FAB M2 Subtype Research Articles

ARNT/HIF-1β: An AML Biomarker?

AbstractAbstract 2903 Background:Despite improvements in chemotherapy protocols and bone marrow transplantation techniques, AML remains a lethal disease. Although patients with AML typically respond well to initial therapy, the majority relapse and eventually succumb to the disease. This relapse is thought to be the result of the resistance of leukaemic stem cells to the effects of chemotherapy. Some mechanisms have been discovered which contribute to resistance, but this phenomenon remains incompletely understood and is a significant barrier to improving patient outcomes. TEL is a member of the ETS transcription factor gene family and is essential for developmental processes such as haematopoiesis. TEL is frequently targeted by chromosomal translocations in human malignancies, resulting in the expression of oncogenic TEL gene fusions. A TEL-ARNT gene fusion was reported in a single case of AML, FAB subtype M2, in a patient whose leukaemic cells contained the balanced translocation t(1;12)(q21;p13). ARNT (also known as HIF-1β) is a nuclear protein and functions as part of a heterodimer, whose partners include HIF-1α and the aryl hydrocarbon receptor (AhR). HIF-1 expression is commonly deregulated in cancer and AhR signalling has been linked to leukaemogenesis. Materials and Results:Immunoblot analysis of patient bone marrow mononuclear cell lysates revealed that 5 of 29 AML specimens (∼17%) had elevated levels of ARNT protein, whereas 0 of 19 MDS specimens showed ARNT expression. In addition, AML specimens that had elevated ARNT protein levels also had elevated levels of phosphorylated Akt, while AML specimens that did not have elevated ARNT protein expression did not have activation of Akt signaling. Intriguingly, ARNT expression was noted in one patient who had progressed from MDS to AML. We then studied the expression of ARNT in HL60 cells, which are highly sensitive to induction of apoptosis by troglitazone (TG), and in U937 cells, which are TG resistant. In HL60 cells, ARNT mRNA levels remained constant following TG treatment and ARNT protein levels markedly decreased, while in U937 cells, ARNT mRNA levels increased and ARNT protein levels remained constant. We then tested the effect of exogenous expression of ARNT on the sensitivity of HL-60 cells to apoptosis induced by TG, daunorubin and hydrogen peroxide. HL-60 cells transduced with a retrovirus expressing ARNT became resistant to the induction of apoptosis by all these agents. These cells also had constitutive activation of Akt signaling, and treatment of these cells with a specific inhibitor of Akt signaling reversed their resistance to TG-induced apoptosis. Conclusions:The expression of ARNT confers resistance to apoptosis in AML cell lines and activates Akt signalling. In addition, ARNT is overexpressed in a significant subset of AML patients, in whom it activated Akt signaling, and ARNT expression correlates with progression of MDS to AML. ARNT may play an important role in chemoresistance and may be useful as a predictive or prognostic biomarker. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-116: GSK2334470: A potent and highly selective inhibitor of PDK1

Abstract Phosphoinositide-dependent protein kinase-1 (PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway which is the most commonly deregulated signaling pathway across all cancers. In vivo studies have shown that AKT, p70S6K, RSK and protein kinase C are key mediators of PDK1 function, regulating diverse cellular processes. Activation of these substrates by PDK1 leads to an increase in glucose uptake, protein synthesis, and inhibition of pro-apoptotic proteins. Conversely, knockdown of PDK1 protein levels in tumor cells leads to decreased tumor cell proliferation and increased apoptosis. Furthermore, a hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN+/- mice. Taken together, these observations suggest that an inhibitor of PDK1 could be beneficial in treating cancer. Therefore, we initiated a medicinal chemistry program to discover PDK1 inhibitors as potential anticancer agents. We have identified an aminoindazole PDK1 inhibitor, GSK2334470, derived from a fragment screening hit which we optimized using structure-based design and PDK1 protein crystallography. Potency and kinase selectivity were attributed to key interactions within the ATP binding site between the inhibitor and the back pocket and glycine-rich loop of PDK1. GSK2334470 is a potent inhibitor of PDK1 kinase (IC50 = 0.5 nM) with a high level of specificity for PDK1 based on test results from in an in vitro kinase selectivity panel of more than 280 kinases. In PC-3 cells, GSK2334470 effectively inhibited phosphorylation of AKTT308 (IC50 = 113 nM) and RSKS221 (IC50 = 293 nM) but not AKTS473 (IC50 > 30,000 nM). These results are consistent with the high specificity of GSK2334470 for PDK1 inhibition. In a panel of approximately 300 cell lines, GSK2334470 demonstrated modest antiproliferative activity overall, with low micromolar activity seen in several breast cancer cell lines and in a variety of hematological cancer cells. Interestingly, GSK2334470 displayed sub-micromolar antiproliferative activity against AML cell lines that are clinically classified as M4 and M5 FAB-subtypes. Therefore, we evaluated the pharmacodynamics of GSK2334470 in mice implanted with OCI-AML2 xenografts. GSK2334470 dosed i.p. at 100 mg/kg resulted in 58 and 29% inhibition of AKTT308 phosphorylation at 3 and 6 h, respectively and 57 and 71% inhibition of RSKS221 phosphorylation at 3 and 6 h, respectively. There was no observed change on AKTS473 phosphorylation. Our results demonstrate that GSK23334470 is a potent, highly selective PDK1 inhibitor which can decrease PDK1 signaling in a tumor xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-116.

