Letters to the Editor 1360 1 2 1 1 2 M Maffioli , A Genoni , D Caramazza , B Mora , A Bussini , 7 Rumi E, Pietra D, Guglielmelli P, Bordoni R, Casetti I, Milanesi C et al. Acquired 1 3 2 1 M Merli , T Giorgino , R Casalone and F Passamonti copy-neutral loss of heterozygosity of 1p as a molecular event 1 Division of Hematology, Department of Medicine, University associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms. Hospital Ospedale di Circolo e Fondazione Macchi, Varese, Italy; Blood 2013; 121: 4388–4395. 2Genetic Unit, Medical Genetic and Cytogenetics Laboratory, SSD, 8 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. SMEL, University Hospital Ospedale di Circolo e Fondazione Macchi, Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med Varese, Italy and 2013; 369: 2379–2390. 3Institute of Biomedical Engineering, National Research Council of 9 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2. N Engl J Italy (ISIB-CNR), Padua, Italy Med 2013; 369: 2391–2405. E-mail: [email protected] 10 Landgren O, Goldin LR, Kristinsson SY, Helgadottir EA, Samuelsson J, Bjorkholm M. Increased risks of polycythemia vera, essential thrombocythemia, and myelofi- brosis among 24 577 first-degree relatives of 11 039 patients with myeloproli- REFERENCES ferative neoplasms in Sweden. Blood 2008; 112: 2199–2204. 1 Passamonti F, Maffioli M, Caramazza D, Cazzola M. Myeloproliferative neoplasms: 11 Rumi E, Passamonti F, Della Porta MG, Elena C, Arcaini L, Vanelli L et al. Familial from JAK2 mutations discovery to JAK2 inhibitor therapies. Oncotarget 2011; 2: chronic myeloproliferative disorders: clinical phenotype and evidence of disease 485–490. anticipation. J Clin Oncol 2007; 25: 5630–5635. 2 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of- 12 Malak S, Labopin M, Saint-Martin C, Bellanne-Chantelot C, Najman A. Long term function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: follow up of 93 families with myeloproliferative neoplasms: life expectancy and 1779–1790. implications of JAK2V617F in the occurrence of complications. Blood Cells Mol Dis 3 Tefferi A, Lasho TL, Abdel-Wahab O, Guglielmelli P, Patel J, Caramazza D et al. 2012; 49: 170–176. IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast- 13 Olcaydu D, Rumi E, Harutyunyan A, Passamonti F, Pietra D, Pascutto C et al. The phase essential thrombocythemia, polycythemia vera or myelofibrosis. Leukemia role of the JAK2 GGCC haplotype and the TET2 in familial myeloproliferative 2010; 24: 1302–1309. neoplasms. Haematologica 2011; 96: 367–374. 4 Vannucchi AM, Lasho TL, Guglielmelli P, Biamonte F, Pardanani A, Pereira A et al. 14 Saint-Martin C, Leroy G, Delhommeau F, Panelatti G, Dupont S, James C et al. Mutations and prognosis in primary myelofibrosis. Leukemia 2013; 27: 1861–1869. Analysis of the ten-eleven translocation 2 (TET2) gene in familial myeloproli- 5 Passamonti F, Elena C, Schnittger S, Skoda RC, Green AR, Girodon F et al. ferative neoplasms. Blood 2009; 114: 1628–1632. Molecular and clinical features of the myeloproliferative neoplasm associated 15 Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA et al. Proposals and with JAK2 exon 12 mutations. Blood 2011; 117: 2813–2816. rationale for revision of the World Health Organization diagnostic criteria for 6 Tefferi A, Pardanani A, Lim KH, Abdel-Wahab O, Lasho TL, Patel J et al. polycythemia vera, essential thrombocythemia, and primary myelofibrosis: TET2 mutations and their clinical correlates in polycythemia vera, essential recommendations from an ad hoc international expert panel. Blood 2007; 110: thrombocythemia and myelofibrosis. Leukemia 2009; 23: 905–911. 1092–1097.

