Association of Gene Mutations with Response to Arsenic-Containing Compound Qinghuang Powder (复方青黄散) in Patients with Myelodysplastic Syndromes

PURPOSE Patients with myelodysplastic syndromes (MDS) have a survival that can range from months to decades. Prognostic systems that incorporate advanced analytics of clinical, pathologic, and molecular data have the potential to more accurately and dynamically predict survival in patients receiving various therapies. METHODS A total of 1,471 MDS patients with comprehensively annotated clinical and molecular data were included in a training cohort and analyzed using machine learning techniques. A random survival algorithm was used to build a prognostic model, which was then validated in external cohorts. The accuracy of the proposed model, compared with other established models, was assessed using a concordance (c)index. RESULTS The median age for the training cohort was 71 years. Commonly mutated genes included SF3B1, TET2, and ASXL1. The algorithm identified chromosomal karyotype, platelet, hemoglobin levels, bone marrow blast percentage, age, other clinical variables, seven discrete gene mutations, and mutation number as having prognostic impact on overall and leukemia-free survivals. The model was validated in an independent external cohort of 465 patients, a cohort of patients with MDS treated in a prospective clinical trial, a cohort of patients with paired samples at different time points during the disease course, and a cohort of patients who underwent hematopoietic stem-cell transplantation. CONCLUSION A personalized prediction model on the basis of clinical and genomic data outperformed established prognostic models in MDS. The new model was dynamic, predicting survival and leukemia transformation probabilities at different time points that are unique for a given patient, and can upstage and downstage patients into more appropriate risk categories.

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Increased incidence of mitochondrial cytochrome c-oxidase gene mutations in patients with myelodysplastic syndromes

British Journal of Haematology ◽

10.1046/j.0007-1048.2001.03323.x ◽

2002 ◽

Vol 116 (3) ◽

pp. 564-575 ◽

Cited By ~ 45

Author(s):

Poluru L. Reddy ◽

Vilasini T. Shetty ◽

Diya Dutt ◽

Aaron York ◽

Saleem Dar ◽

...

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Myelodysplastic Syndromes ◽

Gene Mutations ◽

Mitochondrial Cytochrome ◽

Oxidase Gene ◽

Mitochondrial Cytochrome C

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Hemochromatois-Associated Gene Mutations in Patients with Myelodysplastic Syndromes with Refractory Anemia and Ringed Sideroblasts.

Blood ◽

10.1182/blood.v108.11.1541.1541 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1541-1541

Author(s):

Zachary P. Nearman ◽

Bianca Serio ◽

Hadrian Szpurka ◽

Ilka Warshawsky ◽

Alan Lichtin ◽

...

Keyword(s):

Clinical Features ◽

Myelodysplastic Syndromes ◽

Phenotypic Variability ◽

Gene Mutations ◽

Differential Susceptibility ◽

Control Population ◽

Refractory Anemia ◽

Hfe Gene ◽

Jak2 Mutation ◽

Ringed Sideroblasts

Abstract Complex interaction between a multitude of genetic variants may be responsible for differential susceptibility to specific diseases, and be responsible for phenotypic variability and heterogeneity of clinical presentations. Such a variability in clinical features confounded for many years investigations into the pathogenesis of myelodysplastic syndromes (MDS). We made a curious observation of increased ferritin levels in some newly diagnosed patients with MDS RARS (refractory anemia with ringed sideroblasts) in whom transfusional iron-overload was unlikely due to very low transfusion burden. Hence, we hypothesized that RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this particular subset of MDS. We studied a cohort of 109 MDS patients; 42 with RARS, and 67 with other forms of MDS (18 RA, 12 RAEB, 7 RAEB-T, 1 CMML, and 29 MDS/MPD overlap). All patients were genotyped using restriction fragment length polymorphism (RFLP) method, designed to detect presence of C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygozity for the C282Y mutation in 21% of RARS patients (vs 9% in control population, n=2016, p= 0.017) while H63D genotype was not increased. The possible pathogenic role of this finding in RARS was supported by the normal distribution of mutant HFE alleles in patients with other forms of MDS (5% vs. 9%, p =0.35). Interestingly, 3/7 patients with RA not fulfilling the RARS criteria, but having increased numbers of ringed sideroblasts (<15%) also showed heterozygozity for either C282Y or H63D allele. To correlate the presence of C282Y allele with clinical features of RARS patients, we have performed a subset analysis. Within this group we have included patients with a rather nebulous and rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt); 7 of these patients (n=10) were found to have either C282Y or H63D allele resulting in a frequency of 30% and 40% of C282Y or H63D allele, respectively. The combined prevalence of either of these alleles in the control population is 33% (vs. 70% in RARSt, p=.01). Previously, we have demonstrated that RARSt patients are characterized by a high prevalence of the V617F JAK2 mutation (Szpurka et al, Blood 2006) suggestive of the pathophysiologic derivation of this syndrome from MPD rather than MDS. Consequently, we have tested the frequency of HFE gene variants associated with hemochromatosis in patients with MPD and Jak2 mutations. Of note is that patients with RARS harbored more C282Y alleles than those with other forms of MDS or MPD with Jak2 mutation (except for those with RARSt; (21% vs 5% and 3%, p =0.036 and .012, respectively). We conclude that hemochromatosis associated mutations may contribute to the pathogenesis of RARS. In patients with MPD and Jak2 mutation, concomitant presence of hemachromatosis-predisposing HFE variants may result in the unusual presentation associated with ringed sideroblasts.

