Myelofibrosis With Myeloid Metaplasia Research Articles

Primary myelofibrosis (PMF), post polycythemia vera myelofibrosis (post-PV MF), post essential thrombocythemia myelofibrosis (post-ET MF), blast phase PMF (PMF-BP): Consensus on terminology by the international working group for myelofibrosis research and treatment (IWG-MRT)

The International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) is comprised of hematologists, hematopathologists, and laboratory scientists and its main goal is to provide a forum for scientific exchange and collaboration. During its first general meeting in April 2006, the IWG-MRT established uniform treatment response criteria for chronic idiopathic myelofibrosis (CIMF); also known as agnogenic myeloid metaplasia (AMM), myelofibrosis with myeloid metaplasia (MMM), and many other names in the hematologic literature. This document summarizes the proceedings from the second meeting of the IWG-MRT, in November 2006, where the group discussed and agreed to standardize the nomenclature referring to CIMF: (i) the term primary myelofibrosis (PMF) was chosen over several other designations including CIMF, AMM, and MMM, (ii) myelofibrosis that develops in the setting of either polycythemia vera (PV) or essential thrombocythemia (ET) will be referred to as post-PV MF and post-ET MF, respectively, and (iii) “leukemic” transformation will be recognized as blast phase disease (PMF-BP, post-PV/ET MF in blast phase).

Growth factor‐independent 1B gene (GFI1B) is overexpressed in erythropoietic and megakaryocytic malignancies and increases their proliferation rate

Growth factor-independent 1B (GFI1B) is a transcription factor essential for the development and differentiation of erythroid and megakaryocytic lineages. We evaluated the GFI1B expression in erythroleukaemia and megakaryocytic leukaemia, as well as in patients with other subtypes of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), myelodysplastic syndrome (MDS), severe aplastic anaemia (SAA), myelofibrosis with myeloid metaplasia (MMM) and in healthy volunteers. GFI1B expression was increased at least threefold in patients with erythroleukaemia (P < 0.01 compared with controls) and megakaryocytic leukaemia (P < 0.05) as well as in their corresponding leukaemic cell lines HEL, K562, CMK and M-07e. Patients with undifferentiated or monocytic AML, ALL, MMM, MDS and CML had no significantly altered GFI1B expression, whereas GFI1B expression was decreased 10-fold in patients with SAA (P < 0.0001 compared with controls). Silencing GFI1B by transfection with small interfering RNA (siRNA) markedly reduced the proliferation rate in the leukaemic cell lines HEL, K562 and NB4 (P < 0.01). Concomitantly, we observed a two- to threefold increase in the apoptosis rate in these cells after transfection with siRNA towards GFI1B. Our data indicate that GFI1B plays a major role in AML-M6 and AML-M7 and qualifies as a target for anti-leukaemic strategies in these malignancies.

Phenotypic Differences amongst Subgroups of Patients with Myelofibrosis with Myeloid Metaplasia (MMM): Analysis of 458 MMM Patients.

Background: Debate continues as to the clinical relevance of subclassifying patients with the myeloproliferative disorder (MPD) MMM into the subgroups of agnogenic myeloid metaplasia (AMM), post-polycythemic myeloid metaplasia (PPMM), and post-thrombocythemic myeloid metaplasia (PTMM). We looked to validate previously described phenotypic variability amongst subtypes, and explore relevant and differences in constitutional symptoms.Methods: An detailed analysis of patients with MMM whom responded to an international web based survey of MPD patients which registered responses regarding diagnosis, disease course, complete blood counts, co-morbidities (Charlson Co-morbidity index), symptoms, and fatigue (Fact-An and Brief Fatigue Inventory (BFI)).Results: A total of 456 patients self described as MMM responded of which 32.3% had AMM (n = 148), 38.0% % had PPMM (n = 174), and 29.7% had PTMM (n = 136). Detailed breakdown of differences in demographics, clinical course, current laboratory studies, treatment history, and current symptoms are outlined in the table below.Phenotypic Variability Amongst 458 MMM PatientsCategoryAMM (N=148; 32.3%)PPMM (N=174; 38.0%)PTMM (N=136; 29.7%)MMM Total (N=458)Δ between groups (p = )Age (Median)615757580.09Sex (% Males)60%49%30%47%<0.01Yrs from MPD Dx5686<0.01Labs/ Prior HistoryHb (g/dL)11.013.711.612.1<0.01% with RBC Tx43%6%11%20%<0.01Leukocytes (×10(9)/L)8.07.67.27.70.64Platelets (×10(9))/L)200352396321<0.01% with Splenomegaly76%49%41%54%<0.01% with Prior Thrombosis10%22%31%21%<0.01% with Prior Bleed16%20%31%22%0.01Treatment History for the MMM% with Prior Tx for MPD64%88%77%77%<0.01% Medical Therapy57%70%75%67%<0.01# Meds (Median (Range))1 (0–8)2 (0–6)2 (0–6)2 (0–8)<0.01% with Splenectomy8%5%6%6%0.48% with SCT6%1%1%3%<0.01MMM Associated Constitutional Symptoms% with Fatigue85%81%85%84%0.56% with Bone Pain51%46%44%47%0.51% with Fevers19%17%18%18%0.87% with Pruritus39%69%39%50%<0.01% with Night Sweats55%58%53%56%0.67% with Weight Loss30%15%16%20%<0.01Hb = Hemoglobin; SCT = Stem Cell TransplantOverall the data suggests patients with AMM are more likely male, with greater burdens of anemia, splenomegaly, and weight loss. However they are less likely to have experienced thrombohemorrhagic complications and have received fewer MPD medications. In contrast PPMM patients (not unsurprisingly) have little trouble from anemia, but have the most pruritus. Patients with PTMM are most likely female, with the highest platelet counts, and the greatest likelihood of thrombohemorrhagic complications. Additionally, all three subgroups of MMM had significant fatigue compared to published controls for both fatigue instruments (P<0.0001) yet there was no difference between MMM subtypes for the severity of fatigue.Conclusions: A large unselected series of MMM patients supports that although constitutional symptoms are significant and similar amongst MMM subgroups, these patients have variable burdens of other MPD manifestations.

