Myelofibrosis With Myeloid Metaplasia Patients Research Articles

PB2195 ASSESSMENT OF PLATELET MEMBRANE ALTERATION IN PATIENTS WITH MYELOID METAPLASIA WITH MYELOFIBROSIS

Background:Patients with Myeloid Metaplasia with Myelofibrosis (MMM) present at the onset or during evolution thrombotic complications. In the assessment of thrombosis risk are important the presence of JAK2 mutation as well as platelet function. Receptors that define the status of activated platelets (CD62P, CD36) are better expressed in chronic myeloproliferative neoplasms (MPNs) patients with thrombotic complications. Numerous studies have shown a high oxidative status in MPNs patients, a situation that correlates with a higher thrombotic risk.Aims:The aim of our study was to determine if resting membrane potential could be correlated with alterations in expression of platelet receptors and reactive species production (ROS)MethodsThis retrospective study included 35 patients with MMM (16 male and 19 female), median age 61.1 (95% CI of median 60,71‐72) as well as 15 healthy volunteers. The diagnosis of MMM was made according to World Health Organization (WHO) classification of MPNs. We evaluated the flow cytometry markers of platelet adhesion (CD42a, CD42b), aggregation (CD41, CD61) and CD36. Production of reactive species (ROS) was examined using fluorescence method with DCFDA and was assessed by aria under curve (AUC) in serial measurements during 900 sec. The determination of platelet membrane potential was done by fluorescence method using 3,3′‐dipropyl‐2,2′‐thiadicarbocyanine iodide (DiSC3‐5)Results:The ROS production is significantly higher in MMM group: median value of AUC patient group 12780882862,5000 compared with controls 11621365017,5000, p= 0.04. Patients in advanced phase (grade 5 Hackett of splenomegaly) has high level of ROS production compared with patients diagnosed in early stage (grade 1 or 2 Hackett of splenomegaly) median value: 13178014970 vs 12303298585, p = 0.001. There are no differences between groups that was splitted by type of treatment Ruxolitinib/Hydroxiureea or type of mutation diagnosed JAK2V617F/CALR/no mutation. Patients with MMM present higher resting membrane potential (RMP) compared with controls (median value ‐ 63 mV vs–57.5 mV, p = 0.05). The expression of CD36 receptor was higher in MMM group vs controls: median value 27.91 vs 20.91, p = 0.003. Number of microparticle (MP) derivated from platelet, erythrocyte and endothelial cells are no different between patient and control group. We obtain only lower receptor expression on MP surface for CD41/CD61, CD42b and GlyA in patient group compared with control group, p < 0.05. There are significant correlation between resting membrane potential and expression of CD42b (r ‐0.45, p = 0.04) and GlyA receptors (r ‐0.45, p = 0.04).Summary/Conclusion:Patients with MMM especially those with have a higher level of ROS production especially in advanced phase. The hyperpolarisation of cell macrophages membrane could be associated with high ROS production, no reports are regarding platelet membrane. In our study we did not obtain any significant correlation between RMP and ROS production. The expression of CD 36 is higher in MMM patients but was not correlated with ROS production. It was proved that generation of intracellular ROS is associated with CD36 signaling and represent one start point for thrombosis. Low expression of CD42b and GlyA of microparticle surface is associated with lower RMP that correspond to activated status of platelet membrane. We have to verify these results in higher number of patients in order to find significant correlations

Bone morphogenetic protein antagonist gene NOG is involved in myeloproliferative disease associated with myelofibrosis

In a case with secondary myelofibrosis occurring after essential thrombocythemia, cytogenetic analysis revealed an isolated translocation t(X;17)(q27;q22) in all cells. We found that a bacterial artificial chromosome (BAC) encompassing the breakpoint on chromosome 17 long arm contained only one gene, NOG. We therefore investigated the occurrence of this rare breakpoint in myeloproliferative disorders (MPDs). We identified three more patients with a 17q abnormality in MPDs: myelofibrosis with myeloid metaplasia (MMM); chronic myeloid leukemia positive for t(9;22)(q34;q11) with additional t(4;17)(p15;q22) at diagnosis; and myelofibrosis complicating polycythemia vera. All three cases exhibited a split of BACs containing NOG. The protein encoded by NOG, noggin, acts as an antagonist to bone morphogenetic secreted protein 2 and 4 (BMP2 and BMP4). A comparative analysis of gene expression on Agilent 22K oligonucleotide microarrays in purified CD34+ cells from the blood of MMM patients showed significant downregulation of BMPR2, BMPR1B, BMP2, and BMP8; upregulation of BMP3 and BMP10; and a trend to lower expression of NOG. Thus, given that expression and release of BMPs are important in the induction of osteosclerosis and angiogenic activity, the observed BMP deregulations could be triggered by potential NOG genetic alterations in the four cases here described, and may contribute to the myelofibrotic process characterized by bone marrow stromal reaction including collagen fibrosis, osteosclerosis, and angiogenesis.

