Spectrum of Gene Mutations and Clinical Significance in Myeloid Malignancies Patients with ASXL1 Mutations

Abstract Abstract Introduction: With the development of next generation sequencing (NGS), the interrelation between genetic and epigenetic abnormality in myeloid malignancies has attracted significant attention. Clinical reports provide strong evidence that while the specific gene mutations are the initial event for the myeloid malignancies, the concomitant gene mutations contribute to the disease progression. Although ASXL1 mutations have been found in myeloid malignancies, the impact of co-mutation with ASXL1 on the disease progression remains largely unknown. In the current study, we aim to investigate the clinical significance of the association between ASXL1 mutations and a spectrum of gene mutations in a large cohort of patients with myeloid malignancies. Methods: Targeted sequencing including 112 hematopoietic malignancy-related genes was used to analyze the gene mutations in patients with ASXL1 mutations. The impact of gene mutations on clinical characteristics and prognosis was further analyzed. The correlation between clinical/laboratory features and the gene mutations was performed by the χ2 test, and differences in values and in ranks were assessed by Student t-tests. Overall survival rate was assessed by the Kaplan-Meier method and calculated by the Log-rank test. Results: A cohort of 138 myeloid malignant patients harboring ASXL1 mutations was recruited to the current study, including patients with myelodysplastic syndromes (MDS) (37.68%, n = 52), myeloproliferative neoplasms (MPN) (21.01%, n = 29), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (7.25%, n = 10), and acute myeloid leukemia (AML) (34.06%, n = 47). In addition, to ASXL1 mutations, 89 genes were mutated in these patients, and 96.4% (133) of the patients were accompanied by at least one gene mutation. Among those mutated genes, 55.8% (77/138) was epigenetic genes, 65.9% (91/138) was signal transduction pathway genes, 28.2% (39/138) was spliceosome related genes, 36.9% (51/138) was transcription factor genes, and 18.8% (26/138) was cell cycle and apoptosis related genes. The most common co-mutated genes were RAS pathway related genes (25.4%, 35/138) and SETBP1 (21.7%, 30/138). Patients with ASXL1 and RAS pathway co-mutations (ASXL1mutRASmut) had significantly lower levels of hemoglobin and platelets compared to ASXL1 mutated patients without RAS pathway mutation (ASXL1mutRASwt) (hemoglobin 81 (33-152) g/L vs. 96 (18-195) g/L, P=0.012; and platelets (51 (8-695)×109/L vs. 75 (3-3149) × 109/L, P=0.032, respectively). Importantly, MDS patients with ASXL1mutRASmut were more likely to be associated with high International Prognostic Scoring System (IPSS) scores (P=0.016). Moreover, the median survival time of these ASXL1mutRASmut patients (mean = 17 months, 1-35 months) was significantly shorter than that of ASXL1mutRASwtpatients (mean = 21 months, 2-75 months) (P=0.031). Conclusions: Our study provides a comprehensive overview of the association between the clinical features and prognosis with genes co-mutated with ASXL1 in patients with myeloid malignancies. We conclude that concomitant mutations of ASXL1 with RAS pathway genes associate with high risk of myeloid transformation and lower overall survival rates. Disclosures No relevant conflicts of interest to declare.

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The Impact of Epigenetic Modifications in Myeloid Malignancies

International Journal of Molecular Sciences ◽

10.3390/ijms22095013 ◽

2021 ◽

Vol 22 (9) ◽

pp. 5013

Author(s):

Deirdra Venney ◽

Adone Mohd-Sarip ◽

Ken I Mills

Keyword(s):

Disease Progression ◽

Dna Methyltransferase ◽

Therapeutic Potential ◽

Current Knowledge ◽

Myeloproliferative Neoplasms ◽

Isocitrate Dehydrogenase 1 ◽

Myeloid Malignancies ◽

Broad Term ◽

Critical Components ◽

The Impact

Myeloid malignancy is a broad term encapsulating myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Initial studies into genomic profiles of these diseases have shown 2000 somatic mutations prevalent across the spectrum of myeloid blood disorders. Epigenetic mutations are emerging as critical components of disease progression, with mutations in genes controlling chromatin regulation and methylation/acetylation status. Genes such as DNA methyltransferase 3A (DNMT3A), ten eleven translocation methylcytosine dioxygenase 2 (TET2), additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2) and isocitrate dehydrogenase 1/2 (IDH1/2) show functional impact in disease pathogenesis. In this review we discuss how current knowledge relating to disease progression, mutational profile and therapeutic potential is progressing and increasing understanding of myeloid malignancies.

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Cooperative Epigenetic Regulation By ASXL1 and NF1 Loss on Leukemogenesis

Blood ◽

10.1182/blood-2018-99-110964 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 652-652

Author(s):

Peng Zhang ◽

Fuhong He ◽

Jie Bai ◽

Shi Chen ◽

Stephen D. Nimer ◽

...

Keyword(s):

Disease Progression ◽

Signaling Pathway ◽

Mouse Models ◽

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Mapk Pathway ◽

Gene Mutations ◽

Rna Seq ◽

Ras Pathway ◽

Myeloid Malignancies

Abstract Introduction: Additional sex combs-like 1 (ASXL1) is frequently mutated in a wide range of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasm, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). Notably, ASXL1 mutations are generally associated with the poor clinical outcomes. We and others have established Asxl1 mouse models and demonstrated that loss of Asxl1 leads to MDS-like disease, which can transform to myeloid leukemia in aged mice. These studies suggest that additional mutations may cooperate with Asxl1 loss to induce the leukemia transformation. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Methods: To identify cooperating events with ASXL1 mutations in myeloid malignancies, we recruited a cohort of 138 ASXL1 mutated patients and performed targeted exome sequencing. We then characterized the hematopoietic features using mouse models. A serial hematopoietic phenotypic analyses were used, including peripheral blood counts, flow cytometry, colony assay, morphology and transplantation assays. To decipher the molecular mechanisms by which loss of Asxl1 and Nf1 cooperate in promoting myeloid leukemia transformation, we performed RNA-seq and ChIP-seq to identify the differentially expressed genes and their associated histone modifications in four genotypes of mice, WT, Asxl1+/-, Nf1+/-, and Asxl1+/-;Nf1+/- mice. Finally, the leukemic mice were treated with pharmacologic inhibitors targeting both MAPK pathway and BET bromodomain in vivo as a proof-of-concept approach. Results: We analyzed the gene mutation profiles of 138 ASXL1 mutated patients based on targeted sequencing and found that 35 of these patients have gene mutations involving in RAS/MAPK signaling pathway, including NF1, NRAS, KRAS, PTPN11 or CBL. The incidence of AML was significantly higher in patients with RAS pathway mutations (48.6%) than in the cases without RAS pathway mutations (29.1%, p = 0.036, chi-square test). These data suggest that concomitant mutations of ASXL1 and RAS pathway genes associate with poor prognosis in myeloid malignancies. To validate the functional significance of cooperative mutations of ASXL1 and NF1, a negative regulator of the RAS signaling pathway, in the disease progression of myeloid leukemia, we generated Asxl1+/-;Nf1+/- and Mx1Cre;Asxl1fl/fl;Nf1fl/fl (Asxl1Δ/Δ;Nf1Δ/Δ) mice and performed hematopoietic phenotypic analyses. Asxl1 loss cooperates with haploinsufficiency of Nf1 to accelerate the development of myeloid leukemia in mice. Asxl1Δ/Δ;Nf1Δ/Δ mice displayed a rapid, progressive leukocytosis with severe anemia and thrombocytopenia 3-6 months after pIpC injection, indicative of aggressive myeloid leukemia with a 100% penetrance. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells lead to transcriptional activation of multiple pathways critical for leukemogenesis, such as MYC, NRAS, and BRD4. Convergent analysis of RNA-seq and ChIP-seq data reveal that the hyperactive MYC and BRD4 transcription program is correlated with elevated H3K4 tri-methylation at the promoter regions of genes involving these pathways. Importantly, pharmacological inhibition of both MAPK pathway and BET bromodomain prevents leukemia initiation and inhibits disease progression in Asxl1Δ/Δ;Nf1Δ/Δ mice, and significantly prolonged the survival of leukemic Asxl1+/-;Nf1+/- mice. Conclusion: This study sheds lights on the understanding of the cooperative effect between epigenetic alterations and signaling pathways in accelerating the progression of myeloid malignancies and provides a rationale therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations. Disclosures No relevant conflicts of interest to declare.

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Triple Negative Myeloproliferative Neoplasms (MPNs) Patients Show Low MPN10 Score and Lower Grades of Bone Marrow Fibrosis

Blood ◽

10.1182/blood.v128.22.5477.5477 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5477-5477

Author(s):

Yasser Elnahass ◽

Hossam K Mahmoud ◽

Mervat Mattar ◽

Omar Fahmy ◽

Mohamed A. Samra ◽

...

