NPM1-mutated Acute Myeloid Leukemia Research Articles

PF229 CLUSTER ANALYSIS IDENTIFIES A SPECIFIC IMMUNOPHENOTYPIC PROFILE ASSOCIATED WITH HIGH RISK OF PRIMARY INDUCTION FAILURE AND SHORTER OVERALL AND DISEASE‐FREE SURVIVAL IN NPM1‐MUTATED AML

Background:NPM1‐mutated acute myeloid leukemia is a clinically heterogeneous disease entity encompassing different risk categories; despite growing knowledge on the impact of concurrent mutations, part of this biological and clinical heterogeneity is still unexplained. To date, a comprehensive description of the immunophenotypic landscape in NPM1mut AML has never been reported; specifically, the association between specific immunophenotypic patterns and clinical outcome in this AML subset has never been investigated.Aims:We performed a systematic analysis on an NPM1mut AML cohort using a large panel of immunophenotypic parameters in order to identify immunophenotypic clusters sharing common and recurrent features; we then investigated the clinical relevance of the so defined clusters in terms of outcome prediction.Methods:We identified 64 NPM1mut AML cases treated between 2010 and 2018 with intensive chemotherapy. Flow cytometry was performed on diagnostic BM samples in order to fully characterize the immunophenotypic profiles of leukemic populations. We focused our attention on myeloid cells in the CD45dim SSClow blast area. Based on CD34 expression we identified two main cell subsets: CD34+/CD45dim vs CD34‐/CD45dim. The two subgroups were further analyzed by using a comprehensive antigen panel. Parameters were expressed as percentage of positive cells and/or as mean fluorescence intensity; 6 BM samples from healthy donors were used as controls to set normal references.Results:Cluster analysis led to the identification of 3 main immunophenotypic clusters (C1, C2, C3; Fig 2). Distinctive features of C1 were expression of immature antigens (CD117, HLA‐DR) on both CD34+/CD45dim and CD34‐/CD45dim cell compartments as well as recurrent asynchronous CD11b expression and cross‐lineage CD7 and CD25 expression. In C2 a CD34+/CD45dim cell subset was detectable in 46% of patients without significant aberrations. Furthermore, CD34‐/CD45dim population showed an acute promyelocytic leukemia‐like phenotype (CD117‐/+, HLA‐DR‐) albeit with low SSC and CD13dim. In C3 the leading trait was differentiation to monocytic lineage, with low frequency of aberrant antigen expression. Survival analysis by cluster revealed significant differences in terms of DFS and OS (P = 0.006; P = 0.05, respectively; Figure 1A‐B). Most significantly, patients from C1 had a higher relapse risk and a relatively poor outcome when compared to patients classified as C2 and C3. Since the latter categories had a substantially superimposable outcome, we compared patients from C1 to the rest of the population as a unique entity: belonging to C1 cluster correlated with significantly shorter survival (Figure 1C‐D). Along with FLT3‐ITD and age, C1 phenotype emerged as an independent predictor for DFS and OS (Tab 1). While CR rates after first induction cycle were comparable between C1 and Non‐C1 patients, C1 was enriched with patients not achieving CR after 2 or 3 induction cycles (36% vs 11%; P = 0.016; OR 2.024). In multivariate analysis, a C1 profile was the sole predictive factor for not achieving a CR anytime; only 1 patient from C1 could achieve a CR after being refractory to first induction.Summary/Conclusion:Our analysis led to the identification of 3 distinctive immunophenotypic profiles significantly correlated to clinical outcome. Specifically, C1 identified patients at high risk of refractoriness and shorter DFS and OS. These findings shed a new light on the clinical heterogeneity of NPM1mut AML and warrant further investigation from a clinical and biological perspective.image

Clonal evolution patterns in acute myeloid leukemia with NPM1 mutation

Mutations in the nucleophosmin 1 (NPM1) gene are considered founder mutations in the pathogenesis of acute myeloid leukemia (AML). To characterize the genetic composition of NPM1 mutated (NPM1mut) AML, we assess mutation status of five recurrently mutated oncogenes in 129 paired NPM1mut samples obtained at diagnosis and relapse. We find a substantial shift in the genetic pattern from diagnosis to relapse including NPM1mut loss (n = 11). To better understand these NPM1mut loss cases, we perform whole exome sequencing (WES) and RNA-Seq. At the time of relapse, NPM1mut loss patients (pts) feature distinct mutational patterns that share almost no somatic mutation with the corresponding diagnosis sample and impact different signaling pathways. In contrast, profiles of pts with persistent NPM1mut are reflected by a high overlap of mutations between diagnosis and relapse. Our findings confirm that relapse often originates from persistent leukemic clones, though NPM1mut loss cases suggest a second “de novo” or treatment-associated AML (tAML) as alternative cause of relapse.

Next-generation sequencing in the diagnosis and minimal residual disease assessment of acute myeloid leukemia.

