ALBERT

Description: Abstract 3841 Poster Board III-777 Background and aims p53 gene mutations or deletions are rare in multiple myeloma (MM). Thus, strategies to induce p53 activation in myeloma cells with wild type (wt) p53 may have therapeutic promise. However, p53 signaling and functional consequences are not clearly understood in MM. In this study we used a small molecule MDM2 antagonist; nutlin, to explore the molecular mechanisms associated with nutlin-mediated activation of the p53 signaling pathway resulting in cell cycle arrest and/or apoptosis. Methods MM1.S and H929 cell lines harboring wt p53, LP1 and U266 cell lines expressing mutant (mt) p53, and five primary samples from patients with MM were treated with different concentrations of nutlin or DMSO control. Nutlin-induced cells were assessed for cell viability, induction of p53 and its downstream targets, cell cycle analysis, apoptosis assays and gene expression profile at different time periods. Results Treatment of MM cells harboring wt p53 with nutlin led to a dose-dependent increase in the expression of p53 and its downstream targets, p21 and MDM2. After 24 hours incubation with 10 μM nutlin, p53 protein levels increased approximately 4 to 6-fold in MM1.S and H929 cell lines and in two primary MM samples. Induction of p53 downstream targets was strictly correlated with induction of p53. In contrast, nutlin did not modulate the expression of these proteins in the cells with mt p53. Proliferation of the cells was also affected by nutlin in MM cells harboring wt p53 but not mt p53. Seventy-two hours after incubation with 10 μM nutlin, the viability of the cells (MM cell lines and primary MM samples) with wt p53 declined to 30% as assessed by MTT assay. Nutlin caused up-regulation of several pro-apoptotic targets, PUMA, Bax, and Bak, and down-regulation of two anti-apoptotic targets, Bcl2 and survivin in MM1.S cells. Furthermore, nutlin effectively arrested cell cycle progression in wt p53 MM cells, depleting the cells in S-phase compartment and increasing the cells in G1 and G2/M phase compartment, indicating G1 and G2 arrest. Annexin-V staining and flow cytometry (FCM) studies showed that there was a dose- and time-dependent increase in annexin-V binding in MM1.S cells but not in LP1 or U266 cells. These results suggest that nutlin-induced apoptosis is p53-dependent. At 72 hours following treatment with 10 μM nutlin, annexin-V binding was increased from 22% (1.0 μM) to 80% in MM1.S cells. Western blot (WB) analysis of nutlin-induced cells revealed activation of both caspase-8 and caspase-9 followed by activation of caspase-3 suggesting the association of both extrinsic and intrinsic pathways of apoptosis. In addition, direct inhibition of endogenous survivin by siRNA further enhanced nutlin-induced apoptosis, as measured by WB analysis for activation of caspase-3 and FCM assay for annexin-V binding. Forty-eight hours after nutlin treatment, 60% of MM1.S cells transfected with survivin siRNA were annexin-V positive, whereas control siRNA-transfected cells were 30% annexin-V positive. Moreover, selective blocking of p53 transcription by a p53 inhibitor, pifithrin-α (25 μM), inhibits nutlin-induced up-regulation of p53-transcriptional target genes p21, PUMA, and Bax in MM1.S cells, suggesting a transcription-dependent apoptosis. Studies by confocal microscopy and WB analysis of the fractionated samples revealed accumulation of p53 in both nuclear and cytoplasmic fractions in nutlin-treated MM1.S cells. Since transcriptional activation of p53-target genes occurs in the nucleus, while cytoplasmic p53 mediates transcription-independent apoptosis, the accumulation of nuclear and cytoplasmic p53 suggests that activated p53 used both transcription-dependent and transcription-independent pathways to induce apoptosis in MM cells. Finally, the consequence of the p53 activation in MM was validated by gene expression profiling of MM1.S cells with or without nutlin stimulation, demonstrating up-regulation of p53 and its downstream targets p21, MDM2, and BAX. Conclusion Nongenotoxic activation of the p53 pathway by nutlin sensitized MM cells harboring wt p53 to transcription-dependent and transcription-independent apoptosis. Our studies provide the preclinical framework for the evaluation of nutlin as a novel therapeutic approach in the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3390 Poster Board III-278 Introduction: While the outcome of children with acute lymphoblastic leukemia (ALL) has steadily improved, the prognosis for those who relapse (rALL) remains poor. Partly this has been due to the lack of a standardised approach to children with rALL. The international collaborative trial ALLR3 stratified patients into standard (SR), intermediate (IR) and high risk (HR) groups based on the time from first diagnosis to relapse, site of relapse and immunophenotype. All patients received 3 blocks of therapy, with a minimal residual disease (MRD) assessment at the end of blocks 1 and 3. Allogeneic stem cell transplantation (allo-SCT) was offered to all HR and those IR who had a MRD of ≥10-4 at week 5. In children in whom MRD was unavailable, allo-SCT was offered to those who relapsed within 24 months of stopping therapy. All other patients continued on chemotherapy for 24 months. A day 1 and 2 randomisation was performed between mitoxantrone and idarubicin. Results: 239 patients were enlisted, of whom 216 were randomised and 109 idarubicin and 103 mitoxantrone patients are analysable. There were non-significant differences between the groups with regards to time to relapse, site of relapse and cytogenetic subtypes.108 (51%) were in CR2 at a median follow up of 36 months (range 1-70) and Progression Free Survival (PFS) at 3 years was 50.3% (95% CI 42.9%, 57.3%). Forty four percent of idarubicin and 40% of mitoxantrone patients had disease levels of ≥10-4 at week 5 (p=0.90) at the end of block 1. The PFS at 3 years for those who received idarubicin was 35.9% (25.9%, 45.9%) and mitoxantrone 64.6% (54.2%, 73.2%) (p=0.0004). Adjusted for differences in risk group, country, age, gender and cytogenetic subtype, this difference continues to be significant (p=0.003). Overall mitoxantrone was better tolerated and less toxic than idarubicin. A competing risks model showed that the difference between the two drugs is primarily related to disease control (p=0.02), rather than toxicity of treatment (p=0.09). In the patients who were transplanted, 35% and 5% of patients who received idarubicin (n = 48) or mitoxantrone (n = 44) respectively have relapsed. Based on the intention to treat analyses, there were non significant differences in patients treated without an allo-SCT in both arms. IR patients who were transplanted did worse (p = 0.01) in the idarubicin group but had comparable results in the mitoxantrone group to those who received chemotherapy. As a result the randomisation in ALLR3 has now been closed, though the study continues to accrue to better answer the MRD stratification question. Conclusions: Mitoxantrone is a well tolerated and highly effective drug for the treatment of children with rALL. While MRD at week 5 failed to predict the differences in outcome for the two randomised groups, it identified that children in the IR group with

Description: The BIOV-111 study was a European multicentre phase II non-randomised, open-label study of the novel purine nucleoside analogue, clofarabine (Evoltra ®). The study recruited patients ≤ 21 years old at initial diagnosis with primary refractory or relapsed/refractory acute lymphoblastic leukemia (ALL). The dose of clofarabine was 52mg/m2 daily x 5 days, every 28–42 days. Patients were evaluable after one complete course. The primary endpoint was overall response rate (OR) defined as either a complete response (CR) or CR without platelet recovery (CRp). Pharmacokinetic parameters and molecular responses were assessed in a sub-group of cases. The study enrolled a total of 74 patients. 65 were evaluable for response. We report the final efficacy and safety data on these evaluable patients. In total 120 courses of clofarabine were administered to 65 patients from 25 centres. The median age was 10 yrs (range 0.5–23 yrs). The median number of prior treatments was 2 (range 1–5) and 22 patients (34%) had been previously transplanted. The OR was 26% (6 CR, 11 CRp). In addition 1 PR was observed. 11/18 (61%) responders had a prior transplant and 3 of these patients received a further transplant post clofarabine. Of the 7 patients proceeding to transplant post-clofarabine; 3 patients had achieved a CR and 4 achieved a CRp. The updated median duration of response and survival will be presented. The pharamockinetic analyses showed plasma clofarabine concentration did not correlate with treatment outcome, however the ratio of day2/day 1 end of infusion intracellular clofarabine triphosphate (cloTP) levels were higher in responding pts. Serious adverse events included febrile neutropenia 50.8%, hepatic events (raised bilirubin, raised ALT/AST) 6.2%, renal failure 6.2%, palmar-plantar erythrodysaesthesia 4.6% and bone pain 4.6%, seizures 7.7% and cardiac failure 1%. In conclusion, this study demonstrated that clofarabine can achieve significant, durable response rates in heavily pre-treated paediatric patients with relapsed/refractory ALL and intracellular cloTP accumulation may be predictive of response. The response rates in this study are consistent with a previous clofarabine study (CLO-212) in relapsed/refractory pediatric ALL, however in BIOV-111 a lower incidence of adverse events was observed, including hepatic and renal adverse events, possibly attributable to fewer prior treatments compared to CLO-212 (median 2 vs 3 respectively). The reported episodes of seizure and cardiac failure were associated with pre-existing co-morbidities and/or sepsis in the majority of cases. The safety profile of clofarabine was manageable and acceptable and does not preclude subsequent HSCT.

Description: Background. In a previous retrospective analysis of 326 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) treated by 10 study groups or large institutions between 1986 and 1996, we documented that bone marrow transplantation (BMT) from a HLA-matched related donor, but not from unrelated donor, was superior to chemotherapy alone in terms of disease-free survival and survival (N Engl J Med2000;342:998–1006). To evaluate the impact of recent improvements in chemotherapy and BMT, we performed a second survey of 762 children and young adults with Ph+ ALL treated by 14 study groups or large institutions between 1995 and 2005 to determine the optimal treatment. We limited the study to the 640 evaluable patients who had not received tyrosine kinase inhibitors so that the results can serve as baseline data for future comparison with those of patients treated with tyrosine kinase inhibitors. Methods. Treatment outcome between the current and the previous cohort was compared with log-rank test. The DFS and survival of patients treated with BMT or chemotherapy were compared in a Cox regression analysis, accounting for the time to transplantation. Early response was evaluated by blast count in the peripheral blood on day 8 or in the bone marrow on day 8 or 15 of remission induction. Follow-up time of the current cohort ranged from 0.1 to 11.5 (median, 6) years. Results. The 640 patients in current cohort ranged in age from 1.0 to 17.7 years (median 7.9). Their presenting features were comparable to those of the previous cohort. Complete remission (CR) was achieved in 89% of patients in the current and 82% in the previous cohort. The overall 7-year EFS and survival of the current cohort were superior to those of the previous cohort: 31.2% (SE 2.0) vs. 25.0% (3.0) [p=0.007], and 44.2% (2.2) vs. 36.0% (3.0) [p=0.017], respectively. In the current cohort, 7-year DFS for the 264 patients undergoing BMT in CR1 (adjusted by time to transplant) was 41.2% (3.2) and compared favourably with the 33.0% (3.4) in the 307 patients treated with chemotherapy alone; unsurprisingly, the advantage of BMT was more apparent later in time among patients who survived the early toxicities of treatment (hazard ratio at 3 years from CR: 0.60; CI 0.28–1.30; p=0.002). Importantly, unlike in the previous analyses, the outcome of the 138 patients who had BMT from an unrelated donor did not differ significantly from that of the 65 patients who had a matched sibling donor: DFS 52.0% (4.5) vs 40.9% (6.2) (p=0.16). However, 7-year survival rates did not differ significantly between patients treated with BMT or chemotherapy alone: 51.7% (3.3) vs 45.9% (3.2) [HR 0.9; CI 0.7–1.15; p=0.42], suggesting that patients treated with chemotherapy were more likely than transplanted ones to be salvaged after relapse. Based on presenting age and WBC count, patients could be divided into three prognostic subgroups: group 1 [WBC

Description: Abstract 1583 Poster Board I-609 Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits’ in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity 〉75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver’ CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL. Relapse type Remission duration (years) 〈 2 2 - 5 〉 5 1 • • 2 • ••••••• •• 3 •• •• ••• 4 •• Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. Disclosures No relevant conflicts of interest to declare.

Description: Fenretinide (4HPR) is a relatively safe neoclassical retinoid analog that inhibits growth of various tumors through increased intracellular ceramide and ROS, induction of tumor cell apoptosis and inhibition of angiogenesis. 4HPR has been successfully tested as a chemopreventive and chemotherapeutic agent in clinical trials on various malignancies. In contrast to retinoic acid, 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs in different tumor cell types. The biological effect and therapeutic value in multiple myeloma (MM) has not been investigated. The aim of this study was to investigate the anti-MM effect and mechanism of action of 4HPR using 3 stroma-dependent and 2 stroma-independent MM cell lines established in our laboratory, CD138-selected primary MM cells and co-culture systems of these cells with human osteoclasts and mesenchymal stem cells (MSCs) as previously described (Yaccoby et al., Cancer Res 2004). MM cell apoptosis detected by annexin V flow cytometry and TUNNEL, tumor growth by MTT assay, changes in caspase 3, 8 and 9 activity using Western blotting and ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. 4HPR inhibits growth of all tested MM cells in a dose- and time-dependent manner. The IC50 after 48 hrs in serum-containing media was 10 μM using MTT assay. 4HPR (3 μM) increased percent of apoptotic MM cells by 2.5±0.4 folds (p

Description: Abstract 3027 Poster Board II-1003 Introduction Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D-ligands, including MIC A/B, which are known to be expressed on epithelial tumors, resulting in tumor cytotoxicity (Ayello/Cairo et al, BBMT, 2006). The expression of MIC A/B on these tumors can be induced by in vitro exposure of these cells to HDACi, specifically Romedepsin (RM) (Skov et al, Cancer Res, 2005). Glycogen synthase kinase-3 (GSK-3), a constitutively active serine-threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, has also been shown to be an important regulatory enzyme in the expression of MIC A/B in response to RM (Doble et al, J cell Sci 2003; Frame et al, Biochem J 2001; Skov et al, Cancer Res, 2005). Objective We sought to determine of the expression of MIC A/B in response to RM in various leukemia and lymphoma cells (LL), its influence on NK cell mediated cytotoxicity and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. Methods LL cells (106/ml, RS 4:11 [MLL-ALL], REH [pre-B cell ALL], Jurkat [T-cell ALL], Toledo [DLBCL], Ramos [Burkitt's Lymphoma]) were exposed to RM (10 ng/mL) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B antibody to determine surface expression of MIC A/B. Peripheral blood NK cells (CD3-/56+) were isolated via magnetic separation followed by IL-2 activation (3000IU/ml, 18 hrs). LL cells exposed to RM (generously supplied by Gloucester Pharmaceuticals) were subjected to NK cell mediated cytotoxicity assays (using an europium assay) at effector:target (E:T) ratio of 10:1, as we had previously described (Ayello/Cairo et al, BBMT 2006). LL cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity (Davies et al, Biochem J, 2000), to determine the role of this regulatory enzyme in the RM mediated expression of MIC A/B in these LL cells. Finally, blocking studies were also performed with anti-NKG2D receptor blockers to determine the specific role of NKG2D signal transduction pathway in NK cell mediated cytotoxicity. Results MIC A/B expression significantly increased in LL cells in response to RM ([RS4:11 0.2% vs 19.2%, p〈 0.0001], [REH 0.2% vs 46%, p= 0.0003], [Jurkat 1.12% vs 44.7%, p〈 0.0001], [Toledo 0.5% vs 15.8%, p=0.0001], [Ramos 0.57% vs 33.6%, p=0.0003]). In addition, the expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl (Jurkat [RM vs RM+LiCl] 85% vs 18%, p

Description: BIOV-111 is a European phase II non-randomised, open-label study of a next generation purine nucleoside analogue, clofarabine (Evoltra®), in pediatric patients with relapsed/refractory ALL. We report on the efficacy and safety data. Eligible patients (pts) had either primary refractory (2 pts) or relapsed/refractory (51 pts with ≥ 2 prior lines of treatment) ALL. Clofarabine 52mg/m2 daily × 5 days was given every 28–42 days (1 course). The primary endpoint is overall response rate (ORR) defined as either a complete response (CR) or CR without platelet recovery (CRp) after 2 courses. Adverse events (AEs) were graded according to NCI CTC (v3). Plasma, urine and intracellular clofarabine pharmacokinetics were also investigated. To date, 96 courses have been administered to 53 pts from 25 centres. The median number of prior treatments was 2 (range 1–5) and 20 pts (38%) had been previously transplanted. 8/29 pts receiving ≥2 courses responded (1CR, 7CRp) giving an ORR of 28%. Responses were observed in 14/53 (26%) pts receiving at least one course of clofarabine (6CR, 7CRp, 1 PR ). Eight (57%) responders had a prior transplant and 1 of these patients was transplanted post clofarabine. One pt with a prior transplant remains in remission at 20+ months. Four pts (1CR, 3CRp) have proceeded to transplantation. Serious adverse events (n=103) included febrile neutropenia (51/103), seizures (4/103), streptococcal septicaemia (3/103), palmo-plantar erythrodysaesthesia (2/103) and bone pain (2/103). Three hepatic events occurred (raised bilirubin, raised ALT/AST), 1 renal failure and 1 cardiac failure. AEs occurred in 4 pts. The renal and cardiac failure AE occurred in a pt with known anthracycline cardiac myopathy and renal impairment at study entry. Median end of infusion plasma clofarabine concentration (n=25) on days 1 and 2 were 1.5 (0.5–3.2) uM vs 2.0 (0.1–5.1) uM respectively (p=0.01) and were not different in pts achieving a response. Urinary clofarabine recovery on days 1, 5 and 6 (drug -free) was 48±18 %, 46±12 % and 5±3 %. Clofarabine TP analyses are ongoing. Response rates in this ongoing BIOV-111 study are consistent with the pivotal clofarabine study (CLO-212) in relapsed/refractory pediatric ALL. Notably, BIOV-111 has a lower incidence of AEs, including hepatic and renal AEs; (4% and 1% respectively vs 10% and 8% in CLO-212), possibly attributable to fewer prior treatments compared to CLO-212 (median 2 vs 3 respectively). Clofarabine achieves a significant response rate in this heavily pre-treated patient population and durable responses have been observed which may confer a survival advantage with longer follow-up.

Description: The efficacy of the key anti-leukaemic agent E.coli L-Asparaginase (L-Asp) is limited by variable therapeutic activity and development of antibodies/hypersensitivity. Observing more frequent clinical hypersensitivity in children with high risk acute lymphoblastic leukaemia (ALL), we speculated that L-Asp was proteolytically degraded by lymphoblasts, generating immunogenic fragments. On incubation with whole cell lysate from a lymphoblastic cell line, L-Asp was cleaved into at least three fragments. This cleavage was blocked only by MV026630, a specific inhibitor of the cysteine protease Asparaginyl Endopeptidase (AEP). Incubation of L-Asp with purified recombinant AEP produced an identical cleavage pattern at the carboxy termini of specific asparagine and aspartate residues. The level of AEP expression at diagnosis in 148 childhood ALL patients was measured using U133A, Exon 1.0 ST arrays and quantitative PCR. High levels of expression were detected in 38 patients (25%), including all 6 patients with clinical hypersensitivity. Elevated AEP expression was more commonly observed in high-risk disease (42% v 21%). Molecular modelling of AEP cleavage sites predicts that the N-terminal cleavage site (C1) provides tetramer stability while the highly-conserved second cleavage site (C2) is critical for tetramer formation and enzyme activity. This model was confirmed by analyses of recombinant L-Asp variants mutated to resist AEP cleavage. Cleavage is sequential from the N terminus. A product resistant to cleavage at C1 is not degraded by AEP, forms a tetramer and retains enzymatic activity comparable to the wild-type. The C2 mutated product shows little activity and is rapidly degraded by AEP. Known antigenic epitopes of L-Asp are retained within AEP-cleaved fragments. The contribution of AEP to antigenic processing of L-Asp is being investigated by T-cell activation assays using a synthetic peptide library and T cells from patients with known hypersensitivity. Thus patients with AEP-overexpressing lymphoblasts may not benefit from L-Asp therapy either due to inactivation, the development of antibodies or both. Replacement of a single amino acid of L-Asp can prevent this process without loss of activity. This product potentially has a longer half-life and less antigenicity. Our investigations have identified a hitherto unknown pathway for L-Asp degradation and a novel mechanism of drug resistance in childhood ALL. These observations require further validation by correlation of AEP expression with asparaginase activity and antibody formation in children receiving L-Asp during treatment for ALL. Further potential improvements in therapy could include screening for AEP expression at diagnosis and the use of non E Coli asparaginase or preferably a modified recombinant L-Asp and/or AEP inhibitors for those with high levels of expression.

Description: Megakaryocytes are often expanded in bone marrow (BM) areas infiltrated with myeloma (MM) cells while thrombocytopenia is uncommon in untreated MM patients. The aim of this study was to investigate the reciprocal interaction between primary megakaryocytes and MM cells. Cultures of megakaryocytes and their precursors were prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (〉90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. We initially demonstrated that purified MM cell-conditioned media from 7 patients increased migration of megakaryocyte precursors across a 8 μM pore size membrane 3.6±0.9 fold (p=0.001), an effect that was inhibited by anti-CXCR4 neutralizing antibody (p