Single Cell Sequencing of Pediatric Acute Myeloid Leukemia Reveals Clonal Evolution to Relapse on Combination Chemotherapy with Sorafenib

Introduction: Relapse of pediatric acute myeloid leukemia (AML) remains a leading cause of childhood cancer mortality, and leukemias with activation of the Fms-like tyrosine kinase 3 (FLT3) are particularly susceptible to relapsed disease. Risk-directed therapy to prevent relapse is based both on genetic changes known to drive drug resistance, and measurable residual disease (MRD) at the end of induction therapy (EOI). In adult AML, resistance to type II FLT3-inhibitors, like sorafenib, is primarily driven by on-target FLT3 kinase domain (KD) mutations. However, the resistance mechanisms for pediatric leukemias, which are treated on combination therapies, have not been fully elucidated. MRD is considered the among the most predictive markers of future relapsed disease. It has been assumed that the major clone at the time of MRD assessment will predict the majority clone at relapse. However, this assumption has not been proven. The definition of the most specific genetic and MRD markers of relapse are essential to prognosticate and personalize therapy to prevent relapsed disease. Methods: We performed single cell sequencing (SCS) with a high-throughput DNA sequencing platform, Mission Bio Tapestri, on bone marrow or peripheral blood samples from 24 samples from 8 pediatric patients treated on COG AAML1031 with serial samples from diagnosis, EOI, and relapse. Results: We analyzed a total of 94,833 cells from 8 pediatric patients (median cells per patient 12,428) all treated on AAML1031. SCS revealed a sensitive and specific description of clonal evolution on the combination of sorafenib with cytotoxic chemotherapy. The FLT3 internal tandem duplication (ITD) was controlled by the therapy in only half of the patients. In five of the patients, the FLT3-ITD was present in multiple clones. The FLT3-ITD co-mutated with additional mutations (NRAS, SH2B3, WT1, TET2, or NPM1) in half of the patients. However, the presence of a co-mutation did not necessarily correlate with whether or not the ITD-containing clone persisted at the time of relapse. Of the leukemias whose relapse was not driven by FLT3, the most likely mutational driver of resistance was NRAS. Notably, however, despite the fact that FLT3 KD mutations make up the bulk of mutational resistance to type II FLT3i such as sorafenib in adult patients, there were no on-target FLT3 mutations found in any of these pediatric patients. Further, SCS allows for an unprecedented depth of analysis of the genetic complexity of pediatric AML. Phylogenic analysis revealed that the same mutations may arise independently in different cells (NPM1 W288fs, NRAS G60E). Additionally, the same gene may be mutated twice within the same cell (WT1, TET2). These data, consistent with our prior work, suggest that some leukemias may have a predilection to mutations within specific loci. Finally, although there is a standing assumption that the dominant MRD population will proliferate into relapsed disease, in 3/8 patients, the dominant MRD clone did not predict the dominant relapse clone. Conclusions: SCS allows for direct measurement of clonal hierarchy and evolution, phylogeny, co-mutational status, and zygosity, which can only be inferred through traditional bulk NGS. The mutational mechanisms of resistance seen in adult leukemias treated with sorafenib monotherapy are not necessarily relevant to the pediatric population; rather than on-target FLT3 mutations, off target mutations including NRAS are found. This corroborates prior findings that off-target RAS pathway mutations may drive resistance to FLT3i. Non-RAS off-target mutations found in this cohort do not necessarily predict sorafenib resistance, so may be passenger mutations. The lack of consistent resistance mutations suggests that other mechanisms of resistance such as epigenetic modifications may also drive resistance to combination chemotherapy with FLT3i in pediatric leukemia. Further, SCS exposes more genetic complexity in pediatric AML than has previously been appreciated: the same mutation may independently arise in more than one cell or the same cell may have multiple mutations within the same gene. Finally, the sensitivity of SCS reveals that the major clone at the time of MRD assessment is not necessarily the major clone at relapse. This suggests a benefit of more frequent MRD monitoring to track clonal evolution in real time. Disclosures Smith: Daiichi Sankyo: Consultancy; Revolutions Medicine: Research Funding; AbbVie: Research Funding; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

FDA Cleared Companion Diagnostics (CDx) Tests (IDH1, IDH2 and FLT3) for Acute Myeloid Leukemia (AML) Patient Care