The Effect of Anabolic Steroids on Anemia in Myelofibrosis with Myeloid Metaplasia: Retrospective Analysis of 39 Patients in Japan

Between 1999 and 2005, 285 patients received new diagnoses of myelofibrosis with myeloid metaplasia (MMM) in Japan. Anemic symptoms were present in 162 patients, and hemoglobin (Hb) concentrations were <10 g/dL in 197 patients. Fifty-five MMM patients were treated with anabolic steroids, and their effect on anemia during MMM was evaluated in 39 patients. A "good" response was defined as an Hb increase of >or=1.5 g/dL, cessation of transfusion dependence, and an Hb concentration of >10 g/dL maintained for at least 8 weeks. A "minimum" response was defined as an Hb increase of >or=1.5 g/dL and transfusion independence for at least 8 weeks. Both good and minimum responses were considered "favorable." Favorable responses were achieved in 17 patients (44%, 8 good and 9 minimum responses). None of the pretreatment variables, such as the lack of transfusion dependence, a higher Hb concentration at the start of treatment, or the absence of cytogenetic abnormalities, were associated with a response to anabolic steroid therapy. Adverse events associated with anabolic steroid therapy were moderate and transient. Two patients required definitive withdrawal of treatment. Thus, anabolic steroids are well tolerated and effective for the treatment of anemia in a subset of MMM patients.

The expression of CXCR4 is down-regulated on the CD34+ cells of patients with myelofibrosis with myeloid metaplasia

Purpose: We studied the expression of the chemokine receptor CXCR4 on circulating CD34+ cells of patients with myelofibrosis with myeloid metaplasia (MMM), and examined its relationship to the severity of disease. Patients and methods: Surface and intracellular CXCR4 expression were measured flow cytometrically in 84 consecutive MMM patients, 16 patients with polycythemia vera (PV), and 20 healthy subjects. In 23 MMM patients, CXCR4 gene expression level was also quantitated by real time-RT-PCR in CD34+ cells. Results: The expression of CXCR4 on circulating CD34+ cells was significantly reduced in patients with MMM ( P < 0.001) as compared to normal controls and patients with PV ( P = 0.01). The levels of CXCR4 mRNA in CD34+ cells were lower in patients with MMM as compared with normal subjects, and were directly correlated with the degree of CXCR4 surface expression, demonstrating that transcriptional defects were the major cause for receptor down-regulation. No statistical association was found between JAK2V617F mutational status and the extent of CXCR4 down-regulation. CXCR4 expression on CD34+ cells inversely correlated with the number of circulating CD34+ cells ( R = − 0.55; P < 0.001), and was severely down-regulated in high risk patients and patients with a high “myelodepletion severity index”. CXCR4 down-regulation was associated with advanced patient age, the presence of severe anemia, thrombocytopenia, and degree of bone marrow fibrosis. Conclusions: Reduced expression of CXCR4 by CD34+ cells is a characteristic of MMM which is associated with the constitutive mobilization of CD34+ cells and occurs in patients with advanced forms of the disease.

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.

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.

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.

Evaluation of the In Vitro Activity of the Multikinase Inhibitor BAY 43-9006 (Sorafenib) in Primary Cells from Patients with Myelofibrosis with Myeloid Metaplasia (MMM).

Background: Myelofibrosis with myeloid metaplasia (MMM) currently has limited therapeutic options. BAY 43-9006 (sorafenib; Bayer Pharmaceuticals) is a oral multikinase inhibitor with both inhibitory properties against the Raf/ MEK survival pathway and VEGF that are a potential therapeutic targets for the myeloproliferation and apoptotic resistance seen in this disorder. We evaluated the activity of this agent through inhibition of aberrant in vitro myeloid colony growth, as we have previously successfully utilized in this disorder (Leukemia 2003; 17(5):849).Methods: Peripheral blood mononuclear cells (PBMC) were isolated and plated on the Methocult™ (StemCell Technologies) colony assay media. Dilutions of BAY 43-9006 (0–10 μM) (Phase I trial sustained concentrations of 10 μM (JCO 2005; 23:965) was added for either a 24 hour incubation, or for 14 days. Myeloid colonies were then quantified according to standards established by the manufacturer. Subsequent assays were performed combining BAY 43-9006 with the pro-apoptotic agents arsenic trioxide (ATO) or adaphostin (ADA) (both at 1.25μM). Finally, immunoblotting was performed on protein extracted from treated primary MMM cells to quantify the downregulation of Mcl-1 (shown to be crucial for the pro-apoptotic effects of BAY 43-9006; Yu et. al. Oncogene 2005).Results: PBMC were isolated from 12 MMM patients and 10 controls (normal n=5, other myeloproliferative (MPD) n=5). Twenty-four hour exposure of cells to the agent was insufficient to suppress aberrant colony growth. Continuous exposure (for 14 days) of the agent led to a median inhibitory concentration-50% (IC50) of 12 μM for MMM (compared to 7.1μM for normals, and 5μM for MPD controls). Although no specificity could be deduced from these latter results, 4 MMM patients had an IC50 of <10μM (range 1.25 to 8.6μM). In contrast, the other 8 patients were quite resistant IC50 of > 10μM (higher than clinically achievable ranges). Subsequently we evaluated whether there might be benefit to use of BAY43-9006 in combination with a pro-apoptotic agent (i.e. ATO or ADA). No significant activity was seen combining ADA with BAY43-9006. When BAY43-9006 was combined with ATO, the median IC 50 for the BAY compound decreased to 2μM (range 1.25 to 16), however there was not a significant increase in colony inhibition over ATO alone. In order to evaluate whether the lack of activity of BAY43-9006 observed in colony formation assays was secondary to a lack of downregulation of the established target Mcl-1, we immunoblotted for Mcl-1 (after in vitro incubation) in a subset of MMM patients. Western blots demonstrated successful downregulation of Mcl-1 by Bay 43-9006 in MMM samples, even in samples with clear in vitro resistance to the drug (i.e. IC50 > 10 μM).Conclusions: The multi-kinase inhibitor BAY43-9006 appears to have minimal activity against primary cells isolated from MMM patients. Although, suppression of aberrant colony growth was noted in a subset of patients the sensitivities would not support specificity when compared to controls. Although BAY 43-9006 was observed to down regulate the anti-apoptotic target Mcl-1, in patient samples, no detectable inhibition of colony formation was observed in 8/12 MMM samples. These preliminary results do not appear to support a clinical trial of BAY 43-9006 in MMM, however combination of this multi-kinase inhibitor with other pro-apoptotic agents should be evaluated.

