Higher Bone Marrow Blast Percentage Research Articles

Risk factors for leukemia transformation in patients with myelodysplastic syndromes

Objective: To explore the risk factors in leukemia transformation (LT) in those with myelodysplastic syndromes (MDS) . Methods: From January 2012 to December 2020,data on 320 patients with newly diagnosed primary MDS were gathered from the MDS center. The clinical features and molecular characteristics are explored. Additionally, a retrospective analysis of risk factors for the development of acute leukemia from MDS was done. Results: The median follow-up was13.6 (0.4-107.3) months. 23.4% (75/320) of the MDS patients had LT group. Significant differences between the LT group and non-LT group can be seen in age (P<0.001) , bone marrow blast percentage (P<0.001) , bone marrow fibrosis (P=0.046) , WHO classification (P<0.001) , IPSS-R (P<0.001) and IPSS-R karyotype group (P=0.001) . The median number of mutation of LT group was 1 (1, 3) , that in non-LT group was 1 (0, 2) ,which had a statistical difference (P=0.003) .At the time of the initial diagnosis of MDS, the LT group had higher rates of the TP53 mutation (P=0.034) , DNMT3A mutation (P=0.026) , NRAS mutation (P=0.027) and NPM1 mutation (P=0.017) . Compared with the mutations at first diagnosis and LT of six patients, the number of mutations increased and the variant allele frequencies (VAF) increased significantly in LT patients. Higher bone marrow blast percentage (Refer to <5% , 5% -10% : HR=4.587, 95% CI 2.214 to 9.504, P<0.001, >10% : HR=9.352, 95% CI 4.049 to 21.600, P<0.001) , IPSS-R cytogenetic risk groups (HR=2.603, 95% CI 1.229-5.511, P=0.012) , DNMT3A mutation (HR=4.507, 95% CI 1.889-10.753, P=0.001) , and NPM1 mutation (HR=3.341, 95% CI 1.164-9.591, P=0.025) were all independently associated with LT in MDS patients, according to results of multivariate Cox regression. Conclusion: Bone marrow blast percentage, IPSS-R cytogenetic risk groups, DNMT3A mutation, and NPM1 mutation are independent risk factors in LT for MDS patients.

Hyperfibrinogenemia as a Poor Prognostic Indicator in Myelodysplastic Syndrome.

BackgroundMyelodysplastic syndrome (MDS) is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells. It has been demonstrated that fibrinogen (FIB) is associated with disease risk in several cancer types. Coagulation and fibrinolysis problems are widespread in MDS patients. Therefore, FIB might be one of these indicators. We thus examined the role of FIB levels in the prognosis of MDS.MethodsA cohort of 198 MDS patients were retrospectively analyzed to explore the prognostic value of the plasma FIB levels at diagnosis. Patients were divided into the high FIB group and low FIB group. The prognostic significance of FIB was determined by univariate and multivariate Cox hazard models.ResultsIn our cohort, the FIB levels in 198 MDS patients were higher than those in 100 healthy donors (3.9 g/L vs 2.9 g/L, P < 0.0001). MDS patients with high FIB levels had significantly shorter overall survival (OS; P = 0.001) and decreased leukemia-free survival (LFS; P = 0.036). Multivariate cox proportional hazards regression analysis indicated that, in addition to older age, gender, lower HB, poorer karyotype for OS, lower NE, and higher bone marrow blast percentage for OS and LFS, elevated FIB level was also an independent adverse prognostic factor for OS (P = 0.045) but not for LFS (P = 0.188).ConclusionElevated FIB levels may be associated with mortality risk among MDS patients and could predict disease progress and patient prognosis. Thus, assessment of FIB levels may promote the determination of the prognosis of MDS patients.

The Prognostic Value of Pretherapy Peripheral Blood Inflammatory Indices in Myelodysplastic Syndromes.

BackgroundInflammation appears to have a critical role in carcinogenesis tumor growth according to emerging research. The platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), and plasma C-reactive protein (CRP) are considered to reflect the systemic inflammatory response and clinical prognosis. The prognostic value of inflammatory indices in myelodysplastic syndrome (MDS) patients remains unclear.MethodsA total of 213 MDS patients were enrolled for the study. Univariate and multivariate analyses were performed to determine the prognostic significance of various indicators, including PLR, NLR, and CRP.ResultsMDS patients with higher PLR, NLR, and CRP levels had significantly shorter overall survival (OS). Based on univariate analysis, age (≥60 years), gender (men), lower hemoglobin level (<10 g/dl), higher bone marrow blast percentage (>5%), poorer karyotype, and higher Revised International Prognostic Scoring System (IPSS-R) score were significantly associated with shorter OS. Patients with higher CRP levels had shorter leukemia-free survival (LFS, P = 0.041). However, higher PLR and NLR had no significant influence on LFS (P > 0.05). Multivariate Cox proportional hazards regression analysis indicated that high PLR and CRP were also independent adverse prognostic factors for OS in MDS.ConclusionsElevated PLR and CRP predict poor prognosis independent of the IPSS-R and provide a novel evaluation factor for MDS patients.

