Refractory Anemia With Excess Blasts Research Articles

Azacitidine in Myelodysplastic Syndromes: Multicenter Retrospective Study of 34 Long-Responder Patients

AbstractAbstract 4951 Introduction.Azacitidine (AZA) has proven effective in myelodysplastic syndromes (MDS). The duration of haematological response is limited (median 13. 6 months) (Fenaux, 2009), although some patients (pts) show a prolonged response. The French Group (Itzykson 2011) identified some clinical and haematologic parameters (poor ECOG performance status, IPSS intermediate and poor risk cytogenetics, circulating blasts, high transfusion need) independently associated with a a poorer outcome, and these 4 criteria were integrated in a 3-group prognostic score, validated in other cohorts (van der Helm 2011; Breccia 2012). Moreover, a complex karyotype was also predictive of a shorter duration of response. However long term survival was also observed in some of the pts with poor risk features (Itzykson 2012). Methods.These data prompted us to retrospectively analyse our MDS pts treated with AZA who showed a favourable long-lasting response to AZA (i. e: duration of response ≥ 20 months), in order to enucleate the clinical and haematologic features of long-responder pts. The type of response was defined according to IWG criteria (Cheson 2006): Complete Remission (CR); Partial Remission (PR) and Hematologic Improvement (HI). The response duration was measured from the date of achievement of a first response (HI, PR or CR), (also in pts who subsequently achieved a higher response category) until the date of disease progression or death. Overall Survival (OS) was measured from the start of AZA treatment. Moreover, as some of us (Follo, 2009) demonstrated that phosphoinositide-phospholipase C (PI-PLC) beta1 may represent a target for AZA, in a subgroup of pts we quantified the degree of PI-PLCbeta1 methylation and gene expression before and during AZA administration. Results.Thirty-four pts (M/F: 20/14), from eight Institutions, with a median age of 72 (range 52–84) yrs, showed a response duration ≥ 20 months. At AZA onset, WHO diagnosis was: refractory anemia (RA): 1 pt; refractory cytopenia with multilineage dysplasia (RCMD): 1 pt; RCMD with ringed sideroblasts (RCMD-RS): 1 pt; refractory anemia with excess blasts (RAEB)-1: 8 pts; RAEB-2: 15 pts; cronic myelomonocytic leukemia(CMML): 4 pts; AML with 20–30% blasts: 3 pts, MDS with fibrosis (MDS-F): 1 pt. Four pts had therapy-related MDS. IPSS risk was: low: 3 pts; intermediate-1: 6 pts; intermediate-2: 20 pts, high: 5 pts. IPSS cytogenetic risk was: low: 21 pts (61. 8%); intermediate: 8 pts (23. 5%); high: 5 pts (14. 7%) (3 with complex karyotypes and 2 with isolated -7 or 7q-). ECOG-PS was poor (≥ 2) in 2 pts (5. 8%) and < 2 in the other 32 pts. Transfusion need was high (≥ 4 RBC units/8 weeks) in 17 pts (50%), and low or absent in the remaining 17 pts. Three pts (8. 8%) presented circulating blasts. Following Itzykson’s AZA prognostic scoring system, the risk was low in 12 pts (35. 3%), intermediate in 21 pts (61. 8%), and high in 1 pt (2. 9%), respectively. Time from diagnosis to AZA onset was < 6 months in 21 pts, and > 6 months in 13 pts. The pts received a median of 22 cycles of AZA (range: 8–52). The median number of cyles to any first response was 4 (range: 2–10). Twenty pts (58. 8%) showed an at least two-fold increase of platelets after the first cycle of AZA. The best response achieved was: CR in 23 pts (67. 7%), PR in 2 pts (5. 8%), and HI in 9 pts (26. 5%). Cytogenetic remission was achieved in 7 pts (20. 6%). The median duration of response was 24. 5 (range: 23–88) months. A significant toxicity (grade > 2) was observed in 5 (14. 7%) pts. Twenty-two pts (64. 8%) are still maintaining hematologic response, 6 pts (17. 6%) are still alive but discontinued treatment because of disease progression, and 6 pts died, for AML (2 pts), infection (1 pt), haemorrhage (1 pt), myocardial infarction (1 pt), cachexy (1 pt), respectively. Median OS from the start of AZA was 35. 5 (range: 22–120) months. In a subgroup of pts, we observed an increase in PI-PLCbeta1 expression, that was maintained along with the hematologic response. Moreover, PI-PLCbeta1 early increase (during the cycles 1 to 3) was significantly associated with a higher duration of response. Conclusions.Although our data confirm the finding of other Authors, as the majority of long-responder patients showed pre-treatment favourable prognostic factors, a long-lasting hematologic response can be achieved even in a significant fraction of pts presenting one or more poor risk features (IPSS intermediate or high risk cytogenetics, high transfusion need). Disclosures:Finelli:Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Speakers Bureau.

SF3B1 Mutations Are Associated with Favorable Cytogenetics in Patients with Myelodysplastic Syndromes with Ring Sideroblasts

AbstractAbstract 3823 Background:Somatic mutations of splicing factor 3B, subunit 1 (SF3B1) are prevalent in myelodysplastic syndromes (MDS) with ring sideroblasts (RS). Little is known about the clinical phenotype associated with these mutations. Initial studies have reported an excellent event-free survival (EFS) in patients with SF3B1 mutations but subsequent reports did not confirm that observation. Therefore, its clinical significance remains uncertain. Our objective was to determine if the SF3B1 mutational status predicts distinctive morphological, cytogenetic or clinical characteristics in patients with MDS with RS. Methods and patients:Twenty-seven patients diagnosed with MDS and ≥ 15% RS in bone marrow aspiration between 2000 and 2011 at our institution and other hospitals affiliated to the Quebec Leukemia Cell Bank (BCLQ) and with available material for genetic analyses were included in this study. Morphology of samples was revised. After DNA extraction, polymerase chain reaction was performed to amplify SF3B1 mutational hotspots in exons 13 to 15. Amplicons were sequenced by the Sanger method. Clinical and laboratory data were collected retrospectively from hospital medical records and BCLQ database. Results:Point mutations of SF3B1 were found in 48% (13/27) with the following distribution: refractory anemia with RS (RARS): 7/10, refractory cytopenia with multilineage dysplasia (RCMD): 5/10 and refractory anemia with excess blasts (RAEB): 1/7. All mutations were heterozygous nonsense mutations and K700E exon 15 mutations accounted for 85% (11/13) of mutations. The remaining mutations were located in exon 14 at position 666.In the entire cohort, SF3B1 mutations were associated with an older median age at diagnosis (69 vs 60 years, p= 0.007). They were associated with favorable cytogenetics whereas unmutated SF3B1 was associated with unfavorable cytogenetics (8 cases vs 0 with favorable cytogenetics and 0 cases vs 11 with unfavorable cytogenetics for mutated and wild type SF3B1 respectively, p < 0.0001). Median overall survival (OS) was similar in the 2 groups (not reached vs 27 months, p=0.7) but median EFS was higher in patients with mutated SF3B1 (not reached vs 5 months, p=0.02).To evaluate the influence of higher bone marrow blasts on outcomes, analyses were restricted to the RARS and RCMD-RS categories. In that subgroup, SF3B1 mutations were correlated with higher median platelet counts at diagnosis (201 vs 70 × 109/L, p=0,04). In addition, SF3B1 mutations remained associated with favorable cytogenetics, and unmutated SF3B1 with unfavorable cytogenetics (p=0.004). Moreover, SF3B1 mutations were more frequently detected within favorable or intermediate-1 categories of the International Prognostic Scoring System (IPSS) compared to wild type SF3B1 (p=0.04). However, in patients with < 5% bone marrow blasts, EFS was similar between mutated and unmutated SF3B1 (not reached vs 46 months, p =0.4). Conclusion:In this series, SF3B1 mutations are frequent in MDS with RS and they are strongly associated with favorable cytogenetics. They are mostly detected among low-risk groups of the World Health Organisation classification (RARS and RCMD-RS), but they are not independently associated with an increased overall or event-free survival. Disclosures:No relevant conflicts of interest to declare.

