NPM1-mutated Acute Myeloid Leukemia Patients Research Articles

Circular RNAs of the Nucleophosmin 1 (NPM1) Gene in Acute Myeloid Leukemia Patients

Background: The nucleophosmin 1 (NPM1) gene is not only commonly mutated in acute myeloid leukemia (AML), but also encodes several linear splice isoforms, one of which was recently shown to be of prognostic importance. Furthermore, circular RNAs (circRNAs) are transcribed from the NPM1 gene which demands further investigation with regard to function in normal hematopoiesis and impact on leukemogenesis.Aims: We aimed to investigate circRNAs derived from NPM1 and gain insights into their regulation and function. Additionally, we wanted to determine changes in the circular RNAome in the course of hematopoietic differentiation and leukemic transformation.Methods: Circular NPM1 transcripts were detected by PCR and sequenced in leukemic cell lines (n=7) and healthy control samples (n=3, peripheral blood-derived mononuclear cells). Expression of hsa_circ_0075001 and total NPM1 was measured in a cohort of 23 NPM1 wildtype (NPM1wt) and 23 NPM1 mutated (NPM1mut) AML patients via quantitative real-time PCR (qPCR), and Affymetrix U133plus2 microarray data was set in relation to the expression levels. Principal component analysis (PCA) was conducted to identify groups with similarities in gene expression patterns and differentially expressed genes were subjected to pathway analysis. Next, ribosomal RNA-depleted RNA-seq was performed for 5 NPM1mut and 5 NPM1wt AML cases, as well as 10 healthy control samples derived from 4 FACS-sorted myeloid differentiation stages (myeloblasts, promyelocytes, metamyelocytes and neutrophils). PCA and unsupervised hierarchical clustering were performed based on circRNA expression.Results: We detected and sequenced multiple circular NPM1 transcripts (n=23) in leukemic as well as in healthy control cells. As hsa_circ_0075001 showed differential expression between different AML cell lines in a semi-quantitative PCR analysis, quantification in 46 AML patients via qPCR was performed. This analysis revealed that total NPM1 and hsa_circ_0075001 expression were independent of the NPM1 mutational status. Furthermore, the hsa_circ_0075001 expression status defined distinct leukemia subgroups characterized by similarities in gene expression as determined by PCA. For example, differentially expressed genes between high versus low hsa_circ_0075001 expression groups (dichotomized at the median) were significantly enriched in components of the Toll-like receptor (TLR) signaling pathway, which was downregulated in patients with high hsa_circ_0075001 expression. Expression of hsa_circ_0075001 correlated positively with total NPM1 expression, and RNA-seq analysis further revealed a global correlation of circRNA and parental gene expression. In total, in our cohort circRNAs were found for 19 % of all expressed genes. PCA based on circRNA expression illustrated that immature and mature hematopoietic cells, as well as NPM1wt and NPM1mut AML samples, exhibit distinct circRNA signatures (Figure 1). Thus, circRNA expression seems to play a role during differentiation of normal hematopoietic cells, but also seems to be severely deregulated in AML.Figure 1: Altered circular RNA expression in AML patients compared to healthy control samples. Principal component analysis (PCA) of circRNA expression data of 5 NPM1mut patients (red), 5 NPM1wt patients (green), and 10 healthy control samples, of which 4 were derived from immature (blue) and 6 from more mature myeloid differentiation stages (purple). Data was generated via RNA-Seq and reads derived from circRNAs were aligned and quantified using STAR, and normalized and transformed using DESeq2. PCA was performed based on 500 genes with the highest variance of circRNA expression across all samples.Conclusions: circRNAs transcribed from the NPM1 gene showed differential expression in AML cell lines and healthy cells, and higher hsa_circ_0075001 expression defined an AML subgroup characterized by downregulation of the TLR signaling pathway. These findings provide evidence for the relevance of circular NPM1 transcripts and add another level of complexity to the multifaceted gene NPM1. In general, circRNA expression seems to be involved in the regulation of hematopoietic differentiation, which is in line with previous observations, but, based on distinct circRNA expression profiles in AML, they might also play a significant pathogenic role in leukemic transformation. [Display omitted] DisclosuresPaschka:Celgene: Honoraria; Pfizer Pharma GmbH: Honoraria; Bristol-Myers Squibb: Honoraria; Medupdate GmbH: Honoraria; Novartis: Consultancy; ASTEX Pharmaceuticals: Consultancy.