Decreased serum apolipoprotein A1 level predicts poor prognosis of patients with de novo myelodysplastic syndromes

BackgroundMyelodysplastic syndromes (MDS) is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells. It has been demonstrated that apolipoproteins A1(ApoA1) are associated with disease risk in many cancer types. However, there still lacks evidence regarding the link between ApoA1 and MDS. This study was designed to investigate the prognostic value of pretreatment ApoA1 levels in MDS patients.MethodsWe retrospectively analyzed a cohort of 228 MDS patients to explore the prognostic value of the serum ApoA1 levels at diagnosis. Patients were divided into the high ApoA1 group and the low ApoA1 group. The prognostic significance was determined by univariate and multivariate Cox hazard models.ResultsMDS patients with low ApoA1 levels had significantly shorter overall survival (OS, P < 0.0001) along with a higher frequency of TP53 mutation (P = 0.002). Based on univariate analysis, age (≥ 60 years), gender (male), lower levels of hemoglobin (< 10 g/dl), HDL (≤0.91 mmol/L), higher bone marrow blast percentage (> 5%), higher IPSS-R scores and poorer karyotype were significantly associated with decreased OS. However, low ApoA1 level did not influence leukemia-free survival (LFS, P = 0.367). Multivariate Cox proportional hazards regression analysis indicated that low ApoA1 level (≤ 1.02 g/L) was also an independent adverse prognostic factor for OS in MDS (P = 0.034).ConclusionsDecreased ApoA1 level predicts a poor prognosis of MDS patients and thus provides a novel evaluation factor for them that is independent of the IPSS-R system.

BSG (CD147) Serum Level and Genetic Variants Are Associated with Overall Survival in Acute Myeloid Leukaemia

Basigin (BSG, CD147) is a multifunctional protein involved in cancer cell survival, mostly by controlling lactate transport through its interaction with monocarboxylate transporters (MCTs) such as MCT1. Previous studies have found that single nucleotide polymorphisms (SNPs) in the gene coding for BSG and MCT1, as well as levels of the soluble form of BSG (sBSG), are potential biomarkers in various diseases. The goal of this study was to confirm BSG and MCT1 RNA overexpression in AML cell lines, as well as to analyse soluble BSG levels and selected BSG/MCT1 genetic variants as potential biomarkers in AML patients. We found that BSG and MCT1 were overexpressed in most AML cell lines. Soluble BSG was increased in AML patients compared to healthy controls, and correlated with various clinical parameters. High soluble BSG was associated with worse overall survival, higher bone marrow blast percentage, and higher white blood cell count. BSG SNPs rs4919859 and rs4682, as well as MCT1 SNP rs1049434, were also associated with overall survival of AML patients. In conclusion, this study confirms the importance of BSG/MCT1 in AML, and suggests that soluble BSG and BSG/MCT1 genetic variants may act as potential AML biomarkers.

Elevated mature monocytes in bone marrow accompanied with a higher IPSS-R score predicts a poor prognosis in myelodysplastic syndromes

BackgroundMyelodysplastic syndromes (MDS) is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells. Clinically, elevated mature monocyte in bone marrow is often observed, but its clinical value still remains unclear.MethodsWe retrospectively analyzed a cohort of 216 MDS patients to explore the prognostic value of the percentage of mature monocyte in bone marrow (PMMBM). All patients were divided into elevated PMMBM group and the normal group by 6% PMMBM as the cut-off value.ResultsOur results showed that PMMBM> 6% was associated with inferior overall survival (OS) (P = 0.026) along with higher-risk IPSS-R (P = 0.025) and higher frequency of IDH2 mutation (P = 0.007). Multivariate analyses showed that besides older age (> 60 years) for OS, gender (male) for OS, lower neutrophil count (< 0.8 × 109/L) for OS, higher bone marrow blast percentage (> 5%) for OS and LFS, poorer karyotype for OS, elevated PMMBM was also an independent adverse prognostic factor for OS in MDS (P < 0.0001) but not for LFS (P = 0.736).ConclusionsThese findings indicate that increased PMMBM may assists Revised International Prognostic Scoring System (IPSS-R) to predict a poor outcome and provide a novel evaluation factor for MDS patients especially when their karyotype analyses fail.

Clinico-Genomic Profiling and Clonal Dynamic Modeling of Spliceosome-Mutant Myelodysplastic Syndrome and Acute Myeloid Leukemia