The clinical significance of ABCC3 as an imatinib transporter in chronic myeloid leukaemia

Leukemia (2014) 28, 1360–1363; doi:10.1038/leu.2014.38 Radich et al.8 showed that ABCC3 expression was high in CML–BC patients with disease recurrence. None of these studies performed any functional ex vivo or in vitro work. This study was performed The advent of imatinib has revolutionised the treatment of chronic to further investigate the expression and function of ABC myeloid leukaemia (CML), but in contrast to clinical trial data B transporters involved in the cellular efflux of imatinib and their population studies have demonstrated that 40% of these clinical significance in CML. patients will develop resistance to imatinib. A proportion of these 1 A panel of ABC transporters was analysed in peripheral blood will progress to accelerated phase and/or blast crisis (BC). One leukocytes from four normal individuals and 90 newly diagnosed cause of therapeutic resistance to imatinib is the emergence of chronic-phase (CP) CML patients before imatinib treatment. The BCR–ABL1 kinase domain mutations and gene amplification, study was approved by the Liverpool Central Committee of the UK but there are also BCR–ABL1-independent mechanisms of 2 National Research Ethics Service and all individuals gave informed resistance including altered drug uptake and/or efflux. The consent. Patients were classified into four groups based on their ATP-binding cassette (ABC) transporters actively transport a response to imatinib treatment at 12 months according to the variety of substances/drugs, and their physiological role as a European Leukemia Net 2009 recommendations as follows:9 defence mechanism against xenobiotics has been extensively investigated.2,3 We and others have previously shown that imatinib is a substrate for the ABC efflux transporters ABCB1  Optimal (n ¼ 35); patients who achieve a complete cytogenetic (MDR1, p-glycoprotein) and ABCG2 (BCRP, breast cancer resistance response (CCR) on imatinib. ), whereas its uptake is dependent on the expression/  Suboptimal (n ¼ 23); patients who have achieved a major activity of the influx transporter SLC22A1 (hOCT1, human organic cytogenetic response but no CCR. cation transporter-1).3–6 However, additional transporters can  Failure (n ¼ 18); patients who fail to achieve a major cytogenetic affect treatment outcome in leukaemia, and an interplay response and/or switch to a second generation tyrosine kinase between influx and efflux transporters could account for the inhibitor. observed inter-patient variability to drug response. A study by Hu  BC (n ¼ 14); patients initially diagnosed in CP who subsequently et al.,7 using microarray analysis in acute myeloid leukaemia cell progress to BC. lines showed that hOCT1 mRNA expression was interrelated to the expression of the efflux ABCB1 (MDR1), ABCC3 (MRP3), ABCC4 RNA was extracted using the Qiagen RNA extraction kit (MRP4), ABCG2 (BCRP) and the influx OATP1A2 transporters. (Qiagen ltd, Manchester, UK), and cDNA was synthesised as

Accepted article preview online 20 January 2014; advance online publication, 11 February 2014

Leukemia (2014) 1341 – 1379 & 2014 Macmillan Publishers Limited Letters to the Editor 1361 previously described.5,6 The mRNA expression of ABC transporters Hs00358656_m1, ABCC4 (MRP4): Hs00988734_m1, ABCG2: was assessed by single TaqMan assays for Hs01053787 (Life Technologies, Paisley, UK). The real-time PCR ABCB1: Hs00184491_m1, ABCC1: Hs00219905_m1, ABCC3: amplifications were run in an ABI Prism 7900HT (Life Technologies)

Figure 1. Gene expression of ABC transporters in relation to clinical outcome in CML. The Mann–Whitney two-tail distribution test was used. Only the ABCC3 transcripts levels (RQ) correlated to imatinib treatment failure (P ¼ 0.0002). Statistical analysis was performed by GraphPad Prism 5 software.

18.00 MDCKII A-B 18.00 MDCKII-ABCC3 A-B 15.00 MDCKII B-A 15.00 MDCKII-ABCC3 B-A MDCKII-ABCC3 A-B+Probenecid 12.00 12.00 MDCKII-ABCC3 B-A+Probenecid 9.00 9.00 6.00 6.00 3.00 3.00 MDCKII monolayers Imatinib transport across 0.00 Imatinibtransport across 0.00 0 50 100 150 200 250 300MDCKII-ABCC3 monolayers 0 50 100 150 200 250 300 Times (min) Times (min)