Thrombopoietin/Bcl-Xl Signaling Is Essential for the Self-Renewal Phenotypes of AML1-ETO Expressing Human Hematopoietic Cells.

Abstract 1424Poster Board I-447AML1-ETO (AE) fusion protein is generated as a result of translocation (8;21), accounting for 40% of the FAB M2 subtype of acute myeloid leukemia (AML). This group of patients has a 5-year survival rate of approximately 50% due to disease relapse. Retroviral gene transfer of AE in CD34+ human umbilical cord blood (hUBC) cells enhances self-renewal phenotypes, making this model a powerful tool to study cellular pathophysiology in response to fusion protein expression during the early stage of disease formation. Based on the current understanding of t(8;21) AML as a stem cell disease, these AE-expressing hUBC cells may also represent the chemotherapy-non-targetable leukemic stem cells that are responsible for the relapsing disease. In the current study, we identified thrombopoietin (TPO) as an essential cytokine for the growth of AE cells. Whereas normal CD34+ hUBC cells cultured in the absence or presence of TPO showed similar growth rates, withdrawing TPO in AE cell cultures resulted in a gradual proliferative defect that was associated with loss of colony forming ability and long-term culture-initiating cells (LTC-IC) frequency. Adding TPO to methylcellulose media leads to an increase in colony numbers and an enhanced replating ability, further confirming that TPO is particularly critical for the growth of the clonogenic AE progenitor cells. Moreover, TPO withdrawal caused a decrease in the percentage of CD34+ cells, and an increase in the percentages of both CD11b+ and CD14+ cells. AE cells incubated in media without TPO adapted a more mature morphology, including nuclear segmentation and cytoplasmic granule formation. At the molecular level, various genes involved in hematopoietic cell self-renewal are downregulated in AE cells cultured without TPO. Taken together, these results indicate that TPO signaling is a critical mediator of the self-renewal phenotypes of the AE cells. Interestingly, AE cells displayed addiction to TPO-mediated signaling as early as one week after AE expression. Therefore, we further hypothesized that AE is directly involved in this process. Indeed, we found that the c-Mpl gene, which encodes the Janus Kinase 2 (JAK2)-activating TPO receptor, is upregulated in AE cells. Additionally, a chromatin immunoprecipitation assay provided evidence of direct AE binding to the promoter region of c-Mpl. These data indicate that AE directly upregulates c-Mpl gene expression in hematopoietic stem and progenitor cells. In the study of the downstream effectors of the TPO signaling, we found that Bcl-xL, an essential anti-apoptotic protein in AE cells, is maintained at high levels by TPO, mainly in a post-translational manner, suggesting that an important component of TPO signaling is to promote cell survival. In fact, recent evidence on the opposing effects of DNA damage and JAK2 activation on Bcl-xL post-translational modifications (deamidation) led us to speculate that the negative effects from increased DNA damage associated with AE expression may be counterbalanced by TPO signaling. In conclusion, our data provide a novel therapeutic approach for t(8;21) AML treatment by targeting the TPO signaling pathway, thereby augmenting conventional chemotherapy in the prevention of disease relapse. Disclosures:No relevant conflicts of interest to declare.

