Marion E. Frew

Aberrant methylation of the negative regulators RASSFIA, SHP-1 and SOCS-1 in myelodysplastic syndromes and acute myeloid leukaemia.

Mutations in the receptor tyrosine kinase (RTK/RAS) signalling pathway frequently provide a proliferative signal in myeloid malignancies. However, the role of RASSF1A, SHP‐1 and SOCS‐1, negative regulators of RTK/RAS signalling, has not been extensively investigated in the myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). This study employed methylation‐specific polymerase chain reaction (MS‐PCR) to determine if aberrant promotor methylation of RASSF1A, SHP‐1 and SOCS‐1 is involved in the pathogenesis of myeloid malignancies. Patients with MDS (n = 107), AML (n = 154) and juvenile myelomonocytic leukaemia (JMML, n = 5) were investigated, together with 15 normal controls. Primers were located in the promotor region of each gene as well as within exon 2 of SOCS‐1. Methylation of RASSF1A was found in five of 55 (9%) MDS cases, but not in any of 57 AML cases studied. RASSF1A methylation was present in one case (20%) of JMML. SHP‐1 methylation was present in 13 of 121 (11%) AML cases but was not found in MDS or JMML. SOCS‐1 promoter methylation was present in eight of 74 (11%) MDS patients but was not seen in JMML or AML. Importantly, RAS mutations and RASSF1A and SOCS‐1 methylation were mutually exclusive indicating that approximately 30% of MDS cases had a defect of the RTK/RAS pathway and its negative regulation. Finally, SOCS‐1 exon 2 methylation may not be pathogenetically relevant, since it was detected in samples from normal individuals and did not correlate with promotor methylation.

JAK2 V617F Mutation is uncommon in chronic myelomonocytic leukaemia

Recently, four independent groups have reported an acquired somatic point mutation in the JAK2 gene (V617F) in approximately 90% of patients with polycythaemia vera as well as nearly half of patients with idiopathic myelofibrosis (IMF) and essential thrombocythaemia (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005). In addition, a report by Steensma et al (2005) suggests that the V617F mutation is infrequent in myelodysplastic syndromes. The JAK2 mutation is likely to contribute to the myeloproliferative state, as cellular expression leads to growth factor independence (James et al, 2005). In this study, we assessed the pathogenetic relevance of the JAK2 mutation in chronic myelomonocytic leukaemia (CMML), a related disorder that has distinctive myeloproliferative and myelodysplastic features (Harris et al, 1999). Mutational analysis, using allele-specific polymerase chain reaction as previously described (Baxter et al, 2005), was undertaken on genomic DNA obtained from the peripheral blood of 47 CMML patients after informed consent [32 males, 15 females, mean age 74 years (range 34– 90 years)], mean white blood cell count (WCC) 19Æ1 · 10/l (range 2Æ7–94 · 10/l). RAS mutations were present in 11% (4/35) (Johan et al, 2005; unpublished data). In addition, 18 cases of IMF and 25 normal samples were analysed. Only one of 47 (2%) CMML patients (74-year-old female, WCC 7Æ5 · 10/l) possessed the JAK2 V617F mutation. This patient also lacked a RAS mutation. In contrast, 8/21 (38%) patients with IMF were positive, in support of previous reports (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005). All 25 normal samples were wild-type for exon 12 JAK2. In conclusion, therefore, our data confirms that the JAK2 V617F mutation is uncommon in CMML (2%) and, in contrast to the classical Ph-negative chronic myeloproliferative disorders, is unlikely to play a significant pathogenetic role.

Mutations in PTPN11 are uncommon in adult myelodysplastic syndromes and acute myeloid leukaemia