Keyword(s):

Bone Marrow ◽

Gene Mutation ◽

Disease Burden ◽

Triple Negative ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Janus Kinase ◽

Driver Gene ◽

The Impact

Abstract Introduction: The vast majority ofmyeloproliferative neoplasms (MPNs) patients are characterized by a molecular genetic background and by variable symptoms reflecting disease burden that may correlate with prognosis. Aim: To study the impact of triple negative status of driver gene mutations: Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) on disease burden and its correlation with symptom severity (MPN10 score) and degree of bone marrow (BM) fibrosis in MPNs patients. Patients and Methods: MPN Symptom Assessment Form Total Symptom Score (MPN-SAF TSS) was assessed as median of 10 items: fatigue, concentration, early satiety, inactivity, night sweats, itching, bone pains, abdominal discomfort, weight loss and fever. JAK2V617F and MPL W515exon 10 mutations were performed by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) while CALR exon 9 insertion/deletion was detected by high-resolution melting (HRM) curve analysis. Results: 100 MPNs patients (54 males and 46 females): 18 polycythemia vera (PV), 41 essential thrombocythemia (ET), 24 primary myelofibrosis (PMF), 10 Post-ET/PV-myelofibrosis (post-ET/PV-MF) and 7 myelodysplastic/myeloproliferative neoplasms (MDS/MPN) were included. Median age at diagnosis was 55 years (17-75) and was lower in ET than PV and PMF patients; 44 (19-75) years vs. 56 (34-70) years and 56 (20-75) years, respectively (p=0.06). JAK2 mutation was positive in 15 (85%) PV patients, 14 (34%) ET patients, 15 (62%) PMF patients, 8(80%) post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.001). CALR mutation was positive in zero (0%) PV patients, 10 (24%) ET patients, 4 (17%) PMF patients, zero (0%) Post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.05). MPL mutation was positive in zero (0%) PV patients, 2 (5%) ET patients, 1(4%) PMF patients, zero (0%) Post ET/PV-MF patients and zero (0%) MDS/MPN patients. Twenty four/93 (26%) patients were triple negative; 15 ET (16%), 3 PV (3%), 6 PMF (6%). Median MPN10 score was 21 (4-45) in ET versus 37.5 (25-56) in PV, 54 (15-80) in PMF and 59 (45-75) in Post-ET/PV-MF (p<0.001). Median MPN10 score was 25 (10-50) in triple negative patients vs. 40 (4-80) in MPNs patients showing at least one driver mutation positivity (p<0.001). BM fibrosis was present in 6 (15%) patients with triple negative vs. 33 (85%) patients showing at least one molecular marker positivity (p=0.007). Out of 52 patients having splenomegaly; seven (13.5%) patients were triple negative vs. 45 (87%) patients with at least one gene mutation (p<0.001). Out of the 24 triple negative patients, 19 (80%), 4 (16%), 1 (3%) and 0(0%) had BM fibrosis grades 0, 1, 2 and 3 vs. 36 (52%), 7 (10%), 12 (17%), 14 (20%) out of 69 patients with at least one gene mutation, respectively (p=0.002). After a median follow-up period of 16 months (3-151), overall survival (OS) was 95%. OS in PV and ET patients was 100 % versus 83 % in PMF patients (p=0.08). OS in triple negative group was 100% versus 94% in the gene mutations group (p=0.387). Conclusion: Driver gene mutations show an impact on disease symptoms and burden. Triple negative MPNs patients in our cohort have significantly low MPN10 score and less BM fibrosis which may indicate a more indolent disease course. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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EZH2 in Myeloid Malignancies

Cells ◽

10.3390/cells9071639 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1639 ◽

Cited By ~ 3

Author(s):

Jenny Rinke ◽

Andrew Chase ◽

Nicholas C. P. Cross ◽

Andreas Hochhaus ◽

Thomas Ernst

Keyword(s):

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Residual Disease ◽

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Loss Of Function ◽

Disease Pathogenesis ◽

Catalytic Core ◽

Myeloid Malignancies ◽

Treatment Free Remission

Our understanding of the significance of epigenetic dysregulation in the pathogenesis of myeloid malignancies has greatly advanced in the past decade. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic core component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for gene silencing through trimethylation of H3K27. EZH2 dysregulation is highly tumorigenic and has been observed in various cancers, with EZH2 acting as an oncogene or a tumor-suppressor depending on cellular context. While loss-of-function mutations of EZH2 frequently affect patients with myelodysplastic/myeloproliferative neoplasms, myelodysplastic syndrome and myelofibrosis, cases of chronic myeloid leukemia (CML) seem to be largely characterized by EZH2 overexpression. A variety of other factors frequently aberrant in myeloid leukemia can affect PRC2 function and disease pathogenesis, including Additional Sex Combs Like 1 (ASXL1) and splicing gene mutations. As the genetic background of myeloid malignancies is largely heterogeneous, it is not surprising that EZH2 mutations act in conjunction with other aberrations. Since EZH2 mutations are considered to be early events in disease pathogenesis, they are of therapeutic interest to researchers, though targeting of EZH2 loss-of-function does present unique challenges. Preliminary research indicates that combined tyrosine kinase inhibitor (TKI) and EZH2 inhibitor therapy may provide a strategy to eliminate the residual disease burden in CML to allow patients to remain in treatment-free remission.

