Disease In Acute Myeloid Leukemia Research Articles

Single-Cell Transcriptomics for Residual Disease Detection in Acute Myelogenous Leukemia Post Allogeneic Hematopoietic Cell Transplantation

Detection of residual disease is a critical component of modern, risk-adapted therapy for Acute Myeloid Leukemia (AML). However, the genetic and phenotypic diversity of AML has made the development of a universal assay for disease assessment particularly challenging. While purely mutation-based tests promise high sensitivity, they are not broadly applicable given molecular heterogeneity and complex clonal evolution. Single-cell approaches, such as multiparameter flow cytometry (MFC), are more broadly applicable and increasingly accepted as the standard in clinical care. However, the limited number of leukemia-specific cell-surface markers and high numbers of shared markers between malignant myeloid blasts and healthy progenitors make MFC data extremely challenging to interpret. Motivated to develop a broadly applicable assay that can provide a more confident assessment of residual disease, we developed a platform using droplet-partitioned single-cell RNA sequencing accompanied by a computational pipeline specifically tailored to quantify residual disease after allogeneic HCT (alloHCT).With bone marrow samples from an 11-year-old patient with suspected post-alloHCT relapse of AML, we interrogated three methods of sample processing, 1) RBC lysis, 2) Ficoll-centrifugation, and 3) Ficoll-centrifugation combined with CD34+ immunomagnetic selection. The samples were further split to separately capture the 3' or 5' end of polyadenylated transcripts. The six resulting libraries were sequenced using standard short-read sequencing, and reads were demultiplexed and counted using common workflows. Data from the samples were combined, and sub-populations were visualized using UMAP (see Figure).This study demonstrated the feasibility of real-time single-cell sequencing for clinical utility. It is possible to process, capture, and sequence a patient's sample in approximately three working days (A). By integrating our data with single-cell expression profiles from an atlas of healthy human bone marrow, we were able to identify cells with gene-expression programs distinct from those of normal hematopoietic cells (B). With these integrated data, we could clearly identify populations of cells that embed away from healthy atlas cells (yellow circle, B), defining a different than normal single-cell profile. This “malignant” profile also included several genes whose expression is usually restricted to healthy hematopoietic progenitors (Panel C), suggesting these cells had a severely dysregulated transcriptome. As this patient was post-alloHCT, we interrogated the abundance of single-nucleotide-polymorphisms (SNPs) in the sequence data. We quantified these SNPs in single cells to distinguish each cell as either of donor or recipient origin using a method we have previously validated for genotyping RNA sequence in single cells. We clearly demonstrate that those cells identified as “different than normal” have a distinct SNP profile suggesting they are of recipient origin. Further analysis revealed that this malignant population was highly enriched for a population of cells expressing a previously described set of “AML-restricted genes” (Huang, B. et al., ASH 2021). (Panel E). Finally, from the Ficoll-processed sample, we quantified a level of 9.8% residual disease (243 malignant cells from a total of 2487). Notably, the number of abnormal myeloid progenitors determined by MFC was 2.0% which increased to 13% on a subsequent marrow sample drawn one week later. Incidentally, we observed only minimal differences across the two single-cell sequencing chemistries (3' vs. 5').Taken together, our data strongly argue that droplet-based, single-cell RNA sequencing is a feasible and powerful tool for the ascertainment of residual disease in AML. Given the robust nature of the platform and the ability to incorporate SNP integration into the analytic pipeline, it allows confident detection of residual disease in the post-alloHCT setting. By combining genomic quantification of transcripts with the power of SNP-based genotyping all at the level of the single cells, we believe this technology can substantially improve our diagnosis of post-alloHCT AML relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Venetoclax with Azacitidine or Low Dose Cytarabine As an Alternative to Intensive Chemotherapy in Fit Adults during the COVID19 Pandemic: Real World Data from the UK National Health Service

Venetoclax with Azacitidine or Low Dose Cytarabine As an Alternative to Intensive Chemotherapy in Fit Adults during the COVID19 Pandemic: Real World Data from the UK National Health Service

A Temporal and Multinational Assessment of Acute Myeloid Leukemia (AML) Cancer Incidence, Survival, and Disease Burden