The Cell of Origin in NPM1 Positive Acute Myeloid Leukemia (AML) Is Prognostic

Abstract Mutations in the nucleophosmingene (NPM1) are common in patients with cytogenetically-normal acute myeloid leukemia (AML) and have been associated with positive outcomes. Although most leukemic stem cells (LSCs) arise from CD34+ primitive progenitors, our recent data suggest that a fraction arise from less primitive CD34- progenitors and seem to have a better prognosis (Gerber et al. Haematologica 2016). Conversely, poor-risk AMLs such as those harboring internal tandem duplications of the FMS-like tyrosine kinase 3 (FLT3-ITD) or with poor-risk cytogenetics appear to arise from LSCs with a primitive hematopoietic stem cell (HSC) phenotype. NPM1-mutated AMLs can be either CD34- or CD34+, and have been associated with variable prognoses. We hypothesize that the cell of origin, CD34- or CD34+, of NPM1-mutated AML has prognostic implications. A retrospective cohort analysis was conducted using electronic medical record data from patients aged 18 or older with newly-diagnosed NPM1-mutated AML treated with intensive induction chemotherapy at Johns Hopkins Hospital from 2008-2015. NPM1 and FLT3-ITD mutations were determined from PCR assays on diagnostic samples. We (Gerber et al. Haematologica2016) and others have shown that CD34+ cells represent normal, non-leukemia progenitors when they comprise <1% of mononuclear cells (MNCs); thus, >/= 1% MNCs was the cut-off used to distinguish CD34+ from CD34- AMLs. Clinical outcomes including complete remission (CR) rate were compared using Chi-squared tests and event-free survival (EFS) and overall survival (OS) were estimated per the Kaplan-Meier method, using Wilcoxon tests to determine the impact of CD34 status. Of 46 consecutive, newly diagnosed NPM1-positive patients treated between 2008-15, 36 (78%) had normal cytogenetics and 37 (80%) had de novo AML. 26/46 (57%) had CD34+ disease; of these, 18/26 (69%) had FLT3-ITD mutations, compared to 9/20 (45%) with CD34- AML (p=0.10). In the CD34- patients with FLT3-ITD, the mean allelic ratio (mutant to wild type allele) ±SEM was 0.26±.08 compared to 2.3±1.0 in the CD34+ patients (p=0.07). Comparing CD34+ and CD34- patients, there were no significant differences in mean age at diagnosis (52 vs 59), sex, initial hemoglobin, platelet count, or lactate dehydrogenase, but CD34+ patients had significantly higher baseline white blood cell count (mean 78 vs 38 x 103 cells/mm3; p=0.04). There was a trend toward lower CR rates following induction for CD34+ patients (69 vs 89%; p=0.11), and similar proportions of patients underwent bone marrow transplantation (BMT) (50% vs 37%, p=0.38). However, CD34+ patients had significantly shorter EFS (figure 1; median 8.0 vs 34.3 months; p=0.04), and a trend toward inferior OS (15.4 vs 35.4 months; p=0.36). The lack of statistical significance in OS may reflect effective salvage by allogeneic transplantation. In conclusion, in this small retrospective cohort analysis of NPM1-mutated AML patients, CD34 expression was associated with significantly reduced EFS and, among FLT3-ITD mutated patients, a trend toward higher allelic ratios. Hence, CD34 positivity may be an early indicator of poor prognosis in NPM1+ AML and requires further characterization to determine its full prognostic and predictive value in guiding AML therapy. These data support evidence that the biology of AML is not only a function of genetic mutations, but also in part a functionof the stage of hematopoietic differentiation at which the leukemogenic mutations develop. Figure 1 EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Figure 1. EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Disclosures No relevant conflicts of interest to declare.

Pretransplant NPM1 MRD levels predict outcome after allogeneic hematopoietic stem cell transplantation in patients with acute myeloid leukemia.