Acute myeloid leukemia (AML) is one of the most lethal blood cancers from which nearly 10,000 people die in the United States each year. While therapies for other blood cancers have made some progress, the standard of care for AML, a combination of toxic chemotherapies, has changed very little over the past four decades. In recent years the US Food and Drug Administration (FDA) has been very active in approving targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations. Additionally, the Leukemia and Lymphoma Society's Beat AML Master Clinical Trial has shown that waiting for molecular results prior to treatment decision leads to better outcomes. Versiti Blood Center of Wisconsin Diagnostics laboratory which is certified under the Clinical Laboratory Improvement Amendments (CLIA) and qualified to perform high complexity clinical laboratory testing has performed the verification studies and offers two companion diagnostics tests for IDH1 and IDH2 mutations for AML patients. Also, in collaboration with Invivoscribe Inc., the AML patient can be tested for Leukostrat CDx FLT3 mutations assay so that the same AML patient can get three CDx test results leading to available drug therapy treatment decision making by physicians. IDH1 CDX Test: IDH1 CDx is indicated as an aid in identifying AML patients with an IDH1 mutation for treatment with ivosidenib (TIBSOVO®). Mutations in codon R132 of IDH1 can be found in 6% to 10% of AML patients. The IDH1 CDx test detects five IDH1 mutations R132H (CAT), R132C (TGT), R132G (GGT), R132S (AGT), and R132L (CTT) using PCR technology with homogeneous real-time fluorescence detection. The assay sensitivity for these five IDH1 mutations is 100% at variant allele frequencies of 2% and higher and 98% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170041). IDH2 CDx: IDH2 CDx is indicated as an aid in identifying AML patients with an IDH2 mutation for treatment with IDHIFA® (enasidenib). Mutations in the R140 and R172 codons of IDH2 8% to 19% of AML patients.The IDH2 CDX test detects nine IDH2 mutations (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W) using PCR technology with real-time fluorescent detection. The assay sensitivity for these nine IDH2 mutations is 99.8% or greater at variant allele frequencies of 2% and higher or 93.5% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170005). FLT3 CDx: The FLT3 Leukostrat® CDx Assay is the FDA approved (PMA number P160040) predictive test for the efficacy of midostaurin (RYDAPT®) therapy in all AML patients, regardless of cytogenetics and efficacy of gilteritinib (XOSPATA ® ) therapy in relapsed or refractory AML patients. FLT3 is one of the most commonly mutated genes in AML with 30% of patients at the time of diagnosis 1. The most prevalent type of FLT3 mutation is an internal tandem duplication (ITD) in the juxtamembrane domain. The second most common mutation type in the FLT3 gene is a tyrosine kinase domain (TKD) point mutation in the codon for an aspartate (D835) or an isoleucine (I836) residue. The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based, in vitro diagnostic test designed to detect internal tandem duplication (ITD) mutations and the tyrosine kinase domain (TKD) mutations D835 and I836 in genomic DNA extracted from mononuclear cells obtained from peripheral blood or bone marrow aspirates of patients diagnosed with AML. Versiti Blood Center sends the patient specimens to Invivoscribe Inc. where the LeukoStrat® CDx FLT3 Mutation Assay is performed and the interpretive comments are included in the patient report by Versiti. From our experience pathologists and treating physicians want molecular test results as fast as possible, especially for the actionable gene mutations in IDH1, IDH2 and FLT3. The IDH1 CDx, IDH2 CDx and FLT3 CDx tests are highly sensitive and Versiti provides average turn around time of 3 business days which enable rapid decision making on the recently available drug therapies for AML patients. We strongly recommend that the IDH1 CDx, IDH2 CDx and FLT3 CDx tests should be performed on all AML patients for better care. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: In recent years the US Food and Drug Administration (FDA) approved targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations.

Clinical Characteristics and Outcomes of Patients with Newly Diagnosed De Novo Acute Myeloid Leukemia (AML) during the COVID-19 Pandemic

