Pathophysiology Of Myelodysplastic Syndromes Research Articles

PHF6 Somatic Mutations and Their Functional Role in the Pathophysiology of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Recurrent somatic nonsense PHF6 mutations have been reported in patients with T-acute lymphocytic leukemia, AML and chronic myeloid leukemia in blast crisis. Germ line (GL) PHF6 mutations are responsible for Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and dysmorphic features. PHF6 is a highly conserved 41kDa protein with ubiquitous expression in hematopoietic cells, including CD34+ cells.We screened patients (N=1166) with myeloid neoplasms by targeted multi-amplicon deep NGS targeting all ORFs of PHF6 to determine the prevalence and distribution and molecular context of PHF6 gene alterations. In total, we identified and verified 52 cases with somatic PHF6 mutations, 32 of which were frameshift or nonsense mutations and with a strong male predominance (76%). Mutations were distributed almost equally between 2 DNA binding domains. Previously, PHF6 has been included in other screening panels (Haferlach et al. 2014 and Papaemmanuil 2013) with somatic mutations found in 24/944 and 21/738 MDS cases, respectively. SNP-array karyotyping showed that microdeletions involving the PHF6 locus were present in about 1.2% of myeloid neoplasms, but affected only female patients.The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.018). The most commonly associated somatic mutations included RUNX1 (P=.001) and IDH1 (P=.008) but not IDH2 (P>.1). There was no impact on overall survival with respect to PHF6 mutant status in total or within individual risk groups (low risk (RA,RARS) vs. high-risk (RAEB1/2). Concomitant PHF6 and RUNX1 mutations were associated with particularly poor prognosis. RUNX1 mutational status correlated with PHF6 expression levels and PHF6 expression inversely correlated with RUNX1 mRNA levels. Subsequent analysis of clonal architecture using VAF calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene in 18% of cases. PHF6 variant allelic frequency (VAF) varied between disease subtypes, with the highest clonal burden found in AML patients (P<.01). Within MDS patients we also found lower expression of PHF6 mRNA in CD34+ cells in MDS overall vs. controls (P<.01), as well as lower expression of PHF6 in advanced myeloid neoplasms (P<.05). Lower expression (defined as mean+1SD of controls) was found in 12% and 23% of patients with lower- or higher- risk MDS, respectively.Recent studies have proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and DNA damage. Following shRNA knockdown, hematopoietic cell lines showed only moderately accelerated growth and increased response to growth factors, while EPO-dependent UT7, did not result in growth factor autonomy. To delineate the possible pathophysiological pathway involving PHF6, we compared transcriptional expression profiles of 5 different cell lines with shPHF6 to WT counterparts. We then studied the consequences of PHF6 knockdown on transcriptional profiles. We have found 1020 transcripts differentially expressed (with at least 1.5x change up/down) in the context of shPHF6 knock down. Concordant results among all 5 cell lines resulted in 354 genes that were upregulated and 766 that were down-regulated. Analyses with primary patient data derived from low PHF6 expressors and mutant cases found a concordance of 71 upregulated genes and 80 genes that were downregulated.The most significant functional group of transcripts that was found to be modulated was a family belonging to ribosomal biogenesis pathway (pFDR<1x10-6). Mass spec fingerprinting found protein-protein interaction partners that were found to be dysregulated on a transcriptional level. This finding of protein interaction/transcriptional dependence might suggest feedback mechanisms on a transcriptional level.In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1/IDH1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. DisclosuresSekeres: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. Makishima:The Yasuda Medical Foundation: Research Funding.

Dysregulation of ANKRD11 Influenced Hematopoisis By Histone Acetylation-Mediated Gene Expression in Myelodysplastic Syndrome

Background and Object In addition to histone deacetylation, the importance of histone over-acetylation induced oncogene transcription in initiation and progression of myelodysplastic syndrome (MDS) has been proposed recently. Our previous whole-exome sequencing identified a new somatic mutation, ANKRD11, an important factor in histone acetylation regulation. Its roles in MDS pathophysiology need to be clarified.Methods The next generation target sequencing (Including ANKRD11) was carried out in 320 patients with MDS using the MiSeq Benchtop Sequencer. ANKRD11 mRNA expression in bone marrow of MDS was measured by real-time PCR. Loss and gain of function assay were carried out in myeloid cell lines K562, MEG-01£¬or SKM-1 to observe the influence on cell proliferation and differentiation . The levels of histone acetylation at H3 and H4 were detected by Western blot.Results Target sequencing in a cohort of 320 MDS patients identified 14 of ANKRD11 mutations (4.38%, Fig.1), which were confirmed by Sanger sequencing. Meanwhile, no ANKRD11 mutations in 100 normal controls were defined. ANKRD11 mutations occurred frequently in exons 10 and 9. The mRNA expression levels of ANKRD11 were significantly decreased in MDS patients, especially in ANKRD11mutant patients (Fig.2). ANKRD11 knockdown in K562 and MEG-1 resulted in growth inhibition, cell cycle arrest and erythroid/megakaryocytic differentiation retardant. In MDS cell line SKM-1, the arrested differentiation was rescued by over-expression of ANKRD11. Consistent with a role for ANKRD11 in histone acetylation, ANKRD11 KD increased acetylation of histones H3 and H4 at H3K14 and H4K5 and resulted in the upregulation of genes involved in differentiation inhibilation (SOX6, P21, et al). Finally, the ANKRD11 KD-mediated influence on cell proliferation and differentiation were reversed by inhibiting histone acetyltransferase activity.Conclusion Our assay defined that ANKRD11 was a crucial chromatin regulator that suppress histone acetylation and then decrease gene expression during myeloid differentiation, providing a likely explanation for its role in MDS pathogenesis. This study further support histone acetylase inhibitor as a potential treatment in MDS. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic and Epigenetic Drug Targets in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders of hematopoietic system, characterized by genetic, epigenetic or microenvironmental alterations of aging hematopoietic stem cells. Pathophysiology of MDS comprises the suppression of normal hematopoiesis and reduced myeloid progenitor cells differentiation, with the main consequence of peripheral cytopenias and increased risk to evolution in acute myeloid leukemia (AML). This review summarizes the evolving understanding of the role of genetic and epigenetic alterations involved in pathogenesis and current and future strategies for therapeutic targeting in myelodysplastic syndromes. In addition to molecular characteristics, immune and microenvironmental factors in bone marrow of MDS patients may further modify the MDS manifestations, its clinical presentation, disease course, risk of transformation to AML and prognosis of MDS, as well as response to therapy. Current clinical response to therapy approaches are exerted both by epigenetic alterations and by induction of apoptosis. Future treatment strategies in preclinical and clinical investigations are directed towards new dosing schedules of existing drugs, new genetic and epigenetic targets and combination of different agents, including hypomethylation agents and histone deacetylase inhibitors.

