Search

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remain incompletely understood. Here, using integrated whole genome and transcriptome sequencing of 124 iAMP21-ALL patients, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL according to patterns of copy number alteration and structural variation. This large dataset enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which are differentially expressed compared to non-iAMP21-ALL cases, and including multiple genes implicated in the pathogenesis of acute leukemia: CHAF1B, DYRK1A, ERG, HMGN1 and RUNX1. Using multimodal single cell genomic profiling, including single cell whole genome sequencing of two cases, we documented clonal heterogeneity and genomic evolution, formally demonstrating that acquisition of the iAMP21-chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

PURPOSE Acute lymphoblastic leukemia (ALL) is the most prevalent cause of childhood cancer and requires a long course of therapy consisting of three primary phases with interval intensification blocks. Although these phases are necessary to achieve remission, the primary chemotherapeutic agents have potentially serious toxicities, which may lead to delays or discontinuations of therapy. The purpose of this study was to perform a comprehensive pharmacogenomic evaluation of common antileukemic agents and develop a polygenic toxicity risk score predictive of the most common toxicities observed during ALL treatment. METHODS This cross-sectional study included 75 patients with pediatric ALL treated between 2012 and 2020 at the University of Florida. Toxicity data were collected within 100 days of initiation of therapy using CTCAE v4.0 for toxicity grading. For pharmacogenomic evaluation, single-nucleotide polymorphisms (SNPs) and genes were selected from previous reports or PharmGKB database. 116 unique SNPs were evaluated for incidence of various toxicities. A multivariable multi-SNP modeling for up to 3-SNP combination was performed to develop a polygenic toxicity risk score of prognostic value. RESULTS We identified several SNPs predictive of toxicity phenotypes in univariate analysis. Further multivariable SNP-SNP combination analysis suggest that susceptibility to chemotherapy-induced toxicities is likely multigenic in nature. For 3-SNPscore models, patients with high scores experienced increased risk of GI ( P = 2.07E-05, 3 SNPs: TYMS-rs151264360/FPGS-rs1544105/GSTM1-GSTM5-rs3754446), neurologic ( P = .0005, 3 SNPs: DCTD-rs6829021/SLC28A3-rs17343066/CTPS1-rs12067645), endocrine ( P = 4.77E-08, 3 SNPs: AKR1C3-rs1937840/TYMS-rs2853539/CTH-rs648743), and heme toxicities ( P = .053, 3 SNPs: CYP3A5-rs776746/ABCB1-rs4148737/CTPS1-rs12067645). CONCLUSION Our results imply that instead of a single-SNP approach, SNP-SNP combinations in multiple genes in drug pathways increases the robustness of prediction of toxicity. These results further provide promising SNP models that can help establish clinically relevant biomarkers allowing for greater individualization of cancer therapy to maximize efficacy and minimize toxicity for each patient.

PURPOSE To establish a patient-specific polygenic score derived from cytarabine (ara-C) pathway pharmacogenomic evaluation to personalize acute myeloid leukemia (AML) treatment. MATERIALS AND METHODS Single nucleotide polymorphisms (SNPs) in the ara-C-pathway genes were analyzed with outcome in patients from the multicenter-AML02 trial (N = 166). Multi-SNP predictor modeling was used to develop 10-SNP Ara-C_SNP score (ACS10) using top SNPs predictive of minimal residual disease and event-free survival (EFS) from the AML02-cohort and four SNPs previously associated with ara-C triphosphate levels in the AML97 trial. ACS10 was evaluated for association with outcomes in each clinical trial arms: the standard low-dose ara-C (LDAC, n = 91) and augmented high-dose ara-C (HDAC, n = 75) arms of AML02 and the standard Ara-C, daunorubicin and etoposide (ADE) (n = 465) and the augmented ADE + gemtuzumab ozogamicin (GO; n = 466) arms of AAML0531 trial. RESULTS In the standard LDAC-arm of AML02 cohort, the low-ACS10 score group (≤ 0) had significantly worse EFS (ACS10 low v high hazard ratio [HR] = 2.81; 95% CI, 1.45 to 5.43; P = .002) and overall survival (OS; HR = 2.98; 95% CI, 1.32 to 6.75; P = .009) compared with the high-ACS10 group (score > 0). These results were validated in the standard-ADE arm of AAML0531, with poor outcome in the low-ASC10 group compared with the high-ACS10 group (EFS: HR = 1.35, 95% CI, 1.04 to 1.75, P = .026; OS: HR = 1.64, 95% CI, 1.2 to 2.22, P = .002). Within the augmented arms (AML02-HDAC and AAML0531-ADE + GO), EFS and OS did not differ between low- and high-ACS10 score groups. In both cohorts, patients with low-ACS10 consistently showed a 10-percentage point improvement in 5-year EFS with augmented therapy (AML02-HDAC or AAML0531-ADE + GO arms) than with standard therapy (AML02-LDAC or AAML0531-ADE arms). CONCLUSION Patients with low-ACS10 score experienced significantly poor outcome when treated on standard regimen. Augmentation with either high-dose ara-C or GO addition improved outcome in low-ACS10 group. A polygenic ACS10 score can identify patients with unfavorable pharmacogenetic characteristics and offers a potential for an elective augmented therapy option.

Cytarabine arabinoside (Ara-C) has been the cornerstone of AML chemotherapy for decades. Following cellular uptake, it is phosphorylated into its active triphosphate form (Ara-CTP), which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in the Ara-C metabolic pathway have been shown to impact intracellular abundance of Ara-CTP and thus its therapeutic benefit. Recently, SAMHD1 (SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1) has emerged to play a role in Ara-CTP inactivation, development of drug resistance and consequently, clinical response in AML. Despite this the impact of genetic variations in SAMHD1 on outcome in AML has not been investigated in depth. In this study, we evaluated 25 single nucleotide polymorphisms (SNPs) within SAMHD1 gene for association with clinical outcome in 400 newly diagnosed pediatric AML patients from two clinical trials- AML02 and AML08. Three SNPs, rs1291128, rs1291141, and rs7265241 located in the 3' region of SAMHD1 were significantly associated with at least one clinical outcome endpoint: minimal residual disease (MRD) after induction I, event free survival (EFS), or overall survival (OS) in the two cohorts. In an independent cohort of patients from COG-AAML1031 trial (n=854), rs7265241 A>G remained significantly associated with EFS and OS. In multivariable analysis, adjusting for other prognostic factors such as race, age, risk group, and white blood cell count, all the SNPs remained independent predictors of clinical outcome endpoints. These results highlight the relevance of the SAMHD1 pharmacogenomics in context of response to Ara-C in AML and warrants the need for further validation in expanded patient cohorts.

Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.

Gemtuzumab ozogamicin (GO) is an anti-CD33 monoclonal antibody linked to calicheamicin, a DNA damaging agent, and is a well-established therapeutic for treating acute myeloid leukemia (AML). In this study, we used LASSO regression modeling to develop a 10-gene DNA damage response gene expression score (CalDDR-GEx10) predictive of clinical outcome in pediatric AML patients treated with treatment regimen containing GO from the AAML03P1 and AAML0531 trials (ADE+GO arm, N = 301). When treated with ADE+GO, patients with a high CalDDR-GEx10 score had lower complete remission rates (62.8% vs. 85.5%, P = 1.77*10(−5)) and worse event-free survival (28.7% vs. 56.5% P = 4.08*10(−8)) compared to those with a low CalDDR-GEx10 score. However, the CalDDR-GEx10 score was not associated with clinical outcome in patients treated with standard chemotherapy alone (ADE, N = 242), implying the specificity of the CalDDR-GEx10 score to calicheamicin-induced DNA damage response. In multivariable models adjusted for risk group, FLT3-status, white blood cell count, and age, the CalDDR-GEx10 score remained a significant predictor of outcome in patients treated with ADE+GO. Our findings present a potential tool that can specifically assess response to calicheamicin-induced DNA damage preemptively via assessing diagnostic leukemic cell gene expression and guide clinical decisions related to treatment using GO.

Left ventricular assist device (LVAD) implantation is increasingly utilized in patients with advanced heart failure and morbid obesity. Laparoscopic sleeve gastrectomy (LSG) can facilitate weight loss in this population and can ultimately change the pharmacokinetics of heart failure therapeutics. In this study, we aimed to explore the changes in cardiovascular pharmacotherapy post LSG intervention. We conducted a retrospective observational cohort study of morbidly obese LVAD patients between 2013 and 2019 at the University of Florida with available pharmacotherapeutic data at 1 and 6 months. Thirteen post-LSG patients and 13 control subjects were included in the final analysis. In the post-LSG group, the mean body mass index decreased significantly (44 ± 5 vs. 34 ± 4.9, P0.001), and 7 patients were successfully bridged to cardiac transplantation. Only 3 patients required adjustment of their LVAD speed. Mean return to flow decreased by 8 mm Hg, despite a 45% reduction in the mean number of vasodilators per patient (1.2 vs. 0.7, P = 0.03). Mean weekly warfarin dose decreased by 35% after 6 months (32.9 ± 20.9 vs. 50.7 ± 26.6, P = 0.01). The use of diuretics, vasodilators, and beta-blockers was significantly reduced by 50%, 45%, and 35%, respectively. None of these changes were observed in the control group at 6-month follow-up post LVAD. In this single-center experience, weight loss post LSG is associated with decreased vasodilator, diuretic, and anticoagulant medication requirements in LVAD patients.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Recently, mRNA-expression signature enriched in LSCs was used to create a 17-gene leukemic stem cell (LSC17) score predictive of prognosis in adult AML. By fitting a Cox-LASSO regression model to the clinical outcome and gene-expression levels of LSC enriched genes in 163 pediatric participants of the AML02 multi-center clinical trial (NCT00136084), we developed a six-gene LSC score of prognostic value in pediatric AML (pLSC6). In the AML02 cohort, the 5-year event-free survival (EFS) of patients within low-pLSC6 group (n = 97) was 78.3 (95% CI = 70.5–86.9%) as compared with 34.5(95% CI = 24.7–48.2 %) in patients within high-pLSC6 group (n = 66 subjects), p