Introduction The COVID-19 pandemic disrupted non-urgent and preventive medical care. During the early peak of the pandemic, an estimated 41% of US adults delayed or avoided medical care (Czeisler et al, CDC, 2020). While there were documented declines in the number of emergency department visits for myocardial infarction, stroke and hyperglycemia, similar data is not available related to acute myeloid leukemia (AML) (Lange et al, CDC, 2020). A delay in the diagnosis of AML could lead to presentation when patients are less able to withstand chemotherapy or have a higher disease burden which could compromise overall survival (OS). In this retrospective analysis, we aim to elucidate if there was a difference in clinical, cytogenetic, or molecular presentations and if there was an effect on early mortality as determined by overall survival at 1 and 6 months. Methods We compared the clinical, cytogenetic, and baseline molecular genetics of consecutive adult patients diagnosed with de novo AML at Dana-Farber Cancer Institute/Brigham and Women's (DFCI/BWH) Hospital from March 23, 2020, the date of the Massachusetts COVID State of Emergency, to August 23, 2020 to a historical cohort of similar patients between presenting between March 23, 2017 and August 23, 2020. Data was obtained from the Hematological Malignancy Data Repository and via review of the medical record. Patients were excluded from this cohort if they were diagnosed with acute promyelocytic leukemia, had known antecedent myeloid malignancy, or if they did not have DFCI/BWH 96-gene next-generation sequencing panel (RHP) performed at the time of diagnosis. Baseline clinical, laboratory, cytogenetic, and molecular characteristics and outcomes were compared between the pre-pandemic and pandemic cohorts using chi-squared, Fisher's exact, and Wilcoxon rank sum analyses (where appropriate) at a significance of p<0.05. Results Thirty-eight AML patients presented during the COVID-19 pandemic (PAN) and 308 in the pre-pandemic (PREPAN) period. There was no statistically significant difference in the monthly rate of new patients presenting in PREPAN and PAN cohorts (8 vs. 6 new patients/month, p=0.73). The median age at presentation (64 PREPAN vs. 65 PAN, p=0.77), sex, and therapeutic approach (intensive, non-intensive, supportive care, other) were not statistically different between cohorts. Presenting white blood cell count, platelet count, and fibrinogen were not different between cohorts, while hematocrit was significantly lower in the PAN cohort (23.8% vs. 26.0%, p=0.001). There was a trend for a higher median blast percentage (28.5% vs. 13%, p=0.09) in the PAN cohort. There were no differences between the cohorts in the median number of cytogenetic abnormalities, nor in the incidence of complex karyotype, (25.3% vs. 23.7%) across PREPAN and PAN respectively. There were also no significant differences in the European LeukemiaNet (ELN) risk classification scores across the PREPAN and PAN time periods, with 57.8% vs. 52.6% of total patients presenting with adverse risk disease respectively. When specific mutations of TP53, NPM1, and FLT3 were evaluated, only FLT3 demonstrated a statistical difference with a higher proportion in the pandemic group (p=0.04). OS at 1-month (97.4% and 93.2%, p=0.15) and 6-months (71.1% and 75.0%, p-0.87) were not statistically different in the PREPAN and PAN cohorts, respectively. Conclusion These data represent a novel analysis of the presenting clinical, cytogenetic and molecular characteristics of de novo AML during the COVID-19 pandemic. In contrast to other diseases, we did not see fewer de novo AML presentations during the peak of the COVID pandemic. While the reasons are unknown and require validation in large cohorts, the symptoms of leukemia including symptomatic anemia (low hematocrit) and higher WBC and blast count possibly driven by FLT3 mutations may drive patients to seek emergent clinical evaluation despite COVID pandemic barriers. The lack of difference in cytogenetic or other prognostic entities may demonstrate a lack of symptom correlation causing patients to present for care. The higher incidence of FLT3 mutations and lower hematocrit could reflect more symptomatic presentation of AML during the COVID pandemic. Since these differences may be a surrogate for a higher disease burden, it will be important to compare outcomes at longer time points. Figure 1 Figure 1. Disclosures DeAngelo: Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Incyte: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Takeda: Consultancy; Glycomimetrics: Research Funding; Blueprint: Research Funding; Abbvie: Research Funding; Servier: Consultancy. Stone: Bristol Meyers Squibb: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Boston Pharmaceuticals: Consultancy; Innate: Consultancy; Foghorn Therapeutics: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Glaxo Smith Kline: Consultancy; Celgene: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; OncoNova: Consultancy; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Agios: Consultancy, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Aprea: Consultancy; Arog: Consultancy, Research Funding; Jazz: Consultancy; Macrogenics: Consultancy; Novartis: Consultancy, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Garcia: AstraZeneca: Research Funding; Prelude: Research Funding; Pfizer: Research Funding; Genentech: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Winer: Abbvie: Consultancy; Takeda: Consultancy; Novartis: Consultancy.

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. Figure 1 Figure 1. Disclosures Pollard: 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.