The objective was to evaluate the prognostic impact of pre-transplant minimal residual disease (MRD) as determined by real-time quantitative polymerase chain reaction in 67 adult NPM1-mutated acute myeloid leukemia patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twenty-eight of the 67 patients had a FLT3-ITD (42%). Median age at transplantation was 54.7 years, median follow-up for survival from time of allografting was 4.9 years. At transplantation, 31 patients were in first, 20 in second complete remission (CR) and 16 had refractory disease (RD). Pre-transplant NPM1 MRD levels were measured in 39 CR patients. Overall survival (OS) for patients transplanted in CR was significantly longer as compared to patients with RD (P=0.004), irrespective of whether the patients were transplanted in first or second CR (P=0.74). There was a highly significant difference in OS after allogeneic HSCT between pre-transplant MRD-positive and MRD-negative patients (estimated 5-year OS rates of 40 vs 89% P=0.007). Multivariable analyses on time to relapse and OS revealed pre-transplant NPM1 MRD levels >1% as an independent prognostic factor for poor survival after allogeneic HSCT, whereas FLT3-ITD had no impact. Notably, outcome of patients with pre-transplant NPM1 MRD positivity >1% was as poor as that of patients transplanted with RD.

Clonal Evolution in NPM1 Mutated Acute Myeloid Leukemia (AML)

Background: Mutations in the nucleophosmin (NPM1) gene represent one of the most common gene mutations in AML, and are considered a founder event in the pathogenesis of AML. Recently, we gained novel insights into clonal architecture and clonal evolution in 53 NPM1 mutated (NPM1mut) AML patients (pts). Here, we showed the highest stability for DNMT3A mutations (DNMT3Amut) suggesting that these mutations represent an early event in leukemogenesis as well as an increase in genomic complexity.Aims: To validate our previous results on clonal evolution in a larger cohort of NPM1mut AML pts and to define relapse specific mutation patterns.Methods: Paired samples at diagnosis and relapse from 129 NPM1mut AML pts were assessed for additional mutations by comprehensive mutation analysis (FLT3-ITD, FLT3-TKD, DNMT3A, IDH1/2, NRAS, ASXL1, TP53, MLL-PTD). In addition, 11 AML cases with loss of NPM1mut at the time of relapse and 12 cases with persisting NPM1mut were subjected to whole exome sequencing (WES).Results: At diagnosis, the incidence of concurrent gene mutations was as follows: FLT3-ITDmut 32% (40/126), FLT3-TKDmut 19% (22/118), DNMT3Amut 69% (78/113), NRASmut 19% (22/117), IDH1mut 21% (26/123) and IDH2mut 19% (22/118). None of the pts analyzed exhibited a TP53 (n=53), MLL-PTD (n=63) or ASXL1 (n=59) mutation. At relapse, a significant shift in the genetic pattern was found in 74 pts (57%). FLT3-ITDmut was lost in 10 pts and newly acquired in 24 pts,16 pts lost FLT3-TKDmut. DNMT3Amut was lost in 2 pts and gained in 1 pt. NRASmut was lost in 12 pts and gained in 4 pts. IDH1mut was lost in 3 pts and acquired in 4 pts, while IDH2mut was lost in 2 pts and gained in only one pt. ASXL1mut and TP53mut were gained in 2 and 1 pts, respectively; gain of MLL -PTDmut (n=4) was restricted to pts with loss of NPM1mut at the time of relapse. Based on these findings we calculated the following stabilities: FLT3-ITDmut 75%, FLT3-TKDmut 27%, DNMT3Amut 97%, NRASmut 45%, IDH1mut 88% and IDH2mut 91%, respectively. In total, 11 pts (9%) lost NPM1mut at relapse while DNMT3Amut was present in 9/11 pts at diagnosis and remained stable at relapse; 4/11 pts gained MLL-PTDmut, one pt ASXL1mut and 3 pts acquired RUNX1mut.By WES we observed a persistence of mutations known to be involved in clonal hematopoiesis, such as DNMT3A and TET2 mutations. These were usually seen during all analyzed time points (diagnosis, remission and relapse). In addition, we found distinct mutational patterns at the time of relapse compared to the time of diagnosis. For example, relapse- and diagnosis-specific mutations in NPM1mut loss cases were significantly enriched for different signaling pathways.Conclusions: 74 (57%) of 129 NPM1mut AML pts showed clonal evolution at the time of relapse. DNMT3Amut demonstrated the highest stability (97%) confirming our previous findings that DNMT3Amut constitutes an early event which persists in preleukemic hematopoietic stem cells. High-resolution sequencing of selected cases is ongoing and will further unravel the clonal hierarchy in NPM1mut AML.Those authors equally contributed to work: SKS, SC, LB and KD. DisclosuresHeuser:Karyopharm: Research Funding.

