Risk Myelodysplastic Syndrome Patients Research Articles

Predicted costs of iron‐chelators in myelodysplastic syndromes: A 10‐year analysis based on actual prevalence and red cell transfusion rates

Consideration of iron-chelation (IC) in transfusion-dependent patients is recommended in most clinical-practice guidelines on myelodysplastic syndromes (MDS). The financial impact of IC on health-care systems is predicted through economic modeling, but an analysis based on actual prevalence is lacking. Here, we have investigated the potential drug-costs and need for IC in a cohort of 189 United Kingdom-based MDS patients diagnosed from 2000 to 2010. Patients with low or intermediate-1 IPSS scores were identified as eligible for IC if ≥24 red cell units (RCU) had been transfused over 12 consecutive months or the transfusion-intensity averaged ≥2 RCU per month. Drug-costs were calculated from the time patients qualified for IC until death or last follow-up. In 159 patients with low/intermediate-1 MDS, survival was superior with a low IPSS score (P = 0.014), age <70 years (P = 0.043), transfusion-independence at diagnosis (P = 0.0056) and transfusion-intensity of <2 RCU per month (P = 0.009). Reflecting the time elapsed since diagnosis, longer survival was observed with a cumulative red cell load of ≥75 U (P = 0.046). By logistic-regression analysis, transfusion-intensity independently predicted survival (P = 0.0035) in low and intermediate-1 risk MDS patients. Forty-one patients fulfilled criteria for consideration of IC. Of these, 6 patients died within 1 month; 35 patients survived for a median of 16 months (range 1-61). Had patients commenced IC, the anticipated drug-costs alone would have been ~$526,880-$2,064,800 over 10 years. The lack of association between cumulative transfusion-load and survival calls for a prospective evaluation of the cost-utility of IC in patients surviving long-term, to enable evidence-based recommendations in MDS management.

Identification of a risk dependent microRNA expression signature in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self-renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down-regulation of members of the leukaemia-associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate-1) and higher risk (Intermediate-2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.

Prognostic factors for response and overall survival in 282 patients with higher-risk myelodysplastic syndromes treated with azacitidine

AbstractPrognostic factors for response and survival in higher-risk myelodysplastic syndrome patients treated with azacitidine (AZA) remain largely unknown. Two hundred eighty-two consecutive high or intermediate-2 risk myelodysplastic syndrome patients received AZA in a compassionate, patient-named program. Diagnosis was RA/RARS/RCMD in 4%, RAEB-1 in 20%, RAEB-2 in 54%, and RAEB-t (AML with 21%-30% marrow blasts) in 22%. Cytogenetic risk was good in 31%, intermediate in 17%, and poor in 47%. Patients received AZA for a median of 6 cycles (1-52). Previous low-dose cytosine arabinoside treatment (P = .009), bone marrow blasts > 15% (P = .004), and abnormal karyotype (P = .03) independently predicted lower response rates. Complex karyotype predicted shorter responses (P = .0003). Performance status ≥ 2, intermediate- and poor-risk cytogenetics, presence of circulating blasts, and red blood cell transfusion dependency ≥ 4 units/8 weeks (all P < 10−4) independently predicted poorer overall survival (OS). A prognostic score based on those factors discriminated 3 risk groups with median OS not reached, 15.0 and 6.1 months, respectively (P < 10−4). This prognostic score was validated in an independent set of patients receiving AZA in the AZA-001 trial (P = .003). Achievement of hematological improvement in patients who did not obtain complete or partial remission was associated with improved OS (P < 10−4). In conclusion, routine tests can identify subgroups of patients with distinct prognosis with AZA treatment.

Lipid Peroxidation Products In Bone Marrow Plasma (BMp) and Peripheral Blood Plasma (PBp) From Patients with Low Risk Myelodysplastic Syndrome (MDS)

AbstractAbstract 2924Patients with Low-risk MDS have ineffective hematopoiesis, which results in refractory cytopenias requiring frequent blood transfusions for anemia treatment, nevertheless iron overload can occur even without transfusions. Recent research showed that reactive oxygen species were higher while reduced glutathione GSH was lower in RBC and platelets of MDS patients,when compared to normal individuals. The presence of highly reactive free radicals may play a role in the peroxidation of membrane lipids. Indirect evidence supports the role of oxidative DNA damage in the pathogenesis of MDS. Studies of the expression of antioxidant enzymes suggest that many CD34+ progenitors have inadequate oxidative stress defence. In this study we evaluated lipid peroxidation products measuring the thiobarbituric acid reactive substances (TBARS) in bone marrow plasma (BMp) and peripheral blood plasma (PBp) in low risk MDS patients. Materials and Methods:BMp and PBp for TBARS determination were obtained from 15 Low and Int-1 Risk MDS patients: 4 males, 11 females, mean age 65.5 years (range 50–83) at least 30 days from the last transfusion. Ferritin (FE), Transferrin (Tf), Tf Saturation (Tfs) were performed on the same day. As a control for TBARS determination we used (BMp) and (PBp) from 15 healthy bone marrow adult donors for transplant, age range 20– 55 years. Plasma levels of lipid peroxidation products were estimated according to the method of Ohkawa et al. as modified by Yagi. Lipid peroxides were expressed as nmol of malondialdehyde (MDA) per ml of plasma. Statistical analysis were performed using unpaired and paired Student's t-test and Pearson correlation was used to establish the relationship between variables (SAS V.9.2). Results:In MDS patients BMp and PBp TBARS levels were significantly increased: 10.59±0.84 and 5.25±0.55 respectively compared with controls: 4.49±0.47 and 1.89±0.26 (p<0.0001). Eighty-six percent of the patients (13/15) showed high serum FE concentration, mean 1341.3 ng/ml (range 631–3000ng/ml) We detected a positive and significant correlation between TBARS in PBp and BMp with FE levels (r=0.68, p=0.0049) and (r=0.54, p=0.0363) respectively. Additionally, both TBARS levels were highly correlated with the Tfs: r=0.81 (p=0.0025) and r=0.87 (p=0.0005) for BMp and PBp respectively. Also the patients showed high correlation between Tfs and FE (r=0.73, p=0.011).When patients were grouped according to the amount of blood transfusions in greater or less than 20, FE levels were 1793.5±224 and 824±188 (p=0.0063), and Tfs were 72.3%±3 and 45%±8 (p=0.0036) respectively. However, in both groups TBARS in BMp and PBp did not show significant differences. TBARS levels showed no significant correlations with haemoglobin concentration nor platelet number. Conclusions:To date, the role of oxidative stress in MDS has not been fully elucidated. The current results show elevated levels of TBARS in BMp and PBp in patients with low risk MDS, as an evidence of increased oxidative stress, probably caused by the cellular alterations observed in MDS. We can speculate that iron overload may be a contributing factor, as we found a positive correlation between FE and Tfs with TBARS in both BMp and PBp in our patient population. Disclosures:No relevant conflicts of interest to declare.

Treatment of Higher Risk Myelodysplastic Syndrome Patients Unresponsive to Hypomethylating Agents with ON 01910.Na

AbstractAbstract 4010Patients with IPSS intermediate or high risk myelodysplastic syndrome (MDS) have few treatment options once their disease fails to respond to hypomethylating agent therapy. The styryl sulfone mitotic inhibitor ON 01910.Na, inhibits Polo-1 kinase, PI3-kinase and AKT pathways, and the drug has shown promising results in patients (pts) with advanced solid tumors (Jimeno et al, J Clin Onc 26:5504, 2008) and in Phase I/II studies of MDS pts, including those with trisomy 8 (Sloand at al, Proc ASH 2007, #822, Proc ASH 2008, #1651). In an ongoing Phase II clinical trial, we have treated 10 MDS pts unresponsive to at least 4 cycles (range 4–12 cycles) of hypomethylating agent therapy (5 post-azacytidine, 4 post-decitabine, and 1 pt treated with both agents) with ON 01910.Na. The pts had IPSS Intermediate-1 (n=3), Intermediate-2 (n=4) and High (n=3) risk MDS. The study cohort comprised pts with RAEB-1 (4 pts), RAEB-2 (3 pts) and RAEB-T (3 pts), with a median age of 80 years (range 65–86) and 2.3 year median (range 0.4–5.4) prior duration of MDS. Their cytogenetic profile included 5 pts with Good, 4 with Intermediate [t(8,10), t(14,18), +8, and (8+, 19+)], 1 with Poor risk cytogenetics (+8, 5q-, 1p-). At baseline, all pts were red blood cell transfusion-dependent. After the initial 2 pts were treated with 800mg/m2/day × 2day continuous IV infusion (CIVI)/week × 3weeks/month, the subsequent 8 patients received 1800mg/day × 3day CIVI q2weeks/month × 2 months, then monthly. Patients underwent bone marrow sampling and evaluation after every other cycle of treatment. To date, 7 pts have completed at least 2 cycles of therapy, with 2 pts having completed the full treatment course of 7 monthly cycles (median 4.7 for all pts). Responses according to IWG 2006 criteria were: Marrow CR (mCR) (2), Partial response (1), stable disease (SD) with hematologic improvement (HI) (2; 1 HI-E, N, 1 HI-P, N) = 5/10 overall responses (50%). Four pts had SD without HI, 1 pt progressed to AML. mCRs occurred in those with <10% marrow blasts. The 3 patients with trisomy 8 alone or with additional cytogenetic abnormalities achieved 1 mCR, 2 SD, associated with decrements in proportions of +8 karyotypes from 80% and 65% to 15% in the 2 pts with additional abnormalities. Grade 3/4 non-hematologic drug-related or possibly related toxicities occurred in 4 pts: 1 GI, 1 dysuria, 1 fatigue, 1 epistaxis; 2 pts had pulmonary infections unrelated to study drug. Two pts discontinued treatment early due to drug toxicity (1) or AML progression (1). No hematologic toxicities occurred. Nine of 10 pts remain alive (5.4 month median, range 2.5–13+) after study entry. To determine if a biologic response to ON 01910.Na treatment could be quantified, a novel nanoscale immunoassay (NIA) was used to measure changes in AKT kinase family activation in CD34+ cells isolated from the bone marrow specimens collected before and during treatment. In the subset of specimens analyzed to date, phosphorylation of AKT2 decreased by 36% in pts who had a response to therapy, suggesting that ON 01910.Na treatment altered oncogenic signaling in the MDS pts. Completion of NIA analysis will demonstrate whether changes in individual AKT isoforms (implicated in cell cycle arrest and apoptosis) occur uniquely in pts who have a clinical response to treatment. These preliminary data demonstrate encouraging efficacy and drug tolerance with ON 01910.Na in a substantial portion of higher risk MDS patients whose disease had been unresponsive to hypomethylating agent therapy. Accrual and continuing therapy for trial completion are ongoing. Disclosures:Off Label Use: The drug ON01910.Na is an investigational agent. Felsher:Cell Bioscience: On The Advisory Board. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership. Greenberg:Onconova Therapeutics Inc: Research Funding.

