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This paper is about the issue of students’ monologic speech, examining possible differences in fluency, production over levels of proficiency (as represented by TOEIC scores), and how this speech improves over an academic year. The data, which is based on the speech of 12 participants, was taken from the corpus Monologic and Dialogic Corpus 2019 (20,368 words and 42 subjects), and from the second corpus Monologic and Dialogic Corpus 2020 (16,997 words and 29 participants). Research questions focused on characterizing the speech in monologues of higher and lower proficiency Japanese EFL (English as a Foreign Language) learners in regards to the fluency variables of speaking time, articulation rate, mean length runs (MLRs), number of words, and percentage of silence. Furthermore, research aims focused on how acoustic, lexical and syntactical dysfluency might change over the year. Results showed that the average time of monologic speech was higher in the high-proficiency group than in the low-proficiency group in 2019 and 2020, but this difference was not statistically significant. In the high-proficiency group, there was a significant difference between 2019 and 2020 in meaningless syllables, whereas the other acoustic, lexical, and syntactical dysfluency variables showed no significant difference over the academic year 2019/2020. Further research will be conducted on how direct feedback might impact students’ output.

OBJECTIVES Del Nido cardioplegia (DNC) has been shown to be safe in adults with normal coronary arteries who are undergoing valve surgery. This study compared the effects of DNC versus traditional blood-based cardioplegia on postoperative complications in patients who underwent coronary artery bypass grafting (CABG). METHODS A retrospective analysis was performed on 863 patients who underwent CABG with DNC (n = 420) or control cardioplegia (CC) (n = 443) between 2014 and 2017. The full cohort of DNC and CC recipients, as well as propensity score-matched pairs, was compared regarding preoperative risk variables and outcomes. RESULTS The DNC and CC groups showed no significant differences in mean cardiopulmonary bypass time (53.09 vs 52.10 min, P = 0.206) or aortic cross-clamp time (32.82 vs 33.28 min, P = 0.967). The groups also showed no difference in operative mortality (2.1% vs 2.5%, P = 0.734); however, DNC use resulted in lower rates of overall infections (1.7% vs 4.3%, P = 0.024), total sternal infections (0.9% vs 3.2%, P = 0.023), postoperative atrial fibrillation (23.8% vs 30.7%, P = 0.023) and postoperative ventricular tachycardia (0.5% vs 3.4%, P = 0.002). A propensity-matching analysis (n = 335 pairs) showed similar statistically significant decreases with DNC. CONCLUSIONS In this large cohort of CABG patients, DNC was shown as a safe alternative to CC and was associated with lower postoperative dysrhythmia and infection rates. These findings show that DNC is safe and effective in patients whose operative interventions may require only single-dosing cardioplegia; its use in longer cases should be further explored given its low complication rate.

OBJECTIVES Provision of reproductive health preventive services to adolescents is critical given their high rates of sexually transmitted infections and unintended pregnancies. Pediatricians are well positioned to provide these services but often face barriers. With this project, we aimed to build quality improvement (QI) capacity within pediatric practices to improve adherence to national guidelines for adolescent reproductive health preventive services. METHODS In 2016, an accountable care organization overseeing health care delivery for low-income children in the Midwestern United States used practice facilitation, a proven approach to improve health care quality, to support pediatric practices in implementing reproductive health QI projects. Interested practices pursued projects aimed at providing (1) sexual risk reduction and contraceptive counseling (reproductive health assessments [RHAs]) or (2) etonogestrel implants. QI specialists helped practices build key driver diagrams and implement interventions. Outcome measures included the proportion of well-care visits with RHAs completed and number of etonogestrel insertions performed monthly. RESULTS Between November 1, 2016, and December 31, 2019, 6 practices serving >7000 adolescents pursued QI projects. Among practices focused on RHAs, the proportion of well-care visits with completed RHAs per month increased from 0% to 65.8% (P < .001) within 18 months. Among practices focused on etonogestrel implant insertions, overall insertions per month increased from 0 to 8.5 (P < .001). CONCLUSIONS Practice facilitation is an effective way to increase adherence to national guidelines for adolescent reproductive health preventive services within primary care practices. Success was driven by practice-specific customization of interventions and ongoing, hands-on support.

The blast-induced motion of spheres has been studied experimentally where the shock wave is rapidly decaying during the period that quasi-steady acceleration would be developed in the case of a step-function shock wave as considered in most shock-tube studies. The motion of sphere models ranging from 39 to 251 mm in diameter and having a range of densities was assessed using the “free-flight” method in a simulator specially designed to replicate the decaying shock wave profile of spherical blast including negative phase and positive entropy gradient. A standardized blast-wave simulation of 125 kPa and 6-ms positive-phase duration was applied for all experiments. In all cases, there are three phases to the motion: a relatively low “kickoff” velocity from the shock diffraction, acceleration or deceleration during the positive duration, then deceleration through the negative phase and subsequent quiescent air. The unexpected deceleration of larger spheres after their kickoff velocity during the decaying yet high-speed flow of the blast wave seems associated with the persistence of a ring vortex on the downstream side of the sphere. The flow is entirely unsteady with initial forces dominated by the shock diffraction; therefore, the early motion of spheres under such conditions is not governed by quasi-steady drag as in classical aerodynamics. The work will help establish scaling rules for model studies of blast-induced motion relevant to improvised explosive devices, and preliminary results are shown for motion imparted to a human skull surrogate.

© 2016 Diabetes Technology Society. The purpose of developing mobile applications for diabetes is generally to: (1) provide enhanced access to timely information for patients, health care professionals, and researchers; (2) facilitate remote monitoring and diagnosis of patients, often based on information delivered by wearable devices; (3) provide decision support to assist patients in selecting treatment; or (4) deliver timely recommendations for treatment to increase adherence to prescribed therapy. There is a perception that mobile applications can provide meaningful clinical benefits, however, there is only sparse convincing evidence to support this belief at the present time. Compounding this problem is the short life span of digital software, such that if a traditional type of randomized controlled trial is conducted on a product, by the time the study has been designed, approved by an IRB, conducted, and analyzed, the product might have significantly changed to a next generation system. Because of great interest in establishing what are the potential benefits, metrics of success, and appropriate components of mobile applications for diabetes, Diabetes Technology Society and William Sansum Diabetes Center launched the Digital Diabetes Congress, March 7-8, 2017, in San Francisco. This report contains summaries of the meeting's 12 sessions. Each summary was written by the session's moderator who helped develop the session prior to the event and keep it on track during the event. This meeting report presents a summary of how 57 panelists, speakers, and moderators, who are leaders in digital health, see the current and future landscape of digital health tools applied to diabetes.

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

Follicular differentiation can be exhibited by a variety of epidermal lesions. We report an example of panfolliculoma purely confined to the epidermis. The lesion presented as a verrucous plaque on the leg of a 55-year-old man. Histopathologic sections showed an acanthoma with follicular differentiation including areas mimicking the follicular germ, matrix, root sheath and infundibular components.

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. The MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 μg mL−1 without interference from other algae acidic hydrolyzate components.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Childhood cancers represent distinct clinical entities, often with unique genomic alterations and therapeutic responses that differ from cancers arising in adults. Pediatric acute myeloid

Chronic obstructive pulmonary disease (COPD) is a progressive, smoking-related, inflammatory lung disease in which tumor necrosis factor-alpha is overexpressed and has been suggested to play a pathogenic role.To determine if infliximab, an anti-TNF-alpha antibody, results in clinical benefit and has an acceptable safety profile in patients with moderate to severe COPD.In a multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-finding study, subjects with moderate to severe COPD received infliximab (3 mg/kg [n = 78] or 5 mg/kg [n = 79]) or placebo (n = 77) at Weeks 0, 2, 6, 12, 18, and 24. Efficacy, health status, and safety were assessed through Week 44.Infliximab was generally well tolerated, but showed no treatment benefit as measured by the primary endpoint, Chronic Respiratory Questionnaire total score. Similarly, there was no change in secondary measures, including prebronchodilator FEV(1), 6-min walk distance, SF-36 physical score, transition dyspnea index, or moderate-to-severe COPD exacerbations. Post hoc analysis revealed that subjects who were younger or cachectic showed improvement in the 6-min walk distance. Malignancies were diagnosed during the study in 9 of 157 infliximab-treated subjects versus 1 of 77 placebo-treated subjects. No opportunistic infections were observed, and there were no differences in the occurrence of antibiotic-requiring infections, although the incidence of pneumonia was higher in infliximab-treated subjects. No infection-related mortality was observed. Higher proportions of infliximab-treated subjects discontinued the study agent due to adverse events (20-27%) than did placebo-treated subjects (9%).Subjects with moderate to severe COPD did not benefit from treatment with infliximab. Although not statistically significant, more cases of cancer and pneumonia were observed in the infliximab-treated subjects. The impact of infliximab on malignancy risk in patients with COPD needs to be further elucidated.

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (

A variation of the Wickens' Task was performed to examine the assumption that people can detect certain stimuli on their periphery without decreasing the performance of the primary task. Participants were instructed to respond to a change in a peripheral stimulus without shifting their gaze from a primary task in the center of their visual field. Our data suggests that both type and magnitude of change have a significant effect on detection rate and reaction time. The data also suggests that the performance of the primary task did not decay after the change of the stimuli occurred. Based on these findings, we argue that people can detect various types of changes without shifting gaze and without degrading task performance. Therefore, an interface particularly designed for peripheral vision is possible, and it will potentially provide benefits to both productivity and safety.

In efforts to discover genes uniquely expressed in childhood AML, we performed transcriptomesequencing (RNA-Seq) in pediatric AML and contrasted the expression signature to that in normal marrow hematopoiesis. This effort led to the discovery of over 200 genes that lack expression in normal hematopoietic cells, but are variably expressed in pediatric AML cells. Mesothelin(MSLN) was discovered to be one of the most highly expressed genes in a subset of childhood AML cases (p In this study, RNA obtained from diagnostic bone marrow specimens from childhood AML (N=434) and normal marrow (N=20) was subjected to wholetranscriptomesequencing and MSLN expression was quantified and normalized and reported as reads perkilobaseof exon per million reads mapped (RPKM). Similar data was obtained from adult TCGA AML database. Quantitative RT-PCR (qRT-PCR) and multidimensional flowcytometry(MDF) was used for confirmation of the transcript and cell surface protein expression. TARGET AML methylation data was used for correlation with transcript expression. Of the 434 specimens analyzed, MSLN mRNA expression was variably expressed (RPKM range 0-618.8), with expression detected in 119 patients (27%). Confirmatory studies by qRT-PCR on specimens with and without MSLN expression (N=137) showed correlation between RNA-Seqand PCR data. Cell surface MSLN expression was assessed by MDF using a PE-conjugated MSLN antibody (PE-mesoAb) and verified expression of MSLN protein on the leukemic cell surface in every case with MSLN transcript expression (Figure 1A). Evaluation of CD34+/CD38- hematopoietic progenitor cells by PE-mesoAbdemonstrated lack of MSLN expression by MDF. Evaluation of matched diagnostic and relapse specimens from MSLN-expressing patients (n=27) confirmed that MSLN expression was largely stable (R2=0.87), thus substantiating its expression in the major AML clone. Comparison of MSLN expression in pediatric vs. adult AML demonstrated a higher prevalence in pediatric AML (TARGET: 27% vs. TCGA: 11%)(Figure 1B). Evaluation of the clinical and biologic features in MSLN expressing (MSLN+) and non-MSLN expressing (MSLN-) pediatric patients revealed that MSLN expression was rarely observed in patients with normal karyotype (p We further inquired about the mechanism by which MSLN expression might be regulated. Whole genome sequencing data failed to identify any genomic alterations in MSLN that could result in high expression. Therefore, we interrogated the possibility of epigenetic regulation of MSLN expression. Integration of the expression and methylation profiling cases with matching RNA-Seqand methylation data (N=246) demonstrated thathypomethylationof the MSLN promoter significantly correlated with high MSLN expression, implicating epigenetic regulation in the expression of MSLN in AML. Mesothelin, a therapeutic target in solid tumors, is highly expressed in biologically distinct subsets of childhood AML. High expression on leukemic blasts and lack of expression in normal hematopoiesis makes this antigen an ideal target for therapeutic intervention in AML. As a cell surface protein, this antigen avails itself for immune targeting by antibody drug conjugates, CAR-T cells, and T cell receptor mediated targeting. Figure 1 Mesothelin expression in childhood AML. Figure 1. Mesothelin expression in childhood AML. Disclosures Loken: Hematologics: Employment, Equity Ownership. Pardo:Hematologics, Inc: Employment.

