Endogenous Erythroid Colony Growth Research Articles

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 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...

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.

Endogenous Erythroid Colony Formation in Chronic Myeloid Leukemia: A Recurrent Finding Associated with Persistent Minimal Residual Disease Under Imatinib

In vitro endogenous erythroid colony (EEC) formation is a common finding in BCR-ABL-negative myeloproliferative neoplasms. The aim of the present study was to determine the prevalence and the clinical significance of EEC growth in chronic myeloid leukemia (CML). Results of clonogeneic progenitor cell assays from 52 patients with newly diagnosed CML were correlated with disease characteristics at presentation and molecular response to imatinib. EECs (median 7 per dish, range 1-39) were detectable in 16 patients (31%). The proportion of patients with a high-risk Sokal score was lower in the EEC group (7% vs. 30%, respectively). The cumulative incidence of achieving a major molecular response after 2 years of imatinib was similar for both groups. However, patients with EECs were less likely to achieve a more profound decline of BCR-ABL transcripts. After 6 years of imatinib, the cumulative probability [95% CI] of reaching a ≥4 log reduction of BCR-ABL was 48% [16%; 92%] for patients of the EEC group and 84% [63%; 97%] for patients of the No EEC group. The probability [95% CI] of achieving a >4.5 log reduction of BCR-ABL after 7 years was 13% [2%; 61%] for patients with EECs and 52% [30%; 78%] for patients without EECs. In vitro EECs disappeared after achievement of a major molecular response in all evaluable patients. The data indicate that EEC formation is a recurrent finding in patients with CML which deserves further attention as a possible biomarker predicting the degree of molecular response to imatinib.

ConcomitantJAK2V617F-positive polycythemia vera and B-cell chronic lymphocytic leukemia in three patients originating from two separate hematopoietic stem cells

Rare concurrent PV and CLL patients have been reported(6–8). In these patients, the JAK2 V617F mutation, a marker of PV, was absent in the CLL B-cell lineage. These earlier findings did not rule out a possibility that these two distinct hematological disorders had not arisen from the same primitive HSC that was the subject of somatic mutation(s) leading to two different clonal diseases. We studied three such subjects: Patient 1, a 79-year-old Caucasian female, had PV diagnosed in 1998 and CLL in 2000. Patient 2, a 67-year-old African American female, was diagnosed with PV in 1997 and with B-CLL in 2001. Patient 3, is a 78-year-old Caucasian female whose PV and CLL were diagnosed over 10 years ago. The PV diagnoses were supported by growth of endogenous erythroid colonies (EECs), a hallmark of PV, and the presence of JAK2 V617F mutation in granulocytes. The results are summarized in Table 1. Patient 1 had a JAK2 V617F allelic burden of 50% in the PV clone (granulocytes and monocytes) but not in T- or B-lymphocytes. We also investigated the JAK2 V617F-positive cells by genotyping individual EEC colonies; 10 EEC were heterozygous, 1 was homozygous, and 2 were without the JAK2 V617F mutation, consistent with an earlier finding that the JAK2 mutation in PV is not the primary disease-initiating event(9–11). Similarly, patients 2 and 3 had the JAK2 V617F mutation in their granulocytes, but none in CD19-positive B- and CD-3-positive T-cells. PV and CLL lineages of all three patients utilized different active X-chromosomes (see Table 1). Table 1 The JAK2 V617F mutation and X chromosome clonality analyses in myeloid and lymphoid lineages. We have analyzed T- and B-lymphocytes, granulocytes, monocytes and platelets from three such female patients for specific CD markers of lineage commitment and ... Thus, the hematopoietic neoplasms in these individuals with CLL and PV arose from distinct HSC. Earlier reports did not rule out the possibility that these disorders had not arisen from the same HSC that was the subject of further somatic mutation(s) leading to a clone (pre-JAK2 V617F clone/pre-leukemic CLL clone) that preceded the subsequent JAK2 V617F PV mutation. This possibility is now conclusively ruled out by the fact that the PV and CLL clones of these females utilized different active X-chromosomes, i.e. their CLL and PV arose from separate HCS. Nevertheless, it is well possible that the presence of as yet-undefined germ-line mutation(s) may predispose all hematopoietic stem cells to somatic events leading to two different hematological malignancies. Such evidence exists for familial clustering of PV and other myeloproliferative disorders(12), and is also suggested from the increased risk of lymphoproliferative neoplasms in MPN(13, 14).

