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Abstract Background Deletions and partial losses of chromosome 7 (chr7) are frequent in acute myeloid leukemia (AML) and are linked to dismal outcome. However, the genomic landscape and prognostic impact of concomitant genetic aberrations remain incompletely understood. Methods To discover genetic lesions in adult AML patients with aberrations of chromosome 7 [abn(7)], 60 paired diagnostic/remission samples were investigated by whole-exome sequencing in the exploration cohort. Subsequently, a gene panel including 66 genes and a SNP backbone for copy-number variation detection was designed and applied to the remaining samples of the validation cohort. In total, 519 patients were investigated, of which 415 received intensive induction treatment, typically containing a combination of cytarabine and anthracyclines. Results In the exploration cohort, the most frequently mutated gene was TP53 (33%), followed by epigenetic regulators (DNMT3A, KMT2C, IDH2) and signaling genes (NRAS, PTPN11). Thirty percent of 519 patients harbored ≥ 1 mutation in genes located in commonly deleted regions of chr7—most frequently affecting KMT2C (16%) and EZH2 (10%). KMT2C mutations were often subclonal and enriched in patients with del(7q), de novo or core-binding factor AML (45%). Cancer cell fraction analysis and reconstruction of mutation acquisition identified TP53 mutations as mainly disease-initiating events, while del(7q) or −7 appeared as subclonal events in one-third of cases. Multivariable analysis identified five genetic lesions with significant prognostic impact in intensively treated AML patients with abn(7). Mutations in TP53 and PTPN11 (11%) showed the strongest association with worse overall survival (OS, TP53: hazard ratio [HR], 2.53 [95% CI 1.66–3.86]; P

Abstract Introduction IDH2 mutation is an unfavorable prognostic factor in patients with primary myelofibrosis (PMF) but its effect on myelofibrosis (MF) remains largely unclear. Methods In this study, we aimed to elucidate the roles of IDH2 mutation in the development and progression of MF by transcriptomic and molecular techniques using the Idh2R172K transgenic mice. Results We found that thrombopoietin (TPO)‐overexpressed Idh2R172K (Idh2R172K + TPO) mice had accelerated progression to MF, compared with TPO‐overexpressed Idh2‐wild (WT + TPO) mice, showing activation of multiple inflammatory pathways, among which nuclear factor κB (NFκB) was the most significantly enhanced. Single‐cell transcriptomes of the marrow cells in early MF showed that S100a8/a9 expression was mainly confined to neutrophil progenitors in the WT + TPO mice, but highly expressed in several types of myeloid precursor cells, including the megakaryocyte progenitors in the Idh2R172K + TPO group. Furthermore, Idh2R172K mice at age of 18 months had larger spleens, increased S100a8/a9‐Tlr4 expression, and elevated serum S100a8/a9 levels compared with WT mice. PMF patients with IDH2 mutations had higher bone marrow plasma S100A8/A9 levels than those without IDH2 mutations. Conclusion Overall, our findings showed that IDH2 mutation induced proinflammatory effects, which further exacerbated MF, as evidenced by the increase in S100a8/a9 levels and NFκB hyperactivation in Idh2R172K + TPO mice.

Abstract We conducted a retrospective population-based, matched cohort study using the National Health Insurance Research Database to estimate healthcare resource utilisation (HRU) and costs in patients with newly diagnosed AL amyloidosis in Taiwan. Cases were matched 10:1 by age, sex, and area of residence to patients without AL amyloidosis (comparators) randomly selected from the database during the same time period. Annual all-cause HRU and costs for 3 years were quantified. AL amyloidosis-attributable costs were obtained by subtracting all-cause HRU costs incurred by comparators from cases. The mean age of all patients was 60.78 years and 59.07% were male. Co-morbidities were more frequent in cases than comparators. By 6 months after diagnosis, 12.1% of cases had died versus 0.9% of comparators. In the first year, cases had 103% more outpatient visits, 177% more emergency room visits, were hospitalised 4-times more frequently, and spent 5.5-times more days in hospital than comparators, and total healthcare costs were > sixfold higher. Costs incurred during the first year after diagnosis accounted for 55% of the 3-year cumulative cost. High HRU costs associated with delayed diagnosis and end-organ damage indicate a need for earlier diagnosis and more effective treatments for AL amyloidosis.

Abstract The European Leukemia Net recommendations provide valuable guidance in treatment decisions of patients with acute myeloid leukemia (AML). However, the genetic complexity and heterogeneity of AML are not fully covered, notwithstanding that gene expression analysis is crucial in the risk stratification of AML. The Stellae-123 score, an AI-based model that captures gene expression patterns, has demonstrated robust survival predictions in AML patients across four western-population cohorts. This study aims to evaluate the applicability of Stellae-123 in a Taiwanese cohort. The Stellae-123 model was applied to 304 de novo AML patients diagnosed and treated at the National Taiwan University Hospital. We find that the pretrained (BeatAML-based) model achieved c-indexes of 0.631 and 0.632 for the prediction of overall survival (OS) and relapse-free survival (RFS), respectively. Model retraining within our cohort further improve the cross-validated c-indexes to 0.667 and 0.667 for OS and RFS prediction, respectively. Multivariable analysis identify both pretrained and retrained models as independent prognostic biomarkers. We further show that incorporating age, Stellae-123, and ELN classification remarkably improves risk stratification, revealing c-indices of 0.73 and 0.728 for OS and RFS, respectively. In summary, the Stellae-123 gene expression signature is a valuable prognostic tool for AML patients and model retraining can improve the accuracy and applicability of the model in different populations.