Arsenic trioxide and all-trans retinoic acid target NPM1 mutant oncoprotein levels and induce apoptosis in NPM1-mutated AML cells

AbstractNucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (∼30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)–retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1 mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics.

The NPM1 mutation type has no impact on survival in cytogenetically normal AML.

NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML.

Analysis Of Leukemic Stem Cell Population Comparing NPM1wt and NPM1mut AML Patients and Potential Therapeutic Targets

Treatment of acute myeloid leukemia (AML) patients became more effective, yet the relapse rate is still high and cure rate still low. Different therapeutic approaches, based on molecular targeting are offering new treatment strategies using markers like FLT3, NPM1, cKIT and DNMT3A. Many others are currently under investigation. Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSC represent a critical target for further therapeutic options. AML with mutated nucleophosmin (NPM1) gained attention because of its distinctive molecular, clinical, and prognostic features. NPM1 mutation is also an interesting target for specific immunotherapy (Greiner et al., Blood 2012), however LSC of this new entity have not been completely characterized. Differentially expressed genes might influence molecular and immunological relevant pathways which could have an impact on the better overall survival of the mutated patients.In this study, we were interested in expression differences of the LSC enriched cell fraction descendent of NPM1wt and NPM1mut primary AML patients. We suggest that expression differences between the patients groups could be a factor for the better overall survival of NPM1mut patients. In addition we aimed to study new targets on NPM1mut LSC for new therapeutical purposes.We enriched the CD34+CD38- fraction of primary AML peripheral blood mononuclear cells (PBMC), by Fluorescence-activated cell sorting (FACS) and Magnetic-activated cell sorting (MACS), comparing both methods in efficiency and feasibility. We chose FACS for further enrichment assays, as it resulted in a mean purity of 92% vs 88% using MACS, and a higher cell number after sorting (9% more) in the first pilot trials. We sorted 21 AML patient samples; 12 NPM1wt and 9 NPM1mut. We also enriched healthy donor samples for HSC purification using FACS, in order to compare expression levels. We showed that enriched CD34+CD38- cells in NPM1mut AML samples harbor cytoplasmic NPM1 via immunocytochemical staining, indicating that these cells belong to the leukemic clone. The cell number and RNA quality was sufficient for further Microarray studies in which we analysed the CD34+CD38- enriched compartments descendent from NPM1mut and NPM1wt patients. Those showed significant differences in gene expression patterns which noticeably are immunologically coined, for example: immunoglobulin superfamily, member 10 (p = 0.0003405; fold change: 6.22) and the interleukin 12 receptor, beta 1 (p = 0.000834, fold change: 1.87). This impression was confirmed by pathway analysis indicating deregulation of pathways like the NO2-dependent IL 12 Pathway in NK cells and the Th1/Th2 Differentiation and the Platelet Amyloid Precursor Protein Pathway.Functional assays, confirming the biological importance of these factors have been performed for example an assay to test the effect of IL12 on AML cellines.Furthermore we screened our data for new potential therapeutic target structures, specifically on enriched CD34+CD38- cells of NPM1mut patients, comparing the expression level of target genes to NPM1wt CD34+CD38- cells, and the expression level on HSC. Amongst others, our most promising candidates are SERPINA1 (p = 0.0062344, fold change: 14.32), OSCAR (p = 6.11E-05, fold change: 9.03) and several further interesting genes. These genes could be used in order to target CD34+CD38- cells of NPM1mut patients in a therapeutic manner.Taken together, we successfully enriched the CD34+CD38- fraction of NPM1mut and NPM1wt AML patients and performed Microarray analysis, unraveling gene expression differences between the two patients groups, which seem to be of an immunological nature. This could be a factor, amongst others, for the better overall survival of the NPM1mut patient group. Furthermore we described new potential targets structures on CD34+CD38- cells for NPM1mut patient's treatment. Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease (MRD) Monitoring in NPM1 Mutated Acute Myeloid Leukemia (AML): Impact of Concurrent FLT3-ITD and DNMT3A Mutations on MRD Kinetics and Clinical Outcome