Aim: Cytarabine (Ara-C), a mainstay of acute myeloid leukemia (AML) treatment, is a prodrug requiring activation to ara-CTP for its antileukemic activity. Aim of this study was to evaluate impact of genetic variants in the key genes involved in ara-C metabolism on the leukemic cell intracellular levels of ara-CTP. Method: We investigated SNPs in 14 ara-C metabolic-pathway genes, for association with intracellular ara-CTP levels, in leukemic cells obtained post-initiation of cytarabine infusion in pediatric AML patients (n = 68). Results: Nine SNPs were significantly associated with leukemic cell intracellular concentration of ara-CTP. A comprehensive ara-CTP-SNP-score (ACSS) was further developed from top four SNPs identified in regression model. Patients were classified into three groups based on ACSS: high-ACSS (score >0), intermediate-ACSS (score = 0) and low-ACSS (score

Acute myeloid leukemia (AML) is a heterogeneous disease characterized in part by genetic and epigenetic alterations. Cytarabine arabinoside (Ara-C) has been the cornerstone of chemotherapy treatment for patients diagnosed with AML for decades. Following cellular uptake, ara-C is phosphorylated into its active metabolite, ara-CTP, which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in ara-C activation/inactivation pathway (Fig 1A), can impact intracellular abundance of ara-CTP and thus its therapeutic benefit. Recently, deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) was shown to limit the efficacy of ara-C by intracellularly hydrolyzing its active metabolite (PMID: 27991919). Other nucleoside analogs, such as clofarabine, fludarabine, and gemcitabine have also been shown to be substrates of SAMHD1 (PMID:30305425). Among adult AML patients, higher SAMHD1 expression in the leukemic cells has been found to correlate with poor outcome (PMID: 30341277). However, whether genetic variation in SAMHD1 has any impact on the clinical outcomes in pediatric AML patients has not been evaluated in depth. In this study, we looked into 25 single nucleotide polymorphisms (SNPs) within SAMHD1 gene for their association with clinical outcome of newly diagnosed pediatric AML patients in 2 cohorts (multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=231] clinical trials). Briefly, genotypes for the 25 SNPs within SAMHD1 genes were obtained from Illumina 2.5 Omni data of AML patients. Association analysis was conducted using logistic regression models comparing minimal residual disease (MRD) positivity after treatment with chemotherapy using an unadjusted model and an adjusted model stratified by initial risk assignment of the patient determined at diagnosis (i.e. low, standard, and high). MRD positivity was defined as one or more leukemic cell per 1000 mononuclear bone-marrow cells. Cox proportional hazard models were used to examine the association of the SNPs with EFS and OS and considered three modes of inheritance. Cox regression models were performed with or without adjusting for provisional risk group assignment at time of diagnosis. Significance levels for association of SNP with clinical outcome were set at p < 0.05. Three top SNPs significantly associated with clinical outcomes were all localized in the 3'UTR region of SAMHD1(Fig 1B). Presence of the variant allele of rs7265241 was associated with a lower EFS and OS in both AML02 and AML08 cohorts. Figure 1C shows the results for OS in AML02 (HR = 2.24, 95% CI (1.05-3.55), p=0.02) and AML08 (HR = 1.52, 95% CI (1.04-1.99), p=0.01). Another 3'UTR SNP, rs1291128 was associated with poor OS in AML02 and in the clofarabine plus ara-C treatment arm in AML08 trial. For rs1291141, T allele was associated with higher MRD positivity (OR: 2.12, p=0.01) in AML02 and poor OS (HR = 1.55, 95% CI (1.12-2.14), p=0.008) in AML08(Fig 1D). Consistent with the clinical outcome results, rs1291141 T allele was also associated with higher SAMHD1 expression in the whole blood (p=8.1e-14) and several other tissues in the GTEx database. https://gtexportal.org/home/ Our results suggest that genetic variation in SAMHD1 is associated with clinical outcomes in pediatric AML patients. Future studies are aimed at looking at haplotypes and SNP-SNP combinations to establish the impact SAMHD1 pharmacogenomics on its expression within leukemic cells and subsequent response to nucleoside analogs in AML patients. SAMHD1 has also been implicated as a tumor suppressor; thus, future studies will evaluate its role both as a proliferation regulator and in drug resistance. Acknowledgements: NIH-R01-CA132946 (Lamba and Pounds), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC), and American Society of Hematology Bridge funding (Lamba) funded the study. We thank Drs. Campana, Coustan-Smith, and Raimondi for MRD and cytogenetic data. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Despite cure rates in acute lymphoblastic leukemia (ALL) exceeding 90% in clinical trials, morbidity due to drug toxicities is high. Genetic polymorphisms can influence gene expression and activity, impacting pharmacokinetics and causing inter-individual variation in drug levels, which contributes to toxicity if levels are high or relapse if levels are low. We hypothesize that pharmacogenomic testing will identify patient specific variations in genes involved in metabolism of cytotoxic agents. This knowledge will allow clinicians to optimize therapy by providing pharmacogenomics based biomarkers related to increased toxicities. Data has shown that treatment interruptions and omissions due to toxicities affect outcomes and morbidities in children with cancer. Objective : To correlate pharmacogenomic biomarkers with toxicity phenotypes in children receiving therapy for ALL. Methods: This cross-sectional study involved subjects at a tertiary academic center (Fig. 1A). Subjects aged 1 year to 26 years with ALL treated after May 2012 were eligible. A total of 75 patients treated between 2012 and 2020 were included. Pharmacogenomic testing was performed on peripheral blood. Genomic DNA was tested for 118 single-nucleotide polymorphisms (SNP) in 55 genes for transport and metabolism of cytarabine, vincristine, methotrexate, dauno/doxorubicin, and mercaptopurine/thioguanine were analyzed using the Sequenom-based genotyping that uses MALDI-TOF based chemistry. SNPs were tested using logistic regression models for association with toxicities in additive, dominant, and recessive modes of inheritance. CTCAE v4.0 was used for grading all toxicities during the first 100 days of therapy. For endocrine (endo) and neurological (neuro) toxicities, 25 patients exhibited between grade 1-3 toxicities. For gastrointestinal (GI) toxicities, 25 patients exhibited between grade 2-3 toxicities. For hematological (heme) toxicities, 11 patients exhibited between grade 2-4 toxicities. Odds ratio and 95% confidence interval were calculated for each test and SNPs with association P-value 0, 0 or Results: For a GI toxicity score derived from 3 SNPs (TYMS-rs151264360, FPGS-rs1544105, and GSTM5-rs3754446), patients with >0 score had 79% incidence of GI toxicity (N=67) as compared to 10% in patients with score of 0 and 8% in patients with score 0; no toxicity was observed in patients with neurotoxicity score of 0 (p=4.7E-08, Fig. 1E). None of the patients with a score of Discussion: We identified germline SNPs predictive of toxicity phenotypes in a cohort of 75 subjects with ALL. The results of our multivariable SNP combination analysis suggest susceptibility to chemotherapy-induced toxicities is likely multigenic in nature. Instead of a single SNP approach, identification of combinations of mutations in drug pathways increases the robustness of predicting a patient's response to chemotherapy. Our results provide promising SNP models that can help establish clinically relevant biomarkers allowing for individualization of cancer therapy to optimize treatment for each patient. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Nucleoside analog, cytarabine (ara-C) is the mainstay of acute myeloid leukemia (AML) chemotherapy. Cytarabine and other nucleoside analogs require activation to the triphosphate form (ara-CTP). Intracellular ara-CTP levels demonstrate significant inter-patient variation and have been related to therapeutic response in AML patients. Inter-patient variation in expression levels of drug transporters or enzymes involved in their activation or inactivation of cytarabine and other analogs is a prime mechanism contributing to development of drug resistance. Since microRNAs (miRNAs) are known to regulate gene-expression, the aim of this study was to identify miRNAs involved in regulation of messenger RNA expression levels of cytarabine pathway genes. We evaluated miRNA and gene-expression levels of cytarabine metabolic pathway genes in 8 AML cell lines and The Cancer Genome Atlas (TCGA) data base. Using correlation analysis and functional validation experiments, our data demonstrates that miR-34a-5p and miR-24-3p regulate DCK, an enzyme involved in activation of cytarabine and DCDT, an enzyme involved in metabolic inactivation of cytarabine expression, respectively. Further our results from gel shift assays confirmed binding of these mRNA-miRNA pairs. Our results show miRNA mediated regulation of gene expression levels of nucleoside metabolic pathway genes can impact interindividual variation in expression levels which in turn may influence treatment outcomes.

