FLT3-ITD Allelic Ratio Research Articles

Use of Machine Learning in 2074 Cases of Acute Myeloid Leukemia for Genetic Risk Profiling

Use of Machine Learning in 2074 Cases of Acute Myeloid Leukemia for Genetic Risk Profiling

Effect of gilteritinib on survival in patients with FLT3-mutated (FLT3mut+) relapsed/refractory (R/R) AML who have common AML co-mutations or a high FLT3-ITD allelic ratio.

7000 Background: The FLT3 inhibitor, gilteritinib, showed superior response and overall survival (OS) compared with salvage chemotherapy (SC) in patients (pts) with FLT3mut+ R/R AML in the phase 3 ADMIRAL study. We analyzed the impact of baseline co-mutations and FLT3-ITD allelic ratio (AR) on response and OS. Methods: A total of 37 recurrently mutated genes in AML (Archer Core Myeloid Panel) were analyzed by next-generation sequencing; the cutoff for co-mutation positivity (co-mut+) was ≥0.027. Baseline FLT3-ITD AR ( FLT3-ITD to FLT3 wild-type DNA) was measured by the LeukoStrat CDx FLT3 Mutation Assay. The median FLT3-ITD AR value of 0.77 was used to define high (≥0.77) vs low (<0.77) FLT3-ITD AR. Results: Analysis of 361 FLT3mut+ pts identified four major co-mutation cohorts, each with ≥10% of pts: NPM1 (n=173; 47.9%), DNMT3A (n=115; 31.9%), DNMT3A/NPM1 (n=86; 23.8%) , and WT1 (n=65; 18.0%). In addition, seven pts (1.9%) had all three co-mutations (ie, NPM1, DNMT3A, and WT1). The gilteritinib arm had superior response rates and OS across all four major co-mutation cohorts, with the greatest survival benefit in pts with DNMT3A/NPM1 co-mut+ (Table). In FLT3-ITD AR analyses (n=335), gilteritinib conferred longer OS than SC in pts with a high or low FLT3-ITD AR (gilteritinib: high FLT3-ITD AR, 7.1 mos vs low FLT3-ITD AR, 10.6 mos; SC: high FLT3-ITD AR, 4.3 mos vs low FLT3-ITD AR, 6.9 mos). In both arms, OS was longer in the low FLT3-ITD AR cohort than the high FLT3-ITD AR cohort but the difference in the gilteritinib arm was not statistically significant (gilteritinib: HR=1.341, P=0.0712; SC: HR=2.01, P=0.0021). Conclusions: The ADMIRAL trial shows that the clinical benefit of gilteritinib in FLT3mut+ R/R AML is maintained regardless of NPM1, DNMT3A, DNMT3A/ NPM1, or WT1 co-mut+ or high FLT3-ITD AR. Clinical trial information: NCT02421939. [Table: see text]

Prognostic impact of calculation methods for FLT3-ITD allelic ratio and NPM1 status in AML patients

Acute myeloid leukaemia (AML) is a heterogeneous disease requiring genetic profiling for risk stratification and personalised treatment. The 2017 European LeukaemiaNet recommendations initiated the use of FLT3-ITD allelic ratio (AR) in combination with NPM1 status.

Re-Examination of 2017 ELN Risk Classification By a Cohort of 739 De Novo aml Patients in Taiwan: Co-Occurring Poor-Risk Mutations May Further Predict Outcome in FLT3-ITD Patients

IntroductionRecent advances in the discovery of the genomic landscape in AML prompts necessity to re-examine the 2017 European LeukemiaNet (ELN) recommendation. In this study we aimed to validate the usefulness of 2017 ELN risk stratification in a large Taiwan cohort with special focus on the prognostic relevance of FLT3-ITD allelic ratio and its interaction with other mutations.MethodsWe retrospectively included 1040 de novo non-M3 AML patients. AML was risk-stratified according to the 2017 ELN recommendation. 739 (71.1%) patients who received standard chemotherapy were included for survival analysis. Allogeneic hematopoietic stem cell transplantation (HSCT) was performed in 293 (39.6%) patients. Mutational analyses of fifteen genes, including CEBPA, NPM1, FLT3, RUNX1, ASXL1, TP53, splicing factors (SF), such as SRSF2, U2AF1, and SF3B1, as well as KIT, NRAS, KRAS, DNMT3A, TET2, and WT1 were performed. FLT3-ITD/wild allelic ratios were calculated as the ratio of the area under the curve by fragment analysis. High FLT3-ITD allelic ratio (FLT3-ITDhigh) was defined as ³ 0.5 and low allelic ratio (FLT3-ITDlow) defined as < 0.5.ResultsAccording to the 2017 ELN risk classification, favorable, intermediate and adverse categories comprised 34.6%, 29.2% and 36.2% patients, respectively. NPM1 mutations and FLT3-ITD, the most common mutations in this cohort, were detected in 217 (20.9%) and 216 (20.8%) patients, respectively, with a significant association between each other. The median value of the FLT3-ITD/wild ratio was 0.68 without difference between NPM1-mutated and NPM1-wild group. Of note, patients with FLT3-ITDhigh had higher WBC count and LDH level than those with FLT3-ITDlow.Overall, the CR rate and relapse rate were 74.2% and 54.7%, respectively and 5-year overall survival (OS) was 43.2±1.9%. The CR rate (92.3%) was higher in the 2017 ELN favorable risk group than in the intermediate (73.0%) and adverse groups (52.0%, P<0.001). Similarly, favorable-risk patients had lower relapse rate, longer disease-free survival (DFS) and OS compared to those with intermediate- and adverse-risk features (all P<0.001).As to the prognostic impact of FLT3-ITD, we showed that FLT3-ITD patients had significantly lower CR rate, higher relapse rate, reduced DFS and OS than those without. There was a strikingly difference in treatment response between the low and high FLT3-ITD allelic ratio groups: CR rate (80.7% vs. 63.6%, P=0.0319), relapse rate (56.5% vs. 66.2%, P=0.329), DFS (14.2 vs. 4.6 months P=0.011) and OS (24.0 vs. 11.9 months, P=0.048). Interestingly, patients with FLT3-ITDhigh had a better OS if they received allogeneic HSCT than those who did not.Among the 2017 ELN favorable-risk category, we found that patients with mutated NPM1 and FLT3-ITDlow had significantly shorter OS (median, not reached vs. 31.6 months, P=0.003, Figure. 1A) and a trend of shorter DFS (median 14.9 months vs. 93.9 months, P=0.089, Figure. 1B) compared to other ELN favorable subgroups. To find the cause of the difference, we investigated the concurrent mutations in the patients with mutated NPM1 and FLT3-ITDlow. 46.2% of them had concurrent poor-risk mutations, such as ASXL1, RUNX1, TP53, WT1, TET2, DNMT3A, and SF mutations. Similarly, among the 2017 ELN intermediate-risk category, patients with mutated NPM1 and FLT3-ITDhigh had more unfavorable outcomes compared to those with wild-type NPM1 and without FLT3-ITD (DFS, median 3.7 vs. 11.6 months, P=0.028 and OS, median, 11.4 vs. 26.5 months, P=0.067). Presence of concurrent poor-risk mutations were also identified in 72.9% of these patients. Based on these findings, we postulated that concomitant poor-risk genetic alterations at least partially affected the prognosis of FLT3/ITD patients. In the cohort of FLT3-ITD patients, patients harboring poor-risk mutations had shorter DFS and OS than those without (P=0.028 and P=0.031, respectively). Further, co-occurrence of FLT3-ITDhigh and poor-risk mutations that predicted a worst outcome, seemed to define a highly adverse prognostic group.ConclusionsWe showed that ELN 2017 risk classification could well stratify AML patients in Taiwan. The prognostic relevance of FLT3-ITD may further depend on the presence or absence of co-occurring poor-risk genetic alterations, which seemed to add an adverse effect in patients with FLT3-ITD. These observations warrant confirmation in other prospective and large-scale studies. [Display omitted] DisclosuresKo:Roche: Research Funding; GNT Biotech & Medicals Crop.: Research Funding; Abbevie: Research Funding; Mumdipharma Taiwan: Consultancy.

