Cytogenetically Normal Acute Myeloid Leukemia Research Articles

Overexpression of CTNNB1: Clinical implication in Chinese de novo acute myeloid leukemia

Activation of Wnt/β-catenin signaling played a crucial role in tumorigenesis, and β-catenin (CTNNB1) overexpression has been identified in numerous solid tumors. The present study was designed to determine CTNNB1 expression and its clinical significance in Chinese de novo acute myeloid leukemia (AML) patients. Real-time quantitative PCR was carried out to detect the pattern of CTNNB1 expression in 140 AML patients and 46 controls. The level of CTNNB1 transcript in AML patients was significantly up-regulated compared with controls (P < 0.001). CTNNB1high patients showed significantly older age than CTNNB1low patients (P < 0.05). The frequency of high CTNNB1 expression was significantly observed in patients with intermediate/poor karyotypes. CTNNB1high patients had a significantly lower complete remission (CR) rate than CTNNB1low patients (P = 0.004). Among cytogenetically normal AML (CN-AML), CTNNB1high patients presented significantly shorter overall survival (OS, P = 0.004) and leukemia-free survival (LFS, P = 0.038) than CTNNB1low patients. Multivariate analysis confirmed that CTNNB1 expression was an independent prognostic factor for OS among CN-AML. Moreover, CTNNB1 expression level significantly decreased after CR stage (P = 0.032) and increased in relapsed stage (P = 0.015). Our findings suggest that CTNNB1 is overexpressed and confers a poor prognosis in AML, and could be used as a biomarker in monitoring disease recurrence.

Overexpression of indoleamine 2,3-dioxygenase correlates with regulatory T cell phenotype in acute myeloid leukemia patients with normal karyotype.

BackgroundProduction of immunosuppressive enzymes such as indoleamine 2,3-dioxygenase (IDO) is one of the strategies employed by hematologic malignancies, including acute myeloid leukemia (AML), to circumvent immune surveillance. Moreover, IDO has the ability to convert CD4+CD25− conventional T cells into regulatory T cells (Tregs). In this study, we evaluated the expression of IDO in cytogenetically normal acute myeloid leukemia (CN-AML) patients and its correlation with the Treg marker, FOXP3, as well as clinical and laboratory parameters.MethodsThirty-seven newly diagnosed CN-AML patients were enrolled in our study along with 22 healthy individuals. The expression of the IDO and FOXP3 genes was analyzed by SYBR Green real-time PCR.ResultsBoth IDO and FOXP3 were highly upregulated in CN-AML patients compared to control groups (P=0.004 and P=0.031, respectively). A positive correlation was observed between IDO and FOXP3 expression among AML patients (r=0.512, P=0.001). Expression of IDO and FOXP3 showed no significant correlation with laboratory parameters such as white blood cell and platelet counts, hemoglobin levels, bone marrow blast percentage, gender, and FLT3 mutation status (P>0.05).ConclusionHigher IDO expression in CN-AML patients may be associated with an increased Treg phenotype which may promote disease progression and lead to poor prognosis of CN-AML patients.

Prognostic utility of six mutated genes for older patients with acute myeloid leukemia.

Approximately 50% of older patients with acute myeloid leukemia (AML) do not obtain chromosomal abnormalities as an effective risk-stratification, and present cytogenetically normal AML (CN-AML). To develop a reliable prediction model for stratifying the risk of these elderly patients, we conducted a study with a discovery and validation design. As a result, we found the top 6 mutated genes in the discovery cohort of 26 case by the whole exome sequencing, and verified as recurrent mutations in the large cohort of 329 patients by Sanger sequencing. The top 6 genes were NPM1, FLT3-ITD, DNMT3A, CEBPA double allele, IDH1 and IDH2 mutations, and the frequency of each gene in the combining cohort was 36.8%, 19.8%, 20.1%, 5.8%, 14.9% and 22.5%, respectively. In addition, clinical variables such as age, white blood cell counts, genes of IDH1 and DNMT3A mutations, European LeukemiaNet genotype (NPM1 mutations and lacking FLT3-ITD or CEBPA double allele mutations) and treatment protocols were independent factors for predicting the probabilities of overall and event-free survival. The prediction nomograms based on these significant factors showed accurate discrimination. In conclusion, we developed a reliable prediction model for stratifying the risk of elderly patients with CN-AML.

Residual disease detection using targeted parallel sequencing predicts relapse in cytogenetically normal acute myeloid leukemia.

Despite achieving complete remission after intensive therapy, most patients with cytogenetically normal (CN) AML relapse due to the persistence of submicroscopic residual disease. In this pilot study, we hypothesized that detection of leukemia-specific mutations following consolidation treatment using a targeted parallel sequencing approach predicts relapse. We included 34 AML patients of whom diagnostic material and remission bone marrow slides after at least one cycle of consolidation were available. Isolated DNA was screened for mutations in 19 genes using an Ion Torrent sequencing platform. Furthermore, the variant allelic frequency of distinct mutations was validated by digital PCR and sequencing using a barcoding approach. Twenty-seven out of 34 patients could be analyzed for mutation clearance. We identified 68 somatic mutations at diagnosis (median, 3 mutations per patient; range 1-5) and 22 of these were still detected in 16 patients after consolidation therapy with a reliable sensitivity of 0.5% (median, 1 mutation; range 0-3). The most frequent noncleared mutations were found in DNMT3A. However, as persistence of these mutations has recently been shown to be without any impact on relapse risk, we performed survival and relapse risk analysis excluding DNMT3A mutations. Importantly, persistence of non-DNMT3A mutations was associated with a higher risk of AML relapse (7/8 pts versus 6/19 pts; P = .013) and with a shorter relapse-free survival (333 days vs. not reached; log-rank P = .0219). Detection of residual disease by routine targeted parallel sequencing proved feasible and effective as persistence of somatic mutations other than DNMT3A were prognostic for relapse in CN AML.