Abstract In 2022, two novel classification systems for myelodysplastic syndromes/neoplasms (MDS) have been proposed: the International Consensus Classification (ICC) and the 2022 World Health Organization (WHO-2022) classification. These two contemporary systems exhibit numerous shared features but also diverge significantly in terminology and the definition of new entities. Thus, we retrospectively validated the ICC and WHO-2022 classification and found that both systems promoted efficient segregation of this heterogeneous disease. After examining the distinction between the two systems, we showed that a peripheral blood blast percentage ≥ 5% indicates adverse survival. Identifying MDS/acute myeloid leukemia with MDS-related gene mutations or cytogenetic abnormalities helps differentiate survival outcomes. In MDS, not otherwise specified patients, those diagnosed with hypoplastic MDS and single lineage dysplasia displayed a trend of superior survival compared to other low-risk MDS patients. Furthermore, the impact of bone marrow fibrosis on survival was less pronounced within the ICC framework. Allogeneic transplantation appears to improve outcomes for patients diagnosed with MDS with excess blasts in the ICC. Therefore, we proposed an integrated system that may lead to the accurate diagnosis and advancement of future research for MDS. Prospective studies are warranted to validate this refined classification.

Abstract Acute myeloid leukemia (AML) with CEBPA bZIP in-frame mutations (CEBPA bZIP-inf) is classified within the favorable-risk group by the 2022 European LeukemiaNet (ELN-2022). However, heterogeneous clinical outcomes are still observed in these patients. In this study, we aimed to investigate the mutation profiles and transcriptomic patterns associated with poor outcomes in patients with CEBPA bZIP-inf. One hundred and thirteen CEBPA bZIP-inf patients were identified in a cohort of 887 AML patients homogeneously treated with intensive chemotherapy. Concurrent WT1 or DNMT3A mutations significantly predicted worse survival in AML patients with CEBPA bZIP-inf. RNA-sequencing analysis revealed an enrichment of interferon (IFN) signaling and metabolic pathways in those with a shorter event-free survival (EFS). CEBPA bZIP-inf patients with a shorter EFS had higher expression of IFN-stimulated genes (IRF2, IRF5, OAS2, and IFI35). Genes in mitochondrial complexes I (NDUFA12 and NDUFB6) and V (ATP5PB and ATP5IF1) were overexpressed and were associated with poorer survival, and the results were independently validated in the TARGET AML cohort. In conclusion, concurrent WT1 or DNMT3A mutations and a dysregulated immune and metabolic state were correlated with poor survival in patients with CEBPA bZIP-inf, and upfront allogeneic transplantation may be indicated for better long-term disease control.

Abstract Myelodysplastic syndromes (MDS) have varied prognoses and require a risk-adapted treatment strategy for treatment optimization. Recently, a molecular prognostic model (Molecular International Prognostic Scoring System [IPSS-M]) that combines clinical parameters, cytogenetic abnormalities, and mutation topography was proposed. This study validated the IPSS-M in 649 patients with primary MDS (based on the 2022 International Consensus Classification [ICC]) and compared its prognostic power to those of the IPSS and revised IPSS (IPSS-R). Overall, 42.5% of the patients were reclassified and 29.3% were up-staged from the IPSS-R. After the reclassification, 16.9% of the patients may receive different treatment strategies. The IPSS-M had greater discriminative potential than the IPSS-R and IPSS. Patients with high, or very high-risk IPSS-M might benefit from allogeneic hematopoietic stem cell transplantation. IPSS-M, age, ferritin level, and the 2022 ICC categorization predicted outcomes independently. After analyzing demographic and genetic features, complementary genetic analyses, including KMT2A-PTD, were suggested for accurate IPSS-M categorization of patients with ASXL1, TET2, STAG2, RUNX1, SF3B1, SRSF2, DNMT3A, U2AF1, and BCOR mutations and those classified as MDS, not otherwise specified with single lineage dysplasia/multi-lineage dysplasia based on the 2022 ICC. This study confirmed that the IPSS-M can better risk-stratified MDS patients for optimized therapeutic decision-making.

Background/purpose: The S100 family proteins are involved in a variety of important biological processes, most notably immune and inflammatory responses. Their dysregulation also plays a role in the pathogenesis of human cancers. S100A4, also known as metastasin, has long been regarded as a biological marker in tumor progression and metastasis in multiple solid cancers, but its clinical significance in acute myeloid leukemia (AML) has not been extensively studied. Methods: We retrospectively studied the association between S100A4 gene expression and the clinical characteristics, mutational and transcriptomic profiles of 227 AML patients treated with standard intensive chemotherapy. Genetic mutations of myeloid disease associated genes were analyzed by Sanger sequencing. Microarray-based transcriptomic gene expression profiling was performed on archived bone marrow mononuclear cells. Bioinformatic analyses, including differential gene expression and gene set enrichment analysis, were conducted to delineate the underlying pathogenic mechanisms. Results: Higher S100A4 expression was associated with older age, monocytic differentiation of leukemic cells, and adverse clinical outcome. S100A4 high-expressors had inferior overall survival and disease-free survival; this finding could be validated in the TCGA AML cohort (both the microarray and RNA-seq platforms). Multivariate Cox regression analysis supported S100A4 as an independent prognostic factor. Bioinformatic analysis showed that AML with higher S100A4 expression was enriched for the interferon, NLRP3 inflammasome, and epithelial–mesenchymal transition pathways. Conclusion: This study provides evidence that S100A4 overexpression serves as a poor prognostic biomarker in AML, holds potential to guide treatment planning in the clinic, and indicates novel therapeutic directions.

