Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

Saikosaponin d (SSd) is one of the main active ingredients in Radix Bupleuri. In our study, network pharmacology databases and metabolomics were used in combination to explore the new targets and reveal the in-depth mechanism of SSd. A total of 35 potential targets were chosen through database searching (HIT and TCMID), literature mining, or chemical similarity predicting (Pubchem). Out of these obtained targets, Neuropilin-1 (NRP-1) was selected for further research based on the degree of molecular docking scores and novelty. Cell viability and wound healing assays demonstrated that SSd combined with NRP-1 knockdown could significantly enhance the damage of HepG2. Metabolomics analysis was then performed to explore the underlying mechanism. The overall difference between groups was quantitatively evaluated by the metabolite deregulation score (MDS). Results showed that NRP-1 knockdown exhibited the lowest MDS, which demonstrated that the metabolic profile experienced the slightest interference. However, SSd alone, or NRP-1 knockdown in combination with SSd, were both significantly influenced. Differential metabolites mainly involved short- or long-chain carnitines and phospholipids. Further metabolic pathway analysis revealed that disturbed lipid transportation and phospholipid metabolism probably contributed to the enhanced anti-hepatoma effect by NRP-1 knockdown in combination with SSd. Taken together, in this study, we provided possible interaction mechanisms between SSd and its predicted target NRP-1.

Recently, sperm-associated antigen 6 (

BACKGROUND: To analyze for genetic mutations which may presage peritoneal metastasis by using targeted next-generation sequencing (NGS).
MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded primary tumor specimens were obtained from 10 patients with small obstructing colorectal cancer and peritoneal metastasis (group A) and five with large non-obstructing colorectal cancer and no recurrence (group B). DNA was extracted for the sequencing of 409 cancer genes. The distribution of genetic mutations was compared between the two groups to find genetic mutations related to peritoneal metastasis.
RESULTS: When the samples were sorted based on similarity of gene expression by hierarchical clustering analysis, the samples were well divided between the two study groups. Mutations in AT-rich interactive domain-containing protein 1A (ARID1A), polycystic kidney and hepatic disease 1 (PKHD1), ubiquitin-protein ligase E3 component n-recognin 5 (UBR5), paired box 5 (PAX5), tumor protein p53 (TP53), additional sex combs like 1 (ASXL1) and androgen receptor (AR) genes were detected more frequently in group A.
CONCLUSION: A number of somatic mutations presumed to be relevant to colorectal cancer with peritoneal metastasis were identified in our study by NGS.

Cancer testis (CT) antigens are expressed in various types of tumors and represent the potential targets for T cell-based immunotherapy. Analysis of CT gene expression and DNA methylation have indicated that certain CT genes are epigenetically regulated and studies have confirmed that certain CT antigens are regulated by DNA methylation. In this study, we explored the epigenetic regulation of MAGE-A3 and improved the clinical outcome of MAGE-A3-specific T cell therapy in esophageal squamous cell carcinoma (ESCC). We used molecular profiling datasets in The Cancer Genome Atlas to analyze CT gene expression in ESCC and its regulation by DNA methylation. We performed quantitative reverse transcription PCR (qRT-PCR), immunohistochemistry and bisulfite sequencing in ESCC cell lines and ESCC tissues. Functional assays, such as flow cytometry, cytotoxicity assays and ELISA, were performed to determine the demethylation agent, decitabine (5-aza-2'-deoxycytidine, DAC)-treated cancer cell improved antigen specific T cells response. ESCC tumor cell-xenograft mouse model and enzyme-linked immunospot (ELISPOT) assays were used to determine the function of DAC treatment in enhancing anti-MAGE-3 T cell responses in ESCC. Furthermore, we performed qRT-PCR and flow cytometry in the peripheral blood mononuclear cells (PBMC) of myelodysplastic syndromes (MDS) patients. MAGE-A3, one of the CT antigens, expressed at various levels in ESCC and was interfered by DNA methylation. We observed an efficient increase in MAGE-A3 expression in tumor cells and tissues after the treatment of decitabine and the expression of MAGE-A3 was affected by DNA methylation. Functional assays showed enhanced secretion of IFN-γ and cytolysis of MAGE-A3 antigen-specific T cells by DAC-treated target cells. In the tumor cell-xenograft mouse model and ELISPOT assays, DAC increased the expression of MAGE-A3 and T cell mediated tumor clearance in ESCC as well. Notably, the proportions of MAGE-A3-responsive T cells were elevated in DAC-treated patients with MDS, indicating DAC dismissed the epigenetic inhibition of MAGE-A3. DAC would probably improve the clinical outcome of MAGE-A3-specific T cell therapy by augmenting the expression of target gene.

Core-binding factor acute myeloid leukemia (CBF-AML) data in Asian countries has been rarely reported. We analyzed 392 patients with CBF-AML [281 with t(8;21), 111 with inv.(16)/t(16;16)] among data from 3041 patients with AML from the Korean AML Registry. Interestingly, del(9q) was less frequently detected in Korean than in German patients with t(8;21) (7.5% vs. 17%), and del(7q) was more frequently detected in Korean patients with inv(16). Overall survival (OS) was similar between patients in the first complete remission (CR) who received allogeneic (alloSCT) and autologous stem cell transplantation (ASCT) for CBF-AML. OS of t(8;21) patients was poor when undergoing alloSCT in second/third CR, while OS of inv(16) patients in second/third CR was similar to that in first CR. Patients with > 3-log reduction of RUNX1/RUNX1T1 qPCR had improved 3-year event-free survival (EFS) than those without (73.2% vs. 50.3%). Patients with t(8;21) AML with D816 mutation of the c-Kit gene showed inferior EFS and OS. These poor outcomes might be overcome by alloSCT. Multivariate analysis for OS in patients with t(8;21) revealed older age, > 1 course of induction chemotherapy to achieve CR, loss of sex chromosome, del(7q), and second/third CR or not in CR before SCT as independent prognostic variables. Especially, del(7q) is the most powerful prediction factor of poor outcomes, especially in patients with t(8;21) (hazard ratio, 27.23; P < 0.001). Further study is needed to clarify the clinical effect of cytogenetics and gene mutation in patients with CBF-AML, between Asian and Western countries.

KIT D816 mutations (KIT D816

Chromosomal rearrangements involving one donor chromosome and two or more recipient chromosomes are called jumping translocations. To date only few cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with jumping translocations have been described and the underlying mechanisms remain unclear. Here, we analyzed 11 AML and 5 MDS cases with jumping translocations. The cases were analyzed by karyotyping, FISH, telomere length measurement, and next-generation sequencing with an AML/MDS gene panel. Cases with jumping translocations showed significantly (P < .01) shorter telomeres in comparison to healthy age-matched controls. Additional neo-telomeres were found in two cases. In total, eight cases showed recipient chromosomes with a breakpoint in the centromeric region all of them harboring a pathogenic variant in the TP53 gene (n = 6) and/or a loss of TP53 (n = 5). By contrast, no pathogenic variant or loss of TP53 was identified in the six cases showing recipient chromosomes with a breakpoint in the telomeric region. In conclusion, our results divide the cohort of AML and MDS cases with jumping translocations into two groups: the first group with a telomeric breakpoint of the recipient chromosome is characterized by short telomeres and a possibly telomere-based mechanism of chromosomal instability formation. The second group with a centromeric breakpoint of the recipient chromosome is defined by mutation and/or loss of TP53. We, therefore, assume that both critically short telomeres as well as pathogenic variants of TP53 influence jumping translocation formation.

RATIONALE: The gene deletion (5)(q22q35) is reported in 10-20% of myelodysplastic syndrome (MDS) cases and is associated with response to lenalidomide and favorable prognosis. The authors report here a clinical case of MDS transformation to B-cell acute lymphocytic leukemia (B-ALL) with an associated accrual of an additional mutation following treatment with lenalidomide.
PATIENT CONCERNS: A 69-year-old man presented with progressive anemia, normal white blood cell count, and thrombocytopenia consistent with MDS. He was administered lenalidomide for 27 months, then developed acute B-cell lymphocytic leukemia and acquired a previously unreported mutation in the gene enhancer of zeste homolog 2 (EZH2).
DIAGNOSES: After 27 months of therapy with lenalidomide, a surveillance bone marrow aspiration (BMA) revealed 90% cellularity with persistent multilineage dysplasia and a population of blasts comprising 54% of all bone marrow elements by morphology, consistent with B-ALL, even though the patient was asymptomatic. Conventional karyotype showed no signs of del(5)(q22q35) MDS, however bone marrow next-generation sequencing (NGS) demonstrated the accrual of a nonsense mutation (c.211del pL71*) in exon 3 of EZH2. A confirmatory BMA yielded 70% blasts and clinical features indicative of B-ALL.
INTERVENTIONS: Mini-hyper-CVD (cyclophosphamide and dexamethasone at 50% dose reduction, no anthracycline, methotrexate at 75% dose reduction, cytarabine at 0.5 g/m × 4 doses) was administered for 21 days.
OUTCOMES: A follow-up BMA was performed 2 months after mini-hyper-CVD therapy, showing dysplastic features with 25% ring sideroblasts, but no evidence of B-ALL. The patient is currently receiving monthly-low dose decitabine, ofatumumab, and dexamethasone, and is transfusion independent and asymptomatic after 7 cycles.
LESSONS: The present study shows an extremely rare progression of del(5)(q22q35) MDS to B-ALL with accompanying NGS data and a newly described acquisition of an EZH2 frameshift mutation. This case highlights the importance of NGS as a diagnostic and surveillance tool for MDS.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor widely used to treat MDS and AML, yet the impact of AZA on the cell-surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome, the transcriptome, and the cell-surface proteome. Untreated AML cell lines showed substantial overlap at all three omics levels; however, while AZA treatment globally reduced DNA methylation in all cell lines, changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteome analysis found no commonly regulated proteins. Gene set enrichment analysis of differentially regulated RNA and surface proteins showed a decrease in metabolic pathways and an increase in immune defense response pathways. As such, AZA treatment led to diverse effects at the individual gene and protein levels but converged to common responses at the pathway level. Given the heterogeneous responses in the four cell lines, we discuss potential therapeutic strategies for AML in combination with AZA.

BACKGROUND: Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology.
METHODS: Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR.
RESULTS: Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D.
CONCLUSIONS: In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

In our previous study, the 4‑aminobutyrate aminotransferase (ABAT) gene was screened and selected as a target gene that may affect the prognosis of myelodysplastic syndrome (MDS). The present study aimed to determine the prognostic value of ABAT in 152 patients with MDS, 29 patients with acute myeloid leukemia (AML) and 40 controls, by detecting the expression and methylation levels of the ABAT gene. In patients with MDS, the expression levels of ABAT were significantly reduced compared with in the controls (P<0.0001), and the degree of DNA methylation was increased in MDS subjects (P<0.0001). Age, hemoglobin level, marrow blasts, International Prognostic Scoring System karyotype, and the expression and methylation levels of ABAT were associated with overall survival (OS), as determined by univariate analysis. Multivariate analysis revealed that older age, higher marrow blasts and higher methylation percentage were independent risk factors for OS. In addition, a functional study demonstrated that ABAT gene silencing increased cell apoptosis and blocked the G1/S phase in SKM‑1 and THP‑1 human leukemia cells. A γ‑aminobutyrate aminotransferase inhibitor also blocked the G1/S phase; however, it had no effect on cell apoptosis. In conclusion, the present study demonstrated that ABAT methylation served an essential role in the progression of MDS and therefore may be considered an indicator of poor prognosis for hematological malignancies.

Now that the spectrum of somatic mutations that initiate, propagate, and drive the progression of myeloproliferative neoplasms (MPNs) has largely been defined, recent efforts have focused on integrating this information into clinical decision making. In this regard, the greatest progress has been made in myelofibrosis, in which high-molecular-risk mutations have been identified and incorporated into prognostic models to help guide treatment decisions. In this chapter, we focus on advances in 4 main areas: (1) What are the MPN phenotypic driver mutations? (2) What constitutes high molecular risk in MPN (focusing on

The inv(3)(q21q26.2) altering a single chromosome 3 homolog is an established myeloid malignancy-associated entity. Comparatively, double inv(3) cases involving both homologs are exceedingly rare with 13 reports across AML, CML and MDS. This scarcity was confirmed by finding only 2 new cases out of 34,898 bone marrows collected during a 55 year period at a large medical center (0.0005%). The double inv(3) was detected by karyotype and confirmed by FISH on both homologs in a 41 year old female and a 72 year old male with AML. In the latter case, a 2.26-fold increase in MECOM RNA level was found using an NGS myeloid gene panel. Chromosomal microarray analysis identified segmental copy-neutral loss-of-heterozygosity (CN-LOH) at 3q21 extending to near the q-arm terminus. This is the third report of distal 3q CN-LOH, substantiating that the double inv(3) arises through somatic repair of acquired segmental LOH. Long term clinical and genetic evaluation revealed no discernible morphologic difference between single and double inv(3) cases, conventional chemotherapy resistance and rapid dominance of the double inv(3) clone. The two new cases are consistent with relatively longer survival of double inv(3) patients in the absence of concurrent chromosome 7 loss compared to those with both abnormalities. Importantly, the first known outcome data of bone marrow transplantation in double inv(3) AML is also presented.

Transformation of myelodysplastic syndromes (MDS) into secondary acute myeloid leukemia (sAML) is defined by an arbitrary boundary of ≥20% bone marrow blasts but does not necessarily reflect a defined biological transition. The more obvious distinction lies between MDS patients that have an isolated bone marrow failure phenotype and those with excess blasts. Subtyping of MDS might be more accurately stratified into clonal cytopenias and oligoblastic leukemias, using the degree of dysplasia and blast percentage as risk features, respectively, rather than as diagnostic criteria. Transformation from MDS to sAML often involves clonal evolution or expansion of existing subclones that can be assessed by changes in variant allele frequencies of the somatic mutations that define them. There are a number of predictors for transformation that have been identified: these include mutations of genes in growth signaling pathways (NRAS, KRAS, PTPN11, FLT3), mutations in genes more commonly observed in AML (NPM1, WT1, IDH2), certain cytogenetic abnormalities (monosomy 7, complex karyotype, loss of 17p). Gene expression profiles that divide MDS into two major categories identify a progenitor gene signature subtype associated with a high risk of AML transformation. Assessing for these genetic abnormalities may better identify MDS patients at greatest risk of transformation.

BACKGROUND: Monitoring of measurable residual disease (MRD) in patients with advanced myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML) who achieve a morphological complete remission can predict haematological relapse. In this prospective study, we aimed to determine whether MRD-guided pre-emptive treatment with azacitidine could prevent relapse in these patients.
METHODS: The relapse prevention with azacitidine (RELAZA2) study is an open-label, multicentre, phase 2 trial done at nine university health centres in Germany. Patients aged 18 years or older with advanced MDS or AML, who had achieved a complete remission after conventional chemotherapy or allogeneic haemopoietic stem-cell transplantation, were prospectively screened for MRD during 24 months from baseline by either quantitative PCR for mutant NPM1, leukaemia-specific fusion genes (DEK-NUP214, RUNX1-RUNX1T1, CBFb-MYH11), or analysis of donor-chimaerism in flow cytometry-sorted CD34-positive cells in patients who received allogeneic haemopoietic stem-cell transplantation. MRD-positive patients in confirmed complete remission received azacitidine 75 mg/m
FINDINGS: Between Oct 10, 2011, and Aug 20, 2015, we screened 198 patients with advanced MDS (n=26) or AML (n=172), of whom 60 (30%) developed MRD during the 24-month screening period and 53 (88%) were eligible to start study treatment. 6 months after initiation of azacitidine, 31 (58%, 95% CI 44-72) of 53 patients were relapse-free and alive (p<0·0001; one-sided binomial test for null hypothesis p
INTERPRETATION: Pre-emptive therapy with azacitidine can prevent or substantially delay haematological relapse in MRD-positive patients with MDS or AML who are at high risk of relapse. Our study also suggests that continuous MRD negativity during regular MRD monitoring might be prognostic for patient outcomes.
FUNDING: Celgene Pharma, José Carreras Leukaemia Foundation, National Center for Tumor Diseases (NCT), and German Cancer Consortium (DKTK) Foundation.

How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDSs) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPNs or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.

BACKGROUND: Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients.
METHODS: MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2'deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34
RESULTS: MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors.
CONCLUSIONS: MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.

Myelodysplastic syndromes (MDS) and related myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) are clonal stem cell disorders, primarily affecting patients over 65 years of age. Mapping of the MDS and MDS/MPN genome identified recurrent heterozygous mutations in the RNA splicing machinery, with the SF3B1, SRSF2, and U2AF1 genes being frequently mutated. To better understand how spliceosomal mutations contribute to MDS pathogenesis in vivo, numerous groups have sought to establish conditional murine models of SF3B1, SRSF2, and U2AF1 mutations. The high degree of conservation of hematopoiesis between mice and human and the well-established phenotyping and genetic modification approaches make murine models an effective tool with which to study how a gene mutation contributes to disease pathogenesis. The murine models of spliceosomal mutations described to date recapitulate human MDS or MDS/MPN to varying extents. Reasons for the differences in phenotypes reported between alleles of the same mutation are varied, but the nature of the genetic modification itself and subsequent analysis methods are important to consider. In this review, we summarize recently reported murine models of SF3B1, SRSF2, and U2AF1 mutations, with a particular focus on the genetically engineered modifications underlying the models and the experimental approaches applied.

BACKGROUND: Mesenchymal stromal cells (MSCs) are an essential element of the bone marrow (BM) microenvironment, playing a crucial function in regulating hematopoietic stem cell proliferation and differentiation. Recent findings have outlined a putative role for MSCs in hematological malignancy development. So far, conflicting results have been collected concerning MSC abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In particular, a considerable amount of evidence has been accumulated strongly supporting a permissive role of MSCs in malignancy evolution to MDS, while a potentially causative or promoting function performed by MSCs in AML has not yet been fully clarified. Here, we compared MSCs isolated from healthy, MDS, and AML subjects to investigate MSC alterations and to emphasize putative common and/or diverse features.
METHODS: We isolated and expanded MSCs from AML patients (AML-MSCs) and MDS patients (MDS-MSCs), and we analyzed and compared their phenotypic and functional properties with respect to each other and versus healthy donor-derived MSCs (HD-MSCs).
RESULTS: We found that stable MSC cultures could be easily established from HD and MDS mononuclear BM-derived cells, while a substantial fraction (25%) of AML patients failed to yield MSCs. Nevertheless, isolated MDS-MSCs and AML-MSCs, as well as HD-MSCs, contained the basic features of MSCs. Indeed, they displayed similar surface marker expression and efficient capacity to differentiate versus osteogenic and adipogenic lineage in vitro. We also proved that MDS-MSCs and AML-MSCs, analyzed by fluorescence in-situ hybridization, did not harbor leukemic cell cytogenetic abnormalities. Moreover, MDS-MSCs and AML-MSCs were similar in terms of ability to sustain AML cell viability and immune-regulatory capacity. However, we were also able to detect some differences between AML-MSCs and MDS-MSCs. Indeed, we found that the frequency of rescued MSCs was lower in the AML group than in the HD and MDS groups, suggesting that a reduced number of MSC precursors could inhabit AML BM. Instead, MDS-MSCs showed the lowest proliferative capacity, reflecting some intrinsic and particular defect.
CONCLUSIONS: Overall, our results elucidated that MDS-MSCs and AML-MSCs did not show macroscopic and/or tumor-related defects, but both displayed functional features potentially contributing to favor a leukemia-protective milieu.

PURPOSE OF REVIEW: Although BCR-ABL1-negative myeloproliferative neoplasms (MPN) are chronic, clonal hematopoietic stem cell (HSC) disorders marked by proliferation of one or more myeloid lineages, a substantial proportion of patients transform to acute myeloid leukemia. Leukemic transformation (LT) from a pre-existing MPN carries a dismal prognosis. Here, we review recent genetic, biological, and clinical data regarding LT.
RECENT FINDINGS: In the last decade, DNA sequencing has revolutionized our understanding of the genomic landscape of LT. Mutations in TP53, ASXL1, EZH2, IDH1/2, and SRSF2 are significantly associated with increased risk of LT of MPNs. Preclinical modeling of these mutations is underway and has yielded important biological insights, some of which have therapeutic implications. Recent progress has led to the identification of recurrent genomic alterations in patients with LT. This has allowed mechanistic and therapeutic insight into the process of LT. In turn, this may lead to more mechanism-based therapeutic strategies that may improve patient outcomes.

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent knock-in alleles of

Myeloid hematological malignancies are clonal bone marrow neoplasms, comprising of acute myeloid leukemia (AML), the myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), the myeloproliferative neoplasms (MPN) and systemic mastocytosis (SM). The field of epigenetic regulation of normal and malignant hematopoiesis is rapidly growing. In recent years, heterozygous somatic mutations in genes encoding epigenetic regulators have been found in all subtypes of myeloid malignancies, supporting the rationale for treatment with epigenetic modifiers. Histone deacetylase inhibitors (HDACi) are epigenetic modifiers that, in vitro, have been shown to induce growth arrest, apoptotic or autophagic cell death, and terminal differentiation of myeloid tumor cells. These effects were observed both at the bulk tumor level and in the most immature CD34⁺38

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous

Acute myeloid leukemia (AML) is a clonal myeloid neoplasm that typically arises de novo; however, some cases evolve from a preleukemic state, such as myelodysplastic syndrome (MDS). Such secondary AMLs and those with typical MDS-related clinical features are known as AMLs with myelodysplasia-related changes (AML-MRC). Because patients with AML-MRC have poor prognosis, more accurate diagnostic approaches are required. In this study, we performed targeted sequencing of 54 genes in 3 cell populations (granulocyte, blast, and T-cell fractions) using samples from 13 patients with MDS, 16 patients with clinically diagnosed AML-MRC, 4 patients with suspected AML-MRC but clinically diagnosed as AML not otherwise specified (AML-NOS), and 11 patients with de novo AML. We found that overlapping mutations, defined as those shared at least by the blast and granulocyte fractions, were significantly enriched in patients with MDS and AML-MRC, including those with suspected AML-MRC, indicating a substantial history of clonal hematopoiesis. In contrast, blast-specific nonoverlapping mutations were significantly enriched in patients with de novo AML. Furthermore, the presence of overlapping mutations, excluding

We analyzed the clinical significance and genetic features of ASXL2 and ZBTB7A mutations, and the alternatively spliced isoform of the RUNX1-RUNX1T1 transcript, which is also called AML1-ETO9a (AE9a), in Japanese CBF-AML patients enrolled in the JALSG AML201 study. ASXL2 and ZBTB7A genes were sequenced using bone marrow samples of 41 AML patients with t(8;21) and 14 with inv(16). The relative expression levels of AE9a were quantified using the real-time PCR assay in 23 AML patients with t(8;21). We identified ASXL2 (34.1%) and ZBTB7A (9.8%) mutations in only AML patients with t(8;21). ASXL2-mutated patients had a significantly higher WBC count at diagnosis (P = 0.04) and a lower frequency of sex chromosome loss than wild-type patients (33 vs. 76%, respectively, P = 0.01). KIT mutations were the most frequently accompanied with both ASXL2 (36%) and ZBTB7A (75%) mutations. Neither ASXL2 nor ZBTB7A mutations had an impact on overall or event-free survival. Patients harboring cohesin complex gene mutations expressed significantly higher levels of AE9a than unmutated patients (P = 0.03). In conclusion, ASXL2 and ZBTB7A mutations were frequently identified in Japanese AML patients with t(8;21), but not in those with inv(16). Further analysis is required to clarify the detailed biological mechanism of AE9a regulation of the cohesin complex.

Therapeutic options for patients with lower-risk myelodysplastic syndrome (MDS) who have failed prior therapies are limited particularly after hypomethylating agent. Several studies have indicated that deregulation of innate immunity signaling is critical in the pathogenesis of MDS. This process involves Toll-like receptor stimulation, cytokine overexpression, and nuclear factor-kB (NF-kB) activation. Since ruxolitinib, a JAK1/JAK2 inhibitor, suppresses NF-kB expression, we conducted a phase 1 dose-escalation study to determine the safety and efficacy of ruxolitinib in previously treated lower-risk MDS patients with evidence of NF-kB activation. Nineteen patients, 8 with chronic myelomonocytic leukemia and 11 with MDS, were enrolled. No dose limiting toxicity was observed and the maximum tolerated dose was 20 mg twice daily. Responses were restricted to MDS patients with an overall response rate of 22% [hematological improvement in platelets (HI-P) = 2, hematological improvement in erythrocytes (HI-E) = 1, partial cytogenetic response (PCyR) = 1]. Of these patients, 2 relapsed (HI-P and PCyR) and 2 continue to be in HI-P and HI-E, respectively, with ongoing therapy. Meaningful improvement in bone marrow dysplasia was only seen in a patient who achieved HI-E. Phosphorylated p65 (pp65) decreased in 6 of 15 patients (40%) including the 2 patients with continued response to treatment and increased in a patient who relapsed after a short-lived HI-P. This suggests potential correlation between reduction in pp65 expression and response duration. In conclusion, ruxolitinib was well-tolerated in previously treated lower-risk MDS patients with evidence of NF-kB activation and resulted in low but significant frequency of responses. (NCT01895842).

ASXL1 (additional sex combs like 1) is a gene that is mutated in a number of hematological neoplasms. The most common genetic alteration is c.1934dupG p.Gly646fs. Previous publications have shown that ASXL1 mutations have a negative prognostic impact in patients with MDS and AML, however, controversy exists regarding the molecular testing of ASXL1 c.1934dupG as polymerase splippage over the adjacent homopolymer could lead to a false-positive result. Here, we report the first study to systematically test different targeted next generation sequencing (NGS) approaches for this mutation in patients with hematologic neoplasms. In addition, we investigated the impact of proofreading capabilities of different DNA polymerases on ASXL1 c.1934dupG somatic mutation using conventional Sanger sequencing, another common method for ASXL1 genotyping. Our results confirm that ASXL1 c.1934dupG can be detected as a technical artifact, which can be overcome by the use of appropriate enzymes and library preparation methods. A systematic study of serial samples from 30 patients show that ASXL1 c.1934dupG is a somatic mutation in haematological neoplasms including MDS, AML, MPN and MDS/MPN and often is associated with somatic mutations of TET2, EZH2, IDH2, RUNX1, NRAS and DNMT3A. The pattern of clonal evolution suggests that this ASXL1 mutation might be an early mutational event that occurs in the principal clonal population and can serve as a clonal marker for persistent/relapsing disease.

As diagnosing therapy-related myeloid neoplasms (t-MN) is often challenging, we reviewed clinicopathological features of t-MN patients. Medical records of 138 patients, diagnosed with t-MN between 1995 and 2017, were reviewed. Of 138 patients, 80 had t-MDS, 53 t-AML, and 5 t-MDS/MPN (age, 22-88 years; median 64 years; male/female ratio, 0.8). The median latency time was 6 years and 5 months. Of 115 patients, 56 patients received cytotoxic-/radiotherapy for a solid tumor, 56 for hematological malignancy, and 3 for an auto-immune disorder, respectively. Another 21 patients had a combination of 2 disorders. Moreover, 2 patients had 3 previous malignancies. Breast cancer was the most prevalent tumor, followed by low-grade B non-Hodgkin lymphoma. Immunophenotyping and immunohistochemistry showed aberrant expression of B-, T-, or NK-cell markers in 21% and 6%, respectively. In 90% of the patients, dysplasia in ≥ 1 lineage was found. KMT2A fusion gene transcripts were seen in 5%. Cytogenetic analysis showed complex karyotypes (31%) and chromosome 5 and/or 7 abnormalities (40%). Almost 82% of the patients died and the median overall survival was about 1 year. Our study confirms that previous therapy for breast cancer is the most important cause of t-MN. KMT2A fusion genes are prevalent and complex karyotypes and/or chromosomes 5 and/or 7 abnormalities are common.

Breast cancer is one of the most common cancers among women and increased expression of some polymorphic genes, which is rare within families, enhances the risk of breast cancer incidence. The correct identification of the functional SNPs of such genes is important for characterizing the functional aspect of these SNPs which can be assessed by evaluating their significant influence on the structure and function of proteins. Since the presence of SNPs in these genes affects the quality of life of a breast cancer patient, thus, the associated diagnostic markers have a reliable potential for assessing the prognosis of breast cancer. ATP-binding cassette (ABC) genes have been shown to obstruct the treatment of breast cancer by providing resistance to malignant cells from anti-cancer drugs. Some allelic variants of ABCG2 and ABCB1 are also associated with occurrence of skin toxicity during the treatment of breast cancer with anti-cancer drugs. The present study has incorporated comprehensive bioinformatics analysis to explore the possible disease-associated mutations of ABCB1 gene, a gene that resulted from gene-environment interaction study, and understand their consequential effect on the structural and functional behavior of P-glycoprotein. Two gene variants (R538S and M701R) of P-glycoprotein were selected as potentially detrimental point mutations, and these variants were modeled. Molecular dynamic simulation (MDS) studies unraveled the atomic interactions and motion trajectories of the native as well as the two mutant (R538S and M701R) structures and were predicted to have a deleterious effect on breast cancer associated P-gp. Thus, the present study may broaden the way to design novel potent drugs for overcoming the problems associated with multidrug resistance (MDR) resulting from a change in protein conformation due to a mutation in ABCB1 gene.