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• W1900543102 abstract "Patients with chronic myeloproliferative neoplasms (MPN) are a group of late onset, progressive haemopoietic disorders characterized by an increased output of mature myeloid cells, bone marrow (BM) fibrosis and risk of transformation to acute myeloid leukaemia (AML) (Abdulkarim et al, 2009). Somatic mutations of Janus Kinase 2 (JAK2), most commonly JAK2V617F, occur at high frequency in MPN patients, with almost full penetrance in polycythaemia vera (PV) patients in particular. Although murine in vivo studies indicate the acquisition of a JAK2 mutation is sufficient for disease development, it is evident from human studies that additional genes are involved (reviewed in Tefferi, 2010). The RUNX1 (AML1) gene product is a transcription factor essential for normal haemopoiesis, acting as a key regulator of haemopoietic-specific genes and is one of the most frequently deregulated genes in AML (Pabst & Mueller, 2007). RUNX1 has been shown to be over-expressed in granulocytes and primary erythroid progenitors of chronic phase MPN patients (Wang et al, 2010) and is one of several genes in which mutations have been identified in blasts from MPN patients who have progressed to leukaemia, suggesting a role for RUNX1 in MPN disease progression (Ding et al, 2009; Beer et al, 2010). Previous RUNX1 mutational screens in small numbers of patients have not identified mutations in chronic phase-MPN. Harada et al (2003) screened 12 PV, 21 essential thrombocythaemia (ET) and 13 idiopathic myelofibrosis (IMF) patients for mutations in RUNX1 exons 3–5; a later study (Harada et al, 2004) screened 13 PV, 21 ET and 15 IMF patients for mutations in exons 6–8 of RUNX1. However these studies, undertaken in the pre-JAK2 V617F era, did not encompass all RUNX1 coding exons (Harada et al, 2003, 2004). To investigate whether disruption of RUNX1 activity may be an early, or possible initiating event in MPN we have performed a comprehensive mutational screen of all RUNX1 exons in genomic DNA isolated from mononuclear cells (MNC) of 47 chronic phase MPN patients with confirmed JAK2 mutant-positive PV [consisting of 45 patients with the JAK2V617F mutation and 2 patients with a JAK2I540-E543delinsMK (JAK2 exon 12) mutation]. Further, we have found previously that mutational changes in MPN patients are not always detectable in heterogeneous cell populations, such as BM and peripheral blood (PB) MNC, but may only be detected by analysis of clonal populations, such as erythroid blast-forming units (BFU-E) (Butcher et al, 2008). Therefore, to facilitate the detection of RUNX1 mutations that may co-exist with JAK2 mutations at low frequency, we screened JAK2 mutation-positive and erythropoietin-independent BFU-E (eBFU-E) isolated from a subset of 16 patients. MPN patients fulfilled World Health Organization criteria for the diagnosis of PV. Approval for the study was obtained from the Human Research Ethical Review Committees of the Royal Adelaide Hospital, The Queen Elizabeth Hospital and Walter and Eliza Hall Institute of Medical Research. MNC were isolated from BM or PB by standard Ficoll-Paque PLUS™ density centrifugation procedure (GE Healthcare Inc., Uppsala, Sweden). BFU-E, eBFU-E and myeloid colony-forming units (CFU-myeloid) were isolated from 12 to 14 day cultures of MNC as described (Butcher et al, 2008). Mesenchymal stromal cells (MSC) were isolated from BMMNC seeded at 2·4 × 105 cells/ml in α-minimal essential medium supplemented with 20% fetal bovine serum and 100 μmol/l l-ascorbate-2-phosphate and incubated at 37°C, 5% CO2 for 24 h. Following removal of non-adherent cells, MSC were further maintained in culture over several weeks with three passages by trypsinization to ensure the removal of residual, adherent haemopoietic cells. The JAK2V617F mutation was detected in PV patient genomic DNA by single nucleotide primer extension and quantification using the SNaPshot Multiplex kit (Applied Biosystems, Carlsbad, CA, USA). The JAK2 exon 12 mutation was detected by polymerase chain reaction (PCR) amplification and sequencing as previously described (Butcher et al, 2008). The known coding exons of RUNX1 and flanking intronic regions were screened using High Resolution Melt analysis followed by PCR amplification and sequencing of samples