Twenty-four hip replacements were performed with use of a medial protrusio technique to stabilize the fit of a hemispherical metal shell in the acetabulum in nineteen patients who had dysplasia of the hip. All of the hips were followed for a minimum of five years (average, seven years; range, five to thirteen years). Six of the hips were type I, seven were type II, eight were type III, and three were type IV according to the criteria of Crowe et al. The acetabular cup was implanted with the medial aspect of its dome beyond the Kohler line (drawn from the ischium along the ilioischial line) in all hips. An autogenous graft sculpted from the femoral head was used to cover 15 to 30 percent of the superolateral portion of the cup in one type-I hip, four type-III hips, and one type-IV hip. The need for these six bone grafts could have been avoided by reaming two to three millimeters more medially or by allowing 20 percent of the superolateral portion of the cup to be uncovered. Sixty to 84 percent of each bone graft was resorbed, effectively leaving the superolateral portion of the cup uncovered. The amount of the surface of the cup that was beyond the Kohler line averaged 41 percent for the six type-I hips, 43 percent for the seven type-II hips, 41 percent for six of the type-III hips, and 44 percent for one of the type-IV hips. Crossing of the ilioischial and iliopubic lines was noted on the radiographs of two type-III and two type-IV hips. Radiographs of two type-I hips and one type-II hip showed 7 to 17 percent of the surface of the dome of the cup through the internal pelvic wall (beyond the iliopubic line). None of the twenty-four metal shells were revised. A reoperation was performed on two hips to exchange a worn polyethylene insert, and three femoral components that had been fixed without cement were revised because of mechanical loosening. Wear averaged 0.26 millimeter per year in the fourteen hips that had a titanium femoral head and 0.09 millimeter per year in the nine hips that had a cobalt-chromium femoral head. The remaining hip had a ceramic femoral head, and the wear rate was 0.09 millimeter per year. The medial protrusio technique is a predictable, reproducible method for obtaining fixation of a porous-coated, hemispherical acetabular component in a dysplastic acetabulum. The technique permits the use of a porous-coated (bone-ingrowth) component; avoids the use of support bone graft and thereby reduces the operative time; facilitates rehabilitation by permitting earlier weight-bearing of the hip; and permits the use of a modular bearing surface, which may allow future exchange of only this surface rather than revision of the entire acetabular component because of excessive wear.

The cleavage of radical anions of substituted α-phenoxyacetophenones, X-C6H4COCH2OPh, IIa−k, has been studied in DMF by voltammetric and coulometric techniques. The standard potentials (E°) for formation of and rate constants, k, for the cleavage of the radical anions were determined using linear sweep voltammetry, LSV, together with digital simulation and previously reported laser flash photolysis data. The rate constants cover a range of almost eight orders of magnitude (0.4 s-1 for X = p-MeCO- to 1.3·107 s-1 for X = p-MeO−). The relative driving forces, ΔΔG°het(RX•-), for the heterolytic cleavage of the radical anions (to give R• + X-) were estimated from thermochemical cycles. A combined plot of log(k) versus ΔΔG°het(RX•-) for the radical anions of IIa−k and of α-aryloxyacetophenones gave a curve with α = 0.5 at high driving forces and α = 1 at low driving forces, where α = ∂Δ /∂ΔG°. The plot was analyzed using a model in which reversible cleavage of the radical anions takes place inside the solvent c...

Introduction In light of increasing patient demands and expectations in TKA, a detailed understanding of bone morphology may be the key to optimizing tibial component performance. Current tibial components in TKA fail to reproduce native human geometry. Accurate measurements and ratios of proximal tibia dimensions are important as components move towards more anatomic designs. Methods A consecutive series of TKAs were performed at one center. Proximal tibial measurements were obtained following proximal tibial resection at the time of TKA. These were compared with demographic parameters including height, weight, and gender. Results 145 consecutive primary TKAs (21 bilateral) in 124 patients (85 females, 60 males) were analyzed for this study. Statistical analysis revealed multiple correlations and trends. The tibial dimensions increase linearly in males and females as tibial component size increases. The AP/ML ratio (aspect ratio) of the proximal tibia is fairly constant over the range of bone sizes for both males and females. There is no correlation between aspect ratio and tibial size, nor are there statistical differences between the aspect ratios in males and females. Tibial measurements and component size are positively correlated with both patient height and weight for males and females, although a stronger correlation exists for male patients. On average, the asymmetry between the lateral and medial condyles increases as a function of tibial size. This asymmetry increases more dramatically in males than females. Conclusions These important clinical observations of tibial morphology can be used in designing component lines that more closely resemble native human anatomy, possibly improving performance. Summary Sentence Proximal tibial morphological measurements and ratios provide the design rationale for advanced, conforming components, possibly improving implant positioning, performance and longevity.