Flow Cytometric Score Correlates with Clinical Response to Azacitidine In Intermediate-2 and High Risk Myelodysplastic Syndrome Patients

AbstractAbstract 441New treatment strategies that potentially change the natural course of intermediate (int)-2 and high risk myelodysplastic syndromes (MDS), such as azacitidine, are emerging. Recently, we reported that flow cytometric analysis of bone marrow (BM) in low and int-1 risk MDS is instrumental to identify clinically relevant subgroups. (Westers et al, Blood 2010) Moreover, it was reported that a flow cytometric scoring system (FCSS) is predictive for worse outcome in MDS. (Wells et al, Blood 2003, van de Loosdrecht et al, Blood 2008) The FCSS is a scoring system that allows for a numerical display of immunophenotypic aberrancies in the (im)mature myelo-monocytic lineage. Scores are generated by enumerating abnormalities; e.g. high scores reflect a high number of aberrancies. The current study aimed to investigate the role of this flow cytometry-based scoring system to assess and monitor response to treatment in int-2 and high risk MDS patients treated with azacitidine. Bone marrow aspirates were analyzed by flow cytometry in 18 MDS patients who were treated with azacitidine. Aspirates were drawn before treatment and after every third cycle of azacitidine. Response to treatment was evaluated using IWG-2006 criteria. The median age was 71 (range 50–78). Distribution over WHO 2001 categories was RCMD-RS n=2, RAEB-2 n=7, AML with 20–30% blasts n=6 and MDS/MPD n=3. International prognostic scoring system (IPSS) categories comprised int-2 n=8 and high n=5. In 5 patients the IPSS score could not be assessed due to lack of cytogenetics, these patients were at least int-2 MDS patients. Flow cytometric follow up was available in 12 patients due to short follow up, i.e. in 4 responders, 4 progressive disease (PD) and 4 stable disease (SD) patients; 3 patients stopped due to non-hematologic toxicity, 4 patients died of PD. Median follow up was 7 months (range 3–12). Median pre-treatment Hb was 6.7 mmol/L, platelets 35.5*10e9/L and absolute neutrophil count (ANC) 0.82*10e9/L. Responders had a significant increase in Hb (median 7.8 mmol/L, p=0.04), platelets (291.5 *10e9/L, p=0.03) and ANC (1.4*10e9/L, p=n.s. compared with baseline values). SD and PD patients had a median Hb of 6.5 mmol/L, platelets 69*10e9/L and ANC 0.77*10e9/L. The median pre-treatment flow score was 6 (range 3–8). Interestingly, responders had a significant decrease in flow score from median 5 to median 2 (range 1–3, p=0.005) after 3 months of treatment. No change in flow scores was seen in PD and SD patients after 3 months of treatment (median=6, range 4–8 and pre-treatment FCSS median=6.5, range 3–8). A sustained decrease in flow score was seen in 4 responders after 6 months of treatment (median 2, range 1–3) parallel to a further increase in median Hb (9.0 mmol/L), platelets (278.5 *10e9/L) and ANC (1.7*10e9/L). After 9 cycles of azacitidine, 3 patients were still responsive to treatment (median Hb 9.9 mml/L, platelets 169, ANC 4.07, median flow score 5, range 2–4). The majority of SD and PD MDS patients had aberrant marker expression on myeloid progenitors, such as CD5, CD7 and/or CD56 compared with responsive MDS patients, (63% (5/8) vs 25% (1/4). Moreover, initial loss of aberrant marker expression on myeloid progenitors was detected in one patient who responded to azacitidine treatment. At 9 months, this initially responsive patient and a patient from the stable disease group developed progressive disease. Interestingly, the initially responsive patient showed an increase in the percentage of myeloid progenitors with an aberrant immunophenotype at 6 months; of note, the total percentage of CD34+ cells was less than 3% by flow cytometry. The initial loss of aberrant marker expression on myeloid progenitors might be caused by a relative increase of normal progenitors and decrease of malignant progenitors caused by azacitidine treatment. In conclusion, our data indicate that flow cytometry identifies MDS patients who may benefit from azacitidine treatment by detection of aberrant marker expression on myeloid progenitors. Moreover, the data indicate that the FCSS may be instrumental in selection of SD patients who may benefit from prolonged treatment with azacitidine. Disclosures:Ossenkoppele:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Microsatellite Instability (MSI) In High Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML) Cells Promotes Frameshift Mutations In DNA Repair Genes: Indications for PARP Inhibitor Therapy.

AbstractAbstract 1194Despite major advances in the biology and pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) identification of the most effective and safest form of treatment continue to present a formidable challenge particularly in older patients. Older patients (>70 years) that constitute the majority of MDS/AML patients are often resistant to chemotherapy, achieve short lived remission and are not candidates for stem cell transplantation. Therefore the emphasis is to prolong survival or improve the quality of life. Currently, a number of therapeutic strategies are being evaluated and these include treatment with DNA methyltransferase or histone deacetylase inhibitors. Seminal work in breast cancer have shown that inhibitors of poly ADP ribose polymerase (PARP) activity can selectively target tumour cells through exploitation of inherent DNA repair defects. MDS/AML are characterized by genomic instability (GI) and single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and it has been suggested that the underlying cause of this GI is a defect in double strand DNA repair. We have demonstrated that non homologous end joining, a major pathway for the repair of double strand DNA breaks is overactive and associated with extensive joining errors in primary AML cells. Hence, potentially MDS/AML patients are candidates for PI therapy. We have also shown more recently, that 15% of MDS/AML primary patient cells and cell lines are sensitive to PARP inhibitors (PI) through exploitation of homologous recombination DNA repair defects. To further elucidate the mechanisms that underlie PI sensitivity in MDS/AML we tested for microsatellite instability (MSI) in MDS/AML cell lines and high risk MDS patients and the presence of frameshift mutations in specific DNA repair genes that confer PI sensitivity. MSI is a change in length of a microsatellite allele caused by insertion or deletion of nucleotides that are misincorporated during DNA replication and not removed by the mismatch repair pathway. Using fluorescent PCR analysis, PI sensitive cell lines, P39, KG-1 and Molm-13 showed MSI-high (instability at ≥ 2 loci) at 5 mononucleotide microsatellites, in contrast to 12 PI insensitive cell lines that showed no MSI at these loci. We also show using fluorescent PCR and DNA sequencing that these MSI positive cell lines demonstrate MSI (monoallelic 1–2 base pair [bp] deletion) in the coding region microsatellites of DNA repair genes, Ataxia telancgiectasia mutated gene (ATM), CTiP, and MRE11. Monoallelic 1–2 bp base pair deletions at these loci produced frameshift mutations that induced aberrant gene splicing transcripts in ATM and MRE11 and a markedly truncated CTiP gene transcript. No MSI was detected in DNA repair genes CHK1, RAD50, PTEN, BLM and ATR in these cell lines and no mutations were observed at any DNA repair gene microsatellite in the 13 PI insensitive cell lines. We then determined MSI in high risk MDS patients with or without monosomy 7 (-7/del7q). 13 of 63 (21%) high risk MDS patients showed MSI (9 MSI-low and 4 MSI-high). Of the 13 MSI positive patients, 7 (4 MSI-high, 3 MSI-low) had monosomy 7 and other complex chromosomal abnormalities (Group 1, 54%), 2 (MSI-low) patients had isolated monosomy 7 (Group 2, 15%) whilst 4 patients (MSI-low) had normal cytogenetics (Group 3, 30%). Constitutional DNA from these patients did not show MSI at these loci. Significantly, however, Group 3 with MSI and normal cytogenetics all had widespread UPD and cryptic chromosome changes determined by SNPA. Strikingly, thus all 13 patients with MSI possessed chromosomal abnormalities, both gross and cryptic. Furthermore, 12 patients (19%) found to be cytogenetically normal and lacking UPD and genomic aberrations by SNPA did not show MSI. We have also identified that 3 patients with MSI-high (Group 1) and 1 patient with MSI-low (Group 3) had a monoallelic 1 bp deletion in the CTiP exon coding microsatellite. 1 bp deletion within the coding exon of CTiP resulted in an abbreviated CTiP gene transcript. In conclusion, we have made the important correlation between MSI and subsequent frameshift mutations in specific DNA repair genes with the gross and cryptic chromosomal changes observed in MDS/AML. Identification of a cohort of MDS/AML patients with MSI would herald a significant advancement for the selection of candidates for PI therapy. Disclosures:No relevant conflicts of interest to declare.

Thrombocytopenia Predicts for Poor Survival In Patients with Lower Risk Myelodysplastic Syndromes (MDS)

AbstractAbstract 4021The incidence of thrombocytopenia in MDS is reported to be as high as 40–60% in various reports and is more commonly seen in patients presenting with higher risk disease (∼80%) by International Prognostic Scoring System (IPSS) compared to Int-1 (∼50%) or low risk MDS (12%). Thrombocytopenic MDS patients are at an increased risk of bleeding and hemorrhagic complications have been reported in 3– 50% patients with fatal hemorrhage occurring in 14–20% cases. The prognostic significance of thrombocytopenia in lower risk MDS patients (low+Int-1 risk) has not been clearly defined. To determine whether thrombocytopenia in lower risk MDS patients affects survival, we performed a retrospective study of 1,402 patients diagnosed with MDS between 1987 and 2010. Of these, 687 patients were classified by IPSS as follows: 22%(151/687) were identified as having low risk, 44% (301/687) had Int-1 risk, 4% 26/387) had Int-2, and 30% (209/687) had high risk disease. The overall incidence of thrombocytopenia (platelets <100,000/ul) across all groups was 54% (373/687) with the highest incidence being in the higher risk groups (46%), followed by Int-1 (43%) and low risk (7.5%). Of the 452 lower risk patients, 41% were thrombocytopenic (187/452). Overall, 256 lower risk patients had platelets of <150,000 and these were further divided into three subgroups based on platelet counts ranging between: 1) 100–149,000/ul; 2) 50–99,000/ul; 3) less than 50,000/ul. Median overall survival (OS) was obtained for each group. Statistical significance was calculated using Kruskal-Wallis rank sum test of equality of population. Non-thrombocytopenic lower risk patients showed a statistically significant higher median OS compared to thrombocytopenic patients (3.2 vs. 1.3 yrs p < 0.0005). Median OS progressively decreased with declining platelet range (2.4, 1.6 and 1.2 years) below 150,000/ul. Patients whose platelet counts were less than 50,000/ul had survival rates that were comparable to Int -2 patients (1.2 yrs). We conclude that the presence of thrombocytopenia predicts for poor survival in lower risk MDS patients and this information may be helpful in making therapeutic decisions. Disclosures:No relevant conflicts of interest to declare.

A Phase I Pilot Study of Bystander Vaccine and Lenalidomide Immune Augmentation In Patients with Myelodysplastic Syndrome (MDS)

AbstractAbstract 2925 Introduction:Antineoplastic vaccine strategies aimed at augmenting immuno-surveillance have been limited by the lack of known tumor antigens and the inability to sustain an immune response. A “bystander” vaccine approach has been tested with a K562-GMCSF transfected cell line in combination with autologous cells in acute myeloid leukemia (AML) and in combination with imatinib in chronic myeloid leukemia (CML). Lenalidomide, a thalidomide analogue approved for the treatment of deletion 5q MDS, has stimulatory effects on CD8+ T cells and augments vaccine response in a colon cancer and melanoma model. We present results of a phase I, dose escalation study using a K562-GMCSF-CD40 Ligand transfected, “bystander” vaccine in combination with lenalidomide in International Prognostic Scoring System (IPSS) intermediate and high risk MDS patients who failed hypomethylating agents. Methods:The study was a standard “3+3”, dose escalation phase I design. Eligibility was restricted to patients with a diagnosis of MDS subtypes RAEB-1, and RAEB-2 by World Health Organization classification (WHO), or RAEB-t by FAB criteria. An ANC greater than 500/μl and platelet count greater than 20,000/μl were required. Prior therapy with lenalidomide, secondary MDS, and a diagnosis of proliferative CMML were excluded. GM-CSF and CD40L constructs were transfected into the K562 cell line. Co-transfected clones were selected and subcutaneously injected in the axillary nodal basin of subjects on days 8 and 22 of a 28-day cycle for a total of four cycles. Lenalidomide was administered at a daily dose of 10mg on days 1 through 21 every 28 days until disease progression or limiting toxicity. Toxicities were monitored weekly and BM biopsy repeated after 2 & 4 cycles and at discontinuation of vaccine therapy. The dose escalation of vaccine was 10 × 106 cells in cohort 1, 30 × 106 in cohort 2, 60 × 106 in cohort 3 and 120 cells × 106 in cohort 4. MTD was defined as the highest dose level at which <1 out of 6 subjects experience a DLT. Responses were measured using the international working group criteria (IWG) 2006 for MDS. Results:Between March 2009 and August 2010, eleven patients were enrolled after signing informed consent. The median age was 74 years, with a male predominance (n=9). According to WHO classification, 4 patients had RAEB-1, 6 had RAEB-2, and 1 had AML (RAEB-t by FAB). The IPSS score at study entry was int-1 (4), int-2 (3), and high risk (4). After four cycles of therapy, no treatment related grade 3 or 4 toxicities were seen in cohorts 1–3 (10-60 × 106 cells). Two participants in cohort 2 (30 × 106) developed grade 3 myelosuppression deemed disease related by the investigator. One participant in cohort 2 died while on this study secondary to a non-treatment related pneumonia. Dose limiting toxicity (DLT) was reached in cohort 4 (120 × 106). One patient developed grade 3 dyspnea and fatigue after one injection of vaccine. This cohort will be expanded to a total of 6 patients. Grade 1 toxicities in all cohorts included rash, myelosuppression, nausea, vomiting, and fatigue. The IWG response rates were complete response (CR) in 2/11 (18%), marrow CR (1/11) (9%), and partial response (PR) in 1/11 (9%). The overall response rate was 4/11 (36%). In all patients who achieved a CR the median duration of response was 367 days (136-490). Conclusions:Although the MTD has not been reached, this cell-based immunotherapy was well tolerated. Response rates and the duration of these responses in patients who failed hypomethylating agents is encouraging and warrants further investigation. Accrual to the study is ongoing and updated data will be presented. Disclosures:Lancet:Eisai: Consultancy; Celgene: Honoraria. List:Celgene: Research Funding.

Alterations In the Signaling Profile of Leukemic Progenitors Can Predict the Response of Myelodysplastic Syndrome (MDS) Patients to Azacytidine

AbstractAbstract 2921STAT3 and STAT5 regulate fundamental cellular processes and comprise the most studied signaling molecules of both normal and malignant hematopoiesis. Deregulation of STAT signaling contributes to leukemogenesis and may serve as a treatment target. In leukemic progenitors (LPs), the clustering of STAT3 and STAT5 phosphorylation patterns, both basal and after growth factor stimulation, can be achieved by flow cytometry, leading to the identification of distinct signaling profiles (SPs). In acute myeloid leukemia patients, SPs reflect the biological behavior of the LPs and can distinguish patient subgroups with worse prognosis and/or resistance to treatment. As epigenetic defects of genes involved in cell signaling are frequently observed in cancer cells we investigated the alterations in the SPs of MDS progenitors during azacytidine treatment and their correlation with response, cytogenetics and transfusion requirements.Bone marrow samples of 24 high risk MDS patients were obtained before and 15 days after the initiation of azacytidine in order to assess potential changes in SP before the disappearance of the LPs. According to the IWG response criteria patients were divided into group A (CR, PR and HI, n=10) and group B (stable disease and failure, n=14). Immunomagnetically purified LPs were either left untreated or stimulated with G-CSF and GM-CSF for 15' and then stained intracellularly with monoclonal antibodies against STAT3 and STAT5.The comparisons of basal and potentiated responses before and 15 days after azacytidine initiation were made with Mann-Whitney U-test. Clustering of SPs was performed with hierarchical cluster analysis and was correlated with treatment response, cytogenetics and transfusion dependence by using Chi square or Fisher Exact tests as appropriate. All analyses were performed using SPSS 14.0 software (SPSS Science, Chicago, IL).By clustering the SPs before and 15 days after the initiation of azacytidine we distinguished two subgroups of patients based on both the basal levels and potentiated response to growth factors. Patients with generally weak expression of STAT3 and STAT5 had significantly better response to azacytidine compared to those with strong expression of the same molecules (p=0.035), whereas there were no correlation of SPs with the karyotype (p=0.45) and transfusion rate (p=0.39). In line with the above, we further identified a STAT3+STAT5+ double positive population of MDS progenitors whose pretreatment levels after G-CSF and GM-CSF stimulation were inversely associated with treatment response (figure 1). Additionally, SP kinetics were following the disease course and response to therapy. In two late-stage MDS patients who achieved complete remission the SP was restored to early-stage MDS levels in day 15 after azacytidine initiation (figure 2A). In contrast, the SPs in the majority of non-responding patients remained unaltered (figure 2B), whereas the SP of a relapsing patient reverted to pretreatment levels after an initial restoration to early-stage MDS levels (figure 3). [Display omitted] [Display omitted] [Display omitted] In conclusion, we demonstrate that SP alterations of MDS progenitors during azacytidine treatment can predict clinical response. Moreover, it appears that azacytidine can restore the leukemic signaling in MDS by modifying both the basal and potentiated expression of STAT3 and STAT5. Our findings advocate the differentiating activity of hypomethylating agents, potentially via epigenetic reprogramming of pivotal signaling networks of leukemic progenitors. Disclosures:No relevant conflicts of interest to declare.

