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PURPOSE: Pediatric oncology and bone marrow transplant patients are at high risk of infection, and limitations to dental expertise among medical providers render patients vulnerable to central line–associated bloodstream infections from oral pathogens. Traditionally, oral health maintenance relied on patients and bedside nurses; however, routine methods are often suboptimal to prevent central line–associated bloodstream infection in high-risk patients. Limited overlap of medical and dental expertise, and limited dental resources in typical oncology units, prevent optimal oral care for children with cancer, requiring novel solutions to better integrate specialties. METHODS: Here, we outline the creation of a novel Pediatric oncodental team to address oral-systemic infection prevention strategies for high-risk patients. RESULTS: Our oncology and dental teams created a systematic approach for increasing oral surveillance and treatment in select high-risk patients. Supervised pediatric dental residents participated in scheduled oncology rounds, and a permanent oral health educator with a background in dental hygiene was also hired as a dedicated dental professional within our oncology department. CONCLUSION: Our pediatric oncodental team aims to sustain optimal oral complication prevention strategies to reduce the risk of infection, provide education on the significance of the oral-systemic link in cancer care, and improve access and continuity of care.

The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions. Supplementary Information The online version contains supplementary material available at 10.1007/s12094-021-02621-w.

Pediatric acute myeloid leukemia (AML) is a devastating disease with a high risk of relapse. Current risk classification designates patients as high or low risk (LR) based on molecular features and therapy response. However, 30% of LR patients still suffer relapse, indicating a need for improvement in risk stratification. Cytokine levels, such as IL-6 and IL-10, have been shown to be prognostic in adult AML but have not been well studied in children. Previously, we reported elevated IL-6 levels in pediatric AML bone marrow to be associated with inferior prognosis. Here, we expanded our investigation to assess cytokine levels in diagnostic peripheral blood plasma (PBP) of pediatric AML patients and determined correlation with outcome. Diagnostic PBP was obtained from 80 patients with LR AML enrolled on the Children’s Oncology Group AAML1031 study and normal PBP from 11 controls. Cytokine levels were measured and correlation with clinical outcome was assessed. IL-6, TNFα, MIP-3a, and IL-1β were significantly higher in AML patients versus controls when corrected by the Bonferroni method. Furthermore, elevated TNFα and IL-10 were significantly associated with inferior outcomes. Our data demonstrate that in diagnostic PBP of LR pediatric AML patients, certain cytokine levels are elevated as compared to healthy controls and that elevated TNFα and IL-10 are associated with inferior outcomes, supporting the idea that an abnormal inflammatory state may predict poor outcomes. Studies are needed to determine the mechanisms by which these cytokines impact survival, and to further evaluate their use as prognostic biomarkers in pediatric AML.

Young males have a unique but rare predilection to develop mediastinal nonseminomatous germ cell tumors (NSGCTs) and concomitant acute megakaryoblastic leukemia (AMKL). Common cytogenetic and molecular abnormalities such as isochromosome 12p and somatic Tumor Protein P53(TP53) and Phosphatase And Tensin Homolog (PTEN) mutations have been reported in the presumed mutual neoplastic clones of origin. We report the case of a 17-year-old male who presented with a mediastinal NSGCT with high-grade sarcomatous transformation and a diagnosis of AMKL approximately 4 months later. Next-generation sequencing revealed identical KRAS Proto-Oncogene, GTPase (KRAS) p.Ala146Thr, TP53 p.Leu257Pro, and PTEN p.Leu181Pro missense mutations at similar variant allele frequencies in both the NSGCT and AMKL samples. Cytogenetic and microarray analyses detected shared copy gains in all chromosomes except chromosomes 9, 13, and Y. Multiple additional clonal chromosomal alterations were detected in the AMKL sample when compared with the NSGCT. This case emphasizes the shared clonal origins of these malignancies and identifies KRAS and other copy number alterations as potential molecular drivers in a subset of these combined diseases.

Current therapies for relapsed/refractory (R/R) pediatric myeloid neoplasms are inadequately effective. Real-world data (RWD) can improve care by augmenting traditional studies and include individuals not eligible for clinical trials. The Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) consortium recently completed T2016-003, a phase 1 study of decitabine, vorinostat, fludarabine, cytarabine, and granulocyte colony-stimulating factor (G-CSF) in R/R acute myeloid leukemia (AML), which added epigenetic drugs to a cytotoxic backbone. We report results of RWD from six centers that treated 28 pediatric patients (26 with AML, two with other myeloid neoplasms) identically to the TACL study but who were not enrolled. This allowed unique analyses and the ability to compare data with the 35 TACL study patients. The overall response rate (ORR) (complete response [CR] plus CR with incomplete count recovery) among 26 RWD evaluable patients was 65%. The ORR of 13 patients with relapsed AML with epigenetic alterations was 69% (T2016-003 + RWD: 68%, n = 25), of eight patients with refractory AML was 38% (T2016-003 + RWD: 41%, n = 17) and of five patients with therapy-related AML (t-AML) was 80% (T2016-003 + RWD: 75%, n = 8). The mean number of Grade 3/4 toxicities experienced by the T2016-003-eligible RWD population (n = 22) (one per patient-cycle) was not meaningfully different than those (n = 6) who would have been TACL study-ineligible secondary to comorbidities (two per patient-cycle). Overall, this therapy was well tolerated and effective in pediatric patients with R/R myeloid neoplasms, particularly those with epigenetic alterations, t-AML, and refractory disease.

