Identification of Defects in the Transcriptional Program during Differentiation of CD34+Cells Selected from Patients with Both, Low- and High-Risk Myelodysplastic Syndromes.

Abstract The development of MDS is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells. To detect alterations within the transcriptional program in MDS derived CD34+ cells during lineage-specific differentiation, CD34+ bone marrow cells were selected from healthy individuals (n=3) and patients with low-risk (IPSS, n=3) or high-risk (n=4) MDS and stimulated in vitro with EPO, TPO or G/GM-CSF to induce lineage-specific differentiation. Lineage-determined cells were harvested and if necessary purified by immunomagnetic beads at days 4, 7 and 11 for gene expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA). The experiments were done in triplicates for each of the time points and each of the conditions. First, we identified 260 genes with a significant expression pattern associated with normal lineage-specific differentiation. These continuously up- or down-regulated genes are considered to be part of a specific genetic program of normal hematopoietic cells during lineage-specific differentiation. In MDS, 57% of these genes showed a different expression from the normal transcriptional pattern. Thirteen of 24 genes up-regulated during normal erythropoiesis were opponently expressed in MDS containing putative new erythro-specific genes like two GTPase activator proteins, RAP1GA1 and ARHGAP8, which regulate small Rho GTPases. Fourteen of 22 continuously up-regulated genes during normal granulopoietic development displayed a significantly different expression in MDS containing the putative candidate desmocollin 2, a gene which is involved in intercellular cell-adhesion. Delta-like 1 (DLK1) is known to be overexpressed in stem cells from patients with myelodysplastic syndrome. The role of DLK1 in normal hematopoiesis is still not defined. We found DLK1 with increasing expression during normal megakaryopoiesis but reverse expression during megakaryopoiesis in MDS. Interestingly, in erythropoiesis from both, high- and low risk MDS we found overexpression of Bladder cancer overexpressed (BLOV1) and Apoptosis inhibitor 5 (API5, which acts as a cellular survival factor by inhibiting apoptosis after growth factor withdrawal). These genes are not expressed in normal erythropoiesis. Furthermore, we identified the gene for a novel v-maf-like protein F, MafF-like (v-maf: musculoaponeurotic fibrosarcoma oncogene homolog F) to be significantly downregulated exclusively in low-risk MDS. MafF belongs to a basic leucine-zipper(bZIP)-transcription factor family normally involved in multiple physiological processes including hematopoiesis and stress responses. Our data provide the first comprehensive transcriptional analysis of differentiating human CD34+ cells derived from normal individuals compared to MDS. It gives new insights to understand the alteration of differentiation and proliferation of MDS derived CD34+ cells. In particular, the study could identify the gene encoding for the MafF-like protein that acts as a transcriptional regulator of normal hematopoiesis to be significantly down-regulated in low-risk MDS.

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Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow

Blood ◽

10.1182/blood.v100.10.3553 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3553-3560 ◽

Cited By ~ 166

Author(s):

Wolf-K. Hofmann ◽

Sven de Vos ◽

Martina Komor ◽

Dieter Hoelzer ◽

William Wachsman ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

High Risk ◽

Bone Marrow Cells ◽

Low Risk ◽

Control Group ◽

Expression Data ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Marrow Cells

Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.

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Myelodysplastic Syndromes Are Characterized by Gene Expression Changes in Hematopoietic Stem Cells and Alterations in Hematopoietic Stem Cell and Myeloid Progenitor Composition.

Blood ◽

10.1182/blood.v114.22.1762.1762 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1762-1762

Author(s):

Christopher Y. Park ◽

Wendy W Pang ◽

Peter L Greenberg ◽

Irving L. Weissman

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

High Risk ◽

Low Risk ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Transcriptional Changes ◽