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Increased Expression of Macrophage Migration Inhibitory Factor (MIF) Receptor CD74 Is Associated with Inferior Outcome in Younger Patients (Pts) with Cytogenetically Normal Acute Myeloid Leukemia (CN-AML): a Cancer and Leukemia Group B (CALGB) Study.

Blood ◽

10.1182/blood.v114.22.1616.1616 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1616-1616 ◽

Cited By ~ 1

Author(s):

Eyal C. Attar ◽

Kati Maharry ◽

Krzysztof Mrózek ◽

Michael D. Radmacher ◽

Susan P. Whitman ◽

...

Keyword(s):

Overall Survival ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Advanced Pancreatic Cancer ◽

Disease Free Survival ◽

Prognostic Impact ◽

Free Survival ◽

Expression Levels ◽

The Impact ◽

Disease Free

Abstract Abstract 1616 Poster Board I-642 CD74 is a type II integral membrane protein receptor that binds its ligand MIF to induce phosphorylation of the extracellular signal-regulated kinase-1/2 (ERK-1/2) and drive cellular proliferation via nuclear factor-kappa B (NF-kB) activation. CD74 expression has been identified in human solid tumors, and its expression is associated with adverse prognosis in advanced pancreatic cancer. As CD74 is expressed and NF-kB constitutively activated in myeloblasts, we hypothesized that CD74 expression might also be associated with adverse outcome in AML. To investigate the prognostic impact of CD74 expression in the context of other predictive molecular markers in CN-AML, we assessed CD74 expression levels by Affymetrix HG-U133 Plus 2.0 microarray in 102 younger [<60 years (y)] adults with primary CN-AML, treated on the front-line CALGB 19808 trial with an induction regimen containing daunorubicin, cytarabine, etoposide and, in some cases, the inhibitor of multidrug resistance valspodar, and consolidation with autologous stem cell transplantation. Microarray data were analyzed using the Robust Multichip Average method, making use of a GeneAnnot chip definition file, which resulted in a single probe-set measurement for CD74. At diagnosis, CD74 expression, when assessed as a continuous variable, was significantly associated only with extramedullary disease involvement (P=.006) among clinical features, and with none of the molecular prognostic variables tested, including NPM1, WT1, CEBPA, FLT3 (FLT3-ITD and FLT3-TKD) mutations, MLL partial tandem duplication, or differential BAALC and ERG expression levels. Although CD74 expression levels were not associated with achievement of complete remission (CR; 83% vs 81%), higher levels of CD74 were associated with shorter disease-free survival [DFS; P=.046, hazard ratio (HR) 1.85, 95% confidence interval (CI) 1.12-3.08] and with shorter overall survival (OS; P=.02, HR 1.32, CI 1.04-1.67). In multivariable analyses, higher CD74 expression was independently associated with shorter DFS (P=.045, HR 1.98, CI 1.16-3.40), after adjusting for WT1 mutations (P<.001) and FLT3-TKD (P=.04), and shorter OS (P=.01, HR 1.58, CI 1.11-2.25) after adjusting for FLT3-TKD (P=.02), WT1 mutations (P=.007), BAALC expression levels (P=.02), white blood counts (P=.007), and extramedullary involvement (P=.04). As quartiles 2-4 had similar expression levels distinct from the lowest quartile, to display the impact of CD74 expression levels on clinical outcome only, pts were dichotomized into low (the lowest quartile) and high (the top three quartiles) CD74 expressers. The Kaplan-Meier curves for DFS and OS (Figures 1 and 2) are shown below. In conclusion, our study identifies elevated CD74 expression as associated with adverse prognosis in younger CN-AML pts. Since we previously reported that higher CD74 expression was favorably associated with achievement of CR in AML patients receiving chemotherapy plus bortezomib, an inhibitor of the proteasome and NF-kB (Attar et al., Clin Cancer Res, 2008;14:1446-54), it is possible that in future studies elevated CD74 levels can be used not only for prognostication, but also to stratify CN-AML pts to study of bortezomib-containing chemotherapy regimens. Figure 1 Disease free survival Figure 1. Disease free survival Figure 2 Overall survival Figure 2. Overall survival Disclosures No relevant conflicts of interest to declare.

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Impact of TET2 mutations On Responsiveness to Demethylating Agents in MDS.

Blood ◽

10.1182/blood.v114.22.1606.1606 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1606-1606

Author(s):

Anna M. Jankowska ◽

Mohammed Shaik ◽

Heather Cazzolli ◽

Rebecca Ganetzky ◽

Mikkael A. Sekeres ◽

...