Study objective Early bleeding is a common source of morbidity associated with left ventricular assist device (LVAD) implantation. Our objective was to identify potential predictors of peri-implant bleeding. Methods We conducted a retrospective cohort study of LVAD implants at our institution between January 2010 and November 2018. A total of 210 patients were included. Data were collected for the duration of implant hospitalization, including perioperative invasive hemodynamics, echocardiography and operative details, antiplatelet and anticoagulant use, bleeding events and blood product use, and thromboembolic events. Peri-operative bleeding was defined as a transfusion requirement of >4 units of packed red blood cells in the intraoperative and first 7 days postoperative period, or a major 7-day post-implant overt bleeding event requiring procedural intervention. Results Perioperative bleeding occurred in 32% of patients and required surgical re-exploration in 9%. Multivariable logistic regression analysis identified history of previous sternotomy (OR 2.63, 95% CI 1.29 to 5.35, p-value 0.008), preoperative glomerular filtration rate 13 mm Hg (OR 2.36, 95% CI 1.19 to 4.67, p-value 0.014) and concomitant tricuspid valve repair (OR 2.48, 95% CI 1.23 to 5.01, p-value 0.011) as independent predictors of perioperative bleeding. In-hospital thromboembolic events occurred in 5% of patients, but there were no significant predictors for them. Conclusions Elevated right atrial pressure appears to be a reversible risk factor for early bleeding that should be targeted during pre-implant optimization of LVAD candidates.