IntroductionIn a recent update on MRD monitoring in 407 NPM1 mutated (NPM1mut) AML patients (pts) we could confirm the results from our previous study showing that achievement of RQ-PCR negativity after double induction (DI), after completion of therapy (CT) as well as during the follow-up period (FUP) is significantly associated with a lower cumulative incidence of relapse (CIR) and superior overall survival (OS) [Döhner K, Annals of Hematol; 2013;Suppl.1,92:S39]. In addition, in pts with concurrent FLT3-ITD (FLT3-ITDmut) or DNMT3A (DNMT3Amut) mutations, we also showed that the median NPM1mut transcript levels after each treatment cycle were significantly higher. AimTo evaluate the impact of concurrent FLT3-ITD and DNMT3Amut on MRD kinetics and clinical outcome in NPM1mutAML pts. MethodsFor this analysis we included all pts enrolled on one of two AMLSG treatment trials [AMLHD98A (NCT00146120) n=46; AMLSG 07-04 (NCT00151242) n=199] for whom the FLT3-ITD and DNMT3A mutation status at the time of diagnosis was determined. MRD levels (ratio NPM1mut/ABL1 transcripts x 104) were detected by NPM1mut specific RQ-PCR using TaqMan technology; the sensitivity of the assays was 10-5 - 10-6. DNMT3A and FLT3-ITD mutation status was assessed by standard PCR-based methods ResultsIn total, 1588 samples [bone marrow n=1564; peripheral blood n=24] from 245 NPM1mut pts were analyzed [at diagnosis, n= 240; during therapy, n= 807; during FUP, n= 541]. FLT3-ITD and DNMT3A mutation status was available in 245/245 (FLT3-ITDmut n=94) and in 234/245 (DNMT3Amut n= 122) pts, respectively. Pre-treatment NPM1mut transcript levels did not correlate with clinical characteristics, DNMT3A or FLT3-ITD mutation status and had no impact on event-free survival, relapse-free survival and OS. Multivariable analyses stratified for FLT3-ITD mutation status after DI and CT revealed RQ-PCR negativity as a significant factor for longer remission duration (hazard ratio (HR) 15.15 and 8.95, respectively) and better OS (HR 6.13 and 4.27, respectively); DNMT3A mutation status had no significant impact in these models. Subgroup analyses showed that the proportion of pts achieving RQ-PCR negativity after DI, after CT and during FUP was significantly lower in DNMT3Amut compared to the DNMT3A wildtype pts (8.6% vs 33.3%, p=<0.001; 36,3% vs 61.9%, p=0.009; 33% vs 51%, p=0.04, respectively) whereas for FLT3-ITDmut pts this effect was only significant after DI (8.3% vs 25%, p=0.022). Based on these findings we further investigated the impact of RQ-PCR negativity in the context of concurrent FLT3-ITD and DNMT3A mutations. After DI, there was no significant difference in CIR and OS for RQ-PCR negative pts with respect to FLT3-ITD or DNMT3A mutation status. After CT, RQ-PCR negative pts with DNMT3Amut had a significantly higher CIR compared to DNMT3A wildtype pts (34% vs 8% at 4 years; p=0.007). This adverse prognostic impact was consistently seen during the FUP (CIR 21% vs 3% at 4 years; p=0.01); there was no difference in CIR rates between pts with and without FLT3-ITD mutations. ConclusionsWe demonstrate a significant correlation between the DNMT3A mutation status and the achievement of RQ-PCR negativity at all clinically relevant time points i.e. after DI, and CT, and during FUP while this strong correlation was not observed for FLT3-ITDmut. Within the NPM1mut RQ-PCR negative group the presence of DNMT3Amut allows the identification of pts at high risk of relapse. Based on our findings DNMT3A mutation status should be determined in NPM1mut pts to further refine MRD monitoring. The establishment of DNMT3Amut specific MRD assays might provide additional information on MRD status in these pts. Disclosures:Schlegelberger:Celgene: Consultancy. Lübbert:Johnson and Johnson: Advisory Board Other. Kindler:Novartis: Membership on an entity's Board of Directors or advisory committees. Germing:Celgene: Honoraria, Research Funding. Schlenk:Novartis: Research Funding; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Chugai: Research Funding; Ambit: Honoraria; Celgene: Honoraria, Research Funding.

