Search

JAK2V617F is a gain of function point mutation that occurs in Myeloproliferative Neoplasm (MPN) patients and deranges their hemopoiesis at cellular level. We speculate that hyperfunctioning JAK2 can modify osteoclast (OCL) homeostasis in MPN patients. We studied 18 newly diagnosed MPN patients and four age-matched normal donors (ND). Osteoclast forming assays started from selected monocytes also and under titrated concentrations of the JAK2 Inhibitor AG-490 (Tyrphostin). Genomic DNA was extracted from the formed osteoclasts, and the JAK2V617F/JAK2WT genomic DNA ratio was calculated. OCLs formed from monocytes derived from heterozygous (Het) for the JAK2V617F mutation MPN patients, were three times more compared to those from JAK2 wild type (WT) MPN patients (p=0,05) and from ND as well (p=0,03). The ratio of JAK2V617F/JAK2WT genomic DNA was increased in OCLs compared to the input monocyte cells showing a survival advantage of the mutated clone. In comparison to ND and JAK2 WT MPN patients, OCLs from patients JAK2V617F (Het) were more susceptible to JAK2 inhibition. These alterations in osteoclast homeostasis, attributed to mutated JAK2, can deregulate the hemopoietic stem cell niche in MPN patients.

PET/CT in primary mediastinal large B-cell lymphoma responding to rituximab-CHOP: An analysis of 106 patients regarding prognostic significance and implications for subsequent radiotherapy

Data derived from clinical trials consistently show a prolongation of overall survival of late-stage MDS patients with the introduction of azacytidine. Nevertheless, the applicability of the above results to real-world clinical settings may be questionable due to the strict design, the controlled medical environment, and the limited patient sample of explanatory studies. We retrospectively compared the outcome of two well-balanced groups of late-stage MDS patients. The first consisted of 46 patients treated with azacytidine (AZA cohort) and the second of 41 patients treated with other agents (non-AZA cohort). Patients in the AZA cohort displayed superior survival compared to the non-AZA ones. However, subgroup analysis revealed that azacytidine conferred a significant survival advantage only in patients with AML–MDS and those who attained a CR at any time after treatment initiation, while all other patients displayed comparable outcome with the non-AZA cohort. Larger series are needed to determine which patients benefit most from azacytidine therapy.

Background/Aims: Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) share the same acquired lesion JAK2V617F and may exhibit substantial overlap. Variability in JAK activation and allele burden, complemented by host, genetic and non-genetic modifiers, determine the phenotype. The aim of this study was to investigate the presence of the JAK2 mutation in association with the ratio of metallopeptidases inhibitors (TIMPs) to tissue metallopeptidases (MMPs) in MPNs, where inhibitory rather than proteolytic activity in marrow microenvironment appears to predominate. Methods: 94 patients with polycythemia vera, essential thrombocythemia and primary myelofibrosis, and 102 healthy individuals were evaluated. Allele-specific PCR and RFLP were used to detect JAK2 and genomic status. Serum concentrations of MMP and TIMP were measured by ELISA. The parameters were assessed with covariance analysis, and adjusted for gender, age and co-morbidity. Results: Mutation frequency was 81.91%. Abnormal TIMP/MMP ratios were identified in all three diseases. JAK2 mutation was correlated with significant changes in TIMP concentrations. Conclusions: Identification of an abnormal TIMP/MMP ratio in all three diseases, regardless of the JAK2 status, indicates invariable marrow remodeling. In this particular group of patients, presence of a JAK2V617F mutation, being associated with even higher ratios, appears to be a concurring participant in bone marrow-reforming processes. Additional research may delineate correlates with the JAK2 allelic burden.

CD4(+)CD25(+)FOXP3(+) T regulatory cells (T(regs)) prevent autoimmunity by restricting overexuberant immune responses, but the same subpopulation can incur detrimental effects on antitumor responses. In both cases, the suppressor potential of T(regs) appears to be strongly influenced by their compartmentalization. In myelodysplastic syndromes (MDS), immune deregulation and autoimmunity in the early stages might lead to ineffective hematopoiesis and bone marrow (BM) failure, whereas late-stage disease is characterized by the immune escape of the malignant clone. We show that these two stages of MDS are associated with differential T(reg) activity. Specifically, we found that in early stage MDS, compared with normal hematopoiesis and late stage MDS, T(regs) are dysfunctional and their BM homing through the CXCL12/CXCR4 axis is seriously impaired as a result of CXCR4 downregulation. Conversely, in late stage MDS, T(regs) are systemically and locally expanded and retain their function and migratory capacity. Moreover, T(reg) levels follow the disease course and are significantly reduced in treatment responding patients. Our findings indicate T(reg) involvement in the pathophysiology of MDS; defective suppressor function and BM trafficking of T(regs) may be important in the autoimmune process of early MDS, but increased T(reg) activity could favor leukemic clone progression in late stage disease.