Reduced CD26 Dipeptidylpeptidase IV Membrane Expression and Activity of CD34+ Cells from Patients with Myelofibrosis with Myeloid Metaplasia.

Myelofibrosis with Myeloid Metaplasia (MMM) is characterized by an abnormal constitutive mobilization of CD34+ stem/progenitor cells. The SDF-1alpha axis chemoactracts hematopoietic stem and progenitor cells and is thought to play a crucial role in the homing/mobilization of these cells from the bone marrow, interacting with its unique receptor CXCR4. CD26 (dipeptidylpeptidase IV/DPPIV) is an extracellular peptidase, present both as membrane-bound and as a catalitically active soluble form in plasma. It has the ability to cleave SDF-1alpha at its position-2 proline inducing significant changes in its receptor binding and/or functional activation, thus inhibiting normal SDF-1alpha induced migration. In this study we evaluated the expression of CD26/DPPIV in patients with MMM (N=63) and its functional activity (N=8). CD26/DPPIV cell surface expression was measured by flow cytometry on peripheral blood mononuclear cells (PBMNC) or on immunoselected circulating CD34 + cells. The CD26/DPPIV activity was measured in 96-well microplates using the chromogenic substrate gly-pro-p-nitoanilide. The proteolytic activity was determined by measurements of the amount of nitoanilide (pNA) formed in the supernatant at 405 nm. Absorbance was measured at 405 nm and the picomoles of pNA formed were calculated by comparison with a pNA standard curve. The CD26 expression on the membrane of PBMNC of patients with MMM was comparable to that found on PBMNC of normal controls (median 29.5 %, range 0.72–61.1; median 33.1 %, range 20.3–39.2, respectively). However, the percentage of circulating CD34+CD26+ cells was significantly reduced (P<0.05) in patients with MMM (median, 0.2 %, range 0–48.4%) with respect to normal controls (median 10.3 %, range 0–30.2%). The CD26/DPPIV activity evaluated on purified circulating CD34+ cells from patients with MMM was significantly lower (p<0.03) than that of CD34+ cells from normal controls (median 14.2 pmol, range 0–45.6; median 67.2 pmol, range 34.5–81.1, respectively). The same activity, evaluated on CD34− circulating cells was comparable between patients with MMM and normal controls. In addition, the peptidase activity mediated by the sCD26 was significantly lower (p<0.03) in patients with MMM (median value of absorbance, 409; range 281–681) than in controls (median 518, range 494–550). In summary, our data show that both CD26 expression and its peptidase activity are lower in circulating CD34+ cells of patients with MMM than in CD34+ cells of normal controls; similarly, a significant reduction of the activity of sCD26 peptidase, derived from all the CD26+ peripheral blood cells, was found in patients with MMM compared with controls. These novel findings suggest that not only modifications of CXCR4 expression but also an altered regulation of SDF-1 activity can affect the correct function of the CXCR4/SDF-1 alpha axis in patients with MMM.

Phase II Study of the Combination of Low-Dose Thalidomide, Prednisone, and Oral Cyclophosphamide (TPC Regimen) in the Treatment of Anemia, Splenomegaly, and Constitutional Symptoms Associated with Myelofibrosis with Myeloid Metaplasia (MMM).