Equity Ownership ◽

Myeloid Progenitors

Abstract Abstract 1762 Poster Board I-788 The myelodysplastic syndromes (MDS) represent a heterogeneous group of disorders characterized by peripheral cytopenias due to impaired hematopoietic differentiation. To date, most data characterizing the immature hematopoietic compartment in MDS have relied on evaluation of CD34+ bone marrow cells, which are a heterogeneous population containing a predominance of oligo- and unilineage-potent progenitors and few hematopoietic stem cells (HSC). In this study we show that MDS are disorders of HSC, evidenced by the presence of recurrent cytogenetic alterations, including -5q, -7, and -20q, in highly purified HSC (Lin-CD34+CD38-CD90+CD45RA-) by FISH. Because MDS HSC harbor cytogenetic changes, we sought to better characterize the molecular basis of MDS HSC function by performing whole transcriptome analysis of highly purified HSC and committed myeloid progenitor populations from low-risk (n=8) and high-risk (n=2) MDS patients. When compared to control HSC from healthy patients (n=10), MDS HSC showed broad transcriptional changes. Using the significance analysis of microarrays (SAM) algorithm and Ingenuity Pathways Analysis software, we identified 3,258 differentially expressed genes (FDR < 0.1) with increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). Interestingly, while MDS common myeloid progenitors (CMP, Lin-CD34+CD38+CD123+CD45RA-) showed increased expression of cell death-related genes when compared to normal CMP (p < 0.001), neither MDS HSC nor multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-) showed significant differential expression of these genes when compared to their normal counterparts. To assess the cellular and developmental correlates of HSC/committed progenitor transcriptional changes, we evaluated by flow cytometry the frequency of HSC and committed myeloid progenitors in bone marrow aspirates from 35 low-risk MDS, 6 high-risk MDS and 32 healthy patient samples (range 4-84 yo). Low-risk MDS bone marrow samples showed significantly increased numbers of HSC compared to normal bone marrow samples (+3-fold change, p < 0.03). In addition, myeloid progenitor composition was frequently altered in low-risk MDS patients, with decreased percentages of granulocyte-macrophage progenitors (GMP, Lin-CD34+CD38+CD123+CD45RA+) when expressed as a percentage of total myeloid progenitors [including GMP, CMP and megakaryocyte-erythroid progenitors (MEP, Lin-CD34+CD38+CD123loCD45RA-)] (-2.3-fold change, p < 1e-6). This altered myeloid progenitor profile was highly specific to MDS, even when MDS patient samples were compared to a group of control bone marrow samples from non-MDS patients exhibiting at least one cytopenia (n=34, p < 1e-5), allowing for the distinction of MDS samples from non-MDS cytopenias with 0.89 sensitivity and 0.89 specificity. Together, these data indicate that MDS HSC exhibit significantly altered gene expression profiles and suggest that gene expression changes in MDS HSC induce the altered developmental fate decisions and transcriptional changes observed in MDS committed myeloid progenitors. These data also demonstrate that the changes in MDS myeloid progenitor composition may provide a novel, flow cytometric method for distinguishing MDS from other hematologic conditions that mimic MDS. Finally, these studies indicate that molecular characterization of MDS phenotypes may require evaluation of purified hematopoietic progenitors in order to account for the differential effect of MDS-associated changes on specific hematopoietic progenitor populations. Disclosures Weissman: Amgen: Equity Ownership; Cellerant Inc.: Founder; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

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Let-7a, Mir-17 and Mir-20a Expression Levels in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndromes (MDS) Are Associated with Established Prognostic Factors, Supporting Their Implication in the Pathogenesis of the Disease,

Blood ◽

10.1182/blood.v118.21.3792.3792 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3792-3792 ◽

Cited By ~ 1

Author(s):

Christos K Kontos ◽

Vassiliki Pappa ◽

Diamantina Vasilatou ◽

Maria-Angeliki S Pavlou ◽

Frida Kontsioti ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Prognostic Factors ◽

High Risk ◽

Mirna Expression ◽

Who Classification ◽

Low Risk ◽

Expression Levels ◽

Cd34 Cells ◽

Bone Marrow Blasts

Abstract Abstract 3792 Introduction: MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose: In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods: We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results: In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion: To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures: No relevant conflicts of interest to declare.

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Reduced TET2 expression in Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2822.2822 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2822-2822

Author(s):

