Lesions In Acute Myeloid Leukemia Research Articles

Ubtf tandem Duplications Define a Novel Subtype of Acute Myeloid Leukemia Associated with Younger Age, WT1 Mutations and HOXA9 Marked Overexpression

Background Upstream Binding Transcription Factor ( UBTF) gene plays an important role in ribosomal RNA transcription. UBTF tandem duplications (TD) have been recently described as a recurrent genetic lesion in acute myeloid leukemia (AML). UBTF-TDs appear to be mutually exclusive with other class-defining lesions in AML and display a unique comutational signature. UBTF-mutated AML affects predominantly younger patients and is associated with trisomy 8, internal tandem duplication of FLT3 ( FLT3-ITD), mutations in WT1 and poor prognosis. Moreover, HOX and MEIS genes seem to be overexpressed in AML with UBTF-TD similarly to NPM1-mutated ( NPM1m) or KMT2A-rearragend ( KMT2Ar) AML, suggesting potential sensitivity to menin inhibitors. In this study we sought to characterize UBTF-TDs in our AML cohort. Methods We studied 456 adult patients (median age 54 years; range 18-70) diagnosed with de novo AML and included in AML-12 protocol from CETLAM group. All patients from the 2012-2017 period (n=403) were consecutively studied and we also selected 53 cases of WT1-mutated AML from the 2017-2020 period, given the strong association with UBTF-TD. Screening for UBTF-TD was performed on bone marrow (BM) or peripheral blood (PB) genomic DNA samples. Exon 13 of UBTF gene was amplified and analysed by Polymerase chain reaction (PCR) and subsequent fragment length analysis by capillary electrophoresis. Mutated samples were confirmed through Sanger sequencing. Further, RNA from 9 UBTF-TD cases was available and HOXA9 and MEIS1 gene expression analysis was performed by Real-Time Polymerase Chain Reaction (RT-qPCR). Clinical and biological data were obtained from CETLAM registry. Results UBTF-TD was found in 13 patients from our cohort whose characteristics are detailed in Table 1. UBTF-TDs range from 45 base pairs (bp) to 365 bp, being the 48 bp the most recurring size. As described, although small UBTF-TDs are in frame insertions, the largest ones are not multiple of 3. Notably, most UBTF-TDs presented small insertions and deletions among the duplication. Presence of UBTF-TD was associated with younger age (36 vs 55 years; p<0.001) and BM dysplastic changes (80% of U BTFMUT). UBTF-TD was particularly frequent (8.9% of all cases) in patients below 40 years, compared to other lesions as CEBPA mutations (1.1%). Eleven (85%) U BTFMUT patients presented intermediate cytogenetics, being trisomy 8 (5), normal karyotype (3) and 9q deletion (2) the most recurrent findings. Two cases of adverse karyotype with MECOM-rearrangements were found. As previously described, there was a strong association between UBTF-TD and of FLT3-ITD (38.5%). Other recurrent genetic lesions in AML like NPM1 and CEBPA mutations or core-binding factor (CBF) rearrangements were exclusive of UBTF wild type patients. Further, we found that UBTFMUT leukemias showed marked overexpression of HOXA9 compared with other AML subtypes (22.1-fold vs 5.0-fold; p<0.001; Fig. 1) and similar to NPM1m AML (22.3-fold). On the other hand, MEIS expression did not differ significantly among groups. Conclusions In our cohort of adult AML patients, UBTF-TDs are found in 1.8% of cases and are especially frequent in younger individuals in absence of other class-defining genetic lesions. The presence of UBTF-TD is strongly associated with cytomorphological dysplasia and co-occurrence of FLT3-ITD and WT1 mutations, while NPM1 and CEBPA mutations and CBF rearrangements appear to be mutually exclusive. HOXA9 upregulation could be a leukemogenic event in UBTF-TD AML, as described in NPM1-m and KMT2A-r AML, suggesting potential utility of menin inhibitors in this subset of patients. Screening for UBTF-TD is going to be incorporated in our molecular diagnostic panel for new AML cases.

The Genomic Landscape and Its Clinical Implication in NPM1-Mutated AML Patients: A Study within the AMLSG 09-09 Clinical Trial