Introduction: Ara-C (cytarabine), daunorubicin, and etoposide (ADE) remain key chemotherapies used in remission induction and consolidation therapy of pediatric acute myeloid leukemia (pAML). However, multiple drug resistance is a major cause of therapeutic failure in pAML. Several ADE-significant pharmacokinetic/pharmacodynamic (PK/PD) genes have been identified. However, it is unknown which microRNAs (miRNA) have a significant impact on the regulation of gene (mRNA) expression levels involved in ADE's pharmacology and the treatment outcomes of pAML patients. Methods: pAML patients with available bone marrow mRNA and miRNA expression levels from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database were included in the analysis. For step 1, we assembled a list of significant PK/PD genes (n=67) to ADE. In step 2, correlation analyses between mRNA expression levels of target PK/PD genes and miRNA expression levels were performed using mRNA-seq and miRNA-seq data from the TARGET database. miRNA-mRNA pairs with significant anti-correlation for expression levels using Spearman's rho (FDR < 0.05) were included for further analysis. In step 3, the least absolute shrinkage and selection operator (LASSO) was used to fit the Cox regression model with overall survival (OS) as the outcome variable on miRNAs with significant negative correlation with PK/PD genes from step 2. A thousand bootstraps of LASSO Cox regression were performed to identify miRNAs represented in at least 75% of the models. Coefficients from select miRNAs were used to generate a miRNA signature equation for the ADE response (ADEmiR) score. In step 4, patients were classified into low or high score groups according to the median value from the ADEmiR score, and analysis was performed for association with event-free survival (EFS), OS, minimal residual disease after the end of induction 1 (MRD1), and complete remission after the end of induction1 (CR1). Lastly, multivariate analysis was conducted using confounding factors such as age, clinically identified risk groups, FLT3 mutation status, and white blood cell count (WBC). The ADEmiR score equation was then validated using miRNA expression levels data from 164 adult AML patients from The Cancer Genome Atlas (TCGA) database. Results: 219 patients were identified with a mean age of 10±6 years, and 54% were male. Within TARGET cohort, the high ADEmiR score group was associated with significantly inferior EFS (HR=3.26, p Conclusions: The ADEmiR signature scoring system utilizing miRNA could serve as a prognostic tool to predict treatment outcomes in AML patients receiving ADE. Ongoing studies conducted by our group are focused on validating the ADEmiR score in other cohorts as well as its integration with our other gene-expression based prognostic tools such as ADE-Response Score (ADE-RS), and pediatric leukemic stem cell (pLSC6) score to predict optimal ADE treatment outcomes in pediatric AML. Disclosures No relevant conflicts of interest to declare.

Introduction Morbid obesity of class II or higher (BMI ≥ 35) is common in the heart failure population and is associated with poor outcomes after advanced heart failure therapies. Only 15% of obese left ventricular assist device (LVAD) patients are able to achieve adequate weight loss which permits heart transplantation. It has been reported that laparoscopic sleeve gastrectomy (LSG) is promising in this population given efficacy and minimally invasive nature. We sought to describe how a decrease in BMI post LSG impacts medical therapy with the hypothesis it may improve response to heart failure medical therapy. Methods We retrospectively reviewed LVAD patients who underwent LSG 2013 and 2019 at the University of Florida. Inclusion criteria included age > 18 and continued LVAD support for ≥ 6 months post LSG. Clinical data at 6 months follow-up was analyzed. Results We found 14 patients meeting the inclusion criteria. Mean age was 41 years and 61% were men. No major acute complications were noted post operatively from LSG. Mean BMI decreased from 44 to 34 (P Conclusions In this single center experience, in addition to providing succesful reduction in BMI conducive to heart transplantation, LSG was feasible and associated with significant reduction in antihypertensive and anticoagulation requirements.

Introduction: Cytarabine, daunorubicin and etoposide (ADE) are commonly used for remission and intensification of pediatric acute myeloid leukemia (AML). However, development of drug resistance is a major cause of treatment failure. In this study, we performed a comprehensive evaluation of expression levels of genes of pharmacological significance (pharmacokinetic /pharmacodynamic) to ADE and derived a drug response score predictive of treatment outcomes in pediatric AML patients. Methods: This study included 163 cases (median age=8.79 year, range= (0.013-21.1)) with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression and clinical data available. We used a penalized LASSO regression algorithm (glmnet R-package) to fit a cox regression model on diagnostic leukemic cell gene expression levels of 66 genes of pharmacological significance to ADE. We performed 1000 bootstraps of LASSO regression with event free survival (EFS) as the outcome variable and the five genes represented in at least 95% of the models were included to build an ADE-Response Score (ADE-RS) equation. Patients were classified into low or high score groups using recursive portioning implemented in Rpart-R package and evaluated for association with minimal residual disease after induction I (MRD1), EFS and overall survival (OS). ADE response score equation was further validated using RNA-Seq gene-expression data obtained from diagnostic samples of 432 pediatric AML patients enrolled in Children's Oncology Group (COG) AAML0531 and AAML03P1 treatment protocols. Results: After applying LASSO regression, we defined the equation: ADE-RS = (0.128 x DCTD) - (0.0993 x TOP2A) + (0.212 x ABCC1) - (0.113 x MPO) - (0.126 x CBR1) to develop ADE-response score (ADE-RS), followed by classifying patients into low (60%; 98 patients) or high (40%; 65 patients) score groups. Patients in the high ADE-RS group had significantly worse EFS (HR=4.07(2.43-6.84), P < 0.0001; Figure 1A) and OS (HR= 4.54(2.42-8.49), P In a multivariable cox-regression models that included pLSC6-ADE response score groups, MRD1 status, risk groups, WBC at diagnosis and age in AML02 cohort, high pLSC6-ADE score group was found significantly associated with poor EFS (HR=6.02(2.71-13.2), P Discussion: In this study, we defined a pharmacological response score focused on key genes of PK/PD significance to ADE. We further integrated LSC score with the ADE response score to improve our ability to predict treatment outcome in AML patients across different clinical trials. ADE-RS was composed of five genes: DCTD, which is a deaminase, involved in ara-C inactivation; CBR1, a carbonyl reductase involved in inactivation of daunorubicin (DNR); MPO, myeloperoxidase, an etoposide activator; ABCC1, an efflux transporter of DNR and etoposide; and TOP2A, DNA topoisomerase II alpha, which is a target for DNR and etoposide. Integrated pLSC6 and ADE-RS has a potential to predict treatment outcomes using diagnostic gene expression levels and accordingly develop treatment strategies to improve treatment outcome. Figure 1 Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding.

