ALBERT

Description: Background: Gene expression profiles have been associated with prognosis in myelodysplastic syndromes. WT1 is a tumor-suppressor gene coding for a transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms’ tumor. In normal bone marrow, WT1 expression is low or undetectable, whereas it is aberrantly expressed in hematological malignancies. Evidence indicates that WT1 is important in the lineage-specific differentiation of hematopoietic cells and leukemogenesis. In myelodysplastic syndromes (MDS), WT1 expression has prognostic significance: it is directly correlated with the type of MDS, with IPSS score and with disease progression. Recent data demonstrate that WT1 is a potent activator of the EPO gene under normoxia and it is suggested that WT1 may regulate paracrine EPO synthesis in a tissue-specific manner. Bmi-1 is a transcriptional repressor gene which may be expressed restrictedly in stem cells and progenitors and is required to regulate the adult self-renewing hematopoietic and leukemic stem cells. It appears that it also plays an important role in providing cells the potential for proliferation. A number of reports on Bmi-1 provide perspectives on the close association of its expression with the progression of hematopoietic malignancies. Furthermore, flow cytometry has shown that Bmi-1 positivity in CD34+ cells is positively correlated with IPSS score. Introduction: We have designed a study to evaluate changes in gene expression profiles of bone marrow mononuclear cells of primary low and intermediate-1 IPSS risk MDS patients receiving erythropoetic growth factors (darbepoetin or high-dose rHuEpo alpha). Associations with response, changes in Hb, in percentage of CD34+ and apoptotic cells are evaluated. We present preliminary results in 6 patients. Methods: Bone marrow samples were obtained before and after 12 weeks of treatment. Mononucleated cells were cryopreserved and later thawed for total RNA extraction and cDNA synthesis. Gene expression profiling and the expressions of WT1 and Bmi-1 by RT-PCR were evaluated. Results: Baseline median Hb was 9,5 g/dL (interquartile range 8,7 10,1). Baseline mean Bmi-1 was 14011 (± SD 3927). All patients had a major erythroid response to treatment. Preliminary results demonstrate a significant decrease in WT1 from baseline median 1635 (interquartile range 1251–2150) to 1192 (interquartile range 1005–1365, P=0.046). Fig. 1. Changes in WT1 expression during therapy. Fig. 1. Changes in WT1 expression during therapy. Discussion: Though few patients have yet been studied, it is suggested that WT1 decreases in patients responding to erythropoetic growth factors. Further evaluation with stratification for erythropoetic response in the extended study may furnish novel associations between gene expression, erythropoetic growth factors and prognosis in MDS patients.

Description: Dasatinib was approved for use in the treatment of patients (pts) with chronic myeloid leukaemia (CML) and resistance to Imatinib. We applied a rescue treatment based on Dasatinib therapy to achieve a pharmacological immunomodulation in a setting of CML-relapsed allogenic stem cell transplantated (A-HSCT) pts. Patients were required to have both resistance to Imatinib and unresponsiveness to cellular therapy such as Donor Lymphocyte Induction (DLI). We hypothesized that Dasatinib could potentially improove the disease by immunomodulatory action. Primary aim of this therapeutic design was to address, in single institution trial, therapeutic force of a innovative pharmacologic strategy to induce the cytogenetic response followed by DLI. Therefore, we investigated Dasatinib ability to achieve immuno-effects by targeting key mediators of Th1, Th2, and Treg response. Biological effects were examined on conventional diagnostic parameters such as haematological chimerism, cariotype and Bcr-Abl gene transcript. Herein, we present interim results of a pilot group of 3pts. Patients received dasatinib 70 mg twice daily(140 mg total daily dose). Dose modifications were allowed for the management of toxicity. Treatment was performed until complete cytogenetic, molecular response and haematological full donor chimerism. Materials and Methods: To investigate the immunological changes, we used a TaqMan® Low Density Array, based on comparative CTdd CT method on Applied Biosystems 7900HT, to perform relative quantification of cDNA derived from peripheral venous blood specimens harvested after DLI, before and after starting dasatinib therapy. Assumed that normal control values of all transcripts were = 1, we evaluated over or down regulation of gene expression profile (GEP) of a panel of 48 genes involved in immune response. Results: clinical changes after third month of dasatinib therapy. Case 1: responsive patient, maintained a mixed haematologic chimerism, but showed a complete cytogenetic and molecular remission. Following, patient restarted with DLI therapy. Case 2: responsive patient, showed nearly full-donor haematologic chimerism with complete cytogenetic and molecular remission. Case 3: patient no evalutable because brief treatment (only 1 month). Dasatinib caused early haematological toxicity. Patient maintained a low level of donor T cells with presence of Philadelphia chromosome associated to elevated p210 molecular signal. Gene expression profiles post-dasatinib therapy: According to in vitro experiments (Blood October 25, 2007), in all cases we observed a down regulation of IL-2 and IL-12B (Th1), IL-6 and IL- 18, IL-10 (Th2) cytokines and mediators of apoptosis such as EGR2, EGR1. By contrast, multiple pro-inflammatory factors were up-expressed: IFN-g, IL-17, IL-7. Only in case number 1, TNF-a and IFN-g molecular pathways were not influenced by the drug. In fact their elevated expression was preserved as compared to pre-dasatinib levels. Noterworthy among cases number 2 and 3 (with mixed chimerism), Dasatinib improved a marked inhibition of Th1 effectors in addition to down-regulation of several important molecular transcripts: SERPIN B3–B4, BCL2A1, SELP, PIAS 1, IRF8, IRF1, CCL7, CCL5, CXCL9, CCR4, ICAM. Regards to T regulatory cells, Foxp3 was strongly up-regulated in case number 1 and down-regulated in case2and 3. Conclusion: We think that Dasatinib represent a possibility of cure for for CML pts relapsed after A-HSCT and unresponsive to alternative treatments. For imatinib-resistant CML patients, such as in this study, there are few currently available effective options. The present results strongly emphasize the importance of immune response control to achieve the desired clinical effects.

