Letters to the Editor 1570 1 1 1 1 1 A Tefferi , TL Lasho , C Finke , AA Belachew , EA Wassie , 4 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR 2 3 1 RP Ketterling , CA Hanson and A Pardanani mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 1 Division of Hematology, Mayo Clinic, Rochester, MN, USA; 2013; 369: 2391–2405. 2Division of Cytogenetics, Mayo Clinic, Rochester, MN, USA and 5 Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD et al. JAK2 3Division of Hematopathology, Mayo Clinic, Rochester, MN, USA or CALR mutation status defines subtypes of essential thrombocythemia with E-mail: [email protected] substantially different clinical course and outcomes. Blood 2013; e-pub ahead of print 23 December 2013. 6 Vannucchi AM, Lasho TL, Guglielmelli P, Biamonte F, Pardanani A, Pereira A et al. Mutations and prognosis in primary myelofibrosis. Leukemia 2013; 27: 1861–1869. 7 Gangat N, Caramazza D, Vaidya R, George G, Begna K, Schwager S et al. DIPSS plus: REFERENCES a refined Dynamic International Prognostic Scoring System for primary myelofi- 1 Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH et al. CALR vs brosis that incorporates prognostic information from karyotype, platelet count, and JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and transfusion status. J Clin Oncol 2011; 29: 392–397. molecular comparisons. Leukemia 2014; 28: 1472–1477. 8 Cervantes F, Dupriez B, Pereira A, Passamonti F, Reilly JT, Morra E et al. 2 Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L et al. New prognostic scoring system for primary myelofibrosis based on a study of the Impact of calreticulin mutations on clinical and hematological phenotype International Working Group for Myelofibrosis Research and Treatment. Blood and outcome in essential thrombocythemia. Blood 2013; e-pub ahead of print 2009; 113: 2895–2901. 26 December 2013. 9 Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A et al. 3 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114: 2013; 369: 2379–2390. 937–951.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

A multi-centre phase 2 study of azacitidine in chronic myelomonocytic leukaemia

Leukemia (2014) 28, 1570–1572; doi:10.1038/leu.2014.85 effusions). Of 74 patients pre-screened for eligibility, 32 were registered for trial entry (see CONSORT Flow Diagram, 2 Therapeutic options for chronic myelomonocytic leukaemia Supplementary Figure 1). AZA was administered (75 mg/m for (CMML) remain limited. Azacitidine (AZA) is approved for CMML-2 7 days, 5-2-2 schedule) every 28 days, for a minimum of six cycles. lacking proliferative features (white cell count (WCC) o13 Â 109/l); Responders and patients with stable disease were allowed to however, AZA registration studies for myelodysplasia (MDS) continue therapy until loss of response/progressive disease or contained small numbers of CMML patients (n ¼ 30), preventing development of unacceptable toxicities. Assessment of spleen size definitive conclusions with regards to toxicity and efficacy.1,2 was by clinical examination. Toxicities were determined as per Published retrospective case series of AZA therapy for CMML National Cancer Institute-Common Toxicity Criteria (Version 3.0). document overall response rates (ORR) and complete remission Patients remained on follow-up until 12 months after the last (CR) rates of 39–60% and 11–40%, respectively,3–5 according to patient’s last dose of treatment (median follow-up 12.8 months, International Working Group (IWG) 2006 criteria.6 On behalf of the range 0.6–30.3). UK National Cancer Research Network (UKNCRN) MDS Trial ORR was defined (by central review) as the sum of clinical Subgroup, we conducted a multi-centre phase 2 trial of AZA in remission, good response and minor response determined accord- 8 CMML patients with features of advanced disease, incorporating ing to Wattel et al. (Supplementary Table 1), at day 28 of the sixth mutation analysis and methylation status. or last cycle of AZA (whichever occurred first). These response Between January and August 2010, 32 patients with