Introduction: As US and global populations continue to age, a corresponding rise in incidence of age-associated cancers including acute myeloid leukemia (AML) remains persistent (Kraywinkel & Spix, 2017; Reedijk et al., 2019; Siegel, Miller, & Jemal, 2020; Yi et al., 2020). Characterizing the current and future epidemiologic impact of AML is critical for assessing therapeutic demand and informing allocation of health care resources. However, very few recent publications have reported international estimates of AML diagnoses and none have projected future cases. In this study, we estimate current and future incidence of AML (2021-2040) in 29 countries from 4 continents. Next, we describe survival trends in the US over the last 4 decades and by age at diagnosis. Finally, we assess the societal burden of AML in 29 countries by calculating average years of life lost (AYLL) due to AML-related death in 2021.Methods: Incidence rates were age-standardized to the World Health Organization (WHO) New World Population. For these calculations, age-specific rates were estimated using data from the International Agency for Research on Cancer's (IARC's) Cancer Incidence in Five Continents Volume XI (CI5-XI) database for 28 countries outside of the US (11 in Asia, 9 in Europe, 6 in the Americas, and 2 in Oceania). For US rates, data from National Cancer Institute's (NCI's) Surveillance, Epidemiology, and End Results (SEER) 21 were assessed. To project crude (unstandardized) incidence rates and the number of incident cases forward to 2040, age- and sex-specific incidence rates were applied to each country's age- and sex-specific population projections using data from the United Nation's World Population Prospects. Five-year Kaplan-Meier survival curves reflecting observed survival by year of diagnosis and age at diagnosis were derived using SEER 9 and SEER 18 databases, respectively. AYLL calculations utilized US age- and sex-specific mortality rates from the National Center for Healthcare Statistics (NCHS); non-US mortality rates were estimated by applying the incidence to mortality ratio from the US. The WHO's Global Health Observatory provided life expectancy data to estimate AYLL for each of the 29 countries.Results: Age-standardized incidence rates ranged from 0.70 cases per 100,000 persons (Costa Rica) to 3.23 cases per 100,000 persons (United States) with a median of 2.28 cases per 100,000 persons (interquartile range (IQR): 1.61-2.71). Projected growth in incident AML cases varied from +3% (Russia) to +51% (Colombia) over the next 20 years; median growth in incident AML cases was +32% (IQR: 27%-43%). AML survival after diagnosis more than doubled in the US over the last 40 years with the following median survival times observed: 4 months (1978-1987) vs. 11 months (2008-2017). Five-year survival in the US was likewise extended during this period: 8% (1978-1987) to 28% (2008-2017). Disparate survival was observed when patients were stratified by age. During the period 2008 to 2017, US cases diagnosed 60 years and older (which represented 67% of all new AML cases) had a median survival of 3 months with a five-year observed survival (OS) of 10%; this was far lower than their younger counterparts (< 60 years) who experienced a median survival of 60 months and a 5-year OS of 51%. The multinational assessment of societal disease burden due to premature AML death estimated a median AYLL of 19.6 years (IQR: 17.3-23.3 years).Conclusion: This epidemiologic assessment of AML disease burden projected significant growth in new AML diagnoses over the next 20 years. In fact, we estimated the growth of AML cases to outpace population growth in most countries - a phenomenon that can be attributed in part to the disproportionate increase in the elderly population where AML is most persistent. Our analysis additionally confirmed differences in survival by age with younger patients exhibiting more favorable survival compared to older adults. This stark contrast in prognosis may be partly explained by the current treatment approach using stem cell transplant for younger, fit patients and the limited therapeutic options among older patients. Despite the noted improvements in survival over the last 4 decades, median survival among all AML patients remains poor highlighting the unmet medical need for novel therapeutic approaches. [Display omitted] DisclosuresAnderson: Amgen: Current Employment, Current equity holder in publicly-traded company. Girguis: Amgen: Current Employment, Current equity holder in publicly-traded company. Kim: Amgen: Current Employment, Current equity holder in publicly-traded company. Shewale: Amgen: Current Employment, Current equity holder in publicly-traded company. Braunlin: Amgen: Current Employment, Current equity holder in publicly-traded company. Werther: Amgen: Current Employment, Current equity holder in publicly-traded company. Hidalgo-Lopez: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Zaman: Amgen: Current Employment, Current equity holder in publicly-traded company. Kim: Amgen: Current Employment, Current equity holder in publicly-traded company.

Myeloid Sarcoma Reveals a Unique Tumor Micro-Environment and Differential Prognosis Based on Transplant Status and Type of Treatment Received

BackgroundMyeloid sarcoma (MS) is the rare extramedullary proliferation of myeloid blasts that disrupts the normal architecture of various tissues in patients with Acute myeloid leukemia (AML). MS can present concurrently with untreated AML or present as relapsed AML. Based on our clinical observations and emerging immune checkpoint inhibitor (ICI) studies in AML (Davids et al NEJM 2016), MS appears to behave differently based on transplant status and tends to have better ICI sensitivity. One of the prominent mechanisms of relapse post-transplant is immune escape via the loss of leukemia recognition by the graft. This is mediated by down regulation of HLA expression. We hypothesize that the immune signatures in the bone marrow (BM) microenvironment are distinct from those from the local MS microenvironment. Thus, we aimed to comprehensively explore the MS microenvironment at single-cell level, providing clues for more effective therapies including ICI, and insights into the molecular pathogenesis, clinicopathological features and outcomes of MS.MethodsThis is an investigator-initiated study where we collected paired fresh MS tissues and BM samples from patients with MS as well as retrospective collection of paired MS and BM samples that are already sectioned and paraffin-embedded. Eligible patients were consentable adults > 18 years old and have MS lesions whether at diagnosis or at relapse of their AML disease. We excluded patients who have multiple malignancies or patients who are unable to provide samples. Accepted samples are 10-millimeter tissue or 1 million cells per milliliter. The fresh samples were cryopreserved and used for single cell RNA sequencing and ex vivo immune cellular assays. The sectioned tissues were used for multiplex immunohistochemical staining (mIHC) that tested a panel of 6 antibodies (CD3, CD68, CD4, CD33, PDL-1 and HLA-DR). We sought to further perform spatial transcriptome analysis (Visium) that complements mIHC for in-depth characterization of the MS microenvironment and immunopathogenesis. The clinical and genomic annotations for these samples were abstracted from the patients' charts. We have utilized Next generation sequencing (NGS) panels that interrogated 24- 180 oncogenes, including FLT3, IDH, CALR, NPM1, KRAS, NRAS, and TP53. Survival estimates using K-M curves and multivariate analysis were performed via SPSS.ResultsOur study recruited 30 patients so far. Mean age at diagnosis was (57.6 ± 14 years); 63.3% of the patients were males (n=19) and 80% of the patients were Caucasian (n=24). In our cohort, 46.6% of the patient had MS diagnosed as the cause of relapse after stem cell transplant (n=14) while the rest had MS at the initial diagnosis of AML. The most common cytogenetic abnormality in our cohort was complex karyotype in 30% of the patients. Molecular landscape of MS at diagnosis and relapse was done in 90% of the patients and shown in figure 1A,B. MS conferred a poor prognosis if diagnosed on initial presentation vs a new presentation at relapse post stem cell transplant with a median OS (4 months vs 25 months p 0.003) (HR 3.9 - 95% CI 1.4-11.1) figure 2. Interestingly, we have noticed that patients who received Venetolcax based therapies have poor prognosis compared to other therapies (IDH/FLT3 inhibitors, conventional chemotherapies, or ICI); median OS (9.3 months vs 24 months p 0.02) (HR 3.9 - 95% CI 1.2-12.6) figure 3. In a prelim analysis using validated mIHC, we have observed abundant HLA-DR expression in both blast cells and nonmalignant stromal cells of the MS microenvironment (n=4) figure-4. By contrast, PD‐L1 was expressed mainly on a number of stromal cells rather than blast cells in the MS microenvironment. Further special localization analysis of tumor immune infiltrates (TILs) is being conducted to disclose clinical outcomes and biological significance of PD-L1, HLA-DR and TILs in MS. Spital transcriptomics are in process and will be presented.ConclusionMS diagnosed concurrently with untreated AML appears to confer a poor prognosis and behave differently than MS diagnosed as relapse post-transplant. MS appears to be resistant to Venetoclax based therapies and patients tends to do worse on this therapy. In contrast to BM microenvironment, blast cells within MS appears to have abundant expression of HLA-DR in both neoplastic tumor cells and nonmalignant stromal cells promoting immune recognition which might explain their sensitivities to ICI. [Display omitted] DisclosuresAbaza: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinner: Pfizer: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria. Altman: Glycomimetics: Other: data monitoring committee; ALX Oncology Inc.: Research Funding; Loxo: Research Funding; ImmunoGen: Research Funding; Abbvie: Honoraria, Research Funding; Amgen: Research Funding; Aprea: Research Funding; Astellas: Honoraria, Research Funding; Aptos: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Research Funding; Biosight: Membership on an entity's Board of Directors or advisory committees, Other: reimbursement for travel, Research Funding; Kura Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kartos Therapeutics: Research Funding.

Prevalence and Risk Factors of Antibodies to Class I and II Human Leukocyte Antigens in Haploidentical Allograft Candidates: A Prospective Study on 3805 Subjects

The aim of this study is to investigate the prevalence and risk factors of anti-human leukocyte antigen (HLA) antibodies in haploidentical candidates.This study was completed at Peking University People's Hospital, Beijing China. We performed a prospective analysis of patients with hematological diseases concerning the prevalence and risk factors of anti-HLA antibodies. Patients were enrolled between July 2015 - December 2019. Serum was collected for PRAs test within 1 month before haploidentical transplantation. The risk factors, such as age, sex, total transfusion, red blood cell (RBC) transfusion, platelet (PLT) transfusion, pregnancy, disease duration and diagnosis were collected. Univariate and multivariate logistic regression analyses were performed to evaluate the risk factors of anti-HLA antibodies.Six hundred and eighty (17.9%) patients were positive for panel reactive antibodies (PRA)-class I, 360 (9.5%) for class II, 768 (20.2%) class I or II, and 272 (7.1%) positive for class I and II both. Multivariate analysis indicated that female was related to higher risk of having PRAs for class I (P = 0.011), class I or II (P = 0.009), anti-HLA-A (P = 0.015), anti-HLA-DP (P = 0.048) and also for having higher mean fluorescence intensity (MFI) (2000 or more) of PRAs in class I (P = 0.020) and class I or II (P = 0.005). Compared to patients with myelodysplastic syndrome (MDS), patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), aplastic anemia (AA) had a lower incidence for PRAs in class I, class II, class I or II, class I and II, anti-HLA-A. anti-HLA-B, anti-HLA-C, anti-HLA-DQ, anti-HLA-DR, anti-HLA-DP (Table 1). Prior pregnancy was a risk factor for PRAs (P < 0.001), and no previous pregnancy group having lower MFI of PRAs in class I (P = 0.001) and class I or II (P = 0.004). PLT transfusion (more than 4 times) rleted with a higher prevalence of PRAs (P < 0.001), and also had a higher MFI of PRAs in class II (P < 0.001), class I and II (P < 0.001). Patients with RBC transfusion (more than 3 times) had a higher prevalence of PRAs in class I (P = 0.001), class II (P = 0.029), class I or II (P < 0.001), anti-HLA-A (P = 0.001), anti-HLA-B (P < 0.001), anti-HLA-C (P = 0.007), anti-HLA-DQ (P < 0.001) and anti-HLA-DR (P = 0.011). In addition, diseases duration (8 months or more) was also associated with higher MFI of PRAs in class I (P = 0.023) and class I or II (P = 0.004).Subgroup analysis showed that 11.7% of pediatric patients were positive for PRAs in class I; 19.2% of adults, 17.9% of elder patients; 12.4% of males; 26.1% of females; 21.0% of patients with AML; 10.5% of patients with acute lymphoblastic leukemia (ALL); 18.9% of patients with AA; 30.3% of patients with MDS; 16.6% of patients with other hematological diseases. The positive rate of class II PRAs in children was 4.3%; 11.1% for adults; 9.5% for elder patients; 5.5% for males; 15.4% for females; 11.4% for patients with AML; 5.2% for patients with ALL; 10.3% for patients with AA; 17.2% for patients with MDS; 6.6% of patients with other hematological diseases. Multivariate analysis showed that, in children, PLT transfusion and diagnosis were the two main risk factors of PRAs in class I and class II (P < 0.001, P = 0.017). In adults, diagnosis (P = 0.003), transfusion (P < 0.001) and pregnancy (P < 0.001) were the three main factors associated with PRAs in class I and transfusion (P < 0.001) and pregnancy (P < 0.001) were the two main factors associated with PRAs in class II. In males, PLT transfusion (P < 0.001) and diagnosis (P < 0.001) were the two main factors associated with PRAs in class I and class II. In ALL subgroup, gender (P = 0.026, P = 0.048), pregnancy (P < 0.001) and transfusion (P < 0.001) were the three main factors associated with PRAs in class I and II. In AA subgroup, gender (P = 0.004) and PLT transfusion (P < 0.001) were risk factors for class I PRAs, pregnancy (P = 0.008) and PLT transfusion (P = 0.003) were risk factors for class II PRAs. In elder patients, females, AML, MDS and other diseases subgroup, transfusion and pregnancy were the two main factors associated with PRAs in class I and class II.Our results indicated that female sex, diagnosis, pregnancy, transfusion, disease duration were independent risk factors of anti-HLA antibodies in haploidentical allograft candidates, which provided evidence for best haploidentical donor selection. The risk factors of anti-HLA antibodies were different among total patients and those of cases in different subgroups. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transcriptomic Features of Immune Exhaustion and Senescence Predict Outcomes and Define Checkpoint Blockade-Unresponsive Microenvironments in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML.In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases).We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D).Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. [Display omitted] DisclosuresRadojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.

