The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

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 remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, 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 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the 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.

Impact of Laparoscopic Sleeve Gastrectomy on Cardiovascular Pharmacotherapy in Left Ventricular Assist Device Patients.

ABSTRACT: 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, P < 0.001), 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.

Pharmacogenomic Score Effectively Personalizes Treatment of Acute Myeloid Leukemia.

PURPOSE: Cytarabine (also known as ara-C) has been the backbone of acute myeloid leukemia (AML) chemotherapy for more than five decades. Recent pharmacogenomics-based 10-SNP ara-C (ACS10) scores showed low ACS10 (≤0) to be associated with poor outcomes in patients with AML treated with standard chemotherapy. Here, we evaluated the ACS10 score in the context of three different induction I regimens in patients with pediatric AML. EXPERIMENTAL DESIGN: ACS10 score groups (low, ≤0, or high, >0) were evaluated for association with event-free survival (EFS) and overall survival (OS) by three randomized treatment arms in patients treated on the AML02 (NCT00136084) and AML08 (NCT00703820) clinical trials: AML02 low-dose ara-C (LDAC arm, n = 91), AML02 + AML08 high-dose ara-C (HDAC arm, n = 194), and AML08 clofarabine + ara-C (Clo/ara-C arm, n = 105) induction I regimens. RESULTS: Within the low-ACS10 score (≤0) group, significantly improved EFS and OS were observed among patients treated with Clo/ara-C as compared with LDAC (EFS, HR = 0.45; 95% CI, 0.23-0.88; P = 0.020; OS, HR = 0.44; 95% CI, 0.19-0.99; P = 0.048). In contrast, within the high-ACS10 score group (score >0), augmentation with Clo/ara-C was not favorable as compared with LDAC (Clo/ara-C vs. LDAC, EFS, HR = 1.95; 95% CI, 1.05-3.63; P = 0.035; OS, HR = 2.10; 95% CI, 0.96-4.59; P = 0.063). Personalization models predicted 9% improvement in the outcome in ACS10 score-based tailored induction (Clo/ara-C for low and LDAC for high-ACS10 score groups) as compared with nonpersonalized approaches (P < 0.002). CONCLUSIONS: Our findings suggest that tailoring induction regimens using ACS10 scores can significantly improve outcomes in patients with AML. Given the SNPs are germline, preemptive genotyping can accelerate matching the most effective remission induction regimen.

Statistical Methods Inspired by Challenges in Pediatric Cancer Multi-omics.

Pediatric cancer multi-omics is a uniquely rewarding and challenging domain of biomedical research. Public generosity bestows an abundance of resources for the study of extremely rare diseases; this unique dynamic creates a research environment in which problems with high-dimension and low sample size are commonplace. Here, we present a few statistical methods that we have developed for our research setting and believe will prove valuable in other biomedical research settings as well. The genomic random interval (GRIN) method evaluates the loci and frequency of genomic abnormalities in the DNA of tumors to identify genes that may drive the development of malignancies. The association of lesions with expression (ALEX) method evaluates the impact of genomic abnormalities on the RNA transcription of nearby genes to inform the formulation of biological hypotheses on molecular mechanisms. The projection onto the most interesting statistical evidence (PROMISE) method identifies omic features that consistently associate with better prognosis or consistently associate with worse prognosis across multiple measures of clinical outcome. We have shown that these methods are statistically robust and powerful in the statistical bioinformatic literature and successfully used these methods to make fundamental biological discoveries that have formed the scientific rationale for ongoing clinical trials. We describe these methods and illustrate their application on a publicly available T-cell acute lymphoblastic leukemia (T-ALL) data set. A companion github site ( https://github.com/stjude/TALL-example ) provides the R code and data necessary to recapitulate the example data analyses of this chapter.

Polygenic Pharmacogenomic Markers as Predictors of Toxicity Phenotypes in the Treatment of Acute Lymphoblastic Leukemia: A Single-Center Study.

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.

SAMHD1 single nucleotide polymorphisms impact outcome in children with newly diagnosed acute myeloid leukemia.

Cytarabine arabinoside (Ara-C) has been the cornerstone of acute myeloid leukemia (AML) chemotherapy for decades. After 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 has been shown to affect 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 the SAMHD1 gene for association with clinical outcome in 400 pediatric patients with newly diagnosed AML from 2 clinical trials, AML02 and AML08. Three SNPs, rs1291128, rs1291141, and rs7265241 located in the 3' region of SAMHD1 were significantly associated with at least 1 clinical outcome: minimal residual disease after induction I, event-free survival (EFS), or overall survival (OS) in the 2 cohorts. In an independent cohort of patients from the COG-AAML1031 trial (n = 854), rs7265241 A>G remained significantly associated with EFS and OS. In multivariable analysis, all the SNPs remained independent predictors of clinical outcome. 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.

Genome-wide CRISPR/Cas9 screen identifies etoposide response modulators associated with clinical outcomes in pediatric AML.

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.

Polygenic Ara-C Response Score Identifies Pediatric Patients With Acute Myeloid Leukemia in Need of Chemotherapy Augmentation.

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.

A ten-gene DNA-damage response pathway gene expression signature predicts gemtuzumab ozogamicin response in pediatric AML patients treated on COGAAML0531 and AAML03P1 trials.

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 = 1.7 7 * 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.

The genomic landscape of pediatric acute lymphoblastic leukemia.

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.

Predictors of perioperative bleeding in left ventricular assist device implantation.

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 <60 ml/min (OR 2.58, 95% CI 1.34 to 4.94, p-value 0.004), preoperative right atrial pressure >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.

A six-gene leukemic stem cell score identifies high risk pediatric acute myeloid leukemia.

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 < 0.00001. pLSC6 remained significantly associated with EFS and overall survival (OS) after adjusting for induction 1-MRD status, risk-group, FLT3-status, WBC-count at diagnosis and age. pLSC6 formula developed in the AML02 cohort was validated in the pediatric AML-TARGET project data (n = 205), confirming its prognostic value in both single-predictor and multiple-predictor Cox regression models. In both cohorts, pLSC6 predicted outcome of transplant patients, suggesting it as a useful criterion for transplant referrals. Our results suggest that pLSC6 score holds promise in redefining initial risk-stratification and identifying poor risk AML thereby providing guidance for developing novel treatment strategies.

Correction: A six-gene leukemic stem cell score identifies high risk pediatric acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNAs Mediated Regulation of Expression of Nucleoside Analog Pathway Genes in Acute Myeloid Leukemia.

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.

Comprehensive Ara-C SNP score predicts leukemic cell intracellular ara-CTP levels in pediatric acute myeloid leukemia patients.

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 <0). ACSS designation was significant predictor of intracellular ara-CTP levels (p = 0.00012), suggesting a cumulative or synergistic effect of the significant SNPs. CONCLUSION: ACSS score designation holds promise in definfing ara-C dose. Validation of the clinical utility of ACSS score in other independent cohorts will help identification of patients with potentially lower or higher levels of the ara-CTP in leukemic cells, thereby opening up opportunities for dose management to reduce toxicity and enhance efficacy.