Thrombocythemia and polycythemia in patients younger than 20 years at diagnosis: clinical and biologic features, treatment, and long-term outcome

AbstractSixty-four patients < 20 years of age, investigated for a suspicion of Philadelphia-negative myeloproliferative disease (MPD), were retrospectively evaluated to characterize the different forms and to examine the treatments used and long-term outcome. JAK2 mutations, endogenous erythroid colony growth, and clonality were investigated in 51 children. Mutations of thrombopoietin, the thrombopoietin receptor (MPL), and the erythropoietin receptor and mutations of other genes involved in the pathogenesis of MPD were investigated in JAK2 wild-type patients. Based on our criteria for childhood MPD, we identified 34 patients with sporadic thrombocythemia (ST), 16 with hereditary thrombocytosis (HT), 11 with sporadic polycythemia (SP), and 3 with hereditary polycythemia (HP). JAK2V617F mutations were present in 47.5% of ST and in no HT. The MPLS505A mutation was detected in 15/16 HT patients and in no ST (P < .00001). The JAK2V617F mutation occurred in 27% of SP patients diagnosed according to the Polycythemia Vera Study Group or World Health Organization 2001 criteria. Children with ST received more cytoreductive drugs than those with HT (P = .0006). After a median follow-up of 124 months, no patient had developed leukemia or myelofibrosis and 5% had thrombosis; the miscarriage rate in thrombocythemic patients was 14%. The low complication rate in our population suggests that children with MPD may be managed by tailored approaches.

Clinical and Biological Features, Treatment and Long-Term Outcome of 65 Children with Ph- Myeloprolipherative Disorders (MPD).