Phase I Trial Results for SL-401, a Novel Cancer Stem Cell (CSC) Targeting Agent, Demonstrate Clinical Efficacy at Tolerable Doses In Patients with Heavily Pre-Treated AML, Poor Risk Elderly AML, and High Risk MDS

AbstractAbstract 3298 Background and Rationale:SL-401 (DT388IL3) is a novel cancer stem cell (CSC)-directed recombinant biologic agent that targets the interleukin-3 receptor (IL-3R). IL-3R is over-expressed on acute myeloid leukemia (AML) stem cells relative to normal hematopoietic stem cells. IL-3R is also over-expressed on AML blasts. In pre-clinical studies, SL-401 targets and eradicates CSCs and tumor bulk in both in vitro and in vivo assays, and prolongs survival of immunosuppressed mice harboring human AML xenografts. Study Design:A Phase I dose escalation study was undertaken in 74 patients with relapsed or refractory adult AML (n=56), de novo poor risk elderly AML (n=11), or high risk myelodysplastic syndrome (MDS) (n=7). Thirty-three patients were >/= 2nd salvage. Cytogenetics were unfavorable (36%), intermediate (59%), favorable (0%), and not determined (5%). The median age was 65 years (range, 24–84). Patients received SL-401 via a 15 minute intravenous infusion in one of two dosing regimens for one cycle to determine the maximum tolerated dose (MTD) and to evaluate clinical activity. In Regimen A, 45 patients received doses ranging from 4 to 12.5 ug/kg every other day for up to six doses. In Regimen B, 29 patients received doses ranging from 7.1 to 22.1 ug/kg daily for up to 5 doses. Safety:SL-401 was well-tolerated at clinically active doses. Grade 1–2 hypoalbuminemia was common (68%) but manageable and not dose-limiting. Other less common grade 1–2 adverse events (AEs) included fever and chills, largely infusion-related. Grade >/= 3 AEs included transaminase elevations (20%), mostly transient, and vascular leak (4%) which was the dose limiting toxicity (DLT) at 22.1 ug/kg daily × 5. The MTD was established at 16.6 ug/kg daily for 5 doses; no MTD was defined for the every other day schedule. Notably, there was no treatment-related delayed recovery of the bone marrow. This is consistent with the known over-expression of the drug's target, IL-3R, on leukemia versus normal hematopoietic stem cells. Absolute neutrophil count and hemoglobin remained stable, and near or above baseline pre-treatment levels, throughout therapy and up to day 30 post-infusion. Mean platelet counts were 58,000/uL pre-treatment, 30,000/uL at day 15, and back to baseline by day 30. Efficacy:Overall, we observed 2 complete remissions (CRs), 4 partial responses (PRs) and 14 minor responses (MRs) including 4 with >50% reduction in blasts. In addition, anti-tumor activity, defined as blast reductions or disease stabilization, was seen in 46% of patients with relapsed or refractory AML, 55% of poor risk elderly AML, and 43% of high risk MDS patients. Durable complete remissions (CRs) were induced in 2 patients with chemorefractory AML. One patient, who sustained a CR of >15 months duration, had a previous history of refractory AML, including two stem cell transplants, and had relapsed for a third time prior to entry onto this study. The second patient had a sustained CR of 8 months duration, after failing standard AML induction chemotherapy. This is consistent with the dual mechanism of action of SL-401 targeting both blasts and leukemia stem cells. In addition, the median survival of >/= 2nd salvage AML patients was 3.2 months (95% CI: 2.0, 8.4) and the 12 months overall survival was 22% (95% CI: 5.1, 45.7) which is favorable compared with historical 12-months survival of 2–8%. Also of note, SL-401 demonstrated a clinical anti-CSC effect as evidenced by a decrease in CSC activity in patient bone marrows (n=3), as determined by an ex vivo colony formation assay pre- and post-SL-401 treatment. Conclusions:SL-401 demonstrated clinical activity, including durable CRs as well as blast reductions and disease stabilization. Preliminary signals for a clinical anti-CSC effect and survival benefit in heavily pre-treated patients were also observed. SL-401 had a favorable safety profile and exhibited no myelosuppression. Given these promising results, Phase II studies of SL-401 are planned in patients with advanced AML and MDS. In parallel, SL-401 will be studied in additional Phase I trials as both single agent and in combination with other agents in IL-3R+ leukemias including chronic myeloid leukemias (CML). Disclosures:Konopleva:Stemline: Research Funding. Cirrito:Stemline: Employment, Equity Ownership, Patents & Royalties. Niecestro:Stemline: Consultancy, Equity Ownership. Bergstein:Stemline: Board of Directors, Employment, Equity Ownership, Patents & Royalties. Frankel:Stemline: Research Funding.

Circulating T Cells Derived From Patients with Low Risk Myelodysplastic Syndromes Show Altered Chemokine Receptor Expression