High bone marrow miR-19b level predicts poor prognosis and disease recurrence in de novo acute myeloid leukemia

Oncogenic role of miR-19 family has been identified in human cancers especially in lymphoid malignancies. However, to date, little studies investigated the role of miR-19 family in myeloid malignancies. Herein, we examined miR-19a/b expression and explored its clinical significance in de novo acute myeloid leukemia (AML). The detection of miR-19a/b expression was performed by real-time quantitative PCR in bone marrow mononuclear cells of 113 patients and 42 healthy donors. Both miR-19a/b levels were significantly increased in AML patients in contrast to controls. Patients with miR-19a/b overexpression were more frequently occurred in female, and had an older age. Moreover, cases with miR-19a overexpression had a higher frequency of U2AF1, C-KIT and CEBPA mutations, whereas miR-19b overexpressed cases harbored U2AF1 and IDH1/2 mutations. There was no significant association of miR-19a overexpression with complete remission (CR) rate and overall survival (OS) among whole-cohort AML, non-M3 AML, and cytogenetically normal AML (CN-AML). However, although miR-19b overexpression was not correlated with CR rate, patients with miR-19b overexpression presented significantly shorter OS in whole-cohort AML and a trend in non-M3 AML and CN-AML patients. Importantly, our data also showed that miR-19a/b expression level at CR phase was lower than diagnosis time, and was returned to primary level even higher when at relapse phase. Our findings revealed that miR-19a/b overexpression were frequent events in de novo AML patients. Moreover, up-regulation of miR-19b expression was associated with poor prognosis and disease recurrence in AML.

Overexpression of miR-216b: Prognostic and predictive value in acute myeloid leukemia.

Accumulating studies have shown that miR-216b acted as a tumor suppressor and was down-regulated in solid tumors. However, little studies revealed the role or clinical implication of miR-216b in blood cancers. Herein, we reported miR-216b expression and its clinical significance in patients with acute myeloid leukemia (AML). In the current study, we analyzed bone marrow (BM) miR-216b expression in 115 de novo AML patients examined by real-time quantitative PCR. Notably, BM miR-216b expression was significantly up-regulated in AML patients, and could serve as a potential biomarker distinguishing AML from controls. No significant correlations of BM miR-216 expression were found with sex, age, white blood cells, hemoglobin, platelets, BM blasts, French-American-British classifications, and karyotypes. Significantly, patients with high miR-216b expression tended to have a lower frequency of FLT3-ITD mutation and higher incidence of U2AF1 and IDH1/2 mutations. Moreover, complete remission (CR) rate and overall survival were negatively affected by BM miR-216b overexpression among cytogenetically normal AML (CN-AML). Cox regression analyses showed that high BM miR-216b expression may act as an independent risk factor in CN-AML patients. Among the follow-up patients, BM miR-216b level in CR phase was markedly lower than in diagnosis time, and was returned in relapse phase. Collectively, our findings indicated that miR-216b overexpression was a frequent event in de novo AML, and independently conferred a poor prognosis in CN-AML. Moreover, miR-216b expression was a valuable biomarker correlated with disease recurrence in AML.

KRAS overexpression independent of RAS mutations confers an adverse prognosis in cytogenetically normal acute myeloid leukemia.

The prognostic value of RAS mutations has been systematically investigated in acute myeloid leukemia (AML). However, clinical significance of RAS expressions in AML remains poorly determined. To explore the clinical significance, we analyzed KRAS and NRAS expressions in 143 de novo AML patients by real-time quantitative PCR. KRAS and NRAS expressions were significantly up-regulated in AML patients. KRAS and NRAS mutations were identified in 4% (6/143) and 8% (12/143) of these patients, respectively. However, no significant association was observed between RAS mutations and expressions. High KRAS expression was associated with older age, higher white blood cells, and a tendency of higher platelets, whereas high NRAS expression was only correlated with older age. Complete remission (CR) rate and overall survival of AML patients were adversely affected by KRAS overexpression, but not NRAS overexpression. Multivariate analysis revealed that KRAS acted as an independent prognostic predictor in cytogenetically normal AML (CN-AML). Moreover, the prognostic value of KRAS expression was validated using the published data from Gene Expression Omnibus datasets. In the follow-up patients, KRAS expression rather than NRAS expression in CR time tended to decrease compared to newly diagnosis time, and both KRAS and NRAS expressions were significantly increased when in relapse time. Our findings revealed that RAS overexpression and mutations were common events in AML with potential therapeutic target value. KRAS overexpression independent of RAS mutations conferred an adverse prognosis in CN-AML.

E-26 Transformation-specific Related Gene Expression and Outcomes in Cytogenetically Normal Acute Myeloid Leukemia: A Meta-analysis.