Despite the use of intensive chemotherapeutic regimens containing ara-C, daunorubicin and etoposide (ADE), in combination with stem cell transplantation for high-risk group patients, approximately significant patients experience relapse. Resistant and relapsed disease remains the most prevalent forms of clinical failure in treating this disease. The current scientific challenge is to identify candidate genes that are truly essential to drug resistance and thus may be pharmacologically targeted in a clinically effective manner. The development of the CRISPR/cas9 genome editing tool has dramatically improved the capabilities for functional screening in multiple systems including AML where genome-wide drop-out screens in AML cancer cell lines identified AML-essential genes. None of the studies have unfortunately integrated the CRISPR/cas9 screening tool with patient outcome data. In this study, we have successfully performed Genome scale Crispr knock out GeCKO -CRISPR/cas9 screening using Brunello library (targeting 19,114 genes) in K562 cell lines followed by treatments with etoposide, dauno and cytarabine. We performed a large-scale transduction into ~30 x 106 cells at a low multiplicity of infection (0.3). After puromycin selection the mutant cell library was exposed in triplicate to IC30 concentration of chemotherapeutic agents for up to 18 days with collection of samples at day 4 (ara-C, dauno, etoposide) days 12 and 18 (dauno and etoposide). A vehicle-only exposure was similarly carried out for 18 days. Direct sequencing of PCR amplified sgRNA guides from the pooled cells by short read sequencing (Illumina) was used to quantify the representation of each knockout clone in the pooled population (schema shown in Fig 1). The relative enrichment/depletion of each knockout clone was determined by ratio of the abundance of each clone between two samples. The FASTX-Toolkit, was used to extract the unique sgRNA sequences which were assembled into a Burrows-Wheeler index using the Bowtie build-index function and number of uniquely aligned reads for each sgRNA were calculated. The MaGeCK algorithms was used to analyze the read count data and perform statistical comparisons. The results for the etoposide screening are shown in Fig 2, overall 17 genes showed significant association with responsiveness to etoposide at all the three different time points (4, 12 and 18 days). Further investigation of these candidate genes in AML patients treated on AML02 trial demonstrated consistent and significant association between expression levels of several genes identified in CRISPR screening and clinical outcome. Fig 3 shows a representative result for 2 of the genes identified in etoposide screening, ABCC1and RAD54L2 both were associated with etoposide resistance in CRISPR screening and concordantly high expression was associated with greater induction 1 MRD, inferior event free survival (EFS, Fig 3) and overall survival (OS) in the AML02 cohort. ABCC1 is a drug efflux transporter that has been implicated in resistance to etoposide and daunorubicin as well as has been associated with poor prognosis in AML consistent with our results. RAD54L2, is a helicase involved in DNA damage repair response pathway. Other genes of interest identified in etoposide screening with significant association with clinical endpoints as MRD, EFS and OS included TKT, involved in pentose phosphate pathway and implicated in regulation of metabolic switch in cancer. A nucleotide excision repair gene, RAD23B, implicated in breast cancer progression as well as in CML and ALL. Similarly, for daunorubicin and cytarabine CRISPR/cas9 synthetic lethal screening identified target genes of clinical relevance that will be discussed in the presentation. Our approach shows that using CRISPR/cas9 targeted synthetic lethal screening as a reliable approach to not only identify and establish genes predictive of drug resistance and poor outcome but also potential targets for novel drug development that could be used in combination with currently approved agents. Disclosures No relevant conflicts of interest to declare.