Risk-stratification of acute myeloid leukemia (AML) based on recurrent somatic abnormalities has evolved substantially in recent years, as illustrated by the current 2017 European LeukemiaNet (ELN) risk stratification.1 These 2017 ELN AML risk stratification recommendations are based on (cyto)genetic aberrations, including hotspot mutations such as those in NPM1, but also small insertions, deletions and point mutations that occur throughout TP53, RUNX1 and ASXL1, the latter being associated with adverse outcome.1 Next-generation sequencing (NGS) enables reliable detection of patient-specific mutations covering complete genes in molecularly heterogeneous diseases such as AML. NGS should, therefore, be incorporated in the routine work-up of preferably bone marrow specimens for accurate risk stratification in AML. Since risk assessment according to 2017 ELN recommendations only requires knowledge of the status of a handful of well-known driver mutations,1 targeted NGS, easily reaching a sensitivity of 1-2%, is currently the most appropriate and cost-effective approach for routine testing in AML. Targeted NGS using a variety of gene panels has been successfully introduced in routine clinical laboratories; however, several challenges remain. Gene panels A number of commercially available gene panels focusing on genes frequently mutated in myeloid malignancies have been introduced, e.g., the Illumina TruSight Myeloid panel, the Archer VariantPlex Core Myeloid panel, the Human Myeloid Neoplasms QIASeq DNA Panel and the AmpliSeq for Illumina Myeloid panel among many others. As expected, these panels contain all genes relevant for the 2017 ELN classification and show an enormous overlap in additional mutational hotspots and complete coverage of genes frequently mutated in myeloid diseases. In addition to these commercial panels, gene panels can be easily configured to meet local requirements. For instance, if AML patients are classified locally according to 2017 ELN, only NPM1, CEBPA, FLT3, RUNX1, ASXL1, and TP53 need to be included in a small and cost-effective gene panel. These types of NGS-based assays are now emerging.2 Importantly, both commercial NGS-based assays and those developed in-house as well as downstream analyses should be thoroughly validated locally before implementation in daily practice can be considered. Some genes are particularly difficult to sequence with NGS using gene panels. Bi-allelic mutations in CEBPA characteristically confer a favorable outcome in patients with AML.1 CEBPA is a GC-rich gene which is notoriously difficult to amplify by polymerase chain reaction (PCR) and sequence, and should be given special attention when incorporated in a gene panel. Although some commercial NGS gene panel protocols do now successfully include this single exon gene, other NGS approaches, such as capture-based NGS or custom panels for CEBPA mutation detection could be considered. FLT3 internal tandem duplications (ITD) can be reliably determined by fragment-length PCR following standardized protocols;3 however, the size of the FLT3 ITD and the duplication itself make it challenging to sequence the variably-sized amplicons appropriately by NGS and subsequently to analyze the FLT3 ITD by sequence alignment to reference sequences. Moreover, the 2017 ELN recommendations require assessment of the size of the FLT3 ITD clone.1 NPM1-mutant AML cases with high FLT3 ITD/FLT3 wildtype ratios (>0.5) are considered at intermediate risk, whereas NPM1 wildtype AML cases with high FLT3 ITD/FLT3 wildtype ratios are seen as adverse. Standardized NGS-based protocols need to be developed not only for the detection of FLT3 ITD, but also for the quantification of FLT3 ITD/FLT3 wildtype ratios. Examples of sensitive and specific custom-made FLT3 ITD NGS-based mutation detection assays have been published.4,5

Characterization and dynamics of specific T cells against nucleophosmin-1 (NPM1)-mutated peptides in patients with NPM1-mutated acute myeloid leukemia

Nucleophosmin(NPM1)-mutated protein, a leukemia-specific antigen, represents an ideal target for AML immunotherapy. We investigated the dynamics of NPM1-mutated-specific T cells on PB and BM samples, collected from 31 adult NPM1-mutated AML patients throughout the disease course, and stimulated with mixtures of 18 short and long peptides (9-18mers), deriving from the complete C-terminal of the NPM1-mutated protein. Two 9-mer peptides, namely LAVEEVSLR and AVEEVSLRK (13.9–14.9), were identified as the most immunogenic epitopes. IFNγ-producing NPM1-mutated-specific T cells were observed by ELISPOT assay after stimulation with peptides 13.9–14.9 in 43/85 (50.6%) PB and 34/80 (42.5%) BM samples. An inverse correlation between MRD kinetics and anti-leukemic specific T cells was observed. Cytokine Secretion Assays allowed to predominantly and respectively identify Effector Memory and Central Memory T cells among IFNγ–producing and IL2–producing T cells. Moreover, NPM1-mutated-specific CTLs against primary leukemic blasts or PHA-blasts pulsed with different peptide pools could be expanded ex vivo from NPM1-mutated AML patients or primed in healthy donors. We describe the spontaneous appearance and persistence of NPM1-mutated-specific T cells, which may contribute to the maintenance of long-lasting remissions. Future studies are warranted to investigate the potential role of both autologous and allogeneic adoptive immunotherapy in NPM1-mutated AML patients.

Glycolytic Enzyme PKM2 Mediates Autophagic Activation to Promote Cell Survival in NPM1-Mutated Leukemia.

