La mutazione di tipo A della nucleofosmina nella diagnosi e nel follow-up della leucemia mieloide acuta

Acute myeloid leukaemias (AML), which account for about 80% of the acute leukaemias in adult, represent a phenotypically and genetically heterogeneous group of clonal haematopoietic diseases in which, failure to differentiate and over proliferation in the stem cell/progenitor compartment, result in accumulation of non-functional myeloblasts. Nucleophosmin-1 (NPM1) mutations is the most frequent gene alteration in AML (30%), and are particularly frequent in AML with normal karyotype (AML-NK) (60%). These alterations have been shown to carry prognostic significance because they seem to identify patients with better response to chemotherapy. As a consequence, the analysis of NPM1 mutational status is now recommended for inclusion in the genetic routine characterization of AML (Gorello P. et al., Leukemia 2006; Ammatuna E. et al., Clin Chem 2005; Nelida I.N. et al., Leukemia 2005; Scholl S. et al., Leuk Res 2007; Lin LI et al., Leukemia 2006). Finally, given their high prevalence and stability over the course of the disease, NPM1 mutations may serve as an ideal target for minimal residual disease (MRD) assessment, particularly for patients with AML-NK (Gorello P. et al., Leukemia 2006). In early 2005, Falini et al. (Falini B. et al., New Engl J Med 2005) reported that NPM1 gene mutations, mainly consisting of small nucleotide insertion/deletions, occur frequently in AML and are strongly associated with NK. Over 40 different types of mutations in the NPM1 (NPM1m) locus have been described so far, which result in the formation of different mutant proteins. This alterations consist of the insertion/deletion of short nucleotide stretches (4 or 10 bps) after nucleotide position 956 in NPM1 exon 12 that lead to an ORF shift resulting in different protein variants containig novel C-terminus portions. The most common NPM1 mutation is the type A (NPM1 mut-A), accounting for 75% - 85% of cases, and consist of duplication of a TCTG tetranucleotide at position 956 to 959 of the reference sequence (GenBank accession number NM_002520). Available methods to detect nucleophosmin mutations are costly, require sophisticated equipment, and are often less sensitive. The aim of this work was to develop two assays that enable rapid and sensitive detection of NPM1 mut-A (Ottone T. et al., Genes Chromosomes Cancer 2009): a fragment analysis method and an RT-ASO-PCR (Reverse Trascriptase Allele Specific Oligonucleotide Polymerase Chain Reaction) method. Furthermore a semi-nested ASO-PCR method was also designed in order to increase the sensitivity of our assay for the monitoring of MRD. After informed consent, bone marrow and peripheral blood cells were collected at presentation from 107 patients with AML diagnosed at the Institute of Hematology, Department of Biopathology of the University Tor Vergata of Rome. For each patient reverse trascribed cDNA was divided into three aliquotes used for capillary elettrophoresis, sequencing and ASO-PCR. Semi-nested ASO-PCR experiments were carried out using RT-ASO-PCR products. For capillary electrophoresis fluorescent labeled PCR products were used to screen for the presence of NPM1 mutations using the CEQTM 8000 Genetic Analysis Sistem (Beckman Coulter) instrument. Electroferogram showed 348 bp and 352 bp fragment size, corresponding to NPM1wt and NPM1m, respectively. OCI-Aml3 cell line was used as a positive control. The sensitivity of method was tested using the following serial dilutions of the mutated with wild type allele: 0.01, 0.10, 0.25, 0.50, 0.75, 1.00 (mut/wt). To confirm fragment analysis results, 17 NPM1m and 10 NPM1wt samples were sequenced: cDNA were amplified and the products, after purification, were prepared for sequencing using the CEQTM 8000 instrument. With the aim of analyzing the presence of type A mutation in NPM1 exon 12, an RT-ASO-PCR strategy was used. A forward primer was designed to specifically amplify NPM1 exon 12 only if the mutation A is present; this primer in fact contains an intentional mismatch at third nucleotide from 3’ end to improve specificity. The amplified region includes the insertion of a TCTG tetranucleotide and corresponds to a 320 bp amplified product. As internal PCR control the ABL housekeeping gene was also amplified in each sample. In order to increase the sensitivity for the assay for MRD monitoring, we set up a semi-nested ASO-PCR; for this purpose an internal forward primer NPM-AN for NPM1 mut-A was designed, corresponding to a 319 bp amplified fragment. Fragment analysis allowed to identify 17 NPM1m samples. All the NPM1m samples were heterozygous confirming that NPM1m allele is negative dominant. As concerning sensitivity method enabled to detect the NPM1 mutations present in concentration of 10-2. Sequencing analysis confirmed in all instances NPM1m and NPM1wt cases, showing that 12 samples harboured the most frequent type A mutation, the remaining mutated samples showing type B (3 cases), type D (1 case) and type K mutation (1 case). RT-ASO-PCR assay confirmed the capillary electrophoresis and sequencing results, showing a concordance in 100% of cases; in fact this method identified the 12 out of 17 NPM1m samples as NPM1 mut-A, with an overall frequency of 70%. The semi-nested ASO-PCR assay was carried out to increase the sensitivity of this assay for the monitoring of MRD. The sensitivity calculated for both methods, RT-ASO-PCR and semi-nested ASO-PCR, indicated that this assays could detect the NPM1 mut-A present in concentration of 10-2 and 10-5, respectively. Out of 107 patients, cytogenetic data were available for 99 patients, divided into 2 groups: a group (n=15) consisting of NPM1m and a group (n=84) with NPMwt. Twenty seven out of 99 had normal karyotype. Amongst twenty seven, 13 were NPM1m with an incidence of 48%. The capillary electrophoresis assay developed in this work proved to be a fast, reproducible and easy method applicable to NPM1m diagnosis, capable to discriminate 1 mutated cell out of 100. RT-ASO-PCR and the more sensitive semi-nested ASO-PCR were developed for specifically detect NPM1mut-A, the most frequent NPM1 mutation. These methods were capable to detect 1 mutated cell out of 100 and out of 100.000, respectively. Monitoring more patients by these new assays in MRD longitudinal studies could be relevant for better assess molecular remission and the risk of relapse in AML-NK.