Relapse Prediction Post Allogeneic Hematopoietic Cell Transplant for Myelodysplastic Syndromes Is Not Improved with Use of the More Stringent Blast Percentage Categories in the Revised International Prognostic Scoring System

AbstractAbstract 2011▪▪This icon denotes a clinically relevant abstractResidual disease at the time of allogeneic hematopoietic cell transplant (HCT) has been shown to adversely affect outcomes in patients with acute myelogenous leukemia (AML). Assessment of the impact of MDS disease burden at HCT on post-transplant outcomes is less well defined. While blast percentage is easily quantified on morphology and flow cytometry, the blast percentage captures only a fraction of the true MDS clone. Consequently, improved measures of residual disease in MDS patients are needed to improve post HCT outcome prediction. We hypothesized that a more stringent blast percentage cutoff as well as an assessment of recognizable residual cytogenetic disease burden would better predict outcome post HCT. Disease burden at HCT was characterized as reported in the Revised International Prognostic Scoring System (IPSS-R) (<2%, >2-<5%, 5–10% and >10%) and by assessment of cytogenetic residual disease by calculating the percent of metaphases with ongoing cytogenetic abnormalities immediately prior to transplant (% positive metaphases <25%, 25–50%, and > 50%). Metaphase data was unavailable in n=7. Those patients with normal cytogenetics (n=26) were classified in the <25% category.One hundred consecutive patients with MDS undergoing allogeneic HCT at the University of Minnesota between 1995 –2011 were studied. Their median age was 52 (18–69). At diagnosis, the majority of patients had either refractory cytopenia of multilineage dysplasia (RCMD) (25%) or refractory anemia with excess blasts (RAEB) 1 or 2 (49%) and were INT-1 (37%) or INT-2/High risk (60%). Donor type was related donor (52%), matched unrelated (13%) and umbilical cord blood (35%). Half received myeloablative conditioning and Karnofsky Performance Status (KPS) > 80 in 95%. Results:(Table)Both higher blast percentage at HCT and high percentage of residual cytogenetically positive cells correlated with inferior overall survival as well as more frequent NRM. Relapse incidence was similar in those with blasts up to 10%. Percentage of abnormal metaphases was not predictive of relapse. Conditioning intensity was the only factor that impacted the 2 year relapse rate with decreased relapse seen with myeloablative conditioning.Table% of Patients Per StrataStrata2 year Overall Survival (OS)% (CI 95%)P value2 year Non-Relapse Mortality (NRM) % (CI 95%)P value2 year Relapse % (95% CI)P valuePercentage Blasts at HCT49%≤2%46% (32–59%)<0.0133% (20–46%)0.0229% (15–42%)0.8928%>2–<536% (19–53%)46% (27–66%)21% (6–37%)16%5–10%50% (25–71%)38% (14–61%)25% (11–56%)7%>10%0%86% (55–100%)NEPercentage Abnormal Metaphases At HCT42%<25%56% (39–70%)0.0326% (12–39%)0.0631% (16–46%)0.3311%25–50%50% (18–75%)40% (11–69%)10% (0–27%)47%>50%28% (16–42%)51% (35–67%)25% (12–38%Conditioning IntensityMyeloablative50%42% (28–55%)0.9948% (33–63%)0.3314% (4–24%)0.02Reduced Intensity50%39% (26–53%)34% (21–48%)37% (22–51% Conclusion:These data suggest that survival and relapse prediction are not improved by using the more stringent IPSS-R blast cutoff of < 2% versus <5% at HCT. Additionally, our data suggest that use of a disease burden measure as represented by % residual positive metaphases at HCT may serve as another predictor of outcome. Survival and NRM were worse with >10% blasts or > 50% residual positive metaphases. These patients require additional therapy or new strategies to improve post HCT outcomes. A refined measure of disease burden at HCT using both blast count and % residual abnormal metaphases may allow for more comprehensive prediction of post HCT outcomes in MDS. Disclosures:No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) with Erythroid Predominance Exhibits Clinical and Molecular Characteristics that Differ from Other Types of AML

The clinical importance of erythroid predominance in bone marrow of patients with acute myeloid leukemia (AML) is controversial. These cases represent a heterogeneous group of diseases that historically have been classified into different categories. We studied 313 AML patients and specifically compared the clinical, cytogenetic, and molecular features of cases of AML with erythroid predominance, arbitrarily defined as ≥50% erythroid precursors, to AML cases without erythroid predominance. We also assessed 51 patients with a high-grade myelodysplastic syndrome (MDS), refractory anemia with excess blasts (RAEB). All neoplasms were classified according to the World Health Organization classification. With the exception of therapy-related AML/MDS, the presence of erythroid predominance in variously classified categories of AML was associated with a survival advantage. In addition, AML with erythroid predominance had a lower frequency of cytogenetic abnormalities as well as a lower frequency of mutations involving NPM1, NRAS and FLT3 as compared with AML without erythroid predominance. We conclude that the clinical, cytogenetic, and molecular features of AML with erythroid predominance in the non-therapy-related setting are much closer to those of a high-grade myelodysplastic syndrome than they are to other types of AML.

