Inés Gómez-Seguí

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.

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].

Single-Nucleotide Polymorphism Array-Based Karyotyping of Acute Promyelocytic Leukemia

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.

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

Abstract 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 (SF3B1mutDNMT3Amut). 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, SF3B1mutDNMT3Amut 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 SF3B1mutDNMT3Awt patients. In conclusion, DNMT3A mutations are present in a significant proportion of SF3B1mut patients with a negative clinical impact.

Absence of mutations in the activation loop and juxtamembrane domains of VEGFR-1 and VEGFR-2 gene in chronic myelomonocytic leukemia (CMML)

Angiogenesis is recognized as a main feature of many human eoplasms. Vascular endothelial growth factor (VEGF) is a potent ngiogenic peptide with exerts its biologic effects by interaction ith either of 2 high-affinity tyrosine kinase receptors, VEGFR-1 FLT1) and VEGFR-2 (KDR). Several studies [1] have shown that oth monocytes and myeloid precursors are able to produce and ecreted VEGF and, commonly, co-express one or both VEGF recepors. On the other hand, overexpression and secretion of VEGF have een observed in 72% of patients with acute myeloid leukemia AML), with the corresponding mRNA of the genes FLT1 or KDR eing expressed in 60% and 19% of AML patients, respectively [2]. Mutations in the activation loop (AL) and/or juxtamembrane JM) domains of FLT1 and KDR genes could potentially lead to contitutive tyrosine kinase activity. In this regard, a comprehensive utational screening of 298 exons of the tyrosine kinome, includng FLT1 and KDR, which was carried out by direct sequencing in cohort of 26 CMML, has found no mutations in tyrosine kinase enes other than JAK2 and RAS [3]. To elucidate the potential role of FLT1 and KDR in MDS, especially n those subgroups characterized by monocytic differentiation, we ave analyzed by direct sequencing AL and JM domains of FLT1 nd KDR in a series of 40 samples of CMML diagnosed according to orld Health Organization criteria. Genomic DNA was obtained from bone marrow samples at the ime of diagnosis. DNA from healthy donors was used as negative ontrol. Direct sequencing was carried out using specific primers anking the AL and JM domains of both genes (exons 16 and 17, espectively) [4] and performed on an ABIPRISM 310 DNA Analyzer Applied Biosystems, Foster City, CA). Sequence analysis was coroborated with RefSeqGene NG 012004.1 for KDR and NG 012003.1 or FLT1. No mutations in AL and/or JM domains of FLT1 and KDR genes ere detected in any of the 40 samples analyzed. In spite of direct equencing techniques have a relatively low sensitivity for detecion of acquired mutations (roughly 20%), the probability of false egative results in this setting is unlikely. CMML is a hematopoietic tem cell disorder and the proportion of mutated clonal cells should lways be far above the sensitivity threshold of the technique. In act, 9 of 40 patients (22%) with available karyotype had a clonal ytogenetic abnormality and the median percentage of abnormal etaphases in these cases was 47% (range, 8–100%). Although our tudy does not rule out the occurrence of mutations in other regions f the FLT1 and KDR genes, the incidence of FLT1 and KDR AL and M domain mutations in patients with CMML is, if any, extremely are.