Background: Farnesyltransferase inhibitors (FTIs) target proteins needing prenylation for functioning. Tipifarnib (Zarnestra®), a potent and specific inhibitor of farnesyltransferase, showed considerable activity in phase I and II studies in myelodysplastic syndrome (MDS), but the optimal regimen achieving high response rates with minor myelosuppression remains to be determined. Additionally, a direct effect on purified human MDS progenitors has not yet been shown. Methods: MDS and normal CD34+ cells isolated by using immunomagnetic beads were plated for short-term cultures in semisolid media or liquid cultures for flow-cytometric assessment of apoptosis in the presence of either DMSO or various FTI concentrations. Results: Tipifarnib exerted selective in vitro toxicity against clonal MDS hematopoiesis at concentrations less than 10 nM the effect being more prominent in white cell progenitors. This action was not due to apoptosis induction as both normal and MDS progenitors displayed equivalent DiOC3 and annexin V expression up to 72 h after exposure to tipifarnib. Conclusion: The leukemic clone is more susceptible in tipifarnib than normal progenitors. Since myelosuppression represents the main obstacle in the clinical use of tipifarnib in MDS, further reduction of the currently employed dose will potentially result in a more tolerable regimen without compromising its antileukemic action.

Invariant natural killer T cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. On activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete T helper 1 and 2 (Th1/2) cytokines but also cytokines with hematopoietic potential such as GM-CSF. Here, we show that the myeloid clonogenic potential of human hematopoietic progenitors is increased in the presence of glycolipid-activated, GM-CSF–secreting NKT cells; conversely, short- and long-term progenitor activity is decreased in the absence of NKT cells, implying regulation of hematopoiesis in both the presence and the absence of immune activation. In accordance with these findings, iNKT-cell–deficient mice display impaired hematopoiesis characterized by peripheral-blood cytopenias, reduced marrow cellularity, lower frequency of hematopoietic stem cells (HSCs), and reduced early and late hematopoietic progenitors. We also show that CD1d is expressed on human HSCs. CD1d-expressing HSCs display short- and long-term clonogenic potential and can present the glycolipid α-galactosylceramide to iNKT cells. Thus, iNKT cells emerge as the first subset of regulatory T cells that are required for effective hematopoiesis in both steady-state conditions and under conditions of immune activation.

The purpose of this retrospective study, the largest unselected series in our country, was to illustrate the clinicopathological features of non-Hodgkin’s lymphoma (NHL) classified according to the World Health Organization (WHO) classification of lymphoid neoplasms. A retrospective analysis was conducted and clinical features of histological subtypes were established in 810 patients (age ≧15 years) with NHL who were treated at 8 major centers representative of Greece. There were 435 males and 375 females 95% of them aged >30 years. B symptoms were present in 34% of the patients, while 45.3% had stages I–II and 54.6% had stages III–IV. LDH was increased in 37% of the patients. B cell lymphomas formed 88% of the cases whereas T cell lymphomas formed 12% of the total. Indolent lymphomas accounted for 31.1%, aggressive ones for 66.7% and very aggressive ones for 2.4% of all NHLs. Among indolent lymphomas extranodal ones (MALT B cell lymphoma) were the most common subset while follicular lymphoma grade I and II and small lymphocytic ones presented with equal frequency. Among the aggressive lymphomas diffuse large cell lymphoma (DLCL) was the most common subtype; this entity along with large-cell immunoblastic lymphomas accounted for 45.2% of all B cell lymphomas. Among the T cell lymphomas, peripheral T cell lymphomas and anaplastic large cell lymphomas of the T/null-cell type were the most common subtypes. The most common extranodal presentation was the gastrointestinal tract (GI). Next in frequency were primary extranodal NHL of the head and neck region. MALT B cell lymphomas were found in almost half of the patients with GI tract NHL, whereas in all other extranodal places DLCL was the predominant histological subtype. The median survival for indolent and aggressive NHL was 123.5 and 55.5 months, respectively. This is the first report of a large series of malignant lymphomas in Greece using the WHO classification. It appears that there are no significant differences between NHL in Greece and other large series as far as clinical and extranodal presentation is concerned. The frequency of follicular lymphoma in the current study is comparable to that reported from Asian countries and mainland Europe, but lower than that of US and Northern European series. There were no important differences in the incidence of the remaining histological subtypes between Greece and other European countries.

Lymph node infiltration by monoclonal plasma cells can occur either in aggressive forms of myeloma or may represent regional extension of extramedullary plasmacytomas, whereas lymph node plasmacytoma presenting as a solitary extramedullary plasmacytoma is very unusual. We report two cases of lymph node plasmacytomas without systemic disease diagnosed after surgical excision. Clinical remission was achieved after local radiotherapy although one patient relapsed with multifocal extramedullary plasmacytomas 20 months after radiotherapy.

To evaluate the activity of thalidomide in Waldenstrom's macroglobulinemia (WM), 20 patients were treated on a dose schedule that escalated from 200 mg/d to 600 mg/d. On an intention-to-treat basis, five (25%) patients achieved a partial response, which was noted within 3 months of treatment. Adverse effects were common and prevented dose escalation of thalidomide in 75% of patients and led to premature discontinuation of treatment in 35%. We subsequently evaluated the oral combination of clarithromycin (500 mg twice per day), low-dose thalidomide (200 mg once daily), and dexamethasone (40 mg once per week). Our preliminary analysis on 12 previously treated patients indicate activity of this regimen in WM: three patients achieved a partial response and two patients demonstrated monoclonal protein reduction of greater than 25%. This combination was associated with a variety of side effects due not only to thalidomide, but also to corticosteroids and to clarithromycin. Our preliminary data indicate that this combination may be a useful salvage regimen for some patients with heavily pretreated macroglobulinemia.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder characterized by pancytopenia, hemolysis, and thrombosis. Abdominal vein thrombosis is a life-threatening manifestation of this disease. We present a patient with complete spleen necrosis due to thrombosis of the splenic vessels. After splenectomy, other causes of thrombophilia were excluded and the diagnosis of PNH was established. The patient was put on anticoagulation but despite the prophylactic international normalized ratio maintained over the last 18 months of follow-up, he had another episode of intrahepatic thrombosis which was treated with tissue plasminogen activator thrombolysis.