A new recurrent chromosomal translocation t(3;11)(q13;q14) in myelodysplastic syndromes associated with overexpression of the ILDR1 gene

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by ineffective hematopoiesis and an increased risk of evolution to acute myeloid leukemia (AML). In this study, the combination of conventional cytogenetic, FISH studies and molecular techniques allowed us to unveil a novel recurrent t(3;11)(q13;q14) causing the overexpression of the immunoglobulin-like domain-containing receptor (ILDR1) gene. The analysis of gene expression was extended to Refractory Anemia (RA) and Refractory Anemia with excess blasts (RAEB) cases revealing ILDR1 overexpression in 36% of RAEB subgroup. The biological implications of the ILDR1 overexpression in MDS pathogenesis and its potential prognostic significance should be further investigated.

SF3B1 mutations are prevalent in myelodysplastic syndromes with ring sideroblasts but do not hold independent prognostic value

SF3B1 mutations were recently reported in myelodysplastic syndromes (MDSs), especially in the presence of ring sideroblasts (RSs). We sought to define the interaction between SF3B1 mutations, morphology, karyotype, and prognosis in MDS with more than or equal to 15% RS (MDS-RS). We studied 107 patients with MDS-RS, including 48 with refractory anemia with RS (RARS), 43 with refractory cytopenia with multilineage dysplasia (RCMD)-RS, 11 with refractory anemia with excess blasts-1 (RAEB1)-RS, and 5 with RAEB2-RS. SF3B1 mutations were detected in 53 (∼ 50%) patients: 35 RARS (73%), 16 RCMD-RS (37%), and 2 RAEB1-RS (18%). In univariate analysis, the presence of SF3B1 mutations was associated with better overall (P < .01) and leukemia-free (P < .01) survival; however, in both instances, significance was completely accounted for by World Health Organization morphologic risk categorization. In other words, when RARS and RCMD-RS were analyzed separately, there was no additional prognostic value from the presence or absence of SF3B1 mutations.

Identification of genes underlying different methylation profiles in refractory anemia with excess blast and refractory cytopenia with multilineage dysplasia in myelodysplastic syndrome

BackgroundMyelodysplastic syndrome (MDS) is a preleukemic condition that transforms into acute myeloid leukemia. However, the genetic events underlying this transformation remain poorly understood. Aberrant DNA methylation may play a causative role in the disease and its prognosis. Thus, we compared the DNA methylation profiles in refractory anemia with excess blast (RAEB) to those in refractory cytopenia with multilineage dysplasia (RCMD).MethodsBone marrow samples were collected from 20 patients with primary MDS (9 with RAEB and 11 with RCMD), and peripheral blood samples were collected from 4 healthy controls. These samples were assessed using a commercial whole genome-wide methylation assay. Methylation-specific polymerase chain reaction (PCR) was used to detect the methylation of candidate gene promoters in RAEB and RCMD.ResultsMicroarray data revealed significant hypermethylation in 69 genes within RAEB but not RCMD. Candidate genes were mapped to 5 different networks, and network 1 had the highest score due to its involvement in gene expression, cancer, and cell cycle. Five genes (GSTM5, BIK, CENPH, RERG, and ANGPTL2) were associated with malignant disease progression. Among them, the methylated promoter pairs of GSTM5 (55.5% and 20%), BIK (20% and 0%), and ANGPTL2 (44.4% and 10%) were observed more frequently in RAEB.ConclusionDNA methylation of GSTM5, BIK, and ANGPTL2 may induce epigenetic silencing and contribute to the increasing blasts and resulting MDS progression; however, the functions of these genes were not determined. Further study focusing on epigenetic silencing using various detection modalities is required.

Sole Trisomy 22 Not Associated with inv(16) in Myelodysplastic Syndrome

Trisomy 22 is closely associated with inv(16) or t(16;16) and could be a marker of cryptic rearrangement of CBFB/MYH11 in acute myeloid leukemia (AML). Trisomy 22 not associated with CBFB/MYH11 rearrangement is a rare event. Here, we report a case diagnosed as refractory anemia with excess blasts-2 (RAEB-2) with sole trisomy 22 in the absence of CBFB/MYH11 rearrangement. The cytogenetic study of bone marrow cells disclosed trisomy 22 in 10% of metaphase cells analyzed. The other chromosomal abnormalities were not found. Fluorescence in situ hybridization (FISH) using CBFB/MYH11 probe to detect cryptic inv(16)(p13q22) showed negative result. We also excluded rearrangements of chromosome 5, 7, 8, 20, and ETV6 by FISH. Sole trisomy 22 not associated with inv(16) is a true entity. (Ewha Med J 2012;35(1):62-64)

P4-12-11: Myelodysplastic Syndrome Post Primary Breast Cancer Treatment: Cases from a Community Cancer Center: 1990–2010.

Abstract Background: Myelodysplastic syndrome (MDS) has not been well documented or described post breast cancer (BC) treatment. Methods: A cohort of all breast cancer patients from 1989 to 2010 at our institution were followed for incidence of blood disorders of any type by our registry system and by linkage to the Surveillance, Epidemiology and End Results (SEER) registry for our area. All cases of post breast cancer leukemia were reviewed for incidence of MDS from a cohort of 9846 cases diagnosed between 1990 and 2010. Coding of cases was assigned by the SEER registry using ICD-0 codes for type of leukemia from pathology reports. 20 cases of MDS were identified and confirmed using SEER coding for MDS and death certificate review if applicable. Variables of interest included age, race, treatment received, recurrence and treatment for recurrence, time interval from breast cancer diagnosis to leukemia diagnosis and time from leukemia diagnosis to follow up or death. Cytogenetic studies were available on 5 of the 20 patients. Results: Mean age of the patients was 56 years, range 33–79, with 65% &amp;lt; age 65. 90% of the patients were white and 10% Asian. TNM BC stage distribution was stage 0=2, stage I=8, stage II=8, and stage III=2. MDS was diagnosed at a median time post BC diagnosis of 65 months, range 8–205 months including 5 patients treated for recurrence and median 47 months, range 8–205 months excluding patients with recurrence. Nine patients had initial chemotherapy treatment, two without initial adjuvant therapy received chemotherapy for recurrence, and 9 received radiation only. Eight of eleven patients who received chemotherapy were treated with doxorubicin. All eleven received cyclophosphamide. Five of the 20 cases had MDS, not otherwise specified (NOS) (9989), 4 cases had MDSaAML, 3 MDS treatment related (9987), 3 refractory anemia (RA) (9980), 3 refractory anemia with excess blasts (RAEB) (9983), 1 RA with sideroblasts (9982), and 1 MDS with 5q deletion. Three of the five patients with cytogenetics available had chromosome 7 abnormalities and one had 11q21,23. Eleven of the 20 patients (55%) died of MDS or MDSaAML with a mean survival of 17 months, range 0.5-72 months. Six patients were alive at follow up with mean survival 56 months, range 43–89. Two of the surviving patients had stem cell transplants. The remaining three patients died of other causes with mean survival 66 months, range 7–109 months. Mean survival of all patients was 36.26 months, range .53-109 months with a significant difference in survival time between the patients that died of MDS, those alive at follow up and those that died of other causes (F statistic 6.56, p=.008). Discussion: MDS is a significant complication of breast cancer treatment with radiation and/or chemotherapy with a high rate of associated mortality. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-12-11.

Mutations of the Spliceosome Complex Genes Occur In Adult Patients but Are Very Rare In Children with Myeloid Neoplasia

Abstract 2797Somatic point mutations occur frequently in adult patients with myelodysplastic syndromes (MDS) and are associated with distinct clinical features such as overall survival, genomic aberrations and clonal transformation. Recently a number of new genes had been identified which are involved in methylation and chromatin regulation (TET2, DNMT3A, ASXL1). Although mutations of these genes are present at high frequency in adult cohorts, they are very rare events in children with myeloid disease. Based on the results from a large-scale whole exome sequencing study of adult patients with myeloid neoplasia, a new group of genes involved in the formation of spliceosome complex was discovered to be recurrently somatically mutated in approximately 40% of patients with MDS and 55% of chronic myelomonocytic leukemia (CMML) cases (Yoshida, et al., unpublished data). Spliceosomes are macromolecular RNA-protein complexes that remove noncoding introns from precursor mRNA. We hypothesized that the disruption of the spliceosome complex might play a driving role in the leukemogenesis in pediatric MDS. Using targeted re-sequencing we investigated the 3 exclusive hotspots of 2 spliceosome genes that were found to be mutated in adult MDS: U2AF1 (S34), U2AF1 (Q157) and SFRS2 (P95).We analyzed a total of 339 WHO-defined pediatric cases: juvenile myelomonocytic leukemia (JMML), n=112; secondary CMML, n=12; Down syndrome-associated MDS, n=6; de novo acute myeloid leukemia (AML), n=12, MDS-related AML, n=50; refractory anemia with excess blasts (RAEB), n=52; RAEB in transformation (RAEB-T), n=11; and refractory cytopenia (RC), n=84.We found two heterozygous missense mutations: SFRS2 c.284C>T/P95L in a patient with JMML, and U2AF1 c.101C>A/S34Y in a patient with refractory cytopenia. The first patient presented with non-syndromic JMML with somatic mutation of PTPN11 c.226G>C/E76Q, normal cytogenetics, increased HbF(80%), splenomegaly, monocytosis, and was 4.5 years old at diagnosis. The patient underwent successful SCT. The analysis of buccal-swab DNA confirmed the somatic status of the SFRS2 mutation. The second patient was diagnosed at age of 17 years with systemic mastocytosis positive for c-kit D816V mutation, and with associated clonal hematological non-mast cell lineage disease: refractory cytopenia with normal cytogenetics. SCT was also performed with success on this patient. To ensure that the low rate of mutations found in our pediatric cohort was not due to technical issues, we also sequenced major mutation sites of U2AF1 and SFRS2 genes in 19 adult patients with MDS: refractory anemia (RA), n=8; RC with multilineage dysplasia (RCMD), n=3; RAEBI/II, n=5; CMML, n=3. We found 4 heterozygous changes: SFRS2 missense mutation c.284C>T/P95L in two patients with RA and RAEBII; SFRS2 deletion c.284_307/P95RfsX120 in one RA patient, and U2AF1 c.470A>C/Q157P in one RA patient. Interestingly, 3 out of 4 patients with a mutated spliceosome gene harbored a mutation of the ASXL1 gene, but none was mutated for DNMT3A.In summary, re-sequencing of 3 spliceosome gene hotspots revealed the presence of heterozygous mutations in 2/339 children and 4/19 adults with myeloid disease. The analysis of another gene-hotspot implicated in the MDS whole exome study, SF3B1 K700 is ongoing, but all pediatric cases analyzed to date were negative. The drastically reduced frequency of spliceosome mutations in pediatric compared to adult myeloid malignancies suggests a different pathogenetic mechanism in childhood disease, and fits well with previous reports that somatic mutations of non-Ras-pathway genes, such as DNMT3A, are less prevalent in pediatric cohorts. The functional impact of spliceosome mutations on leukemogenesis warrants further study. Disclosures:No relevant conflicts of interest to declare.

Single Cell Network Profiling (SCNP) Reveals Age-and Disease-Based Heterogeneity in Healthy Individuals and in Patients with Low Risk (LR) Myelodysplastic Syndrome (MDS)

Abstract 2788 Background:Normal hematopoiesis changes with age through unknown mechanisms. Low risk (LR) myelodysplasia (MDS) is characterized by cytopenias arising through inefficient hematopoiesis. We hypothesized that both of these differences might result from changes in responsiveness to external signaling. To test this hypothesis, we utilized SCNP, a multiparametric flow cytometry-based assay that simultaneously measures both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators, quantitatively at the single cell level (Kornblau et al. Clin Cancer Res 2010). Methods/Objective:SCNP was applied to examine baseline and intracellular signaling responses induced by the extracellular modulators EPO and GCSF in bone marrow (BM) mononuclear cells (BMMC) derived from healthy donors (n=15) and MDS (n=9) patients. The effects of donor age on signaling profiles in healthy BMMC were compared between samples collected by BM aspirate from 6 subjects aged 23–43 years (“younger”) and from the BM present in hip replacement samples from 9 subjects aged 54–82 years (“older”). Signaling profiles were also determined for 9 LR MDS patients aged 53–83 years and compared to the age-matched healthy “older” control. Metrics used for analysis included fold change, total phosphorylation levels, and the Mann-Whitney U statistic model. Results:There were no differences in the frequency of CD34+ cells (R2= 0.006, p= 0.78) between “younger” and “older” healthy donor samples, suggesting any differences observed in signaling would likely be due to donor age rather than sample source. There was no age-related difference in functional signaling ability in response to GCSF-induced p-STAT1, p-STAT3, & p-STAT5 levels; however, early erythroblasts and normoblasts from healthy “older” donors were significantly less responsive to EPO, as measured by induced phospho (p)-STAT5 levels than those derived from healthy “younger” donors (e.g. R2= 0.654 p=0.008 for erythroblasts and R2=0.628 p=0.0004 for normoblasts). Signaling profiles classified Refractory Anemia with Excess Blasts (RAEB) patients into 2 categories based on differences in EPO- and GCSF-induced signaling (Table 1). Compared to age-matched healthy “older” controls, one subset was characterized by a high % of RBC precursors (CD45lo nRBC) and increased p-STAT5 levels in response to EPO and the other subset by a high % of myeloid cells with robust GCSF-induced p-STAT3 & p-STAT5 responses in both total myeloid and CD34+ cells. By contrast, patient samples with Refractory Anemia with Ringed Sideroblasts (RARS) had a high % of CD45lo nRBC but lacked robust p-STAT5-induced signaling after modulation with EPO. Conclusions:Overall, these data show the feasibility of using the SCNP assay in BM samples to functionally characterize signaling pathways simultaneously in different cell subsets of healthy donors and patients with MDS. In healthy individuals, age-related differences in EPO signaling were discovered. In LR MDS, differences in signaling were observed between cases and in comparison to the data from healthy controls. Deciphering signaling profiles in healthy donor versus MDS patient samples may result in improved, biologically-based disease classification that informs more effective patient management. The clinical relevance of these findings in terms of disease course and treatment is currently under investigation.Table 1Healthy Younger (n=6)Healthy Older (n=9)RAEB (n=5)RARS (n=2)MPD/MDS (n=2)Median (Range)Pt 003Pt 008Pt 004Pt 006Pt 013Pt 005Pt 010Pt 011Pt 014CD45lo nRBC%14 (8–22)31 (24–53)62697576055188Myeloid38 (26–45)24 (20–42)2021848092721747EPO→p-STAT5 nRBCUu0.68 (0.60–0.73)0.57 (0.53–0.62)0.770.680.540.610.520.620.510.660.54GCSF→p-STAT3Myeloid0.58 (0.55–0.61)0.56 (0.50–0.61)0.600.550.890.810.860.500.530.640.51CD340.78 (0.73–0.79)0.70 (0.64–0.80)NDND0.920.880.910.510.680.900.64GCSF→p-STAT5Myeloid0.60 (0.57–0.62)0.58 (0.55–0.64)0.630.560.840.820.870.510.530.620.50CD340.85 (0.80–0.86)0.76 (0.72–0.86)NDND0.860.900.940.530.740.930.68 Disclosures:Cleary Cohen:Nodality Inc.: Consultancy, Equity Ownership. Huang:Nodality Inc.: Consultancy, Equity Ownership. Cesano:Nodality: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership. Ware:Nodality Inc.: Employment, Equity Ownership.

Next-Generation Deep-Sequencing Enables a Quantitative Monitoring of RUNX1 Mutations in 534 Patients with Myelodysplastic/Myeloproliferative Neoplasms and Myelodysplastic Syndromes

Abstract 2792 Introduction:Somatic mutations of key candidate genes have gained interest as biomarkers predicting poor survival in myelodysplastic/myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX1 (runt-related transcription factor 1) deregulations constitute such a disease-defining molecular aberration and are usually tested applying a combination of denaturing high-performance liquid chromatography and direct Sanger sequencing. Patient-specific RUNX1 mutations were proposed to represent clinically useful molecular alterations to follow disease progression from MDS to s-AML. Study design: Using genomic DNA obtained from mononuclear cells a next-generation amplicon deep-sequencing (NGS) assay targeting the complete coding region of RUNX1 was developed on a longitudinal series of 116 retrospective samples obtained from 25 patients collected between 11/2005 and 6/2010 (454 Life Sciences, Branford, CT). Subsequently, this assay was applied to characterize an unselected prospectively collected MPN/MDS patient cohort during their course of disease. Results: Here, we present analyses on a cohort of 534 patients (females: 200; males 334). The median age was 72.0 years (25.2–95.7 years). The cohort included 149 chronic myelomonocytic leukemias (CMML), 11 cases with 5q- syndrome, 10 cases with refractory cytopenia with unilineage dysplasia (RCUD), 15 cases with refractory anemia with ring sideroblasts (RARS), 105 cases with refractory cytopenia with multilineage dysplasia (RCMD), 135 cases with refractory anemia with excess blasts-1 (RAEB-1), 87 cases with refractory anemia with excess blasts-2 (RAEB-2), and 22 cases with t-MDS, respectively. In total, 130 RUNX1 mutations were observed in 17.8% (95/534) of these patients. The mutational clone size ranged from 1.7% to 94% and amounted to a median of 31%. In comparison to our data from an AML cohort, i.e. 460 patients at diagnosis with 112 (24.3%) cases mutated, the median clone size was about 10% lower in MPN/MDS. In detail, 74.7% (71/95) of patients harbored one mutation, whereas 25.3% (24/95) of cases harbored two (17.9%; 17/95) or >=3 (7.4%; 7/95) mutations. The 130 RUNX1 mutations were characterized as follows: 29% frame-shift mutations, 42% missense, 14% nonsense, 13% exon-skipping, and 2% in-frame insertion/deletion alterations, respectively. The following codons were recurrently mutated: Arg174 (8/95 patients; 9.4%), Arg177 (6/95 patients; 7.0%), and Arg135 (5/95 patients; 5.3%). The mutations were predominantly located in the RHD domain (55%) and TAD domain (13%) and in cases with 2 or more alterations only 15% (4/24) harbored mutations outside of these regions. In all cases with 3 concomitant mutations both domains were affected (4/4 patients). Further, cases with >1 RUNX1 mutation were more frequently observed in CMML (33.3%; 8/24 mutated), RAEB-1 (17.2%; 5/29 mutated) and RAEB-2 (34.5%, 10/29 mutated) as compared to other disease groups, respectively. In subsequent serial analyses including 56 samples from 22 cases the amplicons carrying the respective known alteration were analyzed with increased coverage for disease status monitoring (in median 833 reads/amplicon were sequenced; leading to a sensitivity of ∼1:800). With a median time span of 2.5 months between the molecular analyses a total of 2 to 4 samples per patient were analyzed. In 5/22 patients, this assay then allowed to monitor the treatment success of allogeneic stem cell transplantation: in 3 cases the mutations known before transplantation became undetectable; in 2 cases the same mutated clones still remained detectable at a level of 0.2% and 23%, respectively. Further, in 17 patients quantitative assessment of mutated RUNX1 read counts was used to monitor stable disease (n=12) or allowed to follow an increasing clone size in 3 patients that progressed into s-AML (39% -> 53% increase; 31% -> 42% increase; 7% -> 37% increase). Summary: Unbiased techniques such as deep-sequencing provide the required diagnostic specificity and sensitivity to enable classification and individualized monitoring of disease progression. We here demonstrate that amplicon-based NGS is a suitable method to accurately detect and quantify the broad spectrum of molecular RUNX1 aberrations with high sensitivity. It is therefore suitable for therapy guidance. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Harbich:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Allogeneic Hematopoietic Stem Cell Transplantation for Children with Myelodysplastic Syndrome: A Report of a Korean Single Center Experience