Bloodstream infections (BSIs) cause morbidity and mortality in pediatric patients with leukemia. Antibiotic prophylaxis during periods of chemotherapy-induced neutropenia may reduce the incidence of BSIs.A levofloxacin prophylaxis guideline was implemented for pediatric patients with acute myeloid leukemia and relapsed acute lymphoblastic leukemia. We conducted a retrospective cohort study over 4 years (2 years pre and 2 years post implementation) of the practice guideline to assess the impact on central line-associated bloodstream infections (CLABSI) and BSI events. Secondary outcomes included incidence of Clostridioides difficile-associated diarrhea, bacteremia due to multidrug-resistant organisms (MDRO), and bacteremia due to levofloxacin nonsusceptible organisms. STATA was used for data analysis.Sixty-three and 72 patients met inclusion criteria for the pre- and postimplementation cohorts, respectively. Demographics were similar between the groups. We observed 60 BSI events in the pre-group versus 49 events in the post-group (p = .1). Bacteremia due to Gram-negative rods (risk ratio [RR] 0.37 [0.21, 0.66], p .001) and National Healthcare Safety Network (NHSN) CLABSIs (RR 0.62 [0.44, 0.89], p = .01) were significantly reduced in the postimplementation group. The incidences of C. difficile-associated diarrhea and MDRO bacteremia were similar between groups. However, we observed an increase in the incidence of BSI due to Gram-negative rods that were nonsusceptible to levofloxacin (RR 3.38 [0.72, 6.65], p .001).Following implementation of a levofloxacin prophylaxis guideline, we observed a significant decrease in BSIs due to Gram-negative rods and NHSN CLABSIs. Vigilant monitoring of outcomes post guideline implementation is critical to track emergence of resistant organisms.

Acute myeloid leukemia in a child with familial platelet disorder and a cryptic runx1 intragenic deletion

Acute myeloid leukemia (AML) is a heterogeneous disease that accounts for ~20% of all childhood leukemias, and more than 40% of children with AML relapse within three years of diagnosis. Although recent efforts have focused on developing a precise medicine-based approach towards treating AML in adults, there remains a critical gap in therapies designed specifically for children. Here, we present ex vivo drug sensitivity profiles for children with de novo AML using an automated flow cytometry platform. Fresh diagnostic blood or bone marrow aspirate samples were screened for sensitivity in response to 78 dose conditions by measuring the reduction in leukemic blasts relative to the control. In pediatric patients treated with conventional chemotherapy, comprising cytarabine, daunorubicin and etoposide (ADE), ex vivo drug sensitivity results correlated with minimal residual disease (r = 0.63) and one year relapse-free survival (r = 0.70; AUROC = 0.94). In the de novo ADE analysis cohort of 13 patients, AML cells showed greater sensitivity to bortezomib/panobinostat compared with ADE, and comparable sensitivity between venetoclax/azacitidine and ADE ex vivo. Two patients showed a differential response between ADE and bortezomib/panobinostat, thus supporting the incorporation of ex vivo drug sensitivity testing in clinical trials to further evaluate the predictive utility of this platform in children with AML.

Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3 has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven challenging. This paper describes synthetic studies and biological assays for a naphthalene sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation in cellulo and induce apoptosis in AML cells. We describe two different approaches to inhibitor design: First, variation of substituents on the naphthalene sulfonamide core allows improvements in anti-STAT activity and creates a more thorough understanding of anti-STAT SAR. Second, a novel approach involving hybrid sulfonamide–rhodium(II) conjugates tests our ability to use cooperative organic–inorganic binding for drug development, and to use SAR studies to inform metal conjugate design. Both approaches have produced compounds with improved binding potency. In vivo and in cellulo experiments further demonstrate that these approaches can also lead to improved activity in living cells, and that compound 3aa slows disease progression in a xenograft model of AML.

A case of a 19-year-old female with low-risk acute myeloid leukemia is presented who was diagnosed with idiopathic hyperammonemic encephalopathy following the development of abrupt neurologic decline, respiratory alkalosis, and elevated plasma ammonia levels of unknown etiology. Delayed symptom recognition of this exceedingly rare condition contributes to the often fatal outcomes of idiopathic hyperammonemic encephalopathy. As illustrated by this case, prompt diagnosis and utilization of a variety of ammonia-modulating treatment modalities can result in remarkable clinical recovery. This case provides guidance to clinicians in counseling families about the possibility of neurologic recovery in similar clinical scenarios.

Barrier island lagoons are the most common type of estuary in the world and can be prone to eutrophication as well as the formation and closure of ocean inlets via severe storm activity. This study describes the biological, chemical, and physical changes that occurred along the south shore of Long Island, NY, USA, following the formation of a new ocean inlet in eastern Great South Bay (GSB) by Hurricane Sandy in October of 2012. Time series sampling and experiments were performed at multiple locations within GSB and neighboring Moriches Bay from 2013 through to 2018. Historical comparisons to prior water quality monitoring data, fecal coliform concentrations, and hard clam growth rates were also made. Measurements indicated that the New Inlet provided asymmetrical ocean flushing. Within locations north (Bellport Bay) and east (Narrow Bay, western Moriches Bay) of the New Inlet, water residence times, summer water temperatures, total and dissolved nitrogen, chlorophyll a, the harmful brown tide alga, Aureococcus anophagefferens, pigments associated with diatoms and dinoflagellates (fucoxanthin and peridinin), and fecal coliform bacteria levels all significantly decreased, while salinity, dissolved oxygen, and water clarity significantly increased. In contrast, waters west of the New Inlet within the center of GSB experienced little change in residence times, significant increases in chlorophyll a and harmful brown tides caused by A. anophagefferens, as well as a significant decrease in water clarity and summer dissolved oxygen levels. Growth rates of juvenile hard clams (Mercenaria mercenaria) near the New Inlet increased compared to before the inlet and were significantly higher than in central GSB, where growth rates significantly declined compared to before the inlet. Hence, while enhanced ocean flushing provided a series of key ecosystem benefits for regions near the New Inlet, regions further away (> 10 km) experienced more frequent HABs and poorer performance of bivalves, demonstrating that enhanced ocean flushing provided by the breach was not adequate to fully restore the whole GSB ecosystem.

