CSF3R T618I Is a Highly Prevalent and Specific Mutation in Chronic

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 WT 34.3 85 0 0 7 72 M Yes No M696T WT WT 34.0 78a 801 64 M Yes Yes WT WT WT 28.7 90 0 0 1 71 F Yes Yes WT WT WT 48.4 82 5 0 5 52 M Yes Yes WT WT WT 27.8 90 0 0 3 68 F Yes Yes WT WT WT 138.0 95 0 0 4 83 M Yes Yes WT WT WT 114.3 84 7 0 5 81 M Yes Yesb WT WT V617F 74.6 93 2 1a 1 59 F No No M696T WT WT 24.1 65 24 1 4 73 M No Yes WT WT V617F 16.7 85 4 1 1 78 F No Yes WT WT WT 47.4 73 14 3 1 71 M No Noc WT WT WT 26.9 82 2 4 0 80 M No Nod WT WT 12.8 80 0 0 13 71 M No No WT WT WT 167.8 65 20 3 9 81 M No No WT WT WT 38.1 89 2 0 6 21 M No No WT WT WT 54.3 76 15 0 1 90 M No No WT G870S WT 18.8 83 8 0 3 59 F No No WT WT WT 22.4 90 0 0 1 74 F No No WT WT WT 70.1 74 18 0 1 63 M No No WT WT WT 44.9 74 15 0 0 68 F No No WT WT V617F 11.8 71 1 0 3 72 M No No WT WT WT 15.9 78 2 0 7 76 M No No WT G870S WT 64.2 69 21 0 0 70 M No No WT WT WT 62.8 55 36 0 0 83 M No No WT WT WT 48.2 64 24 0 0 Abbreviations: CSF3R, colony-stimulating factor 3 receptor; F, female; JAK, Janus kinase; M, male; MGUS, monoclonal gammopathy of uncertain significance; PMN, polymorphonuclear; SETBP1, SET binding protein 1; WBC, white blood cells; WHO, World Health Organization; WT, wild type. aOccasional values were out of range; however, majority of test results fit WHO definition of chronic neutrophilic leukemia. bLow-grade follicular lymphoma. cCD5-negative lymphoproliferative disorder. dMantle cell lymphoma. Cytogenetic studies among the 12 WHO-defined CNL cases were all normal, at time of diagnosis.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1870 – 1873 CSF3R mutations in chronic neutrophilic leukemia A Pardanani et al 1872 strictly met WHO criteria for such diagnoses, was performed Clinical correlations before the results of mutation analysis became available. We found no significant correlation between the three CNL groups Accordingly, five broad patient groups could be defined: (i) CNL- (WHO-defined vs MG-associated vs unconfirmed) and either age, suspected cases meeting WHO criteria for CNL and without gender or leukocyte count (P40.05). Similarly, there was no concomitant monoclonal gammopathy (MG) or lymphoid neo- significant correlation between presence of CSF3R mutation and plasm (WHO-defined CNL; n ¼ 12); (ii) CNL-suspected cases either age, gender or leukocyte count (P40.05). After a median meeting WHO criteria for CNL but with associated MG or lymphoid follow-up of 18 months (range 1–146), 23 deaths (66%) were neoplasm (MG-associated CNL; n ¼ 6); (iii) CNL-suspected cases recorded in the overall cohort of clinically suspected CNL. The not meeting WHO criteria for CNL (n ¼ 17); (iv) aCML-suspected median survival of patients with MG-associated CNL was cases meeting WHO criteria for aCML (n ¼ 9); and (v) aCML- significantly (Po0.05) longer than the other two CNL subgroups suspected cases not meeting WHO criteria for aCML (n ¼ 10). (Figure 1). Considering all 35 cases of clinically suspected CNL, the Among the six patients with MG-associated CNL, five had presence or absence of CSF3R mutation did not affect survival monoclonal gammopathy with uncertain significance (MGUS) and (Figure 2; P ¼ 0.83), whereas there was a trend for shortened one a low-grade lymphoproliferative neoplasm. One patient each survival among SETBP1-mutated patients (Figure 3; P ¼ 0.1). with MGUS subsequently developed overt multiple myeloma and smoldering multiple myeloma. Of the 17 patients with unconfirmed CNL, 2 had MGUS (12%) and 1 a lymphoproliferative DISCUSSION neoplasm (6%). The reasons for the 17 cases of ‘CNL’ not meeting The current study confirms the seminal observation by Maxson WHO criteria included the presence of concurrent infection/ et al.11 regarding the association between CNL and CSF3R inflammatory condition (n ¼ 4), circulating immature cells 410% mutations and clarifies a number of clinically relevant issues (n ¼ 6), circulating blasts X1% (n ¼ 2), leukocyte count 9 including mutational frequency, location and specificity in strictly o25 Â 10 /l (n ¼ 2), dysplastic changes (n ¼ 1), or alternative WHO-defined CNL. We show an apparently invariable association hematological diagnosis (CMML or polycythemia vera; n ¼ 2). between these mutations and WHO-defined CNL and a high Relevant clinical data for the 35 clinically suspected cases of CNL degree of specificity. The most frequent CSF3R mutation we are summarized in Table 1. Among the 19 patients with clinically suspected aCML, central review disclosed 9 cases of WHO-defined aCML. The other 10 were confirmed to have BCR–ABL1-positive CML (n ¼ 2), CMML (n ¼ 3), 1 myelodysplastic syndrome/myeloproliferative neoplasm-unclassi- fied (MDS/MPN-U; n ¼ 2) PMF (n ¼ 1), systemic mastocytosis 0.8 P=0.045 associated with MDS/MPN-U (n ¼ 1), and MPN-U (n ¼ 1). The 170 cases of CMML or PMF were all confirmed to meet WHO 0.6 diagnostic criteria.