Background: In September 2017, the FDA announced re-approval of gemtuzumab ozogamicin (GO), a CD33-directed antibody-drug conjugate, for treatment of newly diagnosed and relapsed/refractory AML setting the pace for a new era of personalized therapeutic options for AML. While the future of GO as a therapy in AML is bright, studies have shown that clinical response to GO is subject to interpatient variability (Gbadamosi 2018). Hitherto, efforts to understand the interpatient variation have focused on profiling CD33 expression levels (Pollard et al. 2016) and more recently on genetic variation in CD33 and ABCB1 predicative of GO response (Chauhan et al. 2019, Rafiee et al. 2019). However, efforts to understand interpatient variability in regards to calicheamicin, the DNA-damaging cytoxin linked to the antibody portion of GO, have been limited. Thus, we hypothesized that interpatient differences in expression levels of genes involved in pharmacodynamic effects of calicheamicin may impact GO response/resistance. Methods: Herein, our group has used the least absolute shrinkage and selection operator (LASSO) regression analysis method to identify critical genes with expression levels predictive of clinical outcomes in response to GO. Using data from the TARGET database, the expression levels of 18 genes, selected for their role in calicheamicin induced DNA-damage response, were extracted for AML patients treated with GO in the AAML03P1 and AAML0531 clinical trials (N=128; Table 1). Using a penalized LASSO regression algorithm (glmnet R-package), a Cox regression model was fit on to the expression levels of the selected gene. A thousand bootstraps of LASSO regression were performed and genes included in greater than 95% of the models were further selected. Average coefficients from the LASSO model were used to generate a gene signature equation designated as the DNA-damage response score (DDRS) given the nature of the genes included. Patients were classified into high (DDRS High) or low (DDRS Low) values for DDRS according to the median value and evaluated using Cox-proportional hazard model for survival data analysis, while the Chi-square test was used to examine the differences between categories, and the Wilcoxon rank-sum test was used to assess the differences in averages where appropriate. Results: The DDRS equation was defined as DDRS = (AKT1*-0.070) + (CASP9*-0.091) + (H2AFX*-0.160) + (XRCC4*0.373) where gene expression levels are multiplied by the coefficients obtained from the LASSO regression (Figure 1). Patients in the DDRS High group had significantly worse event free survival (EFS; HR = 2.22, P < 0.001; Figure 2A), lower complete remission rates (67.7% vs 94.6%, P < 0.001; Figure 3A), and a trend towards worse overall survival (OS; HR = 1.70, P = 0.058) as compared to patients within DDRS Low group. DDRS score was also significantly different amongst patients that were MRD+ve vs. MRD-ve after induction 1 therapy (21% difference, P = 0.038; Figure 3B). Consistent results were seen within standard risk group patients where patients in the DDRS High group had significantly worse EFS (HR = 2.29, P = 0.01; Figure 2B) as compared to those in the DDRS Low group. In preliminary multivariate Cox regression analysis, the DDRS remained a significant predictor of EFS amongst age, risk group, FLT3 status, and WBC (HR 2.42, P < 0.001; Table 2). Conclusion: Our preliminary investigation using LASSO regression model defined DDRS, a gene signature predictive of clinical response in patients treated with GO. The model included four genes well known for their involvement in DNA-damage response: AKT1, a kinase that regulates cell growth and division; CASP9, The initiator caspase involved in apoptosis; H2AFX, a DNA-damage marker that recruits other DNA-damage response proteins to damaged loci; and XRCC4, a ligase for DNA damage repair. Our future work focuses on expanding this investigation in bigger cohorts of patients representing different risk groups of AML as well as in vitro mechanistic studies. Once validated for its sensitivity and specificity to calicheamicin response, our results hold promise towards developing strategies for understanding interpatient variability in GO response and personalizing GO therapy based on diagnostic gene expression signatures. Disclosures No relevant conflicts of interest to declare.

Introduction Perioperative bleeding in the setting of LVAD implantation adversely impact outcomes. Hypothesis This project aims to determine pre-operative and intraoperative risk factors for this complication. Methods A retrospective cohort study was conducted including 90 consecutive advanced heart failure patients who underwent LVAD implantation as a bridge to transplantation or destination therapy from 2008 to 2017. By protocol, all anti-platelet therapies other than aspirin were discontinued 7 days pre-implantation. Aspirin was continued if clinically indicated. Additionally, any patients on anti-coagulant therapy were transitioned to intravenous heparin. Heparin infusion was discontinued 6 hours pre-operatively. Perioperative hemodynamics, laboratory values, cardiopulmonary bypass parameters, and blood product utilization were collected. Results Observable bleeding within 7 days of implant occurred in 15 patients (16%). This was significantly associated with the pre-operative use of heparin (OR 6.4; 95% CI 1.8-23.2; p=0.005) and its incidence increased for each 1 mmHg increase in right atrial pressure during pre-operative right heart catheterization (OR 1.1; 95% CI 1.0-1.2; p=0.03). Aspirin use within 3 days of implant increased the need of packed RBC transfusion both intra-operatively (OR 3.2; 95% CI 1.3-8.2; p=0.01) and post-operatively (OR 3.0; 95% CI 1.1-8.4; p=0.04) as well as increased the risk of hemoglobin drop ≥3 gm/dl on post-operative day 1 or the need of more than 2 packed RBC units peri-operatively (OR 4.2; 95% CI 1.6-11.4; p=0.004). Cardiopulmonary bypass time > 60 minutes increased the need for packed RBC transfusion post-operatively (OR 3.07; 95% CI 1.05-9.01; p=0.04). Conclusions Pre-operative aspirin and heparin are reversible factors that may affect the need for peri-operative blood products transfusion. This may indicate that these agents may need to be withheld longer or alternative antithrombotic preoperative agents should be considered.