Background: Mutations in the Nucleophosmin 1 gene ( NPM1 mut ) represent one of the most common genetic lesions in acute myeloid leukemia (AML).Based on its characteristic clinico-pathologic features, NPM1 mutAML has been recognized as a distinct entity among the category “AML with recurrent genetic abnormalities”. According to the ELN 2022 genetic risk-stratification, NPM1 mut AML, in the absence of FLT3-ITD mutation, is associated with a favorable prognosis. However, a significant proportion of these patients (pts) relapse after intensive therapy suggesting that other co-mutations may have an impact on outcome. Aims: To comprehensively characterize the genomic landscape and leukemogenic trajectoriesin a large cohort of NPM1 mut AML pts and to investigate its prognostic and predictive impact on outcome. Methods: Targeted DNA sequencing (mean read depth: 1817) on the entire coding region of 263 genes was performed in 568 NPM1 mut AML pts (median age: 58.7 years; 18-60 years, n=317; >60 years, n=251). All pts were enrolled in the randomized open-label Phase 3 AMLSG 09-09 trial [NCT00893399; Döhner H et al. Lancet Haematol 2023]. In this trial, pts were assigned to intensive chemotherapy plus all- trans retinoic acid with or without gemtuzumab ozogamicin; none of the pts received midostaurin. Results: In total n=3,058 variants (variant allele frequency of ≥1%) were identified in 195/263 genes. The median number of co-mutations was 3 (range 0-11). The most common co-mutated genes were DNMT3A (49.5%), FLT3-TKD (42.8%) PTPN11 (24.8%), NRAS (22.7%) TET2 (21.7%), IDH2 (21.3%), IDH1 (18%), and FLT3-ITD (17.3%). DNMT3AR882 hotspot mutations occurred more frequently in younger pts (36.6% vs 17.1%), while there was no difference for DNMT3AnonR882 mutations between the two age groups (21.8% and 21.9%). An age-dependent difference was also identified for mutations in myelodysplasia-related genes ( ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2) as defined by the ICC, occurring with a higher incidence in older pts (30.3% vs 12.3% in younger pts). Analyzing the mutational pattern of co-mutations, we found statistically significant tertiary gene-gene interactions: e.g., NPM1- NFE2- STAG2 (p<.001), NPM1- IDH2- SRSF2 (p<.001), NPM1- CEBPA- TET2 (p<.001), NPM1- DNMT3AR882- NRAS (p=.002), and NPM1- ASXL1- SRSF2 (p=.004); mutual exclusivities were identified for NPM1- DNMT3AR882- DNMT3AnonR882 (p<.001), NPM1- IDH2- TET2 (p<.001), NPM1- DNMT3AR882- SRSF2 (p<.001), NPM1- IDH1- TET2 (p<.001), and NPM1- FLT3-ITD- KRAS (p<.002). Correlating co-mutation data with outcome, we found that DNMT3AR882 hotspot mutations confer inferior event-free (EFS) and overall survival (OS) only in younger pts (EFS, p<.001 vs p=.11, Figure 1a; OS, p=.003 vs p=.8), whereas DNMT3AnonR882 mutations did not impact prognosis within the two age groups. We also found a negative prognostic impact of IDH1 mutations which was restricted to younger pts (EFS, p=.05), whereas IDH2 mutations were associated with superior EFS in older pts (p=.04) and superior OS in both groups (p=.05 and p=.03). Of note, co-mutations occurring in one or more of the myelodysplasia-related genes did not impact EFS or OS (Figure 1b). In multivariable analysis (all pts) including age, WBC, LDH, allogeneic transplantation in CR1, and mutations with an incidence of at least 3% as covariables, age (HR,1.03; p<.001), DNMT3AR882 (HR, 1.86; p<.001), FLT3-ITD (HR, 1.54; p=.012), IDH1 (HR, 1.48; p=.009), MYC (HR, 1.83; p=.032), and WT1 (HR, 1.73; p=.012), were associated with an inferior EFS, while SMC3 mutation showed favorable EFS (HR, 0.44; p=.019). To further study the leukemogenic trajectories, we used an oncogenetic tree modeling algorithm, which yielded a tree with several main branches including DNMT3AR882, DNMT3AnonR882, FLT3-TKD , IDH1, IDH2, PTPN11, and TET2. These mutations might represent initiating events which predispose to additional events with further distinct branches. Conclusions: Our study provides comprehensive data on the genomic landscape and its clinical impact in pts with NPM1mut AML fit for intensive chemotherapy. The co-mutational pattern clearly differs between younger and older NPM1 mutAML pts. Using this large dataset allowed the identification of secondary and tertiary gene-gene interactions with significant impact on outcome. Further data analysis is ongoing and will be presented at the meeting.

Molecular and Clinical PHF6 Mutant Myeloid Neoplasia Provides Clues As to Their Pathogenesis and Therapeutic Targeting

Congenital PHF6 mutations (PHF6MT) cause the X-linked neurodevelopmental disorder, Börjeson-Forssman-Lehmann syndrome (BFLS). In contrast, acquired PHF6MT are frequently detected in hematologic neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL). Indeed, PHF6 is highly expressed in brain/developing CNS as well as in hematopoietic subpopulations suggesting its role in neurogenesis and hematopoiesis consistent with the neurologic BFSL presentation of the germline and leukemias with somatic mutations of this gene. This study focuses on involvement of PHF6 lesions in AML. Here, we hypothesize that there are functional links between lymphoid and myeloid neoplasia (MN) with PHF6-deficiency that can be instructive in terms of the impact of this gene as a tumor suppressor gene (TSG). In total, we analyzed sequencing results of 6886 AML cases, including 616 cases from open sources such as The Beat AML Master dataset (Tyner JW , Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-531). We first investigated the clinical phenotype associated with PHF6MT. We detected a total of 111 (1.6% of cases) PHF6MT. While frameshifts were found across all the coding regions, missense and stop-gains were concentrated in the two PHD-type zinc finger domains and were overlapping between other MN and the previously reported T-ALL cases. PHF6MT were significantly more common in male cases (2.3 vs. 0.8%; p<.001) similar to other X-genes such as UBA1, ZRSR2, and STAG2 but different from ATRX, PIGA, and KDM6A (UTX). When comparing primary AML (pAML) with secondary AML (sAML), sAML had more PHF6MT than pAML (2.9 vs. 1.4%; p<.001). While PHF6MT were not associated with chromosomal abnormalities in male cases, female cases with X chromosome deletion (delX) showed higher frequency of PHF6MT compared to females with diploid X chromosome (6.5 vs. 0.8%; p=.0019). Consequently, when delX were included, a total of 2.3% of female patients had PHF6 hits with 10% of them in a homo/hemizygous configuration. While RUNX1, U2AF1, and ASXL1 mutations coincided with PHF6 hits in males, in female cases, delX and ASXL1 mutations were the most common associated defects (Fig.1). Interestingly, RUNX1 mutations (RUNX1MT) in females were enriched in cases with delX. As a result, PHF6MT were enriched into two distinct subclasses: while PHF6MT were enriched in male cases with sAML and RUNX1MT (8.8%), all female cases with sAML, RUNX1MT, and delX also acquired PHF6MT (Fig.2). A higher frequency of PHF6MT in advanced MN than early diseases (e.g., low-risk MDS), suggestive of a secondary rather than a founder role of PHF6MT prompted us to study the clonal architecture of PHF6MT cases using variant allelic frequencies. RUNX1MT were dominant/co-dominant to PHF6 hits in the clonal evolution of AML in contrast to T-ALL, in which PHF6MT appears to be an earlier event. Similar relationship was found with regard to PHF6 hits and other myeloid gene mutations (e.g., ASXL1, DNMT3A, or SRSF2) suggesting a lineage-restricted TSG function of PHF6 in AML following myeloid commitment (consistent with the physiologic expression of PHF6 in pre-B, pre-T, and erythroid progenitors during hematopoietic ontogeny). PHF6 mutations also affected AML patients’ outcome. Overall, PHF6MT showed worse outcome in male cases, but not in female cases. However, if PHF6MT cases also had RUNX1 mutations, the outcome was extremely low in both sexes. Unsupervised RNAseq analysis of PHF6 mutational status vs. expression pattern revealed that PHF6MT cases showed higher mRNA levels of several lymphoid genes (GCSAM, LY9, or BLNK). This finding was also confirmed by RNAseq of in-house PHF6 knock-out K562 and U-937 clones. Immunohistochemistry unveiled that LY9 was positive in illustrative PHF6MT cases and flow cytometry revealed the presence of lymphoid markers in 2/3 PHF6MT cases. Our study confirms that PHF6MT are more common in male AML. Also we demonstrate the intricate relationships between PHF6MT and RUNX1MT in male sAML patients which extend also to female cases with delX. In AML, PHF6MT are a late event compared to T-ALL, in which they occur early in leukemogenesis. This is also in line with higher frequency of PHF6MT in sAML. Some PHF6MT cases have higher expression of lymphoid genes, which may be candidate for molecular targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract P195: Omacetaxine mepessucinate plus venetoclax show strong synergistic anti-leukemic activity in nucleophosmin (NPM1)-mutated AML patient-derived xenograft (PDX) models to support a phase 1 clinical trial

