Publications by authors named "Mariam Ibañez"

33 Publications

A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms.

Cancers (Basel) 2021 Apr 18;13(8). Epub 2021 Apr 18.

Hematology Research Group, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain.

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.
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http://dx.doi.org/10.3390/cancers13081947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072643PMC
April 2021

Case Report: Partial Uniparental Disomy Unmasks a Novel Recessive Mutation in the Gene in a Patient With a Severe Phenotype of Chédiak-Higashi Syndrome.

Front Immunol 2021 31;12:625591. Epub 2021 Mar 31.

Department of Hematology, Hospital Universitario y Politécnico La Fe, Barcelona, Spain.

Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive (AR) immune disorder that has usually been associated to missense, nonsense or indels mutations in the gene. In this study, we describe for the first time the case of a CHS patient carrying a homozygous mutation in the gene inherited as a result of a partial uniparental isodisomy (UPiD) of maternal origin. Sanger sequencing of the cDNA and single nucleotide polymorphism (SNP)-arrays were performed to identify the causative mutation and to explain the molecular mechanism of inheritance, respectively. Partial-UPiD leads to a copy neutral loss of heterozygosity (CN-LOH) of the telomeric region of chromosome 1 (1q41q44), unmasking the potential effect of the mutation detected. The mutation (c.8380dupT) is an insertion located in exon 32 of the gene resulting in a premature stop codon and leading to the loss of all the conserved domains at the C-terminal of the LYST protein. This would account for the severe phenotype observed. We also reviewed the only two previously reported cases of CHS as a result of a uniparental disomy. In this study, we show that the combination of different strategies, including the use of SNP-arrays, is pivotal to fine-tune the diagnosis of rare AR disorders, such as CHS. Moreover, this case highlights the relevance of uniparental disomy as a potential mechanism of CHS expression in non-consanguineous families.
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http://dx.doi.org/10.3389/fimmu.2021.625591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044466PMC
March 2021

Analysis of SNP Array Abnormalities in Patients with DE NOVO Acute Myeloid Leukemia with Normal Karyotype.

Sci Rep 2020 04 3;10(1):5904. Epub 2020 Apr 3.

Hematology Service, Hospital Universitario y Politécnico La Fe, Valencia, Spain.

Nearly 50% of patients with de novo acute myeloid leukemia (AML) harbor an apparently normal karyotype (NK) by conventional cytogenetic techniques showing a very heterogeneous prognosis. This could be related to the presence of cryptic cytogenetic abnormalities (CCA) not detectable by conventional methods. The study of copy number alterations (CNA) and loss of heterozygozity (LOH) in hematological malignancies is possible using a high resolution SNP-array. Recently, in clinical practice the karyotype study has been complemented with the identification of point mutations in an increasing number of genes. We analyzed 252 de novo NK-AML patients from Hospital La Fe (n = 44) and from previously reported cohorts (n = 208) to identify CCA by SNP-array, and to integrate the analysis of CCA with molecular alterations detected by Next-Generation-sequencing. CCA were detected in 58% of patients. In addition, 49% of them harbored CNA or LOH and point mutations, simultaneously. Patients were grouped in 3 sets by their abnormalities: patients carrying several CCA simultaneously, patients with mutations in FLT3, NPM1 and/or DNMT3A and patients with an amalgam of mutations. We found a negative correlation between the number of CCA and the outcome of the patients. This study outlines that CCA are present in up to 50% of NK-AML patients and have a negative impact on the outcome. CCA may contribute to the heterogeneous prognosis.
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http://dx.doi.org/10.1038/s41598-020-61589-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125150PMC
April 2020

Acute Promyelocytic Leukemia: A Constellation of Molecular Events around a Single Fusion Gene.

Cancers (Basel) 2020 Mar 8;12(3). Epub 2020 Mar 8.

Department of Hematology, Hospital Universitario y Politécnico La Fe, 46026 Valencia, Spain.

