Letters to the Editor 1363 3 Steinbach D, Legrand O. ABC transporters and drug resistance in leukaemia: was 10 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative

P-gp nothing but the first head of the Hydra? Leukaemia 2007; 21: 1172–1176. CT method. Nat Protoc 2008; 3: 1101–1108. 4 Burger H, van Tol H, Boersma AW, Brok M, Wiemer EA, Stoter G et al. 11 Jordanides NE, Jorgensen HG, Holyoake TL, Mountford JC. Functional ABCG2 is mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ overexpressed on primary CD34 þ cells and is inhibiteb by imatinib mesylate. ABCG2 drug pump. Blood 2004; 104: 2940–2942. Blood 2006; 108: 1370–1373. 5 Thomas J, Wang L, Clark RE, Pirmohammed M. Active transport of imatinib into 12 Porro A, Iraci N, Soverini S, Diolaiti D, Gherardi S, Terragna C et al. c-Myc and out of cells: implications for drug resistance. Blood 2004; 104: 3739–3745. oncoprotein dictates transcriptional profiles of ATP-binding cassette transporter 6 Wang L, Giannoudis A, Lane S, Williamson P, Pirmohamed M, Clark RE. Expression genes in chronic myeloid . Mol Cancer Res 2011; 8: 1054–1066. of the uptake drug transporter hOCT1 is an important determinant of the 13 Tang L, Bergevoet SM, Gilissen C, de Witte T, Jansen JH, van der Reijden BA et al. response to imatinib in chronic myeloid leukaemia. Clin Pharm Therapeut 2008; Hematopoietic stem cells exhibit a specific ABC transporter gene 83: 258–264. expression profile clearly distinct from other stem cells. BMC Pharmacol 2010; 10: 7 Hu S, Franke RM, Filipski KK, Hu C, Orwick SJ, de Bruijn EA et al. Interaction of 1–9. imatinib with human organic ion carriers. Clin Cancer Res 2008; 14: 3141–3148. 14 Giannoudis A, Davies A, Lucas C, Harris RJ, Pirmohamed M, Clark RE. Effective 8 Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B et al. Gene expression uptake may occur without human organic cation transporter 1 (hOCT1): changes associated with progression and response in chronic myeloid leukemia. implications for the treatment of imatinib-resistant chronic myeloid leukemia. Proc Natl Acad Sci USA 2006; 103: 2794–2799. Blood 2008; 112: 3348–3354. 9 Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J et al. Chronic 15 Zelcer N, Saeki T, Reid G, Beijnen JH, Borst P. Characterisation of drug transport by myeloid leukemia: an update of concepts and management recommendations of the human multidrug resistance protein 3 (ABCC3). J Biol Chem 2001; 276: European Leukemia Net. J Clin Oncol 2009; 27: 6041–6051. 46400–46407.

Chronic neutrophilic leukemia with concurrent CSF3R and SETBP1 mutations: single colony clonality studies, in vitro sensitivity to JAK inhibitors and lack of treatment response to