Insight into the molecular pathophysiology of myelodysplastic syndromes: targets for novel therapy.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by abnormal cellular differentiation and maturation with variable progression to acute leukemia. Over the last decade, scientific discoveries have unraveled specific pathways involved in the complex pathophysiology of MDS. Prominent examples include aberrations in cytokines and their signaling pathways (such as tumor necrosis factor-alpha, interferon-gamma, SMAD proteins), mutations in genes encoding the RNA splicing machinery (SF3B1, SRSF2, ZRSR2, and U2AF1 genes), mutations in genes disrupting the epigenetic machinery (TET2, DNMT3A, DNMT3B, EZH2, ASXL1). In addition, abnormalities in regulatory T-cell dynamics and atypical interactions between the bone marrow microenvironment, stroma and progenitor cells, and abnormal maintenance of telomeres are also notable contributors to the complex pathogenesis of MDS. These pathways represent potential targets for novel therapies. Specific therapies include drugs targeting aberrant DNA methylation and chromatin remodeling, modulating/activating the immune system to enhance tumor-specific cellular immune responses and reduce anomalous cytokine signaling, and blocking abnormal interaction between hematopoietic progenitors and stromal cells.

Tumor suppressor microRNAs are downregulated in myelodysplastic syndrome with spliceosome mutations.

Spliceosome mutations are frequently observed in patients with myelodysplastic syndromes (MDS). However, it is largely unknown how these mutations contribute to the disease. MicroRNAs (miRNAs) are small noncoding RNAs, which have been implicated in most human cancers due to their role in post transcriptional gene regulation. The aim of this study was to analyze the impact of spliceosome mutations on the expression of miRNAs in a cohort of 34 MDS patients. In total, the expression of 76 miRNAs, including mirtrons and splice site overlapping miRNAs, was accurately quantified using reverse transcriptase quantitative PCR. The majority of the studied miRNAs have previously been implicated in MDS. Stably expressed miRNA genes for normalization of the data were identified using GeNorm and NormFinder algorithms. High-resolution melting assays covering all mutational hotspots within SF3B1, SRSF2, and U2AF1 (U2AF35) were developed, and all detected mutations were confirmed by Sanger sequencing. Overall, canonical miRNAs were downregulated in spliceosome mutated samples compared to wild-type (P = 0.002), and samples from spliceosome mutated patients clustered together in hierarchical cluster analyses. Among the most downregulated miRNAs were several tumor-suppressor miRNAs, including several let-7 family members, miR-423, and miR-103a. Finally, we observed that the predicted targets of the most downregulated miRNAs were involved in apoptosis, hematopoiesis, and acute myeloid leukemia among other cancer- and metabolic pathways. Our data indicate that spliceosome mutations may play an important role in MDS pathophysiology by affecting the expression of tumor suppressor miRNA genes involved in the development and progression of MDS.

Myelodysplastic Syndrome and Minimal Change Nephrotic Syndrome treated with Steroid Challenge

Glomerulonephritis associated with malignancy is deemed to be paraneoplastic glomerulonephritis. Myelodysplastic syndrome (MDS) is a group of heterogeneous clonal hematopoietic stem cell disorders characterized by impaired hematopoietic cell differentiation and cytopenia. The pathophysiology of MDS ...

Pathophysiology of MDS: genomic aberrations.

Myelodysplastic syndromes (MDS) are characterized by clonal proliferation of hematopoietic stem/progenitor cells and their apoptosis, and show a propensity to progress to acute myelogenous leukemia (AML). Although MDS are recognized as neoplastic diseases caused by genomic aberrations of hematopoietic cells, the details of the genetic abnormalities underlying disease development have not as yet been fully elucidated due to difficulties in analyzing chromosomal abnormalities. Recent advances in comprehensive analyses of disease genomes including whole-genome sequencing technologies have revealed the genomic abnormalities in MDS. Surprisingly, gene mutations were found in approximately 80-90% of cases with MDS, and the novel mutations discovered with these technologies included previously unknown, MDS-specific, mutations such as those of the genes in the RNA-splicing machinery. It is anticipated that these recent studies will shed new light on the pathophysiology of MDS due to genomic aberrations.

