (2015) 4(1), 5–8 The chronic myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) are a unique group of hematopoietic stem cell disorders, characterized by the increased production of morphologically normal red cells, white cells and platelets alone or in combination and a tendency to develop myelofibrosis, extramedullary hematopoiesis with bone marrow failure or leukemic transformation, albeit at varying frequencies. These shared clinical features not only result in phenotypic mimicry among these three disorders but also between them, their companion myeloid neoplasms (MN) and certain benign disorders of hematopoiesis as well. In this regard, although they are clonal stem cell disorders, the chronic MPN differ from their companion MN because with supportive therapy alone, survival in PV, ET and PMF is usually measured not in years but decades. This makes diagnostic accuracy essential. However, until the 2005 discovery of a mutation (V617F) in JAK2 [1], a tyrosine kinase essential for hematopoietic progenitor cell proliferation and differentiation, the diagnosis of a specific MPN was largely based on clinical phenotype and, in particular exclusion of absolute erythrocytosis [2], the hall mark of PV, the most common MPN and the one in which the risk of thrombosis or hemorrhage is greatest. The development of a clinical assay for JAK2 V617F and subsequently, for mutations in JAK2 exon 12, redefined the MPN on a molecular basis with approximately 95% of PV patients expressing JAK2 V617F and 3% expressing JAK2 exon 12 mutations. By contrast, only 50–60% of ET and PMF patients expressed JAK2 V617F; the discovery of MPL mutations primarily in 4% of ET and 8% of PMF patients still left approximately 30–40% of MPN patients, including some with PV, without an identified molecular basis for their disease. In 2007, taking advantage of these molecular findings, the WHO proposed new diagnostic criteria for the MPN, relying on the use of molecular assays for JAK2 and MPL mutations together with stipulated values for the hemoglobin or hematocrit value, the use of the serum erythropoietin (EPO) level, endogenous erythroid colony formation, bone marrow histology, cytogenetics and flow cytometry

Usefulness of BCSH Criteria for Diagnosing Japanese Polycythemia Vera

Objectives: The WorldHealth Organization (WHO) classification (2008) is wid ely usedto d iagnose polycythemia vera (PV). However, some patients clinically suspected of PV may not be definitely diagnosed because they do not have a high hemoglobin (Hb) level, one of the main andessential d iagnostic criteria, or because of the lack of or unfilledminor criteria such as bone marrow biopsy, endogenous erythroid colonies, and serum erythropoietin. The British Committee for Standards in Haematology (BCSH) employed hematocrit (Ht), but not Hb, as an indicator in the PV diagnostic guidelines. Furthermore, if the presence of the JAK2 gene mutation is demonstrated, PV can be diagnosed with Ht only by BCSH. Therefore, we evaluated the usefulness of BCSH criteria for diagnosing PV in Japanese patients. Methods: We determinedhematological andclinical differences in 99 patients who met the BCSH criteria (Group A) and69 PV patients who met the WHO criteria (Group B). Results: All patients in Group B also met the BCSH criteria. Thirty patients in Group A (43%) did not fulfill the WHO criteria. No significant differences were observedin white bloodcell andplatelet counts, serum erythropoietin levels, the JAK2V617F allele burd en, or the risk of thrombosis andtransformation into myelofibrosis between Groups A andB. Conclusions: PV cases diagnosedby the BCSH criteria showedthe same clinical picture as those diagnosedby the WHO criteria. In view of actual clinical settings, the diagnostic criteria of BCSH criteria may be useful for the diagnosis of PV in Japanese patients.

Three Phenotypes of JAKV617F Mutated Essential Thrombocythemia and Early Thrombocythemic Stage of Polycythemia Vera in 10 Patients: Important to Differentiate because Natural History Differ