Reduced Expression of CXCR4 on Circulating CD34+ Cells Is Associated with Hematopoietic Progenitor Cells (HPC) Mobilization in Patients with Myelofibrosis with Myeloid Metaplasia (MMM).

Spontaneous mobilization of HPC is a key feature of MMM. We investigated the CXCR4/SDF-1 (stromal derived factor-1) axis in 52 consecutive MMM patients. The percentage of circulating CD34+ cells co-expressing CXCR4 at the cytofluorimetric analysis was significantly lower (P=0.02) in MMM patients (median, 37%, range, 0 to 92%) than in the normal controls (n=12; median, 75%; range, 39 to 91%). CXCR4 mean fluorescence intensity was also highly significantly lower in MMM patients (median 0.73; range, 0.06 to 7.05) than in normal controls (median, 1.84; range, 0.41 to 6.69; P=0.004). By analyzing the association between CXCR4 expression and the mobilization of progenitor cells, an inverse correlation was found between CXCR4 expression and the number of circulating CD45+/CD34+ HPC (R=−0.40; P=0.005). By restricting the relation analysis of CXCR4 expression and clinical features to those patients who were not receiving cytoreductive treatment (n=41), there was no correlation between CXCR4 expression and patients' sex, hemoglobin concentration, WBC, prefibrotic stage. By contrast, CXCR4 expression was inversely correlated with clinical characteristics that portrayed an advanced disease, such as older age (R=−0.31; P=0.026), longer disease duration (R=−0.37; P=0.008), larger splenomegaly (r=−0.33; P=0.015) and lower platelet count (r=0.42; P=0.007). The levels of CXCR4 mRNA, measured in peripheral blood CD34+ cells of patients with MMM were lower as compared with those of cells purified from normal peripheral blood (ΔCT by real time RT-PCR using GAPDH as housekeeping reference gene: 5.33 ± 0.49 vs. 1.53 ± 0.79, mean ± SD; P=0.003). SDF-1 plasma levels were significantly increased in patients with MMM but no correlation was documented with CXCR4 expression. We conclude that reduced expression of CXCR4 on CD34+ cells is a mechanism for the constitutive mobilization of HPC in MMM.

Glivec/STI571 Treatment Stimulates Megakaryopoiesis and Normalizes PDGF Receptor beta Kinase Expression in Thrombocytopenic Patients with Myeloid Metaplasia with Myelofibrosis.

Myeloid Metaplasia with Myelofibrosis (MMM) is a Philadelphia negative chronic myeloproliferative disorder associated with myeloid metaplasia in the spleen and liver, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ hematopoietic progenitors (up to 200 folds) with a prominent amplification of dystrophic megakaryocytes (Mk). Alteration of tyrosine kinase signals is suspected to play a key role in the myeloproliferation and in the increased sensitivity of hematopoietic progenitors to growth factors. Glivec is a small-molecule tyrosine kinase inhibitor of the ABL fusion gene, platelet derived growth factor receptor beta (PDGFRB) and SCF receptor (c-kit). In this study, we investigated the effect of Glivec/STI571 on the megakaryopoiesis and the kinase expression of 11 MMM patients enrolled in a European EUMNET clinical trial. We showed that 50 percents of the treated patients exhibited a tendency to a decrease in the CD34+ cell number from 261/μL to 56/μL with a normalization of the CD34+ cell count (1–10/μL) in 20 percents of these patients (2/10). We also observed a reduction of a CD34+CD41+ Mk subpopulation co-expressing the CD9 tetraspanin that is suggested to be a marker of MMM. Normalization of the CD9 gene expression level was further confirmed by quantitative RT-PCR in MMM mononuclear cells. In contrast, 55 percents of MMM patients (5/9 patients) showed an increase in their platelet count from 191 x 109/L to 282 x 109/L during treatment with Glivec. Interestingly, an increase in the platelet count was also observed in three of the five thrombopenic patients who exhibited an increase greater than 50 x 109/L (p= 0.02) being associated with an augmentation in the number of clonogenic megakaryocytic progenitors (CFU-Mk). The effect of Glivec treatment on megakaryopoiesis was further illustrated by a morphometric analysis on bone marrow histological sections showing a normalization of the size and shape of the dystrophic MMM Mk. We also analyzed by quantitative RT-PCR the effect of Glivec treatment on receptor kinase gene expression in circulating mononuclear cells from MMM patients and demonstrated a normalization of the receptor beta of the PDGF, a cytokine involved in Mk differentiation. Actually, the PDGFB expression that was down-regulated in MMM PBMC before Glivec as compared to normal PBMC (−1.14 +/− 0.76 and − 0.14 +/− 0.42, respectively; p= 0.02) was significantly increased during therapy, reaching a normal level in 60% of tested patients. In conclusion, our results show that Glivec treatment reduced the CD34+ cell number and stimulated the megakaryopoiesis of MMM thrombopenic patients, probably by restoring Mk progenitor differentiation.

99mTc-BW250/183 Bone Marrow Immunoscintigraphy Is Correlated to Clinical and Biologic Features of Myelofibrosis with Myeloid Metaplasia.

BACKGROUND: Myelofibrosis with myeloid metaplasia (MMM) is a rare chronic myeloproliferative disease characterized by both myeloproliferative and myelodepletive features: myeloproliferation typically includes enhanced spontaneous mobilization of hematopoietic progenitor cells (HPC) from the bone marrow (BM) and their homing into extramedullary sites (mainly spleen); myelodepletion results from exhaustion of both BM and extramedullary hemopoiesis.AIMS: To investigate the extent and distribution of hematopoiesis in MMM patients and to capture its relationship with BM fibrosis, HPC mobilization and clinical severity.METHODS: Immunoscintigraphy employing a dual-head camera was performed 120–260 minutes (median 180 minutes) after administration of 553–830 MBq (median 700 MBq) 99mTc-BW250/183, corresponding to 0.3–0.5 mg. Hematopoietic function in the central compartment (sacrum) was described by “subtypes” (normal, increased or decreased) and by the sacrum-to-soft tissue uptake ratio (UR) (Huic at el, J Nucl Med 1997). The degree of peripheral BM displacement (limbs) was described through 5 “types” (I to V) (Huic at el, J Nucl Med 1997).RESULTS: Twenty-three MMM patients (13 males, median age 55 years) were studied. Eleven patients showed a reduced uptake by the central BM compartment: they had a higher WHO fibrosis grade (p=0.008), lower hemoglobin values (p=0.012) and lower platelet counts (p=0.007) than patients with a preserved central compartment. Patients with an exhausted central compartment at immunoscintigraphy also showed a significantly higher mobilization of HPC into peripheral blood, as documented by higher values of the following parameters: CD34+ count (0.86% vs 0.12%; p=0.029), immature myeloid cells or blasts (9.5% versus 0.3%; p=0.005), spleen size (9.3 vs 2.4 centimeters from costal arc; p=0.007). Among the patients with a depressed central compartment, those who also lost peripheral BM function (type V) showed a more severe myelodepletion and more intense HPC mobilization. On the opposite side, among the 12 patients with a preserved central compartment, the 8 ones with a mild peripheral BM displacement (type I–II) showed absent or mild fibrosis (WHO 0-1), elevated platelet counts, normal to high hemoglobin values, minimally enlarged spleens and no hints of increased HPC mobilization (CD34+ <0.1%; no blasts; <=1% immature myeloid cells). From these data it appears that progressive fibrosis and exhaustion of central BM is accompanied by a gradual displacement of hemopoiesis into the peripheral bone compartment and the spleen and by derangement of HPC trafficking.CONCLUSIONS: BM immunoscintigraphy accurately tracks MMM clinical features and may help staging MMM patients, understanding MMM biology and targeting therapiesClinical and immunoscintigraphy parameters (means and ANOVA p values)TypeI-IIIII–IVVpSubtypenot reducedreducedreducedUR9.392.531.220.041Fibrosis (grade)1.02.02.70.001Hb (g/dl)13.511.19.60.023PLT *10(9)613283860.04WBC *10(9)8.96.26.90.059Spleen (cm)1.97.610.50.011Immature myeloid cells (%)0.33.69.70.0005Blast cells (%)0.00.23.80.048CD34+ cells (%/μl)0.1 / 6.40.4 / 22.11.3 / 155.50.009 / 0.12LDH (UI/l)56695613810.014Severity Score§2.43.03.80.036Disease duration (mo)4330740.45§ Barosi et al, Leuk Lymph 2002

Incidence and Clinical Profile of JAK2 V617F Mutation in Myelofibrosis with Myeloid Metaplasia.

