Granulocyte JAK2 (V617F) Mutation Status in Myeloid Neoplasms with Ringed Sideroblasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 854-854 ◽  
Author(s):  
Luca Malcovati ◽  
Matteo G. Della Porta ◽  
Daniela Pietra ◽  
Anna Galli ◽  
Erica Travaglino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
X Chromosome ◽  
Jak2 V617f ◽  
X Chromosome Inactivation ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Mutation Status ◽  
Ringed Sideroblasts ◽  
Myeloid Neoplasms ◽  
V617f Mutation

Abstract The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts (RARS). We have previously demonstrated that mitochondrial iron accumulation in this condition is in the form of mitochondrial ferritin (MtF). A gain-of-function mutation of JAK2 is found in most patients with chronic myeloproliferative disorders. A high frequency of this mutation has been also reported in RARS associated with marked thrombocytosis (RARS-T), a provisional entity characterized by marked increase in platelet count, hypercellular marrow with increased megakaryocytes, and ringed sideroblasts. In this study, we investigated the granulocyte JAK2 (V617F) mutation status in 73 patients receiving a diagnosis of myeloid malignancy with ringed sideroblasts at the Division of Hematology, University of Pavia Medical School & IRCCS Policlinico San Matteo Pavia, Italy between 2001 and 2006. According to the WHO classification of the myeloid neoplasms, 23 patients had RARS, 17 had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), 16 had refractory anemia with blasts excess, four had MDS with isolated del(5q), and 13 fulfilled the criteria for RARS-T. JAK2 (V617F) mutation status was analyzed on peripheral blood granulocytes through a quantitative real time PCR-based allelic discrimination assay. We compared clinical and biological features of patients with RARS-T with those of patients with refractory anemia or cytopenia with ringed sideroblasts, and found that RARS-T patients had higher neutrophil and platelet counts, lower frequency of cytogenetic abnormalities and higher incidence of the JAK2 (V617F) mutation (P values ranging from <.001 to .02). JAK2 (V617F) was detected in six out of 63 evaluable cases (9.5%), one being diagnosed as MDS with isolated del(5q) and five as RARS-T, resulting in an incidence of mutation in the latter group of 45%. The proportion of mutant alleles ranged between 2.8% and 18.4%, values commonly observed by us in essential thrombocythemia [Blood. 2006 May 1;107(9):3676–82]. Focusing the analysis on RARS-T, a significantly higher hemoglobin level at diagnosis was found in mutated (median value 11.2 g/dL, range 10.1–15.4) compared with non mutated patients (median value 9 g/dL, range 6–9.9) (P=.009). JAK2-positive patients also showed a significantly lower percentage of ringed sideroblasts in the bone marrow (P=.01), and an increased marrow reticulin fibrosis (P=.03). We then evaluated the clonality of hematopoiesis in female patients through analysis of X-chromosome inactivation patterns (XCIPs) in circulating granulocytes and bone marrow CD34-positive cells. Twenty-one out of 23 informative female patients with ringed sideroblasts (91%), as well as 5/6 RARS-T (83%) displayed a clonal pattern of X-chromosome inactivation. These observations suggest that refractory anemia with ringed sideroblasts with marked thrombocytosis is a clonal stem cell disorder significantly associated with the JAK2 (V617F) mutation. This disorder shows both dysplastic and proliferative features, the presence of the mutation being associated with a predominant myeloproliferative phenotype. The recognition of a heterogeneous genetic background in myeloid neoplasms with ringed sideroblasts suggests that different mechanisms might be involved in the induction of mitochondrial ferritin expression in these disorders.

RARS-T without Thrombocytosis?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4607-4607
Author(s):  
Christophe Ravoet ◽  
Marie-Agnès Azerad ◽  
Vanessa Delrieux ◽  
Nathalie Meuleman ◽  
Dominique Bron
Keyword(s):  
Bone Marrow ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Trisomy 8 ◽  
Ringed Sideroblasts ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Transfusion Requirements ◽  
V617f Mutation