NPM1 Variant Allele Frequency and Outcomes in AML

Background: Mutations in the NPM1 gene are among the most common genetic alterations in patients with acute myeloid leukemia (AML). NPM1 mutations predominantly occur in patients with normal or intermediate-risk abnormal cytogenetics, and define a distinct subgroup of AML patients recognized in the 2016 WHO classification. Overall, mutated NPM1 associates with favorable response to induction chemotherapy and relatively favorable overall survival. However, this prognostic impact is modulated by the presence of other gene mutations including FLT3 internal tandem duplications (ITD) and DNMT3A mutations. Recently, Patel and colleagues reported that a high variant allele frequency (VAF) of mutated NPM1 at the time of initial diagnosis associates with unfavorable outcomes in de novo AML (Blood 131:2816-25). This interesting and unexpected observation prompted us to investigate the association between NPM1 VAF and outcomes in a large AML patient cohort.Patients and Methods: We studied NPM1 mutated AML patients who had been enrolled on two successive multicenter phase III trials of the German AML Cooperative Group (AML-CG 1999, NCT00266136; AML-CG 2008, NCT01382147) and genetically characterized by amplicon-based targeted next-generation sequencing (NGS, Agilent Haloplex; Metzeler et al., Blood 128:686-98 and unpublished data). All patients had received induction chemotherapy containing cytarabine with either daunorubicin plus thioguanine or mitoxantrone. The minimum VAF for calling of insertion/deletion variants was 0.05, and samples with NPM1 coverage <100-fold (n=17) were excluded. FLT3 internal tandem duplication (ITD) status and FLT3 ITD-to-wild type allelic ratio were determined by PCR and fragment analysis from gDNA.Results: We identified 417 NPM1-mutated patients (type A mutations, 316; type B, 28; type D, 35; and other types, 38). Median patient age was 56 years (range, 19 - 86 years), and 31/414 patients (7.5%) with cytogenetic data had abnormal karyotypes. The median NPM1 VAF was 0.43 (range, 0.05 to 1.0). Type A NPM1 mutations had significantly higher VAFs than non-type A mutations (median, 0.43 vs. 0.41; P=.0002), while type B mutations had lower VAFs than non-type B mutations (median, 0.34 vs. 0.43; P=.0025) (Figure A). Age or karyotype did not associate with NPM1 VAF.NPM1 VAF, as a continuous variable, did not associate with response to induction chemotherapy (P=.6) or relapse-free survival (P=.22). A higher NPM1 VAF did, however, associate with shorter OS (hazard ratio for an increase in NPM1 VAF equal to the interquartile range, 1.14; 95% confidence interval, 1.00-1.30; P=.049). In particular, patients in the lowest quartile of NPM1 allele burden ('low NPM1 VAF') had longer OS than patients with allele burdens above the 25th percentile ('high NPM1 VAF') (median OS, 63.7 vs. 27.0 months; 5-year OS, 51% vs 42%; P=.05; Figure B).Patients with high NPM1 VAF had higher leukocyte counts (median, 46000/µl vs. 9300/µl; P<.0001) and bone marrow blast percentages (median, 85% vs. 80%; P=.0004) than patients with low NPM1 VAF. On the genetic level, patients with high NPM1 VAF more frequently had concomitant FLT3-ITD (47% vs. 37%; P=.07), and particularly FLT3-ITD with a high (≥0.5) mutant-to-wild type ratio (33% vs. 17%; P=.007), compared to patients with low NPM1 VAF. DNMT3A co-mutation was also more frequent in patients with high vs. low NPM1 VAF (63% vs. 46%; P=.002). In multivariable analyses adjusting for FLT3-ITD allelic ratio and/or DNMT3A mutation status, only the latter genetic alterations but not NPM1 VAF remained associated with OS.Conclusion: Our study confirms the recent report that adult AML patients with high NPM1 mutant allele burden have shorter survival. In our cohort, however, higher NPM1 VAF also associated with higher leukocyte counts and marrow blast percentage, and with prognostically adverse FLT3-ITD and DNMT3A mutations. After adjusting for these confounders, NPM1 allelic burden did not independently predict survival in our analysis. We therefore suspect that high NPM1 VAF may be a surrogate marker of highly proliferative AML subsets, for example those with high allelic ratio FLT3-ITD, rather than a novel independent prognostic factor. [Display omitted] DisclosuresPrassek:Jannsen: Other: Travel support; Celgene: Other: Travel support. Subklewe:Roche: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy.

RNA-based FLT3-ITD allelic ratio is associated with outcome and ex vivo response to FLT3 inhibitors in pediatric AML

AbstractControversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters ARspearman = 0.62 (95% confidence interval, 0.22-0.87) and ITDlengthspearman = 0.98 (95% confidence interval, 0.90-1.00), only AR ≥ 0.5 and length ≥48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: Plogrank = .008; ITDlength: Plogrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR ≥ 0.5 compared with ITD-AR-low and ITD− patient samples (P < .001). RNA-based FLT3-ITD measurements are recommended for risk stratification, and the relevance of AR regarding eligibility for FLT3-targeted therapy warrants further study.

Impact of Specimen Heterogeneity on Biomarkers in Repository Samples from Patients with Acute Myeloid Leukemia: A SWOG Report.

Current prognostic models for acute myeloid leukemia (AML) are inconsistent at predicting clinical outcomes for individual patients. Variability in the quality of specimens utilized for biomarker discovery and validation may contribute to this prognostic inconsistency. We evaluated the impact of sample heterogeneity on prognostic biomarkers and methods to mitigate any adverse effects of this heterogeneity in 240 cryopreserved bone marrow and peripheral blood specimens from AML patients enrolled on SWOG (Southwest Oncology Group) trials. Cryopreserved samples displayed a broad range in viability (37% with viabilities ≤60%) and nonleukemic cell contamination (13% with lymphocyte percentages >20%). Specimen viability was impacted by transport time, AML immunophenotype, and, potentially, patients' age. The viability and cellular heterogeneity in unsorted samples significantly altered biomarker results. Enriching for viable AML blasts improved the RNA quality from specimens with poor viability and refined results for both DNA and RNA biomarkers. For example, FLT3-ITD allelic ratio, which is currently utilized to risk-stratify AML patients, was on average 1.49-fold higher in the viable AML blasts than in the unsorted specimens. To our knowledge, this is the first study to provide evidence that using cryopreserved specimens can introduce uncontrollable variables that may impact biomarker results and enrichment for viable AML blasts may mitigate this impact.