Somatic CNVs and LOH in Acute Myelomonocitic Leukemia

In acute myeloid leukemias (AML) chromosomal aberrations, detectable by conventional cytogenetics or targeted molecular techniques, provide the basis for a classification with prognostic relevance. However, cases with normal cytogenetics and undefined prognosis still constitute the single largest group. Recent advances in genome-wide analysis of submicroscopic DNA segment copy number variations (CNVs) may allow the identification of novel molecular tumor-associated abnormalities in the normal cytogenetics group (somatic CNVs). However, CNVs are also present physiologically in the normal population (germline CNVs) (Redon et al., 2006) and can represent potential predisposition factors in disease. Indeed, CNVs can have dramatic phenotypic consequences as a result of altering gene dosage, disrupting coding sequences, or perturbing long-range gene regulation. We used the last generation of Affymetrix single nucleotide polymorphism (SNP)/CNV microarrays (SNP Array 6.0) containing probes for the detection of CNVs and SNPs, with an inter-marker distance of 680 bases and a resolution power of 100 kb. SNP Array 6.0 Assay kit (Affymetrix, Santa Clara, CA) is able to assess copy number changes (CNVs) at a resolution comparable with data obtained using oligonucleotide-array-comparative genomic hybridization (aCGH) and provides also information on loss of heterozygosity (LOH) of the allelic imbalance and copy number neutral type. In the present communication we report preliminary results of a study aimed to test the ability of such arrays to distinguish tumor-associated somatic CNVs and LOHs from germ-line ones by comparing bone marrow samples from AML patients at diagnosis (>90% blasts) and at the remission phase. So far, 8 M4–M5 FAB subtype AML patients have been studied, 6 females, 2 males (median age 38 years, range 25–51). At diagnosis 4 cases with normal karyotype, 2 cases with trisomies (respectively trisomy 13 in 25% and trisomy 22 in 80% of 20 metaphases), 1 inversion (inv (16) (p13q22)) and 1 balanced translocation (t (6;14) (q27;q23)) were detected by conventional cytogenetic analysis. We obtained arrays with quality control (QC) call rates in excess of 90% in all cases (>95% in 8/13 cases) and MAPD <0.4 (<0.35 in 7/13 arrays), using Genotyping Console Version 2.1 for signal intensity analysis, as recommended by Affymetrix. To obtain copy number and LOH calls we used a predefined reference model file, obtained from 270 healthy individuals (HapMap collection). All samples that were regarded as normal karyotype by chromosomal banding had detectable submicroscopic abnormalities by the SNP/CNV array assay. Results obtained are reported in table 1. We found 13 somatic gains not in overlap with known CNVs deposited in the Toronto Database of Genomic Variants. The only recurrent somatic CNV (2/8 patients) was a gain of 109kb in 7q22.1, where genes MGC57359 and GATS map. Five recurrent germline CNVs have been detected, both at diagnosis and remission samples, which could represent regions determining susceptibility to AML. The trisomy 13 case showed a whole chromosome somatic LOH at chromosome 21. 3/8 patients had an interstitial somatic LOH in 19q13.12 in correspondence with adhesion molecules genes (CEACAM1, MEGF8, PSG 1-6-7, ZNF 526). Finally, we detected an interstitial germline LOH, common to all samples, in 16q22.1, where CBFB (core binding factor beta) gene maps, involved in FAB subtypes evaluated in this study. Although this is an ongoing study, with preliminary results, we think that such genome-wide characterization of sub-microscopic DNA alterations might contribute to the discovery of new markers and target genes, with diagnostic, prognostic or therapeutic relevance.CNV or LOH per sampleNormal karyotypeAbnormal karyotypemedianrangemedianrangeCNV (diagnosis)3816–527918–163ratio gain/loss at diagnosis76–7154–22CNV (remission)2416–324920–167ratio gain/loss at remission11–291–20germline CNV1814–21337–77somatic CNV162–30513–86LOH (diagnosis)299285–314316307–317LOH (remission)291285–297295282–318germline LOH283278–289287287–308somatic LOH1612–19208–30Table 1

Prognostic value of dysmyelopoietic features in de novo acute myeloid leukaemia: a report on 132 patients

The prognostic value of cytological features was assessed in 132 patients with de novo acute myeloid leukaemia (AML) treated by anthracycline-cytosine-arabinoside combination chemotherapy. Of these patients, 98 (75%) achieved complete remission (CR). A significantly lower CR rate was seen in patients with trilineage dysmyelopoiesis (TDMP) (P = 0.003), but not in patients with dyserythropoiesis and/or dysgranulopoiesis without abnormal megakaryocytes. Other unfavourable factors were age greater than 50 years (P = 0.042), leucocyte count greater than 100 x 10(9)/l (P = 0.006), M5 FAB subtype (P = 0.013), presence of complex cytogenetic rearrangement or abnormalities of chromosome 5 and/or 7 (P = 0.001). Bone marrow eosinophilia greater than 3% was significantly associated with a higher CR rate (P = 0.04). In a multivariate analysis, a low CR rate was best predicted by the presence of a complex karyotype or abnormalities of chromosome 5 and/or 7 (P = 0.0001) and by the TDMP (P = 0.0036). Median actuarial disease-free survival (DFS) was 24 months. Actuarial DFS was significantly shorter in patients with TDMP (P = 0.0001) and an elevated leucocyte count (P = 0.02). Age, FAB subtype and karyotype had no significant incidence on DFS. Presence of TDMP appears to be an important prognostic factor in de novo AML. This could be used as one of the guidelines to therapy.