Abstract 1889Poster Board I-912 Introduction:MPD rarely occur in children and published data of pediatric patients (pts) are limited. In adults, current diagnostic criteria are those of the WHO2008 and the therapeutic options are supported by controlled studies. From our experience, we suggested that the biological markers show a clinical relevance in Ph- MPD children and may influence their management. Patients and methods:We retrospectively analyzed 65 pts aged ≤20 years (yrs) at diagnosis (dx) with Ph-negative MPD, observed between December 1981 and March 2009. Dx was performed according to the PVSG or WHO criteria. Since 2002, 52/65 pts were studied for JAK2 mutations, polycytemia rubra vera-1 (PRV-1) RNA expression, thrombopoietin (TPO) and its receptor (c-MPL) mutations, erythropoietin receptor (EPO-r) gene mutations, spontaneous endogenous erythroid colony (EECs) growth, clonality on female pts. Results:Thirty-nine females and 26 males with a median age at dx of 14 yrs were classified as follow: 34 (52%) essential thrombocythemia (ET), 16 (24.5%) familial thrombocythemia (FT), 11 (17%) polycythemia vera (PV), 3 (4.5%) familial polycythemia (FP) and 1 idiopathic myelofibrosis (IM). Clinical and biological data, treatment and outcome are reported. Pts with familial disease were younger than those with the sporadic form (p<.05). Hematocrit (Hct) values were higher in ET than in FT pts (p .0047). Fifty pts underwent a bone marrow aspirate and biopsy (15 FT and FP children were excluded). No chromosome abnormalities were found. Overt fibrosis was present in 1 pt. Symptoms at dx were recorded in 21/64 pts (33%) and splenomegaly was present in 13/64 (20%), more frequently in sporadic disease pts. The JAK2V617F mutation was found in 47.5% of ET and 27% of PV. Clonal myelopoiesis was present in 7/11 (63.5%) ET and 2/3 PV females. None of the FT and FP pts showed JAK2V617F mutations or clonality. EECs grew in 58% of ET, in 42% of FT and in 40% of PV. PRV-1 RNA overexpression was found in 29/44 pts (66%). No EECs growth or EPO-r gene mutation was found in FP pts. The median EPO level was 5 mU/ml (1.1–16.4) in polycythemic pts. MPLS505A mutations were detected in15/16 FT pts and a novel HIF2A mutation in 1 FP pt. Treatment was modified during follow-up (f-up): 15 children did not undergo any treatment and 16 pts received more than one cytoreductive drugs. Phlebothomies were carried out in 9 polycythemic pts. Overall, 26 pts were treated with different cytoreductive drugs (hydroxyurea, interferone-alpha, anagrelide and pipobroman). Because of the persistently higher platelet (PLT) count, ET children needed more cytoreductive drugs than FT pts (p .0006). Anti-platelet agents were used in 40 pts and stopped in 30. No hemorrhagic events were recorded; 3 pts (5%) developed thromboses. Eight pts had 11 pregnancies (4 abortions: 2 spontaneous). Five pts showed progressive splenomegaly, 2 (1 PV and 1 ET, untreated) of them developed IM. Two pts developed a cancer. All pts are alive after a median f-up of 10 yrs. Conclusions:This represents the largest reported series of pediatric MPD. A broad familial work-up combined to molecular analyses allowed us to characterize the MPD disorders in our pediatric population, to plan suitable treatments and to better manage their disease.ET (34 pts)FT (16 pts)PV (11 pts)FP (3 pts)IM (1 pt)M/F10/247/98/31/2FAge at Dx (yrs)15.53 (5.24–19.75)11.91 (0.22–17.45)16.47 (3.68–18.8)11.18 (11.18–14.16)19WBC × 109/L9,88 (5,62–22,22)8,43 (5,17–16,06)6,73 (4,86–28,67)6,76 (5,4–6,77)13,35Hct %41.65 (29–53)36.15 (32.3–42)53.9 (50–72.5)53.20 (42.4–54.5)31PLT × 109/L1109 (633–2800)990,5 (611–2950)217 (166–945)207 (202–271)995Symptoms12/33 (36%)4/16 (25%)5/11 (45%)0/3noSplenomegaly8/34 (23.5%)2/15 (13%)3/11 (27%)0/3noAnti-platelet agents27/34 (79.4%)9/16 (56%)4/11 (36%)0/3noCytoreductive drugs23/34 (68%)2/16 (12.5%)1/11 (9%)0/3noEECs pos11/19 (58%)5/12 (42%)4/10 (40%)0/3noPRV-1 RNA overexpr14/19 (74%)6/12 (50%)6/10 (60%)3/3 (100%)yesJAK2 V617F mut10/21 (47.5%)0/133/11 (27%)0/3noMonoclonality7/11 (63.5%)0/72/3 (66%)0/2yesMPLS505A mut0/1515/16 (94%)–––Thromboses1/34 (3%)2/16 (12.5%)0/110/3noPregnancies92–––F-up (yrs)10.02 (0.37–27.28)11.02 (0.86–26.96)9 (0.25–17.14)2 (2–9)12 Disclosures:No relevant conflicts of interest to declare.

Clinical and hematological presentation of children and adolescents with polycythemia vera

Polycythemia vera (PV) in children and adolescents is very rare. Data on clinical and laboratory evaluations as well as on treatment modalities are sparse. Here, we report the long-term clinical course of a PV patient first diagnosed more than 40 years ago at age 12. In addition, after a systematic review of the scientific medical literature, clinical and hematological data of 35 patients (19 female and 17 male) from 25 previous reports are summarized. Three patients developed PV following antecedent hematological malignancies. Budd-Chiari syndrome was diagnosed in seven patients indicating a particular risk of young patients of developing this disorder. One patient presented with ischemic stroke, one patient with gangrene, and three patients with severe hemorrhage. Three patients died from disease-related complications. Hematocrit levels and platelet counts were not correlated with disease severity. Leukocytosis >15 x 10(9)/L was present in 9/35 patients and associated with a thromboembolic or hemorrhagic complication in seven patients. The few available data on molecular genetics and endogenous erythroid colony growth indicate changes comparable to those detectable in adult patients. Treatment varied enormously. It included aspirin, phlebotomy, hydroxycarbamide, busulfan, melphalan, pyrimethamine, and interferon-alpha. Two patients successfully underwent stem cell transplantation. Currently, it is impossible to treat an individual pediatric PV patient with an evidence-based regimen.