In 10 JAK2V617F positive patients with early myeloproliferative essential thrombocythemia (ET) or polycythemia vera (PV) we sequentially used the Polycythemia Vera Study Group (PVSG), the Thrombocythemia Vera Study Group (TVSG) and the 2008 WHO criteria for the clinical diagnosis of essential thrombocythemia (ET) ad polycythemia vera (PV). Subsequently, we evaluated bone marrow features and laboratory and molecular markers including endogenous erythroid colony (EEC), serum erythropoietine (EPO) mutation for the classification of early MPD. Six symptomatic patients had a delayed diagnosis of slow onset latent (masked) ET and 4 patients presented with rapid onset PV associated with thrombocythemia. We could clearly distinguish three phenotypes of ET when the 2008 European clinical, molecular and pathological (2008 WHO-ECMP) criteria for ET and PV are applied. These include ET phenotype 1, ET phenotype type 2 with features of early polycythemia vera (PV) and normal red cell mass, and ET phenotype 3 associated with megakaryocytic granulocytic myeloproliferation (ET-MGM) in the absence of characteristic features of primary myelofibrosis (PMF). Through the presentation of a number of highly illustrative clinical cases during long-term follow-up we could demonstrate that diagnostic differentiation of three phenotypes of ET is important, because natural history clearly differ.

Revisiting the IGF-1R Signaling Pathway in Myeloproliferative Neoplasms: Quantification of IGF-1 Receptor May be Useful in the Differential Diagnosis of Polycythemia

Endogenous erythroid colony formation in vitro is one of the important criteria for the diagnosis of polycythemia vera (PV). Extensive studies performed in the last decade suggest that IGF-1 plays a significant role in the erythropoiesis and endogenous colony formation in PV. Therefore, IGF-1R signal pathway is important in the pathogenesis of PV. We elected to revisit IGF-1R pathway by flow cytometry technique in PV and other myeolproliferative neoplasms (MPNs). 32 MPN patients (including 11 PV, 19 myelofibrosis (MF), 12 essential thrombocytopenia (ET), 8 secondary PV , and 17 age-matched normal controls were studied. Methods: 1) plasma IGF-1, IGFBP-1, IGFBP-3 were assayed by ELISA(R&D Systems, Minneapolis, MN) ; 2) quantification of IGF-1R . Peripheral blood mononuclear cells (MNC) harvested from Ficoll-Paque separation were stained with phycoerythrin conjugated antibody against IGF-1 receptor (R&D Systems), and assayed by flow cytometry using FACS Calibur system (BD Biosciences, San Jose, CA; 3) quantification of IGF-1R phosphoration (pIGF-1R) after IGF-1 stimulation. MNC cells were suspended in serum-free α-MEM medium for 2 hours, then starved cells were treated with or without 10 nM IGF-1 for 3 min and assayed for pIGF-1R by flow cytometry using Alexa Fluor 647 conjugated antibody against phosphorylated IGF-1 receptor (BD Biosciences); 4) Interferon alpha (IFNα) Treatment. IFN-α (Schering Corporation, Kenilworth, NJ) was added to the cell culture at 100 unit per ml. Cells were incubated with IFN-α for either 2 hours or 3 days and then collected for further pIGF-1R analysis. The results showed 1) No difference in plasma IGF-1 levels in MPN and controls; IGFBP-1 and IGFBP-3 were significantly elevated in MPN than controls but the change of IGFBP-3 in MF patients was not significant. 2) the PV group had significantly elevated IGF-1R expression than secondary PV (Fig 1). Mean ± SE (MFI expression) were 529 ± 84 and 90 ± 30 in PV versus secondary PV, respectively (p=0.004). 3) The untreated PV group had significantly higher IGF-1R than MF, ET, and controls. MF and ET were not different from controls, but 8/19 (43%) in MF and 6/12 (50%) in ET have elevated IGF-1R than controls. 4) No difference in IGF-1R expression in Jak2 (+) versus Jak2 (-) in MPN patients. Mean ± SE (MFI) were 293.2 ± 62.1 and 164.8 ± 47.7 in JAK2(+) versus JAK2 (-) respectively (p=NS). 5) Preliminary studies showed PV has a significant elevated pIGF-1R than controls in unstimulated PB CD34+ cells, but not in MNC cells. 6) IFN-α decreased IGF-1R expression and pIGF-IR about 10% in the PV group.Conclusions: 1) IGF1R signal pathway is more active in PV than MF, ET and controls. 2) Quantification of IGF-1R expression by flow cytometry may be helpful in differential diagnosis in primary versus secondary PV. 3) IGF-1R expression is not correlated with JAK2 status. 4) IGF-1R phosphorylation is more elevated in unstimulated CD34+ cells of PV patients than in controls suggesting autonomous erythroid colony formation in PV may be associated with IGF-1R activation. 5) interferon-α has some inhibitory effects in IGF-1R expression and phosphorylation but is unlikely to be the main mechanism of its induced remission of JAK2 –mutant allele-burden effects. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.