Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations
Introduction
Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). As the JAK2 V617F mutation is present in a significant proportion of RARS-T patients [1], [2], [3], [4], [5], [6], [7], many investigators consider this entity to be more closely related to MPN, as reflected in the newest version of the 2008 revised WHO classification of myeloid neoplasms [8]. Some cases of RARS-T revealed the MPL W515L mutation typical for classical MPN [9], [10]. In addition, we have previously postulated that an aberrant STAT5 phosphorylation pattern may be a characteristic feature of true RARS-T, while negative cases may result from an unrelated etiology [11]. Consequently, a significant minority of patients with RARS-T that does not harbor JAK2/MPL mutations may simply represent RARS cases with other thrombocytosis-inducing conditions, or be due to unrelated molecular lesions producing a similar phenotype. Conversely, morphologically indistinguishable phenotypes may be due to different gene mutations functionally connected by convergent networks.
Using single nucleotide polymorphism (SNP) arrays we have recently observed a frequent area of somatic uniparental disomy (UPD) at 4q24, most commonly encountered in patients with chronic myelomonocytic leukemia (CMML), mixed MDS/MPN, some typical MDS, secondary acute myeloid leukemia (sAML) and one report on RARS-T [12], [13], [14], [15], [16], [17], [18]. Overlapping microdeletions on 4q24 pointed towards the TET2 gene, in which mutations were identified by us and other groups in different myeloid malignancies, most significantly MPN and MDS/MPN [15], [16], [19]. Recent studies have also identified mutations of ASXL1 (additional sex combs like 1) gene in MDS and MPN [20], [21] that is located in the chromosomal region 20q11. ASXL1, along with TET2, is a member of WNT pathway and possibly regulates histone modifications [21]. Based on the association with MDS/MPN, and the established correlation of RARS-T with JAK2 V617F and MPL W515L mutations, we therefore sought to evaluate the mutational status of TET2 and ASXL1 in RARS-T patients.
Section snippets
Patients
Bone marrow and blood samples were collected from 23 patients with RARS-T. Informed consent was obtained according to protocols approved by the IRB of the Cleveland Clinic. The RARS-T patient characteristics are presented in Table 1. Metaphase cytogenetic (MC) analysis was performed on marrow aspirates according to standard methods.
DNA extraction
The Genomic DNA Purification Kit (Gentra Systems, Inc., MN) was used for DNA isolation according to manufacturer instruction.
TET2 and ASXL1 sequencing. JAK2 V617F and MPL W515L mutation detection by ARMS-PCR
The status of the JAK2 V617F and MPL
Results and discussion
The previously reported cohort of 20 patients with RARS-T [11] has been extended to newer cases and all patients were analyzed for the presence of ASXL1 and TET2 mutations. The extended group of 23 patients with phenotypic features consistent with RARS-T (Table 1) was subjected to MC and SNP-A analysis. All patients showed the presence of ring sideroblasts (>15%), some degree of reticulin fibrosis and varying degrees of thrombocytosis (>450 × 109/L). SNP-A facilitated detection of previously
Conflict of interest
None.
Acknowledgements
This work was supported by NIHR01 HL082983 (JPM), U54 RR019391 (JPM), K24 HL077522 (JPM), Award from AA & MDS International Foundation and a charitable donation from the Robert Duggan Cancer Research Fund.
Contributions. HS designed and performed molecular assays, wrote the manuscript; AMJ performed molecular assays and analyzed results; HM performed molecular assays, JB performed immunohistochemistry, NB classification of patients and database; EDH reviewed pathologic specimens, interpretation
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