Philadelphia Chromosome-negative Chronic Myeloproliferative Disorders Research Articles

Gene expression profiling with principal component analysis depicts the biological continuum from essential thrombocythemia over polycythemia vera to myelofibrosis

The recent discovery of the Janus activating kinase 2 V617F mutation in most patients with polycythemia vera (PV) and half of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF) has favored the hypothesis of a biological continuum from ET over PV to PMF. We performed gene expression profiling of whole blood from control subjects (n = 21) and patients with ET (n = 19), PV (n = 41), and PMF (n = 9) using DNA microarrays. Applying an unsupervised method, principal component analysis, to search for patterns in the data, we demonstrated a separation of the four groups with biological relevant overlaps between the different entities. Moreover, the analysis separates Janus activating kinase 2-negative ET patients from Janus activating kinase 2-positive ET patients. Functional annotation analysis demonstrates that clusters of gene ontology terms related to inflammation, immune system, apoptosis, RNA metabolism, and secretory system were the most significantly deregulated terms in the three different disease groups. Our results yield further support for the hypothesis of a biological continuum originating from ET over PV to PMF. Functional analysis suggests an important implication of these gene ontology clusters in the pathogenesis of these neoplasms and in disease evolution from ET over PV to PMF.

Congress report: 49th Annual Meeting of the American Society of Haematology, Georgia World Congress Center, December 8–11 2007, Atlanta, Georgia

This report dwells on selected abstracts presented at the 49th Annual Meeting of the American Society of Hematology (ASH), which was held at the Georgia World Congress Center in Atlanta, Georgia in December 2007. The aim of this report is not to discuss the very details of distinct scientific questions or clinical problems but rather to inform the practising haematologist about relevant clinical and scientific advances in the broad field of haematology. This selection of studies is of course a subjective one as the abstracts presented herein were judged upon their significance solely by the reflection of authors' personal interests. The presentation of the first pilot studies exploring the efficacy and safety of JAK-2 inhibitors in Philadelphia-chromosome negative chronic myeloproliferative disorders was certainly one of the most notable topics of the meeting. Concerning other haematologic malignancies, we felt that there were no groundbreaking developments as in recent years when novel forms of treatment such as proteasome inhibitors, tyrosine kinase inhibitors or thalidomide and its analogues were presented. But with large and often randomized trials with sufficient follow-up periods, the progress suggested by smaller phase II trials was further consolidated allowing the physician to recognize not only the true benefit but also the potential harm of these novel therapies in broad clinical practice.

Hypercoagulability and Tissue Factor Gene Upregulation in Hematologic Malignancies

Thrombotic complications in patients with hematologic malignancies are as frequent as in those with solid tumors and significantly affect morbidity and mortality. In acute leukemia, thrombosis and bleeding manifestations may occur concomitantly as a part of the same thrombo-hemorrhagic syndrome. In patients with Philadelphia chromosome-negative chronic myeloproliferative disorders (i.e., essential thrombocythemia [ET] and polycythemia vera [PV]), a thrombosis rate as high as 40% has been recorded. A hypercoagulable state is present in virtually all of these patients, even without clinical manifestations. In this review, we focus on the pathogenic mechanisms underlying the hypercoagulable state of these two hematologic malignancies. Although the pathogenesis of hypercoagulability is complex, a central role is played by the fundamental molecular changes of both the leukemic cells and of the progeny arising from the hematopoietic progenitor cells that have undergone clonal rearrangement. These cells overexpress procoagulant factors, as well as adhesion molecules and cytokines capable of inducing procoagulant changes in the vascular wall and stimulating increased cellular interactions. Recent molecular studies in experimental models of human tumors have demonstrated for the first time that oncogene- and repressor gene-mediated neoplastic transformation induces activation of blood coagulation. Similarly, in cells from patients with acute promyelocytic leukemia, the T15-17 translocation induces hyperexpression of tissue factor (TF) and renders the patient hypercoagulable. Furthermore, in blood cells from patients with PV or ET, the presence of the JAK2V617F mutation translates into activation of hemostasis, with increased expression of platelet-associated TF microparticles and the formation of increased platelet/neutrophil aggregates. Understanding the pathophysiology of hypercoagulability is critical to the design of appropriate measures for intervention in these hematologic disorders to prevent thromboembolic complications.

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

VEGF Expression Correlates With Microvessel Density in Philadelphia Chromosome–Negative Chronic Myeloproliferative Disorders

We examined microvessel density (MVD) and immunohistochemical expression of vascular endothelial growth factor (VEGF) in the bone marrow biopsy specimens of 98 patients with Philadelphia chromosome-negative (Ph-) chronic myeloproliferative disorders (CMPDs). There were significantly more MVD "hot spots" in chronic idiopathic myelofibrosis (CIMF; mean +/- SD, 25.6 +/- 6.3) and polycythemia vera (PV; 20.7 +/- 10.2) cases than in essential thrombocythemia (ET) cases (10.1 +/- 4.5) and normal control (NC) samples (7.5 +/- 3.6) (P < .05). Similar results were found using a semiquantitative method (P < .0001). A calculated VEGF index (VEGF(i)) was higher in CIMF (0.29 +/- 0.15) and PV (0.28 +/- 0.20) cases than in ET (0.12 +/- 0.05) and NC (0.08 +/- 0.04) cases (P < .0001). MVD and VEGF(i) were higher in the myelofibrotic phases of CIMF and PV. There was a direct correlation between VEGF(i) and MVD when considering the Ph- CMPDs together (r = 0.67; P < .001) and when considering PV (r = 0.79; P < .001) and CIMF (r = 0.40; P = .013) as individual entities. Our data could provide a rationale for directly targeting VEGF or endothelial cells in CIMF and PV.

Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Expression Is Increased in Ph−Chronic Myeloproliferative Disorders and Correlates with the Microvessel Density and VEGF.

Angiogenesis is a dynamic process regulated by a number of pro- and anti-angiogenic molecules. The most important pro-angiogenic factor is vascular endothelial growth factor (VEGF), which is capable of stimulating mitogenic activity and the proliferation of endothelial cells, increasing vascular permeability, and inducing vasodilation. VEGF carries out many of its functions by means of the VEGF receptor 1 (VEGFR-1) and receptor 2 (VEGFR-2). Increased microvessel density (MVD) has been demonstrated in many haematological malignancies, including Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph−CMPDs). Recently, we firstly reported a direct correlation between VEGF immunohistochemical expression and MVD in the bone marrow biopsies (BMBs) of Ph−CMPDs patients. Increased protein and RNA expression of VEGFR-1 and VEGFR-2 have been documented in acute myeloid leukemia and myelodysplastic syndrome but, at present, no data have been reported on their expression in Ph−CMPDs. In this study we examined MVD and the immunohistochemical expression of VEGF, VEGFR-1 and VEGFR-2 in BMBs of a large series of Ph−CMPDs. The study population included 83 consecutive patients (52 males, 31 females, M/F=1.6/1; median age 61 years, range: 18–85), 27 affected by essential thrombocythemia (ET), 21 by polycythemia vera (PV) and 35 by chronic idiopathic myelofibrosis (CIMF), according to the WHO classification. The normal controls (NCs) consisted of 10 BMBs obtained for staging purposes which were free of neoplasias and other abnormalities. MVD was analysed using the “hot-spot” method with the anti-CD34 (QB-END/10) antibody. Immunohistochemical VEGF and VEGFR-1 immunoreactivity was expressed as index, based on the formula [n (i) = (% of BMB cellularity x % n-positive cells)/104]. VEGFR-1(i) resulted significantly higher in Ph−CMPDs than in NCs (0.25 ± 0.17 vs 0.07± 0.03; p=0.001) and, in particular, in PV (0.30±0.19) and CIMF (0.31±0.17) (Bonferroni's LSD test; p<0.05). On the contrary, we found significantly reduced VEGFR-2 expression in Ph−CMPDs than in NCs (10.43±13.27 vs 58.56±10.11; p<0.001). A direct correlations was demonstrated between the VEGF(i) and MVD (r:0.71; p<0.001), VEGFR-1(i) and MVD (r: 0.57; p<0.001) and between VEGFR1(i) and VEGF(i) (r:0.58; p<0.001) in Ph−CMPDs considered as a whole. We did not find any correlation considering the VEGFR-2. In conclusion, our study demonstrates increased expression of the VEGFR-1 and decreased expression of VEGFR-2 in Ph−CMPDs. The increased VEGFR-1 parallels the expression of VEGF and a direct correlations between this pro-angiogenic factor and its receptor has been found. Our data, support the hypothesis of an autocrine VEGF/VEGFR-1 loop in the pathogenesis of Ph−CMPDs.

Serglycin proteoglycan in hematologic malignancies: a marker of acute myeloid leukemia

Serglycin is the major cell-associated proteoglycan of hematopoietic cells. Previous work has demonstrated that serglycin may be involved in targeting some proteins to granules of cytotoxic lymphocytes, mast cells and neutrophils. We characterized the expression of serglycin in various hematologic malignancies by immunohistochemistry and ELISA. Serglycin expression was found to distinguish acute myeloid leukemia (AML) from acute lymphoblastic leukemia. In contrast to myeloperoxidase, serglycin was found to be a selective marker for immature myeloid cells, distinguishing AML from Philadelphia chromosome-negative chronic myeloproliferative disorders.

Stem Cell Defects in Philadelphia Chromosome Negative Chronic Myeloproliferative Disorders: A Phenotypic and Molecular Puzzle?

Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph(-) CMPD) comprise a group of heterogenous haematological stem cell disorders. These diseases harbour a pathological bone marrow stem cell which overwhelms normal stem cells due to sustained and uncontrolled proliferation. By clonal evolution, acute leukaemia or bone marrow fibrosis evolve in a proportion of cases with as yet unknown underlying mechanisms. Previously, groundbreaking investigations in Ph(-) CMPD detected an acquired mutation in the Janus kinase 2 (JAK2) in the majority of patients with polycythaemia vera (PV) and in up to 50% of patients with essential thrombocythaemia (ET) and chronic idiopathic myelofibrosis (CIMF). Unlike the stem cell defect in Philadelphia chromosome-positive chronic myeloid leukaemia only a subfraction of clonally proliferating haematopoiesis may be affected by the JAK2 mutation. More recently, another mutation in the juxtamembrane domain of the thrombopoietin receptor Mpl was discovered in about 5% of patients with CIMF and ET. In accordance with the uncontrolled Abl kinase activity in Ph(+) chronic myloid leukaemia these mutations in Ph(-) CMPD apparently represent a key to unlock some of the as yet unknown basic molecular defects and this raises hope for an upcoming efficient targeted therapy. However, neither the JAK2(V617F) nor the Mpl(W515L/K) provide the initiating molecular events. Moreover, apart from distinction between reactive and neoplastic lesions, detection of these mutations does not allow a clear-cut discrimination between the particular subtypes. This review will focus on previous and recent findings in the field of molecular defects in Ph(-) CMPD.

The Gain-of-Function JAK2 V617F Mutation Shifts the Phenotype of Essential Thrombocythemia and Chronic Idiopathic Myelofibrosis to More "Erythremic" and Less "Thrombocythemic": A Molecular, Histologic, and Clinical Study