Prognostic significance of myeloperoxidase expression in childhood acute myeloid leukemia

The percentage of myeloperoxidase (MPO)-positive blast cells is associated with prognosis in adult acute myeloid leukemia (AML), but this association is unsubstantiated in pediatric AML. We retrospectively compared cytochemical MPO results with outcome in 154 patients younger than 21 years treated on three consecutive institutional protocols for newly diagnosed AML (1987-2001). Patients with FAB M0 and M7 AML (no MPO expression) or M3 AML (100% MPO expression) and Down's syndrome were excluded. Median MPO expression was higher in FAB M2 subtype than in other subtypes (P < 0.0001) and differed significantly across cytogenetic risk groups (P = 0.002) with highest MPO expression among those with favorable karyotypes. The percentage of MPO-positive blasts was not significantly associated with the probability of complete remission (P = 0.97), event-free survival (P = 0.72), or survival (P = 0.76) in multivariate analyses that accounted for age, FAB subtype, presenting WBC count, cytogenetic and protocol treatment risk group. In analysis limited to patients with intermediate-risk cytogenetics, higher MPO expression appeared to be associated with improved EFS (P = 0.06) but was not associated with remission induction rate (P = 0.16) or overall survival (P = 0.38). The percentage of MPO-positive blast cells is related to FAB subtype in pediatric AML but has limited prognostic relevance.

Roles of p15 Ink4b and p16 Ink4a in myeloid differentiation and RUNX1-ETO-associated acute myeloid leukemia

Inactivation of p15 Ink4b expression by promoter hypermethylation occurs in up to 80% of acute myeloid leukemia (AML) cases and is particularly common in the FAB-M2 subtype of AML, which is characterized by the presence of the RUNX1-ETO translocation in 40% of cases. To establish whether the loss of p15 Ink4b contributes to AML progression in association with RUNX1-ETO, we have expressed the RUNX1-ETO fusion protein from a retroviral vector in hematopoietic progenitor cells isolated from wild-type, p15 Ink4b or p16 Ink4a knockout bone marrow. Analysis of lethally irradiated recipient mice reconstituted with RUNX1-ETO-expressing cells showed that neither p15 Ink4b or p16 Ink4a loss significantly accelerated disease progression over the time period of one year post-transplantation. Loss of p15 Ink4b alone resulted in increased myeloid progenitor cell frequencies in bone marrow by 10-month post-transplant and a 19-fold increase in the frequency of Lin −c-Kit +Sca-1 + (LKS) cells that was not associated with expansion of long-term reconstituting HSC. These results strongly suggest that p15 Ink4b loss must be accompanied by additional oncogenic changes for RUNX1-ETO-associated AML to develop.

Cytogenetic, morphological, and immunophenotypic patterns in Omani patients with de novo acute myeloid leukemia

Chromosome aberrations observed at diagnosis are considered to be the most valuable prognostic factors in acute myeloid leukemia (AML). Some specific aberrations vary in frequency among different geographical areas and ethnic groups. There are only limited studies on the role of such variability in AML patients. Here, we report the results of a cytogenetic study on 63 ethnic Omani patients with de novo AML: 18 children (≤16 years) and 45 adults. By sex, 41 were male and 22 female; median age at diagnosis was 25 years. The morphological diagnosis was based on the French–American–British (FAB) WHO criteria. Chromosome abnormalities were present in 39 of 63 patients (62% overall, or 44% for adults and 18% for children). Karyotypes with a sole abnormality accounted for 20 of 63 patients (32%). Chromosome abnormalities were more common in patients with the FAB-M2 subtype (15 of 22; 68%), which was also the most frequent subtype observed (22 of 63; 35%). Among the normal karyotypes (24 of 63; 38%), M2 subtype was the also most frequent (7 of 24; 29%), followed by M4 (4 of 24; 17%). Balanced translocations, t(8;21) and t(15;17) were observed in 7 of 63 (11%) and 6 of 63 (10%), respectively. Inv(16) was seen in 2 of 63 (3%). Trisomy 8 was the most frequent numerical anomaly, found in 7 of 63 (11%). Monosomy 7 was seen in 3 of 63 (5%). The patterns in our study were similar to those reported from Saudi Arabia and Kuwait, but the frequency of abnormalities varied. Our population differed morphologically, with the M2 subtype as most common, whereas M4 and M3 were more commonly in those reports. A comparison of our findings was made with other geographic and ethnic groups. This is the first systematic cytogenetic study of an ethnic Omani population.

A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy

AbstractThe 8;21 chromosomal translocation occurs in 15% to 40% of patients with the FAB M2 subtype of acute myeloid leukemia (AML). This chromosomal abnormality fuses part of the AML1/RUNX1 gene to the ETO/MTG8 gene and generates the AML1-ETO protein. We previously identified a C-terminal truncated AML1-ETO protein (AEtr) in a mouse leukemia model. AEtr is almost identical to the AML1-ETO exon 9a isoform expressed in leukemia patients. Here, we describe a novel function of AEtr in the development of aneuploidy through spindle checkpoint attenuation. AEtr cells had a reduced mitotic index following nocodazole treatment, suggesting a failure in a subset of cells to arrest in mitosis with a functional spindle checkpoint. Additionally, primary leukemia cells and cell lines expressing AEtr were aneuploid. Moreover, AEtr cells had reduced levels of several spindle checkpoint proteins including BubR1 and securin following treatment with the spindle poison nocodazole. These results suggest that inactivation of the spindle checkpoint may contribute to the development of aneuploidy described in t(8;21) leukemia patients.

