Chronic Idiopathic Myelofibrosis Patients Research Articles

Polycythemia Vera and Chronic Idiopathic Myelofibrosis CD34+ Cells Preferentially Traffic to Splenic Niches

AbstractAbstract 1750 Background:Polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF) are both clonal disorders of CD34+ hematopoietic stem and progenitor cells (HSPC) that undergo inappropriate expansion. The pathological CD34+ clones from both disorders are found at increased levels in the peripheral blood of patients, indicating that the interactions between these CD34+ cells and tissue HSPC niches are different from normal HSPC. Other studies have implicated deficiencies in the CXCR4 signaling axis as a possible reason for the abnormal trafficking of HSPCs in PV and CIMF. We set out to functionally test the importance of CXCR4 for the interactions of HSPCs collected from peripheral blood of patients with PV and CIMF with the bone marrow and other tissue microenvironments. Methods:We purified CD34+ cells from peripheral blood of patients with PV or CIMF, labeled them with fluorescent trackers, and engrafted them into unmanipulated SCID mice. Using video rate, scanning confocal microscopy we tracked CD34+ cell interactions with bone marrow, spleen and liver. To functionally test the role of CXCR4 in the homing of patient CD34+ cells to different microenvironments, we treated CD34+ cells with a blocking antibody prior to engraftment. Results:Flow cytometry revealed that PV CD34+ cells had significantly lower surface expression of CXCR4 than control HSPCs (PV mean: 32.93% positive, range: 14.8–55.1; Control mean: 58.85% positive, range: 33.5–78.7; p value = 0.018) while CIMF samples had scattered CXCR4 expression (mean 61.2% positive range: 0.6–95.4) PV CD34+ cells migrated toward SDF-1 in migration assays, demonstrating that although they had lower surface CXCR4 expression, they were still able to respond to signals from the environment through this receptor. In vivo imaging of mice after engraftment revealed that PV and CIMF CD34+ cells had decreased BM homing compared to control HSPCs. However, CD34+ cells from PV and CIMF patients homed to similar areas of bone marrow vasculature as control cells. Treating CD34+ cells with a blocking antibody against CXCR4 before engraftment significantly reduced the number of PV and CIMF cells that homed to the bone marrow, indicating that their bone marrow homing still relied on CXCR4. Interestingly, there were an increased number of PV and CMF cells that homed to the spleen at baseline compared to control HSPC, suggesting that a distinct homing mechanism favored PV and CMF cell trafficking to the spleen over the BM. The number of cells that homed to the spleen increased upon antibody blockade of CXCR4, indicating that splenic homing of CD34+ cells did not depend on CXCR4/SDF-1 interactions. Conclusion:CD34+ HSPC from PV and CIMF patients and controls homed to similar areas of the BM, although PV and CIMF cells had significantly decreased BM homing. Conversely, PV and CIMF CD34+ cells homed to the spleen in significantly higher numbers than control cells. PV, CIMF and control CD34+ cells were all dependent on CXCR4 for maximal BM homing. Splenic homing was not CXCR4 dependent, however, and PV and CIMF cells homed to the spleen in higher numbers when BM homing was inhibited by CXCR4 blockade. Taken together, we conclude that unique signaling mechanisms regulate trafficking of CD34+ cells to the BM vs. the splenic microenvironments. PV and CIMF CD34+ cells have an abnormal response to these tissue signals, resulting in their preferential homing to the spleen. Molecularly targeted therapies with the Jak2 inhibitors significantly decrease splenomegaly in CIMF patients, although they do not reverse the BM disease process. Recent evidence in a mouse model suggests that the BM is in fact a sanctuary site for disease during treatment with these agents. Whether PV and CIMF CD34+ cells in patients can escape apoptosis during treatment by trafficking to more favorable microenvironments is unknown. Our study suggests that an increased understanding of the mechanisms that these cells use to engage different tissue niches could aid the treatment of these diseases. Disclosures:No relevant conflicts of interest to declare.

Assessment of Immature Platelet Fraction in Patients with Ph-Negative Myeloproliferative Disorders and Thrombocytosis.

