Types Of Myelodysplastic Syndromes Research Articles

Bone Marrow Levels of PARP1 mRNA Predict Response to Treatment with 5-Azacytidine in Patients with Myelodysplastic Syndrome

IntroductionPoly [ADP-ribose] polymerase 1 (PARP1) is the major member of a family of nuclear enzymes that participate in DNA repair and gene transcription, by recruiting various proteins involved in DNA repair to sites of DNA damage. PARP1 overexpression has been correlated to poor treatment response in children with acute lymphoblastic leukemia, while PARP1-driven apoptosis is important in patients with chronic lymphocytic leukemia. Moreover, PARP1 inhibitors have been tested in vitro in lymphoid malignancies, acute myeloid leukemia, myelodysplastic syndrome (MDS) and acute promyelocytic leukemia.We have recently shown that bone marrow PARP1 mRNA levels correlate to the prognosis of patients with MDS (Diamantopoulos et al. Blood Cancer 2017). Moreover, PARP1 has been related to hypermethylation through DNA methyltransferase-1 (DNMT1) gene upregulation and protein activation (Zampieri et al. PLoS One. 2009). This correlation with DNA methylation is very important, since hypermethylation is a major event in the pathogenesis of high risk MDS, and hypomethylating agents, such as 5-azacytidine, are the most effective available treatment option for patients with higher risk MDS.AimBased on the relation of PARP1 with hypermethylation, we investigated the correlation of bone marrow PARP1 mRNA levels of patients with MDS with treatment response to 5-azacytidine.MethodsBone marrow samples were collected from patients with MDS at diagnosis. Only samples from patients that were eventually treated with 5-azacytidine were selected for analysis. RNA extraction and reverse transcription were performed using standard protocols. Primers for PARP1 and beta-actin were designed using the primer3 software (University of Massachusetts, USA) and a SYBR green based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). PARP1 mRNA levels were expressed as a PARP1/beta-actin ratio. IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA) was used for the analysis of the results.ResultsSixty-seven patients diagnosed with MDS per the WHO 2008 classification and treated with 5-azacytdine were included in the study. The basic characteristics of the patients are shown in Table 1. The median follow-up of the patients was 25.8 (2.6-117.0) months. Response to treatment with 5-azacytidine per the International Working Group (IWG) criteria is shown in Table 1. The median PARP1 mRNA level was 0.0793 (range (0.0000424 - 3.43). Response to treatment differed between patients with high and low PARP1 mRNA levels (Independent samples Kruskal-Wallis test, 2-sided p=0.009). More precisely, the median level of PARP1 mRNA was 4 times higher in responders (partial response, complete response, hematologic improvement) than in non-responders (failure and stable disease), Independent samples Mann-Whitney U test, 2-sided p=0.006. We found no correlation of any other of the studied factors (MDS type, IPSS, IPSS-R, WPSS, karyotype risk, number of cytopenias, marrow blast percentage, hemoglobin level, neutrophil and platelet count, dose reduction of 5-azacytidine) to response to treatment in this cohort of patients. Although there was no correlation of PARP1 mRNA levels with overall survival, survival of patients after 5-azacytidine treatment tended to be longer in patients with higher PARP1 mRNA levels (38.7 versus 19.8 months, log rank test, p=0.136).DiscussionWe found that patients with MDS and high PARP1 mRNA levels have a better response to treatment with 5-azacytidine. This correlation has a theoretical basis, since upregulation of PARP1 is correlated to DNA hypermethylation, and hypomethylating agents may be more effective in cells with higher methylation levels. Moreover, in our cohort of patients, PARP1 level was the only factor affecting treatment response, although this response was not translated into a survival benefit.PAPR1 is the only biological factor and one of very few identified factors to predict response to treatment with hypomethylating agents - low hemoglobin and platelet count were found to be an independent factor of poor response to hypomethylating agents (Jung et al. Oncotarget 2016), and absence of aberrant myeloid progenitor cells were found to predict response to treatment (Alhan et al. Cytometry B Clin Cytom 2014). A possible synergy between PARP1 inhibitors and hypomethylating agents in MDS should be further investigated. [Display omitted] DisclosuresVassilakopoulos:Amgen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene/GenesisPharma: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Gender and Marital Status Influence Survival of Myelodysplastic Syndrome and Risk of Secondary Acute Myeloid Leukemia: A Population-Based Study of Patients in the Surveillance, Epidemiology, and End Results (SEER) Database