Dasatinib inhibits progenitor cell proliferation from polycythaemia vera

A mutation of Janus kinase 2 V617F is present in most patients with polycythaemia vera (PV). However, it is generally believed that JAK2(V617F) is not the sole molecular abnormality in PV. Since dasatinib is currently evaluated in patients with PV, it is of interest to study the effects of dasatinib on the growth of clonal progenitor cells in vitro. Peripheral blood mononuclear cells from patients with PV, chronic myeloid leukaemia (CML) and controls were exposed to dasatinib (0.1 to 500 nm mL(-1)). Colony growth was stimulated by interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin. Endogenous erythroid colony (EEC) growth was investigated without exogenous cytokines. Real-time PCR was performed to assess the percentage of JAK2(V617F) cells. 10 nm of dasatinib suppressed EEC growth from PV by 89% (P = 0.002). This inhibition was dose dependent and occurred at pharmacological concentrations. Erythroid and myeloid colony growth was also significantly suppressed in the presence of exogenous cytokines. When compared to PV the inhibition of stimulated colony growth was significantly less pronounced in controls but tended to be more vigorous in CML. Interestingly, despite the potent inhibition of PV cells real-time PCR revealed that the numbers of JAK2(V617F) transcripts did not decrease upon exposure to dasatinib. This study shows a marked inhibition of the proliferative capacity of progenitor cells from PV. Although JAK2(V617F) transcript levels did not decrease upon exposure to dasatinib, the drug might suppress PV progenitors through inhibition of a yet undefined molecular target.

LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells

The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial.

The JAK2 Kinase Inhibitor LS104 Induces Growth-Arrest and Apoptosis in JAK2V617F Positive Cells.

The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase inhibitor. Ba/F3 cells stably transfected with EpoR and either the V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT) were treated with LS104. Apoptosis assays as well as immunoblotting of JAK2 and of downstream signalling pathways were performed. The effects of LS104 on kinase activity were determined in an in vitro JAK2 kinase assay. A combination of LS104 with JAK-inhibitor I, which acts via the ATP-binding site, was tested in apoptosis assays. Finally, growth of endogenous erythroid colonies (EECs) obtained from patients with V617F-positive MPD was tested upon addition of LS104. LS104 selectively and dose dependently induced apoptosis in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT control cells. By immunoblotting we found inhibition of JAK2 autophosphorylation and of downstream targets as STAT5, AKT and ERK upon treatment with LS104. Activation of these targets by JAK2 was confirmed in experiments using JAK2 siRNA. The IC50 for JAK2 in an in vitro kinase assay using LS104 was <5μM, and this effect was not reversible using increased ATP-doses. Combination treatment of Ba/F3-EpoR-VF cells using LS104 plus JAK-inhibitor I lead to significantly increased apoptosis as compared to each substance alone. Furthermore, we observed 89% inhibition of in vitro formation of EECs at 10μM LS104 whereas growth of CFU-GM obtained from normal controls was virtually unaffected. Taken together, our data show that LS104 specifically inhibits JAK2 kinase activity and JAK2 downstream signals and thereby induces apoptosis in V617F-positive cells. Our data suggest, that LS104 either irreversibly binds to the ATP-binding site or acts as a substrate kinase inhibitor and may be combined with ATP-competitive JAK2 inhibitors to enhance treatment efficacy. Growth of EECs from patients with MPD is shown to be significantly suppressed by LS104. Based on these data, a phase I/II clinical trial of LS104 for patients with JAK2V617F-positive MPD has been initiated recently.

Efficacy of TG101348, a Selective JAK2 Inhibitor, in Treatment of a Murine Model of JAK2V617F-Induced Polycythemia Vera.

The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

EEC Growth, JAK2 Mutation, PRV-1 Overexpression and X-Chromosome Inactivation Pattern: Predicting Values on Polycythemia Vera Evolution and Risk of Thrombosis in Idiopathic Thrombocytosis.