We report the case of a 3-year-old girl diagnosed with acute megakaryoblastic leukemia, who presented after >1 year of bilateral leg pain. At times the pain was severe enough to prevent ambulation, prompting visits to her primary care provider. However, it was not until acute respiratory failure occurred with subsequent hospitalization in the pediatric intensive care unit that severe anemia and thrombocytopenia were discovered and the diagnosis of acute myeloid leukemia was made. Bilateral lower extremity swelling was noted on admission and radiographs showed diffusely abnormal appearance of the long bones of her lower extremities with periosteal reaction and echogenic debris in the subperiosteal space, thought to represent leukemic cells. This case highlights the importance of recognizing atypical signs and symptoms of myelodysplastic syndrome progressing to acute leukemia in the context of abnormal bone pain and radiographic changes.

Purpose of Review Acute myeloid leukemia (AML) in children remains a challenging disease to cure with suboptimal outcomes particularly when compared to the more common lymphoid leukemias. Recent advances in the genetic characterization of AML have enhanced understanding of individualized patient risk, which has also led to the development of new therapeutic strategies. Here, we review key cytogenetic and molecular features of pediatric AML and how new therapies are being used to improve outcomes. Recent Findings Recent studies have revealed an increasing number of mutations, including WT1, CBFA2T3-GLIS2, and KAT6A fusions, DEK-NUP214 and NUP98 fusions, and specific KMT2A rearrangements, which are associated with poor outcomes. However, outcomes are starting to improve with the addition of therapies such as gemtuzumab ozogamicin and FLT3 inhibitors, initially developed in adult AML. Summary The combination of advanced risk stratification and ongoing improvements and innovations in treatment strategy will undoubtedly lead to better outcomes for children with AML.

Transient abnormal myelopoiesis (TAM) is a common and potentially fatal neonatal complication of newborn babies with Down syndrome (DS). Children born with mosaic DS are also at risk of developing TAM. However, due to their variable phenotypes, early identification of patients with mosaic DS may be difficult; thus, early diagnosis of TAM is just as challenging. In this report, we describe a case of a phenotypically normal newborn who presented with concerns for neonatal leukemia. The diagnosis of mosaic DS and TAM was confirmed with abnormal GATA1 mutation testing, highlighting the importance of early GATA1 mutation testing in newborn leukemia with high suspicion for TAM.