Follicular differentiation can be exhibited by a variety of epidermal lesions. We report an example of panfolliculoma purely confined to the epidermis. The lesion presented as a verrucous plaque on the leg of a 55-year-old man. Histopathologic sections showed an acanthoma with follicular differentiation including areas mimicking the follicular germ, matrix, root sheath and infundibular components.

We propose a novel approach for ICU patient risk stratification by combining the learned “topic” structure of clinical concepts (represented by UMLS codes) extracted from the unstructured nursing notes with physiologic data (from SAPS-I) for hospital mortality prediction. We used Hierarchical Dirichlet Processes (HDP), a non-parametric topic modeling technique, to automatically discover “topics” as shared groups of co-occurring UMLS clinical concepts. We evaluated the potential utility of the inferred topic structure in predicting hospital mortality using the nursing notes of 14,739 adult ICU patients (mortality 14.6%) from the MIMIC II database. Our results indicate that learned topic structure from the first 24-hour ICU nursing notes significantly improved the performance of the SAPS-I algorithm for hospital mortality prediction. The AUC for predicting hospital mortality from the first 24 hours of physiologic data and nursing text notes was 0.82. Using the physiologic data alone with the SAPS-I algorithm, an AUC of 0.72 was achieved. Thus, the clinical topics that were extracted and used to augment the SAPS-I algorithm significantly improved the performance of the baseline algorithm.

Abnormalities of epigenetic regulatory genes including DNMT3A, IDH1, IDH2 and TET2 are common in adults with AML. The prevalence of these abnormalities appears to increase with older age, but their impact in pediatric AML is less certain. The Children's Oncology Group (COG) has previously reported that mutations of DNMT3A, IDH1 and IDH2 are very rare in pediatric patients (Ho et al Leukemia 2010; Ho et al Pediatric Blood and Cancer 2011). In the current study we examined the prevalence and prognostic significance of TET2 gene mutations in a large cohort of pediatric patients with AML. We screened for genomic mutations in the TET2 gene using DNA extracted from diagnostic specimens of 949 pediatric de novo AML patients treated on the COG studies AAML03P1 (N=226) and AAML0531 (N=723). The COG trial AAML03P1 was a phase III pilot study with non-random assignment to gemtuzumab ozogamicin in combination with multi-agent chemotherapy. AAML0531 was a phase III trial with randomization to gemtuzumab ozogamicin. The entire coding sequence of TET2 and was amplified and sequenced. Mutational data was correlated with clinical characteristics and outcome data in both univariable and multivariable analyses. Mutations of the TET2 gene were found in 26 of 949 samples (2.7%). Mutations were found across TET2 gene exons 3-11 from amino acid 121 to 1920. There were 14 missense mutations, 8 nonsense mutations, 2 insertion/deletion (in/del) resulting in frame shift and 2 samples with multiple mutations (1 with missense mutation and in/del frameshift; 1 with nonsense mutation and in/del frameshift). There was no significant difference in the age, gender, race or ethnicity of patients with or without TET2 mutations. Patients with TET2 mutations had a higher prevalence of normal cytogenetics than those without a TET2 mutation (44% vs. 22%, P=0.011). There was also a significant association of TET2 mutations with core binding factor leukemia, although the direction of the association differed between t(8;21) and inv(16). Patients with TET2 mutations compared to those without these mutations were more likely to have t(8;21) (53% vs. 17%, P= 0.021) but less likely to have inv(16) (0% vs. 21%, P=0.039). Molecular mutations currently used in risk stratification of pediatric AML were evaluated. There was no association with FLT3/ITD or mutations of CEBPA, but there was a strong association with NPMc+ which is known to confer a favorable prognosis. TET2 mutant patients had a 32% prevalence of NPMc+ compared to only 7% NPMc+ among TET2 non-mutant (P= Comparing patients with and without TET2 mutations, there was no significant difference in overall survival (OS) (5 yr: 77% vs. 65%, P=0.194), event free survival (EFS) (5 yr: 58% vs. 50%, P=0.411) or relapse risk (40% vs. 37%, P=0.772). We performed multivariable analysis of OS and EFS for TET2 mutation status, risk grouping, NPMc+ status and treatment exposure to gemtuzumab ozogamicin. TET2 mutation status was not predictive of outcome in this multivariable analysis. However, genetic risk group was significantly associated with both OS and EFS and treatment with gemtuzumab ozogamicin was associated only with EFS (HR 0.824 for patients treated with gemtuzumab ozogamicin). Due to the strong association of TET2 mutation and NPMc+, we further evaluated Kaplan Meier estimates of EFS of the 4 groups of patients stratified by both TET2 mutation and NPMc+ status and determined that co-occurrence of TET2 mutation does not modify the clinical significance of NPMc+. (Figure 1) This study demonstrates that TET2 gene mutations are not common events in childhood AML but are highly associated with NPMc+ and may hint at potential cooperation of genetic and epigenetic factors that mediate myeloid leukemogenesis. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures Off Label Use: Arsenic Trioxide in pediatric patients with newly diagnosed APL. Gemtuzumab Ozogamicin in Pediatric AML..

Mutations in the FLT3 gene are among the most common somatic events in AML, with a higher prevalence in adults than in children. The most common activating mutations of FLT3 include internal tandem duplications (FLT3/ITD) in the juxtamembrane domain (JMD) or missense mutations in the tyrosine kinase domain (TKD) at the D835/I836 positions (FLT3/ALM). To date, much of the data on FLT3 mutations has been derived from adult studies and comprehensive sequencing of the FLT3 gene from recent TCGA analysis demonstrated that FLT3 activating mutations were limited to the FLT3/ITD in the JMD and D835/I836 hotspots. As part of the Children's Oncology Group (COG)/NCI TARGET AML initiative, we interrogated the genomic landscape of pediatric AML and identified and verified novel FLT3 activating events that appear to be unique to childhood AML and could provide a target for therapeutic intervention. Whole genome sequencing was performed in a discovery cohort (N=200) followed by validation with targeted exome capture for a total of 799 diagnostic specimens from children treated on contemporary COG trials. In addition to the known FLT3 mutations (FLT3/ITD, N=128 and D835/I836, N=37), we identified novel point mutations (PM; N=49) and novel insertion-deletions (in-dels; N=12). We observed a prevalence of 7.6% of novel PMs and in-dels, in addition to the FLT3/ITD and D835/I836 mutations. The total prevalence of all FLT3 mutations was 28%. In contrast to adult AML, where virtually all non-ITD activating mutations are limited to the D835/I836 region, FLT3PMs in the pediatric cohort occurred in TKD domain (N=44), but commonly occurred in the transmembrane domain (TMD) and JMD. Twelve somatic mutations were identified at distinct positions within the JMD, the region involved in regulation of activity of the kinase. Among the JMD mutations, 9 occur at novel pediatric specific sites with significant activating potential (E573D/G, L576R, T582N, D586Y, Y589H, E596K/G, E598D, Y599C, D600G). Crystal structure analysis of FLT3 variants was used to assess the potential functional significance of the newly discovered variants. This structural modeling indicated that many of the mutations within the TMD (e.g. A680V) and TKD1 (L616R, M664I, M665L) were predicted to cause JMD destabilization, resulting in dysregulated activation of FLT3. Additionally, almost all JMD mutations have the potential to significantly disrupt the auto-inhibitory conformation, resulting in constitutive FLT3 activation. Mutations causing excessive activation of the kinase may have significant oncogenic capacity. In order to assess functional implications of the mutations, we cloned and expressed 6 of the most common novel variants (E573D, L576R, Y599C, D600G, F612L, and A680V) for functional assessment. Of the 6 mutations tested, 5 (E573D, L576R, Y599C, D600G, and A680V) resulted in auto-phosphorylation of FLT3, demonstrating dysregulated and enhanced kinase activation. Acquired mutations following tyrosine kinase inhibitor (TKI) exposure are heavily implicated in resistance and are almost exclusively confined to the two TKD regions, and the D835 position is a hotspot for resistance conferring mutations. It is important to identify patients at diagnosis who harbor dual mutations as this could indicate de novo resistance to TKIs, and exposure to these agents would have no efficacy, but may result in unnecessary toxicity. The presence of dual FLT3/ITD and FLT3/ALM mutations at diagnosis has been reported to occur at a very low prevalence. We analyzed the presence of co-occurring FLT3/ITDand FLT3 PMs in our pediatric cohort. Of the 128 patients with FLT3/ITD, 18 (14%) harbored a secondary FLT3 PM. Mutations in the JMD (N=8) and TMD (N=2) accounted for 56% of co-occurring mutations. Only 22% (N=4) mutations occurred at the D835/I836 sites, with an additional 22% (N=4) located at other TKD sites. Identified TKD diagnostic mutations included F612L, F616R, N676K/S, and N841K, a few which have only been reported in the setting of post TKI relapse. We identified novel pediatric specific FLT3 mutations with significant functional capacity. Importantly, these activating mutations may be uniquely susceptible to FLT3 inhibition and provide a therapeutic target in pediatric AML. Further work is ongoing to completely understand the oncogenic potential of each unique mutation and their prognostic and therapeutic implications. Disclosures No relevant conflicts of interest to declare.

