Philadelphia-negative Myeloproliferative Disorders Research Articles

RUNX1 Expression Characterizes the Endothelial Cells from the Spleen and Bone Marrow of Patients with Primary Myelofibrosis

Background.Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph−) myeloproliferative disorder characterized by extramedullary haematopoiesis and abnormal neoangiogenesis in both the bone marrow (BM) and the spleen. We previously provided evidence that endothelial cells (ECs) from either the spleen or the splenic vein of PMF patients frequently share the JAK2V617F mutation with the hematopoietic malignant cells. More recently, we confirmed this observation also in BM-derived ECs of PMF patients. The mechanism underlying this phenomenon remains, however, not yet clarified. RUNX1 is a critical regulator of hematopoiesis, required for hematopoietic stem cell (HSC) generation and function. In human embryo, it is expressed in all emerging HSCs and progenitors and it is a necessary transcription factor for endothelial to hematopoietic transition. In the adult humans it is expressed in all blood cells, in decreasing intensity according to the maturation status, except erythrocytes. In angiogenesis, it induces endothelial differentiation and maturation as well as vascular network formation by promoting expression of VE-cadherin. Finally, it is involved in retinal aberrant neoangiogenesis.Aim. To assess if neoangiogenetic activity observed in spleen and BM of PMF patients is associated with RUNX1 expression in ECs.Patients and Methods. Paraffin-embedded BM sections from patients with MF (n=3), and from patients with lymphomas (named as CTRLs), who underwent BM biopsy for disease staging (n=2), were used for immunostaining analysis. Spleens samples were collected from 3 patients with PMF, who received splenectomy for clinical reasons, and from 2 healthy subjects (HS), who underwent surgery after traumatic damage. Fresh spleen samples were embedded in OCT, and then snap-frozen and stored in liquid nitrogen. Endothelial colony forming cells (ECFCs) were obtained from peripheral blood (n=1 PMF, n=1 HS), PMF spleen tissue (n=1) and cord blood (n=1), according to Ingram et al (Blood 2004;104:2752) and cytospun on slides at confluence. ECFCs, BM and spleen sections were stained with antibodies directed against RUNX1 and VE-cadherin. The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGEJ software. We evaluated 10 fields for each BM and spleen section and measured the number of RUNX1-positive vessels/total vessels. We considered positive a vessel that contained at least one RUNX1-positive cell. The results are shown as mean ± SD.Results. Immunofluorescence staining of spleen and BM sections confirmed the presence of increased neoangiogenetic processes in samples from PMF patients with respect to CTRLs. The staining of BM and spleen sections obtained from PMF patients with antibodies anti-RUNX1 and anti-VE-cadherin (endothelial marker) allowed the detection of RUNX1+VE-cadherin- cells in the parenchima of BM and spleen (Figure 1A, 1C), occasionally in perivascular position (Figure 1A, arrowhead). Interestingly, in all patients analyzed, we could detect RUNX1+VE-cadherin+ cells in both the parenchima and in the vessels both in the BM and the spleen (Figure 1A, 1C, arrow). In BM, 22 ± 12% of the vessels had at least one double positive cell in the microvessel wall; in spleen tissue the percentage of RUNX1+ ECs increased to 60 ± 13%.We detected RUNX1+VE-cadherin- cells only in the BM parenchima of CTRLs but not in the spleen of HS, whereas RUNX1+VE-cadherin+ cells were never observed in the vessels of neither the spleen nor the BM (Figure 1B, 1D) of HS and CTRLs, respectively.The circulating ECFCs obtained from both PMF and HS were, as expected, VE-cadherin+ but did not express RUNX1; however, 30% of spleen-derived-ECFCs of the PMF patient were RUNX1+. In cord blood-derived ECFCs a small percentage (3%) of RUNX1+ cells was observed.Conclusions.Our data show a selective expression of RUNX1 in splenic- and BM-ECs of PMF patients, suggesting that activation of RUNX1 expression could be associated with the neoangiogenetic processes that characterize the disease. The expression of RUNX1 in ECFCs from spleen but not in their circulating counterpart suggests a role for the splenic microenvironment in determining RUNX1 expression in ECs. Further studies are ongoing to assess the genotype of RUNX1+ ECs of PMF patients. DisclosuresNo relevant conflicts of interest to declare.

Calreticulin: Challenges Posed by the Intrinsically Disordered Nature of Calreticulin to the Study of Its Function.

Calreticulin is a Ca2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca2+, calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.

The IL-31/IL-31 receptor axis: general features and role in tumor microenvironment.