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New Insight Into the Biology, Risk Stratification, and Targeted Treatment of Myelodysplastic Syndromes

American Society of Clinical Oncology Educational Book ◽

10.1200/edbk_175397 ◽

2017 ◽

pp. 480-494

Author(s):

Mintallah Haider ◽

Eric J. Duncavage ◽

Khalid F. Afaneh ◽

Rafael Bejar ◽

Alan F. List

Keyword(s):

Risk Stratification ◽

Myelodysplastic Syndromes ◽

Rna Splicing ◽

Somatic Mutations ◽

Prognostic Significance ◽

Gene Mutations ◽

International Prognostic Scoring System ◽

Prognostic Assessment ◽

Sequencing Part ◽

Gene Panels

In myelodysplastic syndromes (MDS), somatic mutations occur in five major categories: RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modifiers. Although many MDS cases harbor more than one somatic mutation, in general, there is mutual exclusivity of mutated genes within a class. In addition to the prognostic significance of individual somatic mutations, more somatic mutations in MDS have been associated with poor prognosis. Prognostic assessment remains a critical component of the personalization of care for patient with MDS because treatment is highly risk adapted. Multiple methods for risk stratification are available with the revised International Prognostic Scoring System (IPSS-R), currently considered the gold standard. Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS. In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R. Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS. Examples include deletion 5q, spliceosome complex gene mutations, and TP53 mutations.

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Deciphering the Key Pharmacological Pathways and Targets of Yisui Qinghuang Powder That Acts on Myelodysplastic Syndromes Using a Network Pharmacology-Based Strategy

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/8877295 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Zijian Han ◽

Luping Song ◽

Kele Qi ◽

Yang Ding ◽

Mingjun Wei ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Target Genes ◽

Network Pharmacology ◽

Analysis Tool ◽

Antitumor Effects ◽

Active Compounds ◽

Pharmacological Mechanism ◽

Putative Target ◽

Target Disease ◽

Qinghuang Powder

Background. Yisui Qinghuang powder (YSQHP) is an effective traditional Chinese medicinal formulation used for the treatment of myelodysplastic syndromes (MDS). However, its pharmacological mechanism of action is unclear. Materials and Methods. In this study, the active compounds of YSQHP were screened using the traditional Chinese medicine systems pharmacology (TCMSP) and HerDing databases, and the putative target genes of YSQHP were predicted using the STITCH and DrugBank databases. Then, we further screened the correlative biotargets of YSQHP and MDS. Finally, the compound-target-disease (C-T-D) network was conducted using Cytoscape, while GO and KEGG analyses were conducted using R software. Furthermore, DDI-CPI, a web molecular docking analysis tool, was used to verify potential targets and pathways. Finally, binding site analysis was performed to identify core targets using MOE software. Results. Our results identified 19 active compounds and 273 putative target genes of YSQHP. The findings of the C-T-D network revealed that Rb1, CASP3, BCL2, and MAPK3 showed the most number of interactions, whereas indirubin, tryptanthrin, G-Rg1, G-Rb1, and G-Rh2 showed the most number of potential targets. The GO analysis showed that 17 proteins were related with STPK activity, PUP ligase binding, and kinase regulator activity. The KEGG analysis showed that PI3K/AKT, apoptosis, and the p53 pathways were the main pathways involved. DDI-CPI identified the top 25 proteins related with PI3K/AKT, apoptosis, and the p53 pathways. CASP8, GSK3B, PRKCA, and VEGFR2 were identified as the correlative biotargets of DDI-CPI and PPI, and their binding sites were found to be indirubin, G-Rh2, and G-Rf. Conclusion. Taken together, our results revealed that YSQHP likely exerts its antitumor effects by binding to CASP8, GSK3B, PRKCA, and VEGFR2 and by regulating the apoptosis, p53, and PI3K/AKT pathways.

