NPM1 Mutation Status Research Articles

Dual Inhibition of Bruton's Tyrosine Kinase and BCL2: A Promising Therapeutic Strategy for Myeloid and Lymphoid Leukemias

Background: The identification of effective therapies based on targeted interventions for human cancers faces the challenges of genetic and epigenetic heterogeneity underlying the disease. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse.Methods: We used a functional ex vivo screening assay to identify small-molecule targeted inhibitors and inhibitor combinations demonstrating selective efficacy across broad categories of leukemia. Primary mononuclear cells isolated from leukemia patients (n=588) were plated in the presence of a panel of graded concentrations of over 120 single-agent inhibitors or combinations spanning multiple drug classes. Leukemia specimens from 5 diagnosis subgroups included acute myeloid leukemia (AML; n=293), acute lymphoblastic leukemia (ALL; n=83), chronic lymphocytic leukemia (CLL; n=140), chronic myeloid leukemia (CML; n=26) and myeloproliferative neoplasms/myelodysplastic syndrome (MPN or MDS/MPN; n=46) patients. The combinations were designed as drug pairs that target non-overlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. IC50 and AUC values were derived from probit-based regression for each response curve. Mutational status for FLT3-ITD and NPM1 were compiled from clinical labs or by capillary electrophoresis using a QiaXcel instrument. Disease status was obtained from clinical chart review. Single and combination drug treatment IC50 and AUC values were compared within groups by Friedman test, across groups by Kruskal-Wallis test, and with continuous variables by Spearman rank correlation.Results: Among 588 unique leukemia patients evaluated, we observed that the combination of venetoclax (a BCL2 inhibitor) with ibrutinib(a BTK inhibitor) showed enhanced benefit above that for either single agent in both myeloid and lymphoid malignancies, including AML, ALL, and CLL. Expanded analyses of combination sensitivity in AML patients showed no significant associations with age, gender, or ELN prognostic risk. Comparison of AML samples by disease treatment status indicated a slightly greater sensitivity in relapsed/refractory (n=42) versus newly diagnosed (n=115) AML samples by both AUC and IC50 (p=0.028 and 0.026, respectively). Among AML samples, FLT3-ITD and NPM1 mutation status was available for 204 and 188 patients, respectively. Venetoclax + ibrutinib was significantly more effective in patient samples harboring FLT3-ITD or NPM1 mutations by both AUC (p=0.0002 and p=0.0032, respectively) and IC50 (p=0.0002 and p=0.021, respectively). Furthermore, among 112 AML samples with RNAseq data available, sensitivity to the combination (AUC) was significantly correlated with gene expression for BCL2 and BTK (Spearman r: -0.48 and -0.21, respectively). Evaluation of the combination using an expanded 7x7 concentration matrix on AML cell lines revealed synergy for the majority of drug-pair concentrations by the Bliss independence model. Analysis to align additional clinical and genetic features for leukemia patient samples with drug sensitivities is in progress and results with be presented at the meeting.Conclusion: Both myeloid and lymphoid-derived primary leukemia cells show sensitivity to combined inhibition of BCL2 and BTK with the combination of venetoclax and ibrutinib, suggesting this drug pair may have broad therapeutic indications. [Display omitted] DisclosuresTyner:Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Constellation: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Incyte: Research Funding; Array: Research Funding; Aptose: Research Funding. Danilov:TG Therapeutics: Consultancy; Takeda Oncology: Research Funding; Aptose Biosciences: Research Funding; Genentech: Consultancy, Research Funding; Astra Zeneca: Consultancy; Gilead Sciences: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Verastem: Consultancy, Research Funding. Druker:Aileron Therapeutics: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Oregon Health & Science University: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Monojul: Consultancy; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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.

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.

Predictive Response Biomarkers for BET Inhibitors in AML

BACKGROUND: Despite mounting knowledge, the management of patients with acute myeloid leukemia (AML) can be challenging, particularly for relapsed/refractory patients who lack innovative, effective treatments. Recent preclinical studies of AML have demonstrated promissory therapeutic activity of the novel class of inhibitors of bromodomain and extra terminal (BET) family proteins. To identify AML patients that may benefit from BET inhibitor treatment, we sought to discover and evaluate predictive molecular biomarkers for sensitivity towards BET inhibition, focusing on the prototype BET inhibitor JQ1.METHODS: Following written informed consent and according to the Declaration of Helsinki, bone marrow, peripheral blood, and skin biopsy samples were obtained prospectively from AML patients (n=139) and analyzed for their whole exome profiles and ex vivo responses to JQ1 exposure, as part of an Individualized Systems Medicine (ISM) study of AML (Pemovska et al., 2013). Consequently, associations between molecular features and drug responses were investigated using logistic regression models for data integration. Novel signals emerging from our cohort of patients were carried forward for reproducibility assessment in publicly available cell line datasets (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer for genomic data, and Cancer Therapeutics Response Portal and Genomics of Drug Sensitivity in Cancer for drug response data). In addition, literature-reported indicators of sensitivity were examined in the AML cohort in order to evaluate their clinical translatability.RESULTS: Enabling a genome-wide search for predictive biomarkers for BET inhibitor response, our bioinformatics pipeline returned a set of signals significantly associated with JQ1 treatment.Frequently mutated in AML, isocitrate dehydrogenases IDH1 and IDH2, tet methylcytosine dioxygenase TET2, and Wilms' tumor WT1 each appeared to confer resistance upon mutation to the samples harboring them. Participating in a common biochemical pathway, the 4 genes have been shown to be mutated following a mutual exclusivity pattern in AML (Scourzic et al., 2015), which was successfully replicated in our cohort. Accordingly, grouping of samples with mutation in any of the genes yielded a significantly more resistant group to JQ1 treatment in comparison to wild-type samples. Incorporation of TET2 and WT1 deletions from the samples' copy number variation data supported this hypothesis. Importantly, this observation was also successfully validated in cell line drug sensitivity data (Figure 1A).Moreover, FLT3-mutated samples in our cohort returned significantly higher sensitivity scores to JQ1 compared to wild-type samples, irrespective of their NPM1 mutation status. The superior response trend observed within this group was maintained independently of whether the mutation was an internal tandem duplication (FLT3-ITD) or in the tyrosine kinase domain (FLT3-TKD) of the gene (Figure 1B).Finally, and in contrast to other studies, AML samples harboring mutation to NPM1 were not significantly more sensitive to BET inhibition with respect to NPM1 wild-type samples.CONCLUSION: Taken together, the results suggest novel insights for the stratification of AML patients for treatment using BET inhibition therapy, taking this malignancy's heterogeneity into account. Particularly, while FLT3 mutations are to be considered as sensitizing factors to BET inhibitors, samples with mutations in IDH1, IDH2, TET2, or WT1 were resistant, and thus, patients harboring these gene mutations may not benefit. These observations also propose drug partners that are worth investigating in combination with BET inhibitors in the defined patient subpopulations, namely FLT3 inhibitors and hypomethylating agents (or IDH1/2 inhibitors), respectively. Furthermore, the variability of responses observed among samples harboring NPM1 mutations highlights the molecular heterogeneity existing within this group of patients, stressing the need for identifying additional companion diagnostics for a more effective stratification of this population. Although our findings require additional validation in a larger cohort and through clinical investigations, this study provides evidence of molecular indicators that could help identify AML patients that would benefit from BET inhibitor therapy. [Display omitted] DisclosuresTsallos:Novartis: Research Funding. Wennerberg:Novartis: Research Funding. Porkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.