The myelodysplastic syndromes (MDS) are a cluster of clonal hematopoietic disorders with diverse origins. MDS frequently progresses to secondary acute myeloid leukemia (sAML). sAML is unique from de novo AML and is associated with a poorer response to induction chemotherapy and decreased survival (Granfeldt Ostgard, et al 2015). To remedy this situation, we conducted a retrospective study of the National Cancer Institute's (NCI) Surveillance, Epidemiology, and End Results (SEER) database. A logistic regression analysis was performed to determine the significant risk factors associated with transformation of MDS into sAML. In addition, a Cox proportional hazard regression model was used to analyze the prognostic factors for survival of the included patients. Leukemic transformation was fitted in the Cox model as a time-dependent covariate.A total of 36,558 patients with primary, newly-diagnosed MDS were identified in the SEER database during the study period (January 1, 2001 to December 31, 2013) and grouped on the basis of age (≤ 40 and > 40 years). This population is among the largest of any population based-study of MDS or sAML. Table 1 presents a summary of patient demographics and clinical characteristics for the 2 groups of patients, who differed significantly in gender, race, marital status, WHO classification, type of follow-up, and rate and time interval of leukemic transformation. Transformation occurred more frequently (3.7% vs. 2.5%) and more quickly (median time of transformation: 4 vs. 13 months) for patients in the under 40 group. Other studies have also identified a detrimental effect of age on the progression of MDS to sAML (Hulegardh, et al 2015, Shi, et al 2004). Table 2 summarize our analysis of overall survival (OS) and cancer-specific survival (CSS), respectively. For both groups of patients, refractory anemia with excess blasts had the worst prognosis for OS and CSS. Most histological types of MDS including therapy-related myeloid neoplasms were all significant indicators of poor survival when compared to refractory anemia, with the exception of refractory thrombocytopenia. We also found that the negative impact on survival of a subtype other than refractory anemia was greater for patients under 40, particularly for CSS.Regarding demographic factors, older age and female gender were predictors of shorter and longer survival, respectively, with similar effects for each age group. Black race was positively associated with survival, when compared to being white and over 40 (the influence of race was not examined in the under 40 group). This study is among the first to assess the influence of marital status on the outcomes of patients with MDS. Our analysis revealed that being married was positively associated with OS and CSS for patients older than 40, when compared to being single, separated, divorced, or widowed.However, the effect of marital status could be related to race, as Martinez et al found that non-married white patients in the California Cancer Registry (CCR) had a greater risk of mortality than did non-married black, Hispanic, or Asian/Pacific Islander patients (Martinez, et al 2016).In conclusion, we have conducted a retrospective analysis of the SEER cancer registry database. Our results indicate that younger patients not only had a greater likelihood of developing sAML than did older patients but also had a shorter time interval to transformation. Survival among older patients is influenced by demographic factors including age, gender, race, and marital status. Accordingly, the accuracy of current risk assessment systems may be improved by including these types of data.*Correspondending author information: Dr. Jian Huang, Department of Hematology, The fourth Affiliated Hospital, College of Medicine, Zhejiang University. Email: househuang@zju.edu.cn.Funding: Social development project of public welfare technology research, Zhejiang Provincial Department of science and technology, Zhejiang Province, China (2016C33160). Public technology research projects of Yiwu City, Zhejiang Province, China (2016-S-05). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activation Levels of b-Catenin Signaling in Osteoblasts Define a Threshold for the Development of Myeloid Neoplasia of Variable Severity

A growing body of evidence indicates that osteoblasts have a direct pathogenetic role in the development of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and transformation of MDS to AML. Models of osteoblast-induced MDS or AML (OIAML) include b-catenin activation in osteoblasts inducing AML in mice and seen in more than 30% of MDS and AML patients; deletion of Dicer1 in osteoblast progenitors leading to MDS in mice; Noonan syndrome mutation (PTPN11) in stromal cells driving MPN in mice; and Schwachman-Diamond syndrome mutation in stromal cells driving MDS in mice and predicting AML progression in patients. To further understand the mechanism of OIAML we examined whether a defined threshold of AML-inducing signal by osteoblasts is required for disease development. For this purpose we constitutively activated b-catenin in osteoblasts using three different lines of 2.3Col1 a 1-Cre transgenic mice characterized by different recombination efficiencies in bone. Line 1, in which recombination efficiency was 80%. Line 2 was the same original line in which after several inter-breedings we detected a reduction in recombination efficiency to 20%. Line3, which has 75% recombination efficiency. Activation of b-catenin in osteoblasts using Line 1, led to the development of MDS which rapidly transformed to full-blown AML characterized by multi-organ infiltration with myeloid blasts, hematopoietic deregulation and lethality by 6 weeks of age. In contrast, activation of b-catenin in osteoblasts using Line 2 did not lead to AML development. Mice remained healthy and survived until at least 10 months of age, the entire time they were observed. Activation of b-catenin in osteoblasts using Line 3 led to anemia, with the appearance of myeloid blasts in the blood, marrow and spleen and segmented neutrophils in blood. In addition to what was observed with Line 1, Pelger Huet neutrophils and Howell-Jolly bodies were detected in the blood. The latter are RBCs with inclusions of nuclear chromatin remnants (damaged chromosome fragments) indicating genomic instability and a dysfunctional spleen, unable to clear them from the circulation, due to dense infiltration with blasts and megakaryocytes. A spectrum of atypical immature monocytes with abundant pale blue agranular cytoplasm with vacuoles were observed in the blood. Taken together these features resemble characteristics of human MDS/CMML. Mice die between 4 and 14 weeks of age, when they undergo “blast crisis” indicating transformation to AML. To examine whether and how these observations relate to the human disease, we compared the levels of b-catenin activation in osteoblasts (calculated as a function % osteoblasts (Lin-/CD34-/RUNX2+ cells) expressing activated b-catenin) with the different MDS diseases subtypes as those are defined by the 2008 World Health Organization (WHO) classification system. We found that increased levels of b-catenin activation in osteoblasts correlate with the severity of the MDS type and with conversion to AML. Collectively, these observations suggest that a minimal activation level of a key leukemogenic signal from the stroma, is required to initiate cancer development, and provides mechanistic insight into the formation and progression of preleukemic stem cells in AML and the transformation of MDS to AML. DisclosuresRaza:Onconova Therapeutics: Research Funding, Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Janssen R&D: Research Funding; Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding.