BACKGROUND: We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone (THAL-PRED) for the treatment of both anemia and thrombocytopenia in MMM (Blood 2003; 101:2534), with response rates of 62% and 75% for each cytopenia respectively. Alkylators have long been shown to have palliative benefit in MMM, however are used judiciously as long-term use could increase likelihood of leukemic transformation. Additionally, oral cyclophosphamide has been shown to synergize with thalidomide and steroids in the treatment of myeloma (Garcia-Sanz et. al. Leukemia 2004; 18:856–863). In the current study, we investigated the value of adding oral cyclophosphamide to THAL-PRED for MMM (TPC).METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin < 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide (THAL) daily, oral cyclophosphamide (CTX) 25 mg daily for three months, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide alone as maintenance. Responses were assessed by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria for MMM clinical responses (Tefferi et. al. Blood 2006; epub May 2006).RESULTS: 13 patients were enrolled to the trial (median age 73 years, range, 46–80; 7 males). Six patients (46%) completed the planned three cycles (i.e. 12 weeks) of treatment, the remaining seven patients discontinued treatment early for the following reasons: progression (n=3), patient choice (n=2), other medical problem (n=1) and died on study (n=1). After the initial 3 months of treatment, 3/13 (23%) patients evidenced a response (n = 1) or stable disease (n = 2) and continued on an additional three months of THAL alone. At enrollment, nine patients (69%) were anemic (8 of whom (62%) were red cell transfusion dependent). Ten patients (77%) were at least moderately thrombocytopenic (platelet count < 100 x 109//L). Eight patients (62%) had palpable hepatomegaly/splenomegaly, and five patients (38%) had severe constitutional symptoms (night sweats). According to IWG-MRT criteria for MMM the best responses observed from therapy was a CI (clinical improvement) in 2 individuals (one each for anemia (1/9 patients (11%); and thrombocytopenia (1/10 patients (10%)). No clear responses in organomegaly or constitutional symptoms were observed. The regimen overall was well tolerated with toxicities in excess of grade 2 all being related to myelosuppression (anemia (n=2), neutropenia (n=2), and thrombocytopenia (n=3)).CONCLUSIONS: This phase II study of combination therapy with thalidomide-prednisone-cyclophosphamide in patients with MMM strongly suggests that the addition of the alkylator cyclophosphamide largely negates the clinical benefits of THAL-PRED. This observation supports the potential immunologic mechanism of THAL-PRED benefit in MMM, and cautions against potential combinations of immunological and cytotoxic therapy in this disorder.

Phase II Study of Liposome Encapsulated Doxorubicin Citrate Complex, NPLD (Myocet™) in Myelofibrosis with Myeloid Metaplasia (MMM).

MMM is a chronic myeloproliferative disorder accompanied by an intense bone marrow stromal reaction that includes collagen fibrosis, osteosclerosis, and angiogenesis.The molecular pathogenesis of MMM has not been fully characterized despite the recent discovery of an activating JAK2 mutation (JAK2V617F) in approximately half of the patients. Conventional drug therapy in MMM not improves survival and is used for palliative purposes only.Myocet™ is a proprietary liposomal formulation of doxorubicin designed to enhance delivery of the drug to tumor cells and reduce side effects while maintaining equivalent anti-tumor efficacy.Moreover liposomes are avidly removed from circulation by the macrophages of the RES or MPS and are taken up in the spleen, liver and bone marrow were show both a cytotoxic and anti-angiogenic activityWe thus designed a phase II study for patients with primary MMM. The treatment program consisted of 25 mg total dose of Myocet™ given I.V. every two weeks for six months. Study eligibility criteria included a histologically confirmed diagnosis of MMM, platelet count ≥ 100 × 109/L, and neutrophil count ≥ 1 × 109/L, PS ≤ 2, LVEF > 50%, normal liver and renal function.8 patients (median age, 66 years; range, 60–73) were enrolled to the trial. Median duration of disease prior to protocol treatment was 45 months (range, 24–144). All pts (100%) had received prior therapy for MMM, including thalidomide 3 (37%), hydroxyurea 8 (100%), interferon 2 (25%)), anagrelide 2 (25%), and erythropoietin 8 (100%). 3 patients (37%) were RBC transfusion-dependent and 1 had severe constitutional symptoms (night sweats or disease related fever).Baseline median (range) values for palpable spleen size, leukocyte count, platelet count, CD34 cell count, and serum lactate dehydrogenase (LDH) were 10 cm BCM (median, range 6 to 30), 8.5 × 109/L (3–36.2), 250 × 109/L (160–950), 151.1 × 106/L (1.9–356), and 680 U/L (316–868), respectively. 8/8 pts had JAK2 V617F mutation. In addition, pre-treatment and post-treatment quantitative assessment by real-time quantitative polymerase chain reaction (RQ-PCR) of WT1 transcripts in the peripheral blood were performed and the values were expressed as WT1 copies every 104 copies of ABL. 7/8 pts are evaluable for response and toxicity (i.e., received six months of therapy) 1 pt discontinued because of progression.Clinico-hematologic responses have been observed in 7/7 (100%) pts and were evaluated according to European Myelofibrosis Network criteria These include 1 CR, 5 Major Response,1 Minor Response1 of 3 transfusion-dependent pts have become transfusion independent. The median time to response was 12 weeks (range, 4 to 18). Therapy has been well tolerated. Only a Grade 3 thrombocytopenia was seen.Striking WT1 gene expression levels in samples that were obtained after treatment were found to be significantly lower (median 480 range 210–520) than those before (median 1000, range 560–1230) (P < .0001, Mann-Whitney U test) The median post-treatment follow up at the time of this writing was 10 months (range 8–13)We conclude that Myocet has clinical activity in a subset of patients with myelofibrosis with an acceptable toxicity profile.

Responses to Thalidomide Combined with Prednisone Therapy for Myelofibrosis with Myeloid Metaplasia.

Myelofibrosis with myeloid metaplasia (MMM) is currently classified as a classic (i.e. not yet molecularly defined) myeloproliferative disorder (MPD), along with essential thrombocythemia (ET) and polycythemia vera (PV). All three MPDs represent stem-cell-derived clonal myeloproliferation that, in the case of MMM, is accompanied by an intense bone marrow stromal reaction that includes collagen fibrosis, osteosclerosis, and angiogenesis. To present the results of a long-term analysis of 2 sequential phase 2 trials of thalidomide (alone or in combination) for palliation of myelofibrosis with myeloid metaplasia (MMM). We analyzed (March 2003 to August 2005) initial and long-term outcomes from 33 patients with symptomatic MMM who had enrolled in either our thalidomide single-agent trial (n=12) or our trial of low-dose thalidomide (50 mg/d) combined with prednisone (n=21). Among the 33 study patients, 18 (54%) showed some improvement in their clinical course. Response rates for specific end points included improvements in anemia (12 of 33 [36%]), thrombocytopenia (8 of 12 [66%]), or splenomegaly (5 of 30 [17%]). The combination of low-dose thalidomide and prednisone, as opposed to single-agent thalidomide, was better tolerated and more efficacious. After a median follow-up of 17 months (range, 9–27 months), 10 of 33 patients (33%) showed an ongoing response, including 8 patients in whom protocol treatment has been discontinued for a median of 21 months (range, 16–27 months). Durable treatment responses were documented for only anemia and thrombocytopenia. Treatment response was not affected by the baseline status of bone marrow fibrosis, angiogenesis, osteosclerosis, cytogenetics. Unusual drug effects, all reversible, included leukocytosis (8 patients) and/or thrombocytosis (6 patients). Thalidomide (alone or combined with prednisone) is an effective first-line treatment of symptomatic anemia or thrombocytopenia in MMM. Thalidomide-based therapy has the potential to produce durable responses in MMM-associated cytopenias, even after discontinuation of the drug.

The Burden of Fatigue and Quality of Life in Myeloproliferative Disorders (MPDs): An International Internet Based Survey of 1179 MPD Patients.

Background: Scant data exists regarding the burden of fatigue and constitutional symptoms in patients with myeloproliferative disorders (MPDs) (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis with myeloid metaplasia (MMM)).Methods: An Internet based survey of MPD patients regarding clinical course, blood counts, constitutional symptoms, and fatigue (FACT-AN and Brief Fatigue Inventory (BFI)).Results:Patient Demographics: A total of 1179 MPD patients (median age 56; 41.4% male) internationally (76% USA: 50 states, 30 countries, 6 continents) responded to the survey. Self reported diagnoses were PV (35%), ET (26%), or MMM (39%).Clinical History: 1025 patients (87%) had undergone MPD therapy (drugs 70.5%, phlebotomy 44.1%, other 16%). Thrombosis (n=261, 22%), hemorrhage (N=272; 23%), and splenomegaly (N=478 (43%) were frequently reported. Blood tests (available in 917) showed 39% were anemic, 7% transfusion dependent.Symptoms: Fatigue, pruritus, night sweats and bone pain were present across the majority of MPD patients (see table). Additionally it was observed that these symptoms appeared with nearly equal frequency in PV as in MMM patients. However MMM patients displayed more advanced features (fevers, night sweats and weight loss).MPD Associated Constitutional Symptoms in 1179 PatientsSYMPTOMPV (N=405)ET (N=304)MMM (N=456)MISSING (N=14)TOTAL (N=1179)P VALUEFatigue344 (84.9%)220 (72.4%)381 (83.6%)7945 (81.1%)<0.0001Pruritus265 (65.4%)120 (39.5%)228 (50.0%)3613 (52.6%)<0.0001Night Sweats199 (49.1%)123 (40.5%)254 (55.7%)4576 (49.4%)0.0002Bone Pain174 (43.0%)125 (41.1%)214 (46.9%)4513 (44%)0.2480Fevers53 (13.1%)26 (8.6%)81 (17.8%)2160 (13.7%)0.0013Weight Loss39 (9.6%)22 (7.2%)93 (20.4%)0154 (13.2%)<0.0001Spleen Pain17 (4.2%)27 (8.9%)30 (6.6%)474 (6.3%)<0.0001Fatigue (82% MPD linked) was significantly increased across all MPD subtypes compared to published controls for both the BFI (P<0.0001) and Fact-An (P<0.0001). Additionally 69% curtail social activity because of fatigue, 35% need assistance with activities of daily living, and 11% reported MPD associated medical disability. Fatigue strongly correlated with multiple MPD disease features including: anemia, bone pain, constitutional symptoms, pruritus, splenic pain, smoking, thrombosis, and hemorrhagic events (all p<0.0001). Interestingly, even in “asymptomatic” MPD patients (those without anemia, splenomegaly, or thrombohemorrhagic complications) (n=279)) 24% (41% PV, 31% ET, 28% MMM) described significant fatigue worse than published controls.Conclusions: Fatigue and other constitutional symptoms are a significant burden to MPD patients, despite current therapies employed. Additionally fatigue is also a significant problem even in MPD patients lacking other advanced disease features. Clearly, direct attempts to improve fatigue (through pharmacologic and non-pharmacologic interventions) are necessary.

Long-Term Survival of Patients with Myelofibrosis Treated with Allogeneic Hematopoietic Stem Cell Transplantation.

Myelofibrosis with myeloid metaplasia (MMM) is a disorder characterized by clonal myeloproliferation, ineffective erythropoiesis, extramedullary hematopoiesis and bone marrow fibrosis. Between 4/1998 and 1/2006, 17 patients with MMM were treated with allogeneic hematopoietic stem cell transplantation. Patients included 8 males and 9 females with a median age of 51.8 years (range 37–66). All patients were transfusion-dependent at the time of transplantation, 3 (18%) had abnormal cytogenetics and 4 (24%) had a prior splenectomy. Six patients (35%) received a reduced intensity conditioning regimen consisting of either fludarabine and busulfan (1 pt), fludarabine and low dose total body irradiation (1 pt), fludarabine and melphalan (3 pts) or fludarabine and cyclophosphamide (1 pt). Eleven patients (65%) received an ablative regimen of either busulfan and cyclophosphamide (10 pts) or cyclophosphamide and total body irradiation (1 pt). Patients with unrelated donors also received ATG as part of their conditioning. The hematopoietic cell source was peripheral blood in 12 pts (71%) and bone marrow in 5 pts (29%). Six pts (35%) received cells from an HLA-identical sibling, 1 pt (6%) had a 1 Antigen mismatch sibling and 10 pts (59%) received stem cells from an unrelated donor. Graft-versus-host disease prophylaxis consisted of tacrolimus in 15 pts (88%) and cyclosporine in 2 pts (12%). Transplant-related mortality was 17% (sepsis 1 pt, VOD 1 pt, acute GVHD 1 pt) all occurring in the group treated with a myeloablative regimen. Of the remaining 14 patients, all achieved hematological recovery and transfusion independence. There were 2 relapses and one patient died of disease recurrence; both relapses occurred in the group receiving a reduced intensity regimen. The other 12 patients remain alive and in remission. The 6-year actuarial survival is 67% with no statistical difference between the groups who received a reduced intensity versus a myeloablative regimen. In conclusion, Myelofibrosis with Myeloid Metaplasia can be successfully treated with allogeneic hematopoietic stem cell transplantation with a significant proportion of patients achieving long-term survival. Hematopoietic recovery can be achieved despite the presence of marrow fibrosis. The optimal preparative regimen remains to be determined with more relapses observed in the reduced intensity regimen group and a higher mortality seen in the myeloablative group. A risk-stratification strategy for regimen selection should be considered in future studies.

Concomitant Myelofibrosis and Plasma Cell Disorders: The Mayo Clinic Experience.

BACKGROUND: Bone marrow fibrosis might represent either a primary myeloid malignancy such as myelofibrosis with myeloid metaplasia (MMM) or a reactive phenomenon associated with other malignancies or inflammatory/infectious processes. The development of myelofibrosis in the context of a plasma cell dyscrasia is uncommon and incompletely understood from the standpoint of both pathogenesis and clinical implications. We sought to determine the origin and clinical ramifications of this phenomenon based on a consecutive series of patients with plasma cell disorders (PCD).METHODS: A retrospective search of institutional databases was performed for clinical cases of PCD with the concomitant observation of myelofibrosis. Bone marrow aspirates and trephines from the time of diagnosis of both PCD and myelofibrosis were reviewed. Clinical history was abstracted for clinical manifestations as well as disease course. DNA from archived cytogenetic pellets, when available, from the time of diagnosis, was screened for the presence of JAK2V617F.RESULTS: Among 7587 patients with a PCD seen over the last 30 years at our institution, 29 (0.3%) displayed concomitant myelofibrosis (median age 59 years, range 27–77; 52% males). The specific PCD was multiple myeloma in 25 patients, smoldering myeloma in 2, plasmacytoma in 1, and heavy chain disease in 1. Four patients (14%) had organomegaly and bone marrow histological features most consistent with MMM. Otherwise, myeloproliferative disorder (MPD)-characteristic features were infrequent and included leukocytosis and/or thrombocytosis in only 2 patients. None of the patients displayed thrombohemorrhagic complications. Archived DNA was available in 7 cases without either clinical or bone marrow histological evidence of a MPD and yet revealed the presence of JAK2V617F in 6 of the 7 cases (86%). In addition, one other patient without MPD phenotype displayed a monosomy 5 abnormality. To date, 2 patients, one with and one without a MPD phenotype underwent leukemic transformation. All other causes of death were related to PCD-associated complications. Median survival (Kaplan-Meier) for the patients with multiple myeloma was 50 months. Survival stratified by the myeloma international staging system (ISS) was similar or better than expected (published controls of 62, 44, and 29 months): median survivals for this series of 73, 51 and 37 months for stage I, II, and III disease, respectively.CONCLUSIONS: Myelofibrosis that accompanies a PCD often represents an underlying MPD and does not negatively impact the natural history of the associated PCD.

Genetic Profiling of Myeloproliferative Disorders by Single Nucleotide Polymorphism Oligonucleotide Microarray.