Protein Carbonylation in Patients with Myelodysplastic Syndromes

Introduction:Myelodysplastic syndromes (MDS) are a heterogenous group of oncohematologic diseases, characterized by bone marrow hypercellularity, dysplasia of myeloid lineage cells, peripheral cytopenias, and an increased risk of evolution to acute myeloid leukemia (AML). Pathophysiology of MDS is not fully understood, however it has been described that oxidative stress plays an important role in initiation and disease progression. Oxidative stress is defined as an imbalance between prooxidative and antioxidative processes that prefers the production of reactive oxygen species (ROS) over an antioxidant defence. The association between MDS and oxidative stress was discussed in several studies, where functional mitochondrial abnormalities in MDS patients have been demonstrated as one of the possible sources of oxidative stress. Another mechanism could be associated with mitochondrial dysfunction via iron overloading, mitochondrial DNA mutation, systemic inflammation, and bone marrow stromal defects. Protein carbonylation is ROS-mediated protein oxidation and it is defined as an irreversible post-translational oxidative modification. The detection of protein carbonyl groups is a specific useful marker of oxidative stress.The aim of this study was to determine the levels of carbonylated proteins in plasma of MDS patients resp. in plasma of individual subtype of MDS patients and evaluate changes of carbonylated plasma proteome in MDS patients compared with healthy controls.Methods:The content of protein carbonyls was measured after derivatization with dinitrophenylhydrazine by monitoring absorbance between 250 and 500nm. Carbonylated proteins of plasma samples were purified after reaction with biotin hydrazide. The negative controls were processed using the exact same procedure, except biotin hydrazide labeling. The dialyzed biotin hydrazide labeled samples and the negative controls were isolated using monomeric avidin resin. All the samples were prepared in doublets. One part of samples was used for the on bead tryptic digestion and the second part of samples was separated by SDS-PAGE after eluation with Laemmli buffer. The obtained carbonylated proteins were identified by mass spectrometry (LC-MS/MS).Results:We have examined 32 patients with different MDS subtype (10 patients RAEB-1,2; 12 RCMD; 10 RARS) and 20 healthy controls. Diagnoses of different subgroups of MDS were established according to the WHO classification criteria. The median age of patients group was 64,5 and presented 17 males (57 %); the median age of healthy controls group was 43 and presented 10 males (50 %).We have found significantly (P <0.02) increased level of protein carbonyls in plasma of MDS patients compared to healthy controls, the increased level of protein carbonyls was determinated in all tested MDS subtypes. The difference was elevated significantly in RARS patients (45,8 ± 0,5 %, P <0.005) in comparison with healthy controls. We also observed proteome changes in isolated carbonylated proteins of RARS patients and healthy controls.Conclusion:Protein carbonylation also plays an important role in signalling processes. Therefore protein carbonylation in MDS disease could lead not only to the loss of protein function by oxidative modification but also can influence protein expression playing significant role in MDS disease. We suggest that protein carbonylation is not "only" a consequence of oxidative stress in MDS disease, but also plays an active role as an important cause of pathogenic changes related to angiogenesis, myelomonocytic lineage development and complement impairment in MDS.Acknowledgment:This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. DisclosuresNo relevant conflicts of interest to declare.

Increased Expression of HERG1 K+ Channels Contribute to MDS Progression and Display Correlation with Prognosis Stratification

