Thalidomide plus prednisone with or without danazol therapy in myelofibrosis: a retrospective analysis of incidence and durability of anemia response

Low-dose thalidomide and prednisone alone or combined are effective therapies in some persons with primary myelofibrosis (PMF) and anemia with or with RBC transfusion dependence. Danazol is also effective in some persons with PMF and anemia. Responses to these drugs are typically incomplete and not sustained. It is unclear whether adding danazol to thalidomide and prednisone would improve ef ficacy. We retrospectively compared the outcomes of 88 subjects with PMF and anemia receiving thalidomide and prednisone without ( n = 46) or with danazol ( n = 42). The primary end point was anemia response, which was 71% (95% con fidence interval (CI), 57, 85%) in subjects receiving thalidomide/prednisone/danazol compared with 46% (32, 60%; P = 0.014) in those receiving thalidomide/ prednisone. Response rates in subjects who were RBC transfusion dependent was also higher in the danazol cohort (61% (38, 84%)) vs. 25% (6, 44%); P = 0.024). Time to response was rapid (median, 2 months (range, 1 –11 months)) and similar between the cohorts. Response duration was longer in the thalidomide/prednisone/danazol cohort (HR 2.18 (1.18–5.42); P = 0.019). Adverse effects were mild and similar between the cohorts. In conclusion, thalidomide/ prednisone/danazol seems superior to thalidomide/prednisone in persons with PMF and anemia. Our conclusion requires con firmation in a randomized trial.

About one-third of persons with primary myelo fibrosis (PMF) have anemia at diagnosis and it develops in most others as the disease evolves 1,2. Anemia and red blood cell (RBC) transfusion dependence are independent adverse prognostic variables for survival 1–3. Erythropoiesis- stimulating agents (ESAs), androgenic steroids, thalidomide, lenalidomide, splenectomy, and prednisone are only modest activity in reversing anemia and ruxolitinib, pacritinib and fedratinib typically worsen anemia 4–7. New effective therapies are needed. Thalidomide is active in PMF because of its anti- angiogenic, cytokine regulatory, and immune-modulating properties 8,9. Thalidomide, 100 –400 mg/day, is reported to improve anemia in 20 –60% of subjects 10–12. However, these doses are associated with substantial toxicity and are poorly tolerated 10,11. The combination of low-dose thali- domide, 50 mg/day, and prednisone is better tolerated and

in slightly higher responses than thalidomide alone13. Androgenic steroids reverse anemia by stimulating erythropoietin, increasing iron use and reversing telomere © The Author(s) 2018 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution andreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a lin kt ot h eC r e a t i v eCommons license,and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicatedotherwise in a credit line to the material. If