Leukemia (2014) 28, 1363–1365; doi:10.1038/leu.2014.39 the presence or absence of CSF3R mutation did not appear to affect survival, whereas there was a trend for shortened survival among SETBP1-mutated patients. In the current study, we investigated the clonal distribution of Colony-stimulating factor 3 receptor gene (CSF3R) encodes the mutant CSF3RT618I and SETBP1 in a CNL patient expressing both cell surface, transmembrane receptor for granulocyte colony- mutations and describe our observations regarding the effect of stimulating factor (G-CSF). Nonsense somatic mutations involving JAK inhibitors, both in vitro and in vivo. The study patient was a 66- the intracytoplasmic domain of CSF3R have been described in year-old women with history of radiation therapy after lumpect- B41% of patients with severe congenital neutropenia (SCN) and omy for breast cancer in 1997. In October 2012, the discovery of arise at the stem cell level.1 SCN-associated CSF3R mutations three synchronous lesions in the left breast necessitated are acquired and might contribute to leukemic transformation, mastectomy. White blood cell count (WBC) was approximately possibly through cooperation with other oncogenes.2,3 13 Â 10 À 9/l at the time. Subsequently, her WBC gradually A germline CSF3R transmembrane mutation (C–A substitution at increased to 180 Â 10 À 9/l. Bone marrow examination on 15 nucleotide 2088; T617N) had been described in autosomal March 2013 showed predominantly granulocytic proliferation with dominant hereditary neutrophilia,4 before the seminal report by over 95% cellularity and no dysplastic features or monocytosis. Maxson et al.5 that established activating CSF3R mutations as a Cytogenetic studies and mutation screening for JAK2V617F and unique molecular marker in chronic neutrophilic leukemia (CNL). BCR-ABL1 were negative. A working diagnosis of CNL was made These authors subsequently reported that CSF3RT618I induced a and the patient was placed on hydroxyurea therapy. lethal myeloproliferative disorder in a murine bone marrow Under Mayo institutional review board-approved protocol, we transplant model.6 Treatment with ruxolitinib was reported to obtained both peripheral blood and buccal cells from the study have a salutary effect both in this mouse model and a patient with patient. Mononuclear and granulocyte cell fractions were enriched CSF3RT618I mutation.5 Primary cells from the latter patient were for by double-ficoll density gradient centrifugation. We used reportedly sensitive to inhibition by ruxolitinib (IC50, 127 nM). positive selection antibody-labeled magnetic bead separation Subsequent to the report by Maxson et al.,5 we confirmed the to further purify CD3 þ and CD34 þ cell fractions from the high frequency of CSF3R mutations in World Health Organization mononuclear cell fraction (Stem Cell Technologies, Vancouver, CA, (WHO)-defined CNL. In our study, CSF3RT618I occurred exclusively USA). DNA sequencing was used to screen for CSF3R and SETBP1 in WHO-defined CNL with mutational frequency of 83%, while the mutations, as previously described.8 For detection of CSF3RT618I, mutation was absent in WHO-defined atypical chronic myeloid we used primers: (CSF3R forward) 50-GTCTGGGAAGCCACAAGAAG-30, leukemia (aCML), monoclonal gammopathy-associated CNL and (CSF3R reverse) 50-GACCAGGGGATTCAAAGTCA-30, and for all other cases of unconfirmed CNL or aCML. Similar exon 14 SETBP1D868N, we used primers: (SETBP1 forward) 50-ACCTGGA mutations were absent in chronic myelomonocytic leukemia AGCTGTCTCCACCCA-30, (SETBP1 reverse) 50-CGGTGGCCATGCCG or primary myelofibrosis.7 We also discovered that 40% of the GTTCTT-30. Products were visualized on a 1.3% agarose gel and patients with CSF3RT618I-mutated CNL also expressed other DNA Sanger sequencing method was used to confirm the mutations in SET binding protein-1 (SETBP1).8 Although the presence of CSF3R and SETBP1 mutations. number of informative cases were too small to make definitive Single colonies were obtained by plating mononuclear cells in conclusions, considering all 35 cases of clinically suspected CNL, duplicate with complete hematopoietic cytokine-enriched

Accepted article preview online 21 January 2014; advance online publication, 11 February 2014