Abstract 4980 BackgroundMyeloproliferative disorders (MPD) in general result from proliferation of a clone of myeloid cells derived from a neoplastic pluripotent precursor or from connective tissue elements in bone marrow. This leads to increased numbers in one or more blood cell lines in peripheral blood. Some MPDs can be associated with thrombocytosis (MPD-T): essential thrombocythaemia (ET), polycythaemia vera (PV) and early stages of chronic idiopathic myelofibrosis (CIMF). Usually in MPD-T the thrombocytosis is caused by increased platelet production from proliferating mature megakaryocytes, especially in ET. Elevated platelet counts in these patients are often associated with both thromboembolic events and bleeding. One of the goals of MPD treatment is the control of platelet count. Immature platelets mirror the platelet production in bone marrow. In certain automated blood count analyzers it is possible to measure Immature platelet fraction (IPF) from the routine CBC samples sent to haematology lab. Reference range for IPF parameter for method used in this study is 1,1-6,1%. ObjectiveMeasurement of IPF parameter by fully automated analyzer (XE-2100, Sysmex, Kobe, Japan) in optical channel and analyzing it ( software IPF Master) in patients treated for Ph-negative MPD. We enrolled 85 pts- 67 ET (79%), 10PV(12%) and 8 CIMF (9%) patients; 57 were women and 28 men; median age 56 years ranging from 20 up to 83 years. We analyzed and evaluated IPF in whole group as well as in subgroups depending on diagnosis, gender, age, JAK-2 mutation and platelet count. ResultsAt the time of assessment the majority of our pts were already commenced on treatment for their MPD. Platelet counts (plt) in whole cohort ranged from 164 up to 2148 ×109/l, with median 374 ×109/l. Thirty eight pts (45%) had plt < 350 ×109/l. Plt <450 ×109/l (WHO 2008 recommended cut off level for thrombocytosis) were found in 59 pts (69%). IPF median in whole cohort was 5,9% (0,7-14,4%). When comparing IPF in subgroups mentioned above statistically significant differences (p<0,05) was found only between subgroups with normal and abnormal plt counts: IPF median 7,45% (0,8-14,4%) resp. 4,6% (0,7-11,9%), (p=0,002) and between subgroups with less and more than 450×109/l plt (IPF median 6,6% (0,8-14,4%) resp. 3,85% (0,7-9,6%), (p<0,001). Fact, that patients with higher plt had lower IPF and vice versa, was confirmed also by Spearman correlation coefficient. When correlating results of plt a IPF in the whole cohort, we found the trend to indirect dependence (rs= –0,386). ConclusionsMPD-T patients in our cohort did not have marked elevation of IPF parameter, neither those with high platelet counts (so far untreated pts). Thus we can speculate, that increased number of Plts in peripheral blood is caused by increased number of mature megacaryocytes in bone marrow which produce adequate numbers of platelets without increase of immature fraction rather than increased number of immature platelets released from megacaryocytes appearing in normal numbers in bone marrow Thus it seems that negative feedback of increased Plt counts on releasing of Plts from megacaryocytes is maintained also in patients with MPD-T. Further assessment is needed and should further determine, what is the clinical relevance (if any) of measurement of IPF in patients treated for MPD-T. DisclosuresBlatny:Sysmex, Czech Republic: Consultancy.

Bone Marrow Phospho-STAT5 Expression in Non-CML Chronic Myeloproliferative Disorders Correlates With JAK2 V617F Mutation and Provides Evidence of In Vivo JAK2 Activation

The recently described JAK2 V617F mutation, present in a substantial proportion of nonchronic myelogenous leukemia chronic myeloproliferative disorders (non-CML CMPDs), is changing the way we conceptualize and diagnose these diseases. We hypothesized that the activation of this tyrosine kinase might result in activation of downstream mediators such as STAT5, which would be detectable in bone marrow biopsies. We examined the expression of activated STAT5 (nuclear phospho-STAT5) in 73 bone marrow biopsies from patients with CMPDs [20 essential thrombocythemia (ET), 26 chronic idiopathic myelofibrosis (CIMF), and 27 polycythemia vera] and 39 controls. We compared the results with the JAK2 mutational status and clinical parameters. The frequency of the JAK2 V617F was 73% (85% in PV, 65% in ET, and 65% in CIMF). All patients with the JAK2 V617F showed abnormal nuclear megakaryocytic phospho-STAT5 (nMEG pSTAT5) expression. In the JAK2 wild-type group, nMEG pSTAT5 was observed in 2/7 ET, and 3/9 CIMF patients. nMEG pSTAT5 staining was 100% sensitive and 88% specific for JAK2 V617F. Clinically, nMEG pSTAT5+ patients seemed to require cytoreductive therapy more often than those without nMEG p-STAT expression. pSTAT5 immunohistochemistry is a useful diagnostic test in bone marrow biopsies from suspected non-CML CMPD patients. It identifies most of the patients with the JAK2 V617F but also other JAK2 wild-type CMPD patients. The presence of nMEG pSTAT5 in a subset of CMPD patients lacking the mutation suggests that alternate tyrosine kinase/phosphatase pathways may be involved and warrant further investigation. Phosphoprotein detection represents a new area for diagnostic pathology that exploits specific functional characteristics of cells within the context of a tissue section.