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a distinct leukemia entity in the 2016 updated WHO classification of myeloid neoplasm. Our previous report showed that autophagic activity was elevated in NPM1-mutated AML, but the underlying molecular mechanisms remain elusive. Mount of study provides evidence that glycometabolic enzymes are implicated in the autophagic process. Pyruvate kinase isoenzyme M2 (PKM2), a key glycolytic enzyme, has been recently reported as a tumor supporter in leukemia. However, little is known about the roles of PKM2 in autophagic activity in NPM1-mutated AML. In this study, PKM2 highly expressed in NPM1-mutated AML, and partially, high levels of PKM2 were upregulated by PTBP1. Further experiments demonstrated that PKM2 mediated autophagic activation and increased the phosphorylation of key autophagy protein Beclin-1. Importantly, functional experiments demonstrated that PKM2 contributed to cell survival via autophagic activation. Ultimately, high PKM2 expression was associated with short overall and event-free survival time in NPM1-mutated AML patients. Our findings indicate for the first time that glycolytic enzyme PKM2 mediates autophagic activation and further contributes to cell survival in NPM1-mutated AML, suggesting that PKM2 may serve as a promising target for treatment of NPM1-mutated AML.

Up-regulation of EMT-related gene VCAN by NPM1 mutant-driven TGF-β/cPML signalling promotes leukemia cell invasion.

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) is acknowledged as a distinct leukemia entity in the 2016 updated World Health Organization (WHO) classification. NPM1-mutated AML patients are correlated with higher extramedullary involvement. Epithelial-mesenchymal transition (EMT) is one of the key steps which cause distant metastasis in tumor. However, whether EMT-related programs contribute to cell invasion in NPM1-mutated AML remains unclear. In this study, we identified the EMT-related gene versican (VCAN) in NPM1-mutated AML across three patient datasets. Further experiments validated the elevated VCAN expression in NPM1-mutated AML primary blasts and OCI-AML3 cells with NPM1 mutation. Mechanistic studies revealed that increased VCAN expression was at least partially regulated by NPM1 mutant via TGF-β/cPML/Smad signalling. Functional evaluations showed that silencing VCAN by shRNA significantly suppressed cell migration and invasion capacity, whereas increased VCAN by overexpressing NPM1-mA enhanced migration and invasion ability of leukemia cells. Finally, we found that high expression of VCAN was associated with poor prognosis in AML patients. These findings provide insights into the involvement of EMT-related gene VCAN in the pathogenesis of NPM1-mutated leukemia, which suggests that VCAN is an attractive target for novel diagnostic and therapeutic strategies in NPM1-mutated AML.

Prognostic Impact of the Interaction between DNMT3A mutation and Internal Tandem Duplication of the FLT3 Gene (FLT3-ITD) in Patients with De Novo Mutated NPM1 (NPM1mut) acute Myeloid Leukemia

IntroductionThe association of NPM1mut and FLT3-ITD in de novo acute myeloid leukemia (AML) with intermediate-risk cytogenetics has different prognostic impact depending on the FLT3 allelic burden. Previous studies published by our cooperative group showed that patients with de novo AML of intermediate-risk cytogenetics with NPM1mut and FLT3-ITD low ratio (<0.5, FLT3low) at diagnosis presented an overall survival and relapse rate similar to those with NPM1mut and FLT3wt. Therefore, in the CETLAM-2012 protocol, patients with FLT3low NPM1mut AML are not considered for allogenic hematopoietic stem cell transplant (allo-HSCT) in first complete remission (CR1). Recent studies suggest that the co-occurrence of DNMT3A mutation in FLT3-ITD NPM1mut AML patients confers a worse prognosis regardless of FLT3-ITD ratio.We analysed our data to determine whether these findings were confirmed in our cohort, specifically in the low FLT3-ITD ratio patients, since this could have therapeutic implications.Methods and patientsA total of 163 patients with de novo AML, intermediate-risk cytogenetics and NPM1mut were analysed (median age 53 years (18-72); male:female 72:91 (0.79)). Eighty patients (49%) harboured an FLT3-ITD, with a high allelic ratio in 42 of 76 patients with available ITD/wt ratio (55%). They were included in the AML-2003 (n=49) and AML-2012 (n=114) CETLAM protocols. Proportion of patients undergoing alloHSCT in CR1 is detailed in table 1.Bone marrow samples from diagnosis were studied for DNMT3A mutations as previously described. The definition of complete remission (CR), overall survival (OS), leukemia-free survival (LFS) and risk of relapse (RR) followed recommended ELN criteria. The Kaplan-Meier method was used to estimate the distribution of LFS and OS, for RR cumulative incidence was used.ResultsOut of the 163 patients with AML of intermediate risk cytogenetics and NPM1mut, 78 presented DNMT3A mutations (48%). Of these, 62 (79%) presented mutations in codon R882 or corresponded to DNA insertions/deletions while 16 (21%) harboured missense mutations. Presence of DNMT3A mutation did not associate with FLT3-ITD (ITD/85 DNMT3Awt vs ITD/78 DNMT3Amut, p=0.394). In the entire cohort, 5-year OS, LFS and RR were 58±4.5%, 59±4.6% and 27±13.9%. FLT3-ITD ratio confirmed its prognostic impact when analysing FLT3wt (n=83) vs FLT3low (n=34) vs FLT3high (n=42) patients (5-year OS of 68±6% vs 62±8.7% vs 37±8.6%; p=0.002; and 5-year RR of 18±9.4% vs 27±16.1% vs 41±23.2%; p=0.023). On the contrary, DNMT3Amut did not exert any effect on overall outcome (5-yr OS DNMT3Awt vs DNMT3Amut 61±6.2% vs 55±6.2%; p=0.234)When DNTM3A mutational status was considered, the impact of FLT3-ITD on outcome was mitigated in wild-type DNMT3A population. Thus, we found that DNMT3Awt patients presented no statistical differences in OS according to FLT3 mutational status or ratio: FLT3wt (n=46) vs FLT3-ITD (n=39) was 67±8.5% vs 57±8.2%; p=0.122, whereas FLT3wt (n=46) vs FLT3low (n=18) vs. FLT3high (n=19) was 67±8.5% vs. 66±11.5% vs 46±11.8%; p=0.088 (image 1A).This was also seen in relation to LFS and RR according to FLT3 ratio: 5-yr LFS of FLT3wt vs FLT3low vs FLT3high was 72±7.9% vs 61±12.6% vs 51±13.4%; p=0.244 and 5-year RR of the same groups: 19±8.8% vs 26±12.5% vs 27±21.9%; p=0.724 (image 2A).In the DNMT3Amut group, patients with FLT3-ITD (n=41) presented shorter OS than those with FLT3wt (n=37) with an OS of 37±10.7% vs 69±7.8%; p=0.028. When FLT3 ratio was considered, FLT3wt (n=37) vs FLT3low (n=16) vs FLT3high (n=23) showed an OS of 69±7.8% vs. 58±13.2% vs 27±13.1%; p=0.038 (image 1B).Similar results were seen in LFS according to FLT3 ratio (FLT3wt (n=29) vs FLT3low (n=16) vs FLT3high (n=20) 71±8.6% vs 53±12.9% vs 18±13.8%; p=0.012).Finally, we observed significant differences in the 5-year RR when considering DNMT3Amut patients in relation to FLT3 ratio (FLT3wt vs FLT3low vs FLT3high 18±10.6% vs 27±20% vs 54±28.8%; p=0.021)(image 2B).ConclusionsIn this study, patients with NPM1mut and FLT3-ITDlow presented a similar outcome to patients with NPM1mut and FLT3wt regardless of DNMT3A mutational status. These results support the modification of alloHCST policy in CR1 in CETLAM-2012, which do not consider alloHSCT for patients with FLT3low. On the other hand, concurrence of DNMT3A mutation may have an added negative effect in patients with NPM1mut and FLT3-ITDhigh, which should be further confirmed in larger studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gemtuzumab Ozogamicin in NPM1-Mutated Acute Myeloid Leukemia (AML): Results from the Prospective Randomized AMLSG 09-09 Phase-III Study

Gemtuzumab Ozogamicin in NPM1-Mutated Acute Myeloid Leukemia (AML): Results from the Prospective Randomized AMLSG 09-09 Phase-III Study

Homozygous Loss of a Critical CTCF Recognition Sequence in the HOXA gene Cluster Eliminates CTCF Binding but Does Not Appreciably Reduce HOXA9 expression in NPM1 Mutated AML Cells