Mutated Nucleophosmin 1 (NPM1) Is an Immunogenic Target and Patients with NPM1mut Acute Myeloid Leukemia (AML) Showed High Expression of Different Leukemia-Associated Antigens (LAAs)

Abstract 3592Nucleophosmin gene 1 mutations (NPM1mut) are one of the most frequent molecular alterations in AML and distinct immune responses might contribute to the favorable prognosis of AML patients with NPM1mut. Recently, we showed specific T cell responses of CD4+ and CD8+ T cells against epitopes derived from mutated regions of NPM1 (Greiner et al., Blood. 2012 May 16, Epub). In the present study, we investigated clinical parameters and the clinical outcome of NPM1mut AML patients in accordance to their immune responses against different NPM1 epitopes. Moreover, we examined the quantitative expression of different leukemia-associated antigens (LAAs) in NPM1mutAML patients.In ELISpot analysis of 33 healthy volunteers and 27 AML patients, we detected T cell responses of CD4+ and CD8+ T cells against epitopes derived from the mutated region of NPM1. We performed further tetramer assays against the most interesting epitopes and chromium release assays to show the cytotoxicity of peptide-specific T cells. Microarray analysis was performed to analyze the expression of different LAAs in NPM1mut and NPM1wtAML patients.Two epitopes (peptide #1 and #3) derived from NPM1mut induced CD8+ T cell responses. 33% of the NPM1mut AML patients showed immune responses against peptide #1 and 44% against peptide #3. NPM1mut AML patients showed a significantly higher frequency of T cell responses against peptide #3 in contrast to HVs (p=0.046), whereas for peptide #1 the frequency of T cell responses of AML NPM1mut patients and HVs was not significantly different. Specific lysis of pulsed T2 cells but also NPM1mut leukemic blasts was detected in chromium release assays. Therefore, overlapping peptides (OL) were analyzed in ELISpot assays and the peptide called OL8 showed favorable results to activate both CD8+ and CD4+ T cells.We performed survival analysis for these 33 NPM1mut patients analyzed by ELISpot comparing cases with or without specific T cell responses. Our data suggest a trend to a better overall survival (OS) for patients with specific T cell responses against peptide #1 or #3. However, the patient numbers are small and the data have to be interpreted carefully. Analyses with material from larger controlled clinical trials with a high number of patients with NPM1mut AML have to be performed. Our microarray analysis of 30 AML patients showed a high expression of different LAAs like RHAMM, WT-1 and BCL-2 in all subtypes of cells of NPM1mutAML patients, also in leukemic progenitor cells. This demonstrates that NPM1 is an AML subtype suitable for poly-targeted immunotherapeutic trials.Taken together, NPM1mut might constitute an interesting target structure for individualized immunotherapeutic approaches in NPM1mut AML patients. We hypothesize that immune responses to NPM1 mutation may contribute to the favorable prognosis. Disclosures:No relevant conflicts of interest to declare.

Molecular Minimal Residual Disease After Induction Is Related to ABC Proteins' Activity and Associated with Survival in 26 Patients with NPM1 Mutated Acute Myeloid Leukemia

AbstractAbstract 4909 Background:A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. The role of ABC proteins, and specially ABCB1 (PgP/mdr1), in this resistance has been well established, and higher ABC proteins’ activity, assessed with functional tests, has been associated with poorer complete remission rates and poorer overall prognosis (Marzac et al, Haematologica, 2011). Furthermore, the evaluation of molecular minimal residual disease (MRD), using mutated nucleophosmin (NPM1)expression quantification has been related to patients’ global prognosis (Krönke et al, J. Clin. Oncol., 2011), and to response to treatments. In this study, we evaluate the impact of ABC proteins’ activity on MRD after one course of induction chemotherapy, in 26 patients with NPM1 mutated AML. Material and methods:We retrospectively identified 26 AML patients with NPM1 mutation treated in our center and with MRD data. MRD was evaluated as the ratio of NPM1 mutated allele and total NPM1, using PCR DNA quantification and the delta delta Ct method. MRD was measured at the time of diagnosis and after one course of anthracycline-based induction chemotherapy. ABC proteins’ activity was evaluated at the time of diagnosis using JC1 +/− cyclosporine A assay (Legrand et al, Blood, 2001). Correlations between ABC proteins’ activity and the level of post induction MRD were evaluated with the Mann-Whitney test. Survival was evaluated using the Cox model. For all analyses, P values were considered significant when lower than 0. 05. Results:Median age at diagnosis was 53 years old. Twenty-two patients had normal cytogenetic analysis at diagnosis, and the other 4 patients had intermediate prognosis cytogenetic analysis. Nine patients harboured FLT3-ITD mutation.Median ABC proteins’ activity was 0. 11 (0 – 0. 77). After one course of induction chemotherapy, 3 patients did not reach cytological complete remission. In 17 patients MRD level after induction therapy was inferior to 1 %, in 11 patients MRD was inferior to 0. 1 % and in 7 patients MRD was inferior to 0. 01 %. Overall, higher MRD level after induction (defined by MRD level higher than 0. 1 %) was associated with poorer prognosis for disease free survival (HR= 4. 25 [95% CI 1. 049–17. 27]; p=0. 04), and for overall survival HR=11. 25 [95% CI 1. 22–103. 23]; p=0. 03). Higher ABC proteins’ activity was associated with higher MRD levels post induction, and patients who did not reach MRD level lower than 0. 1 % had significantly higher ABC proteins’ activity than other patients (p=0. 008). ABC proteins’ activity was also associated with overall survival in our patients (p=0. 04). Conclusion:Higher ABC proteins' activity is associated with higher MRD levels after one course of induction chemotherapy in 26 NPM1 mutated AML patients, and is also associated with poorer overall survival. The poorer prognosis associated with high ABC proteins' activity in AML seems to be in part related to direct resistance to chemotherapy. These data should be confirmed in larger studies. Disclosures:No relevant conflicts of interest to declare.