Scoring System To Predict the Risk of Early Death during Induction Chemotherapy for Primary Acute Myeloid Leukemia (AML).

Complications during induction chemotherapy (CT) for AML cause mortality in 5–20% of cases. It is relevant to define the risk of early death in this setting. In patients with a high probability of dying due to toxicity, the intensification of the supportive measures and/or alternative antineoplastic approaches could be appropriate. The aim of this study was to detect the features associated with lethal complications during induction CT for AML. We defined early deaths (ED) as those occurring before 42 days after the start of induction in the absence of evident leukemia. We analyzed all consecutive patients diagnosed with AML between June 1998 and February 2006 in 20 Spanish hospitals. These cases were treated according two multicenter trials CETLAM 99 (n=326) and CETLAM 2003 (n=248). Both schemes included idarubicin 12 mg/m2 days 1,3 and 5, etoposide 100 mg/m2 days 1–3 and cytarabine 500 mg/m2/12 hours days 1, 3, 5 and 7 as front-line treatment. In the CETLAM 2003, G-CSF (150 mg/m2 days 0–7) was added as priming therapy. The series included 574 patients, 248 (43%) female, with a median age of 48 years. Creatinine level was elevated (> 1,2 mg/dL) in 11% of patients. The overall mortality rate during induction (ED) was 12% (n=69). 335 (58%) patients achieved a complete remission with a single course of CT, 108 (19%) a partial remission and 62 (11%) were refractory. The most common causes of death were infection (n= 28, 46%), bleeding (n=7, 11%), pulmonary failure not due to infection (n=6, 10%), and multiorgan failure (n=4, 7%). Univariate analysis showed that age older than 50 years old, male gender, M4 or M5 FAB subtype, leukocyte count higher than 100x109/L, blasts in the marrow >70% and creatinine level above 1,2 mg/dL were associated with more frequent ED. In multivariate analysis, elevated creatinine level [hazard ratio (HR) 2.6 (1.3–5.2); P=0.009], leukocytosis >100x109/L [HR 2.3 (1.1–4.6) P=0.021] and age > 50 years old [HR 2.1 (1.4–3.9) P=0.018] were independent risk factors. The remaining parameters, including among others the use of G-CSF, gender, blasts in the marrow, treatment protocol, cytogenetics at diagnosis and FLT-3 mutational status were not associated with higher incidence of ED. Taking into account the three significant variables identified as risk factors, we developed a scoring system to predict the probability of ED during induction. The probability of ED in the low risk (none risk factor, n=212), intermediate risk (1 risk factor, n=239) and high risk (2 or 3 risk factors, n=57) categories were 3%, 11% and 28% respectively (P<0.001). The HR of ED for patients in the intermediate and high risk groups were 3.6 (1.5–8.1, P=0.003) and 7.9 (3.1–19.7; P<0.001) as compared with the low risk group. In conclusion, impaired kidney function, advanced age and hyperleukocytosis are the relevant variables increasing mortality during induction CT for AML. With these three factors it is possible to define a scoring system which predicts the probability of ED. A different approach for patients in the high-risk group should be investigated.

In Vivo Analysis of the Role of C/EBPα in Acute Promyelocytic Leukemia Genesis.