Introduction: The identification of driver mutations has dramatically changed the management of myeloid malignancies. More recently, mutations have been identified in RNA splicing machinery in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) (Yoshida et al. 2011). Recurring missense mutations have been observed in the RNA splicing factor 3B subunit 1 (SF3B1), serine and arginine-rich splicing factor (SRSF2) and U2 small nuclear RNA auxiliary factor 1 (U2AF1). In this study, we sought to decipher the disease-specific features and clinical outcomes of individual spliceosome mutations in patients with MDS and AML. Methods: We conducted a retrospective observational study of 40 patients with one or more mutations in 3 spliceosome genes (SRSF2, U2AF1, SF3B1) from the University of Massachusetts (UMass) Leukemia Registry. Demographics, peripheral blood data, clinical parameters such as treatment offered including bone marrow transplant and response to chemotherapy was longitudinally recorded. We performed clonal dynamic modeling to identify clonal evolution in individual patients with spliceosome-mutant MDS or AML. Results: Longitudinal data was available for 83% (33/40) patients with an average follow up duration of 91 weeks. Average age at diagnosis was 75±4 years which was consistent across the 3 mutational cohorts. A mortality of ~35% was observed in our cohort (Table 2). MDS was diagnosed in 48% (19/40), AML and AML-MRC were diagnosed in 33% (13/40) and 13% (5/40), respectively (Table 1). SRSF2 was the most common spliceosome mutation (50%) and SF3B1 and U2AF1 were equally divided in the remainder. Cytogenetic abnormalities were found in 5/33 (15%) in SRSF2, 4/33 (12%) in U2AF1, and 1/33 (3%) in the SF3B1 groups. Of the 16 patients with SRSF2-mutant hematological conditions, 7/16 (40%) had persistent disease (PD) which was similar across the other two spliceosome genes. Compared to U2AF1- and SF3B1-mutant patients, SRSF2-mutant patients presented with a higher WBC count (mean 45.9 x 103/mcl ± 13.6 x 103/mcl), higher bone marrow (BM) cellularity (73% ± 4.4%), and higher BM blast percentage (24% ± 6.9%) at the time of diagnosis (Table 3), which was independent of the FLT3 status. The most commonly recurring co-occurring mutants with SRSF2 were TET2 (occurred 8/16 times along with SRSF2 with a rate of 50%), IDH2 (31%), STAG2 (25%) and RUNX1 (25%). In the U2AF1-mutant cohort, RUNX1 (30%) and TET2 (20%) were the most frequent co-mutants. In the SF3B1 cohort, BCOR (29%) and KRAS (29%) were the most frequently mutated and shared few co-occurring mutations with SFSR2-mutant and U2AF1-mutant cohorts as demonstrated in the circosplots (Figure 3). The heatmap further elaborates this association and alludes to a possible genetic signature associated with favorable or adverse clinical outcomes (Figure 2). Conclusion: This study deciphers among SRSF2-, U2AF1-, and SF3B1-mutant MDS/AML and demonstrates that SRSF2-mutant MDS/AML presents with higher WBC count, BM cellularity, and BM blast burden. SRSF2-mutant MDS/AML carries the most dismal prognosis amongst the spliceosome mutations. The co-mutational landscape of SF3B1-mutant MDS/AML is distinctly different from SRSF2 and U2AF1 cohort. While we only identified 1 individual with SF3B1 mutation with longitudinal data and hence no meaningful conclusion is possible, SRSF2 cohort shows poor response to standard chemotherapy in 2/3 individuals (Figure 1). We also found that BRAF and CEBPA are not co-occurring mutations within the spliceosome-mutant cohort, suggesting mutual exclusivity (Figure 2). While our understanding of MDS/AML expands, new drugs are constantly under development and have led to significant improvements in the disease outcome in younger patients. Seiler et al recently studied a new oral drug H3B-8800, which binds the SF3B complex in a competitive manner and has demonstrated potent inhibition and lethality of the spliceosome-mutant cells (Seiler et al. 2018). After demonstration of survival advantage in vivo in murine models, the drug is now in Phase 1 clinical trial which is pending completion soon. Our dataset provides clinical correlates of spliceosome-mutant AML/MDS in a longitudinal fashion and also alludes to a possible genetic signature associated with favorable or adverse outcomes in these patients. Disclosures No relevant conflicts of interest to declare.

Clinical Significance of Monosomal Karyotype in MDS

To investigate the frequency, karyotype characteristics and prognosis significance of monosomal karyotype (MK) in newly diagnosed MDS patients. The clinical, laboratorial and follow-up data of 202 MDS patients received the chromosome karyotype test in Department of Hematology, Ningbo Hospital of Zhejiang University from 2009 to 2018 were analyzed retrospectively, the monosomal karyotype features, clinical characteristics and their effects on the prognosis of MDS patients also were analyzed. Among 202 cases of MDS, 25 (12.38%) confirmed to be the MK. The abnormality of chromosome 5 (60.00%), 7 (56.00%), 17 (56.00%), 15 (56.00%), 13 (40.00%) and 20（40.00%）were common in monosomal karyotype. MK+-MDS (MDS with monosomal karyotype) patients had higher bone marrow blast percentage than MK--MDS (MDS without monosomal karyotype) patients, the median are 6.25% and 3.00% (P＜0.01) respectively, but there were no difference in age, sex, hemoglobin level, white blood cell count, neutrophile granulocyte percentage, platelet count, blood blast percentage, serum ferritin, folic acid and vitaminB12 between MK+-MDS and MK--MDS. The overall survival time of MK+-MDS and MK--MDS patiens with chromosome 3, 5, 7, 13, 15, 17 abnormalities was significantly shorter than MK--MDS and AK+MK--MDS patients (MDS with abnormal karyotype but without monosomal karyotype) , the MK+-MDS patients had a median survival time of 7.33 months, but the median survival time had not been reached in MK--MDS and AK+MK--MDS patients had not been reached by the end of the follow-up, and could not be assessed (P＜0.01). The monosomal karyotype is a poor prognosis factor for newly-diagnosed MDS patients. The poor prognosis suggested by monosomal karyotype may be related with the abnormality of 3, 5, 7, 13, 15 and 17 chromosome.

Factors Associated with Early Therapy Initiation in Patients (pts) with Myelodysplastic Syndromes (MDS) in the Connect® MDS/AML Disease Registry

Factors Associated with Early Therapy Initiation in Patients (pts) with Myelodysplastic Syndromes (MDS) in the Connect® MDS/AML Disease Registry

Uniparental Disomies (UPD) of Chromosome 13q Is Associated with Shorter Disease-Free Survival in Adult Patients (Pts) with De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Approximately 45% of adult AML pts do not have cytogenetic abnormalities visible by microscopic karyotype analysis and are classified as CN-AML. However, these pts can still have one or more UPD (the loss of a chromosome or its segment and duplication of the remaining chromosome or segment), since UPDs are not detectable by typical karyotyping methods. Because UPDs have been shown to contribute to malignant transformation and outgrowth in various cancers and leukemias, we sought to identify recurrent UPDs in CN-AML pts and explore their associations with disease presentation, gene mutations and outcome. We used Infinium OmniExpress SNP arrays to detect UPDs in a cohort of 425 pts, who were similarly treated with intensive chemotherapy on Alliance for Clinical Trials in Oncology therapeutic trials. Pts having early death (<30 days) were excluded. The mutation status of 80 genes were previously assessed by targeted sequencing (Leukemia, 2017;31(10):2211-8).UPDs of 10 different chromosome arms were detected in >1% of pts (Figure A). The most commonly occurring UPDs were UPD of 13q (present in 7.5% of pts), UPD of 6p (in 2.4% of pts) and UPD of 11p (in 2.4% of pts). We examined associations between UPDs of 13q, 6p and 11p with demographic and clinical diagnostic characteristics. UPD of 6p was associated with higher peripheral blood (PB) blast percentage (median 86% vs 70%, P=.02); UPD of 11p was associated with lower platelet counts (41 vs. 60 x 109/L, P=.03) and higher PB blast percentage (85% vs 60%, P=.002); and UPD of 13q was associated with higher white blood cell count (53 vs 29 x 109/L, P=.02), higher PB blast percentage (77% vs 58%, P=.004) and higher bone marrow blast percentage (80% vs 69%, P=.005). We also examined the associations between UPDs and gene mutations present in >2% of CN-AML pts in our sample series. 13q UPD significantly co-occurred with FLT3-internal tandem duplication (ITD) (P<.001), and UPD of 11p significantly co-occurred with bi-allelic CEBPA mutations (P<.001). The co-occurrence of FLT3-ITD with 13q UPD likely has biologic relevance since FLT3 is located on 13q, whereas the association between 11p UPD with CEBPA mutation is less clear, as CEBPA is on 19q.Assessment of the association between UPDs and outcome was performed. Because of differences in induction therapies between pts younger and older than 60 years (y), outcome analyses must be performed separately in these two age groups. Based on sample size limitations we were only able to assess the impact of 11p UPD and 13q UPD on achievement of complete remission (CR), disease-free survival (DFS) and overall survival (OS) in the 315 pts younger than 60 y. UPD of 11p was associated with longer OS (3 y rates, 83% vs 45%, P=.02), and UPD of 13q was associated with shorter DFS and OS (3 y rates, 0% vs 38%, P<.001; 17% vs 49%, P<.001) (Figure B). To examine the combined effect of mutations and UPDs on outcome we performed multivariable analyses (MVAs). We found that UPD of 13q was significantly associated with shorter DFS (P=.009) after adjusting for hemoglobin levels (P=.02), FLT3-ITD status (P <.001), and mutation of DNMT3A (P<.001), RUNX1 (P=.002) and WT1 (P=.03) (Table). UPD of 13q did not impact on CR or OS when considered in a MVA, and UPD of 11p did not remain significant in MVA.Finally, since FLT3-ITD and UPD of 13q often co-occurred and were both found to be associated with shorter DFS in the MVA of adult younger pts, we examined younger pts who harbored FLT3-ITD for differences in DFS associated with UPD of 13q. We found those pts with 13q UPD had significantly shorter DFS (P=.003) compared to those without (Figure C), which further demonstrates 13q UPD status is useful for prognostic stratification independent from its association with the known prognostic marker FLT3-ITD.These data show the incorporation of 13q UPD might improve current genetic risk stratification models. Follow-up studies investigating the mechanistic roles of these recurrent UPDs, especially the relationship between UPD of 11p and bi-allelic CEBPA mutation, could shed light on novel pathways underlying leukemic transformation and proliferation in CN-AML. [Display omitted] DisclosuresPowell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria.

Predictive and Prognostic Markers in Adults With Acute Myeloid Leukemia: A Single-Institution Experience

BackgroundAcute myeloid leukemia (AML) is a heterogeneous malignancy with diverse genetic abnormalities, clinical presentations, and outcomes. Known predictive and prognostic factors in AML include age, performance status, comorbidities, cytogenetics, and molecular mutations. Identifying prognostic and predictive factors can inform the choice of induction therapy and outcomes prediction. Patients and MethodsA retrospective review was performed of 137 adult AML patients from 2010 to 2015. Predictors of complete remission (CR) and overall survival (OS) were determined for patients treated with 3+7 (3 days of anthracycline and 7 days of cytarabine) or hypomethylating agent. Variables associated with CR or OS were assessed using univariate Cox regression and a multivariate Cox model. ResultsThe average age was 65 years and 91 patients (66%), sample size is 137 patients had primary AML. Patients in the 3+7 induction group were younger, had a higher bone marrow blast percentage, and more de novo AML compared with those in the hypomethylating agent group (P < .001, P < .001, P = .005, respectively). Univariate logistic regression for CR showed a significant association between age (P < .001), choice of induction (P < .001), and monosomy (P = .015), although only induction with 3+7 (P < .001) and absence of monosomy (P = .042) remained significant in multivariate analysis. Univariate Cox regression indicated that age (P = .003), AML status (de novo or secondary; P = .0277), choice of induction (P = .030), and monosomy (P = .010) had a significant association with OS. Only younger age (P = .018) and absence of monosomy (P = .022) were predictive of OS in multivariate Cox analysis. ConclusionPositive predictors of CR in adult AML include absence of monosomy and induction treatment with 3+7; whereas positive predictors of OS are younger age and absence of monosomy.

Establishment and validation of anovel risk model for estimating time to first treatment in 120 patients with chronic myelomonocytic leukaemia.

SummaryChronic myelomonocytic leukaemia is a rare disease and data on the treatment are often extrapolated from myelodysplastic syndrome studies. Although several scores exist for the prognosis of overall survival in chronic myelomonocytic leukaemia, so far there is no designated score for the prediction of the time to first treatment. We tested clinical parameters and cytogenetic information for their ability to predict the time to first treatment in our single center cohort of 55 unselected consecutive chronic myelomonocytic leukaemia patients. In multivariate analysis we identified elevated lactate dehydrogenase (≥223 U/l), higher bone marrow blast percentage (≥7.5%) and thrombocytopenia (<55 G/l) at initial diagnosis as the most relevant parameters for the time to first treatment. Using these three parameters we developed a risk score that efficiently estimates the time to treatment initiation with azacitidine or hydroxyurea (p < 0.001; log-rank). In the high-risk group (≥2 risk factors) 85% of patients required treatment within 1 year, whereas this was the case in 48% in the intermediate-risk (1 risk factor) and in 0% in the low-risk group (0 risk factors). Our risk model was validated in an external test cohort of 65 patients and may serve as a simplified and easily applicable tool for identifying patients who may not require early treatment initiation.

Bone marrow miR-10a overexpression is associated with genetic events but not affects clinical outcome in acute myeloid leukemia

BackgroundAccumulating studies have linked the disruptions of microRNA-10 (miR-10) to acute myeloid leukemia (AML) with NPM1 mutation. However, miR-10 expression and its clinical implication in AML remain poorly defined. Although a recent report showed high serum level of miR-10a was associated with adverse prognosis in AML, herein, we found bone marrow (BM) miR-10 overexpression was not a prognostic biomarker in AML. MethodsBM miR-10 expression was examined by real-time quantitative PCR in BM mononuclear cells in 115 de novo AML patients and 45 controls. ResultsBM miR-10 (miR-10a/b) expression was significantly up-regulated in AML patients, and was positively correlated with each other. Overexpression of miR-10a was associated with lower percentage of BM blasts, whereas miR-10b overexpression tended to correlate with higher percentage of BM blasts. Importantly, miR-10a overexpression was significantly associated with FAB-M3/t(15;17) subtypes and NPM1 mutation, meanwhile, overexpression of miR-10b was correlated with NPM1 and DNMT3A mutations. However, miR-10a/b overexpression was not associated with complete remission rate, and did not have an impact on both leukemia free survival and overall survival time in non-M3 AML patients without NPM1 mutation. ConclusionsBM miR-10 overexpression is associated with genetic events but not affects clinical outcome in AML.

Longitudinal Tracking of MDS Patients Using Next Generation Sequencing Provides a Predictive Measure for Azacitidine Response and AML Progression

▪Introduction:Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by dysplastic changes in one or more cellular lineages causing impaired bone marrow function. One third of patients diagnosed with MDS progress to secondary acute myeloid leukemia (sAML). These patients have significantly worse prognoses than de novo AML patients. Azacitidine (AZA), a hypomethylating agent is commonly used to treat MDS patients as a frontline therapy. Although its survival benefits over supportive care in a randomized trial has been demonstrated, the underlying genetics and clonal dynamics upon AZA response/AML progression have not been well examined. Using next generation sequencing (NGS) technology, we attempted to assess the clinical relevance of somatic mutations and their dynamics as they relate to AZA treatment in MDS patients using longitudinal samples.Patients and Methods:Ninety-five MDS patients (56 lower risk and 39 higher risk MDS based on the revised IPSS scoring system) were enrolled in this study. The median age of the 95 patients is 67 years (range of 31 Ð 84) and median follow-up duration was 747 days (range of 137-3328 days). We performed targeted deep sequencing (entire exon region of a panel of 84 myeloid genes, Agilent custom probe set) on 285 bone-marrow samples including the longitudinal samples taken at diagnosis (n=95) and post-AZA treatment, (median 4 cycles) as well as T-cell fraction (CD3+). We multiplexed and sequenced the samples using an Illumina Hiseq 2000. After read mapping and variant calling, hierarchical clustering, pathway and survival analyses were performed in R.Results:Targeted sequencing on the myeloid gene panel revealed 176 mutations in 68 patients (68/95, 71.6%) with a median of 2 mutations per patient (ranges 2-6). The average on-target coverage for 285 sequenced samples was 1205x. Twenty-five of 44 mutated genes were recurrently mutated. ASXL1 was the most frequently mutated in the cohort (21%), followed by TET2 (15%), DNMT3A (11%), and SRSF2 (11%). Mutated genes were then grouped into 8 biological pathways, defined in The Cancer Genome Atlas (TCGA) AML study. The most frequent biological pathway with mutated genes at diagnosis was DNA methylation (28.4%), followed by spliceosome (25.2%), chromatin modifiers (22.1%), myeloid transcription factors (TFs) (11.6%), activated signaling (11.6%), tumor suppressors (12.6%), and cohesin complex (6.3%).When assessing the differences in patterns of variant allele frequency (VAF), we found significant VAF reduction in responders compared to non-responders (p = 0.007, repeated measures using general linear model, Figure A). Multivariate analyses revealed that mutation burden in different genes and biological pathways have distinct impact on AZA response, AML transformation, and overall survival. Higher bone marrow blast percentage (5%) was associated with all three measures (Figure B). Most significantly, mutations in activated signaling pathway genes are associated with AML progression (p=0.002). In addition, we could not detect decreased VAFs in activated signalling pathway genes even in responders (Figure C-D). Patients with SRSF2 mutations tend to respond to AZA (OR 14.084, p=0.003). Mutations in tumor suppressors (HR 4.825, p<0.001) and myeloid TFs (HR 3.070, p=0.020) were adverse prognostic factors in overall survival. Of interest, mutations in DNA methylation pathway were not independent prognostic factor for AZA response, AML transformation, or overall survival.Conclusion:These data and analyses show that reduction in mutation burden is correlated with AZA response. Mutations in different genes and biological pathways are associated with distinct clinical measures that tumor suppressors and myeloid TFs were identified as poor prognostic factors in terms of OS. Persistent mutation burden in activated signaling pathways is a strong predictor for AML transformation. In summary, longitudinal tracking of MDS patients using NGS may improve criteria for AZA response and early detection of AML progression. [Display omitted] DisclosuresJang:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Research Funding.

BRD4 Short Isoform Is a Myelodysplasia Prognostic Marker Involved in Inappropriate DNA Damage Response

INTRODUCTION: Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. BRD4 is a BET member of bromodomain-containing proteins, known as epigenome readers, exerting key roles in chromatin remodeling and transcriptional regulation. BRD4 gene encodes two major isoforms, short (BRD4S) and long (BRD4L). Recently, Floyd SR et al (Nature, 2013) described BRD4S as an endogenous inhibitor of the DNA damage response (DDR). Under normal conditions, damaged DNA induces histone phosphorylation of H2AX at Ser 139 (γ-H2AX) and activates a protective signaling network that blocks cell cycle and recruits DNA repair factors. Despite having been described as a therapeutic target in acute myeloid leukemia (AML) by Zuber J et al (Nature, 2011), BRD4 has never been studied in myelodysplastic syndrome (MDS) and its role in the pathogenesis is currently unknown.AIMS: To evaluate BRD4L and BRD4S expression in MDS and AML patients, correlating with clinical data, progression and survival. We also explored the BRD4 role in DDR signaling using human leukemia cell line models.MATERIAL AND METHODS: Diagnostic total bone marrow (BM) samples from 24 healthy donors (HD) and 99 patients, including 48 MDS (22 higher-risk MDS and 36 lower-risk MDS) and 51 AML (16 of them with MDS-related changes, AML-MRC), were collected. We also isolated CD34+ cells from 7 HD, 5 de novo AML, 4 AML-MRC and 14 MDS (6 higher-risk and 8 lower-risk MDS). BRD4L and BRD4S gene expressions were assessed through q-PCR and expressed as median (minimum-maximum). MDS patients were stratified according IPSS, WHO classification, R-IPSS and cytogenetic risk. Samples were age-adjusted when significant differences were observed, using ANOVA and Tukey’s test. Progression-free and overall survival curves were estimated by Kaplan-Meier method and were analyzed by the Wilcoxon´s test and Cox regression. JQ1, a specific BRD4 inhibitor, was kindly provided by James Bradner. A panel of human myeloid leukemia cell lines (KG1a, HEL, HL60, U937) in exponential growth was treated with increasing doses of JQ1 for 48 hours and cell growth (MTT colorimetric assay), apoptosis (annexin-V/PI) and cell cycle (flow cytofluorometric analysis detecting nuclear PI incorporation) were evaluated. We also determined the expression of p-γH2AX (DDR signaling) by western blot, after 12 hours of JQ1 treatment.RESULTS: A higher expression of BRD4S was observed in total BM cells from AML (4.01 [0.33-2.58], P=.01) and MDS patients (4.21 [0.01-56.17], P=.01) compared with HD (2.11 [0.04-10.32], P=.01). When stratified according WHO classification, AML-MRC (4.5 [0.33-25.22], P=.04) and higher-risk MDS (4.66 [0.17-56.17], P=.04) subgroups showed higher BRD4S expression. In CD34+ cells, BRD4S expression was increased in de novo AML (0.28 [0.21-0.45]) compared with lower-risk MDS (0.02 [0.00-0.44], P=.01). BRD4L mRNA expression was not modulated in total BM and CD34+ cells from any subgroup. With median follow-up time of 34.4 months, we found that higher BRD4S gene expression was a worse prognostic factor for MDS transformation and survival, along with IPSS, R-IPSS, low hemoglobin (less than 10g/dL) and higher BM blast percentage. After multivariate analysis, BRD4S gene expression and higher-risk (very high, high and intermediate) R-IPSS remained as independent prognostic factors for MDS progression and overall survival. KG1a and U937 cells showed greater resistance to JQ1, with lower apoptosis rate and proliferation (IC50 not reached, over 3000nM), whereas HEL and HL60 were more responsive (IC50 under 800nM in both cell lines). JQ1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and apoptosis and caused a progressive increase in histone phosphorylation of γ-H2AX, indicating activation of DDR signaling.CONCLUSIONS: MDS is a clonal myeloid neoplasm characterized by profound epigenetic modifications. Our data establishes BRD4S as a novel MDS prognostic factor, related to aggressive phenotype, higher progression rate and shorter survival. Biologically, BRD4S plays a role in the inappropriate DNA Damage Response of MDS, favoring the disease towards genetic instability and clonal evolution. DisclosuresNo relevant conflicts of interest to declare.