Acute Myeloid Leukemia in Western Kenya: A Snapshot

Introduction: Steady advances in the treatment of acute myeloid leukemia (AML) have improved outcomes in high-resource settings, with a 5-year overall survival rate of 29% and rising in the United States. In contrast, a diagnosis of AML in many resource-limited settings automatically confers a less than 10% one-year survival rate. To better understand this significant disparity, as well as how to narrow it, it is important to gather data illustrating the current landscape of AML management in resource limited-settings, including patient characteristics, disease-related and treatment-related factors. Here we examine the population of patients with AML at a single large academic medical center in Western Kenya that serves a catchment area of more than 20 million people.Objectives: To describe characteristics of patients presenting with AML at Moi Teaching and Referral Hospital (MTRH) hematology and oncology clinic between 2014 and 2020, to help identify areas of need to inform future interventions.Methodology: Retrospective, cross-sectional chart review study of all newly diagnosed patients (age 15 years and older) with AML presenting to the MTRH adult hematology and oncology clinic from January 2014 to December 2020.Results: We reviewed the charts of 113 patients with AML. The median age at diagnosis was 40 years (range 15-86 years), with an average age of 42 years. Forty-nine percent (n=55) were female. Thirty-five patients did not have French American British (FAB) subtypes documented (this method remains the main form of AML disease classification in this resource-limited setting). M2 subtype was the most common (n= 24). Seven patients had acute promyelocytic leukemia (APML), of which 4 died due to bleeding complications and lack of access to ATRA. Three APML patients who had access to ATRA were alive more than 1 year after diagnosis. White blood count (WBC) at diagnosis ranged between 600/cm3 and 336,000/cm3 with neutrophil predominance. Mean hemoglobin at presentation was 7g/dl (range 2.6g/dl-16g/dl). Most patients had been transfused with red blood cells prior to presentation and continue to require more transfusion. Platelet counts ranged between 4,000/cm3 to 782,000/cm3 with 36% of patients (n=41) having a count of less than 50,000/cm3. Fifty patients with AML received low dose subcutaneous cytarabine (20mg subcutaneous twice a day for 10 days every 4 to 6 weeks) and 3 patients had etoposide added to their treatment (50mg/m2 intravenous once a day for 7 days). No patient was treated with standard intensive induction chemotherapy, (7+3), due to lack of adequate supportive care. Only 5 of 63 (7.9%) non-APML patients whose outcomes were established survived for more than 12 months. The median overall survival at after diagnosis was 45 days. Thirteen percent of patients were lost to follow up (n= 15) and 1 patient was referred to another facility for possible induction with 7+3.Conclusion: AML remains a disparately lethal disease in resource-limited settings, where it impacts a relatively healthy, young patient population. In well-resourced settings, many of these patients would have a reasonable chance at long term survival and potential cure, but in Western Kenya most patients die within a few months of diagnosis. Due to the lack of adequate supportive care resources, even the younger patients mostly did not receive standard-of-care intensive induction therapy. The outdated FAB classification system is still in use. Lack of access to improved diagnostics, appropriate supportive care (antimicrobials and transfusion products) and limited availability of newer, effective, and less toxic treatment regimens are the main impedance to care. More efforts are needed to improve the management of acute leukemia in under-resourced countries. DisclosuresLeBlanc: Pfizer: Consultancy, Other: Advisory Board; Otsuka: Consultancy, Honoraria, Other; Daiichi-Sankyo: Consultancy, Honoraria, Other: Advisory board; AbbVie: Consultancy, Honoraria, Other: Advisory board; Travel fees, Speakers Bureau; Flatiron: Consultancy, Other: Advisory board; Helsinn: Consultancy, Research Funding; Duke University: Research Funding; Agios: Consultancy, Honoraria, Other: Advisory board; Travel fees, Speakers Bureau; Amgen: Consultancy, Other: travel; BMS/Celgene: Consultancy, Honoraria, Other: Travel fees, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria, Other: Advisory board; American Cancer Society: Research Funding; Heron: Consultancy, Honoraria, Other: advisory board; AstraZeneca: Consultancy, Honoraria, Other: Advisory board, Research Funding; Seattle Genetics: Consultancy, Other: Advisory board, Research Funding; CareVive: Consultancy, Other, Research Funding; NINR/NIH: Research Funding; UpToDate: Patents & Royalties.