Abstract Despite different novel targeted drugs against specific genetic lesions have been developed for acute myeloid leukemia (AML) treatment, therapy in many AML subtypes should still rely on drugs possibly targetting alternative pathways essential for leukemia survival. Different biological and clinical evidences point to nucleophosmin (NPM1) gene mutations (the most frequent genetic lesion in AML) among strong predictors of sensitivity to venetoclax, a small molecule drug inhibitor of the B-cell leukemia/lymphoma-2 (Bcl-2) anti-apoptotic protein, highly expressed in most AML. Venetoclax appeared to be extremely efficacious in combination with hypomethylating agents (HMA) as first-line therapy, particularly in NPM1-mutated AML. Omacetaxine mepessucinate is a synthetic form of the plant cephalotaxine alkaloid homoharringtonine (HHT). HHT binds to the A-site cleft of ribosomes preventing the initial elongation step of protein synthesis and leading to a transient but profound inhibition of protein synthesis especially of those with a short half-life, including the antiapoptotic protein myeloid cell leukemia-1 Mcl-1, acting as mechanism of resistance following Bcl-2 inhibition. Besides its action as ‘Mcl-1 inhibitor’ (targeting its protein levels), we focused on HHT because i) it is a FDA-approved drug, in the setting of chronic myeloid leukemia (CML); ii) it showed some activity in clinical trials in AML; iii) it was shown to be a safe treatment, when evaluated also in association with chemotherapy, either standard intensive or low-dose regimen, in AML; iv) it is administered subcutaneously in its FDA-approved formulation; v) it has never been used in combination with venetoclax. Here, we sought to explore the therapeutic potential of omacetaxine mepessucinate and venetoclax in pre-clinical in vivo models, and used 2 different NPM1-mutated AML PDX (one, NPM1/FLT3-ITD double mutant; the other, DNMT3A/NPM1/FLT3-ITD triple mutant) edited to express luciferase allowing to track the disease by in vivo bioluminescence imaging (BLI) during treatment and evaluate drug response. HHT was given at 1 mg/Kg by subcutaneous administration for 2 consecutive weeks (5 days/week) and venetoclax at 100 mg/kg by gavage for 4 weeks (5 days/week), in cycles of 28 days. The anti-leukemic activity of the treatment was measured by evaluating AML growth/tissue infiltration on sacrificed animals by immunohistochemistry or flow cytometry and in vivo by BLI. Strikingly, on overt disease combinatorial HHT and venetoclax treatment resulted in a strong and synergic anti-leukemic effect with no leukemia detection in a cohort of mice sacrificed and analyzed after the first cycle, and in a significant advantage in a cohort analyzed for survival compared with single-drug or vehicle-treated animals. These data gave the support to design a pilot phase 1 clinical trial aimed at assessing safety and preliminary efficacy of omacetaxine mepessucinate plus venetoclax in relapsed/refractory NPM1-mutated AML patients. The trial is now open to patients accrual at our Hematology department. Citation Format: Federica Mezzasoma, Valeria Cardinali, Ilaria Gionfriddo, Francesca Milano, Sofia Sciabolacci, Alessio Ferrari, Marcella Sabino, Serenella Silvestri, Serena Donini, Valentina Tini, Giulio Spinozzi, Brunangelo Falini, Maria Paola Martelli. Omacetaxine mepessucinate plus venetoclax show strong synergistic anti-leukemic activity in nucleophosmin (NPM1)-mutated AML patient-derived xenograft (PDX) models to support a phase 1 clinical trial [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P195.