Background Relapse free survival of pediatric AML remains only 60%. Current standard myelosuppressive therapy has been maximized, so novel therapies with minimal toxicities are needed to improve outcomes. Previously, we found atovaquone (AQ), an FDA-approved drug that treats pneumocystis jiroveci pneumonia (PJP), reduces AML burden - by suppressing oxidative phosphorylation (OXPHOS) - in xenograft mice. Clinically achievable concentrations of AQ for anti-PJP are 40-80µM, but the anti-leukemia effects are observed as low as 10µM (Stevens et al, Bld Adv, 2019). This makes AQ an ideal drug to incorporate into AML treatment. AQ is a daily administered oral medication, and plasma levels depend on patient compliance, absorption, and entero-hepatic recirculation, which can be compromised due to the patient population and adverse events (AE) of chemotherapy. Here we investigated the feasibility of incorporating AQ into standard pediatric AML treatment. Methods Patients with de novo AML were enrolled at two children's hospitals in the USA. Daily administration of AQ at established PJP prophylaxis dosing was combined with standard chemotherapy for AML, based on the Medical Research Council (MRC) backbone of cytarabine 100mg/m2 q12h x 10 days, and daunorubicin 50mg/m2/dose on days 1, 3, and 5. As it was unclear if our AQ dosing would provide adequate PJP prophylaxis, it was left to provider discretion to give additional PJP protection. AQ compliance, AEs (per NCI CTCAE v5), parent/caregiver ease of administration score (scale: 1-5, 1=very difficult, 5=very easy to administer) and peripheral blood/bone marrow pharmacokinetics (PK) were collected during Induction 1. Real time AQ plasma concentration results were not provided. To address feasibility of achieving adequate levels, all gastrointestinal (GI) AEs ≥ grade 2 were collected, in addition to grade 4 or greater AEs. Patients who took at least 85% of planned doses and missed less than 2 consecutive doses of AQ were eligible for analyses. Correlative biology studies assessed AQ induced apoptosis at 30uM, effects on OXPHOS and relevant signaling activities. Patient derived xenografts (PDX) were established and treated with AQ. This trial is registered with ClinicalTrials.gov (NCT03568994). Results A total of 26 pediatric AML patients enrolled (ages 8 months - 19.7 years, mean 10.7 years); 24 patients were evaluable for this study. Two patients had Grade ≥ 3 GI toxicities that prohibited enteral administration so they were excluded from AQ PK and ease of administration analyses. We found that 14/24 (58%) patients achieved plasma levels above the target anti-leukemia concentration (10µM) by day 11. At the end of induction, 19/24 (75%) patients achieved plasma levels above 10µM, but only 7/24 (29%) patients achieved adequate levels for PJP prophylaxis (40µM). Only 1 patient achieved levels above 40µM throughout the trial [FIG A]. Mean ease of administration score was 3.8. For the youngest patients (x ≤ 2.6years), the average score was 3.4 which was not significantly different from older patients (ANOVA, p > 0.05) [FIG B]. Ease of administration scores showed no association with plasma levels (Pearson's correlation, p > 0.05). Finally, correlative biology studies in patient samples demonstrated robust AQ-induced apoptosis, OXPHOS suppression, and prolonged survival in a PDX model receiving AQ [FIG C]. Conclusion Our data demonstrate the feasibility of combining AQ with traditional chemotherapy for pediatric AML. Patients of all ages were able to tolerate AQ and no AEs were attributable to AQ administration. The target anti-leukemic concentration of AQ in the plasma (> 10uM) was frequently achieved, but concentrations of > 40uM at standard dosing were rare. Low plasma levels of AQ did not correlate with the presence of GI related AEs or weight loss, so plasma levels should be monitored to ensure sufficient PJP prophylaxis. Our correlative biology results support suppression of OXPHOS as the primary mechanism of action by which AQ exerts its anti-leukemia effect, and AQ may be an active anti-leukemia agent for pediatric AML patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background : Although in recent clinical trials the 5-year event-free survival rates for pediatric Acute Myeloid Leukemia (AML) range between 49% and 64%, relapse still occurs in up to one-third of cases. Long-term outcomes of children with relapsed or refractory disease remain poor, with overall survival under 40%. Novel drugs directed toward distinct pathways and new molecular targets are required to continue improving outcomes for this aggressive hematological disease. Use of the BCL-2 inhibitor, venetoclax, has improved overall and event-free survival in adult patients with newly diagnosed, intensive-chemotherapy ineligible AML, however, its effects in pediatric AML patients remain unclear. We reviewed our multi-institutional experience with venetoclax in this population. Methods : We performed a retrospective review of patients ages 0 to 23 y/o with AML and treated with at least one cycle of venetoclax at either MD Anderson Cancer Center or Texas Children's Hospital prior to May 2021. Flow cytometry was used to assess morphology and response to therapy. Adverse events (AEs) associated with venetoclax were graded according to the Common Terminology Criteria for Adverse Events version 5.0. Descriptive statistics were used to report efficacy and toxicity data. Results : 46 patients with AML who received venetoclax-containing regimens were identified. There was a similar proportion of males and females (52% and 48%) and most were Caucasian (48%). The median age was 21 years (range, 1-23) with eight patients (17%) Conclusion : Our findings suggest that in pediatric and early young adults, venetoclax is well-tolerated with a variety of cytotoxic agents and has a safety profile similar to that in adults. Patients should be monitored closely for prolonged myelosuppression and febrile neutropenia. As dosing varied significantly in our cohort, more studies are needed to establish an optimal dose in the pediatric population. Longer follow-up is expected to provide more insight on the improvement venetoclax may have on overall and progression-free survival. Figure 1 Figure 1. Disclosures Kadia: Sanofi-Aventis: Consultancy; Dalichi Sankyo: Consultancy; Genentech: Consultancy, Other: Grant/research support; Genfleet: Other; AstraZeneca: Other; Astellas: Other; Pulmotech: Other; Cure: Speakers Bureau; Cellonkos: Other; Jazz: Consultancy; Pfizer: Consultancy, Other; Ascentage: Other; Novartis: Consultancy; Liberum: Consultancy; BMS: Other: Grant/research support; Amgen: Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support. Konopleva: Cellectis: Other: grant support; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; KisoJi: Research Funding; Calithera: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding. DiNardo: Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Forma: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Daver: Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Novartis: Consultancy; Astellas: Consultancy, Research Funding; Glycomimetics: Research Funding; Novimmune: Research Funding; Hanmi: Research Funding; Genentech: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; FATE Therapeutics: Research Funding; ImmunoGen: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Short: AstraZeneca: Consultancy; Jazz Pharmaceuticals: Consultancy; NGMBio: Consultancy; Novartis: Honoraria; Astellas: Research Funding; Takeda Oncology: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Issa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Ravandi: Amgen: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria, Research Funding; Prelude: Research Funding; Astex: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Kantarjian: Ascentage: Research Funding; Astra Zeneca: Honoraria; Pfizer: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; KAHR Medical Ltd: Honoraria; Ipsen Pharmaceuticals: Honoraria; NOVA Research: Honoraria; Novartis: Honoraria, Research Funding; Immunogen: Research Funding; Astellas Health: Honoraria; BMS: Research Funding; Aptitude Health: Honoraria; Jazz: Research Funding; AbbVie: Honoraria, Research Funding; Precision Biosciences: Honoraria; Amgen: Honoraria, Research Funding; Taiho Pharmaceutical Canada: Honoraria.