HOX genes encode homeodomain transcription factors that play a central role in hematopoietic stem/progenitor cell (HSPCs) self-renewal and the development of acute myeloid leukemia (AML). HOXA and HOXB genes display canonical expression patterns in primary AML samples that correlate with specific recurrent mutations, including expression of only HOXA genes in AMLs with MLL rearrangements and both HOXA and HOXB gene expression in normal karyotype AMLs with mutations in NPM1 (i.e., the NPMc mutation). Although regulation of HOXA genes in MLL-rearranged AML cells has been studied extensively, the regulatory mechanisms that govern HOXA expression in other AML types remains unclear. The chromatin organizing factor CTCF contributes to HOXA gene regulation by controlling chromatin looping and three-dimensional structure in a variety of cell types, including AML cells, and a recent study demonstrated that expression of HOXA9 in MLL-rearranged AML cell lines is dependent on a critical CTCF-binding site between HOXA7 and HOXA9 (CBS7/9). However, whether this site is required for HOXA gene expression in AMLs with NPMc remains unknown.The AML cell line OCI-AML3 contains the NPMc mutation (in addition to a DNMT3AR882C mutant allele) and expresses both HOXA and HOXB genes. We therefore used this cell line to investigate the role of CTCF in regulating HOXA expression in AML cells with this canonical stem cell expression pattern. ChIP-seq for CTCF via ‘ChIPmentation’ identified eight CTCF binding sites across the HOXA cluster in OCI-AML3 cells, including CBS7/9, which is consistent with previous data in AML cells. These sites were identical to those identified in MLL-rearranged AML cell lines, suggesting that CTCF binding is conserved between these AML types. Similar to previous work, ChIP-seq for histone methylation marks associated with active (H3K4 trimethylation) and repressed (H3K27 trimeylation) chromatin demonstrated distinct chromatin domains at the HOXA locus on either side of CBS7/9, indicating that CTCF acts as a barrier between these domains. Using a computational prediction tool for CTCF binding, we identified two potential CTCF-binding sequences at the CBS7/9 locus, including one bidirectional major site (CBS7/9-A; 20 bp) with a high binding score, separated by only 6 bp from a secondary unidirectional site (CBS7/9-B; 9 bp).To define the precise sequence important for CTCF binding in OCI-AML3 cells, we used CRISPR-Cas9 to generate a library of mutant cells with deletions of different sizes at the CBS7/9 locus, and then performed ChIP-seq on the mutant cells to measure CTCF binding. We found that mutants with deletions at CBS7/9-A were depleted relative to wild-type cells. Further analysis demonstrated that deletions as small as 10 bp affecting the CBS7/9-A site were sufficient to reduce CTCF binding to background levels. CRISPR-Cas9 generated deletions in the MLL-rearranged THP-1 cell line that spanned the same sequence also eliminated CTCF ChIP-seq signal, indicating that it is equally important for CTCF binding in MLL-rearranged and NPM1-mutant AML cells.Our collection of mutant OCI-AML3 cell lines included five with biallelic deletions of the CBS7/9-A site, which completely eliminated all CTCF binding at this locus, but did not affect cellular proliferation or survival. Surprisingly, preliminary analysis of expression in the five homozygous mutant OCI-AML3 cells demonstrated that HOXA9 gene expression by RT-qPCR remained largely intact compared to wild-type OCI-AML3 cells, and was not statistically different from three OCI-AML3 cell lines with a heterozygous deletion of the CBS7/9 site.In summary, we have mapped the precise recognition sequence for a critical CTCF binding site in the HOXA locus in human AML cells. Deletion of both alleles of this regulatory sequence in NPM1-mutated OCI-AML3 cells does not affect cell viability and appears to have only modest effects on expression of the adjacent HOXA9 gene. This is in contrast to a recent report suggesting that homozygous deletion of this CTCF binding site is critical for maintaining HOXA gene expression and may be required for cell viability in MLL-rearranged AML cell lines. These differences suggest that maintenance of HOXA expression in NPM1-mutated vs. MLL-rearranged AML cells may occur through distinct regulatory mechanisms. DisclosuresNo relevant conflicts of interest to declare.

CD97 Is Associated with Poor Overall Survival in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a heterogeneous, hematologic malignancy characterized by clonal proliferation of myeloid precursors. Overall survival for patients with AML remains dismal (<50% for younger patients and <10% for older patients) due to high relapse rates, necessitating the identification of novel therapeutic targets. CD97 is a glycoprotein belonging to the EGF-TM7 molecule family, a subgroup of adhesion G-protein coupled receptors. CD97 was shown to play a role in cell migration, adhesion and interaction with cell surface proteins and components of the extracellular matrix. In several solid cancers, CD97 association with integrin was shown to regulate invasion, migration, angiogenesis, and poor survival outcome. Even though CD97 is normally expressed on myeloids and leukocytes, there is sparse data related to its expression on myeloid leukemia cells. In this study, we characterized CD97 expression in patients with AML using publically available data sets to determine its association with patient's clinical and molecular characteristics and clinical outcome.Methods:CD97 mRNA (RNA Seq V2 RSEM) expression and Z-score data was downloaded from TCGA. We generated Kaplan-Meier survival curves to compare overall (OS) and event-free (EFS) survival between patients with CD97 high (Z≥1) and CD97 low (Z<1) expression after stratification by age, cytogenetic status, transplant status and NPM1 mutation status. Association between CD97 expression and patient clinical and molecular characteristics was performed by Mann-Whitney U's non-parametric t-test and Fisher's exact test. STATA 12.0 SE was used to perform a Cox Proportional Hazards Model.Results: Patients with cytogenetically normal AML (CN-AML) had significantly higher CD97 expression than cytogenetically abnormal AML (CA-AML) (1.31 fold, p=0.023). Patients with high CD97 expression had significantly higher WBC count (median: 56.1 vs 13.1, p=0.004) and % bone marrow blasts (median: 83 vs 71%, p=0.019). CD97 was significantly higher in patients with NPM1 mutation (n=48) compared with patients with NPM1 wild type (n=125) (1.56 fold, p<0.000). CD97 was also significantly higher in patients with FLT3 mutation (ITD and point mutations) (n=49) compared with patients with FLT3 wild type (n=124) (1.4 fold, p=0.0008). Additionally, CD97 was significantly lower in the patients with RUNX1 mutation (n=17) compared with wildtype gene (n=156) (42.1% lower; p=0.0002).The OS of the CD97 high was significantly shorter than that of the CD97 low patients (median: 7.35 vs. 24.1 months; p=0.0015). Patients with CD97 high expression had significantly shorter EFS than that of CD97 low (median: 5.35 vs. 12 months; p=0.0015). Furthermore, in CN-AML CD97 high patients had shorter OS than CD97 low patients in CN-AML (median: 7.5 vs 20.5 months; p=0.0045; Figure 3B). In multivariate survival analysis, CD97 high expression was associated with shorter OS when adjusted for age, cytogenetic risk and transplant status (HR= 1.96; 95% Cl: 1.19-3.24; p=0.009).Because CD97 expression was associated with NPM1 mutation in AML, we stratified patients according to NPM1 mutational status and found that in patients with wildtype NPM1, CD97 high expression was associated with significantly shorter OS (Median survival: 6.35 vs 22.3 months; P= 0.0081) and EFS (Median survival: 5.35 vs 13.4 months; P=0.0006). In patients with NPM1 mutation, a similar but not significant association was observed with CD97 high expression and shorter OS (median survival: 7.5 vs 24.1 months; P=0.1) and EFS (median survival: 5.8 vs 11.1 months; P=0.27).Furthermore, in patients ≥ 60; CD97 high expression was associated with shorter OS compared with CD97 low (median survival 3.3 vs 11 months; p=0.0019) and EFS (Median survival 2.9 vs 9.2 months; p=0.0092). Similar but not significant association was observed in in patients<60 (Median OS: 27.5 vs 53.9; p=0.2) and (Median EFS 8.5 vs 53.9; p=0.2).Conclusion: Patients with high CD97 expression had shorter OS and EFS. CD97 was higher in CN-AML compared with CA-AML patients. Additionally, high CD97 was also associated with NPM1 mutation. Our findings demonstrate that CD97 expression contributes to the clinical outcome of patients with AML, particularly in older patients. This study provides rationale for further functional and mechanistic studies aiming to understand the role of CD97 in AML. DisclosuresNo relevant conflicts of interest to declare.

Expression of PD-L1 in Leukemic Progenitor Cells Defines NPM1 Mutated AML As a Potential Subgroup for PD1/PD-L1 Directed Immunotherapy

Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1(NPM1mut) may constitute an immunogenic leukemia subtype. NPM1mut AML generally correlates with a better prognosis, however the underlying mechanisms still need to be clarified. Checkpoint inhibition targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer treatment including the treatment of hematological malignancies.Expression of CD34/CD38/CD274 was evaluated in 20 NPM1mut versus 20 wild-type (NPM1wt) AML patient samples via flow cytometry analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor and stem cells (LSC). We also investigated the influence of the anti-PD-1 antibody Nivolumab® on the antigen-specific immune responses in ELISpot assays. Additionally, we assessed the effect of Nivolumab in colony forming unit (CFU) immunoassays.Many AML cases showed relevant expression of PD-L1. Bulk cells of NPM1mut AML showed a significantly higher PD-L1 expression in comparison to NPM1wtAML patients (median of 1.5%, range 0.0-8.5%, versus 0.3%, range 0.1-1.1%). Importantly, PD-L1 expression was detected at a higher level in leukemic progenitor cells (CD34+CD38-) of NPM1mut than of NPM1wtAML (median of 3.3%, range 0.0-17.2%, versus 0.3%, range 0.0-3.0%). In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC fraction.CFU immunoassays showed a significant inhibition of CFU when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. Immune effects increased adding Nivolumab to the CTL for several days before starting CFU immunoassays.In summary, we detected higher PD-L1 expression in NPM1mut patients, especially in the leukemic progenitor compartment. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via expression of PD-L1. Immunogenicity of neoantigens derived from NPM1mut with higher PD-L1 expression constitute promising target structures for individualized immunotherapeutic approaches. DisclosuresSchrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Bullinger:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria.

Assessment of Treatment Effects By Measurable Residual Disease Monitoring in NPM1-Mutated AML Patients Randomized for Gemtuzumab-Ozogamicin (GO) within the AMLSG 09-09 Trial of the German-Austrian AML Study Group (AMLSG)

Assessment of Treatment Effects By Measurable Residual Disease Monitoring in NPM1-Mutated AML Patients Randomized for Gemtuzumab-Ozogamicin (GO) within the AMLSG 09-09 Trial of the German-Austrian AML Study Group (AMLSG)

More Than 10% of NPM1-Mutated AML Relapses Occur after 5 Years from Complete Remission

