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ABSTRACT: The present study evaluated the expression profile of 19 genes previously reported in microarray studies and associated with resistance or sensitivity to vincristine (RPLP2, CD44, TCFL5, KCNN1, TRIM24), prednisolone (F8A, CDK2AP1, BLVRB, CD69), daunorubicin (MAP3K12, SHOC2, PCDH9, EGR1, KCNN4) and l-asparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3). We studied 140 bone marrow samples at diagnosis from children with acute lymphoblastic leukemia (ALL) treated according to the Brazilian Childhood Leukemia Treatment Group (GBTLI) ALL-99 protocol. The expression profiles of the genes listed above were analyzed by real-time quantitative polymerase chain reaction (PCR) and then related to the clinical and biological prognostic factors. The results showed significant associations (p ≤ 0.05) between the expression levels of genes GPR56, BLVRB, IGFBP7 and white blood cell (WBC) count at diagnosis; GATA3, MAN1A1, CD44, MAP3K12, CLEC11A, SHOC2 and CD10 B-lineage ALL; TCFL5 and bone marrow status at day 14; MAP3K12 and TRIM24 and bone marrow status at day 28; and CD69, TCFL5 and TRIM24 genes and ETV6/RUNX1 positive ALL. The up-regulation of SHOC2 was also associated with better 5-year event-free survival (EFS) in univariate and multivariate analysis (p = 0.02 and p = 0.03, respectively). These findings highlight genes that could be associated with clinical and biological prognostic factors in childhood ALL, suggesting that these genes may characterize and play a role in the treatment outcome of some ALL subsets.
Leukemia & lymphoma 08/2012; · 2.40 Impact Factor
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Priscila P Zenatti,
Daniel Ribeiro,
Wenqing Li,
Linda Zuurbier,
Milene C Silva,
Maddalena Paganin,
Julia Tritapoe,
Julie A Hixon,
André B Silveira,
Bruno A Cardoso, [......],
Maria L Toribio,
Jörg Kobarg,
Martin Horstmann,
Rob Pieters, Silvia R Brandalise,
Adolfo A Ferrando,
Jules P Meijerink,
Scott K Durum,
J Andrés Yunes,
João T Barata
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ABSTRACT: Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.
Nature Genetics 09/2011; 43(10):932-9. · 35.53 Impact Factor
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Letícia F Leal,
Lívia M Mermejo,
Leandra Z Ramalho,
Carlos E Martinelli,
José A Yunes,
Ana Luiza Seidinger,
Maria José Mastellaro,
Izilda A Cardinalli, Silvia R Brandalise,
Ayrton C Moreira,
Luiz G Tone,
Carlos A Scrideli,
Margaret Castro,
Sonir R Antonini
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ABSTRACT: CTNNB1/β-catenin mutations and activation of Wnt/β-catenin pathway are frequent in adult adrenocortical tumors (ACT), but data on childhood ACT are lacking.
The aim of the study was to investigate the presence of Wnt/β-catenin pathway abnormalities in childhood ACT.
Clinicopathological findings and outcome of 62 childhood ACT patients were analyzed regarding CTNNB1 mutations and the expression of Wnt-related genes (CTNNB1; WNT4, a Wnt ligand; SFRP1, DKK3, and AXIN1, Wnt inhibitors; TCF7, a transcription factor; and MYC and WISP2, target genes) by quantitative PCR and immunohistochemistry.
CTNNB1-activating mutations were found in only four of 62 ACT (6%), all of them harboring TP53 mutation. There was association between the presence of CTNNB1 mutations and death (P = 0.02). Diffuse β-catenin accumulation was found in 71% of ACT, even in ACT without CTNNB1 mutations. Compared to normal adrenals, ACT presented increased expression of CTNNB1 (P = 0.008) and underexpression of Wnt inhibitor genes: DKK3 (P < 0.0001), SFRP1 (P = 0.05), and AXIN1 (P = 0.04). With regard to Wnt/β-catenin target genes, ACT presented increased expression of WISP2 but lower expression of MYC. Higher overall survival was associated with underexpression of SFRP1 (P = 0.01), WNT4 (P = 0.004), and TCF7 (P < 0.01).
CTNNB1 mutations are not common in childhood ACT but appear to associate with poor prognosis. Nevertheless, most ACT exhibit increased expression of β-catenin and WISP2 and reduced expression of Wnt inhibitor genes (DKK3, SFRP1, and AXIN1). Thus, in addition to CTNNB1 mutations, other genetic events affecting the Wnt/β-catenin pathway may be involved in childhood adrenocortical tumorigenesis.
The Journal of clinical endocrinology and metabolism 08/2011; 96(10):3106-14. · 6.50 Impact Factor
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Silvia R Brandalise,
Vitória R Pinheiro,
Simone S Aguiar,
Eduardo I Matsuda,
Rosemary Otubo,
José A Yunes,
Waldir V Pereira,
Eny G Carvalho,
Lilian M Cristofani,
Marcelo S Souza,
Maria L Lee,
Jane A Dobbin,
Maria S Pombo-de-Oliveira,
Luiz F Lopes,
Katharina N T Melnikoff,
Algemir L Brunetto,
Luiz G Tone,
Carlos A Scrideli,
Vera L L Morais,
Marcos B Viana
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ABSTRACT: PURPOSE To describe event-free survival (EFS) and toxicities in children with low-risk acute lymphoblastic leukemia (ALL) assigned to receive either continuous 6-mercaptopurine (6-MP) and weekly methotrexate (MTX) or intermittent 6-MP with intermediate-dose MTX, as maintenance treatment. PATIENTS AND METHODS Between October 1, 2000, and December 31, 2007, 635 patients with low-risk ALL were enrolled onto Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI) ALL-99 protocol. Eligible children (n = 544) were randomly allocated to receive either continuous 6-MP/MTX (group 1, n = 272) or intermittent 6-MP (100 mg/m(2)/d for 10 days, with 11 days resting) and MTX (200 mg/m(2) every 3 weeks; group 2, n = 272). RESULTS The 5-year overall survival (OS) and EFS were 92.5% +/- 1.5% SE and 83.6% +/- 2.1% SE, respectively. According to maintenance regimen, the OS was 91.4% +/- 2.2% SE (group 1) and 93.6% +/- 2.1% SE (group 2; P = .28) and EFS 80.9% +/- 3.2% SE (group 1) and 86.5% +/- 2.8% SE (group 2; P = .089). Remarkably, the intermittent regimen led to significantly higher EFS among boys (85.7% v 74.9% SE; P = .027), while no difference was seen for girls (87.0% v 88.8% SE; P = .78). Toxic episodes were recorded in 226 and 237 children, respectively. Grade 3 to 4 toxic events for groups 1 and 2 were, respectively, 273 and 166 for hepatic dysfunction (P = .002), and 772 and 636 for hematologic episodes (P = .005). Deaths on maintenance were: seven (group 1) and one (group 2). CONCLUSION The intermittent use of 6-MP and MTX in maintenance is a less toxic regimen, with a trend toward better long-term EFS. Boys treated with the intermittent schedule had significantly better EFS.
Journal of Clinical Oncology 03/2010; 28(11):1911-8. · 18.37 Impact Factor
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Juliana F Mata,
Vanessa S Silveira,
Elvis C Mateo,
Maria A A Cortez,
Rosane G P Queiroz,
José Andrés Yunes,
Maria Lúcia M Lee,
Sílvia R C Toledo,
Antonio Sérgio Petrilli, Silvia R Brandalise,
Luiz G Tone,
Carlos A Scrideli
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ABSTRACT: Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL).
The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test.
A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P < 0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P = 0.01) and low risk group classification (P = 0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P = 0.03, P = 0.02 and P = 0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P = 0.02).
These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL.
Pediatric Blood & Cancer 03/2010; 55(1):100-7. · 1.89 Impact Factor
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Bruno K L Duarte,
Isabella Valente,
Afonso C Vigorito,
Francisco J P Aranha,
Gislaine Oliveira-Duarte,
Eliana C M Miranda,
Irene Lorand-Metze,
Katia B Pagnano,
Marcia Delamain,
José F Marques Junior, Silvia R Brandalise,
Márcio Nucci,
Carmino A De Souza
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ABSTRACT: We evaluate the effectiveness and toxicity of high-dose sequential chemotherapy (HDS) as salvage therapy in patients with advanced-stage Hodgkin lymphoma.
We performed a retrospective analysis on 77 patients receiving HDS between 1998 and 2006. Patients enrolled were in disease progression or relapsed disease, or did not achieve a complete remission after first-line treatment. HDS consisted of the sequential administration of cyclophosphamide and granulocyte colony-stimulating factor with stem cell harvesting, followed by methotrexate plus vincristine and etoposide.
The majority of patients had stage III/IV (64%) and B symptoms (71.4%). Disease status improvement after HDS was observed in 24 of 57 patients (42%) previously in disease progression or relapse. HDS-related deaths occurred in 8 of 77 patients (10.4%). Four patients (5.2%) developed acute myeloid leukemia/myelodysplastic syndrome. Overall, disease-free and progression-free survival was 27%, 57%, and 25%, respectively.