Renata Scopim-Ribeiro ◽

Joao Machado-Neto ◽

Paula de Melo Campos ◽

Patricia Favaro ◽

Adriana S. S. Duarte ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

High Risk ◽

Myeloid Leukemia ◽

Research Funding ◽

Erythroid Differentiation ◽

Low Risk ◽

High Risk Disease ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 2822 Introduction: Acquired mutations in TET2 and DNMT3A have been found in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and may predict a worse survival in these diseases. TET2 mutations are considered to be a loss-of-function mutation and results in decreased 5-hydroxymethylcitosine (5-hmc) levels. In normal CD34+ cells, TET2 silencing skews progenitor differentiation towards the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. Dnmt3a participates in the epigenetic silencing of hematopoietic stem cell regulatory genes, enabling efficient differentiation. Here, we attempted to evaluate the expression of TET2 and DNMT3A in total bone marrow cells from normal donors, patients with MDS and AML, and in CD34+ cells from MDS and normal controls during erythroid differentiation. Materials and Methods: The study included normal donors (n = 21), patients with MDS (n = 43) and AML (n = 42) at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. MDS patients were stratified into low and high-risk according to WHO classification (RCUD/RCMD/RARS=31 and RAEB1/RAEB2=12). TET2 and DNMT3A mRNA expression was assessed by quantitative PCR. CD34+ cells from normal donors (n = 9) and low-risk MDS patients (n = 7) were submitted to erythroid differentiation. Cells were collected and submitted to immunophenotyping for GPA and CD71 (days 6 and 12) and q-PCR for TET2 and DNMT3A expression (days 6, 8 and 12). Results of gene expression in normal donors and patients are presented as median, minimum-maximum, and were compared using Mann-Whitney test. Student t test was used for comparison of gene expression during CD34+ erythroid diferentiation. Overall survival was defined from the time of sampling to the date of death or last seen. Univariate analysis for overall survival was conducted with the Cox proportional hazards model. Results: TET2 expression was significantly reduced in both AML (0.62; 0.01–32.69) and MDS (1.46; 0.17–21.30) compared to normal donors (2.72; 0.43–31.49); P<0.0001 and P=0.01, respectively. TET2 expression was also significantly reduced in AML compared to MDS (P=0.0007). MDS patients were stratified into low and high-risk disease, and we still observed a significant reduction in TET2 expression in high-risk (0.73, 0.17–7.25) when compared to low-risk (1.58; 0.48–21.30; P=0.02) patients, but no difference was noted between normal donors vs. low-risk MDS, and high-risk MDS vs. AML. In MDS cohort, the median overall survival was 14 months (range 1–83), increased TET2 expression was associated with a longer survival (HR, 0.44; 95% CI, 0.21–0.91, P=0.03), and, as expected, WHO high-risk disease was associated with a shorter survival (HR, 10.16; 95% CI, 3.06–33.72, P<0.001), even though the confidence interval (CI) was large. TET2 expression did not impact survival in our cohort of AML patients. The erythroid differentiation was effective in cells from normal donors and MDS patients, as demonstrated by the flow cytometry analyses of GPA and CD71. TET2 expression was significantly increased on day 12 of erythroid differentiation, P<0.05. On the other hand, DNMT3A expression was similar between normal donors (0.74; 0.22–1.53), MDS (0.78; 0.26–3.46) and AML (0.95, 0.15–6.46), and during erythroid differentiation, with no impact on survival. Conclusion: These data suggest that decreased TET2 expression may participate in leukemogenesis, and supports the participation of TET2 in the erythroid differentiation of MDS. DNMT3A was not differentially expressed in AML and MDS, indicating that the presence of mutations in this gene may be the predominant mechanism of changes in protein function. We thus suggest that decreased TET2 expression may explain the reduced levels of 5-hmc found in TET2 wild type patients, and may become a predictive marker for outcomes in MDS and other myeloid diseases. Further studies would be necessary to better elucidate the clinical relevance and biologic significance of our findings, and whether the decreased TET2 expression results in hypermethylation in these diseases. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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IRS2 Is Dowregulated In Primary MDS Cells and During MDS Erythroid Differentiation

Blood ◽

10.1182/blood.v116.21.1886.1886 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1886-1886

Author(s):

Joao Machado-Neto ◽

Patricia Favaro ◽

Mariana Lazarini ◽

Adriana S S Duarte ◽

Letícia Fröhlich-Archangelo ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