Associating Ex Vivo Drug Sensitivity with Differentiation Status Identifies Effective Drug Combinations for Acute Myeloid Leukemia

The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p<0.0001). This broad sensitivity of the VEN+DORA combination was not significantly associated with an array of clinical, genetic, and mutational features in the patient samples tested, in contrast to single agents, particularly VEN. Analysis of blood cell differential counts of patient samples tested identified increased monocyte levels were significantly correlated with sensitivity to DORA and resistance to VEN as single agents (Spearman r = -0.3 and 0.6; p<0.0001), associations that were not apparent with the combination. For patient samples with accompanying FAB differentiation state-based designations (n=108), sensitivities of the combination were similar across classifications of undifferentiated (M0/M1) through monocytic (M4/M5) acute leukemia. In contrast, single-agent VEN was significantly more sensitive in undifferentiated compared to monocytic specimens, whereas DORA sensitivity showed the reverse trend (though to a lesser degree). These differences in sensitivity were further validated by immunophenotyping data where available (n=105), which showed surface markers associated with resistance to VEN (CD11b, CD14, CD16, CD56, CD64, HLADR; Wilcoxon Rank Sum, p<0.001 to p=0.007) or sensitivity to VEN (CD117; p=0.001) or DORA (CD14; HLADR; p=0.004). By contrast, none of these associations significantly distinguished sensitivity for the VEN+DORA combination. Expression levels of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were concordant with their respective drug sensitivities associated with FAB classification; that is, significantly higher levels of BCL2 in M0/M1 leukemias and MAPK14 in M4/M5 cases (Mann-Whitney test; p<0.0001; n=145). Further dissection of transcriptomic and drug sensitivity data revealed strong correlation and gene set enrichment for DORA and VEN sensitivities with monocyte-like and progenitor-like signatures, respectively (n=225), for cell differentiation states previously described for AML (van Galen 2019), and these associations diminished for the combination treatment. Lastly, the VEN+DORA combination enhanced efficacy and synergistic inhibition was confirmed using human AML cell line models tested with a matrix of potential dose concentrations. Taken together, these findings suggest that exploiting distinct, complementary sensitivity profiles of targeted therapies with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers an opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML. DisclosuresDruker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

PARP Inhibition with Talazoparib Enhances DNA Damage and Anti-Leukemic Activity of Venetoclax in Preclinical Human Acute Myeloid Leukemia (AML) Models

Background: BCL-2 is an anti-apoptotic protein associated with chemotherapy resistance and survival of acute myeloid leukemia (AML) cells following cell stress or DNA damage. Venetoclax (Ven) is a BH3-mimetic that competitively binds to BCL-2, allowing BH3 proteins to bind to pro-apoptotic proteins (BAX, BAK) and induce cell death. The combination of Ven with Azacitidine (Aza), is now standard of care therapy for older patients with AML. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent the repair of single stranded DNA breaks by blocking the nicotinamide adenine dinucleotide (NAD) catalytic domain of the PARP protein and preventing dissociation of PARP from the DNA (termed PARP trapping). The PARP inhibitor, talazoparib (Tala) displays 10,000-fold increased PARP trapping as compared to other clinical PARP inhibitor agents in tumor models. We hypothesized that Tala could enhance the anti-leukemic activity of Ven on AML cells specifically by increasing DNA damage.Methods: Human AML (Molm13) cells were continuously exposed to increasing doses of Ven (1nM - 10mM), Tala (1nM - 10mM), and Aza (0.1-5mM) alone and in combination. Synergy reports were generated using CompuSyn software. Cell viability was measured using a WST colorimetric assay. Apoptosis (annexin /PI), DNA damage (pH2AX), and cell cycle arrest (propidium iodide) were quantified by flow cytometric analysis. DNA damage was also evaluated using the Fast-Halo assay. In vivo efficacy was assessed in NSG mice systematically engrafted with stably luciferase transfected Molm13 BLIV cells via tail vein injection. Cohorts of mice (5-10) were treated with vehicle, Ven (100 mg/kg given 5 days/week for 3 weeks), Tala (0.33 mg/kg for 5 days/week over 3 weeks), Ven + Tala (same doses), and Ven+ Aza (1.25 or 2.5 mg/kg daily for 7 days). Treatment effects on leukemia burden, toxicity, and overall survival were determined using weekly whole animal bioluminescent imaging, total animal weights, and time to morbidity, respectively.Results: Human AML cells treated with Ven displayed dose and time-dependent induction of DNA damage, apoptosis, and cell death. Similarly, treatment with single agent Tala resulted in dose-dependent anti-proliferative effects and PARP trapping. Combination in vitro therapy with Ven (0.1 - 1 μM) + Tala (10- 100 nM) or Ven (same doses) + Aza (1 μM) resulted in significant and synergistic anti-leukemic effects improving upon monotherapy with Ven, Tala, or Aza alone. However, in contrast to Ven+Aza, increased levels of DNA damage (as determined by phosphorylated H2AX and Fast Halo assays) were detected as well as induction of apoptosis, cell cycle arrest, decreased viability, and increased cell death vs vehicle and single agent therapy.We then evaluated the effects of in vivo treatment with vehicle, Ven, Tala, Ven + Tala, and Ven +Aza in NSG mice systemically engrafted with human luciferase-tagged AML cells (Molm13 BLIV). Single agent Ven or Tala therapy decreased systemic AML burden but did not prolong overall survival over vehicle treated mice. Combination Ven + Tala significantly prolonged overall survival of leukemia xenografted mice over vehicle, Ven, or Tala monotherapy. Of note, Ven + Tala treatment appeared to result in similar disease burden reduction and prolonged overall survival as Ven + Aza treated animals (Figure 1). Preliminary experiments of iv vivo triple therapy with Ven + Aza + Tala in AML xenografted mice resulted in further reduction in AML disease burden but was associated with marked weight loss leading to early morbidity and shortened overall survival (data not shown). Additional doses and schedules of this triplet approach are under investigation.Conclusions: Our results demonstrate that addition of the potent PARP inhibitor, talazoparib, to the BCL-2 inhibitor, venetoclax, enhances DNA damage and results in potent in vitro and in vivo activity in preclinical human AML models. Further evaluation of this combination in primary AML samples with varying genetic backgrounds as well as in confirmatory primary xenograft models is underway. These data strongly support future clinical investigation of PARP inhibitors as a novel class of agents with the potential to significantly enhance the efficacy of novel targeted therapies for acute leukemia. [Display omitted] DisclosuresWang: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; AbbVie: 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; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; DAVA Oncology: Consultancy, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Advisory Board; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.

WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Background:The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo.Methods:Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline.Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry.Human AML cell lines were studied in vitro for comparison.Results:In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays.Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS.Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models.In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A.Conclusion:We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. DisclosuresNo relevant conflicts of interest to declare.

The Krüppel-like Factor 4 Sustains a Leukemic Gene Expression Profile in MLL-AF9-Driven Acute Myeloid Leukemia

Among hematological malignancies, acute myeloid leukemia (AML) confers poor prognosis and limited progress has been made in the translation of decades of research into improved clinical outcomes. The current paradigm is that eradication of leukemia stem cells (LSCs) represents an avenue for overcoming relapse and refractory disease, but therapy focusing on eradicating this leukemic population has not been developed to-date. Further studies of unique signaling pathways and vulnerabilities in LSCs are warranted to design targeted therapies that could impact patient outcomes. To evaluate whether the stemness transcription factor Krüppel-like Factor 4 (KLF4) is important in the progression of AML, we retrovirally transduced MLL-AF9 into Klf4 fl/fl(fl/fl)and Klf4 fl/flVav-Cre (Δ/Δ) lineage − Sca-1 + c-Kit + (LSK) bone marrow cells and transplanted into C57BL/6 recipients. Here we report that the KLF4 promotes disease progression in the MLL-AF9-driven syngeneic AML mouse model. Strikingly, Δ/Δ AMLs exhibited improved disease latency and penetrance, and a seven-fold reduction in leukemia-initiating cell frequency in a secondary transplantation study. Δ/Δ LSCs, defined as leukemic granulocyte macrophage progenitors (L-GMP), demonstrated lessened clonogenicity in methylcellulose cultures and reduced representation of cells in the G 2/M phase of the cell cycle. RNAseq analysis of L-GMP revealed decreased expression of hematopoietic and leukemic stemness gene sets such as RAS signaling, and induction of inflammatory response gene (TNF-α, IFNα, IFNβ) pathways in Δ/Δ LSCs. To evaluate human relevance, we used CRISPR-Cas9 based targeted deletion of the human KLF4 gene in a MLL-AF9 PDX line and observed improved survival and defects in expansion as seen in the syngeneic mouse model . Lastly, to correlate KLF4-associated signaling present in murine AML LSCs with human AML, we used CRISPR-Cas9-based targeted deletion of KLF4 in MOLM-13 (KO) to generate two validated clones. MOLM-13 KO cells showed reduced cell proliferation in vitro and in vivo. Further, RPPA analysis revealed reduced RAS pathway activity (IR-β, β-Raf), accumulation of proteins associated with the S and G 1 phases (e.g., CDKN2A, p21, Histone H3, CENP-A), and decrease expression in regulators of the G 2/M checkpoint (e.g., Aurora A, B, Chk1, Plk1, Wee1, Cyclin B, pCDK1). Collectively, our data suggest a mechanism in which KLF4 contributes to AML disease by establishing a gene expression profile supporting stemness of AML LSCs. DisclosuresNo relevant conflicts of interest to declare.

IER3 (IEX-1) dysregulation serves as a potential prognostic factor in acute myeloid leukemia patients.

IntroductionImmediate early response 3 (IER3) has association with hematological malignancies’ risk and prognosis, such as myelodysplastic syndrome, while its relation to acute myeloid leukemia (AML) is not clear. This study aimed to explore the correlation of IER3 with AML risk, clinical characteristics, complete remission (CR), event‐free survival (EFS), and overall survival (OS).MethodsA total of 93 de novo AML patients were included in this study. In addition, 30 patients with non‐hyperplasia hematologic malignancies requiring bone marrow testing (as disease controls) and 30 health donors (as health controls) were also recruited. Bone morrow samples of AML patients (before treatment), disease controls (before treatment), and health controls (at donation) were collected. IER3 in bone marrow mononuclear cells was detected by reverse transcription‐quantitative polymerase chain reaction.ResultsIER3 was increased in AML patients compared with disease controls and health donors (both P < .001), and receiver operating characteristic (ROC) curve showed that IER3 had certain capability of distinguishing AML patients from disease controls (area under curve (AUC): 0.735, 95% confidence interval (CI): 0.650‐0.820), and health donors (AUC: 0.789, 95% CI: 0.712‐0.866). Meanwhile, IER3 was correlated with FLT3‐ITD mutation (P = .030) and poor NCCN risk stratification (P = .031) in AML patients. Moreover, IER3 had negative association with CR in AML patients (P = .022), and showed certain potential in discriminating CR patients from non‐CR patients (AUC: 0.655, 95% CI: 0.533‐0.777). Besides, IER3 was negatively associated with EFS (P = .033), but not OS (P = .083) in AML patients.ConclusionIER3 dysregulation serves as a potential prognostic factor in AML patients.