Description: Abstract 4541 Introduction: Limited data demonstrate to what extent preventing fungal exposures is effective in preventing infection and disease. Further studies are needed to determine the optimal duration of fluconazole prophylaxis in allogeneic recipients to prevent invasive disease with fluconazole-susceptible Candida species during neutropenia. Oral, nonabsorbable antifungal drugs might reduce superficial colonization and control local mucosal candidiasis, but have not been demonstrated to reduce invasive candidiasis. Anti-fungal prophylaxis is recommended in a subpopulation of autologous recipients with underlying hematologic malignancies with prolonged neutropenia and mucosal damage. Methods: This is a retrospective study of 1007 SCT performed in our center between 1992 and 2009 in 809 consecutive patients, irrespective of diagnosis. HEPA filter and environmental monitoring (air, water, surfaces) are attributes of our transplant center. Results: The main characteristics of the patients are reported in Table 1. Systemic prophylaxis was used according to the guidelines (Table 2): fluconazole in the nineties, then itraconazole and from 2004 was either abolished or substituted with non-adsorbable prophylaxis in transplants with standard risk. Secondary prophylaxis was prescribed for high risk patients (with infectious fungal history, suggestive iconography, positive fungal biomarker). In 17 years our Center has never been colonized by mould. Only 3 probable aspergillosis infections and 4 proven fungal infections (fusarium, mucor and 2 aspergillosis) were diagnosed, all in allogeneic patients (2 haplotipical, 1 singenic, 1 sibiling, 1 MUD and 2 mismatched), resulting in death in all cases. No infection was documented in autologous setting, while the infection rate in allogenic setting was 3.6% with an incidence rate of 1.1 infection per 10000 transplants/year. These results are significantly lower than published reports. Conclusion: Systemic antifungal prophylaxis should not be performed in autologous SCT patients. The abuse of systemic prophylaxis targeting yeasts has influenced the change of epidemiology in the transplant setting with prevalence of mould infections. The identification of high risk patients is useful to select patients for systemic antifungal or secondary prophylaxis to reducing overtreatment, incidence of resistant strains and costs. Disclosures: No relevant conflicts of interest to declare.

Description: The aim of our perspective observational study was identify a fast molecular biomarker of tumorigenic-proliferative haematological disorders at on set using a non-invasive method. At this end, to better discriminate between myeloproliferative or lymphoproliferative hematological disease, from May to July 2014, we collected peripheral blood mononuclear cells (PBMCs) from patients at the first medical examination and without drug therapy. The patients were divided into groups on the basis of the diagnosis. Group 2 (n=8) included patients suffered from mixed disorders such as myeloproliferative neoplasms (MPD) associated to monoclonal gammopathy of undetermined significance (MGUS). Group 3 (n=8) included patients with only MGUS, group 4 included 9 patients with only primary myelofibrosis (M). Healthy donors (Group 1, n=16) were considered as normal subject or calibrator in molecular analysis. Their PBMCs were used to perform relativegene expression profile of a transcriptome involved in apoptotic control, stem-cell differentiation, immune network, inflammation and leukemogenesis, in order to detect whether these may serve to label the patient groups. A multigene expression assay (47 genes) was carried out with the TaqM,an® Low Density Array Fluidic card. In Group 3 (MGUS), 6 genes were differentially expressed (BMI-1, FLT3, FZD1, FZD5, ICAM1, IMP3). Also, we compared variance, main value for each gene in each group and T-test that showed a significant differential expression pattern (p