Measurable residual disease monitoring by NGS before allogeneic hematopoietic cell transplantation in AML

AbstractMolecular measurable residual disease (MRD) assessment is not established in approximately 60% of acute myeloid leukemia (AML) patients because of the lack of suitable markers for quantitative real-time polymerase chain reaction. To overcome this limitation, we established an error-corrected next-generation sequencing (NGS) MRD approach that can be applied to any somatic gene mutation. The clinical significance of this approach was evaluated in 116 AML patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) in complete morphologic remission (CR). Targeted resequencing at the time of diagnosis identified a suitable mutation in 93% of the patients, covering 24 different genes. MRD was measured in CR samples from peripheral blood or bone marrow before alloHCT and identified 12 patients with persistence of an ancestral clone (variant allele frequency [VAF] >5%). The remaining 96 patients formed the final cohort of which 45% were MRD+ (median VAF, 0.33%; range, 0.016%-4.91%). In competing risk analysis, cumulative incidence of relapse (CIR) was higher in MRD+ than in MRD− patients (hazard ratio [HR], 5.58; P < .001; 5-year CIR, 66% vs 17%), whereas nonrelapse mortality was not significantly different (HR, 0.60; P = .47). In multivariate analysis, MRD positivity was an independent negative predictor of CIR (HR, 5.68; P < .001), in addition to FLT3-ITD and NPM1 mutation status at the time of diagnosis, and of overall survival (HR, 3.0; P = .004), in addition to conditioning regimen and TP53 and KRAS mutation status. In conclusion, NGS-based MRD is widely applicable to AML patients, is highly predictive of relapse and survival, and may help refine transplantation and posttransplantation management in AML patients.

Prognostic value of genetic mutations in adolescent and young adults with acute myeloid leukemia.

Clinical outcomes and the genetic background of acute myeloid leukemia (AML) in adolescent and young adults (AYAs) are known to differ in younger children and older adults. To clarify the impact of genetic mutations on clinical outcomes of AYAs with AML, we analyzed data from the JPLSG AML-05 and JALSG AML201 studies. AYAs aged 15-39years (n=103) were included. FLT3-ITD, KIT, CEBPA, NRAS, KRAS, WT1, MLL-PTD, and NPM1 mutations were analyzed. Overall survival (OS) of the AYAs was 61% and event-free survival was 38% at 3years. FLT3-ITD (HR 2.10; 95% CI 1.07-4.12; p=0.031) and NPM1 (HR 0.24; 95% CI 0.06-1.00; p=0.050) mutations were associated with risk of overall mortality in multivariate analysis. OS was significantly different according to FLT3-ITD and NPM1 mutation status (p=0.03). Survival was 100% with NPM1 mutations in the absence of FLT3-ITD and 35% (95% CI 14-57%) with FLT3-ITD in the absence of NPM1 mutations. The OS of AYAs, children (n=413) and older adults (n=124) of the AML-05 and AML201 participants were significantly different (p<0.0001). This is the first report to combine clinical and genetic data of AYA AML from the major Japanese pediatric and adult study groups.

Circular RNAs of the nucleophosmin (NPM1) gene in acute myeloid leukemia.

In acute myeloid leukemia, there is growing evidence for splicing pattern deregulation, including differential expression of linear splice isoforms of the commonly mutated gene nucleophosmin (NPM1). In this study, we detect circular RNAs of NPM1 and quantify circRNA hsa_circ_0075001 in a cohort of NPM1 wild-type and mutated acute myeloid leukemia (n=46). Hsa_circ_0075001 expression correlates positively with total NPM1 expression, but is independent of the NPM1 mutational status. High versus low hsa_circ_0075001 expression defines patient subgroups characterized by distinct gene expression patterns, such as lower expression of components of the Toll-like receptor signaling pathway in high hsa_circ_0075001 expression cases. Global evaluation of circRNA expression in sorted healthy hematopoietic controls (n=10) and acute myeloid leukemia (n=10) reveals circRNA transcripts for 47.9% of all highly expressed genes. While circRNA expression correlates globally with parental gene expression, we identify hematopoietic differentiation-associated as well as acute myeloid leukemia subgroup-specific circRNA signatures.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10−4). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

Use of Wilms Tumor 1 Gene Expression as a Reliable Marker for Prognosis and Minimal Residual Disease Monitoring in Acute Myeloid Leukemia With Normal Karyotype Patients.

Acute myeloid leukemia with normal karyotype (AML-NK) represents the largest group of AML patients classified with an intermediate prognosis. A constant need exists to introduce new molecular markers for more precise risk stratification and for minimal residual disease (MRD) monitoring. Quantitative assessment of Wilms tumor 1 (WT1) gene transcripts was performed using real-time polymerase chain reaction. The bone marrow samples were collected at the diagnosis from 104 AML-NK patients and from 34 of these patients during follow-up or disease relapse. We found that overexpression of the WT1 gene (WT1high status), present in 25.5% of patients, was an independent unfavorable factor for achieving complete remission. WT1high status was also associated with resistance to therapy and shorter disease-free survival and overall survival. Assessment of the log reduction value of WT1 expression, measured in paired diagnosis/complete remission samples, revealed that patients with a log reduction of< 2 had a tendency toward shorter disease-free survival and overall survival and a greater incidence of disease relapse. Combining WT1 gene expression status with NPM1 and FLT3-ITD mutational status, we found that the tumor behavior of intermediate patients (FLT3-ITD-/NPM1- double negative) with WT1high status is almost the same as the tumor behavior of the adverse risk group. WT1 expression status represents a good molecular marker of prognosis, response to treatment, and MRD monitoring. Above all, the usage of the WT1 expression level as an additional marker for more precise risk stratification of AML-NK patients could lead to more adapted, personalized treatment protocols.