An aberrant tyrosine kinase signalling due to non-receptor tyrosine kinase JAK2 V617F mutation has been highlighted in the pathogenesis of polycythemia vera (PV). In myelofibrosis with myeloid metaplasia (MMM) this mutation has been reported to be present in approximately 50% of the patients and its pathogenetic role is not elucidated. Here we report the results of JAK2 V617F mutation in a retrospective analysis of blood samples from 170 patients with MMM. Search for JAK2 mutation was performed by an allele specific PCR from DNA purified from granulocytes. To evaluate whether the mutation was carried in the homozygous or heterozygous state, digestion of PCR products with BsaXI restriction enzyme was performed. The overall frequency of JAK2 V617F mutation was 60% and homozygosity for the mutation was found in 39.2% of mutant samples. Disease duration was similar in JAK2 mutated and wild type patients. Patients who harboured an homozygous mutation had an higher myeloproliferative severity score (that indexed leukocytosis, thrombocytosis and splenomegaly) than patients who had a wild type or heterozygous genotype. In post-PV MMM, the mutant gene was present in 22/22 (100%) of patients, and the frequency of homozygosity was 59% of the mutated cases. In post-ET MMM (n=13), the mutant gene was present in 46% of the patients, and the frequency of homozygosity was 16% of the mutant samples. In idiopathic MMM (n=135), the incidence of the mutational state was 54.8%, with 35.1% of homozygote mutation. Patients who had received a diagnosis of prefibrotic myelofibrosis (WHO classification) had an incidence of the mutation significantly lower than patients who were diagnosed in the fibrotic stage (6/18, 33% vs 68/112, 60.7%; P=0.001). By considering only patients not receiving cytoreductive or disease modifying agents, patients who had an heterozygote mutation, had a mean Hb value higher than wild type patients (9.4 g/dL vs. 11.4 g/dL; P=0.004). Moreover, patients who had an homozygote mutation had the myeloproliferative severity score higher than both heterozygote and wild type patients (2.26 vs 1.59, P=0.008, and 2.26 vs.1.52, P=0.001, respectively). We conclude that JAK2 V617F mutation is significantly represented in MMM patients. It is a necessary event in the transformation from PV to MMM while not in the transformation from ET to MMM. Patients who harboured an heterozygote state maintained higher Hb values than patients with a wild type genotype, while the homozygote mutation was associated with leukocytosis, thrombocytosis and splenomegaly.

JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood 2003; 102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM.Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes.Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026).Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

X-Linked Clonality Analysis and Quantitative JAK2V617F Assessment Reveals a Strong Association between Clonality and JAK2V617F in PV but Not in ET/MMM, and Identifies a Subset of JAK2V617F Negative ET and MMM Patients with Clonal Hematopoiesis.

Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are categorized as a set of phenotypically related myeloproliferative disorders (MPD). Recent work has identified a somatic mutation in the JAK2 tyrosine kinase (JAK2V617F) in the majority of PV and in a subset of ET and MMM. As most, but not all, patients with MPD have clonal granulocytes, we sought to determine if there was a relationship between granulocyte clonality and JAK2V617F mutational status using a quantitative clonality assay for the human androgen receptor gene (HUMARA) and a quantitative real-time PCR assay for JAK2V617F. 168 of 190 female MPD patients were informative for the HUMARA polymorphism, and 121 patients (72%) had allele skewing consistent with clonal granulopoiesis including 80% of MMM, 75% of PV, and 67% of ET. The quantitative real-time PCR assay for JAK2V617F detected the JAK2V617F allele in 98.8% of PV, 69% of ET, and 33% of MMM, and was more sensitive than direct sequencing. A significant correlation between the degree of allele skewing and quantitative JAK2V617F allelic ratio was observed in PV (p<0.0001) but not in ET (p= 0.2) or MMM (p=0.771). In addition, a subset of patients with ET and MMM with clonal granulocytes had JAK2V617F present in some, but not all, clonal granulocytes. These data suggest acquisition of the JAK2V617F mutation may be sufficient for the development of PV, but additional genetic events are necessary to cooperate with JAK2V617F in ET and MMM. Most importantly, we identified a subset of patients with ET (23%) and MMM (17%), but not PV, with clonal granulocytes who were JAK2V617F negative. A high-throughput screen for novel activating mutations in signal transduction pathways in JAK2V617F negative ET and MMM is warranted.

Microcytosis in Myelofibrosis with Myeloid Metaplasia: Prevalence and Clinical Correlates.

Background:Microcytosis is a characteristic laboratory feature for both iron deficiency anemia and thalassemia. Microcytosis is also infrequently seen with “anemia of chronic disease”. A partial list of the latter include rheumatoid arthritis, polymyalgia rheumatica, diabetes mellitus, connective tissue disease, chronic infection, Hodgkin's lymphoma, Castleman's disease, and renal cell carcinoma. In the current study, we show that microcytosis is a frequent laboratory feature in myelofibrosis with myeloid metaplasia (MMM) and investigate its clinical relevance in the particular setting.Methods:In order to minimize the confounding effects of nutritional deficiencies, red blood cell transfusion, and drug therapy on mean corpuscular volume (MCV), the current study was limited to young patients (age < 60 years) that had not received either red blood cell transfusion or drug therapy for MMM during the time of their initial laboratory investigation.Results:The study population consisted of 102 consecutive patients with MMM (median age 51 years, range 18–60) including 60 males. At diagnosis, the median (range) values for hemoglobin, leukocyte count, and platelet count were 11 gm/dL (7.4–14.9), 8.3 x 109/L (1–40) and 357 x 109/L (14–1512), respectively. Spleen measurements were documented in 78 patients (median 6 cm below the left costal margin, range 0–24) and the incidence of hypercatabolic symptoms was 18% (12 of 67 evaluable patients). The Dupriez prognostic score distribution included low-risk (n=73), intermediate-risk (n=19), and high-risk (n=10) disease categories.The overall incidence of microcytosis (MCV < 80 fL) was 28%; median MCV was 84.3 fL (range, 60.6–99.1). Among the 28 patients with microcytosis, MCV was less than 70 fL in 4 patients. Concomitant serum ferritin levels were available in 44 patients (median, 92 μg/L; range, 4–6440) and the levels were below 30 μg/L in 7 patients including 4 with microcytosis. Bone marrow iron stains were sporadically performed and showed normal iron stores in the majority of evaluable cases. The study cohort was followed for a median of 70 months (range 0–300). Multivariate analysis of variable that were measured in all study patients identified anemia, thrombocytopenia, and age but not MCV as independent prognostic indicators of inferior survival. The addition of variables that were measured in a variable proportion of the study population disclosed additional prognostic value for hypercatabolic symptoms and abnormal cytogenetics. Interestingly, microcytosis was significantly associated with the absence of cytogenetic abnormalities (p=0.04); among the 10 patients with abnormal cytogenetic findings, 9 had an MCV of ≥ 80 fL. Microcytosis did not correlate with several other clinical and laboratory features including gender, presence of hypercatabolic symptoms, spleen size, hemoglobin level, platelet count, leukocyte count, serum lactate dehydrogenase level, and the Dupriez prognostic score.Conclusion:Microcytosis with a normal or elevated serum ferritin level is a frequent laboratory feature in MMM but does not appear to influence survival. The observation regarding the low incidence of cytogenetic abnormalities in MMM patients with microcytosis is intriguing but requires validation by a prospective study. Whether or not MMM-associated microcytosis represents an acquired defect of globin synthesis, as might be the case in some patients with myelodysplastic syndrome, remains to be elucidated.

TGF-β1 Affects the Proliferation of Normal Human Bone Marrow Fibroblasts and Its Expresssion of Smad3 and Smad7 mRNA.

Current drug therapy in myelofibrosis with myeloid metaplasia (MMM) still can not get great progress, the development of the myelofibrosis eventually causes bone marrow failure. The histopathology of MMM patients showed that the bone marrow tissue were full of marrow fibroblasts and an extensive deposition of collagens. Further researches suggested that the proliferation of the fibroblasts was controlled by many factors. One of the most important factor was TGF-β1, and its effects largely depended on Smad pathway. In order to understand the effects of TGF-β1 on the proliferation of marrow fibroblasts, we stimulated the normal human bone marrow fibroblasts with different concentrations of TGF-β1 in the current study. The proliferation test with direct cell counting and MTT assay revealed that different concentrations of TGF-β1 had various effects and it could get the maximal proliferation effect at 0.1ng/ml. Then we detected the Smad3 mRNA and Smad7 mRNA in one normal human bone marrow fibroblasts with RT-PCT methods at above concentration (0.1ng/ml). We found that the expression level of Smad3 mRNA decreased with the time, and that the expression level of Smad7 mRNA reached the highest at 30 min after stimulation by TGF-β1, then it began to reduce. These results suggested that TGF-β1 activate the Smads pathway in normal human marrow fibroblasts, and its proliferation effect require the activation of this pathway. We also noticed that the changes of the smad3 mRNA and smad7 mRNA were similar in skin fibroblasts stimulated by TGF-β1. These changes were considered as a feedback modulation mechanism. Whether the over-proliferation of marrow fibroblasts in MMM patients is correlated with the imbalance of this feedback warrants further research. It may provide a more specific and targeted therapy for blocking myelofibrosis. [Display omitted]

JAK2 V617F Mutational Status in Myelofibrosis at and before Disease Progression Including Leukemic Transformation: A Longitudinal Study in 68 Patients.

Background: An acquired point mutations of JAK2 (V617F) was recently described in a substantial proportion of patients with myeloproliferative disorders (MPD), including myelofibrosis with myeloid metaplasia (MMM). The current study examines i) JAK2 V617F mutational status at time of leukemic transformation (LT) of MMM and its clinical correlates, ii) the possibility of changes over time in mutational status in MMM patients that have either undergone leukemic transformation (LT) or experienced disease progression.Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from either peripheral blood mononuclear cells (PBMC) (i.e. fresh specimens) using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Fluorescent dye chemistry sequencing was performed using the same primers used for amplification.Results: The study population consisted of 68 patients including 33 patients with disease transformation into AML.i. MMM patient cohort with LT (n=33). The JAK2 V617F mutant allele was detected in 21 of the 33 patients (64%; homozygous in 6%) at the time of LT. As expected, PPMM was over-represented in the group with the mutation but the presence of the mutation affected neither the clinical presentation at time of LT or overall outcome. Furthermore, in 9 patients (7 heterozygotes and 2 with wild-type allele), archived bone marrow that was collected at a median of 23 months (range, 3–105) before LT was available and disclosed no change in mutational status.ii. MMM patient cohort with disease progression without LT (n=35; 19 AMM, 9 PPMM, 7 PTMM): Each patient in this group had at least two bone marrow or peripheral blood samples that were collected at two different time points separated by a median of 18.2 months (range (3–82). Of the total 35 patients in this group, 32 (91%) showed no change in JAK2 V617F mutational status over time including 3 patients in whom the first sample was collected before they transformed into MMM from antecedent PV or ET. In the remaining 3 patients, a switch from wild-type to heterozygous state (1 patient), and heterozygous to homozygous (2 patients), was documented after 78, 43, and 83 months, respectively.Conclusions: In most instances, JAK2 V617F mutational status in MMM remains unchanged over time including during leukemic transformation. Furthermore, the presence of the mutation in MMM-related LT does not appear to affect clinical outcome. These observations undermine the role of JAK2 V617F in either disease progression or LT in MMM.

Clinical Classification of Myelofibrosis with Myeloid Metaplasia (MMM): Cluster Analysis of 861 Patients Enrolled into a Nationwide Prospective Registry (RIMM).

BACKGROUND: MMM is a rare chronic myeloproliferative disease with heterogeneous clinical presentation, natural history and prognosis. Knowledge about MMM is burdened by limited sample size and selection bias of the available studies.AIM: To get a clinical classification of MMM for biological and clinical studies.METHODS: The database of the RIMM, a population-based prospective cohort started in 1999, was enquired and data of 861 patients at diagnosis were extracted. The EM-algorithm (WEKA software) was used for a non-supervised data-mining based on 11 variables: age, spleen size, constitutional symptoms, hemoglobin level, platelet and blood cell count, LDH and percentages of erythroblasts, immature myeloid cells, blasts, and basophils. Differences among clusters were first tested with ANOVA or n-way Chi-square and subsequently investigated with t-test or 2x2 Chi-square. Survival differences among clusters were tested through Cox univariate and multivariate regression analysis.RESULTS: Five clusters (C) were identified: C1 identified younger patients with thrombocytosis and scarce myeloproliferation. C2 also identified patients with a high platelet count, but older and anemic. C3 identified patients with intense myeloproliferation (marked leukocytosis and splenomegaly), while C4 (5% of the patients) collected patients with an even more severe myeloproliferation, associated with a high number of circulating immature myeloid cells and blasts. C4 was also characterized by frequent chromosome abnormalities, anemia and thrombocythopenia. Finally, C5 identified patients with bilinear/trilinear cytopenias. The clusters also differed for variables not incorporated by the data-mining process, i.e. CD34+ cell count (p=0.028); i.e. the rate of homozygous V617F mutation of JAK2 gene, which was significantly higher in C3 and C4 (32.4% vs 12.5%, p=0.019). The clusters also significantly differed for overall survival (p<0.0001), independently of patients' age. Finally, referral patterns differed among clusters, with C5 and C1 patients being mostly referred to Internal Medicine and Hematology units (p<0.01), respectively.CONCLUSIONS: Thanks to a large and unbiased repository of cases, we identified 5 classes of MMM patients with specific clinical presentation. The biologic and prognostic value of the clusters deserves further investigations at longer follow-up periods.Clinical parameters in the different clusters (means)CLUSTERAGE (yrs) *HB (g/l) */PLT*10(9)*WBC *10(9) *SPLEEN (cm) *BLASTS*(%)CD34 %/μlSEVERITY SCORE§ *156.713.3 /58311.53.50.210.85 /631.25272.110.6 /48218.32.70.821.12 /2972.04365.110.8 /30318.39.91.471.7 /3132.54467.68.4 /11027.513.45.662.72 /2754.30568.28.4 /1344.26.80.782.92 /523.36All65.510.7 /36213.56.51.181.78 /2002.36* p<0.0001 § The score considers HB, WBC, PLT and SPLEEN (Barosi, Leuk Lymph 2002).

Myeloablative Conditioning of Patients with Myelofibrosis with Myeloid Metaplasia Using i.v. Treosulfan and Autologous Peripheral Blood Progenitor Cell Transplantation (PBPCT) with High Doses of CD34+ Cells Results in Hematologic Responses - 32 Months Follow-Up of Three Patients and Short-Term Results of a New Study.