FLT3-ITD DNA allelic burden, but not mRNA levels, influences the biological characteristics of AML patients

Abstract FMS-like tyrosine kinase 3 gene internal tandem (FLT3-ITD) mutations represent one of the most frequent genetic lesions in acute myeloid leukemia (AML) and imparts a negative prognostic. For an optimal patient management, current clinical guidelines recommend the evaluation of the allelic ratio (AR), expressed as the DNA FLT3-ITD/WT mutational burden. We sought to evaluate the differences between the AR and FLT3-ITD/WT mRNA ratio (RR) and their respective impact on the biological characteristics of AML patients. A total of 32 DNA and mRNA samples from AML patients with FLT3-ITD were evaluated. There was a good correlation between the AR and RR (Spearman’s rho= 0.652, P &lt;0.001). None of the biological characteristics were influenced by the RR values, whereas patients with high AR values (≥0.5) had higher WBC counts (Mann-Whitney, P= 0.01), LDH levels (Mann-Whitney, P= 0.037), and circulating blasts levels (Mann-Whitney, P= 0.023) than patients with low AR values (&lt;0.5). Also, there was a good correlation between AR values and WBC count (Spearman’s correlation, P= 0.001), and LDH levels (Spearman’s correlation, P= 0.007). In our study population the AR, but not the RR, influenced the biological characteristic of patients suggesting a dose-independent effect of FLT3-ITD mutations.

Survival differences and associated molecular signatures of DNMT3A-mutant acute myeloid leukemia patients

Acute myeloid leukemia (AML) is a very heterogeneous and highly malignant blood cancer. Mutations of the DNA methyltransferase DNMT3A are among the most frequent recurrent genetic lesions in AML. The majority of DNMT3A-mutant AML patients shows fast relapse and poor survival, but also patients with long survival or long-term remission have been reported. Underlying molecular signatures and mechanisms that contribute to these survival differences are only poorly understood and have not been studied in detail so far. We applied hierarchical clustering to somatic gene mutation profiles of 51 DNMT3A-mutant patients from The Cancer Genome Atlas (TCGA) AML cohort revealing two robust patient subgroups with profound differences in survival. We further determined molecular signatures that distinguish both subgroups. Our results suggest that FLT3 and/or NPM1 mutations contribute to survival differences of DNMT3A-mutant patients. We observed an upregulation of genes of the p53, VEGF and DNA replication pathway and a downregulation of genes of the PI3K-Akt pathway in short- compared to long-lived patients. We identified that the majority of measured miRNAs was downregulated in the short-lived group and we found differentially expressed microRNAs between both subgroups that have not been reported for AML so far (miR-153-2, miR-3065, miR-95, miR-6718) suggesting that miRNAs could be important for prognosis. In addition, we learned gene regulatory networks to predict potential major regulators and found several genes and miRNAs with known roles in AML pathogenesis, but also interesting novel candidates involved in the regulation of hematopoiesis, cell cycle, cell differentiation, and immunity that may contribute to the observed survival differences of both subgroups and could therefore be important for prognosis. Moreover, the characteristic gene mutation and expression signatures that distinguished short- from long-lived patients were also predictive for independent DNMT3A-mutant AML patients from other cohorts and could also contribute to further improve the European LeukemiaNet (ELN) prognostic scoring system. Our study represents the first in-depth computational approach to identify molecular factors associated with survival differences of DNMT3A-mutant AML patients and could trigger additional studies to develop robust molecular markers for a better stratification of AML patients with DNMT3A mutations.

Prognostic Impact of a Composite Genetic Profile Defined By Cytogenetics and Next Generation Sequencing at Diagnosis on Treatment Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation in Acute Myeloid Leukemia

Prognostic Impact of a Composite Genetic Profile Defined By Cytogenetics and Next Generation Sequencing at Diagnosis on Treatment Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation in Acute Myeloid Leukemia

Using singscore to predict mutation status in acute myeloid leukemia from transcriptomic signatures.

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.

Using singscore to predict mutation status in acute myeloid leukemia from transcriptomic signatures

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.

Using singscore to predict mutation status in acute myeloid leukemia from transcriptomic signatures

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.

Using singscore to predict mutations in acute myeloid leukemia from transcriptomic signatures.

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.

CLI24-001: First-in-human study of SEL24/MEN1703, an oral dual PIM/FLT3 kinase inhibitor, in patients with acute myeloid leukemia.

TPS7062 Background: FLT3-ITD is one of the most common genetic lesions in acute myeloid leukemia (AML). PIM kinases are oncogenic FLT3-ITD targets expressed in AML cells and increased PIM kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. In addition, inhibition of PIM kinases restores sensitivity to FLT3 inhibitors and dual FLT3/PIM inhibition eradicates FLT3-ITD+ cells including primary AML cells. SEL24/MEN1703, a potent PIM/FLT3 dual inhibitor, demonstrates a significantly broader spectrum of activity in AML cell lines and primary AML blasts, irrespective of FLT3 status, compared to monotherapy with either FLT3 or PIM inhibitors such as quizartinib or AZD1208. Methods: CLI24-001 is a First in Human, open label, non-randomized, multi-center, Phase I/II dose-escalation and cohort expansion study of SEL24/MEN1703 in AML patients (excluding APL) not suitable for chemotherapy. SEL24/MEN1703 is given orally, QD, for 14 days in a 21-day cycle with cycles repeated until disease progression or unacceptable toxicity. Dose escalation follows a 3+3 design to identify the recommended phase 2 dose (RP2D). In the phase 2 part/cohort expansion, subjects will receive SEL24/MEN1703 at the RP2D, to further investigate the safety profile and signs of antileukemic activity. In both study parts, patients are eligible regardless of mutational status and/or prior exposure to FLT3 inhibitors; prior treatment with PIM inhibitors is not allowed. Main inclusion criteria comprise a white blood count (WBC) of ≤30 x 109/L (hydroxyurea/leukoapheresis permitted to lower WBC). Key secondary objectives include pharmacokinetics (PK) and single agent efficacy. The study is enrolling at 5 US sites and will be extended, both in US and EU, in the cohort expansion part. This is the first trial testing a dual PIM/FLT3 inhibitor with the potential to be active in AML regardless of FLT3 status andwith a potential to overcome FLT3 inhibitor resistance. (Sci Adv. 2015;1:e1500221; Oncotarget. 2018 Mar 30;9(24):16917-16931) Clinical trial information: NCT03008187.