A targeted next-generation sequencing in the molecular risk stratification of adult acute myeloid leukemia: implications for clinical practice.

Conventional cytogenetics can categorize patients with acute myeloid leukemia (AML) into favorable, intermediate, and unfavorable‐risk groups; however, patients with intermediate‐risk cytogenetics represent the major population with variable outcomes. Because molecular profiling can assist with AML prognosis and next‐generation sequencing allows simultaneous sequencing of many target genes, we analyzed 260 genes in 112 patients with de novo AML who received standard treatment. Multivariate analysis showed that karyotypes and mutation status of TET2,PHF6,KIT, and NPM1 mutation/FLT3‐ internal tandem duplication (ITD)negative were independent prognostic factors for the entire cohort. Among patients with intermediate‐risk cytogenetics, patients with mutations in CEBPA double mutation, IDH2, and NPM1 in the absence of FLT3‐ITD were associated with improved Overall survival (OS), similar to those with favorable‐risk cytogenetics; patients with mutations in TET2,RUNX1,ASXL1, and DNMT3A were associated with reduced OS, similar to those with unfavorable‐risk cytogenetics. We concluded that integration of cytogenetic and molecular profiling improves prognostic stratification of patients into three groups with more distinct prognoses (P < 0.001) and significantly reduces the number of patients classified as intermediate risk. In addition, our study demonstrates that next‐generation sequencing (NGS)‐based multi‐gene sequencing is clinically applicable in establishing an accurate risk stratification system for guiding therapeutic decisions.

A Comprehensive Panel of Molecular Mutations Notably Improves a Cytogenetic Prognostication System in Routine AML Diagnostics

Background: Based on sequencing studies the molecular landscape of AML has been unraveled. Novel prognostic scores combining molecular mutations and karyotype have been proposed (Grimwade et al. Blood, 2016, Döhner et al. NEJM 2015). However, these proposed classification systems differ in several aspects and yet no consensus has been established which genetic information is required for prognostication in AML today.Aims: 1) Test the prognostic value of a panel of molecular markers in addition to a cytogenetic score in a large cohort of AML patients. 2) Determine the proportion of patients with a suitable molecular marker for disease monitoring (MRD) applying molecular genetics.Patients and Methods: 867 de novo AML cases younger than 60 years were investigated (median age: 48 years, median follow up of 41 months). All patients were evaluated for karyotype, KMT2A-PTD, FLT3-ITD and in addition for mutation status of ASXL1, CEBPA, DNMT3A, NPM1, RUNX1 and TP53 according to the proposal by Grimwade et al. Blood 2016.Results: First, AML were classified according to the refined MRC cytogenetic classification with AML with t(15;17)/PML-RARA regarded as a separate group (n=89 (10%), 90% overall survival (OS) at 5 years). 89 cases (10%) were assigned to the favourable risk group (t(8;21)/RUNX1-RUNX1T1: n=42; inv(16)/t(16;16)/CBFB-MYH11: n=47), 570 (68%) to the intermediate risk group and 119 (14%) to the adverse risk group. OS at 5 years was 66%, 53% and 28%, respectively, and differed significantly between all four subgroups (for all comparisons p<0.001). Next, the following subgroups were separated: CEBPA double mutated (dm) cases (n=44 (5%); OS at 5 years: 83%), NPM1mut/FLT3-ITD- AML (n=181 (21%); OS at 5 years: 62%), and NPM1mut/FLT3-ITD+ AML (n=137 (16%); OS at 5 years: 47%; for all comparisons between these 3 groups p<0.001). Thus, prognosis of CEBPAdm cases was comparable to PML-RARA+ AML. OS in NPM1mut/FLT3-ITD- AML is comparable to CBF-leukemias. In NPM1mut AML no prognostic impact of DNMT3Amut was found. In all these 3 groups defined on molecular genetics no prognostic impact of additional karyotype information on OS was observed. Next, in the remaining cases of the cytogenetic intermediate risk group (n=209) the prognostic impact of mutations in ASXL1, DNMT3A, RUNX1, TP53, KMT2A-PTD and FLT3-ITD was evaluated. In multivariate Cox regression analysis mutations in TP53 (relative risk (RR): 3.5; p=0.04), ASXL1 (RR: 2.2, p=0.004), and FLT3-ITD (RR: 1.8; p=0.04) were independently associated with shorter OS. OS at 5 years was 25% in cases carrying at least one of these mutations compared to 54% in cases with none of these mutations (p=0.001). Within the adverse cytogenetic risk group cases with either a complex karyotype (n=27) or a KMT2A (n=25) or MECOM rearrangement (n=14) had the worst outcome compared to the remaining cases (OS at 5 yrs: 19% vs 54%, p=0.02). In the remaining cases the presence of at least one mutation in either ASXL1, TP53 or FLT3-ITD was associated with worse outcome (OS at 5 yrs: 33% vs 74%, p=0.04). Thus, AML with complex karyotype, KMT2A or MECOM rearrangements had the worst prognosis, while cases with adverse cytogenetics and at least one mutation in either ASXL1, TP53 or FLT3-ITD have a slightly better outcome which is comparable to AML with intermediate risk cytogenetics harbouring one of these mutations. OS in AML with adverse cytogenetics without mutations in ASXL1, TP53 or FLT3-ITD is not worse than in AML with intermediate cytogenetics also lacking these mutations.A fusion gene or a molecular mutation as target for MRD monitoring was present in 791 patients (91%) when all genes analysed were taken into account. If only markers showing prognostic relevance, i.e. fusion genes, mutations in NPM1, CEBPA, FLT3-ITD, ASXL1 and TP53 were considered a MRD marker was still available in 726 cases (84%).Conclusions: 1) In AML a prognostication system is feasible based on the identification of t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv(16)/t(16;16)/CBFB-MYH11, 11q23/KMT2A rearrangements, 3q26/MECOM rearrangements, complex karyotype, and mutation status of NPM1, CEBPA, ASXL1, TP53, and FLT3-ITD (figure 1). 2) The analysis of these parameters allows to identify an MRD marker in 84% of patients. 3) The analysis of additional genes may be required in a comprehensive AML work-up as soon as novel targeted treatment strategies will become available. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Absolute Quantification of Pre-microRNA-155 Copy Numbers By Digital Droplet PCR Identifies Acute Myeloid Leukemia (AML) Patients with Adverse Outcome

