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Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored. Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors. Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype. Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

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.

Foxp3(+) T regulatory cells (Tregs) and Th17 cells accumulate synchronously at tumor sites during cancer progression, where their interplay is apparently affecting the efficiency of the antitumor response. In myelodysplastic syndromes, a hematopoietic malignancy of myeloid origin, Tregs are highly increased in the late stages of the disease (L-MDS), but the mechanisms driving Treg expansion and the interaction between Treg and Th17 cell dynamics are still unknown. We demonstrate that the proliferative capacity of Tregs is deficient during the early MDS stages (E-MDS), while in L-MDS it returns to normal levels. In addition, synchronously to Treg expansion, L-MDS patients exhibit increased numbers of functionally competent bone marrow IL-17(+) and FOXP3(+)/IL-17(+) cells, in contrast to E-MDS patients, where Th17 cells are significantly decreased and hypofunctional. Our findings suggest similar kinetics of Treg and Th17 cells between MDS and solid tumors, indicating a common immune pathogenetic pathway between diverse cancer types.

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.

Chronic myeloproliferative disorders (cMPDs) are clonal hemopoietic malignancies arising at the multipotent stem cell level. These conditions are characterized by increased blood count, marrow hyperplasia and extramedulary hemopoiesis. Vascular events might complicate their course, and transformation to either acute leukemia or myelofibrosis can finally occur. Among cMPDs, Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) belong to the group of Ph-negative cMPDs. Although they share common pathogenetic features, these entities have a quite different prognosis. The common pathogenetic basis of Ph-negative cMPDs was recognized long ago, and it was suggested that a stimulating factor might enhance bone marrow hemopoietic activity. Hemopoietic progenitors from cMPDs show hypersensitivity to low levels of a variety of hemopoietic cytokines. The independency of erythroid precursors from erythropoietin became the first surrogate marker of an abnormal hemopoietic clone. This clone is characterized by increased proliferation and survival, as well as by decreased apoptosis, leading to the accumulation of mature blood cells that additionally show a phenotype of activated cells. Recently four independent groups have described an activating point mutation in the JAK2 kinase as a key pathogenetic event in Ph-negative cMPDs. JAK2 is a tyrosine kinase that acts as a second intracellular messenger for many hemopoietic cytokine receptors. It is now believed that jacking up hemopoiesis can explain many features of myeloproliferation. Interestingly, some features are associated with intracellular levels of mutated JAK2 (the "dosage hypothesis"). The mutation in JAK2 kinase is not an example of a genetic defect leading to a single disease, since it occurs in many other myeloid disorders, and probably represents a secondary hit in a multistep ongogenetic process. Nevertheless, it has changed the way we approach cMPD patients and has clarified many aspects of their biology.

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.

beta-Thalassaemia major is a severe, transfusion-dependent anaemia that also causes infertility due to iron deposition to endocrine organs. Very few pregnancies have been reported among such patients. In this report we describe the evolution and successful outcome of pregnancy in 5 Greek women with beta-thalassaemia major. There were four full-term and one preterm deliveries of two normal and three small for the date neonates. Cardiovascular changes related to gestation may aggravate the underlying multiorgan damage of the pregnant mother and predispose to poor fetal growth and development. All five patients followed a strict transfusion regimen in order to maintain the haemoglobin level above 10 g/dl. The inadvertent administration of desferrioxamine in one patient until the 8th gestational week did not seem to have any serious effects on the development and well-being of the fetus. Although pregnancy is not contraindicated in beta-thalassaemia major, intensive individualized care is required if it is to be safe for the mother, and have a reasonably good chance of producing a healthy child.

Immunophenotyping and genotyping are usually helpful in defining cell lineage. However, aberrant immunohistochemical expression is seen in a small proportion of B and T cell lymphomas.1 ,2 Immunohistochemical studies may be supplemented by a molecular search for immunoglobulin heavy ( IGH) chain and T cell antigen receptor ( TCR) rearrangements. It has been reported that simultaneous rearrangements of both IGH and TCR are not rare and cross-lineage rearrangements are encountered in about 10% of mixed lymphomas.3 These cases may correspond either to dual genotype lymphomas with two distinct monoclonal populations or to bigenotypic lymphomas with IGH and TCR rearrangements in the same cell. We describe an extremely rare case of a patient with lymphoma expressing both B and T cell antigens and showing both IGH and TCR rearrangements. The patient presented with fever, generalised lymphadenopathy and multiple maculopapular skin eruptions. The patient had a history of erythema nodosum diagnosed 1 year before. Laboratory investigation showed a worsening pancytopenia, hypogammaglobulinaemia with IgG levels of 562 mg/dL, high lactate dehydrogenase (981 IU/L) and β2-microglobulin 10.7 mg/L (range 0.8–2.4). The anaemia (Hct 28%, Hb=9.5 g/dL) was microcytic with features of anaemia of chronic disease. HIV antibodies, anti-HCV and HBsAg were negative whereas anti-HBs and anti-HBc were positive. CT scans revealed enlarged cervical, mediastinal, hilar, axillary, mesenteric, retroperitoneal, iliac and femoral lymph nodes. An axillary lymph node biopsy along with bone marrow and skin biopsies were performed. Histological examination revealed complete effacement of lymph node architecture by …

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.