Impact of Genetic Profile on Clinical Outcomes in Adults ≥60 with AML: The Princess Margaret Cancer Centre Experience

Acute myeloid leukemia (AML) is a clinically and biologically heterogeneous disease. Traditionally, cytogenetic analysis has been the backbone for prognostication and treatment decisions. Outcomes vary between age groups with older adults generally having a poorer prognosis. Next Generation Sequencing (NGS) has expedited the discovery of novel genetic lesions in AML to better predict response to intensive chemotherapy and overall survival (OS).The aims of our study were to describe the genetic profile of older adults with AML and to determine its impact on treatment response and survival.We included all new patients with a diagnosis of AML (≥20% blasts in peripheral blood or bone marrow; acute promyelocytic leukemia and myeloid sarcoma were excluded), treated at Princess Margaret Cancer Centre between February 2015 and August 2017.NGS was performed on DNA isolated from peripheral blood or bone marrow samples at diagnosis. Analysis was performed using the TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA) on the MiSeq benchtop genome sequencer (Illumina). Of the 54 genes included in the panel, the complete coding region was sequenced in 15, with hotspot region coverage provided for 39.Demographic and clinical data were obtained from the Princess Margaret Cancer Centre Registry.We identified 454 patients with a new diagnosis of AML in whom frontline NGS was performed. Of these, 300 were 60 years (range 60-92) or older. Demographic and clinical data are shown in Table 1. The median age overall at presentation was 67 yrs (range 18-92) and 57% were male. de novo AML was diagnosed in 329 patients (72%), secondary AML in 17%, and therapy related myeloid neoplasm in 11%. Secondary AML (sAML) was more frequently seen in the ≥60 vs the <60 population (21% vs 8%). Cytogenetic risk stratification by MRC was favorable in 6%, intermediate in 51%, and adverse in 27%. Unsuccessful cytogenetic testing occurred in 12%.In total 283 patients (62%) received intensive chemotherapy (95% in the younger group and 45% in the older cohort). 216 patients (76%) achieved a response (CR/CRi/morphologic leukemia free state). No statistically significant difference was seen between age groups (P=0.73)NGS detected 1655 variants. Of these, 1353 classified as oncogenic and 302 as variants of unknown significance (the latter were excluded from subsequent analysis). 14 patients had no mutations identified by NGS.Forty three genes were recurrently mutated in the study cohort: 36 were mutated in >1% of patients, and 11 genes were mutated in >10% of patients. The most commonly mutated genes included DNMT3A (25%), NPM1 (21%), TET2 (19%), RUNX1 (16%), TP53 (16%), ASXL1 (15%), IDH2 (15%), SRSF2 (15%), NRAS (12%), and IDH1 (11%). Older patients had a greater number of mutations overall compared to younger adults (median: 3 vs 2; P = 0.001). In total 986 oncogenic variants were identified in the older group (median 3, range 1-12), while 367 oncogenic variants (median 0, range 0-2) were present in patients < 60 years old (P < 0.001). The older group more commonly harbored mutations in TET2 (25 vs 10%), ASXL1 (21 vs 4%), RUNX1 (20 vs 8 %), SRFS2 (20 vs 6 %), STAG2 (11 vs 6%) and U2AF1 (8 vs 2%).Patients with PTPN11 and NPM1 mutations had longer OS, while patients carrying mutations in ASXL1, JAK2, RUNX1, TP53 and SRSF2 had a shorter OS (Table 2). No differences in OS between age groups (P= 0.2). Multivariate Cox regression analysis showed that male sex (P= 0.0057), sAML (P<0.001), MRC adverse cytogenetic analysis (P<0.002), ASXL1 mutations (P= 0.071) and TP53 mutations (P= 0.089), were associated with shorter OS (Table 3).We then analyzed the correlation between mutations and response rate in patients receiving intensive treatment. PTPN11 and NPM1 mutations were associated with a greater likelihood of achieving a response, while patients with mutations in BCOR, TP53, SF3B1 and U2AF1 had a lower chance of response. Multivariate Cox regression analysis of mutations known to affect response did not show any differences by age, gender, or diagnosis. Mutations in BCOR (P= 0.045), TP53 (P= 0.032) and U2FA1 (P=0.019) were significantly associated with primary induction failure.Overall, older AML patients harbored a greater number of mutations than did their younger counterparts. No age-related differences were observed in mutations known to affect response and/or OS. TP53 mutations, adverse cytogenetics and sAML, were poor prognostic factors regardless of age. [Display omitted] DisclosuresSchimmer:Medivir AB: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Yee:Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding; Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding. Maze:Novartis: Consultancy, Honoraria. Schuh:Novartis: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Teva: Consultancy; Celgene: Consultancy; Amgen Inc.: Consultancy; Shire: Consultancy; Jazz: Consultancy.