Impact of Genetic Abnormalities and Measurable Residual Disease Levels on Outcome in Patients with MDS/AML Pre-Emptively Treated with Azacitidine: Correlative Results of the Prospective RELAZA2 Trial

Impact of Genetic Abnormalities and Measurable Residual Disease Levels on Outcome in Patients with MDS/AML Pre-Emptively Treated with Azacitidine: Correlative Results of the Prospective RELAZA2 Trial

MDS-107: MDS Patients' Needs from Online Discussion Forums: An Artificial Intelligence and Natural Language Processing Analysis of 20,000 Posts in US, UK, Canada, and China

Context: Online patient discussion forums are important in helping myelodysplastic syndrome (MDS) patients and caregivers navigate the disease and treatment journey. These forums can uncover patient information gaps or needs. Access to therapies and cultural backgrounds influence perceptions of health and disease management approaches. Patient interactions may also identify similarities and cultural nuances of MDS patients across countries. Objective: To understand what conversations from online forums of MDS patients and their caregivers convey about their needs as they navigate the disease and treatment journey. Design: We leveraged Artificial Intelligence and Natural Language Processing (NLP) to algorithmically analyze over 20,000 public, anonymous online comments from 2011 to 2019 to understand the motivation of MDS patients and caregivers from the US, UK, Canada, and China. The NLP algorithm generates a Network that organizes large volumes of unstructured data into topics based on semantic similarity. Results: Seven overarching motivations were extrapolated. “Emotional” refers to receiving/providing emotional support to other patients. “Treatments” relates to treatments, including regimes and decisions. “Transplants” includes the benefits, risks, and experience of transplants. “Clinical” relates to diagnosis, progression, and monitoring. “Education and Logistics” focuses on learning about the disease and care options. “Physical” highlights management of symptoms and treatment side effects. “Diet and Lifestyle” focuses on diet changes and other aspects of living with MDS. The analysis of 20,000 posts detected different motivations but also universal similarities in MDS narratives across countries. In the US, the top conversation driver indicated that patients seek out those with similar cytogenetics profiles, MDS types, or diagnoses. In contrast, in the UK, emotional support was a top motivational driver for patients and caregivers. In Canada, the conversation was driven by patients asking others for specific treatment experiences to evaluate their treatment options. Conversely, patient posts in China indicated that patients are more focused on understanding the disease. Conclusions: The findings suggest that there are unmet information and support needs among MDS patients and caregivers. These include disease/treatment information, support, adequate information sources, and overall guidance to navigate patients through their journey. Greater healthcare professional awareness of these gaps may improve patient care.

Anti- Inflammatory Treatment in Pediatric Advanced Myelodysplastic Syndrome Associated with Inflammatory Manifestations May Reverse Progression and Bridge to a Successful HSCT

Introduction Inflammatory Manifestations (IM) are well described in 10-30% of adult patients with Myelodysplastic Syndrome (MDS). Most case series do not report correlation between cytogenetic or MDS type. Different treatment modalities have been tried, but their role in relation to HSCT has not been established. Data for the pediatric MDS is scant. Methods We report here 4 pediatric cases of advanced MDS associated with IM who were diagnosed over a one year period in a tertiary pediatric center. Apart from routine MDS diagnostic methods, we tested patients for genetic predisposition, somatic mutations and cytokine levels to better characterize this entity. Results One patient was diagnosed with severe congenital neutropenia prior to MDS diagnosis, while the other 3 had primary MDS with no genetic background. Two of them were tested for somatic mutations and both harbored a somatic mutation in the PTPN11 gene. IM included Fever (N=4), Significant weight loss (N=3), Upper GI Ulcers (N=2), colitis (N=1), arthritis (N=1) and pulmonary congestion (N=2). All patients had Lansky score Systemic steroid treatment was initiated for all patients with at least partial clinical response observed in all. One patient was treated additionally with Anti IL 6 (Tocilizumab) monoclonal antibody (his serum IL6 was >log of the normal limit) and ATG. Bone marrow aspiration post treatment showed significant reduction in blast count in 3 patients, and complete normal hematopoiesis with no evidence of MDS in one patient. This patient remained in remission for 3 month and then his MDS relapsed with IM. Three patients had a matched sibling donor, and one patient had a 9/10 HLA matched unrelated donor. Myeloablative conditioning (Busulfan- Cyclophosphamide and Melphalan) was given to 3 patients, while one patient received reduced toxicity conditioning due to liver injury from ATG. All patients had neutrophil engraftment and 100% donor chimerisim. At a median follow up of 7 months, 3 patients are alive with no evidence of disease, and one patient died from HSCT complications. Conclusion MDS with IM is a significant entity in the pediatric population, demonstrating a heterogeneous phenotype and genetic background. Anti-inflammatory treatment may improve clinical symptoms and decrease blast count, bridging to a successful HSCT. Larger prospective studies with longer follow up are needed in order to better characterize this entity and its optimal treatment.

Characteristics of Long-Term Survival of Patients with MDS Treated with 5-Azacytidine. Results from the Hellenic 5-Azacytidine Registry