Mutated regions of nucleophosmin 1 elicit both CD4+ and CD8+ T-cell responses in patients with acute myeloid leukemia

AbstractMutations in the nucleophosmin gene (NPM1mut) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1mut. In the present study, we were able to demonstrate both CD4+ and CD8+ T-cell responses against NPM1mut. Ten peptides derived from wild-type NPM1 and NPM1mut were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr51-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR–binding epitopes were used to test the role of CD4+ T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1mut induced CD8+ T-cell responses. A total of 33% of the NPM1mut AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4+ T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8+ and CD4+ T cells. The results of the present study show that NPM1mut induces specific T-cell responses of CD4+ and CD8+ T cells and therefore is a promising target for specific immunotherapies in AML.

High CD33 expression levels in acute myeloid leukemia cells carrying the nucleophosmin (NPM1) mutation

The CD33 antigen is expressed on the blast cells of most cases of acute myeloid leukemia and represents a suitable tumor-associated target antigen for antibody-based therapies. The aim of this study was to investigate the relationship between the CD33 levels quantified by mean fluorescence intensity and antibody binding capacity, and the presence/absence of NPM1 and FLT3 gene mutations in 99 newly diagnosed acute myeloid leukemia cases. The CD33 intensity evaluated as mean fluorescence intensity and antibody binding capacity was significantly higher in the NPM1-mutated acute myeloid leukemia cases compared to the NPM1-unmutated cases (P=0.0001 and P=0.0088, respectively). On the contrary, FLT3 gene mutations did not influence the levels of CD33 expression on the leukemic cells. These results establish a rational basis for the therapeutic use of anti-CD33 antibodies in NPM1-mutated acute myeloid leukemia patients.

Acute myeloid leukemia with mutated nucleophosmin (NPM1): is it a distinct entity?

AbstractAfter the discovery of NPM1-mutated acute myeloid leukemia (AML) in 2005 and its subsequent inclusion as a provisional entity in the 2008 World Health Organization classification of myeloid neoplasms, several controversial issues remained to be clarified. It was unclear whether the NPM1 mutation was a primary genetic lesion and whether additional chromosomal aberrations and multilineage dysplasia had any impact on the biologic and prognostic features of NPM1-mutated AML. Moreover, it was uncertain how to classify AML patients who were double-mutated for NPM1 and CEBPA. Recent studies have shown that: (1) the NPM1 mutant perturbs hemopoiesis in experimental models; (2) leukemic stem cells from NPM1-mutated AML patients carry the mutation; and (3) the NPM1 mutation is usually mutually exclusive of biallelic CEPBA mutations. Moreover, the biologic and clinical features of NPM1-mutated AML do not seem to be significantly influenced by concomitant chromosomal aberrations or multilineage dysplasia. Altogether, these pieces of evidence point to NPM1-mutated AML as a founder genetic event that defines a distinct leukemia entity accounting for approximately one-third of all AML.