Despite the treatment-related mortality, HDS is feasible, with satisfactory response rates, even in patients with poor prognosis.
Clinical Lymphoma & Myeloma 12/2009; 9(6):449-54. · 1.13 Impact Factor
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ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. Here, we compared patients with or without NOTCH1 mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of Notch transcriptional and CK2 post-translational inactivation of PTEN, we treated T-ALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL.
Haematologica 12/2009; 95(4):674-8. · 6.42 Impact Factor
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ABSTRACT: Increased activity of multidrug resistance (MDR) genes has been associated with treatment failure in acute leukemias, although with controversial reports. The objective of the present study was to assess the expression profile of the genes related to MDR: ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP in terms of the clinical and biological variable and the survival of children with acute lymphoblastic leukemia (ALL).
The levels of mRNA expression of the drug resistance genes ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP were analyzed by quantitative real-time PCR using the median values as cut-off points, in consecutive samples from 140 children with ALL at diagnosis.
Expression levels of the ABCG2 gene in the patient group as a whole (P = 0.05) and of the ABCG2 and ABCC1 genes in patients classified as being at high risk were associated with higher rates of 5-year event-free survival (EFS) (P = 0.04 and P = 0.01). Expression levels of the ABCG2 gene below the median were associated with a greater chance of death related to treatment toxicity for the patient group as a whole (P = 0.009) and expression levels below the median of the ABCG2 and ABCC1 genes were associated with a greater chance of death due to treatment toxicity for the high-risk group (P = 0.02 and P = 0.03, respectively).
The present data suggest a low participation of the drug efflux genes in treatment failure in patients with childhood ALL. However, the low expression of some of these genes may be associated with a higher death risk related to treatment toxicity.
Pediatric Blood & Cancer 09/2009; 53(6):996-1004. · 1.89 Impact Factor
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Marcia R. Amorim,
Alexandre B. C. Figueiredo,
Alessandra Splendore,
Isis Q. Magalhães,
Maria S. Pombo-d´e-Oli˙veira,
Bcsgail,
Kênia B. El-Jaick,
Maria Lydia D'andrea,
Jozina Aquino,
Dora Márcia Alencar, [......],
Maria Lucia M. Lee,
Luis Fernando Lopes,
Carmen M. Mendonça,
Flávia Nogueira,
Flávia Pimenta,
Vitória P. Pinheiro,
Denise Bousfield Da Silva,
Elaine Sobral,
Fernando R. Vargas,
Fernando Werneck
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ABSTRACT: Denaturing high-performance liquid chromatography (dHPLC) was developed to screen DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. In this study, we have evaluated the dHPLC screening method and direct sequencing for the detection of GATA1 mutations in peripheral blood and bone marrow aspirates samples from children with Down syndrome (DS). Cases were ascertained consecutively as part of an epidemiological study of DS and hematological disorders in Brazil. A total of 130 samples corresponding to 115 children with DS were analysed using dHPLC and direct sequencing methods to detect mutations in GATA1 exons 2, 3 and 4 gene sequences. The overall detection rate of sequencing and dHPLC screening methods was similar. Twenty mutations were detected in exon 2 and one mutation in exon 3 (c.231_232 dupGT) sequences of acute megakaryoblastic leukemia and transient leukemia samples. Four GATA1 mutations were newly described [c.155C > G; c.156_178 del23 bp; c.29_30 del GG; c.182C > A and c.151A > T,c.153_162 del 10 bp). Out of four, three had single nucleotide change. In conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.
07/2009; 50(5):834-840.
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Carlos A Scrideli,
Juliana G Assumpção,
Mônica A Ganazza,
Marcela Araújo,
Silvia R Toledo,
Maria Lúcia M Lee,
Elisabete Delbuono,
Antonio S Petrilli,
Rosane P Queiróz,
Andrea Biondi,
Marcos B Viana,
José A Yunes, Silvia R Brandalise,
Luiz G Tone
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ABSTRACT: Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise.
We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements.
At least one marker was detected by polymerase chain reaction in 96.4% of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor.
This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.