Introduction: During the last few years epirubicin (E) and mitoxantrone (M) (Novantrone) have been used in the treatment of non-Hodgkin's lymphoma (NHL), because of their favorable principal profile. In particular, M has less severe non-hematological toxicity. Patients and methods: A randomized multicenter phase III study was conducted in order to compare the efficacy and toxicity of CEOP and CNOP in intermediate- and high-grade NHL, CEOP (arm A) consisted of cyclophosphamide 1000 mg m 2 , vincristine 2 mg, E 70 mg m 2 on day 1 and prednisone 60 mg on days 1 7. The CNOP regimen (arm B) was identical to CEOP except for replacement of E by M at a dose of 12 mg m -2 . Randomization was stratified according to stages I IV. From September 1993 to March 1999, 249 patients registered for the trial. Patient characteristics were equally distributed in the two arms, except for age and International Prognostic Index (IPI) groups. Results: There were no significant differences between the two groups in the rates of complete (CR) and partial response (PR). The overall response rate was 78% in arm A (57% CR, 21% PR) and 82% in arm B (60% CR, 22% PR). With a median follow-up time of 47.3 months, the median survival was not reached in arm A, while it was 39.5 months in arm B (P = 0.09). Three-year survival rates were 62.5% for CEOP and 51.5% for CNOP. There was no significant difference regarding the time to progression between the two groups (29.7 vs. 18.5 months); furthermore the median duration of CRs was 71.6 and 49 months for CEOP and CNOP, respectively (P = 0.07). The therapeutic efficacies of both regimens were equivalent among the four IPI groups. More alopecia was observed in arm A. WHO grade >2 neutropenia was more frequent in arm B. Supportive treatment with G-CSF was given to 22 and 24 patients, respectively. Conclusion: There were no significant differences in terms of overall response rates, overall survival and time to progression between CEOP and CNOP in the treatment of intermediate- and high-grade NHL. Patients with low or low intermediate IPI risk treated with either CEOP or CNOP showed significantly better survival, response rates and time to progression than those with high intermediate or high IPI risk. Therefore, new improved therapeutic approaches should be developed for the treatment of high IPI risk patients.

Non-African Burkitt's lymphoma is a rare disease among adults without AIDS. Among 1352 Greek adult patients with non-Hodgkin's lymphoma, 24 cases (1.8%) were classified as Burkitt's (BL) or Burkitt-like (BLL) lymphoma. Eleven cases fulfilled the criteria of BL and 13 of BLL. No statistical differences were found in the general characteristics of the two groups at the time of diagnosis. Extranodal involvement was a common finding in both groups and bulky disease (>10 cm) was observed in almost one half of the patients. The majority of the patients were treated with intensive, although different, protocols. After induction treatment, complete remission (CR) was achieved in 14 patients (60.8%). CR was reached in all cases with stage I–II, while in stage IV the CR rate was 30.4%. The median overall survival was 27 months. The median survival for BL was 13 months compared to 27 months in the BLL group ( P =0.34). The data of the present retrospective analysis, indicated that there were not significant clinical differences between BL and BLL variants. Since BLL is still a non-reproducible category in the REAL classification, all BL variants must be treated uniformly with intensive protocols.

We analyzed the expression of CD1d, an antigen-presenting molecule, on peripheral blood leukemic cells of cases of chronic lymphocytic leukemia (CLL) by flow cytometry. We demonstrated variable expression of CD1d on leukemic lymphocytes and an association between high expression of CD1d with shorter time to treatment and overall survival of patients. CD1d was positively associated with CD38 expression, but not with unmutated heavy chain variable (VH) mutational status or adverse cytogenetics of leukemic lymphocytes. Our findings support that CD1d expression is a prognostic marker for CLL.

Myelodysplastic syndrome (MDS) patients with renal impairment (RI) were not assessed in the approval trials of 5-azacytidine, thus the optimal use of 5-azacytidine in such patients is currently undefined. We retrospectively analyzed 42 IPSS intermediate-2 and high-risk patients with moderate, mild or no RI undergoing 5-azacytidine therapy in a non-trial setting. We demonstrate that patients in all three groups achieved comparable responses and had similar overall and event-free survival. Likewise, both treatment toxicity and dose adjustments were not significantly influenced by renal function status. A transient but reversible decline in glomerular filtration rate was observed in patients either with or without RI, without affecting the therapeutic schedule. Our results provide the first evidence that 5-azacytidine is effective and well-tolerated in patients with mild and moderate RI and, if confirmed by prospective randomized studies, advocate that such patients can be managed in an analogous fashion to patients with normal renal function.

