Mutational analysis of bone marrow mesenchymal stromal cells in myeloid malignancies

Francesco D'Alo', Emiliano Fabiani, Giulia Falconi, Luana Fianchi, Francesco Guidi, Silvia Bellesi, Maria Teresa Voso, Giuseppe Leone

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1 Citazioni (Scopus)

Abstract

Microenvironment is actively involved in the pathophysiology of hematopoietic malignancies. Mesenchymal stromal cells (MSC) support hematopoiesis through the production and secretion of cytokines, cell–cell interactions and immunomodulating properties. These cells are defined, plastic-adherent, growing cells, are known to express CD105, CD73, and CD90, lack common hematopoietic antigens, and differentiate into osteoblasts, adipocytes, and chondroblasts in vitro [1]. Anomalies of multiple MSC features have been described in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These include significantly reduced growth, proliferative and differentiating capacity, premature replicative senescence, abnormal expression of surface molecules and chemokines, and reduced ability to support hematopoietic stem and progenitor cell growth in long-term culture assays [2]. The molecular basis of MSC dysfunction in MDS and AML is still under investigation. Previous studies have reported the occurrence of non-clonal chromosomal aberrations in bone marrow MSC isolated from patients with MDS and AML, which only very rarely correspond to the cytogenetic markers observed in the hematopoietic leukemic clone of the same individual [3]. Somatic mutations of multiple genes have been described in myeloid malignancies and often concur to identify distinct leukemia subtypes or prognostic subgroups. Recently, deep sequencing approaches have identified new recurrent mutations of genes involved in epigenetic and spliceosome machineries in AML and MDS samples 4, 5 and 6. We investigated the frequency of recurrent mutations of epigenetic and spliceosomal genes, and of FLT3 and NPM1 genes, in matched bone marrow hematopoietic cells and MSC isolated from 41 patients with myeloid malignancies. The study population included 9 patients with de novo AML, 9 with MDS, 7 with chronic myeloproliferative neoplasms (MPN), 3 with secondary AML (sAML, 2 evolved from MDS, 1 from MPN), and 13 with therapy-related myeloid neoplasms (7 t-AML and 6 t-MDS). Bone marrow mononuclear cells (BM-MNC) were isolated at the time of diagnosis by Ficoll gradient centrifugation. MSC were expanded using Mesencult medium (Stem Cell Technologies, Voden Medical Instruments, Milan, Italy) in plastic-adherent cultures up to the second passage. Flow cytometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive, CD105 positive) in more than 99% of the MSC population. DNA was extracted from BM-MNC and MSC using the QIAamp DNA Mini Kit (Qiagen, Milan, Italy). The following hot-spot mutations were studied on genomic DNA by Sanger sequencing (ABI PRISM 3100; Applied Biosystems/Life Technologies, Milan, Italy): IDH1 R132, IDH2 R140 and R172, DNMT3A R882, U2AF1 S34 and R35, SF3B1 exons 13–14 and 15–16, and SRSF2 exon 1 [7]. In addition, FLT3 and NPM1 mutations were analyzed in patients with de novo or therapy-related AML. FLT3 internal tandem duplication and tyrosine kinase domain mutations were studied by reverse transcription polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism RT-PCR, respectively, whereas NPM1 exon 12 mutations were detected by RT-PCR with high-resolution melting curve analysis, followed by Sanger sequencing of positive cases. In BM-MNC, FLT3 internal tandem duplication and FLT3 tyrosine kinase domain mutations were found in two patients (one patient with de novo AML and one patient with a t-AML), whereas NPM1 exon 12 mutations were present in six AML patients (4 de novo, 3 COSM158604 and 1 COSM1319219; 1 sAML, COSM158604; 1 t-AML, COSM28937). No FLT3 and NPM1 mutations were found in MSC compartment in all studied patients. Mutations of IDH1 R132 were found in BM-MNC isolated from one patient with de novo AML (R132C) and in two patients with t-AML (one R132H and one R132L). One t-AML patient had an IDH2 R140Q mutation in BM-MNC, whereas no m
Lingua originaleEnglish
pagine (da-a)731-733
Numero di pagine3
RivistaExperimental Hematology
DOI
Stato di pubblicazionePubblicato - 2014

Keywords

  • Mesenchymal stromal cells
  • Mutational analysis
  • Myeloid malignancies

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