We investigated the prevalence of the JAK2 V617F gain-of-function mutation in patients with Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph- MPD) and explored the links between JAK2 mutational status and the clinicopathologic picture of essential thrombocythemia (ET), chronic idiopathic myelofibrosis (CIMF), and polycythemia vera (PV). Allele-specific polymerase chain reaction results for 59 ET, 18 CIMF, and 9 PV cases were compared with values for clinical variables at presentation and last follow-up and with the diagnostic trephine bone marrow biopsy pictures. JAK2 V617F was found in 38 (64%) of ET cases, 7 (39%) of CIMF cases, and 9 (100%) of PV cases. The ET patients with the mutant JAK2 showed significantly higher (although not overtly polycythemic) red blood cell parameter values, lower platelet counts, and higher white blood cell counts. Similar trends were found in CIMF. Megakaryocyte clustering was much less pronounced in the CIMF cases with mutant JAK2, with an analogous trend occurring in the ET cases. Bone marrow cellularity values and the numbers of CD34+ and CD117+ blasts in the ET and CIMF groups did not differ. Fibrosis was slightly less marked in the ET cases with mutant JAK2. The mutation did not significantly influence the clinical course during the follow-up in either disease in the short term (median follow-up, 22 months). The JAK2 V617F mutation is prevalent in all Ph- MPD and may skew their presenting phenotype, including bone marrow histology, toward a more "erythremic" and less "thrombocythemic" phenotype.

The JAK2 V617F mutation involves B‐ and T‐lymphocyte lineages in a subgroup of patients with Philadelphia‐chromosome negative chronic myeloproliferative disorders

The JAK2 V617F mutation is a frequent genetic event in the three classical Philadelphia-chromosome negative chronic myeloproliferative disorders (Ph(neg.)-CMPD), polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Its occurrence varies in frequency in regards to phenotype. The mutation is found in the majority of patients with PV and about half of the patients with ET and IMF. These diseases are clonal stem cell disorders arising in an early stem cell progenitor. The level in the stem cell hierarchy on which the initiating genetic events and the JAK2 V617F mutation occurs is not known. The mutation has so far been detected in all cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In this study, we detected the JAK2 V617F mutation by real-time quantitative PCR (qPCR) in both B-lymphocytes and T-lymphocytes in a subgroup of patients with Ph(neg.)-CMPDs. These results demonstrate the origin of the JAK2 V617F positive disorders in an early stem cell with both lymphoid and myeloid differentiation potential.

Quantitative PCR Assessment of JAK2 Mutational Status in Philadelphia-Chromosome Negative Chronic Myeloproliferative Disorders - The JAK2 V617F Mutation Is an Event in an Early Stem Cell and the Clonal Involvement of Different Haematopoietic Cells Varies between Individual Patients.

Introduction: The JAK2 V617F mutation is believed to occur in an early stem cell. Analysis of the JAK2 mutational status of patients suspected of a chronic myeloproliferative disorder has already been widely introduced into the diagnostic procedure. The ability to determine the size of the mutated clone and to monitor its evolution over time would be of great interest. Most methods are based on sequencing and qualitative or semi-quantitative PCR methods with limited sensitivity We present a highly sensitive real-time quantitative PCR assay, and have determined the percentage of mutated alleles in granulocytes, monocytes, T-lymphocytes and B-lymphocytes.Methods: Peripheral blood from 13 patients (PV=10, IMF=2, ET=1) with a known JAK2 mutation was collected. CD66-FITC, CD14-PE-Cy7, CD19-PE and CD3-APC were used to label granulocytes, monocytes, B-lymphocytes and T-lymphocytes, respectively. The cells were collected by a four-way FACS sort with a specificity of 99%. DNA was extracted by standard procedure. A real-time quantitative assay with a common forward primer, a Taqman probe and a wildtype specific reverse primer or a mutation specific reverse primer, was used to determine the ratio of mutated vs. wildtype alleles. The sensitivity of the PCR assay was determined by dilution series to be better than 1:10000. In the cell fractions we defined a minimum of 5 % mutated alleles to be significant.Results: All 13 patients had JAK2 V617F clonal granulocytes with a median proportion of mutated alleles of 60%, ranging from 8–96%. Eight patients (PV= 6, IMF=2) had more than 50 % mutated alleles in their granulocytes indicating homozygocity. Twelve (PV=10, IMF=2) out of 13 patients had clonal monocytes, median proportion of mutated alleles 35%, range (2–84%). Five patients had homozygote monocytes. Median proportion of mutated alleles in T-lymphocytes was 3%, range (0.24–83%), and four patients (PV=2, IMF=1, ET=1) had T-lymphocytes with more than 5% mutated alleles, 2 of them being homozygote. In B-lymphocytes only 2 patients had more than 5 % mutated alleles, none were homozygote. Median proportion of mutated alleles 3.5 %, range (1–46%).Conclusions: By a highly sensitive realtime quantitative PCR assay we have demonstrated that the JAK2 V617F mutation is an event in an early stem cell with the potential of both myeloid and lymphoid differentiation. The fraction of mutated alleles varies not only within the different cell types, but also to a great extend between individual patients. As expected all 13 patients harboured the mutation in their granulocytes Twelve, five and two patients had clonal JAK2 mutated monocytes, T-cells and B-cells respectively. The ratio of mutated vs. wt alleles allowed us to determine homozygocity in 8 cases within a specific cell type, mostly granulocytes, but homozygocity was also detected in monocytes and T-cells. In conclusion this highly sensitive quantitative assay may detect more JAK2 V617F patients than previously reported assays and may be useful in monitoring the size of the mutated clone during cytoreductive therapy.

The 2001 World Health Organization and Updated European Clinical and Pathological Criteria for the Diagnosis, Classification, and Staging of the Philadelphia Chromosome-Negative Chronic Myeloproliferative Disorders

The clinical criteria according to the Polycythemia Vera Study Group (PVSG) do not distinguish between essential thrombocythemia (ET), thrombocythemia associated with early-stage polycythemia vera (PV) and prefibrotic chronic idiopathic myelofibrosis (CIMF). The criteria only classify the advanced stage of PV with increased red cell mass. The classification of myeloproliferative disorders (MPDs), proposed by the World Health Organization (WHO) in 2001, is a compromise of the clinical PVSG and WHO bone marrow criteria, and excludes early stages of ET and PV. The updated European clinical and pathological criteria combine the WHO bone marrow criteria with established and new clinical, laboratory, biological, and molecular MPD markers. This allows clinicians and pathologists to diagnose early-stage MPD and to differentiate ET, PV, and prefibrotic chronic idiopathic myelofibrosis (CIMF). Depending on laboratory tests and diagnostic criteria used, the population of the MPD patients defined as ET, PV, and CIMF are heterogeneous at the clinical, laboratory, and biological and pathological levels. The recent discovery of the JAK2 V617F mutation, which is the cause of a distinct trilinear MPD in its manifold clinical manifestations during long-term follow-up, increases the specificity of a positive JAK2 V617F polymerase chain reaction (PCR) test for the diagnosis of MPD (near 100%), but only half of the ET and CIMF patients according to the PVSG (sensitivity 50%) and the majority of PV patients (sensitivity 95%) are JAK2 V617F positive. A comparison of the laboratory features of JAK2 V617-positive and JAK2 wild-type ET patients clearly showed that JAK2 V617-positive ET is characterized by higher values for hemoglobin, hematocrit, and neutrophil counts; lower values for serum erythropoietin (EPO) levels, serum ferritin, and mean corpuscular volume; and by increased cellularity of the bone marrow in biopsy material. This indicates that JAK2 V617-positive ET patients, diagnosed according to the PVSG criteria, represent a "forme fruste of PV" consistent with early PV mimicking ET (JAK2 V617F trilinear MPD). In contrast, the JAK2 wild-type ET patients had significantly higher platelet counts and usually had a clinical picture of ET with normal serum EPO levels, PRV-1 expression, and leukocyte alkaline phosphatase score, and a typical WHO ET bone marrow picture. The clinical and pathological data on JAK2 V617F-positive MPD patients suggest that the JAK2 V617F mutation defines one disease entity with several sequential steps of ET, PV, and secondary myelofibrosis during long-term follow-up, and that the wild-type JAK2 MPDs may represent another distinct entity with a related but different molecular etiology. MPD-specific markers such as serum EPO, endogenous erythroid colony formation (EEC), and JAK2 V617F have high specificities, but the sensitivities are not high enough to detect the early stages of the MPDs, ET, PV, and prefibrotic CIMF. Bone marrow histopathology in addition to clinical, laboratory, biological, and molecular markers, including the JAK2 V617 PCR test, serum EPO, PRV-1, EEC, LAP score, peripheral blood parameters, and spleen size on echogram will detect the early stages of MPD and allows diagnostic differentiation of the three primary MPDs (ET, PV, and CIMF) in both JAK2 V617F-positive and JAK2 wild-type MPD patients.

Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

The recent discovery of a single point mutation in the JH2 pseudokinase domain of Janus kinase 2 (JAK2) in a considerable fraction of patients has shed light on the molecular pathomechanism in Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph- CMPDs). We established a robust and reliable method for detection of the JAK2 mutation in bone marrow cells derived from archival bone marrow trephines based on polymerase chain reaction and subsequent restriction site analysis. In a series of proven Ph- CMPDs classified according to World Health Organization criteria (n = 79), we detected the JAK2 mutation in 90% of polycythemia vera, 22% of cellular prefibrotic chronic idiopathic myelofibrosis, 60% of advanced chronic idiopathic myelofibrosis, and 27% of essential thrombocythemia. JAK2 mutation was not detected in Ph+ chronic myeloid leukemia (n = 5), acute myeloid leukemia (n = 10), acute lymphoblastic leukemia (n = 10), secondary erythrocytosis (n = 10), or normal bone marrow (n = 10). Restriction site analysis was also suitable for unfixed cell populations derived from peripheral blood and bone marrow aspirates. Besides providing support in the differential diagnosis of reactive versus neoplastic myeloproliferations, this newly developed assay reveals considerable overlaps between histologically different disease entities, indicating that additional genetic alterations might be responsible for the established differences of CMPD subentities.

Bone marrow CD34+ progenitor cells in Philadelphia chromosome-negative chronic myeloproliferative disorders – A clinicopathological study on 575 patients

Contrasting the circulating CD34+ hematopoietic progenitor cells (HPCs) in chronic myeloproliferative disorders (CMPDs), scant knowledge is available regarding their quantity in the bone marrow (BM). Therefore, a clinicopathological study was performed on trephine biopsies in 575 patients with CMPDs focused on chronic idiopathic myelofibrosis (CIMF). A comparison with 25 healthy subjects revealed no significant differences in the numbers of HPCs (6 +/- 3/mm2) in polycythemia vera, essential thrombocythemia and advanced fibro-osteosclerotic stages of CIMF. Pre-fibrotic and early-stage CIMF displayed 16 +/- 11 precursors per mm2 BM. Sequential biopsies in this disorder showed a decline in HPCs (10 +/- 6/mm2) with evolving myelofibrosis-myeloid metaplasia (MMM), while in terminal stages acceleration generated an increase (24 +/- 25/mm2). A significant association between the quantity of HPCs and the development of myelofibrosis, splenomegaly, and anemia as well as an increase in peripheral blasts was recognizable in CIMF. Moreover, in all subtypes of CMPDs, a favorable prognosis was significantly associated with a higher number of HPCs in the BM. In conclusion, enhanced inflow of precursors from the BM with subsequent trapping, self-renewal and mobilization by the spleen is assumed to indicate a progressive generalization and worsening of the outcome. This putative pathomechanism is significantly associated with the evolution of MMM.

The Incidence of Myelofibrosis in Essential Thrombocythaemia, Polycythaemia vera and Chronic Idiopathic Myelofibrosis: A Retrospective Evaluation of Sequential Bone Marrow Biopsies

The incidence of myelofibrosis (MF) among the three major Philadelphia chromosome-negative chronic myeloproliferative disorders, i.e. essential thrombocythaemia (ET), polycythaemia vera (PV) and chronic idiopathic myelofibrosis (CIMF), is not well documented since the diagnostic criteria have recently been redefined by the WHO. Therefore we performed a retrospective analysis of follow-up biopsies of 275 patients with ET, PV and CIMF according to the WHO classification of chronic myeloproliferative disorders. In the diagnostic bone marrow biopsies, MF was observed in 57 of the 136 CIMF patients (42%), 4 of the 73 PV patients (5%) and none of the 66 patients with ET. Within a median observation time of 2.9 years, 34 of the 79 patients with CIMF (43%), 13 of the 69 patients with PV (19%) and 1 of the 66 patients with ET (1.5%) – each initially without MF – developed MF regardless of myelosuppressive therapy.

Chronic myeloproliferative disorders.

The Philadelphia chromosome-negative chronic myeloproliferative disorders (CMPD), polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (IMF), have overlapping clinical features but exhibit different natural histories and different therapeutic requirements. Phenotypic mimicry amongst these disorders and between them and nonclonal hematopoietic disorders, lack of clonal diagnostic markers, lack of understanding of their molecular basis and paucity of controlled, prospective therapeutic trials have made the diagnosis and management of PV, ET and IMF difficult. In Section I, Dr. Jerry Spivak introduces current clinical controversies involving the CMPD, in particular the diagnostic challenges. Two new molecular assays may prove useful in the diagnosis and classification of CMPD. In 2000, the overexpression in PV granulocytes of the mRNA for the neutrophil antigen NBI/CD177, a member of the uPAR/Ly6/CD59 family of plasma membrane proteins, was documented. Overexpression of PRV-1 mRNA appeared to be specific for PV since it was not observed in secondary erythrocytosis. At this time, it appears that overexpression of granulocyte PRV-1 in the presence of an elevated red cell mass supports a diagnosis of PV; absence of PRV-1 expression, however, should not be grounds for excluding PV as a diagnostic possibility. Impaired expression of Mpl, the receptor for thrombopoietin, in platelets and megakaryocytes has been first described in PV, but it has also been observed in some patients with ET and IMF. The biologic basis appears to be either alternative splicing of Mpl mRNA or a single nucleotide polymorphism, both of which involve Mpl exon 2 and both of which lead to impaired posttranslational glycosylation and a dominant negative effect on normal Mpl expression. To date, no Mpl DNA structural abnormality or mutation has been identified in PV, ET or IMF. In Section II, Dr. Tiziano Barbui reviews the best clinical evidence for treatment strategy design in PV and ET. Current recommendations for cytoreductive therapy in PV are still largely similar to those at the end of the PVSG era. Phlebotomy to reduce the red cell mass and keep it at a safe level (hematocrit < 45%) remains the cornerstone of treatment. Venesection is an effective and safe therapy and previous concerns about potential side effects, including severe iron deficiency and an increased tendency to thrombosis or myelofibrosis, were erroneous. Many patients require no other therapy for many years. For others, however, poor compliance to phlebotomy or progressive myeloproliferation, as indicated by increasing splenomegaly or very high leukocyte or platelet counts, may call for the introduction of cytoreductive drugs. In ET, the therapeutic trade-off between reducing thrombotic events and increasing the risk of leukemia with the use of cytoreductive drugs should be approached by patient risk stratification. Thrombotic deaths seem very rare in low-risk ET subjects and there are no data indicating that fatalities can be prevented by starting cytoreductive drugs early. Therefore, withholding chemotherapy might be justifiable in young, asymptomatic ET patients with a platelet count below 1500000/mm(3) and with no additional risk factors for thrombosis. If cardiovascular risk factors together with ET are identified (smoking, obesity, hypertension, hyperlipidemia) it is wise to consider platelet-lowering agents on an individual basis. In Section III, Dr. Gianni Tognoni discusses the role of aspirin therapy in PV based on the recently completed European Collaboration on Low-dose Aspirin in Polycythemia Vera (ECLAP) Study, a multi-country, multicenter project aimed at describing the natural history of PV as well as the efficacy of low-dose aspirin. Aspirin treatment lowered the risk of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke (relative risk 0.41 [95% CI 0.15-1.15], P =.0912). Total and cardiovascular mortality were also reduced by 46% and 59%, respectively. Major bleedings were slightly increased nonsignificnsignificantly by aspirin (relative risk 1.62, 95% CI 0.27-9.71). In Section IV, Dr. Giovanni Barosi reviews our current understanding of the pathophysiology of IMF and, in particular, the contributions of anomalous megakaryocyte proliferation, neoangiogenesis and abnormal CD34(+) stem cell trafficking to disease pathogenesis. The role of newer therapies, such as low-conditioning stem cell transplantation and thalidomide, is discussed in the context of a general treatment strategy for IMF. The results of a Phase II trial of low-dose thalidomide as a single agent in 63 patients with myelofibrosis with meloid metaplasia (MMM) using a dose-escalation design and an overall low dose of the drug (The European Collaboration on MMM) will be presented. Considering only patients who completed 4 weeks of treatment, 31% had a response: this was mostly due to a beneficial effect of thalidomide on patients with transfusion dependent anemia, 39% of whom abolished transfusions, patients with moderate to severe thrombocytopenia, 28% of whom increased their platelet count by more than 50 x 10(9)/L, and patients with the largest splenomegalies, 42% of whom reduced spleen size of more than 2 cm.

Clinical and scientific advances in the Philadelphia-chromosome negative chronic myeloproliferative disorders.

The chronic myeloproliferative disorders are clonal hematopoietic stem cell disorders and include chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and agnogenic myeloid metaplasia (AMM). These diseases are characterized by clonal expansion of the myeloid compartment, increased marrow angiogenesis, and varying risks for blastic transformation. A clear molecular abnormality exists (t(9;22) leading to the fusion of BCR-Abl) only for CML, which led to effective targeted therapy (STI-571). Since no similar pathogenetic mechanism has been discovered for the t(9;22) negative chronic myeloproliferative disorders, their respective diagnosis is currently based on a variety of rather cumbersome diagnostic criteria. Polycythemia vera is distinguished from reactive erythrocytosis through erythropoietin independent growth of erythroid progenitors in vitro, suppressed levels of endogenous erythropoietin, possible overexpression of PRV-1 (polycythemia rubra vera-1), decreased c-Mpl expression on megakaryocytes, as well as overexpression of bcl-xL, and potentially aberrant activity of the Jak-Stat pathway. ET is defined by thrombocytosis and is distinguished from reactive states by decreased megakaryocyte c-Mpl expression, and a propensity for thrombosis. AMM has been associated with a variety of observations including increased concentrations of pro-fibrotic cytokines, increased angiogenesis, and myeloid expansion. AMM is often indistinguishable clinically and prognostically from the advanced phases of other CMPD (specifically post-polycythemic and post-thrombocythemia myeloid metaplasia), all of which are subentities of a diagnosis of myelofibrosis with myeloid metaplasia (MMM). The management of CMPD patients is quite varied given the broad range of disease severity and survival observed. The role of stem cell transplantation is limited by the age and comorbidities encountered in CMPD patients. Since no broadly applicable therapy effects the mortality of the CMPD, management currently focuses on the prevention/palliation of disease morbidity (i.e. vascular complications, pruritus, organomegaly, constitutional symptoms). Palliative strategies which currently focus on non-specific myelosuppresion, will hopefully be soon replaced by targeted therapies as insight into pathogenetic mechanisms of these diseases evolves.

Molecular detection of c-mpl thrombopoietin receptor gene expression in chronic myeloproliferative disorders.

BACKGROUND: Chronic myeloproliferative disorders (CMPD) originate from a pluripotent haematopoietic progenitor cell but show a marked degree of heterogeneity, especially between Philadelphia chromosome positive and negative disease entities. Abnormal megakaryopoiesis...