Monitoring of measurable residual disease and chimerism in patients with JAK2 V617F-positive myelofibrosis after allogeneic hematopoietic cell transplantation

Monitoring of measurable residual disease and chimerism in patients with JAK2 V617F-positive myelofibrosis after allogeneic hematopoietic cell transplantation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Monitoring of measurable residual disease and chimerism in patients with JAK2 V617F-positive myelofibrosis after allogeneic hematopoietic cell transplantation Jong-Mi Lee, Ari Ahn, Eun Jeong Min, Sung-Eun Lee, Myungshin Kim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2654645/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract A significant portion of patients with myelofibrosis suffer from relapse after allogeneic hematopoietic stem cell transplantation (allo-HCT). Recognition of early relapse is key to guiding immunotherapeutic intervention, albeit particularly challenging due to complex disease dynamics. In practice, measurable residual disease (MRD) and chimerism are routinely assessed, but their clinical utilities are undefined. Here, we performed intensive molecular testing to measure JAK2-V617F burden (JAK2-MRD) and donor chimerism. Serially collected samples were obtained from 34 consecutive patients at +30, +100, +180 and +360 days after reduced-intensity allo-HCT. Approximately half of the patients harbored persistent JAK2-MRD during 1 year of monitoring. Overt relapse occurred in six patients at median 7.5 months after allo-HCT. Increased JAK2-MRD ratio (≥3-fold than day +30) at day +100 was the most sensitive and earliest indicator for overt relapse. Mixed chimerism (MC; donor chimerism ≤95%) was observed in 10 patients. Intermediate MC (77%–95%) frequently converted to full chimerism after early tapering of immunosuppressive therapy, but high-level MC (≤77%) at day +180 was only seen in relapsed patients. Cytogenetic changes presented in five patients and were mostly found at the time of relapse. Ultimately, comprehensive and longitudinal assessment of molecular monitoring is beneficial to manage myelofibrosis in allo-HSCT settings. Health sciences/Medical research/Genetics research Health sciences/Diseases/Haematological diseases/Haematological cancer/Myeloproliferative disease Health sciences/Risk factors Health sciences/Diseases/Cancer/Haematological cancer/Myeloproliferative disease Figures Figure 1 Figure 2 Introduction Myelofibrosis (MF), including primary MF (PMF), post-essential thrombocythemia MF (post-ET/MF), and post-polycythemia MF (post-PV/MF), is the most severe form of MPN. Allogeneic hematopoietic stem cell transplantation (allo-HCT) is the only known curative option for MF, but the use of this therapy is typically limited by age-related comorbidities, and a significant portion of allo-HCT recipients suffers from relapse [1, 2]. A complex interplay between the fibrotic marrow niche, engraftment, and disease clone influences clinical courses after allo-HCT. Therefore, it is challenging to define and predict relapse in patients with MF after allo-HCT. In 2013, the International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MPN) and European LeukemiaNet (ELN) suggested the definition of complete remission (CR) and relapse in MF [3]. This criterion is widely used by researchers and clinicians so far; however, it has not yet been validated in the post-allo-HCT setting. It also does not consider chimerism and the variable resolutions of fibrosis. Given the usual complications of transplants, relying on clinical parameters or conventional strategies may lead to a significant delay in effective interventions post-transplant [4]. In 2021, the European Society of Blood and Marrow Transplantation (EBMT) group proposed the definitions and methodologies for relapse, focusing on MF after allo-HCT [5]. This recommendation emphasized the role of molecular assays, including measurable residual disease (MRD) and chimerism. Although efforts were made to reflect real-world scenarios, they still need to be validated and supported by the accumulated data. In this current study, we conducted a single-center cohort study that included consecutive patients harboring JAK2 V617F who underwent allo-HCT for MF. We measured MRD and chimerism to define relapse and investigated their prognostic impacts in these patients after allo-HCT. Together with the bone marrow and other hematological and clinical parameters, we explored the patho-histological and molecular biological dynamics in MF. We then further investigated the optimized threshold and time points for the molecular assays (MRD and chimerism) to predict early relapse. Methods Patients and samples We enrolled 34 primary and secondary patients with MF and with mutated JAK2 V617F [6] who received allo-HCT at Seoul St. Mary’s Hospital between December 2012 to November 2021. A total of 150 samples were obtained at the time of allo-HCT (n=33) and at 30 d (n=32), 100 d (n=31), 180 d (n=30), and 360 d (n=24) after allo-HCT. Bone marrow morphology and medical records were thoroughly reviewed to determine clinical courses and relapse status. This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, South Korea (KC22RISI0120) and was conducted in accordance with the tenets of the Declaration of Helsinki. Transplantation procedures All of the patients received a reduced-intensity conditioning regimen, which consisted of fludarabine (30 mg/m 2 for 5 days) and busulfan (3.2 mg/kg for 2 days) with total body irradiation (TBI) 200–400 cGy [7]. Graft-Versus-Host disease (GVHD) prophylaxis consisted of anti-thymocyte globulin (ATG; Thymoglobulin ® )/calcineurin inhibitor/methotrexate (MTX). ATG was administered at a dose of 2.5–7.5 mg/kg according to the donor types (≥5 mg/kg in mismatched donor transplantation). MTX (5 mg/m 2 ) was used on days +1, +3, +6, and +11, along with calcineurin inhibitor (cyclosporine for matched sibling donors and tacrolimus for unrelated donors and haploidentical familial donors). The calcineurin inhibitor dose was tapered gradually starting on day 100–120 after allo-SCT in the absence of acute GVHD. Early tapering of immunosuppressive therapy (IST) was done for the patients who were clinically suspected to relapse. The other general transplantation procedures were performed as described previously [8, 9]. Definitions for relapse The relapse status was established based on the EBMT definition [3, 5]. The morphological and clinical criteria include the following: an increase in age-adjusted cellularity with an abnormal Myeloid:Erythroid ratio; typical megakaryocytic abnormalities; increase in grade of myelofibrosis; and/or development of myelodysplasia, monocytosis, or increased blast count, accompanied by irreversible cytopenia (hemoglobin <100 g/L, neutrophil count <1x10 9 /L, and platelet count <100 x10 9 /L) or an increased immature myeloid cell count in the peripheral blood. In addition to the overt relapse by morphological and clinical criteria, the prognostic relevance of cytogenetic relapse or evolution, molecular relapse, and chimerism relapse were also investigated. Cytogenetic relapse was defined as the appearing preexisting cytogenetic abnormality, and cytogenetic evolution, the new development of any abnormality, which were confirmed by repeated testing. Molecular relapse and chimerism relapse were assessed by JAK2 -MRD and chimerism testing, respectively. The optimized threshold and time points for molecular relapse and chimerism relapse were subjects to be investigated. MRD monitoring using JAK2 V617F quantification DNA was extracted from bone marrow or from peripheral blood using QIAsymphony DSP DNA kits with QIAsymphony instrument (Qiagen, Hilden, Germany). Quantification was performed using Qubit dsDNA Broad Range Assay kits (Thermo Fisher Scientific, Waltham, MD, USA). MRD monitoring for JAK2 V617F was performed using real-time PCR (JAK2 MutaQuant kit, Ipsogen; Qiagen) according to the manufacturer’s instructions. Briefly, a short amplicon