AbstractAbstract 4018Several T cell abnormalities have been described in myelodysplastic syndromes (MDS), and such abnormalities may become important for identification of patients who will benefit from T cell targeting immunosuppressive treatment. Chemokine receptor repertoires are important in the regulation of both T cell migration and function and may therefore be important in the development of MDS. Materials and Methods:The chemokine receptor expression by circulating T cells were investigated by multicolor flow cytometry for patients with newly diagnosed low risk MDS (N=16) and for healthy controls (N=18). CD3+CD8+ and CD3+CD8- cells were examined for expression of CCR2-7, CXCR3-4 and CX3CR1 (only 7 unselected patients examined for CX3CR1 expression). CCR6 and CXCR4 expression was also investigated for specific T cell subsets defined by CD62L and CD45RA expression (naïve, central memory, effector memory and terminal effector). CX3CR1 expression was stratified for CD8+ and CD8- subpopulations according to high, low and negative expression of CCR5. Results:Chemokine receptor profiles showed several differences between low risk MDS patients and controls. Total CD8+ T cells from MDS patients showed increased expression of CCR3 (p=0.005) and decreased expression of CCR7 and CCR4 (p=0.023 and p=0.036 respectively), whereas the CD8- T cells from MDS patients showed increased expression of CX3CR1 (p=0.043). In contrast, CCR6 expression was increased only by CD8+ central memory T cells (p=0.044). Finally, CD8+CCR5- and CD8-CCR5high cells from MDS patients showed increased expression of CX3CR1 compared with the controls (p=0.011 and p=0.049). Discussion:CCR7 is mainly expressed by central memory and naïve T cells whereas CX3CR1 is especially expressed by cytotoxic effector lymphocytes independent of the lymphocyte subclass (i.e. CD4, CD8, delta/gamma and NK cells). The observed changes are in line with a shift from naïve/central memory to effector/effector memory dominance, especially in the CD8+ population. A similar shift has been described previously using CD45RA and CD62L (Zou et al, Leukemia 2009). Our median values are in line with such a shift. CCR6 expression is associated with IL17 production both for CD8+ and CD4+cells, and increased levels of circulating CD4+ IL17 producing cells in low risk MDS have been described. Conclusions:The chemokine receptor profiles of circulating T cells differ between low risk MDS patients and healthy controls, especially for the CD8+ T cell subset. These differences may be important for T cell trafficking and disease development, and they may reflect a shift from naïve to effector/effector memory cell dominance in low risk MDS. Disclosures:No relevant conflicts of interest to declare.

Immunomodulatory Effects of Azacitidine Treatment In High Risk Myelodysplastic Syndrome

AbstractAbstract 1860Immune editing is recognized as being important in the pathogenesis of myelodysplastic syndrome (MDS). In low risk MDS, inappropriate immune reactions are thought to contribute to the initiation and progression of the disease. IL-17 producing CD4+ T-helper cells have been associated with various autoimmune diseases and are found in increased frequencies in low-risk MDS. These increased Th17 frequencies are accompanied by a reduced FoxP3+ T-regulatory (Treg) frequency. On the other hand, in high-risk MDS, the elevated number of bone marrow blasts was associated with increased numbers of Treg. These increased Treg may inhibit immune responses directed against the dysplastic blasts and as such permit progression of the disease. The demethylating agents '5-aza-2'-deoxycytidine and '5-azacitidine (Aza) have shown significant clinical benefits in the treatment of MDS. Although aberrant DNA methylation patterns appear to play a role in the pathogenesis of MDS, clinical responses to DNA demethylating agents could not be related to changes in DNA methylation patterns. Cytokine production and FoxP3 expression are also regulated by epigenetic mechanisms, and FoxP3 expression is increased upon in vitro treatment with Aza. Since cytokines and FoxP3+ Treg may play a role in the pathogenesis of MDS, we set out to investigate the effects of Aza (Vidaza®) treatment on CD4+ T-helper subset frequencies in high-risk MDS patients. Nine patients treated with 75 mg/m2/day subcutaneously for 7 consecutive days in a 28 day cycle were included in this study. Peripheral blood samples were drawn before, 15 days after start of treatment and at the end of the third cycle. There was no change in absolute or relative CD3+ and CD3+CD4+ T-cell numbers during treatment. The proportion of Treg was slightly but not significantly increased (paired student's T-test, p=0.08) on day 15 (11.9 % of CD4+) compared to levels before treatment (9.6 % of CD4+) while numbers dropped to pretreatment levels after the third cycle(9.6 % of CD4+). Cytokine producing cells were enumerated after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin in the presence of Brefeldin A. No significant changes in IFNγ (23.8%, 22.5% and 17.9% of CD4+ cells pretreatment, 15d post treatment and after the third cycle respectively) TNFα (38.3, 35.9 and 39.3% of CD4+ cells) or IL-4 (0.6, 1.0 and 0.6% of CD4+ cells) producing cells were observed. However, the proportion of IL-17 producing cells progressively decreased during treatment (1.1, 0.7 and 0.6% of CD4+ cells; ANOVA, p=0.006). In conclusion, Aza may modulate the immune response and in particular reduce IL-17 responses. This effect of Aza may provide a rationale for expanding the group of patients eligible for Aza treatment with low risk MDS patients, because low risk MDS has been associated with elevated, potential pathogenic, Th17 cells. Disclosures:Ossenkoppele:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Cost effectiveness of deferasirox compared to desferrioxamine in the treatment of iron overload in lower-risk, transfusion-dependent myelodysplastic syndrome patients

Objective:The study evaluated the cost effectiveness of deferasirox (Exjade) compared to non-proprietary desferrioxamine (DFO) for the control of transfusional iron overload in lower risk myelodysplastic syndromes (MDS) patients. A UK National Health Service perspective was adopted.Methods:Recent clinical evidence has demonstrated the efficacy and safety of deferasirox in transfusion-dependent MDS patients with elevated serum ferritin levels. An economic model was used to extrapolate the clinical benefits of iron chelation therapy (ICT) in a cohort of lower risk MDS patients. Costs for drug acquisition, drug administration and monitoring, and quality of life (utility) outcomes associated with mode of drug administration were derived from a variety of sources. The incremental cost per QALY gained for deferasirox was estimated. Costs and outcomes were discounted at 3.5% in line with UK standards.Results:The base-case cost effectiveness of deferasirox versus DFO was estimated to be £20,822 per QALY gained, the key driver being the additional quality of life benefits associated with a simpler mode of administration for deferasirox. A mean survival benefit for both forms of ICT of 4.5 years was estimated. The results were sensitive to drug dose, days of DFO administration, and patient weight.Conclusions:In the UK, a cost per QALY below £20,000–30,000 is considered cost effective. Hence, the results from this economic analysis suggest deferasirox is cost effective in lower risk, transfusion-dependent, MDS patients. Limitations with the analysis include a lack of comparative randomised controlled trial evidence, in particular to differentiate survival and clinical outcomes for deferasirox and DFO.