Background:The E-26 transformation-specific related gene (ERG) is frequently expressed in cytogenetically normal acute myeloid leukemia (CN-AML). Herein, we performed a meta-analysis to investigate the relationship between the prognostic significance of ERG expression and CN-AML.Methods:A systematic review of PubMed database and other search engines were used to identify the studies between January 2005 and November 2016. A total of 667 CN-AML patients were collected from seven published studies. Of the 667 patients underwent intensive chemotherapy, 429 had low expression of ERG and 238 had high expression of ERG. Summary odds ratio (OR) and the 95% confidence interval (CI) for the ERG expression and CN-AML were calculated using fixed- or random-effects models. Heterogeneity was assessed using Chi-squared-based Q-statistic test and I2 statistics. All statistical analyses were performed using R.3.3.1 software packages (R Foundation for Statistical Computing, Vienna, Austria) and RevMan5.3 (Cochrane Collaboration, Copenhagen, Denmark).Results:Overall, patients with high ERG expression had a worse relapse (OR = 2.5127, 95% CI: 1.5177–4.1601, P = 0.0003) and lower complete remission (OR = 0. 3495, 95% CI: 0.2418–0.5051, P < 0.0001). With regard to the known molecular markers, both internal tandem duplications of the fms-related tyrosine kinase 3 gene (OR = 3.8634, 95% CI: 1.8285–8.1626, P = 0.004) and brain and acute leukemia, cytoplasmic (OR = 3.1538, 95% CI: 2.0537–4.8432, P < 0.0001) were associated with the ERG expression. In addition, the results showed a statistical significance between French-American-British (FAB) classification subtype (minimally differentiated AML and AML without maturation, OR = 4.7902, 95% CI: 2.7772–8.2624, P < 0.0001; acute monocytic leukemia, OR = 0.2324, 95% CI: 0.0899–0.6006, P = 0.0026) and ERG expression.Conclusion:High ERG expression might be used as a strong adverse prognostic factor in CN-AML.

High CD200 expression is associated with poor prognosis in cytogenetically normal acute myeloid leukemia, even in FlT3-ITD-/NPM1+ patients

Overexpression of CD200, a trans-membrane protein belonging to the immunoglobulin superfamily, has been associated with poor prognosis in patients with acute myeloid leukemia (AML). As few data are available in the subset of cytogenetically-normal (CN) AML, we retrospectively evaluated the correlations between CD200 expression and response to therapy in a series of 139 adults with CN-AML.CD200 was expressed in 67/139 (48%) cases; 18 of them (28%) expressed CD200 at high intensity. No differences in CD200 expression rate were observed according to age, WBC count, type of leukemia, FLT3 or NMP1 mutation, and CD56 expression. A higher incidence of CD200 expression was observed in CD34+ cases (P<0.0001) and in BCL2+ patients (P=0.04).Complete remission (CR) was evaluable achieved in 98 patients (70%): 56/71 (79%) in CD200- and 47/67 (63%) in CD200+ patients (P=0.03), with a lower CR rate in patients with high CD200 intensity (9/18, 50%). CD200 expression had a negative impact on long-term outcome. CD200 expression, per se, did not impact on disease-free survival (DFS), but cases with high CD200 expression had a lower 3-year DFS compared to CD200-negative and low-expressing ones (0% vs 65% vs 68%, P=0.019). Three-year overall survival (OS) was 51% in CD200- and 27% in CD200+ patients (P=0.01), with a significant difference among cases with low or high CD200 expression (35% vs 0%, P=0.001). CD200 high expression defined a group with very poor DFS and OS also among the 37 FLT3-/NPM1+: 3-year DFS and OS were 88% and 60% in CD200-, 50% and 32% in CD200 low and 0% and 0% in CD200 high patients, respectively (P=0.01 for DFS and P=0.05 for OS).Our data suggest a negative impact of CD200 expression in CN-AML, with a further worsening in high-expressing cases, also in the subset of FLT3-/NPM1+ patients.

Gene and protein analysis reveals that p53 pathway is functionally inactivated in cytogenetically normal Acute Myeloid Leukemia and Acute Promyelocytic Leukemia

BackgroundMechanisms that inactivate the p53 pathway in Acute Myeloid Leukemia (AML), other than rare mutations, are still not well understood.MethodsWe performed a bioinformatics study of the p53 pathway function at the gene expression level on our collection of 1153 p53-pathway related genes. Publically available Affymetrix data of 607 de-novo AML patients at diagnosis were analyzed according to the patients cytogenetic, FAB and molecular mutations subtypes. We further investigated the functional status of the p53 pathway in cytogenetically normal AML (CN-AML) and Acute Promyelocytic Leukemia (APL) patients using bioinformatics, Real-Time PCR and immunohistochemistry.ResultsWe revealed significant and differential alterations of p53 pathway-related gene expression in most of the AML subtypes. We found that p53 pathway-related gene expression was not correlated with the accepted grouping of AML subtypes such as by cytogenetically-based prognosis, morphological stage or by the type of molecular mutation. Our bioinformatic analysis revealed that p53 is not functional in CN-AML and APL blasts at inducing its most important functional outcomes: cell cycle arrest, apoptosis, DNA repair and oxidative stress defense. We revealed transcriptional downregulation of important p53 acetyltransferases in both CN-AML and APL, accompanied by increased Mdmx protein expression and inadequate Chk2 protein activation.ConclusionsOur bioinformatic analysis demonstrated that p53 pathway is differentially inactivated in different AML subtypes. Focused gene and protein analysis of p53 pathway in CN-AML and APL patients imply that functional inactivation of p53 protein can be attributed to its impaired acetylation. Our analysis indicates the need in further accurate evaluation of p53 pathway functioning and regulation in distinct subtypes of AML.