Background: Myelodysplastic syndromes (MDS) are defined as a heterogeneous group of clonal hematopoietic stem cell (HSC) malignant disorders which are characterized by bone marrow failure and dysplasia of blood cells. As continually terminated in AML, MDS are treated as the "pre-leukemia" condition. HERG K+ channels, being three subtypes: HERG1, HERG.2, HERG3, are expressed transiently at early stages of cells such as progenitor and stem cells, disappearing at later stages of cells like mature cells. Our previous work and others demonstrated that HERG1, as an oncoprotein, was over expressed in AML cells and played crucial roles in SDF-1induced leukemia cell migration. The expression and functional role of HERG1 K+ channels in MDS development is not reported. We investigated the HERG1 K+ channels expression and explored the functional link between HERG1 K+ channels and MDS progressionMethods: The expression of HERG1 K+ channels in untreated MDS, AML patients and normal control was detected by flow cytometry. The roles of HERG1K+ channels in regulation of SKM-1 cell proliferation, apoptosis and cell cycle by CCK-8 assay and flow cytometry, respectively.Results: We observed that the expression of HERG1 K+ channels on bone marrow (BM) mononuclear cells (MNCs) in MDS patients was significantly higher than that in the controls (42 ± 7.62% vs 19.8 ± 2.79%, p < 0.01) , but was lower than that in AML (42 ± 7.62% vs 52.18 ± 9.72%, p < 0.01). MDS subtypes mainly contained refractory cytopenia unilineage dysplasia (RCUD), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blast I (RAEB-I), refractory anemia with excess blasts II (RAEB-II) and MDS-unclassified (MDS-U). We next analyzed percentage of HERG1 K+ channels in MDS subtypes and found that level of HERG1 K+ channels on the MNCs in each subtypes of MDS was significant higher than that in the control group (Control: 19.8 ± 2.79%, MDS-RCUD: 30.91 ± 1.48%, MDS-RCMD: 39.06 ± 2.47%, MDS-RAEB-I: 44.76 ± 5.54%, MDS-RAEB-II: 49.69 ± 3.28%, p < 0.01), suggesting that HERG1 K+ channels expression might be positively associated with malignancy degree of MDS.HSCs played important roles in the pathophysiology of MDS. Our results also revealed that with the increase of malignancy degree, the percentage of HERG1K+ channels on CD34+CD38- derived cells from MDS subtypes tended to elevate in corresponding MDS subtypes (MDS-RCUD: 61.21 ± 9.46%, MDS-RCMD: 65.66 ± 4.57%, MDS-RAEB-I: 72.35 ± 9.38%, MDS-RAEB-II: 75.71 ± 4.24%, p < 0.05), which supported the notion in other way that MDS are HSC malignant disorders. The over-expression of HERG1 K+ channels on CD34+CD38- cells in MDS patients might be correlated with the oncogenesis of MDS.In addition, the prognosis stratification of MDS patients was performed according to International Prognostic Scoring System (IPSS) scores and the untreated MDS cohort was categorized as four risk groups: Low-R, Int-1-R, Int-2-R and High-R. Our results showed that there was a positively correlation between HERG1 level and IPSS scores of patients (Low-R: 31.93 ± 3.47%, Int-1-R: 39.95 ± 5.76%, Int-2-R: 45.94 ± 6.34%, High-R: 49.05 ± 3.04%, p < 0.01). This showed that expression level of HERG1 K+ channels was helpful for predicting the prognosis of de novo MDS.Furthermore, we analyzed HERG1 K+ channel role on MDS cell proliferation and apoptosis. Incubation with 0, 10, 20 uM E-4031 ( HERG K+ channels inhibitor) with SKM-1 cells (MDS cell line) for 48 h, the results showed that blockage of HERG1 decreased the proliferation of SKM-1 cells but had rarely effects on cell apoptosis and cell cycle distribution. In consistent with other studies, HERG1 K+ channels had already been shown to be necessary for growth of cancer cells through specific activities independent of cell cycle such as interacting with TNFR1 protein which could activate NF-κB to facilitate cell proliferation and favoring transduction of growth signals by MAP kinase/c-fos pathway. These findings showed that physiological activity of HERG1 K+ channels was crucial for MDS cell proliferation and HERG1 K+ channels may be a potential therapeutic target for MDS.Conclusion: Briefly, our study firstly showed that HERG1 K+ channels were aberrantly over-expressed on MDS stem cells, mononuclear cells, and positively associated with malignancy degree of MDS. HERG1 K+ channels functionally contribute to MDS progression and may be a potential therapeutic target for MDS. DisclosuresNo relevant conflicts of interest to declare.

HIF-1a Pathway, As a Signal Funnel for Genetic, Epigenetic, and Metabolic Aberrations, Is Sufficient and Essential for MDS Development

Myelodysplastic Syndromes (MDS) are heterogeneous clonal hematopoietic disorders, which are characterized by ineffective hematopoiesis and uni- or multi-lineage dysplasia. Despite the fact that a variety of genetic, epigenetic, and metabolic aberrations have been identified, the clinical features for MDS remain similar, indicating that there is a common underlying mechanism for MDS pathogenesis. Accumulating clinical and research evidence has shown the involvement of systemic inflammation and activated immune signaling in MDS pathogenesis. Hypoxia inducible factor-1α (HIF-1α) is a critical transcription factor for the hypoxic response, angiogenesis, and cancer development. Importantly, HIF-1α is also a key regulator for immune cell activation.To determine whether MDS patients have an activated HIF-1α signature, we analyzed a public gene expression array data set of CD34+ BM cells from a large cohort of MDS patients (n = 183). Although HIF-1α mRNA remains unchanged, some HIF-1α target genes relating with survival/cell growth, glucose metabolism, and invasion/metastasis were significantly activated in the broad spectrum of MDS. We further determined HIF-1α expression in the BM biopsies from MDS patients (n = 39) using immunohistochemistry staining. We found a high frequency of HIF-1α expressed cells in both low- and high-risk IPSS groups. Consistent with this human data, deficiency of frequently mutated genes in MDS resulted in an activated HIF-1α signature in mice. We found that the levels of Hif-1α protein in the c-Kit+ BM cells from Dnmt3a-KO, Runx1-KO, and Mll-partial tandem duplication (PTD) knock-in (MllPTD/WT) mice were significantly upregulated compared with those from WT controls. Although the expression level of Hif-1α protein in the c-Kit+ BM cells from Tet2-KO mice was not changed, they had an activated HIF-1α signature. IDH1/2 mutations are also known to elevate Hif-1α proteins. These results suggest that the HIF-1α pathway is widely activated in MDS patients by MDS-genic mutations through different mechanisms.To determine whether HIF-1α is sufficient for developing MDS phenotypes, we generated blood specific inducible HIF-1α transgenic mice. Using Vav1-Cre/Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-1α can be induced in a doxycycline dependent manner. HIF-1α-induced mice developed thrombocytopenia, leukocytopenia, and macrocytic anemia. We found micromegakaryocyte and multi-segmented megakaryocyte formations which are a major diagnostic criteria for MDS. In the BM of anemic mice, we found significant reduction of basophilic and polychromatic erythroblasts. The central macrophages are known to form erythroblastic islands that play a critical role in the late stage of erythropoiesis. Both the frequency and absolute number of F4/80+ Ter119+ BM erythroblastic islands were significantly decreased in HIF-1α-induced mice compared to the control mice. We also found activation of both innate and adaptive immunity in HIF-1α-induced mice. Lineage specific HIF-1α induction revealed the cell-intrinsic effect of HIF-1α on aberrant megakaryocytopoiesis and cell-extrinsic effect of HIF-1α on macrocytic anemia development.We have previously shown that MllPTD/WT mice develop several MDS-associated features, such as increased self-renewal and apoptosis in HSPCs, expansion of myeloid progenitors, and skewed myeloid differentiation. Synergistic effects between MLL-PTD and RUNX1 mutant (S291fs) or Runx1 deletion, further accumulated the Hif-1α protein and caused a variety of MDS features that phenocopy the human MDS. To understand the role of HIF-1α in the context of MDS pathogenesis, we took genetic and pharmacologic approaches. Hif-1α deletion significantly abrogated disease development and rescued macrocytic anemia and thrombocytopenia. This is not due to the elimination of donor derived cells. After the deletion of Hif-1α, a majority of donor derived cells in PB were still CD11b+ cells. HIF-1α inhibition significantly reduced colony formation of MllPTD/WT/RUNX1-291fs cells and MllPTD/WT/Runx1D/D cells in vitro, and prolonged survival of MDS mice in vivo.In conclusion, we identified the sufficient and essential role of HIF-1α in developing MDS. These findings implicate that HIF-1α pathway may be the common underlying mechanism of MDS pathophysiology and could be an effective therapeutic target for a broad spectrum of MDS patients. DisclosuresShih:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Association of XPD (Lys751Gln) and XRCC1 (Arg280His) gene polymorphisms in myelodysplastic syndrome