Objectives/Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with high mortality. To date, there is no comprehensive population-based analysis of patients with AML in Asia, including Taiwan. Material and methods: This is a retrospective cohort study using three population-based databases, namely, the Taiwan Cancer Registry, Taiwanese National Health Insurance Research Database, and Taiwan Death Registry, between 2001 and 2015 to provide detailed information on patients with AML and relevant clinical variables, such as sex, age, year of diagnosis, socioeconomic status (SES) level, hospital level, treatment location, and Deyo-Charlson Comorbidity Index (Deyo-CCI) score. Results: Patients with newly diagnosed AML (n = 9949) were included in the study. The median age was 60 years, and the overall age-adjusted AML incidence over 15 years was 2.44 per 100,000 person-years. The median overall survival (OS) of patients younger than 65 years was 18 months, whereas the OS of patients older than age 65 was only 5 months. AML patients with a prior cancer history had the worst outcomes, and the acute promyelocytic leukemia subtype predicted better survival. Patients who were older, male and a higher Deyo-CCI score had a significantly higher risk of death. In contrast, patients with a higher SES level and receiving treatment in a medical center had a lower risk of mortality than their respective counterparts. Conclusion: Our study results could enable clinicians to obtain a comprehensive picture of the epidemiology, survival outcomes and unmet medical needs of AML patients in Taiwan.

Abstract Objective The optimal treatment for vitreoretinal lymphoma (VRL) remains a challenge, as central nervous system (CNS) relapse occurs frequently, leading to the worst impact on survival. We previously proposed combined intravitreal methotrexate and systemic high‐dose methotrexate therapy for this disease. This study aimed to report the long‐term outcomes of patients with VRL using this combination treatment. Methods We conducted a retrospective cohort study on patients with VRL at a tertiary referral center between 2003 and 2018. Results Thirty‐two patients were included, of whom 23 had primary VRL (PVRL) and nine had concurrent intraocular and CNS diseases. The treatment was well tolerated. Twenty‐six (81.3%) patients achieved complete response (CR). After a median follow‐up time of 103.5 months, the 5‐year survival rate was 73.3%, whereas the 5‐year progression‐free survival (PFS) rate was 29.9%. Twenty‐four (75%) patients relapsed, including 12 with isolated intraocular relapses at first relapse and a total of 17 with CNS/systemic relapses. The development of CNS/systemic relapse negatively affected survival, but intraocular relapse did not. The median CNS/systemic PFS was 69.5 months, but the risk of CNS/systemic relapse increased steadily with a cumulative incidence rate at 2, 5, and 10 years being 22.6%, 44.2%, and 65%, respectively. Multivariate analysis identified concurrent CNS disease at diagnosis as the only poor‐risk factor for CNS/systemic relapse. Conclusions This study confirms good efficacy and acceptable toxicities of the combination approach. However, incorporation of further intensive consolidation strategies into the treatment protocol to effectively prevent subsequent CNS/systemic relapse deserves to be considered.

Abstract A set of myelodysplasia-related (MDS-R) gene mutations are incorporated into the 2022 European LeukemiaNet risk classification as adverse genetic factors for acute myeloid leukemia (AML) based on their poor prognostic impact on older patients. The impact of these mutations on younger patients (age

Abstract RUNX1 mutations are frequently detected in various myeloid neoplasms and implicate unfavourable clinical outcomes in patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). On the other hand, high expression of RUNX1 is also correlated with poor prognosis in AML patients. However, the clinical relevancy of RUNX1 expression in MDS patients remains elusive. This study aimed to investigate the prognostic and biologic impacts of RUNX1 expression in MDS patients. We recruited 341 MDS patients who had sufficient bone marrow samples for next‐generation sequencing. Higher RUNX1 expression occurred more frequently in the patients with Revised International Prognostic Scoring System (IPSS‐R) higher‐risk MDS than the lower‐risk group. It was closely associated with poor‐risk cytogenetics and mutations in ASXL1, NPM1, RUNX1, SRSF2, STAG2, TET2 and TP53. Furthermore, patients with higher RUNX1 expression had significantly shorter leukaemia‐free survival (LFS) and overall survival (OS) than those with lower expression. Subgroups analysis revealed that higher‐RUNX1 group consistently had shorter LFS and OS than the lower‐RUNX1 group, no matter RUNX1 was mutated or not. The same findings were observed in IPSS‐R subgroups. In multivariable analysis, higher RUNX1 expression appeared as an independent adverse risk factor for survival. The prognostic significance of RUNX1 expression was validated in two external public cohorts, GSE 114922 and GSE15061. In summary, we present the characteristics and prognosis of MDS patients with various RUNX1 expressions and propose that RUNX1 expression complement RUNX1 mutation in MDS prognostication, wherein patients with wild RUNX1 but high expression may need more proactive treatment.

Abstract The incidence rate of AL (light-chain) amyloidosis is not known in Asia. We conducted a retrospective cohort study using the Taiwan National Healthcare Insurance Research database and Death Registry to estimate incidence and all-cause case fatality rates, and characteristics of patients with AL amyloidosis in Taiwan. All patients with confirmed, newly diagnosed AL amyloidosis from 01-Jan-2016 until 31-Dec-2019 were enrolled and followed up until dis-enrolment, death or study end (31-Dec-2019). There were 841 patients with newly diagnosed AL amyloidosis with median age of 61.4 years and 58.7% were men. At diagnosis, cardiac, renal and liver-related diseases were present in 28.54%, 23.19% and 2.14% of patients, respectively. AL amyloidosis age-adjusted annual incidence was 5.73 per million population in 2016 and 5.26 per million population in 2019. All-cause case fatality ranged from 1.7 to 2.9% over the study period and was highest (~10%) in patients ≥ 80 years. Survival was significantly lower in patients with co-morbid cardiac, renal, or liver-related diseases which could indicate organ involvement. The incidence of AL amyloidosis in Taiwan appears to be similar to Western countries. The poor prognosis in patients with co-morbid diseases highlights the need for earlier diagnosis.