covering the JAK2 V617F region was amplified using 25 ng of purified DNA. Positive and negative calibrators at four different concentrations were included in each run to obtain standard curves. All of the samples were tested in duplicates, and the mean cycle threshold (Ct) values were transformed to copy numbers of JAK2 V617F and wild-type using the prepared standard curves. The JAK2 -MRD was expressed as the variant allele frequency (VAF) as a percentage of JAK2 V617F copy numbers for total JAK2 ( JAK2 V617F plus JAK2 wild-type) copy numbers. We developed the other JAK2 -MRD marker, which represented the change of JAK2 V617F, through calculating the ratio of VAF at each time point to the previous VAF ( JAK2 -MRD ratio). Chimerism monitoring We monitored % donor chimerism using both next-generation sequencing-based assay (NGS chimerism) and short tandem repeat-based assay (STR chimerism). NGS chimerism was analyzed using Devyser chimerism NGS kits (Devyser, Stockholm, Sweden). In a single tube, 24 insertion–deletion mutation markers were sequenced using MiSeq (Illumina, San Diego, CA, USA). Data analysis was performed using a dedicated program. STR chimerism was assessed using AmpFlSTR Identifier PCR Amplification (Applied Biosystems, Warrington, UK) as previously reported [10, 11]. Briefly, 16 STR markers were amplified. PCR was performed using a C1000 Touch™ Thermal Cycler (Bio-Rad laboratories Inc., Hercules, CA, USA). Amplified PCR products were analyzed via capillary electrophoresis using an ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). GeneMapper ID Software Version 4.1 (Applied Biosystems, Foster City, CA, USA) was used for automated genotyping and the quantification of peak areas. Statistics The endpoints were morphological/clinical relapse (called “overt relapse” hereafter) and death. The patients’ characteristics were expressed as median and range for continuous variables and frequencies for categorical variables. Categorical data were compared by Fisher’s exact test or the χ 2 test, and continuous data were compared by the Wilcoxon test. We analyzed the predictive power of clinical and molecular factors for relapse, non-relapse mortality (NRM), relapse-free survival (RFS), and overall survival (OS). We defined RFS as the time from the allo-HCT to relapse or death and OS as the time from the allo-HCT to death from any cause. The medical records were tracked until October 2022. Receiver operating characteristic (ROC) analysis was performed to determine the optimal threshold and time points of JAK2 -MRD and chimerism for predicting relapse. The obtained thresholds and time points were validated via time-dependent ROC analysis in the competing risks setting using time ROC package in R [12]. Relapse and NRM were considered competing risk events. Area-under-the-curve (AUC) values were computed at days +300 and +500 after allo-HCT. The RFS and OS were estimated using the Kaplan–Meier method. A competing risk analysis was performed to estimate the probability of a cumulative incidence of relapse (CIR) and NRM. The CIR was compared across groups using the Gray test and cmprsk module in R [13, 14]. The RFS and OS were compared using the Cox proportional hazards regression. The continuous values of JAK2 -MRD and chimerism at different time points were evaluated as time-dependent covariates. Statistical analyses were performed using MedCalc version 19.1.7 (MedCalc Software; Ostend, Belgium) and R software version 4.1.3 (R Foundation for Statistical Computing, Vienna, Austria). Results Baseline characteristics and clinical outcomes The patient demographics are shown in Table 1. The median age was 62.5 (range: 57–67) years at allo-HCT. They comprised 23 patients (67.6%) with primary MF, 5 patients (14.7%) with post-polycythemia vera-MF, and 6 patients (17.6%) with post-essential thrombocythemia MF. Five patients (14.7%) had more than 10% of blast at the time of allo-HCT. Six patients suffered from overt relapse at a median of 7.5 months (range: 3.3-14.7 months) after allo-HCT. Eight patients died due to relapse (n=3), infection (n=3), chronic GVHD (n=1), and other causes (n = 1). The median follow-up duration was 20.4 months (95% confidence interval [CI]: 15.0–81.3 months) after allo-HCT. The 2-year OS was 28.0% (95% CI: 14.6–49.1%). The median RFS was 18.9 months (95% CI: 14.0–42.3 months). The 1-year CIR and NRM were 14.7% (95% CI: 5.4–28.5%) and 11.8% (95% CI: 3.7–24.9%), respectively Dynamics of JAK2 -MRD JAK2 -MRD was positive in 93.9% (31/33) patients with a median VAF of 52.5% (95% CI: 32.9–71.7%) at the time of allo-HCT (Figure 1A). Approximately half of the patients showed positive JAK2 -MRD during 1 year after allo-HCT; 62.5% at day +30, 48.4% at day +100, 46.7% at day +180, and 50% at day +360. We then compared the JAK2 -MRD between relapsed and non-relapsed patients. The JAK2 -MRD VAF was higher in relapsed patients than in non-relapsed patients at days +100 and +180 ( P = 0.005 and 0.011, respectively) (Figure 1B). ROC analysis indicated that JAK2 -MRD VAF at day +100 was the significant predictor of overt relapse ( P <0.001). The optimal JAK2 -MRD VAF threshold was 0.021%, and the AUC value was 0.877 with 100% sensitivity and 70% specificity (Figure 1C, dotted line). As measured by the JAK2 -MRD ratio, the optimal threshold was ≥ 3-fold increase at day +100, and the AUC value increased up to 0.983 with 100% sensitivity and 91.3% specificity (Figure 1C, solid line). In the analysis of time-dependent ROC for overt relapse with competing risk (NRM), JAK2 -MRD ratio at day +100 showed the best performance with AUC value 1.000 at day +300 and 0.986 at day +500 (Supplementary table 1, Supplementary figures 1A and B). Dynamics of chimerism A total of 117 samples obtained after allo-HCT were measured for chimerism using the NGS and STR methods. We defined mixed chimerism (MC) as patients with less than 95% donor chimerism. Those with MC were of 3.1% (3.1%), 22.6% (19.4%), 23.3% (23.3%), and 12.6% (16.0%) at day +30, +100, +180, and +360, as confirmed by NGS and STR (in parentheses) (Figures 1D and E). When we compared the chimerism data between relapsed and non-relapsed patients, NGS chimerism at day +180 was significantly different between them ( P = 0.018) (Fig.1-F, G). ROC analysis also confirmed that NGS chimerism at day +180 was the significant predictor of overt relapse ( P = 0.001). The optimal NGS chimerism threshold was 77%, and the AUC value was 0.840 with 100% sensitivity and 60% specificity (Figure 1H and I). Time-dependent ROC analysis also revealed that NGS chimerism at day +180 was the best predictor for over relapse with AUC value 0.932 at day +300 and 0.834 at day +500 (Supplementary table 1, Supplementary figures 1C and D). Prognostic impact of JAK2 -MRD and NGS chimerism In the survival analysis, none of the baseline characteristics, including age (≥65 years), diagnosis, conventional risk status, donor type, ABO compatibility, and GVHD, were found to be significant predictors for relapse and survival (Table 2). Increased JAK2 -MRD ratio (≥ 3-fold) at day +100 and high-level MC (≤77%) at day +180 were significantly associated with a CIR, RFS, and OS (Supplementary Figure 3). MC (≤95%) at day +180 was significantly associated with CIR and RFS but not with OS. Increased blast (≥10%) at the time of allo-HCT was associated with NRM risk. Markers for overt relapse prediction Of the 34 patients, increased JAK2 -MRD ratio (≥ 3-fold), MC (≤95%), high-level MC (≤77%), and cytogenetic relapse/evolution were presented in 14, 10, 4, and 5 patients, respectively, during the monitoring period. The respective positive predictive values of each maker were 42.9% (6/14), 60% (6/10), 100% (4/4), and 80% (4/5), and their respective negative predictive values were 100% (20/20), 100% (24/24), 93.3% (28/30), and 93.1% (27/29). The cytogenetic changes that appeared in relapse were mainly cytogenetic evolution (80%, 4/5) when cytogenetic relapse was observed in only one patient (#23 in Figure 2). Next, we tried to identify the utility of those markers to predict overt relapse after allo-HCT. Figure 2 and Supplementary Figure 2 depict scenarios of patients (n = 15) who showed emerging molecular markers of relapse. Increased JAK2 -MRD ratio (≥ 3-fold) appeared first in five of 6 relapsed patients (83%), which preceded 134 ± 130 days before overt relapse. In a patient (#8) presenting overt relapse with cytogenetic evolution at day +270, increased JAK2 -MRD ratio and MC (≤95%) caught up late at day +360. These results collectively indicated that increased JAK2 -MRD ratio (≥ 3-fold) was the most powerful marker for the prediction of overt relapse. Nine patients did not progress to overt relapse after they presented molecular markers of relapse. The emerging molecular makers of these patients were as follows: increased JAK2 -MRD ratio only (n = 5), MC only (n = 2), MC followed by increased JAK2 -MRD ratio (n = 1), and MC followed by increased JAK2 -MRD ratio and cytogenetic evolution (n = 1). We observed that early tapering of IST was done in 10 patients presenting molecular markers of relapse. Of note, a significant number of the 10 patients did not progress to overt relapse (n = 9, P = 0.0001) except a patient (#2) who discontinued IST after high-level MC (≤77%) at day +180. Discussion In this study, we performed a comprehensive analysis for JAK2 -MRD and chimerism using longitudinal samples from 34 consecutive patients with MF and investigated their impact on the early detection of relapse and prognosis after allo-HCT. JAK2 -MRD persisted in approximately half of the patients during 1 year after allo-HCT, which was in line with previous reports [15, 16, 17]. As long-lasting JAK2 -MRD was common, particularly in reduced intensity allo-HCT, positive JAK2 V617F at certain time points has limited significance in our patients with MF. Therefore, we determined the JAK2 -MRD ratio to be a new molecular marker that reflected the change (increase or decrease) of the JAK2 V617F and determined that increased JAK2 -MRD ratio (≥ 3-fold) at day +100 was the best indicator for overt relapse. Compared to previous studies that pointed out the critical time point for JAK2 -MRD as +180 days [4, 16, 17], the JAK2 -MRD ratio (≥ 3-fold) at day +100 was a superior marker to predict relapse. Moreover, it is also feasible in routine schedule according to the EBMT guideline, which recommended to assess MRD monitoring at 30, 100, 180, 270, and 360 days after allo-HCT [5]. Along with MRD, chimerism monitoring is essential in assessing the degree of engraftment and risk of relapse in MF [18]. However, strategies for relapse prediction in early stages still represent a need to be addressed [19, 20]. We carried out concurrent chimerism analysis using both the STR and NGS methods. With enhanced sensitivity and accuracy [21], NGS chimerism showed a better performance in relapse prediction compared with STR chimerism. An estimated one-third of patients developed MC (≤95%) during the follow-up period, but this showed little significance in relapse prediction. It was notable that high-level MC (≤77%) at day +180 was a significant marker in predicting overt relapse with 100% specificity. In patients with overt relapse, an increased JAK2 -MRD ratio was the first sign to appear, followed by MC (≤95%) in a short period, which was supported by a previous study [19]. Monitoring MRD and engraftment using these molecular markers was significant because they provided not only the information for early relapse but also the depth of disease remission in order to guide therapeutic interventions, including the early tapering of IST or donor lymphocyte infusion [18, 22, 23]. The present study further showed that intermediate MC, whose donor chimerism was between 77% and 95%, frequently converted to full chimerism after the early tapering of IST. On the other hand, patients with high-level MC (≤77%) eventually developed overt relapse. These results supported that early tapering of IST upon the persistence or emerging molecular markers might prevent relapse through the strong graft-versus-tumor (GVT) effect of MF [18, 24]. Taken together, we carefully suggest that MC status has a role not only as a valuable predictor for relapse but also a significant marker in considering additional interventions, such as tapering IST or donor lymphocyte infusion. Although a number of studies indicated the prognostic significances of JAK2 -MRD [1, 15, 16, 17, 24] or MC [19], the utility of molecular markers remains unclear [25, 26]. Our findings support that molecular monitoring is beneficial for predicting relapse and survival and guiding treatment decisions. Cytogenetic changes were observed during the time of overt relapse. Most of the cytogenetic changes were cytogenetic evolutions, which was in line with our previous study showing that cytogenetics changes in MPN were associated with disease progression [27]. Because the prognostic relevance of the cytogenetic relapse and evolution has not yet been clarified [5, 28], it should be further validated with a large number of cases over a long follow-up period. This study had several limitations. First, the statistical power of this study was limited by a small sample size. In addition, our study could not assess the lineage-specific chimerism, especially T cell-lineage chimerism, which would have better reflected the GVT effect and heralded impending relapse in a reduced-intensity allo-HCT setting [19, 29, 30]. Despite these limitations, this present study demonstrated that the JAK2 -MRD and chimerism assessment is beneficial in defining and predicting relapse after allo-HCT. Based on our experience, the comprehensive and longitudinal assessment of molecular, cytogenetic, and clinical factors is required to properly manage patients with MF in an all-HSCT setting [31]. In summary, JAK2 -MRD was found to be a sensitive and early detector for relapse, but it frequently remains detectable for over a year. Therefore, serial assessment at short intervals would be beneficial, and an optimal threshold need to be established. In this study, the JAK2 -MRD ratio ≥ 3-fold at days +100 was related to relapse. Chimerism was a specific marker for relapse, especially on 180 days after allo-HCT. Although we included limited patients, our results support the prognostic relevance of JAK2 -MRD and chimerism. Declarations Acknowledgements: The authors wish to thank the Catholic Genetic Laboratory Center for their contribution to the experiments. This study was partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (No. 2021R1F1A1058613). Author Contributions: J.M.L. was responsible for designing and directing the project, collecting and analyzing data, interpreting results, and writing the manuscript. A.A. aided in interpreting clinical data. E.J.M provided statistical contributions. Y.K. provided critical feedback on the report. S.E.L. and M.K.conceived the study and provided overall direction and planning. 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Cytogenetic evolution in myeloid neoplasms at relapse after allogeneic hematopoietic cell transplantation: association with previous chemotherapy and effect on survival. Biology of Blood and Marrow Transplantation. 2017;23:782-9. Valcarcel D, Martino R, Caballero D, Mateos M, Perez-Simon J, Canals C, et al. Chimerism analysis following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning. Bone marrow transplantation. 2003;31:387-92. Lee HC, Saliba RM, Rondon G, Chen J, Charafeddine Y, Medeiros LJ, et al. Mixed T lymphocyte chimerism after allogeneic hematopoietic transplantation is predictive for relapse of acute myeloid leukemia and myelodysplastic syndromes. Biology of Blood and Marrow Transplantation. 2015;21:1948-54. McLornan DP, Sirait T, Hernandez-Boluda JC, Czerw T, Hayden P, Yakoub-Agha I. European wide survey on allogeneic haematopoietic cell transplantation practice for myelofibrosis on behalf of the EBMT chronic malignancies working party. Curr Res Transl Med. 2021;69:103267. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementary.docx Supplementary materials Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 05 Apr, 2023 Review # 2 received at journal 04 Apr, 2023 Reviewer # 2 agreed at journal 23 Mar, 2023 Review # 1 received at journal 17 Mar, 2023 Reviewer # 1 agreed at journal 17 Mar, 2023 Reviewers invited by journal 15 Mar, 2023 Editor assigned by journal 07 Mar, 2023 Submission checks completed at journal 07 Mar, 2023 First submitted to journal 06 Mar, 2023 Unknown event 06 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2654645","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":181488899,"identity":"0fd26425-941e-4831-b08e-74bad2e7e356","order_by":0,"name":"Jong-Mi Lee","email":"","orcid":"","institution":"Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Jong-Mi","middleName":"","lastName":"Lee","suffix":""},{"id":181488900,"identity":"8da2c813-1874-49b0-94c7-001d679bebc6","order_by":1,"name":"Ari Ahn","email":"","orcid":"https://orcid.org/0000-0003-3408-767X","institution":"Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Ari","middleName":"","lastName":"Ahn","suffix":""},{"id":181488901,"identity":"3a6b7c37-b42d-44b3-bbd4-4bf28f9ca169","order_by":2,"name":"Eun Jeong Min","email":"","orcid":"","institution":"College of Medicine, The Catholic University of Korea, Seoul, South Korea","correspondingAuthor":false,"prefix":"","firstName":"Eun","middleName":"Jeong","lastName":"Min","suffix":""},{"id":181488902,"identity":"1d3ee7ee-ac35-4362-972c-70471d6e5782","order_by":3,"name":"Sung-Eun Lee","email":"","orcid":"","institution":"Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Kore","correspondingAuthor":false,"prefix":"","firstName":"Sung-Eun","middleName":"","lastName":"Lee","suffix":""},{"id":181488903,"identity":"7f6cae25-d555-4eb6-9e75-e38bceafd16a","order_by":4,"name":"Myungshin Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYDACZhBxQIKBH8K24IEIHyBCi2QDA2MzA4MEEVpgCgwOQLQgRHAB3XbmZx8+nLGINj5++PnjggoJGf4G5ocfGM7cw6nF7DCb8cwZNyRyt51JM2yecUaCR+IAm7EEw41iPFp4mJl5PgC13GAwbOZtk+AxAAoyMHxIIKxl8wz2j1At7N+I0AJ02AYJHpgtPEBbbuDTwmbMCPRC7owzOYWzeUB+OcxTLJFwBo+W84cfM3w4Vpfb3358w2eeCht7/vb2jR8+HMOtBQsARS5JGkbBKBgFo2AUYAAAY0lO0MXwcosAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8632-0168","institution":"Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea","correspondingAuthor":true,"prefix":"","firstName":"Myungshin","middleName":"","lastName":"Kim","suffix":""},{"id":181488904,"identity":"04b81899-3c8c-4edc-879a-1aa96670be85","order_by":5,"name":"Yonggoo Kim","email":"","orcid":"","institution":"College of Medicine, The Catholic University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Yonggoo","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2023-03-04 11:15:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2654645/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2654645/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34548824,"identity":"665f2ecf-fa7f-44a4-bf1b-79ffb3a970dd","added_by":"auto","created_at":"2023-03-20 22:09:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":232027,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of best-performing molecular markers for overt relapse. (A) \u003cem\u003eJAK2\u003c/em\u003e-MRD detection rates during 1 year of follow-up. (B) Comparison of \u003cem\u003eJAK2\u003c/em\u003e-MRD VAF between relapsed and unrelapsed patients at different time points. (C) ROC curves of the \u003cem\u003eJAK2\u003c/em\u003e-MRD VAF and \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio at D100. The \u003cem\u003eJAK2\u003c/em\u003e-MRD VAF at D100 (dotted line) showed an AUC value of 0.877 and optimal threshold of 0.021% with 100% sensitivity and 70% specificity (P\u0026lt;0.001). The \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio at D100 (solid line) showed the best discriminative power for overt relapse (AUC: 0.983) at an optimal threshold of 2.877% with 100% sensitivity and 91.3% specificity (P\u0026lt;0.001). (D-E) MC (donor chimerism \u0026lt;95%) rates by NGS and STR during 1 year of follow-up. (F-G) Comparison of donor chimerism measured by NGS and STR according to the relapse occurrences at different time points. Significant difference of the donor chimerisms between the relapsed and unrelapsed patients was found in only NGS-chimerism at D180. (H) NGS-chimerism at D180 AUC value of 0.840 and optimal threshold of 76.63% with 60% sensitivity and 100% specificity (P=0.001), but (I) STR-chimerism D180 showed no significant AUC values (P=0.073).\u003c/p\u003e\n\u003cp\u003eAbbreviations: VAF, variant allele frequency; ROC, receiver operating characteristic; AUC, area under the curve; MC, mixed chimerism; NGS, next-generation sequencing-based assay; STR, short tandem repeat-based assay\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2654645/v1/0b03ee565e60a994c3fac2fe.jpg"},{"id":34547947,"identity":"1c3b4ad6-dd25-45aa-9574-834dbc5162f2","added_by":"auto","created_at":"2023-03-20 22:01:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261726,"visible":true,"origin":"","legend":"\u003cp\u003eChronology of emerging relapse evidences.\u003c/p\u003e\n\u003cp\u003eSwimmer plot of the 15 patients with relapse evidences and their outcomes, including 6 relapsed and 9 unrelapsed patients. The values of donor chimerism and \u003cem\u003eJAK2\u003c/em\u003e-MRD VAF are presented in supplementary figure 3.\u003c/p\u003e\n\u003cp\u003eAbbreviations: R-pt, relapsed patient; UR-Pt, unrelapsed patient; D, day; MRD, measurable residual disease; MC, mixed chimerism; IST, immunosuppressive therapy; VAF, variant allele frequency\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2654645/v1/ccf9737905a880c990533351.jpg"},{"id":34548826,"identity":"ea2828ff-87df-47da-bea9-87091585a559","added_by":"auto","created_at":"2023-03-20 22:09:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":475024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2654645/v1/dbb171ee-7519-4321-8ac2-459d5293b973.pdf"},{"id":34547948,"identity":"c67f9245-1887-4421-8610-7f28264fd175","added_by":"auto","created_at":"2023-03-20 22:01:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":729560,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-2654645/v1/275d1c84861aad35677c07ad.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Monitoring of measurable residual disease and chimerism in patients with JAK2 V617F-positive myelofibrosis after allogeneic hematopoietic cell transplantation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyelofibrosis (MF), including primary MF (PMF), post-essential thrombocythemia MF (post-ET/MF), and post-polycythemia MF (post-PV/MF),\u0026nbsp;is the most severe form of MPN. Allogeneic hematopoietic stem cell transplantation (allo-HCT) is the only\u0026nbsp;known\u0026nbsp;curative option for MF, but\u0026nbsp;the use of this therapy is typically limited by age-related comorbidities,\u0026nbsp;and a significant portion of allo-HCT recipients suffers from relapse [1, 2]. A complex interplay between\u0026nbsp;the\u0026nbsp;fibrotic marrow niche, engraftment, and disease clone influences clinical courses after allo-HCT. Therefore, it is challenging to define and predict relapse in patients\u0026nbsp;with\u0026nbsp;MF after allo-HCT. In 2013,\u0026nbsp;the\u0026nbsp;International\u0026nbsp;Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MPN) and European LeukemiaNet (ELN) suggested the definition of complete remission (CR) and relapse in MF [3]. This criterion is widely used by researchers and clinicians so far; however, it has\u0026nbsp;not\u0026nbsp;yet\u0026nbsp;been validated in the post-allo-HCT setting. It also does not consider chimerism and\u0026nbsp;the\u0026nbsp;variable resolutions\u0026nbsp;of fibrosis. Given the usual complications of transplants, relying on clinical parameters or conventional strategies may lead to a significant delay in effective interventions post-transplant [4]. In 2021,\u0026nbsp;the\u0026nbsp;European Society of Blood and Marrow Transplantation (EBMT) group proposed the definitions and methodologies for relapse, focusing on MF after allo-HCT [5]. This recommendation emphasized the role of molecular assays,\u0026nbsp;including measurable residual disease (MRD) and chimerism. Although efforts were made to reflect real-world scenarios, they still need to be validated and supported by the accumulated data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this current study, we conducted a single-center cohort study that included consecutive patients harboring \u003cem\u003eJAK2\u003c/em\u003e V617F who underwent allo-HCT for MF. We measured MRD and chimerism to define relapse and investigated their prognostic impacts in\u0026nbsp;these\u0026nbsp;patients after allo-HCT. Together with the bone marrow and other hematological and clinical parameters, we explored the patho-histological and molecular biological dynamics in MF.\u0026nbsp;We\u0026nbsp;then\u0026nbsp;further investigated the optimized threshold and time points for the molecular assays (MRD and chimerism) to predict early relapse.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe enrolled 34 primary and secondary patients\u0026nbsp;with\u0026nbsp;MF\u0026nbsp;and\u0026nbsp;with mutated \u003cem\u003eJAK2\u003c/em\u003e V617F\u0026nbsp;[6]\u0026nbsp;who received allo-HCT at Seoul St. Mary\u0026rsquo;s Hospital between December 2012 to November 2021. A total of 150 samples were obtained at the time of allo-HCT (n=33)\u0026nbsp;and at\u0026nbsp;30\u0026nbsp;d\u0026nbsp;(n=32), 100\u0026nbsp;d\u0026nbsp;(n=31), 180\u0026nbsp;d\u0026nbsp;(n=30), and 360\u0026nbsp;d\u0026nbsp;(n=24) after allo-HCT. Bone marrow morphology and medical records were thoroughly reviewed to determine clinical courses and relapse status. This study was approved by the Institutional Review Board of Seoul St. Mary\u0026rsquo;s Hospital, The Catholic University of Korea, Seoul,\u0026nbsp;South\u0026nbsp;Korea (KC22RISI0120) and was conducted in accordance with the tenets of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransplantation procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;of the\u0026nbsp;patients received\u0026nbsp;a reduced-intensity conditioning regimen,\u0026nbsp;which consisted of fludarabine (30 mg/m\u003csup\u003e2\u003c/sup\u003e for 5 days) and busulfan (3.2 mg/kg for 2 days) with total body irradiation (TBI) 200\u0026ndash;400 cGy\u0026nbsp;[7]. Graft-Versus-Host disease (GVHD) prophylaxis consisted of anti-thymocyte globulin (ATG;\u0026nbsp;Thymoglobulin\u003csup\u003e\u0026reg;\u003c/sup\u003e)/calcineurin inhibitor/methotrexate (MTX). ATG was administered at a dose of 2.5\u0026ndash;7.5 mg/kg according to the donor types (\u0026ge;5\u0026nbsp;mg/kg in mismatched donor transplantation). MTX (5 mg/m\u003csup\u003e2\u003c/sup\u003e) was used on days +1, +3, +6, and +11, along with calcineurin inhibitor (cyclosporine for matched sibling donors and tacrolimus for unrelated donors and haploidentical familial donors). The calcineurin inhibitor dose was tapered gradually starting on day 100\u0026ndash;120 after allo-SCT in the absence of acute GVHD. Early tapering of immunosuppressive therapy (IST) was done for the patients who were clinically\u0026nbsp;suspected to\u0026nbsp;relapse.\u0026nbsp;The other general transplantation procedures were performed as described previously [8, 9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDefinitions for relapse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relapse status was established based on the EBMT definition [3, 5].\u0026nbsp;The morphological and clinical criteria include\u0026nbsp;the following: an\u0026nbsp;increase in age-adjusted cellularity with\u0026nbsp;an\u0026nbsp;abnormal Myeloid:Erythroid ratio;\u0026nbsp;typical megakaryocytic abnormalities;\u0026nbsp;increase in grade of myelofibrosis;\u0026nbsp;and/or development of myelodysplasia, monocytosis,\u0026nbsp;or increased blast\u0026nbsp;count, accompanied by irreversible cytopenia (hemoglobin \u0026lt;100\u0026nbsp;g/L, neutrophil count \u0026lt;1x10\u003csup\u003e9\u003c/sup\u003e/L,\u0026nbsp;and\u0026nbsp;platelet count \u0026lt;100 x10\u003csup\u003e9\u003c/sup\u003e/L) or\u0026nbsp;an\u0026nbsp;increased immature myeloid cell\u0026nbsp;count\u0026nbsp;in\u0026nbsp;the peripheral blood.\u0026nbsp;In addition to the overt relapse by morphological and clinical criteria, the prognostic relevance of cytogenetic relapse or evolution, molecular relapse, and chimerism relapse were also investigated. Cytogenetic relapse was defined as the appearing preexisting cytogenetic abnormality,\u0026nbsp;and cytogenetic evolution, the\u0026nbsp;new development of any abnormality, which were confirmed by repeated testing. Molecular relapse and chimerism relapse were assessed by\u0026nbsp;\u003cem\u003eJAK2\u003c/em\u003e-MRD\u0026nbsp;and chimerism testing, respectively. The optimized threshold and time points for molecular relapse and chimerism relapse\u0026nbsp;were subjects to be investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRD monitoring using \u003cem\u003eJAK2\u003c/em\u003e V617F quantification\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from bone marrow or\u0026nbsp;from\u0026nbsp;peripheral blood\u0026nbsp;using\u0026nbsp;QIAsymphony DSP DNA kits with QIAsymphony instrument (Qiagen, Hilden, Germany). Quantification was performed\u0026nbsp;using\u0026nbsp;Qubit dsDNA Broad Range Assay kits\u0026nbsp;(Thermo Fisher Scientific, Waltham, MD, USA). MRD monitoring for \u003cem\u003eJAK2\u003c/em\u003e V617F was performed using real-time PCR (JAK2 MutaQuant kit, Ipsogen;\u0026nbsp;Qiagen) according to the manufacturer\u0026rsquo;s instructions. Briefly, a short amplicon covering the \u003cem\u003eJAK2\u003c/em\u003e V617F region was amplified using 25\u0026nbsp;ng of purified DNA. Positive and negative calibrators at\u0026nbsp;four\u0026nbsp;different concentrations were included in each run to obtain standard curves. All\u0026nbsp;of the\u0026nbsp;samples were tested in duplicates,\u0026nbsp;and the mean cycle threshold (Ct) values were transformed to copy numbers of \u003cem\u003eJAK2\u003c/em\u003e V617F and wild-type using the prepared standard curves. The \u003cem\u003eJAK2\u003c/em\u003e-MRD was expressed as the variant allele frequency (VAF) as a percentage of \u003cem\u003eJAK2\u003c/em\u003e V617F copy numbers for total \u003cem\u003eJAK2\u003c/em\u003e (\u003cem\u003eJAK2\u003c/em\u003e V617F plus \u003cem\u003eJAK2\u003c/em\u003e wild-type) copy numbers. We developed the other \u003cem\u003eJAK2\u003c/em\u003e-MRD marker,\u0026nbsp;which represented the change of \u003cem\u003eJAK2\u003c/em\u003e V617F, through calculating the ratio of VAF at each time point to the previous VAF (\u003cem\u003eJAK2\u003c/em\u003e-MRD ratio).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChimerism monitoring\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe monitored % donor chimerism using both\u0026nbsp;next-generation sequencing-based assay (NGS chimerism) and short tandem repeat-based assay (STR chimerism). NGS chimerism was analyzed using Devyser chimerism NGS kits\u0026nbsp;(Devyser, Stockholm, Sweden).\u0026nbsp;In a single tube, 24 insertion\u0026ndash;deletion mutation markers were sequenced using MiSeq (Illumina, San Diego, CA, USA). Data analysis\u0026nbsp;was\u0026nbsp;performed using a dedicated program.\u003c/p\u003e\n\u003cp\u003eSTR chimerism was assessed using AmpFlSTR Identifier PCR Amplification (Applied Biosystems, Warrington, UK) as previously reported [10, 11]. Briefly, 16 STR markers were amplified. PCR was performed using a C1000 Touch\u0026trade; Thermal Cycler (Bio-Rad laboratories Inc., Hercules, CA, USA). Amplified PCR products were analyzed\u0026nbsp;via\u0026nbsp;capillary electrophoresis using an ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). GeneMapper ID Software Version 4.1 (Applied Biosystems, Foster City, CA, USA) was used for automated genotyping and\u0026nbsp;the\u0026nbsp;quantification of peak areas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe endpoints were\u0026nbsp;morphological/clinical relapse (called \u0026ldquo;overt relapse\u0026rdquo;\u0026nbsp;hereafter) and death.\u0026nbsp;The patients\u0026rsquo; characteristics were expressed as median and range for continuous variables and frequencies for categorical variables. Categorical data were compared\u0026nbsp;by\u0026nbsp;Fisher\u0026rsquo;s exact test or the \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003etest,\u0026nbsp;and continuous data were compared\u0026nbsp;by the\u0026nbsp;Wilcoxon test. We analyzed the predictive power of clinical and molecular factors for relapse, non-relapse mortality (NRM), relapse-free survival (RFS), and overall survival (OS). We defined RFS as the time from the allo-HCT to relapse or death and OS as the time from the allo-HCT to death from any cause. The medical records were tracked until October 2022. Receiver\u0026nbsp;operating characteristic (ROC) analysis was performed to determine the optimal threshold and time points of \u003cem\u003eJAK2\u003c/em\u003e-MRD and chimerism for predicting relapse.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe obtained thresholds and time\u0026nbsp;points were validated\u0026nbsp;via\u0026nbsp;time-dependent ROC analysis in the competing risks setting using time ROC package in R [12]. Relapse and NRM were considered competing risk events.\u0026nbsp;Area-under-the-curve (AUC)\u0026nbsp;values were computed at days +300 and +500 after allo-HCT. The RFS and OS were estimated using the Kaplan\u0026ndash;Meier method. A competing risk analysis was performed to estimate the probability of a cumulative incidence of relapse (CIR) and NRM. The CIR was compared across groups using the Gray test and cmprsk module in R [13, 14]. The RFS and OS were compared using the Cox proportional hazards regression. The continuous values of \u003cem\u003eJAK2\u003c/em\u003e-MRD and chimerism at different time points were evaluated as time-dependent covariates. Statistical analyses were performed using MedCalc version 19.1.7 (MedCalc Software; Ostend, Belgium) and R software version 4.1.3 (R Foundation for Statistical Computing, Vienna, Austria).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBaseline characteristics and clinical outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient demographics are shown in Table 1. The median age was 62.5 (range: 57\u0026ndash;67) years at allo-HCT. They comprised 23 patients (67.6%) with primary MF, 5 patients (14.7%) with post-polycythemia vera-MF, and 6 patients (17.6%) with post-essential thrombocythemia MF. Five patients (14.7%) had more than 10% of blast at the time of allo-HCT. Six patients suffered from overt relapse at\u0026nbsp;a\u0026nbsp;median\u0026nbsp;of\u0026nbsp;7.5 months (range: 3.3-14.7 months) after allo-HCT. Eight patients died due to relapse (n=3), infection (n=3), chronic GVHD (n=1),\u0026nbsp;and other\u0026nbsp;causes\u0026nbsp;(n = 1). The median follow-up duration was 20.4 months (95% confidence interval\u0026nbsp;[CI]: 15.0\u0026ndash;81.3 months) after allo-HCT. The 2-year\u0026nbsp;OS\u0026nbsp;was 28.0% (95% CI: 14.6\u0026ndash;49.1%). The median\u0026nbsp;RFS\u0026nbsp;was 18.9 months (95% CI: 14.0\u0026ndash;42.3 months). The 1-year\u0026nbsp;CIR\u0026nbsp;and NRM were 14.7% (95% CI: 5.4\u0026ndash;28.5%) and 11.8% (95% CI: 3.7\u0026ndash;24.9%), respectively\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamics of\u003cem\u003e\u0026nbsp;JAK2\u003c/em\u003e-MRD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJAK2\u003c/em\u003e-MRD was positive in 93.9% (31/33) patients with\u0026nbsp;a\u0026nbsp;median VAF of 52.5% (95% CI: 32.9\u0026ndash;71.7%) at the time of allo-HCT (Figure 1A).\u0026nbsp;Approximately\u0026nbsp;half of the patients showed positive \u003cem\u003eJAK2\u003c/em\u003e-MRD during 1 year after allo-HCT; 62.5% at day +30, 48.4% at day +100, 46.7% at day +180, and 50% at day +360.\u0026nbsp;We\u0026nbsp;then\u0026nbsp;compared the \u003cem\u003eJAK2\u003c/em\u003e-MRD between relapsed and non-relapsed patients. The\u003cem\u003e\u0026nbsp;JAK2\u003c/em\u003e-MRD\u0026nbsp;VAF\u0026nbsp;was higher in relapsed patients than in non-relapsed patients at days\u0026nbsp;+100 and +180 (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.005 and 0.011, respectively) (Figure 1B). ROC analysis indicated that \u003cem\u003eJAK2\u003c/em\u003e-MRD VAF at day +100 was the significant predictor of overt relapse (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001). The optimal \u003cem\u003eJAK2\u003c/em\u003e-MRD VAF threshold was 0.021%, and the AUC value was 0.877 with 100% sensitivity and 70% specificity (Figure 1C, dotted line). As measured by\u0026nbsp;the\u003cem\u003e\u0026nbsp;JAK2\u003c/em\u003e-MRD ratio, the optimal threshold was\u0026nbsp;\u0026ge;\u0026nbsp;3-fold increase at day +100, and the AUC value increased up to 0.983 with 100% sensitivity and 91.3% specificity (Figure 1C, solid line). In the analysis of time-dependent ROC for overt relapse with competing risk (NRM), \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio at day +100 showed the best performance with AUC value 1.000 at day +300 and 0.986 at day +500 (Supplementary table 1, Supplementary figures\u0026nbsp;1A\u0026nbsp;and\u0026nbsp;B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamics of chimerism\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 117 samples obtained after allo-HCT were measured for chimerism using\u0026nbsp;the\u0026nbsp;NGS and STR methods. We defined mixed chimerism (MC) as patients with less than 95% donor chimerism.\u0026nbsp;Those with\u0026nbsp;MC\u0026nbsp;were\u0026nbsp;of 3.1% (3.1%), 22.6% (19.4%), 23.3% (23.3%), and 12.6% (16.0%) at day +30, +100, +180, and +360, as confirmed by\u0026nbsp;NGS and STR (in parentheses) (Figures\u0026nbsp;1D\u0026nbsp;and\u0026nbsp;E). When we compared the chimerism data between relapsed and non-relapsed patients, NGS\u0026nbsp;chimerism at day +180 was significantly different between them (\u003cem\u003eP\u003c/em\u003e = 0.018) (Fig.1-F, G). ROC analysis also confirmed that NGS\u0026nbsp;chimerism at day +180 was the significant predictor of overt relapse (\u003cem\u003eP\u003c/em\u003e = 0.001). The optimal NGS\u0026nbsp;chimerism threshold was 77%, and the AUC value was 0.840 with 100% sensitivity and 60% specificity (Figure 1H\u0026nbsp;and\u0026nbsp;I). Time-dependent ROC analysis also revealed that NGS\u0026nbsp;chimerism at day +180 was the best predictor for over relapse with AUC value 0.932 at day +300 and 0.834 at day +500 (Supplementary table 1, Supplementary figures\u0026nbsp;1C\u0026nbsp;and\u0026nbsp;D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrognostic impact of \u003cem\u003eJAK2\u003c/em\u003e-MRD and NGS\u003c/strong\u003e\u003cstrong\u003echimerism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;the\u0026nbsp;survival analysis, none of the baseline characteristics,\u0026nbsp;including age (\u0026ge;65 years), diagnosis, conventional risk status, donor type, ABO compatibility, and GVHD,\u0026nbsp;were\u0026nbsp;found to be significant predictors\u0026nbsp;for relapse and survival (Table 2). Increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (\u0026ge; 3-fold) at day +100 and high-level MC (\u0026le;77%) at day +180 were significantly associated with a CIR, RFS,\u0026nbsp;and OS (Supplementary Figure 3). MC (\u0026le;95%) at day +180 was significantly associated with CIR and RFS but not\u0026nbsp;with\u0026nbsp;OS. Increased blast (\u0026ge;10%) at the time of allo-HCT was associated with NRM risk.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarkers for overt relapse prediction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the 34 patients, increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (\u0026ge;\u0026nbsp;3-fold), MC (\u0026le;95%), high-level MC (\u0026le;77%), and cytogenetic relapse/evolution were presented in 14, 10, 4,\u0026nbsp;and 5 patients,\u0026nbsp;respectively,\u0026nbsp;during the monitoring period. The\u0026nbsp;respective\u0026nbsp;positive predictive values of each maker were 42.9% (6/14), 60% (6/10), 100% (4/4), and 80% (4/5), and their\u0026nbsp;respective\u0026nbsp;negative predictive values were 100% (20/20), 100% (24/24), 93.3% (28/30),\u0026nbsp;and 93.1% (27/29). The cytogenetic changes\u0026nbsp;that\u0026nbsp;appeared in relapse were mainly cytogenetic evolution (80%, 4/5) when cytogenetic relapse was observed in only one patient (#23 in Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we tried to identify the utility of those markers to predict overt relapse after allo-HCT. Figure 2 and Supplementary Figure 2 depict scenarios of patients (n = 15) who showed emerging molecular markers of relapse. Increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (\u0026ge; 3-fold) appeared first in five of 6 relapsed patients (83%),\u0026nbsp;which preceded 134 \u0026plusmn; 130 days before overt relapse. In a patient (#8) presenting overt relapse with cytogenetic evolution at day +270, increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio and MC (\u0026le;95%) caught up late at day +360. These results collectively indicated that increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (\u0026ge; 3-fold) was\u0026nbsp;the\u0026nbsp;most powerful marker for\u0026nbsp;the\u0026nbsp;prediction of overt relapse.\u003c/p\u003e\n\u003cp\u003eNine patients did not progress to overt relapse after they presented molecular markers of relapse. The emerging molecular makers of these\u0026nbsp;patients were as follows:\u0026nbsp;increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio only (n = 5), MC only (n = 2), MC followed by increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (n = 1), and MC followed by increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio and cytogenetic evolution (n = 1). We observed that early tapering of IST was done in 10 patients presenting molecular markers of relapse. Of note, a significant number of the\u0026nbsp;10 patients\u0026nbsp;did not progress to overt relapse (n = 9, \u003cem\u003eP\u003c/em\u003e = 0.0001) except a patient (#2) who discontinued IST after high-level MC (\u0026le;77%) at day +180.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we performed\u0026nbsp;a\u0026nbsp;comprehensive analysis for \u003cem\u003eJAK2\u003c/em\u003e-MRD and chimerism using longitudinal samples from 34 consecutive patients\u0026nbsp;with\u0026nbsp;MF and investigated their impact on\u0026nbsp;the\u0026nbsp;early detection of relapse and prognosis after allo-HCT. \u003cem\u003eJAK2\u003c/em\u003e-MRD\u0026nbsp;persisted in\u0026nbsp;approximately\u0026nbsp;half of the patients during 1\u0026nbsp;year\u0026nbsp;after allo-HCT,\u0026nbsp;which was in line with previous reports [15, 16, 17]. As\u0026nbsp;long-lasting \u003cem\u003eJAK2\u003c/em\u003e-MRD was\u0026nbsp;common,\u0026nbsp;particularly\u0026nbsp;in reduced intensity allo-HCT, positive \u003cem\u003eJAK2\u003c/em\u003e V617F at certain time points\u0026nbsp;has limited significance in our patients\u0026nbsp;with\u0026nbsp;MF. Therefore, we\u0026nbsp;determined the\u0026nbsp;\u003cem\u003eJAK2\u003c/em\u003e-MRD\u0026nbsp;ratio\u0026nbsp;to be\u0026nbsp;a new molecular marker that reflected the change (increase\u0026nbsp;or\u0026nbsp;decrease) of the \u003cem\u003eJAK2\u003c/em\u003e V617F and determined that increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (\u0026ge;\u0026nbsp;3-fold) at day +100 was the best indicator for overt relapse. Compared to previous studies\u0026nbsp;that pointed out\u0026nbsp;the critical time point for \u003cem\u003eJAK2\u003c/em\u003e-MRD as +180 days [4, 16, 17],\u0026nbsp;the \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio (\u0026ge; 3-fold) at day +100 was\u0026nbsp;a\u0026nbsp;superior marker to predict relapse. Moreover, it is also feasible in routine schedule according to\u0026nbsp;the EBMT guideline,\u0026nbsp;which recommended to assess MRD monitoring at 30, 100, 180, 270,\u0026nbsp;and 360 days after allo-HCT [5].\u003c/p\u003e\n\u003cp\u003eAlong with MRD, chimerism monitoring is essential\u0026nbsp;in\u0026nbsp;assessing\u0026nbsp;the degree of engraftment and risk of relapse in MF [18]. However, strategies for relapse prediction in early stages still represent a need to be addressed [19, 20]. We carried out concurrent chimerism analysis using both\u0026nbsp;the\u0026nbsp;STR and NGS methods. With enhanced sensitivity and accuracy [21], NGS\u0026nbsp;chimerism showed\u0026nbsp;a\u0026nbsp;better performance in relapse prediction compared with STR\u0026nbsp;chimerism.\u0026nbsp;An estimated\u0026nbsp;one-third of patients developed MC (\u0026le;95%) during\u0026nbsp;the\u0026nbsp;follow-up\u0026nbsp;period, but\u0026nbsp;this\u0026nbsp;showed little significance\u0026nbsp;in\u0026nbsp;relapse\u0026nbsp;prediction. It was notable that high-level MC (\u0026le;77%) at day +180 was\u0026nbsp;a\u0026nbsp;significant marker\u0026nbsp;in\u0026nbsp;predicting\u0026nbsp;overt relapse with 100% specificity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn patients with overt relapse,\u0026nbsp;an\u0026nbsp;increased \u003cem\u003eJAK2\u003c/em\u003e-MRD ratio was the first\u0026nbsp;sign\u0026nbsp;to appear, followed by MC (\u0026le;95%) in a short period, which was supported by\u0026nbsp;a\u0026nbsp;previous study [19]. Monitoring MRD and engraftment using these molecular markers was significant because they provided not only the information for early relapse but also the depth of disease remission in order to guide therapeutic interventions,\u0026nbsp;including\u0026nbsp;the\u0026nbsp;early tapering of IST or donor lymphocyte infusion\u0026nbsp;[18, 22, 23]. The present study further showed that intermediate MC, whose donor chimerism was between 77% and 95%, frequently converted to full chimerism after\u0026nbsp;the\u0026nbsp;early tapering of IST. On the other hand, patients with high-level MC (\u0026le;77%) eventually\u0026nbsp;developed\u0026nbsp;overt relapse. These results supported that early tapering of IST upon the persistence or emerging molecular markers might prevent relapse through\u0026nbsp;the\u0026nbsp;strong graft-versus-tumor (GVT) effect of MF [18, 24]. Taken together,\u0026nbsp;we carefully suggest that MC status has a role not only as a valuable predictor for relapse but also a significant marker\u0026nbsp;in\u0026nbsp;considering additional interventions,\u0026nbsp;such as tapering IST or donor lymphocyte infusion.\u003c/p\u003e\n\u003cp\u003eAlthough a number of studies indicated the prognostic significances of \u003cem\u003eJAK2\u003c/em\u003e-MRD [1, 15, 16, 17, 24]\u0026nbsp;or MC [19], the utility of molecular markers remains unclear\u0026nbsp;[25, 26]. Our findings support that molecular monitoring is beneficial for predicting relapse and survival and guiding treatment decisions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Cytogenetic changes were observed\u0026nbsp;during\u0026nbsp;the time of overt relapse. Most of the cytogenetic changes were cytogenetic evolutions,\u0026nbsp;which was in line with our previous study showing that cytogenetics changes in MPN were associated with disease progression [27]. Because the prognostic relevance of the cytogenetic relapse and evolution has not\u0026nbsp;yet\u0026nbsp;been clarified [5, 28], it should be further validated with a large number of cases\u0026nbsp;over\u0026nbsp;a long follow-up period.\u003c/p\u003e\n\u003cp\u003eThis study had several limitations. First, the statistical power of this study was limited by a small sample size. In addition, our study could not assess the lineage-specific chimerism, especially T cell-lineage chimerism, which\u0026nbsp;would have\u0026nbsp;better reflected the\u0026nbsp;GVT effect and heralded\u0026nbsp;impending relapse in\u0026nbsp;a\u0026nbsp;reduced-intensity allo-HCT\u0026nbsp;setting\u0026nbsp;[19, 29, 30]. Despite these limitations, this present study demonstrated that the \u003cem\u003eJAK2\u003c/em\u003e-MRD and chimerism assessment is beneficial\u0026nbsp;in\u0026nbsp;defining\u0026nbsp;and predicting\u0026nbsp;relapse after allo-HCT.\u0026nbsp;Based on our experience,\u0026nbsp;the\u0026nbsp;comprehensive and longitudinal assessment of molecular, cytogenetic,\u0026nbsp;and clinical factors is required to properly manage patients\u0026nbsp;with\u0026nbsp;MF in\u0026nbsp;an\u0026nbsp;all-HSCT setting [31].\u003c/p\u003e\n\u003cp\u003eIn summary, \u003cem\u003eJAK2\u003c/em\u003e-MRD was\u0026nbsp;found to be a\u0026nbsp;sensitive and early detector for relapse, but it frequently remains detectable for over a year. Therefore, serial assessment at short intervals would be beneficial, and\u0026nbsp;an\u0026nbsp;optimal threshold need to be established. In this study, the\u0026nbsp;\u003cem\u003eJAK2\u003c/em\u003e-MRD ratio \u0026ge; 3-fold at days +100 was related to relapse. Chimerism was a specific marker for relapse,\u0026nbsp;especially on 180 days after allo-HCT. Although we included limited patients, our results support the prognostic relevance of \u003cem\u003eJAK2\u003c/em\u003e-MRD and chimerism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e The authors wish to thank\u0026nbsp;the Catholic Genetic Laboratory Center for their contribution to the experiments.\u0026nbsp;This study was partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT,\u0026nbsp;and Future Planning (No. 2021R1F1A1058613).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eJ.M.L. was responsible for designing and directing the project, collecting and analyzing data, interpreting results,\u0026nbsp;and writing the manuscript. A.A.\u0026nbsp;aided in\u0026nbsp;interpreting\u0026nbsp;clinical data. E.J.M provided statistical contributions. Y.K. provided critical feedback on the report. S.E.L. and M.K.conceived the study and provided overall direction and planning. All\u0026nbsp;of the\u0026nbsp;authors discussed the results and contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcLornan DP, Szydlo R, Robin M, van Biezen A, Koster L, Blok HJP, et al. 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Blood, The Journal of the American Society of Hematology. 2013;122:872-84.\u003c/li\u003e\n\u003cli\u003eGerds AT, Gotlib J, Ali H, Bose P, Dunbar A, Elshoury A, et al. Myeloproliferative neoplasms, version 3.2022, NCCN clinical practice guidelines in oncology. Journal of the National Comprehensive Cancer Network. 2022;20:1033-62.\u003c/li\u003e\n\u003cli\u003eKim Y, Park J, Jo I, Lee GD, Kim J, Kwon A, et al. Genetic\u0026ndash;pathologic characterization of myeloproliferative neoplasms. Experimental \u0026amp; Molecular Medicine. 2016;48:e247-e.\u003c/li\u003e\n\u003cli\u003eErtz-Archambault N, Kosiorek H, Slack JL, Lonzo ML, Greipp PT, Khera N, et al. Cytogenetic evolution in myeloid neoplasms at relapse after allogeneic hematopoietic cell transplantation: association with previous chemotherapy and effect on survival. Biology of Blood and Marrow Transplantation. 2017;23:782-9.\u003c/li\u003e\n\u003cli\u003eValcarcel D, Martino R, Caballero D, Mateos M, Perez-Simon J, Canals C, et al. Chimerism analysis following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning. Bone marrow transplantation. 2003;31:387-92.\u003c/li\u003e\n\u003cli\u003eLee HC, Saliba RM, Rondon G, Chen J, Charafeddine Y, Medeiros LJ, et al. Mixed T lymphocyte chimerism after allogeneic hematopoietic transplantation is predictive for relapse of acute myeloid leukemia and myelodysplastic syndromes. Biology of Blood and Marrow Transplantation. 2015;21:1948-54.\u003c/li\u003e\n\u003cli\u003eMcLornan DP, Sirait T, Hernandez-Boluda JC, Czerw T, Hayden P, Yakoub-Agha I. European wide survey on allogeneic haematopoietic cell transplantation practice for myelofibrosis on behalf of the EBMT chronic malignancies working party. Curr Res Transl Med. 2021;69:103267.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"blood-cancer-journal","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"bcj","sideBox":"Learn more about [Blood Cancer Journal](http://www.nature.com/bcj/)","snPcode":"41408","submissionUrl":"https://mts-bcj.nature.com/cgi-bin/main.plex","title":"Blood Cancer Journal","twitterHandle":"@bloodcancerjnl","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2654645/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2654645/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"A significant portion of patients with myelofibrosis suffer from relapse after allogeneic hematopoietic stem cell transplantation (allo-HCT). Recognition of early relapse is key to guiding immunotherapeutic intervention, albeit particularly challenging due to complex disease dynamics. In practice, measurable residual disease (MRD) and chimerism are routinely assessed, but their clinical utilities are undefined. Here, we performed intensive molecular testing to measure JAK2-V617F burden (JAK2-MRD) and donor chimerism. Serially collected samples were obtained from 34 consecutive patients at +30, +100, +180 and +360 days after reduced-intensity allo-HCT. Approximately half of the patients harbored persistent JAK2-MRD during 1 year of monitoring. Overt relapse occurred in six patients at median 7.5 months after allo-HCT. Increased JAK2-MRD ratio (≥3-fold than day +30) at day +100 was the most sensitive and earliest indicator for overt relapse. Mixed chimerism (MC; donor chimerism ≤95%) was observed in 10 patients. Intermediate MC (77%–95%) frequently converted to full chimerism after early tapering of immunosuppressive therapy, but high-level MC (≤77%) at day +180 was only seen in relapsed patients. Cytogenetic changes presented in five patients and were mostly found at the time of relapse. Ultimately, comprehensive and longitudinal assessment of molecular monitoring is beneficial to manage myelofibrosis in allo-HSCT settings.","manuscriptTitle":"Monitoring of measurable residual disease and chimerism in patients with JAK2 V617F-positive myelofibrosis after allogeneic hematopoietic cell transplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-20 22:00:58","doi":"10.21203/rs.3.rs-2654645/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-04-05T09:56:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-04-04T08:40:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-03-24T02:51:11+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-03-17T20:06:13+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-03-17T17:40:29+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-03-15T13:10:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-07T11:19:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-03-07T11:14:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Blood Cancer Journal","date":"2023-03-06T13:15:06+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2023-03-06T12:05:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"blood-cancer-journal","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"bcj","sideBox":"Learn more about [Blood Cancer Journal](http://www.nature.com/bcj/)","snPcode":"41408","submissionUrl":"https://mts-bcj.nature.com/cgi-bin/main.plex","title":"Blood Cancer Journal","twitterHandle":"@bloodcancerjnl","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6c0618b-ab23-47b3-834e-9c5a79b05ef9","owner":[],"postedDate":"March 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":19728256,"name":"Health sciences/Medical research/Genetics research"},{"id":19728257,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Myeloproliferative disease"},{"id":19728258,"name":"Health sciences/Risk factors"},{"id":19728259,"name":"Health sciences/Diseases/Cancer/Haematological cancer/Myeloproliferative disease"}],"tags":[],"updatedAt":"2023-05-16T14:11:48+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-20 22:00:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2654645","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2654645","identity":"rs-2654645","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}