500 p=0.017

400 p=0.016 p=0.006 Optimal p=0.028 Failure 300

200 ng/million of cells 100 Imatinib uptake/retention assay; 0 Imatinib Imatinib Probenecid Probenecid 1/2h 4hs 1/2h 4hs Figure 2. Efflux of imatinib across MDCKII (a) and MDCKII–ABCC3 (b) monolayers. There was an increased transport of imatinib in the basal to apical (B–A) direction in MDCKII–ABCC3 (efflux ratio 8.99) cells in comparison with MDCKII (efflux ratio 2.49). This effect is abolished by the ABCC3 inhibitor probenecid (efflux ratio 1.26). (c) Optimal patients had a better uptake of imatinib within 30 min of drug exposure than those failing treatment (P ¼ 0.028). This effect was lost at the 4-h time point indicating efflux of imatinib by that time. Pre-incubation with probenecid lead to a marked retention of the drug even at 4 h (optimals: P ¼ 0.006, failures: P ¼ 0.016). Unpaired two-tail distribution t-test was used. Statistical analysis was performed by GraphPad Prism 5 software.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1341 – 1379 Letters to the Editor 1362 system using manufacturer’s conditions and the relative Figure 2 shows the results from three separate monolayers. expression level of a particular gene of a given sample was Figure 2a presents the transport of imatinib across parental calculated by the comparative Ct method using the 2 À DDCt MDCKII cells that were used as a control. There was no significant formula to achieve results for relative quantification (RQ).10 A pool difference between basal to apical (B–A) and apical to basal (A–B) of cDNA from four normal individuals was used as calibrator, and transport, indicating minimal polar transport of the drug across all the samples were normalised to GAPDH (Hs99999905_m1, those cells. However, in MDCKII–ABCC3-transfected cells, there endogenous control). The RQ Manager software supported by the was a significantly increased transport of imatinib in the B–A ABI Prism 7900HT System was used for data analysis. The Mann– direction with an efflux ratio of 8.99 (Papp MDCKII–ABCC3 (B–A)/ Whitney two-tail distribution test was used to correlate ABC Papp MDCKII–ABCC3 (A–B), where Papp is the apparent perme- transporter mRNA expression with clinical response using the ability in cm/s). This effect was abolished by the ABCC3 inhibitor GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA, USA) probenecid (efflux ratio: 1.26), indicating that imatinib is effluxed statistical software. by ABCC3 (Figure 2b). In order to reconfirm our mRNA data on the The ABC mRNA expression results varied among patients. functional level and correlate the transcript levels to ABCC3 ABCG2 amplified in only 1/14 patients and was not included in the activity, we performed an uptake/retention assay on patients with rest of the study. This is in agreement with previous reports high (average RQ 40.412±0.06) and low ABCC3 (average RQ showing that ABCG2 is expressed in CD34 þ CML cells but not on o0.412 þ 0.06) mRNA and no differences on their hOCT1 mRNA their mature counterpart.11,12 ABCB1 showed the lowest levels of levels. Radiolabelled imatinib was used in the presence and mRNA expression. whereas ABCC1 had the highest with absence of the efflux transporter inhibitor probenecid in primary intermediate levels for ABCC3 and ABCC4 (Figure 1). This variation peripheral blood mononuclear cells from 12 (six optimal and six could be attributed to the transcriptional or epigenetic regulation failure) CML patients at diagnosis. These data are represented in of the ABC . Porro et al.12 showed that c-Myc can regulate Figure 2c and demonstrate an increased imatinib uptake in the transcription of several ABC transporter genes, prevalently in samples from optimal responders in comparison with those failing CML CD34 þ cells by binding to their promoter. Interestingly, treatment at 30 min (P ¼ 0.028) but not at 4 h (P ¼ 0.451). This c-myc does not bind to ABCG2 promoter when the latter is occurs because by the later time point, imatinib is already methylated failing to drive its transcription.12 Tang et al.13 effluxed out of the cell. Pre-incubation of cells with probenecid observed that ABCC3 was lower in haematopoietic than showed a better cellular retention of imatinib that was more unrestricted somatic and mesenchymal stem cells. Both studies marked at the 4 h time point (P ¼ 0.006 and 0.016 between highlight further the differences in the transporter expression optimal responders and failures, with and without inhibitor, levels of CD34 þ versus the mature blood