Description: Abstract 3631 Introduction: The erythroid differentiation defect observed in 5q– syndrome has been attributed to the RPS14 gene located within the CDR of the long arm of chromosome 5. We have recently demonstrated that RPS14 expression increases during lenalidomide treatment. However, haploinsufficiency of RPS14, which encodes ribosomal protein S14, does not explain clonal dominance. The expression of miRNAs, miR-145 (5q33.1) and miR-146a (5q33.3), in CD34+ bone marrow (BM) cells of individuals with MDS with deletion of the long arm of chromosome 5 (del(5q)) is lower compared to normal controls (Starczynowski et al, Nature Medicine, 2010). miRNAs are small noncoding RNAs that post-transcriptionally repress specific messenger RNA targets through interaction with the 3′ untranslated region (UTR). Loss of noncoding transcripts encoding miRNAs within the CDR may result in haploinsufficiency by loss of inhibition of their targets. Concurrent loss of both miR-145 and miR-146a resulted in activation of innate immune signalling through elevated expression of their respective targets, TIRAP and TRAF6. Furthermore, knockdown of miR-145 and miR-146a or overexpression of TRAF6 in mouse HSPC (Hematopoietic stem and progenitor cells) recapitulated features of 5q– syndrome, such as bone marrow dysplasia, anemia and thrombocytosis. We present preliminary results of changes in miRNA expression in IPSS lower-risk MDS with del(5q) during treatment with lenalidomide. Methods: A prospective single-arm trial investigating the efficacy and safety of lenalidomide in 46 patients with MDS with del(5q) with/without additional cytogenetic abnormalities and Hb 〈 10 g/dL. Lenalidomide was administered orally at a starting dose of 10 mg/day for a maximum of 12 months. When necessary, dosing was reduced to 5 mg/day or 5 mg on alternate days. Bone marrow assessments were performed at baseline and every 3 months, thereafter. For the evaluation of miRNA-145 and miRNA-146a in patient samples, 300 ng/μl of miRNAs were isolated in each purified BM sample by using mirVana™ miRNA Isolation Kit-Ambion and TaqMan miRNA Array Analysis was performed to determine the expression of miRNAs (7900HT Sequence Detection System Applied Biosystems). Patient BM-miRNAs were calibrated with miRNAs from BM of healthy volunteer donors. It was assumed that BM expression value of each calibrator miRNA was 1 unit. RPS14 gene assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results: Four patients have been evaluated (1 M, 3 F; ages 65, 66, 73 and 76 years, respectively) at baseline and after 12 weeks. At baseline, 2 patients were RBC-transfusion dependent. One patient had one additional cytogenetic abnormality (+8 in 15% metaphases). All patients obtained an erythroid response by week 12: mean Hb values significantly increased from 8.4 ± 0.9 at baseline to 11.6 ± 0.9 g/dL (p=0.01). All patients obtained a cytogenetic response, 2 of which were complete. miRNA-145 and miRNA-146a expression were both low at baseline and significantly increased by week 12 (Table). Conclusions: Preliminary results confirm that, in IPSS lower-risk MDS with del(5q), miRNA-145 and miRNA-146a expression is low. Lenalidomide treatment is associated with erythroid responses and cytogenetic remissions concurrent with significant increases in miRNA-145 and miRNA-146a expression. Disclosures: Oliva: Celgene: Consultancy.

Description: Bortezomib (Velcade) is used widely for the treatment of various human cancers; however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that bortezomib induces proteasome-independent degradation of the TRAF6 protein, but not mRNA, in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of bortezomib due to down-regulation of the proteasomal subunit α-1 (PSMA1). To determine the molecular consequences of loss of TRAF6 in MDS/AML cells, in the present study, we applied gene-expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe herein novel mechanisms by which TRAF6 is regulated through bortezomib/autophagy–mediated degradation and by which it alters MDS/AML sensitivity to bortezomib by controlling PSMA1 expression.