Spliceosome Gene Mutations Are Frequently Found In JAK2 Negative Myelofibrosis and Associated With Worse Clinical Outcomes

Myelofibrosis (MF), a Philadelphia chromosome negative myeloproliferative neoplasm (MPN) requires clonal markers for accurate diagnosis according to the 2008 World Health Organization classification scheme. However, molecular mutations in Janus Kinase 2 (JAK2) are found in only 50% of MF patients (pts). Various somatic mutations identified in other myeloid cancers have also been found in MF with pre-existing JAK2 V617F mutations. We previously reported in 57 pts diagnosed with Triple Negative MF (TN-MF: JAK2 exon12/14 and wild type (WT) MPL) the absence of mutations in genes commonly identified in other myeloid malignancies, specifically TET2, DNMT3A, CBL, IDH1/2, SH3B2 (LNK), N/KRAS and EZH2. Only somatic mutations in ASXL1 were identified in a small cohort of TN-MF (16%). Mutations in components of the RNA splicing machinery have been recently identified in varying frequencies in different myeloid malignancies. We performed RNA-sequencing on 2 samples (JAK2 mutant (MUT) and 1 JAK2 WT) and found that 2 spliceosome genes (U2AF1 and SF3B1) had higher mRNA expression levels in the JAK2 WT (MFC=0.76 and 0.82, respectively) compared to JAK2 MUT patient. We hypothesized that somatic mutations in RNA splicing factor genes occur in TN-MF pts and may be helpful in the biological characterization of this group of pts. Therefore, we isolated DNA from bone marrow (BM) or peripheral blood mononuclear cells from a cohort of MF pts (N=132; JAK2 MUT:75 and JAK2 WT:57) and performed Sanger sequencing for SF3B1 (exons 13-16), U2AF1 (exons 2, 6 and 7), and SRSF2 (exons 1 and 2). Baseline characteristics of pts and clinical data including hematologic parameters, BM results, and presence of splenomegaly by palpation were collected. Pts were stratified based on the Dynamic International Prognostic Scoring System-Plus risk as high=40, Int-2=51, Int-1=11 and low-risk=5 pts. The mean duration of follow-up was 16.5 months in JAK2 MUT and 12.8 months in JAK2 WT. Spliceosome mutations were found in 35/132 (27%) (SRSF2=17%, U2AF1=8% and SF3B1=2%) of pts. Interestingly, the frequency of spliceosome mutations in JAK2 WT was higher (31%; SRSF2=26%, U2AF1= 5%, SF3B1=0) compared to JAK2 MUT (18%; SRSF2=10%, U2AF1=6%, SF3B1=2%) pts. Of note, the 2 pts with SF3B1 mutations (K700E) had ring sideroblast in the BM (occasional and 50%). A large number of WT JAK2 patients (50%) had concomitant SRSF2 and ASXL1 mutations. Both SRSF2 and ASXL1 have been associated with poor prognosis in MF. Indeed, in our JAK2 WT cohort, pts who harbored SRSF2 mutations had a higher mean percentage of BM blasts compared to the ones that were SRSF2 WT (5.6% vs 1.8%; P=.006). Moreover, JAK2 WT pts carrying spliceosome mutations had more severe anemia as shown by lower hemoglobin (8.98 g/dL vs 10.5) and higher leukocyte counts (27.5 x109/L vs 20.5) compared to WT cases. Furthermore, we noticed that they also had higher frequency of RBC (Red Blood Cells) transfusions compared to WT (60% vs 40%) cases. Spleen examination by palpation below the left sub-costal margin showed that JAK2 WT cases carrying spliceosome mutations had larger splenomegaly compared to WT cases [10.1 cm (range 4-26), vs 8.7cm (range 2-20); P=0.05]. In conclusion, molecular alterations in the spliceosome machinery are frequently found in MF pts who are WT for JAK2/MPL and are associated with higher BM blast percentage, more severe anemia, higher leukocyte counts, transfusion dependence and more prominent splenomegaly. The observation of appreciable frequency of spliceosome mutations in JAK2 WT MF pts opens the possibility of using spliceosome inhibitors in the management of this disease group. Disclosures:No relevant conflicts of interest to declare.

Complex, Not Monosomal, Karyotype Is the Cytogenetic Marker of Poorest Prognosis in Patients With Primary Myelodysplastic Syndrome

Complex karyotype (CK) is the poorest risk factor in patients with myelodysplastic syndrome (MDS). It has recently been reported that monosomal karyotype (MK) worsens the prognosis of patients with CK. PATIENTS AND METHODS; We analyzed 1,054 adult patients with MDS with an abnormal karyotype from the Spanish Registry of MDS. The aim of the study was to describe the incidence, characteristics, and prognosis of MK; the main end points were overall survival (OS) and leukemia-free survival. MK was identified in 172 patients (16%), most of whom (88%) presented with CK. Variables significantly associated with OS were age (hazard ratio [HR], 1.90; P < .001), bone marrow (BM) blast percentage (HR, 1.05; P < .001), hemoglobin level (HR, 1.71; P < .001), platelet count (HR, 1.41; P < .001), karyotype complexity (CK [three abnormalities]: HR, 1.81; P = .003; very CK [> three abnormalities]: HR, 2; P < .001), and abnormalities of chromosome 5 and/or 7 (HR, 1.89; P < .001). Variables significantly related to the risk of transformation to acute myeloid leukemia (AML) were higher BM blast percentage (HR, 1.12; P < .001) and karyotype complexity (CK: HR, 2.53; P = .002; very CK: HR, 2.77; P < .001). After accounting for karyotype complexity, MK was not associated with OS or evolution to AML. In conclusion, these results demonstrate that the prognostic value of MK in MDS is not independent and is mainly the result of its strong association with number of chromosomal abnormalities.