The Potential Advantage of Targeting Both PD-L1/PD-L2/PD-1 and IL-10-IL-10R Pathways in Acute Myeloid Leukemia.

Tumor cells promote the suppression of host anti-tumor type 1 T cell responses by various mechanisms, including the upregulation of surface inhibitory molecules such as programmed death ligand (PD-L)-1, and the production of immunosuppressive cytokines such as interleukin-10 (IL-10). There are over 2000 trials investigating PD-L1 and/or its receptor programmed-death 1 (PD-1) blockade in cancer, leading to the approval of PD-1 or PD-L1 inhibitors in several types of solid cancers and in hematological malignancies. The available data suggest that the molecule PD-L1 on antigen-presenting cells suppresses type 1 T cell immune responses such as cytotoxicity, and that the cytokine IL-10, in addition to downregulating immune responses, increases the expression of inhibitory molecule PD-L1. We hypothesize that the manipulation of both the co-inhibitory network (with anti-PD-L1 blocking antibodies) and suppressor network (with anti-IL-10 blocking antibodies) is an attractive immunotherapeutic intervention for acute myeloid leukemia (AML) patients ineligible for standard treatment with chemotherapy and hematopoietic stem cell transplantation, and with less severe adverse reactions. The proposed combination of these two immunotherapies represents a new approach that can be readily translated into the clinic to improve the therapeutic efficacy of AML disease treatment.

Impact of CMV reactivation on relapse of acute myeloid leukemia after HCT is dependent on disease stage and ATG

AbstractCytomegalovirus (CMV) reactivation is a frequent complication after allogeneic hematopoietic cell transplantation (HCT), whose impact on clinical outcome, in particular on leukemic relapse, is controversial. We retrospectively analyzed 687 HCT recipients with acute myeloid leukemia (AML) and ciclosporin-based immunosuppression to better understand the differential impact of CMV on transplant outcomes depending on AML disease stage and in vivo T cell depletion with antithymocyte globulin (ATG). Without ATG, CMV reactivation associated with significantly reduced relapse, yet its effect was more pronounced for advanced disease AML (P = .0002) than for patients in first complete remission (CR1, P = .0169). Depending on the disease stage, ATG exposure abrogated relapse protection following CMV reactivation in advanced stages (P = .796), while it inverted its effect into increased relapse for CR1 patients (P = .0428). CMV reactivation was associated with significantly increased nonrelapse mortality in CR1 patients without ATG (P = .0187) but not in those with advanced disease and ATG. Following CMV reactivation, only patients with advanced disease had significantly higher event-free survival rates as compared with patients without CMV. Overall, our data suggest that both ATG and disease stage modulate the impact of post-HCT CMV reactivation in opposite directions, revealing a level of complexity that warrants future studies regarding the interplay between antivirus and antitumor immunity.

Rational biomarker development for the early and minimally invasive monitoring of AML

AbstractRecurrent disease remains the principal cause for treatment failure in acute myeloid leukemia (AML) across age groups. Reliable biomarkers of AML relapse risk and disease burden have been problematic, as symptoms appear late and current monitoring relies on invasive and cost-ineffective serial bone marrow (BM) surveillance. In this report, we discover a set of unique microRNA (miRNA) that circulates in AML-derived vesicles in the peripheral blood ahead of the general dissemination of leukemic blasts and symptomatic BM failure. Next-generation sequencing of extracellular vesicle-contained small RNA in 12 AML patients and 12 controls allowed us to identify a panel of differentially incorporated miRNA. Proof-of-concept studies using a murine model and patient-derived xenografts demonstrate the feasibility of developing miR-1246, as a potential minimally invasive AML biomarker.

Measurable Residual Disease in AML

Introduction Acute myeloid leukemia (AML) is a notoriously heterogeneous hematological malignancy with a relatively poor outcome depending on the risk classification, which is determined by white blood cell count (WBC), age, cytogenetics, and specific mutations.1 Although the majority of patients achieve a morphological complete remission (CR, defined as

Clinical implications of SRSF2 mutations in AML patients undergoing allogeneic stem cell transplantation.

The SRSF2 mutations are frequently found in acute myeloid leukemia (AML) and mostly affect the P95 residue. Mutations in this splicing factor mediate abnormal splicing associated with exon skipping events, including EZH2 as a crucial target. While SRSF2 mutations are enriched in secondary AML and associated with worse outcomes following chemotherapy consolidation, very little is known about the associated biological and clinical implications in AML patients consolidated with allogeneic hematopoietic stemcell transplantation (HSCT). Here we retrospectively analyzed 263 adult AML patients who received an allogeneic HSCT regarding the biological and clinical implications of the SRSF2 mutation status at diagnosis and in morphologic remission at HSCT. We found 12.5% of the patients to be SRSF2 mutated at diagnosis. Mutated patients had increased EZH2 missplicing events with P95H likely driving this pathobiology most effectively. However, the amount of EZH2 missplicing events, as a functional surrogate marker did not associate with relevant biological or clinical characteristics. We observed a persistence of mutations in remission before HSCT in the majority (93%) of SRSF2 mutated AML patients. Importantly, the variant allele frequency (VAF) levels of SRSF2 mutations in remission at HSCT did not correlate with outcomes following HSCT consolidation, limiting the applicability of SRSF2 mutations as a marker for residual AML disease. Following allogeneic HSCT SRSF2 mutated AML patients experienced a 2-year overall survival of 77%, indicating that SRSF2 mutated AML patients may benefit from HSCT consolidation.

