Expression In Acute Myeloid Leukemia Research Articles

Altered miRNA and gene expression in acute myeloid leukemia with complex karyotype identify networks of prognostic relevance

Recently, the p53-miR-34a network has been identified to have an important role in tumorigenesis. As in acute myeloid leukemia with complex karyotype (CK-AML) TP53 alterations are the most common known molecular lesion, we further analyzed the p53-miR-34a axis in a large cohort of CK-AML with known TP53 status (TP53(altered), n=57; TP53(unaltered), n=31; altered indicates loss and/or mutation of TP53). Profiling microRNA (miRNA) expression delineated TP53 alteration-associated miRNA profiles, and identified miR-34a and miR-100 as the most significantly down- and upregulated miRNA, respectively. Moreover, we found a distinct miR-34a expression-linked gene expression profile enriched for genes belonging to p53-associated pathways, and implicated in cell cycle progression or apoptosis. Clinically, low miR-34a expression and TP53 alterations predicted for chemotherapy resistance and inferior outcome. Notably, in TP53(unaltered) CK-AML, high miR-34a expression predicted for inferior overall survival (OS), whereas in TP53(biallelic altered) CK-AML, high miR-34a expression pointed to better OS. Thus, detailed molecular profiling links impaired p53 to decreased miR-34a expression, but also identifies p53-independent miR-34a induction mechanisms as shown in TP53(biallelic altered) cell lines treated with 15-deoxy-Δ(12,14)-prostaglandin. An improved understanding of this mechanism might provide novel therapeutic options to restore miR-34a function and thereby induce cell cycle arrest and apoptosis in TP53(altered) CK-AML.

Heritable polymorphism predisposes to high BAALC expression in acute myeloid leukemia

Overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is implicated in myeloid leukemogenesis and associated with poor outcome in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia patients. Additionally, high BAALC expression occurs in glioblastoma, melanoma, and childhood gastrointestinal stroma tumors, suggesting an oncogenic role for BAALC. However, the mechanisms underlying the deregulated expression are unknown. We hypothesized that a common heritable genetic feature located in cis might account for overexpression of BAALC in an allele-specific manner. By sequencing the genomic region of BAALC we identified nine informative single nucleotide polymorphisms (SNPs) and tested them for a possible association with BAALC expression levels. We show that BAALC overexpression occurs in the presence of the T allele of SNP rs62527607[GT], which creates a binding site for the activating RUNX1 transcription factor in the BAALC promoter region. The mechanism is demonstrated experimentally in vitro using luciferase reporter assays and electrophoretic mobility shift assay (EMSA) analysis. The association of high BAALC expression with the T allele and its correlations with RUNX1 expresser status are shown in vivo in a test set (n = 253) and validation set (n = 105) of samples from cytogenetically normal AML patients from different populations. Thus, we identify a heritable genomic feature predisposing to overexpression of an oncogene, thereby possibly leading to enhanced AML leukemogenesis. Our findings further suggest that genomic variants might become useful tools in the practice of personalized medicine.

A critical appraisal of tools available for monitoring epigenetic changes in clinical samples from patients with myeloid malignancies

Research over the past decade has confirmed that epigenetic alterations act in concert with genetic lesions to deregulate gene expression in acute myeloid leukemia and myelodysplastic syndromes. Epigenetic alterations may serve as markers of disease, and may potentially be used for classification, prognostication and to monitor minimal residual disease. In addition, we now have the capability to pharmaceutically target epigenetic modifications, and there is an urgent need for early validation of the efficacy of the drugs. Also, an improved understanding of the functionality of epigenetic modifications may further pave the road towards individualized therapy. The recent advances in biotechnology and bioinformatics provide a plethora of novel tools for characterizing the epigenome in clinical samples, but at this point the practical, clinical utility of these methodologies needs further exploration. Here, we provide the pros and cons of the currently most feasible methods used for characterizing the methylome in clinical samples, and give a brief introduction to novel approaches to sequencing that may revolutionize our abilities to characterize the genomes and epigenomes in acute myeloid leukemia and myelodysplastic syndrome patients.