Abstract 4500 Purpose:Accounting for 4% of childhood hematological malignancy, myelodysplastic syndrome (MDS) is very rare in children than in adults. Therapy of these disorders has been associated with intensive treatment related toxicities and a high risk of relapse. Children with refractory anemia (RA)/refractory anemia with excess blast (RAEB) usually do not respond to acute myeloid leukemia (AML)- type chemotherapy, and chemotherapy without hematopoietic stem cell transplantation (HSCT) resulted in survival rates below 30%. Since failure rates after HSCT are lower in this group when treated at diagnosis, HSCT should be strongly considered, especially when a fully matched sibling donor (MSD) is available. The optimal treatment for children with RA/RAEB without matched sibling donor is presently undetermined. Some of them have stable disease course and require no therapy for years. However, aggressive treatment with HSCT using alternative donors should be considered for severe cytopenia. We analyzed the results of allogeneic HSCT in 16 children who were diagnosed with de novo MDS in a single Korean center. It is the first report about the outcome of HSCT for Pediatric MDS in Korea. Patients and Methods:Between November 1997 and June 2011, 16 children with MDS underwent allogeneic HSCT at Asan Medical Center. These patients had RA (n=7), and RAEB (n=9). Peripheral blood smears, bone marrow aspirates and biopsies of all patients were assessed by central morphology review according to the WHO classification. HSCT was recommended if the patients developed neutropenia (ANC<500/μ L) or transfusion dependency. Median age at transplant of 9 males and 7 females was 8.9 years (range 1.6–20.3). Cytogenetic analysis at diagnosis was available in 15 patients. Nine of them had a normal karyotype and 6 patients had miscellaneous chromosomal changes, but nobody had monosomy 7. Six patients (33%) were grafted from an MSD, whereas the remaining were transplanted from an unrelated donor (UD) (n=6), umbilical cord blood (n=3) or a haplo-identical family donor (n=1). All patients received myeloablative conditioning including busulfan (n=13) or total body irradiation (n=3). The median number of transfused CD34 cells was 8.2×106/kg for bone marrow or peripheral blood stem cell transplantation, and 1.3×105/kg for umbilical cord blood transplantation (UCBT). Graft-versus-host disease (GVHD) prophylaxis with cyclosporine and methotrexate was used for 14 patients, and cyclosporine and MMF were used for UCBT. Results:Sustained neutrophil engraftment was achieved in 11 patients, and five patients experienced graft failure. One patient died of subarachnoid hemorrhage on day 1 before engraftment, and 4 patients experienced primary graft failure; all patients were regrafted from haplo-identical related donors. Three patients are alive without disease, but one patient died 103 days after third HSCT from CMV pneumonia. The cumulative incidence of grade II-IV acute GVHD in the 15 evaluable patients was 0.27, and they were all manageable. Two patients developed extensive chronic GVHD; one patient died from disease progression to AML 579 days after HSCT, the other died from fungal pneumonia and intracranial hemorrhage 113 days after HSCT. With a median follow-up of 6.0 years (range 0.1–14), the cumulative incidence of treatment related mortality (TRM) and relapse was 0.19 and 0.20, respectively. The Kaplan-Meier estimates of overall survival (OS) and event free survival (EFS) at 5 years were 0.64 and 0.41. The diagnosis of RAEB was positively associated with relapse (P=0.02) and significantly decreased OS (P=0.024). Donor type was not significantly associated with TRM, graft failure, relapse and outcome. While acute GVHD did not affect the outcome, chronic GVHD significantly affected OS (P=0.0013). Conclusions:Though our results showed considerable early engraftment failure rates (31.3%), most of all were successfully regrafted form haplo-identical related donors, and showed comparable treatment outcome. Allogeneic HSCT following a myeloablative conditioning can offer a high probability of cure for children with MDS, however, efforts to further reduce the rejection, relapse and TRM in patients undergoing allogeneic HSCT are needed. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Early Hematopoietic Stem Cell Transplant Is Associated with Improved Outcomes in Children with MDS,

Abstract 4126 Background:Childhood myelodysplastic syndrome (MDS) is a rare, heterogeneous disorder that is clinically distinct from adult MDS. Hematopoietic stem cell transplant (HSCT) is the treatment of choice, but there is no consensus regarding patient, disease, or treatment-related factors that predict outcomes after HSCT.Materials and Methods: We performed a retrospective review of 37 consecutive pediatric patients who received allogeneic HSCT for MDS at the University of Minnesota Amplatz Children’s Hospital between 1990 and 2010. The median age at transplant was 11 years (range 1–21 years). Twenty patients had primary (de novo) MDS and 17 had secondary MDS (4 treatment-related, 8 with preceeding idiopathic aplastic anemia, 3 with Schwachman Diamond syndrome, and 2 familial). Those with Fanconi Anemia were excluded. Cytogenetics at diagnosis included monosomy 7 (n=21), trisomy 8 (n=7), normal/other (n=8). Thirty-one had refractory cytopenia (RC) and 6 had refractory anemia with excess blasts (RAEB) according to the modified WHO MDS classification. Patients were scored according to the International Prognostic Scoring System as low risk (n=1), intermediate-1 (Int-1; n=15), intermediate-2 (Int-2; n=21), or high risk MDS (n=0). Six patients received pre HSCT chemotherapy. Immediately prior to transplant, 27 had <5% bone marrow (BM) blasts and 10 had ≥ 5% blasts. Time from diagnosis to transplant was <140 days in 18 patients and ≥140 days in 19. Donor sources included umbilical cord blood (UCB; n=9), HLA-matched related donor (MRD; n=15), HLA-matched unrelated donor (MURD; n=7), and HLA-mismatched unrelated donor (MMURD; n=6). All patients received myeloablative conditioning prior to transplant (Cy/TBI n=35, Bu based n=2). The majority (70%) received cyclosporin based GVHD prophylaxis. Results:Neutrophil engraftment occurred in 89% (95%CI 77–97%) of patients at a median of 23 days (range 12–40). Patients transplanted after year 1999 were more likely to engraft (RR 2.27; 95% CI 1.06–4.88, p=.04). Overall survival (OS) was 70% (95%CI 53–82%) and 53% (95% CI 36–68%) at 1 and 3 years. In multivariate analysis (MVA), OS at 1 year was increased in patients who did not receive pre HSCT chemotherapy (RR of death 0.04; 95% CI 0–0.50, p=.01) and decreased in those with an IPSS score of Int-2 (RR of death 11.96; 95%CI 1.29–110.74, p=.03). Disease free survival (DFS) was 62% (95%CI 44–75%) and 48% (95% CI 31–63%) at 1 and 3 years. In MVA, factors associated with improved DFS at 3 years include having secondary MDS (RR of death or relapse 0.13; 95% CI 0.02–0.69 p=.02), undergoing HSCT after 1999 (RR 0.06; 95% CI 0.01–0.70, p=.02), not receiving pre HSCT chemotherapy (RR 0.06, 95% CI 0.01–0.36, p<.01), and a short interval (<140 days) from diagnosis to transplant (RR 0.21; 95% CI 0.05–0.85, p=.03). Those with an IPSS score of Int-2 had a significantly lower DFS (RR 3.96; 95% CI 1.12–14.00, p=.03). WHO classification, cytogenetics and pre HSCT blast percentage had no significant impact on either OS or DFS. The relapse rate at 2 years was 20% (95% CI 733%). Factors associated with decreased relapse include having secondary MDS (RR 0.04; 95% CI 0.01–0.21, p<.01) and not receiving pre HSCT chemotherapy (RR 0.21; 95% CI 0.05–0.85, p=0.03). Treatment-related mortality (TRM) was 25% (95%CI 11–39%) at 1 year. The risk of TRM was increased in patients with a pre HSCT blast count ≥ 5% (RR 6.65; 95% CI 1.60–27.67, p= 0.01) and was decreased in patients who did not receive pre HSCT chemotherapy (RR 0.07; 95% CI 0.01–0.69, P=.02). At 100 days the cumulative incidence of grades II-IV and III-IV acute graft versus host disease (GVHD) was 41% (95% CI 24–57%) and 16% (95% CI 5–28%), respectively. The incidence of chronic GVHD at one year was 19% (95% CI 6–32%). Conclusions:Our results suggest that in order to achieve optimal outcomes, children with MDS should be referred for allogeneic HSCT soon after diagnosis and that unlike in adult MDS, pre HSCT chemotherapy does not appear to improve outcomes. Disclosures:No relevant conflicts of interest to declare.