Myelodysplastic syndromes (MDSs) are heterogeneous hematopoietic disease characterized by ineffective haematopoiesis that frequently transforms into acute leukaemia. Alterations in many individual biologic pathways have been reported in MDS pathophysiology. Disease progression along the MDS, acute myeloid leukemia (AML) continuum is believed to be a consequence of stepwise accumulation of DNA mutations which infers a defect in DNA repair. The present study investigated the association between DNA repair genes (XRCC1, XRCC3, OGG1, XPD and RAD51) and the risk of developing MDS. The study was carried out in 92 primary MDS patients. The genotyping study was carried out by PCR-RFLP technique. We have studied seven single-nucleotide polymorphisms (SNPs) of five DNA repair genes (XRCC1 (Arg194Trp, Arg280His, Arg399Gln), XRCC3, XPD, RAD51 and OGG1). Significantly, a high frequency of DNA repair gene XRCC1 (Arg280His) (p=0.05) and XPD (Lys751Gln) (p=0.01) polymorphism was observed in MDS patients compared to controls. The distribution of polymorphisms in MDS subgroups showed a significant association of XRCC1 with RAEB I compared to other subgroup. Though a high frequency of XRCC1 gene polymorphism was observed in farmers and tobacco chewers, it was not statistically significant. Our study suggests that XRCC1 (Arg280His) and XPD polymorphisms are associated with risk of MDS and XRCC1 polymorphism strongly associated with advanced MDS subgroup. Hence, these polymorphisms can be used as a prognostic marker in MDS.

Pediatric myelodysplastic syndromes

Pediatric myelodysplastic syndromes (MDS) are a group of rare clonal hematopoietic stem cell disorders characterized by varying degree of cytopenias, ineffective and dysplastic hematopoiesis, and the risk of leukemic transformation. The clinical, laboratory, and histologic presentation of pediatric MDS shares significant overlap with other inherited and acquired bone marrow failure (BMF) disorders. Given that the majority of pediatric patients with MDS present with a hypocellular bone marrow, the histopathologic diagnosis is challenging and usually requires ancillary molecular studies. While rapid advancements have been made in the field of adult MDS, the underlying genetics and pathophysiology of pediatric MDS are still poorly understood. The recent discovery of germline mutations in GATA2 leading to MDS suggests that some pediatric and young adult patients with MDS have an underlying genetic predisposition. Nevertheless, the molecular underpinnings remain poorly understood in most cases. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment option. Optimal timing of HSCT is not often straightforward, as some pediatric patients with low-grade MDS have an indolent disease course while others show rapid progression to advanced MDS and leukemia. Lastly, the classification of pediatric MDS has evolved over the years and is different from the terminology currently used in adult MDS. This review will focus on the current classification schemes of pediatric MDS and address clinical, laboratory, and pathologic features, as well as diagnostic criteria, genomic advances, and therapeutic options and prognosis.

Genetics factors associated with myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are a clinically and cytogenetically heterogeneous group of clonal diseases. Clonal chromosomal abnormalities are observed in 30-50% of patients with MDS. The deletions are among the most common alterations, and often involve the long arms of chromosomes 5, 7, 8, 13, and 20 and the short arms of chromosomes 12 and 17. The advent of new technologies for the detection of genetic abnormalities led to the description of a new set of recurrent mutations, leading to new insights into the pathophysiology of MDS. The recent recognition that genes involved in the regulation of histone function (EZH2, ASXL1, and UTX) and DNA methylation (DNMT3A, IDH1/IDH2, and TET2) are frequently mutated in MDS, has led to the proposal that there is an important link between genetic and epigenetic alterations in this disease. In fact, regulatory factors have also been considered as miR-143/miR-145, miR-146a, miR-125a and MiR-21. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems. In recent years research has brought new insights into these diseases, but few of the findings are sufficiently robust to be incorporated into the clinical routine at this time. Thus, the aim of this study was to review the role of genetic factors involved in the diagnosis and development of the different phenotypes of MDS.

Non-t(6;9) and Non-Inv(3) Balanced Chromosomal Rearrangements Are Associated With Poor Survival Outcomes in Myelodysplastic Syndromes.

Cytogenetics is an important predictor of survival in patients with myeloid malignancies including myelodysplastic syndromes (MDS). The roles of balanced chromosomal rearrangements (BCR) specifically balanced translocations and inversions in MDS are less established. We hypothesized that BCR are commonly found in MDS and might confer important prognostic and therapeutic effect. Cytogenetic, hematologic, clinical, and survival data from a total of 302 MDS patients seen at the Cleveland Clinic between the years 2002 and 2011 were collected. Direct sequencing for genes relevant in MDS pathophysiology was performed on patients with BCR. Categorical data were analyzed using the χ2 test and survival outcomes were analyzed using the Kaplan-Meier method. Twenty-three patients (8%) carried non-t(6;9) and non-inv(3) BCR. These patients had short overall survival (OS) similar to patients with poor risk cytogenetics, defined according to the International Prognostic Scoring System, which was not altered by new single-nucleotide polymorphism arrays or acquired somatic uniparental disomy lesions. Among the detected somatic mutations, only patients harboring mutations in the RNA splicing gene serine/arginine-rich splicing factor 2 (SRSF2) conferred worse outcomes in this group of patients. Therapeutically, patients who received allogeneic hematopoietic cell transplantations had better OS compared with patients treated with pharmacologic therapies or best supportive care only. BCR are not common in MDS and are associated with poor survival, which might be influenced by the presence of SRSF2 mutation.

The inflammatory microenvironment in MDS.

Myelodysplastic syndromes (MDS) are a collection of pre-malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and a tendency to evolve into leukemia. Among MDS's pathogenic mechanisms are genetic, epigenetic, apoptotic, differentiation, and cytokine milieu abnormalities. Inflammatory changes are a prominent morphologic feature in some cases, with increased populations of plasma cells, mast cells, and lymphocytes in bone marrow aspirates. Accumulating evidence suggests that the bone marrow microenvironment contributes to MDS disease pathology, with microenvironment alterations and abnormality preceding, and facilitating clonal evolution in MDS patients. In this review, we focus on the inflammatory changes involved in the pathology of MDS, with an emphasis on immune dysfunction, stromal microenvironment, and cytokine imbalance in the microenvironment as well as activation of innate immune signaling in MDS patients. A better understanding of the mechanism of MDS pathophysiology will be beneficial to the development of molecular-targeted therapies in the future.

Usefulness of Adalimumab for Treating a Case of Intestinal Behçet's Disease With Trisomy 8 Myelodysplastic Syndrome.

Behçet's disease (BD) is a systemic vasculitis, while myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematologic disorders characterized by ineffective hematopoiesis. Some studies suggest a relationship between MDS and BD, especially intestinal BD, and trisomy 8 seems to play an important role in both diseases. There are several reports on patients with BD comorbid with MDS involving trisomy 8 that frequently have intestinal lesions refractory to conventional medical therapies. Tumor necrosis factor (TNF)-α is strongly involved in the pathophysiology of several autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and BD. In addition, TNF-α plays an important role in the pathophysiology of MDS by inhibiting normal hematopoiesis and inducing the programmed cell death of normal total bone marrow cells and normal CD34+ cells. Recent clinical reports demonstrate the favorable effect of TNF-α antagonists in patients with refractory intestinal BD and in those with MDS. We present the case of a patient with intestinal BD and MDS involving trisomy 8 who was successfully treated with adalimumab.

Hematopoietic Cell Kinase (HCK) Is Involved in Myelodysplastic Syndromes and Acute Leukemia Pathophysiology By Modulating PI3K Pathway and Enhancing Erythroid Differentiation