Abstract Background: Recently, a provisional entity called “refractory anemia with ringed sideroblasts with thrombocytosis” (RARS-T) has been characterized in the MDS/MPD overlap category of the WHO classification. Diagnostic criteria include RARS features in association with sustained platelet counts > 600 (500) x 109/l and exclusion of cytogenetic abnormalities such as 5q−, t(3,3) and inv(3). Reticulin fibrosis in the bone marrow has been significantly associated with RARS-T. Splenomegaly also seems more frequent than in other RARS subtypes. Among these patients, approximately two thirds present with a JAK2 V617F mutation. On the contrary, JAK2 V617F mutation appears extremely rare in patients with typical RARS (Szpurka H. Blood 2006, Cabello AI. Leuk Research 2005, Malcovati L. ASH 2006, Remacha AF. Haematologica 2006). Case report: In 2001, we observed a 67 years old patient with RARS (22 to 45% ringed sideroblasts). Conventional cytogenetics revealed a trisomy 8 and deletion of chromosome Y. A mild to moderate fibrosis was observed in the bone marrow. At diagnosis, the patient was anaemic but had normal platelet counts which remained strictly normal during the next 6 years. After failure of various treatments, the transfusion requirements increased due to progressive alloimmunization and probably a moderate splenomegaly which had appeared 3 years after the diagnosis (splenic volume evaluated by ultrasonography was completely normal until 2004). In July 2006, the haemoglobin level was 6.1 g/dl despite the transfusion of 11 RBC units in the previous month. In the aim to overcome the suspected hypersplenism, a splenectomy was performed. In the subsequent days following splenectomy, we observed a rapid and persistent increase in platelet count with a peak of 2550 x 109/l. A JAK2 V617F mutation was demonstrated. The normalization and maintenance of platelets was obtained with small doses of anagrelide (0.5mg four doses/week) with RBC transfusion requirements decreasing by 50%. Conclusion: This patient presented most of the main characteristics of RARS-T except thrombocytosis (note that trisomy 8 has been described in association with RARS-T). Our observation suggests that thrombocytosis could be absent in some patients presenting with a very similar pattern of features as described in RARS-T. We postulate that hypersplenism (and possibly other particular biological characteristics) could, in some cases, mask the complete picture of RARS-T.

The JAK2-V617 Mutation Status Identifies Subtypes of Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2695-2695
Author(s):  
Annette Schmitt-Graeff ◽  
Soon-Siong Teo ◽  
Manfred Olschewski ◽  
Andreas Kirn ◽  
Petra Reinecke ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Initial Diagnosis ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Mutation Status ◽  
Allelic Ratio ◽  
Positive Group ◽  
Ringed Sideroblasts ◽  
Platelet Counts ◽  
V617f Mutation

Abstract Sideroblastic erythropoiesis is a feature of pure refractory anemia with ringed sideroblasts (RARS), but may also be associated with megakaryocytic proliferation and marked thrombocytosis (RARS-T). Recently, the identification of a JAK2-V617F mutation in small series of patients has suggested that RARS-T is a JAK2 mutation-related disorder. The aim of our study was to correlate the presence of JAK2-V617F with clinical and hematological features in a larger cohort of patients with RARS-T. An allele-specific PCR for JAK2-V617F genotyping was carried out on DNA samples extracted from bone marrow biopsies obtained from 28 patients (mean age 71 years, mean follow-up 72 months). Our assay measures the allelic ratio of the mutated JAK2 allele, i.e. the percentage of JAK2 loci with the mutated dT-nucleotide (%T). We detected the JAK2-V617F mutation in 12/28 patients (43%) with a predominance of the female gender (8/12). In 7 patients, we found %T<50% (range 16 to 43) at first presentation compatible with a heterozygous JAK2-V617F mutation. Since we are analyzing a mixture of cells, we cannot exclude the presence of a small population of cells that are already homozygous for JAK2-V617F. In 4 cases, the mutant allele predominated (%T> 50%), demonstrating cells homozygous for JAK2-V617F at initial diagnosis. In one patient the allelic ratio of mutated JAK2 increased from 0 to 43 and 98 when biopsies obtained 8 and 16 months after the initial diagnosis were analyzed. At the time when JAK2 V617F was negative, the patient fulfilled the criteria of RARS but not yet of RARS-T since the platelet count was < 500 x 10^9L. The acquisition of the JAK2V617 mutation was accompanied by a progression into RARS-T. In another patient, the ratio increased from 19 to 72 within 14 months. In both cases, the increased ratio of mutated JAK2 was associated with rising platelet counts. On histological evaluation, all JAK2-V617F positive cases displayed a megakaryocyte morphology in keeping with a typical myeloproliferative phenotype. In 4 of the negative cases, a predominance of small hypolobated forms indicated myelodysplastic features. With regard to the mutation status, no differences were found for platelet counts, spleen size, marrow fiber score or vascular events. However, V617-positive patients had significantly higher erythrocyte values (p = 0,003), lower MCV (p = 0,0003) and higher leukocyte counts (p = 0,034). The mean survival time of patients in the positive group was 5.14 years versus 6.43 years in the negative group. The mutation-positive patients had a relative death risk which was 82.3% lower than that of the positive group (relative risk of JAK2-V617F positive versus negative 0.177; 95% confidence interval: 0.040–0.789; p = 0,023). Our results suggest that RARS-T may be divided into two biologically distinct subgroups according to the JAK2-V617F mutation status with a more favorable prognosis for the positive disease. An evolution from V617 heterozygosity to homozygosity during the course of RARS-T may occur in a subset of patients.