GRP94 rewires and buffers the FLT3-ITD signaling network and promotes survival of acute myeloid leukemic stem cells.

Internal tandem duplications in the tyrosine kinase receptor FLT3 (FLT3-ITD) are among the most common lesions in acute myeloid leukemia and there exists a need for new forms of treatment. Using ex vivo drug sensitivity screening, we found that FLT3-ITD+ patient cells are particularly sensitive to HSP90 inhibitors. While it is well known that HSP90 is important for FLT3-ITD stability, we found that HSP90 family members play a much more complex role in FLT3-ITD signaling than previously appreciated. First, we found that FLT3-ITD activates the unfolded protein response, leading to increased expression of GRP94/HSP90B1. This results in activation of a nefarious feedback loop, in which GRP94 rewires FLT3-ITD signaling by binding and retaining FLT3-ITD in the endoplasmic reticulum, leading to aberrant activation of downstream signaling pathways and further inducing the unfolded protein response. Second, HSP90 family proteins protect FLT3-ITD+ acute myeloid leukemia cells against apoptosis by alleviating proteotoxic stress, and treatment with HSP90 inhibitors results in proteotoxic overload that triggers unfolded protein response-induced apoptosis. Importantly, leukemic stem cells are strongly dependent upon HSP90 for their survival, and the HSP90 inhibitor ganetespib causes leukemic stem cell exhaustion in patient-derived mouse xenograft models. Taken together, our study reveals a molecular basis for HSP90 addiction of FLT3-ITD+ acute myeloid leukemia cells and provides a rationale for including HSP90 inhibitors in the treatment regime for FLT3-ITD+ acute myeloid leukemia.

Gene Mutations as Emerging Biomarkers and Therapeutic Targets for Relapsed Acute Myeloid Leukemia

It is believed that there are key differences in the genomic profile between adult and childhood acute myeloid leukemia (AML). Relapse is the significant contributor of mortality in patients with AML and remains as the leading cause of cancer death among children, posing great challenges in the treatment of AML. The knowledge about the genomic lesions in childhood AML is still premature as most genomic events defined in children were derived from adult cohorts. However, the emerging technologies of next generation sequencing have narrowed the gap of knowledge in the biology of AML by the detection of gene mutations for each sub-type which have led to the improvement in terms of prognostication as well as the use of targeted therapies. In this review, we describe the recent understanding of the genomic landscape including the prevalence of mutation, prognostic impact, and targeted therapies that will provide an insight into the pathogenesis of AML relapse in both adult and childhood cases.

Abstract 4087: Development of a potent, dual pan-PIM/FLT3 inhibitor for the treatment of heme malignancies

Abstract Despite huge effort spent on understanding the pathogenesis of acute myeloid leukemia (AML), current standards of care are still based on the same chemotherapy agents as two decades ago - combinational treatment of cytarabine with an anthracycline. Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in AML. Although FLT3 inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. Selvita has developed a potent and selective first-in-class, dual PIM/FLT3 kinase inhibitor, the SEL24-B489 compound, and profiled its activity for in vitro and in vivo AML models showing significantly broader anti-tumor activity of SEL24-B489 than selective FLT3-ITD or PIM inhibitors. We compared SEL24-B489 head-to-head with a selective PIM inhibitor (AZD1208) and a selective FLT3-ITD inhibitor (AC220) in a panel of AML cell lines with FLT3-ITD or unmutated kinase (FLT3-WT) as well as peripheral AML cells and CD34+ bone marrow blasts. SEL24-B489 exhibited a significantly broader activity, irrespective of FLT3 status, than either of the selective inhibitors. Since PIM kinases have emerged as important mediators of FLT3-inhibitor resistance, we hypothesized that the dual specificity of SEL24-B489 might overcome the phenotype of resistance. We utilized previously developed MOLM-14 cells transduced with either FLT3-WT or FLT3 alleles containing TKD point mutations to show that neither of the these mutations decreased the cellular sensitivity to SEL24-B489. Higher cellular activity and biomarker response of SEL24-B489 than competitive inhibitors was shown by inhibition of specific biomarkers such as S6 and STAT5 phosphorylation at nanomolar concentrations in both FLT3-ITD positive and FLT3-WT cell lines in vitro. We have also demonstrated SEL24-B489 superior potency of SEL24-B489 in xenograft models in vivo. Consistent with the experiments in vitro showing marked synergy between SEL24-B489 and AraC, a combination of these agents resulted in almost completely blocked tumor growth in vivo. Most importantly, SEL24-B489 has been selected as a clinical candidate and is currently in phase I clinical trials. Citation Format: Wojciech Czardybon, Renata Windak, Aniela Gołas, Michał Gałęzowski, Aleksandra Sabiniarz, Izabela Dolata, Magdalena Salwińska, Paweł Guzik, Magdalena Zawadzka, Ewelina Gabor-Worwa, Bożena Winnik, Małgorzata Żurawska, Ewa Kolasińska, Ewelina Wincza, Marta Bugaj, Monika Danielewicz, Grzegorz Dubin, Ewa Jabłońska, Maciej Szydłowski, Tomasz Sewastianik, Bartosz Puła, Anna Szumera-Ciećkiewicz, Monika Prochorec-Sobieszek, Elżbieta Mądro, Ewa Lech-Marańda, Krzysztof Warzocha, Jerome Tamburini, Przemysław Juszczyński, Krzysztof Brzózka. Development of a potent, dual pan-PIM/FLT3 inhibitor for the treatment of heme malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4087. doi:10.1158/1538-7445.AM2017-4087

A Machine-Learning Analysis Suggests That FLX925, a FLT3/CDK4/6 Kinase Inhibitor, Is Potent Against FLT3-Wild Type Tumors Via Its CDK4/6 Activity