Introduction: Src family non-receptor tyrosine kinase regulates diverse cellular responses mainly by activating PI3K signaling, an important pathway that contributes to many cancers pathology. HCK, a Src family member, is restricted expressed in hematopoietic cells; however, high levels of HCK have been reported in solid tumors and hematologic neoplasms and have been associated with a worse prognosis. Identification of unbalanced pathways and upregulated proteins and a better knowledge of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) pathogenesis could help identify potential druggable targets and new prognostic biomarkers. The clinical and biological role of HCK in MDS and AML pathophysiology has not yet been elucidated and may be a novel therapeutic approach.Aims: To evaluate HCK expression in hematopoietic stem cells (HSC/CD34+) and in total bone marrow (BM) cells from healthy donors and MDS and AML patients. We further attempted to analyze the HCK knockdown and drug inhibition on differentiation, proliferation, apoptosis and migration in HSC/CD34+ and in a panel of human leukemia cell lines (KG1a, U937, HL60, P39).Materials and Methods: A total of 34 healthy donors and 139 patients at diagnosis (MDS=75 [low-risk=46, high-risk=29], AML with Myelodysplastic related changes (AML-MRC)=21 and de novo AML=43), were included in the study. HCK gene and protein expression was analyzed by qPCR and Western blot. Results are expressed as median (minimum–maximum) after appropriate statistical analysis. For functional analysis, HCK was inhibited with specific lentiviral vector system in HSC/CD34+ (isolated from umbilical cord blood units and BM), KG1a, U937, HL60 and P39 cells. Apoptosis was evaluated by Annexin-V/PI, cell growth by CellTiter assay, migration by Transwell, CXCR4 expression and actin polymerization by flow cytometry. We also verified the effects of a HCK specific inhibitor, ASN05260065, kindly provided by ASINEX and Dr Maurizzio Bottas.Results: HCK mRNA were significantly increased in HSC/CD34+ from patients with MDS (4.87 [0.03-14.75]; P=.003) and AML (8.36 [0.70-24.47]; P=.003) compared to healthy donors (1.19 [0.06-3.02]). When patients were stratified according to WHO classification, HCK expression was significantly higher in low-risk MDS compared to high-risk and AML-MRC (8.10 [2.30–14.75] vs 3.22 [0.03–6.33] and 1.02 [0.70–2.78], P<.001). Similar results were found in BM cells: HCK transcripts were significantly increased in MDS compared to healthy donors, and in low-risk compared to high-risk MDS (P<.0001). Interestingly, 5 of 5 MDS patients showed a significant decrease in HCK expression after disease progression. Also, a negative correlation was observed between BM blast percentage and HCK expression in MDS patients. In HSC/CD34+ cells, BFU-E colony number was significantly lower after HCK inhibition. HCK depletion reduced cell death in HSC/CD34+, P39 and KG1a cells and diminished proliferation in KG1a and U937 cells. Moreover, HCK knockdown decreased the phosphorylation of AKT, mTOR (PI3K pathway members) and ERK and treatment of HCK silenced cells with PI3K inhibitors had no synergic effect. Confirming the HCK lentivirus silencing results, HCK specific inhibitor treatment downregulated PI3K and ERK signaling. In addition, HCK inhibition significantly reduced migration towards CXCL12 chemoattractive stimuli by regulating CXCR4 expression and F-actin polymerization. Attempts have been made to transduct CD34+ from low-risk MDS patients however more than 80% cells were apoptotic and impossible to perform further assays.Conclusions:HCK is overexpressed in HSC/CD34+ and BM cells from MDS, mainly in low-risk MDS, and de novo AML. During MDS progression, HCK expression reduction correlates with increasing blast percentage and could indicate less dependence on microenvironment. HCK inhibition reduced BFU-E colony formation and our previous results showed that HCK expression increases during erythroid differentiation of non-transduced low-risk MDS CD34+ cells. Thus, HCK seems to play a role as a downstream effector in erythropoietin (Epo) signaling which could explain the higher HCK expression in low-risk MDS. Besides, HCK is an upstream regulator of PI3K and MAPK pathway that enhances cell apoptosis, survival and proliferation. Our study described new insights to the role of HCK in Epo/PI3K signaling and in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

A Detailed Analysis of Myelodysplastic Syndrome Complicated By Autoimmune or Inflammatory Disorders: A Possible Efficacy of Low-Dose Lenalidomide

[Background] Approximately 10-20 % of the patients with myelodysplastic syndrome (MDS) are known to be complicated by autoimmune or inflammatory disorders (ADs) such as vasculitis. However, most of the reports on such MDS were published before 2000, in which the diagnosis of MDS was made with previous criteria; about 20% (range 9-43%) of the patients in these reports may not be diagnosed as MDS with the present criteria. In addition, many of these reports do not offer sufficient information on cytogenetic analysis, a presently known critical factor that affect pathophysiology of MDS. These facts could make difficult to discuss its characteristics, prognosis and treatment options. Therefore, we made a detailed retrospective analysis on clinical features and treatment outcomes of MDS complicated by ADs based on more recent knowledge.[Results] A total of 102 Japanese patients with MDS (70 males and 32 females, median age of 73), diagnosed between June 2007 and June 2014, were evaluated. The diagnosis was made with WHO (2008) classification. Bone marrow G-band chromosome analysis was available in 95 % of the patients. "Very High" risk category of IPSS-R criteria comprised 29% of the patients. As summarized in Table 1, 14 episodes of AD were observed in 13 (13%) patients; 4 with Sweet syndrome, 2 each with Bechet like symptoms, interstitial pneumonia, large vessel vasculitis and myelitis, and 1 each with HPS and seronegative arthritis. As in the previous reports, the patients who developed ADs (AD group) were characterized by younger age and higher frequency of complex cytogenetics compared with those who did not (non-AD group). In addition, patients of AD group showed lower neutrophil count and higher frequency of trisomy 8 (p=.034 and <.01, respectively). Of note, despite no significant difference in median IPSS-R score, the progression rate to AML in AD group was more than two-times higher compared with non-AD group (p=.01). Survival rates were not significantly different between two groups.Corticosteroids were given to 8 patients of AD group and 6 of them (75 %) showed a certain degree of responses. Six patients with relapsed or refractory AD after the steroid therapy were treated with low-dose Lenalidomide (LEN) (10mg/day for 21 days). Interestingly, 5 of them responded to LEN with 3 achieving complete remission. Furthermore, the patients of AD group received LEN showed better survival compared with those treated without LEN (1.2y vs. 3.1y, p=.017) (Figure 1).[Conclusion] The present observation indicated that MDS with AD may be associated with cytogenetic abnormalities including trisomy 8 and higher progression rate to AML. Of note, LEN might be a possible treatment option for those resistant to steroid therapy. [Display omitted] [Display omitted] DisclosuresNakamae:Novartis: Honoraria, Research Funding, Speakers Bureau, Travel/accomidations/meeting expenses Other. Hino:Nippon Shinyaku CO.,LTD: Research Funding; astellas CO.,LTD: Research Funding.

