Low-risk Myelodysplastic Patients Research Articles

RUNX1 Mutation Accompanied with Dysregulated Cellular Senescence in Lower-Risk Myelodysplastic Syndrome Patients Is Associated with Disease Progression

Background and Aims A part of lower-risk myelodysplastic (LR-MDS) patients progress to higher-risk MDS or acute myeloid leukemia (AML). The progression may be predicted in some of these patients by determining mutations associated with myeloid malignancies at the time of diagnosis. We focused to find out dysregulated pathways caused by a well-defined mutation associated with disease progression. Methods We examined 158 samples from LR-MDS patients at diagnosis using TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to identify the mutational profile. We applied RNA-seq (NEB; Illumina) on bone marrow CD34+ cells of 5 LR-MDS patients with RUNX1 mutation and 6 LR-MDS patients without mutations. We performed differential gene expression analysis, gene set enrichment analysis (GSEA), Reactome biological pathway and Gene Ontology (GO) annotations. Results Forty out of 158 patients (25%) progressed during the follow-up period (median: 45.1 months). Mutation in RUNX1 gene was detected in 10 patients. Using RNA-seq, significantly dysregulated expression was observed in 986 genes (FDR<0.05; FC >2). Patients with mutation in RUNX1 gene had significant down regulation of expression genes involved in cellular senescence induced by DNA Damage/Telomere Stress (FDR=1.4e-11) and Oxidative Stress (FDR=3.6e-9), HDACs deacetylate histones (FDR =1.1e-13) and Senescence Associated Secretory Phenotype (FDR=1.4e-11) according to Reactome pathway database. There were 21 genes involved in the regulation of these pathways, of which 10 were part of all described pathways and belonged to the Nucleosome Assembly according to GO biological process with 33 down regulated genes (FDR=6.3e-28). According to GSEA, GO chromatin silencing gene set (Figure 1) was down regulated in the patients with RUNX1 mutation (p<0.001; NES=-1.8) same as Reactome apoptosis (p<0.001; NES-1.7). In these patients, expression of 750 genes was up-regulated (FDR<0.05; FC >2); however, they did not form a specific pathway. The up-regulated genes were classified as so-called leukemia-associated antigens (LAAs) such as PRAME (adjusted p=6.9e-09; logFC=6.8), WT1AS (adjusted p=4.4e-07; logFC=5.7), BAALC (adjusted p=3.5e-06; logFC=2.1), WT1 (adjusted p=6.8e-07; logFC=3.2), FLT3 (adjusted p=0.004; logFC=1.3) and LEF1 (adjusted p=5.3e-08; logFC=-4.2). Conclusions To study the transcriptome of the hematopoietic stem cells of LR-MDS patients, we used gene expression profiling and identified biological processes in relation to mutations in RUNX1 gene that were detected in patients with disease progression. According to our results, we suppose that cells of LR-MDS patients with no mutations are maintained in senescent state or apoptosis in opposite to RUNX1 mutated cells. Finding out of dysregulated senescence was reinforced by other dysregulated pathways such as HDACs deacetylate histones, chromatin structural changes and senescence-associated secretory phenotype. About cellular senescence is known that may act as a strong tumor suppression mechanism that restrains proliferation of cells at risk for malignant transformation. We suppose that in precancerous cells of LR-MDS patients apoptosis or cellular senescence limits the replicative capacity of cells, thus preventing the proliferation. The disturbed state of cellular senescence in RUNX1 gene mutated hematopoietic stem cell can escape proliferation control and then can contribute to the disease progression in these patients. Supported by grants NV18-03-00227 and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Mutations in RUNX1 and TP53 Genes Predict Progression in Patients with Lower-Risk Myelodysplastic Syndrome

Background and AimsA part of lower-risk myelodysplastic (LR-MDS) patients progress to higher-risk MDS or acute myeloid leukemia. These patients have more aggressive type of disease than is expected based on their classification according to the International Prognostic Scoring System. Alongside, recent advances in DNA sequencing have enabled the identification of a large number of mutated genes in more than 80% of MDS patients. We focused on the identification of mutational profile that would be predictive of disease progression in LR-MDS patients.MethodsWe examined samples from 73 LR-MDS patients by TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to determinate the mutational profile. In 32 (44%) patients, paired samples processed at the time of diagnosis and during progression were analyzed. The median age of patients was 64.5 years (range, 29-83 years). The median observation period was 33.4 months (range, 4.5-195.9 months) from diagnosis, 36 (49%) patients remained alive including 8 who underwent HSCT. The FASTQ files were analyzed by NextGENe V2.4.2.2 (SoftGenetics). The survival distributions were estimated using the Kaplan-Meier method and prognostic factor analysis was performed with Cox's multivariate regression method. The Mann-Whitney test was used to determine the group independence.ResultsIn total, 70% of patients had a recurrent mutation in at least one gene of the panel (87.5% of patients with progression and 56% without progression). The patients, who progressed, carried on average of 2.4 mutations and unprogressed patients 1.9 mutations. Most of the mutations detected in LR-MDS were in SF3B1 (30.1% of patients) and DNMT3A (24.7% of patients) genes but had no effect on overall survival (OS). Multivariate analysis demonstrated that RUNX1 and TP53 mutations were the strongest independent prognostic factors for OS (HR: 14.9; p=0.0001/HR:6.3; p=0.0007) at the time of diagnosis and also for progression-free survival (PFS) (HR: 3.6; p=0.05/HR:3.3; p=0.05). Moreover, mutations in these genes were detectable in 59.4% of patients with disease progression and only in 4.8% of patients without progression, who however were followed shortly (4.7 months). The median OS was 27.9 and 39.6 months in the patients with mutations of RUNX1 and TP53 genes compared to 84.2 months (p <0.0001) in patients without these mutations. The new mutations acquired during progression were present most in NRAS and PTPN11 genes. The variant allele frequency (VAF) of other mutations detected at the time of diagnosis increased during disease progression. Presence of mutations in TP53 gene (p=0.0006), RUNX1 gene (p=0.003), IPSS-R score (p=0.01) and hemoglobin level (p=0.05) were independent variables between patients with and without progression.ConclusionsThe goal of the study was to detect mutations in relevant genes that might predict disease progression up to 6 years from the time of diagnosis in LR-MDS patients and comparison of mutant allele frequencies in selected cases between the time of diagnosis and disease progression. We found that 60% of patients who progressed, carried mutations in RUNX1 and TP53 genes at the time of diagnosis. On the contrary, other mutations detectable at the time of diagnosis had no effect on OS and PFS. The VAF of mutations detected at the diagnosis increased at the time of progression. The NRAS and PTPN11 genes contained most of the new mutations acquired during progression. In conclusion, the results indicate that routine monitoring for mutations in LR-MDS should be performed to refine the risk prediction of disease progression.Supported by AZV grants (16-33485A, 16-31689A, 17-31398A and NV18-03-00227), RVO-VFN64165 and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. DisclosuresNo relevant conflicts of interest to declare.

Perl's Stain Grade in the Bone Marrow Aspirate Correlates with Overall Survival in Low Risk Myelodysplastic Patients

BackgroundMyelodysplastic syndromes (MDS) are heterogeneous group of acquired clonal hematopoietic stem cell disorders characterized by atypical stem cells maturation and genetic instability leading to an enhanced risk of progression to acute myeloid leukemia. Low risk MDS patients have a lower probability to evolve in leukemia but are commonly characterized by dyseritropoiesis. These patient are incline to long term accumulation of iron in the organs due mostly to red blood cell transfusion (RBC) but iron overload may also occur in MDS patients who do not receive RBC transfusions due to the ineffective erythropoiesis. It is well known the effect of oxidant-mediated tissue's injury through the formation of free toxic iron species in the liver and heart site, but recent knowledges assumes that this mechanism is effective also in the bone marrow nice, where oxidative stress seems to impaired the haematopoietic stem cells growth. At this moment microscopic examination of the stainable iron in the bone marrow is considered the gold standard for determining the iron stores. The effect of bone marrow's iron overload on overall survival in the low risk MDS has been a matter of unresolved debate. We aimed to investigate the predictive value of bone marrow iron accumulation as demonstrated by Perl's staining on outcome in such patients.DesignWe retrospectively analyzed all low risk,intermediate-I MDS patients who had diagnosed in our institution in the last 20 years (since 1998). Diagnosis of MDS was made according to WHO criteria. Patients were stratified based on International Prognostic Scoring System (IPSS). Patients had undergone bone marrow aspiration as part of the diagnostic work up for their MDS. Two different experienced hematologist analyzed all samples. Bone marrow aspiration slides with at least seven fragments were considered suitable. Perl's Prussian blue stain was used to stain bone marrow, assessed by modified Gale's grading (Tab. 1) and then correlated with outcome.Patients and methodsMarrow staining of one hundred and fourteen consecutive MDS patients were revised and analyzed. Median age was 70 years (range 32-93). Eighty three patients were IPSS low- risk and 30 Intermediate I. All patients were evaluated for bone marrow iron stores with Perl's stain. Twenty-seven patients had grade 1 (+), 31 grade 2 (++) and 56 grade 3 (+++). Patients had never or minimally received RBC . None of these patient had received iron chelation before marrow examination. Probability of overall survival (OS) was estimated by the Kaplan-Meier method and the significance was assessed by the log-rank test.Results20-year OS was significantly lower in patients with higher Perl's score (median = 80 ±7 months in grade 3; median = 70 ±17 months in grade 2; median = 144 ±18 months in grade 1 , P=0.011); Fig. 1ConclusionsWe evaluated retrospectively the bone marrow aspirate from 114 consecutive new MDS low-risk, Intermediate-I IPSS patients with Perl's stain for iron detection. Although Perl's grading is a qualitative method, it is still the gold standard to detect iron storage in the bone marrow. Our results correlate Perl's stain at diagnosis with long term outcome in MDS patients. We show how higher grade of iron storage at diagnosis can impact on outcome in these patients. We conclude that Perl's stain, together with Ferritin and blood transfusional burden could be another marker at diagnosis of iron-related toxicity that predict overall survival. DisclosuresPilo:Novartis Italy: Honoraria. Angelucci:Jazz Pharmaceuticals Italy: Other: Local ( national) advisory board; Vertex Pharmaceuticals Incorporated (MA) and CRISPR CAS9 Therapeutics AG (CH): Other: Chair DMC; Roche Italy: Other: Local (national) advisory board; Novartis: Honoraria, Other: Chair Steering Comiittee TELESTO Protocol; Celgene: Honoraria, Other: Chair DMC.

Transfusion-dependent low-risk myelodysplastic patients receiving deferasirox: Long-term follow-up

Myelodysplastic syndromes (MDSs) are characterized by ineffective hematopoiesis that results in peripheral cytopenias. Anemia is the most common symptom of MDS and the majority of patients become transfusion-dependent with the risk of iron overload, which may lead to cardiac, hepatic and endocrine complications. Deferasirox is an orally available iron chelator administered once-daily in transfusion-dependent patients with various chronic anemias. Its efficacy has been established in controlled clinical trials. In the present study, we describe our experience with 55 consecutive MDS patients [International Prognostic Scoring System risk score of low (n=32) or intermediate-1 (n=23)] treated with deferasirox in a routine clinical setting following Consensus Guidelines on Iron Chelation Therapy. According to WHO classifications, patients had refractory anemia (n=30), refractory anemia with ringed sideroblasts (n=16), refractory cytopenia with multilineage dysplasia (n=8) or refractory cytopenia with multilineage dysplasia and ringed sideroblasts (n=1). The median monthly transfusion requirement at baseline was 3 units. Patients received a starting dosage of 10 mg/kg/day, subsequently titrated according to serum ferritin (SF) levels which were measured monthly. Safety assessment included monitoring of liver and renal parameters and recording adverse events (AE) during treatment. At the baseline, the mean ± SD SF level was 2,362±172 ng/ml and after 24 months, the mean ± SD decrease in SF was 1,679±209 ng/ml. Sixteen patients had sustained hematological improvement meeting International Working Group 2006 criteria. One patient became transfusion-independent. No severe AE were reported. In conclusion, deferasirox therapy was effective and safe in reducing transfusional iron overload and it reduces transfusion requirement in a subset of patients.

Azacitidine Low-Dose Schedule In Low-Risk Myelodysplastic Syndromes. Preliminary Results of a Multicenter Phase II Study

AbstractAbstract 4029▪ Background:Azacitidine (AZA) at a dose of 75 mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates and prolongation of survival in high-risk MDS patients (pts) (Fenaux, 2009). However few data are hitherto available concerning the efficacy and safety of Aza in lower risk MDS. A lower dose regimen, AZA 5 (75 mg/mq daily, subcutaneously, for 5 consecutive days every 4 weeks) have shown to induce response rates consistent with the currently approved schedule (Lyons, 2009), however in this study pts were not classified according to IPSS risk. Aim:The use of AZA in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial, AZA 5 regimen was administered to IPSS low-or-intermediate-1 risk pts, for a total of 8 courses, in order to evaluate its efficacy and safety. Furthermore pharmacogenomic studies (GEP, SNP) cytokine network and PI-PLC-beta1 methylation and gene expression, before and at the end of 4th and 8th course of Aza treatment, were planned to identify new biological markers to predict the response. Methods:From September 2008 to February 2010, 34 patients (24 males, 10 females), with a median age of 71 (56-84) yrs, with symptomatic transfusion-dependent anemia, previously unresponsive to erythropoietin (EPO) or not expected to respond to EPO, or with severe neutropenia or thrombocytopenia, were enrolled into the study. According to WHO classification, 15 pts had RA, 6 RARS, 7 RCMD and 6 RAEB-1. Results:At present time 30/34 pts are evaluable: 23/30 pts (77%) completed the treatment plan (8 courses), 3/30 pts (10%) are ongoing and 4/30 (13%) died during the treatment period. According to the 2006 International Working Group criteria, overall response rate (ORR) was 60,9 % (14/23 pts): 5 pts (21,7%) achieved complete remission (CR), while 9 pts (39,1%) showed an hematologic improvement (HI) (7 erythroid responses, 1 erythroid/platelet response and 1 neutrophil/platelet response). 9/23 pts (39%) maintained a stable disease (SD). Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%). 4 pts (11,7%) died during treatment (2 pts after the 1th cycle and 2 pts after the 4th course) because of septic shock, gastrointestinal hemorrage, pneumonia, and respiratory distress, respectively. The median duration of response was 3,5 months (range 1–14 months). Surprisingly, 3/14 patients (2 CR and 1 HI erythroid) showed a long duration of response (11, 13 and 14 months, respectively), still ongoing, after discontinuation of AZA. Preliminary data on the lipid signalling pathways suggested a direct correlation between the demethylating effect on PI-PLC-beta1 and responsiveness to treatment. Conclusion:Our study shows that AZA low-dose schedule may be a feasible and effective treatment for low-risk MDS pts and may induce durable responses. Despite AZA safety, extreme caution is needed in pts with age-related comorbidities and/or with severe neutropenia or thrombocytopenia, especially in low-risk MDS. Furthermore, PI-PLC–β1 demethylation and gene expression could represent a new biological marker to predict the clinical response to AZA Disclosures:Off Label Use: In Italy the use of Azicitidine for Low-Risk Myelodysplastic patients is off-label. The use of azacitidine in our study is part of a Phase II clinical trial. Finelli:Celgene: Consultancy.

Expression of Toll-Like Receptors on Peripheral Blood Cells After Allogeneic Stem Cell Transplantation: Ongoing Results of a Prospective Study

AbstractAbstract 4704 Background and aim of the study.Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, in the pathogenesis of autoimmune diseases and graft-versus-host disease (GVHD). In the cross-talk between innate and adaptive immune system, pattern recognition receptors such as Toll-like receptors (TLRs) play a key role. TLRs recognize common protein, carbohydrate or DNA/RNA pattern motifs leading to signaling for cytokine production and T cell and dendritic cell maturation. These receptors may act as tuner of inflammatory and immunologic reactions. Very little is known about the expression and the function of TLRs in vivo in patients who underwent to allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression and the function of TLRs on lymphocytes and monocytes in relation to infection (CMV and HHV-6, especially) and the onset of GVHD. Patients and methods.The expression of TLRs on T cells and monocytes was analyzed by flow cytometry at day +30, +90, +180 after SCT and at the onset of GVHD. The expression of receptors for lipid-based pathogen-associated molecular patterns (PAMPs: TLR 1,2,4 and 6 surface receptors), receptors for nucleic acid based PAMPs (TLR 3,7,8 and 9 located in cytoplasmic compartments), TLR5 and, TLR10 (surface receptors) was evaluated as mean fluorescence intensity (MFI). Ex vivo induction of cytokines (TNFalpha, MCP1, IFNgamma, IL-10) by TLR ligands was analyzed in the cell supernatant by ELISA. Since the beginning of the study, we have analyzed data of 12 healthy donors and 14 patients. Median age was 46 years (range, 25–64) and 7 patients were male. Results.Acute GVHD developed in 7 patients. Patients without acute GVHD after SCT and healthy donors showed different MFI of TLR3 on T cells (5,8±1,4 vs 4,2±1,05 p=0,02), of TLR4 on monocytes (26,1±1,01 vs 15,8±4,9 p=0,004), and of TLR6 on T-lymphocytes (7,3±3,2 vs 4,6±1,1 p=0,02) and monocytes (27±12,1 vs 14,9±4,6 p=0,01). TLR3 expression was significantly decreased on T-lymphocytes and monocytes in patients with acute GVHD in comparison to those without GVHD (4,06±0,8 vs 5,8±1,4 p=0,02; 9,3±7,2 vs 38,02±30 p=0,04). The levels of TLR5 on T cells and monocytes were higher in patients with acute GVHD compared to healthy donors (8,4±2,1 vs 6,4±1,6 p=0,04; 54,2±20,2 vs 33,2±16,5 p=0,04). An increased induction of IFNgamma upon TLR1 ligand activation was observed in patients without GVHD in comparison to healthy donors and patients with GVHD (p=0,04). TLR6 ligand induced significantly the production of IFN gamma in patients with GVHD in comparison to controls and the other patients (p=0,03). Patients without GVHD showed a trend toward a decreased induction of MCP1 upon TLR4 ligand activation (p=0,07). The rate of infections (especially CMV reactivation), clinical and transplant characteristic were not significantly different between patients with and without GVHD. ConclusionIn our study, a different expression profile of TLRs was found in healthy donors, in patients after SCT without acute GVHD and in those with GVHD. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. The assessment of a larger number of patients would be useful to understand the complex interplay between pathogens, self or non-self DNA and RNA and the immune system after SCT. Disclosures:Off Label Use: In Italy the use of azacitidine for Low-risk Myelodysplastic patients is off label. The use of azacitidine in our study is part of a Phase II clinical trial.

Deferasirox is a powerful NF-kappaB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging.

Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-kappaB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes. We evaluated deferasirox activity on nuclear factor-kappaB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 muM deferasirox for 18h. Nuclear factor-kappaB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate. Nuclear factor-kappaB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.

P087 Analysis of iron homeostasis and erythroid activity in a cohort of low-risk myelodysplastic patients

P087 Analysis of iron homeostasis and erythroid activity in a cohort of low-risk myelodysplastic patients

Non Invasive Assessment of Hepatic Fibrosis by Measurement of Liver Stiffness in Post Transfusional Iron Overload: Preliminary Results in 15 Patients.

Background: The prognosis of liver iron overload is highly dependent on liver iron content (LIC) and the extent of liver fibrosis. Liver biopsy is the invasive reference method for fibrosis evaluation but with several limitations. Non-invasive biological methods (FibroTest, APRI) are not applicable in blood diseases. Transient elastography (TE) is a new non-invasive and rapid bedside method used to measure liver stiffness.This technique has been extensively studied with success in evaluation of liver fibrosis in HCV infection (Ziol, Hepatology 2005; Castera, Gastroenterology 2005). Until now, no data are available on evaluation of liver fibrosis by TE in highly transfused patients with iron overload.Methods: We assessed liver stiffness (kPa) by elastography measurement (Fibroscan; Echosens, Paris, France). Ishak's and Metavir's scales were used to stage fibrosis and hepatitis activity; LIC was measured by atomic absorption spectometry in a central laboratory (Rennes, France) and by MRI procedure according to Gandon (Lancet 2004). Cardiac MRI T2* (indirect estimation of iron heart content) was also measured by MRI according to Anderson (Eur Heart J 2001). Serum ferritin level and HCV serology were assayed in all cases. Correlation statistical analysis used Spearman's Rho and Pearson tests and mean comparisons were done by a non-parametric Mann-Whitney test.Results: 15 liver samples (weight >1mg except 1) from monthly transfused patients (13 major beta Thalassemia, 1 sickle cell disease, 1 myelodysplastic syndrome) were analyzed. Median age was 37 years (7–75). Histological results were: 3 cirrhosis (METAVIR F=4 (F4), or Ishak's Staging =6 (IS6)) among 5 patients with severe fibrosis (F3,F4) or (IS4-6) and 10 patients with no or mild fibrosis (F0-2) or (IS0-3). Mean ferritin was 2579 ng/ml CI 95% [1372–3786]. 2 patients were HCV positive. Correlation between LIC and ferritin was 0.82 (p<0,05); between LIC and MRI LIC: 0.88 (p<0,05); LIC and MRI Heart T2*: 0.72(p<0,05); METAVIR F score and Ishak's grading score: 0.86 (p<0,05). A correlation was also observed between METAVIR F and elastography: 0.60(p<0,05); and mean values of elastography were significally different in patients with severe fibrosis: 9.1kPa CI95% [4.5–13.7] vs 5.9kPa CI95% [4.6–7.2] in those without sever fibrosis (p<0,05) (fig). A value of elastography above 6.25kPa (Se=80%; Sp=70%; AUROC=0.820) identified patients at risk for severe fibrosis (F3,F4 or IS 4–6) (Negative Predictive Value = 88%; Positive Predictive Value = 57%).Conclusion: A significant correlation between Metavir Fibrosis scale and elastography values was found. This new non-invasive method would be helpful to evaluate liver fibrosis and to determine patients who could avoid invasive procedures in particular in regularly transfused low risk myelodysplastic patients at risk of bleeding due to abnormal platelet function. These preliminary results will have to be confirmed in a larger population. [Display omitted]