Myeloproliferative disorders (MPD) are clonal hematopoietic diseases including polycythemia vera (PV), essential thrombocytosis (ET) and myelofibrosis with myeloid metaplasia (MMM). Single-nucleotide polymorphism DNA microarray (SNP-Chip) was used to analyze 43 MPDs. All five cases of PV having homozygous JAK2V617F had uniparental disomy (UPD), thus duplicating the allele with the mutated JAK2 and eliminateing the normal allele. Concerning MMM, either the Rb1 (13q14) or NF1 (17q11) gene was deleted in five of the 9 cases that had no Jak2 mutations. Two of these cases had UPD at 1p. Sequencing of the target genes within this region of UPD revealed a point mutation of the MPL gene (S240F or W515L). Each of these cases also had a deletion of Rb suggesting that the mutated MPL may co-operate with an aberrant Rb. The 9 cases of MMM with JAK2 mutations had only infrequent (2/9 cases) additional genomic alterations. Interestingly, No genomic alterations were noted in any of the cases (10) of ET having a normal Jak2. Two of 7 ET cases with JAK2 mutations had deletion of either 5q or trisomy 9. In summary, UPD is the predominant mechanism by which MPD develops homozygous JAK2 mutations. 1p UPD is a signpost that MPL is mutated in cases of MMM having wild-type JAK2; and these cells often have loss of Rb. ET cases with no JAK2 mutations are surprisingly free of genomic alterations.

Atiprimod Inhibits the Growth of JAK2 Tyrosine Kinase Over-Expressing Cells “In Vitro” and “Ex Vivo”.

Background:A novel somatic single point non-synonymous mutation (1849 G to T) in the tyrosine kinase JAK2, rendering the enzyme constitutively active, has been reported in classical myeloproliferative disorders (MPD), specifically, in greater than 90% of patients with polycythemia vera (PV), and 50% of patients with essential thrombocythemia (ET) and myelofibrosis with myeloid metaplasia (MMM). Consequently, JAK2 tyrosine kinase has become a significant target for the development of specific inhibitors of its activity with possible therapeutic benefits for patients with classical MPD. We report here the results of a study using atiprimod, an anti-inflammatory and anti-cancer compound belonging to the azaspirane class of cationic amphiphilic drugs in JAK2 over-expressing cell lines and patient samples. In contrast to TKI inhibitors, this drug is causing downregulation of JAK2 protein by facilitating its degradation, possibly through the ubiquitin-proteosome pathway (Faderl, S. et al, Leukemia Research 2006 in press).Methods and Results:We have examined the growth inhibitory effect of atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R) and, either wild-type JAK2 (JAK2WT) or mutant JAK2 (JAK2V617F). Atiprimod growth inhibition, measured using 72 hours MTS assay, was significant against both mutant JAK2V617F cells (IC50 0.42μM) and wild-type JAK2WT cells (IC50 0.65μM). Atiprimod was found to induce apoptosis in cell lines as evidenced by an increase in the mitochondrial membrane potential and augmented annexin V positivity by flow cytometry. Mononuclear cells, isolated from peripheral blood of PV patients (JAK2V617F positive), were cultured in cytokine supplemented serum free media for expansion of erythroid progenitors. Expansion spanned a three week, three step sequential treatment of the erythroid progenitor cells with specific combinations of cytokines. Finally, treatment of expanded erythroid progenitor cells with atiprimod for 24 and 48 hours resulted in increased annexin V positive cells, in both, a dose and time dependent manner.Conclusion:Our preliminary pre-clinical results suggest that atiprimod might be a beneficial medication for MPD patients carrying the JAK2V617F mutation. Atiprimod is currently being tested in a phase I clinical study.

A Phase II Trial of Combination Therapy with Thalidomide, Arsenic Trioxide, Dexamethasone, and Ascorbic Acid (TADA) in Patients with Chronic Idiopathic Myelofibrosis (CIMF) or Overlap Myelodysplastic/Myeloproliferative Diseases (MDS/MPD).