Somatic Mutations of the Breast Cancer Amplified Sequence-1 (BCAS1), a Novel Leukemogenic Driver in Myelodysplastic Syndromes with Del(20q)

Deletion of the long arm of chromosome 20 [del(20q)] is a recurrent clonal abnormality seen in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML). Del(20q) is believed to originate in a pluripotent stem cell of both myeloid and lymphoid cells and contribute to clonal evolution by the loss/inactivation of ≥1 tumor suppressor genes. Commonly deleted regions (CDRs) of del(20q) contain genes that are overexpressed in hematopoietic progenitor cells of del(20q) patients (pts). Although associated with good prognosis when present as a sole abnormality in MDS, the natural history may vary within similar disease subtypes. Next-generation sequencing technologies have accelerated the discovery of genetic mutations implicated in MDS pathogenesis. To date, the vast majority of these mutations are neither specific to a disease subtype nor to a cytogenetic abnormality. We hypothesized that molecular mutations are the main drivers of leukemic progression in MDS with del(20q). Whole exome sequencing (WES) was performed on 6 pts with del(20q): MDS=3, MPN=3. DNA (3ug) was used for WES. 20 million reads were run on an Illumina HiSeq2000 sequencer. A bioinformatic algorithm filtered all tumor variants based on variation score (≥30) and coverage (30X). Tumor nucleotide variants were counted if unique to the tumor excluding SNPs and germline variants. We detected 42 candidate genes. Only 1 nucleotide variation was detected on the chromosome (chr) 20 of 1 high risk MDS with del(20q) pt. The alteration resulted in a proline to a glutamine substitution at position 488 of exon 11 (P488Q) of a gene called Breast Cancer Amplified Sequence-1 (BCAS1). BCAS1 gene is located in chr 20q13.2 region. BCAS1 overexpression has been implicated in aggressive breast and colon cancer subtypes but the mechanisms for this overexpression have not been clarified. Of note, BCAS1 mutations have not been previously identified in any hematologic malignancy. A larger cohort of pts: N=395; AML=70, MPN=205, MDS=90, other bone marrow failure diseases=30, and hematologic cell lines were tested for BCAS1 mutations. We found recurrent BCAS1P488Q mutation in 10/56 (18%) of MDS with del(20q) pts who evolved to high risk disease and AML (sAML). All BCAS1 mutant (BCAS1MUT) pts have an associated del(20q) abnormality. BCAS1MUT pts have a median age of 67 yrs (range, 45-87); male/female=9:1, had more leukopenia (3 vs. 11 x109/L, P=.04), and neutropenia (1.3 vs. 8.3 x109/L, P=.04) compared to other del(20q) pts without mutation. Metaphase cytogenetics, FISH and SNP-A showed that the CDRs of the 20q region of all BCAS1MUT pts did not contain the 20q13.2 region. Screening for 12 genes relevant to MDS pathophysiology showed that BCAS1MUT pts harbored unfavorable genetic mutations: SRSF2 (1/10), TP53 (2/10) and U2AF1 (3/10) with 1 pt having both TP53 and U2AF1. BCAS1P488Q was mutually exclusive in 4 pts. BCAS1MUT had a dismal prognosis compared to BCAS1WT (PFS: 5.1 vs. 15.9 mos, P=.03). Bioinformatic analysis of publicly available gene expression data showed increased BCAS1 mRNA level in AML, CML and in the K562 cells. RT-PCR found a higher (5-fold) BCAS1 mRNA level in the index case compared to one WT pt and no expression in healthy subjects. K562 and CML primary cells (N=6) were used as positive controls. Presence of BCAS1 protein was confirmed by immunohistochemistry and western blotting. CGH revealed that 10% of breast tumors have copy-number gains of a 1-Mb region of 20q13 where BCAS1 maps. Amplification of this region was seen in aggressive subtypes of squamous cell carcinoma. We applied FISH analysis for BCAS1 finding no amplification of the 20q13 region in MUT vs WT (2 vs 2) pts suggesting that other mechanisms possibly other genetic mutations lead to BCAS1 overexpression in sAML. In the K562 cells, 3 copies of BCAS1 were identified which may explain increased BCAS1 expression in these cells. In sum, recurrent BCAS1 mutations are found specifically in MDS with del(20q) pts who transform to AML. They are exclusively found at the time of AML transformation. mRNA and protein overexpression are observed in BCAS1MUT pts and lead to inferior survival outcomes and poor response to chemotherapy. This is the first genetic mutation solely found in a specific MDS cytogenetic subset and may represent a novel mechanism for leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of patients with de novo myelodysplastic syndromes (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.