We have previously demonstrated that endogenous erythroid colony (EEC) growth could predict PV evolution in idiopathic thrombocytosis (IT) (Blood 83:744, 1994). We also showed that female patients with IT and monoclonal X-chromosome inactivation pattern (XCIP) had a high risk of vascular complications (Blood 100:1596, 2002). Recently, PRV-1 overexpression and JAK2V617F mutation have been described and implicated in the diagnosis of Ph(-) chronic myeloproliferative disorders. In a large cohort of patients with IT, we aimedto determine the correlation among the molecular markers of EEC growth, JAK2 mutation status, PRV-1 level and XCIP, andto assess the predicting values of these biomarkers on PV evolution and risk of thrombosis.Allele-specific PCR assay was used to detect JAK2V617F in peripheral blood granulocytes (n=178) or bone marrow cells (n=75) from patients with IT. EEC assay was performed on 235 patients in a serum-free culture system at initial diagnosis. PRV-1 mRNA expression in granulocytes from 187 patients was measured by real-time RQ-PCR assay and expressed as the copy number of PRV-1 normalized to GAPDH gene (NCN). Clonality analysis of XCIP with HUMARA-PCR assay was performed on 123 female patients. JAK2V617F was detected in 57.7% of patients with IT. Forty-one percent of IT patients had EEC growth at initial presentation. Of those analyzed for PRV-1 levels, 138 patients had not received platelet-lowering agents at time of analysis; 72 (38.5%) had PRV-1 overexpression with NCN ≥ 1 (NCN of 30 normal subjects, mean±SE: 0.32±0.04, range: 0.04–0.97). Seventy-six female patients had monoclonal XCIP and 28 had polyclonal XCIP; the remaining 19 patients had ambiguous or homozygous pattern of XCIP. EEC growth was strongly correlated with JAK2 mutation (P<0.0001) and PRV-1 overexpression (P<0.0001). PRV-1 overexpression was highly associated with JAK2 mutation (P<0.0001) and monoclonal XCIP (P=0.003). The frequency of JAK2 mutation or EEC growth was not different between monoclonal and polyclonal XCIP. Thirty-four patients subsequently developed PV (IT-PV), ranging from 1.5 months to 90 months. All 34 IT-PV patients had EEC growth and all but one harbored JAK2 mutation at initial presentation. Patients with IT-PV had a significantly higher frequency of EEC(+) (P<0.0001), JAK2(+) (P<0.0001), and PRV-1(+) (P<0.0001) than those without PV evolution, whereas there was no difference in age (P=0.914) or XCIP (P=0.550) between IT with and without PV evolution. One hundred and sixty-nine patients had all biomarkers of JAK2/EEC/PRV-1 analyzed, 57 patients were JAK2(+)/EEC(+)/PRV-1(+) and 55 were JAK2(-)/EEC(-)/PRV-1(-). Eighteen patients with all 3 biomarkers-positive developed PV later compared with none of patients with all 3 biomarkers-negative (P<0.0001). Eighty-two patients experienced thrombotic complications during their disease courses. The occurrence of thrombosis was strongly associated with EEC growth (P=0.0002), JAK2 mutation (P=0.007), PRV-1 level (P=0.004), and XCIP (P=0.034). The present study showed that EEC growth and JAK2 mutation were predictors of PV evolution; EEC growth, JAK2 mutation, PRV-1 overexpression and monoclonal XCIP predicted the risk of thrombosis in patients with IT.

Current diagnostic criteria for the chronic myeloproliferative disorders (MPD) essential thrombocythemia (ET), polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF)

The clinical criteria for the diagnosis of essential thrombocythemia (ET) according to the polycythemia vera study group (PVSG) do not distinguish between ET and thrombocythemia associated with early stage PV and prefibrotic chronic idiopathic myelofibrosis (CIMF). The clinical criteria of the PVSG for the diagnosis of polycythemia vera (PV) only detects advanced stage of PV with increased red cell mass. The bone marrow criteria of the World Health Organization (WHO) are defined by pathologists to explicitly define the pathological criteria for the diagnostic differentiation of ET, PV, and prefibrotic and fibrotic CIMF. As the clinical PVSG and the pathological WHO criteria show significant shortcomings, an updated set of European Clinical and Pathological (ECP) criteria combined with currently available biological and molecular markers are proposed to much better distinct true ET from early PV mimicking ET, to distinguish ET from thrombocythemia associated with prefibrotic CIMF, and to define the various clinical and pathological stages of PV and CIMF that has important therapeutic and prognostic implications. Comparing the finding of clustered giant abnormal megakaryocytes in a representative bone marrow as a diagnostic clue to MPD, the sensitivity for the diagnosis of MPD associated with splanchnic vein thrombosis was 63% for increased red cell mass, 52% for low serum EPO level, 72% for EEC, and 74% for splenomegaly indicating the superiority of bone marrow histopathology to detect masked early and overt MPD in this setting. The majority of PV and about half of the ET patients have spontaneous EEC, low serum EPO levels and PRV-1 over-expression and are JAK2 V617F positive. The positive predictive value for the diagnosis of PV of spontaneous growth of endogenous erythroid colonies (EEC) of peripheral blood (PB) and bone marrow (BM) cells is about 80-85% when either PB or BM EEC assays, and up to 94% when BM and PB EEC assays were performed. The diagnostic impact of low serum EPO levels (ELISA assay) in a large study of 186 patients below the normal range (<3.3 IU/l) had a sensitivity specificity and positive predictive value of 87%, 97% and 97.8%, respectively, for the diagnosis of PV. There is a significant overlap of serum EPO levels in PV versus control and controls versus SE. The specificity of a JAK2 V617F PCR test for the diagnosis of MPD is high (near 100%), but only half of ET and MF (50%) and the majority of PV (up to 97%) are JAK2 V617F positive. The use of biological markers including JAK2 V617 PCR test, serum EPO, PRV-1, EEC, leukocyte alkaline phosphatase score and peripheral blood parameters combined with bone marrow histopathology has a high sensitivity and specificity (almost 100%) to diagnose the early and overt stages of ET, PV and CIMF in JAK2 V617F positive and negative MPDs.