newly criteria incorporate assessment of proliferative disease, including diagnosed or previously treated CMML7 (hydroxycarbamide and/or leukocytosis, extramedullary involvement and spleen size. Patients supportive care only) were enroled after written informed consent. who received o1 cycle were not considered evaluable. þ Safety and tolerability, and ORR (as defined by Wattel et al.8) were Mutations were analysed (using DNA from CD14 marrow co-primary end points. Secondary end points included response mononuclear cells) in TET2, ASXL1, EZH2, CBL and NRAS by high- according to modified IWG criteria (incidence of CR, partial resolution melt and/or direct sequence analysis (n ¼ 21) as 9–11 remission and haematological improvement),6 overall and previously described. Global levels of DNA methylation were progression-free survival (OS, PFS), time to transformation or determined for eight patients analysed at baseline and post death and biological correlates (mutation analysis of TET2, ASXL1, course six using Illumina Infinium human 454 BeadChip oligo EZH2, CBL and NRAS genes, and gene methylation status). The trial arrays, which determine the % DNA methylation at 450 000 was approved by the UK National Research Ethics Service (EudraCT individual CG sequences. Analyses were performed by Gen-Probe 2008-006349-23 and ISRCTN 21428905). Inclusion criteria were: Life Sciences (Manchester, UK). OS, PFS, time to acute (1) CMML-2 and (2) CMML-1 patients with symptomatic marrow myelogenous leukemia (AML) transformation and death, and failure or proliferative disease, intermediate or high Dusseldorf duration of response were based on available data on 21st score (for WCC 412 Â 109/l) or int-2 or high IPSS score (for WCC January 2013. OS was defined as the time from registration to o12 Â 109/l), and/or symptomatic splenomegaly or systemic death from any cause or last follow-up. Patients discontinuing symptoms (including weight loss of 10% from baseline over the protocol treatment were still followed for OS. Analyses were previous 6 months with no other explanation) or symptomatic carried out using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). extramedullary disease (including skin infiltration and serous Baseline characteristics are summarized in Table 1.

Accepted article preview online 26 February 2014; advance online publication, 18 March 2014

Leukemia (2014) 1543 – 1572 & 2014 Macmillan Publishers Limited Letters to the Editor 1571 Table 1. Baseline patient characteristics Table 2. Response to treatment as determined by the criteria of Wattel et al. (2A) or the IWG (2B) Characteristic (n ¼ 30) Value Total, n ¼ 30 (%) Median age (range) 70 years (57–85) Sex, M/F, n (%) 20 (67)/10 (33) (A) Response according to Wattel et al. Prior hydroxycarbamide, n (%) Yes: 13 (43.3), no: 17 (56.7) CLR 1 (3%) CMML-1 or -2a, n (%) 21/30 (70%), 8/30 (27%) GR 2 (7%) WHO performance status, n (%) 0–8 (26%) MR 10 (33%) 1–16 (53%) Stable disease 2 (7%) 2–6 (20%) Progression 11 (37%) Extramedullary diseaseb, n (%) 5 (17%) Not evaluable (died before completion of last cycle) 2 (7%)a c Splenomegaly , n (%) 14 (47%) Not evaluable (o1 cycle) 2 (7%) Abnormal karyotype, n (%) 5 (17%) ORR 43% (25%, 63%) Median Hb, g/dl (range) 10.1 (6.6–14.3) Median WCC, Â 109/l (range) 15.9 (5.0–124.7) (B) Response using IWG criteria Median plt count, Â 109/l (range) 43 (15–462) CR 2 (7%) Median blood monocytesd, Â 109/l (range) 2.7 (0.3–21.9) PR 0 (0%) Median blood neutrophils, Â 109/l (range) 7.9 (1.2–71.7) Marrow CR 2 (7%) Median marrow blasts, % (range) 4.5 (0–15) Marrow CR, HI-P 1 (3%) Red cell transfusion dependent, n (%) 17 (57%) Stable disease 5 (17%) Median red cell units transfused, n (range)e 6 (0–34) Fail 8 (27%)a Plt transfusion dependent, n (%) 5a (17%) Progressive disease 9 (30%) Median plt units transfused, n (range)e 3 (1–15) HI-E, HI-P 1 (3%) TET2, n mutated/n evaluable (%) 17/27 (63) Not evaluable (o1 cycle) 2 (7%) ASXL1, n mutated/n evaluable (%) 8/21 (38) Incidence of CR/PR 7% (0.8–22) EZH2, n mutated/n evaluable (%) 1/25 (4) CBL, n mutated/n