Background Pediatric acute myeloid leukemia (AML) is a rare disease with roughly 600 cases diagnosed in the United States each year with minimal improvement in clinical outcomes over the last few decades. We previously demonstrated that an ex vivo drug sensitivity assay (DSA) can predict clinical response in myelodysplastic syndrome (Spinner et al. Blood Adv 2020). Here we investigated whether the DSA performed on pre-induction pediatric AML samples correlates with clinical response and can identify potent novel drug combinations. Methods Pre-induction blood or bone marrow samples were assayed from 20 de novo pediatric AML patients diagnosed at Texas Children's between 5/2015 and 10/2020. All patients consented to research (82% enrolled in clinical trial identifier NCT03568994) and received ADE (Cytarabine, Daunorubicin, and Etoposide), and next-generation sequencing was done as part of clinical care. Risk stratification was per AAML1831 guidelines. Drug sensitivity data was analyzed from 13/20 samples that passed quality control with matched treatment conditions: 9/13 (69%) patients had M1/M2 histology, 3/13 (23%) were M4/M5 and 1/13 (8%) was M7 with a median age of 12.3 years. For the ex vivo DSA, samples were incubated in conditioned media and treated with a single dose of up to 25 unique compounds and up to 149 drug combinations. After 72 hours, changes in tumor blast populations were assessed by flow cytometry using an 11-marker panel to identify blasts. For each treatment condition, drug sensitivity was calculated based on the number of blasts remaining after treatment compared to DMSO control. Clinical response data, including minimal residual disease (MRD) percentage by flow cytometry, and 1-year relapse-free survival (RFS), were correlated with drug sensitivity results. Log odds ratios (OR) were calculated with the Haldane-Anscombe correction. ORs were used to quantitatively measure the association between clinical attributes and the DSA to the clinical response data. For evaluation of ORs, a normalized blast score of 70% viability was used to maximize the separation between high and low drug sensitivity. Results Ex vivo drug sensitivity correlated with both MRD (r=0.63) and 1-year RFS (r=0.59) in the de novo patient subset (Fig A). Three patients with an MRD >1% exhibited low ex vivo sensitivity to ADE, and among these 3 patients, 2 did not achieve 1-year RFS. Results from the DSA predicted increased odds of having an MRD >1% compared to demographic and mutational clinical attributes that showed weaker associations with MRD (Fig B). Of the 77 treatment conditions that were tested in 13 patient samples, Bortezomib in combination with Panobinostat (B/P) was the most efficacious treatment in the DSA, where drug sensitivity ranged from low (>100% blast viability) to high (0% blast viability). Separation of patient samples into two distinct low and high DSA response groups was observed with B/P, whereas ADE and single agents showed a graded distribution (Fig C). Within these response groups, pAML3 showed low sensitivity to ADE in the ex vivo DSA and the patient did not respond to ADE. In contrast, pAML8 showed high sensitivity to ADE ex vivo and the patient responded to ADE treatment. While pAML3 and pAML8 showed similar ex vivo sensitivity to B/P as for ADE (Fig D), pAML4 showed preferential sensitivity to ADE and not B/P, and conversely pAML6 showed sensitivity to B/P and not ADE. Conclusion Ex vivo drug sensitivity to ADE correlates with both MRD and 1-year RFS in a cohort of 13 de novo pediatric AML patients. These results suggest that clinical response in pediatric AML may be assessed prior to treatment using an ex vivo drug sensitivity assay. Compared to demographic and mutational clinical characteristics queried, ex vivo drug sensitivity to ADE has the potential to be a more predictive measure compared to clinical attributes alone. Combining genomics with functional ex vivo drug sensitivity data could further enhance precision medicine and biomarker discovery in pediatric AML. The DSA also highlights Bortezomib/Panobinostat as a potential novel drug combination for pediatric AML, and the ability to identify a patient sample that is insensitive to ADE and sensitive to Bortezomib/Panobinostat ex vivo supports the use of the DSA to not only predict clinical response but also to possibly inform treatment decisions for pediatric AML patients. Figure 1 Figure 1. Disclosures Strachan: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Gu: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Kita: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Richardson: Notable Labs: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Anderson: Notable Labs: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Santaguida: Notable Labs: Consultancy, Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Patents & Royalties.

Acute promyelocytic leukemia (APL) is a rare disease accounting for only 5%−10% of pediatric acute myeloid leukemia (AML) and fewer than 1000 cases occur annually in the United States across all age groups. Characterized by t (15; 17), with a resultant PML-RARA gene fusion driving leukemia development, advances in therapy have improved outcomes for APL significantly in the past several decades, now making APL the most curable form of AML in both children and adults. Cure rates in APL are now comparable to pediatric B-lymphoid leukemias. The success of APL treatment is due, in part, to the breadth of understanding of the driver PML-RARA mutation as well as collaborative efforts to quickly introduce and maximize the benefit of new therapies. Here, we review the presentation, clinical features, pathogenesis, and treatment advances in pediatric APL.

10032 Background: Pediatric acute myeloid leukemia (AML) is a rare disease with roughly 500 cases diagnosed in the United States each year and has had minimal improvement in clinical outcomes over recent decades. Novel treatment development to improve outcomes may be enhanced with an accompanying test for predicting treatment response. We previously demonstrated that an ex vivo drug sensitivity assay (DSA) can predict clinical response in myelodysplastic syndrome. Here we investigated the use of the DSA in pediatric AML patients, including a subset participating in a clinical trial of atovaquone. Atovaquone is an FDA-approved anti-parasitic drug that was associated with lower relapse rates in adult AML patients. Adding atovaquone and other standard of care combination treatments into the DSA, we investigated whether the assay, performed on pre-induction samples, correlated with measures of clinical response. Methods: We assayed pre-induction blood or bone marrow samples from 22 de novo pediatric AML patients diagnosed at Texas Children’s between 5/2015 and 10/2020 who consented to research (82% enrolled in clinical trial identifier NCT03568994). We subsetted this analysis to patients who received ADE (Cytarabine, Daunorubicin, Etoposide) (n = 20) induction, with the majority additionally receiving atovaquone (n = 16). For the DSA, samples were incubated with up to 25 compounds, including the treatment drug combinations and each of the compounds individually. After incubation, changes in tumor blast populations were assessed by flow cytometry. For each drug condition, drug sensitivity was calculated based on the number of blasts remaining after treatment. After quality control, downstream analyses were limited to 13 samples. Clinical response data, including minimal residual disease (MRD) percentage by flow cytometry and one year relapse-free survival, were correlated with the drug sensitivity results. Results: For the de novo subset analysis, we observed correlations between ex vivo drug sensitivity with both MRD percentage (r = 0.63) and one year relapse-free survival (RFS1, AUC = 0.92). The 3 patients with lowest ex vivo sensitivity had the highest MRD percentages (mean 21%). 2 of the 3 patients who did not achieve one year relapse-free survival had the lowest ex vivo sensitivity. Drug combination sensitivity correlated more with MRD and RFS1 than the single agents alone (single agent mean MRD r = 0.39). Conclusions: In our cohort, ex vivo DSA for ADE and atovaquone in pediatric AML cases correlated with both MRD and one year relapse-free survival. This suggests that clinical response in pediatric AML may be assessed prior to treatment using a DSA. This study also suggests that the DSA can be used to test drug combinations, and thus may be used for investigating novel treatment combinations. Further development of the DSA may benefit treatment decisions and prioritization of drug development.