A dual inhibitor overcomes drug-resistant FLT3-ITD acute myeloid leukemia

FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor prognosis. Multiple FLT3 inhibitors are in various stages of clinical evaluation. However, resistance to FLT3 inhibitors resulting from acquired point mutations in tyrosine kinase domain (TKD) have limited the sustained efficacy of treatments, and a “gatekeeper” mutation (F691L) is resistant to most available FLT3 inhibitors. Thus, new FLT3 inhibitors against both FLT3 internal tandem duplication (FLT3-ITD) and FLT3-TKD mutations (including F691L) are urgently sought. Herein, we identified KX2-391 as a dual FLT3 and tubulin inhibitor and investigated its efficacy and mechanisms in overcoming drug-resistant FLT3-ITD-TKD mutations in AML. KX2-391 exhibited potent growth inhibitory and apoptosis promoting effects on diverse AML cell lines harboring FLT3-ITD mutations and AC220-resistant mutations at the D835 and F691 residues in TKD and inhibited FLT3 phosphorylation and its downstream signaling targets. Orally administered KX2-391 significantly prolonged the survival of a murine leukemia model induced by FLT3-ITD-F691L. KX2-391 also significantly inhibited the growth of 4 primary AML cells expressing FLT3-ITD and 2 primary AML cells expressing FLT3-ITD-D835Y. Our preclinical data highlight KX2-391 as a promising FLT3 inhibitor for the treatment of AML patients harboring FLT3 mutations, especially refractory/relapsed patients with F691L and other FLT3-TKD mutations.

Dual Mechanism Inhibitor Control and Pharmacological Utility of Drug Resistance FLT3-ITD in Acute Myeloid Leukemia Cells

FLT3 mutations are among the most common genetic alterations in acute-myeloid leukemia (AML). They are associated with poor prognosis. Multiple FLT3 inhibitors have been in clinical evaluation at various stages. Resistance to FLT3 inhibitors due to acquired point mutations in the tyrosine-kinase domain (TKD), have limited the effectiveness of treatments. A “gatekeeper” mutation (F691L), is also resistant to most FLT3 inhibitors. New therapies are therefore needed. FLT3 inhibitors are needed to protect against FLT3-TKD mutations and FLT3 internal tandem duplicate (FLT3–ITD). We identified KX2-391, a dual FLT3/tubulin inhibitor, and examined its efficacy and mechanisms for overcoming drug-resistant FLT3ITD-TKD mutations. KX2-391 had potent growth inhibitory effects and apoptosis promoting effects on AML cell lines that harbor FLT3-ITD mutations. KX2-391 orally administered significantly prolonged the survival time of a murine model with leukemia caused by FLT3ITD-F691L. KX2-391 also inhibited growth of primary AML cells that express FLT3ITD-F691L and 2 primary cells that are FLT3ITD-D835Y. Preclinical data suggest that KX2-391 is a promising FLT3 inhibitor. The treatment of AML patients with FLT3 mutations, particularly refractory/relapsed patients suffering from F691L or other FLT3TKD mutations.

Combining Mass Spectrometry-Based Phosphoproteomics with a Network-Based Approach to Reveal FLT3-Dependent Mechanisms of Chemoresistance

FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor clinical outcome, relapse and chemotherapeutic resistance. Elucidating the molecular mechanisms underlying FLT3-dependent pathogenesis and drug resistance is a crucial goal of biomedical research. Given the complexity and intricacy of protein signaling networks, deciphering the molecular basis of FLT3-driven drug resistance requires a systems approach. Here we discuss how the recent advances in mass spectrometry (MS)-based (phospho) proteomics and multiparametric analysis accompanied by emerging computational approaches offer a platform to obtain and systematically analyze cell-specific signaling networks and to identify new potential therapeutic targets.

TERT rs2853669 as predictor for overall survival in patients with acute myeloid leukemia

IntroductionObjectiv. To investigate the contribution of TERT rs2736100 and rs2853669 gene polymorphisms in defining the genetic predisposition to AML, their association with different prognostic markers and their impact on survival, outcome and the prognosis of affected patients. Also, we investigated the association of TERT SNPs in AML in the presence or absence of DNMT3A (R882), NPM1 and FLT3 mutations.Material and methodsA total of 509 participants were enrolled in our study, consisting of 146 AML patients and 363 healthy participants, with no history of malignancy. TERT rs2736100 and rs2853669 polymorphisms were genotyped by using TaqMan SNP genotyping assays FLT3 (ITD, D835), DNMT3A (R882) and NPM1 c.863_864insTCTG (type A) mutation status was analyzed in each AML case.ResultsTERT rs2736100 and rs2853669 were not associated with AML risk in the codominant, dominant, recessive or allelic models. Multivariate Cox regression showed that TERT rs2853669 was a significant predictor for overall survival in AML patients. After adjusting for age, gender, cytogenetic risk group, ECOG status, FTLT3, DNM3A or NMP1 mutation, AML subtype and treatment, the estimated adjusted hasard ratio (HR adjusted=1.54, 95%CI:[1.01;2.35]) showed that the TERT rs2853669 variant genotype had a negative influence on survival time.ConclusionsTERT rs2853669 and rs2736100 polymorphisms were not risk factors for developing AML in the Romanian population, but TERT rs2853669 variant genotype had a negative effect on AML patients overall survival in the presence of other known prognostic factors.