evaluable (%) 2/26 (8) Abbreviations: CR, complete remission; CLR, clinical remission; E, erythroid; NRAS, n mutated/n evaluable (%) 1/21 (5) GR, good response; HI, haematological improvement; IWG, international working group; MR, minor response; ORR, overall response rate; P, platelets; Abbreviations: CMML, chronic myelomonocytic leukaemia; F, female; PR, partial remission. (A) Response to azacitidine (AZA) according to the Hb, haemoglobin; M, male; Plt, platelets; WCC, white cell count. Overall 32 criteria of Wattel et al.8 (see Supplementary Table 1 for definitions). patients were registered for the study. Two patients received no treatment B: response to AZA according to modified IWG 2006 criteria.6 Response at and were not included in the final analysis (one was found to be in day 28 of the sixth or last cycle of AZA is shown. ORR for Wattel et al. criteria transformation to acute myelogenous leukemia before therapy and another is calculated from the combination of CLR, GR and MR (brackets represent had an unexpected rise in creatinine precluding treatment). Median time 95% confidence interval). The majority of MR represented leucoreduction from diagnosis to trial entry was 6.7 months. aInformation unknown for one (by 450%) and/or splenic reduction, with five of seven patients who were patient. bExtramedullary disease included skin lesions in all five cases, with no on hydroxycarbamide at trial entry able to stop this drug. Of the five ascites, pleural or pericardial effusions recorded at diagnosis. cSplenomegaly patients with documented extramedullary disease (all had skin lesions), defined as 40 cm palpable spleen below costal margin (information only one patient experienced a resolution (classified as a GR according to unknown for two patients in this category). Of 14 patients with splenomegaly, Wattel et al.). (B) The ORR for the IWG criteria (incorporating CR, PR, marrow the median spleen size was 10.5 cm below the costal margin (range 3–25). CR and HI) was 20%. 33% of patients underwent transformation to acute dTwo patients had monocyte counts o1.0, both of whom were being treated myelogenous leukemia during the follow-up period. aIncludes two patients with hydroxycarbamide at trial entry. eIn the 8 weeks before trial entry. who died before day 28 of first and third cycles.

Fourteen patients stopped AZA between registration and the sixth and follow-up in cycle 1). Median survival was 16 months from or last course (whichever was the earliest); 24 had dose delays and/ registration (95% confidence interval (10, not reached)). or modifications. Median number of cycles was seven. A summary of A number of parameters were considered for their AZA administration (including number of cycles and reasons for predictive value for response (data not shown). These included stopping) is given in Supplementary Table 2. One patient had a mutation status (summarized in Table 1), CMML-1 vs CMML-2, grade 3/4 non-haematological adverse reaction (respiratory tract presence of cutaneous lesions, karyotypic abnormalities and infection) during the first six cycles. No AZA-related deaths were methylation status. Small numbers in these groups precluded reported during or beyond this time. Two patients (7%) discontinued meaningful statistical analyses and no conclusions could be AZA because of toxicity in the first six cycles (one after cycle 1 and drawn. one after cycle 6 of treatment). The most common toxicities Of eight patients in whom follow-up samples were available, (haematological and non-haematological, all grades) are shown in seven had a reduction in DNA methylation levels after 6 months of Supplementary Table 3. Overall non-haematological toxicity was treatment, and on average, the proportion of methylated CGs fell manageable (predominantly Grade 1 or 2), and haematological (from 53.1 to 50.9%, P ¼ 0.02, data not shown). Similar trends were toxicity was similar to or less than previous AZA studies. observed when data for individual CGs were visualized as heat Responses are shown in Table 2. Twenty patients were red cell