Multilineage dysplasia has no impact on biologic, clinicopathologic, and prognostic features of AML with mutated nucleophosmin (NPM1)

AbstractNPM1-mutated acute myeloid leukemia (AML) is a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. The significance of multilineage dysplasia (MLD) in NPM1-mutated AML is unclear. Thus, in the 2008 WHO classification, NPM1-mutated AML with MLD is classified as AML with myelodysplasia (MD)–related changes (MRCs). We evaluated morphologically 318 NPM1-mutated AML patients and found MLD in 23.3%. Except for a male predominance and a lower fms-related tyrosine kinase 3–internal tandem duplication (FLT3-ITD) incidence in the MLD+ group, no differences were observed in age, sex, cytogenetics, and FLT3-–tyrosine kinase domain between NPM1-mutated AML with and without MLD. NPM1-mutated AML with and without MLD showed overlapping immunophenotype (CD34 negativity) and gene expression profile (CD34 down-regulation, HOX genes up-regulation). Moreover, overall and event-free survival did not differ among NPM1-mutated AML patients independently of whether they were MLD+ or MLD−, the NPM1-mutated/FLT3-ITD negative genotype showing the better prognosis. Lack of MLD impact on survival was confirmed by multivariate analysis that highlighted FLT3-ITD as the only significant prognostic parameter in NPM1-mutated AML. Our findings indicate that NPM1 mutations rather than MLD dictate the distinctive features of NPM1-mutated AML. Thus, irrespective of MLD, NPM1-mutated AML represents one disease entity clearly distinct from AML with MRCs.

AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features

Acute myeloid leukemia (AML) with mutated NPM1 usually carries normal karyotype (NK), but it may harbor chromosomal aberrations whose significance remains unclear. We addressed this question in 631 AML patients with mutated/cytoplasmic NPM1. An abnormal karyotype (AK) was present in 93 of 631 cases (14.7%), the most frequent abnormalities being +8, +4, -Y, del(9q), +21. Chromosome aberrations in NPM1-mutated AML were similar to, but occurred less frequently than additional chromosome changes found in other AML with recurrent cytogenetic abnormalities according to WHO classification. Four of the 31 NPM1-mutated AML patients karyotyped at different time points had NK at diagnosis but AK at relapse: del(9q) (n = 2), t(2;11) (n = 1), inv(12) (n = 1). NPM1-mutated AML with NK or AK showed overlapping morphologic, immunophenotypic (CD34 negativity), and gene expression profile (down-regulation of CD34 and up-regulation of HOX genes). No difference in survival was observed among NPM1-mutated AML patients independently of whether they carried a NK or an AK, the NPM1-mutated/FLT3-ITD negative cases showing the better prognosis. Findings in our patients point to chromosomal aberrations as secondary events, reinforce the concept that NPM1 mutation is a founder genetic lesion, and indicate that NPM1-mutated AML should be clinically handled as one entity, irrespective of the karyotype.

Minimal residual disease levels assessed by NPM1 mutation–specific RQ-PCR provide important prognostic information in AML

AbstractNucleophosmin (NPM1)–mutated acute myeloid leukemia (AML), which is recognized as a provisional entity in the World Health Organization 2008 classification of myeloid neoplasms, accounts for 30% of AML. We analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients with 17 different NPM1 mutation–specific real-time quantitative polymerase chain reaction (RQ-PCR) assays. Paired diagnostic/relapse samples of 84 patients revealed stable NPM1 mutations in all cases, suggesting that they are pathogenetically early events and thus applicable for minimal residual disease detection. A total of 47 relapses were predictable because of an NPM1 mutation level (%NPM1/ABL1) increase of at least 1 log or in 15 cases because of NPM1 mutation levels not decreasing less than 3 log ranges. A high prognostic value of NPM1 levels was shown for 4 different intervals after therapy was initiated. Furthermore, thresholds of 0.1 and 0.01%NPM1/ABL1 during/after treatment discriminated between prognostic subgroups. Univariate analyses, including age, white blood cell count, blast count, CD34 positivity, FLT3 mutations status, FAB type, karyotype, NPM1 mutation type, and pretreatment NPM1 mutational level, showed that, besides NPM1 mutation level, only age and FLT3-LM mutation status were prognostically significant for EFS. Multivariate analysis, including age, FLT3-LM status, and NPM1 mutation level at different time points, demonstrated that NPM1 level was the most relevant prognostic factor during first-line treatment. Similar results were obtained in patients undergoing second-line chemotherapy or allogeneic stem cell transplantation.