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Profiling of Multiple Gene Mutations In Myelodysplastic Syndromes Using High-Throughput Resequenceing Combined with Barcode Labeling

Blood ◽

10.1182/blood.v116.21.4011.4011 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4011-4011

Author(s):

Yasunobu Nagata ◽

Masashi Sanada ◽

Kenichi Yoshida ◽

Takeo Nakaya ◽

Aiko Matsubara ◽

...

Keyword(s):

Candidate Genes ◽

High Throughput ◽

Myelodysplastic Syndromes ◽

Sanger Sequencing ◽

Large Scale ◽

Conflicts Of Interest ◽

Single Case ◽

Gene Mutations ◽

Multiple Gene ◽

Chain Reactions

Abstract Abstract 4011 Myelodysplastic syndromes (MDS) are a highly heterogeneous group of myeloid neoplasms characterized by ineffective hematopoiesis and a predisposition to acute myeloid leukemia, where a model of multisteponcogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations involved in the development of MDS, including mutations of RAS, RUNX1, CEBPA, TET2, CBL, EZH2 and TP53, where multiple gene mutations frequently harbor in a single case. However, the entire profiles of these multiple gene mutations with their relationship with WHO classification, chromosomal alterations, and clinical pictures have not been explored in a large series of MDS cases. Screening possible gene mutations in dozens of candidate genes in a large number of samples using Sanger sequencing were a time-consuming and labor-intensive task. So in order to overcome this issue and to obtain comprehensive registries of gene mutations in known candidate genes in a total of 170 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 80 candidate genes using Genome Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of individual samples. Briefly, each fragmented genomic DNA was frist amplified by single-primer polymerase-chain reactions (PCR), from which target sequences were concentrated using the SureSelect-system® (Agilent). Captured targets were primed with 6-base barcode sequences to discriminate the sample, which were subjected to high-thoughput resequencing using Genome Analizer®(Illumina). All 170 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS and KRAS, c-CBL and TET2 had been determined by Sanger sequencing, and thus were considered to an ideal sample set for this study, in which genome-wide copy numbers were characterized in detail and the known mutations works as a control to measure the performance of the barcode resequencing. Targeted 80 genes consisted of exons with total length of ∼500Kb, and included genes which were known to be mutated in MDS and related disorders, and other candidate targets of mutations. We were able to analyze up to 80 samples per 1 run and efficiently detected mutations in targeted genes by the high average coverage obtained from these sequences. On average 80% of targeted regions were covered with >20 depths of reading. In this meeting, we will present the result of our large-scale mutation study in MDS and related disorders and discuss the genetic basis of MDS in terms of multiple gene mutations as well as copy number alterations. Disclosures: No relevant conflicts of interest to declare.

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Dysregulation of RUNX1 Plays a Critical Role in the Progression of Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v126.23.4108.4108 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4108-4108

Author(s):

Hiroko Sakurai ◽

Yuka Harada ◽

Hirotaka Matsui ◽

Hideaki Nakajima ◽

Toshio Kitamura ◽

...

Keyword(s):

Splice Site ◽

Myelodysplastic Syndromes ◽

Critical Role ◽

Gene Mutations ◽

Splicing Factor ◽

Expression Level ◽

Dominant Negative Effect ◽

Rapid Progression ◽

Myeloid Neoplasms ◽

Cd34 Cells

Abstract RUNX1/AML1 mutations have been frequently detected in patients with myeloid neoplasms, especially myelodysplastic syndromes (MDS) and chronic monocytic leukemia (CMML). Although the mutations have been analyzed thoroughly, its expression level has not been investigated. Therefore, we attempt to clarify the expression of RUNX1 in the pathogenesis of myeloid neoplasms. The study was approved by the institutional review board and patients gave written informed consent for the study, according to the Declaration of Helsinki. Several isoforms of RUNX1 mRNA are known and we analyzed RUNX1a (including exon 7a which has stop codon) and RUNX1b (skipping exon 7a and including exon 7b and 8). Expression levels of full length isoform (RUNX1b) and short isoform (RUNX1a which has a dominant negative effect on RUNX1b) in CD34+ cells from patients with myeloid neoplasms were examined. A part of patients with MDS or myelodysplastic syndrome / myeloproliferative neoplasms (MDS/MPN) including CMML showed RUNX1a overexpression. Average of relative RUNX1a expression level in MDS patients (n=34) and MDS/MPN patients (n=20) was 7.4-fold and 8.6-fold of the level in normal bone marrow (BM), respectively, whereas most of these patients showed almost same or slight increase of expression level of RUNX1b compared with normal BM. Interestingly, some patients showed high expression of RUNX1a and repression of RUNX1b. In both disease categories, patients with excess blasts displayed a significantly higher expression level of RUNX1a compared with normal BM and patients without excess blasts. During the disease progression in a single patient with MDS or MDS/MPN, the expression of RUNX1a became higher, while azacitidine treatment reduced RUNX1a expression. Genomic mutations of RUNX1 were also examined. RUNX1 mutations were detected in 16% of MDS and 35% of MDS/MPN. Surprisingly, a part of patients had both RUNX1 gene mutation and RUNX1a overexpression, and they showed rapid progression of disease. To evaluate the effects of RUNX1a overexpression, RUNX1a was transduced into CD34+ cells from MDS patients with low expression level of RUNX1a. RUNX1a-transduction resulted in cell proliferation on MS5 stromal cells. These results indicate that overexpression of RUNX1a may add growth advantage to CD34+ cells in patients with MDS or MDS/MPN. We next analyzed the mechanism of RUNX1a overexpression. Gene mutations affecting exon recognition were examined in the patients. Splicing factor mutations, SRSF2 and U2AF1, were detected frequently in MDS (15%) and MDS/MPN (50%). Patients with splicing factor mutations showed higher RUNX1a expression than patients without the mutations. To confirm that the splicing factor mutations affect the expression of RUNX1a, we performed enforced expression of SRSF2 p.P95H mutant using pMYs.IRES.EGFP retrovirus vector in a MDS-derived cell line, TF-1. After a single cell sorting, independent 13 expanding clones were analyzed. Most of the clones demonstrated higher expression of RUNX1a than mock cells, whereas RUNX1b expression was reduced in all clones. Increase of RUNX1a expression in SRSF2 mutant-transduced TF-1 cells was also confirmed by Western blot. Moreover, the clones with higher GFP intensity showed higher expression level of RUNX1a, suggesting that SRSF2 p.P95H expression level may affect the expression level of RUNX1a. Furthermore, SRSF2 mutant-transduced TF-1 cells showed phenotypic changes of higher CD11b and CD14 than mock TF-1 cells, suggesting that SRSF2 mutant may induce monocytic differentiation via RUNX1a overexpression. Gene mutations of RUNX1 in intron 6 and exon 7a were also analyzed. A 5' splice site change just after exon 6 was detected in a CMML patient with RUNX1a overexpression, which may be another mechanism of RUNX1a overexpression. Mutations of exon 7a or changes in 3' splice site just before exon 7a have not been detected yet. In conclusion, our data suggest that overexpression of RUNX1a may play a critical role in the progression of MDS and MDS/MPN, in addition to RUNX1 mutations. Splicing factor mutations are suspected to contribute to the mechanism of the dysregulation of RUNX1. Disclosures No relevant conflicts of interest to declare.

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Clonal Evolution of Gene Mutations in AML Transformation from Patients with De Novo Myelodysplastic Syndromes Carrying Spliceosome Mutations: An Analysis of 71 Paired Samples

Blood ◽

10.1182/blood.v128.22.1969.1969 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1969-1969

Author(s):

Ming-Chung Kuo ◽

Po-Nan Wang ◽

Po Dunn ◽

Tung-Liang Lin ◽

Jin-Hou Wu ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndromes ◽

De Novo ◽

Direct Sequencing ◽

Gene Mutations ◽

Initial Diagnosis ◽

Detection Sensitivity ◽

Cohesin Complex ◽

Allele Burden ◽

Epigenetic Regulators

Abstract Background and Purpose: The molecular pathogenesis of progression of myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) remains incompletely understood. We studied genemutations involving spliceosome in the transformation of MDS to sAML by comparing matched paired MDS/sAML bone marrow samples to determine the roles of SRSF2, U2AF1 SF3B1, and ZRSR2 mutations in the evolution of MDS to sAML. Patients and Methods: One hundred and forty-nine de novo MDS patients (2 RCUD, 5 RARS, 27 RCMD, 52 RAEB-1, and 63 RAEB-2) were examined forspliceosome mutations at initial diagnosis and 93 patients progressed to sAML with a median follow-up of 16.4 months. Seventy one of them had paired MDS/sAML bone marrow samples available for comparative analyses. Mutational analyses of spliceosome were performed by pyrosequencing with a detection sensitivity of 5% for U2AF1 (exons 2 and 6) and SRSF2 (P95 of exon 1) mutations and by direct sequencing for SF3B1 (exons13-16) and ZRSR2 (whole coding exons 1-11) mutations. Additional 29 gene mutations known to involve in myeloid neoplasms were also analyzed by direct sequencing or next-generation sequencing (NGS, Ion Torrent PGM) followed by Sanger sequencing validation; NGS was mainly used for mutation detection of epigenetic regulators and cohesin complex. The allele burden of targeted genes was determined by pyrosequencing and/or NGS. Results: The frequencies of U2AF1, SRSF2, SF3B1, and ZRSR2 mutations in the 149 MDS patients were 14.8% (22/149), 12.8% (19/149), 12.1% (17/141), and 7.5% (10/133), respectively. Together, spliceosome mutations occurred in 51.5% of MDS patients at initial diagnosis and were mutually exclusive. All the 5 RARS patients had SF3B1 mutations. Co-existed mutations with epigenetic regulators were detected in 36 patients (52.9%), with RUNX1 in 13 (19.1%), with cohesin complex including STAG2, SMC3, RAD21,or SMC1A, in 13 (19.1%), with signaling pathways including RAS, PTPN11, JAK2, or FLT3-TKD in 4 (5.9%), BCOR in 2, and one each with CEBPa and SETBP1. There were no differences in blood counts, percentage of blasts in bone marrow and blood, WHO subtype, cytogenetic risk group, and IPSS-R between spliceosome-mutated and -unmutated patients. Of the patients carrying spliceosome mutations, only SRSF2 mutation had prognostic impact on predicting a higher risk of sAML transformation (P = 0.025) and sAML-free survival (median 10.8 months, 95% CI 5.1-16.5 months) compared to SRSF2-unmutated patients (median 17 months, 95% CI 8.5-25.5 months, P = 0.050). Of the 71 paired MDS/sAML samples, 37 (52.1%) had spliceosome mutations at diagnosis; the mutational status and patterns remained unchanged in all the 37 matched sAML samples but allele burden was apparently increased in the sAML samples with SRSF2 (P = 0.017) or SF3B1 (P = 0.015) mutations. In addition, one ZRSR2 mutant clone and two SRSF2 mutant clones evolved during sAML progression. Acquisition of other mutated genes was found in 37 spliceosome-mutated patients at sAML phases, including RUNX1 in 5, N-RAS in 5, CEBPa in 4, K-RAS in 3, FLT3-ITD in 3, and one each with ASXL1, TET2, STAG2, WT1, PTPN11, CBL, FLT3-TKD, and C-FMS. Notably, of the 3 patients acquiring spliceosome mutations, none gained other mutated genes during sAML transformation.Clonal expansion of other mutated genes were observed in 4 cases with RUNX1, in 2 with TET2, and one each with N-RAS, CEBPa, ASXL1, SMC1A, and STAG2. Conclusions: Our results showed that spliceosome mutations occurred in more than half of de novo MDS patients at initial diagnosis. Clonal expansion, evolution, or unchanged allele burden of spliceosome mutations might occur during sAML transformation with frequent acquisition of additional mutated genes. SRSF2 mutation predicted a higher risk and more rapid sAML transformation. (Grants support: NHRI-EX103-10003NI, MOHW103-TD-B-111-09 and CMRPG3D1532) Disclosures No relevant conflicts of interest to declare.

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