Survival 0.4 CSF3R and SETBP1 mutation results 0.2 A total of 14 CSF3R mutations were identified in 13 patients, all of whom belonged to the group with either WHO-defined CNL (n ¼ 12) or unconfirmed CNL (n ¼ 1). The overall CSF3R mutational 0 frequency was 100% in WHO-defined CNL vs 0% in MG-associated 020406080 100 120 140 160 CNL vs 6% in CNL-suspected cases not meeting WHO criteria for Months CNL vs 0% in WHO-defined aCML vs 0% in aCML-suspected cases Monoclonal gammopathy (MG)-associated CNL: n=6; median 60 months not meeting WHO criteria for aCML (Po0.0001). CSF3RT618I was WHO-defined CNL without MG: n=12; median 21 months the most frequent CSF3R mutation, occurring in 10 (all with WHO- Unconfirmed CNL (not meeting WHO criteria): n=17; median 14 months defined CNL) of the 13 patients with CSF3R mutations; the Figure 1. Kaplan–Meier survival curves for patients with WHO- remaining 3 patients harbored CSF3RI598I (WHO-defined CNL) or defined CNL without MG (n ¼ 12), MG-associated CNL (n ¼ 6), and CSF3RM696T (one case each with WHO-defined CNL and unconfirmed CNL (not meeting WHO diagnostic criteria; n ¼ 17). unconfirmed CNL). CSF3RT618I occurred exclusively in WHO-defined CNL with mutational frequency of 83% and was absent in WHO-defined 1 aCML, MG-associated CNL or all other cases of unconfirmed CNL or aCML. Furthermore, similar exon 14 mutations were absent in 0.8 another 170 patients with CMML or PMF. One WHO-defined CNL patient with CSF3RT618I (extracellular domain mutation) also 0.6 P=0.83 harbored another CSF3R exon 17 mutation (2341_2342insC; cytoplasmic domain), which introduces stop codon 7 residues

Survival 0.4 from the insertion site. Among all 34 cases with clinically suspected CNL, six harbored SETBP1 mutations (G870D/S ¼ 4; G872R ¼ 1; and D868N ¼ 1); 4 of 0.2 the 6 patients, all with WHO-defined CNL, also harbored CSF3RT618I mutations. Overall, SETBP1 mutational frequencies in 0 WHO-defined CNL, aCML, CMML and PMF were 33, 0, 7 and 3%, 0 20406080100 120 140 160 respectively. JAK2V617F screening among all clinically suspected Months CNL patients disclosed only three positive cases (one had MG- CSF3R-unmutated cases of clinically suspected CNL: n=22; median 19 months associated CNL and two had unconfirmed CNL). Among the 10 CSF3R-mutated cases of clinically suspected CNL: n=13; median 22 months patients with unconfirmed aCML, one patient whose diagnosis Figure 2. Kaplan–Meier survival curves for patients with clinically was revised to CMML displayed SETBP1 mutation (D868N) and two suspected CNL who were found to harbor CSF3R mutations (n ¼ 13) with revised diagnoses of CMML or PMF harbored JAK2V617F. versus those without CSF3R mutations (n ¼ 22).

Leukemia (2013) 1870 – 1873 & 2013 Macmillan Publishers Limited CSF3R mutations in chronic neutrophilic leukemia A Pardanani et al 1873 1 AUTHOR CONTRIBUTIONS A Pardanani and A Tefferi designed the study, contributed patient samples, 0.8 analyzed the data, and wrote the paper. J Tyner contributed to data analysis and writing of the paper. MA Elliott contributed patient samples. CA Hanson reviewed bone marrow histology. RA Knudson and RP Ketterling reviewed the 0.6 P=0.1 cytogenetics data. TL Lasho and R Laborde performed the molecular studies.

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