Acute promyelocytic leukemia (APL) is characterized by the infiltration of bone marrow (BM) and peripheral blood (PB) by leukemic cells presenting a block of differentiation at the stage of promyelocytes. At the cytogenetic level, APL is associated with the t(15;17) which causes the fusion of two genes: Retinoic Receptor α (RARα) and Promyelocytic Leukemia (PML), on chromosomes 17 and 15, respectively. C/EBPα is a leucine zipper transcription factor essential to normal granulopoiesis. Mutations in C/EBPα gene are detected in 6–10% of Acute Myeloid Leukemia (AML) cases and C/EBPα activity is down-regulated by AML1-ETO fusion protein associated with FAB M2 subtype. We decided to test whether PML-RARα interferes with C/EBPα function, thus contributing to the blockage of differentiation characteristic of APL. We generated mutant mice expressing PML-RARα and haploinsufficient for C/EBPα (PR C/EBPα+/−) by crossing hCG PML-RARα transgenic mice (PR TM) and C/EBPα+/−. Leukemia was not detected in WT (n=415) and C/EBPα+/− (n=47) mice after 800 days of follow up. In contrast, 8.2% PR TM (19/233) and 14.9% (13/87) PR C/EBPα+/− mice developed a form of leukemia that closely resembled human APL and identical to that developed by PR TM. The leukemia-free survival was significantly shorter in PR C/EBPα+/− compared to PR TM (644.3 days; 95% Confidence Interval, 95%C.I.: 586.4 – 702.2 vs 718.4 days; 95%C.I.: 689.3 – 747.5, P=0.02). Both groups presented a long latency for the development of the disease with a mean age (95%C.I.) at diagnosis of 399.9 days (184–673) and 495.8 days (215–757) in PR and PR C/EBPα+/−, respectively. PR and PR C/EBPα+/− leukemic mice presented similar WBC counts (107.1 ± 82.65x103vs 63.55 ± 57.82x103cells/ml, P=0.26), hemoglobin concentrations (10.87 ± 3.69 vs 9.92 ± 2.39 g/dl, P =0.57) and platelet counts (283.8 ± 188.7x103vs 177.8 ± 149.5x103platelets/ml, P =0.24). In both groups, the leukemic cells resembled promyelocytes, and expressed the phenotype CD11b+ Gr1+ CD34± c-Kit+, which represented 46.65 ± 26.89%; 32.72 ± 15.16% and 1.91 ± 1.42% of the spleen cells from PR; PR C/EBPα+/− and WT, respectively. In order to isolate PML-RARα leukemic cells and their normal counterparts to gene expression assessment, BM samples from WT mice (pooled from 2 mice), non leukemic PR C/EBPα+/− mice (pooled from 2 mice), and spleen cells from WT and leukemic PR C/EBPα+/− mice were first submitted to red cells lyses, stained with previously conjugated antibodies. Cells expressing CD16/32, CD11b, c-kit and CD34, but neither CD3 nor CD45/B220 were isolated. These cells presented medium to large size with a granular cytoplasm. C/EBPα and PML-RARα expression was analyzed by qRT-PCR in the sorted cells. Compared to WT cells, promyelocytes from leukemic and non leukemic PR C/EBPα+/− mice expressed significantly less C/EBPα, with the lowest levels detected in leukemic samples. As expected, PML-RARα was not detected in WT samples. Comparison between cells suspensions containing similar numbers of promyelocytes revealed that PML-RARα expression was higher in leukemic compared to non leukemic PR C/EBPα+/− mice. In conclusion, our data strongly suggest that PML-RARα fusion protein acts as a dominant negative product on C/EBPα gene expression in a specific subset of early myeloid cells and contribute to the pathogenesis of APL.

AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.

Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.

A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis

The t(8;21)(q22;q22) translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in 15% of all cases of AML, including 40-50% of FAB M2 subtype and rare cases of M0, M1 and M4 subtypes. The most commonly known AML1-ETO fusion protein (full-length AML1-ETO) from this translocation has 752 amino acids and contains the N-terminal portion of RUNX1 (also known as AML1, CBFalpha2 or PEBP2alphaB), including its DNA binding domain, and almost the entire RUNX1T1 (also known as MTG8 or ETO) protein. Although alterations of gene expression and hematopoietic cell proliferation have been reported in the presence of AML1-ETO, its expression does not lead to the development of leukemia. Here, we report the identification of a previously unknown alternatively spliced isoform of the AML1-ETO transcript, AML1-ETO9a, that includes an extra exon, exon 9a, of the ETO gene. AML1-ETO9a encodes a C-terminally truncated AML1-ETO protein of 575 amino acids. Expression of AML1-ETO9a leads to rapid development of leukemia in a mouse retroviral transduction-transplantation model. More importantly, coexpression of AML1-ETO and AML1-ETO9a results in the substantially earlier onset of AML and blocks myeloid cell differentiation at a more immature stage. These results indicate that fusion proteins from alternatively spliced isoforms of a chromosomal translocation may work together to induce cancer development.

High WT1 Expression After Induction Therapy Predicts High Risk of Relapse and Death in Pediatric Acute Myeloid Leukemia

To determine whether minimal residual disease (MRD) measured by Wilms' tumor gene 1 (WT1) expression is a prognostic marker in pediatric acute myeloid leukemia (AML), we quantified WT1 transcript by real-time quantitative-polymerase chain reaction in 92 AML at diagnosis and during follow-up. Patients (median age, 6 years; cytogenetics, favorable 27%, intermediate 59%, poor 13%) were treated between 1995 and 2002 and enrolled in Leucémie aiguë Myéloblastique Enfant (LAME) 89/91, LAME 99 pilot study and Acute Promyelocytic Leukemia French collaborative protocols. With a median follow-up of 26 months, event-free survival was 56% with a standard deviation (SD) of 5% and overall survival of 62.5% with an SD of 6%. WT1 copy number was normalized by TATA box binding protein gene transcripts and expressed as WT1/TBP x 1,000 ratio. Median WT1 ratio in normal patient controls was 12 (range, 0 to 57). A level over two SD than normal bone marrow controls (ie, WT1 ratio > 50), was considered as significant overexpression. At diagnosis, WT1 overexpression was detected in 78% of patients (72 of 92 patients; median copy ratio, 2231). The WT1 values were significantly higher (P = .01) in favorable cytogenetics and lower (P < .0001) in M5-FAB subtype, 11q23 rearrangements (P < .001), and infants (P = .003) and demonstrate a strong correlation with fusion transcript AML1-ETO, PML-RARalpha expression. After induction treatment, WT1 ratio was analyzed in 46 of 72 patients and found above 50 in nine of 36 patients and five of 25 patients at D35-50 and 3 to 5 months, respectively. WT1 ratio > 50 after induction is an independent prognostic risk factor of relapse (P = .002) and death (P = .02). WT1 quantification is an informative molecular marker for MRD in pediatric AML and is now performed as prospective analysis in ELAM02 protocol.

Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.

The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.

G1 Cell-Cycle Arrest and Apoptosis by Histone Deacetylase Inhibition in MLL-AF9 Acute Myeloid Leukemia Cells Is MLL-AF9 Independent.

Acute myeloid leukemia (AML) with MLL rearrangements (MLLmut), found mainly in M5 or M4 FAB subtypes, is frequent in infants and secondary leukemias. The most common MLL translocation gives rise to MLL-AF9. MLL protein interacts with histone deacetylases (HDACs) -1 and -2 through the MLL repression domain.We report the effects of HDAC inhibition by valproic acid (VPA) in MLL-AF9 AML-M5 cells (THP-1, MM6 and MOLM-13) and MLLmut AML-M5 blasts. VPA led to histone hyper-acetylation, strong cell-growth inhibition, G1 cell-cycle arrest and apoptosis. Combined treatment with all-trans-retinoic-acid (ATRA) did not substantially improve these effects. VPA increased MLL-AF9 transcription, indicating that VPA overcomes the cell-growth promoting activity and resistance to apoptosis conferred by MLL-AF9 in AML-M5 cells, even with increased MLL-AF9. A small number of genes were significantly affected by VPA in p53-absent THP-1 cells (GeneChip analysis), and the majority of these were up-regulated. VPA potently induced p21 and cyclin G2 (CG2) expression. p21 and CG2 targeted inhibition demonstrated that p21 acts as a key regulator in the VPA-inducted G1 cell-cycle arrest, while induction of CG2 has no effect.These data suggest that these poor prognosis patients may benefit from HDAC inhibitor therapy.

MRP3, BCRP, and P-Glycoprotein Activities are Prognostic Factors in Adult Acute Myeloid Leukemia

P-Glycoprotein (Pgp) is associated with poor outcome in acute myeloid leukemia (AML). We have investigated other ATP-binding cassette proteins such as BCRP, MRP1, MRP2, MRP3, and MRP5 for their potential implication in chemoresistance. Eighty five AML patient samples were analyzed in this study. First, MRP3 function was higher in patients which had a high level of leukocytes (P = 0.01), a M5 FAB subtype (P = 0.04), and an intermediate or poor cytogenesis (P = 0.05). BCRP activity was not correlated with clinical or biological variables, but high Pgp activity was correlated with the following variables: CD34 expression (P = 0.002), FAB subtype (P = 0.002), intermediate or poor cytogenesis (P = 0.02), and elderly patients (P = 0.03). Second, Pgp, MRP3, and BCRP activities were correlated with complete remission (P = 0.02, P = 0.04, and P = 0.04, respectively), disease-free survival (P = 0.02, P = 0.03, and P = 0.25, respectively), and overall survival (P = 0.04, P = 0.04, and P = 0.05, respectively) in multivariate analysis. The patient samples expressing one or none of these Pgp, MRP3, or BCRP functional proteins have a better prognosis than the patients expressing two or three of these functional proteins (complete remission, P = 0.02; disease-free survival, P = 0.01; overall survival, P < 0.001). BCRP and MRP3 may also be involved in chemoresistance in AML, especially MRP3 in patients with M5 FAB. Additional modulation of BCRP or MRP3 to Pgp modulation may be necessary in some patients in order to improve the treatment outcome.

WT1 Overexpression after Induction Therapy in Children AML Is Associated with Higher Risk of Relapse.