High Risk ◽

Erythroid Differentiation ◽

Low Risk ◽

Leukemic Cells ◽

Granulocytic Differentiation ◽

Expression Levels ◽

Cd34 Cells

Abstract Abstract 1886 Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The abnormal differentiation of myeloid and erythroid cells is probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling, from upstream activators, to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P<0.0001). Western blot analysis corroborated these findings. According to FAB and WHO classifications, real time RT-PCR demonstrated a significantly lower expression of IRS2 in high-risk MDS samples when compared with low-risk: FAB, 0.34 [0.15–1.56] vs. 1.05 [0.19–4.06], P=0.0172; WHO, 0.30 [0.15–1.44] vs. 1.00 [0.19–4.06], P=0.0204. Based on the IPSS classification and cytogenetic risk group, the expression levels of IRS2 were similar between the low and high-risk groups. During erythroid differentiation, we evaluated IRS2 gene expression of CD34+ cells on days 6, 8 and 12 of culture. On day 12 of normal CD34+ erythroid differentiation, there was an 8.25-fold increased in IRS2 expression, compared to day 6. Interesting, MDS CD34+ cells showed a lower increment in IRS2 transcripts at the same time point (3.89-fold increase only). In conclusion, the down regulation of IRS2 in primary MDS cells and the lower increase in its expression during MDS erythroid differentiation, suggest a role of IRS2 to maintain the effective hematopoiesis. Moreover, IRS2 lower expression in high-risk group, suggests that IRS2 plays a role in the MDS pathophysiology and disease progression. Supported by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

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Altered Expression of the Repair Genes in CD34+ Cells May be Responsible for Formation and Accumulation of Mutations in MDS Patients

Blood ◽

10.1182/blood.v126.23.4119.4119 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4119-4119

Author(s):

Jan Valka ◽

Monika Belickova ◽

Jitka Vesela ◽

Eliska Stara ◽

Barbora Pejsova ◽

...

Keyword(s):

Gene Expression ◽

Dna Repair ◽

High Risk ◽

Expression Profile ◽

Low Risk ◽

Control Group ◽

Dna Repair Genes ◽

Altered Expression ◽

Cd34 Cells ◽

Repair Genes

Abstract Background and Aims Recent studies have demonstrated that in most cases of myelodysplastic syndrome (MDS) at least one mutation has been detected, suggesting that abnormal DNA repair may represent both cause and consequence of malignant transformation. In this study we investigated a possible role of different alterations in DNA repair genes in pathogenesis of MDS. Methods Gene expression of CD34+ cells was measured by RT² Profiler PCR Arrays (Qiagen). Polymorphisms and mutations were studied by targeting next generation sequencing (SeqCap EZ System, NimbleGen). Expression analysis of 84 DNA repair genes was performed in 18 MDS patients and subsequent analysis of selected genes was performed on a cohort of 80 patients. The enrichment resequencing of 84 genes was done in 16 patients. Results Five differentially expressed genes between CD34+ cells of patient and control samples were identified (p<0.05). The increased expression was detected in MPG and XPC genes and decreased expression in RAD51, RPA3 and XRCC2 genes. RAD51 gene showed significantly higher expression in the patients with low-risk MDS forms (RA, RARS, del5q) compared to control group (p=0.0005) and to contrast down-regulated expression was detected (p=0.0002) in high-risk MDS patients (RAEB-1 and -2, AML with myelodysplasia). The group of patients with RCMD showed the average expression at the level of the control group. The expression profile shown a gravity-related decreasing trend after dividing the patients according to IPSS-R, IPSS-R cytogenetic groups and blasts count. Expression profile of XRCC2 gene has similar characteristics as RAD51 (p<0.0001). The expression of RPA3 gene was generally decreased (p<0.0001) with a decreasing trend depending on the disease severity according to MDS forms, IPSS-R and cytogenetics. The RAD51, XRCC2 and RPA-3 genes are related to homologous recombination mechanism, with XRCC2 and RPA3 as a supporting factors for RAD51. The survival curves for all the three genes shown significant differences between groups with over-, intermediate- and down-expressed gene (p=0.0001 for RAD51; 0.0022 for XRCC-2 and 0.0054 for RPA3). MPG gene expression was up-regulated by all MDS types with no significant difference between each other (p=0.0015). Deeper testing of XPC gene expression demonstrated the up-regulation only in low-risk MDS group (p=0.0084). Gene-based analysis showed seven candidate SNPs significantly associated with the disease susceptibility using the HapMAP-CEU population from NCBI PubMed database as control group. With the presence of MDS, these polymorphisms are significantly connected: rs4135113 (p=0.03), rs12917 (p=0.003), rs2230641 (p=0.01), rs2228529 and rs2228526 (p=0.04, respectively p=0.03) and rs1799977 (p=0.04). Within the distribution of tested patient groups according to cytogenetics, we observed significant increase of dependency of these polymorphisms (OR 4.1-9.7, p<0.0001) and the presence of MDS in the group of patients with very-high risk cytogenetics. Conclusions DNA repair mechanisms are responsible for correcting DNA damage and preserving genomic integrity. Our study demonstrates, that altered expression of the repair genes in CD34+ cells may be responsible for the formation and accumulation of mutations in hematological malignancies. Furthermore, we have identified genetic variants that might contribute to the pathogenesis of MDS by modifying individual risk for the disease. Supported by grant (NT/13899, NT/14377, and NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