maps by hierarchical clustering. There was no relationship and/or platelet transfusion dependent at trial entry; six became between the clinical response or TET2 mutations and the transfusion independent during treatment (30%). Transfused units reduction in methylation. of red cells fell from a mean of 3 per patient (s.d. 3.6, n ¼ 29, 1 In this phase 2 study of AZA in CMML we have demonstrated missing) during the first cycle to a mean of 1.1 (s.d. 1.6, n ¼ 20, 1 good tolerability, but only modest response rates. Haematological missing) by cycle 6. Median duration of response for those toxicity has not previously been reported in detail,3,4 however, the patients included in the ORR (n ¼ 13) was 7.5 months (range 2–32, incidence in our study compared very favourably with that 2 missing). Univariate landmark analysis of responders vs non- described in MDS studies (Grade 3/4 anaemia, neutropenia and responders showed no difference in survival (log-rank test, thrombocytopenia in 20, 30 and 33% as compared with 57, 91 and P ¼ 0.7). As of 9th January 2013, 4 patients remain on therapy 85%, respectively, in the AZA001 trial).1 The lower incidence of (at cycles 18, 27, 34 and 34), 6 patients are alive but off therapy neutropenia in particular may well relate to the frequent and 19 patients have died (one patient withdrew from treatment leukocytosis associated with CMML.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1543 – 1572 Letters to the Editor 1572 Definition of response in CMML is difficult because of the 1Beatson West of Scotland Cancer Centre and University of Glasgow, coexistence, often in the same patient, of both cytopenias and Glasgow, UK; proliferative features. ORR rate in this study by the criteria of 2Department of Hematology, East Kent Hospitals, Canterbury, UK; Wattel et al. was 43%. Most responses (10/13) were however 3Leeds Institute for Cancer Studies and Pathology, University of minor by these criteria, predominantly a reduction in WCC by Leeds, Leeds, UK; 450%. Although AZA clearly demonstrated cytoreductive ability, 4Worcestershire Acute Hospitals NHS Trust and University of how clinically meaningful (or cost-effective) such responses are is Birmingham, Birmingham, UK; debatable, as no improvement in marrow parameters were 5Clinical Trials Research Unit, University of Leeds, Leeds, UK; observed in this subgroup, and similar responses may have 6Institute of Biomedical Research, University of Birmingham, arguably been achieved by using alternative cytoreductive agents. Birmingham, UK; Responses also appeared less durable than that in previous 7Faculty of Medicine, University of Southampton, Southampton, UK; MDS studies with a median response duration of 7.5 months 8Department of Hematology, University Hospital of Wales, Cardiff, UK and (as compared with 13.6 months in the AZA001 study). By IWG 9Department of Hematology, St James’s Institute of Oncology, Leeds, UK criteria, our ORR was 20%, with the incidence of CR/marrow CR E-mail: [email protected] 17% (as compared with 29% in the AZA001 study of all MDS subtypes).1 These results also compare less well with a phase 2 study of decitabine in CMML, which demonstrated a CR/marrow CR rate of 31% and ORR of 38%.12 Overall, a median of seven cycles of AZA were delivered, less than the nine cycles delivered in REFERENCES AZA001, but comparable to single-centre series. Our outcomes 1 Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A et al. with AZA in this prospective, multi-centre setting are also inferior Efficacy of azacitidine compared with that of conventional care regimens in the to those reported in retrospective series of CMML.3–5 A partial treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 2009; 10: 223–232. explanation for this may relate to the incorporation of higher 2 Kaminskas E, Farrell A, Abraham S, Baird A, Hsieh LS, Lee SL et al. Approval numbers of poor-risk patients, as only CMML-2 or CMML-1 summary: azacitidine for treatment of myelodysplastic syndrome subtypes. patients with symptomatic or significant marrow failure or Clin