Abstract 4878: Sensitive and specific DNA and RNA sequencing techniques for detecting minimal residual disease

Abstract The goal of this study was to assess the clinical applicability of molecular genetic techniques in the study of minimal residual disease (MRD) in pediatric acute myeloid leukemia (AML) via DNA and RNA-based methods. Currently, multi-parametric flow cytometry (MPFC) for surface immunophenotypes is the gold standard for MRD with a limit of detection between 0.001-0.002. However, approximately one-third of children with no detectable MRD by MPFC after first induction still relapse and suffer inferior outcomes. One potential reason is that a majority of refractory or recurrent AML clones will present with altered immunophenotypes compared to diagnostic specimens. In contrast, DNA mutations present at diagnosis will remain in refractory disease and therefore, a gene-based MRD platform surveying many genes could enable targeted, gene-specific therapy. Recently, the heterogeneous and unique genomic landscape of pediatric AML was characterized in the TARGET project and highlights the potential for leveraging companion molecular screens in the analysis of AML MRD. Contrary to adult AML, pediatric AML is not primarily characterized by single nucleotide variants (SNVs), but rather by complex structural variants (SV) that are difficult to detect using standard next generation sequencing techniques and analysis pipelines. The need to detect these complex SV and SNVs, all of which are at low allelic ratios (AR) at the remission state, demands assays that are capable of analyzing both DNA and RNA molecules at levels of detection comparable to MPFC. As proof of principal, we leveraged a unique capture technique (ArcherDx; CoreAML) to detect an important SV in pediatric AML, the internal tandem duplication in Fms related tyrosine Kinase (FLT3-ITD). Via a serial dilution of cells with known FLT3-ITD allelic ratio (MV4-11 cells), to determine a limit of detection, we were able to detect the mutation as low as 0.001. Next, we bench-marked the technique using diagnostic and relapse samples, and successfully detected FLT3-ITD at AR &amp;lt;0.01. To complement the DNA-based approach, we compared two error-corrected (EC) RNA assays (a Druley lab developed protocol and the ArcherDx; HemeV2 panel) to further assay SVs that cause novel gene fusions and aberrant exon usage, which are not detectable via DNA assays. Compared to existing cytogenetic and RNA-seq data, this new platform was capable of detecting known and novel cryptic translocations. Finally, we integrated the results from the FLT3-ITD assay and the RNA assay with our custom error-corrected sequencing data to create a novel bioinformatics workflow for assessing the biological implications of MRD clones detected. Collectively, our data support that EC sequencing, at both the DNA and RNA level, enable accurate detection of low allelic variants that could be used for improved clinical MRD diagnostics, prognostication and therapeutic selection. Citation Format: Erin L. Crowgey, Nitin Mahajan, Wing H. Wong, Edward A. Kolb, Todd Druley. Sensitive and specific DNA and RNA sequencing techniques for detecting minimal residual disease [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 4878. doi:10.1158/1538-7445.AM2017-4878

Improved Prognostic Significance of Genomic and Transcriptional Biomarkers By Examining Enriched Populations of AML Blasts: A SWOG Report

INTRODUCTION. Most studies of AML biomarkers have examined cryopreserved mononuclear cells (MNCs) obtained from repositories. These MNCs typically include non-leukemic cells (e.g., lymphocytes) and heterogeneous populations of viable and nonviable AML blasts at various stages of differentiation. We hypothesize that these variables negatively impact the prognostic power of current biomarkers. In an attempt to improve the prognostic power of such biomarkers, we examined enriched populations of viable AML blasts from 190 randomly selected AML patients on SWOG trials. To our knowledge, this study represents the largest examination of the quality of cryopreserved AML blasts and the potential prognostic benefit of enriching for viable leukemic blasts.METHODS. Cryopreserved bone marrow (BM, N=124) and peripheral blood (PB, N=116) samples from 190 AML patients on SWOG trials (SWOG-9031, SWOG-9333, S0106, and S0112) were randomly selected. The samples were thawed and sorted for viable AML blasts using a combination of fluorochrome-conjugated antibodies and DAPI. DNA and RNA were extracted and evaluated from both the unsorted AML samples (A-MNCs) and viable AML blasts (A-Blasts). FLT3-ITD allelic ratio (AR) was quantified by fragment analyses. Expression of 13 potentially prognostic transcripts was quantified via quantitative RT/PCR: BAALC, CCNA1, CEBPA, ERG1, EVI1, FLT3, GATA2, IL3RA, JAG1, KIT, MN1, RUNX1, and WT1. Fold expression differences were computed by the comparative CT method. Associations between quantitative biomarkers (FLT3-ITD AR and transcript expression) and survival (overall, OS and relapse-free, RFS) were analyzed using Cox proportional hazards regression, with interaction terms for biomarker by cell population (A-MNCs vs. A-Blasts) or CD34 expression (separately for A-MNCs and A-Blasts). A predetermined FLT3-ITD AR cutoff of 0.5 was utilized based on results by Schneider et al. (Blood, 2012) and others. For paired samples, BM was used.RESULTS. Lymphocyte percentage varied (median 7.1%, range 0.4 - 70.1%) and was correlated with patients’ pre-treatment blast percentages (rs = -0.29, P = 0.0002). Viability by DAPI varied widely (median 66.5%, range 5.2 - 95.6%), and AML blasts displayed inter- and intra-sample immunophenotypic heterogeneity, with the slight majority expressing CD34 (54%) using a predetermined immunofluorescence cut-off > 104. For OS, there was no significant difference (P=0.67) in the effect of FLT3-ITD AR between A-Blasts (HR=0.91, P=0.78) and A-MNCs (HR=1.05, P=0.88). However, for RFS, the effect of FLT3/ITD AR differed significantly (P=0.025) between A-Blasts (HR=1.93, P=0.14) and A-MNCs (HR=0.87, P=0.73). Kaplan-Meier curves of RFS by FLT3-ITD AR for A-MNCs (Figure 1A) and A-Blasts (Figure 1B) also suggest this trend. A similar, but nonsignificant, trend towards higher HRs in A-Blasts was displayed in analyses restricted to NPM1 mutated patients (Table 1). Similar analyses did not show such a striking interaction between cell population and transcript biomarkers, but we found that most transcripts displayed significant associations with immunophenotype (data not shown). Therefore, we examined if CD34 expression might impact the prognostic value of transcripts, separately by cell population. The expression of two genes (CCNA1 and GATA2) displayed significant interactions with CD34 expression in relation to clinical outcomes (Table 1). Moreover, the interaction between biomarker and CD34 expressions differed depending on which cell population was examined, such that the interaction was significant in the A-Blasts but not A-MNCs.CONCLUSION. Cryopreserved samples vary widely in percentages of non-leukemic, dying cells and differentiation stage of leukemic blasts. These factors impact the measurement of quantitative biomarkers and may also impact the significance and prognostic value of these biomarkers. Future studies must consider the effects that sample viability, composition, and differentiation stage may have on quantitative biomarkers. In addition, we are examining the potential impact that mutations in other genes (e.g., ASXL1, DNMT3A, RUNX1, etc.) may have on our results.ACKNOWLEDGEMENT. The authors wish to gratefully acknowledge the important contributions of the late Dr. Stephen H. Petersdorf to SWOG and to study S0106.SUPPORT. NIH/NCI grants CA160872,CA180819, CA180828, and CA180888. [Display omitted] [Display omitted] DisclosuresWood:Seattle Genetics: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement. Erba:Jannsen: Consultancy, Research Funding; Sunesis: Consultancy; Gylcomimetics: Other: DSMB; Agios: Research Funding; Celgene: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Incyte: Consultancy, DSMB, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; Astellas: Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; Celator: Research Funding; Juno: Research Funding. Othus:Celgene: Consultancy; Glycomimetics: Consultancy. Radich:Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy; Gilliad: Consultancy.