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder. Myeloablation with high-dose treosulfan and autologous PBPCT provides a palliative approach. A minimum of 5 x 106 CD34+ cells/kg was collected in all patients after G-CSF-priming (16 μg/kg/d for 4 days). Myeloablation consisted of treosulfan (total dose 42 g/m2). To date we have transplanted 3 patients, all female on an individual basis. We observed a prolonged time to reconstitution of leucocytes > 1/nl post transplantation of 28 days (#1, #2) and 38 days (#3). The observation period post transplantation is 32 months now. Patient #1, who required erythrocyte transfusions twice weekly pretransplant received her last erythrocyte transfusion on day 56; her last Hb-value is 9.5 g/dl. The second patient (#2) recovered to platelet counts higher than pre transplantation (58/nl) at 3 months (143/nl) and had 125/nl at last follow-up. Pat. #3 showed a marked reduction of max. spleen size and a rise in Hb from 9 g/dl to 12 g/dl after 12 months, the last Hb value at 26 months was 7.6 g/dl and she received about 3 erythrocyte transfusions per month again. She then underwent allogeneic transplantation from her sister and is currently alive and well. Three male patients were treated in a new one-armed, multicenter study applying the aforementioned protocol. In two patients we observed again a prolonged reconstitution period of 28 (S1) and 44 (S2) days. In one patient (S1) leukocytosis and thrombocytosis resolved while transfusion dependent anemia persisted. In the second patient (S2) transfusion dependent anemia persisted. He received a backup transplantation but still remains transfusion-dependent. Patient S3 had a critical thrombocytopenia of 16/nl before transplantation and proved to be refractory to thrombocyte transfusions post-transplantation. He developed cerebral hemorrhage and died. The conditioning regimen was well tolerated and despite the prolonged aplasia period neutropenic fever was rare (median 1 day, range 0–6 days). The overall response rate was 60%, the mortality rate was 17% which is both compatible with previous data of autologous transplantation in MMM patients undergoing busulfan conditioning. This multicenter study is continuing to recruit patients (http://leukaemie.krebsinfo.de/kn_home/Studien/studie_101.html ).

A Case of Multiple Myeloma(MM) with Myelofiblosis(MF) Undergone Autologous Peripherial Blood Stem Cell Transplantation.

Myelofibrosis with myeloid metaplasia(MMM) is a progenitor cell colony disease. The mechanism is not very clean. The prognosis is poor in MMM patients of the traditional treatment with Hydroxyurea, Interferne, Prednisone, transfusion. Thalidomide is a new anti-angiogenesis drug of inhibiting the genesis of fibrosis. Some reports presented that the cure method only is stem cell transplantation includes autologous and allogeneic bone marrow transplantation.Patient Sui, Man, 55 years, Chinese. He was hospitalized on Aug fifth, 2004, because he has had pale, severe weakness, for 5 months. T 36.C, HR 70, R 18/min, BP114/59 mmHg. Hb56g/L, WBC 5.7x109/L, Plt 72 x109/L.There were 0.66 plasma cell in bone marrow and his bone marrow aspiration with dry draw at the diagnosis,. The result of bone marrow biopsy was fibrosis. He received 4 courses of VAD regimens (VCR 0.4mg,d1–4; ADM 10mg,d1–4, DXM 40mg d1–4) before autologous peripheral stem cell (PBSCs) transplantation. The plasma cell count decreased from 0.66 before to 0.025 after the first course of VAD regimen in the bone marrow. The stem cell mobilization regimen was chemotherapy (cyclophosphamide, Cy, 2g/m2), high-dose methylprednisolone (MP), and G-CSF 250 ug, bid d1–5. The nucleated cell count of collection showed the a minimum of 4.17x108/kg per kg of body weight, CD34+ was 0.834x106/kg.The conditional regimen was melphalan 200mg/kg divided 2 times a day and cyclophosphamide 600mg VD. He received re-transfused PBSCs on March 25,2005. His neutrophil cell decreased 0 at +4 day after transplantation. He received platelet transfusion of single donor two times when his platelet decreased 7 x 109/L. his hemotopoietic recovered at +11d. He left transplantation ward +13 d. His platelet, WBC were normal, Hb 108g/L At+d. Bone marrow draw was no dry. There was 0.07 of plasma cell in his bone marrow. The result of reticulum protein stain was degree 3 after and 4 before PBSCT at +70d.Discussion: MM is incurable tumor because the tumor cells polluted in graft of bone marrow or peripheral blood stem cells. Some author considered the pollution tumor cell decreases with high-dose MP mobilization. Mobilization of stem cell in advanced MMM is difficulty, so we (1) increased the 3 times of mobilization, (2) added high-dose of MP to mobilization regimen of combination of Cy and G-CSF. The patient had received pre-transplantation of four courses of VAD chemotherapy regimen and the combination of west and Traditional Chinese Medicine to manage during stem cell transplantation. The patient had chronic gastric ulcer so we afraid of the condition result in relapse of it so we given the patient intravenous melphalan. Some researchers studied 21 cases of multiple myeloma were treated by autologous bone marrow transplantation. The condition regimen was melphalan 16 mg/kg. The role of allogeneic BMT as compared with autologous BMT is not yet defined. The patient had been followed up for 4 months, the result showed his plasma cell was 0.07 in his bone marrow. The degree of myeloid metaplasia had changed from degree 4 to 2. Conclusion: Intensive treatment with marrow rescue could induce complete remission of long duration in some patients and provided the basis for increased attempts to cure myeloma by intentive chemoradiotherapy and BMT. It is effective in multiple myeloma treatment with autologous stem cell transplantation. The curable method is only allogeneic stem cell maybe.