Abstract Myeloproliferative neoplasms (MPNs) are characterized by upregulation of proinflammatory cytokines and immune dysregulation, which provide a reasonable basis for immunotherapy in patients. Megakaryocytes are crucial in the pathogenesis of primary myelofibrosis (PMF), the most clinically aggressive subtype of MPN. In this study, we aimed to explore PD‐L1 (programmed death‐ligand 1) expression in megakaryocytes and its clinical implications in PMF. We analyzed PD‐L1 expression on megakaryocytes in PMF patients by immunohistochemistry and correlated the results with clinicopathological features and molecular aberrations. We employed a two‐tier grading system considering both the proportion of cells positively stained and the intensity of staining. Among the 85 PMF patients, 41 (48%) showed positive PD‐L1 expression on megakaryocytes with the immune‐reactive score ranging from 1 to 12. PD‐L1 expression correlated closely with higher white blood cell count (p = 0.045), overt myelofibrosis (p = 0.010), JAK2V617F mutation (p = 0.011), and high‐molecular risk mutations (p = 0.045), leading to less favorable overall survival in these patients (hazard ratio 0.341, 95% CI 0.135–0.863, p = 0.023). Our study provides unique insights into the interaction between immunologic and molecular phenotypes in PMF patients. Future work to explore the translational potential of PD‐L1 in the clinical setting is needed.

Background/purpose: To investigate the clinical characteristics of intraocular lymphoma and to evaluate two protocols of intravitreal methotrexate injection. Methods: A retrospective chart review was conducted of newly-diagnosed intraocular lymphoma patients between January 2013 and January 2018 at National Taiwan University Hospital. Patients were divided into two groups. In Group A, intravitreal methotrexate was administered weekly for the initial 8 weeks, every 2 weeks for the following 12 weeks, and then monthly for 7 months. In Group B, intravitreal methotrexate was administered twice a week for the initial 2 weeks, weekly for the subsequent 2 weeks, once every 2 weeks for the next 1 month, and monthly for the last 10 months. Results: A total of 12 patients were analyzed in the study; seven of these patients were allocated to Group A. Differences in the overall survival and progression-free survival between the two groups did not yield statistical significance. The median visual acuity was improved from LogMAR 0.46 to LogMAR 0.30 with borderline significance in Group A (p = 0.053). Two of seven patients in Group A and five of five patients in Group B developed punctate keratitis during intravitreal methotrexate injection treatment. Conclusion: Intravitreal methotrexate is an effective and repeatable treatment for intraocular lymphoma. A new protocol with reduced frequency of intravitreal injections as shown in this study could potentially produce similar results without a worse prognosis, along with a decrease in the incidence of keratitis.

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.

NLRP3 inflammasome and IFN-stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndromes (MDSs). Gene mutations involving mRNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDSs through undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS BM specimens, we showed accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor cyclic GMP-AMP synthase. Cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling caused ISG induction, NLRP3 inflammasome activation, and maturation of the effector protease caspase-1. Deregulation of RNA polymerase III drove cytosolic R-loop generation, which upon inhibition, extinguished ISG and inflammasome response. Mechanistically, caspase-1 degraded the master erythroid transcription factor, GATA binding protein 1, provoking anemia and myeloid lineage bias that was reversed by cGAS inhibition in vitro and in Tet2–/– hematopoietic stem and progenitor cell–transplanted mice. Together, these data identified a mechanism by which functionally distinct mutations converged upon the cGAS/STING/NLRP3 axis in MDS, directing ISG induction, pyroptosis, and myeloid lineage skewing.

Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

Abstract Limited real-world data are available regarding treatment practices and outcomes of multiple myeloma (MM) in Asia. We conducted a retrospective cohort study using the Taiwan National Healthcare Insurance Research database and Taiwan Death Registry and used a Markov model to describe disease progression and outcomes in 4092 patients newly diagnosed with MM from 01-Jan-2007 to 31-Dec-2015. We observed marked differences in the characteristics, length and outcome of the clinical journey between patients who did/did not receive autologous stem cell transplant, and between patients initiated on novel agents versus other treatment regimens. In transplant recipients, initiation with combined thalidomide + bortezomib increased over time (12.2–77.5%). Progression-free survival after first-line treatment improved and a lower percentage of patients died. Lenalidomide in second and third-line regimens increased (15.5–31.5%). In non-transplanted patients, initiation with novel agents increased (17.5–54.6%), but death rates remained high (60.3%). The treatment landscape of MM in Taiwan has evolved, with increased use of combined bortezomib + thalidomide for first-line and lenalidomide for second/third-line but many patients die before receiving second-line treatment. Novel agents with different modes of action should be used as early as possible to maximize their benefits. Improved MM treatments remains a critical medical need.

Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy in terms of clinical features, underlying pathogenesis and treatment outcomes. Recent advances in genomic techniques have unraveled the molecular complexity of AML leukemogenesis, which in turn have led to refinement of risk stratification and personalized therapeutic strategies for patients with AML. Incorporation of prognostic and druggable genetic biomarkers into clinical practice to guide patient-specific treatment is going to be the mainstay in AML therapeutics. Since 2017 there has been an explosion of novel treatment options to tailor personalized therapy for AML patients. In the past 3 years, the U.S. Food and Drug Administration approved a total of eight drugs for the treatment of AML; most specifically target certain gene mutations, biological pathways, or surface antigen. These novel agents are especially beneficial for older patients or those with comorbidities, in whom the treatment choice is limited and the clinical outcome is very poor. How to balance efficacy and toxicity to further improve patient outcome is clinically relevant. In this review article, we give an overview of the most relevant genetic markers in AML with special focus on the therapeutic implications of these aberrations.

Abstract Philadelphia chromosome‐negative (Ph−) myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal disorders of the bone marrow, and are associated with a high disease burden, reduced quality of life (QOL), and shortened survival. This multinational, multicenter, non‐interventional registry “MERGE” was initiated with an objective to collect data on the epidemiological indices of classical Ph‐MPNs, existing treatment patterns, and impact of MPNs on health‐related QOL in various countries/regions in Asia, including the Middle East, Turkey, and Algeria. Of the 884 eligible patients with MPNs, 169 had myelofibrosis (MF), 301 had polycythemia vera (PV), 373 had essential thrombocythemia (ET), and 41 had unclassified MPNs. The median age was 58 years (range, 47‐66 years), and 50% of patients were males. The prevalence and incidence of MPNs were estimated to be 57‐81 and 12‐15 per 100 000 hospital patients per year over the last 4 years, respectively, in these countries. Total symptom score (mean [standard deviation; SD]) at baseline was highest in patients with MF (23.5 [17.47]) compared with patients with ET (14.6 [14.26]) and PV (16.6 [14.84]). Patients with ET had a lower mean (SD) number of inpatient visits (0.9 [0.77] days), and patients with MF had more outpatient visits (5.2 [3.17] days) on an average, compared with the entire MPN group. The study showed that patients with MPNs have a severe disease burden and reduced QOL. A discordance between physician and patient perception of symptom assessment was observed in this study (International clinical trials registry ID: CTRI/2014/05/004598).

Abstract: Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies. Known predisposing factors to adult MDS include rare germline mutations, cytotoxic therapy, age-related clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. To date, no published studies characterizing MDS-associated germline susceptibility polymorphisms exist. We performed a genome-wide association study of 2 sample sets (555 MDS cases vs 2964 control subjects; 352 MDS cases vs 2640 control subjects) in non-del(5q) MDS cases of European genomic ancestry. Meta-analysis identified 8 MDS-associated loci at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2) that approached genome-wide significance. Gene expression for 5 loci that mapped within or near genes was significantly upregulated in MDS bone marrow cells compared with those of control subjects (P < .01). Higher PLA2G4A expression and lower EYA2 expression were associated with poorer overall survival (P = .039 and P = .037, respectively). Higher PLA2G4A expression is associated with mutations in NRAS (P < .001), RUNX1 (P = .012), ASXL1 (P = .007), and EZH2 (P = .038), all of which are known to contribute to MDS development. EYA2 expression was an independently favorable risk factor irrespective of age, sex, and Revised International Scoring System score (relative risk, 0.67; P = .048). Notably, these genes have regulatory roles in innate immunity, a critical driver of MDS pathogenesis. EYA2 overexpression induced innate immune activation, whereas EYA2 inhibition restored colony-forming potential in primary MDS cells indicative of hematopoietic restoration and possible clinical relevance. In conclusion, among 8 suggestive MDS-associated loci, 5 map to genes upregulated in MDS with functional roles in innate immunity and potential biological relevance to MDS.

Abstract: Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

Abstract Background Long non-coding RNAs (lncRNAs) represent the majority of cellular transcripts and play pivotal roles in hematopoiesis. However, their clinical relevance in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains largely unknown. Here, we investigated the functions of HOXB-AS3, a lncRNA located at human HOXB cluster, in the myeloid cells, and analyzed the prognostic significances in patients with AML and MDS. Methods shRNAs were used to downregulate HOXB-AS3 in the cell lines and the effect was evaluated by quantitative polymerase chain reaction. The proliferation of the cell lines was illustrated by proliferation and BrdU flow assays. Further, we retrospectively analyzed the HOXB-AS3 expression in 193 patients with AML and 157 with MDS by microarray analysis, and evaluated its clinical importance. Results Downregulation of HOXB-AS3 suppressed cell proliferation. Mechanistically, HOXB-AS3 potentiated the expressions of several key factors in cell cycle progression and DNA replication without affecting the expressions of HOX genes. In AML, patients with higher HOXB-AS3 expression had shorter survival than those with lower HOXB-AS3 expression (median overall survival (OS), 17.7 months versus not reached, P

Background: Expression of long non-coding RNAs (lncRNAs) has recently been recognized as a potential prognostic marker in acute myeloid leukemia (AML). However, it remains unclear whether incorporation of the lncRNAs expression in the 2017 European LeukemiaNet (ELN) risk classification can further improve the prognostic prediction. Methods: We enrolled 275 newly diagnosed non-M3 AML patients and randomly assigned them to the training (n = 183) and validation cohorts (n = 92). In the training cohort, we formulated a prognostic lncRNA scoring system composed of five lncRNAs with significant prognostic impact from the lncRNA expression profiling. Findings: Higher lncRNA scores were significantly associated with older age and adverse gene mutations. Further, the higher-score patients had shorter overall and disease-free survival than lower-score patients, which were also confirmed in both internal and external validation cohorts (TCGA database). The multivariate analyses revealed the lncRNA score was an independent prognosticator in AML, irrespective of the risk based on the 2017 ELN classification. Moreover, in the 2017 ELN intermediate-risk subgroup, lncRNA scoring system could well dichotomize the patients into two groups with distinct prognosis. Within the ELN intermediate-risk subgroup, we found that allogeneic hematopoietic stem cell transplantation could provide better outcome on patients with higher lncRNA scores. Through bioinformatics approach, we identified high lncRNA scores were correlated with leukemia/hematopoietic stem cell signatures. Interpretation: Incorporation of lncRNA scoring system in 2017 ELN classification can improve risk-stratification of AML patients and help clinical decision-making. Fund: This work was supported Ministry of Science and Technology, and Ministry of Health and Welfare of Taiwan. Keywords: lncRNA, AML, Risk stratification, Prognostication

Background: Multicolor flow cytometry (MFC) analysis is widely used to identify minimal residual disease (MRD) after treatment for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, current manual interpretation suffers from drawbacks of time consuming and interpreter idiosyncrasy. Artificial intelligence (AI), with the expertise in assisting repetitive or complex analysis, represents a potential solution for these drawbacks. Methods: From 2009 to 2016, 5333 MFC data from 1742 AML or MDS patients were collected. The 287 MFC data at post-induction were selected as the outcome set for clinical outcome validation. The rest were 4:1 randomized into the training set (n = 4039) and the validation set (n = 1007). AI algorithm learned a multi-dimensional MFC phenotype from the training set and input it to support vector machine (SVM) classifier after Gaussian mixture model (GMM) modeling, and the performance was evaluated in The validation set. Findings: Promising accuracies (84·6% to 92·4%) and AUCs (0·921–0·950) were achieved by the developed algorithms. Interestingly, the algorithm from even one testing tube achieved similar performance. The clinical significance was validated in the outcome set, and normal MFC interpreted by the AI predicted better progression-free survival (10·9 vs 4·9 months, p

Abstract Background DNMT3A gene mutation has been associated with poor prognosis in acute myeloid leukemia, but its clinical implications in myelodysplastic syndrome (MDS) and dynamic changes during disease progression remain controversial. Results In this study, DNMT3A mutation was identified in 7.9% of 469 de novo MDS patients. DNMT3A-mutated patients had higher platelet counts at diagnosis, and patients with ring sideroblasts had the highest incidence of DNMT3A mutations, whereas those with multilineage dysplasia had the lowest incidence. Thirty-one (83.8%) of 37 DNMT3A-mutated patients had additional molecular abnormalities at diagnosis, and DNMT3A mutation was highly associated with mutations of IDH2 and SF3B1. Patients with DNMT3A mutations had a higher risk of leukemia transformation and shorter overall survival. Further, DNMT3A mutation was an independent poor prognostic factor irrespective of age, IPSS-R, and genetic alterations. The sequential study demonstrated that the original DNMT3A mutations were retained during follow-ups unless allogeneic hematopoietic stem cell transplantation was performed, while DNMT3A mutation was rarely acquired during disease progression. Conclusions DNMT3A mutation predicts unfavorable outcomes in MDS and was stable during disease evolutions. It may thus be a potential biomarker to predict prognosis and monitor the treatment response.

Abstract: Long noncoding RNAs (lncRNAs) not only participate in normal hematopoiesis but also contribute to the pathogenesis of acute leukemia. However, their clinical and prognostic relevance in myelodysplastic syndromes (MDSs) remains unclear to date. In this study, we profiled lncRNA expressions in 176 adult patients with primary MDS, and identified 4 lncRNAs whose expression levels were significantly associated with overall survival (OS). We then constructed a risk-scoring system with the weighted sum of these 4 lncRNAs. Higher lncRNA scores were associated with higher marrow blast percentages, higher-risk subtypes of MDSs (based on both the Revised International Prognostic Scoring System [IPSS-R] and World Health Organization classification), complex cytogenetic changes, and mutations in RUNX1, ASXL1, TP53, SRSF2, and ZRSR2, whereas they were inversely correlated with SF3B1 mutation. Patients with higher lncRNA scores had a significantly shorter OS and a higher 5-year leukemic transformation rate compared with those with lower scores. The prognostic significance of our 4-lncRNA risk score could be validated in an independent MDS cohort. In multivariate analysis, higher lncRNA scores remained an independent unfavorable risk factor for OS (relative risk, 4.783; P < .001) irrespective of age, cytogenetics, IPSS-R, and gene mutations. To our knowledge, this is the first report to provide a lncRNA platform for risk stratification of MDS patients. In conclusion, our integrated 4-lncRNA risk-scoring system is correlated with distinctive clinical and biological features in MDS patients, and serves as an independent prognostic factor for survival and leukemic transformation. This concise yet powerful lncRNA-based scoring system holds the potential to improve the current risk stratification of MDS patients.

Abstract Background Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia-like disease in mice. However, actual effects of a “physiological” dose of mutant ASXL1 remain unexplored. Methods We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system. Results Heterozygotes (Asxl1 tm/+ ) marrow cells had higher in vitro proliferation capacities as shown by more colonies in cobblestone-area forming assays and by serial re-plating assays. On the other hand, donor hematopoietic cells from Asxl1 tm/+ mice declined faster in recipients during transplantation assays, suggesting compromised long-term in vivo repopulation abilities. There were no obvious blood diseases in mutant mice throughout their life-span, indicating Asxl1 mutation alone was not sufficient for leukemogenesis. However, this mutation facilitated engraftment of bone marrow cell overexpressing MN1. Analyses of global gene expression profiles of ASXL1-mutated versus wild-type human leukemia cells as well as heterozygote versus wild-type mouse marrow precursor cells, with or without MN1 overexpression, highlighted the association of in vivo Asxl1 mutation to the expression of hypoxia, multipotent progenitors, hematopoietic stem cells, KRAS, and MEK gene sets. ChIP-Seq analysis revealed global patterns of Asxl1 mutation-modulated H3K27 tri-methylation in hematopoietic precursors. Conclusions We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies.

Purpose: Nowadays, myelofibrosis can be cured only by allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the transplantation experience is very limited worldwide. We retrospectively investigated the treatment outcome of myelofibrosis patients post allo-HSCT at National Taiwan University Hospital. Results: Eleven myelofibrosis patients received allo-HSCT. The median age at transplantation was 51 years old and the median time from diagnosis to transplantation was 10.1 months. All patients were classified as intermediate-2 or high risk based on the International Prognostic Scoring System (IPSS). Conditioning regimens were myeloablative in 5 and reduced-intensity in 6 patients. All patients achieved full donor chimerism, and post-transplantation bone marrow biopsies showed significant improvement of myelofibrosis in 3 patients. The transplant-related mortality at 100 days post transplantation was 9%. Four patients had grade III-IV acute graft-versus-host disease. Six patients died, and the causes of death were GVHD (n=3), infection (n=2) and secondary graft failure (n=1). The estimated overall survival at two years was 45.5%. Conclusions: Allogeneic hematopoietic stem cell transplantation is effective in treating myelofibrosis and is associated with modest toxicity and post-transplantation complications. In order to improve the treatment outcome, we have to manage GVHD and infection more carefully.

Homeodomain-only protein homeobox (HOPX) is the smallest homeodomain protein. It was regarded as a stem cell marker in several non-hematopoietic systems. While the prototypic homeobox genes such as the HOX family have been well characterized in acute myeloid leukemia (AML), the clinical and biological implications of HOPX in the disease remain unknown. Thus we analyzed HOPX and global gene expression patterns in 347 newly diagnosed de novo AML patients in our institute. We found that higher HOPX expression was closely associated with older age, higher platelet counts, lower white blood cell counts, lower lactate dehydrogenase levels, and mutations in RUNX1, IDH2, ASXL1, and DNMT3A, but negatively associated with acute promyelocytic leukemia, favorable karyotypes, CEBPA double mutations and NPM1 mutation. Patients with higher HOPX expression had a lower complete remission rate and shorter survival. The finding was validated in two independent cohorts. Multivariate analysis revealed that higher HOPX expression was an independent unfavorable prognostic factor irrespective of other known prognostic parameters and gene signatures derived from multiple cohorts. Gene set enrichment analysis showed higher HOPX expression was associated with both hematopoietic and leukemia stem cell signatures. While HOPX and HOX family genes showed concordant expression patterns in normal hematopoietic stem/progenitor cells, their expression patterns and associated clinical and biological features were distinctive in AML settings, demonstrating HOPX to be a unique homeobox gene. Therefore, HOPX is a distinctive homeobox gene with characteristic clinical and biological implications and its expression is a powerful predictor of prognosis in AML patients.

Hematopoietic stem cell transplantation (HSCT) can either cause or eliminate autoimmune disease. Here, we report two cases. One was a 33-year-old woman with myelodysplastic syndrome (refractory anemia) who received bone marrow transplantation from her human leukocyte antigen (HLA)-identical sister who had a history of Graves' disease. Antithyroid antibodies, including antimicrosomal antibody and antithy-roglobulin antibody, appeared 4 months after transplantation. Clinical hyperthyroidism appeared 7 months after transplantation, and a hypothyroid state was noted 2 months later. The other case was a 50-year-old woman with Sjögren's syndrome and hypothyroidism who was diagnosed with peripheral T cell non-Hodgkin's lymphoma. She received allogeneic peripheral blood stem cell transplantation (PBSCT) from her histocompatible sister owing to only partial response to traditional chemotherapy. Cure of lymphoma and remission of Sjögren's syndrome was noted 4 years after PBSCT. These two illustrative cases, one of acquisition of hyperthyroidism and the other of remission of Sjögren's syndrome after transplantation, highlights that HSCT can induce adoptive autoimmune disease or cure coincidental autoimmune disease. Donor selection and attentive monitoring is required in such circumstances.

Invasive fungal infections (IFIs) is an important complication for acute myeloid leukemia (AML) patients receiving induction chemotherapy. However, the epidemiological information is not clear in Southeastern Asia, an area of potential high incidences of IFIs. To clarify it, we enrolled 298 non-M3 adult AML patients receiving induction chemotherapy without systemic anti-fungal prophylaxis from Jan 2004 to Dec 2009, when we applied a prospective diagnostic and treatment algorithm for IFIs. Their demographic parameters, IFI characters, and treatment outcome were collected for analysis. The median age of these patients was 51 years. Standard induction chemotherapy was used for 246 (82.6%) patients, and 66.8% of patients achieved complete remission (CR) or partial remission. The incidence of all-category IFIs was 34.6% (5.7% proven IFIs, 5.0% probable IFIs and 23.8% possible IFIs). Candida tropicalis was the leading pathogen among yeast, and lower respiratory tract was the most common site for IFIs (75.4%, 80/106). Standard induction chemotherapy and failure to CR were identified as risk factors for IFIs. The presence of IFI in induction independently predicted worse survival (hazard ratio 1.536 (1.100-2.141), p value = 0.012). Even in those who survived from the initial IFI insults after 3 months, the presence of IFIs in induction still predicted a poor long-term survival. This study confirms high incidences of IFIs in Southeastern Asia, and illustrates potential risk factors; poor short-term and long-term outcomes are also demonstrated. This epidemiological information will provide useful perspectives for anti-fungal prophylaxis and treatment for AML patients during induction, so that best chances of cure and survival can be provided.

Robo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear.We investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort.Higher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA.BM Robo4 expression can serve as a new biomarker to predict clinical outcomes in AML patients and Robo4 may serve as a potential therapeutic target in patients with higher Robo4 expression.