Study on Clinical and Laboratory Characteristics of Chronic Eosinophilic Leukemia CEL/Hypereosinophilic Syndrome (CEL/HES).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4637-4637
Author(s):  
Zhijian Xiao ◽  
Yue Zhang ◽  
JianXiang Wang ◽  
Yushu Hao
Keyword(s):  
Bone Marrow ◽  
Hypereosinophilic Syndrome ◽  
Jak2 V617f ◽  
Break Point ◽  
Jak2 V617f Mutation ◽  
Sequencing Analysis ◽  
Mutation Status ◽  
Chronic Eosinophilic Leukemia ◽  
Clonal Proliferation ◽  
V617f Mutation

Abstract Hypereosinophilic syndrome (HES) was a group of diseases associated with persistent eosinophilia without unknown causes, companied with tissue and organ impairments. In 2001, WHO classification of hematopoietic and lymphoid neoplasms classified chronic eosinophilic leukemia (CEL) / HES into the category of chronic myeloproliferative disease, and proposed that the principal basis for the identification of CEL and HES was whether to have the evidence of eosinophils clonal proliferation: CEL had the evidence of eosinophils clonal proliferation, while HES was lack of the evidence of eosinophils clonal proliferation. In this study, 20 cases of CEL/ HES patients were analyzed retrospectively, and nested RT-PCR was used to detect FIP1L1-PDGFRA (F/P) fusion gene; Allele-specific PCR (ASP) conjoint sequencing analysis was used to detect JAK2 V617F, and PCR-RFLP was adopted to detect the mutation status of JAK2 V617F; and PCR is applied to detect TCRγ rearrangement. The clinical and laboratory characteristics of CEL and HES were compared. The ratio of male and female in the 20 cases of patients was 19:1, and the median age was 33 (20–57). F/P detection was positive for 12 cases, and the sequencing confirmed that FIP1L1 break point was at intron 10–12, while PDGFRA break point was at exon 12. There was 1 case of patient found that had JAK2 V617F, and the mutation status analysis showed that it was the mutation on heterozygote. 6 cases were detected having TCRγ gene rearrangement, of which 4 cases were CEL patients, and other 2 cases were HES patients. Most of CEL patients had respiratory symptoms in the early stage, easily companied with circulatory systematic impairment and nervous systematic symptoms. The incidence of splenomegaly of CEL patients was obviously higher than that of HES patients (92.5% vs 42.5%, p=0.031), so did the incidence of anemia and myelofibrosis. There was no difference in EO, WBC, PLT and EO% in peripheral blood as well as bone-marrow eosinophils percentage and bone-marrow primitive cells’ percentage between two groups. Abnormal morphology of eosinophils was often found in CEL patients, with the main manifestation of eosinopenia, basopenia and plasma vacuoles. Our data showed that Eosinophilia is often caught by male, mainly by the young; CEL patients have the main manifestation of the circulatory systematic, respiratory and gastrointestinal symptoms, and have a high incidence of anemia and thrombocytopenia. The routine examination has a little significance for the identification o CEL and HES, while the bone marrow smears morphological examination has a certain help for the diagnosis of CEL; Some HES patients have JAK2 V617F mutation, and further studies on the effect of JAK2 V617F mutation on the pathogenesis of HES can contribute to the diagnosis of such patients in the future and the development of the new targeted drug therapy; Some CEL patients have TCRγ rearrangement, while the relationship of CEL and TCRγ needs a further study.

Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1643-1643
Author(s):  
Hadrian Szpurka ◽  
Lukasz P Gondek ◽  
Sanjay R Mohan ◽  
Eric D Hsi ◽  
Karl S Theil ◽  
...  
Keyword(s):  
Clonal Selection ◽  
Neutral Loss ◽  
Significant Proportion ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Molecular Defect ◽  
Ringed Sideroblasts ◽  
V617f Mutation

Abstract Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (&gt;600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis (RARS-t): A Clinicopathological Entity with Questionable Pathogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5252-5252
Author(s):  
Anupma Nayak ◽  
Abha Goyal ◽  
Judith Brody ◽  
Peihong Hsu ◽  
Steven Savona ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Normal Karyotype ◽  
Jak2 V617f ◽  
Clinicopathological Features ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Simultaneous Occurrence ◽  
Jak2 Mutation ◽  
Ringed Sideroblasts ◽  
V617f Mutation

Abstract Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-t) is a clinicopathological entity with features of both myelodysplastic syndrome and myeloproliferative disorder. The cases of RARS-t reported in literature are few and do not confirm whether this disease represents a variant of Refractory anemia with ringed sideroblasts (RARS), a variant of Essential Thrombocythemia (ET), or the simultaneous occurrence of two separate disorders (RARS and ET). Because of this ambiguity, RARS-t has been assigned to MDS/MPD- Unclassifiable subcategory as a provisional entity in the WHO (2001) classification. Recent studies have demonstrated association of RARS-t with JAK2 V617F mutation in up to 67% of cases, suggesting its proximity to MPD. In a few studies, presence of JAK2 mutation was related with better prognosis. Based on this evidence, there are chances that this provisional entity may be clubbed with the group of MPD disorders in the revised WHO classification. We present here 4 cases of RARS-t diagnosed within a period of 1 year (2007–2008) at our institution. Clinicopathological features of these cases are mentioned in the attached table (Table-1). Two of these cases were not associated with anemia at the time of presentation, however could be diagnosed as RARS-t based on the presence of dyserythropoetic features and &gt;15% ringed sideroblasts in marrow on iron stains. Other causes of reactive ring sideroblasts and thrombocytosis were ruled out in these cases. 2/4 cases were positive for JAK2 V617F mutation, however no correlation was found between the JAK2 positivity and the clinicopathological features including prognosis. All the 4 patients were given hydroxyurea and pyridoxine therapy. In contrast to other studies, one of our JAK2 positive cases had to be maintained on the transfusion support alone due to the multiple treatment failures. Our data adds to the literature supporting the association of RARS-t with JAK2 mutation. However, we do not agree with the notion that JAK2 positivity renders a better prognosis. Also, lack of any correlation of clinicopathological features in our cases with the JAK2 mutation drives us to think that it might be only a secondary association rather a primary pathogenesis event. Review of the literature reminds us that the pathogenetic events resulting in myelodysplastic component of this overlap syndrome have only been rarely addressed. In future, more studies focusing on the mutations responsible for genesis of ringed sideroblasts such as ALAS2, ABCB7, FECH and gene expression profiles of bone marrow progenitor cells are required for better understanding of this common but under reported entity. Table 1 Case Age Sex Hb. (g/dL) Hct. MCV (fL) RS% Platelet (× 109/L) WBC (× 109/L) JAK2mutation Other cytogenetics Fibrosis Organomegaly Case#1 84 F 12.9 38.9 94.6 &gt;15% 709 8.3 Negative normal karyotype 1+ reticulin Absent Case#2 85 M 10.3 28.3 131 &gt;15% 1072 7.2 Negative normal karyotype 1+ reticulin Absent Case#3 68 M 6.9 25 101.3 &gt;15% 634 5.4 Positive normal karyotype 1+ reticulin Absent Case#4 63 M 13.7 41.5 78.7 &gt;15% 1272 17.7 Positive normal karyotype 1+reticulin Absent

scholarly journals Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2173-2181 ◽  
Author(s):  
Hadrian Szpurka ◽  
Ramon Tiu ◽  
Gurunathan Murugesan ◽  
Samer Aboudola ◽  
Eric D. Hsi ◽  
...  
Keyword(s):  
Melting Curve ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Melting Curve Analysis ◽  
Jak2 Mutation ◽  
Ringed Sideroblasts ◽  
Polymerase Chain ◽  
V617f Mutation ◽  
Reaction Sequencing

