Leukemia (2013) 27, 1870–1873 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE CSF3R T618I is a highly prevalent and specific mutation in chronic neutrophilic leukemia A Pardanani1, TL Lasho1, RR Laborde1, M Elliott1, CA Hanson2, RA Knudson3, RP Ketterling3, JE Maxson4,5, JW Tyner5,6 and A Tefferi1 Truncation mutations of the receptor cytoplasmic domain for colony-stimulating factor 3 (CSF3R) are frequently seen in severe congenital neutropenia, whereas activating missense mutations affecting the extracellular domain (exon 14) have been described in hereditary neutrophilia and chronic neutrophilic leukemia (CNL). In order to clarify mutational frequency, specificity and phenotypic associations, we sequenced CSF3R exons 14–17 in 54 clinically suspected cases of CNL (n ¼ 35) or atypical chronic myeloid leukemia (aCML; n ¼ 19). Central review of these cases confirmed WHO-defined CNL in 12 patients, monoclonal gammopathy (MG)-associated CNL in 5 and WHO-defined aCML in 9. A total of 14 CSF3R mutations were detected in 13 patients, including 10 with CSF3RT618I (exon 14 mutation, sometimes annotated as CSF3R T595I). CSF3RT618I occurred exclusively in WHO- defined CNL with a mutational frequency of 83% (10 of 12 cases). CSF3R mutations were not seen in aCML or MG-associated CNL. CSF3RT618I was also absent among 170 patients with primary myelofibrosis (PMF; n ¼ 76) or chronic myelomonocytic leukemia (CMML; n ¼ 94). SETBP1 mutational frequencies in WHO-defined CNL, aCML, CMML and PMF were 33, 0, 7 and 3%, respectively. Four CSF3RT618I-mutated cases co-expressed SETBP1 mutations. We conclude that CSF3RT618I is a highly sensitive and specific molecular marker for CNL and should be incorporated into current diagnostic criteria. Leukemia (2013) 27, 1870–1873; doi:10.1038/leu.2013.122 Keywords: myeloproliferative; G-CSF; severe congenital neutropenia; hereditary neutrophilia INTRODUCTION acquired extracellular domain mutations are infrequently reported 8,10 11 Colony-stimulating factor 3 receptor gene (CSF3R), mapping to in acute myeloid leukemia. Most recently, Maxson et al. made chromosome 1p34.3, encodes the trans-membrane receptor for the seminal observation regarding the association between CSF3R granulocyte colony-stimulating factor (G-CSF; CSF3), which pro- mutations and chronic neutrophilic leukemia (CNL). The current vides the proliferative and survival signal for granulocytes and also study was undertaken to determine the frequency, location and contributes to their differentiation and function.1 CSF3R harbors 17 specificity of CSF3R mutations in CNL and the closely related exons and its protein 813 amino acids. The cytoplasmic domain of atypical chronic myeloid leukemia (aCML). CSF3R is functionally assigned to proliferation (proximal region) and differentiation/regulation of proliferation (distal region).2 Nonsense somatic mutations affecting the cytoplasmic domain PATIENTS AND METHODS of CSF3R and leading to carboxyl-truncation have been described Patients and samples in B40% of patients with severe congenital neutropenia, where The current study was approved by the Mayo Clinic institutional review they are acquired and occur in conjunction with other inherited board. Patients were primarily identified through search of hematopathol- mutations (for example, ELANE and HAX1).3 Such mutations appear ogy databases for a diagnosis of ‘CNL’ or ‘aCML’. Inclusion to the current to be stem cell-derived,3 associated with but not essential for study required availability of archived bone marrow or peripheral blood severe congenital neutropenia -associated acute myeloid granulocytes for DNA extraction, as well as bone marrow morphology and 4,5 cytogenetic information at the time of first referral to the Mayo Clinic. The leukemia, promote STAT5-mediated clonal advantage in diagnoses of CNL, aCML, chronic myelomonocytic leukemia (CMML) and mouse progenitor cells6 and co-operate with other oncogenes 5,7 primary myelofibrosis (PMF) were confirmed by World Health Organization to induce acute myeloid leukemia. Severe congenital (WHO) criteria.12 CMML and PMF patients were selected from databases of neutropenia -associated CSF3R mutations occasionally affect the patients previously annotated for other mutations.13,14 Patient information extracellular domain of the receptor.4,8 was updated through review of patient histories and correspondence, A germline CSF3R mutation (C-to-A substitution at nucleotide social security death index or a telephone call to the patient or their local 2088; T617N) was recently described in autosomal dominant physician. hereditary neutrophilia associated with splenomegaly and 9 increased circulating