Genomic Profiling Identifies Novel Mutations and Fusion Genes in Newly Diagnosed and Relapsed Pediatric FLT3-ITD-Positive AML

Pediatric cancers are distinct from adult cancers in both their genomic alterations and therapeutic responses. Fms-like tyrosine kinase 3 (FLT3) mutations, especially internal tandem duplications (ITD), are among the most common mutations in acute myeloid leukemia (AML). FLT3-ITD mutations occur in approximately 15% of pediatric and 25-30% of adult AML, and are generally associated with poor prognosis. However, a number of studies have suggested that FLT3-ITD-positive(+) AML requires additional cooperative mutations. The objective of this study was to characterize the mutational landscape in a cohort of FLT3-ITD+ pediatric AML patients (median age,12.6 years; range, 2.8-19.2 years) enrolled to the AML02 and AML08 trials using samples obtained at diagnosis (n=34) and paired diagnosis/relapse samples (n=5). Children with promyelocytic leukemia were excluded. Samples were analyzed by RNASeq, a targeted 95 gene next generation sequencing (NGS) panel, and whole exome sequencing (WES).At diagnosis, 58.8% of the samples contained fusion genes; 41.2% were NUP98-NSD1, 11.8% were novel fusions (NSD1-CAPRIN1, NSD1-RALBP1, RUNX1-BCL11B, ZEB2-BCL11B), and 5.9% were previously reported fusions (CBFB-MYH11, DEK-NUP214). The NGS panel identified that WT1 and NPM1 were routinely mutated at a frequency of 32.4% and 20.6% respectively. While the NPM1 mutation was either a 4bp insertion at amino acid (a.a.) 287 or 288, WT1 mutations were heterogenenous with missense mutations, insertions and deletions all being reported. WT1 mutations and NUP98-NSD1 co-associated in 7 patients, 1 patient also harbored a TYK2 mutation; in the remaining 7 patients with NUP98-NSD1 fusions, a mutation in RAD21 or NRAS was observed in 2 patients. For samples with other fusions (n=6), we detected an average of 1 additional mutation per sample, which included mutations (variant allele frequency; VAF) in DNMT3A (0.44), IDH2 (0.49), KIT (0.37), NPM1 (0.51), PLCG2 (0.44), RAD21 (0.55), and SMC1A (0.47). No fusion genes were observed in 13 patients. In this latter subset, mutations in NPM1 (n=6) and WT1 (n=3) were observed. Other alterations that were identified in these samples included mutations in DNMT3A, IDH2, PLCG2, and PRKCB, which co-occurred with NPM1 mutations. Three patients did not harbor a fusion gene or a gene mutation by our analysis. When looking at cumulative incidence of relapse or resistant disease, our study results are concordant with previous reports where a NUP98-NSD1 fusion associated with worse prognosis (hazard ratio [HR] = 3.2, p = 0.02), but FLT3-ITD allelic ratio >0.4 was not prognostic (HR = 1.1, p=0.87). NPM1 mutations were not significantly associated with better prognosis (HR = 0.2, p = 0.11).We next sought to identify relapse specific alterations by analysis of paired diagnosis/relapse samples by RNASeq, NGS panel, and WES. Notably, the FLT3-ITD mutation was maintained at relapse in all samples. From the NGS panel, we observed the emergence of a MED12 mutation (P1751Q, VAF 0.37) and WT1 mutation (p.S152*, VAF 0.19) at relapse; a mutational switch in WT1 from diagnosis to relapse was also observed (5bp insertion at a.a. 157 to 2bp insertion at a.a. 158). By RNASeq analysis, we found a novel relapse specific fusion gene, LUZP6-OSBL1A. From exome sequencing, mutations in transcription factors were observed at relapse such as CREBBP, GLI3, and TBX20. Our analysis of relapse specific genes showed recurrent mutations in HUWE1, OGT, NACAD, and UNC13A. Intriguingly, both OGT and HUWE1 have been implicated in cancer metabolic reprogramming, and regulate MYC transcriptional programs. OGT is an O-Linked β-N-acetylglucosamine (O-GlcNAc) transferase involved post-translational modification of serine and threonine residues. HUWE1 is an E3 ubiquitin ligase that has been established as a tumor suppressor and previously reported to be mutated in AML.In conclusion, we demonstrate that additional genomic alterations are observed in the majority of pediatric FLT3-ITD+ AML samples evaluated, with a high proportion of samples containing fusion genes, WT1 and NPM1 mutations. We also identified novel fusion genes and mutations that have not been previously reported in pediatric FLT3-ITD+ AML, including relapse specific mutations. These results provide further biological insight into the genomic heterogeneity of pediatric FLT3-ITD+ AML, warranting further investigations in larger patient cohorts. DisclosuresInaba:Arog: Research Funding.

Clonal Selection in Acute Myeloid Leukemia Following Intensive Induction Correlates with Differences in Differentiation Capacity of Leukemic Sub-Clones

Abstract Introduction: Leukemic sub-clones which survive chemotherapy during induction period may remain dormant and subsequently lead to relapse in patients with acute myeloid leukemia (AML). No mechanism linking clonal selection to a specific leukemia-associated genetic aberration is known. Clonal selection is a common phenomenon; therefore, any suggested mechanism should be applicable to all kinds of leukemia-specific genetic aberrations. We hypothesized that clonal selection might result from differences in differentiation capacity among sub-clones and that chemo-resistance is not necessarily directly affected by specific mutations. Patients and Methods: We tested this hypothesis in vitro using the kasumi-1 leukemia cell line, which undergoes limited differentiation while growing and is composed of both CD34+ and CD34- sub-populations. Kasumi-1 cells were sorted by fluorescence-activated cell sorting (FACS-Aria IIIu, BD Biosciences) to CD34+CD117+ and CD34-CD117+ sub-populations. Each sub-population was separately exposed to escalating doses of daunorubicin (DNR) and cytarabine (ARA-C) for 66 hours. Cell viability was determined by alamarBlue assay (Bio-Rad, USA); apoptosis was assessed by Annexin-V and propidium iodide (PI) staining. In vivo experiments included BM samples derived from three AML patients, with both NPM1 and FLT3 gene mutations, who received intensive induction and subsequently relapsed. Blast cells were sorted to CD34+CD117+ and CD34-CD117+ sub-populations. Targeted gene sequencing was conducted using Ion Torrent™ Personal Genome Machine® (PGM) System (Life Technologies, USA). Allelic frequency of NPM1 and DNMT3A mutations in CD34+ and CD34-sub-populations was measured. FLT3-ITD was sequenced using theGeneScan method and theallelic ratio (AR) was calculated. Results: Following sorting,cultured kasumi-1 CD34+ cells partly differentiated into CD34- cells and several weeks later, the phenotype became similar to the original kasumi-1 phenotype (a mixture of 34+/- cells). In contrast, the CD34- sub-population maintained its phenotype. Immediately after sorting by CD34 expression, both sub-populations were exposed to increasing doses of DNR or ARA-C. Only the CD34+ sub-population was found to be resistant to ARA-C (at all tested concentrations), as determined by the cell viability assay (fig 1a). The CD34- sub-population exhibited a 4-fold higher apoptosis rate than CD34+ cells after exposure to 0.16 µM ARA-C. DNR (0.01 µM) resulted in 2.5-fold higher apoptosis in the CD34- compared to CD34+ sub-population (fig 1b). The sub-clonal composition of BM blasts obtained from three AML patients at diagnosis and relapse was compared. NPM1 mutation was identified in all patients at diagnosis. FLT3-ITD mutation was identified in one patient at diagnosis and in the other two at relapse. To explore the differentiation capacity of each sub-clone, blasts were sorted to well-defined CD34+ and CD34- subgroups. At diagnosis, most of the leukemic blasts presented with the CD34- phenotype, while at relapse, the CD34+ population grew in all patients. The FLT3-ITD allelic ratio was higher in CD34+ cells. In the first patient, FLT3-ITD was not detected at diagnosis, while at relapse, the wild-type allele of FLT3 was lost and FLT3-ITD was solely present in the CD34+ sub-population. In CD34- cells, the AR was low (0.55) at relapse. In the second patient, the dominant clone at diagnosis was NPM1mutFLT3wtDNMT3Awt and &gt;95% cells were CD34-. At relapse, the FLT3-ITD DNMT3Awt clone became dominant and was mostly located within the rising CD34+ population. Another sub-clone, exhibiting both FLT3-ITD and DNMT3A R882C mutation, was identified and was found to reside only within the CD34+ population. Similar findings were also observed in the third patient. We screened additional eight patients presenting with FLT3-ITD and in 4 of them, while both CD34+/- blasts were observed, the FLT3-ITD carrying clones resided only in one sub-population. Conclusions: Chemo-resistance and survival of a specific AML sub-clone correlate with its phenotype and differentiation level. Even genetic aberrations which have no direct interaction with chemotherapy biological effect, may give rise to clonal selection. Sub-clonal differentiation capacity may affect its sensitivity to chemotherapy. Disclosures No relevant conflicts of interest to declare.

Postinduction Minimal Residual Disease Predicts Outcome and Benefit From Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia With NPM1 Mutation: A Study by the Acute Leukemia French Association Group.

Purpose This study assessed the prognostic impact of postinduction NPM1-mutated ( NPM1m) minimal residual disease (MRD) in young adult patients (age, 18 to 60 years) with acute myeloid leukemia, and addressed the question of whether NPM1m MRD may be used as a predictive factor of allogeneic stem cell transplantation (ASCT) benefit. Patients and Methods Among 229 patients with NPM1m who were treated in the Acute Leukemia French Association 0702 (ALFA-0702) trial, MRD evaluation was available in 152 patients in first remission. Patients with nonfavorable AML according to the European LeukemiaNet (ELN) classification were eligible for ASCT in first remission. Results After induction therapy, patients who did not achieve a 4-log reduction in NPM1m peripheral blood-MRD (PB-MRD) had a higher cumulative incidence of relapse (subhazard ratio [SHR], 5.83; P < .001) and a shorter overall survival (OS; hazard ratio [HR], 10.99; P < .001). In multivariable analysis, an abnormal karyotype, the presence of FLT3-internal tandem duplication (ITD), and a < 4-log reduction in PB-MRD were significantly associated with a higher relapse incidence and shorter OS. In the subset of patients with FLT3-ITD, only age, white blood cell count, and < 4-log reduction in PB-MRD, but not FLT3-ITD allelic ratio, remained of significant prognostic value. In these patients with nonfavorable AML according to European LeukemiaNet, disease-free survival and OS were significantly improved by ASCT in those with a < 4-log reduction in PB-MRD. This benefit was not observed in those with a > 4-log reduction in PB-MRD, with a significant interaction between ASCT effect and PB-MRD response ( P = .024 and .027 for disease-free survival and OS, respectively). Conclusion Our study supports the strong prognostic significance of early NPM1m PB-MRD, independent of the cytogenetic and molecular context. Moreover, NPM1m PB-MRD may be used as a predictive factor for ASCT indication.

Comparative Value of Post-Remission Treatment in Cytogenetically Normal AML Subclassified by NPM1 and FLT3-ITD Allelic Ratio

Comparative Value of Post-Remission Treatment in Cytogenetically Normal AML Subclassified by NPM1 and FLT3-ITD Allelic Ratio

Allogeneic Transplantation in First Remission Improves Outcomes Irrespective of FLT3-ITD Allelic Ratio in FLT3-ITD–Positive Acute Myelogenous Leukemia

The adverse prognosis of internal tandem duplication in the FMS-like tyrosine kinase 3 gene(s) (FLT3-ITD) in patients with acute myelogenous leukemia (AML) may depend on allelic burden. We compared postremission treatment with chemotherapy and hematopoietic stem cell transplantation (HSCT) in 169 FLT3-ITDmut intermediate cytogenetic risk AML patients with allelic ratio evaluable at diagnosis who achieved first complete remission (CR1) with induction therapy. To minimize selection bias, the analysis was limited to patients who remained in CR1 for at least 4 months (median time to HSCT) after achieving CR1, and propensity score matching was implemented. Sensitivity analysis including patients who remained in CR1 for at least 3 months was applied as well. HSCT in CR1 was associated with longer relapse-free survival (RFS) and overall survival (OS), with 3-year estimated rates of 18% and 24%, respectively (P < .001), for patients receiving chemotherapy and 46% and 54%, respectively (P < .001), for those undergoing HSCT. Multivariate regression models showed that HSCT remained statistically significant with improved RFS and OS independent of FLT3-ITD allelic ratio and NPM1 status. Irrespective of postremission therapy, relapse remains the main reason for treatment failure, with a 3-year incidence of 68% in chemotherapy recipients versus 41% in HSCT recipients. Allogeneic HSCT improved disease outcomes compared with chemotherapy after propensity score matching was applied. The improvement observed for RFS (hazard ratio [HR], 0.55; P = .09) and OS (HR, 0.58; P = .10) with HSCT as postremission therapy in patients who remained in CR1 for at least 4 months did not reach statistical significance; however, the sensitivity analyses including patients who remained in CR1 for at least 3 months showed significant improvement in both RFS (HR, 0.31; P = .002) and OS (HR, 0.27; P = .02) after propensity score matching. Our results indicate that HSCT in CR1 for AML FLT3-ITDmut patients is associated with longer RFS and OS. Innovative transplantation strategies to improve relapse incidence are urgently needed.