is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visithttp://creativecommons.org/licenses/by/4.0/. Correspondence: Zhijian Xiao ( [email protected]) 1MDS and MPN Centre, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China 2State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China Full list of author information is available at the end of the article Xueping Luo, Zefeng Xu, and Bing Li contributed equally to this work. Blood Cancer Journal 12345678901234567890 loss14,15. Danazol, a synthetic androgen with reduced masculinizing activity, is reported to reverse anemia and thrombocytopenia in persons with PMF 16,17. Combining therapies with different mechanisms of action might be even more effective in reversing anemia. Herein, we report ef ficacy, safety, and long-time outcome of therapy with low-dose thalidomide and prednisone with or with- out danazol in subjects with PMF and anemia with or without RBC transfusion dependence. Subjects and methods Subjects This study was approved by the Ethical Committee of Institute of Hematology, CAMS and PUMC according to guidelines of the Declaration of Helsinki. From March 2006 to September 2016, 88 consecutive subjects and anemia with or without RBC transfusion dependence were enrolled. Eligibility criteria were: (1) PMF according to the WHO 2016 criteria 18; (2) age ≥ 18 years; (3) hemoglobin concentration <100 g/L or RBC transfusion dependence19; (4) no exposure to ESAs, androgens, tha- lidomide, lenalidomide, or corticosteroids <12 weeks pre- enrollment; (5) creatinine ≤2 mg/dL, direct bilirubin <2 times upper limit of normal (ULN) and alanine amino- transferase (ALT)/aspartate aminotransferase (AST) ≤3 ULN. Subjects receiving ruxolitinib during the past sev- eral years were excluded. Subjects had a pre-therapy physical examination, baseline laboratory assessment of serum chemistries and blood hematologic parameters, bone marrow aspirate and biopsy, and cytogenetic ana- lyses. Prognostic cohort was assigned using the Dynamic International Prognostic Scoring System (DIPSS) 2 for all subjects and DIPSS-plus 3 for those with cytogenetics data. Bone marrow fibrosis was graded using European con- sensus guidelines 20. Therapy Subjects received thalidomide, 50 mg p.o. at bed time continuously. Prednisone, 0.5 mg/kg/day, was given for 1 month, 0.25 mg/kg/day, for the next month, 0.125 mg/ kg/day for the third month and tapered thereafter. Danazol, 600 mg/day p.o. was given continuously. Patients with neutrophil count <1.0 × 10E + 9/L and/or platelet count <80 × 10E + 9/L were assigned to thalidomide/ prednisone/danazol therapy, the others were assigned to thalidomide/prednisone therapy. Laboratory studies were performed weekly for 12 weeks. Responders continued on their assigned therapy, whereas others stopped. Packed RBCs were transfused for a hemoglobin concentration <60 g/L or symptoms of anemia. Evaluation of response and adverse events (AEs) The primary study outcome was anemia response. In subjects with splenomegaly or thrombocytopenia, spleen and platelet responses were also analyzed. Anemia and spleen responses were assessed according to the revised International Working Group for Myelo fibrosis Research and Treatment (IWG-MRT) consensus criteria 21. Thrombocytopenia response was de fined as a platelet increase >50 × 10E + 9/L in subjects with baseline pla- telets <100 × 10E + 9/L. Toxicity was assessed by the National Cancer Institute Common Toxicity Criteria for Adverse Events, Version 4 22. Statistical analyses Follow-up was to death or 10 June 2017. Quantitative data are expressed as median and range and qualitative data as percent. Baseline variables were compared between cohorts using χ 2 test for categorical variables and Mann–Whitney U-test for continuous variables. Clinical responses were compared between cohorts with χ2 test. Logistic regression was used to assess relationships between baseline variables and outcomes. Variables sig- nificant in univariate analyses were included in the mul- tivariate analysis. Response duration was de fined as the interval from anemia response to loss of response, change of therapy, or death. Response duration was calculated using the Kaplan –Meier method and compared by the log-rank test. P-values are two-sided and statistical sig- nificance de fined as P < 0.05. Statistical analyses were performed using the IBM SPSS 22.0 package (SPSS, Chicago, IL, USA).

Subject- and disease-related variables Eighty-eight subjects with PMF and anemia were eval- uated. Forty-six received thalidomide and prednisone and 42 received thalidomide, prednisone, and danazol. Base- line variables were similar (Table 1). Median age was 53 years (range, 21 –77 years). Forty-six (52%) were male. Thirty-eight subjects (43%) were RBC transfusion dependent 19 and 49 (56%) had platelets <100 × 10E + 9/ L. Median hemoglobin concentration and median white blood cell (WBC) and platelet levels were 71 g/L, 3.49 × 10E + 9/L and 84 × 10E + 9/L, respectively. Spleens were palpable a median of 4 cm (range, 0 –24 cm) below the left costal margin (LCM). Fourteen of 54 subjects (26%) with evaluable cytogenetics had unfavorable karyotypes according to DIPSS-plus 3. Thirty-one of 70 subjects tested had JAK2V617F. Forty- five subjects (51%) were inter- mediate-1, 38 (43%) were intermediate-2 and 5 (6%), high- risk according to the DIPSS 2. Thirty-one subjects received prior therapy(ies) includ- ing 9, thalidomide; 20, androgenic steroids; 5, recombi- nant human erythropoietin; 3, corticosteroids; 4, hydroxyurea; 3, interferon; and 1, melphalan. Median interval from diagnosis to study entry was 0 month (range, 0–62 months). Althought on-study, nine subjects received Luo et al . Blood Cancer Journal (2018) 8:9 Page 2 of 5 Blood Cancer Journal hydroxycarbamide (hydroxyurea) alone ( N = 7) or with interferon ( N = 2) and two received melphalan. Responses and outcome The anemia response rate for all subjects was 58% (95% confidence interval (CI) 48, 68%). Subjects receiving thalidomide/prednisone/danazol had a signi ficantly higher response rate compared with those receiving tha- lidomide/prednisone (71% (57, 85%) vs. 46% (32, 60%); P = 0.014). Response rates in subjects who were RBC transfusion dependent were also higher in the danazol cohort (61% (38, 84%) vs. 25% (6, 44%); P = 0.024). There is no signi ficant correlations between anemia response rate and JAK2 V17F mutation state ( P = 0.238). Sixty-eight percent (52, 84%) of JAK2V617F subjects responded compared with 54% (38, 70%) JAK2 wild-type subjects ( P = 0.238). In subgroup analysis, 63% (39, 87%) of JAK2 V617F subjects responded compared with 42% (20, 64%) JAK2 wild-type subjects among patients receiving thalidomide/prednisone ( P = 0.229), 73% (51, 95%) of JAK2V617F subjects responded compared with 65% (44, 86%) JAK2 wild-type subjects among patients receiving thalidomide/prednisone/danazol ( P = 0.875). In multivariate analyses, only thalidomide/prednisone/ danazol therapy (odds ratio (OR) = 3.39 (1.29, 8.89); P = 0.013) and not being RBC transfusion dependent (OR = 2.90 (1.11, 7.61); P = 0.03) were signi ficantly associated with response (Table 2). Responses occurred rapidly: median time to response was 2 months (range, 1–11 months) and did not differ between the cohorts. Interval to response varied: 61% of responders did so by 3 months, 94% by 6 months and only 6% after 6 months. The minimum duration of treatment was 3 months, with a median of 25 months (range 3 –117+ months). Subjects receiving thalidomide/prednisone/danazol had significantly longer response durations than those Table 1 Baseline characteristics of patients Variable Thalidomide/ prednisone Thalidomide/ prednisone/ danazol P-value No. 46 42 Age (years) 53 (21, 77) 53 (26, 72) 0.776 Male n (%) 24 (52.2%) 22 (52.3%) 0.985 Previous therapy 13 (28.3%) 18 (42.9%) 0.152 Spleen size below left costal margin (cm) 3 (0,18) 5 (0, 24) 0.606 Constitutional symptoms 18 (39.1%) 11 (26.2%) 0.197 Hemoglobin (g/L) 71 (31, 99) 71 (38, 99) 0.854 WBC counts (×10 9/L) 4.07 (0.75, 22.36) 3.22 (1.51, 25.63) 0.335 Platelets counts (×109/L) 100 (3, 1064) 80 (14, 540) 0.631 Serum EPO (mU/mL) 504 (21, 774) 652 (76, 774) 0.845 RBC transfusion dependency 20 (43.5%) 18 (42.9%) 0.953 Blood blasts% 0 (0,17) 0 (0, 2) 0.100 Marrow blasts% 0 (0,7) 0 (0,5) 0.202 Bone marrow fibrosis 0.328 MF-2 35 (76.1%) 28 (66.7%) MF-3 11 (23.9%) 14 (33.3%) Cytogeneticsa 0.777 Unfavorable 9 (27.3%) 5 (23.8%) Favorable 24 (72.7%) 16 (76.2%) JAK2V6A7F mutationb 0.810 Positive 16 (46%) 15 (43%) Negative 19 (54%) 20 (57%) DIPSS risk group 0.217 Intermediate-1 20 (43.5%) 25 (59.5%) Intermediate-2 22 (47.8%) 16 (38.1%) High 4 (8.7%) 1 (2.4%) WBC white blood cell, EPO erythropoietin, DIPSS Dynamic International Prognostic Scoring System aCytogenetic information was available in 54 patients bJAK2V6A7F mutation status was available in 70 patients Table 2 Multivariate analysis of anemia response Variable OR (95% CI) Multivariate analysis, P Treatment group 0.013 Thalidomide/prednisone 1 Thalidomide/prednisone/ danazol 3.39 (1.29 –8.89) Gender 0.161 Male 1 Female 2.01 (0.76 –5.31) RBC transfusion dependent 0.03 ≥6U/12W 1 <6U/12W 2.90 (1.11 –7.61) Palpable spleen length 0.225 LCM <5 cm 1 LCM ≥5 cm 1.82 (0.69 –4.79) OR odds ratio, CI confidence interval, LCM left costal margin Luo et al . Blood Cancer Journal (2018) 8:9 Page 3 of 5 Blood Cancer Journal receiving thalidomide/prednisone (hazard ratio (HR) 2.18, 95%CI (1.18 –5.42), P = 0.019; Fig. 1). Median response duration was 27 months (95% CI, 15 –39 months) overall, 30 months (10 –49 months) in the thalidomide/pre- dnisone/danazol cohort compared with 11 months (0–30 months) in the thalidomide/prednisone cohort. Twenty-two of 49 subjects (45% (31, 59%)) with baseline platelets 50 × 10E + 9 L including 58% (39, 77%) of subjects receiving tha- lidomide/prednisone/danazol vs. 30% (11, 49%; P = 0.06) of subject in the thalidomide/prednisone cohort. Median time to platelet response was 3 months (range, <1–9 months) and was similar between the cohorts as was response duration (21 months (95% CI, 7 –35 months)). There was a spleen response in 16 of 41 evaluable subjects (39% (24, 54%)) with similar response rates between the cohorts. Adverse events AEs were dose dependent and reversible with similar incidences (save ALT/AST increases) and severities in the cohorts. No subject discontinued therapy because of drug-related AEs. Leukocytosis and thrombocytosis occurred in 19% (11, 27%) and 24% (15, 33%) of subjects. There was no thrombo-embolic event. The most frequent non-hematologic AE was increased ALT/AST in 19% (8, 31%) of subjects receiving thalidomide/prednisone/dana- zol compared with 4% (3, 22%; P = 0.07) of subjects receiving thalidomide/prednisone. Other non- hematologic AEs were less frequent and did not differ significantly between the cohorts (Table 3) including increased bilirubin in four, constipation in six, hypergly- cemia in six; rash in five; edema in six, neurological symptoms in seven; abdominal distention in four; hyper- tension in three, somnolence in two, and creatinine ele- vation in two. There was no case of prostate cancer or hepatic adenoma.