CD34-positive myeloid progenitors; Mutation screening functional studies suggested enhanced receptor dimerization For CSF3R mutation analysis, exons 14–17 were amplified for all clinically and signaling that was abrogated by JAK2 inhibition and suspected cases of CNL or aCML using standard PCR conditions. Primers induction of neutrophilia and splenomegaly in mice.9 Similar but for CSF3R were as follows: 14 forward: 50-CCACGGAGGCAGCTTTAC-30, 1Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA; 2Division of Hematopathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA; 3Division of Cytogenetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA; 4Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA; 5Knight Cancer Institute, Portland, OR, USA and 6Department of Cell and Development Biology, Oregon Health and Science University, Portland, OR, USA. Correspondence: Professor A Tefferi, Division of Hematology, Department of Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA. E-mail: [email protected] Received 12 April 2013; accepted 16 April 2013; accepted article preview online 22 April 2013; advance online publication, 17 May 2013 CSF3R mutations in chronic neutrophilic leukemia A Pardanani et al 1871 14 reverse: 50-AAATCAGCATCCTTTGGGTG-30; 15 forward: 50-TGACTTTGAA distribution of continuous variables between categories were analyzed by TCCCCTGGTC-30, 15 reverse: 50-TGAGGTTCCCTGTGGGTG-30; 16 forward: either Mann–Whitney (for comparison of two groups) or Kruskal–Wallis 50-AAAATGGAAAGATCGGAGGG-30, 16 reverse: 50-CTTGGCTTCAGAAGG (comparison of three or more groups) test. Patient groups with nominal TGTCC-30, and 17 forward: 50-CTGTCACTTCCGGCAACAT-30, 17 reverse: variables were compared by w2 test. Overall survival was calculated from 50-TGGCCCAAAGACACAGTCGT-30. Following amplification, the PCR pro- the date of first referral to the date of death (uncensored) or last contact ducts were sequenced via standard capillary electrophoresis by Applied (censored). Survival curves were prepared by the Kaplan–Meier method Biosystems 3730 series DNA Analyzers (Carlsbad, CA, USA), and results and compared by the log-rank test. The Stat View (SAS Institute, Cary, NC, were analyzed using Sequencher software (Gene Codes Inc., Ann Arbor, MI, USA) statistical package was used for all calculations. USA). Patients with CMML and PMF were screened for exon 14 mutations only. PCR and Sanger sequencing was used for SETBP1 mutation screening in RESULTS PMF, CNL and aCML patients (forward primer 50-ATGCACCCACTTTCAA CACA-30 and reverse primer 50-AAAAGGCACCTTTGTCATGG-30 to generate CSF3R mutation screening included exons 14 through 17 for all 54 sequence for the amino-acid region 825–1013). For the CMML cohort, clinically suspected cases of ‘CNL’ (n ¼ 35) or ‘aCML (n ¼ 19). An we used the ViiA7 quantitative RT-PCR platform (qPCR) and MeltDoctor additional 170 patients with CMML (n ¼ 94) or PMF (n ¼ 76) were high-resolution melting assay (Life Technologies, Grand Island, NY, USA) screened for mutations involving CSF3R exon 14 only. In addition, using forward primer 50-GCGAGATTGGCTCCCTAAAG-30 and reverse primer SETBP1 mutation screening was performed in all the cases. All 0 0 5 -CCAGGGAGCAGAAATCAAAA-3 to generate sequence for the amino- study patients underwent bone marrow biopsy with cytogenetic acid region 860–1000. Targeted cases were validated using Sanger assessment and presence of BCR–ABL1 was excluded in every case sequencing to confirm the presence of a mutation. of suspected CNL by FISH and/or PCR analysis. Statistical analysis All statistical analyses considered clinical and laboratory parameters Confirmation of diagnosis according to WHO criteria obtained at the time of first referral, which coincided in most instances Central review of clinicopathological data for the 54 clinically with the time of bone marrow /granulocyte collection. Differences in the suspected cases of CNL or aCML, in order to identify those who Table 1. Mutational status and other clinical and laboratory characteristics of 27 patients with clinically suspected chronic neutrophilic leukemia Age Sex WHO- MGUS/ CSF3R SETBP1 JAK2 WBC PMN Immature Blast Monocyte defined lymphoma mutation (type) mutation mutation ( Â 109/l) (%) cells (%) (%) (%) 37 F Yes No T618I WT WT 72.8 94 3 0 1 78 M Yes No T618I G870D WT 29.4 78a 11a 00 55 M Yes No T618I WT WT 81.3 85 5 0 2 79 F Yes No T618I, WT WT 38.0 91 0 0 1 2341_2342insC 68 M Yes No T618I D868N WT 49.2 85 7 0 2 79 M Yes No T618I WT 84.7 93 4 0 2 73 M Yes No T618I G870D WT 58.7 82 0 0 10a 80 M Yes No T618I WT WT 21.7 90 0 0 1 47 M Yes No T618I G872R WT 22.6 87 5 0 1 63 F Yes No T618I WT WT 65.9 92 1 0 5 26 F Yes No I598I WT