Abstract JAK2 V617F mutation recently was identified as a pathogenic factor in typical chronic myeloproliferative diseases (CMPD). Some forms of myelodysplastic syndromes (MDS) show a significant overlap with CMPD (classified as MDS/MPD), but the diagnostic assignment may be challenging. We studied blood or bone marrow from 270 patients with MDS, MDS/MPD, and CMPD for the presence of JAK2 V617F mutation using polymerase chain reaction, sequencing, and melting curve analysis. The detection rate of JAK2 V617F mutants for polycythemia vera, chronic idiopathic myelofibrosis, and essential thrombocythemia (n = 103) was similar to the previously reported results. In typical forms of MDS (n = 89) JAK2 V617F mutation was very rare (n = 2). However, a higher prevalence of this mutation was found in patients with MDS/MPD-U (9 of 35). Within this group, most of the patients harboring JAK2 V617F mutation showed features consistent with the provisional MDS/MPD-U entity refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T). Among 9 RARS-T patients, 6 showed the presence of JAK2 V617F mutation, and in 1 patient without mutation, aberrant, positive phospho-STAT5 staining was seen that is typically present in association with JAK2 V617F mutation. In summary, we found that RARS-T reveals a high frequency of JAK2 V617F mutation and likely constitutes another JAK2 mutation-associated form of CMPD.

Relationship between JAK2 V617F Mutation Status and Constitutive Mobilization of CD34-Positive Cells into Peripheral Blood in Patients with Chronic Myeloproliferative Disorder.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2586-2586
Author(s):  
Francesco Passamonti ◽  
Elisa Rumi ◽  
Emanuela Boveri ◽  
Daniela Pietra ◽  
Laura Vanelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Count ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Myeloproliferative Disorder ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Mutation Status ◽  
Cell Counts ◽  
V617f Mutation

Abstract A gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently reported in patients with polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Abnormal trafficking of CD34-positive cells with increased numbers in the peripheral blood is found in CIMF and in advanced stages of other myeloproliferative disorders. To determine whether the unique JAK2 V617F mutation affects the mobilization of CD34-positive cells into peripheral blood, we studied the relationship between JAK2 mutation status, bone marrow and circulating CD34-positive cells in 72 patients diagnosed according to the WHO criteria. A quantitative real-time polymerase chain reaction (PCR)-based allelic discrimination assay was used for the quantitative detection of the JAK2 V617F alleles in circulating granulocytes. Bone marrow CD34-positive cells were quantitatively assed on paraffin immunostained sections, while circulating CD34-positive cells were enumerated by flow cytometry using a single-platform assay. Overall, 57% of the patients studied carried the JAK2 V617F mutation. Within these patients, median values for JAK2 V617F alleles in circulating granulocytes were as follows: 29% in PV, 4% in ET, 12% in prefibrotic CIMF, 27% in fibrotic CIMF, and 99% in post-PV myelofibrosis. The vast majority of circulating granulocytes were homozygous for the mutation in all but one of patients with post-PV myelofibrosis. Decreased numbers of bone marrow CD34-positive cells and increased counts of circulating CD34-positive cells were detected in patients with fibrotic bone marrow. The higher the degree of fibrosis, the higher the circulating CD34-positive cell count (P&lt;0.001) and the lower the bone marrow CD34-positive cell count (P&lt;0.01). All patients with PV, ET and prefibrotic CIMF, and 7 out of 21 patients with fibrotic CIMF had circulating CD34-positive cell counts lower than 10 x 106/L. Conversely, all patients with post-PV myelofibrosis had counts higher than 10 x 106/L. In univariate analysis, there was an inverse relationship between percentage of JAK2 V617F alleles and bone marrow CD34-positive cells (r=−0.35, P&lt;0.01), and a direct relationship between percentage of JAK2 mutant alleles and circulating CD34-positive cells (r=0.46, P=0.001). Multivariate analysis showed that disease category (P=0.0008) and percentage of JAK2 V617F alleles (P=0.03) were independently related to circulating CD34-positive cell counts. These observations suggest that the JAK2 V617F mutation might be involved in the constitutive mobilization of CD34-positive cells into peripheral blood that is found in patients with myeloproliferative disorder. Nonetheless, constitutive mobilization is present in a considerable portion of patients who do not carry the JAK2 mutation, pointing to additional pathogenetic mechanisms. Findings on patients with PV suggest that transition form heterozygosity to homozygosity for JAK2 V617F may represent an important step in the progression of PV to myelofibrosis. Thus, sequential evaluation of the percentage of JAK2 mutant alleles and enumeration of circulating CD34-positive cells may be useful for disease monitoring in PV.

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