Frequency of the JAK2V617F Mutation in Platelets from Essential Thrombocythemia (ET) Patients.

The JAK2V617F mutation has been recently reported in patients with polycythemia vera (PV) and a proportion of patients with ET and myelofibrosis with myeloid metaplasia. This acquired point mutation constitutively activates JAK2 tyrosine kinase and is believed to underlie growth factor hypersensitivity displayed by hematopoietic progenitors in these disorders. Identification of this genetic abnormality may be useful for diagnostic purposes although its prognostic implication remains to be determined. The prevalence of this mutation in granulocytes from ET patients ranges from 23 to 57% depending on the detection technique used. ET is characterized by predominant involvement of the megakaryocytic lineage and increased platelet production. In order to evaluate whether the frequency of the V617FJAK2 mutation in platelets from ET patients differs from that previously reported in granulocytes, we amplified a fragment of JAK2 cDNA from leukocyte-depleted platelets by RT-PCR followed by digestion with BsaXI restriction endonuclease (PCR-RFLP). The JAK2V617F mutation abolishes a BsaXI restriction site present in the wild-type sequence and generates a different band pattern. In addition, allele-specific RT-PCR (ASPCR) was performed with a primer pair common to both wild-type and mutant alleles and a third primer complementary to the mutant allele. Forty four patients with ET diagnosed according to the PVSG criteria were included, median age 39 years old (14–81), 10 were men; 21 were studied before treatment, 10 during treatment with anagrelide or hydroxyurea and 13 both before and during treatment. Twenty-two (50%) patients were found to harbour the JAK2V617F mutation by both PCR-RFLP and ASPCR, 3 were homozygous. No change in genotype was found in repeated samples from patients studied both before and during treatment. Hemoblobin levels and the frequency of thrombosis were significantly higher in patients with the mutation respect to those without (141 ±14 g/L vs. 129 ± 11 g/L, p=0.009, and 41% vs. 4.5%, p=0.004, respectively). In addition, patients carrying the mutation were significantly older (51 vs. 33 years old, p=0.002) and had lower platelet counts at diagnosis (1.070 x 109/L vs. 1.500 x 109/L, p=0.02). Interestingly, 2 patients harbouring the mutation developed PV on follow-up. In conclusion, the prevalence of the JAK2V617F mutation in platelets from ET patients did not differ from that previously reported in granulocytes. The increase in the frequency of thrombosis found in patients carrying the mutation should be confirmed in a larger study. Besides, the finding of higher hemoglobin levels in this group of patients is of interest since JAK2V617F-positive ET patients have been reported to display PV markers such as endogenous erythroid colony growth and PRV-1 overexpression, suggesting they may have larger involvement of the erythroid lineage. Further studies will help clarify whether the risk of developing PV is higher in this subset of patients.

Neutrophil PRV-1 mRNA Overexpression in Chronic Idiopathic Myelofibrosis May Be Associated with Poor Prognosis.