Oxidative Stress Is Increased in Chronic Idiopathic Myelofibrosis (CIMF) Patients, and Is Associated with High Levels of Homocysteine (Hcy) and Depletion of Holotranscobalamin (holoTC).

In patients with myeloproliferative disorders, the increased cellular turnover may lead to hyperhomocysteinemia (HH), caused by depletion of folate and/or cobalamin (VitB12). HH represents a cause of oxidative stress, associated with a number of diseases, among which cardiovascular events play an important role. Recently, holoTC, that consist of VitB12 linked to its transport molecule, transcobalamin, has been proposed as a more reliable marker than the dosage of total serum VitB12 to detect the deficiency of this vitamin.The aim of the study was to investigate the clinical role of oxidative stress in CIMF. We performed in 67 CIMF patients (M/F: 38/29; median age 63, range 34–79), the dosage of reactive oxygen species (ROS), total antioxidant capacity (TAC), Hcy, VitB12, folates and holoTC, and correlated the results with the following clinical variables: age, sex, cytogenetics, white blood cells and platelets counts, hemoglobin and LDH levels, splenomegaly and hepatomegaly, hypertension, diabetes, smoking habit, cardiovascular events, secondary tumors, and presence of blasts in peripheral blood. Serum concentrations of VitB12, folates and holoTC were determined by AxSYM B12 or Folate microparticle enzyme immunoassay (MEIA) and new Axis-Shield HoloTC MEIA, respectively; plasma Hcy levels were measured by fluorescence polarization immunoassay (FPIA). All the panel was performed on the Abbott AxSYM analyser. CIMF patients presented increased oxidative stress (lower levels of TAC and higher levels of ROS), compared to a panel of 53 healthy volunteers (Mann Whitney U test, p<0.0001). Higher values of Hcy were detected in the group of CIMF presenting holoTC deficiency (Mann Whitney U test, p=0.03). Consistently, holoTC directly correlated with TAC (Spearman correlation, p=0.01). Interestingly, levels of holoTC and TAC were significantly lower in the group of CIMF patients with blasts in the peripheral blood (Mann Whitney U test, p=0.01 and p=0.009, respectively). Notably, levels of VitB12 and folates did not correlate with Hcy, holoTC, TAC or ROS. No correlations were found between the levels of Hcy, holoTC, TAC, ROS, VitB12 or folates and the remaining clinical variables considered. In conclusion, we demonstrated that CIMF patients present an increase in oxidative stress compared to healthy controls. We observed that holoTC depletion is associated to HH and reduced TAC, consistently with an increased oxidative potential. These findings indicate the presence of a clinically significant VitB12 deficiency, detectable only by holoTC dosage, in the presence of normal levels of total serum VitB12. The other interesting finding is the association of holoTC and TAC deficiency with the presence of blasts in the peripheral blood of the patients, suggesting a role of oxidative damage in CIMF progression. Therefore, our findings suggest that, in CIMF patients, the dosage of ROS, TAC, holoTC, and Hcy, may represent a valuable clinical aid in the diagnosis of unapparent VitB12 deficiency, and provide a rational basis to prevent and/or correct the increase in oxidative potential associated with this disease.

Immunohistochemical Expression of Vascular Endothelial Growth Factor Correlates with Microvessel Density in Ph− Chronic Myeloproliferative Disorders.