Prognostic Impact of Monosomal Karyotype in Patients with Myelodysplastic Syndrome and Abnormal Karyotype. A Report From the Spanish Group of MDS (GESMD)

Abstract 1724Cytogenetic abnormalities (CA) are the most important prognostic factor in patients with myelodysplastic syndromes (MDS). Monosomal karyotype (MK) defined as the presence of at least two autosomal monosomies or one monosomy plus other structural CA, has been associated with poor prognosis in acute myeloid leukemia (AML) but its significance in MDS remains unclear. The aim of our study was to analyze the prognostic impact of MK in adult patients with MDS and CA.Patients from Spanish Registry of MDS diagnosed with MDS by WHO 2008 criteria and with CA detected by conventional cytogenetics have been included in the study. Statistical analysis consisted of Kaplan-Meyer univariate analysis (UA) including all known variables associated with prognosis in MDS and a Cox-regression multivariate analysis (MA) in which we included only those variables with a P<0.1 in UA.There were 1054 patients, 478 (45.3%) women. The median age was 71 (range 16–96) years. Median follow-up for survivors was 24.1 (range 0–210) months. There were 609 (57,8%) refractory anemia (RA) and 445 (42.8%) refractory anemia with excess of blasts (RAEB). The IPSS was low, intermediate-1, intermediate-2 and high in 18.3%, 39.3%, 25%, and 10.6%, respectively (6.7% were unclassifiables). Complex karyotype (CK) and MK was observed in 203 (19.3%) and 172 (16.3%) patients, respectively. Patients with MK showed worse prognostic characteristics than those without MK: More Intermediate-2 and high risk patients (50% vs. 20% and 30% vs. 6.7%; P<0.001); more frequent CK: (87.2% vs. 6%; P<0.001), higher median bone marrow (BM) blast count (7% vs. 3%; P<0.001) and lower hemoglobin (Hb) level (89 vs. 96 g/L; P<0.001).Median OS for the whole group was 32.7 months. In the UA, the variables associated with lower OS were: Male sex, RAEB subtype (vs. RA), higher IPSS, CK, MK, older age, higher peripheral blood (PB) and bone marrow (BM) blast percentage and lower Hb, platelets and neutrophil count. In the MA the variables associated with lower OS were: Age>60 years (HR 1.7; P<0.001), CK (HR 2.19; P<0.001), higher BM blast (HR 1.07; P<0.001), Hb<100 g/L (HR 1.788; P<0.001) and platelet count <100×109/L (HR 1.62; p<0.001). MK did not reach statistical significance in the MA (HR 1.45; P=0.059).In the group of patients with CK the UA showed that the presence of MK was associated with a trend to lower OS (Log rank 2.8, P=0.092) while the presence of ≥4 vs. 3 CA was associated with lower OS (Log rank 7.5; P=0.006). Other variables associated with lower OS in UA in this subset of patients were: RAEB (vs RA), presence of abnormalities of 5 and/or 7 chromosome, higher IPSS group, older age, higher BM blast percentage, and lower Hb and platelet count. In MA the variables associated with lower OS in patients with CK were: age>60 years (HR 1.734, P=0.008), RAEB vs. RA (HR 1.542, P=0.02) Hb <100 g/L (HR 2.1, P<0.001) platelets <100×109/L (HR1.886; P=001) and the presence of abnormalities of both 5 and 7 chromosomes (HR 2.058; P=0.001). The presence of MK did not reach statistical significance in the MA.At last follow-up, 221(21%) patients had shown AML evolution at a median of 9 months (0–125 months) for a 1 and 4 years probability of AML evolution of 14.2% (95% CI 11.8–16.6) and 28.6% (95 CI: 24.8–32.4). The variables associated with higher risk of AML evolution in the UA were: RAEB, higher IPSS, CK, MK, higher PB and BM blast percentage and lower hemoglobin, platelet and neutrophil count. In multivariate analysis the only variables that retained statistical significance were BM blasts (HR 1.13; P<0.001) and CK (HR 3.2; P<0.001).In conclusion, our study shows that the presence of MK is highly associated with CK and other high-risk features of MDS. In our population, the MK was not an independent risk factor for OS while the presence of CK was the main risk factor associated with poorer OS in MDS patients with abnormal karyotype. Disclosures:No relevant conflicts of interest to declare.

Having a higher blast percentage in circulation than bone marrow: clinical implications in myelodysplastic syndrome and acute lymphoid and myeloid leukemias

Determining the percentage of peripheral blood (PB) and bone marrow (BM) blasts is important for diagnosing and classifying acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Although most patients with acute leukemia or MDS have a higher percentage of BM blasts than PB blasts, the relative proportion is reversed in some patients. We explored the clinical relevance of this phenomenon in MDS (n = 446), AML (n = 1314), and acute lymphoblastic leukemia (ALL) (n = 385). Among patients with MDS or ALL, but not AML, having a higher blast percentage in PB than in BM was associated with significantly shorter survival. In multivariate analyses, these associations were independent of other relevant predictors, including cytogenetic status. Our findings suggest that MDS and ALL patients who have a higher percentage of PB blasts than BM blasts have more aggressive disease. These data also suggest that MDS classification schemes should take into account the percentage of blasts in PB differently from the percentage of blasts in BM.