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Identifying and Validating an Acidosis-Related Signature Associated with Prognosis and Tumor Immune Infiltration Characteristics in Pancreatic Carcinoma

Journal of Immunology Research ◽

10.1155/2021/3821055 ◽

2021 ◽

Vol 2021 ◽

pp. 1-19

Author(s):

Pingfei Tang ◽

Weiming Qu ◽

Dajun Wu ◽

Shihua Chen ◽

Minji Liu ◽

...

Keyword(s):

Gene Expression ◽

High Risk ◽

Pancreatic Carcinoma ◽

Cox Regression ◽

Risk Group ◽

Immune Dysfunction ◽

Low Risk ◽

Excellent Performance ◽

Kaplan Meier ◽

Prognostic Prediction

Background. Acidosis in the tumor microenvironment (TME) is involved in tumor immune dysfunction and tumor progression. We attempted to develop an acidosis-related index (ARI) signature to improve the prognostic prediction of pancreatic carcinoma (PC). Methods. Differential gene expression analyses of two public datasets (GSE152345 and GSE62452) from the Gene Expression Omnibus database were performed to identify the acidosis-related genes. The Cancer Genome Atlas–pancreatic carcinoma (TCGA-PAAD) cohort in the TCGA database was set as the discovery dataset. Univariate Cox regression and the Kaplan–Meier method were applied to screen for prognostic genes. The least absolute shrinkage and selection operator (LASSO) Cox regression was used to establish the optimal model. The tumor immune infiltrating pattern was characterized by the single-sample gene set enrichment analysis (ssGSEA) method, and the prediction of immunotherapy responsiveness was conducted using the tumor immune dysfunction and exclusion (TIDE) algorithm. Results. We identified 133 acidosis-related genes, of which 37 were identified as prognostic genes by univariate Cox analysis in combination with the Kaplan–Meier method ( p values of both methods < 0.05). An acidosis-related signature involving seven genes (ARNTL2, DKK1, CEP55, CTSV, MYEOV, DSG2, and GBP2) was developed in TCGA-PAAD and further validated in GSE62452. Patients in the acidosis-related high-risk group consistently showed poorer survival outcomes than those in the low-risk group. The 5-year AUCs (areas under the curve) for survival prediction were 0.738 for TCGA-PAAD and 0.889 for GSE62452, suggesting excellent performance. The low-risk group in TCGA-PAAD showed a higher abundance of CD8+ T cells and activated natural killer cells and was predicted to possess an elevated proportion of immunotherapeutic responders compared with the high-risk counterpart. Conclusions. We developed a reliable acidosis-related signature that showed excellent performance in prognostic prediction and correlated with tumor immune infiltration, providing a new direction for prognostic evaluation and immunotherapy management in PC.

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Integrated Transcriptomic Analysis of Necrosis-related Gene in Diffuse Gliomas

Journal of Neurological Surgery Part A Central European Neurosurgery ◽

10.1055/s-0039-1683448 ◽

2019 ◽

Vol 80 (04) ◽

pp. 240-249

Author(s):

Jiajia Wang ◽

Jie Ma

Keyword(s):

Gene Expression ◽

High Risk ◽

Differentially Expressed Genes ◽

Expression Profiles ◽

Prognostic Index ◽

Gene Expression Profiles ◽

Risk Groups ◽

Low Risk ◽

Differentially Expressed ◽

Low Grade

Glioblastoma multiforme (GBM), an aggressive brain tumor, is characterized histologically by the presence of a necrotic center surrounded by so-called pseudopalisading cells. Pseudopalisading necrosis has long been used as a prognostic feature. However, the underlying molecular mechanism regulating the progression of GBMs remains unclear. We hypothesized that the gene expression profiles of individual cancers, specifically necrosis-related genes, would provide objective information that would allow for the creation of a prognostic index. Gene expression profiles of necrotic and nonnecrotic areas were obtained from the Ivy Glioblastoma Atlas Project (IVY GAP) database to explore the differentially expressed genes.A robust signature of seven genes was identified as a predictor for glioblastoma and low-grade glioma (GBM/LGG) in patients from The Cancer Genome Atlas (TCGA) cohort. This set of genes was able to stratify GBM/LGG and GBM patients into high-risk and low-risk groups in the training set as well as the validation set. The TCGA, Repository for Molecular Brain Neoplasia Data (Rembrandt), and GSE16011 databases were then used to validate the expression level of these seven genes in GBMs and LGGs. Finally, the differentially expressed genes (DEGs) in the high-risk and low-risk groups were subjected to gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes pathway, and gene set enrichment analyses, and they revealed that these DEGs were associated with immune and inflammatory responses. In conclusion, our study identified a novel seven-gene signature that may guide the prognostic prediction and development of therapeutic applications.