Although acute promyelocytic leukemia (APL) is one of the most characterized forms of acute myeloid leukemia (AML), the molecular mechanisms involved in the development and progression of this disease are still a matter of study. APL is defined by the rearrangement as a consequence of the translocation t(15;17)(q24;q21). However, this abnormality alone is not able to trigger the whole leukemic phenotype and secondary cooperating events might contribute to APL pathogenesis. Additional somatic mutations are known to occur recurrently in several genes, such as , , and , whereas mutations in other common AML genes are rarely detected, resulting in a different molecular profile compared to other AML subtypes. How this mutational spectrum, including point mutations in the fusion gene, could contribute to the 10%-15% of relapsed or resistant APL patients is still unknown. Moreover, due to the uncertain impact of additional mutations on prognosis, the identification of the APL-specific genetic lesion is still the only method recommended in the routine evaluation/screening at diagnosis and for minimal residual disease (MRD) assessment. However, the gene expression profile of genes, such as , and once combined with the molecular events, might improve future prognostic models, allowing us to predict clinical outcomes and to categorize APL patients in different risk subsets, as recently reported. In this review, we will focus on the molecular characterization of APL patients at diagnosis, relapse and resistance, in both children and adults. We will also describe different standardized molecular approaches to study MRD, including those recently developed. Finally, we will discuss how novel molecular findings can improve the management of this disease.
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http://dx.doi.org/10.3390/cancers12030624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139833PMC
March 2020

Do next-generation sequencing results drive diagnostic and therapeutic decisions in MDS?

Blood Adv 2019 11;3(21):3454-3460

Department of Hematology, Hospital Universitario y Politécnico La Fe, Valencia, Spain.

This article has a companion Point by Thol and Platzbecker.
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http://dx.doi.org/10.1182/bloodadvances.2019000680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855105PMC
November 2019

Spanish Guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia.

Br J Haematol 2020 03 16;188(5):605-622. Epub 2019 Oct 16.

Department of Haematology, Hospital Universitari i Politècnic La Fe, València, Spain.

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.
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http://dx.doi.org/10.1111/bjh.16175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064979PMC
March 2020

Clinical and molecular characterization by next generation sequencing of Spanish patients affected by congenital deficiencies of fibrinogen.

Thromb Res 2019 Aug 25;180:115-117. Epub 2019 Jun 25.

Unidad de Hemostasia y Trombosis, Servicio de Hematología, Hospital Universitario y Politécnico La Fe, Valencia, Spain.

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http://dx.doi.org/10.1016/j.thromres.2019.06.015DOI Listing
August 2019

RNA Sequencing Analysis for the Identification of a PCM1/PDGFRB Fusion Gene Responsive to Imatinib.

Acta Haematol 2019 14;142(2):92-97. Epub 2019 May 14.

Hematology Department, University Hospital La Fe, Valencia, Spain.

The platelet-derived growth factor receptor β (PDGFRB) gene translocations lead to a spectrum of chronic myeloid neoplasms, frequently associated with eosinophilia. Clinical heterogeneity is associated with a molecular one. Here, we report a novel case of a patient harboring a t(5;8)(q33;p22) translocation, resulting in the PCM1/PDGFRB fusion. Conventional cytogenetics and RNA sequencing were performed to identify the chromosomes and the genes involved in the rearrangement, respectively. This study shows that the combination of different strategies is pivotal to fine-tune the diagnosis and the clinical management of the patient. After 1 year of treatment with imatinib, the patient achieves hematological and molecular remission. We present an attractive strategy to identify novel and/or cryptic fusions, which will be relevant for clinicians dealing with the diagnosis of the patients with myelodysplastic syndrome/myeloproliferative diseases with atypical manifestations.
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http://dx.doi.org/10.1159/000497348DOI Listing
February 2020

Clinical Utility of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia Diagnostics.

J Mol Diagn 2019 03 19;21(2):228-240. Epub 2018 Dec 19.

Molecular Biology Unit, Clinical Pathology Service, La Fe University and Polytechnic Hospital, Valencia, Spain; La Fe Health Research Institute, Hematology Research Group, Valencia, Spain; CIBERONC, Carlos III Health Research Institute, Madrid, Spain. Electronic address:

Next-generation sequencing (NGS) has redefined the genetic landscape of acute myeloid leukemia (AML), providing new molecular markers for diagnostic and prognostic classifications. However, its application in the clinical setting is still challenging. We hypothesized that a 19-gene AML-targeted NGS panel could be a valid approach to obtain clinically relevant information. Thus, we assessed the ability of this panel to classify AML patients according to diagnostic and prognostic indexes in a cohort of 162 patients. The assay yielded a median read depth >2000×, with 88% of on-target reads and a mean uniformity >93% without significant global strand bias. The method was sensitive and specific, with a valid performance at the clinical variant allele frequency cutoff of 3% for point mutations and 5% for insertions or deletions (INDELs). Three-hundred thirty-nine variants were found (36% INDELs and 64% single nucleotide variants). Concordance between NGS and other conventional techniques was 100%, but the NGS approach was able to identify more clinically relevant mutations. Finally, all patients could be classified into one of the 2016 World Health Organization diagnostic categories and virtually all into the recently proposed prognostic indexes (2017 European LeukemiaNet and Genomic classification). To sum up, we validate a reliable and reproducible method for AML diagnosis and demonstrate that small, well-designed NGS panels are sufficient to guide clinical decisions according to the current standards.
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http://dx.doi.org/10.1016/j.jmoldx.2018.09.009DOI Listing
March 2019

The modular network structure of the mutational landscape of Acute Myeloid Leukemia.

PLoS One 2018 10;13(10):e0202926. Epub 2018 Oct 10.

Hematology Service, Hospital Universitario y Politécnico La Fe, Valencia, Spain.

Acute myeloid leukemia (AML) is associated with the sequential accumulation of acquired genetic alterations. Although at diagnosis cytogenetic alterations are frequent in AML, roughly 50% of patients present an apparently normal karyotype (NK), leading to a highly heterogeneous prognosis. Due to this significant heterogeneity, it has been suggested that different molecular mechanisms may trigger the disease with diverse prognostic implications. We performed whole-exome sequencing (WES) of tumor-normal matched samples of de novo AML-NK patients lacking mutations in NPM1, CEBPA or FLT3-ITD to identify new gene mutations with potential prognostic and therapeutic relevance to patients with AML. Novel candidate-genes, together with others previously described, were targeted resequenced in an independent cohort of 100 de novo AML patients classified in the cytogenetic intermediate-risk (IR) category. A mean of 4.89 mutations per sample were detected in 73 genes, 35 of which were mutated in more than one patient. After a network enrichment analysis, we defined a single in silico model and established a set of seed-genes that may trigger leukemogenesis in patients with normal karyotype. The high heterogeneity of gene mutations observed in AML patients suggested that a specific alteration could not be as essential as the interaction of deregulated pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202926PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179200PMC
March 2019

Negative impact on clinical outcome of the mutational co-occurrence of SF3B1 and DNMT3A in refractory anemia with ring sideroblasts (RARS).

Leuk Lymphoma 2017 07 24;58(7):1686-1693. Epub 2016 Oct 24.

a Department of Hematology , University Hospital La Fe , Valencia , Spain.

The incidence of SF3B1 mutations in patients with RARS is high. Recently, it has been shown that SF3B1 and DNMT3A mutations overlap more often than expected, although it is not clear how this could affect the disease. We studied SF3B1 and DNMT3A in 123 RARS patients: 101 out of 123 samples (82%) had somatic mutations in SF3B1, and 13 of them (13%) showed a co-mutation (SF3B1DNMT3A). All co-mutated patients had a normal karyotype, and 12 of them (92%) were lower-risk patients (IPSS and IPSS-R). Despite their favorable profile, SF3B1DNMT3A patients showed a higher RBC transfusion dependency (92% versus 48%, p = .007), a shorter overall survival (OS) (median, 30 versus 97 months, p = .034), and a higher risk of progression to acute myeloid leukemia (AML) at 5 years (25% versus 2%, p = .023) than SF3B1DNMT3A patients. In conclusion, DNMT3A mutations are present in a significant proportion of SF3B1 patients with a negative clinical impact.
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http://dx.doi.org/10.1080/10428194.2016.1246725DOI Listing
July 2017

The Mutational Landscape of Acute Promyelocytic Leukemia Reveals an Interacting Network of Co-Occurrences and Recurrent Mutations.

PLoS One 2016 17;11(2):e0148346. Epub 2016 Feb 17.

Hematology Service, Hospital Universitario y Politécnico La Fe, Valencia, Spain.

Preliminary Acute Promyelocytic Leukemia (APL) whole exome sequencing (WES) studies have identified a huge number of somatic mutations affecting more than a hundred different genes mainly in a non-recurrent manner, suggesting that APL is a heterogeneous disease with secondary relevant changes not yet defined. To extend our knowledge of subtle genetic alterations involved in APL that might cooperate with PML/RARA in the leukemogenic process, we performed a comprehensive analysis of somatic mutations in APL combining WES with sequencing of a custom panel of targeted genes by next-generation sequencing. To select a reduced subset of high confidence candidate driver genes, further in silico analysis were carried out. After prioritization and network analysis we found recurrent deleterious mutations in 8 individual genes (STAG2, U2AF1, SMC1A, USP9X, IKZF1, LYN, MYCBP2 and PTPN11) with a strong potential of being involved in APL pathogenesis. Our network analysis of multiple mutations provides a reliable approach to prioritize genes for additional analysis, improving our knowledge of the leukemogenesis interactome. Additionally, we have defined a functional module in the interactome of APL. The hypothesis is that the number, or the specific combinations, of mutations harbored in each patient might not be as important as the disturbance caused in biological key functions, triggered by several not necessarily recurrent mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148346PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757557PMC
July 2016

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

Leuk Lymphoma 2016 Feb 19;57(2):429-435. Epub 2015 Jun 19.

a Department of Medical Pathology , Hospital Universitario y Politécnico La Fe , Valencia , Spain.

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.
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http://dx.doi.org/10.3109/10428194.2015.1049167DOI Listing
February 2016

Single-nucleotide polymorphism array-based karyotyping of acute promyelocytic leukemia.

PLoS One 2014 24;9(6):e100245. Epub 2014 Jun 24.

Hematology Department, Hospital Universitari i Politècnic La Fe, Valencia, Spain; Department of Medicine, University of Valencia, Valencia, Spain.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or FLT3 mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100245PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069034PMC
March 2015

WT1 isoform expression pattern in acute myeloid leukemia.

Leuk Res 2013 Dec 22;37(12):1744-9. Epub 2013 Oct 22.

Department of Hematology, Hospital Universitari i Politècnic La Fe, Valencia, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.leukres.2013.10.009DOI Listing
December 2013

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

Leuk Res 2013 Dec 21;37(12):1690-6. Epub 2013 Sep 21.

Department of Medical Pathology, Hospital Universitario La Fe, Valencia, Spain.

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.
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http://dx.doi.org/10.1016/j.leukres.2013.09.015DOI Listing
December 2013

Novel real-time polymerase chain reaction assay for simultaneous detection of recurrent fusion genes in acute myeloid leukemia.

J Mol Diagn 2013 Sep 25;15(5):678-86. Epub 2013 Jun 25.

Laboratory of Molecular Biology, Department of Clinical Chemistry, University Hospital La Fe, Valencia, Spain.

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.
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http://dx.doi.org/10.1016/j.jmoldx.2013.04.003DOI Listing
September 2013

Response to lenalidomide in myelodysplastic syndromes with del(5q): influence of cytogenetics and mutations.

Br J Haematol 2013 Jul 25;162(1):74-86. Epub 2013 Apr 25.

Laboratori de Citogenètica Molecular, Laboratori de Citologia Hematològica, Servei de Patologia, Hospital del Mar, Barcelona, Spain.

Lenalidomide is an effective drug in low-risk myelodysplastic syndromes (MDS) with isolated del(5q), although not all patients respond. Studies have suggested a role for TP53 mutations and karyotype complexity in disease progression and outcome. In order to assess the impact of complex karyotypes on treatment response and disease progression in 52 lenalidomide-treated patients with del(5q) MDS, conventional G-banding cytogenetics (CC), single nucleotide polymorphism array (SNP-A), and genomic sequencing methods were used. SNP-A analysis (with control sample, lymphocytes CD3+, in 30 cases) revealed 5q losses in all cases. Other recurrent abnormalities were infrequent and were not associated with lenalidomide responsiveness. Low karyotype complexity (by CC) and a high baseline platelet count (>280 × 10(9) /l) were associated with the achievement of haematological response (P = 0·020, P = 0·013 respectively). Unmutated TP53 status showed a tendency for haematological response (P = 0·061). Complete cytogenetic response was not observed in any of the mutated TP53 cases. By multivariate analysis, the most important predictor for lenalidomide treatment failure was a platelet count <280 × 10(9) /l (Odds Ratio = 6·17, P = 0·040). This study reveals the importance of a low baseline platelet count, karyotypic complexity and TP53 mutational status for response to lenalidomide treatment. It supports the molecular study of TP53 in MDS patients treated with lenalidomide.
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http://dx.doi.org/10.1111/bjh.12354DOI Listing
July 2013

Analysis of SNP rs16754 of WT1 gene in a series of de novo acute myeloid leukemia patients.