The growth of myeloma cells depends on bone marrow (BM) stroma consisting of stromal cells, secreted cytokines and the extracellular matrix (ECM). Decorin, a small leucine-rich proteoglycan in the ECM, is a signaling ligand and native anti-tumor agent. However, the role of decorin in patients with myeloma is not clear. We evaluated the correlation between the decorin levels measured by enzyme-linked immunosorbent assay in BM plasma from 121 patients with newly diagnosed myeloma based on their clinical features and treatment response. The median decorin levels in the patients and the normal control group were 12.31 ng/mL [standard deviation (SD), 7.50 ng/mL; range, 2.45 to 44.46 ng/mL] and 10.31 ng/mL (SD, 2.42 ng/mL; range, 4.85-15.14 ng/mL), respectively (P < 0.001). Using 15.15 ng/mL as a cut-off, 46 patients (38%) exhibited higher decorin levels (H-DCN), whereas the other patients exhibited normal to lower decorin levels (NL-DCN). Except for the median age, which was significantly younger in the H-DCN than in the NL-DCN group (60.6 ± 14.0 vs. 65.8 ± 12.2 years, respectively; P = 0.034), there were no differences between the two groups. However, in 79 patients who had received novel agent-based induction, the overall response rate was significantly better in the H-DCN than in the NL-DCN (97 vs. 63%, respectively; P < 0.001), as was the depth of responses (P = 0.008), which were not observed in those who had received chemotherapeutic agents alone. Progression-free survival (PFS) was significantly longer in H-DCN than NL-DCN (not reached vs. 19.5 mo, respectively; P = 0.0003). Multivariate analyses indicated that H-DCN, as a significantly independent factor, was associated with better treatment response (odds ratio, 20.014; 95% CI, 2.187-183.150; P = 0.008) and longer PFS (hazard ratio, 0.135; 95% CI, 0.051-0.361; P < 0.001). These findings disclose the potential role of decorin in myeloma and provide a basis for further study on possible synergistic anti-myeloma effects between decorin and the novel agents that target BM stroma.

CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical implications of bone marrow CXCR4 expression in patients with acute myeloid leukemia showed not only higher CXCR4 expression was an independent poor prognostic factor, irrespective of age, white blood cell counts, cytogenetics, and mutation status of NPM1/FLT3-ITD and CEBPA, but also showed CXCR4 expression was inversely associated with mutations of CEBPA, a gene encoding transcription factor C/EBPα. Patients with wild-type CEBPA had significantly higher CXCR4 expression than those with mutated CEBPA. We hypothesized that CEBPA might influence the expression of CXCR4. To test this hypothesis, we first examined endogenous CXCR4 expression in 293T and K562 cells over-expressing wild-type C/EBPα p42 and demonstrated that CXCR4 levels were increased in these cells, whilst the expression of the N-terminal mutant, C/EBPα p30, diminished CXCR4 transcription. We further showed p42 was bound to the CXCR4 promoter by the chromatin immunoprecipitation assays. Induction of p42 in the inducible K562-C/EBPα cell lines increased the chemotactic migration. Moreover, decreased expression of C/EBPα by RNA interference decreased levels of CXCR4 protein expression in U937 cells, thereby abrogating CXCR4-mediated chemotaxis. Our results provide, for the first time, evidence that C/EBPα indeed regulates the activation of CXCR4, which is critical for the homing and engraftment of acute myeloid leukemia cells, while p30 mutant impairs CXCR4 expression.

Somatic Nucleophosmin (NPM1) mutation frequently occurs in acute myeloid leukemia (AML), but its role in leukemogenesis remains unclear. This study reports the first "conventional" knock-in mouse model of Npm1 mutation, which was achieved by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG) to mimic human mutation without any "humanized" sequence. The resultant mutant peptide differed slightly different from that in humans but exhibited cytoplasmic pulling force. Homozygous (Npm1(c+/c+)) mice showed embryonic lethality before day E8.5, wheras heterozygous (Npm1(wt/c+)) mice appeared healthy at birth and were fertile. Approximately 36% of Npm1(wt/c+) mice developed myeloproliferative disease (MPD) with extramedullary hematopoiesis. Those Npm1(wt/c+) mice that did not develop MPD nevertheless gradually developed monocytosis and showed increased numbers of marrow myeloid precursors. This second group of Npm1(wt/c+) mice also showed compromised cobblestone area formation, suggesting pathology in the hematopoietic niche. Microarray experiments and bioinformatic analysis on mice myeloid precursor cells and 227 human samples revealed the expression of CXCR4/CXCL12-related genes was significantly suppressed in mutant cells from both mice and humans. Thus, our mouse model demonstrated that Npm1 mutation can result in MPD, but is insufficient for leukemogenesis. Perturbation of hematopoietic niche in mutant hematopoietic stem cells (implied by underrepresentation of CXCR4/CXCL12-related genes) may be important in the pathogenesis of NPM1 mutations.

Recently, dynamic contrast-enhanced magnetic resonance imaging has been shown to be a non-invasive technique that provides global and functional imaging of bone marrow angiogenesis in acute myeloid leukemia.To assess the clinical implication of changes in angiogenesis shortly after induction chemotherapy, dynamic contrast-enhanced magnetic resonance imaging was performed prospectively before treatment (day 0) and on day 7 in 80 patients with de novo acute myeloid leukemia.We demonstrated that a post-therapeutic reduction in Peak (negative ΔPeak) compared with the day 0 value was significantly associated with a higher chance of achieving complete remission, and better overall and disease free survival (P=0.022, 0.003 and 0.007, respectively). Cox’s multivariate analysis also identified negative ΔPeak value as an independent good prognostic factor for overall and disease free survival. Our findings provide evidence that the change of Peak on day 7 relative to pre-treatment levels may be a relevant biomarker for early identification of patients who may fail conventional induction chemotherapy (ClinicalTrials.gov Identifier: NCT00172562).