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Ninety Percent Sustained Complete Response (CR) Rate Projected 4 Years after Onset of CR in Gene Expression Profiling (GEP)-Defined Low-Risk Multiple Myeloma (MM) Treated with Total Therapy 3 (TT3): Basis for GEP-Risk-Adapted TT4 and TT5

Blood ◽

10.1182/blood.v112.11.162.162 ◽

2008 ◽

Vol 112 (11) ◽

pp. 162-162 ◽

Cited By ~ 5

Author(s):

Bart Barlogie ◽

Elias J. Anaissie ◽

John D. Shaughnessy ◽

Frits van Rhee ◽

Mauricio Pineda-Roman ◽

...

Keyword(s):

Gene Expression ◽

High Risk ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Complete Response ◽

Low Risk ◽

Event Free Survival ◽

Free Survival ◽

Response Onset ◽

Dose Dense

Abstract We have previously reported on the remarkable activity of the TT3 program that incorporated both bortezomib (V) and thalidomide (T) into the up-front management of 303 patients. TT3 consisted of 2 cycles each of induction prior to and of dose-reduced consolidation therapy with VTD-PACE (cisplatin, doxorubicin, cyclophosphamide, etoposide) after melphalan 200mg/m2 (M200)-based tandem transplants, followed by maintenance therapy for 3 years with VTD and, in later stages, VRD (substituting T for lenalidomide, R). Characteristics included a median age of 59yr (range, 33–75yr), B2M >=4mg/L in 37%, albumin <3.5g/dL in 26%, ISS stages II and III in 33% and 21%, cytogenetic abnormalities (CA) in 33% and gene expression profiling (GEP)-defined high-risk MM in 15% of the 275 patients with such data. With a median follow-up of 39 months, 4-yr overall survival (OS) and event-free survival (EFS) estimates were 78% and 71%, respectively, including 84% and 77% among the 85% with GEP-defined low-risk MM contrasting with 43% and 33% in the remainder with high-risk MM (both p<0.0001). Near-CR and CR, attained in 86% and 63%, were sustained at 4 years from response onset in 78% and 87%, which pertained to 83% and 90% with low-risk MM but to only 44% and 57% with high-risk MM (all p <0.0001). These results were corroborated in a TT3 extension trial (TT3E) that enrolled 175 additional patients, comprising higher proportions of CA (42%) and GEP-defined high-risk MM (21%). Two-year estimates of OS and EFS are 85% and 85%, with 94% and 92% in low-risk patients versus 61% and 62% in high-risk MM (p=0.0001, p=0.0003); the 2-yr estimate of remaining in CR is 93% including 100% in low-risk and 77% in high-risk MM (p=0.01). Multivariate analysis of features linked to OS in TT3 included GEP-defined high-risk, CA, B2M and LDH elevation, collectively accounting for 41% of outcome variability by R2 statistics; the corresponding R2 values for EFS and n-CR duration were 38% and 39%. Compared to the predecessor trial, TT2, that evaluated the role of T in a randomized trial design in 668 patients, TT3 data were superior for OS (p=0.08), EFS (<0.0001), n-CR duration (p<0.0001) and CR duration (p=0.0002). In the low-risk subgroup, EFS (p=0.0001), n-CR duration (p<0.0001) and CR duration (Figure 1a; p=0.0002) all were superior in TT3 versus TT2; whereas, in the high-risk MM group, outcomes remained poor also with TT3 despite superior EFS (Figure 1b; p=0.03). Based on these data, we have now started a GEP-risk-based algorithm of assigning separate therapies to good-risk (TT4) and poor-risk MM (TT5). As the TT3 results for low-risk are difficult to improve upon, TT4 randomizes patients between standard TT3 and TT3-LITE that employs only 1 cycle each of induction and consolidation (with anticipated further improvement in compliance) and 4-day-fractionated M50×4 to enable the addition of VTD and thus exploit synergistic drug interactions to occur. In order to sustain tolerable effective therapies for at least 3 years and prevent recurrence from previous drug-free or insufficiently effective phases in TT3, TT5 for high-risk MM employs less dose-intense and more dose-dense highly synergistic combination therapy, utilizing M10-VTD-PACE for induction, M80 (in 4 daily fractions of M20) plus VRD-PACE tandem transplants, separated by 2 cycles of M20 (in 4 daily fractions of M5) plus VTD-PACE, and followed by 2 years of monthly alternating R-VD and M-VD. Figure 1a: Superior CR duration with TT3 v TT2 in GEP-low-risk MM: Figure 1a:. Superior CR duration with TT3 v TT2 in GEP-low-risk MM: Figure 1b: Superior event-free survival with TT3 v TT2 in GEP-high-risk MM: Figure 1b:. Superior event-free survival with TT3 v TT2 in GEP-high-risk MM:

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MDM2 SNP309 and TP53 SNPArg72Pro Polymorphisms in Myelodysplastic Syndrome.

Blood ◽

10.1182/blood.v114.22.1745.1745 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1745-1745

Author(s):

João Agostinho Machado Neto ◽

Fabiola Traina ◽

Paula Melo Campos ◽

Marilisia Andreoli ◽

Irene Lorand Metze ◽

...

Keyword(s):

Overall Survival ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Disease Progression ◽

Low Risk ◽

Healthy Controls ◽

Mdm2 Snp309 ◽

P53 Pathway ◽

Hematopoietic Stem ◽

High Risk Disease

Abstract Abstract 1745 Poster Board I-771 Introduction Myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The progression of the disease may be associated with genetic or epigenetic alterations and a possible change in protein function. MDM2/P53 pathway plays an important role in the control of apoptotic and proliferation mechanisms. Single nucleotide polymorphisms (SNPs) were identified in the TP53 and MDM2 genes. MDM2 SNP309 results in higher levels of MDM2 and attenuates p53 pathway. The SNP in codon 72 of the TP53 gene results in either a C or G nucleotide and leads to either Proline (Pro) or Arginine (Arg), respectively. The Arg variant has been shown to be more potent in apoptosis induction and the Pro variant has been shown to be better in inducing cell-cycle arrest and to have a greater ability to repair damaged-DNA. The aim of the present study was to investigate the incidence of MDM2 and TP53 polymorphisms in MDS patients and to correlate the frequency of these SNPs with age, neutrophis and platelets at diagnosis, low risk versus high risk disease according to FAB (RA and RARS versus AREB and AREBt) and IPSS (Low and Int-1 versus Int-2 and high), cytogenetic risk (low versus intermediate and high risk), disease progression and overall survival. Patients and Methods We studied 103 healthy controls and 63 patients with MDS. According to FAB, patients were distributed as follows: 43 RA, 10 RARS, 7 RAEB, 1 RAEBt and 2 CMML. The median follow-up time was 40 months (range 2 – 159 months). Samples were obtained from peripheral blood or bone marrow and were screened for the presence of polymorphisms MDM2 SNP309 and TP53 SNPArg72Pro, by PCR analysis with specific primers and appropriate restriction enzyme. Appropriate statistical analyses were used for each test. Results The frequencies of genotypes for MDM2 SNP309 and TP53Pro7Arg were similar between MDS and healthy controls; MDM2 SNP309: 51% vs 53%, for TT, 38% vs 32% for TG, and 11% vs 15% for GG, TP53Pro7Arg: 47.5% vs 44%, for Arg/Arg, 47,5% vs 42% for Pro/Arg, and 5% vs 14% for Pro/Pro. No differences were observed between MDS patients with presence or absence of the polymorphisms in relation to age, neutrophis and platelets at diagnosis, low risk versus high risk disease according to FAB, IPSS and cytogenetic risk, disease progression and overall survival. Conclusions MDM2 and TP53 polymorphisms have been described to affect the risk for cancer, onset age and overall survival in solid tumors and leukemias. This was the first study to report these SNPs in MDS and leads to believe that these polymorphisms are not associated with risk for the disease and with clinical data. Keywords: MDM2, p53, myelodysplasia, polymorphisms Disclosures No relevant conflicts of interest to declare.

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