Introduction: Resistance and relapse remain major obstacles in the treatment of acute myeloid leukemia (AML). Pre-existence and persistence of drug resistant leukemia stem cells (LSCs) is considered one of the major causes of relapse. A previous study (Ng et al., 2016) reported a prognostic signature of 17 genes (LSC17 score) differentially expressed in LSC+ compared to LSC- cell fractions that predicted outcome in patients with AML thereby classifying patients into high and low risk groups. The goal of this study is to determine the validity of LSC17 score in pediatric AML patients and to enhance its clinical utility by exploring a new score with limited number of stem cell genes. Methods: 150 pediatric patients with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression data and clinical data were included in the study. Since only 14 of the 17 genes were represented on the Affymetrix U133A gene chip we tested the validity of the LSC14 score using the previously defined equation (Ng et al, 2016) with multiple clinical endpoints such as minimum residual disease (MRD), event free survival (EFS) and overall survival (OS). To reduce the model complexity, we applied a penalized regression algorithm called the least absolute shrinkage and selection operator (LASSO) implemented in the glmnet R-package using event free survival (EFS) as an outcome variable. Score of the new equation, which included three genes, was designated as pediatric-LSC3 (pLSC3). pLSC3 was tested in the AML02 cohort for association of high or low pLSC3 (based on the median value) with clinical endpoints mentioned above. pLSC3 score equation was validated using publically available gene-expression data from 117 pediatric relapse enriched AML patient cohort enrolled in Children's Oncology Group (COG) protocol (TARGET database). COX-proportional hazard models and Log rank test were used for survival data analysis. Results: AML02 cohort: Patients with high LSC14 scores (greater than median), had significantly worse MRD (p In a multivariate COX regression model, pLSC3 score groups was the only significant covariate (table 1). It explained 13.1% of variability in EFS and 11.6% of variability in OS, while other prognostic factors such as risk groups, FLT3 status, treatment arm and age collectively explained 15.1 and 12.1 % of variability. Discussion: In summary, our results show validity of a previously defined LSC14 score in a pediatric AML population from the multicenter AML02 clinical trial. To enhance the clinical utility, score equation was further simplified and the final score (pLSC3) was derived from three genes: DNMT3B, which encodes for DNA methyltransferase; CD34, an important cell surface marker for early-undifferentiated LSCs; and GPR56, a G protein coupled receptor of significance in AML. Given that there is need to refine classification of a highly heterogeneous group of patients with standard risk AML, we show that differentiating standard risk patients based on pLSC3 score should be considered in the future. We show the relevance of pLSC3 in two independent cohorts, opening up opportunities to improve treatment outcomes of pediatric patients with AML. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) treatment response remains poorly understood. Although multiple studies have focused on understanding the transcriptomic and epigenetic landscape of AML, a genome-wide analysis of SNPs in pediatric AML has not yet been investigated in depth. Thus, we sought to identify genetic variants predictive of AML response, relapse, and survival in pediatric AML patients. For this study, we generated genome-wide SNP data patients (n=160) treated on the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) using Infinium Omni 2.5M Exome Beadchip. Standard GWAS QC procedure was followed in order to remove SNPs with call rate < 95%, monomorphic SNPs, SNPs with MAF Acknowledgments: We are thankful for funding from NIH R01-CA139246 and ALSAC. Disclosures No relevant conflicts of interest to declare.

This paper presents a variation tolerant driving technique for all-digital self-timed 3-level signaling high-speed SerDes transceivers. The proposed design generates the 3-level signal without a ½VDD driver, thus removing all the overhead and hassle of an additional supply. Moreover, the proposed all-digital scheme uses half the clock frequency while maintaining the same data rate of the conventional scheme. As a result, the proposed design is much more robust across all possible variations. The transceiver is designed for a 3mm long lossy on-chip differential interconnect in TSMC 65nm CMOS technology. The transmitter serializes the parallel 1.9375Gbps 8-bit, then multiplexes it with the 15.5GHz clock to generate the three level signal on the differential TL, and a simple RX detects both the data and the clock from the signal.

This paper proposes a new architecture that multiplexes both data and clock on serial links, reduces Inter symbol interference (ISI) by using a resistive termination technique, and uses two-level Manchester encoding to solve the reduced swing problem and enable the use of power efficient circuitry. Using this signaling scheme makes the system insensitive to jitter accumulation along the transmission line, and avoids the need for a power hungry clock and data recovery (CDR) circuit. A self-calibrating digital-delay line is also implemented inside the decoder to enable the system to operate efficiently across process, voltage and temperature variations. The proposed scheme is implemented for a 3mm long on-chip transmission line in TSMC 65nm technology and simulation results are presented.

Purpose Landmark trial data suggests intra-aortic balloon pump (IABP) use does not reduce mortality in patients with acute myocardial infarction and cardiogenic shock (AMI-CS). However, significant heterogeneity in shock severity exists in AMI-CS cohorts. Predictors of survival in AMI-CS managed with IABP have not been fully studied. Methods We performed a single center retrospective analysis of patients presenting with AMI-CS that underwent IABP insertion between 2014 and 2019. Survival data was obtained using EMR vital status query. We reviewed demographic, vitals, laboratory, EKG, catheterization, and hemodynamic data when available. Wilcoxon rank-sum test was used for continuous variable comparisons between patients who died before discharge and patients who survived to hospital discharge after IABP insertion. Chi-square or fisher exact tests were used to test association of categorical variables with the outcome variable. Results Of the 120 patients included in the study, 84 patients (70%) survived the hospitalization. Univariate analysis showed that survivors to hospital discharge were younger (mean age=62.71±11.85 vs 67.1±12.01; P=0.024), presented with lower INR (mean=1.23± 0.30 vs 1.85±0.75), serum creatinine (mean= 1.42±0.99 vs 1.91±1.15; P=0.002), and lactic acid (mean=2.65±2.15 vs 6.1±5.1; P=0.0007). Patients who survived had higher serum hemoglobin (mean= 12.85±2.57 vs 10.87±3.26 P= 0.0002), and were much less likely to be on inotrope or pressor support prior to IABP insertion (38% vs 83%, p= 1.39 E-05). Conclusion In this single center retrospective study of AMI-CS patients managed with IABP, several clinical variables were significantly associated with survival to hospital discharge and suggested an earlier or less severe stage of shock. Our findings highlight the need for additional studies to define predictors of survival in AMI-CS managed with IABP, which may guide optimal selection of a subset of AMI-CS that has favorable outcomes with IABP support.