Keyword(s):

Conflicts Of Interest ◽

Methylation Status ◽

Epigenetic Silencing ◽

Specific Gene ◽

Aberrant Methylation ◽

Global Methylation ◽

Myeloid Malignancies ◽

Demethylating Agents ◽

Potential Marker ◽

Epigenetic Instability

Abstract Abstract 1606 Poster Board I-632 Aberrant epigenetic silencing of tumor suppressor and differentiation genes constitutes an important mechanism in the pathogenesis of MDS and related myeloid malignancies. Demethylationg agents such as azacitidine and decitabine lead to degradation of DNMT1 and may reverse aberrant methylation. While the drugs demonstrate efficacy in MDS, response rates are variable. Thus, many primarily refractory patients are exposed to these therapies unnecessarily. While search for markers of responsiveness included study of methylation status of potential marker promoters or global methylation patterns, to date predictive tests have not been developed. Similarly, mechanisms of epigenetic instability responsible for wide-spread promoter methylation have not been clarified, preventing development of diagnostic markers. TET2 mutations are frequent events in a variety of MDS subtypes, particular chronic myelomonocytic leukemia (CMML) and other MDS/MPN, as well as AML derived from those conditions. It is likely that TET2 alterations are important in the pathogenesis of myeloid malignancies, but little is known regarding the function of TET2. A recent report indicated that a related family member, TET1, converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (hmC). Hydroxylation of 5mC prevents DNMT1 from homologous methylation of daughter DNA strands during cell division, thus preventing maintenance methylation (Tahiliani et al. Nature 324, 2009). Consequently, closely related TET2 may play a role in epigenetic regulation. As a consequence, TET2 mutations may lead to accumulation of aberrantly methylated CpG islands. Of utmost importance is whether TET2 mutations or resultant epigenetic silencing of specific gene or gene groups affects response to hypomethylating agents. We hypothesized that TET2 mutations play a role in epigenetic instability and may serve as markers of responsiveness/refractoriness to the therapy with demethylating agents. We have determined TET2 mutational status by sequencing all exons in 32 patients with myeloid malignancies (MDS (N=18) and MDS/MPN (N=14)) who underwent therapy with the demethylating agents azacitidine (N=27) or decitabine (N=5). For definition of response we applied International Working Group Criteria in patients who received a sufficient dose and number of cycles to allow assessment of response. Overall response rate (complete+partial responses (CR+PR) + hematologic improvement (HI)) was achieved for 9/32 patients (28%) after 35 cycles, including 4 patients who achieved CR, 2 PR, and 3 CI. In total, 12 TET2 mutations were identified in 9/32 patients (28%), of whom 5 had MDS/MPN (3 with CMML-1/2) and 4 had sAML. Unique compound heterozygosity was found in 3 patients; consequently biallelic inactivation of TET2 was found in 4 patients. For analyses, patients with partial and complete responses were compared with refractory patients and response was correlated with the presence of TET2 mutations. Among TET2-mutated patients, only 1 patient responded to therapy, whereas 8 additional patients, including 3 patients with biallelic inactivation of the TET2 gene, did not show any improvement (11%). In contrast, among patients with WT TET2 gene, responses were seen in 8/23 patients (35%; p=.19). While the cohort of treated patients was small, our preliminary results indicate that the presence of TET2 mutations may represent a negative predictor of response to demethylating agents. Disclosures No relevant conflicts of interest to declare.

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TIMP-1 and TIMP-2 Levels Are Directly Correlated with Neoangiogenesis and Are Prognostic Factors in Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.4866.4866 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4866-4866

Author(s):

Luciana Correa Oliveira de Oliveira ◽

Juliana Alves Uzuelli ◽

Ana Paula Alencar de Lima Lange ◽

Barbara Amelia Aparecida Santana-Lemos ◽

Marcia Sueli Baggio ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Overall Survival ◽