& 2014 Macmillan Publishers Limited Leukemia (2014) 1341 – 1379 Letters to the Editor 1364 methylcellulose with and without the addition of fedratinib (a SETBP1D868N CSF3R T618I JAK2 inhibitor) or a commercially available JAK1 inhibitor. On day 11, erythroid and granulocyte individual colonies were counted and collected at each concentration. Single colonies were screened for mutational contribution of both SETBP1 and CSF3R Granulocytes mutations. The patient harbored both CSF3RT618I and SETBP1D868N mutations in granulocytes, mononuclear cells and CD34 þ myeloid progenitors, while neither mutation was present in buccal or CD3 þ (T cells) (Figure 1). Colony-forming unit assay- Mononuclear Cells derived 30 single colonies (15 erythroid and 15 granulocyte) were analyzed for the presence of CSF3R and SETBP1 mutations, and all (100%) harbored heterozygous SETBP1D868N and 27 (90%) heterozygous CSF3RT618I; two granulocyte colonies were wild-type and one homozygous for CSF3RT618I (Figure 2). Fedratinib and the JAK1 inhibitor revealed activity in suppressing CD34+ colony formation by 450% at a drug concentration of X600 nM (Figure 3). Mutation analysis in post-treatment residual single colonies revealed persistence of both mutations, even under conditions of higher ambient drug concentrations (Figure 4). CD3+ Having failed hydroxyurea therapy, the study patient was subsequently treated with ruxolitinib at 10 mg twice daily starting on 10 June 2013, in addition to 1 g/day of hydroxyurea. At the time, WBC was 86.5 Â 10 À 9/l, Hgb 11.8 g/dl and platelets 122 Â 10 À 9/l. On 17 June 2013, WBC decreased to 43.9 and hydroxyurea was held. On 25 June 2013, WBC increased to Buccal 89.6 Â 10 À 9/l and ruxolitinib was increased to 15 mg twice daily. On 15 July 2013, WBC had further increased to 190 Â 10 À 9/l and hydroxyurea was added to the treatment regimen at 1 g/day. Figure 1. Genomic sequencing of CSF3RT618I (wild type ¼ ACC; On 2 August 2013, WBC was recorded at 91.3 Â 10 À 9/l and mutant ¼ ATC) and SETBP1D868N (wild type ¼ GAC; mutant ¼ AAC) subsequently increased to 140 Â 10 À 9/l, which led to an increase mutations in fractionated cells. in her ruxolitinib dose to 20 mg twice daily on 10 December 2013.

CFU-GM (N=15) BFU-E (N=15 ) SETBP1 D868N

SETBP1 HET CSF3R WT N=2 Heterozygous (HET)

CSF3R T618I 90% of colonies are heterozygous for both SETBP1 HET SETBP1 HET CSF3R HET N=12 CSF3R HET N=15 SETBP1D868N and Wild-type (WT) CSF3R T618I

Heterozygous (HET)

SETBP1 HET CSF3R HO N=1 Homozygous (HO)

Figure 2. Single colonies were genotyped for both SETBP1 and CSF3R mutations. (a) Summary of colony type vs mutation profile. (b) Sequencing traces exhibiting allelic contribution of mutation. HET, heterozygous; HO, homozygous; WT, wild type.

Figure 3. Number and type of hematopoietic colonies (red ¼ BFU-E; gray ¼ CFU-GM) after 11 days of sustained exposure to fedratinib (a JAK2 inhibitor) or a commercially available JAK1 inhibitor. BFU-E, burst forming unit, erythroid; CFU-GM, colony forming unit, granulocyte monocyte; ND, not determined.

Leukemia (2014) 1341 – 1379 & 2014 Macmillan Publishers Limited Letters to the Editor 1365 No Drug 0.05µM 0.1µM 0.6µM

CSF3R T618I CSF3R T618I CSF3R T618I CSF3R T618I

WT HET HO WT HET HO WT HET HO WT HET HO

WT WT WT WT D868N D868N D868N D868N HET HET HET HET SETBP1 SETBP1 SETBP1 SETBP1 HO HO HO HO

CSF3R T618I CSF3R T618I CSF3R T618I CSF3R T618I D868N D868N D868N D868N SETBP1 SETBP1 SETBP1 SETBP1 Figure 4. Summary of clonal distribution of CSF3R and SETBP1 mutations in both BFU-E (R) and CFU-GM (G) and mutational allele contribution of both CSF3RT618I and SETBP1D868N mutations (a, b: no drug; c, d: 0.05 mM; e, f: 0.1 mM; g, h: 0.6 mM).