There is increasing evidence that neovascularization may play an important role in hematological malignancies (particularly lymphomas, acute leukemia and myelodysplastic syndromes), but only a few studies have been performed in Ph− chronic myeloproliferative disorders (CMPDs). Increased angiogenesis has been reported in chronic idiopathic myelofibrosis (CIMF), whereas high serum levels of vascular endothelial growth factor (VEGF) have been described in Ph− CMPDs. These findings have led to the use of new therapeutic approaches aimed at directly targeting endothelial cells or VEGF. The aim of this study was to evaluate microvessel density (MVD) and immunohistochemical VEGF expression in the different categories of Ph− CMPD. We examined the bone marrow biopsies (BMBs) of 98 Ph− CMPD patients, classified as follows on the basis of the WHO criteria: 29 cases of essential thrombocythaemia (ET), 39 cases of CIMF (11 CIMF-0, 11 CIMF-1, 7 CIMF-2 and 10 CIMF-3) and 30 cases of polycythemia vera (PV) (20 polycythemic phase [pp-PV] and 10 post-polycythemic myelofibrosis [post-PVMF]. We also analysed 20 normal BMBs of subjects not suffering from hematological disease. MVD was analysed using the “hot-spot” method and an anti-CD34 antibody. Immunohistochemical VEGF expression was expressed as a VEGF index based on the formula [VEGF(i) = VEGF(+) cells x BMB cellularity/100]. Data were statistically tested at the 5% significance level (p<0.05; one-way ANOVA; Tukey's HSD test; Pearson test). MVD was significantly greater in the CIMF and PV patients (respectively 25.6 ± 6.3 and 20.7 ± 10.2) than in the ET patients (10.1 ± 4.5) or controls (7.5 ± 3.6) (p<0,01). No differences were found in relation to the CMF grading score (CIMF-0: 24.7 ± 6.5; CIMF-1: 26.1 ± 6.4; CIMF-2: 25.3 ± 4.9; CIMF-3: 26.2 ± 7.5), but post- PVMF patients had higher microvessel counts than the pp-PV patients (31.9 ± 7.3 vs 15.1 ± 5.9; p<0.01). There were also significant differences in VEGF(i) between the CIMF and PV patients (respectively 0.29 ± 0.15 and 0.28 ± 0.20) and the ET patients (0.12 ± 0.05) or controls (0.08 ± 0.04). There were no significant differences among the different grades of fibrosis (CIMF-0: 0.25 ± 0.15; CIMF-1: 0.32 ± 0.17; CIMF-2: 0.36 ± 0.16: CIMF-3: 0.27 ± 0.14), but a remarkable difference was found between the pp-PV and post-PVMF patients (0.21 ± 0.15 vs 0.49 ± 0.19; p<0.01). Finally, there was a strong correlation between MVD and VEGF(i) in the patients with CIMF (0.4006; p=0.013) and those with PV (0.7923; p<0.01). In conclusion, our findings show that angiogenesis was not different in ET patients and normal controls, whereas both MVD and VEGF levels were significantly higher in the patients with CIMF or PV, and showed a correlation between them. If confirmed, these results could serve as a rational guide for the anti-angiogenetic therapy of Ph− CMPD.

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.

Essential thrombocythemia or chronic idiopathic myelofibrosis? A single-center study based on hematopoieticbone marrow histology

We reviewed a large series of patients with essential thrombocythemia diagnosed on the basis of the Polycythemia Vera Study Group criteria, and reclassified them by evaluating their major morphologic features and clinical course using the World Health Organization classification. The morphologic review of the bone marrow biopsies of 116 patients (44 males and 72 females; aged 19 – 83 years, median 55 years; median follow-up 121 months) led to 22 cases (19%) being classified as essential thrombocythemia (ET), 24 (21%) as chronic idiopathic myelofibrosis (CIMF)-0, 44 (37%) as CIMF-1, 13 (12%) as CIMF-2, 9 (8%) as latent phase polycythemia vera, and four (3%) as chronic myeloproliferative disorder, unclassifiable. There was a significant difference in the median age of the ET and fibrotic CIMF patients (54.7 ± 13.55 vs. 59.13 ± 15.05 years; P = 0.03). Histologic analysis showed that the simultaneous presence of loose clusters of large/giant megakaryocytes and nuclear hyperlobulation was significantly different between the ET and the prefibrotic CIMF (P<0.01) and fibrotic CIMF patients (P<0.01), and that the association of dense clusters of megakaryocytes with maturation defects and bulbous nuclei also distinguished the prefibrotic CIMF (P<0.05) and fibrotic CIMF patients (P<0.001) from those with ET. The association of cellularity, granulocytic proliferation and reticulin fibers was helpful in distinguishing prefibrotic from fibrotic CIMF (P<0.001).

High Resolution Amplicon Melting Curve Analysis: A Sensitive and Rapid Method To Detect the Janus Kinase 2 (JAK2) Gene Mutation.