Prognostic Factors ◽

Bone Disease ◽

Cox Regression ◽

Conflicts Of Interest ◽

Newly Diagnosed ◽

Significant Difference ◽

The Impact

Abstract Abstract 4866 Background Multiple myeloma (MM) is an incurable malignant disease, characterized by increased angiogenesis in the bone marrow (BM) microenvironment and aberrant BM metabolism. Matrix metalloproteinases (MMP) are a family of zinc-dependent endopeptidases implicated in tumour progression, invasion, metastasis and angiogenesis, via proteolytic degradation of extracellular matrix. MMPs are inhibited by tissue inhibitors of metalloproteinase (TIMP). Although recent studies have implicated MMP 9 in MM bone disease, little is known about the role of the TIMPs. Objectives a) to compare levels of sRANKL, OPG, MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF, bFGF, microvessel density (MVD) between newly diagnosed MM patients and healthy controls; b) to determine the association of these molecules with disease progression, bone disease and neoangiogenesis and c) to evaluate the impact of these variables on survival. Patients and Methods As of July 2009 38 newly diagnosed and untreated multiple myeloma patients were enrolled in the study. The median age was 61years-old (range 39-91) with 24 (63%) males. Patients were diagnosed and categorized according The International Myeloma Working Group criteria and ISS, respectively. Bone involvement was graded according to standard X-ray: patients with no lesions, or with one/ two bones involved or diffuse osteoporosis were classified as low score, whereas patients with lesions in more than two bones or presence of bone fracture were classified as high score. MMP-2 and MMP-9 were determined by PAGE gelatin zymography from plasma as previously described. MMP-9, TIMP-1 and TIMP-2, OPG and sRANKL concentrations were measured by ELISA. The levels of VEGF, bFGF were obtained using cytometric bead array. Ten healthy volunteers were used as controls. Bone marrow MVD measured in hotspots was evaluated in 26 out of 38 patients at diagnosis and 15 patients with Hodgkin Lymphoma stage IA and IIA (used as controls) by staining immunohistochemically for CD34. Comparisons among groups were analyzed by ANOVA and the correlation by the Spearman's correlation coefficient. Cox regression were performed for overall survival (OS) analysis. Results Patients with MM had elevated TIMP-1, TIMP-2 and OPG values compared with controls. No significant difference was found between plasma sRANKL, pro-MMP2, pro-MMP9 and MMP-9 levels. We found that plasma TIMP-1 levels correlated positively with bFGF, VEGF, MVD, beta-2 microglobulin (B2M) and OPG (r: 0.514, p=0,001, r: 0.350, p=0,031; r: 0.610, p<0.0001; r: 0.760, p<0.0001 and r: 0.701, p<0.0001, respectively) and TIMP-2 levels with bFGF, DMV, B2M and OPG (r: 0.512, p=0.002; r: 0.595, p<0.0001; r: 0.587, p<0.0001 and r: 0.552, p<0.0001, respectively). TIMP-1 and TIMP-2 levels correlated with the ISS stage (p<0.0001, p=0.006, respectively). The only variables that correlated with clinical bone disease staging were hemoglobin, B2M and albumin levels, whereas TIMP-1, TIMP-2, bFGF, VEGF and OPG correlated with DMV. On the univariate analyses, age, gender, proMMP2, TIMP-1, TIMP-2, creatinine, B2M and MVD were significantly associated with overall survival. In Cox regression model, TIMP-1, TIMP-2 and B2M levels remained to be significantly associated with OS. In conclusion, our results suggest that TIMP-1 and TIMP-2 levels are strongly associated with neoangiogenesis and are independent prognostic factors in MM. Disclosures No relevant conflicts of interest to declare.

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Impaired Hydroxylation of 5-Methylcytosine In TET2 mutated Patients with Myeloid Malignancies

Blood ◽

10.1182/blood.v116.21.1.1 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1-1

Author(s):

Anna Jankowska ◽

Myunggon Ko ◽

Yun Huang (equal contribution) ◽

Utz J. Pape ◽

Hadrian Szpurka ◽

...

Keyword(s):