Abstract In recent years expression levels of several genes &amp; microRNAs (miR) were identified as strong prognostic markers, capable to refine AML risk stratification. So far technical difficulties, including the limitations of established methods for comparable, absolute quantification &amp; the lack of defined cut points prevented translation of these findings into clinical practice. Innovative digital droplet PCR (ddPCR) is a novel technique with high sensitivity, specificity that allows absolute quantification - without the need for standard curves - &amp; promises better inter-laboratory comparability. In AML pts, high miR-155 expression levels associate with the presence of FLT3-ITD &amp; independently predict inferior outcome. Here, for the first time we applied ddPCR for absolute quantification of pre-miR-155 to define an absolute cut point to discriminate between high &amp; lowexpressers, which was then validated in a second set of AML pts. We analyzed a homogeneous test set of 71 AML pts treated between January 2000 &amp; June 2012 in our institution. All pts received cytarabinebased induction therapies &amp; were consolidated with allogeneic hematopoietic stem cell transplantation (HCT) after non-myeloablativeconditioning (NMA; consisting of fludarabine[30mg / m² at days -4 to -2] &amp; 2Gy total body irradiation [day 0]). At NMA-HCT ptswere in first (n=43; 60.6%) or second complete remission (CR; n=16; 22.5%) or CR with incomplete recovery (n=12; 16.9%). At diagnosis, cytogenetics &amp; mutation status of the NPM1, CEBPA, IDH1, IDH2 &amp; DNMT3A gene &amp; presence of FLT3-ITD or FLT3-TKD mutation were assessed. The expression of the pre-miR-155 stem loop was measured using an EvaGreen-based ddPCR assay &amp; normalized to the absolute copy numbers of ABL1. The R Package OptimalCutpointswas used to determine a cut point of 1.104 copies pre-miR-155 per 100 ABL1 copies to discriminate between high (n =29; 40.8%) &amp; low (n =42; 59.2 %) miR-155 expressers. High miR-155 expressers, more often had a FLT3-ITD (p=.039) &amp; less frequently a mutation in the FLT3-TKD (p=.010). No significant association was found for other clinical or biological markers at diagnosis. In the test set, pts with more than 1.104 copies pre-miR-155 per 100 ABL1 copies at diagnosis had a significant shorter event-free survival (EFS; p=.03, figure 1A) &amp; overall survival (OS; p=.009, figure 1B). To validate these findings, we used a second set of 71 pts (median age 63.4y [range 37.1 to 74.7]) with a median follow-up of 3.7y for pts alive that all received cytarabinebased induction therapies &amp; NMA-HCT as consolidation. The ptsin the validation set also did not differ significantly in the analyzed clinical or molecular characteristics (i.e. white blood count, hemoglobin, platelets, blasts in bone marrow or peripheral blood at diagnosis, remission status at HCT [CR1 vs. CR2 vs. CRi], ELN genetic group, mutational status of FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 &amp; presence of FLT3-ITD). Using the determined cut point of 1.104 copies pre-miR-155 / 100 ABL1 copies in the test set, patients in the validation set were divided in 39 patients (54.9%) with a high miR-155 expression &amp; 32 (45.1%) with a low miR-155 expression. Pts with high miR-155 expression in the validation set had shorter EFS (p=.11, figure 2A) by trend &amp; a significant shorter OS (p=.05, figure 2B). In conclusion, ddPCRis a novel, feasible method that allows absolute quantification of miRexpression. We defined an absolute cut point of 1.104 copies pre-miR-155 per 100 ABL1 copies for the prognosticator miR-155 in AML without the need for standard curves. Pts with pre-miR-155 expression above the cut point had a significant shorter EFS &amp; OS. Remarkably, using a second clinically comparable set, we were able to validate our test set findings. Future studies are planned to confirm the clinical impact of pre-miR-155 expression levels at diagnosis, as well as the identified absolute pre-miR-155 / ABL1 copy number cut point to distinguish high from low miR-155 expressers. Figure 1 Test Set Figure 1. Test Set Figure 2 Validation Test Figure 2. Validation Test Disclosures Poenisch: Mundipharma: Research Funding.

High Expression of ZBTB7A at Diagnosis Associated with Inferior Outcome in Acute Myeloid Leukemia Patients Receiving Hematopoietic Stem Cell Transplantation

