Óscar Fuster

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

Background Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. Design and Methods We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. Results FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. Conclusions FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.

A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute promyelocytic leukemia

Chromosomal translocations in hematological malignancies often result in novel fusion chimeric genes. We report a case of acute myeloid leukemia with a clonal translocation t(11;12)(p15;q13) displaying morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. The gene fused to NUP98 (nucleoporin 98) was detected by comparative genomic hybridization array as the retinoid acid receptor gamma gene (RARG). The involvement of RARG in a chimeric fusion transcript has not been reported previously in human leukemia.

Minimal residual disease detection in acute myeloid leukemia by mutant nucleophosmin (NPM1) : Comparison with WT1 gene expression

BACKGROUND Molecular analysis of minimal residual disease is only applicable in acute myeloblastic leukemia (AML) patients with genetic markers (20-30%). This study analyzes the feasibility of the real-time quantitative polymerase chain reaction (RQ-PCR) assay to detect mutant nucleophosmin (NPM1) during follow-up in AML patients. Moreover, we compare the NPM1 results with those of WT1 expression to MRD assessment. METHODS The study includes 97 samples from 24 AML patients with type A NPM1 mutation at diagnosis. MRD was evaluated simultaneous by RQ-PCR assay to detect NPM1-mutated and WT1 expression. RESULTS The expression levels of WT1 and NPM1 in 93 paired samples showed a strong positive correlation (r=0.81; p<0.0001). However, the kinetics of disappearance were different, WT1 decreased rapidly after induction but maintained these residual levels after treatment in patients in complete remission, whereas NPM1 experienced a mild reduction after induction but was undetectable in long survivor patients. CONCLUSIONS This study shows the feasibility of the RQ-PCR assay to monitor MRD in AML patients carrying NPM1 mutations and its advantage over RQ-PCR assay for WT1. Owing to NPM1-mutated is specific of leukemic cells and shows higher levels at presentation.

Rapid Detection of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia Using High-Resolution Melting Analysis

The most frequent KIT mutations reported in core-binding factor acute myeloid leukemia are point mutations and insertions/deletions in exons 17 and 8. The vast majority of KIT mutation detection procedures are time-consuming, costly, or with a high lower limit of detection. High-resolution melting (HRM) is a gene scanning method that combines simplicity and rapid identification of genetic variants. We describe an HRM method for the simultaneous screening of exons 8 and 17 KIT mutations and report the results obtained in 69 core-binding factor acute myeloid leukemia patients. Mutation detection was compared with sequencing as the gold standard. The HRM method used high-resolution melting master reagents (Roche) and the LightCycler 480 (Roche) platform. HRM was reproducible, showed a lower limit of detection of 1%, and discriminated all patients with mutated KIT from controls without false positive or false negative results. Additionally, most of the mutations were differentiated from the other mutations. KIT mutations were present in 15.9% of patients, showing a higher incidence in inv(16) (25.8%) than in t(8;21) (7.9%). The presence of a KIT mutation was associated with a high white blood cell count, and adult patients with an exon 17 mutation had a higher incidence of relapse. These findings verify that HRM is a reliable, rapid, and sensitive method for KIT mutation screening. Furthermore, our study corroborates the unfavorable prognosis associated with exon 17 KIT mutations.

Adverse prognostic value of MYBL2 overexpression and association with microRNA-30 family in acute myeloid leukemia patients

The MYBL2 gene encodes a transcription factor implicated in cell proliferation and maturation whose amplification or overexpression has been associated with different human malignancies, suggesting that it could be implicated in tumorigenesis. We analyzed MYBL2 expression and its prognostic value in 291 patients with de novo acute myeloid leukemia (AML) and we also evaluated its association with microRNAs 29 and 30 families. MYBL2 expression in AML patients was increased relative to CD34+ cells. Moreover, MYBL2 overexpression was associated with lower expression of miR-30a (P=0.024), miR-30b (P=0.021) and miR-30c (P=0.009). Multivariate analysis showed that MYBL2 expression was an independent factor for disease-free survival (HR 3.0, 95% CI 1.5-6.0, P=0.002) and cumulative incidence of relapse (HR 2.6, 95% CI 1.2-5.6, P=0.015) in patients with an intermediate-risk karyotype. In conclusion, our data showed that MYBL2 expression analysis could be useful to define subgroups of patients with poor prognosis.

Complex Variant t(9;22) Chromosome Translocations in Five Cases of Chronic Myeloid Leukemia

The Philadelphia (Ph1) chromosome arising from the reciprocal t(9;22) translocation is found in more than 90% of chronic myeloid leukemia (CML) patients and results in the formation of the chimeric fusion gene BCR-ABL. However, a small proportion of patients with CML have simple or complex variants of this translocation, involving various breakpoints in addition to 9q34 and 22q11. We report five CML cases carrying variant Ph translocations involving both chromosomes 9 and 22 as well as chromosomes 3, 5, 7, 8, or 10. G-banding showed a reciprocal three-way translocation involving 3q21, 5q31, 7q32, 8q24, and 10q22 bands. BCR-ABL fusion signal on der(22) was found in all of the cases by FISH.

Rapid Screening of ASXL1, IDH1, IDH2, and c-CBL Mutations in de Novo Acute Myeloid Leukemia by High-Resolution Melting

Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1(R132) and 6% with IDH2(R140)), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, [corrected] or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome.

WT1 isoform expression pattern in acute myeloid leukemia

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression.

Study of the S427G polymorphism and of MYBL2 variants in patients with acute myeloid leukemia

Dysregulation of MYBL2 has been associated to tumorigenesis and the S427G polymorphism could induce partial inactivation of MYBL2, associating it with cancer risk. It has previously been shown that MYBL2 was over-expressed in some acute myeloid leukemias (AML), portending poor prognosis. However, to date no studies have investigated the S427G or other genetic variants of MYBL2 in AML. This study analyzed the S427G in 197 AML patients and 179 controls and screened the MYBL2 sequence in patients. In contrast to other studies in solid tumors, the S427G was not associated with the incidence of AML. This study detected four unannotated genetic alterations, of which the Q67X could be involved in MYBL2 dysfunction. Eight polymorphisms were identified, among which the rs73116571, located in a splicing region, was associated with higher incidence in AML and weaker MYBL2 expression, suggesting pre-disposition to AML. Additional functional studies should be performed to verify these genetic variations as possible targets in AML.

Novel Real-Time Polymerase Chain Reaction Assay for Simultaneous Detection of Recurrent Fusion Genes in Acute Myeloid Leukemia

The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML.