ORIGINAL ARTICLE Impact of molecular residual disease post allografting in myelofibrosis patients C Wolschke 1 , A Badbaran 1 , T Zabelina 1 , M Christopeit 1 , F Ayuk 1 , I Triviai 1 , A Zander 1 , H Alchalby 1 , U Bacher 2 , B Fehse 1 and N Kröger 1 We screened 136 patients with myelofibrosis and a median age of 58 years who underwent allogeneic stem cell transplantation (AHSCT) for molecular residual disease for JAKV617F (n = 101), thrombopoietin receptor gene (MPL) (n = 4) or calreticulin (CALR) (n = 31) mutation in peripheral blood on day +100 and +180 after AHSCT. After a median follow-up of 78 months, the 5-year estimated overall survival was 60% (95% confidence interval (CI): 50–70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18–34%) for the entire study population. The percentage of molecular clearance on day 100 was higher in CALR- mutated patients (92%) in comparison with MPL- (75%) and JAKV617F-mutated patients (67%). Patients with detectable mutation at day +100 or at day +180 had a significant higher risk of clinical relapse at 5 years than molecular-negative patients (62% vs 10%, P o0.001) and 70% vs 10%, P o0.001, respectively) irrespectively of the underlying mutation. In a multivariate analysis, high-risk diseases status (hazard ratio (HR) 2.5; 95% CI: 1.18–5.25, P = 0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76–25.30, P o0.001) were significant factors for a higher risk of relapse. Bone Marrow Transplantation (2017) 52, 1526–1529; doi:10.1038/bmt.2017.157; published online 17 July 2017 INTRODUCTION Allogeneic stem cell transplantation can cure a substantial number of patients with myelofibrosis. Relapse is the major case of treatment failure, and depending on disease status and conditioning regimen, it occurs in 10 to 43% of transplanted patients. 1–5 A small study suggested that donor lymphocyte infusions were less effective at clinical relapse than at the time of minimal residual disease (MRD), 6 indicating that early detection of MRD may be important. As JAK2V617F, 7 myeloproliferative leukemia virus (MPL) 8 and more recently, calreticulin (CALR) mutations 9,10 are detectable in ~ 80 to 90% of myelofibrosis patients, these mutations may serve as MRD markers after allogeneic stem cell transplantation. The aim of the study was to evaluate the impact of detectable molecular markers in peripheral blood on day +100 and +180 after SCT on clinical long-term outcome with respect to clinical relapse. PATIENTS AND METHODS We screened 154 patients with primary myelofibrosis (PMF (n = 102), post ET/PV myelofibrosis (n = 46), myelofibrosis in transformation or transformed to acute leukemia (n = 5) and unclassifiable myeloproliferative neoplasms (n = 1) for mutational status. We applied JAK2V617F- 11 and MPL-specific quantitative PCRs (sensitivity for both: 0.01%). 12 CALR mutations were identified by next-generation sequencing and confirmed by Sanger sequencing. For CALR type-2 mutation we made use of a novel digital PCR-based assay described elsewhere. 13 In short, a duplex-digital PCR was designed that simulta- neously detects the CALR p.K385fs*47 (type-2) mutation and the respective wild-type locus. As shown using serial dilutions and buffy-coat cells from healthy donors, the assay combines high sensitivity (0.02%) with excellent specificity. 13 The JAK2V617F mutation was found in 101 patients, MPL and CALR mutations in 4 and 31 patients, respectively, whereas 13 patients were 'triple negative'. For detection and interpretation of MRD, we excluded 'triple-negative' patients and those who received a second allogeneic transplantation (n = 5) from further analysis. Thus, the presented analysis is based on 136 patients (57 females and 79 males) with a median age of 58 years (range: 32–75) and dynamic International Prognostic Scoring System at transplant of low (n = 2), intermediate-1 (n = 15), intermediate-2 (n = 42) or high risk (n = 69); Lille score was low (n = 20), intermediate (n = 65) and high (n = 25). The donor source was HLA-identical sibling (n = 26), and unrelated donor (n = 110) including mismatched unrelated donors (n = 43). All but two patients received PBSCs. All patients underwent busulfan/ fludarabine-based reduced intensity conditioning before allogeneic stem cell transplantation as reported recently. 1 Patients’ characteristics are listed in Table 1. Statistical methods To analyze the impact of residual disease on day +100 and +180 after stem cell transplantation, only those patients who are alive and relapse free with an investigation of the corresponding mutation on days +100 (n = 107) and/or +180 (n = 89) were included. End point was incidence of clinical relapse. Characteristics of patients were expressed as median and range for continuous variables and frequencies for categorical variables. Categorical data were compared using χ 2 test or Fisher's exact test. Survival curves were estimated by the Kaplan–Meier method. The log-rank test was used to estimate the incidence of relapse to account for competing risk. For incidence of relapse, non-relapse mortality after SCT was considered competing events. The Gray test was used to compare the cumulative incidence curves. Univariate analysis was performed using Fine and Gray proportional hazard model: most calculations were performed using SPSS Version 15 (SPSS, IBM Corp, Armonk, NY, USA); the competing risk analyses were carried out using ACCorD (V Gebski, NHMRC Clinical Trials Center and University of Sydney, Camperdown, NSW, Australia). 1 Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany and 2 Department of Hematology and Oncology, University Hospital Göttingen, Göttingen, Germany. Correspondence: Professor N Kröger, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Martinistraße 52, Hamburg D-20246, Germany. E-mail: nkroeger@uke.uni-hamburg.de Received 3 October 2016; revised 9 March 2017; accepted 29 April 2017; published online 17 July 2017 Bone Marrow Transplantation (2017) 52, 1526 – 1529 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0268-3369/17 www.nature.com/bmt