The Wilms' s tumor gene (WT1) is a tumor suppressor gene highly expressed in most acute leukemias. To determine whether WT1 gene expression is a valuable and informative marker for minimal residual disease in pediatric AML, we quantified WT1 transcript amount by RQ-PCR in 92 de novo AML and 20 normal controls. The WT1 transcripts obtained were normalized with respect to the number of TBP transcripts and expressed as WT1 copy numbers by the ratio WT1/TBPx1000. The WT1 levels were extremely low in normal controls, and the median number of WT1 copies was 10 (range 4–30 ). A level above 50 copies was considered as significant. All the patients (aged 2 months-18 years, median age:5.9 years, male/female ratio 0.87) were treated for de novo AML between 1995 and 2003 in two French institutions and enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: MO 5.4%, M1 4.3%, M2 18.5%, M3 14.1%, M4 and M4Eo 12%, M5 33.6 %, M7 10.9% and unclassified 1%. Cytogenetics features according to the MRC classification were favourable, intermediate or poor in 27% (23/83), 59% (49/83) and 13% (11/83) respectively. With a median follow up of 24 months (range 8–97months) OS was 75± 8% and EFS 60 ± 6%. At diagnosis WT1 overexpression was detected in 78.3% (72/92) with a median copy number of 2231 (range 50-429200). The WT1 values were significantly higher (p=0.02) in M2-FAB subtype and lower (p=0.01) in M5-FAB subtype while no correlation was found with WBC count or cytogenetic abnormalities. WT1 quantification for MRD was evaluable in 41/72 pts and positive in 9/32 at D40-50, 5/25 at M3-M5, 5/7at M6-M8. At least one analysis above 50 copies after induction therapy is associated with a significant risk of subsequent relapse 8/11 vs 8/30 ( p=0.007) RR=22 (IC 95%:46–118) and death 10/14 vs 3/30 (p=0.001) RR=7.3 (IC95%: 1–34). Although retrospective analysis may include bias, we conclude that WT1 is a useful and informative molecular marker for MRD in pediatric AML and performed as prospective analysis in ELAM02 protocol.

Early Allogeneic Bone Marrow Transplantation for Young Adults with Acute Myeloid Leukemia in First Complete Remission: The 17 Year Experience of the BGMT Group.

Background. From 1984 and during 17 years, the BGMT inter-group prospectively followed the same strategy of early allogeneic bone marrow transplantation (alloBMT) compared to non allogeneic treatment in 4 consecutive trials, as modality of consolidation for patients (pts) under 45 with an HLA identical sibling donor in first complete remission (CR) of an acute myeloid leukemia (AML). The purpose of this study was to evaluate this strategy by an intent to treat analysis based on donor availability.Pts and methods. In each of these trials, pts received an induction by daunorubicine 60 mg/m2/d 3d + cytarabine 100 mg/m2/d (CI) 10 d. followed by a light consolidation based on cytarabine 50 mg/m2/12h SC 7d + daunorubicine 60 mg/m2/d 2d. Pts under 45 with a sibling donor had an alloBMT (cyclophosphamide 60 mg/kg IV 2d + fractionated TBI 12 Gy) without any further chemotherapy. If not, pts were treated according to a strategy of chemotherapy which evolved the years according to experience and knowledge. The 182 pts with a donor were compared to the 290 who had not. The median age of these pts (M/F = 103/79 vs 154/136) was 34 vs 33.5 years (15–45). Median WBC count was 15 vs 16 (0.2–35) and WBC was ? 30 109/L for 65 vs 94 pts. The FAB distribution was: M1/M2 = 92 vs 150; M3 = 10 vs 14; M4/M5 = 63 vs 110; M0/M6/M7 = 17 vs 16. The cytogenetics risk groups distribution was: low risk = 32 vs 62 (24 vs 28%); intermediate risk = 78 vs 127 (58 vs 57%); high risk = 24 vs 34 (18 vs 15%). CR was achieved in 2 courses for 19 vs 42 pts (10 vs 14 %).Results. AlloBMT was done for 90.7% of the eligible pts. With a median follow-up of 114 months, 10-year cumulative incidence of relapse and non relapse death, overall survival and leukemia free survival (LFS) were 27.2% vs 54.5% (p < .001), 24.4% vs 5.6% (p < .001), 51.2% vs 43.4% (p = .11), and 48.3% vs 39.9% (p < .03), respectively. For all patients, univariate and multivariate analysis found that initial WBC count, FAB subtypes, cytogenetic risk group, number of course to achieve CR, demonstrated a significant prognosis influence. A better LFS was observed in the donor group among patients with M1 to M5 FAB subtypes (52.4% vs 41.9% (p < .01)), among those with WBC > 30 109/L (47.3% vs 30.7% (p < .02)), and among those of the intermediate cytogenetic risk group (58% vs 43% (p < .01)). For low cytogenetic risk group no difference was shown between donor an no donor group (58.6% vs 65.9% (p =.63). In this study, alloBMT presents limitations in the most aggressive diseases such as M0/M6/M7 FAB subtypes (11.8% vs 6.3% (p = .15)) or high cytogenetic risk group (12.5% vs 7.8% (p = .67). In this situation it does not seem to be sufficient enough to control disease by the graft versus leukemia effect alone. For these pts new investigations should be undertaken.

Immunophenotyping of acute leukemias and myelodysplastic syndromes.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain