High Allelic Ratio Research Articles

Biology and Clinical Implications of Complex Landscape of Cooperating Events in FLT3-ITD Acute Myeloid Leukemia

FLT3-ITD mutations are among the most common somatic mutations in acute myeloid leukemia (AML) and are important in prognostic determination as well as therapeutic allocation. Recent studies have demonstrated improved outcomes with the addition of FLT3 inhibitors and for some patients hematopoietic stem cell transplant (HSCT) in first remission (CR1). We have previously demonstrated that the outcome of FLT3-ITD patients can be quite heterogeneous based on the co-occurrence of a few specific risk stratifying mutations, including NPM1 and NUP98-NSD1. We sought to interrogate the complex landscape of cooperating events with FLT3-ITD AML and potential impacts on outcome in the context of contemporary therapies, including the FLT3 inhibitor sorafenib.Of the 1296 children and young adult patients with de novo AML enrolled on COG AAML1031, 229 had FLT3-ITD mutations and were included in this study. Patients with high allelic ratio (HAR; >0.4) FLT3-ITD were allocated to Arm C, received sorafenib in combination with chemotherapy and received HSCT in CR1. Those with low allelic ratio (LAR; £ 0.4) FLT3-ITD were treated on Arm A/B and received chemotherapy, no sorafenib, and did not receive HSCT in CR1 unless they had evidence of residual disease following induction I (MRD³ 0.1%) or a high-risk cytogenetic feature. FLT3-ITD status and allelic ratio were determined by PCR and all samples also underwent karyotyping, FISH, and next generation sequencing in 195 (85%) of cases for determination of comprehensive co-occurring mutational profile.Among the 229 FLT3-ITD positive patients, allelic ratio ranged from <0.1-20.8, with 96 (42%) patients classified as LAR and 133 (58%) patients as HAR. Among the cohort overall, the significant majority of 85% (n=195) harbored a cooperating genomic aberration. The most common co-occurring single gene mutations were: WT1 (31%, n=71), NPM1 (20%, n=46), NRAS (9.2%, n=21), FLT3-TKD (7%, n=16), CEBPA (6.5%, n=15), KMT2A-PTD (5.7%, n=13) (Figure 1A). KMT2A-PTD lesions were significantly more prevalent among FLT3-ITD vs non ITD patients, 5.7% vs. 0.65% (p<0.001). Normal karyotype was detected in 50% of patients. The most common recurring cytogenetic abnormalities were NUP98-NSD1/t(5;11) fusions (19.2%, n=44), trisomy 8 (10%, n=23), DEK-NUP214/t(6;9) fusions (7%, n=16), KMT2A rearrangements (3.9%, n=9)(Figure 1A). In contrast, the other high risk abnormalities (monosomy 5/del5q, monosomy 7) were absent or exceedingly rare, while the low risk lesions t(8;21) and inv(16) were also rare (3%, n=7 each). We have previously reported outcome of the more common and risk stratifying mutations with co-occurring NUP98-NSD1 resulting in dismal prognosis regardless of treatment arm, while outcome for those with WT1 was improved with Arm C treatment and approached that of other FLT3-ITD patients(Figure 1B). Evaluation of the FLT3-ITD/trisomy 8 patients demonstrated those treated on Arm C experienced poor outcomes with an EFS of 30% and was equivalent to 29% for those on Arm A/B (p=0.96, Figure 1C), with a corresponding OS of 40% vs. 34% (p=0.66) respectively. In contrast, evaluation of outcome of the KMT2A-PTD patients demonstrated those treated on Arm C had a favorable 5-year event-free survival (EFS) of 71% vs. 23% (p=0.05) for those on Arm A/B (Figure 1D), with a corresponding 5-year overall survival (OS) of 86% vs. 46% (p=0.15) respectively.Comprehensive sequencing demonstrated the FLT3-ITD samples identified co-occurring genetic mutations or cytogenetic abnormalities in the majority of cases. Although KMT2A-PTD is rarely reported in pediatric compared to adult AML, we found it was enriched in FLT3-ITD patients and this cohort experienced favorable outcomes when treated with transplant and sorafenib. Patients with dual FLT3-ITD/trisomy 8 had suboptimal outcomes similar to other poor risk co-occurring lesions and comparable regardless of AR or treatment arm. While there was some overlap with WT1 mutations in this cohort, further investigation into prognostic impact of this cooperating event is warranted. The prognostic implications FLT3-ITD mutations vary and we provide further data that the comprehensive cooperating mutational profile is critical to understanding the prognostic implications in specific patients, and may also impact response to FLT3 inhibitor therapy. [Display omitted] DisclosuresHylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees.

Significant Improvements in Survival for Patients with t(6;9)(p23;q34)/DEK-NUP214 in Contemporary Trials with Intensification of Therapy: A Report from the Children's Oncology Group

The occurrence of t(6;9)(p22;q34)/DEK-NUP214 is a rare subtype of pediatric AML that commonly co-occurs with FLT3-ITD mutations and is associated with poor outcomes, regardless of FLT3-ITD status. With increased recognition of the inferior prognostic impact of FLT3-ITD and to a lesser degree t(6;9), and with early identification of these lesions, these patients have been allocated to high risk intensive therapy on more contemporary clinical trials. Therefore we sought to interrogate the outcome of children with t(6;9) AML to determine if intensification of therapy, specifically the use of hematopoietic stem cell transplant (HSCT) in CR1, and FLT3 inhibitors for ITD+ patients, may have improved outcomes for this high risk group of patients.We evaluated the outcomes of all patients with t(6;9) detected by karyotype analysis on pediatric patients with de novo AML enrolled on the previous 7 Children's Oncology Group (COG) or its predecessor (POG or CCG) trials over 31 years (1988-2017). A total of 66 cases of t(6;9) AML were identified from CCG-2861 (n=2), CCG-2891 (n=5), POG-9421 (n=7), CCG-2961 (n=10), AAML03P1 (n=7), AAML0531 (n=17), and AAML1031 (n=18). For all studies prior to 2005 (CCG-2861, CCG-2891, POG-9421, CCG-2961) neither t(6;9) nor FLT3-ITD was used as a prognostic factor to adjust treatment, while on AAML0531 post an amendment in 2008 all patients on AAML1031 with high allelic ratio (HAR; >0.4) FLT3-ITD were allocated to HSCT in CR1, which continued on AAML1031 and this group of patients also received sorafenib.Among all 66 t(6;9) patients identified, 45 (68%) had known data for ITD. Among these, 69% (n=31) harbored a co-occurring FLT3-ITD mutation (AR range 0.07-13.35), while 31% (n=14) were FLT3-ITD negative. In the early phase of trials prior to recognition of FLT3-ITD (1988-2002; CCG-2861, CCG-2891, POG-9421, and CCG-2961), the 5-year event-free survival (EFS) and overall survival (OS) for all t(6;9) patients was dismal at 14% and 18%, respectively (Figure 1). Trials from 2006-2010 (AAML03P1, AAML0531), evaluated the addition of gemtuzumab ozogamicin (GO) to chemotherapy as well as the use of HSCT in CR1 for patients with matched related donors, and the 5-year EFS and OS for patients with t(6;9) improved to 48% and 58% (p<0.001 compared to 1998-2002, Figure 1). Among this group of patients, only 16% received HSCT in CR1. The prevalence and clinical implications of FLT3-ITD in childhood AML was reported early and was incorporated as a risk stratifying biomarker following an amendment to 0531 and for all FLT3-ITD patients with HAR disease on AAML1031; this cohort was considered high risk and allocated to intensified therapy, which in many cases significantly impacted the treatment of patients t(6;9) given the significant overlap. In the most recent trial (AAML1031, 2011-2017), the 5-yr EFS and OS for patients with t(6;9) was excellent at 71% and 94%, which was significantly improved compared to other trials (p<0.001; Figure 1). Among the 18 t(6;9) patients on 1031, 72% (n=13) were allocated to HSCT in CR1, the indication for 7 was HAR FLT3-ITD and they also received sorafenib and for 6 was presence of residual disease after induction 1 (4 with LAR FLT3-ITD, 2 ITD negative). The EFS and OS for the t(6;9)/FLT3-ITD HAR and t(6;9)/FLT3-ITD LAR/neg ITD groups who both received HSCT in CR1 were comparable and very good (EFS: 87.5% vs 100%, OS: 87.5% vs 100%, p=NS for both).We show that over the past 3 decades, outcomes of patients with t(6;9) AML have improved significantly on upfront trials. While general improvements in AML treatment and supportive care are an important aspect, our data with excellent EFS on AAML1031 suggest that the intensification of upfront therapy with HSCT, which occurred for many t(6;9) patients due to their co-occurrence with FLT3-ITD, was critical to these improvements. On the current COG phase III AAML1831 trial, all patients with t(6;9) regardless of ITD status are considered high risk and allocated to HSCT in CR1, while those with a FLT3 mutation also receive gilteritinib. Evaluation of the outcome of t(6;9) patients with this therapy will be important as while FLT3-ITD patients have been an example of a group whose poor outcomes have been shown to be abrogated with intensified therapy, it appears that t(6;9) patients may have benefitted from their co-occurrence pattern and are also a group whose outcomes can be significantly improved with an intensified upfront therapeutic approach. [Display omitted] DisclosuresPollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees.

Global Proteomic Profiling Identifies Novel Prognostic Factors in Undifferentiated Leukemia Blasts from Patients with NPM1 Mutations: A Previously Unreported Approach to Biomarker Discovery from the Fred Hutch and SWOG

AML blasts harbor multiple genomic, epigenomic, and transcriptomic abnormalities. Proteins are responsible for much of the functional biology derived from these transforming events, yet few studies have examined the global proteome in specimens from AML patients. To gain insight into the biology and prognostic biomarkers for AML, we examined the genome, transcriptome and proteome in highly enriched less differentiated, viable leukemic blasts (VLBs) from previously untreated patients with NPM1 mutated (NPM1+) AML. We focused on NPM1+ AML because 1) NPM1 mutations are the most common genomic abnormality used for risk stratifying AML patients; 2) clinical outcomes for NPM1+ patients vary substantially; and 3) restricting studies to VLBs from patients with NPM1+ AMLreduces the molecular diversity and confounding interactions that may impede identification of biological and clinically relevant biomarkers.Cryopreserved specimens from patients with NPM1+ AML were obtained from the SWOG Leukemia and Fred Hutch/University of Washington Hematopoietic Disease Repositories (N=178). All patients were treated with dose intensive induction and consolidation with curative intent. VLBs were isolated from specimens using fluorescence-activated cell sorting (FACS). Fragment analysis was used to identify FLT3-ITDs, while other genomic mutations were identified using targeted next generation sequencing (NGS). RNA sequencing (RNAseq) was used to quantify transcript expression, while protein expression was quantified using tandem mass tag labelling followed by liquid chromatograph with tandem mass spectrometry (TMT-LC-MS/MS). Prognostic significance of biomarkers was examined by univariate and multivariate analyses, with the later adjusting for both age and ELN2017 risk.Adequate analyte from VLBs were available for RNAseq and TMT-LC-MS/MS studies in 131 and 71 patients, respectively. Proteomic profiling identified 6712 expressed proteins. Multivariable analyses adjusting for age and ELN2017 risk identified 211, 218, and 313 proteins were significantly associated with CR, EFS, and OS, respectively, of which 116 and 174 had a HR ≥ 2 or ≤ 0.5 for EFS and OS (Fig 1A). Analyses showed an overall modest correlation between RNA and protein expression for the 6297 genes present in both RNA and protein datasets (Fig 1B, R =0.27). A total of 163 of the 174 proteomic candidates for OS had RNAseq data, but RNA expression was not significantly correlated with OS for most of these genes (Fig 1C). Similarly, the expression correlation between RNA and protein was not improved by restricting the analyses to these 163 genes (Fig 1D, R =0.28).We also compared protein expression between the NPM1+ VLBs and CD34 progenitors from healthy donors (NLCD34=5), which identified 747 proteins with significant expression differences between the two cell populations (FDR ≤ 0.1). Seventy-three of the 747 proteins displayed > 3-fold increased expression in NPM1+ VLBs, some of which activate targetable pathways (HOMER->WNT/CTNNB1) and/or amenable to adoptive immunotherapies (e.g., CT45). Additional analyses identified 401 proteins with significantly increased expression in VLBs harboring a high FLT3-ITD allelic ratio (NPM1+/FLT3-ITD-H), a less favorable population of patients. In these less favorable patients, many of the same proteins remained overexpressed (e.g., CT45 with 7.85-fold increased expression, FDR=0.009), but other targetable proteins also emerged. For example, PKCD had a 3.41-fold increased expression (FDR=0.001) in the NPM1+/FLT3-ITD-H VLBs, and targetable inhibitors against this protein (e.g., Parsaclisib) are already in clinical trials for other cancers.This study examines for the first time the global proteome in a large number of VLBs from patients with AML. As with recent publications examining other cancers, the overall expression correlation between RNA and protein was modest, at best, and most of the informative prognostic proteins for OS were not significant in the transcriptome. In addition, the results show the potential to identify novel proteomic targets in subpopulations of AML patients, which may not be otherwise discovered using other means. Thus, the results demonstrate the need to include proteomic profiling in future multi-omics studies seeking to discover novel prognostic biomarkers and therapeutic targets. [Display omitted] DisclosuresErba: AbbVie Inc; Agios Pharmaceuticals Inc; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Incyte Corporation; Jazz Pharmaceuticals Inc; Novartis: Speakers Bureau; AbbVie Inc: Other: Independent review committee; AbbVie Inc; Agios Pharmaceuticals Inc; ALX Oncology; Amgen Inc; Daiichi Sankyo Inc; FORMA Therapeutics; Forty Seven Inc; Gilead Sciences Inc; GlycoMimetics Inc; ImmunoGen Inc; Jazz Pharmaceuticals Inc; MacroGenics Inc; Novartis; PTC Therapeutics: Research Funding; AbbVie Inc; Agios Pharmaceuticals Inc; Astellas; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Daiichi Sankyo Inc; Genentech, a member of the Roche Group; GlycoMimetics Inc; Incyte Corporation; Jazz Pharmaceuticals Inc; Kura Oncology; Nov: Other: Advisory Committee. Moseley: BioSight Ltd: Consultancy. Radich: Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Venetoclax Plus Decitabine for Young Adults with Newly Diagnosed ELN Adverse-Risk Acute Myeloid Leukemia: Interim Analysis of a Prospective, Multicenter, Single-Arm, Phase 2 Trial

Background:The standard of induction therapy for fit patients(pts) with acute myeloid leukemia (AML) has not changed much since 1973, when the 7+3 regimen of cytarabine and anthracycline was born. This combination of agents produces complete remission (CR) in about 60-80% of younger adults and in 40-60% of older adults (60 years) depending on genetic risk group. However, compared with AML in favorable- and intermediate-risk categories, induction therapy for fit AML pts in adverse-risk category had significantly lower CR rates and dismal outcome. Recently, combination therapy with venetoclax and azacitidine or decitabine for the treatment of older pts with de novo AML unsuitable for intensive chemotherapy, has resulted in response rates of 60-70%. Currently, the role of venetoclax in combination with azacitidine or decitabine in young adults with newly diagnosed ELN adverse-risk AML remains unclear. This study aims to evaluate the safety and efficacy of venetoclax plus decitabine as induction therapy in young adults with newly diagnosed ELN adverse-risk AML.Methods:This is an interim analysis of an ongoing phase 2 clinical trial (NCT04752527) of a planned 42 untreated ELN adverse-risk AML pts 18-59. The fast next-generation sequencing (NGS) for the most commonly mutated genes in AML using Oncomine TM myeloid research assay on Ion S5 and Chef platform (Thermo Fisher) was completed within 72 hours, which could screen for the ELN adverse-risk features in AML pts together with multiplex RT-PCR and fluorescence in situ hybridization (FISH). In cycle 1, pts receive decitabine 20mg/m 2 on d1-5 and venetoclax escalated from 100mg to 200mg to 400mg until 28-day cycle is finished. For pts with high FLT3-ITD allelic ratio, sorafenib was optional administered at a dose of 400mg orally twice daily. Bone marrow assessments were performed on d28 before subsequent cycle. Pts who achieve composite complete remission (defined as complete remission [CR], CR with incomplete hematologic recovery [CRi], CR with partial hematological recovery [CRh], and morphologic leukemia free state [MLFS]) receive 1 or 2 cycles of consolidation with high dose of cytarabine followed by allogeneic hematopoietic stem cell transplantation. Non-responders or pts who achieve partial remission (PR) receive 1 additional cycle of combination of venetoclax plus decitabine. The primary objective is to determine the composite complete remission rate (CR+CRi+CRh+MLFS). The endpoint is to show that venetoclax plus decitabine is superior to historical control (HC) of cytarabine combined with idarubicin (12 mg/m 2) in young adults with newly diagnosed ELN adverse-risk AML.Results:From February 1, 2021 to July 31, 2021, a total of 104 newly-diagnosed AML pts underwent screening, and 30 pts were classified as ELN adverse-risk category by NGS (within 72 hours) together with multiplex RT-PCR and FISH. Totally, 19 pts with ELN adverse-risk were enrolled, comprising 14 males and 5 females. 2 (10%) pts had secondary AML (sAML). Median age was 38 years (range, 19-57 years). The baseline patient characteristics are summarized in Table 1. Outcome data were updated as of July 2021, for a median follow-up of 2.7 months. Among 14 evaluable pts, 4 (28.6%) achieved a CR, 5 (35.7%) achieved a CRh and 5 (35.7%) had a PR in cycle 1. Individual pts data of this study (n = 14) was compared to a HC, combining individual pts data of AML with adverse-risk from the SZ3202 registry (n = 42) and the pts treated with IA (IDA:12 mg/m 2) in the same period (n = 18). The venetoclax plus decitabine cohort demonstrated higher rates of CR than the HC cohort, with an observed CR/CRh rate of 64.3%, compared with 38.3% (Figure 1).The regimen was well tolerated, with 4- and 8-week mortality rates of 0% and 0%, respectively. The most frequent nonhematologic adverse events of any grade were neutropenic fever (n=6) and pneumonia (n=3). With a median follow-up of 2.7 months, the median remission duration and overall survival (OS) have not been reached. Tumor lysis syndrome occurred in one patient and resolved with medical management.Conclusions:Preliminary results indicate that venetoclax plus decitabine is an effective, lower-intensity regimen that is well tolerated for young adults with newly diagnosed ELN adverse-risk AML, producing high rates of CR, low rates of infections, and low rates of early death. Recruitment of young adults with newly diagnosed ELN adverse-risk AML pts for this trial is ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Proteogenomic Sequencing Allows Early Detection of Relapse Clone with CN-LOH at FLT3-ITD Locus from Initial Diagnosis in AML

IntroductionFLT3-ITD is one of the most common and clinically relevant mutations in acute myeloid leukemia (AML). A high FLT3-ITD allelic ratio (AR) (>0.5) is strongly associated with poor prognosis. FLT3-ITDs are heterogeneous mutations, varying in sizes and locations with some patients having multiple FLT3-ITDs. Unfortunately, conventional clinical techniques are often not adequate in measuring these characteristics at sufficient resolution. The current study aimed to characterize an AML case with 2 FLT3-ITDs at diagnosis, map the multi-omic evolution of AML clones, and understand genetic patterns underlying AML relapse using single-cell proteogenomic sequencing (ScPGseq).MethodsLeukemic samples from a 46-year old female patient diagnosed with de novo AML obtained at initial diagnosis and relapse were archived and used in this study. Based on the 2017 ELN risk stratification, the patient was classified into a low-risk group due to normal karyotype/low FLT3-ITD AR/NPM1+. The patient achieved complete remission (CR) after induction chemotherapy in combination with midostaurin. The patient relapsed 2 months after 4 cycles of consolidation, with a CR duration of 6 months. After relapse, the disease was refractory despite reinduction and salvage therapies, even with gilteritinib (Fig A). ScPGseq was performed using the Mission Bio's AML panel and antibody oligoconjugates for 16 cell surface proteins. Raw sequencing reads were processed using Mission Bio's Tapestri pipeline and results were exported from Tapestri Insights (v3.0.2). All downstream computational and statistical analyses were performed using R and Python.ResultsConsistent with clinical sequencing (R 2 = 0.997) and PCR, ScPGseq identified 5 mutations in FLT3 (21bp and 39bp ITDs and D835Y), NPM1, and WT1 at diagnosis (2367 cells). At relapse, 1 FLT3-ITD (21bp) and mutations in NPM1 and WT1 were detected while the other FLT3-ITD (39bp) and FLT3 D835Y were absent (2611 cells). Clonal analyses of mutation patterns identified 4 AML clones at diagnosis (C1-4) and 1 at relapse (C3R; Fig B). Two closely related clones, C3 and C3R (WT1+/NPM1+/21bp FLT3-ITD+) differed in zygosity of 21bp FLT3-ITD, where nearly all cells in C3R carries homozygous FLT3-ITD. SNP array confirmed the presence of copy neutral loss of heterozygosity (CN-LOH) in chr13q.Analysis of 16 cell surface proteins along with clonal information identified 3 clusters including one devoted to non-leukemic fractions (i.e., no mutations) (top) and another nearly exclusively consisting of mutant cells from diagnosis, which we termed “monocyte-like signature” (right bottom). The other cluster was composed of nearly all mutant cells from the relapse sample and about 1/3 of mutant cells from diagnosis, which we termed “immature myeloid cell signature” (left) (Fig C).When investigating further, C3 cells with immature myeloid cell signature (98/287 cells, 34.1%) exhibited much higher allelic burden of (mean 73% vs. 56%, adj. p < 1.1e-6) and were significantly enriched with homozygous 21bp FLT3-ITDs compared to C3 cells with monocyte-like signature (189/287 cells, 65.9%) (41/98 cells, 42% vs. 11/189 cells, 5.8%, adj. p = 2.7e-13). No other mutations showed similar patterns. It indicates that C3R existed from the initial diagnosis at low frequency, instead of losing heterozygosity during relapse. Without the single cell proteogenomic analyses, C3R cells, present in <5% cells at the initial diagnosis could have not been detected.The multi-omic information elucidates the complete clonal history of this AML (Fig D). Starting with WT1 and NPM1 mutations (C1), 3 FLT3 mutations (2 ITDs and D835Y) were subsequently acquired in 3 subclones (C2-4). A subset of C3 cells (WT1+/NPM1+/21bp FLT3-ITD+) further gained CN-LOH in chr13q (C3R at diagnosis). When treated, AML cells without homozygous 21bp FLT3-ITD were cleared. Cells with homozygous 21bp FLT3-ITD survived/escaped from the treatment and became the dominant clone at relapse (C3R at relapse).ConclusionThe current study demonstrates that ScPGseq allows1) simultaneous and comprehensive analyses of multiple FLT3-ITDs at the single-cell level2) early detection of relapse clone with subclonal homozygous 21bp FLT3-ITD from the initial diagnosis, which explains one of the mechanisms of relapse in AML cases with low FLT3-ITD AR3) multi-omic clonal analyses, which further refine clonal models relying only on mutation profiles. [Display omitted] DisclosuresMinden: Astellas: Consultancy. Kim: Pfizer: Honoraria, Research Funding; Bristol-Meier Squibb: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Paladin: Honoraria, Research Funding.

KMT2A Partial Tandem Duplications (KMT2A-PTD) Is a Rare, but Recurrent Genomic Event in Childhood AML and Associated with High Rate of Co-Occurring FLT3 Mutations

KMT2A partial tandem duplication (KMT2A-PTD), characterized by a large internal duplication spanning 6-8 exons, has been documented in adult AML with a prevalence of 3-10% and is associated with poor outcomes. Despite the high prevalence of KTM2A fusions and known associated outcomes, prevalence and clinical implications of KMT2A-PTD has not been well characterized in childhood AML. We interrogated the transcriptome and associated clinical and genomic data from pediatric AML patients treated on COG AAML1031 to define the prevalence of KMT2A-PTD, frequency of co-occurring genetic mutations and outcomes associated with this variant.Ribo-depleted RNA seq data from 1,294 patients with AML (age 0-29 years) enrolled on COG AAML1031 with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS), disease-free survival (DFS) and relapse risk (RR).Of the 1,294 patients studied, KMT2A-PTD was identified in 20 patients (1.5%). Evaluation of karyotype demonstrated striking paucity of karyotypic alterations with 75% with normal karyotype; 3 cases (15%) of trisomy 11 were identified. In contrast to KMT2A-rearranged (KMT2A-r) AML, which has the highest prevalence in young patients (median age 3.3 years), the median age with KMT2A-PTD was 13.4 years (p<0.0001). Further analysis demonstrated a high rate of co-occurrence of FLT3 mutations; 15 patients (75%) had FLT3-ITD (N=13; 65%) and/or FLT3 activating mutations (N=5; 25%). The rate of co-occurring FLT3-ITD was significantly higher in the KMT2A-PTD cohort compared to patients without KMT2A-PTD (65% vs. 19%, p<0.001). Prevalence of KMT2A-PTD in patients with FLT3-ITD was 5.7%. Additional co-occurring genetic mutations were identified in WT1 (20%) and the RAS pathway. There was a striking enrichment of mutations that are commonly associated with AML in older adults with 8/20 patients (40%) harboring mutations in IDH1/IDH2, U2AF1, and TP53, far in excess of what is seen in other pediatric AML patients (Figure 1A).We evaluated the significance of KMT2A-PTD on clinical outcome. Patients with KMT2A-PTD had a poor response to induction therapy with a morphologic CR rate of 45% and rate of residual disease (MRD) positivity by flow cytometry after initial course of therapy was 40%. Additionally, high rates of relapse were noted for patients with KMT2A-PTD with a RR of 53%. EFS for patients with and without KMT2A-PTD was 39% vs. 46%, respectively (p=0.54) with a corresponding OS of 58% and 64% (p=0.61). Outcome analysis for the KMT2A-PTD cohort demonstrated dual KMT2A-PTD/FLT3-ITD patients had an EFS of 46% compared to 29% for KMT2A-PTD/non-ITD patients (p=0.62, Figure 1B). We inquired whether hematopoietic stem cell transplant (HSCT) may modify outcomes in patients with KMT2A-PTD. Of the 20 patients with KMT2A-PTD, 7 (35%) proceeded to HSCT in CR1, with the indication in 6 of 7 patients due to FLT3-ITD high allelic ratio (HAR, ≥0.4). Stem cell transplant recipients had a DFS of 83% vs. 0% for those who continued on protocol chemotherapy (p=0.002, Figure 1C) with corresponding OS of 100% vs. 43%, respectively (p=0.05).In this large cohort of childhood AML patients treated on AAML1031, we identified a small subset of patients with KMT2A-PTD and show the high prevalence of co-occurring FLT3 mutations. Although KMT2A-PTD patients with and without FLT3-ITD had similar outcomes, the cohort who received HSCT in CR1 (most with FLT3-ITD) experienced improved outcomes compared to the rest of the cohort, suggesting that intensification of therapy may benefit this group of patients overall. Further research into KMT2A-PTD in pediatric AML will guide future risk-adapted therapy and enhance understanding of biologic implications of this lesion, including whether altered KMT2A may serve as a therapeutic target as it may for KMT2A-r AML. [Display omitted] DisclosuresPollard: Syndax: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees.

Gemtuzumab Ozogamicin (GO) In Children With De Novo Acute Myeloid Leukemia (AML) Improves Event-Free Survival (EFS) By Reducing Relapse Risk – Results From The Randomized Phase III Children's Oncology Group (COG) Trial, AAML0531

▪ BackgroundEscalation of treatment intensity for childhood acute myeloid leukemia has improved EFS but with excessive toxicity and toxic mortality (TM), compelling a search for more targeted less toxic alternatives. ObjectiveTo determine if the addition of GO to standard chemotherapy improves EFS in pediatric AML. MethodsPatients < 30 years of age were randomly assigned to receive standard therapy alone (noGO) or to receive two doses of GO 3 mg/m2/dose on day 6 of Induction I (IndI) and on day 7 of Intensification II (IntII) in a 5 cycle chemotherapy backbone previously piloted by COG (Cooper et al, Cancer 2012). Use of stem cell transplant (SCT) was stratified by overall risk group assignment (based on cytogenetics, FLT3-ITD high allelic ratio (HAR), and IndI response) in which high risk patients were allocated to best allogeneic donor SCT after Int I, low risk (LR) patients received chemotherapy only, and intermediate risk (IR) were assigned to SCT if there was a matched family donor (MFD). ResultsBetween 2006-2010, 1022 of 1070 enrolled non-DS pts were eligible for analysis; characteristics are presented in Table 1. Median follow-up was 3.6 (range 0-6.4) years for those alive.Protocol therapy was well tolerated with a TM of 2% in induction and 5% overall with no difference by study arm. VOD was observed in 3% (severe in 0.6%) with no difference by study arm. From time of enrollment, GO was significantly associated with better overall EFS (hazard ratio (HzR) 0.83 (95% CI: 0.7-0.99; p=.04) and relapse-free survival (HzR 0.74 (0.6-0.93; p=.01)) whereas OS was not significantly improved (HzR 0.91 (0.74-1.13)). At 3 yr, noGO v GO EFS was 47 v 53%, p=.05 and OS was 65 v 69%, p=.18. In multivariate analyses, GO was significantly associated with improved EFS than standard therapy after adjustment for significant adverse risk factors: age <2yr, initial WBC>100,000x109/L, and black race. GO was not associated with significantly better induction complete remission (CR) when compared to standard therapy (88% v 85%; p=NS). Table 2 illustrates the overall results by randomized arm and by overall risk group from time of CR showing a consistent reduction in relapse risk in all risk groups. Further risk group analysis found several unique results. In the LR group, relapse rates (RR) trended lower in the GO arm but the benefit was reduced by TM during Int 2 & 3 that was significantly worse in the GO arm (3 v 10%, p=.02). In the IR group, EFS, RR, and OS trended towards improvement with GO. However when pts were censored at the time of SCT (as treated), the outcomes were not significantly different between arms (log-rank p=.14 EFS and p=.81 OS). Table 1 shows an imbalance for SCT received in assigned IR pts with fewer actually receiving SCT in the noGO arm. As such, Table 2 shows outcomes by intent-to-treat for IR pts with MFD censored and by as treated for IR pts receiving MFD SCT. Noteworthy in the IR group alone who did receive a SCT, GO arm pts had a significantly better DFS than the noGO arm (intent to treat: p=.022; as treated: p=.044). However, for the IR pts randomized to the noGO arm, SCT failed to provide benefit. For HR pts achieving CR they as well trended towards better survival & RR in the GO arm. ConclusionGO improves EFS in children, adolescents and young adults with AML by reducing the risk of relapse among those achieving remission. Disclosures:No relevant conflicts of interest to declare.

Validation of the ELN 2017 Classification for AML with Intermediate Risk Cytogenetics with or without NPM1 -Mutations and High or Low Ratio FLT3-ITD s

IntroductionNucleophosmin 1-mutations (NPM1) and internal tandem duplications in the FMS-like tyrosine kinase 3 receptor gene (FLT3-ITDs) are the most frequently detected molecular abnormalities in patients with AML and are used for risk assessment and stratification of post-remission therapy. In the 2017 ELN recommendations (Döhner et al, Blood 2017) FLT3-ITD mutations are grouped according to the ratio of the mutant (mt) to wild type (wt) alleles into low ratio (LR) and high allelic ratio (HR) mutations using a cut-off of 0.5. AML with intermediate risk (IR) cytogenetic abnormalities and NPM1 -mutant FLT3- wt AML is grouped together with NPM1 -mutant and LR FLT3-ITD as favorable risk AML, only NPM1 -wt HR FLT3-ITD AML is considered adverse risk. Since validation studies have not been published so far for the 2017 ELN recommendations, we analyzed survival of patients with IR cytogenetic abnormalities who were treated in randomized controlled trials on intensive induction chemotherapy of the Study Alliance Leukemia (SAL) in the past two decades.MethodsWe performed a retrospective study on all patients who had received intensive induction chemotherapy for newly diagnosed AML within randomized SAL trials with available data on ELN 2017 classification. All molecular studies were performed centrally in one laboratory. Cox regression analyses were fitted with baseline risk factors including genetic markers for the full analysis set. Interaction tests were used to determine the impact of gene-gene interactions. Extended Cox regression models with a time-dependent covariate modeling the time of allogeneic hematopoietic stem cell transplantation (alloHCT) were used to study the role of alloHCT for patients who had reached first complete remission (CR1).ResultsIn total, data on 1610 IR-patients was evaluable. The median age at first diagnosis of AML was 55 years (range, 18 to 87 years), both sexes were equally represented and 72% of patients had normal karyotype AML. Eighty-three percent of patients had de novo AML, 12% secondary AML and 3% therapy-related AML. The median follow-up was 81 months (interquartile range, 63 to 103 months). Numbers of patients in the respective genetic subgroups defined by the 2017 ELN classifications and 5-year overall survival (5-yr-OS) probabilities from diagnosis were as follows (see also Figure 1): 418 patients with FLT3-ITD wt NPM1 mt AML with 50% 5-yr-OS and 119 patients with LR FLT3-ITD with NPM1 mt AML with 41% 5-yr-OS were classed together as favorable AML, 741 patients with FLT3-ITD wt NPM1 wt AML with 27% 5-yr-OS, 170 patients with HR FLT3-ITD with NPM1 mt AML with 31% 5-yr-OS, and 70 patients with LR FLT3-ITD with NPM1 wt AML also with 31% 5-yr-OS were classed as intermediate risk AML and finally 92 patients with HR FLT3-ITD with NPM1 wt AML with 20% 5-yr-OS were classed as adverse risk AML. In any combination NPM1 mutations had a protective effect. Interaction tests indicated that the combination of an NPM1 -mutation and LR FLT3-ITD increased the risk of mortality compared to the single mutations (Wald-test for interaction, p=0.08).When alloHCT in first complete remission was analyzed in an extended Cox regression analysis as time-dependent covariate, the impact differed among patients assigned to the three risk groups defined by the 2017 ELN recommendations: Results indicated that alloHCT had no impact on overall survival in patients with favorable AML HR 1.0 (95%-CI, 0.8 - 1.2), but a beneficial impact in patients with intermediate risk AML HR 0.8 (95%-CI 0.7 - 0.9) or high risk AML HR 0.7 (95%-CI 0.5 - 1.0).ConclusionsThe risk classification of the 2017 ELN recommendations according to NPM1 and FLT3-ITD genotypes was validated in this large retrospective analysis including data from 1610 patients with newly diagnosed AML with intermediate risk cytogenetic abnormalities or normal karyotype. The time-dependent analysis of alloHCT indicated that patients with favorable AML should not receive alloHCT as post-remission therapy in CR1, whereas patients with intermediate risk or high risk AML would benefit from alloHCT in CR1. [Display omitted] DisclosuresSchetelig:Sanofi Aventis: Consultancy, Research Funding; Roche: Honoraria; Abbvie: Honoraria; Janssen: Consultancy, Honoraria. Rollig:Janssen: Research Funding; Bayer: Research Funding. Kayser:TEVA: Honoraria; Novartis: Honoraria, Speakers Bureau. Stoelzel:medac: Other: Travel support. Bug:Amgen: Honoraria; Janssen: Other: Travel Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel funding; Astellas: Other: Travel Funding; Jazz Pharmaceuticals: Other: Travel Funding. Mayer:Novartis: Research Funding; Eisai: Research Funding. Thiede:Novartis: Consultancy, Speakers Bureau; Roche: Consultancy; Agendix: Employment; Bayer: Consultancy, Speakers Bureau.

Revised Risk Stratification Criteria for Children with Newly Diagnosed Acute Myeloid Leukemia: A Report from the Children's Oncology Group

Introduction: Currently, most phenotypic, cytogenetic or molecular markers identified in children with acute myeloid leukemia (AML) are not used for risk stratification or treatment assignment. Historically, in the Children's Oncology Group (COG) pilot study AAML03P1, patients with core binding fusion (CBF) [t(8;21), inv(16)] or with NPM1 or CEBPA mutations had a more favorable outcome. Patients with FLT3 -ITD with high allelic ratio (FLT3 -ITD-HAR), monosomy 7, or -5/del(5q) had more adverse outcomes. In AAML03P1, 31% of patients were deemed low risk (LR), 9% were high risk (HR) and the remaining 60% were considered standard risk (SR) with event-free survival (EFS) of approximately 50% (Figure 1A). The same risk classification was carried forward to the successor Phase III COG study AAML0531 with similar outcomes. To develop an improved risk stratification strategy, we conducted a comprehensive retrospective evaluation of potential biomarkers that have been reported using available karyotype, immunophenotype and next generation sequencing (NGS) data from patients treated on AAML0531.Methods: AAML0531 enrolled 1,022 patients (age 0-29 years) and randomized to standard therapy with or without gemtuzumab ozogamicin. Centrally reviewed data karyotype and FLT3, NPM1 and CEBPA status were available for this study. An additional 740 individual diagnostic specimens were interrogated by NGS (whole genome, transcriptome or targeted capture sequencing) to identify additional somatic variants including cryptic fusions, single nucleotide (SNV) and copy number variants (CNVs). Post induction response by multidimensional flow cytometry (MDF), an important prognostic modality, was also analyzed for risk stratification assignment in SR patients. Information from genomic and MDF parameters were analyzed for association with event free (EFS) and disease free survival (DFS).Results: Table 1 shows the cytogenetic, molecular, and immunophenotypic markers used in our new risk stratification model. HR fusions detected by karyotype included 5 distinct KMT2A (MLL) fusion types, DEK-NUP214, NPM1 - MLF1, MECOM (3q26.2), and KAT6A (8p11.21). HR cryptic fusions detected by NGS include CBFA2T3-GLIS2, NUP98 family fusions (NUP98-NSD1, NUP98-KDM5A, NUP98-HMGB3 and NUP98-HOXD13) as well as ETS transcription factor family members (ETV6, ERG, FEV). NGS also identified HR MLL, t(6;9) or ETS fusions in 10% of patients who were initially reported as having a normal karyotype. HR stratification by immunophenotype analysis includes the RAM phenotype that was reported in younger patients with overlap with FAB-M7 as well as CBFA2T3-GLIS2 fusions.When karyotype, immunophenotype, and NGS analyses were retrospectively applied to patients treated in AAML0531, the distribution of patients in each risk group changed significantly compared to AAML03P1 (Figure 1A and 1B). According to the revised risk stratification, HR variants constituted 34% of all AML patients and had poor prognosis (EFS-26.3% ± 5.4%). Patients with CBF AML, NPM1 or CEBPA mutations were included in the LR cohort and those with RBM15-MLK1 fusion were relocated to the LR cohort and these collectively constitute 33% of AML cases (EFS-70.4% ± 5.6%). The SR cohort was reduced to 33% in this proposed risk stratification schema (EFS-48.6% ± 6.2%). Minimal residual disease (MRD) by MDF at end of first induction was factored for SR patients in the proposed risk classification (Figure 1C) and found to have prognostic significance; therefore SR patients were assigned to either HR (DFS-28.5% ± 5.2%) or LR (DFS-63.7% ± 4.5%) cohorts.Conclusions: Analysis of refined karyotyping and molecular data and incorporation of validated NGS data for patients in AAML0531 significantly altered the risk stratification used to predict relapse and assign patients to appropriate therapy. This report is the first to study a large number of patients assigned to a clinical trial and apply new standards for karyotyping, molecular and NGS. In future studies, we plan to study all patients on COG trials by NGS methods, validate additional novel biomarkers, and to identify new targets for future precision medicine-based clinical trials. [Display omitted] DisclosuresLoken:Hematologics Inc: Employment, Equity Ownership. Brodersen:Hematologics Inc: Employment.

Genetic identification of patients with AML older than 60 years achieving long-term survival with intensive chemotherapy

AbstractTo design a simple and reproducible classifier predicting the overall survival (OS) of patients with acute myeloid leukemia (AML) ≥60 years of age treated with 7 + 3, we sequenced 37 genes in 471 patients from the ALFA1200 (Acute Leukemia French Association) study (median age, 68 years). Mutation patterns and OS differed between the 84 patients with poor-risk cytogenetics and the 387 patients with good (n = 13), intermediate (n = 339), or unmeasured (n = 35) cytogenetic risk. TP53 (hazards ratio [HR], 2.49; P = .0003) and KRAS (HR, 3.60; P = .001) mutations independently worsened the OS of patients with poor-risk cytogenetics. In those without poor-risk cytogenetics, NPM1 (HR, 0.57; P = .0004), FLT3 internal tandem duplications with low (HR, 1.85; P = .0005) or high (HR, 3.51; P < 10−4) allelic ratio, DNMT3A (HR, 1.86; P < 10−4), NRAS (HR, 1.54; P = .019), and ASXL1 (HR, 1.89; P = .0003) mutations independently predicted OS. Combining cytogenetic risk and mutations in these 7 genes, 39.1% of patients could be assigned to a “go-go” tier with a 2-year OS of 66.1%, 7.6% to the “no-go” group (2-year OS 2.8%), and 3.3% of to the “slow-go” group (2-year OS of 39.1%; P < 10−5). Across 3 independent validation cohorts, 31.2% to 37.7% and 11.2% to 13.5% of patients were assigned to the go-go and the no-go tiers, respectively, with significant differences in OS between tiers in all 3 trial cohorts (HDF [Hauts-de-France], n = 141, P = .003; and SAL [Study Alliance Leukemia], n = 46; AMLSG [AML Study Group], n = 223, both P < 10−5). The ALFA decision tool is a simple, robust, and discriminant prognostic model for AML patients ≥60 years of age treated with intensive chemotherapy. This model can instruct the design of trials comparing the 7 + 3 standard of care with less intensive regimens.

Cup-like blasts in acute myeloid leukaemia with NPM1 and IDH2 mutations.

An 86-year-old woman presented with fever and pneumonia. Her laboratory investigations showed leucocytosis (15 × 10⁹/L) with 92% blasts, anaemia (haemoglobin 5.5 g/dl), thrombocytopenia (platelet 31 × 10⁹/L), and elevation of fibrinogen degradation products (FDPs > 120 μg/ml). The patient refused bone marrow examination. Hypogranular blasts without Auer rods containing nuclear invagination, the so-called “cup-like” or “fish-mouth” appearance, accounted for approximately 17% of the blasts in peripheral blood. Blasts were strongly positive for myeloperoxidase and showed normal female karyotype (46,XX) [Figure 1]. The immunophenotypic features of the blast included expression of CD13(dim), CD33 and CD56 and absence of CD34 and HLA-DR expression, which mimicked those of acute promyelocytic leukaemia (APL) [Figure 2]. Targeted next-generation sequencing using the Oncomine Myeloid Research Assay (Thermo Fisher Scientific, MA, USA) revealed pathogenic variants with allele frequencies greater than 1% in nucleophosmin 1 (NPM1; p.Trp288CysfsTer12), isocitrate dehydrogenase 2 (IDH2; p.Arg140Gln) and Fms-like tyrosine kinase 3 (FLT3; p.Asn676Lys), of which the variant allele frequencies (VAF) were 47.94%, 51.3%, and 3.55%, respectively. The patient was eventually diagnosed with acute myeloid leukaemia (AML) with mutated NPM1. AML with cup-like blasts (CLB-AML) is defined as the presence of more than 10% of blasts with nuclear invaginations that span at least 25% of the nuclear diameter, which are commonly associated with AML M1/2 (myeloid) and M4/5 (myelomonocytic/monocytic) in the FAB classification. NPM-1 and FLT3-ITD mutations have been detected in more than 60% of CLB-AML cases with normal karyotype. NPM1-mutatd AML showing APL-like immunophenotype, negative for both CD34 and HLA-DR, often harbours co-mutations in IDH1/2 or TET2 [1]. Furthermore, high D-dimer/FDP levels, CLB, and APL-like immunophenotype are frequently observed in AML with mutated NPM1 harbouring FLT3 or IDH1/2 co-mutations [2]. Our case is consistent with these clinical, morphological, and molecular features. VAF of FLT3 mutation is less than 5% and much lower compared to that of NPM1 and IDH2, which means that FLT3 mutation may not affect the cup-like morphology and clinical presentation in our case. NPM1-mutated AML carrying concurrent FLT3-ITD mutation especially with high (≥0.5) allelic ratio is well known to be associated with poor prognosis. Molecular analysis is very important in CLB-AML with APL-like phenotype in terms of predicting prognosis and determining the optimal treatment strategy including allogeneic stem cell transplantation or molecular targeted therapies such as FLT3 or IDH1/2 inhibitors. We thank Dr. Makoto Ikejiri, Dr. Kaname Nakatani, and Dr. Keiki Nagaharu at Mie University Hospital for NGS analysis. We thank Gabrielle White Wolf, PhD, from Edanz Group (http://en-author-services.edanz.com/ac) for editing a draft of this manuscript. Y. Sugimoto has received honoraria from Novartis, funding from Takara Bio, and research support from Astellas, Kyowa Kirin, and Ono, and is an accepted researcher from Shojunkai Takeuchi Hospital. All other authors do not have any conflicts of interest to declare.

Trudności w leczeniu chorego na ostrą białaczkę szpikową z mutacją FLT3-ITD i wysokim stosunkiem allelicznym — oporność na standardową chemioterapię indukującą w połączeniu z midostauryną

The development of targeted therapies in AML patients enabling treatment individualization, such as new FLT3 tyrosine kinase inhibitors, is a promising option for improving treatment outcomes and prolonging patient survival. However, the treatment of patients with a high FLT3-ITD allelic ratio (FLT3-ITD high ) associated with an extremely unfavourable prognosis remains a major clinical problem. The study presents a case of a 20-year-old patient with FLT3-ITD high extramedullary AML at diagnosis. Individualized chemotherapy according to the DA ‘3 + 7’ regimen combined with midostaurin was administered. After the induction treatment, complete remission (CR) was not achieved. After second induction chemotherapy, CR1 was achieved with the presence of residua disease. One cycle of consolidation chemotherapy was then administered, and after myeloablative conditioning allogeneic hematopoietic stem cell transplantation from an unrelated donor was performed. The patient has remained in CR with no residual disease for 18 months.

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.

Clinical Outcomes Following Treatment with Gilteritinib or Quizartinib in Patients with Relapsed/Refractory FLT3-ITD+ Acute Myeloid Leukemia

Background: Gilteritinib is approved for patients (pts) with relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML), based on findings from the phase 3 ADMIRAL trial (Perl AE, et al. N Engl J Med. 2019). A phase 3 trial, QuANTUM-R, demonstrated the benefit of quizartinib in pts with R/R AML with FLT3 internal tandem duplication (FLT3-ITD) mutations (Cortes JE, et al. Lancet Oncol. 2019). Although eligibility criteria across both studies were similar, QuANTUM-R was more stringent as to prior therapy intensity and remission duration, which potentially enriched for higher-risk pts. We sought to describe outcomes from ADMIRAL among pts who otherwise met eligibility for QuANTUM-R. Methods: In this post-hoc analysis, a subset of pts from ADMIRAL were matched with R/R FLT3-ITD+ AML pts from QuANTUM-R on the basis of baseline characteristics and prior treatment criteria. Matched pts were either refractory to initial anthracycline-based chemotherapy or had relapsed ≤6 mos after achieving composite complete remission (CRc) with an anthracycline-based regimen. Results: Overall, 218 pts with R/R FLT3-ITD+ AML in the ADMIRAL trial (gilteritinib, n=140; salvage chemotherapy [SC], n=78) were matched with the QuANTUM-R intention-to treat (ITT) population (N=367; quizartinib, n=245; SC, n=122). Proportions of pts preselected for high-intensity SC were 66% (n=143/218) in the matched ADMIRAL ITT population and 77% (n=281/367) in the QuANTUM-R ITT populations. Demographic and baseline characteristics of the matched ADMIRAL ITT population and QuANTUM-R ITT population were similar. Median durations of exposure to gilteritinib and quizartinib were 3.8 mos and 3.2 mos, respectively, and median number of treatment cycles received were five and four, respectively. Rates of hematopoietic stem cell transplantation (HSCT) were similar in pts treated with gilteritinib (35%; n=49/140) or quizartinib (32%; n=78/245), as were the proportions of pts who resumed gilteritinib (23%; n=32/140) or quizartinib (20%; n=48/245) therapy post-HSCT. Median overall survival (OS) in pts treated with gilteritinib or quizartinib was longer than that observed with SC. After a median follow-up of 17.4 mos, median OS was 10.2 mos with gilteritinib versus 5.6 mos with SC (hazard ratio [HR]=0.573 [95% CI: 0.403, 0.814]; one-sided nominal P=0.0008). After a median follow-up of 23.5 mos, median OS with quizartinib was 6.2 mos versus 4.7 mos with SC (HR=0.76 [95% CI: 0.58-0.98]; one-sided P=0.02). After censoring for HSCT, median OS was 9.3 mos with gilteritinib versus 5.5 mos with SC (HR=0.525 [95% CI: 0.356-0.775]; nominal one-sided P=0.0005), and 5.7 mos versus 4.6 mos with quizartinib versus SC, respectively (HR=0.79 [95% CI: 0.59, 1.05]; one-sided P=0.05). In both QuANTUM-R and matched ADMIRAL populations, the survival benefits of quizartinib and gilteritinib compared with SC were maintained across multiple subgroups, including high FLT3-ITD allelic ratio subsets. Compared with SC, high CRc rates were observed in pts treated with either gilteritinib (57%; n=80/140) or quizartinib (48%; n=118/245). The complete remission (CR) rate with gilteritinib was 23% (n=32/140), whereas the CR rate with quizartinib was 4% (n=10/245) (Table). Median time to achieve CRc was 1.8 mos with gilteritinib and 1.1 mos with quizartinib, median duration of CRc was 5.5 mos with gilteritinib and 2.8 mos with quizartinib. The safety profiles of gilteritinib and quizartinib were generally similar, though aspartate or alanine aminotransferase elevations (any grade) were more frequent with gilteritinib (41-44%) than quizartinib (≤13%), whereas neutropenia (14% vs 34%, respectively), fatigue (24% vs 39%, respectively), and prolonged QT intervals (9% vs 27%, respectively) were more frequent with quizartinib. Conclusions: In pts with R/R FLT3-ITD+ AML and similar baseline characteristics, both gilteritinib and quizartinib were generally well tolerated and associated with improved survival and treatment response compared with SC. Responses to gilteritinib and quizartinib, as measured by CRc, were similar; blood count recovery varied between the two FLT3 inhibitors. Although cross-study comparisons have substantial limitations, the findings suggest that while remission is achieved faster with quizartinib, response may be more durable and survival potentially longer with gilteritinib. Disclosures Perl: Syndax: Consultancy, Honoraria; Leukemia &amp; Lymphoma Society, Beat AML: Consultancy; Novartis: Honoraria, Other, Research Funding; Agios: Consultancy, Honoraria, Other; Jazz: Honoraria, Other; FORMA Therapeutics: Consultancy, Honoraria, Other; Daiichi Sankyo: Consultancy, Honoraria, Other: Writing/editorial support, travel costs for meetings, Research Funding; FUJIFILM Pharmaceuticals USA, Inc: Research Funding; New Link Genetics: Honoraria, Other; Arog Pharmaceuticals Inc: Other: uncompensated consulting, travel costs for meetings; Actinium Pharmaceuticals Inc: Consultancy, Honoraria, Research Funding; Biomed Valley Discoveries: Research Funding; Astellas: Consultancy, Honoraria, Other: writing/editorial support, travel costs for meeting presentations related to study, Research Funding; Bayer HealthCare Pharmaceuticals: Research Funding; AbbVie Inc: Consultancy, Honoraria, Other, Research Funding; Takeda: Honoraria, Other: Travel costs for meeting; Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly &amp; Company: Consultancy, Honoraria, Other. Lu:Astellas: Current Employment. Fan:Astellas Pharma: Current Employment. Hasabou:Astellas Pharma: Current Employment. Berrak:Astellas: Current Employment. Tiu:Eli Lilly &amp; Company: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Astellas Pharma Global Development: Current Employment.

Effect of Dexrazoxane on Left Ventricular Systolic Function and Treatment Outcomes in Patients With Acute Myeloid Leukemia: A Report From the Children's Oncology Group.

To determine whether dexrazoxane provides effective cardioprotection during frontline treatment of pediatric acute myeloid leukemia (AML) without increasing relapse risk or noncardiac toxicities of the chemotherapy regimens. This was a multicenter study of all pediatric patients with AML without high allelic ratio FLT3/ITD treated in the Children's Oncology Group trial AAML1031 between 2011 and 2016. Median follow-up was 3.5 years. Dexrazoxane was administered at the discretion of treating physicians and documented at each course. Ejection fraction (EF) and shortening fraction (SF) were recorded after each course and at regular intervals in follow-up. Per protocol, anthracyclines were to be withheld if there was evidence of left ventricular systolic dysfunction (LVSD) defined as SF < 28% or EF < 55%. Occurrence of LVSD, trends in EF and SF, 5-year event-free survival (EFS) and overall survival (OS), and treatment-related mortality (TRM) were compared by dexrazoxane exposure. A total of 1,014 patients were included in the analyses; 96 were exposed to dexrazoxane at every anthracycline course, and 918 were never exposed. Distributions of sex, age, race, presenting WBC count, risk group, treatment arm, and compliance with cardiac monitoring were similar for dexrazoxane-exposed and -unexposed patients. Dexrazoxane-exposed patients had significantly smaller EF and SF declines than unexposed patients across courses and a lower risk for LVSD (26.5% v 42.2%; hazard ratio, 0.55; 95% CI, 0.36 to 0.86; P = .009). Dexrazoxane-exposed patients had similar 5-year EFS (49.0% v 45.1%; P = .534) and OS (65.0% v 61.9%; P = .613) to those unexposed; however, there was a suggestion of lower TRM with dexrazoxane (5.7% v 12.7%; P = .068). Dexrazoxane preserved cardiac function without compromising EFS and OS or increasing noncardiac toxicities. Dexrazoxane should be considered for cardioprotection during frontline treatment of pediatric AML.

Outcome of Childhood Acute Myeloid Leukemia With FLT3-ITD Mutation: The Experience of Children’s Cancer Hospital Egypt, 2007-17

IntroductionThe presence of FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation in pediatric acute myeloid leukemia (AML) is associated with high rates of induction failure and worse survival. Its presence places the patient into a high-risk group. We aimed to describe the outcome of pediatric AML with FLT3-ITD mutation. Patients and MethodsWe performed a retrospective analysis of cases of AML from July 2007 till July 2017 at Children’s Cancer Hospital Egypt. ResultsSeventy-one patients had FLT3 gene mutation out of 687 patients with AML. Sixty-five patients had FLT3 gene mutation with allelic ratio > 0.4; 43 (66.1%) of 65 patients experienced complete remission (CR). Of the 43 patients, 16 patients maintained CR, 18 patients relapsed after first CR, 8 patients died, and 1 patient was lost to follow-up. Patients with relapsing disease died after salvage chemotherapy, except for one patient, who was alive after second CR. Allogeneic bone marrow transplantation (allo-BMT) was performed for 9 (13.8%) of 65 patients in first CR, of whom 8 were alive and in CR, and 1 patient experienced disease relapse and died. Seven patients (10.7%) were alive without allo-BMT. Three years’ overall and event-free survival for patients with FLT3-ITD mutation with high allelic ratio was 26.9% and 22.8%, respectively. Three years’ overall and event-free survival for patients treated with allo-BMT was 77.8% and 78.8%, respectively, versus patients treated without allo-BMT, 16.3% and 12.8%, respectively. ConclusionFLT3-ITD mutation in pediatric AML was associated with poor treatment outcomes, and the survival of relapsing patients was extremely poor. Allo-BMT in first remission was the best treatment option. Alternative donor transplants and FLT3 inhibitors are needed to improve outcome in developing countries.

Sorafenib in Combination with Standard Chemotherapy for Children with High Allelic Ratio FLT3/ITD+ AML Improves Event-Free Survival and Reduces Relapse Risk: A Report from the Children's Oncology Group Protocol AAML1031

INTRODUCTION Sorafenib, a multi-kinase tyrosine kinase inhibitor (TKI) targets FLT3 internal tandem duplication (FLT3/ITD) mutations and has efficacy in adult FLT3/ITD+ AML. High allelic ratio (HAR) FLT3/ITD mutations (allelic ratio of &amp;gt; 0.4, referred to as FLT3/ITD+) confer poor prognosis in de novo pediatric AML. COG AAML1031 Arm C studied the feasibility and efficacy of adding sorafenib to standard chemotherapy and as a single agent maintenance therapy in pediatric HAR FLT3/ITD+ patients. METHODS The AAML1031 primary randomization (Arm A vs. B) has been previously reported (Aplenc et al. Blood 2016;128:899). Patients with FLT3/ITD+ AML were offered secondary enrollment on Arm C and toxicities were monitored on respective treatment arms. An initial safety phase (Cohort 1, C1; N=12) defined the maximum tolerated dose (MTD) of sorafenib when added to induction II chemotherapy and subsequent courses. Following completion, the study was amended (Cohort 2; C2) to start sorafenib on day 11 of induction I and concomitantly with chemotherapy in subsequent cycles. A year of sorafenib maintenance was also added. Due to concerns for cardiac toxicity on interim analyses, the study was amended (Cohort 3; C3) to initiate sorafenib after chemotherapy completion in all courses. Sorafenib pharmacokinetics (PK) and plasma inhibitory activity (PIA) were measured in a subset of patients. Clinical outcome analysis was limited to cohorts 2/3 given lack of induction I sorafenib exposure in C1. Arm C results were compared to N=34 HAR FLT3/ITD+ AML patients on the control arm of COG AAML0531 (Arm A), a group that received comparable chemotherapy without sorafenib. Complete remission (CR), event-free survival (EFS), overall survival (OS) and relapse risk (RR) of patients with HAR FLT3/ITD AML enrolled on the 2 trials were compared to assess the impact of sorafenib use. Cox proportional hazards were used for multivariable analyses. RESULTS In the dose finding phase (C1), 12 patient were enrolled and the MTD of sorafenib defined as 200 mg/m2; dose limiting toxicities included rash (N=2; 1 grade III, 1 grade II), grade II hand foot syndrome and grade III fever. For the evaluation of efficacy, 80 C2/C3 (C2: N=33, C3: N=47) patients were enrolled. 30/80 (37.5%) received at least 1 maintenance cycle; 20/80 (25%) completed all courses. As outcomes were compared to N=34 HAR FLT3/ITD+ AML patients enrolled on the preceding trial AAML0531 Arm A as a historical comparison, clinical characteristics were described for the 2 groups (Table 1). For AAML1031 C2/C3, rates of induction I CR were 73% vs. 56% for AAML0531 Arm A (p=0.078). Following induction II, CR rates were 91% and 70% respectively (p=0.007). 3 year EFS from study entry was 57.5% for Arm C vs. 34.3% for AAML0531 Arm A; (p=0.007) whereas 3 year OS from study entry was not significantly different (63.9% vs. 54.1%, p=0.375, Figure 1A). RR from CR was reduced with sorafenib treatment (Arm C 18.2% vs. historical 52.5%, p=0.006, Figure 1B). The improvement in EFS and RR observed with sorafenib was retained in those with wild-type nucleophosmin (NPM-) FLT3/ITD+ AML but lacked statistical significance compared to historical in NPM+ FLT3/ITD+ disease (Figure 2A/2B). As 21/34 (62%) patients in the historical control did not undergo HSCT there was concern that the improved outcome observed for Arm C reflected increased use of HSCT in AAML1031. Multivariable analysis that adjusted for HSCT resulted in significantly lower EFS and higher RR for FLT3/ITD+ patients on AAML0531, suggesting that increased use of HSCT on Arm C did not explain the improved outcomes observed (EFS: HR 2.04, CI 1.18 - 3.53; p=0.01, RR: HR 3.09, CI 1.28 - 7.49; p=0.012). While dose modifications were more frequent for FLT3/ITD+ patients on Arm C versus arms A/B of AAML1031, targeted toxicity rates were otherwise no higher (Table 2). PIA assays demonstrated median trough FLT3 inhibition of 92% (n=29, 95% CI 79-94%), 91% (n=84, 95% CI 87-94%) and 81% (n=70, 95% CI 76-90%) for induction I, induction II and intensification I, respectively. CONCLUSION Addition of sorafenib to Arm C of AAML1031 was safe and resulted in potent FLT3 inhibition, particularly early in therapy. Sorafenib improved rates of induction II CR as well as 3 year EFS and reduced RR from CR compared to historical controls. These data support use of sorafenib in pediatric patients with HAR FLT3/ITD+ AML. Disclosures Fisher: Pfizer: Research Funding; Astellas: Other: Data Safety Monitoring Board Chair for an antifungal study; Merck: Research Funding. Levine:Viracor: Patents &amp; Royalties: biomarker patent; Biogen: Other: non-financial support; Ironwood: Consultancy; Bluebird Bio: Consultancy; National Cancer Institute: Research Funding; Novartis: Consultancy; Incyte: Consultancy, Research Funding; Kamada: Research Funding. Loken:Hematologics, Inc: Employment, Equity Ownership. OffLabel Disclosure: sorafenib for high allelic ratio FLT3/ITD+ AML

Prospective Evaluation of Allogeneic HSCT at the First Remission for Younger Adults with FLT3-ITD Positive Acute Myeloid Leukemia: The JALSG AML209-FLT3-SCT Study

Prospective Evaluation of Allogeneic HSCT at the First Remission for Younger Adults with FLT3-ITD Positive Acute Myeloid Leukemia: The JALSG AML209-FLT3-SCT Study

Occurrence and Resolution of Anthracycline Cardiotoxicity and Impact on Treatment Outcomes Among Children Treated on the AAML1031 Clinical Trial: A Report from the Children's Oncology Group

Background: Children treated for acute myeloid leukemia (AML) receive high doses of anthracyclines. Anthracycline cardiotoxicity causes significant short and long term morbidity and mortality, thus it is crucial to understand its risk factors, natural history, and impact on treatment outcome. Methods: Between June 2011 and September 2018, AAML1031 enrolled patients aged &amp;lt;30 years for treatment of de novo AML. Echocardiographic evaluations (echo) were required before each treatment course, at the end of protocol therapy, and at yearly intervals during off-protocol follow-up. The lowest shortening fraction (SF) and ejection fraction (EF) in each reporting period were prospectively collected. Cardiotoxicity was defined as SF &amp;lt;28% or EF &amp;lt;55% which represent the protocol-mandated thresholds at which anthracyclines were to be withheld. Higher-grade toxicity (SF &amp;lt;24%, EF &amp;lt;50%) consistent with grade 2 or higher left ventricular systolic dysfunction (grade 2+ LVSD) per CTCAE v3 definitions, and its resolution were also assessed. Resolution was defined as SF returning to baseline or &amp;gt;30% and EF returning to baseline or &amp;gt;60%. Resource utilization (RU) data for patients enrolled at hospitals contributing to the Pediatric Health Information Systems database (PHIS) were merged with AAML1031 study data. Cox regressions were used to assess risk factors for incident cardiotoxicity and recovery among cases, as well as to compare event-free (EFS) and overall survival (OS) in patients with and without cardiotoxicity. Poisson regressions were used to compare on-protocol RU. Cardiotoxicity was treated as a time-varying exposure introduced at time of documented onset in comparisons of EFS, OS and RU. Patients with high allelic ratio FLT3/ITD+ AML were excluded from analyses. Results: 1092 patients were treated on AAML1031 and included in the primary analyses. Echo compliance was high overall (mean ±sd: 74.5% ±19.6), but lower and more variable during off-protocol (mean ±sd: 58.1%, ±31.0) than on-protocol (mean ±sd: 90.6% ±19.5) reporting periods. Over a median follow-up of 3.5 years, 39% (n=428) of patients experienced protocol-defined cardiotoxicity, of whom 54% (n=232) suffered grade 2+ LVSD. Cumulative incidence was higher among patients randomized to receive bortezomib, non-infants, females, and patients with low risk disease who receive more cumulative anthracycline during frontline therapy (Table 1). Patients treated with dexrazoxane were less likely to develop cardiotoxicity. Among patients with grade 2+ LVSD, 49% experienced recovery of cardiac function within a median of 14 months (IQR: 9-20) from initial detection. High risk disease classification and dexrazoxane exposure were associated with LVSD recovery (Table 1). Both EFS (HR 1.23, 95% CI: 1.01-1.50) and OS (HR 1.45, 95% CI 1.15-1.84) were significantly worse in patients with cardiotoxicity meeting per-protocol thresholds for treatment modification. The magnitude of the association with EFS (HR 1.24, 95% CI: 0.98, 1.58) was similar when restricting to grade 2+ LVSD, but more pronounced for OS (HR 1.80, 95% CI 1.38, 2.34). A subset of 389 patients (23% suffered grade 2+ LVSD) were treated at PHIS hospitals and contributed to RU analyses. Cardiotoxicity was associated with greater cardiac ICU requirements, increased use of cardiac-directed interventions including antihypertensives, vasopressors, and heart failure medications, and reduced cardioprotection (Table 2). Conclusions: Echo compliance and cardiotoxicity rates were higher than previously described due in part to more frequent echoes and required reporting of raw echo results. Approximately 20% of pediatric AML patients on AAML1031 developed grade 2+ LVSD, only half of whom had documented resolution of LVSD. Dexrazoxane was strongly associated with reduced LVSD risk and potentially superior recovery. Our results offer further evidence that early cardiotoxicity decreases EFS/OS and that cumulative anthracycline dose is the principal predictor of LVSD. These data argue for comparative effectiveness studies of cardiac-directed interventions after LVSD, and identification of clinical and genetic risk factors for anthracycline cardiotoxicity and its recovery. Such studies may allow for anthracycline de-escalation in select patient populations. Work is ongoing to inform optimal supportive care practices for patients with LVSD using clinical data merged with PHIS. Disclosures Leger: Abbott: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BTG Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Characteristics and Outcome of Patients with Core Binding Factor Acute Myeloid Leukemia and FLT3-ITD: Results from an International Collaboration

Background: Core binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) and is associated with a favorable outcome, particularly if treated with repetitive cycles of high-dose cytarabine as post-remission therapy. Long-time 10-year overall survival (OS) rate was reported of 58% in FLT3-ITD negative patients (pts; Allen et al. Leukemia 2013). Nevertheless, 30-40% CBF-AML pts experience relapse. FLT3-ITD mutations occur in roughly 5-10% of adult CBF-AML. However, their prognostic relevance is still controversial. Aims: To characterize CBF-AML with FLT3-ITD and compare outcomes according to their genetic background. Methods: We retrospectively studied 65 AML pts with CBF-AML and FLT3-ITD (median age at diagnosis, 54 years; range, 22-81 years) diagnosed between 1996 and 2018 within seven study groups/institutions of the US and Europe. Results: Thirty-two (49%) of the 65 pts harbored t(8;21). Median white blood cell and platelet counts at diagnosis of patients with t(8;21) and inv(16) were 18.3/nl (range, 1.8-202/nl) and 31/nl (range, 7-372/nl), respectively. AML diagnoses were de novo in 61 (94%) and therapy-related in 4 (6%) of the pts. Thirty (46%) pts were female. Cytogenetic analysis revealed additional abnormalities (abn) in 38 (58%) pts, most frequently loss of X or Y (n=13; n=12 associated with t(8;21)), complex karyotype (≥3 abn; n=12; n=7 occurring in t(8;21)), trisomy 22 (n=7, all associated with inv(16)) or trisomy 8 (overall n=6, n=5 occurring in inv(16)). Four pts were positive for both mutations, FLT3-ITD as well as FLT3-TKD. Median ITD allelic ratio were 0.44 (range, 0.003-50) and median ITD size 60 bp (range, 3-120 bp). Three older pts (median age, 75.5 years) were treated with either azacitidine + sorafenib, azacitidine + venetoclax or with etoposide + tipifarnib. All three patients receiving non-intensive therapy died within one year and were excluded from further analysis. Complete remission (CR) after anthracycline-based induction therapy was achieved in 98% (n=61/62) of patients fit for intensive treatment including two pts treated with 7+3 ± midostaurin within the RATIFY trial. One patient died during induction. Fifteen (24%) pts underwent allogeneic hematopoietic cell transplantation. Of those, 10 pts were transplanted in 1st and 5 pts in 2nd CR. Median follow-up for the entire cohort was 4.43 years (95%-CI, 3.35-8.97 years). Median and 4-year relapse-free survival (RFS) rates were 3.41 years (95%-CI, 1.26 years - not reached) and 44.9% (95%-CI, 32.9-61.4%). Median and 4-year overall survival rates (OS) were 4.48 years (95%-CI, 2.26 years - not reached) and 51.8% (95%-CI, 39.6.2-67.9%). Neither type of CBF-AML (p=0.60), nor additional chromosomal abn (p=0.80), nor presence of a complex karyotype (p=0.50) had a prognostic impact on OS. Higher age (≥60 years) was an in trend negative prognostic factor on RFS and OS (p=0.07, each). High allelic ratio (≥0.5) had no impact on RFS (p=0.3), but in trend on OS (p=0.10). Conclusions: Despite a high remission rate pts with FLT3-ITD had an inferior outcome as compared to previously published data on CBF-AML without FLT3-ITD. Thus, CBF-AML with FLT3-ITD should not be classified within the low-risk category. CBF pts with FLT3-ITD warrants further study and should be included in FLT3-inhibitor trials. Disclosures Brunner: Astra Zeneca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Novak:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel,Accommodations; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Travel,Accommodations; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stoelzel:Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Thiede:Daiichi Sankyo: Honoraria; AgenDix GmbH: Employment, Equity Ownership; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Diaceutics: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Levis:Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.