Increased CD200 expression in acute myeloid leukemia is linked with an increased frequency of FoxP3+ regulatory T cells

Increased CD200 expression in acute myeloid leukemia is linked with an increased frequency of FoxP3 + regulatory T cells

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

Stem Cell Transplant (SCT) Overcomes the Poor Prognosis Associated with CD25 Expression in Acute Myeloid Leukemia (AML): ARetrospective Single Center Analysis

Stem Cell Transplant (SCT) Overcomes the Poor Prognosis Associated with CD25 Expression in Acute Myeloid Leukemia (AML): ARetrospective Single Center Analysis

Myeloperoxidase Expression in Acute Myeloid Leukemia Helps Identifying Patients to Benefit from Transplant

PurposeDespite extensive study, the use of allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia (AML) vary considerably. The decision of which of these options to choose is complex and depends on both clinical and molecular variables as well as the availability and histocompatability of donor stem cells. So far there is no clear explanation on whether the expression of myeloperoxidase (MPO) relates to the prognosis of AML.Materials and MethodsWe retrospectively analyzed the prognostic significance of the MPO expression in the 140 patients with diagnosed AML treated at a single institution.ResultsIn our study, MPO expression was associated with disease-free survival (DFS) and transplant was beneficial to overcome a negative prognostic effect of MPO-negative at diagnosis based upon the result that the DFS in patients received transplants are not significant between the MPO-positive group and MPO-negative group although DFS in all patients was different according to MPO expression.ConclusionMPO expression at diagnosis helps to choose therapy for each AML patient and can differentiate AML patients who need transplantation.

MicroRNA Expression in Acute Myeloid Leukaemia and Their Effects on the Differentiation of Acute Myeloid Leukaemia Cells

Abstract 2430Acute myeloid leukaemia (AML) is typified by an aberrant halt in maturation of myeloid progenitor cells, leading to uncontrolled proliferation of immature blasts. In the majority of cases of AML with normal karyotype, the underlying cause of the maturation arrest remains unclear. MicroRNAs, small inhibitory RNAs, are known to be dysregulated in cancers and have been postulated to play a causative role in leukaemogenesis.We aimed to investigate the contribution of aberrant microRNA expression to the inhibition of maturation in AML cells. MicroRNA expression profiling was performed on the bone marrow of 28 AML patients with normal karyotype and 8 normal controls. Differential expression was confirmed by qRT-PCR. We found that the expression of a panel of 12 microRNAs was able to accurately separate AML-M1 and AML-M5 subtypes. The AML-M1 subtype represents a more immature cell population when compared to the monoblast morphology observed in AML-M5. Four candidate microRNAs, miR-181a, -146a, -130a and -135b were selected for further investigation based in their putative targeting of key monocytic transcription factors as determined by in silico modelling followed by luciferase assays.In vitro monocyte and macrophage differentiation of HL60 and NB4 cell lines with 1,25-dihydroxyvitamin D3 and/or phorbol-12-myristate-13-acetate treatment resulted in a significant decrease in all four candidate microRNAs (measured by qRT-PCR), supporting the hypothesis that candidate microRNA over-expression in AML-M1 may contribute to its maturation arrest. Over-expression of the candidate microRNAs by transfection with Pre-miR microRNA precursor molecules (Ambion) into the HL60 and NB4 monocyte differentiation model, resulted in the significant suppression of CD14 (Figure, *p <0.05 compared to Scrambled control) and CD15 expression, markers of monocytes and granulocytes respectively. Conversely, knockdown of miR-181a, -146a, -130a and -135b with miRCURY LNA microRNA knockdown probes (Exiqon) induced an increase in CD14 expression in HL60 cells compared to non-targeting Scrambled control.An important regulatory role of these microRNAs in myeloid/monocytic differentiation is strongly suggested by their targeted suppression of the key transcription factors KLF4, MAFB, IRF8, HOXA10, MCL1 and PU.1 which was confirmed by luciferase reporter assay. This study provides evidence that the over-expressed microRNAs discovered in our profiling work between AML-M1 and AML-M5 are biologically relevant microRNAs, which have the potential to affect the maturation potential of AML cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Lack of CD34 Expression in Acute Myeloid Leukemia (AML) Is a Favorable Prognostic Factor in Patients Treated with Stem Cell Transplant Irrespective of the Cytogenetic Risk