AML with Renal Infiltration Manifesting as Acute Renal Failure, Diagnosed with FDG-PET CT Scan: Case Report

Extramedullary disease in acute myeloid leukemia is a known phenomenon with reported incidence of 2.5-9.1 %. However, acute kidney injury due to direct infiltration of malignant cells is reported in only 1% cases of acute leukemia. We report a case of acute myeloid leukemia who developed acute kidney failure at presentation, was diagnosed with renal and pancreatic infiltration by FDG-PET scan and was treated successfully with hypomethylating agent and venetoclax. PET-CT scan can be a non-invasive modality for diagnosing extramedullary disease in acute myeloid leukemia and monitoring of response to therapy in these cases. Early initiation of anti-leukemia therapy in our case lead to complete metabolic response with normalization of the renal functions.

The Degree and Effect Of Weight Loss In Acute Myeloid Leukemia Patients Receiving Induction and Post-Remission Chemotherapy

▪ IntroductionInduction and post-remission chemotherapy for acute myeloid leukemia (AML) is an intensive regimen with significant toxicities performed almost exclusively in inpatient or closely monitored outpatient settings. Extreme weight loss is associated with poor outcomes in cancer. We studied whether treated AML patients (pts) lose a substantial amount of weight and whether weight loss affects overall survival (OS). MethodsAdult pts diagnosed with AML at the Cleveland Clinic from 08/08 - 11/12 were included. All pts completed intensive induction chemotherapy with 7+3 (cytarabine and anthracycline – daunorubicin, idarubicin, or mitoxantrone) as first-line therapy. Pts must have achieved complete remission (CR) by first-line induction or day 14 re-induction chemotherapy. Previously treated pts or those with relapsed or refractory disease were excluded. Co-morbidities and AML disease characteristics were assessed, in addition to weight and body-mass index (BMI) at baseline and throughout AML treatment course until disease relapse, stem cell transplantation (SCT), or loss-to-follow-up/death. The primary endpoints were change in BMI relative to completion of post-remission therapy (PRT), OS, and event-free survival (EFS). OS and EFS were measured from completion of PRT to the date of death (OS) and relapse or need for SCT (EFS). To assess impact of weight recovery on OS and EFS separate linear regression models were fit to each pt to determine rate and direction of change during the first 3 months following the completion of PRT. Pts whose fitted regression lines had slopes>0 were considered to have positive weight trajectories; pts with slopes<0, negative trajectories. Group comparisons were made using the Wilcoxon signed-rank test, logrank test, and proportional hazards models. ResultsOf 126 AML pts who met the inclusion criteria, 43% (n=54) were male and the median age at treatment start was 54 years (range 18-77). Disease characteristics were: acute promyelocytic leukemia 11% (n=14), other AML 89% (n=112); de novo 75% (n=94), secondary 24% (n=31); cytogenetic risk group - favorable 23% (n=29), intermediate 47% (n=59), unfavorable 21% (n=27), unknown 9% (n=11). Co-morbidities potentially impacting BMI and outcome included diabetes (11%), hypertension (42%), hyperlipidemia (28%), alcohol use (65%), use of steroids (13%) or oral contraceptives (16%). Median weight at the start of therapy was 84.9kg (range 37-159.5) and BMI was 29.6 (range 17.6-57.8). BMI groups included normal (18.5-24.9, 26%), overweight-moderately obese (>25-35, 54%), severely obese (>35, 20%). From the start of treatment, median weight loss was: 2% (range -21-53%, p=0.0001) at day 14; 6% (range -19-22%, p<0.0001) at time of CR determination; 4% (range -23-38%, p<0.0001) at end of PRT. With the exception of diabetes (time of CR, p=0.06) and alcohol use (time of CR and end of PRT, p=0.05 in both cases), none of the clinical or demographic factors examined impacted weight loss. In total, 29% of pts (n=37) had >10% weight loss at the completion of PRT, representing an absolute loss of 5.9-20.6kg; 40% of pts (n=50) had a loss<10%, and 31% of pts (n=39) had no change or weight gain. In the group with >10% loss, median time to recovery of >95% pre-treatment weight was 7 months; 11% of pts (n=4) exceeded their pre-treatment weight by >10%. Only age (p=0.06) was associated with weight recovery time, with younger pts recovering weight faster (Figure 1). 60% of pts (n=73) had disease progression and 36% of pts (n=44) had died; estimated EFS and OS are 10.1 (95% CI 6.6-14.7) and 28.2 months (95% CI 17.1-46.4), respectively. The degree of weight loss following completion of PRT was not associated with EFS (p=0.79) or OS (p=0.82). However, there was some indication that pts with positive weight trajectories had longer EFS than pts with negative trajectories, p=0.07 (Figure 2); OS was not impacted significantly (p=0.26). ConclusionAML pts achieving CR with induction therapy experience a median 6% weight loss from start of therapy, but recover by 7 months (median). Those with diabetes or alcohol use lose more weight. Weight changes were not associated with survival; however, there was some suggestion that pts who began to gain weight once PRT was completed had longer EFS than pts whose weight continued to decline. These data can be used to frame pt expectations about weight fluctuations going through AML treatment. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.