cells. It would be respectively). In samples from patients classified as failures, interesting to test this model in our study, but unfortunately we probenecid can therefore increase the intracellular imatinib level lack matching CD34 þ s to their mature CML cells. even at the 30 min time point (P ¼ 0.017). The data suggest that Comparing suboptimal, failure and BC clinical groups to co-administration of probenecid will have a beneficial effect on patients showing an optimal response, there was no statistical the accumulation of imatinib in CML patients, and highlights difference between the mRNA levels of ABCB1, ABCC1 or ABCC4. further the importance of efflux transporters in maintaining However, the mRNA expression levels of ABCC3 were significantly intracellular drug levels. higher in patients failing treatment (P ¼ 0.0002) than in optimal or To our knowledge, this is the first study illustrating that suboptimal responders or the ones who progress to BC. This imatinib is a substrate for the ABCC3 efflux transporter indicates that ABCC3 is possibly having a role in imatinib and highlighting its importance in imatinib-treated patients. treatment failure but not necessarily to disease progression CP-CML patients with high ABCC3 mRNA expression at diagnosis (Figure 1). Spearman’s rank correlation test also showed that were less likely to have a favourable response to imatinib ABCB1 and ABCC3 and to a lesser degree ABCC1 correlated to treatment. Given although that imatinib is transported by a hOCT1 mRNA expression (r ¼ 0.58, Po0.0001; r ¼ 0.51, Po0.0001; number of transporters, it is likely that a complex interplay in the and r ¼ 0.31, P ¼ 0.003, respectively). ABCC4 showed no significant expression and activity or regulation of a number of influx correlation (data not shown). With the exception of the latter, our and efflux transporters will determine both intracellular con- data are in agreement with the study conducted by Hu et al.7 centrations and the likelihood of treatment failure in different regarding the inter-relationship of mRNA expression of influx and imatinib-targeted diseases. efflux transporters. Based on these data, we decided to further investigate whether imatinib is actually a substrate for ABCC3. We therefore used the CONFLICT OF INTEREST well-established monolayer assay using MDCKII cells stably overexpressing ABCC3 in a similar model to that used previously The authors declare no conflict of interest. to study efflux by ABCB1.7,14 Briefly, parental and MDCKII–ABCC3 1 1 1 1 overexpressing cells (kind gift from Professor P Bo¨rst, Amsterdam, A Giannoudis , A Davies , RJ Harris , CM Lucas , 2 1 Netherlands) were seeded at a density of 2 Â 106 cells/ml onto M Pirmohamed and RE Clark 1 24-mm diameter, 0.4-mm pore size, polycarbonate transwell Department of Haematology, Molecular and Clinical Cancer membrane inserts (Appleton Woods, Birmingham, UK), and Medicine, Royal Liverpool University Hospital, Liverpool, UK and 14 2 radiolabelled C-imatinib at 5 mM was added to the Department of Molecular and Clinical Pharmacology, donor compartment (at the apical or the basal side). University of Liverpool, Liverpool, UK Monolayers were incubated with the drugs for 4 h, and at various E-mail: [email protected] time intervals 50 ml of solution was sampled from both compart- ments, mixed with 4 ml scintillation fluid and measured by scintillation counting. At each time point, the transport across REFERENCES the monolayer was calculated from the measured radioactivity in the receiver compartment as a percentage of the initial 1 Francis S, Lucas C, Lane S, Wang L, Watmough S, Knight K et al. A population 7,14 study showing that the advent of second generation tyrosine kinase inhibitors radioactivity in the donor compartment. For the ABCC3 has improved progression-free survival in chronic myeloid leukaemia. Leuk Res inhibition studies, the MDCKII–ABCC3 monolayer was pre-incu- 2013; 37: 752–758. bated with 1 mM probenecid, an ABCC3 inhibitor, in transport 2 Apperley JF. Part I: mechanisms of resistance to imatinib in chronic myeloid 15 buffer for 30 min. leukaemia. Lancet Oncol 2007; 8: 1018–1029.

Leukemia (2014) 1341 – 1379 & 2014 Macmillan Publishers Limited 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. Imatinib 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 leukemia. 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 dasatinib 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 ruxolitinib

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