Abstract 1894 Background:Most of the studies of prognostic factors in acute myeloid leukemia (AML) are focused on predicting outcome after standard chemotherapy treatment. The studies that looked at the importance of these prognostic variables in predicting outcome after hematopoietic stem cell transplantation (HSCT) are very limited. Considering post-HSCT prognostic factors in stratifying AML patients may add a new level of confidence in any risk stratification of these patients. Toward this goal, we investigated the clinical relevance of CD34 expression in the primary leukemic cells on the outcome of allogeneic HSCT. Methods:Data collected from immunophenotyping of 110 patients with AML who were treated with HSCT was reviewed and the expression of CD34 on the blast population as determined by flow cytometry was correlated with clinical behavior and outcome. All the patients were treated with allogeneic HSCT. The median age of these patients was 24 (range: 14–57) and included 88 patients in the intermediate cytogenetic group and 22 in the adverse cytogenetic group. Of the 110 patients 71, (64.5%) were treated with HSCT in first remission (CR1). Results:Twenty-eight (25%) of all AML patients studied did not express CD34 on the surface of the blasts at diagnosis. Patients with CD34 negative blasts had significantly longer overall survival (OS) (P=0.003) as well as longer event free survival (EFS) (P=0.01) when all patients were considered. In the subgroup of patients who received HSCT in CR1, OS and EFS were significantly longer (P=0.017 and P=0.027, respectively) in the CD34-negative patients (N=20). Furthermore, if we consider only patients in the intermediate cytogenetic group at diagnosis, patients with CD34-negative blasts had significantly longer OS (P=0.007) and EFS (P=0.026). Even in patients with adverse cytogenetic abnormalities, OS and EFS were also significantly longer in the CD34-negative patients (P=0.01 for both). This was true when all patients were considered. The same was true when only patients transplanted in CR1 (P=0.05 for EFS in intermediate cytogenetics and P=0.05 for EFS in patients with adverse cytogenetic) were evaluated. Multivariate analysis including CD34 expression and FLT3 mutation status was carried out on a subset of patients (N=63) and showed CD34 expression was an independent prognostic factor for survival and EFS, while FLT3 mutation status became no longer a predictor. Conclusion:Our data suggests that CD34 expression on the blast cells at the time of AML diagnosis may have an adverse prognostic impact even after allogeneic HSCT. Lack of CD34 expression is a powerful independent favorable prognostic factor for AML patients if these patients are treated with HSCT after induction chemotherapy, irrespective if they were in CR1 or in CR2, and irrespective of their cytogenetic risk at diagnosis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abnormal Expression of Ndfip2 and Cbl in Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients: Role of Ubiquitin Proteasome System in Myeloid Neoplasms and Normal Hematopoiesis