Ann Hematol 2012 Dec 16;91(12):1845-53. Epub 2012 Oct 16.

Department of Hematology, Hospital Universitari i Politècnic La Fe, Bulevar Sur s/n, CP 46026, Valencia, Spain.

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.
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http://dx.doi.org/10.1007/s00277-012-1596-xDOI Listing
December 2012

Rapid screening of ASXL1, IDH1, IDH2, and c-CBL mutations in de novo acute myeloid leukemia by high-resolution melting.

J Mol Diagn 2012 Nov 25;14(6):594-601. Epub 2012 Aug 25.

Department of Hematology, University Hospital La Fe, Valencia, Spain.

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.
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http://dx.doi.org/10.1016/j.jmoldx.2012.06.006DOI Listing
November 2012

Prognostic value of cytogenetics in adult patients with Philadelphia-negative acute lymphoblastic leukemia.

Ann Hematol 2012 Jan 21;91(1):19-25. Epub 2011 Sep 21.

Department of Hematology, Hospital Universitario La Fe, Valencia, Spain.

The prognostic value of cytogenetics in adult acute lymphoblastic leukemia (ALL) is not as established as in childhood ALL. We have analyzed the outcome and prognostic value of karyotype in 84 adults diagnosed with Philadelphia-negative ALL from a single institution that received induction chemotherapy and had successful karyotype performed. The most frequent finding was normal karyotype in 35 (42%) cases, followed by aneuploidies in 20 cases (24%) and t(4;11)(q21;q23)/MLL/AF4 in 5 (6%), and the remaining 24(27%) cases carried miscellaneous clonal abnormalities. The group of patients with t(4;11)(q21;q23)/MLL/AF4, hypodiploidy and low hyperdiploidy (less than 50 chromosomes) showed a worse outcome than those with normal karyotype and miscellaneous abnormalities in terms of overall survival (OS) (3 years OS; 47% vs. 13%, p = 0.014) and relapse-free survival (RFS) (3 years RFS; 44% vs. 27%, p = 0.005). Other cytogenetic prognostic classifications reported to date were tested in our series, but any was fully reproducible. In conclusion, karyotype is a useful tool for risk assessment in adult ALL. We have confirmed the bad prognosis of t(4;11)(q21;q23)/MLL/AF4 and hypodiploidy. Besides, low hyperdiploidy could also define a high-risk group of patients who might be candidates for more intensive treatment.
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http://dx.doi.org/10.1007/s00277-011-1331-zDOI Listing
January 2012

CYP2C8 gene polymorphism and bisphosphonate-related osteonecrosis of the jaw in patients with multiple myeloma.

Haematologica 2011 Oct 17;96(10):1557-9. Epub 2011 Jun 17.

Department of Hematology, Hospital Universitario La Fe, Valencia, Spain.

Osteonecrosis of the jaw is an uncommon but potentially serious complication of bisphosphonate therapy in multiple myeloma. Previous studies showed that the presence of one or two minor alleles of the cytochrome P450, subfamily 2C polypeptide 8 gene (CYP2C8) polymorphism rs1934951 was an independent prognostic marker associated with development of osteonecrosis of the jaw in multiple myeloma patients treated with bisphosphonates. The aim of this study was to validate the frequency of SNP rs193451 in 79 patients with multiple myeloma. In 9 (22%) patients developing osteonecrosis of the jaw, a heterozygous genotype was found, in contrast with those who did not develop osteonecrosis of the jaw (n=4, 11%) or healthy individuals (n=6, 13%). We found no differences in the cumulative risk of developing osteonecrosis of the jaw between patients homozygous and heterozygous for the major allele. We were unable to confirm a significant association between this polymorphism and the risk of developing osteonecrosis of the jaw.
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http://dx.doi.org/10.3324/haematol.2011.042572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186320PMC
October 2011

Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

Ann Hematol 2012 Jan 3;91(1):1-7. Epub 2011 May 3.

Laboratory of Molecular Biology, Department of Medical Pathology, Hospital Universitario La Fe, Escuela de enfermería 7ª planta., Avd. Campanar 21, Valencia 46009, Spain.

During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.
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http://dx.doi.org/10.1007/s00277-011-1234-zDOI Listing
January 2012