Neutrophil PRV-1 mRNA is overexpressed in nearly all patients with polycythemia vera (PV) and discriminates PV from secondary polycythemia. In addition, 40– 50% of patients with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (cIMF) overexpress PRV-1. PRV-1 positive ET patients carry a significantly higher risk of thromboembolic complications compared to PRV-1 negative patients. In addition, PRV-1 overexpression and the growth of endogenous erythroid colonies (EECs) are closely correlated in ET patients. A significant proportion of PRV-1 positive/EEC positive ET patients develop PV during follow up, while this has not been observed in PRV-1/EEC negative patients. In contrast, PRV-1 and EEC expression in cIMF has not been analysed in the context of clinical course. We therefore determined PRV-1 expression and EEC formation in a cohort of 21 cIMF patients and compared clinical parameters and risk scores between PRV-1 positive and negative patients.Blood samples were drawn from 21 patients (m/f: 12/9; age: 72, 39–79 years); peripheral blood MNCs and granulocytes were purified concurrently. The former were assayed for EEC growth while the latter were used to measure PRV-1 expression. Diagnosis of cIMF according to the WHO criteria was confirmed by histopathology. The median time from diagnosis was 3 years (0,5–16,5 years). At the time of investigation six patients were receiving imatinib in the setting of a phase II trial and four received hydroxyurea. The nonparametric Wilcoxon rank sum test was used to compare hematocrit, WBC and platelet counts, LDH-levels, age as well as the Cologne risk score between groups.Fourteen of the 21 patients (67%) overexpressed PRV-1. Eight of these also showed autonomous EEC growth. Three of the remaining PRV-1 positive patients were receiving imatinib at the time of analysis and colony growth was suppressed both in the presence and absence of Epo. In the remaining three PRV-1 positive patients EEC growth was not evaluable due to lack of growth with Epo. Of the seven PRV-1 negative IMF patients six did not give rise to EECs, and one was not evaluable due to lack of growth in the presence of Epo. Hence, as previously reported in ET and in five cIMF patients, we find a close correlation between PRV-1 overexpression and EEC formation in our cohort of cIMF patients. These data support the hypothesis that two distinct molecular alterations, one leading to PRV-1 overexpression and EEC formation, the other not, can give rise to the clinical symptoms of cIMF. Because the EEC assay was informative in fewer patients than the PRV-1 assay and because the parameters are closely correlated, patients were classified by PRV-1 expression and the clinical course compared between the two groups. There was no difference between PRV-1 positive and negative patients with respect to WBC- and PLT counts, LDH-levels or highest hematocrit recorded during follow up. In contrast, PRV-1 positive cIMF patients displayed a trend to a poorer Cologne risk score (P=0.095).Thus, PRV-1 overexpressing CIMF may comprise a subgroup of patients with a poorer risk profile. Due to the relatively small sample size the statistical power of our study is limited and verification of the results in a larger sample is required.

In vitro culture growth of erythroid progenitors and serum erythropoietin assay in the differential diagnosis of polycythaemia.

We assessed the in vitro culture growth of erythroid progenitors [burst forming unit-erythroid (BFU-E)] and serum erythropoietin (EPO) levels in different groups of polycythaemia to determine the discriminative power in differential diagnosis of polycythaemia. We used the methylcellulose culture technique to study the growth of endogenous erythroid colonies (EECs) and EPO-dependent BFU-E from bone marrow (BM) and/or peripheral blood (PB) cells from 40 patients with polycythaemia vera (PV), 13 with secondary polycythaemia (SP), 19 with pure erythrocytosis (PE), five with PE and PV evolution later (PE-PV), and 12 with relative polycythaemia (RP). The serum EPO levels were measured by radioimmunoassay before treatment in 47 patients, 23 SP patients, 19 PE patients, five PE-PV patients and 16 RP patients, as well as after treatment in 38 PV patients, five PE-PV patients and 12 PE patients. The results of the erythroid progenitor culture assay showed that the numbers of EPO-dependent BFU-E in BM did not differ significantly among groups. The PB BFU-E were significantly higher in PV than in SP or PE, and no statistical difference were found among patients with SP, PE and RP. There was a correlation between BM BFU-E and PB BFU-E in the individual PV and PE patients. EECs were present in all BM and PB cultures of untreated and phlebotomy-treated PV and PE-PV patients, but were absent in 6 of 17 PV patients who had received cytotoxic therapy. EECs were not found in SP, PE and RP. PB could substitute for BM in the EEC or the BFU-E assay. Both pretreatment and post-treatment serum EPO levels of PV and PE-PV were similar, which were significantly lower than SP, PE or RP. The serum EPO levels in treated PV or PE-PV patients who had normal haematocrit values were not significantly different from those in untreated patients. In contrast, the phlebotomy-treated PE patients had significantly higher serum EPO values than untreated PE patients. In the differentiation between PV and PE, the sensitivity, specificity, positive predictive value and negative predictive value of post-treatment serum EPO levels at a cut-off level of < or = 9 UL-1 were 74%, 92% and 52% respectively. The discriminative power of post-phlebotomy serum EPO levels was even higher with a positive predictive value of 80% and negative predictive value of 92% for the prediction of PV evolution in patients with pure erythrocytosis of unknown origin. The present study showed that apart from EEC assay, the post-phlebotomy serum EPO level was a sensitive and specific parameter in the differential diagnosis of polycythaemia, in particular for the identification of PV among patients with unclassifiable polycythaemia.