BackgroundC-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor (CAR) T-cell therapy. Here, we optimized our CLL-1 CAR for anti-leukemic activity in mouse xenograft models of aggressive AML.MethodsFirst, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vector to coexpress transgenic IL15. We measured the effects of each construct on T cell phenotype and sequential (recursive) co culture assays with tumor cell targets to determine the durability of the anti tumor activity by flow cytometry. We administered CAR T cells to mice engrafted with patient derived xenografts (PDX) and AML cell line and determined anti tumor activity by bioluminescence imaging and weekly bleeding, measured serum cytokines by multiplex analysis. After euthanasia, we examined formalin-fixed/paraffin embedded sections. Unpaired two-tailed Student’s t-tests were used and values of pResultsIn vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (pConclusionCombinatorial treatment with a TNFα blocking antibody and subsequent activation of the caspase-9 control switch increased the expansion, survival and antileukemic potency of CLL-1 CAR T-cells expressing transgenic IL15 while avoiding the toxicities associated with excessive cytokine production and long-term accumulation of activated T-cells.

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

Introduction: C-type lectin 1 (CLL-1, CD371) is highly expressed on the malignant cells from many patients with AML, and CAR T cells directed to this antigen can selectively target both leukemic progenitor cells (LSC) as well as AML blasts whilst sparing normal tissues. We previously showed (1) that such CAR-Ts can recognize and eliminate both AML blasts and primitive AML colony-forming cells in a low tumor-burden model. We have now modified the structure of the CLL-1 CAR and added transgenic expression of IL15 to enhance performance sufficiently for activity even against more extensive disease. Material and Methods: We assessed the phenotype and cytolytic ability of T cells transduced with 5 CLL-1 CAR constructs, varying in their spacer, transmembrane and costimulatory sequences (CD28z-CD8, CD28z-sh, CD28z-CH3, 4-1BBz-sh, 4-1BBz-CH3), and compared these with the effects of our published construct (4-1BBz-CD8)(1). We used flow cytometry to determine the effects of each construct on T cell phenotype and differentiation, and sequential (recursive) co-culture assays with tumor-cell targets to determine the durability of the anti-tumor activity. The most active constructs (CD28z-CD8 and 4-1BBz-CD8) were then evaluated in NOD.SCID IL-2Rg-/- (NSGS) mice engrafted with 1.5x10ˆ6 FFLuc-modified HL 60 AML cells, which received 2x10ˆ6 CLL-1 CAR T cells on day 3. To determine if we could further potentiate the in vivo expansion, persistence and anti-tumor activity of the CLL-1 CAR-T cells, we used a second retroviral vector to co-express transgenic IL15, measuring the effects in vitro and in vivo. Mice engrafted with 1.5x10ˆ6 tumor cells and received 2.5x10ˆ6 CLL-1 CAR T cells on week 3 in patient derived xenograft (PDX) model. We determined antitumor activity by bioluminescence imaging and weekly bleeding and measured serum cytokines by multiplex analysis (Luminex, TX). After euthanasia, we examined formalin-fixed/paraffin embedded sections. Results: Modified CLL-1 CAR constructs were expressed by 70-80% of cells irrespective of CAR sequence, but CD28z-CD8 CAR T cell expansion was significantly higher than CAR T cells with 4-1BBz endodomains (p Conclusion: Addition of transgenic IL15 to CLL-1-CD28z-CD8 CAR augmented activity against AML in a range of cell line and PDX models, and toxicity associated with exuberant CART expansion could be prevented by cytokine blockade and/or an inducible safety switch. References: 1. Tashiro H, et al. Mol Ther. 2017 2.Straathof KC et al. Blood. 2005 Disclosures Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees.

Introduction: New approaches to find and then drug pediatric acute myeloid leukemia (AML)-specific targets are clearly needed to help the nearly 35% of patients who still die from the disease. While RARA is a known druggable target in acute promyelocytic leukemia (APL), the utility of using retinoic acid agonists in non-APL AML has not proven consistently beneficial. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult non-APL AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was detected at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the second-generation retinoic acid agonist tamibarotene which has led to a phase II clinical trial. This study confirms that characterization of the chromatin-defined dependencies in specific cancers can pinpoint targets which can be drugged. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by strong RARA dependence in a majority of pAML. Methods: Three AML cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. For in vivo assessment, 200,000 cells of a RARA SE+ pAML patient sample were injected into each NSGS mouse by tail-vein injection. One week after injection, tamibarotene (6mg/kg) or vehicle treatment was initiated by gavage (n=7 each arm). Peripheral blood monitoring of leukemia burden was determined flow cytometry. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes (Fig 1a), with an overrepresentation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters (Fig 1b). Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes (Fig 1c). Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples suppressed proliferation and increased apoptosis (detected by annexin V+), with minimal effect in Kasumi, a pAML cell line without a RARA SE (Fig 2a). In the RARA SE+ cell lines and samples only, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) (Fig 2b). High RARA mRNA levels confirmed the SE assignments in both cell lines and patient samples (Fig 2c). Tamibarotene induced the transcription of DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial) (Fig 2d). Tamibarotene suppressed colony formation ability in RARA SE+ cell lines. An ongoing RARA SE+ patient-derived pAML xenograft confirmed tamibarotene markedly suppressed disease progression (Fig 3), with vehicle mice requiring euthanasia 42 days after tail-vein injection for significant disease burden, while the tamibarotene treated mice continued to be well-appearing at the same timepoint. Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and seen a high frequency of a RARA SE in pAML. Tamibarotene has anti-proliferative, proapoptotic, and on-target pro-differentiation effects in RARA SE+ pAML in vitro and marked anti-leukemia activity in vivo. Given these positive findings, we are evaluating combinations of other AML-active agents with tamibarotene. Additionally, as there is a range of sensitivity to tamibarotene in RARA SE+ samples, we are also interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Disclosures Wei: NHI NHLBI Grant: Other: received funding . Lin:Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

Emergency departments (EDs) are alert to the possibility of stroke and the need for early interventions to improve long-term clinical outcomes. However, new-onset hemiparesis in pediatric patients with leukemia may be due to a number of different etiologies, including most common side effects from chemotherapeutic agents. We present a case of a 15-year-old boy with pre-B acute lymphoblastic leukemia on chemotherapy, having recently received a high-dose methotrexate infusion in addition to intrathecal methotrexate therapy, who presented to our ED with acute right-sided hemiparesis. He was initially suspected as having a possible ischemic stroke. Magnetic resonance imaging (diffusion-weighted and fluid-attenuated inversion recovery sequence) demonstrated focal areas of diffusion restriction, an early sign of delayed-onset methotrexate neurotoxicity. Our patient received appropriate supportive care and leucovorin rescue with gradual clinical recovery, after a prolonged hospitalization and acute care rehabilitation over the course of several months. Our case illustrates the need for ED providers to consider methotrexate neurotoxicity in pediatric oncology patients presenting with acute neurologic changes.

Background: Currently, pediatric AML risk stratification relies solely on response to therapy and a small number of prognostic genetic factors. With these limited criteria, approximately 75% of children are initially classified as being at a lower risk (LR) of relapse. However, 30% of LR patients relapse, and achieving a second remission is often quite difficult. Therefore, there is an urgent need to understand mechanisms of chemoresistance, in order to identify patients who should receive alternative therapies. We previously showed that increases in IL-6-induced STAT3 activity at relapse were associated with inferior survival [Stevens, et al, Haematologica, 2015]. These findings suggested that the IL-6-induced STAT3 pathway may promote the chemoresistance often seen in relapsed AML. Thus, we hypothesized that IL-6 levels in the bone marrow (BM) niche are dysregulated in pediatric AML and elevated levels are predictive of clinical outcome. We further hypothesized that IL-6 promotes chemoresistance in AML blasts in vitro. Methods: Bone marrow samples from pediatric AML patients diagnosed between 2007-2016 at Texas Children's Cancer Center were studied. Patients were treated similarly (Children's Oncology Group protocols AAML0531 or AAML1031). We obtained plasma from diagnostic BM for 46 patients, and 23/46 had a matched remission sample. For comparison, we obtained normal BM (NBM) plasma from 7 sibling BM donors. Cytokine levels were determined with the 41-plex Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (EMD Millipore). Cytokine and growth factor concentrations between NBM and AML samples were analyzed by the Mann-Whitney U test. Paired diagnostic and remission samples were compared with the Wilcoxon signed-rank test, and correlation of cytokine levels with clinical outcome was assessed by the Kaplan-Meier method. To assess for IL-6-induced chemoresistance, AML cell lines NB4 and THP1 were used. Cells were exposed to IL-6 (50 ng/mL) and soluble IL6 receptor a (sIL-6Rα; 100 ng/mL) or vehicle control (VC) for 24h prior to escalating doses of chemotherapy (mitoxantrone, etoposide, cytarabine). After treatment with chemotherapy for 24h, the percent of Annexin-V+ AML cells was determined by FACS and differences assessed by ANOVA. Results: IL-6 levels were uniformly low in NBM and were elevated at diagnosis in a subset of AML patients. The median in NBM was 1.7pg/mL (25-75 Percentile 1.53-4.54) vs 11.84 pg/mL for AML (25-75 Percentile 4.66-41.47, p 17.5 pg/mL. Patients with high IL-6 (n=11) had relapse free survival (RFS) of 13.6% compared to 61.5% (p=0.007; log-rank test) for patients with low IL-6 (n=18). In addition, we found the following cytokines and growth factors to be significantly higher in AML patients v. controls: IL-8, IL-10, GM-CSF, IL-3, IL-17A, MDC, PDGF-AA, PDGF-BB, IL-1RA, IL-4, and TNFα. Notably, and discrepant from what is seen in adult AML, only IL-6 was found to predict RFS in our cohort. Furthermore, both THP-1 and NB4 cells demonstrated less mitoxantrone-induced apoptosis when pretreated with IL-6 and sIL6Rα, compared to cells treated with VC. Conclusions: Our data demonstrate that in pediatric AML, IL-6 levels in the BM at diagnosis are elevated in a subset of patients and return towards normal levels in the majority of patients at remission. In our cohort, elevated IL-6 levels in the BM at diagnosis identified patients with an inferior outcome. Additionally, we have demonstrated that these inferior outcomes may be due to IL-6-induced chemoresistance. Our results provide support for further development and evaluation of IL-6 as a marker of prognosis and as a potential therapeutic target in pediatric AML. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: We have shown that Stat pathway sensitivity to G-CSF and IL-6, measured by the increase in tyrosine phosphorylated Stat3 (pY-Stat3), is associated with clinical outcome in pediatric AML patients. These results support a possible relationship between chemoresponsiveness and ligand-induced Stat3 response. We hypothesized that consistently altered changes in Stat3 signaling between diagnosis and relapse would represent advantageous adaptations that promote chemotherapy resistance in pediatric AML. Methods: 25 sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the COG frontline trial AAML0531 were analyzed. After thawing, ≥80% viability was confirmed by Trypan exclusion. Constitutively phosphorylated Stats (pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100 ng/ml G-CSF, or 5 or 50 ng/ml IL-6 with 10 or 100 ng/ml soluble IL-6 receptor, respectively, for measurement of ligand-induced pStats. Data were collected on the LSR II (BD) and analyzed with FCS Express 4 (DeNovo). For the ligand-induced pStats, data are expressed as the fold change in mean fluorescence intensity (ΔMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, and TStat3 data are expressed as percent in the positive region, as defined by the upper limit of the signal in the isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse. Results: 24/25 sample pairs had adequate cell numbers and viability for analysis. At both doses of G-CSF, 21/24 pairs demonstrated an increase in pY-Stat3 ΔMFI. At the lower G-CSF dose, the mean ±SEM ΔMFI increased from 1.41±0.13 to 2.36±0.28 (p=0.0001). At the higher dose, ΔMFI increased from 1.65±0.20 to 2.71±0.33 (p=0.0002). Similarly, at both doses of IL-6, 17/24 pairs demonstrated an increase in pY-Stat3 ΔMFI. At the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.25±0.09 to 1.56±0.16 (p=0.0168). At the higher dose, ΔMFI increased from 1.49±0.17 to 2.01±0.24 (p=0.0051). Only 3/24 pairs showed >15% increase in G-CSF receptor expression, while 10/24 had a >15% increase in gp130 expression. No significant changes were seen in constitutive activity of pStats, or TStat3 expression. Conclusions: Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse in pediatric AML. More robust signaling was not due to increased expression of total Stat3 and was rarely associated with increased receptor expression. Our data suggest that ligand-induced Stat pathway activation may be promoting survival in relapsed AML. Our results provide support for further development and evaluation of targeted agents against this pathway in AML. Citation Format: Alexandra M. Stevens, Marcos Ruiz, Michele S. Redell. Increased responsiveness to ligand stimulation of the STAT pathway at relapse in acute myelogenous leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4206. doi:10.1158/1538-7445.AM2014-4206

Abstract 3544 Background: Acute myeloid leukemia (AML) is a very heterogeneous disease and changes in characteristics such as immunophenotype and mutation profiles are known to occur between diagnosis and relapse. Alterations in signaling pathways are likely to occur as well, but have not yet been reported. Our lab has recently shown that Stat3 signaling profiles are associated with outcome in pediatric AML patients. Specifically, patients whose blasts were sensitive to both G-CSF and IL-6, measured by increased tyrosine phosphorylation of Stat3 (pY-Stat3), had significantly improved event-free and overall survival compared to those whose blasts were resistant to one or both of the tested ligands. These results support the importance of ligand responsiveness within the STAT pathway in relation to chemoresponsiveness in AML. We hypothesized that comparison of STAT signaling pathways in paired diagnostic and relapse samples would identify changes that are likely to represent strengthening of pathways involved in chemoresistance. Methods: Twenty two sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the Children's Oncology Group protocol AAML0531 have been analyzed for this study. After thawing, ≥80% viability was confirmed by Trypan blue stain. Constitutively phosphorylated STATs (including pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), pY418-Src, G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100ng/ul G-CSF, or 5 or 50ng/ul IL-6 with 10 or 100 ng/ul soluble IL-6 receptor, respectively, for measurement of ligand-induced pStats. Data were collected on the LSR II (BD) and analyzed with FCS Express 4 (DeNovo). For the ligand-induced pStats, data are expressed as the fold change in mean fluorescence intensity (DMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, TStat3, and pY418-Src data are expressed as percent positive compared to isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse. Results: Of the 22 sample pairs thawed, 21 had adequate cell numbers and viability for analysis. Increases in ΔMFI of pY-Stat3 between diagnosis and relapse were seen in response to both doses of G-CSF. At the lower dose, ΔMFI increased from 1.45±0.14 to 2.30±0.33 (p=0.0023), with increases demonstrated in 16/21 pairs. At the higher dose level, ΔMFI increased from 1.70±0.20 to 2.68±0.38 (p=0.0019) with increases in 16/21 pairs. Similarly, in response to the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.29±0.10 to 1.58±0.18 (p=0.0183), with increases in 14/21 pairs while at the higher dose, ΔMFI increased from 1.53±0.20 to 2.07±0.27 (p=0.0079) with increases in 12/21 pairs. Evaluated parameters that did not change significantly between diagnosis and relapse included G-CSF receptor expression (74.09% to 80.16; p=0.1526), gp130 expression (51.63% to 60.52%; p=0.0569), constitutive activity of pYStat3 (23.60% to 26.63%; p=0.0962), and TStat3 expression (35.32% to 43.96%; p=0.053). Though the changes were small, pY418-Src was found to be significantly increased between paired samples (0.90% to 2.58%; p=0.0011). Conclusions: STAT pathway signaling patterns evolve between diagnosis and relapse in pediatric AML. Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse. This suggests that the resistance seen to chemotherapy in relapsed patients may be in part due to the increased activity of this anti-apoptotic pathway in response to growth factors and cytokines present in the bone marrow niche. The increases in activity within the STAT pathway at relapse provide support for further development and evaluation of targeted agents against this pathway in relapsed patients with AML. Disclosures: No relevant conflicts of interest to declare.