IL-31 is a recently identified cytokine with a well-defined role in the pathogenesis of pruritus. IL-31, whose production is induced by IL-4 and IL-33, binds a heterodimeric receptor (R) composed of the exclusive IL-31RA chain and the shared oncostatin M R. Signaling through the IL-31R involves the MAPK, PI3K/AKT and Jak/STAT pathways. Different variants and isoforms of IL-31RA with different signaling activities have been identified. IL-31 is produced predominantly by circulating Th2 lymphocytes and skin-homing CLA+CD45RO+ T cells. Studies in humans have demonstrated a pathogenic role for IL-31 in atopic dermatitis and allergic asthma. The first demonstration of the involvement of the IL-31/IL-31R axis in cancer came from studies in patients with mycosis fungoides/Sézary syndrome, the most frequent, cutaneous T cell lymphoma. Tumor cells were shown to produce IL-31, whose serum levels correlated with pruritus intensity. Follicular lymphoma (FL) B cells and their counterparts-germinal center B cells-produced IL-31 and expressed IL-31R, which signaled in the former, but not the latter, cells. IL-31 released in association with microvesicles promoted tumor growth through autocrine/paracrine loops. Malignant mast cells from patients with mastocytosis or Philadelphia-negative myeloproliferative disorder produced IL-31, which contributed to pruritus pathogenesis. Finally, patients with endometrial carcinoma displayed high serum levels of IL-31 and IL-33, which may represent promising disease biomarkers. Targeting strategies for the IL-31/IL-31R axis have been developed, including the CIMM331 humanized anti-human IL-31RA antibody recently tested in a phase I/Ib study.

Transient floating thrombus of the internal carotid artery associated with the JAK2V617F mutation

Essential thrombocythemia (ET) is a myeloproliferative disorder (MPD) characterized by sustained elevated platelets. Cerebrovascular disorders can be a complication of ET, with thromboses of cerebral veins occurring more frequent than those of arteries [1, 2]. We describe a 33-year-old woman with no cardiovascular risk factors who experienced an acute ischemic stroke in the left middle cerebral artery territory (MCA). The carotid duplex on admission showed a free-floating thrombus (FFT) in the left internal carotid artery (Fig. 1a, b) that was not producing flow aceleration. However, the thrombus disappeared 2 h later without any clinical deterioration, as shown by a second carotid duplex. The arteriography confirmed these results (Fig. 1c). A brain infarction in the left MCA territory was observed on MRI (Fig. 1d). Peripheral blood showed a platelet count of 505 9 10/L. Additional studies, including an extensive battery of tests for coagulopaties and a complete cardiology study, were negative. The JAK2 mutation was detected in peripheral blood samples by a multiple allelespecific PCR (MAS-PCR). The thrombocytosis decreased spontaneously, as the platelet count dropped from 487 9 10/L at 3 months to 452 9 10/L after 6 months. Bone marrow aspiration showed clustered hyperlobulated megakaryocytes. She was diagnosed with MPD and specifically ET. Antiplatelet treatment was initiated as secondary stroke prevention. The patient presented a good outcome, with progressive neurological improvement. The presence of a mobile FFT in the internal carotid artery as cause of brain ischemia is infrequent and its prevalence and natural history are not well understood. The main associated pathologies are hypercoagulability, carotid dissections, trauma and plaque, although in many cases the association is not identified [3]. Philadelphia-negative MPD, especially ET and polycythemia vera (PV) may be associated with thrombi development, although they are produced more frequently in veins than in arteries [1, 2]. Recent diagnostic criteria for ET have lowered the platelet threshold from 600 9 10/L to 450 9 10/L [4]. Therefore, since the platelet count is not always highly elevated at the time of stroke, the diagnosis of ET is difficult. In our case, a complicated carotid thrombus was the initial presentation of an ET even in presence of mild thrombocythemia. The acquired Janus kinase 2 V617F mutation (JAK2) was recently identified in patients with MPDs [5]. This mutation has been found in more than 90% of patients with PV and in approximately 50% of those with ET, which led to a recent update of the diagnostic algorithms for MPDs, including the JAK2 mutation as a major criterion [4]. The risk of arterial thrombosis is significantly increased in patients with MPDs and the JAK2 mutation (OR 2.49, 95% CI 1.71–3.61) [1] independently of the V617F allele burden [6]. To the best of our knowledge, the presence of mobile floating thrombi in the extracranial carotid arteries associated with the presence of P. Martinez-Sanchez (&) R. Cazorla-Garcia J. Medina-Baez B. Fuentes E. Diez-Tejedor (&) Department of Neurology and Stroke Center, IdiPAZ Research Institute, La Paz University Hospital, Autonoma University of Madrid, Paseo de la Castellana 261, 28046 Madrid, Spain e-mail: patrindalo@hotmail.com

Familial Philadelphia-negative myeloproliferative disorders: clinical and molecular characteristics

Les formes familiales de syndromes myeloproliferatifs (SMP) peuvent etre divisees en deux grands groupes. Le premier groupe correspond aux formes familiales de SMP bcr-abl-negatif, compatibles avec une transmission autosomique dominante a penetrance incomplete. Les caracteristiques cliniques, hematologiques et moleculaires de deux principales cohortes, francaise et italienne, de familles de SMP, sont presentees. A ce jour, le ou les genes impliques dans la survenue de ces formes familiales restent inconnus. Dans cette revue, sont exposes les differents arguments en faveur d’une predisposition genetique et les approches developpees pour rechercher ces facteurs de predisposition. Le second groupe correspond aux entites mendeliennes d’erythrocytoses et de thrombocytoses caracterisees par une penetrance complete et une hematopoiese polyclonale.