▪The majority of relapses in acute myeloid leukemia (AML) patients occur in the first or second year following complete remission. In routine, AML patients are followed during five years because few relapses can occur after three or five years. These late or very late relapses remain poorly described, particularly at the molecular level, with only few consistent series in the literature. (Medeiros B et al., Leuk Lymphoma 2007; Verma D et al., Leuk Lymphoma 2010; Watts J et al., Leuk Res 2014).We retrospectively studied all AML relapses occurring after complete remission (CR) obtained with one or two induction cycles between 2000 and 2012 in Toulouse University Hospital, France. Our analyses focused on late relapses (LR, >3 years from CR) and very late relapses (VLR, >5 years from CR) in comparison to early relapses (ER, ≤3 years from CR).Between 2000 and 2012, out of 636 CR patients, 346 had morphological relapses (54.4%). The median time to relapse was 0.9 years (range, 0.1-11.9 years; interquartile range [IQR], 0.5-1.5 years). There were 198 relapses during the first year (57.2%), 82 during the second year (23.7%), 24 during the third year (6.9%) whereas 42 relapses occurred after 3 years (12.1%) and 16 after 5 years (4.6%). Characteristics at diagnosis, i.e., age, AML status, WBC count, karyotype, FLT3-ITD mutation, CEBPA mutation and induction regimen did not differ between ER and LR or VLR. However, NPM1 mutations were more frequent in LR (NPM1m at diagnosis in relapses >3 years: 46% vs. 28% in relapses <3 years, P=.0532), and in VLR (NPM1m at diagnosis in relapses >5 years: 67% vs. 27% % in relapses <5 years, P=.0070). Allogeneic stem cell transplantation (alloSCT) was more frequently performed in the LR group (24% vs. 14%, P=.0369) and in VLR group (31% vs. 14%, P=.0748). Second CR (CR2) rate and median overall survival from relapse date (OS2) were better in LR and VLR than in ER (CR2ER: 26%, CR2LR: 43%, CR2VLR: 50%; P=.0154; OS2ER: 4.6 months, OS2LR: 10.8 months, OS2VLR: 11.6 months; P=.0024).Among the 142 CR1 patients with NPM1m, 67 relapsed (47.2%). In patients with NPM1m, relapses occurred during the first year in 39 (58.2% of NPM1m relapses), during the second year in 14 (20.9%) and during the third year in 2 (3%) whereas 12 relapses occurred after 3 years (17.9%), 8 occurred after 5 years (11.9%) and 3 after 8 years (4.5%). In NPM1-wild type patients, LR and VLR were significantly less frequent (<3 years: 91.9%; >3 years: 8.1%; >5 years: 2.5%; >8 years: 0.6%; P=.0317, .0037 and .0783 respectively). NPM1m relapses represented one half of LR (48%) and two thirds of VLR (67%). Among them, genotype was NPM1m/FLT3-wild type in most patients (75% in LR and 88% in VLR patients). In LR and VLR, NPM1 mutational status had no impact on CR2 and OS2: CR2LR/NPM1m: 42% vs. CR2LR/NPM1-WT: 38% (P=.8702); CR2VLR/NPM1m: 50% vs. CR2LR/NPM1-WT: 50% (P=1.0000); OS2LR/NPM1m: 7.4 months vs. OS2LR/NPM1-WT: 19.4 months (P=.2019); OS2VLR/NPM1m: 7.8 months vs. OS2VLR/NPM1-WT: 29.8 months (P=.0917).Our data show that LR and VLR are not infrequent in AML patients with NPM1 mutations. Although this finding needs to be validated in updated multicentric cohorts with a very long follow-up, it strongly suggests that AML patients with NPM1 mutations should benefit from a prolonged follow-up beyond 5 years from CR. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal/Measurable Residual Disease Monitoring in NPM1-Mutated Acute Myeloid Leukemia: A Clinical Viewpoint and Perspectives.

Acute myeloid leukemia (AML) with NPM1 gene mutations is currently recognized as a distinct entity, due to its unique biological and clinical features. We summarize here the results of published studies investigating the clinical application of minimal/measurable residual disease (MRD) in patients with NPM1-mutated AML, receiving either intensive chemotherapy or hematopoietic stem cell transplantation. Several clinical trials have so far demonstrated a significant independent prognostic impact of molecular MRD monitoring in NPM1-mutated AML and, accordingly, the Consensus Document from the European Leukemia Net MRD Working Party has recently recommended that NPM1-mutated AML patients have MRD assessment at informative clinical timepoints during treatment and follow-up. However, several controversies remain, mainly with regard to the most clinically significant timepoints and the MRD thresholds to be considered, but also with respect to the optimal source to be analyzed, namely bone marrow or peripheral blood samples, and the correlation of MRD with other known prognostic indicators. Moreover, we discuss potential advantages, as well as drawbacks, of newer molecular technologies such as digital droplet PCR and next-generation sequencing in comparison to conventional RQ-PCR to quantify NPM1-mutated MRD. In conclusion, further prospective clinical trials are warranted to standardize MRD monitoring strategies and to optimize MRD-guided therapeutic interventions in NPM1-mutated AML patients.

Mutant NPM1 Maintains the Leukemic State through HOX Expression

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.

Identification of miRNA-mRNA Network in NPM1 Mutated Acute Myeloid Leukemia

Identification of miRNA-mRNA Network in NPM1 Mutated Acute Myeloid Leukemia

Abstract 519: The long non-coding RNA (lncRNA) HOXB-AS3 regulates transcription of ribosomal RNA (rRNA) in NPM1-mutated (NPM1mut) acute myeloid leukemia (AML)

