Mariam Ibáñez

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

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 × 109/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 × 109/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.

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.

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

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.

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.

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.

Aberrant methylation of tumor suppressor genes in patients with refractory anemia with ring sideroblasts

This study evaluates the incidence and prognostic impact of aberrant methylation of 25 tumor suppressor genes in 40 patients with RARS, a MDS subtype, by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Methylation of at least one gene was detected in 18 patients (45%). The genes methylated were CDKN2B (20%), RASSF1 (18%), RARB (10%), CDH13 (7.5%) and FHIT (5%). Patients with at least one methylated gene had a significantly shorter OS than patients without methylated genes. Aberrant methylation is a frequent event in patients with RARS as in patients with high-risk MDS appears to confer a worse prognosis.

In vitro all-trans retinoic acid sensitivity of acute myeloid leukemia blasts with NUP98/RARG fusion gene

Dear Editor, Retinoic acid receptors (RARs) are nuclear hormone receptors functioning as ligand-activated transcription factors which interact specifically in order to modulate transcription DNA elements. RARs include alpha (RARA), beta (RARB), and gamma (RARG) receptor types. They function as heterodimers with retinoid X receptors (RXRs). Heterodimers formed by RXRA-RARG are necessary for growth arrest and visceral and primitive endodermal differentiation [1, 2]. The RARA is known to be involved in the t(15;17) chromosome translocation uniquely associated with acute promyelocytic leukemia (APL) which generates the RARA/PML fusion [1]. In a few APL cases, RARA is rearranged with partner genes other than PML as a result of variant translocations [3, 4]. In a previous report, we described a novel fusion protein generated by a translocation t(11;12)(p15;q13) that involved the genes NUP98 and RARG in a patient with acute myeloid leukemia (AML) [5]. The involvement of RARG in a chimeric fusion transcript had not been reported previously in human leukemia. Interestingly, the patient blasts displayed morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. As reported, the patient achieved complete remission with standard AML chemotherapy followed by autologous peripheral blood transplantation [5]. Two years later, the patient relapsed with the same morphologic, immunophenotypic and molecular features displayed at diagnosis. The patient achieved second complete remission after the combination of all-trans retinoic acid (ATRA) with standard salvage treatment for relapse, but he died due to an infection complication after umbilical cord blood transplantation. Since it has been previously reported that fusion proteins generated with RARB and RARG also confer responsiveness to ATRA in vitro and in vivo [1, 2], we tested the in vitro sensitivity to ATRA of the patient leukemic blasts. Leukemic cells obtained from the bone marrow were resuspended in RPMI1640 medium, supplemented with 15% FCS and antibiotics at a concentration of 10cells/ml. ATRA (Sigma, St Louis,MO, USA) was added at a final concentration of 10 m. The differentiation effect was assessed by analyzing morphological characteristics (nucleus/ cytoplasm ratio, nuclear shape, granulations, and basophilia) on May-Grünwald-Giemsa stained cytospin slides and by multiparametric flow cytometry using the monoclonal antibodies CD10, CD11b, CD13, CD15, CD16, CD33, CD45, CD117, HLADR, and 7-AAD. Serial studies were performed after incubation at 3 and 5 days for all cell cultures. The experiment was performed in triplicate, and the ATRAsensitive NB4 cell line was used as a control. The analysis did not show changes either in morphological features or differentiation patterns measured by immunophenotype, whereas ATRA-treated NB4 cells showed both morphological differentiation and an increase in CD11b expression as expected (Fig. 1). Since ATRAwas given simultaneously with chemotherapy to our patient, we were unable to evaluate the effect of this agent in vivo. The in vitro studies, however, showed that AML with a NUP98/RARG rearrangement is resistant to Jose Cervera and Miguel A. Sanz senior authors contributed equally to this work. E. Such (*) : L. Cordón :A. Sempere : E. Villamón :M. Ibañez : I. Luna : I. Gómez-Seguí :M. López-Pavía :C. Alonso : J. Cervera :M. A. Sanz Hematology Department, Hospital Universitari i Politècnic La Fe, Valencia, Spain e-mail: such_esp@gva.es

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.

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

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.

Screening for IDH mutations in chronic myelomonocytic leukemia

Recent genome-wide sequencing eff orts have led to the identifi cation of a number of new candidate genes involved in the pathogenesis of myeloid neoplasms. Among them, variations in isocitrate dehydrogenase genes ( IDH1 and IDH2 ) have been identifi ed in several hematologic malignancies. IDH1 and IDH2 mutations have been found in 8 – 10% and 15% of patients with acute myeloid leukemia, res pectively, especially in those having normal karyotype (NK-AML). Both gene mutations were recently also reported in myelodysplastic syndromes and chronic myeloproliferative disorders, and were associated with a high rate of leukemic transformation and poor prognosis [1 – 3]. A few studies have been published on somatic mutations of IDH1 and IDH2 in chronic myelomonocytic leukemia (CMML). Th e reported incidence of these mutations in CMML ranged from 0 to 9% [3 – 7], although their prognostic relevance remains uncertain. All reported IDH1 mutations involve the R132 residue, while IDH2 mutations involve either the R140 or the R172 residue. Mutations in IDH decreased affi nity to isocitrate, displaying a neomorphic catalytic activity toward α -ketoglutarate, producing an accumulation of 2-hydroxyglutarate [2,8 – 10]. It is currently believed that these intracellular changes facilitate oncogenic pathways, including activation of hypoxia-inducible factor-1 α (HIF-1 α ). Nevertheless, their relative contribution is still unclear [2].