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Purpose: Cisplatin-induced hearing loss (CIHL) is common and permanent. As compared with earlier otoprotectants, we hypothesized N-acetylcysteine (NAC) offers potential for stronger otoprotection through stimulation of glutathione (GSH) production. This study tested the optimal dose, safety, and efficacy of NAC to prevent CIHL. Patients and Methods: In this nonrandomized, controlled phase Ia/Ib trial, children and adolescents newly diagnosed with nonmetastatic, cisplatin-treated tumors received NAC intravenously 4 hours post-cisplatin. The trial performed dose-escalation across three dose levels to establish a safe dose that exceeded the targeted peak serum NAC concentration of 1.5 mmol/L (as identified from preclinical models). Patients with metastatic disease or who were otherwise ineligible were enrolled in an observation-only/control arm. To evaluate efficacy, serial age-appropriate audiology assessments were performed. Integrated biology examined genes involved in GSH metabolism and post-NAC GSH concentrations. Results: Of 52 patients enrolled, 24 received NAC and 28 were in the control arm. The maximum tolerated dose was not reached; analysis of peak NAC concentration identified 450 mg/kg as the recommended phase II dose (RP2D). Infusion-related reactions were common. No severe adverse events occurred. Compared with the control arm, NAC decreased likelihood of CIHL at the end of cisplatin therapy [OR, 0.13; 95% confidence interval (CI), 0.021–0.847; P = 0.033] and recommendations for hearing intervention at end of study (OR, 0.082; 95% CI, 0.011–0.60; P = 0.014). NAC increased GSH; GSTP1 influenced risk for CIHL and NAC otoprotection. Conclusions: NAC was safe at the RP2D, with strong evidence for efficacy to prevent CIHL, warranting further development as a next-generation otoprotectant.

Supplementary Fig. 1 Treatment schema & Sampling Timepoints

Supplementary Fig. 4 Evaluation of long-term storage of serum specimens (NAC assay)

Supplementary Fig. 3 Change in serum glutathione concentrations following NAC infusion

Supplementary Fig. 5 Progression-free survival in NAC treated and observation patients

Supplementary Fig. 2 Cumulative incidence of SIOP Grade ≥2 hearing loss

Supplemental Tables and Files

Purpose:Cisplatin-induced hearing loss (CIHL) is common and permanent. As compared with earlier otoprotectants, we hypothesized N-acetylcysteine (NAC) offers potential for stronger otoprotection through stimulation of glutathione (GSH) production. This study tested the optimal dose, safety, and efficacy of NAC to prevent CIHL.Patients and Methods:In this nonrandomized, controlled phase Ia/Ib trial, children and adolescents newly diagnosed with nonmetastatic, cisplatin-treated tumors received NAC intravenously 4 hours post-cisplatin. The trial performed dose-escalation across three dose levels to establish a safe dose that exceeded the targeted peak serum NAC concentration of 1.5 mmol/L (as identified from preclinical models). Patients with metastatic disease or who were otherwise ineligible were enrolled in an observation-only/control arm. To evaluate efficacy, serial age-appropriate audiology assessments were performed. Integrated biology examined genes involved in GSH metabolism and post-NAC GSH concentrations.Results:Of 52 patients enrolled, 24 received NAC and 28 were in the control arm. The maximum tolerated dose was not reached; analysis of peak NAC concentration identified 450 mg/kg as the recommended phase II dose (RP2D). Infusion-related reactions were common. No severe adverse events occurred. Compared with the control arm, NAC decreased likelihood of CIHL at the end of cisplatin therapy [OR, 0.13; 95% confidence interval (CI), 0.021–0.847; P = 0.033] and recommendations for hearing intervention at end of study (OR, 0.082; 95% CI, 0.011–0.60; P = 0.014). NAC increased GSH; GSTP1 influenced risk for CIHL and NAC otoprotection.Conclusions:NAC was safe at the RP2D, with strong evidence for efficacy to prevent CIHL, warranting further development as a next-generation otoprotectant.

Primer sequences used for rtPCR.

Background Pegaspargase (PEG‐ASP) is an integral component of therapy for acute lymphoblastic leukemia (ALL) but is associated with hepatotoxicity that may delay or limit future therapy. Obese and adolescent and young adult (AYA) patients are at high risk. Levocarnitine has been described as potentially beneficial for the treatment or prevention of PEG‐ASP‐associated hepatotoxicity. Methods We collected data for patients age ≥10 years who received levocarnitine during induction therapy for ALL, compared to a similar patient cohort who did not receive levocarnitine. The primary endpoint was conjugated bilirubin (c.bili) >3 mg/dl. Secondary endpoints were transaminases >10× the upper limit of normal and any Grade ≥3 hepatotoxicity. Results Fifty‐two patients received levocarnitine for prophylaxis (n = 29) or rescue (n = 32) of hepatotoxicity. Compared to 109 patients without levocarnitine, more patients receiving levocarnitine were obese and/or older and had significantly higher values for some hepatotoxicity markers at diagnosis and after PEG‐ASP. Levocarnitine regimens varied widely; no adverse effects of levocarnitine were identified. Obesity and AYA status were associated with an increased risk of conjugated hyperbilirubinemia and severe transaminitis. Multivariable analysis identified a protective effect of levocarnitine on the development of c.bili >3 mg/dl (OR 0.12, p = 0.029). There was no difference between groups in CTCAE Grade ≥3 hepatotoxicity. C.bili >3 mg/dl during induction was associated with lower event‐free survival. Conclusions This real‐world data on levocarnitine supplementation during ALL induction highlights the risk of PEG‐ASP‐associated hepatotoxicity in obese and AYA patients, and hepatotoxicity's potential impact on survival. Levocarnitine supplementation may be protective, but prospective studies are needed to confirm these findings., Patients who are adolescents or young adults and/or who are obese are at higher risk for pegaspargase‐induced hepatotoxicity. Levocarnitine supplementation may reduce the risk for hepatoxicity during induction therapy for acute lymphoblastic leukemia.

Being overweight or obese (OW/OB) during B-cell acute lymphoblastic leukemia (B-ALL) induction is associated with chemoresistance as quantified by minimal residual disease (MRD). We hypothesized that caloric and nutrient restriction from diet/exercise could lessen gains in fat mass (FM) and reduce postinduction MRD. The Improving Diet and Exercise in ALL (IDEAL) trial enrolled patients 10 to 21 years old, newly diagnosed with B-ALL (n = 40), in comparison with a recent historical control (n = 80). Designed to achieve caloric deficits ≥20% during induction, reduce fat intake/glycemic load, and increase activity, IDEAL’s end points were FM gain (primary), MRD ≥0.01%, and adherence/feasibility. Integrated biology explored biomarkers of OW/OB physiology. IDEAL intervention did not significantly reduce median FM change from baseline overall (+5.1% [interquartile range [IQR], 15.8] vs +10.7% [IQR, 16.0]; P = .13), but stratified analysis showed benefit in those OW/OB (+1.5% [IQR, 6.6] vs +9.7% [IQR, 11.1]; P = .02). After accounting for prognostic factors, IDEAL intervention significantly reduced MRD risk (odds ratio, 0.30; 95% confidence interval, 0.09-0.92; P = .02). The trial exceeded its adherence (≥75% of overall diet) and feasibility (≥80% completed visits) thresholds. Integrated biology found the IDEAL intervention increased circulating adiponectin and reduced insulin resistance. The IDEAL intervention was feasible, decreased fat gain in those OW/OB, and reduced MRD. This is the first study in any hematologic malignancy to demonstrate potential benefit from caloric restriction via diet/exercise to augment chemotherapy efficacy and improve disease response. A prospective, randomized trial is warranted for validation. These trials were registered at www.clinicaltrials.gov as #NCT02708108 (IDEAL trial) and #NCT01317940 (historical control).

Nearly all international regimens for pediatric acute lymphoblastic leukemia (ALL) incorporate intravenous "high dose" methotrexate (HDMTX, ≥1 gram/m2 ) to penetrate the central nervous system. Dosing is routinely adjusted for body surface area (BSA) but limited data describe the pharmacokinetics of HDMTX, particularly in obese and/or large patients. To understand the impact of body size (BSA) and body fat percentage (BFP) on HDMTX pharmacokinetics, we performed a secondary analysis of 36 children and adolescents 10-21 years old treated for newly diagnosed ALL and who were enrolled in a prospective study examining body composition. All patients received 5 grams/m2 of HDMTX infused over 24 hours. Plasma methotrexate concentrations were measured at +24, +42, and +48 hours. At 48 hours, ≥0.4 μmol/L was defined as "delayed elimination" necessitating prolonged supportive care. BFP was measured using dual-energy x-ray absorptiometry. A nonparametric population pharmacokinetic model was constructed with subsequent simulations to explore effects of BSA and BFP extremes. Despite standard BSA-adjusted dosing, we found significant intra-patient variability in mean MTX concentration (38%, range 1.2-86%). BSA and BFP were not linearly associated with increased area under the curve (AUC, p = 0.74 and p = 0.12), but both larger size (BSA) and greater obesity (BFP) were associated with an approximately twofold higher risk for delayed elimination at 48 hours. HDMTX AUC was not associated with toxicity. MTX pharmacokinetics vary among and even within patients despite BSA-adjusted dosing. Obesity and large size are identified as new risk factors for delayed elimination requiring further investigation. This article is protected by copyright. All rights reserved.

Malnutrition is associated with treatment-related toxicities (TRT) in adults with solid tumors and in children with leukemia. Few studies have assessed whether malnutrition in pediatric patients treated for solid tumors impacts risk for TRT, relapse, and/or survival. To address this knowledge gap, this retrospective study evaluated the association between body mass index (BMI) at diagnosis, and imputed BMI during therapy, on the prevalence of TRT, specific toxicities, relapse, and survival in pediatric patients with solid tumors treated with cisplatin-containing regimens. Kaplan–Meier curves and regression models evaluated the association between patient-specific characteristics (including BMI) and TRT, relapse, and survival. The cohort included 221 patients, of whom 22% were malnourished at diagnosis (10% were underweight and 12% were obese). Most patients (60%) experienced at least one severe TRT, and 30% developed more than one severe TRT. Most patients with obesity at diagnosis remained obese during therapy (62%). In multivariable analysis, obesity at diagnosis was significantly associated with a more than threefold greater risk for developing severe TRT (p = 0.037), specifically for acute or chronic kidney injury (p = 0.014). Obesity at diagnosis and adolescent and young adult age (≥15 years at diagnosis) were associated with worse event-free survival (hazard ratio [HR] 2.32, p = 0.024 and HR 2.28, p = 0.010, respectively) and overall survival (HR 3.69, p = 0.006 and HR 2.6, p = 0.012, respectively). Obese and older patients therefore constitute populations at risk for poorer outcomes. Prospective studies are warranted to gain further insight into the mechanism and role of obesity and adolescence in developing TRT and/or treatment failure.

Summary Background Data to inform surveillance and treatment for leukaemia predisposition syndromes are scarce and recommendations are largely based on expert opinion. This study aimed to investigate the clinical features and outcomes of patients with myelodysplastic syndrome or acute myeloid leukaemia and Shwachman-Diamond syndrome, an inherited bone marrow failure disorder with high risk of developing myeloid malignancies. Methods We did a multicentre, retrospective, cohort study in collaboration with the North American Shwachman-Diamond Syndrome Registry. We reviewed patient medical records from 17 centres in the USA and Canada. Patients with a genetic (biallelic mutations in the SBDS gene) or clinical diagnosis (cytopenias and pancreatic dysfunction) of Shwachman-Diamond syndrome who developed myelodysplastic syndrome or acute myeloid leukaemia were eligible without additional restriction. Medical records were reviewed between March 1, 2001, and Oct 5, 2017. Masked central review of bone marrow pathology was done if available to confirm leukaemia or myelodysplastic syndrome diagnosis. We describe the clinical features and overall survival of these patients. Findings We initially identified 37 patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia. 27 patients had samples available for central pathology review and were reclassified accordingly (central diagnosis concurred with local in 15 [56%] cases), 10 had no samples available and were classified based on the local review data, and 1 patient was excluded at this stage as not eligible. 36 patients were included in the analysis, of whom 10 (28%) initially presented with acute myeloid leukaemia and 26 (72%) initially presented with myelodysplastic syndrome. With a median follow-up of 4·9 years (IQR 3·9–8·4), median overall survival for patients with myelodysplastic syndrome was 7·7 years (95% CI 0·8–not reached) and 0·99 years (95% CI 0·2–2·4) for patients with acute myeloid leukaemia. Overall survival at 3 years was 11% (95% CI 1–39) for patients with leukaemia and 51% (29–68) for patients with myelodysplastic syndrome. Management and surveillance were variable. 18 (69%) of 26 patients with myelodysplastic syndrome received upfront therapy (14 haematopoietic stem cell transplantation and 4 chemotherapy), 4 (15%) patients received no treatment, 2 (8%) had unavailable data, and 2 (8%) progressed to acute myeloid leukaemia before receiving treatment. 12 patients received treatment for acute myeloid leukaemia—including the two patients initially diagnosed with myelodysplastic who progressed— two (16%) received HSCT as initial therapy and ten (83%) received chemotherapy with intent to proceed with HSCT. 33 (92%) of 36 patients (eight of ten with leukaemia and 25 of 26 with myelodysplastic syndrome) were known to have Shwachman-Diamond syndrome before development of a myeloid malignancy and could have been monitored with bone marrow surveillance. Bone marrow surveillance before myeloid malignancy diagnosis was done in three (33%) of nine patients with leukaemia for whom surveillance status was confirmed and 11 (46%) of 24 patients with myelodysplastic syndrome. Patients monitored had a 3-year overall survival of 62% (95% CI 32–82; n=14) compared with 28% (95% CI 10–50; n=19; p=0·13) without surveillance. Six (40%) of 15 patients with available longitudinal data developed myelodysplastic syndrome in the setting of stable blood counts. Interpretation Our results suggest that prognosis is poor for patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia owing to both therapy-resistant disease and treatment-related toxicities. Improved surveillance algorithms and risk stratification tools, studies of clonal evolution, and prospective trials are needed to inform effective prevention and treatment strategies for leukaemia predisposition in patients with Shwachman-Diamond syndrome. Funding National Institute of Health.

The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multicenter cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (

Vitamin D (VD) deficiency is a well-described phenomenon in pediatric patients undergoing hematopoietic stem cell transplant (HSCT). VD modulates inflammation, and deficiency pre-HSCT and at day +100 has been associated with graft-versus-host disease (GVHD) and poorer survival. However, a paucity of data has specifically described the association between VD status and immune-mediated complications including GVHD and veno-occlusive disease (VOD). Additionally, data to guide recommendations for VD monitoring and supplementation during HSCT are scarce. Our primary objective was to evaluate the association between VD and post-HSCT complications. The key secondary aim was to evaluate the routine use and efficacy of VD monitoring and supplementation practices. To our knowledge, this is the largest study of its kind in the pediatric population. This retrospective study evaluated VD level (VDL) before and 1 year after HSCT, VD supplementation practices, and their association with acute GVHD, VOD, and survival in pediatric patients who received autologous and allogeneic HSCT for both malignant and nonmalignant diseases from January 2013 to April 2018. Of 314 HSCTs, 43% of patients (n = 136) had VDL measured before HSCT; 61% of this cohort had pre-HSCT VD insufficiency (30 ng/mL). Neither pre-HSCT nor follow-up VDL was associated with the incidence of GVHD or VOD. Supplementation did not result in significantly different post-HSCT VDL.VDL was correlated with overall survival; every 10-ng/mL increase in VDL was associated with a 28% decreased risk of death (P = .01). Current accepted VD supplementation regimens for pediatric HSCT do not achieve sufficient VDL in most patients after HSCT. VD status was associated with all-cause mortality but not with individual comorbidities; prospective studies are required to establish the connection between VD status, inflammatory-mediated HSCT complications, and potential benefit of VD supplementation before and after HSCT. These studies are needed to inform evidence-based guidelines for monitoring and supplementing VD during HSCT.

Patients undergoing hematopoietic stem cell transplantation (HSCT) are often admitted to the hospital the day they are due to begin their conditioning regimen. Timely initiation of chemotherapy during regular work hours is important for patient safety, because during the night shift fewer physicians and pharmacists are available for urgent or unexpected matters. A review of the data at our institution from October 2017 to August 2018 showed that approximately one-third of our chemotherapy was started during the night shift (after 19:00), and the average time from admission to start of chemotherapy was over 8 hours. There are currently no well-defined benchmarks for timeliness of chemotherapy initiation. The aim of this quality improvement initiative was to increase the percentage of patients who start chemotherapy in the bone marrow transplant unit before 19:00 from 65% to >80% by March 31, 2019. We identified barriers to timely initiation of chemotherapy through process mapping and analysis of failures. The primary barriers were late admissions (after 12:00 pm) and time from admission to preparation of chemotherapy. We addressed mechanisms to mitigate these barriers through Plan-Do-Study-Act testing. Interventions included providing families specific admission times and their rationales and process for notifying pharmacy of admissions immediately on arrival. We used standardized control charts to measure the impact of the interventions on change. We also monitored medication errors before and during the intervention. From September 2018 to March 2019 the percentage of patients who started preparative chemotherapy before 19:00 increased from 65% to 85%, the percentage of patients who were admitted after 12:00 remained similar before (31%) and after the interventions (33%), and the average time from admission to start of chemotherapy decreased from 8.6 hours (513 minutes) to 6.4 hours (382 minutes). Medication errors were similar before (n = 50) and after the interventions (n = 43). Using standardized processes, we demonstrated a substantial decrease in the percentage of HSCT patients starting their preparative regimen after 19:00 without a concurrent increase in errors. We believe these interventions and measurements can be used in all transplant centers and have the potential to influence patient safety and outcomes.

Red cell distribution width (RDW) is an average of the variation in red blood cell (RBC) sizes reported on a complete blood count. An elevated RDW indicates a pathological process that is affecting erythropoiesis. Studies showed that as the severity of disease process increases, the RDW often increases as well. Particularly in resource-limited countries, RDW has been studied as an outcome predictor for conditions in a variety of disciplines and is offered as an adjunct monitoring tool that is cost effective, readily available, and indicative of pathological processes amenable to intervention. Particularly in pediatric critical care settings, RDW has been shown to be a reliable tool for surveillance of disease states such as sepsis. Despite the increased attention of RDW as a marker for disease outcome, collective evaluation on the utility of RDW as a marker for outcome in pediatric critical care settings is lacking. We offer a systematic review and meta-analysis of published studies to assess the ability of RDW to predict illness severity and mortality among pediatric critical care patients. Among eight studies of over 4,800 patients, we found over a two-fold increase in odds for mortality in critically ill children whose RDW was above 15.7%. This is the first systematic review of RDW being used to predict mortality in critically ill children and findings of this study may prompt early intervention in the pediatric critical care setting.

Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is commonly dysregulated in acute lymphoblastic leukemia (ALL). The TACL2014-001 phase I trial of the mTOR inhibitor temsirolimus in combination with cyclophosphamide and etoposide was performed in children and adolescents with relapsed/refractory ALL. Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily on days 1-5. The starting dose of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). PI3K/mTOR pathway inhibition was measured by phosphoflow cytometry analysis of peripheral blood specimens from treated patients. Sixteen heavily-pretreated patients were enrolled with 15 evaluable for toxicity. One dose-limiting toxicity of grade 4 pleural and pericardial effusions occurred in a patient treated at DL3. Additional dose-limiting toxicities were not seen in the DL3 expansion or DL4 cohort. Grade 3/4 non-hematologic toxicities occurring in three or more patients included febrile neutropenia, elevated alanine aminotransferase, hypokalemia, mucositis, and tumor lysis syndrome and occurred across all doses. Response and complete were observed at all dose levels with a 47% overall response rate and 27% complete response rate. Pharmacodynamic correlative studies demonstrated dose-dependent inhibition of PI3K/mTOR pathway phosphoproteins in all studied patients. Temsirolimus at doses up to 25 mg/m2 with cyclophosphamide and etoposide had an acceptable safety profile in children with relapsed/refractory ALL. Pharmacodynamic mTOR target inhibition was achieved and appeared to correlate with temsirolimus dose. Future testing of next-generation PI3K/mTOR pathway inhibitors with chemotherapy may be warranted to increase response rates in children with relapsed/refractory ALL.

7000 Background: Asparaginase is integral to pediatric-inspired regimens (PIR) to treat acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA). However, asparaginase-associated toxicities (AAT) often preclude delivery of planned therapy. Older age, obesity and/or large body surface area (BSA) have been associated with higher risk of AAT in PIR, but data are conflicting, and the impact of dose modification based on these factors is unknown. Methods: We examined induction toxicity data from patients ages 1-30 years enrolled in the frontline Children’s Oncology Group (COG) trials for high-risk B-ALL (AALL0232, 2004-2011) and T-ALL (AALL0434, 2007-2014). During Induction, patients received pegaspargase (2,500 IU/m2 without prescribed dose-capping) plus daunorubicin, vincristine, and prednisone or dexamethasone. AAT were defined as CTCAE v4 hyperbilirubinemia (Grade ≥3), elevated alanine aminotransferase (ALT) (Grade ≥4), thrombosis (any), or pancreatitis (any, included consolidation phase). Obesity was classified using population norms as body mass index (BMI) ≥30 (or ≥95th percentile for age/sex). BSA was analyzed continuously and dichotomized at 1.5 m2 (equivalent to pegaspargase 3,750 IU, the threshold for permissible dose-capping in PIR). The association of AAT with end-Induction minimal residual disease (MRD) ≥0.01% was assessed. Results: Among 4,925 patients, 25% were ≥15 years, 39% had BSA >1.5m2, and 18% had obesity. Multivariable logistic analyses inclusive of BMI and BSA together found increased risk for any AAT in age groups ≥10 years (10-15y, odds ratio (OR) 2.0, 15-20y OR 2.2, ≥21 OR 3.3, p=0.002). Only patients with both obesity and high BSA (>1.5m2) were at additional risk (OR 3.3, p10 years and in those with obesity, but not high BSA alone. Dose capping may not be necessary for children and AYAs with high BSA without obesity. Prospective studies of AAT pharmacogenomics and modifiable risk factors will support safer dosing in PIR. Clinical trial information: NCT00075725, NCT00408005.

BACKGROUND/OBJECTIVE: We prospectively examined neuropsychological outcomes and ototoxicity in children with average-risk medulloblastoma. METHODS: Eligible patients included those treated on COG protocol ACNS0331 who completed audiograms at end of therapy or one-year off-therapy, and neuropsychological assessments between 2- and 5-years post-diagnosis. Conventional pure-tone audiometric evaluations (0.25-8kHz) were assigned an ototoxicity grade based on the International Society of Pediatric Oncology (SIOP) grading scale. Grade for the better hearing ear was used for analyses. Participants were divided into two groups: SIOP grade≥3 hearing loss (HL) versus SIOP grade

BackgroundThere is increasing evidence that adipocytes play an active role in the cancer microenvironment. We have previously reported that adipocytes interact with acute lymphoblastic leukemia (ALL) cells, contributing to chemotherapy resistance and treatment failure. In the present study, we investigated whether part of this resistance is due to adipocyte provision of lipids to ALL cells.MethodsWe cultured 3T3-L1 adipocytes, and tested whether ALL cells or ALL-released cytokines induced FFA release. We investigated whether ALL cells took up these FFA, and using fluorescent tagged BODIPY-FFA and lipidomics, evaluated which lipid moieties were being transferred from adipocytes to ALL. We evaluated the effects of adipocyte-derived lipids on ALL cell metabolism using a Seahorse XF analyzer and expression of enzymes important for lipid metabolism, and tested whether these lipids could protect ALL cells from chemotherapy. Finally, we evaluated a panel of lipid synthesis and metabolism inhibitors to determine which were affected by the presence of adipocytes.ResultsAdipocytes release free fatty acids (FFA) when in the presence of ALL cells. These FFA are taken up by the ALL cells and incorporated into triglycerides and phospholipids. Some of these lipids are stored in lipid droplets, which can be utilized in states of fuel deprivation. Adipocytes preferentially release monounsaturated FFA, and this can be attenuated by inhibiting the desaturating enzyme steroyl-CoA decarboxylase-1 (SCD1). Adipocyte-derived FFA can relieve ALL cell endogenous lipogenesis and reverse the cytotoxicity of pharmacological acetyl-CoA carboxylase (ACC) inhibition. Further, adipocytes alter ALL cell metabolism, shifting them from glucose to FFA oxidation. Interestingly, the unsaturated fatty acid, oleic acid, protects ALL cells from modest concentrations of chemotherapy, such as those that might be present in the ALL microenvironment. In addition, targeting lipid synthesis and metabolism can potentially reverse adipocyte protection of ALL cells.ConclusionThese findings uncover a previously unidentified interaction between ALL cells and adipocytes, leading to transfer of FFA for use as a metabolic fuel and macromolecule building block. This interaction may contribute to ALL resistance to chemotherapy, and could potentially be targeted to improve ALL treatment outcome.

Vitamin D is essential for bone health and has immunomodulatory properties. Most pediatric patients are vitamin D insufficient (30 ng/mL) before hematopoietic stem cell transplantation (HSCT). Standard supplementation strategies fail to achieve vitamin D sufficiency in the acute post-transplantation period, and there are scarce data to support optimal vitamin D supplementation in this patient population. This study aimed to evaluate whether a single, oral, weight-based ultra-high dose of vitamin D (Stoss dosing) was more effective than standard supplementation to achieve pre-HSCT vitamin D sufficiency and reduce the incidence of HSCT-related complications (acute graft-versus-host disease, veno-occlusive disease, and/or transplant-associated thrombotic microangiopathy) that are associated with immune-mediated endothelial damage. Secondary endpoints examined the immunomodulatory properties of vitamin D. We conducted a nonrandomized controlled clinical trial of Stoss-dosed vitamin D in pediatric patients receiving HSCT. The study prospectively enrolled 33 patients, 29 of whom successfully received Stoss-dosed vitamin D and were compared to 136 patients in a historical control. Patient characteristics were compared using Fisher's exact test or t-test. The one-sided Fisher's exact test was used for cohort comparison of the primary endpoints. Logistic regression was used to examine the association between patient-specific factors and total 25-hydroxy vitamin D (25-OHD) levels and the compiled HSCT complications. In the Stoss cohort, 97% (n = 28/29) of patients achieved pre-HSCT vitamin D sufficiency compared to 67% (n = 10/15) of patients in the historical control who were on standard supplementation at the time the total 25-OHD level was assessed (P = .013). The mean total 25-OHD level in the Stoss cohort was significantly higher than patients in the historical control who received standard supplementation (72.2 ng/mL versus 35.8 ng/mL, P.001). Nine patients in the Stoss cohort maintained vitamin D sufficiency throughout the first 100 days after HSCT, and the remaining 19 patients maintained sufficiency for a median of 63 days (range 6-105 days) from the Stoss dose. Patients receiving Stoss-dosed vitamin D developed a lower combined incidence of HSCT-related complications than the historical control (25% [n = 7/28] versus 42% [n = 57/136], P = .055). After Stoss dosing, immunophenotyping studies found a significant decrease in subsets of CD8+ T cells and mononuclear cells (P = .040 and.013, respectively), and, in a subset of cells, larger decreases in phosphoprotein expression were seen with greater increases in total 25-OHD levels. Inflammatory cytokines did not change significantly after Stoss dosing. Stoss dosing is therefore a safe and effective approach to maintain vitamin D sufficiency in the immediate post-HSCT period and may be associated with decreased HSCT-related complications. Randomized studies are warranted to further investigate the efficacy of Stoss-dosed vitamin D to improve bone health and reduce complications in pediatric patients receiving HSCT.

Purpose Cranial radiation therapy (RT) and cisplatin-based chemotherapy are essential to treating many pediatric cancers but cause significant ototoxicity. The objective of this study is to determine the relationship between the RT dose and the risk of subsequent hearing loss in pediatric patients treated with cisplatin. Methods and Materials This retrospective study of cisplatin-treated pediatric patients examined ototoxicity from cranial RT. Ototoxicity was graded for each ear according to the International Society of Pediatric Oncology (SIOP) consensus ototoxicity scale. The RT dose to the cochlea was calculated using the mean, median, maximum, and minimum dose received to determine the most predictive parameter for hearing loss. Multivariable logistic regression models then examined risk factors for hearing loss. Results In 96 children (161 ears) treated with RT + cisplatin, the minimum cochlear RT dose was most predictive of hearing loss. A higher cochlear RT dose was associated with increased hearing loss (odds ratio per 10 Gy dose increase = 1.64; P = .043), with an added risk in those receiving an autologous bone marrow transplantation (hazard ratio = 10.47; P Conclusions This research supports further testing of the minimum cochlear RT dose as a more predictive dose parameter for risk of ototoxicity. The cochlear RT dose was additive to the risk of hearing loss from underlying cisplatin-based chemotherapy. Exposure to autologous bone marrow transplantation was the strongest predictor of developing hearing loss, placing these children at particularly high risk for hearing loss across all cochlear doses. Future prospective studies are crucial to further inform RT dose thresholds and minimize the risk of hearing loss in childhood cancer survivors.

Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia with features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The review examines current definitions and controversies in classification of MPAL, new insights into genomic drivers and pathogenesis, recent evidence to support treatment recommendations, and opportunities for future research. Recent collaborative efforts have made progress in understanding the genomic landscape and optimal therapy for MPAL. The preponderance of retrospective data supports beginning therapy with ALL directed regimens. Differences in prognosis for adult and children with MPAL have led to divergent approaches for therapy intensity, including use of stem cell transplantation consolidation. MPAL remains a challenging leukemia to understand, research, and treat due to low incidence, shifting and subjective approaches to classification, and innate biological heterogeneity. Ongoing research hopes to surmount these obstacles through prospective studies within large cooperative groups to provide new insight into targetable biology and further refine optimal therapy.

Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia with features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The review examines current definitions and controversies in classification of MPAL, new insights into genomic drivers and pathogenesis, recent evidence to support treatment recommendations, and opportunities for future research.Recent collaborative efforts have made progress in understanding the genomic landscape and optimal therapy for MPAL. The preponderance of retrospective data supports beginning therapy with ALL directed regimens. Differences in prognosis for adult and children with MPAL have led to divergent approaches for therapy intensity, including use of stem cell transplantation consolidation. MPAL remains a challenging leukemia to understand, research, and treat due to low incidence, shifting and subjective approaches to classification, and innate biological heterogeneity. Ongoing research hopes to surmount these obstacles through prospective studies within large cooperative groups to provide new insight into targetable biology and further refine optimal therapy.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Background Cisplatin-induced hearing loss (CIHL) is a common and debilitating toxicity for childhood cancer survivors. Understanding provider perspectives is crucial to developing otoprotection studies that are both informative and feasible. Two international trials (ACCL0431 and SIOPEL6) investigated the drug sodium thiosulfate (STS) as an otoprotectant, but definitive interpretation of the findings of these trials has been challenging. Adoption of STS has therefore been uneven, and provider perspectives on its role are unknown. Procedure The Children's Oncology Group (COG) Cancer Control and Supportive Care Neurotoxicity Subcommittee therefore conducted a survey of providers at COG institutions to determine perspectives on pediatric otoprotection practices and research surrounding three major themes: (1) prevalence of routine use of STS with cisplatin-based regimens, (2) application of audiometry to cisplatin therapy, and (3) preferred modalities for otoprotection research. Results Survey respondents (45%, 44/98 surveyed institutions) were of diverse institutional sizes, practice settings, and geographical locations primarily in the United States and Canada. Overall, respondents considered CIHL an important toxicity and indicated strong enthusiasm for future studies (98%, 40/41). Results indicated that while STS was the current or planned standard of care in a minority of responding institutions (36%, 16/44), most sites were receptive to its inclusion in appropriate study designs. Application of audiometry for ototoxicity monitoring varied widely across sites. For otoprotection research, systemic agents were preferred (68%, 28/41) as compared with intratympanic approaches. Conclusion These results suggest that pediatric otoprotection trials remain of interest to providers; the emphasis of these trials should remain on systemic and not intratympanic therapy.

Introduction Acute lymphoblastic leukemia (ALL) therapy confers risk for venous thromboembolism (VTE) and associated acute and long-term morbidity. Obesity increases VTE risk in the general population but its impact on ALL therapy-associated VTE is unknown. Methods In a retrospective cohort of children treated for ALL between 2008 and 2016 (n = 294), we analyzed obesity at diagnosis (body mass index [BMI] ≥95%) and subsequent development of VTE. A subset participated in two concurrent prospective ALL trials studying body composition via dual-energy X-ray absorptiometry (DXA) (n = 35) and hypercoagulability via thromboelastography (TEG) (n = 46). Secondary analyses explored whether precise measurement of body fat and/or global hemostasis ex vivo by TEG could further delineate VTE risk in the obese. Results Overall, we found 27/294 (9.2%) patients developed symptomatic VTE during therapy, 19/27 (70%) occurred during Induction. Study-defined “serious” VTE developed in 4/294 (1.4%) of patients. Obesity but not overweight was strongly predictive of symptomatic VTE (obesity odds ratio = 3.8, 95% confidence interval 1.5–9.6, p = 0.008). In the DXA subset, only 2/35 patients developed symptomatic VTE. However, within those prospectively screened during Induction, 30% (14/46) developed VTE; eight (17%) of these were asymptomatic and found only via screening. Conclusions In this pediatric ALL cohort, obesity conferred more than a three-fold increased risk for symptomatic VTE. In a subgroup of patients who underwent active screening, up to a third were noted to have VTE (symptomatic and asymptomatic). TEG did not predict VTE. Additional studies are necessary to validate these findings and to further refine a risk-stratified approach to thrombo-prevention during ALL therapy.

Children with acute myeloid leukemia (AML) are commonly treated using a daunorubicin-cytarabine ±etoposide (DA/ADE) backbone as developed by the Medical Research Council (MRC) AML 12 trial and since adopted by the Children's Oncology Group (COG). This regimen is an anthracycline-intensive regimen, incorporating mitoxantrone in the consolidation phase, with a stated cumulative cardiotoxic exposure of ~444 mg/m 2 doxorubicin equivalents, though recent data suggests mitoxantrone is more cardiotoxic than previously considered (~10x more cardiotoxic than doxorubicin instead of 3x). The MRC AML15 trial randomized DA/ADE versus fludarabine-cytarabine-idarubicin (FLAG-Ida) and found superior event-free survival (EFS) from FLAG-Ida, with fewer cycles of intensive chemotherapy (4 vs 5), and less than half the cardiotoxic exposure, potentially reducing acute and long-term morbidity. Experience with FLAG-Ida as frontline therapy in children with de novo AML is limited. Following MRC 15, minimal residual disease (MRD) monitoring has become standard of care for risk stratification in AML, but MRD data for the FLAG-Ida regimen in children has not yet been reported. We collected data from 30 pediatric patients (age 0.3-20.0 years) with newly diagnosed de novo AML (no preceding myelodysplastic syndrome, secondary AML, or prior malignancy) and no underlying genetic disease (e.g., Trisomy 21, Fanconi Anemia, Kostmann Syndrome, Shwachman-Diamond Syndrome) treated at our institution with MRC 15-style FLAG-Ida between 2014 and 2021 (Table 1). Each case was characterized by cytogenetics, chromosomal microarray analysis (since 2015, in 29/30), and a proprietary molecular testing panel (since 2017, in 21/30); AML risk category was assigned at diagnosis using the contemporary COG classification (AAML1831). Following Induction I and II, MRD was measured in each patient by multiparameter flow cytometry using a 'difference from normal' approach (MRD+ defined by contemporary COG threshold, MRD≥0.05%). Patients with poor disease response (MRD+) or high-risk cytogenetics proceeded to hematopoietic stem cell transplantation (HSCT) in first remission. Patients routinely received antimicrobial prophylaxis with sulfamethoxazole-trimethoprim, an azole or echinocandin, and levofloxacin. The study was approved by the Institutional Review Board. Following Induction I, 28/30 (93%) patients were MRD negative, and 30/30 (100%) patients following Induction II. Only 1/20 (5%) patients without high-risk AML required HSCT for slow-responding disease; 1/30 (3%) patients were unable to proceed to consolidation (prolonged myelosuppression). No patient developed treatment-related mortality (TRM) during pre-HSCT chemotherapy (2 subsequent to HSCT). Cardiac evaluation in surviving patients was normal (LVSF>28%) post-therapy in 23/24 (96%). Median survival for patients alive at last follow-up was 2.3 years with overall 3-year EFS and overall survival (OS) of 73±9% and 80±8%, respectively (Figure 1A). Survival for patients in the low-risk subset is depicted (Figure 1B, 1C); all low-risk patients were alive at last follow-up. FLAG-Ida was well tolerated and effective in our pediatric AML population with no TRM, excellent early disease response by MRD, and encouraging albeit early EFS and OS. This compares favorably with the adult MRC AML 15 experience as well as with the COG DA/ADE regimen, particularly for MRD response (in AAML0531, ~30% patients were MRD+ following Induction I [Brodersen et al 2020]). These data support our continued use of FLAG-Ida in pediatric AML alone or in combination with targeted and/or molecular therapies. Further study of FLAG-Ida as frontline therapy in children with AML is warranted. Figure 1 Figure 1. Disclosures Gaynon: Takeda pharmaceuticals: Other: member of DSMC committee. Orgel: Jazz Pharmaceuticals: Consultancy.

Mixed phenotype acute leukemia (MPAL) is a rare group of acute leukemias defined by immunophenotypic expression of more than one hematopoietic cell lineage. The World Health Organization (WHO) diagnostic immunophenotypic criteria for MPAL rely on the intensity of lineage-defining antigen expression, predominantly a qualitative assessment, and are often difficult to apply to a phenotypically heterogeneous leukemia. Cases of MPAL defined by isolated myeloperoxidase (isoMPO) expression on otherwise typical acute lymphoblastic leukemia (ALL) are variably diagnosed as MPAL or ALL based on the incompletely defined criteria for assigning MPO expression. We hypothesized that quantitative criteria for antigen intensity could be developed and applied in a uniform manner across flow cytometry instruments, reagents, and analysis software to enable a consistent approach to diagnosing MPAL and better defining isoMPO. We previously reported on a multicenter cohort identified by respective institutions as MPAL with subsequent central review according to 2008 WHO criteria (Oberley et al 2020). Of these, 100 had suitable dot plots for reevaluation (89: B/Myeloid, 10: T/Myeloid, 1: B/T). Antigen expression was concurrently reviewed by two hematopathologists to reach consensus (BLW, AEK). The cohort was divided a priori into randomly selected training and testing cases (n=30/n=70). Classification criteria were generated in the training cohort for WHO lineage-defining antigen expression (myeloid: cMPO, CD64, CD14; B: CD19, T: cCD3) from 10 cases and then refined through review of an additional 20 cases. Positive antigen expression was classified as maximal intensity approaching that of the mature normal counterpart population (NCP) (cMPO: neutrophils; CD64, CD14 and CD11c: monocytes; CD19: B cells; cCD3: T cells) either by 1) range of expression recapitulating maturation of the NCP, irrespective of the percentage on the leukemic population (Figure 1A); or 2) uniform expression above background on a discrete (sub)population (Figure 1B). To account for technical variation within and among laboratories, maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to the maximal intensity of the NCP. An intensity of ≥50% of the NCP above background was defined as sufficient for MPAL lineage assignment and Using this approach, 41/98 (42%) cases previously classified as MPAL remained classified as MPAL: 31 as B/Myeloid (25 by maturational MPO expression, 6 by maturational CD64 and/or CD14 expression); 9 as T/Myeloid (8 by maturational MPO expression, 1 with maturational CD64, CD14 and CD11c expression); and 1 as B/T. No cases in the cohort showed uniform expression ≥50% of the NCP. The remaining 57 showed isolated low-level MPO expression (maximal intensity Novel semiquantitative immunophenotypic criteria applied to a large cohort of acute leukemias originally diagnosed as MPAL was able to objectively identify a large subset as having dim, uniform MPO expression (isoMPO). Our approach emphasizes antigen expression recapitulating maturational expression similar to their non-leukemic cellular counterparts, normalizes absolute intensities to internal positive and negative control populations to minimize technical variability between observers and assays, and as per the 2017 WHO, does not require a specific percent threshold of positivity. While requiring validation, this is a critical first step toward establishing a reproducible delineation of these complex cases to practically implement the WHO classification to support treatment decisions and ongoing investigation into MPAL genomics and outcomes (available for this cohort by ASH). Figure 1 Figure 1. Disclosures Oberley: Caris LIfe Science: Current Employment. Orgel: Jazz Pharmaceuticals: Consultancy.

A Supportive Care Committee was recently developed within the Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Consortium. This was substantiated by the significantly high rate of serious adverse events (SAE) (CTCAE Grade ≥3 toxicity) experienced by patients with relapse/refractory acute leukemia enrolled on our phase I trials. Such treatment-related toxicity has resulted in patients being removed from study and thus potentially not receiving clinical benefit from the novel therapy. In addition, increased treatment-related toxicity may compromise new agents from moving forward in their clinical development. To address these challenges, TACL initiated a Supportive Care Committee to help mitigate the treatment-related toxicity risk that exists in heavily pre-treated patients with relapse/refractory leukemia. This manuscript reviews the mission of the TACL Supportive Care Committee presented at the 2016 TACL Investigators’ Meeting (Los Angeles, CA) and the future direction in providing enhanced supportive care guidelines for all TACL studies.

Adolescents with acute lymphoblastic leukemia (ALL) develop osteopenia early in therapy, potentially exacerbated by high rates of concurrent Vitamin D deficiency. We conducted a randomized clinical trial testing a Vitamin D-based intervention to improve Vitamin D status and reduce bone density decline. Poor adherence to home supplementation necessitated a change to directly observed therapy (DOT) with intermittent, high-dose Vitamin D3 randomized versus standard of care (SOC). Compared to SOC, DOT Vitamin D3 successfully increased trough Vitamin 25(OH)D levels (p = .026) with no residual Vitamin D deficiency, 100% adherence to DOT Vitamin D3, and without associated toxicity. However, neither Vitamin D status nor supplementation impacted bone density. Thus, this adherence-optimized intervention is feasible and effective to correct Vitamin D deficiency in adolescents during ALL therapy. Repletion of Vitamin D and calcium alone did not mitigate osteopenia, however, and new, comprehensive approaches are needed to address treatment-associated osteopenia during ALL therapy.

Asparaginase (ASNase) is an integral part of pediatric induction chemotherapy that has also been shown to improve adult survival rates; however, pegylated (PEG)-ASNase induces severe hepatotoxicity in this population. Recent case reports describe the incorporation of levocarnitine (LC) supplementation into PEG-ASNase-containing induction regimens to prevent or treat hepatotoxicity. Because LC facilitates the metabolism of free fatty acids (FFA), a primary fuel source for ALL cells, LC could potentially interfere with ALL chemotherapy efficacy. To test this, we employed in vitro and in vivo models of ALL. We show in vitro that LC supplementation does not impact cytotoxicity from vincristine, daunorubicin, dexamethasone, or ASNase on human ALL cells nor lead to an increase in ALL cell metabolic rate. In vivo, we demonstrate LC does not impair PEG-ASNase monotherapy in mice with syngeneic ALL. Together, our findings show that LC supplementation is a safe strategy to prevent/reverse ASNase-induced toxicities in preclinical models.

The prevalence of obesity has steadily risen over the past decades, even doubling in more than 70 countries. High levels of body fat (adiposity) and obesity are associated with endocrine and hormonal dysregulation, cardiovascular compromise, hepatic dysfunction, pancreatitis, changes in drug metabolism and clearance, inflammation, and metabolic stress. It is thus unsurprising that obesity can affect the development of and survival from a wide variety of malignancies. This review focuses on acute lymphoblastic leukemia, the most common malignancy in children, to explore the multiple mechanisms connecting acute lymphoblastic leukemia, obesity, and adipocytes, and the implications for leukemia therapy.

Background Optimal chemotherapy for treating mixed-phenotype acute leukemia (MPAL) and the role of hematopoietic stem cell transplantation (HSCT) remain uncertain. Major limitations in interpreting available data are MPAL's rarity and the use of definitions other than the currently widely accepted criteria: the World Health Organization 2016 (WHO2016) classification. Methods To assess the relative efficacy of chemotherapy types for treating pediatric MPAL, the Children's Oncology Group (COG) Acute Leukemia of Ambiguous Lineage Task Force assembled a retrospective cohort of centrally reviewed WHO2016 MPAL cases selected from banking studies for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Patients were not treated in COG trials; treatment and outcome data were captured separately. The findings were then integrated with the available, mixed literature to develop a prospective trial in pediatric MPAL. Results The central review confirmed that 54 of 70 cases fulfilled WHO2016 criteria for MPAL. ALL induction regimens achieved remission in 72% of the cases (28 of 39), whereas AML regimens achieved remission in 69% (9 of 13). The 5-year event-free survival (EFS) and overall survival (OS) rates for the entire cohort were 72% ± 8% and 77% ± 7%, respectively. EFS and OS were 75% ± 13% and 84% ± 11%, respectively, for those receiving ALL chemotherapy alone without HSCT (n = 21). Conclusions The results of the COG MPAL cohort and a literature review suggest that ALL chemotherapy without HSCT may be the preferred initial therapy. A prospective trial within the COG is proposed to investigate this approach; AML chemotherapy and/or HSCT will be reserved for those with treatment failure as assessed by minimal residual disease. Embedded biology studies will provide further insight into MPAL genomics.

Background: Obesity is a worldwide epidemic in children and adolescents. Adult cohort studies have reported an association between higher body mass index (BMI) and increased leukemia-related mortality; whether a similar effect exists in childhood leukemia remains controversial. Objective: We conducted a meta-analysis to determine whether a higher BMI at diagnosis of pediatric acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) is associated with worse event-free survival (EFS), overall survival (OS), and cumulative incidence of relapse (CIR). Design: We searched 4 electronic databases from inception through March 2015 without language restriction and included studies in pediatric ALL or AML (0–21 y of age) reporting BMI as a predictor of survival or relapse. Higher BMI, defined as obese (≥95%) or overweight/obese (≥85%), was compared with lower BMI [nonoverweight/obese (

Introduction: We have reported that mouse and human adipocytes take up and metabolize the anthracycline, daunorubicin (DNR), reducing its concentration in the adipocyte microenvironments. This may contribute to anthracycline resistance for cancers, which reside in adipocyte rich environments such as omentum and bone marrow. Adipocytes express several carbonyl- and aldo-keto reductases (CBRs and AKRs) which metabolize and inactivate anthracyclines, and it is unclear which of these might be important targets to improve treatment outcome. Experimental Procedures: We knocked out AKR1C3 in the human preadipocyte cell line Chub S7 using CRISPR/Cas9 gene editing technology. We chose AKR1C3 first as it is one of the overexpressed enzymes in adipocytes with the highest anthracycline-metabolizing activity. We delivered ribonucleoprotein complexes of CRISPR-Cas9 enzyme plus guide RNAs by nucleofection. Then we established single-cell derived clones and tested for successful KO by Western blot. Finally, we quantified adipocyte lysate AKR activity using a colorimetric assay based on NADPH-dependent reduction of phenanthrenequinone. Data Summary: We chose three Chub S7 preadipocyte clones that demonstrated successful AKR1C3 knockout based on almost undetectable protein expression by western blot. AKR activity was significantly reduced in all three clones; control preadipocytes had 44±4.7, while clones had activity of 28±7.3, 33±3.4, and 35±5.7 pmol/min/µg (p Conclusions: These findings demonstrate that AKR1C3 knockout can be successfully done in human preadipocytes using a CRISPR-Cas9 system. Knockout of AKR1C3 significantly reduces overall aldoketoreductase activity in preadipocyte cells. This implies that a substantial portion of preadipocyte AKR activity is dependent on the isoenzyme, AKR1C3. Future testing is needed to determine whether AKR1C3 KO will reduce the clearance of anthracyclines from the cancer microenvironment, and may represent a treatment target to enhance anthracycline cytotoxicity. Citation Format: Vladislava Paharkova, Etan Orgel, Michael Neely, Stan. Louie, Steven Mittelman. Targeting the anthracycline metabolizing enzyme AKR1C3 in adipocytes to improve cytotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6059.