Down-regulation of pseudogene Vimentin 2p is associated with poor outcome in de novo acute myeloid leukemia.

This study was intended to investigate the expression status of Vimentin 2p (VIM 2p), a pseudogene of Vimentin, and further analyze its clinical significance in AML patients. Real-time quantitative PCR (RQ-PCR) was employed to explore the expression status of VIM 2p in 128 patients with de novo AML and 36 healthy controls. The expression level of VIM 2p was significantly decreased compared with healthy controls (P< 0.001). The patients with low VIM 2p expression were identified in 93 of 128 (73%) of AML patients. No significant differences could be observed in sex, age, blood parameters, FAB/WHO subtypes, karyotype risks and ten gene mutations (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3 A, C/EBPA, N/K-RAS and U2AF1) between VIM 2p low-expressed and high-expressed patients (P> 0.05). Patients with low VIM 2p expression had significantly shorter overall survival (OS) than those with high VIM 2p expression in whole AML cases (median 7 vs. 13 months, respectively, P= 0.032), besides cytogenetically normal AML (CN-AML) and non-M3 AML cohort (P= 0.042 and 0.045, respectively). These findings indicate that VIM 2p down-regulation is a common event in AML and may be associated with poor clinical outcome.

FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with Normal Karyotypes: Mutation Status and Clinical and Laboratory Characteristics.

Objective:In this study, we evaluated the frequency of FMS-like tyrosine kinase 3 (FLT3-ITD and FLT3-TKD) and nucleophosmin (NPM1) mutations in Iranian patients with cytogenetically normal acute myeloid leukemia (CN-AML). The clinical and laboratory characteristics were compared between wild-type and mutant cases.Materials and Methods:Seventy newly diagnosed de novo AML patients were recruited at the time of diagnosis prior to chemotherapy; among them, 54 had CN-AML. For detecting mutations, the FLT3 and NPM1 genes were amplified by the polymerase chain reaction method, followed by direct sequencing.Results:Our results showed that the frequencies of FLT3-ITD, FLT3-TKD, and NPM1 mutations in CN-AML patients were 25.9%, 5.9%, and 20.8%, respectively. The most frequent NPM1 mutation type was the type A mutation. The FLT3-ITD mutation was seen more frequently in non-M3 patients compared with M3 patients. No mutation was observed in either the FLT3-TKD or the NPM1 gene in patients in the M3 French-American-British group. There was no significant association between the presence of FLT3-ITD and NPM1 mutations in CN-AML patients (p>0.05). The frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation was higher in CN-AML patients in comparison with AML patients with cytogenetic aberrations, although the differences were not statistically significant (p>0.05). There were no significant differences in mean white blood cell and platelet counts, serum hemoglobin levels, and bone marrow blast percentages between patients with wild-type and mutant FLT3-ITD and NPM1 genes (p>0.05). No difference was observed in the frequency of FLT3-ITD or NPM1 mutation regarding age or sex (p>0.05).Conclusion:Given the high stability of NPM1 during the disease course, it can be used in combination with FLT3 as well as other known genetic markers to monitor patients, especially for minimal residual disease detection.

Overexpression of the giant FAT1 cadherin gene and its prognostic significance in de novo acute leukaemia patients

FAT1 is a type 1 transmembrane protein belonging to the cadherin superfamily. Many reports showed either overexpression of FAT1 gene or its loss in solid cancers. Limited studies evaluated its importance in leukemogenesis. Real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-PCR) was used to examine the levels of FAT1 mRNA expression in 50 paediatric acute lymphoblastic leukaemia (ALL) and 50 adult cytogenetically normal acute myeloid leukaemia (CN-AML). Each group was compared with 20 age- and gender-matched healthy controls. Peripheral blood (PB) samples were collected at time of diagnosis from all patients and controls. FAT1 mRNA was detected in 66% of the paediatric ALL cases, 50% of the adult CN-AML cases, while not expressed in the PB of the control groups. T cell ALL (T-ALL) cases showed high statistical significant difference in the expression levels of FAT1 when compared to B cell precursor (BCP)-ALL (P = 0.001). Paediatric FAT1high ALL expressors showed poor response to induction therapy than FAT1low ALL (P = 0.001) as well as FAT1pos expressors in adult CN-AML when compared to FAT1neg (P = 0.01). FAT1 plays an important role in leukemogenesis; its expression levels may be used as an independent prognostic indicator in paediatric ALL and may help in distinguishing an important risk group in adult CN-AML.

Epigenetic dysregulation of NKD2 is a valuable predictor assessing treatment outcome in acute myeloid leukemia.