Differential Prognostic Effect of IDH1 Versus IDH2 Mutations in Myelodysplastic Syndromes: A Mayo Clinic Study of 277 Patients

Abstract 971Unlike the case in acute myeloid leukemia, there is limited information on the prognostic impact of IDH mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (six R132S and two R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts (RARS), 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1, and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (p=0.0004; HR 4.0, 95% CI 1.9–8.8), revised International Prognostic Scoring System (IPSS-R) risk category (p<0.0001), and red cell transfusion need (p=0.002) as independent predictors of inferior survival (Figure 1). In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (p=0.001; HR 7.0, 95% CI 2.3–20.8). The presence of IDH2 R140Q did not affect overall (p=0.54) or leukemia-free (p=0.81) survival (Figure 2). The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

SF3B1 Mutations Are Prevalent in Myelodysplastic Syndromes with Ring Sideroblasts but Do Not Hold Independent Prognostic Value

Abstract 460 Background:SF3B1 mutations were recently reported to occur in myelodysplastic syndromes (MDS), especially in the presence of ring sideroblasts (RS) (Papaemmanuil et al. Leukemia Research 2011, Volume 35, Supplement 1, page S18). We sought to accurately define the interaction between SF3B1 mutations, bone marrow morphology, karyotype and prognosis in MDS with ≥15% RS (MDS-RS). Methods:Mayo Clinic databases and cell banks were queried to identify patients with MDS and ≥15% RS (MDS-RS). All study patients were required to have undergone bone marrow examination and cytogenetic evaluation at diagnosis. Pathology slides, including iron stains, were centrally re-reviewed to accurately quantify bone marrow RS percentage and confirm World Health Organization (WHO) morphologic categories. Detailed analysis of clinical and cytogenetic parameters was performed to risk stratify patients according to the revised International Prognostic Scoring System (IPSS-R) (Greenberg et al. Leukemia Research 2011, Volume 35, Supplement 1, page S6). All patients were annotated for their mutational status involving JAK2, MPL and IDH. PCR sequencing was used to identify SF3B1 mutations. Results I: Baseline patient information:The study is currently ongoing; to date, results are availab1e in 88 patients (median age 72 years, 69% males) including 36 (41%) patients with refractory anemia with RS (RARS), 36 (41%) with refractory cytopenia with multilineage dysplasia and ≥15% RS (RCMD-RS), 11 (12%) with refractory anemia with excess blasts (RAEB)-1 and ≥15% RS (RAEB1-RS) and 5 (6%) with RAEB2-RS. Median (range) values were 9.1 g/dL (5.8–15.7) for hemoglobin, 4.1 × 109/L (1.2–35.2) for leukocytes and 164 × 109/L (6–585) for platelets. IPSS-R risk categories were 20% very good, 49% good, 11% intermediate, 3% poor and 16% very poor. Karyotype was normal in 60%, other very good/good risk 16%, intermediate-risk 2%, and poor/very poor in 22%; 14% had monosomal karyotype (MK). Six (7%), four (5%) and zero (0%) patients displayed IDH, JAK2 or MPL mutations. Red cell transfusion need was documented in 27 (31%) patients. Results II: Prevalence and distribution of SF3B1 mutations and clinical correlates:SF3B1 mutations (all heterozygous) were detected in 40 (∼46%) of all 88 study patients: 25 (63%) K700E, 6 (15%) K666N/Q/R, 3 (8%) E622D, 2 (5%) H662D/Q, 2 (5%) Y623C, 1 (3%) R625C, and 1 (3%) T663I. One patient each displayed both SF3B1K700E and IDH2R140Q or SF3B1K700E and JAK2V617F. SF3B1 mutational frequencies were 69% for RARS, 36% for RCMD-RS, 18% for RAEB1-RS and 0% for RAEB2-RS (p=0.0007). SF3B1 mutations clustered with normal (59%) and other very good/good risk (57%) karyotype and were infrequent in poor/very poor karyotype (5%; p=0.0004). Additional significant correlations were noted between SF3B1 mutations and lower IPSS-R risk category (p=0.0004), advanced age (p=0.006), higher platelet count (p=0.0003). Results II: SF3B1 mutations and prognostic relevance in MDS-RS:To date, 68 (∼77%) deaths and 10 (∼11%) leukemic transformations have been documented. Median follow-up for living patients was 82 months. Univariate survival analysis considering all 88 study patients identified the following as strongly significant risk factors: IPSS-R (p<0.0001), WHO morphologic categories (p<0.0001), cytogenetic risk categories (p<0.0001), and transfusion need (p=0.0003). In multivariable analysis, only IPSS-R and transfusion need remained significant. In univariate analysis, the presence of SF3B1 mutations was significantly associated with better overall (p=0.04) and leukemia-free (p=0.03) survival; however, in both instances, significance was completely accounted for by WHO morphologic risk categorization and the morphology-adjusted p values were 0.6 and 0.2, respectively. In other words, when RARS and RCMD were analyzed separately, there was no additional prognostic value from the presence or absence of SF3B1 mutations (Figures 1 and 2; p=0.67 and 0.44, respectively). [Display omitted] [Display omitted] Conclusions:SF3B1 mutations are prevalent in RARS and RCMD-RS but do not provide additional, WHO morphology-independent or IPSS-R-independent, prognostic information. Disclosures:No relevant conflicts of interest to declare.