Background: Both CIMF and MDS/MPD are clonal hematopoietic disorders characterized by cytogenetic abnormalities and myelofibrosis with resultant organomegally. Arsenic Trioxide (ATO) has demonstrated efficacy in MDS in two multicenter trials, including patients with MDS/MPD. Ascorbic acid (AA) enhances the activity of ATO by depleting intracellular glutathione. Thalidomide (Thal), in combination with ATO or steroids, is efficacious in patients with MDS and in patients with CIMF.Methods: We conducted a multicenter trial of Thal (50mg/d PO × 2 weeks, then 100mg/d), ATO (0.25mg/kg IV d1-5 of week 1, then twice weekly weeks 2–12), Dexamethasone (4mg/d PO × 5d every 4 weeks), and AA (1000mg PO 1–2 hours prior to each ATO infusion) over a 12-week cycle in patients with CIMF or MDS/MPD, from 1/05 to 6/06. Patients continued on Thal for an additional 3 months. Bone marrow was assessed at baseline and after week 12, as was spleen size. The primary endpoint was response as defined by the Modified International Working Group (IWG) treatment response criteria for MDS, and the IWG criteria for myelofibrosis with myeloid metaplasia (MMM).Results: Twenty-four patients have been enrolled;18 were evaluable for response. The median age was 66.5 years; 20 patients (83%) had MDS/MPD: 7 with CMML and 13 with MDS/MPD-u, with cytogenetics of: normal (11), +22p (1), +8 (2), −Y (1), +9 (1), complex (2), unknown (2). Four patients (17%) had CIMF with cytogenetics of: normal (2), dup Chr6 (1), unknown (1); Of 14 patients tested, 5 had the JAK2V617F mutation: 1 with CIMF and 4 with MDS/MPD. No patient had the MPL515 mutation. Eleven patients had splenomegally, and 11 received prior therapies, including erythropoietin (8), 5-azacytidine (2), hydroxyurea and anagrelide (1), and prednisone (1). Median baseline lactate dehydrogenase (LDH), leukocyte count, and platelet count were: 329U/L, 13.7k/μL, and 133k/μL, respectively. Reasons for starting therapy included anemia [7 (30%)], thrombocytopenia [8 (33%)], or disease-related symptoms [9 (37%)]. Six patients (25%) discontinued therapy: 3 withdrew consent, 1 had a low platelet count, and 2 due to an adverse event. Responses occurred in 7 patients (39%): 6 (33%) by the IWG MMM criteria, 4 (22%) by the IWG MDS criteria, and 3 (17%) by both. Using the IWG MMM criteria, 1 patient with MDS/MPD had a partial remission and 5 had clinical improvement: 2 with MDS/MPD and 1 with CIMF had hemoglobin responses, and 2 had a spleen response. Using the modified IWG MDS criteria, 1 patient with CIMF and 2 with MDS/MPD had erythroid responses; 1 with MDS/MPD had a platelet response. No patient with a JAK2V617F mutation responded. Grade3/4 non-hematologic toxicities included edema (4), fatigue (3), pleural effusion (2), dyspnea (2), motor and sensory neuropathy (1), pericardial effusion (1), zoster (1), and hematoma (1). There were no thrombotic episodes.Conclusion: The TADA regimen is well-tolerated and yields clinical responses in patients with MDS/MPD or CIMF, even in the absence of the JAK2V617F mutation.

Experimental therapy in myelofibrosis with myeloid metaplasia

Myelofibrosis with myeloid metaplasia (MMM) is a Philadelphia chromosome-negative myeloproliferative disorder that is characterised by constitutional symptoms, progressive anaemia and extramedullary haematopoiesis. There are no curative therapies available for patients with MMM apart from stem cell transplantation, which is associated with significant morbidity and mortality, and for which most patients are not suitable candidates. Traditional pharmacological therapy of MMM has focused on the palliation of symptoms associated with myeloproliferation and correction of cytopoenias. Recently, new findings regarding the molecular basis of MMM and the pathogenesis of the associated bone marrow stromal reaction have provided both basic and clinical researchers with invaluable tools to develop effective targeted therapies for patients with MMM. Several novel treatment strategies are being investigated including antiangiogenic agents, signal transduction inhibitors, inhibitors of fibrogenesis and small-molecule inhibitors of the JAK2V617F mutation. This article reviews the current status of experimental novel therapies for MMM.

MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients

AbstractRecently, a gain-of-function MPL mutation, MPLW515L, was described in patients with JAK2V617F-negative myelofibrosis with myeloid metaplasia (MMM). To gain more information on mutational frequency, disease specificity, and clinical correlates, genomic DNA from 1182 patients with myeloproliferative and other myeloid disorders and 64 healthy controls was screened for MPL515 mutations, regardless of JAK2V617F mutational status: 290 with MMM, 242 with polycythemia vera, 318 with essential thrombocythemia (ET), 88 with myelodysplastic syndrome, 118 with chronic myelomonocytic leukemia, and 126 with acute myeloid leukemia (AML). MPL515 mutations, either MPLW515L (n = 17) or a previously undescribed MPLW515K (n = 5), were detected in 20 patients. The diagnosis of patients with mutant MPL alleles at the time of molecular testing was de novo MMM in 12 patients, ET in 4, post-ET MMM in 1, and MMM in blast crisis in 3. Six patients carried the MPLW515L and JAK2V617F alleles concurrently. We conclude that MPLW515L or MPLW515K mutations are present in patients with MMM or ET at a frequency of approximately 5% and 1%, respectively, but are not observed in patients with polycythemia vera (PV) or other myeloid disorders. Furthermore, MPL mutations may occur concurrently with the JAK2V617F mutation, suggesting that these alleles may have functional complementation in myeloproliferative disease.

Concurrent MPL515 and JAK2V617F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time

MPLW515L/K and JAK2V617F can co-exist in myelofibrosis with myeloid metaplasia (MMM). The chronology of clonal emergence was studied in three such cases using serially stored bone marrow. At diagnosis, a major MPL515 mutant clone was accompanied by a minor JAK2V617F clone in all three instances. At 25 time points over a period of 4-8 years, allele burden fluctuated but remained high for MPLW515L/K and low for JAK2V617F. We conclude that MPLW515L/K and JAK2V617F are both early events in MMM and allele burden, rather than the mere presence of these mutations, might be relevant to phenotypic variation in myeloproliferative disorders.