Rethinking Disease Definitions and Therapeutic Strategies in Essential Thrombocythemia and Polycythemia Vera

The seminal discovery of the JAK2V617F mutation, which is highly prevalent in Philadelphia-negative myeloproliferative disorders, now renamed neoplasms, triggered an almost unprecedented explosion of interest and data in the field. Descriptions of additional mutations in exon 12 of JAK2, at position 515 in MPL, and a number of other mutations at low frequency followed these discoveries. These advances in our understanding of molecular pathogenesis of these conditions coincided with the publication of results from two major clinical studies, ECLAP and PT-1, which contributed important clinical insights and facilitated significant correlative data collection. This article, focusing mainly upon essential thrombocythemia and polycythemia vera, reviews four major themes: the impact upon classification of these disorders considering a radical review of current terminology, and then three areas pertinent to clinical management: the indications for cytoreductive therapy in which the key targets are to reduce thrombohemorrhagic complications, relieve disease-related symptoms, and minimize the risk of transformation to secondary myeloid malignancy such as myelodysplasia, leukemia, and secondary myelofibrosis; and second reviewing current and, last, future therapeutic options, in particular interferon and JAK2 inhibitors.

Abstract A20: Development and validation of a novel acute myeloid leukemia xenograft model that is dependent on the JAK2V617F mutation for growth in vivo

Abstract Background: The Janus-associated kinase (JAK) proteins are a family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo-kinase domain of JAK2 is detected at high frequency in Philadelphia-negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation. Materials and Methods: SET2 cells containing stable doxycycline-inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H-Thymidine or by ATP quantitation. Cell-cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho-STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS. Results: Genetic knockdown using a doxycycline-inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM-1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA-repair enzyme PARP to induce apoptosis in a time- and dose-dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline- JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho-STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression. Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell-cycle arrest and induction of apoptosis in vitro and in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.

ABO blood group does not increase the risk of thrombosis in Philadelphia-negative myeloproliferative disorders

ABO blood group does not increase the risk of thrombosis in Philadelphia-negative myeloproliferative disorders

JAK2V617F Evaluation in Healthy Individuals with a Highly Sensitive Quantitative Assay

The JAK2V617F point mutation has been described in patients with Philadelphia negative myeloproliferative disorders. Improvement in the sensitivity of different methods has lowered the detection threshold of the percentage of mutant allele but currently, there is no consensus on which level of sensitivity is adequate and how to deal with the lowest levels of mutant allele. Moreover, development of JAK2 inhibitors sets the question of monitoring the molecular residual disease. These reinforce the need of the availability of reliable quantitative assays with high sensitivity and of the definition of the limit of significance for a positive result. The aim of this study was thus to establish a threshold for JAK2V617F detection between normal and potential MPD samples. We used the Jak2 MutaQuant™ kit (Ipsogen, Marseille, France), a quantitative highly sensitive allele specific RQ-PCR test on 200 randomly selected healthy individuals (among 366 volunteers). The study was approved by the Institutional Review Board and informed consent was provided according to the Declaration of Helsinki. Exclusion criteria were: age under 18, medical history of deep thrombosis or blood disease, haemoglobin level over 185 g/L for men and over 165 g/L for women, hematocrit over 52% for men and over 48% for women, white blood cell count (WBC) over 12 G/L and platelet count over 400 G/L. Median age was 37 years (19–88), 69% were male, median haemoglobin level was 142 g/L (SD 17.8), median hematocrit was 41.9% (SD 4.8), median WBC was 6.82 (SD 1.8) and median platelet count was 248 (SD 58.2). In parallel, 24 patients presenting with a thrombocytosis or a polycythemia were tested. Genomic DNA was extracted from whole blood. The technology used in this assay allows a sensitive, accurate and highly reproducible detection of single-nucleotide polymorphisms. This technique is based on the use of specific forward primers, for the wild type (WT) and the V617F allele, a common reverse primer, and a double dye TaqMan probe. Only perfect match between primer and target DNA allows extension and amplification in the PCR reaction. This assay benefits from the plasmid technology which allows the precise calibration and normalisation of RQ-PCR results, by generating standard curves based on the known concentration of plasmid dilutions, and precise measurement of the JAK2 WT and V617F alleles copy number in human cell samples without the need of subsequent handling such as capillary electrophoresis or melting curve analysis. Normalized results can be compared between RQ-PCR systems and testing sites. Results are expressed as percentage of JAK2V617F among total JAK2 detected sequences. Limit of blank was determined (95th percentile) on negative samples (n=56) and is approximately equal to 0.04%. Limit of detection was determined (95th percentile) on known low positive samples (n=96) and is approximately equal to 0.2%. As expected, the sensitivity of the test varies according to the total number of JAK2 sequences detectable in 25ng of genomic DNA. In our hands the median sensitivity of the test was 0.1% of mutant alleles (range 0.28–0.038). In all our healthy donor samples the percentage of mutant alleles was lower than 0.1% (% range 0.002–0.085). Among the 24 MPD suspected patients, 11 were positive (range 0.6–62%). Our data contribute to on-going questions regarding the interpretation of JAK2V617F allele burden. Levels of mutation that are significantly above those seen in healthy individuals, even low, should be clearly of clinical interest. They should benefit of a haematological follow up as they might represent a very early stage of a myeloproliferative disorder.