Relapse Mechanism of FLT3-ITD Positive AML and Cell Differentiation Blockage

Introduction: Activating mutation of FLT3 by internal tandem duplications (ITD) is the most common molecular aberration found in AML. FLT3-ITD mutation induces a proliferation signal and is associated with leukocytosis and poor prognosis. The co-existence of FLT3-ITD with translocation t(5:11) that yields fusion transcript of Nucleoporin98 and the nuclear receptor binding SET-domain protein 1 (NUP98/NSD1) was recently reported to be associated with chemo-resistance. Relapse risk of the majority of FLT3-ITD positive AML patients who are negative to NUP98/NSD1 is increased compared to other AML with normal karyotype, despite similar remission rates. The mechanism of FLT3-ITD contribution to relapse development in patients who achieved remission is unknown.Aims: To explore potential mechanisms that allow relapse in FLT3-ITD positive AML in the presence or absence of NUP98/NSD1 fusion gene. In addition, time sequence of the emergence of both mutations is suggested from a case with relapse.Methods: Leukemic blasts derived from newly diagnosed or relapsed FLT3-ITD positive AML patients were enriched for FLT3-ITD mutated sub-clones by sorting according to CD34 expression. Genomic DNA was extracted from each sorted fraction and FLT3-ITD mutation allele load was quantitatively determined by GeneScane. Total RNA was extracted from FLT3-ITD positive patients and RT-PCR was performed to detect NUP98/NSD1 fusion mRNA. All patients received induction with intensive chemotherapy combination of Daunorubicin and Cytarabine and their outcome was recorded. Chemo-sensitivity assays using Ara-C were conducted on different leukemia cell lines sub-populations divided by their CD34 expression.Results: NUP98/NSD1 was identified in 4 of 19 (21%) FLT3- ITD positive adult AML patients with normal karyotype who had a compatible donor and were considered transplant eligible. Patients harboring both NUP98/NSD1 and FLT3-ITD (75%) had higher rate of induction failure than FLT3-ITD patients without NUP98/NSD1 (40%). To explore the correlation between FLT3-ITD and differentiation capacity, primary AML FLT3-ITD positive blasts were sorted into two distinct sub-populations according to CD34 expression (example is shown in fig. 1). In 14/19 patients DNA from CD34+ and CD34- leukemic blasts was successfully extracted and FLT3- ITD allele load was tested and recorded as the ratio between FLT3 normal and mutated alleles. Of these 14 patients, 3 experienced induction failure and 8 (58%) achieved complete remission but unfortunately eventually relapsed. FLT3-ITD allelic ratio (AR) was equally measured in both CD34+ and CD34- sub-populations in 7 patients (50%) while in 5 patients (35.7%) the mutated allele was restricted to the CD34 positive cells and 4/5 (80%) patients experienced relapse. In two patients (14%) who also expressed NUP98/NSD1, the FLT3-ITD mutated allele was restricted to CD34 negative sub-population. In one patient, NUP98/NSD1 was detected in relapse but not in diagnostic specimen. Cytotoxic assay confirmed that differentiation stage as determined by CD34 expression is fundamental for chemo-sensitivity of leukemic cells regardless of their genetic profile. CD34 positive cell lines as well as CD34+ sub-population of Kasumi-1 cell line were resistant to the Ara-C compare to CD34 negative cell lines or the matched Kasumi-1 CD34- sub-population (fig. 2).Conclusions: FLT3-IDT mutation is a late event during leukemogenesis. We observed NUP98/NSD1 that emerged as a new mutation on relapse in a FLT3-IDT positive patient, assuming this may also be a later event. Our strategy to sort blasts according to their CD34 expression enable us to describe the accumulation of FLT3-ITD sub-clones at the primitive stage that express CD34, therefore, these leukemic sub-clones may be enriched with early leukemic precursors. Such differentiating blockage results a higher portion of cells which survive chemotherapy among FLT3-ITD and hence increases relapse risk as an indirect effect. Inducing differentiation in FLT3-ITD positive AML should be further studied as a therapeutic strategy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NPM1 Minimal Residual Disease As Prognostic and Predictive Factor in Young Adults with Acute Myeloid Leukemia: a Study By the French ALFA Group