Haematologica 06/2009; 94(6):781-9. · 6.42 Impact Factor
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Marcia R Amorim,
Alexandre B C Figueiredo,
Alessandra Splendore,
Isis Q Magalhães,
Maria S Pombo-de-Oliveira,
Kênia B El-Jaick,
Maria Lydia D'andrea,
Jozina Aquino,
Dora Márcia Alencar, Silvia R Brandalise, [......],
Maria Lucia M Lee,
Luis Fernando Lopes,
Carmen M Mendonça,
Flávia Nogueira,
Flávia Pimenta,
Vitória P Pinheiro,
Denise Bousfield Da Silva,
Elaine Sobral,
Fernando R Vargas,
Fernando Werneck
[show abstract]
[hide abstract]
ABSTRACT: Denaturing high-performance liquid chromatography (dHPLC) was developed to screen DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. In this study, we have evaluated the dHPLC screening method and direct sequencing for the detection of GATA1 mutations in peripheral blood and bone marrow aspirates samples from children with Down syndrome (DS). Cases were ascertained consecutively as part of an epidemiological study of DS and hematological disorders in Brazil. A total of 130 samples corresponding to 115 children with DS were analysed using dHPLC and direct sequencing methods to detect mutations in GATA1 exons 2, 3 and 4 gene sequences. The overall detection rate of sequencing and dHPLC screening methods was similar. Twenty mutations were detected in exon 2 and one mutation in exon 3 (c.231_232 dupGT) sequences of acute megakaryoblastic leukemia and transient leukemia samples. Four GATA1 mutations were newly described [c.155C > G; c.156_178 del23 bp; c.29_30 del GG; c.182C > A and c.151A > T,c.153_162 del 10 bp). Out of four, three had single nucleotide change. In conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.
Leukemia & lymphoma 06/2009; 50(5):834-40. · 2.40 Impact Factor
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ABSTRACT: The Wilms Tumor gene (WT1) encodes a transcription factor involved in kidney development and malignancy. WT1 expression in a subpopulation of early CD34+ cells has suggested its involvement in hematopoiesis. WT1 is aberrantly expressed in leukemias. High expression of WT1 at diagnosis has been associated with unfavorable prognosis in adult acute myeloid leukemia (AML). The prognostic relevance of WT1 expression in pediatric AML was evaluated in only one study, including 47 patients, which showed that very low levels of WT1 at presentation were associated with an excellent outcome. To test the validity of these findings we measured levels of WT1 in 41 newly diagnosed pediatric AML of the non-M3 FAB subtype.
Patients were treated according to an AML-BFM 83-based protocol in a single institution. Mononucleated cells obtained from presentation BM aspirates were cryopreserved and later thawed and used for total RNA extraction and cDNA synthesis. The quantitative assessment of WT1 transcripts was made by real-time PCR (RQ-PCR). WT1 transcripts values were normalized with respect to the number of ABL transcripts.
WT1 levels were significantly higher in patients bearing favorable chromosome abnormalities, t(8;21) and inv(16) (P = 0.002). Higher levels of WT1 expression were unexpectedly associated with a higher probability of overall survival by Cox regression analysis (P = 0.002). Multivariate regression analysis could not discriminate between the effects of WT1 and cytogenetics on survival.
Higher WT1 expression was associated with favorable cytogenetics subtypes and accordingly with better outcome in children with AML in this study.
Pediatric Blood & Cancer 09/2007; 49(2):133-8. · 1.89 Impact Factor
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Flavio Jose da Costa Ramos,
Maria Tereza Cartaxo Muniz,
Vanessa Cavalcante Silva,
Marcela Araújo,
Ednalva Pereira Leite,
Elizabete Malaquias Freitas,
Crisiane Wais Zanrosso,
Ana Hatagima,
Maricilda Palandi de Mello,
Jose Andrés Yunes,
Terezinha de Jesus Marques-Salles,
Neide Santos, Silvia R Brandalise,
Maria S Pombo-De-Oliveira
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[hide abstract]
ABSTRACT: Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in the metabolism of folate. The presence of polymorphisms that reduce the activity of MTHFR has been linked to the multifactor process of development of acute leukemia. A case control study was conducted on Brazilian children in different regions of the country with the aim of investigating the role of MTHFR C677T and A1298C polymorphisms as risk factors in the development of acute myeloid leukemia (AML). We used the polymerase chain reaction restriction fragment length polymorphism method to genotyping 182 AML and 315 healthy individuals. The genotype 677 CT was associated with decreased risk [odds ratio (OR), 0.37; confidence interval (CI) 95%, 0.14 - 0.92], whereas 1298 AC genotype was linked with an increased risk [OR, 2.90; CI 95%, 1.26 - 6.71] of developing AML in non-white children. Further epidemiological study is needed to unravel the complex multiple gene-environment interactions in the role of the AML leukemogenesis.
Leukemia and Lymphoma 11/2006; 47(10):2070-5. · 2.58 Impact Factor