Telomere exhaustion and increased telomerase activity are associated with the acquisition of aggressive molecular events in a variety of haematological malignancies. In Philadelphia chromosome negative myeloproliferative neoplasms (Ph(neg)MPN's), telomere dynamics during clonal evolution of these diseases have not yet been fully elucidated. Herein we demonstrated that telomere shortening is a global phenomenon in Ph(neg)MPN's, irrespective of disease phenotype, treatment administration and JAK2V617F mutational status but the presence of additional cytogenetic abnormalities further affects them. Consistent with the above finding, TA was upregulated in CD34+ haemopoietic progenitors from almost all Ph(neg)MPN subgroups compared to healthy donors. Moreover, TL below the cut-off value of 27% could predict disease progression in Ph(neg)MPN patients (PFS at 5 years 39% vs 81%). Thus, TL emerges as a new prognostic marker in Ph(neg)MPN, reflecting probably the genetic instability of highly proliferating MPN clones.

Ph-negative chronic myeloproliferative disorders (Ph(neg)cMPD) are treated according to the estimated vascular risk. The recent discovery of V617F point mutation of the JAK2 kinase, which frequently occurs in these diseases, has not changed their management so far. However, emerging data tend to support a prothrombotic role for the mutation, along with a better response of JAK2V617F mutated patients to hydroxyurea treatment. Our data further support this notion.

It is known that the presence of hemoglobin S (HbS) affects the determination of hemoglobin A(2) (HbA(2)) levels in clinical samples. We quantitated this effect using the Menarini HA-8160 analyzer and compared with other instruments (HELENA beta-thal quik column, TOSOH HLC-723G7 and BIORAD Variant II) using the HELENA SAS-MX alkaline gel electrophoresis kit as the reference method. The %HbA(2) values from the HA-8160 analyzer and the alkaline gel electrophoresis show a good linear correlation in the absence of HbS. A strong positive bias in the %HbA(2) values from the HA-8160 is apparent in the presence of HbS in the samples, when compared with the alkaline electrophoresis. The analytical imprecision and bias of the three HPLC instruments are comparable both in the presence and absence of HbS. The manual column method shows a lower bias in the absence of HbS but is more affected when HbS is present in the samples.

Approximately half of essential thrombocythemia (ET) patients and almost all with polycythemia vera (PV) bear the activating JAK2617V>F point mutation, which arises at the multipotent haemopoietic progenitor cell level. Although ET is mainly characterized by megacaryocyte proliferation, the cases that are positive for the JAK2617V>F mutation also show increased bone marrow cellularity and higher erythrocyte and granulocyte counts. After establishing short- and long-term bone marrow cultures we found that the frequency of committed haemopoietic progenitors in the bone marrow, was not increased in JAK2617V>F positive ET compared to the negative ones, whereas in long-term cultures (LTBMC) JAK2617V>F positive ET display a growth pattern more similar to that observed in LTBMC produced by PV marrow cells. Our data support the notion that JAK2617V>F positive ET and PV represents a continuum spectrum of alterations within the same disease.

Invariant NKT cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. Upon activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete Th1/2 cytokines but also cytokines with hematopoietic potential such as IL-3 and GM-CSF. In mice, NKT cells activated by alpha-galactosylceramide (alphaGC), a potent glycolipid ligand, cause an in increase in extramedullary hematopoietic committed progenitor activity through secretion of these cytokines. We tested the role of iNKT cells in regulating hematopoiesis under conditions of activation and in steady state hematopoiesis. We found that GM-CSF-secreting alphaGC -activated iNKT cells enhanced (by 64%, n=5, p In conclusion, iNKT cells when activated enhance the clonogenic capacity of myeloid progenitors and can thus modulate innate immune responses. Additionally, they are required for maintenance of normal hematopoiesis in the absence of immune activation. Our findings offer further evidence for a concerted regulation of the immune and hematopoietic systems and the potential for new therapeutic approaches for the manipulation of hematopoiesis.

Amifostine is a phosphorylated aminothiol that not only protects hematopoietic progenitor cells from chemotherapy and radiotherapy, but also stimulates normal hematopoiesis. The effect of amifostine on the in vitro growth of hematopoietic progenitors derived from B-cell chronic lymphocytic leukemia(B-CLL) was investigated. The colony-forming units (CFU)-granulocyte macrophage (CFU-GM), the burst-forming units-erythroid (BFU-E) and the CFU-granulocyte erythroid macrophage megakaryocytes (CFU-GEMM) increased 38, 20 and 100%, respectively, after the incubation with amifostine. There was no statistical difference in the in vitro progenitor growth of patients grouped according to their disease stage, bone marrow lymphocytic infiltration or therapy. Our data indicate that apart from cytoprotection the parallel use of amifostine and chemotherapy in patients with B-CLL could enhance bone marrow recovery.

T regulatory cells (Tregs) safeguard against autoimmunity, but may compromise antitumor immunity. Aberrant immune signaling via signal transducer and activator of transcription (STAT) proteins is involved in disease pathobiology in numerous malignancies. Azacytidine treatment affects Treg levels, particularly in responders, but its effect on STAT signaling is unknown. We explored the alterations of STAT signaling profile (SP) of Tregs during azacytidine treatment and their association with clinical and biological parameters. Peripheral blood mononuclear cells of 19 late-stage MDS patients were obtained before and 15 days after azacytidine initiation. According to WHO, one had RAEB-I (5%), 9(47%) patients had RAEB-II, 7(37%) CMML-II and 2(10%) AML/MDS. Based on the IWG criteria patients were divided into responders (CR, PR and hematologic improvement, n=8) and non-responders (stable disease and failure, n=11). We applied a modified protocol of phospho-specific flow cytometry to measure either basal (untreated) or potentiated (after stimulation with IL-6 and IL-2 for 15’) phospho-STAT1, STAT3 and STAT5 levels with simultaneous staining for FOXP3. The following potentiated, i.e. target/stimuli, nodes were studied: pSTAT1/IL-6, pSTAT3/IL-6 and pSTAT5/IL-2. Comparisons were performed by paired, unpaired t-test or one-way ANOVA as appropriate. Clustering of SPs was performed with hierarchical cluster analysis and was correlated with response, disease subtype, and cytogenetics by using ÷2 or Fisher Exact tests. The pretreatment levels of Tregs were significantly higher in non-responders compared to responders (8.4%±0.42% vs 5.5%±0.46%, p=0.004). By contrast, azacytidine treatment significantly upregulated Tregs at d15 only in responders (7.1±0.47%, p=0.03), whereas non-responders exhibited a mild, but non-significant increase (9.7%±1.22%, p=0.18). Unsupervised clustering of the Treg SPs revealed 4 clusters. No differences were noticed in the SP of Tregs in relation to treatment response, cytogenetics, IPSS, WPSS and IPSS-R. By contrast, the disease subtypes were nonrandomly distributed among the clusters. Most CMML-2 patients segregated in cluster 4, which displayed high basal levels of phospho-STAT1 and 5, but weak potentiated responses, whereas the cluster 2 was enriched in RAEB-II patients and was characterized by very high basal levels of pSTAT5 and relatively intense response of pSTAT3 in IL-6 (Fig 1). Intriguingly, the pretreatment levels of Helios+ Tregs were significantly higher in patients of cluster 2 (37.9%±7.7% of total Tregs) compared to patients in clusters 1, 3 and 4 (23.1±2.3%, 22.8%±2.4% and 22.2%±2.1%, respectively, p=0.035) indicating a higher level of Treg activity and, potentially, immune suppression in these patients. In addition, the clustering of the ratios of Treg SPs after/before azacytidine treatment revealed a distinct epigenetic modulation of Treg signaling via STATs in CMML-2 compared to all other patients (p=0.036), characterized by downregulation of pSTAT1 and pSTAT5 basal levels and upregulation of potentiated responses at day 15 after first azacytidine administration (Fig 2).Figure 1Pretreatment SPs of Tregs in 19 patients.Figure 1. Pretreatment SPs of Tregs in 19 patients.Figure 2Ratio of the SPs of Tregs 15 days after/before azacytidine initiation in 19 patients.Figure 2. Ratio of the SPs of Tregs 15 days after/before azacytidine initiation in 19 patients. Collectively, consistent with recent data (Constantini B et al, 2012), we observed both arithmetical and functional alterations of Tregs in MDS patients after azacytidine treatment. Moreover, both the pretreatment SP and the differential modulation of STAT signaling in the Tregs of CMML-2 and RAEB-II patients, argue for a potentially diverse epigenetic regulation of tumor immunity among the various MDS subtypes. Disclosures: Tsatalas: Genesis Hellas: Honoraria. Spanoudakis:Genesis Hellas: Honoraria. Kotsianidis:Genesis Hellas: Honoraria, Research Funding.

Expression of CD25 on blasts of AML patients has been shown to hold independent prognostic value for both survival and response to induction therapy. Patients with MDS-related AML have generally higher CD25 expression from de novo AML patients, but though there is paucity of a serviceable biomarker of outcome in MDS patients treated with azacytidine, the prognostic value of CD25 has not yet been investigated. Bone marrow samples of 61 patients with intermediate-2/high risk IPSS, high/very high WPSS and low blast count AML were obtained before and 15 days (D15) after the initiation of treatment. All patients received azacytidine in a non clinical trial setting at an initial dose of 75mg/m2 SC for 7 days on 28-day cycles. CD25 expression was assessed by 4-color flow cytometry on total CD34+ blasts, committed progenitors (Lin-CD38+CD34+) and leukemic stem cells (LSC, Lin-CD38-CD34+). Positivity was defined as a CD25 expression of ≥ 20%. Statistical comparisons were done by ÷2, one-way ANOVA and paired or unpaired t-test as appropriate, and survival with Kaplan-Meier analysis and log-rank test. Overall survival (OS) was defined as the time from azacytidine initiation to death from any cause and event-free survival (EFS) as the time from diagnosis to disease progression, relapse or death. Multivariate survival analysis was based on Cox’s proportional hazards model using a backward stepwise selection procedure with entry and removal criteria of p=0.05 and p=0.10, respectively. As shown in table 1 the cohorts of CD25- and CD25+ patients were well balanced for most known predictive factors and characteristics, except sex. Compared to CD25+ patients the CD25- ones have significantly longer OS (16.2 vs 8.8 months, respectively, p=0.04) and EFS (12.8 vs 6.66 months, p=0.04) in univariate analysis. Multivariate analysis confirmed the independent predictive power of CD25 for OS and EFS (p=0.006 and p=0.009, respectively), whereas heavy transfusion requirements (p=0.003 and p=0.002) and age>75 (p=0.02 and p=0.02) were also independent predictors. The average expression of CD25 in CD34+ blasts of all patients was 21.6%±24%. Compared to committed progenitors, LSCs displayed higher expression (19.4%±23.7% vs 24.1%±28.2%, respectively, p=0.027). Interestingly, on D15 CD25 was downregulated in LSCs (p=0.03) but remained stable in committed progenitors (p=0.8, n=18), indicating a particular sensitivity of the CD25+ subset of LSCs in azacytidine.Table 1Patient characteristics. N/A: not applicable/not available.CD25- (n=36)CD25+ (n=25)p-valueAge72,5 (53,4-83.5)72,9 (52-81.7)0.2 >6531(86%)17(68%) 15%18(50%)9(36%) ≤15%18(50%)16(54%)Transfusions ≥ 4 per month0.48 Yes23(4%)17(4%) No13(4%)8(4%)Response0.4 CR & PR12(33%)4(16%) Hematologic improvement5(14%)4(16%) Stable disease8(22%)5(20%) Failure11(31%)12(48%)Figure 1(A) OS and EFS according to CD25 positivity status. (B) OS and EFS according to transfusion requirements.Figure 1. (A) OS and EFS according to CD25 positivity status. (B) OS and EFS according to transfusion requirements. Collectively, our findings reveal an independent prognostic role for CD25 in MDS patients treated with azacytidine. In addition, the differential expression and epigenetic modulation of CD25 in the LSC compartment support the investigation of therapeutic strategies using monoclonal antibody targeting combined with epigenetic agents. Disclosures: Kotsianidis: Genesis Hellas: Honoraria, Research Funding. Spanoudakis:Genesis Hellas: Honoraria. Tsatalas:Genesis Hellas: Honoraria.