AIM: The present study was aimed to investigate NKD2 expression as well as promoter methylation and further analyze their clinical significance in patients with acute myeloid leukemia (AML).METHODS: Real-time quantitative PCR was carried out to detect the pattern of NKD2 expression in 113 AML patients and 24 controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were carried out to detect NKD2 promoter methylation in 101 AML patients and 24 controls with available DNA.RESULTS: The level of NKD2 transcript in AML patients was significantly down-regulated as compared with controls (P=0.039). NKD2 methylation level in AML patients was significantly higher than controls (P=0.044). Moreover, NKD2 methylation negatively correlated with NKD2 expression in AML patients (R=-0.218, P=0.029). Furthermore, demethylation of NKD2 could increase NKD2 expression in the leukemic cell line THP1 (P<0.05). NKD2 low-expressed and high-expressed patients showed no statistical significance in complete remission (CR) rate among cytogenetically normal AML (CN-AML). However, low NKD2 expression was associated with shorter overall survival (OS) time and acted as independent risk factor in CN-AML according to Kaplan-Meier (P=0.029) and Cox regression analyses (P=0.022). Furthermore, gene expression (GEP) data also confirmed the prognostic value of NKD2 expression in CN-AML patients. Moreover, NKD2 showed significantly increased level in post-CR than initial diagnosis in follow-up AML patients (P=0.024).CONCLUSION: Decreased NKD2 expression inactivated by promoter hypermethylation is a common event in AML and is associated with adverse outcome in CN-AML patients.

Two Cases of Myeloid Sarcoma Presented in Rare Locations Mimicking Lymphoma: Case Reports

Background: Myeloid sarcoma (MS) is a rare malignant neoplasm composed of myeloid blasts occurring at an extramedullary site. In nearly half of the patients, it is misdiagnosed initially, the most common being lymphoma. Here, we reported two cases of MS with monocytic differentiation, morphologically mimicking lymphoma. 1.1.Case presentation 1.1.1. Case 1: A 62-year-old female with 6 year history of chronic myelogenous leukemia (CML), presented with one month history of central nervous system (CNS)symptoms. A magnetic resonance imaging (MRI) scan showed several bilateral frontal and left parietal dural based lesions. Intraoperative consultation for the biopsy from the left frontal dural based lesion favored hematopoietic neoplasm. A limited negative lymphoma panel excluded lymphoma by flow cytometry. MS was diagnosed on the excised left frontal dural based lesion.BCR-ABL1 fusion was detected by fluorescence in situ hybridization (FISH). Intrathecal chemotherapy, however, showed no improvement. 1.1.2. Case 2:A 29-year-old female with no significant past medical history presented with two month history of fatigue, fever, night sweats, weight loss (10 lb in one month), and multiple enlargedlymph nodes.MS was diagnosed on the excised left anterior cervical lymph node that was initially thought to be a large cell lymphoma though. A concurrent bone marrow biopsy showed cytogenetically-normal AML with monocytic differentiation and mutated FLT3 tyrosine kinase domain.After systemic chemotherapy, no residual acute leukemiawas detected. In addition, the sizes of the enlarged lymph nodes were significantly decreased. 1.2.Conclusion We reported two cases of MS with monocytic differentiation, morphologically mimicking lymphoma. One case was a blastic transformation of CML presented in CNS. The other case was MS with concurrent AML presented in lymph nodes. Systemic chemotherapyis the recommended treatment for MS in addition to surgical resection, if surgical resection is necessary.

High Expression of ZBTB7A at Diagnosis Associated with Inferior Outcome in Acute Myeloid Leukemia Patients Receiving Hematopoietic Stem Cell Transplantation

Introduction: The transcription factor ZBTB7A regulates early differentiation of hematopoietic progenitors & has been associated with oncogenic as well as oncosuppressive functions. While it was shown that ectopic overexpression of Zbtb7a in immature lymphocytes leads to the development of an aggressive T-cell lymphoblastic leukemia, high ZBTB7A expression in cytogenetically normal acute myeloid leukemia (CN-AML) is associated with improved outcomes. Furthermore, recently a leukemogenic cooperation between RUNX1/RUNX1T1 &ZBTB7A mutations in t(8;21)-associated AML was suggested.Here, we further evaluated the complex role of ZBTB7A expression in hematopoietic malignancies by assessing its potential prognostic impact in AML pts undergoing hematopoietic stem cell transplantation (HSCT) after non-myeloablative conditioning (NMA).Methods: We analyzed bone marrow (BM) at diagnosis of 140 pts (median age 63 years [y], range 37-75y) treated at our institution between 2000 & 2015. All pts received NMA conditioning (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) followed by HSCT in complete remission with (CR; n=111; 79.3%) or without peripheral hematological recovery (CRi; n=29; 20.7%). Median follow-up for pts alive was 3.5y.Our cohort included pts with CN-AML (n=62, 44.3%), complex karyotype (n=17; P=12.1%) & other cytogenetic abnormalities (n=61; 43.6%). At diagnosis mutations in the genes CEBPA, DNMT3A, IDH1, IDH2, NPM1 & the presence of FLT3-ITD were determined. In diagnostic BM cytogenetics were analyzed using standard techniques for banding & fluorescence in-situ hybridization & the expression of common surface markers was analyzed using flow cytometry. The expression of ZBTB7A was assessed using quantitative RT-PCR & normalized to ABL1 as internal control. As a cut-off the third quartile of normalized gene expression was identified to group high & low ZBTB7A expressers.Results: At diagnosis pts with a high ZBTB7A expression more often had a complex karyotype (P=.02) & were less likely to have core-binding factor AML by trend (P=.18). Additionally, high ZBTB7A levels associated with significantly fewer blasts in peripheral blood (P=.008) & BM (P=.02). The BM mononuclear cells in high ZBTB7A expressers were to a smaller extent positive for myeloid markers (CD38 P=.03; CD33 P=.11; CD13 P=.13) & exhibited a higher percentage of erythroid (Glycophorin A P=.03) as well as monocytic (CD11b P=.04; CD14 P=.01) surface markers. We did not find any statistical associations between ZBTB7A levels & the mutation status of NPM1, CEBPA, IDH1, DNMT3A or the presence of FLT3-ITD. Yet, there was a trend for more IDH2 mutations in the group of high ZBTB7A expressers (P=.18).At diagnosis a high expression of ZBTB7A associated with a significantly higher cumulative incidence of relapse (CIR; P=.002, Figure 1A). This finding also translated into a significantly shorter overall survival (OS; P=.01; Figure 1B) for AML pts with high ZBTB7A levels at diagnosis. When we restricted our analyses to CN-AML, high ZBTB7A expression remained a negative prognostic factor by trend (CIR P=.16; OS P=.11).Conclusion: Expression of ZBTB7A associated with distinct biological features & surface marker pattern in AML. This underlines the results of recent studies which identified ZBTB7A as a novel player in leukemogenesis. However, our findings are in contrast with the previously shown favorable prognostic impact of high ZBTB7A levels in a CN-AML cohort mainly treated with chemotherapy. In contract all pts included in our studies were consolidated with NMA-HSCT. Since this treatment regimen is mainly based on the graft versus leukemia effect a high ZBTB7A expression could potentially interfere with the immunological recognition of the AML blasts resulting in a reduced response to NMA-HSCT.Consequently, future functional studies & clinical trials should aim at further characterize the complex role of ZBTB7A in AML. [Display omitted] DisclosuresPoenisch:Mundipharma: Research Funding.