New treatments for myelodysplastic syndromes.

In the last decade, significant advances have been made in the treatment of patients with Myelodysplastic Syndromes (MDS). Although best supportive care continues to have an important role in the management of MDS, to date the therapeutic approach is diversified according to the IPSS risk group, karyotype, patient’s age, comorbidities, and compliance. Hematopoietic growth factors play a major role in lower risk MDS patients, and include high dose erithropoiesis stimulating agents and thrombopoietic receptor agonists. Standard supportive care should also include iron chelating therapy to reduce organ damage related to iron overload in transfusion-dependent patients. Biologic therapies have been introduced in MDS, as lenalidomide, which has been shown to induce transfusion independence in most lower risk MDS patients with del5q. Hypomethylating agents have shown efficacy in INT-2/high risk MDS patients, reducing the risk of leukemic transformation and increasing survival. Other agents under development for the treatment of MDS include histone deacetylase inhibitors, farnesyltransferase inhibitors, clofarabine and ezatiostat.

Effect of the nonpeptide thrombopoietin receptor agonist eltrombopag on megakaryopoiesis of patients with lower risk myelodysplastic syndrome

Eltrombopag is a nonpeptidyl thrombopoietin receptor agonist. We evaluated the ex vivo effect of eltrombopag on megakaryopoiesis of patients with lower risk myelodysplastic syndromes (MDSs). At a concentration of 0.1 μg/mL, eltrombopag resulted in a significant increase in the number of megakaryocytic colonies in MDS patients and healthy controls compared to baseline. This dose of eltrombopag did not exert any significant change in the proliferation rate or the survival characteristics of patient CD34 + cells that might clinically imply an unfavorable effect on patients’ outcome. These encouraging preclinical data support the rationale of using eltrombopag in the clinic for alleviation of thrombocytopenia in lower risk MDS patients.

5-Azacytidine enhances the anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the poor prognoses for acute myeloid leukemia (AML) and intermediate and high-risk myelodysplastic syndromes (MDS) point to the need for better treatment options. AML and MDS cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, induced meaningful responses in a Phase 1 clinical trial and demonstrated anti-leukemic activity in preclinical models. Recently, it was reported that 5-azacytidine (Vidaza™) prolonged the overall survival of a group of high risk MDS and AML patients. To determine whether the combination of lintuzumab and 5-azacytidine would be beneficial, a mouse xenograft model of disseminated AML was used to evaluate the combination. There was a significant reduction in tumor burden and an increase in overall survival in mice treated with lintuzumab and 5-azacytidine. The effects were greater than that obtained with either agent alone. As the in vivo anti-leukemic activity of lintuzumab was dependent upon the presence of mouse effector cells including macrophages and neutrophils, in vitro effector function assays were used to assess the impact of 5-azacytidine on lintuzumab activity. The results show that 5-azacytidine significantly enhanced the ability of lintuzumab to promote tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytic (ADCP) activities. These results suggest that lintuzumab and 5-azacytidine act in concert to promote tumor cell killing. Additionally, these findings provide the rationale to evaluate this combination in the clinic.

Dose-modified lenalidomide induces sustained hematological response in patients with intermediate to high risk myelodysplasia

No Abstract Available

Cost of the treatment of myelodisplastic syndrome in Brazil

Introduction Myelodysplastic syndrome is an incurable and rare hematological disease that affects the production of blood cells. One aim of treatment is to maintain the blood-cell count to near-normal levels. This is mainly achieved with hematopoietic- growth factors and transfusions. Our objective was to determine the cost of supportive treatment/care for patients with low and intermediate I risk myelodysplastic syndrome in respect to private healthcare plans in Brazil. Method We adapted the National Comprehensive Cancer Network treatment guidelines for intermediate risk myelodysplastic syndrome patients to the Brazilian reality, adopting a decision tree to explore treatment combinations. Then, we calculated the costs for each branch of the tree, according to national prices. We also estimated total costs for a cohort of 100 patients, distributed across treatment combinations according to the expected epidemiology. We assumed a horizon of one year of treatment. Results The mean cost of treatment for low and intermediate I risk myelodysplastic syndrome is US$ 42,758/patient/year. This cost can vary from US$ 24,282 to US$ 121,952, according to patient characteristics and the treatment used. Overall, patients that require immunotherapy with antithymocyte globulins are associated with the highest cost. Those that achieve disease stability solely with the use of erythropoietin were associated with the lowest cost. Conclusion In Brazil, treatment of low and intermediate I risk myelodysplastic syndrome is associated with a mean cost of the order of US$ 42,700/patient/year. New types of therapy have the potential to change this scenario if they can diminish the requirements for supportive care.

Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal disorders characterised by ineffective haematopoiesis with high risk of leukaemia progression. The relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones has been suggested, but valuable criteria to obtain insight into these connections are lacking. This study showed significant increase of CD8 lymphocytes and mature B cells in the bone marrow (BM) compared to peripheral blood (PB) of low risk MDS patients. Different BM levels of Regulatory T cells (Treg) identified two sub-groups in these patients; only the sub-group with lower Treg percentage showed BM recruitment of CD8 lymphocytes. Different levels of CD54 on BM CD8 cells revealed two sub-groups of Intermediate-1 (Int-1) patients. The sub-group with higher CD54 expression on BM CD8 showed high levels of this molecule also on CD4 cells. BM recruitment of CD8 lymphocytes in the low risk group and/or the presence of high CD54 expression on BM CD8 in Int-1 patients were associated with more pronounced dyserythropoiesis and erythropoietin treatment. Our data shed light on the involvement of immune-mediated mechanisms in Low and Int-1 risk MDS patients and suggest that BM versus PB levels of immune effectors could represent useful criteria for a more homogeneous grouping of MDS patients.