Mrna Expression ◽

Genomic Dna ◽

Conflicts Of Interest ◽

Methylation Pattern ◽

Myeloid Malignancies ◽

Cpg Sites ◽

Low Levels ◽

Methylation Patterns ◽

The Impact

Abstract Abstract 1 TET2 mutations are frequently found across broad spectrum of myeloid malignancies but how these mutations contribute to diseases is still unknown. Preliminary results from our laboratory have suggested that TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and consequently, the levels of 5-hmC may be lower in genomes of mutant bone marrow cells. To facilitate study of TET2 function we developed a blot assay to detect 5-hmC in genomic DNA with a specific antiserum to 5-hmC. In a second improved assay with increased sensitivity and precision, we treated genomic DNA with bisulfite in order to convert 5-hmC to cytosine 5-methylenesulfonate (CMS) and measured 5-hmC levels indirectly using a specific anti-CMS serum. Based on the results of this technique we demonstrate here for the first time that indeed TET2 mutations in predicted catalytic residues and other positions compromised TET2 function. We studied 102 patients with various myeloid malignancies (4/28 MDS, 14%, 26/48 MDS/MPN, 54% and 1/4 MPN, 2% and primary 2/11 AML 18% and 3/11 sAML, 27% TET2 mutants, respectively) and compared to wt cases or controls (N=17). Mutations were found throughout the entire coding region and were mostly inactivating (33/45 TET2 mutations). The levels of 5-hmC in genomic DNA from TET2 mutants were significantly decreased in comparison to wt cases and controls (p=4.5e-08 and p=1.8e-09, respectively). Particularly low levels of 5-hmC were found in patients with homozygous (UPD)/hemizygous (deletion) TET2 mutations and those with biallelic mutations. Surprisingly, 18% of all TET2 WT patients also showed low levels of genomic 5-hmC (despite normal TET2 mRNA expression), suggesting that these patients may carry not yet identified variants/lesions in TET2 or other partner proteins involved in TET2-mediated catalysis. To further investigate the impact of TET2 mutations associated with myeloid malignancies we also introduced 9 different missense mutations corresponding to those found in patients into murine Tet2 cells; severe loss of enzymatic activity was observed in 7/9 cases as measured by greatly diminished 5-hmC levels. To study the role of Tet2 in normal hematopoiesis we depleted Tet2 in C57BL/6 mice by retrovirus-mediated transduction of shRNA against Tet2. Tet2 depletion is associated with skewing of hematopoietic differentiation towards the monocyte/macrophage lineage. To further investigate the function of TET2 we transduced the myeloid THP-1 cell line with lentiviral vector containing TET2 cDNA (TET2+) or an empty vector. This manipulation allowed us to select clones showing 19-fold increase in TET2 mRNA expression without significantly alterations of proliferation kinetics. Using this model we studied the impact of TET2 overexpression on resultant methylation pattern of CpG sites. We have applied Illumina Infinium HumanMethylation27 arrays (27,5K CpG sites/14.4K genes). Overexpression of TET2 resulted in a distinct promoter methylation patterns with 169 altered CpG sites with difference of averaged β>0.5 (considered significant as compared to control). Among these differentially methylated loci, 27 promoters were significantly hypomethylated while 42 were hypermethylated as compared to control cells. Change in methylation pattern observed through overexpression of TET2 in vitro prompted us to analyze methylation patterns in patients with and without TET2 mutations or those with decreased 5-hmC levels. Using methylation arrays a total of 62 cases were analyzed. When patients were grouped based on the levels of 5hmC, an associated methylation signature can be clearly discerned with 2512 differentially methylated loci and distinct skewing towards hypomethylation (2510 sites; e.g., TMEM102, ABCC11) vs. hypermethylation (2 sites, AIM2 and SP140), consistent with the observation made in the TET2+ cells line. In sum, our results provide strong evidence for TET2 as the first mutated gene in myeloid malignancies that is involved in conversion of 5-mC to 5-hmC in DNA, indicating the novel role of TET2 in a substantial component of epigenetic deregulation in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

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PDK1 Overexpression In Acute Myeloid Leukemia; Clinical Significance and Potential as a Therapeutic Target

Blood ◽

10.1182/blood.v116.21.2162.2162 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2162-2162

Author(s):

Joanna Zabkiewicz ◽

Lorna Pearn ◽

Robert Hills ◽

Gareth Morgan ◽

Alan Burnett ◽

...

Keyword(s):

Overall Survival ◽

Clinical Significance ◽

Cell Lines ◽

Therapeutic Target ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Cyclin D3 ◽

Significant Heterogeneity ◽

Selective Treatment ◽

Cd34 Cells

Abstract Abstract 2162 PDK1 is a master kinase responsible for regulating at least six kinase groups including AKT, PKC and S6K. Many of these kinases have been shown to be constitutively active in tumour tissues including leukemia suggesting PDK1 is frequently dysregulated. Here we describe the frequency and significance of PDK1 overexpression in AML and determine the potential of PDK1 as therapeutic target. Analysis of 113 AML patients showed that overexpression of PDK1 compared with normal blast cells was frequently observed in 2 groups of patients defined by FAB M1/M0 (38% PDK1Hi) and M4/M5 (44% PDK1Hi). To establish whether overexpression was a property of the entire leukemic population (including putative leukemic stem cells) we carried out multiparameter flow cytometric intracellular staining. In 7/8 patients, we found PDK1 overexpression to be uniformly expressed in all leukemic sub-populations. Ectopic overexpression of PDK1 in normal CD34+ cells promoted their survival in unsupplemented medium (155% ±38, P<0.01, n=6). Similarly, we found that primary AML blasts overexpressing PDK1 displayed significantly higher survival in vitro when cultured in the absence of growth factors than those with normal PDK1 levels (82.4%±10.1 in PDK1Hi vs 64.4%±12.2 in PDK1Norm; P<0.0001, n=71). To determine whether PDK1Hi AML blasts were more dependent on PDK1 activity for their survival, we assessed their sensitivity to PDK1 inhibition compared with PDK1Norm blasts, using a selective inhibitor, BX-795. We found that PDK1Hi blasts showed significantly increased sensitivity to PDK1 inhibition (PDK1Hi 6.4μM +/−3.07 n=30; PDK1Norm 13.4μM +/−7.4 n=37, P=0.001) indicating that oncogene addiction is a feature of PDK1 overexpression in AML. In contrast, normal bone marrow CD34+ cells were resistant to BX-795 suggesting that PDK1 inhibition selectively targets the survival of AML blasts. Western blotting of BX-795-treated AMLs revealed dose dependent knock down of all detectable PDK1 targets substantiating the specificity of this inhibitor; furthermore BX-795 showed synergistic action when used in combination with AraC (CI= 0.7). We next investigated the clinical significance of PDK1 overexpression (using a statistical model to adjust for known prognostic factors) and discovered significant heterogeneity in overall survival between M1/M0 and M4/M5 AMLs (p=0.001 for interaction) with M1/M0 PDK1Hi patients showing significantly improved overall survival (HR 0.14 (0.003-0.61) p=0.004) whereas M4/M5 PDK1Hi patients showed poorer overall survival (although this did not reach significance: HR 2.08 (0.97-4.46) p=0.06). To examine the basis of this differential survival pattern, we surveyed the targets previously associated with PDK1 hyperactivation. We discovered that PDK1 overexpression in M4/M5 patients exclusively suppressed expression of cyclin D3 (R2 =0.675). Ectopic expression of PDK1 in AML cell lines confirmed that PDK1 suppressed D3 expression in monocytic AML cell lines whereas it induced cyclin D3 in AML lines derived from undifferentiated AML. These data indicate that suppression of cyclin D3 expression by PDK1 may reduce the rate of proliferation in M4/M5 AML, decreasing their sensitivity to standard chemotherapeutic treatments (which are most effective in targeting highly proliferative cells). Taken as a whole these data suggest that therapeutic targeting of PDK1 is an effective and selective treatment for AML, but is likely to be most beneficial for M4/M5 patients. Further, these data demonstrate that developmental context can be a significant factor when establishing the role and significance of abnormalities in AML. Disclosures: No relevant conflicts of interest to declare.