HOXB-AS3 Regulates Cell Cycle Progression and Interacts with the Drosophila Splicing Human Behavior (DSHB) Complex in NPM1-Mutated Acute Myeloid Leukemia

Background:The expression and prognostic significance of long non-coding RNAs (lncRNAs) in older (≥60 years) patients with cytogenetically normal acute myeloid leukemia (CN-AML) has recently been reported (Garzon et al, 2014). Among the top up-regulated lncRNAs in NPM1-mutated (NPM1mut) CN-AML, the lncRNA HOXB-AS3 wasidentified. As aberrant expression of HOX genes is associated with NPM1mut AML and HOX-related lncRNAs have been reported to regulate HOX genes (e.g. HOTAIR), we hypothesized that HOXB-AS3 could have a functional role in this disease setting.Methods:HOXB-AS3 expression profiling was performed by qRT-PCR in AML cell lines (Kasumi-1, KG1a, MOLM-13, MV-4-11, OCI-AML3 and THP-1), 12 primary CN-AML patient samples (NPM1mut=6, NPM1-wild-type (WT)=6) and mononuclear cells from healthy donors (n=6). Correlation analysis of mRNA/lncRNA expression was performed using Spearman analysis in 71 older CN-AML patients, profiled by RNA-sequencing. Knock-down (KD) of HOXB-AS3 was performed in vitro (Amaxa-nucleoporation) and in vivo (in a patient-derived xenograft (PDX) model) using LNA-modified gapmers. Comparative proteomic analysis was conducted by applying a modified version of the RNA antisense purification (RAP) protocol (McHugh et al, 2015).Results:Of the cell lines tested, only OCI-AML3, which harborsmutated NPM1, showed high levels of HOXB-AS3 expression. Five- and 3-prime RACE assays identified 3 previously annotated (NR_033201/NR_033203/ENST000491264) and 1 novel variant of HOXB-AS3 in OCI-AML3 cells. NPM1 mutated patient samples exhibited higher expression of HOXB-AS3 in comparison to those with WT NPM1 (P<.01) and healthy donors (P<.01). To gain insights into the function of HOXB-AS3, we derived a HOXB-AS3-related mRNA expression signature; 154 mRNA transcripts correlated positively and 122 correlated negatively with high HOXB-AS3 expression. Gene ontology analysis revealed enrichment of genes involved in the pathway of DNA repair (P<.01) in patients with high HOXB-AS3 expression.To assess the functional impact of HOXB-AS3, HOXB-AS3 KD studies using LNA gapmers were performed. In vitro KD of HOXB-AS3 led to decreased proliferation of OCI-AML3 cells, as measured by BrdU-based cell cycle analysis (S-phase average % in control v KD: 24% v 16%, P=.02). HOXB-AS3 KD also led to a reduction in the number of formed colonies by OCI-AML3 cells (P<.01), whereas it had no significant effect on apoptosis. HOXB-AS3 KD in primary NPM1mut AML samples (n=3) led to a decrease in colony formation (P<.01). Gene set enrichment analysisperformed in HOXB-AS3 silenced OCI-AML3 cells (profiled by RNAseq) revealed activation of the DNA damage response pathway in the HOXB-AS3 expressing cells.To assess the in vivo effects of HOXB-AS3 KD,we treated 10 NSG mice transplanted with the blasts of a NPM1mut AML patient with nanoparticle-formulated anti-HOXB-AS3 gapmers or control gapmers. We obtained RNA after 7 days of treatment and confirmed HOXB-AS3 KD in human cells (P<.01). Survival analysis is currently ongoing but when we compared the leukemia burden at 3- and 6-weeks post-transplant in mice with adequate leukemia engraftment (>30% human CD45+ cells) the percentage of circulating blasts decreased in HOXB-AS3-KD (from 43% to 30%), while it increased in controls (from 36.5% to 62.8%). Last, to identify potential HOXB-AS3-binding proteins we performed RAP experiments in OCI-AML3 cells. HOXB-AS3- and U1-specificRNA-protein complexes were isolated by hybridization with biotinylated probes. Mass spectrometry and comparative analysis of theeluates yielded 22 candidate HOXB-AS3-interacting RNA-binding proteins. These included the core components of the DSHB complex (SFPQ and NONO). RNA-immunoprecipitation experiments validated the interaction of HOXB-AS3 with NONO. The DSHB complex is a major mediator of the DNA damage response and experiments that will elucidate the importance of HOXB-AS3 interaction with NONO in this context are underway.Conclusions: HOXB-AS3 is highly expressed in NPM1mut CN-AML and regulates cell cycle progression of AML blasts. HOXB-AS3 KD decreases cell proliferation and colony formation capacity of leukemia cells in vitro. HOXB-AS3 KD also decreases leukemia burden in an in vivo PDX model. Comparative proteomic analyses identified several binding partners of HOXB-AS3, such as the DSHB complex, which is a key mediator of the DNA repair process. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Significance of IDH1 and IDH2 mutations in Older Patients with Primary Cytogenetically Normal Acute Myeloid Leukemia

IDH1 and IDH2 mutations are frequently identified in older patients with AML.With the development and application of IDH inhibitor, the prognostic significance of these mutations in old patients is of primary importance. To assess the impact on clinic outcome, we analyzed 224 older (> 60 years) patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) similarly treated with intensive induction chemotherapy. Our analysis identified IDH1 and/or IDH2 (IDH1/2), IDH1 or IDH2 mutations in 63(28%), 29(12.9%) and 35(15.6%) patients, respectively. In general, IDH1 and IDH2 mutations were mutually exclusive, with only one patient having both IDH1 R132C and IDH2 R140Q mutations. IDH2 R172 mutations were exclusive with all other mutations analyzed (NPM1, CEBPA, DNMT3A, IDH1 and FLT3-ITD). There was a strong interaction between IDH2 mutation and NPM1 mutational status. Concurrent presence of IDH2 R140 and NPM1 mutations was independently associated with longer overall survival (OS; P=0.038, HR=0.42) and event free survival (EFS; P=0.034, HR=0.46) compared to the wild type counterpart in multivariable analyses. In contrast, IDH1 mutations were independently associated with shorter OS (P=0.004, HR=1.98) and EFS (P=0.014, HR=1.72) in multivariable analyses. Notably, IDH1 R132 mutations harbored higher levels of total 2-hydroxyglutarate compared to IDH2 R140Q mutations. Patients with IDH1 mutations or IDH2 R140 and NPM1 mutations harbored also distinct miRNA-expression signatures. Future studies will establish whether these difference between IDH1 and IDH2 mutations will be relevant with respect to response of the respective subsets of patients to the emerging therapies with IDH1 and IDH2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

EGFL7 Antagonizes NOTCH Signaling, Stimulates Blast Proliferation and Confers Poor Prognosis in Cytogenetically-Normal Acute Myeloid Leukemia (CN-AML)

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein and plays an important role in angiogenesis by regulating the growth, proliferation and migration of endothelial cells. Recent studies in solid tumors have shown that EGFL7 is overexpressed and is associated with a more aggressive disease phenotype. Whether EGFL7 plays a similar role in blood cancers such as acute myeloid leukemia (AML) however, has not been previously reported. To investigate the association of EGFL7 expression with outcome in AML, we measured EGFL7 mRNA expression in newly diagnosed older (≥60 years, n=126) CN-AML patients. In these patients, those with high EGFL7 expression were less likely to achieve CR (52% v 76%, P=.009). Patients with high EGFL7 expression status had shorter event-free survival (5-year rates: 6% v 13%, P=.03) and overall survival (5-year rates: 10% v 16%, P=.009) than patients with low EGFL7 expression status.To validate our clinical data we measured EGFL7 mRNA in primary AML blasts (n=11) compared to normal bone marrow (NBM) (n=5) using RT-PCR, and found a ~2.4 fold-increase in the AML samples, P<0.05. These results were consistent with the protein levels, measured using western blots. Using both ELISA and western blotting, we observed significant increased levels of EGFL7 protein in the medium of cultured primary AML cells (n=3, 31-443 pg/mL) compared to media alone. Importantly, detection of serum EGFL7 levels by ELISA revealed variable but increased levels of EGFL7 in the sera from 3 of 5 (P<0.05) AML patients tested compared with sera from normal healthy controls (n=7). To investigate the functional consequences of EGFL7 stimulation on blast growth we treated primary AML blast cells from three patients with recombinant EGFL7 (rEGFL7) protein and performed colony forming unit (CFU) assays. Treatment with rEGFL7 led to an increase in the number of CFUs (P<0.05) and was associated with increased phosphorylation of AKT. In addition, under starvation conditions, recombinant EGFL7 protein alone promoted the survival and proliferation of MLL-PTD AML cells (n=3) but not normal BM cells (n=3) (P<0.05). To explore the underlying molecular mechanism(s) by which EGFL7 maintains the survival and proliferation of AML cells, we screened for proteins that interact with EGFL7 by using a protein antibody array on human primary AML blasts. This screen revealed that EGFL7 binds to multiple components of key signal transduction pathways important for leukemia, including NOTCH. Previous studies by Lobry C et al., (JExpMed 2013) demonstrated that although NOTCH2 and to a lesser extent, NOTCH1, mRNA was relatively abundant, NOTCH signaling was inhibited in AML blasts. Re-activation of the canonical NOTCH pathway, released a block in differentiation resulting in eradication of the leukemic blasts. Schmidt MH et al., (Nat Cell Biol. 2009) demonstrated that EGFL7 was capable of binding NOTCH receptors and blocking their subsequent activation. Therefore, we hypothesized that one mechanism by which the NOTCH pathway is kept inactive in AML is through autocrine binding of EGFL7 protein to NOTCH receptors. Co-immunoprecipitation assays confirmed binding of EGFL7 to NOTCH2 in both human primary AML cells (n=3, P<0.05) and mouse MLL-PTD AML cells (n=3, P<0.05). Moreover, stimulation of AML blasts with rEGFL7 protein suppressed the cleavage and subsequent activation of NOTCH2, resulting in decreased transcription of NOTCH downstream target genes, HES1 and NRARP (n=3, P<0.05). In summary, we show here that high EGFL7 is associated with worse prognosis in patients with CN-AML, and that EGFL7 is expressed and secreted by the AML cells in an autocrine fashion, promoting their growth in part through antagonizing NOTCH2 activation. Targeting EGFL7 with antibodies to reduce NOTCH binding might represent a novel therapeutic approach to reactivate NOTCH signaling, allowing for blast differentiation and elimination of AML. DisclosuresNo relevant conflicts of interest to declare.

The Cell of Origin in NPM1 Positive Acute Myeloid Leukemia (AML) Is Prognostic

Abstract Mutations in the nucleophosmingene (NPM1) are common in patients with cytogenetically-normal acute myeloid leukemia (AML) and have been associated with positive outcomes. Although most leukemic stem cells (LSCs) arise from CD34+ primitive progenitors, our recent data suggest that a fraction arise from less primitive CD34- progenitors and seem to have a better prognosis (Gerber et al. Haematologica 2016). Conversely, poor-risk AMLs such as those harboring internal tandem duplications of the FMS-like tyrosine kinase 3 (FLT3-ITD) or with poor-risk cytogenetics appear to arise from LSCs with a primitive hematopoietic stem cell (HSC) phenotype. NPM1-mutated AMLs can be either CD34- or CD34+, and have been associated with variable prognoses. We hypothesize that the cell of origin, CD34- or CD34+, of NPM1-mutated AML has prognostic implications. A retrospective cohort analysis was conducted using electronic medical record data from patients aged 18 or older with newly-diagnosed NPM1-mutated AML treated with intensive induction chemotherapy at Johns Hopkins Hospital from 2008-2015. NPM1 and FLT3-ITD mutations were determined from PCR assays on diagnostic samples. We (Gerber et al. Haematologica2016) and others have shown that CD34+ cells represent normal, non-leukemia progenitors when they comprise &lt;1% of mononuclear cells (MNCs); thus, &gt;/= 1% MNCs was the cut-off used to distinguish CD34+ from CD34- AMLs. Clinical outcomes including complete remission (CR) rate were compared using Chi-squared tests and event-free survival (EFS) and overall survival (OS) were estimated per the Kaplan-Meier method, using Wilcoxon tests to determine the impact of CD34 status. Of 46 consecutive, newly diagnosed NPM1-positive patients treated between 2008-15, 36 (78%) had normal cytogenetics and 37 (80%) had de novo AML. 26/46 (57%) had CD34+ disease; of these, 18/26 (69%) had FLT3-ITD mutations, compared to 9/20 (45%) with CD34- AML (p=0.10). In the CD34- patients with FLT3-ITD, the mean allelic ratio (mutant to wild type allele) ±SEM was 0.26±.08 compared to 2.3±1.0 in the CD34+ patients (p=0.07). Comparing CD34+ and CD34- patients, there were no significant differences in mean age at diagnosis (52 vs 59), sex, initial hemoglobin, platelet count, or lactate dehydrogenase, but CD34+ patients had significantly higher baseline white blood cell count (mean 78 vs 38 x 103 cells/mm3; p=0.04). There was a trend toward lower CR rates following induction for CD34+ patients (69 vs 89%; p=0.11), and similar proportions of patients underwent bone marrow transplantation (BMT) (50% vs 37%, p=0.38). However, CD34+ patients had significantly shorter EFS (figure 1; median 8.0 vs 34.3 months; p=0.04), and a trend toward inferior OS (15.4 vs 35.4 months; p=0.36). The lack of statistical significance in OS may reflect effective salvage by allogeneic transplantation. In conclusion, in this small retrospective cohort analysis of NPM1-mutated AML patients, CD34 expression was associated with significantly reduced EFS and, among FLT3-ITD mutated patients, a trend toward higher allelic ratios. Hence, CD34 positivity may be an early indicator of poor prognosis in NPM1+ AML and requires further characterization to determine its full prognostic and predictive value in guiding AML therapy. These data support evidence that the biology of AML is not only a function of genetic mutations, but also in part a functionof the stage of hematopoietic differentiation at which the leukemogenic mutations develop. Figure 1 EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Figure 1. EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Disclosures No relevant conflicts of interest to declare.