Efficacy of low-dose thalidomide/prednisone in persons with PMF and anemia was studied in relatively small series of subjects with variable response criteria and response rates 13,23,24. We con firmed the ef ficacy of thali- domide/prednisone using the current IWG-MRT cri- teria 21. Importantly, adding danazol signi ficantly increased response. In another recent study, danazol alone was reported to have a response rate of 30% (17, 43%) 17. These data suggest therapy with thalidomide/prednisone/ danazol is better than any component therapy. Previous studies reported lower serum EPO levels, smaller spleen size, and lower RBC transfusion frequency were associated with higher anemia response rates 17,25,26. We found such an association only for RBC transfusion dependence, and independence. We also found an Fig. 1 Response duration according to the treatment groups Table 3 Toxicity of the therapeutic regimen Adverse events Thalidomide/ prednisone ( n=46) Thalidomide/ prednisone/danazol (n=42) Hyper-bilirubinemia 1 3 Constipation 4 2 Edema 3 3 Hyperglycemia 3 3 Rash 3 2 Neurological symptoms 52 Abdominal distention 22 Hypertension 1 2 Somnolence 2 0 Creatinine elevation 0 2 Luo et al . Blood Cancer Journal (2018) 8:9 Page 4 of 5 Blood Cancer Journal advantage for combined therapy in subjects who were RBC transfusion dependent. Anemia responses occurred quickly and similarly in the two groups. Median time to response was shorter in our study than a previous study of danazol 17 suggesting thalidomide/prednisone may have accelerated danazol responses. Our data suggest adding danazol to thalidomide/pre- dnisone improves response rates, prolongs response duration in persons with PMF and anemia with and without RBC transfusion dependence. The major limita- tion of our study is that it was a retrospective analysis and not randomized, and that although the cohorts were similar for known predictive variables we cannot be cer- tain they were comparable for unknown predictive vari- ables. As such, our conclusions require con firmation in a randomized trial.

This study was supported in part by National Natural Science Funds (no. 81530008, no. 81370611, no. 81270585, and no. 81470297), Program for Peking Union Scholars and Innovative Research Team and PUMC Youth Fund and Fundamental Research Funds for the Central Universities (no. 3332016089). R.P. G. acknowledges support from the National Institute of Health Research (NIHR) Biomedical Research Centre funding scheme. Author details 1MDS and MPN Centre, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. 2State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. 3Department of Pathology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. 4Divisions of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. 5Haematology Section, Division of Experimental Medicine, Department of Medicine, Imperial College London, London, UK Competing interests R.P.G. is a part-time employee of Celgene Corp. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional af filiations. Received: 6 September 2017 Revised: 10 October 2017 Accepted: 13 October 2017

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