Multilineage hempoietic stem cell defects in Budd Chiari syndrome

Background/Aims: Erythropoietin-independent endogenous growth of erythroid colony from bone marrow cells has been shown in many patients with Budd Chiari syndrome in earlier studies. In another report, increased megakaryocyte colony growth has also been documented in this disease. However, defects in granulocyte-macrophage cell lines in Budd Chiari syndrome have yet to be reported in the literature. Methods: Both in vitro erythroid and granulocyte-macrophage colony cultures from peripheral blood mononuclear cells with and without erythropoietin or granulocyte-macrophage colony stimulating factor, respectively, were studied in 32 patients with Budd Chiari syndrome, along with 20 normal healthy controls and ten patient controls with portal hypertension (five patients each with noncirrhotic portal fibrosis and liver cirrhosis). In 18 patients the occlusion was only in the inferior vena cava, in five patients only in hepatic veins, and in nine patients both inferior vena cava and hepatic veins were blocked. Result: Endogenous erythroid or granulocyte-macrophage colony growth was not observed in any of the normal healthy controls or in patient controls with portal hypertension. However, 22 of the 32 (68.8%) patients showed endogenous erythroid colony growth. Moreover, four of them also showed endogenous growth of granulocyte-macrophage colony, not previously reported in Budd Chiari syndrome. Conclusion: It may be inferred that stem cell defects affecting all three hemopoietic cell lines (erythroid, megakaryocyte and granulocyte-macrophage) occur in Budd Chiari syndrome, which may be the primary defect responsible for the pro-thrombotic state causing venous thrombosis in them.

Erythroid colony growth from peripheral blood and bone marrow in polycythaemia.

Erythroid colony growth in the presence and absence of erythropoietin was compared in 23 patients with primary proliferative polycythaemia (PPP), nine with idiopathic erythrocytosis, 10 with secondary polycythaemia, 15 with pseudopolycythaemia and in 76 normal subjects. Erythroid colonies growing without erythropoietin stimulation (endogenous erythroid colonies) from peripheral blood (BFU-E) were found in 20 of 22 patients with PPP and in two of seven with idiopathic erythrocytosis. None was found in secondary polycythaemia, pseudopolycythaemia, or in normal subjects. Small numbers of endogenous colony forming units-erythroid (CFU-E) (though not BFU-E) were cultured from the bone marrow of three of 24 normal subjects, suggesting that peripheral blood cultures provide a more specific indicator of clonal erythropoiesis. Peripheral blood endogenous erythroid colony growth is an effective and convenient means of distinguishing patients with clonal erythrocytosis and may be of particular value when iron deficiency obscures the diagnosis of PPP on conventional criteria.

The application of X-chromosome gene probes to the diagnosis of myeloproliferative disease.

X-chromosome DNA probes for the phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyl transferase (HPRT) genes were used to study clonality in haemopoietic cells from 63 women with myeloproliferative disease, idiopathic erythrocytosis, secondary erythrocytosis or normal red cell volumes. A total of 25 women (39%) were heterozygous for one of the polymorphisms associated with these genes. Clonality was demonstrated in five out of six patients with polycythaemia vera (PV) and in three other patients with myeloproliferative disease. In all cases of PV, including the patient in whom clonality was not demonstrated, cultures of peripheral blood showed growth of endogenous erythroid colonies.