Chronic myeloproliferative disorders (CMPDs) are clonal hematopoetic stem cell disorders resulting in the increased production of cells of the myeloid lineage. The recently described G to T point mutation at nucleotide 1849 in the JAK2 gene may be a specific molecular marker to aid in the diagnosis of CMPDs, including polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF). Previously described methods of detection for this point mutation include sequencing, allele-specific PCR, and restriction digestion. We report here the development of a novel method to detect the JAK2 point mutation using high resolution amplicon melting curve analysis. Genomic DNA was obtained from peripheral blood samples from patients with PV, ET, CIMF, and suspected CMPD using the Qiagen DNA Blood kit (Valencia, CA). DNA was quantified using PicoGreen (Invitrogen) fluorescent dye followed by spectrophotometry and samples were normalized to a standard concentration. Real time PCR was performed on the Roche Light Cycler in the presence of the double stranded DNA binding dye LCGreen1 using primers that flank the point mutation. High Resolution Melting curve analysis was then performed on the HR-1 instrument (Idaho Technologies), and mutation status assigned by comparing the melting curve position and shape to controls of known mutation status. For comparison, the samples were also restriction digested using BsaX I and analyzed by agarose gel electrophoresis. Mutation status was confirmed by sequencing of the PCR amplicons (Lark Technologies, Houston, TX) for a subset of specimens. Twenty six patients with known CMPD as defined by the WHO criteria (2001) were included in the study. In addition, seven patients with suspected CMPD were studied. The point mutation in JAK2 was identified in 9 (100%) of 9 PV patients, 7 (58%) of 12 ET patients, 1 (50%) of 2 CIMF patients, 3 (100%) of 3 CMPD NOS patients, and 2 (29%) of 7 patients with suspected CMPD. Patients testing homozygous for the JAK2 mutation included 6 patients with PV, 1 patient with ET, and 1 patient with CMPD, NOS; all other positive patients were heterozygous for the mutation. We observed excellent correlation between the melting curve analysis and restriction digest results. High resolution melting curve analysis is a rapid, sensitive, low reagent cost method to assess for the JAK2 point mutation in cases of suspected or known CMPDs. The advantages of this technique include the ability to discriminate heterozygous from homozygous mutations, increased sensitivity as compared to sequencing, rapid turn around time, no post-amplification modification, and the ability to screen for other mutations or polymorphisms within exon 12 of the JAK2 gene.

SDF-1/CXCR4 Interactions in Idiopathic Myelofibrosis.

Interactions between SDF-1 and CXCR4 determine in part the retention, migration and mobilization of hematopoietic stem cells and endothelial progenitor cells during steady state hematopoiesis and following a variety of stimuli. Chronic idiopathic myelofibrosis (CIMF) is characterized by constitutive mobilization of CD34+ hematopoietic stem/progenitor cells as well as endothelial progenitor cells (Barosi et al, Blood 98: 3249, 2001 and J Clin Oncol, in press, 2005). This abnormal cell trafficking has been attributed to dysfunction of the bone marrow (BM) microenvironment and/or the presence of a proteolytic environment which disrupts adhesive interactions which favor retention of CD34+ cells within the BM (Xu et al, Blood 105:4508, 2005). We examined the role of SDF-1/CXCR4 in CD34+ cell trafficking in patients with CIMF. The median level of plasma SDF-1 in CIMF patients (N=27) was 2008 pg/ml (955–2981 pg/ml) which was significantly higher than that found in the plasma of normal individuals (N=13, 1411 pg/ml, 945–1803, p<0.001). Plasma SDF-1 levels in patients with polycythemia vera were intermediate in value (N=10, 1777 pg/ml, 1400–2496 pg/ml). When BM biopsy specimens from normal individuals were immunostained with anit-human SDF-1 monoclonal antibody, SDF-1 was found to be distributed in BM blood vessels of varying sizes, including small arterioles, venules, capillaries and sinusoids. In contrast, the BM biopsies from CIMF patients demonstrated a significant increase in SDF-1 deposition. Increased SDF-1 deposition was found in blood vessels, in areas adjacent to bone trabeculae and within the BM matrix surrounding hematopoietic cells. This abnormal distribution of BM SDF-1 was no longer observed following curative therapy with allogeneic stem cell transplantation. IM PB CD34+ cells were characterized by decreased expression of CXCR4 as well as by decreased in vitro migration in response to SDF-1 (p<0.001). The expression of CXCR4 by IM PB CD34+ cells was, however, significantly increased by incubation with SCF and IL-6 (7.2% to 18.0%). The expression of the cell-bound MMP-2 and MMP-9 by IM CD34+ cells was significantly increased by pre-incubation with SDF-1 in vitro (p<0.01). SDF-1 expression has been previously shown to be regulated by hypoxia inducible factor-1 (HIF-1α). Utilizing Western Blotting, we were able to demonstrate that HIF-1α protein levels were significantly increased in the CD34+ cells of 5/14 patients with CIMF. HIF-1α levels can be regulated by external stimuli such as hypoxia, cytokines or by the constitutive expression of pp60−src (c-src). The elevated HIF-1α expression of the CD34+ cells in a subpopulation of IM were associated with increased expression of c-src. The abnormal distribution of SDF-1 in CIMF, therefore, has multiple biological consequences including: down modulating the CXCR4 expression of CD34+ cells, up regulating CD34+ cell-bound MMP-2 and MMP-9, and promoting the growth and survival of BM stromal cells. These multiple effects of SDF-1 in CIMF likely alter the trafficking of CD34+ cells as well as promote BM fibrosis and osteosclerosis.

Bone Marrow Fibrosis in Chronic Idiopathic Myelofibrosis Is Associated with Megakaryocyte Expression of Osteoprotegerin.

A feature that distinguishes chronic idiopathic myelofibrosis (CIM) from other chronic myeloproliferative disorders is the progression to bone marrow fibrosis and osteosclerosis. Because osteoclast formation can be inhibited by osteoprotegerin (OPG), we investigated its megakaryocytes' expression and serum concentration in patients (pts) with CIM (n=20, median age 64 years, range 46–81) and in 20 controls showing no evidence of neoplastic disease (median age 62 years, range 46–82). The study group comprised 10 pts with prefibrotic cellular CIM and 10 with severe fibrosis. Assessment of OPG concentration in serum was performed using Osteoprotegerin ELISA Kit (Biomedica, Austria). EnVision (DAKO) kit with monoclonal anti-human osteoprotegerin/TNFRSF11B antibody - MAB8051 (R&D Systems, USA) was used to evaluate OPG expression in megakaryocytes. Gomori silver staining technique was applied for semi-quantitative assessment of bone marrow fibrosis according to increased amount of reticulin fibers. OPG serum levels in pts with CIM were significantly higher than in controls (105.78 vs 85.14 pg/ml, p=0.02), and showed positive correlation with age in both groups (r=0.61, p=0.0037 and r=0.55, p=0.012, respectively). Serum levels of OPG in pts with CIM patients were not associated with erythrocyte, leukocyte, platelet numbers and with the Visani, Lille or Cervantes prognostic scores. There was no correlation between serum levels of OPG and its expression in megakaryocytes in pts and controls. In megakaryocytes derived from patients with CIM as well as from controls, OPG expression was detected, but in those derived from CIM hematopoiesis (at any stage of the disease) were significantly higher. OPG expression in megakaryocytes derived from prefibrotic CIM was significantly lower than from advanced stages of the disease (p=0.007).We conclude that OPG appears to be involved in bone marrow fibrosis in CIM through overexpression by megakariocyte.

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 idiopathic myelofibrosis: independent prognostic importance of bone marrow microvascular density evaluated by CD105 (endoglin) immunostaining

Microvascular density (MVD) is substantially increased in bone marrow biopsies of patients with chronic idiopathic myelofibrosis (CIMF). CD105, a useful molecule for assessing MVD in various malignancies, is preferentially expressed by recently formed microvessels. Increased serum-soluble CD105 in patients with chronic myeloproliferative disorders, including CIMF, was documented. CD105 MVD has not so far been investigated in CIMF: to this end, the results in 55 patients with CIMF and 21 controls were compared with the conventional CD34 immunostaining as well as traditional histological and clinical disease features. The MVD mean values estimated by both CD105 and CD34 were significantly higher in CIMF patients than in controls (P<0.00001). In addition, the proportion of CD105-positive megakaryocytes was significantly higher in CIMF than in controls (P<0.0001). A degree of reticulin fibrosis >2 correlated with increased CD105 MVD (P=0.05). A multivariate analysis confirmed that CD105-positive MVD was an independent adverse prognosticator. This study demonstrates that while MVD, as assessed by both CD34 and CD105 immunostaining, is significantly increased in CIMF, only CD105-determined MVD correlates with the degree of fibrosis and is prognostically relevant. These findings provide a rationale for the investigational use of anti-CD105-targeted drugs in CIMF.