Different outcome of allogeneic transplantation in myelofibrosis using conventional or reduced‐intensity conditioning regimens

Allogeneic haematopoietic stem cell transplantation remains the only curative treatment of myelofibrosis with myeloid metaplasia (MMM). Previous reports have indicated significant treatment-related mortality (TRM) for patients transplanted after myeloablative conditioning but superior survival has been reported after reduced-intensity conditioning (RIC). We report the results of a survey of all allogeneic transplantations for MMM performed in Sweden at six transplant units between 1982 and 2004. Twenty-seven patients were transplanted; 17 with a myeloablative conditioning regimen and 10 with RIC. The median age was 50 years (5-63 years) at transplantation. After a median follow up of 55 months, 20 patients are alive. TRM was 10% in the RIC group and 30% in the myeloablative group. There was no difference in survival for high or low-risk patients according to Cervantes score or between sibling and unrelated donor transplantations.

Lenalidomide therapy in myelofibrosis with myeloid metaplasia

AbstractWe present results of 2 similarly designed but separate phase 2 studies involving single-agent lenalidomide (CC-5013, Revlimid) in a total of 68 patients with symptomatic myelofibrosis with myeloid metaplasia (MMM). Protocol treatment consisted of oral lenalidomide at 10 mg/d (5 mg/d if baseline platelet count < 100 × 109/L) for 3 to 4 months with a plan to continue treatment for either 3 or 24 additional months, in case of response. Overall response rates were 22% for anemia, 33% for splenomegaly, and 50% for thrombocytopenia. Response in anemia was deemed impressive in 8 patients whose hemoglobin level normalized from a baseline of either transfusion dependency or hemoglobin level lower than 100 g/L. Additional treatment effects in these patients included resolution of leukoerythroblastosis (4 patients), a decrease in medullary fibrosis and angiogenesis (2 patients), and del(5)(q13q33) cytogenetic remission accompanied by a reduction in JAK2V617F mutation burden (1 patient). Grade 3 or 4 adverse events included neutropenia (31%) and thrombocytopenia (19%). We conclude that lenalidomide engenders an intriguing treatment activity in a subset of patients with MMM that includes an unprecedented effect on peripheral blood and bone marrow abnormalities.

Cytogénétique et génétique moléculaire dans la myélofibrose avec métaplasie myéloïde et dans la polyglobulie de Vaquez

Myelofibrosis with myeloid metaplasia (MMM) is a rare myeloproliferative disorder (MPD) characterized by clonal proliferation of hematopoietic progenitors. 40-50% of karyotypes on blood (or more rarely on bone marrow) revealed at least one abnormality: 30% at diagnosis and 90% in blastic transformation phase. A minority of patients with newly diagnosed polycythemia vera (PV) presented chromosomal abnormalities in their myeloid cells. The most frequent visible alteration in MMM and PV is a 20q deletion, also characterized in other MPDs and myeloid malignancies. Among other chromosomal changes, deletion 13q is more common in MMM than in other MPDs, trisomy 9 and 9p alterations appear more frequent in PV. Cytogenetic studies have disclosed cryptic anomalies and pointed out the high frequency of 9p alterations. JAK2 (V617F) mutation was found in almost all PV patients and near half of MMM patients. This molecular abnormality takes an increased importance in the knowledge of the physiopathology of MPDs, particularly in PV and also in prognosis of MMM patients.

Janus kinase 2 (V617F) mutation status, signal transducer and activator of transcription-3 phosphorylation and impaired neutrophil apoptosis in myelofibrosis with myeloid metaplasia

An activating point mutation in Janus kinase 2 (JAK2 V617F) was recently identified in myelofibrosis with myeloid metaplasia (MMM). To further elucidate the pathogenic significance, we examined the JAK2 mutation burden, phosphorylation of JAK2 substrates and neutrophil apoptotic resistance. Immunoblotting revealed phosphorylation of signal transducer and activator of transcription-3 (STAT3) in all four JAK2 with high V617F mutant allele burden and seven of eight with intermediate mutant allele burden, but only one of eight with wild-type JAK2 (P<0.001). In contrast, STAT5 phosphorylation was undetectable in patient MMM neutrophils; and phosphorylation of Akt and extracellular signal-regulated kinases (ERKs) failed to correlate with JAK2 mutation status. Apoptosis was lower in MMM neutrophils (median 41% apoptotic cells, n=50) compared to controls (median 66%, n=9) or other myeloproliferative disorder patients (median 53%, n=11; P=0.002). Apoptotic resistance in MMM correlated with anemia (P=0.01) and the JAK2-V617F (P=0.01). Indeed, apoptotic resistance was greatest in MMM neutrophils with high mutant allele burden (median 22% apoptosis, n=5) than with intermediate burden (median 39%, n=23) or wild-type JAK2 (median 47%, n=22; P=0.008). These results suggest that mutant JAK2 contributes to MMM pathogenesis by constitutively phosphorylating STAT3 and diminishing myeloid cell apoptosis.