Description: The genetic basis of myelodysplastic syndromes (MDS) is heterogeneous, and various somatic mutations are associated with different phenotypes and clinical outcomes. The only curative treatment for MDS patients is allogeneic hematopoietic stem cell transplantation (HSCT) which is considered as a therapeutic option until the age of 65-70 in eligible patients. Whether the genetic basis influences the outcome of HSCT is currently unclear. Recently, we observed that mutations on ASXL1, RUNX1 and TP53 genes are independent predictors of relapse and overall survival in MDS patients after HSCT, and that the integration of these mutations into currently available predictive models increases the capability to capture prognostic information at individual patient level (Della Porta MG et al. J Clin Oncol, 2016 in press). In this study, we explored the possibility of developing a clinical/molecular predictive model to specifically estimate the outcome after HSCT in patients with MDS or acute myeloid leukemia evolving from MDS (MDS/AML). We studied 401 patients undergoing allogeneic HSCT for primary MDS or MDS/AML between 1997 and 2013 and reported to the GITMO registry. We used massively parallel sequencing to examine tumor samples collected before HSCT for somatic mutations in 34 recurrently mutated genes in myeloid neoplasms. In multivariable analysis with probability of relapse as endpoint the following factors showed independent prognostic value: percentage of marrow blasts (〉10% vs. ≤10%, HR 1.43, P=0.04), cytogenetic risk according to IPSSR (poor/very poor vs. very low/low/intermediate risk, HR 1.85, P=.002), disease status at transplant (refractoriness to induction chemotherapy vs. complete remission, HR 2.4, P40 vs. ≤40 years, HR 1.68, P=.001), comorbidity risk according to HCT-CI (high vs. low/intermediate risk 2.10, P10%, poor/very poor cytogenetic risk according to IPSSR, refractoriness to induction chemotherapy, and driver mutations in ASLX1/RUNX1/TP53 genes. A relapse risk index was calculated as the sum of these weighted scores, and was then categorized into 4 risk groups: low (score=0), intermediate (score=1-2), high (score=3), and very high (score=4). The cumulative incidence of relapse was estimated by a competing risks approach with TRM. In patients receiving standard conditioning, 5-year probability of survival after allogeneic HSCT was 61%, 43%, 39% and 19% for low, intermediate, high and very high risk (P

Description: Abstract 2622 Poster Board II-598 Introduction: Genetic alterations reported in myelodysplastic syndromes (MDS) are not disease-specific and the underlying molecular causes of the disease remain poorly understood. It has been suggested that one or more of the genes mapping within the commonly deleted region of the 5q syndrome, together with other distant genes, may be critical to the development of the 5q syndrome. Potential candidate genes have been identified including the tumor suppressor gene SPARC, and the ribosomial protein gene, RPS14. Haploinsufficiency of RPS14 has been demonstrated and recent evidence indicates RPS14 as a causal gene for the 5q syndrome. Lenalidomide has proven efficacy in MDS patients with del(5q). Rapid and durable responses include transfusion-independence, with a rise in Hb, suppression of the 5q-deletion clone and improvement in bone marrow morphologic features. Methods: In a multicenter Italian phase II trial to evaluate safety, changes in quality of life and efficacy of lenalidomide in primary MDS patients with del(5q) and low or Int-1 risk IPSS, we investigate changes in bone marrow cytogenetics and gene expression patterns during treatment. The starting dose of lenalidomide is 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Dosing is based upon clinical and laboratory findings. Bone marrow cytogenetics and gene expression profiling are performed on study entry and every 12 weeks up to end of study (week 52). Gene expression assays of 51 candidate genes from the published literature and genomic databases have been selected and are carried out with TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative dd CT method, according to manufacturer's instructions. Using an 18S mRNA gene pre-designed assay from Applied Biosystems to detect the expression of the housekeeping gene 18S in each sample, target gene expression is normalized with 18S gene expression derived from a bone marrow pool of normal healthy subjects and for each sample the ratio between the target and 18S are expressed. Results: Baseline values for 23 patients (mean age 73 ± 10 years) are available and 16 have been re-evaluated after 12 weeks. Mean Hb was 8.6 ± 0.9 g/dL and 20 patients were transfusion-dependent. Seven patients had additional cytogenetic abnormalities. At baseline, RPS14 was under-expressed in 19 out of 21 patients evaluated. After 12 weeks RPS14 was re-evaluated in 13 patients: all had erythroid responses and RPS14 increased significantly from 0.07 (IQ Range 0.03–0.13) to 76.1 (0.73– 304.0, p=0.002). SPARC expression was under-expressed in 15/23 patients and variations during treatment were not significant. Baseline FAS gene was under-expressed in all patients and increased above reference values (p=0,006) after 12 weeks in 7/14 cases. IL7R was over-expressed in all patients at baseline (median 3263.3, IQ range 1998.3–5027.1) and was significantly reduced after 12 weeks (median 0.17, IQ range 0.05–2.20, p

Description: Background and Objectives The use of allogenic hematopoietic stem cell transplantation (HSCT) is increasing in older patients because of reduced ablative regimens and improved supportive care. An old patient usually has a old sibling healthy donors (HDs). The safety of G-CSF for mobilization in elderly HDs has not been provided. In our institution a long-term active follow-up study of G-CSF-mobilized healthy volunteer donors has been implemented. Patients and Methods After a median follow up of 94 months (7.8 years) we reviewed and analyzed safety data of our HDs database according to HDs age: HDs-1 (162), patients younger than 50 years old, HDs-2 (62), patients 50-59 years old and HDs-3 (23), patients 60 or more years old. Long-term follow-up included monitoring of neoplastic, cardiac or autoimmune diseases. Results Two hundred and forty-seven successive donors were evaluated and their characteristics are well balanced among age-groups and no statistical differences have been detected: most of them were male (55.9% ), sibling (97.2%) and HLA matching (93.1%). Mobilization failure rate has been 4.3% (7/162), 1.6% (1/62) and 13% (3/23) in HDs-1, HDs-2 and HDs-3, respectively (p=0.075). Short- and long-term safety was not different among age-groups. Bone pain was reported as the most frequent short-term adverse event (76.5%). Other commonly observed short-term symptoms included headache (31.6%), fatigue (28.7%), insomnia (21.5%), nausea (13.0%) and fever (6.1%). For long-term safety surveillance no haematological malignancies were observed. The observed rate of solid tumors, cardiovascular and autoimmune events, was the expected incidence for these diseases in the western countries. Conclusion In our study, mobilization with lenograstim in elderly HDs seems to be as safe as for young HDs safe. This data contribute to the growing body of evidence of the long-term safety of G-CSF for allogenic donor stem cell mobilization also for elderly HDs. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2452 Deletion of chromosome 5q in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients results in loss of miR-146a, which is a negative regulator of the innate immune pathway by targeting TNF receptor associated factor-6 (TRAF6). Therefore, MDS and AML patients with reduced miR-146a expression concomitantly exhibit elevated TRAF6 protein. TRAF6 is an E3 ubiquitin ligase that catalyzes K63-linked polyubiquitin chains on substrates that lead to pathway activation, one of which includes NF-kB. Mice lacking miR-146a, or with overexpression of TRAF6, develop AML- and MDS-like features. Bortezomib (Velcade©), which shows promise alone or in combination with chemotherapy in certain groups of MDS and AML patients, is a selective and reversible inhibitor of the 26S proteasome. Studies on the mechanism of action of Bortezomib have shown that pro-apoptotic proteins are stabilized following proteasome inhibition and contribute to the anti-cancer effect. In this report, paradoxically, we find that Bortezomib induces rapid and complete degradation of TRAF6 protein, but not mRNA, in MDS/AML cell lines and human CD34+ cells. A similar finding was observed when AML cells were treated with MG132, another proteasome inhibitor, indicating that degradation of TRAF6 is secondary to proteasomal inhibition. Interestingly, the reduction in TRAF6 protein coincides with Bortezomib-induced autophagy, as indicated by conversion of LC3B-I to LC3B-II and degradation of SQSTM1/p62, and subsequently with apoptosis in MDS/AML cells. Addition of an autophagy inhibitor (3-methyladenine [3-MA]) to Bortezomib-treated AML cells maintained TRAF6 protein expression and enhanced cell viability. Similarly, TRAF6 degradation was blocked by 3-MA when cells were treated with Rapamycin, an mTOR inhibitor and inducer of autophagy. These findings suggest that a mechanism of Bortezomib-induced cell death in myeloid malignancies involves elimination of TRAF6 protein by autophagosomes. Forced expression of TRAF6 in two AML cell lines partially blocked the cytotoxic effect of Bortezomib, suggesting that TRAF6 is an important target of Bortezomib. To determine whether loss of TRAF6 is sufficient to impede growth of MDS and AML, we used a genetic approach to inhibit TRAF6 in MDS/AML cell lines and bone marrow cells from MDS patients with deletion of chromosome 5q. RNAi-mediated depletion of TRAF6 in MDS and AML samples resulted in impaired malignant hematopoietic stem/progenitor function and rapid apoptosis. To uncover the molecular consequences following loss of TRAF6, we applied gene expression profiling and identified genes relevant to the survival of MDS and AML cells. In summary, these findings implicate TRAF6 in Bortezomib-induced cell death and in the maintenance of myeloid malignancies, and reveal a novel mechanism of TRAF6 regulation through autophagic degradation. Disclosures: Oliva: Celgene: Consultancy.