Introduction: The transcription factor ZBTB7A regulates early differentiation of hematopoietic progenitors & has been associated with oncogenic as well as oncosuppressive functions. While it was shown that ectopic overexpression of Zbtb7a in immature lymphocytes leads to the development of an aggressive T-cell lymphoblastic leukemia, high ZBTB7A expression in cytogenetically normal acute myeloid leukemia (CN-AML) is associated with improved outcomes. Furthermore, recently a leukemogenic cooperation between RUNX1/RUNX1T1 &ZBTB7A mutations in t(8;21)-associated AML was suggested.Here, we further evaluated the complex role of ZBTB7A expression in hematopoietic malignancies by assessing its potential prognostic impact in AML pts undergoing hematopoietic stem cell transplantation (HSCT) after non-myeloablative conditioning (NMA).Methods: We analyzed bone marrow (BM) at diagnosis of 140 pts (median age 63 years [y], range 37-75y) treated at our institution between 2000 & 2015. All pts received NMA conditioning (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) followed by HSCT in complete remission with (CR; n=111; 79.3%) or without peripheral hematological recovery (CRi; n=29; 20.7%). Median follow-up for pts alive was 3.5y.Our cohort included pts with CN-AML (n=62, 44.3%), complex karyotype (n=17; P=12.1%) & other cytogenetic abnormalities (n=61; 43.6%). At diagnosis mutations in the genes CEBPA, DNMT3A, IDH1, IDH2, NPM1 & the presence of FLT3-ITD were determined. In diagnostic BM cytogenetics were analyzed using standard techniques for banding & fluorescence in-situ hybridization & the expression of common surface markers was analyzed using flow cytometry. The expression of ZBTB7A was assessed using quantitative RT-PCR & normalized to ABL1 as internal control. As a cut-off the third quartile of normalized gene expression was identified to group high & low ZBTB7A expressers.Results: At diagnosis pts with a high ZBTB7A expression more often had a complex karyotype (P=.02) & were less likely to have core-binding factor AML by trend (P=.18). Additionally, high ZBTB7A levels associated with significantly fewer blasts in peripheral blood (P=.008) & BM (P=.02). The BM mononuclear cells in high ZBTB7A expressers were to a smaller extent positive for myeloid markers (CD38 P=.03; CD33 P=.11; CD13 P=.13) & exhibited a higher percentage of erythroid (Glycophorin A P=.03) as well as monocytic (CD11b P=.04; CD14 P=.01) surface markers. We did not find any statistical associations between ZBTB7A levels & the mutation status of NPM1, CEBPA, IDH1, DNMT3A or the presence of FLT3-ITD. Yet, there was a trend for more IDH2 mutations in the group of high ZBTB7A expressers (P=.18).At diagnosis a high expression of ZBTB7A associated with a significantly higher cumulative incidence of relapse (CIR; P=.002, Figure 1A). This finding also translated into a significantly shorter overall survival (OS; P=.01; Figure 1B) for AML pts with high ZBTB7A levels at diagnosis. When we restricted our analyses to CN-AML, high ZBTB7A expression remained a negative prognostic factor by trend (CIR P=.16; OS P=.11).Conclusion: Expression of ZBTB7A associated with distinct biological features & surface marker pattern in AML. This underlines the results of recent studies which identified ZBTB7A as a novel player in leukemogenesis. However, our findings are in contrast with the previously shown favorable prognostic impact of high ZBTB7A levels in a CN-AML cohort mainly treated with chemotherapy. In contract all pts included in our studies were consolidated with NMA-HSCT. Since this treatment regimen is mainly based on the graft versus leukemia effect a high ZBTB7A expression could potentially interfere with the immunological recognition of the AML blasts resulting in a reduced response to NMA-HSCT.Consequently, future functional studies & clinical trials should aim at further characterize the complex role of ZBTB7A in AML. [Display omitted] DisclosuresPoenisch:Mundipharma: Research Funding.

Evolutionary Patterns of Cytogenetically Normal Acute Myeloid Leukemia Correlate with Time to Relapse

Even though two-thirds of acute myeloid leukemia (AML) patients respond to induction chemotherapy and achieve complete remission (CR), the majority of these patients will eventually relapse. The time from CR to relapse is an important clinical indicator of disease aggressiveness, as patients relapsing within the first 6 months after initial diagnosis have a poorer prognosis in terms of response to salvage therapy and overall survival compared to patients with a later relapse. To learn about the evolution during the course of disease, we analyzed the somatic mutation patterns from initial diagnosis to relapse in 50 cytogenetically normal (CN) AML patients. Based on the ELN classification, 38% of the patients (n=19) were assigned as "favorable" at diagnosis, all other patients were classified as "intermediate-I". ELN classification was associated with time to relapse as "intermediate-I" patients relapsed earlier than "favorable" patients (median 9.3 months vs. 16.1 months, p=0.008, log-rank test).Somatic alterations were detected by exome sequencing and confirmed by targeted amplicon sequencing of matched diagnostic, remission and relapse samples. FLT3-ITD and NPM1 mutation status were obtained from routine diagnostic tests as the reliable detection of these markers by NGS remains challenging. The vast majority of somatic alterations were present both at diagnosis and at relapse, hereafter referred to as stable mutations (70%, Fig. 1A). All patients in our cohort had ≥1 stable mutation with DNMT3A being the most stably altered gene. In 47 out of 50 patients (94%), we observed mutations that were only found at diagnosis or only at relapse. Based on the mutation patterns, four distinct ’evolutionary’ subgroups of patients were defined (Fig. 1B): (I) patients with an identical mutation profile at diagnosis and at relapse ("stable", n=3, 6%), (II) patients who gained mutations at relapse ("stable + gain", n=24, 48%), (III) patients that lost mutations at relapse ("stable + loss", n=8, 16%), and (IV) patients with both loss and gain of mutations at relapse ("mixed", n=15, 30%). Mutations that were lost during the course of the disease were detected in e.g. PTPN11 or NRAS. Relapse-specific mutations were identified in e.g. IDH1/2, WT1, KPNB1 or KDM6A. Evolutionary subgroups showed differences in time to relapse (Fig. 1C). Patients with "stable + loss" relapsed earlier (median 4.1 months) than patients with gain of mutation at relapse (groups "stable + gain" and "mixed", median 12.2 months). All patients in the category "stable + loss" developed relapse within the first year after complete remission. The "stable" group of 3 patients showed an intermediate time to relapse (median 9.6 months), but was too small for a statistically valid comparison. Ultimately, the genetic evolution of CN-AML patients without gain of new mutations at relapse (categories "stable" and "stable + loss") was associated with significantly earlier relapse compared to patients that gained mutations at relapse (categories "stable + gain" and "mixed", Fig. 1D, p=0.001, log-rank test). Distinct predominant patterns of clonal evolution were observed in the ELN genetic groups, as only one patient of the "stable + loss" group was initially classified as "favorable". Interestingly, applying the ELN classification on relapse samples revealed a switch from "favorable" to "intermediate-I" in six patients, all with gain of mutations at relapse. This points towards more aggressive genetic profiles at relapse in these patients.The acquisition of mutations and/or the outgrowth of a resistant clone during/after chemotherapy might require a longer time or is per se associated with a longer time to relapse and a more favorable prognosis. Loss of mutations at relapse suggest the presence of two clones at diagnosis, with a chemotherapy resistant clone expanding after the eradication of a chemotherapy sensitive clone. As both clones share mutations and only the sensitive clone contains specific alterations, the resistant clone might be an ancestor of the sensitive clone.Taken together, in some patients the AML cells may require additional genetic alterations to become chemotherapy resistant, whereas in other patients the selective eradication of a sensitive clone is a potential mechanism underlying disease progression. Understanding the evolution of AML under selective pressure of chemotherapy is essential to cure or prevent AML relapse. [Display omitted] DisclosuresHiddemann:Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.

Impact of Distinct Genetic and Epigenetic Aberrations on Survival and Response in Acute Myeloid Leukemia Patients Receiving Epigenetic Therapy

Background: Treatment with hypomethylating agents such as decitabine, which results in complete and partial remission rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are no candidates for intensive chemotherapy. While clinical parameters such as reduced performance status, high leukocyte counts, elevated lactate dehydrogenase (LDH) levels as well as poor-risk cytogenetics are associated with lower response rates and shorter overall survival, there exists only limited data on molecular biomarkers that enables for selection of AML patients who are likely to benefit from epigenetic therapy. Shen et al. (JCO 2010) could show that distinct DNA methylation changes are prognostic for overall survival in myelodysplastic syndrome patients, however, response to decitabine therapy could not be predicted. Additionally, mutations in the epigenetic-modifier gene DNMT3A were recently reported to be associated with response and survival in AML patients treated with hypomethylating agents, however, while some of these studies revealed a positive association with outcome, other studies showed no association with response and overall survival (Metzeler et al., Leukemia 2012, DiNardo et al., Leuk Lymph 2014, Coombs et al., Blood 2015).Results: In order to contribute further knowledge regarding prognosis and response to hypomethylating therapy in AML patients, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331, Lübbert et al. Haematologica 2012) to identify potential molecular biomarkers. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, in our cohort patients harboring DNMT3A R882 mutations showed shorter overall survival (hazard ratio (HR): 2.15, 95%-confidence interval (CI): 0.91-5.12, p=0.08). Promoter DNA methylation analyses using pyrosequencing revealed shorter overall survival of patients with higher levels of methylation of ERα (HR: 1.50, CI: 0.97-2.32, p=0.07) and OLIG2 CpG4 (HR: 1.52, CI: 0.96-2.41, p=0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR: 1.74, CI: 1.03-2.96, p=0.04 and HR: 1.61, CI: 0.96-2.71, p=0.07, respectively) whereas the effect of DNMT3A R882 mutations was reduced to HR: 1.94 (CI: 0.79-4.73, p=0.15). In contrast, none of the investigated molecular markers was associated with response to treatment.Conclusion: In addition to the well established adverse prognostic clinical parameters such as patients’ age, reduced performance status and elevated LDH levels, we provide further evidence that pretreatment genetic and especially epigenetic analyses including DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status may be potential molecular biomarkers in AML patients undergoing epigenetic therapy. DisclosuresLübbert:Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding.

Prognostic Significance of IDH1 and IDH2 mutations in Older Patients with Primary Cytogenetically Normal Acute Myeloid Leukemia

IDH1 and IDH2 mutations are frequently identified in older patients with AML.With the development and application of IDH inhibitor, the prognostic significance of these mutations in old patients is of primary importance. To assess the impact on clinic outcome, we analyzed 224 older (> 60 years) patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) similarly treated with intensive induction chemotherapy. Our analysis identified IDH1 and/or IDH2 (IDH1/2), IDH1 or IDH2 mutations in 63(28%), 29(12.9%) and 35(15.6%) patients, respectively. In general, IDH1 and IDH2 mutations were mutually exclusive, with only one patient having both IDH1 R132C and IDH2 R140Q mutations. IDH2 R172 mutations were exclusive with all other mutations analyzed (NPM1, CEBPA, DNMT3A, IDH1 and FLT3-ITD). There was a strong interaction between IDH2 mutation and NPM1 mutational status. Concurrent presence of IDH2 R140 and NPM1 mutations was independently associated with longer overall survival (OS; P=0.038, HR=0.42) and event free survival (EFS; P=0.034, HR=0.46) compared to the wild type counterpart in multivariable analyses. In contrast, IDH1 mutations were independently associated with shorter OS (P=0.004, HR=1.98) and EFS (P=0.014, HR=1.72) in multivariable analyses. Notably, IDH1 R132 mutations harbored higher levels of total 2-hydroxyglutarate compared to IDH2 R140Q mutations. Patients with IDH1 mutations or IDH2 R140 and NPM1 mutations harbored also distinct miRNA-expression signatures. Future studies will establish whether these difference between IDH1 and IDH2 mutations will be relevant with respect to response of the respective subsets of patients to the emerging therapies with IDH1 and IDH2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

INPP4B is a Biomarker of Poor Prognosis in AML Which is Associated with EVI1 Overexpression and a LSC Signature