The PTPN11 gene encodes the ubiquitously expressed nonreceptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2) (Neel, 1993). SHP-2 is a key molecule in the cellular response to growth factors, hormones, cytokines, and cell adhesion molecules and is required for the activation of the RAS/MEK/ERK kinase cascade (Cunnick et al, 2002). Recently, PTPN11 has been identified as the Noonan syndrome disease gene, through use of a positional candidacy approach (Tartaglia et al, 2001). As a result of the rare association of juvenile myelomonocytic leukaemia (JMML) and Noonan syndrome, Tartaglia et al (2003) screened non-syndromic JMML patients for PTPN11 mutations and documented somatic changes in 34% of cases. Furthermore, the same group reported mutations in 10% and 4% of children with myelodysplasic syndromes (MDS) and de novo acute myeloid leukaemia (AML) respectively. These acquired mutations were predicted to cause gain-of-function of SHP-2 through preferential occupation of the activated state of the phosphatase. To assess the pathogenetic relevance of PTPN11 mutations in adult myeloid disorders, we have undertaken mutational analysis of exons 2, 3, 4, 7, 8 and 13 of PTPN11 in MDS and AML. Genomic DNA was obtained from peripheral blood or bone marrow from 107 cases of MDS, refractory anaemia (RA; n 1⁄4 20), RA with ringed sideroblasts (RARS; n 1⁄4 20), RA with excess blasts (RAEB; n 1⁄4 30), RAEB in transformation (RAEBt; n 1⁄4 2) and chronic myelomonocytic leukaemia (n 1⁄4 35). Genomic DNA was also obtained at presentation of 64 cases of AML entered into the Medical Research council (MRC) AML X and XII Trials. The cases were classified according to the French–American–British (FAB) criteria, as: M0 (n 1⁄4 4), M1 (n 1⁄4 8), M2 (n 1⁄4 13), M3 (n 1⁄4 10), M4 (n 1⁄4 13), M5 (n 1⁄4 10), M6 (n 1⁄4 6). Genomic DNA was also prepared from five cases of JMML [four de novo, one associated with neurofibromatosis type 1 (NF1)], and from the peripheral blood of 35 normal individuals using the Nucleon Biosciences BACC II kit. Genomic DNA was amplified using polymerase chain reaction and the coding sequences and intron/exon boundaries corresponding to exons 2, 3, 4, 7, 8 and 13 were screened as described by Tartaglia et al (2001). Conformation sensitive gel electrophoresis (CSGE) was used to screen for mutations and samples displaying abnormal CSGE profiles were purified (Qiagen) and sequenced (MWG Biotech). Screening 64 cases of AML revealed a single missense mutation (1Æ5%). The patient, classified as M2 with normal cytogenetics, exhibited an exon 3 C fi G transition at nucleotide 218 that is predicted to result in a threonine to isoleucine substitution at codon 73. Interestingly, this individual did not possess a RAS (N-, or Ki), c-FMS, FLT3-ITD, FLT3835 or c-KIT816 mutation. A silent change was identified in exon 2 in another AML patient (nucleotide 48A fi G; Ala16Ala). None of the 107 MDS cases exhibited a PTPN11 mutation. However, one of the four de novo JMML cases possessed an exon 3 G fi A transversion at position 226, which is predicted to result in a glutamic acid to lysine substitution at codon 76. A further de novo JMML case possessed an N-RAS12 mutation but was negative for a PTPN11 mutation, while the NF1-associated JMML was negative for both RAS and PTPN11 mutations. All reported mutations affect residues located at the NSH2 and PTP interacting surfaces and result in gain-offunction of SHP-2 through preferential occupation of the phosphatase’s activated state (Tartaglia et al, 2001, 2003). The two mutations identified in this study, which are located in the N-SH2 domain, have previously been associated with de novo and Noonan syndrome-associated JMML, as well as with paediatric MDS (Tartaglia et al, 2003). Our results suggest that PTPN11 mutations are not common in adult AML, occurring in approximately 1Æ5% of cases. Interestingly, the Thr73Ile mutation, which occurred in a case of AML M2, was not associated with a RAS, cFMS, FLT3 or c-KIT mutation, supporting the concept of mutual exclusivity of RTK/RAS pathway mutations. In contrast to the 10% reported frequency of PTPN11 mutations in paediatric MDS (Tartaglia et al, 2003), none of the 107 adult MDS cases possessed a mutation, suggesting a difference in the pathogenesis of adult and paediatric MDS. Finally, the results from our small study of JMML patients supports the findings of Tartaglia et al (2003), that defects in the regulatory components of the mitogen-activated protein kinase cascade, namely RAS, neurofibromin and SHP-2, appear to be mutually exclusive.