Azacitidine Treatment of Lenalidomide-Resistant Myelodysplastic Syndrome with Deletion 5q

Abstract 2774 Background:Lenalidomide is a highly effective treatment for lower risk transfusion dependent myelodysplastic syndrome (MDS) patients with deletion 5q [del(5q)]. Approximately two thirds of patients become transfusion independent for a median duration of 2 years or longer (List et al, NEJM). Effective treatment alternatives after lenalidomide treatment failure are limited. We examined the response to treatment with azacitidine in del(5q) MDS patients after lenalidomide treatment failure. Methods:MDS patients with del(5q) who were treated with azacitidine after lenalidomide failure were identified through the Moffitt Cancer Center (MCC) MDS database and individual charts reviewed. Data collected included demographics, disease baseline characteristics, duration of response to lenalidomide, responses to azacitidine by international working group (IWG) 2006 criteria, transformation to acute myeloid leukemia (AML), and overall survival (OS). Descriptive statistics were used for analysis and Kaplan Meier estimates were used for OS and AML transformation. All analysis was conducted using SPSS version 19.0 statistical software. Results:Between July 2005 and June 2011, 18 del(5q) MDS patients treated with azacitidine were identified. The median age was 66 years (50–83), all patients were Caucasian, and male predominance (67%; 12 patients). ECOG performance status was 0–1 in 94% of the patients. The median duration of follow up from date of azacitidine initiation was 645 days. According to the WHO classification, 7 patients (38.9%) had MDS with isolated del(5q), 7 patients (38.9%) refractory cytopenia with multilineage dysplasia (RCMD), 2 patients (11.1%) refractory anemia with excess blasts 1 (RAEB-1), and 2 patients (11.1%) refractory anemia (RA).The international prognostic scoring system (IPSS) risk category was low in 2 (11.2%), intermediate 1 (int-1) in 14 (77.8%), and int-2 in 2 (11.2%) patients. Based on karyotype, 7 patients (38.9%) had isolated del(5 q), 9 (50%) del 5q +1 abnormality, and 2 (11.2%) with del 5q +2 or more cytogenetic abnormality. All patients were transfusion dependent.The median number of lenalidomide cycles prior to azacitidine was 12 (1–38), with median duration of response 319 days (17–1169). The median number of azacitidine cycles administered was 6 (2–23). The median duration of treatment was 183 days (43–592). The best response to azacitidine by IWG 2006 criteria was one (7.1%) complete response (CR), 2 (5.6%) marrow CR (mCR), 7 (38.9%) hematological improvement (HI), 4 (22.2%) had stable disease, and 4 (22.2%) had progressive disease (PD). The overall response rate was 56%.The HI cell lineage responses were 9 out of 18 (50%) erythroid, 3 out of 13 (23%) HI-P (Platelets), and 2 out 9 (22.2%) HI-N (neutrophils). The median OS was 749 days (95%CI 435–1063) from the time of starting azacitidine. The rate of AML transformation was 27.8% (n=5); median time to AML transformation from azacitidine start was 864 days (95%CI 360–1368). Conclusion:To our knowledge this is the first report demonstrating the activity of azacitidine in patients with del(5q) MDS after lenalidomide treatment failure. Response rates are similar to those reported in non-del(5q) patients providing azacitidine as an effective option for salvage treatment. Larger cohort of patients is needed to confirm these findings. Disclosures:Komrokji:Celgene: Honoraria, Research Funding, Speakers Bureau. Lancet:Celgene: Research Funding. List:Celgene: Consultancy.

Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic Study of 277 patients

Unlike the case with acute myeloid leukemia, there is limited information on the prognostic impact of isocitrate dehydrogenase (IDH) mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (6 R132S and 2 R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts, 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1 and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (P=0.0004; hazard ration 4.0, 95% confidence interval 1.9-8.8), revised International Prognostic Scoring System risk category (P<0.0001), and red cell transfusion need (P=0.002) as independent predictors of inferior survival. In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (P=0.001; hazard ration 7.0, 95% confidence interval 2.3-20.8). The presence of IDH2R140Q did not affect the overall (P=0.54) or leukemia-free (P=0.81) survival. The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS.

Pilot study on combination of azacitidine and low-dose cytarabine for patients with refractory anemia with excess blast

This study analyzed the outcomes of the combination of azacitidine and low-dose cytarabine in patients newly diagnosed with refractory anemia with excess blast (RAEB). Patients were treated with azacitidine 75 mg/m(2) for 7 days subcutaneously and cytarabine 20 mg/m(2) intravenously for 7 days every 28 days. The assigned regimen was repeated for two cycles, then the patients treated with azacytidine alone until progression or allogeneic stem cell transplantation (allo-SCT). Eighteen patients with 5 RAEB-1 and 13 RAEB-2 were enrolled in the current study. After two cycles of the combination therapy, responses were achieved in nine patients (50.0%): four complete response (CR) (22.2%), one partial response (5.6%), two marrow-CR (11.1%), and two hematologic improvement (11.1%). Four patients (22.2%) progressed to acute leukemia during two cycles of the combination therapy. The 1-year overall survival (OS) was 87.5% for the early response group (responses at two cycles) and 0% for the late response group (responses at four cycles, p = 0.042). Plus, the median survival time was 476 days (range, 37-718 days) for the early response group and 221 days (range, 193-249 days) for the late response group. The 1-year OS was 100% for the patients who underwent allo-SCT and 73.4% for those without allo-SCT. In summary, the combination therapy showed promising response rate when compared to treatment with azacitidine alone. However, it was limited in terms of preventing leukemic transformation. Allo-SCT would seem to be the only available treatment that can alter disease progression.

Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

Direct cell–cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain ‘stemness’. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y397], and HSP90α/β and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y397], and HSP90α/β formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y397], and HSP90α/β and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC–MSC interactions. Further, because FAK is an HSP90α/β client protein, these results suggest the utility of HSP90α/β inhibition as a target for adjuvant therapy for myelodysplasia.