Acute myeloid leukemia (AML) is a highly heterogeneous cancer of the bone marrow. To better understand leukemogenesis and improve predictors of patient response to chemotherapy and overall survival (OS), we recently examined the role of inositol polyphosphate-4-phosphatase, type-II (INPP4B) in AML. Normally, INPP4B plays a role in PI3K/Akt signaling by regulating phosphorylation of phosphoinositides (PIs), critical membrane-bound second messenger molecules, by dephosphorylating the 4'-position of PI(3,4)P2 to generate PI(3)P. Because PI(3,4)P2, like PI(3,4,5)P3, is necessary for the activation Akt, INPP4B was first hypothesized and demonstrated to be a tumor suppressor protein, akin to PTEN, in several cancers including breast, prostate and ovarian. Recent work however, including our own study, has demonstrated a paradoxical tumor-promoting role of INPP4B in AML and estrogen receptor positive (ER+) breast cancer. Specifically, we demonstrated that INPP4Bhigh AML patients (25% of patients) had significantly shorter OS, lower response to induction therapy, and shorter event-free survival. Furthermore, INPP4B expression was found to be an independent prognostic marker for OS in AML outperforming FLT3-ITD and NPM1 mutation status. Overexpression of INPP4B in several AML cell lines results in enhanced colony formation potential, chemotherapy drug resistance, and increased proliferation.Though this previous work has shown that INPP4B plays a significant role in AML, it remains unclear why INPP4Bhigh AML differs from INPP4Blow AML, and what causes its upregulation. To address this question, we interrogated gene expression data from three independent datasets (n=942) to identify genes with differential expression between INPP4Bhigh and INPP4Blow AML. Gene expression analysis revealed that INPP4Bhigh AML was associated with differential expression of 233 genes. High INPP4B expression was associated with higher expression of genes related to the hematopoietic lineage, PI3K-Akt signaling, Jak-STAT signaling and ECM-receptor interaction pathways. Specifically, INPP4Bhigh AML has significantly higher expression of leukemic stem cell signature (LSC) genes CD34, RBPMS, GUCY1A3, KIAA0125, SOCS2, SPINK2, HTR1F, PPP1R16B, EVI1 (MECOM), DAPK1, BAALC, ABCB1, and PRKCH. Furthermore, INPP4B was found to be co-expressed with anti-apoptotic genes of the BCL2 family, namely BCL2 and BCL2L1. Further analysis revealed that INPP4B was co-expressed with the transcription factors EVI1, GATA2, NFATC2, ZEB1, GATA3 and ETS1, all of which having predicted binding sites within the INPP4B promoter region.Due to the observed enrichment of hematopoietic lineage/LSC genes in INPP4Bhigh AML, we wanted to validate the potential role of the EVI1 transcription factor in regulating INPP4B expression. Chromatin immunoprecipitation was used to demonstrate that EVI1 binding was enriched in the INPP4B promoter region of both EVI1high OCI/AML-4 and OCI/AML-6 cell lines. In addition, retroviral overexpression of EVI1 in EVI1low U937 cells resulted in subsequent upregulation of INPP4B transcript levels. Moreover, shRNA mediated knockdown of EVI1 in EVI1highOCI/AML-4 and UCSD-1 cells resulted in downregulation of INPP4B expression.Overall, our analysis reveals that INPP4Bhigh AML is characterized by upregulation of genes related to the hemotopoietic lineage, and LSC signature - consistent with the in vitro colony formation phenotype seen in INPP4B overexpressing AML cell lines. Furthermore, we demonstrate that one of the hematopoietic stem cell genes, EVI1 is a potentially key regulator of INPP4B expression in AML. DisclosuresJain:Roche Canada: Research Funding.

Clinical Impact of KMT2C and SPRY4 Expression Levels in Intensively Treated Younger Adult Acute Myeloid Leukemia Patients

Background:Recent publications suggest important roles of lysine methyltransferase 2C (KMT2C, located on 7q) and sprouty 4 (SPRY4, located on 5q) as candidate genes in leukemogenesis of acute myeloid leukemia (AML). The prognostic impact of the gene expression levels (ELs) of both genes on outcome in AML patients (pts) is currently unclear.Aim:To evaluate the prognostic impact of KMT2C and SPRY4 expression in correlation to clinical characteristics and genetic abnormalities assessed at diagnosis in a cohort of intensively treated adult AML pts.Methods: We retrospectively studied 268 AML pts (median age, 48 years; range, 17-60 years) who had been enrolled on 2 AML SHG trials (0295 and 0199, n=148; only normal cytogenetics pts (CN)) and the SAL-AML2003 trial (n=120; only abnormal cytogenetics pts (CA)). Acute promyelocytic and core-binding factor leukemia pts were excluded. Type of AML was de novo in 235 (88%), secondary in 19 (7%) and therapy-related in 14 (5%) of the 268 pts. Regarding baseline characteristics, CN pts had significantly higher white blood counts (WBC; p=0.001) and blast cells in peripheral blood (p=0.02) as compared to CA pts; all other factors were comparable. Cytogenetic analyses could be performed in 263 (98%) of the 268 pts. Cytogenetic risk classification according to ELN guidelines was intermediate-II in 55 (47%) and adverse in 63 (53%) of the CA pts, respectively. Abnormalities (abn) of 5q were present in 21 (18%) and abn of 7q in 16 (14%) of the CA pts. NPM1 and FLT3-ITD were analyzed in 145 (98%) of the CN pts. Of those, 59 (41%) were only NPM1 positive (pos), 12 (8%) were only FLT3-ITD pos, 34 (23%) were double pos and 40 (28%) were double negative (neg).KMT2C and SPRY4 ELs, normalized to ABL1and log2-transformed for analysis, were measured in triplets on cDNA obtained at diagnosis by RT-qPCR. Based on cDNA availability, KMT2C ELs could be analyzed in 143 (97%) of the CN and in all of the 120 CA pts, respectively. SPRY4 ELs could be measured in 30 (21%) of the CN and 107 (89%) of the CA pts, respectively.Results: KMT2C ELs were significantly lower in CN pts with de novo as compared to secondary AML (p= 0.02), whereas there was no difference in CA pts. No significant association was found for SPRY4 and type of AML. KMT2C ELs were significantly lower in FLT3-ITD pos as compared to FLT3-ITD neg CN pts (p=0.046), whereas there was no difference for SPRY4 ELs between the two groups (p=0.57). In addition, there was a significantly lower KMT2C expression in CN pts with intermediate-I risk as compared to NPM1 pos / FLT3-ITD neg pts (p=0.01). Regarding CA pts, there was no difference of KMT2C or SPRY4 ELs in adverse as compared to intermediate-II risk pts (p=0.08; p=0.20, respectively). When focusing on specific subgroups, KMT2C ELs were significantly lower in abn7q CA pts as compared to those without abn7q (p=0.002), whereas there was no difference of SPRY4 ELs in CA pts with or without abn5q (p=0.27). In univariate analysis higher SPRY4 ELs showed a significant favorable impact on relapse-free (RFS, p=0.03) and a trend towards a beneficial impact on overall survival (OS, p=0.06) for CA patients. A similar effect for KMT2C was not observed (RFS, p=0.96; OS, p=0.92). In subgroup analyses of pts with adverse risk cytogenetics, there was no impact of KMT2C or SPRY4 ELs on RFS (p=0.73; p=0.39) or OS (p=0.49; p=0.46), respectively. The same was true for FLT3-ITD pos CN pts (RFS, p=0.73; p=0.37; OS, p=0.91; p=0.36, respectively). In multivariate analyses on RFS and OS in CA pts including age, gender, KMT2C and SPRY4 ELs, logarithm of WBC, blast cells in bone marrow and cytogenetic risk group as variables, only higher age (OS, Hazard ratio (HR),1.28 per 10 years; 95%-confidence interval (CI): 1.02-1.59; p=0.03) and complex karyotype as compared to intermediate-II risk cytogenetics (RFS, HR: 2.25; 95%-CI: 1.20-4.22; p=0.01; OS, HR: 2.97; 95%-CI: 1.65-5.35; p<0.001) had an adverse impact. An effect of KMT2C or SPRY4 on RFS (p=0.84; p=0.16) or OS (p=0.85; p=0.45) was not found in the multivariate setting. In addition, in a multivariate model on CN pts (risk class according to NPM1 and FLT3-ITD mutational status instead of cytogenetic risk class) neither KMT2C nor SPRY4 had an impact on RFS (p=0.13; p=0.39, respectively) or OS (p=0.36; p=0.56, respectively).Conclusions:Lower KMT2C and SPRY4 ELs are associated with distinct genetic risk groups. An impact on prognosis was evident in univariable analyses for SPRY4 but not for KMT2C ELs in CA pts. DisclosuresKayser:Novartis: Consultancy. Platzbecker:Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Heuser:Tetralogic: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding; BerGenBio: Research Funding. Thiede:AgenDix: Employment, Other: Ownership.