Cancer Res 2005; 11: 3604–3608. myeloproliferative disease were included. A majority (57%) failed 3 Costa R, Abdulhaq H, Haq B, Shadduck RK, Latsko J, Zenati M et al. Activity therapy or exhibited progressive disease: of 14 (47%) patients of azacitidine in chronic myelomonocytic leukemia. Cancer 2011; 117: completing six or less cycles, 8 stopped due to disease progression 2690–2696. or transformation to AML. More encouragingly, 15 of 16 patients 4 Thorpe M, Montalvao A, Pierdomenico F, Moita F, Almeida A. Treatment of chronic who were classified as responding or as stable disease continued myelomonocytic leukemia with 5-Azacitidine: a case series and literature review. therapy beyond cycle 6. Of note are the four patients maintaining Leuk Res 2012; 36: 1071–1073. 5 Ades L, Sekeres MA, Wolfromm A, Teichman ML, Tiu RV, Itzykson R et al. Predictive response and continuing therapy for 18 cycles or more. factors of response and survival among chronic myelomonocytic leukemia Modest decreases in global levels of DNA methylation were patients treated with azacitidine. Leuk Res 2013; 37: 609–613. observed although these did not correlate with clinical responses, as 6 Cheson B, Greenberg P, Bennett J, Lowenberg B, Wijermans P, Nimer S et al. 13 observed in other correlative studies of AZA and CG methylation. Clinical application and proposal for modification of the International In summary, despite AZA being licensed for non-proliferative Working Group (IWG) response criteria in myelodysplasia. Blood 2006; 108: CMML-2, our study demonstrated only modest response rates for 419–425. a wider CMML population. Given these findings, we would caution 7 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. WHO Classifi- against use of AZA as ‘standard of care’ in this relatively rare and cation of Tumours of Haematopoietic and Lymphoid Tissues. 4th edn. IARC Press: difficult disorder, but would encourage further clinical trials given Lyon, France, 2008. 8 Wattel E, Guerci A, Hecquet B, Economopoulos T, Copplestone A, Mahe B et al. evidence of good efficacy in a small number of patients. A randomized trial of hydroxyurea versus VP16 in adult chronic myelomonocytic leukemia. Groupe Francais des Myelodysplasies and European CMML Group. Blood 1996; 88: 2480–2487. CONFLICT OF INTEREST 9 Grand FH, Hidalgo-Curtis CE, Ernst T, Zoi K, Zoi C, McGuire C et al. Frequent CBL MWD, JK and DTB have received advisory board and speaker fees from Celgene. The mutations associated with 11q acquired uniparental disomy in myeloproliferative remaining authors declare no conflict of interest. neoplasms. Blood 2009; 113: 6182–6192. 10 Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat ACKNOWLEDGEMENTS Genet 2010; 42: 722–726. This trial was funded by Cancer Research UK (Grant Award Number C24417/A10418) 11 Score J, Hidalgo-Curtis C, Jones AV, Winkelmann N, Skinner A, Ward D et al. and Celgene (UK) Ltd. Celgene (UK) provided study drug. Methylation studies were Inactivation of polycomb repressive complex 2 components in myeloproliferative funded by a grant from Yorkshire Cancer Research (Grant Award Number L335) and and myelodysplastic/myeloproliferative neoplasms. Blood 2012; 119: 1208–1213. on-going support from Leukaemia & Lymphoma Research, UK. We would like to 12 Braun T, Itzykson R, Renneville A, de Renzis B, Dreyfus F, Laribi K et al. Molecular thank staff at the 13 UK centres who recruited patients to this study. predictors of response to decitabine in advanced chronic myelomonocytic leukemia: a phase 2 trial. Blood 2011; 118: 3824–3831. 1 2 3 4 5 13 Kantarjian H, Oki Y, Garcia-Manero G, Huang X, O’Brien S, Cortes J et al. Results MW Drummond , C Pocock , M Boissinot , J Mills , J Brown , of a randomized study of 3 schedules of low-dose decitabine in higher-risk 6 7 5 8 5 6 P Cauchy , NCP Cross , S Hartley , J Kell , A Szubert , PN Cockerill myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood 2007; 9 and DT Bowen 109:52–57.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Leukemia (2014) 1543 – 1572 & 2014 Macmillan Publishers Limited