Abstract 2567 Introduction:Nedd4-like E3 ligases regulate a wide variety of cellular processes, including ubiquitin-mediated trafficking, lysosomal or proteasomal degradation and nuclear translocation of proteins. Ndfip1 and Ndfip2 are endosomal PY-motif containing proteins and are activators of Nedd4 family members. Ndfip proteins are also regulators of PTEN/Akt/mTOR and MAP kinase and are induced upon T-cell activation. Cbl (or c-Cbl) is a highly conserved RING finger ubiquitin ligase (E3) and regulates signaling by multiple tyrosine kinases (TKs) through interaction with their SH2 and SH3 domains. Cbl proteins negatively regulate TK receptors, mediating the ubiquitination and degradation of activated TKs. In addition to its E3-dependent negative role, Cbl mediates positive pro-oncogenic effect when E3 activity is lost. Hematopoietic stem cells (HSC) of Cbl −/− mice exhibit augmented pool size, hyperproliferation and enhanced long-term repopulation capacity. HSC of Cbl −/− mice are also hyper responsive for thrombopoietin and display elevated levels of STAT5 phosphorylation. Aim:The aim was to determine NDFIP1, NDFIP2 and CBL expression in acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) and myelodysplastic syndrome (MDS) patients and to correlate gene expression with clinical features. Furthermore, we analyzed the expression during erythroid, granulocytic and megakaryocytic differentiation, using cell line models. Methods:NDFIP1, NDFIP2 and CBL expression was verified, by q-PCR, in bone marrow aspirates from 15 normal donors, 38 MDS (24 low-risk and 14 high-risk MDS), 28 AML and 5 APL patients, at the time of diagnosis. NB4, a promyelocytic leukemia cell line, was treated with 10−6 M of All-trans retinoic acid (ATRA) for granulocytic differentiation. KU812 was treated with 100μM Hydroxyurea (HU) and 50μM Hemin (HE) for erythroid differentiation. K562 was stimulated with 20nM of Phorbol-13 myristate-12 acetate (PMA) for megakaryocytic differentiation. Results:NDFIP2 gene expression was significantly decreased in AML (0.34 [1.7–0.03] vs. 0.59 [1.98–0.01], P=0.04) and was increased in APL, compared with normal donors (3.33 [4.73–0.32] vs. 0.59 [1.98–0.01], P=0.018) and was also different between each other (P=0.002). CBL was significantly lower in MDS, AML and APL patients, compared with control group (0.36 [3.13–0.05], 0.22 [0.68–0.002], 0.10 [0.18–0.01] vs 0.55 [1.52–0], respectively, with all P<0.01). NDFIP1 expression was not different between all subgroups (P>0.05). In AML, NDFIP1 expression was negatively correlated with peripheral lymphocyte counts (r= −0.39 and P=0.044) and blast counts (r=-0.44 and P=0.02). NDFIP2 expression in AML showed a negative correlation with peripheral lymphocyte counts (r=-0.41, P=0.03) and CBL expression was positively correlated with platelet counts in the same population (r=0.54, P=0.003). In MDS patients, NDFIP1 and NDFIP2 expression was not different from the control group. However, in high-risk MDS, NDFIP1 expression was negatively correlated with bone marrow and peripheral neutrophil counts (r= −0.63, P=0.01; r=−0.60, P=0.002, respectively). During differentiation assay for granulocytic (NB4), erythroid (KU812) and megakaryocytic (K562) lineages, there was an increased expression of NDFIP1 (eight-fold, two-fold and three-fold increase, respectively), NDFIP2 (three-fold, two-fold and four-fold increase, respectively) and CBL (ten-fold, two-fold and three-fold increase, respectively). Conclusions:Ubiquitin-proteasome system (UPS) plays an essential role in the homeostasis of cellular protein traffic and degradation, regulating cell fate, together with autophagy and apoptosis. Disruption of UPS is essential for leukemogenesis and has potential therapeutic implications. NDFIP2 and CBL are important elements of UPS. Our results show that CBL and NDFIP2 are less expressed in AML (except APL) and correlate with platelet, peripheral blasts and lymphocyte counts. The upregulation of these genes during normal hematopoiesis sheds light on their importance in the myeloid differentiation process. The higher expression of NDFIP2 in APL may be related to a better differentiation potential and autophagy activation. Moreover, the correlation of higher levels of NDFIP1 with lower neutrophil counts in high risk MDS suggests that this disease may still have functional response to T-cell activation. Disclosures:No relevant conflicts of interest to declare.

Cancer‐testis antigen expression and its epigenetic modulation in acute myeloid leukemia

Cancer-testis antigens (CTA) represent attractive targets for tumor immunotherapy. However, a broad picture of CTA expression in acute myeloid leukemia (AML) is missing. CTA expression was analyzed in normal bone marrow (BM) as well as in AML cell lines before and after treatment with demethylating agents and/or histone acetylase inhibitors. Presence of selected CTA with a strictly tumor-restricted expression was then determined in samples of patients with AML before and after demethylating therapy. Screening AML cell lines for the expression of 20 CTA, we identified six genes (MAGE-A3, PRAME, ROPN1, SCP-1, SLLP1, and SPO11) with an AML-restricted expression. Analyzing the expression of these CTA in blast-containing samples from AML patients (N = 64), we found all samples to be negative for MAGE-A3 and SPO11 while a minority of patients expressed ROPN1 (1.6%), SCP-1 (3.1%), or SLLP1 (9.4%). The only CTA expressed in substantial proportion of patients (53.1%) was PRAME. Following demethylating treatment with 5'-aza-2'-deoxycytidine, we observed an increased or de novo expression of CTA, in particular of SSX-2, in AML cell lines. In AML patients, we detected increased expression of PRAME and induction of SSX-2 after demethylating therapy with 5-azacytidine. With the exception of PRAME, CTA are mostly absent from AML blasts. However, demethylating treatment induces strong expression of CTA, particularly of SSX-2, in vitro and in vivo. Therefore, we propose that CTA-specific immunotherapy for AML should preferentially target PRAME and/or should be combined with the application of demethylating agents opening the perspective for alternative targets like CTA SSX-2.

Clinical relevance of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 expression in acute myeloid leukemia

Clinical relevance of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 expression in acute myeloid leukemia

Deregulated expression of Kruppel-like factors in acute myeloid leukemia

The known participation of Kruppel-like transcription factors (KLF) in cellular differentiation prompted us to investigate their expression in acute myeloid leukemia (AML) blast cells that are typically blocked in their differentiation. We determined the expression patterns of KLFs with a putative role in myeloid differentiation in a large cohort of primary AML patient samples, CD34+ progenitor cells and granulocytes from healthy donors. We found that KLF2, KLF3, KLF5 and KLF6 are significantly lower expressed in AML blast and CD34+ progenitor cells as compared to normal granulocytes. Moreover, we found markedly increased KLF levels in acute promyelocytic leukemia patients who received oral ATRA. Accordingly, we observed a strong induction of KLF5/6 upon ATRA-treatment in NB4 and HT93 APL but not in ATRA-resistant NB4-R cells. Lastly, knocking down KLF5 or KLF6 in NB4 cells significantly attenuated neutrophil differentiation. In conclusion, we found a significant repression of KLF transcription factors in primary AML samples as compared to mature neutrophils and further show that KLF5 and KLF6 are functionally involved in neutrophil differentiation of APL cells.

Prevalence and clinical characteristics of N-terminally truncated WT1 expression in acute myeloid leukemia

Overexpression of the Wilms’ tumor gene 1 ( WT1) was observed in most leukemia cells. In addition to four major isoforms of WT1, an N-terminally truncated isoforms (sWT1) has been identified. We separately quantified the transcript levels of sWT1 and full-length WT1 (fWT1) in 237 patients with acute myeloid leukemia (AML). sWT1 expression was observed in 45 of 237 (19.0%) AML patients, particularly in acute promyelocytic leukemia (59.3%). Although sWT1 expression was not associated with other genetic mutations and prognosis, fWT1 expression level in sWT1-expressing AML was significantly higher than that in un-expressing AML. These results suggested the possible cooperation of sWT1 and fWT1 in the pathophysiology of AML, while further analysis is required.

CIP2A is over‐expressed in acute myeloid leukaemia and associated with HL60 cells proliferation and differentiation

CIP2A is a newly identified inhibitor of PP2A. It can stabilize c-Myc and promote anchorage-independent cell growth and tumour formation. CIP2A is over-expressed in some solid tumours although its expression in acute myeloid leukaemia (AML) is still unknown. CIP2A mRNA and protein expressions were determined in bone marrow mononuclear cells of both patients with AML and healthy controls using reverse transcription polymerase chain reaction and Western blot, respectively. We used siRNA to knock-down CIP2A expression in HL60 cells and then examined its potential roles during the pathological progression of AML. CIP2A mRNA was present in 54 of 70 (77.14%) patients with newly diagnosed AML and in 11 of 14 (70.86%) patients with relapsed AML, which was significantly higher than complete remission specimens and healthy controls (P<0.001). Knock-down of CIP2A in HL60 cells slowed down cell proliferation, decreased clonogenic activity and promoted cell differentiation. These results suggest that CIP2A is over-expressed in patients with newly diagnosed/relapsed AML and the expression of CIP2A could have potential use as a clinical marker for AML relapse after treatment. The high expression of CIP2A in HL60 cells may be related to active cell proliferation and arrest of cell differentiation. This study may shed light on the molecular function of CIP2A in myeloid leukemogenesis.

Prognostic Importance of Histone MethyltransferaseMLL5Expression in Acute Myeloid Leukemia

To assess the prognostic importance of mixed lineage leukemia 5 (MLL5) expression in acute myeloid leukemia (AML). MLL5 transcript levels from 509 patients with AML who were treated in multicenter trials AML SHG 0199 and AML SHG 0295 and 48 healthy volunteers were analyzed by real-time reverse-transcription polymerase chain reaction in the context of other molecular markers (NPM1, FLT3, CEBPA, IDH1/IDH2, NRAS, KIT, MN1, BAALC, ERG, and WT1). Patients with high (n = 127) compared with low (n = 382) MLL5 expression had a higher complete response rate in multivariate analysis (odds ratio, 1.87; 95% CI, 1.08 to 3.24; P = .026). In multivariate analysis, high MLL5 expression was a favorable prognostic marker for overall survival (OS; hazard ratio [HR], 0.66; 95% CI, 0.49 to 0.89; P = .007) and relapse-free survival (RFS; HR, 0.72; 95% CI, 0.52 to 1.01; P = .057). Patient characteristics, cytogenetic aberrations, and gene mutations were similarly distributed between patients with high and low MLL5 expression except for a higher platelet count in those with high MLL5 expression. MLL5 expression independently predicted prognosis in cytogenetically normal AML patients (n = 268; OS: HR, 0.53; 95% CI, 0.33 to 086; P = .011; RFS: HR, 0.61; 95% CI, 0.38 to 0.99; P = .05) and in patients with core-binding factor leukemias (n = 81; OS: HR, 0.12; 95% CI, 0.02 to 0.91; P = .04; RFS: HR, 0.18; 95% CI, 0.04 to 0.77; P = .02). The prognostic importance of high MLL5 expression was independently validated in 167 patients treated in the AMLSG 07/04 trial (OS: HR, 0.5; 95% CI, 0.27 to 0.92; P = .023; RFS: HR, 0.49; 95% CI, 0.25 to 0.96; P = .033). High MLL5 expression levels are associated with a favorable outcome and may improve risk and treatment stratification in AML.

Prognostic Importance of Histone Methyltransferase MLL5 Expression in Acute Myeloid Leukemia

Prognostic Importance of Histone Methyltransferase MLL5 Expression in Acute Myeloid Leukemia

The Prognostic Value of CD34 Expression In Acute Myeloid Leukemia. A Mystery Solved