▪IntroductionTherapy decision in young adults with acute myeloid leukemia (AML) is mostly guided by pre-therapeutic risk assessment based on cytogenetical and molecular characterizations. Mutations in the nucleophosmin 1 (NPM1 m) gene represent one of the most common gene mutations, found in around one third of AML and associated with normal karyotype in 85% of cases. Because of their homogeneous mutation pattern and their clonal stability, NPM1 mutations are an effective tool for monitoring MRD. The aim of this study was to assess the prognostic impact of post-induction NPM1 MRD in homogeneously treated patients and to address the question of whether NPM1 MRD may be used as a predictive factor of allogeneic stem cell transplantation (SCT) benefit in this subgroup of patients.Materiel and methodsAmong 196 NPM1 m patients treated in the ALFA-0702 trial, 172 achieved complete remission (CR). A MRD was available in 152 patients, on peripheral blood (PB) in 135 patients and on bone marrow (BM) in 135 patients. MRD levels were reported as the normalized values of NPM1 m copy number/ABL copy number x 100 (%). Patients were also screened for FLT3-TKD, FLT3-ITD, CEBPA, DMT3A, IDH1, IDH2, WT1 and TET2 mutations. Patients that did not belong to ELN-favorable group were eligible for allogeneic SCT (N=71).ResultsThe median follow-up was 3.5 years (95%CI: 3.1-3.9). The karyotype was normal in 122 patients (80.3%), and a FLT3-ITD was found in 59/150 (39.3%) with a median FLT3-ITD allelic ratio of 0.385 (range: 0.02-8.0). Fifty out of the 71 patients eligible for SCT were allografted in first CR. The 3-year CIR and OS censored at SCT were 29.3% (95%CI: 21.5-39.4) and 78.6% (95%CI: 69.3-85.5) respectively.At diagnosis, NPM1 baseline levels did not differ between PB and BM (p=.20). However, after induction, the median MRD log reduction was 4.5 in PB (range: 1.7-5.8) and 3.8 in BM (range: 0.01-5.5) (p<.0001). We focused here on the value of PB-MRD log reduction.Patients with a high WBC (>50 G/L) or FLT3-ITD had higher NPM1 baseline but also PB-MRD levels. However, no correlation between PB-MRD log reduction and age, WBC, karyotype, or any of the gene mutations was observed.Patients who did not achieve a 4-log reduction in NPM1 PB-MRD were exposed to a higher risk of relapse and had a shorter survival (3-year CIR censored at ASCT: 65.8% vs. 20.5%, SHR 5.83, 95%CI: 2.78-12.23, p<.001; 3-year OS censored at SCT: 92.3% 40.8%, HR 10.99, 95%CI: 3.97-30.39, p<.001). In multivariable analysis of CIR and OS censored at ASCT, abnormal karyotype, FLT3-ITD, and PB-MRD reduction < 4 log were significantly associated with a higher risk of event.Among the 59 patients with FLT3-ITD, a PB-MRD reduction < 4 log was also predictive of a higher risk of relapse and a shorter survival (SHR 3.74, 95%CI: 1.47-9.53, p=.006; HR 3.26, 95%CI: 1.43-7.44, p=.005) along with age, logWBC, and FLT3-ITD allelic ratio. In multivariable analysis of CIR and OS, only age, logWBC, and PB-MRD reduction < 4 log were significantly associated with a higher risk of relapse or death.Considering allogeneic SCT as a time-dependent variable, DFS but also OS were significantly improved by SCT in non-favorable ELN patients with PB-MRD reduction <4 log (DFS, HR 0.25, 95%CI: 0.06-0.98, p=.047; OS, HR 0.25, 95%CI: 0.06-0.98, p=.047). This benefit was not observed in patients with a favorable PB-MRD response (DFS, HR 1.62, 95%CI: 0.05-5.17, p=.419; OS, HR 2.11, 95%CI 0.57-7.71, p=.261) with a significant interaction between SCT effect and PB-MRD log reduction (DFS, p=.024; OS, p=.027).ConclusionOur study supports the prognostic significance of early NPM1 PB-MRD, independently of cytogenetical and molecular context. Moreover, NPM1 PB-MRD may be used as a predictive factor of allogeneic SCT benefit in non-favorable ELN population. [Display omitted] DisclosuresDe Botton:Agios Pharmaceuticals: Research Funding. Dombret:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Decitabine and sorafenib therapy in FLT3-ITD mutant acute myeloid leukemia- A case series

216 DECITABINE AND SORAFENIB THERAPY IN FLT3-ITD MUTANT ACUTE MYELOID LEUKEMIAA CASE SERIES Monica R. Muppidi1, Elizabeth A. Griffiths1, James E. Thompson1, Laurie A. Ford1, Craig W. Freyer2, Meir Wetzler1, Eunice S. Wang1 1Department of Medicine, 2Department of Pharmacy, Roswell Park Cancer Institute, Buffalo, NY Introduction: Patients with acute myeloid leukemia (AML) characterized by FMS-like tyrosine kinase-3 (FLT-3) internal tandem duplication (ITD) mutations have poor outcomes, especially in the relapsed setting. Although small molecule inhibitors of FLT3 have been explored for these patients, many have demonstrated limited single-agent efficacy with short response durations. Sorafenib, a multi-kinase inhibitor with activity against FLT-3, has previously been evaluated alone and in combination with induction chemotherapy or azacytidine in AML patients. Here we describe our experience with the combination of the DNA hypomethylating agent, decitabine (D), and sorafenib (S) for the treatment of FLT-3 ITD mutant AML. Methods: We retrospectively reviewed records of patients with FLT-3 ITD mutant AML who were treated off protocol with D and S from 2011-present. Descriptive statistics, treatment response, and overall survival were recorded. Results: Six patients were identified. Mean age was 56 (range 34-70) years. Two-thirds (4/6) were female. All patients received at least 1-2 cycles of concurrent D 20 mg/m2 for 10 days and S 200-400 mg twice a day for 28 days. Five patients had relapsed/refractory AML (RR-AML) following 1-3 prior therapies. One patient had de novo AML in complete remission with incomplete count recovery (CRi) and received DS as consolidation. Overall response rate was 83%. Eighty percent (4/5) of patients with RR-AML attained CRi. One patient receiving DS consolidation attained complete remission (CR). Two patients received subsequent allogeneic stem cell transplantation. FLT3-ITD allelic ratio (available on 3 patients) decreased after DS therapy and correlated with CRi. Median overall survival was 111 days (range 62-348) from the initiation of DS to death from any cause or last known follow-up. Two patients developed treatment-related neutropenic sepsis and elevated liver enzymes which did not require dose adjustment. One patient developed heart failure of uncertain etiology. Conclusions: In this single institute case series, we demonstrated that the combination of D(10 days) and S was well tolerated, resulted in high CR/CRi rates (80%), and prolonged overall survival in patients with heavily pretreated relapsed/refractory FLT-3 ITD mutant AML. Further investigation of this regimen in clinical trials is warranted.

Differential impact of allelic ratio and insertion site in FLT3-ITD–positive AML with respect to allogeneic transplantation

AbstractThe objective was to evaluate the prognostic and predictive impact of allelic ratio and insertion site (IS) of internal tandem duplications (ITDs), as well as concurrent gene mutations, with regard to postremission therapy in 323 patients with FLT3-ITD–positive acute myeloid leukemia (AML). Increasing FLT3-ITD allelic ratio (P = .004) and IS in the tyrosine kinase domain 1 (TKD1, P = .06) were associated with low complete remission (CR) rates. After postremission therapy including intensive chemotherapy (n = 121) or autologous hematopoietic stem cell transplantation (HSCT, n = 17), an allelic ratio ≥ 0.51 was associated with an unfavorable relapse-free (RFS, P = .0008) and overall survival (OS, P = .004); after allogeneic HSCT (n = 93), outcome was significantly improved in patients with a high allelic ratio (RFS, P = .02; OS, P = .03), whereas no benefit was seen in patients with a low allelic ratio (RFS, P = .38; OS, P = .64). Multivariable analyses revealed a high allelic ratio as a predictive factor for the beneficial effect of allogeneic HSCT; ITD IS in TKD1 remained an unfavorable factor, whereas no prognostic impact of concurrent gene mutations was observed. The clinical trials described herein were previously published or are registered as follows: AMLHD93 and AMLHD98A, previously published; AML SG 07-04, ClinicalTrials.gov identifier #NCT00151242.

The Outcome Of Allogeneic Hematopoietic Cell Transplantation In Children With FLT3/ITD-Positive Acute Myelogenous Leukemia

FLT3 is a receptor tyrosine kinase expressed on hematopoietic progenitors that regulates hematopoietic development. FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutations whose presence is associated with poor outcome in adults and children with AML. In children with FLT3/ITD who have high allelic ratio (ITD-AR), the progression free survival is estimated to be <20%. Hematopoietic stem-cell transplantation (HSCT) has been a recommended therapeutic option for children who are positive for FLT3. There is limited data on the post transplant outcome for children with FLT3/ITD.We conducted a retrospective study to determine the outcome for all children with FLT3/ITD positive AML who underwent allogeneic HSCT in 4 pediatric centers. Data was collected and analyzed to identify the potential risk factors for overall survival (OS) and event-free survival (EFS). The association with OS and EFS was evaluated by Cox proportional hazard regression with results reported using hazard ratio (95% CI). The survival curve and cumulative incidence were calculated using the Kaplan-Meier method.Between March 2007 and June of 2012, 29 AML patients with FLT3/ITD received HSCT. Median age at HSCT was10.2 years (range 4.5-21.9), 16 were males, and 26 were in first complete remission (CR), 2 with refractory disease and 1 in second CR. Among those with minimal residual disease testing, it was present in 3 (12%) patients in 1stCR. FLT3-ITD allelic ratio at the time of diagnosis was available in 25 patients and among those patient 14 (56%) the ratio was >0.4 (range 0.03-15.99). Eleven patients (38%) received stem-cells from HLA identical siblings while in 18 (62%) an alternative donors stem-cell was utilized (mismatched related donor (n=2), unrelated donor (n=7) or cord blood (n=9)). Eighteen patients (62%) received a total body irradiation (TBI) based preparative regimen while 11 (38%) received a busulfan-based regimen. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitor and mycophenolate for recipients of cord blood HSCT and calcineurin inhibitor plus methotrexate for the rest. Three (10%) patients experienced primary graft failure following cord blood HSCT. All 3 patients received a second graft that resulted in sustained engraftment. The cumulative incidence of neutrophil engraftment at day 42 was 89.6%. Sixteen (55%) patients developed grade 2-4 GVHD and 11 (38%) developed chronic GVHD (6 with severe and 5 mild). None of the patients experienced transplant related mortality. Eleven (38%) patients experienced relapsed disease. The cumulative incidence of relapse at 2 years was 34.7% with 95% CI (20.4%, 54.9%). At 2-years the probability of EFS and OS was 65.3% with 95% CI (45.1%, 79.6%) and 82.2% with 95% CI (58.5%, 91.3%) respectively. Using univariable analysis there was no difference between the EFS of patients who received related donors versus alternative donors, (HR 2.64 (0.79-8.76) p=0.1) and those with residual disease at the time of transplant versus those in complete remission (HR 0.88(0.19-4.09, p=0.8). Patients with higher FLT3/ITD ration at diagnosis had significantly worse EFS (HR 1.42 (1.04-1.93) p=0.03). The use of TBI in the preparative regimen was associated with superior EFS and OS (HR 0.29 (0.08-0.99), p =0.04) and (HR 0.07 (0.01-0.62), p= 0.002) respectively.In conclusion the use of allogeneic HSCT is associated with improved EFS and OS in children with FLT3/ITD positive AML compared to what has been reported in those treated with chemotherapy alone. Disclosures:No relevant conflicts of interest to declare.

Location of FLT3-ITDs within the beta1-Sheet of FLT3 Kinase Confers Resistance to Tyrosine Kinase Inhibitors (TKI) in Vitro.

Abstract 2440 Introduction:In AML, the recently described tyrosine kinase domain-1 (TKD1)-ITDs of FLT3 (Breitenbuecher et al., Blood 2009, Kayser-S et al., Blood 2009) are located within the beta1-sheet, nucleotide binding loop and beta2-sheet of tyrosine kinase domain 1 (TKD1), respectively. Multivariate analysis of clinical data revealed that location of FLT3-ITDs within the beta1-sheet of TKD1 is an unfavorable prognostic factor (Kayser-S et al., Blood 2009). Recently, we uncovered a novel mechanism of primary resistance to FLT3 tyrosine kinase inhibitors (TKIs) in a patient displaying an atypical localization within the beta2-sheet-ITD (A627E). Here, we characterized in-vitro sensitivity to FLT3-TKI in growth factor dependent hematopoietic cell lines expressing a representative panel of FLT3-ITDs isolated from patient material. In particular, we compared sensitivity of beta1-sheet ITDs with typical ITDs located in the juxtamembrane domain of FLT3. Methods:FLT3-ITDs isolated from patient material, were sequenced, subcloned in parallel into two expression vectors (pAL and pMSCV-Puromycin-IRES-GFP), and stably expressed in growth-factor dependent hematopoietic Ba/F3 cells and in parallel in 32D cells. Constitutive phosphorylation of FLT3-ITD receptors and of downstream signaling pathways was analyzed by Western-blotting. Transformation of Ba/F3 and 32D cells was investigated by colony formation assays and by withdrawal of IL-3. Induction of apoptosis in response to various concentrations of FLT3-kinase inhibitors midostaurin (PKC412) and quizartinib (AC220) was measured by flow-cytometry at 24h and 48h, respectively. For statistical analysis of replicates t-test was employed. Results:Biological characteristics of FLT3-ITD mutations located in two different structural domains of FLT3-kinase were characterized: (1) beta1-sheet-ITDs E611V(96nt) and Q613E(99nt) and (2) nucleotide binding loop-ITD A620V(84nt). (Our nomenclature used for description of ITDs indicates position of amino-acid where ITD is located, possible exchange of amino-acid residues at this position and nucleotide length of ITD) Hematopoietic cells (32D and Ba/F3) expressing these ITD mutations showed colony formation in methylcellulose medium, growth-factor independent proliferation upon IL-3 withdrawal and constitutive phosphorylation of FLT3 signaling. Three ‘typical' juxtamembrane domain (JMD) ITDs were used as controls; ITD598/599(36nt), ITD598/599(66nt) and ITDK602R(21nt). As compared to these JM-ITDs, we observed significantly less apoptosis of beta1-sheet-ITDs at all concentrations of FLT3-kinase inhibitors applied. However, this difference in sensitivity gradually decreased when incubating with midostaurin or quizartinib for longer periods of time as 48h. Of note, length of FLT3-ITD mutations did not appear to influence sensitivity to TKI treatment. Conclusion:Investigating representative FLT3-ITD-mutations located within the TKD1 revealed that beta1-sheet-ITDs mediate constitutive activation of FLT3 receptors leading to transformation of hematopoietic cell lines 32D and Ba/F3. In comparison to typical JMD-ITDs, beta1-sheet-ITDs analyzed here revealed resistance to FLT3-inhibitors as midostaurin and quizartinib across two cellular reconstitution models (32D and Ba/F3) and using two different expression vectors (pAL and pMSCV). Our results indicate that differential sensitivity is rather an effect of ITD-localization within a functional domain of FLT3 and not necessarily conferred by the length of ITDs. Taken together, our data provide a rationale to prospectively analyze not only the FLT3-ITD mutation status or FLT3-ITD allelic ratio but also location of ITD-mutations in ongoing clinical trials as this may have direct impact on response to therapy (tyrosine kinase inhibitors, chemotherapy, allogeneic stem cell transplantation) in FLT3-ITD positive AML. Disclosures:Heidel:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fischer:Novartis: Honoraria.