WM is a disease of the elderly with a protracted course in many patients. There are limited data which indicate that several WM patients die due to causes which are not directly related to their underlying malignancy. However, the realization and the estimation of the contribution of unrelated mortality in WM are important for the design of treatment strategy in patients of advanced age. To our knowledge there are no such data published for WM patients. Thus, we analyzed the outcomes of 408 patients with symptomatic WM who received therapy within the centers of the Greek Myeloma Study Group in order to assess disease related survival. In this analysis unrelated death was considered to be a competing risk event. Causes of death other than WM, treatment toxicity or myelodysplasia/transformation were considered as unrelated deaths. Median age of patients was 68 (28-92) years; 21% were >75 years and 9% were ≤50 years of age. Patients who started therapy after 2000 were older (median age 70 vs. 65 years before 2000, p75 years (vs. 13% before 2000). In terms of ISSWM stage, more patients had high and intermediate risk disease after 2000 (41% & 42% vs. 25.5% & 38% before 2000, p Median follow up for all patients was 5.5 years (9 years in the pre-2000 and 4.5 years in the post-2000 group) and 52% of patients have died (77% in the group before 2000 and 40% in the group after 2000). However, 23% of deaths were considered unrelated to WM. Thus, 5-year and 8-year overall survival (OS) was 70% and 54% respectively, with a median OS of all patients of 8.8 years. When we performed survival analysis with unrelated deaths as competing risk, then 5-year risk of WM-related death was 21.4% (95% CI 17-26%) and of unrelated death was 7.6% (95% CI 5-10.5%), while 8-year WM-related death rate was 32% (95% CI 27-37%) and unrelated death 11.5% (95% CI 8-15%). Because older patients are at higher risk of unrelated deaths we performed an age-specific analysis. The median survival of patients >75 years was 5.3 vs. 9.7 years for patients ≤75 years (p75 years, the 5-year death rate due to WM was 22% (95% CI 13-32%) vs. 21% (95% CI 16-26%) for patients ≤75 years (p=0.193), while the 5-year unrelated death rate was 17% (95% CI 10-27%) and 5.1% (3-8%), respectively (p75 years) >40% of deaths are unrelated to WM, while WM-specific death rates were similar for patients >75 or ≤75 years. In patients ≤50 years there were no WM-unrelated deaths. We then evaluated the prognostic significance of IPSSWM, which discriminated 3 groups with 5-year overall survival of 86%, 68% and 51% for low, intermediate and high risk groups, respectively (p In conclusion, this is the first analysis in a large cohort of patients with symptomatic WM in which WM-unrelated death is treated as a competing risk. Many patients of advanced age die of causes unrelated to WM and this fact should be taken into account in the evaluation of long term outcomes and the design of clinical trials in patients with WM, especially since more patients of advanced age are diagnosed and treated for WM. Disclosures: No relevant conflicts of interest to declare.

Renal impairment (RI) is a common complication of myeloma and is associated with increased risk of early death. Accurate assessment of glomerular filtration rate (GFR) in patients with myeloma is important for evaluation of renal function, for drug dosing and for prognostic purposes. The method of assessment of RI and calculation of GFR is important since serum creatinine alone underestimates renal dysfunction, especially in patients of advanced age. The MDRD formula has been widely used for the calculation of GFR but the CKD-EPI formula has been increasingly used among nephrologists since 2009 (Levey et al, Ann Intern Med 2009;150:604-12). However, the performance of these two equations for the estimation of GFR and their relative prognostic ability has not been assessed in large numbers of patients with myeloma. Thus, in order to evaluate the prognostic significance of GFR as calculated by two different formulas, the MDRD and the CKD-EPI, we analyzed the data of 1937 newly diagnosed patients with symptomatic myeloma who were treated within the centers of the Greek Myeloma Study Group. The median age of the patients was 66 years (range 20-96 years); 54% were >65 years of age and 23% >75 years of age; 55% were males and 53% had an ECOG performance status ≤1. Anemia (Hb We examined the prognostic significance in univariate analysis per CKD stage for each calculation formula: median survival for patients with CKD stage 1, 2, 3, 4 and 5, according to MDRD formula was 61, 54, 36, 30 and 23 months, respectively (p In conclusion, for the first time we evaluated two different formulas of estimation of GFR, both based on serum creatinine, age and gender, in a large number of patients with symptomatic myeloma and we found that GFR calculated by the CKD-EPI formula reclassifies the stage of renal impairment of about 9% of patients and has better prognostic ability for survival and predictive ability for early death than the MDRD formula. We propose the broad use of CKD-EPI formula for the estimation of GFR in patients with myeloma. Disclosures: Tsatalas: Genesis Hellas: Honoraria.