IntroductionThe prognosis of patients with myelodysplastic syndromes (MDS) depends on several disease characteristics such as the cytopenias, the percentage of bone marrow blasts, and the cytogenetic profile of the patients. The use of hypomethylating agents (HMAs) has altered the prognosis of patients with higher risk MDS offering a median survival of around 24 months. Nevertheless, the range of survival is wide, with some patients achieving long remissions and high survival rates irrespective of their initial prognostic characteristics. Long-term survivors after 5-azacytidine administration constitute a large group of patients with potentially special characteristics and needs.AimWe analyzed data from a large cohort of patients with MDS treated with 5-azacytidine to describe the hematologic and prognostic characteristics of long-term survivors and compare them to those of patients with shorter survival.Patients and MethodsWe retrospectively recorded through the Hellenic 5-azacytidine registry the main demographic, hematologic and treatment characteristics of adult patients with MDS treated with 5-azacytidine monotherapy. Patient data from 28 centers meeting the 2008/2016 WHO diagnostic criteria for MDS were recorded during a 7-month period.We defined two groups of long-term survivors based on their survival after initiation of treatment with 5-azacytidine (OST). The first group comprised patients with OST above the third quartile (Q3 or 75th percentile) of the whole group (Q3 group) and the second patients with OST above the 90th percentile of the whole group (P90 group). Correlations were made between long- and short-term survivors for both groups.IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA) was used for the statistical analysis of the results.ResultsData from 626 patients was recorded. The Q3 group comprised 157 patients with an OST longer than 24.5 months (median, 43.3 months) and the P90 group 63 patients with an OST longer than 36.4 months (median, 65.7 months). The detailed characteristics of the two groups along with comparisons with the remaining patients with OST below Q3 and P90 respectively are shown in Table 1. Data analysis revealed that the sex, the age, the type of MDS at diagnosis per the 2008/2016 WHO classification, the presence of excess (≥5%) marrow blasts, the number of cytopenias, the hemoglobin, neutrophil and platelet count, and the transfusion needs were not predictive of long-term survival in neither of the groups. On the other hand, the presence of peripheral blood blasts, the karyotype risk, the IPSS, IPSS-R and WPSS classification and response to treatment were predictive of long term survival in both groups (Table 1). Multivariate analysis revealed that response to 5-azacytidine was the strongest determinant of long-term survival (Kaplan Meier, Log Rank, p<0.0001) in a model comprising IPSS, IPSS-R, WPSS and response to treatment. Nevertheless, patients with stable disease were almost equally distributed in the groups of long- and short-term survivors (p=0.795 for the Q3 group and p=0.310 for the P90 group).DiscussionThe use of HMAs in MDS has increased survival rates, hence long-term survival is now a feasible target when managing such patients. One fourth of the patients of this registry achieved an OST over 24.5 months and 10% over 36.4 months. IPSS, IPSS-R and WPSS are powerful prognostic tools for patients with MDS. Among the prognostic components of IPSS and IPSS-R at diagnosis (cytopenias, bone marrow blast count, karyotype risk), the karyotype risk seems to be the stronger determinant of survival. Nevertheless, among long-term survivors there are patients with adverse prognostic characteristics at diagnosis, whose prognosis is altered by the administration of HMAs. Failure to respond to 5-azacytidine is a major determinant of OST, but stable disease was not correlated to survival in this cohort. This result highlights the importance of continuing treatment with hypomethylating agents in patients not achieving an optimal response (PR, CR, HI), since a significant proportion of them may achieve long survival rates. Further search for new clinical and/or molecular prognostic markers is warranted to identify the prognosis of patients with MDS and define those who would benefit from the use of HMAs or other upcoming treatment choices. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SAMD9 and SAMD9L Germline Disorders in Patients Enrolled in Studies of the European Working Group of MDS in Childhood (EWOG-MDS): Prevalence, Outcome, Phenotype and Functional Characterisation

▪Hereditary predisposition has been ever since implicated in the etiology of childhood myelodysplastic syndromes (MDS). Until recently, GATA2 deficiency prevailed as a major germline cause in pediatric primary MDS. In the past 2 years, we and others identified germline mutations in paralogue genes SAMD9 and SAMD9L residing on chromosome 7q21.2 as new systemic diseases with high propensity for MDS with monosomy 7. Although initially, mutations in SAMD9 and SAMD9L genes were associated with MIRAGE and Ataxia-Pancytopenia syndromes, respectively, with recent reports the phenotypes are becoming more intertwined. Nevertheless, the predisposition to MDS with monosomy 7 (-7) remains a common clinical denominator. Both genes are categorized as negative regulators of cellular proliferation and mutations were shown to be activating. Because of their high evolutionary divergence, classical in silico prediction is erratic, thereby establishing in vitro testing as the current gold standard for pathogenicity evaluation. The objectives of this study were to define the prevalence of SAMD9/9L germline mutations in primary pediatric MDS, and to describe the clinical phenotype and outcome. In addition, we aimed to characterize the somatic mutational architecture and develop a functional scoring system.Within the cohort of 548 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, 43 patients (8%) carried SAMD9/9L mutations that were mutually exclusive with GATA2 deficiency and known constitutional bone marrow (BM) failure. MDS type refractory cytopenia of childhood was diagnosed in 91% (39/43), and MDS with excess blasts in 9% (4/43) of mutated cases. Karyotype at diagnosis was normal in 58%, and -7 was detected in 37% of SAMD9/9L cohort. Within MDS subgroup with -7 (n=74), SAMD9/9L mutations accounted for 22% of patients. Notably, the demographics, familial disease, diagnostic blood and BM findings, overall survival (OS) and the outcome after HSCT were not influenced by mutational status in our study cohort (n=548). At the last follow up, 88% (38/43) of SAMD9/9L MDS patients were alive; 35/43 had been transplanted with a 5-year-OS of 85%.Next, we added 26 additional cases with SAMD9/9L mutations diagnosed in Europe within EWOG-MDS studies. In the total cohort of 69 germline mutated patients we found a total of 75 SAMD9/9L mutations, of which 67 were novel. Of those we tested 47 using a HEK293 cell in vitro system and 45/47 mutants inhibited proliferation. While 53/69 patients carried only single germline mutations (missense in 50/53 and truncating in 3/53), in the remaining 16 patients, 11 additional truncating and 7 missense mutations were found. We did not observe an association between germline mutation and phenotype. Immunological issues (e.g. recurring infections, low Ig) were described in 32%/50% of SAMD9/9L-mutated patients, while physical anomalies were very heterogeneous and reported in ~50% of patients in both mutational groups. Intriguingly, genital phenotypes occurred in 40% of SAMD9L, while neurological problems were present in 30% of SAMD9 - mutational subgroups.To elucidate the somatic mutational landscape, we performed whole exome and deep sequencing of 58 SAMD9/9L patients and identified recurrent somatic mutations in known oncogenes that were earlier associated with pediatric MDS: SETBP1 (10%), RUNX1 (7%), ASXL1 (5%), EZH2 (5%), CBL (3%). The identified somatic mutations occurred in association with monosomy 7 background (18/20). Finally, we utilized the results from functional testing of the 47 SAMD9/9L variants as our test cohort to develop combinatorial in silico scoring. The rationale was to decrease the dependency on functional validation. Based on the results of 20 in silico tools we could concatenate a matrix of 5 algorithms to resolve the pathogenicity of >80% of variants. Using this model, all variants predicted as pathogenic showed also growth-restrictive effect in vitro. In summary, pathogenic SAMD9/9L germline mutations account for 8% of primary pediatric MDS and 22% of MDS/-7. The mutations identified are heterogeneous and their effect can be predicted using a combinatorial in silico - in vitro approach. Finally, the clinical outcome and somatic mutational landscape are not influenced by the mutational status. DisclosuresLocatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Niemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Acute pro-B-Cell lymphoblastic leukemia transformed from myelodysplastic syndrome with an ASXL1 missense mutation: A case report with literature review.

The development of acute lymphoblastic leukemia (ALL) from myelodysplastic syndrome (MDS) is a very rare event. The current report presents a rare case of a 33-year-old man who was diagnosed with MDS with multiple-lineage dysplasia (MDS-MLD) that transformed into pro-B-ALL. A missense mutation (S1231F) of the additional sex combs like 1, transcriptional regulator gene was identified, which may have a substantial role in the progression, however does not act as an unfavorable prognostic marker. The patient died during induction chemotherapy. The present study further conducted an analysis on 30 patients to determine progression to ALL. Patients were predominantly male (76.7%, 23/30) with a median age of 56 years (3–90 years). The median time to transformation was 5.5 months (2–50 months). The most common type of MDS with ALL transformation comprised of MDS-excess blasts (MDS-EB; 40%, 12/30), MDS with single-lineage dysplasia (MDS-SLD; 30%, 9/30) and MDS with ring sideroblasts (MDS-RS; 16.7%, 5/30). The majority of the patients transformed to B-cell (66.7%, 16/24) followed by T-cell (33.3%, 8/24) ALL. From the 25 cases where data was available, the complete remission rate was 75% (15/20) with ALL-directed chemotherapy and the median remission duration was 15 months (range 4.5 to 51 months). However, the results indicated that ALL following MDS is characterized by a high rate of early death (20%, 5/25).

Characterization of TP53 mutations in low-grade myelodysplastic syndromes and myelodysplastic syndromes with a non-complex karyotype.

Although commonly associated with high-grade myelodysplastic syndrome (MDS) and MDS with a complex karyotype, TP53 mutations also occur in low-grade MDS and MDS with a non-complex karyotype. In latter cases, their clinicopathological features and the characteristics of TP53 mutations remain poorly characterized. 176 MDS cases with TP53 mutations were stratified and characterized based on their karyotype and histologic subtype. Among 176 cases, 17% had a non-complex karyotype and 24% were low-grade MDS. TP53 mutations often occurred in DNA-binding domains and the majority of cases had only one mutation, irrespective of their karyotype and MDS subtype. The variant allele frequency (VAF), however, was associated with karyotype complexity and the types of MDS with a lower VAF found in cases with a non-complex karyotype and low-grade MDS. A low (<20%) VAF was associated with a better survival, as well as low-grade subtype. In low-grade MDS and MDS with a non-complex karyotype, TP53 mutations showed a lower VAF. Patients with a lower VAF had a better survival. TP53 mutations are not the only prognostic factor in MDS patients with TP53 mutations as the VAF, blast counts and history of prior therapy also play important roles in prognosis.

Mutations of ASXL1 gene in myelodysplastic syndromes

Objective To investigate the mutations of epigenetic regulation factor ASXL1 gene in myelodysplastic syndrome (MDS). Methods Mutation analysis of ASXL1 gene in 53 de novo MDS patients and 20 healthy persons was performed by using polymerase chain reaction (PCR) followed by sequence analysis at DNA level. The clinical and laboratory characteristics were compared in MDS patients with ASXL1 gene mutation and ASXL1 wild type. ASXL1 mutation in mRNA level was detected by using reverse transcription PCR (RT-PCR) followed by sequence analysis. Results ASXL1 gene mutations were observed in 9 cases(16.9 %) of 53 MDS patients.6 mutation types were detected, including 4 frameshift mutations types (2 cases with p.Glu635ArgfsX15, 3 cases with p.Gly646TrpfsX12, 1 case with p.Ala640GlyfsX14 and 1 case with p.Gly790TrpfsX10) and 2 nonsense mutation types (1 case with p.Gln1063X and 1 case with p.Gln695X). All the mutations were heterozygous, and p.Gly790TrpfsX10 and p.Gln695X were new mutation types. In addition, a single nucletide polymorphism (SNP) p.Gly652Ser was also detected in 4 cases with MDS. 5 cases of p.G652S SNP and 1 case of p.Leu1173Leu SNP were detected in 20 healthy people. Frameshift mutation (p.Gly646TrpfsX12) could be detected at mRNA level by using RT-PCR. Differences were not observed in red blood cell counts, white blood cell counts, platelet counts, hemoglobin levels, reticulocyte, neutrophil granulocyte, the peripheral blood lymphocytes percentage, T-cell subsets in the peripheral blood, the proportion of primitive cell in the marrow and MDS types between the patients with ASXL1 gene mutation and ASXL1 wild type patients (P > 0.05). Conclusion There is a high frequency of ASXL1 gene mutation in MDS patients, which can be detected at mRNA level. Key words: Gene, ASXL1; Myelodysplastic syndrome; Mutation; Epigenetic regulation factor

Effects of arsenic disulfide on apoptosis, histone acetylation, toll like receptor 2 activation, and erythropoiesis in bone marrow mononuclear cells of myelodysplastic syndromes patients in vitro

ObjectiveAs the main component of traditional Chinese medicine realgar, arsenic disulfide (As2S2) is widely used in treating myelodysplastic syndromes (MDS). The goal of the current study is to assess the effects of As2S2 on bone marrow mononuclear cells (BMMNC) of MDS. MethodsBMMNCs were obtained from 10 lower risk MDS patients, 5 higher risk MDS patients, and 3 healthy controls. Then, the cells were treated with As2S2 for 48h, using vorinostat (also known as SAHA) as control. Cell proliferation and apoptosis were detected. mRNA and protein levels of histone deacetylase-1 (HDAC1), Toll-like receptor 2 (TLR2), and erythroid transcription factor (GATA-1) were detected by quantitative real-time PCR and western blot analysis. ResultsAfter As2S2 treatment in concentrations ranging from 3.125 to 100μmol/L, cell proliferation was inhibited in both lower risk and higher risk MDS. Fifty percent inhibitory concentrations were 24.4μmol/L and 23.6μmol/L, respectively, for lower and higher risk MDS. Apoptotic cells significantly increased in both types of MDS. mRNA and protein levels of HDAC1 and TLR2 were reduced, whereas GATA-1 was increased in both types of MDS. ConclusionsAs2S2 could inhibit cell proliferation and induce apoptosis through histone acetylation modulation in MDS. Similar to SAHA, As2S2 could reduce TLR2 activation and increase GATA-1 expression. Current data suggest epigenetic and immunological alternations are involved in therapeutic mechanisms of realgar in the treatment of MDS.

An antibody derived from a cured AML patient to identify a unique epitope on CD43 (CD43s) as a novel target for acute myeloid leukemia and myelodysplastic syndrome.

7009 Background: Immunotherapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is hampered by the lack of tumor-specific targets. Methods: We took advantage of the tumor-immunotherapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) and searched the B cell repertoire of a patient with a lasting and potent graft versus AML response for AML-specific antibodies. Results: We identified a donor-derived B cell clone that produced an IgG1 antibody, AT1413, that specifically interacted with AML cell lines, with the patient’s autologous AML blasts, but not with lymphocytes or with cells from liver, colon, skin and other tissues. AT1413 recognized a unique, not previously described, sialylated epitope on CD43 (CD43s). CD43s is overexpressed on all types of AML and MDS, as illustrated by its reactivity with freshly isolated blasts of each of more than 60 randomly selected AML and MDS patients in our clinic, representing all WHO 2008 AML and MDS classes. AT1413 induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) of target cells in vitro. To investigate the effect of AT1413 in vivowe first generated mice populated with human effector cells (NK cells, CTL and myeloid cells) by injecting human hematopoietic stem cells into new born immunodeficient mice. After establishment of a human immune system in these mice we inoculated luciferase labeled AML cells via tail vein injection. Following engraftment of the tumor we dosed the mice biweekly with AT1413 or a control antibody. We observed strongly reduced numbers of AML cells in AT1413- but not in control antibody treated mice. Importantly, AT1413 treatment was tolerated well and did not affect numbers of non-malignant human myeloid cell in these mice. Conclusions: We have obtained an antibody from a cured AML patient which recognizes a unique sialylated epitope on CD43 (CD43s) that is selectively over-expressed on all WHO 2008 types of AML and MDS. This antibody was able to eliminate AML cells in vivo and therefore has high therapeutic potential.

P53 protein expression in patients with myelodysplasia treated with allogeneic bone marrow transplantation.

Tumor protein 53 mutations adversely affect the prognosis of myelodysplastic syndromes (MDS); however, few studies have reported on the prognostic significance of the expression of p53 protein in MDS. The current study investigated p53 immunoreactivity (p53-IR) in bone marrow biopsies (BMBs) obtained at diagnosis from 18 patients (6 females and 12 males; mean age, 50.5 years) with MDS that underwent bone marrow transplantation (BMT) to determine the associations between clinical and histopathological data and outcome. There were 5 refractory cytopenia with multilineage dysplasia (RCMD) and 13 refractory anemia with excess blasts, type 2 (RAEB-2) cases. p53-IR was assessed as the percentage of hematopoietic cells exhibiting intense nuclear staining. The cut off for positivity was 5% of stained cells. A positive p53-IR was detected in 7 patients (38.9%) and was associated with age (P=0.005) and pattern of BM fibrosis (P=0.03). A positive p53-IR was more frequent in females, in highly cellular BMBs and in RAEB-2 cases. Overall survival (OS) was associated with patients' age (P=0.01), hemoglobin level (P=0.04), type of MDS (P=0.05), degree of BM fibrosis (P=0.006) and number of BM blasts (P=0.05). The OS of patients with negative p53-IR tended to be longer compared with that of patients with positive p53-IR, although this difference was not statistically significant (P=0.1). Despite the limitation of the low number of cases, the present results indicate that a positive p53-IR at diagnosis is associated with clinically more aggressive MDS subtypes and adverse histological prognostic factors, such as BM fibrosis. Therefore, the evaluation of p53 expression of BMBs of patients with MDS may be introduced in the histopathological work-up of the disease.

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup.

In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 “new” unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a “clinical warning”; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).

New proposals of the WHO working group (2016) for the diagnosis of myelodysplastic syndromes (MDS): Characteristics of refined MDS types

Based on centrally diagnosed 3528 patients in the Düsseldorf registry, we validated the new proposals for the classification of the MDS by the WHO working group: 256 patients were diagnosed as MDSSLD (7,3%), 978 MDSMLD (27,7%), 227 MDS RS SLD (6,4%); 321 MDS RS MLD (9,1%), 159 MDS del(5q) (4,5%), 481 MDSEB 1 (13,6%), 620 MDSEB 2 (17,6%), and 148 MDS-U (4,2%). 352 patients (16,9% of the non blastic types) changed the category, mainly moving from RCMD to MDS RS MLD, RCUD and RCMD to MDS del(5q). Median survival times of the refined groups differed from more than 60 months in the MDSSLD (RS) groups, 37 months in the MDSMLD (RS) groups, 79 months of the MDS del(5q) group and 21 and 11 months in the MDSEB 1 and 2 groups, respectively. The difference between the groups with regard to the risk of AML evolution was also impressing. No major changes were made with regard to the MDS-U categories. In summary, the proposals of the WHO group for the classification of MDS are thoughtful, taking into account biologic parameters of the diseases, a more precise wording, to some extend pragmatic and feasible.

Poly (ADP-ribose) polymerase 1 mRNA levels strongly correlate with the prognosis of myelodysplastic syndromes

Poly (ADP-ribose) polymerase 1 (PARP-1) has a central role in the repair of DNA breaks and is a promising treatment target in malignancy. We measured PARP1 mRNA levels by a SYBR-green-based PCR in the bone marrow of 74 patients with myelodysplastic syndrome (MDS) and correlated them to their demographic, hematologic and prognostic characteristics. The median PARP1 mRNA levels were correlated to the type of MDS (2008/2016 WHO classification, P=0.005) and to the IPSS score (P=0.002). A correlation was also found with the IPSS-R score (P=0.011) and the cytogenetic risk (P=0.008). In all cases, higher PARP1 levels were correlated with a higher risk category. Moreover, we found a significant survival disadvantage for patients with high PARP1 levels (median survival of 37.4 months versus ‘not reached’ for low PARP1 levels, P=0.0001, and a 5-year survival rate of 29.8 versus 88.9%, respectively). PARP1 mRNA levels were found to be the stronger predictor of survival in multivariate analysis. These correlations have never been reported in the past and may render PARP1 a prognostic factor to be incorporated in the current prognostic systems for MDS, also laying the basis for clinical trials evaluating PARP1 inhibitors in higher-risk MDS.

Clinical, hematological, and cytogenetic profile of adult myelodysplastic syndrome in a tertiary care center.

BackgroundMyelodysplastic syndrome (MDS), a disorder of clonal hematopoiesis, is an important clinical entity, but most of the studies available are conducted among the Western population. Its etiological factors and clinicohematological profile in the Indian population are quite diverse. The information regarding its prognostic factors and cytogenetics is very scarce.Objectives(1) To assess the clinicohematological profile, cytogenetics, prognostic factors, and outcome of MDS and (2) to study its progression to acute myeloid leukemia (AML) in the selected patients over the study period.MethodsA prospective observational study was performed with patients from Department of Medicine and Hematology, Government Medical College, Kozhikode, who were diagnosed with MDS within the study period (from 1 January 2014 to 31 July 2015). Secondary causes of dysplasia were excluded. In possible cases, the international prognostic scoring system was followed. These patients were followed up for an additional 6 months to assess the progression of MDS to AML based on symptoms, signs, hemogram, or repeat peripheral smear/bone marrow studies.ResultsOf the 60 patients, 73% were aged >60 years. Disease was common in males, with a male:female ratio of 7:3. Thirty-five percent of the patients were working in agricultural and allied fields and had pesticide exposure. Patients with prior radiation exposure had significant association with adverse outcome. Fatigue was the prominent symptom and was reported by 90% of the patients. Blasts were >5% in peripheral smear; bone marrow cytopenia and dysplasia at the time of diagnosis had significant association with risk of transforming to AML. Refractory anemia (RA), observed in 22 patients, was the most common type of MDS. Most of the patients with RA with excess blasts type-1 and RA with excess blasts type-2 transformed to AML, and the association was statistically significant. Deletion of short arm of fifth chromosome (5q deletion) was detected in 11 patients. All of them showed good response to treatment with lenalidomide and had a favorable outcome.ConclusionThis study highlights the various etiological factors, and the clinical profile of MDS seen in the Indian population. Cytogenetic analysis and application of the international prognostic scoring system has a significant bearing on the outcome, as exemplified by the response to lenalidomide in patients with 5q deletion. This study also indicates that proper diagnostic and prognostic assessment is necessary to institute appropriate therapeutic options.

Clonal Dynamics of Refractory Aplastic Anemia in Patients Treated with Eltrombopag

Despite documented success of immunosuppressive therapy (IST) in the treatment of AA, a minority of patients remain refractory, most responses are incomplete, and use of hematopoietic cell transplantation (HCT) is limited in older patients or those with significant comorbidities. While the introduction of the cMpl agonist eltrombopag (EPG) as salvage therapy or in conjunction with IST has revolutionized treatment for refractory AA. It may be effective in improving primary response rates to IST, engaging growth factor receptors with agonistic therapeutics (such as EPG) and also has the potential to promote evolution/expansion of mutant clones, thereby increasing the rate of progression to secondary myelodysplastic syndromes (MDS), a serious complication of AA occurring in 10-20% of patients. Clonogenic somatic mutations typical of MDS in patients with AA and PNH may increase the risk of progression to MDS.DNA from marrow samples of primary refractory AA patients was subjected to analysis before and after initiation of EPG to evaluate clonal expansion or evolution using a targeted multi-amplicon deep NGS panel of all ORFs of the top 60 most commonly mutated genes in MDS. In addition to the EPG treatment group, a case control cohort matched for age and duration from AA diagnosis to last clinical follow up (who did not receive EPG), was studied.Among 210 AA patients treated at Cleveland Clinic, we identified 26 who were treated with EPG for IST-refractory AA; median duration of treatment was 56 wks. The overall response rate after 12 weeks of therapy was 58% (15/26), while 31% of patients (8/26) showed stable disease with intermittent transfusions (one of whom underwent HCT). In 3 non-responders, one developed PNH, one had refractory AA/PNH, and one progressed to AML (see below). Expansion of PNH granulocytes after EPG treatment was observed in 23% of patients (6/26). In addition, 15% (4/26) had atypical subclonal chromosomal abnormalities.Prior to EPG, at least a single somatic event was found in 31% of patients (8/26), with 2 patients harboring 2 mutations. Events included CEBPA, EZH2, BCOR/BCORL1, ASXL1, U2AF1/2, TET2, and DNMT3A mutations. Following EPG therapy, acquisition of new somatic mutations was observed in 23% of cases, including RUNX1, U2AF1, BCOR, RIT1, and CEBPA. In cases with pre-existing clones, 6 clones expanded (e.g., BCOR or ASXL1 from VAF of 8 to 21% and 9 to 29%, respectively) despite clinical hematologic response, while in 2 cases clones disappeared (e.g., U2AF2 and BCORL1). In 54% of cases (14/26), we found detectable levels of a PNH clone at the time of diagnosis. Six of those cases had PNH clonal expansion post-EPG treatment, of which two developed clinically significant PNH clonal burden requiring eculizumab therapy.In the case-control cohort, 26 AA patients who received IST but were not treated with EPG, were followed for comparable time periods, and no evidence of progression to MDS was recorded. One patient was noted to have trisomy 15 on cytogenetics at diagnosis. "MDS type" molecular mutations were present in 10 patients similar to EPG cohort. Among these patients, 3 had persistent clones of U2AF1, DNMT3A, and STAT3 over one year without acquisition of any new molecular mutations. . PNH granulocytes expanded in 50% of AA cases, decreased in 30% and stayed stable in 20%. Thus, we did not observe any difference in expansion of PNH clones between those treated and untreated with EPG (p=0.73). Unlike for PNH clones, accounting for both new evolution and expansion of preexisting molecular mutations, the frequency of these clonal events was significantly higher in the EPG treated group (p=0.009).In conclusion, we observed occasional expansion of clones with potentially leukemogenic mutations during treatment with EPG in pts with AA. While higher rates of MDS evolution were not observed in this cohort of EPG treated patients, we found that serial evaluation of somatic mutations can inform clonal evolution and can potentially be used as abiomarker for evaluation of risk for post-AA MDS. Continued use of EPG in such patients should be judicious. DisclosuresCarraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Expression of Poly [ADP-Ribose] Polymerase 1 (PARP-1) and Uridine-Cytidine Kinase (UCK) 1 and 2 in Myelodysplastic Syndrome

IntroductionPoly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA breaks, thus protecting the cell from genomic instability. Overexpression of PARP1 may lead a cell to apoptotic or necrotic death, defining the cell's fate. Genetic defects are important in the pathogenesis of myelodysplastic syndromes (MDS) and the role of PARP1 in the apoptotic pathways seems to be promising, since new agents targeting PARP1 are available. PARP1 expression has never been studied in MDS.Uridine-Cytidine Kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. The UCK family consists of two members, UCK1 and UCK2, both expressed in several tumor cell types. UCK2 in particular has been found to be expressed in pancreatic tumors, neuroblastoma and other malignant cells, but not in normal tissues, although it is normally expressed in human placenta. The selective expression of UCK2 in certain tumor cells enables selective malignant cell targeting with UCK2-dependent pyrimidine analogues, while sparing normal tissues.AimOur aim was to detect PARP1, UCK1 and UCK2 mRNA in bone marrow samples of patients with MDS and investigate its correlations to the hematologic and prognostic characteristics of the patients.Patients and MethodsBone marrow samples were collected from patients with MDS. Quantification of PARP1 mRNA was performed by a SYBR-green based Real-Time PCR, and the results were expressed in correlation to those of the housekeeping gene of beta actin. Quantification of UCK1 and UCK2 was performed by a Real-Time PCR using specific oligonucleotides and TaqMan probes (Applied Biosystems, CA, USA) and all experiments were normalized to the housekeeping gene of glyceraldehyde-2-phosphate dehydrogenase (GAPDH). All reactions were performed on BIORADCFX96 (BIORAD Laboratories, CA, USA).PARP1 protein was also detected by western-blot using both simple films and the Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, CA, USA).ResultsFifty seven (57) patients with MDS were included in the study. The basic demographic and hematologic characteristics of the patients are shown in Table 1. The vast majority of the patients (92.9%) were treatment naïve. PARP1 mRNA levels were measured in 53 patients, while UCK1 and UCK2 mRNA levels were measured in all patients.The median PARP1 mRNA levels were 0.0259 (range 0.0003 - 0.6410) and showed a statistically significant correlation to the type of MDS (according to the WHO classification) (p=0.016) and to the IPSS score (p=0.001) (detailed results in Table). The lowest levels were observed in patients with RA (0.0053 vs 0.1477 for non-RA patients, p= 0.016), as well as in patients with low and intermediate 1 IPSS score (0.0107 vs 0.1831, p=0.01) that had almost 20 times lower PARP1 mRNA levels than patients with intermediate 2 and high risk MDS.The protein could only be detected in samples tested with the Azure c300 Chemiluminescent Western Blot Imaging System. The median value of PARP1 expression was 0.298 (0 - 1.625) and no correlations with the WHO classification or the IPSS were detected. To our knowledge, PARP1 detection by western blotting has never been performed in the past in MDS.Although both UCK1 and UCK2 were detected in the majority of the patients (40/57, 70.2% for UCK1 and 15/57, 73.7% for UCK2), no correlation was found between their levels and the type of MDS or the prognostic factors according to the IPSS or WPSS. The median levels were for UCK1 0.0202 (0 - 2.7431) and for UCK2 0.0169 (0 - 7.270). Detailed results are presented in Table 2.ConclusionsThe correlation of higher levels of PARP1 mRNA with higher risk MDS has never been reported in the past. This correlation, if confirmed by larger studies, may render PARP1 a prognostic factor for patients with MDS. Moreover, this result can lay the basis for the design of clinical trials evaluating the use of PARP1 inhibitors in patients with higher risk MDS. Although we didn't detect any correlation of UCK1 and UCK2 with the prognostic characteristics of MDS, their mere detection in clinical samples of patients with MDS is significant, since these molecules are not detected in every malignancy, but just in a subset of malignant diseases. This finding could be of value since targeted therapies are already available. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.