Abstract 5225: CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing

Abstract Background According to the NCCN guideline, the prognosis of acute myeloid leukemia with normal karyotype (NK-AML) is classified based on the mutational status of NPM1, FLT3-ITD or CEBPA. However, patients with NK-AML without the above mutations, who are in intermediate risk, still show various clinical outcomes. Unfortunately, refractory/relapsed patients with intermediate risk NK-AML cannot be predicted before cytotoxic chemotherapy, yet. With the advent of next generation sequencing technology, we tried to reveal prognostic molecular marker in intermediate risk NK-AML, especially focusing on large structural variations (SVs). Methods A total of nine patients with intermediate risk NK-AML patients were included. Whole genome sequencing (WGS) was performed using a pair of tumor and germline DNA of the patients. Leukemic blasts were obtained from bone marrow aspiration specimens of each patient at the time of diagnosis. As a germline control, epithelial cells were collected from saliva of each patient at the time of complete remission. Then, genomic DNA was massively sequenced using HiSeq X10 system (Illumina Inc., San Diego, CA, USA). Possible SVs including inversion, duplication and translocation were identified with BreakDancer (Washington University School of Medicine, St. Louis, MO, USA), Pindel (The Wellcome Trust Sanger Institute, Cambridge, UK) and Delly (European Molecular Biology Laboratory, Heidelberg, Germany). Possible SVs were considered when they were recurrently identified from the three pipelines. Results This cohort consisted of 7 males and 2 females (median age = 55.6 years old). None of them had secondary AML. Eight patients were relapsed after complete remission and the other patient was refractory to 1st line induction chemotherapy. Through WGS, 25 inversions, 67 duplications and 10 translocations were identified in nine patients. Among them, 4 inversions, 13 duplications and 2 translocations were recurrently mutated. Especially, CDK11B, PTPRN2 and WDPCP were frequently duplicated more than 40% of the patients. Conclusions In this study, we performed WGS to investigate SVs of refractory/relapsed NK-AML patients with intermediate risk. Among various mutations, duplications of CDK11B, PTPRN2 and WDPCP were highly prevalent, so they could be a possible prognostic biomarker in NK-AML with intermediate risk. Further functional validation is required in order to clarify their roles. Citation Format: Jeonghwan Youk, Sunghoon Cho, Daeyoon Kim, Youngil Koh, Inho Kim, Murim Choi, Sung-Soo Yoon. CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5225.

Abstract 368: Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy

Abstract Introduction. Intensive induction chemotherapy in non-M3 young Acute Myeloid Leukemia (AML) patients is represented by the association of an antracycline and Cytarabine. Some treatment regimens including fludarabine or the addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, proved to give a benefit in terms of CR rates. Aims of the study. In a group of 49 patients treated with intensive chemotherapy, we evaluated chromosomal abnormalities with SNP 6.0 and Cytoscan HD (Affymetrix) in order to improve conventional cytogenetic analysis and discover novel chromosomic aberrations related to clinical data and therapy response. Patients and Methods. From 2001 to 2014, 489 patients were treated in our Institution. Among those, in 49 newly diagnosed AML patients (median age 54 (range 19-71)), SNP microarray based-genotyping were performed and then analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team. According to karyotype, FLT3 and NPM1 mutational status, 55.9% of the patients were considered at High Risk (HR) and 4.1% at low risk (LR). Ten patients had secondary AML. Patients were treated with induction schemes including MyFLAI, MyAIE, FLAI, FLAN, FLAG, 3+7 or DAE. Results. The CR rate after induction was 87.8% (43/49 patients). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 1/49. The median OS was 135 months, the 5-years OS in our patients was 55,1%. Patients treated with GO showed a non-statistical trend toward a better OS than patients treated with other regimens (median OS not reached vs 133 months, respectively). We explored the alterations found by SNP array in our patients searching for novel markers of therapy resistance. We found a median of 192,5 total copy number aberrations (range 72- 1071): a median of 145,5 total copy number aberrations in responding patients group (RPG), and a median of 361 total copy number aberrations (p = ns) in non-responding patients group (NRPG). We compared the frequency of detected aberrations in RPG and in NRPG with Fisher's exact test. We found that PIK3CA, Gain chr3:178,927,088-178,929,550 (p = 0,0016), SMAD4, Gain chr18:48,573,154-48,573,255 (p = 0,0166) and several other gene's loci (CASC18, TCF12, UTY, GRB10, ZFY) are significant aberrations in NRPG compared with RPG. Conclusions. We identified a number of genes with significant aberrations in NRPG, particularly PIK3CA, a protein-coding gene involved in cell proliferation and metabolic pathway with interaction with HRAS/KRAS and EGF, and SMAD4, a transcription factor activated by TGF-beta. Those 2 genes were found overexpressed in other solid tumors. We suppose that those genes may be involved in a hyper-proliferative pathway that underlies a mechanism of chemo-resistance. Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universit⁁ 2010-12 (L.Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Maria Chiara Fontana, Giovanni Marconi, Viviana Guadagnuolo, Giorgia Simonetti, Antonella Padella, Simona Soverini, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Silvia Lo Monaco, Marco Manfrini, Elisa Zuffa, Eugenia Franchini, Claudia Venturi, Maria Teresa Bochicchio, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Giovanni Martinelli. Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 368.