In summary, the myelotoxicity of ticlopidine is a well known effect, as has been demonstrated by numerous reports. The real incidence of the most hazardous haematological complication, namely medullary failure of variable severity, has probably been underestimated. It is certain that, with the widespread use of ticlopidine, more cases of marrow aplasia will be reported in the future. The authors believe that until the superiority in terms of efficacy of ticlopidine over aspirin is verified, the first choice of antiplatelet treatment should not include this drug, at least in older patients. Whenever ticlopidine is indicated and used, careful monitoring of the blood count during the initial months of therapy is required, as advised by previous reports.

Advanced stage mycosis fungoides (MF) generally has a poor prognosis, and currently there is no standard treatment available. Here we report the case of a young woman with recalcitrant tumour-stage MF (T3, stage IIb) whose disease was unresponsive to several therapeutic modalities, but who has showed sustained clinical response to pegylated liposomal doxorubucin. No severe infectious complications have been observed. The use of this drug in tumour-stage MF should be investigated further.

Immunophenotyping is indispensable in the differential diagnosis of B-cell chronic lymphoproliferative disorders (B-CLPDs). However, B-CLPDs often show overlapping immunophenotypic profiles and may be diagnostically challenging. CD1d is an HLA class I-like molecule that presents glycolipids to invariant natural killer T cells. Normal mature B cells constitutively express CD1d, but with the exception of some conflicting data, its expression in B-CLPDs is unknown. We demonstrate that in 222 B-CLPD cases, CD1d expression of less than 45% is strongly predictive of CLL (likelihood ratio, 32.3; specificity, 97.4%; sensitivity, 84.1%). In addition, CD1d showed significantly higher staining intensity in splenic marginal zone lymphoma compared with atypical hairy cell leukemia, lymphoplasmacytic lymphoma, and mantle cell lymphoma, thus allowing the discrimination of the former from the latter immunophenotypically overlapping B-CLPDs. It is important to note that in a given patient, CD1d expression on malignant B cells was similar between tissues and remained unaffected by disease stage and treatment status. Our findings strongly argue for the incorporation of CD1d into routine lymphoma panels.

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.

The implication of T cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF) is controversial. CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are pivotal in maintaining immune homeostasis, but their role in IPF pathophysiology has not yet been studied.To explore Treg dynamics and function in IPF.Treg levels and dynamics were analyzed by flow cytometry in the peripheral blood (PB) and bronchoalveolar lavage (BAL) of 21 patients with IPF, 35 patients with lung diseases other than IPF (patients without IPF), 20 patients with collagen vascular diseases with pulmonary parenchymal involvement (CVD-IP), and 28 healthy volunteers. The suppression of autologous CD4(+)CD25(-) cell-proliferative responses and cytokine release by magnetic bead-isolated Tregs was evaluated by proliferation assays and cytometric bead array. Correlations of Treg function and levels with lung function parameters were also performed.In patients with IPF, both BAL and PB Tregs were reduced compared with those of healthy volunteers and patients without IPF, although not always significantly. Treg levels were not affected by the administration of low-dose prednisone in four nonresponding patients. The suppressor potential of BAL and PB Tregs was compromised in patients with IPF and patients with CVD-IP, compared with healthy volunteers and patients without IPF. Similarly, the Treg-induced suppression of helper T-cell type 1 and 2 cytokine secretion was impaired in the BAL of patients with IPF and patients with CVD-IP. Moreover, the defective function of BAL Tregs correlated highly with parameters of disease severity.This study provides the first evidence of global Treg impairment in IPF that strongly correlates with disease severity, suggesting a role for Tregs in the fibrotic process.

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.

A large body of experimental research supports the anti-neoplastic activity of cellular and humoral immunity. Disease and therapy-related immune suppression may be important on the treatment outcome or on the subsequent course of the malignant disease. The aim of the study was to investigate the efficacy of amifostine in preventing the immunological toxicity of post-operative radiotherapy (RT) in breast cancer patients. Using flow-cytometry, we examined comparatively the peripheral blood lymphocytic subpopulations in breast cancer patients undergoing conventional post-operative RT versus a hypofractionated accelerated RT scheme combined with amifostine (HypoARC) administration. Despite the higher radiation dose intensity delivered in the HypoARC group, a significant protection of CD4, CD8, CD19 and CD56 subtypes by amifostine was noted. We further focused on two interesting CD4/CD8 subpopulations involved in cellular apoptosis and trans-endothelial migration, namely the CD95/Fas and CD31 positive lymphocytes. Amifostine protected and induced expansion of these subtypes, which could contribute to the maintenance of a high burden of tumor infiltrating lymphocytes during therapy. It is suggested that amifostine effectively protects lymphocytes against RT, which may enhance the efficacy of the latter. The clinical impact of the CD95(+) and CD31(+) T-cell immunological modulation induced by amifostine requires further investigation.

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.