We assayed granulocyte-macrophage committed progenitor cells (CFU-GM) in the peripheral blood of 34 patients with chronic lymphocytic leukemia (CLL) and 12 normal individuals. The patients were divided into separate groups on the basis of previous therapy (i.e. analysis performed at diagnosis, during and after chemotherapy) and clinical features of the disease (i.e. disease stage, pattern of bone marrow infiltration, peripheral blood lymphocytosis). The mean CFU-GM colony count of the patients was 30 times higher than that of the controls (206.4 ± 197.8 (SD) CFU-GM per 5 times 105 cells plated versus 6.5 ± 3.6). There was no statistical difference in the numbers of circulating CFU-GM between the patients studied at diagnosis (257 ± 215.4 CFU-GM/5 times 105 cells) and those studied during (117.6 ± 169.2 CFU-GM/5 times 105 cells) or after chemotherapy (207.5 ± 105.9 CFU-GM/5 times 105 cells), although a trend towards a higher recovery of myeloid progenitors was observed as a function of time elapsing from the last treatment. In addition, we found no significant difference in the in vitro CFU-GM growth of patients grouped according to their disease stage, pattern of bone marrow infiltration and degree of peripheral blood lymphocytosis. In conclusion, our data indicate that intensification with peripheral blood stem cell support may be feasible in CLL, since progenitor cells of myeloid-monocytic series are markedly increased in the peripheral blood of these patients. Moreover, it is possible to extend this kind of therapy to patients who have undergone previous extensive chemotherapy and who might have persisting bone marrow infiltration.

Abstract 2845 Poster Board II-821 Recently, the IPSS has been proposed as a staging system for patients with WM who require treatment. This system is based on five adverse covariates: age >65 years, hemoglobin '11.5 g/dL, platelet count '100×109/L, beta2-microglobulin >3 mg/L and serum monoclonal protein concentration >7 g/dL. Low risk is defined by the presence of ' 1 adverse characteristic and age '65 years, intermediate risk by the presence of 2 adverse characteristics or only age >65 years and high risk by the presence of >2 adverse characteristics. The aim of our analysis was to independently validate the significance of IPSS not only for overall survival (OS) but also for cause-specific survival (CSS) (i.e deaths unrelated to WM or to complications of treatment are censored). Furthermore, we wanted to assess whether elevated serum LDH may add to the strength of IPSS. From the data base of the Greek Myeloma Study Group, we identified 335 patients with clearly defined criteria for diagnosis and for initiation of treatment who were treated over the last 20 years. Main primary therapies included alkylating agents (43%), CHOP (3%), nucleoside analogues (3%) and rituximab either alone (3%) or in combination with conventional chemotherapy (44%). Before the initiation of treatment the median age of patients was 68 years (range 28 to 89 years). Fifty-nine percent of patients were >65 years, while 75% of patients had hemoglobin levels of 3 mg/L, 33% had lymphadenopathy, 32% splenomegaly, 23% presence of B-symptoms, 12% platelet count of 7 g/dL. Among 152 patients who had died by the time of this analysis, 33 patients (22%) had died due to causes not related to WM, to disease transformation, to myelodysplasia or to complications of treatment. Most frequent causes were second primary solid tumors, celebrovascular accidents, coronary artery disease and congestive heart failure. For the whole group of patients median OS was 105 months and median CSS was 116 months. Patients were divided into low risk (23%), intermediate risk (38%) and high risk (39%), according to IPSS. Median OS was 161 months, 105 months and 64 months respectively (p250 IU/L (normal upper limit 225 IU/L) was found in 18% of patients. Serum LDH was elevated in 16%, 12% and 24% of patients with low, intermediate and high risk, respectively. Median OS according to low or elevated LDH was 109 versus 63 months (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1899 Poster Board I-922 Background-Aim: Ph negative MPNs comprise a heterogeneous group of disorders in which activation of JAK and other downstream signaling pathways can occur in a varying degree, yielding several different phenotypes. The identification of the somatic point mutation in the JAK2 gene (JAK2V617F) as a key pathogenetic mechanism has further assisted in classification and diagnosis, in conjunction with clinical, morphological and biological characteristics of the various disease categories. The aim of this study was to investigate the presence of the mutation in association with the previously recognized as abnormal TIMP/MMP ratio in MPNs. This imbalance appears to favor inhibitory rather than proteolytic activity, resulting in tissue and bone marrow reforming processes to progress towards fibrosis instead of matrix proteins degradation. Subjects and Methods: In a case-control study, 64 consecutive patients (27 males; mean age 61.62 ± 16.64 years) who met WHO and PVSG criteria for polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF), were prospectively included and evaluated. The control group consisted of 74 healthy individuals (54 males; mean age 52.76 ± 15.54 years). Allele specific PCR was used for JAK2 mutation detection in peripheral and/or bone marrow derived DNA. Serum concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 were measured by quantitative sandwich enzyme immunoassay (ELISA). The levels of these parameters were expressed as mean ± SE and assessed with analysis of covariance (ANCOVA), adjusted for gender, age and co-morbidity. Results: Patients were classified as follows: PV n:37, ET n:20, IMF n:7. V617F mutational frequency was 81% [nPV:35 (94%) with exon 12 lesion identified in 2 patients, nET:12 (60%) and nIMF:5 (71%)]. The presence of the mutation was significantly associated with splenomegaly (p=0.018) and age at diagnosis (p=0.004). Regardless of JAK2 status, all patients showed significantly lower MMP-2 levels (181.62 ± 8.00 vs 237.15 ± 7.33, p Conclusions: In the particular group of patients studied, the identification of an abnormal TIMP/MMP ratio in all disease categories, regardless of JAK2 mutation status, supports the notion that BCR ABL negative MPNs are invariably characterised by bone marrow stroma remodelling, more likely as a consequence of continuous inhibition of matrix degradation with subsequent displacement, at a variable degree, by fibrotic tissue. Presence of JAK2V617F, being associated with even lower levels of MMP2, appears to be a concurring participant in this process. Further study will enable to delineate whether abnormal ratios are more prevalent in patients with thrombotic complications and perhaps document changes at different time points during treatment. Possible correlates with V617F allelic burden, especially towards thrombosis or fibrotic transformation, also remain to be elucidated. Disclosures: No relevant conflicts of interest to declare.

NKT cells, a novel class of regulatory T cells, secrete haemopoietic cytokines (GM-CSF, IL-3, IL-6) upon engagement of their TCR. Because of this property we hypothesized that NKT cells are involved in the regulation of haemopoiesis. NKT cells constitute To investigate the effect of cord blood NKT cells on the CFC activity of CD34+ cells, NKT were first activated ex vivo for 10 days in the presence of the CD1d-presented glycolipid a-galactosylceramide and subsequently were purified by flow-sorting using mAbs specific to their TCR a and b chains, i.e., anti-TCR Va24 and Vb11. Purified NKT were co-cultured in a ratio of 10:1 with CD34+ cells. In the absence of exogenous cytokines NKT enhanced the clonogenic capacity of CD34+ cells by 3-fold: 1 CFC per 14 cells (range 10.4–21) in the presence of NKT vs 1 per 43 cells (range 37–55.5) in the absence of NKT (n=4; p=0.024). By contrast, activated or resting autologous T cells co-cultured with CD34+ cells at the same ratio (10:1) had no effect on the CFC frequency, indicating that this enhancing effect on haemopoiesis is a unique property of NKT cells. The effect of NKT in the long-term clonogenic capacity is currently being evaluated. In summary, we have shown that a) CD1d is a novel marker expressed in HSC with long- and short -term clonogenic ability and b) CD1d-restricted NKT cells promote haemopoiesis These findings reveal a novel link between haemopoiesis and the CD1d-NKT axis of immune regulation and set the scene for the study of the role of NKT cells in the processes of engraftment and rejection in HSC transplantation.

CT-associated cognitive dysfunction, which frequently manifests as subtle changes in memory, concentration, and ability to remain focused or organized, can adversely affect cancer pts’ QOL. EPOLYM, a prospective, international, multicenter, open-label, phase IIIb trial has been conducted to investigate the efficacy, safety, and clinical outcomes, including effect on cognitive function, of epoetin alfa 40,000 IU sc QW in anemic (Hb

Abstract 2887 Poster Board II-863 Between November 2002 and April 2006, 72 patients with Waldenstrom's Macroglobulinemia (WM) were enrolled into this multicenter trial of primary treatment with DRC which consisted of dexamethasone 20 mg IV followed by rituximab 375mg/m2 IV on day 1 and oral cyclophosphamide 100 mg/m2 bid on days 1 to 5 (total dose 1000 mg/m2). DRC courses were repeated every 21 days for six courses and then patients without progressive disease were observed without treatment. Patient characteristics, toxicity and response data have been reported previously (Dimopoulos et al, J Clin Oncol 2007; 25:3344): 83% of patients achieved a response including 7% complete, 67% partial and 9% minor responses. In June 2009 we updated this study (minimum follow-up >3 years) in order to assess time to progression, time to next treatment, type of second-line treatment and response to this, overall survival (OS) and cause-specific survival (CSS) in which deaths unrelated to WM or complications of treatment were censored. Second line treatment was administered to patients who experienced progressive disease and also met criteria for treatment requirement based on consensus recommendations (Kyle et al, Sem Oncol 2003; 30:116). As of June 2009, 42 patients fulfilled the criteria for progressive disease (Kimby et al, Clin Lymphoma Myeloma 2006; 6:380) but 14 patients have not yet required second line treatment. The median time to progression was 35 months (95% Confidence Interval: 22-48 months) and the median time to next treatment requirement was 51 months. Among several factors who were analyzed for their possible correlation with shorter time to progression, only lymphadenopathy was significant (p Disclosures: No relevant conflicts of interest to declare.