Abstract Background: A HOXB-locus-embedded lncRNA, named HOXB-AS3 significantly associates with NPM1 mutations in AML. Herein, we evaluate the functional role of HOXB-AS3 expression in NPM1mut AML. Methods: HOXB-AS3 expression was measured by real-time PCR. Knock-down (KD) of HOXB-AS3 was performed in vitro and in vivo with locked nucleic acid-modified gapmers. RNA antisense purification (RAP), RNA-immunoprecipitation (RIP), and Chromatin-immunoprecipitation (ChIP) experiments were performed according to published protocols. Results: Among 7 AML cell lines tested, only OCI-AML3 cells, which harbor NPM1mut, showed detectable HOXB-AS3 expression. HOXB-AS3 was more abundant in NPM1mut AML patient (pt) blasts than blasts of AML pts with wild-type NPM1 (P=.001) and bone marrow samples from healthy donors(P=.001). HOXB-AS3 localized in the nucleus and did not associate with isolated polysomes of OCI-AML3 cells. In vitro HOXB-AS3 KD in OCI-AML3 cells decreased the cells in S phase (P&amp;lt;.001) and increased those in G2/M phase (P=.006). HOXB-AS3 KD reduced the number of formed colonies by OCI-AML3 cells (P=.02). In contrast, overexpression of HOXB-AS3 in K562 cells increased the cells in S phase (P=.02) and decreased those in G0/G1 phase (P=.008). HOXB-AS3 KD in blasts of 3 NPM1mut AML pts decreased the number of formed colonies (P=.03, P=.02, and P&amp;lt;.001). In vivo HOXB-AS3 KD in murine patient-derived xenografts of 2 NPM1mut AML pts prolonged their overall survival (P&amp;lt;.001 and P=.03). RAP-based isolation of HOXB-AS3 and comparative proteomic analyses identified 23 candidate HOXB-AS3-binding proteins. EBP1 was validated as the most avid HOXB-AS3 interactor (P&amp;lt;.001) by RIP experiments. Manipulations of HOXB-AS3 impacted on the (previously reported) EBP1 interaction with NPM1; HOXB-AS3 KD reduced, whereas overexpression of HOXB-AS3 increased the EBP1-NPM1 complex formation. Consequently, HOXB-AS3 KD reduced transcription of rRNA and de novo protein synthesis in OCI-AML3 cells (P&amp;lt;.001 and P=.002) and AML pt blasts (P&amp;lt;.001 and P=.03, respectively). Overexpression of HOXB-AS3 increased rRNA transcription (P&amp;lt;.001), de novo protein synthesis (P=.001). ribosomal DNA (rDNA) promoter occupancy by RNA-Polymerase I (P=.001), and activity of an rDNA promoter-containing luciferase reporter (P=.002) in K562 cells. We hypothesized that HOXB-AS3 guides EBP1 to the rDNA locus. RAP-DNA experiments validated the interaction of HOXB-AS3 with rDNA chromatin (P=.001) and HOXB-AS3 KD decreased the occupancy of the rDNA promoter by EBP1 (P=.002), as shown by ChIP assays. Conclusions: We describe the function of the HOXB-AS3 lncRNA as a compensatory mechanism, which mediates increased rRNA transcription and adequate protein production, in NPM1mut AML. From a therapeutic standpoint, we show that HOXB-AS3-targeting yields anti-leukemic activity in pre-clinical models. Citation Format: Dimitrios Papaioannou, Andreas Petri, Sara Terreri, Charlotte A. Thrue, Deedra Nicolet, Frances A. Collins, Lauren A. Woodward, Prasanthi Kumchala, Malith Karunasiri, Felice Pepe, Marius Bill, Nina Zitzer, Guramrit Singh, Sakari Kaupinnen, Clara D. Bloomfield, Adrienne M. Dorrance, Ramiro Garzon. The long non-coding RNA (lncRNA) HOXB-AS3 regulates transcription of ribosomal RNA (rRNA) in NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 519.

Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients.

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable ("minimal") residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers-also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients' outcomes.

Deep NPM1 Sequencing Following Allogeneic Hematopoietic Cell Transplantation Improves Risk Assessment in Adults with NPM1-Mutated AML

Relapse is the major cause of death in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT). Measurable residual disease (MRD) detected by multiparameter flow cytometry (MFC) before and after HCT is a strong, independent risk factor for relapse. As next-generation sequencing (NGS) is increasingly applied in AML MRD detection, it remains to be determined if NGS can improve prediction of post-HCT relapse. Herein, we investigated pre-HCT MRD detected by MFC and NGS in 59 adult patients with NPM1-mutated AML in morphologic remission; 45 of the 59 had post-HCT MRD determined by MFC and NGS around day 28. Before HCT, MRD detected by MFC was the most significant risk factor for relapse (hazard ratio [HR], 4.63; P < .001), whereas MRD detected only by NGS was not. After HCT, MRD detected by either MFC or NGS was significant risk factor for relapse (HR, 4.96, P = .004 and HR, 4.36, P = .002, respectively). Combining pre- and post-HCT MRD provided the best prediction for relapse (HR, 5.25; P < .001), with a sensitivity at 83%. We conclude that NGS testing of mutated NPM1 post-HCT improves the risk assessment for relapse, whereas pre-HCT MFC testing identifies a subset of high-risk patients in whom additional therapy should be tested.

A distinct immunophenotype identifies a subset of NPM1-mutated AML with TET2 or IDH1/2 mutations and improved outcome.

Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co-occur with FLT3-ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1-mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation ("myeloid AML"), 40 (48%) demonstrated an acute promyelocytic leukemia-like (APL-like) immunophenotype by flow cytometry, with absence of CD34 and HLA-DR and strong myeloperoxidase expression, in the absence of a PML-RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL-like cases. This subset of NPM1-mutated AML was associated with longer relapse-free and overall survival, when compared with cases that were positive for CD34 and/or HLA-DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL-like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1-mutated AML.