Most recent blood count showed a WBC of 107.8 Â 10 À 9/l and REFERENCES À 9 platelet count of 88 Â 10 /l. 1 Germeshausen M, Welte K, Ballmaier M. In vivo expansion of cells expressing In the current report, we describe a double-mutated CNL acquired CSF3R mutations in patients with severe congenital neutropenia. Blood patient (CSF3RT618I and SETBP1D868N) who was refractory to the 2009; 113: 668–670. treatment with both ruxolitinib and hydroxyurea. It is currently 2 Kunter G, Woloszynek JR, Link DC. A truncation mutant of Csf3r cooperates with unknown if the coexpression of SETBP1 in this CSF3RT618I- PML-RARalpha to induce acute myeloid leukemia in mice. Exp Hematol 2011; 39: mutated patient with CNL contributed to the ineffectiveness of 1136–1143. JAK inhibitor therapy both in vivo and in vitro. At least in this 3 Beekman R, Valkhof MG, Sanders MA, van Strien PM, Haanstra JR, Broeders L et al. Sequential gain of mutations in severe congenital neutropenia progressing to particular patient, we observed myeloid cell restriction of the acute myeloid leukemia. Blood 2012; 119: 5071–5077. clone and coexpression of both CSF3RT618I and SETBP1D868N 4 Plo I, Zhang Y, Le Couedic JP, Nakatake M, Boulet JM, Itaya M et al. An activating mutations in erythroid and granulocytic cells; the latter antedated mutation in the CSF3R gene induces a hereditary chronic neutrophilia. J Exp Med the former in order of acquisition, but it would be inappropriate to 2009; 206: 1701–1707. make any conclusions based on a single patient study. 5 Maxson JE, Gotlib J, Pollyea DA, Fleischman AG, Agarwal A, Eide CA et al. Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML. N Engl J Med 2013; 368: 1781–1790. CONFLICT OF INTEREST 6 Fleischman AG, Maxson JE, Luty SB, Agarwal A, Royer LR, Abel ML et al. The authors declare no conflict of interest. The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition. Blood 2013; 122: TL Lasho1, A Mims2, MA Elliott1, C Finke1, A Pardanani1 and 3628–3631. A Tefferi1 7 Kosmider O, Itzykson R, Chesnais V, Lasho T, Laborde R, Knudson R et al. 1 Mutation of the colony-stimulating factor-3 receptor gene is a rare event Division of Hematology, Department of Internal Medicine, with poor prognosis in chronic myelomonocytic leukemia. Leukemia 2013; 27: Mayo Clinic, Rochester, MN, USA and 1946–1949. 2 Division of Hematology and Oncology, Medical University of South 8 Pardanani A, Lasho TL, Laborde RR, Elliott M, Hanson CA, Knudson RA et al. CSF3R Carolina, Charleston, SC, USA T618I is a highly prevalent and specific mutation in chronic neutrophilic leukemia. E-mail: [email protected] Leukemia 2013; 27: 1870–1873.

Impact of on outcome of chronic lymphocytic leukemia patients with relapsed del(17p13.1) karyotype at a single center

Leukemia (2014) 28, 1365–1368; doi:10.1038/leu.2014.42 del(17p13.1) is at least partially linked to malfunction of the tumor suppressor gene TP53 (located on 17pref. 2), which repairs induced by .3–5 Therefore, despite general Chronic lymphocytic leukemia (CLL) patients with del(17p13.1) advances in CLL therapy with chemoimmunotherapy, progress in exhibit short survival once disease progression necessitates therapy this subgroup has been limited. Current guidelines suggest and respond poorly to traditional treatments compared with other allogeneic stem cell transplant as part of initial therapy,6 as cytogenetic subgroups.1 Poor outcome associated with salvage treatment for del(17p13.1) portends an even graver

Accepted article preview online 23 January 2014; advance online publication, 25 February 2014

& 2014 Macmillan Publishers Limited Leukemia (2014) 1341 – 1379