AbstractAbstract 2725Already for decades, controversy exists as to whether CD34 expression predicts for poor prognosis (Kanda et al, Cancer 88:2529, 2000). One of the complicating factors between studies was the use of different cut-off values, ranging from 5% to 25% CD34 (% of WBC). Such cut-offs usually result in arbitrary boundaries between positive (+) and negative (-) cells. Earlier, we have identified a particular subtype of AML with a small (≤1%) CD34+ population present, which was homogeneous in the flowcytometry properties forward- and sideward scatter and CD34 expression (Figure A) and was of non-malignant origin (van der Pol et al, Haematol. 88: 983, 2003). We considered this type of AML as truly negative for CD34+ AML cells (called: CD34- AML). With this definition, patients with >1% CD34+ cells thus have CD34+ AML. In a retrospective analysis of 98 patients with long-term follow up (median= 14.8 months; range 0.4–169), CD34- patients (n=24) had a much better prognosis compared to CD34+ AML patients (n=74): median OS not reached (>160 months) versus 13 months (p<0.005; figure B). For RFS this was >160 months versus 10 months (p=0.009). Importantly, when applying the historical cut-offs of 5%, 10%, 15% and 20%, no significant prognostic value was found, with only trends in some cases. When excluding all CD34- patients, no prognostic impact al all was left for the 4 cut-offs of 5%-20%. This proves that, when applying the cut-off strategy (5%-20%) in the whole patient group (including the CD34- patients) it is the CD34- sub-group herein that is responsible for the better prognosis of patients below these (5-20%) other cut-off levels. The difference in prognosis implies differences in therapy resistance. P-glycoprotein activity is one of the functional factors that best predict therapy resistance and prognosis in AML. Almost all CD34- patients (22/24) had P-glycoprotein activity lower than the median of normal bone marrow (nBM) CD34+ cells. Above 1% CD34 level, increasing CD34 percentages were accompanied by increasing P-glycoprotein activity (38/74 higher than nBM CD34+), as is known from literature too. The prognostic value of CD34-negativity was confirmed in a retrospective setting in a large (n= 394; 85 CD34- and 309 CD34+) group of uniformly treated patients (<60 yrs). The results confirmed the retrospective results: Figure C shows that median OS is not reached (> 41 months) for CD34- patients and 19 months for CD34+ patients (p=0.006). For RFS (n=225) median RFS was not reached but differences were significant ((p=0.003). However, in contrast to the retrospective study, now also the cut-offs of 5–20% had significant, be it low, prognostic impact both for OS and RFS (not shown). This could be explained by the impact of CD34- patients within the patient groups below the cut-offs: when applying the cut-offs of 5%-20% after exclusion of the CD34- group, no prognostic impact at all was left. Altogether these results show that finding a prognostic impact for CD34 percentage using the historical cut-offs strongly depends on the size of the patient group studied and likely also on the size of the CD34- group herein. These phenomena fully explain the discrepancies in literature up to date. NPM1 mutated leukemias are reported as CD34 negative, although without using our strict definition of CD34 negativity. With our definition, 46/68 evaluable CD34- cases were NPM1 mutated, 31/46 with single mutation (group 1) and 15/46 with an additional FLT3-ITD (group 2); 22 patients had neither NPM1 mutations nor FLT3-ITD (group 3) [only 1 patient with cytogenetic good risk aberrancy t(8;21)]. All these 3 CD34 negative subgroups showed similar good prognosis (70% survival after 30–40 months follow up). For comparison, in the CD34+ group, NPM1 single mutation (n=19/260) had good prognosis; NPM1 mutation+FLT3-ITD (n=17/260), similar to FLT3-ITD alone (n=22/260), had a very poor prognosis. In conclusion, we show that a particular group of AML patients, who completely lack malignant CD34 positive cells, have CD34 percentages ≤1 %, with a specific profile on flowcytometer, have a good prognosis compared to CD34+ patients. Cells from CD34- patients show therapy sensitive characteristics and contain both NPM1 wild type and NPM1 mutated cases. The global way in which prognostic impact of CD34 percentage has been applied for decades, thus has prevented the clear statement that CD34 has indeed strong prognostic impact in AML. [Display omitted] [Display omitted] [Display omitted] Disclosure:No relevant conflicts of interest to declare.

The expression of LMO2 protein in acute B-cell and myeloid leukemia

LIM domain only-2 (LMO2) is an important regulator of hematopoietic stem cell development. LMO2 is also expressed in blast cells of different types of acute leukemia. Here, we analyzed the LMO2 protein expression in acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia (B-ALL) and examined whether the LMO2 protein expression can predict outcomes of patients with acute leukemia. Patients with acute B-ALL (22 cases) and AML (57 cases) were examined using immunohistochemistry for LMO2 on paraffin-embedded bone marrow biopsies. We report that LMO2 protein is expressed in a significant proportion of B-ALL and AML and the staining of LMO2 protein does not predict survival in acute leukemia.

Clinical relevance of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 expression in acute myeloid leukemia

The angiogenic-related factors, angiopoietin-1 and angiopoietin-2 and their receptor Tie-2, have wide-ranging effects on tumor behavior that includes angiogenesis and inflammation. These multifaceted pathways present a potential target in developing novel inhibition strategies for cancer therapy. The present work aimed at detecting the prevalence of expression of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 in 56 Egyptian de novo acute myeloid leukemia (AML) patients by conventional RT-PCR to verify the prognostic impact of their expression on the response to induction chemotherapy. Thirty age- and sex-matched healthy volunteers were subjected to the same analysis as a control group. High expression of Ang-1 was detected in the patient group but not the control group. AML patients expressing Ang-2 either solely or in combination with high Ang-1 and/or Tie-2 showed unfavorable response to induction chemotherapy, either failed induction or death during induction. These data provide evidence that the alternation of angiopoietin balance in favor of Ang-2 may play a critical role in the pathophysiology of AML. Furthermore, positive pre-therapeutic expression of Ang-2 indicates viable unfavorable prognostic marker in AML patients and may be used as a prognostic tool in the risk-adaptive management of AML.