Fms-like tyrosine kinase 3 (FLT3) is universally expressed in Acute Myeloid Leukemia (AML) blast cells (Zheng R, et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. Blood. 2004) and activating mutations of FLT3 are amongst the most common genetic lesions in AML (Levis M. FLT3 mutations in acute myeloid leukemia: what is the best approach in 2013? ASH Education Program Book. 2013). FLX925, a small molecule inhibitor of FLT3 would be expected to have antitumor efficacy against FLT3-driven AML tumors. However, FLX925 also inhibits two closely related cyclin-dependent kinases (CDKs), CDK4 and CDK6 (described together as CDK4/6). Since CDK4/6 are important cell cycle genes and CDK4/6 inhibitors have demonstrated anti-tumor activity, the anti-CDK4/6 activity of FLX925 may drive anti-tumor efficacy in additional and potentially overlapping patient populations.To generate hypotheses about which tumors would be sensitive to FLX925 treatment and to understand the relative contributions of FLX925's FLT3- and CDK4/6-inhibitory activities, a panel of XXX tumor-derived cell lines were screened for sensitivity to FLX925. We performed a Gradient Boosting Machine (GBM) analysis, a machine-learning approach, to generate a model that predicts sensitivity to FLX925 based on RNA-Seq expression data. This sensitivity model can predict which cell lines and tumors are likely to be sensitive to FLX925 and also which gene expression patterns contribute to that sensitivity.The GBM analysis was performed on 133 (75%) of the assayed cell lines with a random 45 (25%) of the cell line datasets reserved for model validation. Pearson's correlations of r=0.96 on the test cell lines and r=0.68 on the validation cell lines were observed, indicating good predictive performance with modest overfitting. The two most important predictive genes in the model were Retinoblastoma 1 (RB1) and by Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A). RB1 and CDKN2A are important cell cycle genes immediately downstream and upstream, respectively, of CDK4/6, suggesting that sensitivity to FLX925 in these cell lines is driven by FLX925's CDK4/6 inhibitory activity. To strengthen this conclusion, the modeling was repeated but gene sets covering cell cycle genes or genes in the FLT3/STAT5 signaling pathway were separately excluded from the analysis. While excluding the FLT3/STAT5 pathway genes had no impact on the predictive power of the model, excluding the cell cycle gene set completely abrogated the ability of the GBM to predict sensitivity to FLX925. Thus, we conclude that in this panel of largely FLT3-wild type (FLT3-WT) cell lines, FLX925-sensitivity is driven solely by the compound's CDK4/6 activity.These results suggest that independent, but potentially overlapping, sets of tumors will be responsive to FLX925 due to both its FLT3- and its CDK4/6-inhibitory activities. To predict which tumor types would be most sensitive to FLX925 treatment, we applied our GBM model to the RNA-Seq expression data for 10,537 tumors from The Cancer Genome Atlas (TCGA) consortium. Even though this model is indifferent to FLT3/STAT5-pathway gene expression, it nevertheless identifies AML tumors as having the highest median predicted sensitivity to FLX925. This is true even when all haem/lymph malignancy-derived cell lines are excluded from the training set. Thus, both FLT3-WT and FLT3-activated tumors are expected to have sensitivity to FLX925 treatment, the former due to FLX925's CDK4/6 activity alone and the latter due to the independent FLT3 and CDK4/6 activities of the drug. The anti-tumor activity of FLX925 is currently being investigated in a Ph1/b dose-escalation study in subject with relapsed or refractory AML (NCT02335814). DisclosuresCutler:Amgen, Inc: Equity Ownership; FLX Bio: Employment, Equity Ownership. Fridman:FLX Bio: Employment, Equity Ownership.

Transcriptome Analysis Revealed the Entire Genetic Understanding of Pediatric Acute Myeloid Leukemia with a Normal Karyotype

Background: Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with a current overall survival rate of less than 70%. The pathogenesis of AML is heterogeneous and can be caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations [e.g., t(8;21), inv(16), and MLL-rearrangements] have been well established as diagnostic and prognostic markers of AML. Furthermore, recurrent mutations in FLT3, KIT, NPM1, and CEBPA have been reported in both adult and pediatric AML. Recently, massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for the prediction of the prognosis. However, these mutations are rare in pediatric AML, suggesting that other genetic alterations exist in pediatric AML.In contrast, recent reports have described NUP98-NSD1 fusion as an adverse AML prognostic marker and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harboring NUP98-NSD1 fusion. Intriguingly, PRDM16 overexpression occurs in nearly one-quarter of all children, with AML involving NUP98-NSD1-negative patients. Moreover, this overexpression is enriched in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1, and MLL-PTD).Patients and Methods: To reveal a complete registry of gene rearrangements and other genetic lesions in pediatric AML with a normal karyotype, we performed transcriptome analysis (RNA sequencing) of 61 of 70 de novo pediatric AML patients with a normal karyotype using Illumina HiSeq 2000. We could not perform RNA sequencing in nine patients because of a lack of RNA quantity or quality. Among the 70 AML patients with a normal karyotype, 33 patients overexpressed PRDM16, which was found to be strongly associated with a poor prognosis in our previous studies. All patients were enrolled and treated with AML-05 in the study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG). We also analyzed the known genetic mutations associated with these patients using the data derived from RNA sequencing.Results: A total of 144 candidate gene rearrangements, which were not observed in normal samples, were identified in 51 of 61 samples. Many of the recurrent gene rearrangements identified in this study involved previously reported targets in AML, including NUP98-NSD1, NUP98-JARID1A, CBFA2T3-GLIS2, MLL-MLLT10, and MLL-MLLT3. However, several gene rearrangements were newly identified in the current study, including MLL-SEPT6, HOXA10-HOXA-AS3, PRDM16-SKI, and CUL1-EZH2. We have also performed the validation of these novel gene rearrangements using Sanger sequencing. Most of these gene rearrangements were found in patients with a high expressionof PRDM16. In contrast, CEBPA mutations were frequently observed in patients with a low expression of PRDM16. Known gene alterations, such as FLT3-ITD and MLL-PTD, and mutations of the RAS, KIT, CEBPA, WT1, and NPM1genes were also detected using RNA sequencing.Conclusion: RNA sequencing unmasked a complexity of gene rearrangements and mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from adult counterparts, regarding the spectrum of gene rearrangements and mutations. In the present study, we identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including some novel fusion genes. These findings suggest that gene rearrangements in conjunction with mutations also play essential roles in pediatric AML. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification

The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes.

Landscape of Secondary Genetic Lesions in Acute Myeloid Leukemia with Inv(16)/CBFB-MYH11

Introduction: Acute myeloid leukemia (AML) with inv(16)(p13q22) or t(16;16)(p13;q22) accounts for 5-7% of adult AML and overall is associated with a favorable outcome. However, secondary genetic lesions have been shown to negatively impact on outcome.Aims: To assess the frequency and clinical impact of additional mutations and chromosomal aberrations in AML with inv(16)/CBFB-MYH11.Patients: We analyzed 138 patients (pts) who were referred to our laboratory for diagnosis of de novo AML between 2005 and 2014 (54 females; 84 males; median age 54 years, range: 20-88 years). All patients were proven to have inv(16)(p13q22) or t(16;16)(p13;q22) /CBFB-MYH11 by a combination of chromosome banding analysis, fluorescence in situ hybridization and RT-PCR. All 138 samples were analyzed by next generation sequencing using a 22-gene panel targeting ASXL1, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, RAD21, RUNX1, SF3B1, SMC1A, SMC3, SRSF2, TET2, TP53, U2AF1, and WT1.Results: In total, 127 pts showed an inv(16)(p13q22), 10 pts a t(16;16)(p13;q22). One pt showed a normal karyotype with a cytogenetically cryptic CBFB-MYH11 rearrangement confirmed by RT-PCR.Using standard chromosome banding analysis, additional cytogenetic aberrations (ACA) were observed in 52 pts (38%). The most frequent secondary chromosome aberrations were +8 (15/52; 29%), +22 (15/52; 29%) and +21 (5/52; 10%). With regard to blood counts, cases with sole inv(16) had significantly elevated white blood cell counts compared to patients with inv(16) and ACA (78x109/L vs 20x109/L; p<0.001).112/138 (81%) pts had at least one mutation in addition to CBFB-MYH11, 47/112 (42%) had at least two additional mutations (maximum: four). Most common were mutations in NRAS (35%), KIT (32%), FLT3-ITD and FLT3-TKD(20%) and KRAS (17%). Mutations in other genes (ASXL1, CBL, DNMT3A, RUNX1, SRSF2, TET2 and WT1) were found in less than 10% of cases. Comparing AML with CBFB-MYH11 withthe other core binding factor AML entity, i.e. AML with RUNX1-RUNX1T1 (Krauth et al., Leukemia 2014), the formershowed a higher incidence of additional mutations (81% vs 50%), however, the landscape of mutated genes was comparable. Solely, the frequency of ASXL1 mutations was higher in RUNX1-RUNX1T1 positive AML compared to CBFB-MYH11 positive AML (12% vs <1%). We additionally analyzed concomitant mutations in CBFB-MYH11 positive AML according to functional pathways. Mutations resulting in activated signaling (FLT3- ITDand FLT3- TKD, KRAS, NRAS, KIT) were identified in the majority of cases (n=107/138; 78%), while mutations of tumor suppressors (CBL, TP53, WT1) were detected in 18/138 cases only (13%). Mutations of myeloid transcription factors (CEBPA, RUNX1, ETV6), mutations of genes that modify the epigenetic status (ASXL1, EZH2, TET2, DNMT3A, IDH1/2 and MLL mutations), mutations of cohesin complex genes (SMC1A, SMC3 and RAD21) and spliceosome genes (SF3B1, U2AF1, SRSF2 and ZRSR2) were identified in less than 10% of cases.There was no difference in frequency and types of additional mutations between patients with inv(16) sole and those with inv(16) and ACA with the exception of WT1 mutations, which were more frequent in patients with inv(16) and ACA (8/51; 16% vs 2/84; 2%; p=0.006).Data regarding the prognostic impact of the concurrent genetic lesions, trisomy 22 and KIT mutations, in CBFB-MYH11 AML are controversial. In our cohort, survival analysis revealed no impact of trisomy 22 or concomitant KIT mutations on prognosis of CBFB-MYH11 AML. However, within patients with inv(16) sole those with concomitant KRAS mutations had a significantly worse overall survival (OS) compared to KRAS wild-type patients (2 year OS: 43% vs 23%; p<0.001).Conclusions:1.Secondary genetic lesions are detected in 91% of inv(16)/CBFB-MYH11 positive AML patients.2.NRAS mutations were the most frequent secondary lesions followed by KIT mutations, FLT3-ITD and FLT3-TKD.3.inv(16)/CBFB-MYH11 positive AML show high frequency of mutations resulting in activated signaling.4.Considering controversial studies, trisomy 22 and concomitant KIT mutations had no prognostic impact in our cohort of 132 inv(16)/CBFB-MYH11 AML cases. The only additional genetic marker with a significant adverse prognostic impact on OS was KRAS mutation. DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.