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Cytogenetic Evolution (CE) In Patients (pts) with Low and Intermediate Risk Myelodysplastic Syndromes (MDS) Is Associated with Poor Prognosis

Blood ◽

10.1182/blood.v116.21.2941.2941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2941-2941

Author(s):

Liunan Li ◽

Elias Jabbour ◽

Gautam Borthakur ◽

Stefan Faderl ◽

Tapan Kadia ◽

...

Keyword(s):

Disease Progression ◽

Cytogenetic Analysis ◽

Conflicts Of Interest ◽

Myelogenous Leukemia ◽

Intermediate Risk ◽

Hematopoietic Stem ◽

Increased Risk ◽

Significant Difference ◽

The Impact ◽

Cytogenetic Evolution

Abstract Abstract 2941 Introduction: MDS is a spectrum of abnormalities in the proliferation and differentiation of hematopoietic stem cells that result in peripheral cytopenias, bone marrow dysplasia and increased risk of transformation to acute myelogenous leukemia (AML). Cytogenetic abnormalities occur in more than 50% of patients (pts) and have an impact on survival and risk of transformation to AML. CE, or acquisition of additional clonal chromosomal abnormalities, has been reported to occur in 30 to 50% of primary MDS pts. Their impact on prognosis and transformation into AML among pts with low and intermediate risk MDS is not known. In this study, we analyzed the impact of CE on prognosis in lower risk MDS. Methods: we reviewed 722 pts clinic records of low and intermediate risk MDS pts at MD Anderson Cancer Center (MDACC) from 2000–2010 and conducted a retrospective analysis of all MDS pts with at least two consecutive cytogenetic analysis (365 patients, 50.6%) and compared the cytogenetic evolution group (CE group) with the group without cytogenetic changes (no CE group). Cytogenetic analysis was performed in the Cytogenetics Laboratory at MDACC. Results: CE was detected in 200 pts (55%). Characteristics of patients with CE are: median age 65 years (23-91), IPSS int-1 79%, diploid CG 42%, excess blasts 25%. Pts with CE were more frequently female (p=0.005), and had more frequently abnormalities of chromosome 5 and 7 (p<0.001) at baseline. There were no statistically significant difference between these two groups (p>0.05) regarding age, WBC, platelet, hgb, ANC, BM blasts percent, diagnosis (RA or RAEB), and IPSS score. There were more chr.-5/-7, insufficient metaphases, and other abnormalities, but less diploid cases in CE group compared with no CE group (p<0.001). History of malignancy (p=0.001) and prior chemotherapy exposure were also associated with CE (p=0.001), but this was not as strong for radiation exposure (p=0.066). Also, more CE patients required therapy for MDS compared to no CE patients (p=0.039). Progression free survival was significantly extended in no CE patients (p=0.02). Overall survival was a longer in no CE (34.1months), compared with CE group (26.2 months), although this was not statistically significant. Conclusion: CE is more commonly observed among pts with high-risk features, and is usually associated with disease progression and resistance. Also, prior malignancy and chemotherapy exposure were associated with CE in this study. This data indicates that genomic instability has a role in disease progression in MDS. Further analysis of CE in MDS is needed. Disclosures: No relevant conflicts of interest to declare.

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