displaying an aberrant melting profile, or using direct PCR amplification and sequencing alone. Primer sequences and PCR conditions are available on request. No RUNX1 coding changes were detected in MNC or eBFU-E of the chronic phase PV patients, indicating that mutations are infrequent in this phase of the disease. However, a RUNX1 mutation was detected in BMMNC isolated from a further patient (PV64) who was transforming from PV to leukaemic disease (see Butcher et al, 2008 for patient details). The mutation consisted of a single nucleotide insertion in exon 8 of RUNX1, resulting in a C-terminal frameshift and generation of 196 amino acids of novel sequence (RUNX1L403fsTer599) (Fig 1). Although this mutation has not been previously described, similar mutations have been reported in MDS/AML that result in reduced DNA binding and transactivation potential of RUNX1 (Harada et al, 2004). We have reported previously in patient PV64 a six nucleotide deletion in JAK2 exon 12 (JAK2R541-E543delinsK) that was detected in eBFUE but not in BMMNC (Butcher et al, 2008). To ascertain whether the RUNX1 and JAK2 mutations detected in patient PV64 were present in the same disease clone we used a BM sample obtained during the accelerated phase of disease to generate MSC, and isolate erythroid and myeloid colonies for genotyping. The RUNX1L403fsTer599 mutation was not detectable in MSC indicating that it was somatically acquired. We confirmed the presence of the JAK2 exon 12 mutation in BFU-E; however the RUNX1 mutation was not present in this colony. RUNX1L403fsTer599 was detected in a myeloid colony where it was clearly associated with loss of the normal RUNX1 allele, but not with the JAK2 mutation (Fig 1A). Thus, there are divergent disease clones in this patient, with a JAK2 mutant clone contributing to the erythroid lineage while a RUNX1 mutant clone is producing myeloid progenitors, suggesting the JAK2 and RUNX1 mutations were independently acquired and may have individually contributed to the two disease entities of MPN and acute leukaemia. RUNX1 and JAK2 mutations associated with PV patient PV64 who progressed from MPN to leukaemic disease. (A) JAK2 exon 12 and RUNX1 exon 8 mutations detected by sequence analysis of genomic DNA samples. Sequences were obtained from the identical DNA source as shown. Red arrows indicate a six-nucleotide deletion within JAK2 exon 12 (JAK2R541-E543delinsK) detectable only in BFU-E. Black arrows indicate a T-nucleotide insertion within RUNX1 exon 8 detected in BMMNC. This mutation predicts termination of the normal RUNX1 sequence at amino acid position 402, followed by 196 amino acids of novel sequence (RUNX1L403fsTer599) and is associated with loss of the normal RUNX1 allele as seen in sequence derived from a CFU-myeloid. (B) Schematic representation of the functional protein domain structure of RUNX1 and RUNX1L403fsTer599. Asterisks indicate similar, previously reported C-terminal frameshift mutations of RUNX1 detected in sporadic MDS/AML patients for which functional studies show loss of DNA binding and transactivation potential by RUNX1 (Harada et al, 2004). Solid black areas denote regions of novel amino acid sequence conferred by the frameshift mutations. TA, transactivation domain. These findings are consistent with the hypothesis proposed by others that as yet unidentified genetic lesions contribute to a pre-clinical and unstable haemopoietic phenotype in MPN that is associated with a propensity for the acquisition of mutations in multiple genes, that can drive the development of divergent disease clones (Abdel-Wahab et al, 2010; Tefferi, 2010). The authors would like to thank the Royal Adelaide Hospital Hematology Day Centre staff for valuable assistance with collection of patient samples and Amandine Carmagnac, Peter Brautigan, Milena Stankovic and Glenice Cheetham for assistance with mutation screening. This work was supported by grants from the National Health and Medical Research Council of Australia, the Leukaemia Foundation of Australia and Cancer Council of South Australia and by MedVet Pty Ltd. CLC was supported by a Dora Lush Postgraduate Award." @default.
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• W1900543102 title "RUNX1 mutations are rare in chronic phase polycythaemia vera" @default.
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