A High Sensitivity Detection Technique Reveals JAK2-V617F Mutation in Additional 20% of Patients with Essential Thrombocytemia, but Not in Patients with Primary Myelofibrosis, Considered Negative with a Conventional ASO-PCR

Polycytemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF) are defined as the major Philadelphia-negative myeloproliferative disorders (MPD). The JAK2-V617F gene mutation is a common feature of MPD but it is present in a variable proportion in patients: 95% of PV, 23 to 57% of ET and 30 to 57% of PMF patients. At the present, the most important role of JAK2-V617F mutation testing, over all during the initial evaluation of MPD patients, is that it can definitively confirm the diagnosis. In fact, its specificity permits to discriminate the considerable clinical overlap between reactive cellular proliferations and clonal myeloproliferative disorders. Moreover, because V617F is an acquired mutation that can be present in a small proportion of granulocytic populations, especially for ET, a highly sensitive detection method is essential. In fact, phenotypic effects ascribed to the presence of the JAK2-V617F mutation have been reported over all in studies with ET patients. Different sensitivity of various assays methods partially accounts for the wide range of mutation frequencies reported in literature for ET and PMF. Thus, to try to increase the sensitivity of JAK2 mutational status detection, we developed a Real-Time PCR technique that enhances allele discrimination between mutant and wild-type sequence. A Locked Nucleic Acid (LNA) clamping oligomer was added to the PCR reaction solution. In this macromolecules, the ribose moiety is modified with an extra bridge connecting the 2′ and 4′ carbons. The bridge “locks” the ribose in the 3′-end structural conformation. It binds preferentially the JAK2 wild type sequence preventing from polymerase elongation. The melting curve was used to analyse amplification products, avoiding post-PCR processing and supplying the diagnostic information immediately at the end of the amplification. 236 genomic DNA samples from healthy donors, ET and PMF patients were tested for JAK2-V617F mutation detection with a conventional allelespecific PCR (ASO-PCR, sensitivity: 1–3%). All samples were re-evaluated with a seminested PCR protocol and Real-Time PCR based method in order to improve the sensitivity. The cell line HEL DNA dilutions were used to assess the semi-nested PCR and the Real-Time PCR assay sensitivity level (0,1%). The frequency of JAK2-V617F point mutation increased from 52 to 72% in 168 ET samples. All results obtained with the new technique were confirmed by the semi-nested PCR protocol. In 58 PMF samples tested, 50% of patients were positive for the mutation in ASO-PCR while 55% resulted positive with the semi-nested protocol confirmed by the Real-Time PCR test. The high sensitivity in the JAK2-V617F mutation detection obtained with the semi-nested and the Real-Time PCR revealed that 20% of patients affected by ET and considered JAK2-V617F negative with a conventional ASO-PCR, were effectively positive. The comparison between three different analytical methods revealed that in ET but not in PMF patients, the mutation can be present only in a small proportion of granulocytic populations. Thus, since the prognostic relevance of V617F allele in ET, unlike in PMF, seems to be relevant, our high sensitive detection protocol can be effective for a correct molecular characterization and a diagnostic classification. The finding that a large proportion of ET patients bears a very small amount of JAK2-V617F mutated hematopoiesis further emphasizes the problem of the role of this small clone and warrants longitudinal analysis to understand whether this proportion remains stable or expands over time.

JAK2 V617F, hemostatic polymorphisms, and clinical features as risk factors for arterial thrombotic events in essential thrombocythemia

Dear Editor,ArecentmutationlocatedintheJAK2 gene, JAK2 V617F, hasbeen shown to play a transcendental role in the constitutiveactivation of its tyrosine kinase activity in an importantnumber of cases of Philadelphia negative myeloproliferativedisorders (Ph-MPDs). This mutation is a useful tool fordiagnosing this diseases because different studies show thatthis mutation is highly frequent in patients with polycythemiavera (PV) and is present in around 50% of the patients withessential thrombocythemia (ET) and idiopathic myelofibrosis(IMF) [1]. Among the clinical features and phenotypes thatcharacterize Ph-MPD patients, thrombotic and hemorrhagicepisodes have been shown to be frequent in these patients.Range values for thrombosis at diagnosis fluctuate between34% to 39% for PV and 10% to 29% for ET [ 2]. Forhemorrhage, the values are much more variable [ 3].The mechanisms accounting for the increased risk ofthrombosis and hemorrhage in Ph-MPD patients are notwell understood. It has been accepted that the occurrence ofprevious venous thrombotic events and age increase the riskof thrombosis [4, 5]. The contribution of cardiovasculardisease risk factors in the development of thrombosis inPh-MPD patients is still unclear. Risk factors for hemor-rhage are more elusive and few have been described [2, 4].Studies are limited concerning the impact of hereditarythrombophilia in the development of thrombotic episodes inPh-MPD patients [6, 7]. In addition, the effect of the JAK2F617 allele in the development of thrombotic events is stillcontroversial [2, 8, 9].Thus, we aimed to ascertain the role of JAK2 V617F andother genetic risk factors in the development of thrombosisin patients diagnosed with Ph-MPD. For this purpose, weevaluated the prevalence of JAK2 V617F mutation andnine additional genetic alterations [FV Leiden, PTG20210A, and ZPI R67Stop are polymorphisms associatedwith venous thrombosis; HPA-1 located in the αIIbβ3integrin, HPA-2 located in von Willebrand factor receptorGIb/IX/V, GPIa C807T, and PSGL-1 are polymorphismsassociated with arterial thrombosis; FVII -323 Del/Ins andTUBB1 Q43P located in the megakaryocyte-specifictubulin β1 are two polymorphisms that increase the riskof hemorrhage] in 212 patients [74 PV, 128 ET, and tenIMF] (94 males, 118 females, median age 60.5±16.1, range21–85 years) followed during a 4-year period in HospitalMorales Meseguer (Murcia) and Hospital del Mar (Barce-lona). Thrombotic complications included venous (N=13)and major arterial thromboses (N=42) as well as microvas-cular alterations such as erythromelalgia (N=17). Severalpatients had two episodes of vascular events: five patientshad both venous and major arterial thromboses and twopatients with erythromelalgia developed an episode ofarterial thrombosis. Fifteen patients had hemorrhage. Weconsidered asymptomatic patients those with no thromboticor hemorrhagic episodes. The analysis of the role of genetic

Molecular pathophysiology of Philadelphia-negative myeloproliferative disorders: beyond JAK2 and MPL mutations

The classic Philadelphia-chromosome negative chronic myeloproliferative disorders (MPDs), recently renamed as myeloproliferative neoplasms, that include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), had been largely neglected among hematologic neoplasms

Systemic Mastocytosis with c-KITD816V Mutation Treated with Dasatinib.

Objectives: Systemic mastocytosis (SM) is mainly a clonal disease with a variable clinical outcome. Prognosis is very much related to additional symptoms. If so called c-findings are present, survival is often limited to months. Until recently only Interferon and Cladribine could show some effect on the disease progression. With the introduction of Imatinib some hope grew to treat the disease by acting on c-KIT (CD 117; stem cell factor receptor). However, the substitution of valine for aspartic acid at position 816 in c-KIT (D816V) leads to prolonged mast cell survival and increased proliferation because of constitutive activation of the tyrosine kinase of c-KIT. Between 31% and 100% of patients with SM harbour the c-KIT D816V mutation which is invariably related to Imatinib resistance. Fortunately, the new tyrosinkinase inhibitor Dasatinib (BMS-354825) could show a much higher inhibition of the c-KIT D816V mutated receptor in vitro. Therefore we treated a patient with systemic mastocytosis with associated hematologic clonal non mast cell lineage disease (SM-AHNMD) and c-findings with Dasatinib.Case description: A 69 year old patient was diagnosed 5 years ago with cutaneous mastocytosis. Because of a markedly increased tryptase levels of 130μg/l he was referred to our clinic for further investigation. In a bone marrow biopsy, the classical signs of SM could be found together with a chronic myelomonocytic leukemia (CMML) without any cytogenetic alterations. During 2 years the patient remained clinically stable without any treatment. Subsequently the patient droped weight and got strong lumbar pain. A MRI scan revealed fractures of L2 and L4 without signs for osteoporosis. Additionally, splenomegaly and hepatomegaly have been noticed with enlarged lymph nodes in the retroperitoneal space together with profound thrombocytopenia. Even SM-AHNMD is by definition of the ‘year 2000 Working Conference on Mastocytosis' a distinct entity, the occurring c-findings together with a rapid increase in tryptase levels have been associated with an aggressive disease course. A c-KIT mutation analysis showed a D816V mutation. We decided after approval from the medical council to start the patient on Dasatinib. We started with 50mg daily for 3 days. Because no signs of acute mastcell degranulation we increased the dose to 50mg BID and continued the treatment for 13 weeks. Due to non-hematologic toxicity (fatigue) Dasatinib had to be stopped. Hepatosplenomegaly remained stable, lumbar pain disappeared even after cessation of analgetic therapy and weight increased gradually. However, laboratory follow up (tryptase, soluble interleukin 2 receptor) showed inconsistent results.Conclusion: Our patient with SM-CMML had many signs of systemic aggressive mastocytosis which is a mostly fatal variant of SM. With the introduction of Imatinib, a potent c-kit inhibitor, a novel approach to inhibit mastcell-proliferation was described. However, the most common mutation in CD117 of mastcells (D816V mutation) turned out to be resistant to Imatinib. However, Dasatinib a recently introduced tyrosinkinase inhibitor showed significant efficacy in vitro. A phase II study of Dasatinib in patients with Philadelphia-negative myeloproliferative disorders, including SM has recently been presented by Verstovsek and colleagues showing an overall response rate of 42% in SM. However, these patients were c-KIT mutation negative. This case report shows first evidence of clinical activity of Dasatinib in a patient with systemic aggressive mastocytosis harbouring the c-KIT mutation D816V. Further clinical studies in this patient population are warranted.

JAK2 as a Molecular Marker in Myeloproliferative Diseases

The molecular pathogenesis of the myeloproliferative disorders (MPD) is poorly understood, except for chronic myeloid leukemia (CML). Recently, several groups have discovered a novel recurrent unique acquired clonal mutation in a tyrosine-kinase JAK2 in patients with Philadelphia-negative MPD and other myeloid disorders. It consists in a guanine-to-thymine change encoding a valine to phenylalanine at codon 617 (JAK2 V617F). JAK2 and the other members of the Janus kinase family are tyrosine kinases that function as intermediates between membrane receptors and intracellular signalling molecules. The mutation occurs within the enzymatically inactive JH2 pseudo-kinase domain that regulates the active JH1 kinase domain. The JAK2 activation leads to constitutive JAK and STAT (activators of transcription) hyperactivation with induction of growth factor hypersensitivity and cell transformation. Some authors have found a higher risk of vascular thrombosis and higher platelet activation when the mutation is present. Therefore, the JAK2 mutation offers a molecular target for new drugs investigation in a similar way to bcr/abl rearrangement in CML. For all these reasons, several studies related to JAK2 have arisen in the last year. In this report, we will review the literature and discuss its possible clinical and prognostic significance.

Dasatinib therapy for patients with Philadelphia-negative (ph-) myeloproliferative disorders (MPDs), including systemic mastocytosis

7086 There are no curative medical therapies for Ph- MPDs. Over last several years several of the MPDs have been associated with the abnormal expression of selected tyrosine kinases (e.g. c-kit in SM) and others are suspected to be involved too. Therefore, we engaged in conducting a Phase II study with dasatinib, an orally available multi targeted kinase inhibitor, for patient with Ph- MPDs. Dasatinib is administered at 70 mg PO BID continuously (one month equals one cycle). Response is assessed every 3 cycles, and the therapy is discontinued in those without response after 6 cycles of therapy. Patients are observed for any toxicity; in such cases the dose of dasatinib is adjusted to 50 mg PO BID, then to 40 mg PO BID, or discontinued. The study is ongoing and has enrolled 55 patients; 44 are evaluable for response and toxicity, including 24 with SM (6 with aggressive SM, 4 with SM and associated hematologic non-mast cell disease and 14 with indolent SM with uncontrolled symptoms despite optimal supportive care measures), 10 CIMF, 6 HES, 3 unclassifiable MPD and one PV. Median age is 65 years (range, 27–75); 25 males and 19 females. The overall response rate in SM was 42% (10 pts). Of those, two patients (8%) achieved complete remission, one with SM-CIMF, and one with SM-HES. Both were c-KIT mutation negative and had low, not significant tryptase levels. Both were anemic (Hb 9.4g/dL) and failed erythropoietin therapy, and had abnormal WBC differential; one had low platelets (90×109/L). Symptoms related to SM improved significantly in additional 8 patients, however, no significant response in percentage of bone marrow mast cells or blood tryptase levels have been observed so far. The 6 patients with HES had previously failed imatinib therapy and had no evidence of the FIP1L1-PDGFRA fusion transcript; one achieved complete remission (normalization of blood and bone marrow eosinophil percentage) while others did not respond. No responses have been recorded in patients with CIMF, PV and unclassifiable MPD. No grade 4 toxicity has been observed. Dasatinib is active in SM (overall response rate 42%) mainly by improving symptoms. Updated clinical results on all enrolled patients will be presented. No significant financial relationships to disclose.

Thrombocytémie essentielle. Apport de la mutation V617F de JAK2 pour la stratégie diagnostique, la physiopathologie et les modalités évolutives

An increased platelet number in blood depends on a limited spectrum of causes, which aren't always simple to identify. Secondary thrombocytosis is a reactive process in relation with acute or chronic inflammatory diseases, or asplenia. The infrequent inherited thrombocytoses disorders are suspected when similar cases are observed in the same family. However, the most frequent causes of chronic thrombocytosis in adults are the so-called chronic myeloproliferative syndromes (chronic myelocytic leukaemia, polycythemia vera, primary myelofibrosis, essential thrombocytemia), and to a lesser extent, myelodysplastic syndromes. In the course of these disorders, thrombocytosis is often the first recognized abnormality. Chronic myelocytic leukaemia is easily diagnosed owing to the presence of either the Philadelphia chromosome or the BCR-ABL fusion gene product. The next step still relies upon a distinction according to the PVSG or the WHO criteria of Polycythemia Vera (PV) and Idiopathic myelo fibrosis (IMF) to finally confirm genuine Essential Thrombocythemia (ET). The recent description of the V617F mutation of JAK2 in 90% of PV patients, 43 to 67% with IMF and 50% of ET diagnosed according to either the PVSG or the WHO criteria is a definite characteristic of clonality now accessible in haematology practice. However, this mutation is neither specific nor constant in any of the Philadelphia negative myeloproliferative disorders, which outlines the importance of the WHO criteria of megakaryocytic abnormalities on bone marrow biopsy as the hallmark of Ph negative MPDs. The exclusion of PV and of IMF, including pre fibrotic and early fibrotic forms is still required for the diagnosis of "true" ET. Disease stratification and treatment strategy are targeted on the evaluation and prevention of vascular complications. Acute leukaemia or myelodysplasia, and other clonal progressions like myelofibrotic transformation, are infrequent and delayed events. However, according to the present data, the risk of fibrotic progression or of leukaemic transformation is not related to the mutation status of ET patients.

The first international meeting on V617F JAK2 mutation and its relevance in Philadelphia-negative myeloproliferative disorders

The first international meeting on V617F JAK2 mutation in myeloproliferative disorders (MPD) was held by the PV-Nord group on behalf of the French Society of Hematology and Paris 13 University on November 18, 2005, in Paris (France). Twelve speakers, including representatives of the three European groups who discovered the V617F JAK2 mutation and international experts in the field of Philadelphia-negative MPD, presented original biological and clinical data that allow better insight in the relevance of V617F JAK2 mutation in the pathogenesis and management of those diseases. The role of V617F JAK2 in cytokine receptors trafficking and signaling was described. Follow-up of transgenic mice expressing V617F JAK2 showed that they develop typical features of myelofibrosis. Comparisons of JAK2 mutational status to clonality of hematopoiesis in essential thrombocythemia on the one hand, and to activation of transcription factors in myelofibrosis with myeloid metaplasia on the other hand, suggest that JAK2 mutation could be a second genetic event in a subset of patients. Alternatively, other gene mutation(s) have to be found to explain the development of V617F-negative MPD. In large series of MPD patients presented, clinical characteristics of mutated and non-mutated patients were found different. Finally, the place of V617F JAK2 testing in the diagnosis and management of MPD was discussed.

Clonal lymphocytic populations in Philadelphia-negative chronic myeloproliferative disorders: is the T-cell clonality of ‘undetermined’ significance (TCUS) linked to a worse clinical outcome?

This study examined the clonality of B- and T-cells by PCR in 83 patients with Philadelphia-negative myeloproliferative disorders (Ph-MPD), to investigate its clinical and morphological correlates. Clonal lymphocytic populations were found in 23% of patients (T: n = 20, B: n = 3), with no frequency differences between ET, CIMF and PV. At the presentation, patients with clonal bands were older (58.1±13.8 vs 47.5±14.6, p = 0.0039), but did not differ in other clinical parameters. After the median follow-up of 21 months they were less likely to be asymptomatic (11.8% vs 41.1%, p = 0.029). The T-cell clonality was the strongest predictor of the symptomatic last follow-up by discriminant function analysis, surpassing the patient's age. This surprising negative prognostic impact of lymphocyte clonality in Ph-MPD may result from this phenomenon to be a better measure of the ‘hematopoietic biologic age’ than the metrical age itself.

The presence of the JAK2 V617F mutation is associated with a higher haemoglobin and increased risk of thrombosis in essential thrombocythaemia

Recently, several groups have identified a single point mutation in the JAK2 gene in the Philadelphia-negative myeloproliferative disorders, including polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis with myeloid metaplasia (MMM) (Baxter et al, 2005; James et al, 2005; Jones et al, 2005; Kralovics et al, 2005; Levine et al, 2005). A G → T transversion results in the substitution of valine by phenylalanine at codon 617 (V617F) within the pseudokinase autoinhibitory centre with overall constitutive activation of the tyrosine kinase. The presence of this mutation has been detected in high frequency in patients with PV (65–97%), ET (23–57%) and MMM (35–57%) and suggests that it may have a fundamental role in the pathogenesis of these disorders. Most studies have shown no differences in the clinical phenotype of patients with or without the JAK2 mutation. However, a longer disease duration was associated with the ‘homozygous’ status (Levine et al, 2005) and the mutation correlated with an older age, greater requirement for cytoreductive therapy, and higher frequency of thrombotic, haemorrhagic and fibrotic complications (Kralovics et al, 2005) in myeloproliferative disorders. Only one group have investigated ET patients independently, and they demonstrated a higher haemoglobin and haematocrit in ET patients with the mutation (Antonioli et al, 2005). To address these clinical findings further, we investigated 60 patients with ET for the presence of the JAK2 mutation and possible phenotypic associations. Patient notes were examined for their diagnostic blood counts and thrombotic histories. Thrombotic complications included major thromboses as well as microvascular disturbances. The clinical characteristics for the patients are shown in Table I. We used the previously published allele-specific polymerase chain reaction (PCR) method (Baxter et al, 2005) on whole blood DNA to detect qualitatively the presence or absence of the JAK2 V617F mutation and designed a second allele-specific PCR to confirm the findings independently. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) software, version 11.5, using Wilcoxon's rank sum for continuous variables, Fisher's exact test for nominal variables and binary logistic regression for multivariate analysis with all tests being two-sided and significance taken for values P < 0·05. The JAK2 mutation was identified in 48% of our ET population, which is a similar frequency to those reported by others. There was no difference in age at diagnosis, gender or disease duration between those with and those without the mutation. The proportion of patients receiving some form of cytoreductive therapy was also similar in both the groups. However, we found a significantly higher haemoglobin concentration at diagnosis in ET patients with the mutation (median 14·2 ± 1·5 g/dl vs. 13 ± 1·7 g/dl, P = 0·0426) although no differences were seen in the packed cell volume, white cell or platelet counts. Thrombotic complications were more common in patients with the JAK2 mutation; 18 (62%) vs. 8 (26%), P = 0·009. Although arterial events were more common, overall there was no statistical difference between the two groups, as defined by JAK2 mutational status, for either the type or timing of the thrombotic event. Our data therefore suggests that the presence of the JAK2 mutation in ET defines a subgroup of patients with both a polycythaemic and thrombotic tendency, confirming the results of recent studies. The ease of screening for the V617F mutation makes it a very attractive tool for diagnostic purposes but its role in risk stratification for therapy remains to be further evaluated. Shire Pharmaceuticals for an educational fellowship for Betty Cheung; Special Haematology and Molecular Diagnostics, St Thomas' Hospital for their technical support.

The Presence of the V617F Mutation Is Associated with Higher Haemoglobin, Older Age and an Increased Risk of Thrombosis in Essential Thrombocythemia.

The acquired single nucleotide valine to phenylalanine mutation (V617F) in the JAK2 tyrosine kinase is reported at variable frequencies in patients with the Philadelphia negative myeloproliferative disorders. As yet it is unclear whether its presence has an impact upon the clinical phenotype, complications or prognosis of these conditions. We have screened 87 patients under our care with myeloproliferative diseases (28 Polcythemia Vera [PV], 50 Essential Thrombocythemia [ET] and 9 Idiopathic Myelofibrosis[IMF]) using a modification of previously reported allele specific PCR on whole blood DNA (Baxter,E.J et al 2005 Lancet , 365, 1054–1061) and then correlated these results with clinical data. Overall there were 50 females and 37 males with a median age of 57.4 years (range 11–92) and median follow-up of 61.4 months (1–313). V617F was detected in 26 (93%) patients with PV, 26 (52%) ET and 2 (22%) IMF. Further analysis was performed to correlate V617F status with clinical features in the patients with ET. No survival data is presented for these patients due to the inherent bias in the analysis of fresh rather than archived samples. This will be performed in a prospective manner. However, one of the V617F positive ET patients subsequently died from AML, interestingly remaining heterozygous for this mutation. When comparing ET patients with detectable V617F against those without the mutation, the following significant differences were identified at the time of diagnosis: an older age (median 49.5 vs 39.3 years, p=0.04), higher haemoglobin (median 14 vs 13g/dl, p=0.03) and documented thrombotic events (11/26 vs 2/24, p=0.01). There were no significant differences in gender, platelet count at diagnosis or duration of follow-up. Overall thrombotic events were also more frequent in V617F positive patients (16/26 vs 7/24, p=0.02). There was no difference in thrombotic events following diagnosis (5/26 vs 5/24) or in the type of thromboses (microvasular, arterial or venous). Advanced age is a well established risk factor for thrombosis in ET, hence it is possible that V617F was associated with thrombosis as a consequence of the older age of our ET patients. Multivariate analysis was limited by small patient numbers and failed to demonstrate a significant predictive impact of either age or V617F mutation upon thrombosis. Ongoing studies will hopefully provide larger numbers for further analysis.