Increasing use of high-throughput sequencing technologies has enabled the identification of novel somatic gene alterations in cancers, with important implications in understanding the mechanisms of transformation and potentially in improving therapeutic decision-making. In contrast to many solid tumors, the mutational spectrum of acute myeloid leukemia (AML) has proven bland, with relatively few recurrent somatic mutations per tumor. This result appears more pronounced in pediatric AML, where even mutation of genes commonly mutated in adult disease, such as IDH1/2 or DNMT3A, appear at much lower frequencies. Alterations of ASXL1, a regulator of the polycomb-repressive complex, have been identified in adults with myelodysplastic syndromes, myeloproliferative syndromes, and AML. Mutations in the related gene family member, ASXL2, were recently associated with t(8;21) AML in adults and children. In this study, we evaluated the prevalence and prognostic impact of mutations of these epigenetic regulator genes in pediatric AML patients treated on COG studies AAML03P1 and AAML0531. ASXL1 and ASXL2 (ASXL1/2) were sequenced from genomic DNA isolated by directed capture of the coding sequences of each gene followed by Illumina paired-end sequencing in a total of 769 diagnostic AML specimens. We identified ASXL1/2 mutations in 44/769 pediatric AML cases (5.7%), with ASXL2 altered more frequently (61% of mutated cases) than ASXL1. Consistent with prior reports, the majority of mutations (80%) were nonsense changes involving exons 11-12 of either gene. ASXL1 and ASXL2 mutations were mutually exclusive, with no case demonstrating concurrent mutation of both genes. In comparison of the clinical covariates of cases with ASXL1/2 mutation against those without, there were no differences in age, gender, or ethnicity; however we found an increased proportion of ASXL1/2 mutations in patients of Asian race, constituting 15% of those with mutated ASXL1/2 vs. 5% of those without (p=0.018). Patients possessing ASXL1/2 mutations tended to present with lower peripheral WBC and marrow blast counts (median 20K vs 30.8K, p=0.067 and 49% vs 70%, p=0.003, respectively). In agreement with recent reports, we identified a striking positive association of ASXL1/2 alterations and t(8;21), which was present in 68% of cases with ASXL1/2 vs 12% of those without (p We found no differences in the clinical outcomes of patients with ASXL1/2 mutations in comparison to those without. Complete remission rates were similar for both groups at the end of induction courses 1 and 2 (84 vs 75%, p=0.2 and 91 vs 89%, p=1.0, respectively). 5-year EFS and OS was 59 ± 15% vs 50 ± 4% (p=0.305) and 65 ± 15% vs. 66 ± 4% (p=0.851). Although prior reports have suggested the possibility of enhanced relapse risk with ASXL1/2 mutations, the 5-year RR for pediatric patients treated in these trials was similar at 31 ± 16% vs 40 ± 4% (p=0.407). Given the co-occurrence of ASXL1/2 mutations and t(8;21), we also evaluated outcomes of t(8;21) patients (n=113) with or without ASXL1/2 mutations, however no significant differences in 5-year OS, EFS, or RR were observed in this subset analysis. This work demonstrate that mutations of ASXL1 or ASXL2 are highly prevalent in childhood t(8;21) AML. Although there was no effect on treatment outcomes, this concomitance suggests the possibility of discrete cooperating lesions, even within the group of CBF-translocated AML, which may be amenable to specifically-directed therapy Disclosures No relevant conflicts of interest to declare.

Genetic alterations in the Nucleoporin (NUP) family of genes are involved in myeloid leukemogenesis and are associated with poor prognosis. We previously showed that NUP98-NSD1 is prevalent in acute myeloid leukemia (AML) and is highly associated with FLT3-ITD and dismal outcome. As genetic alterations in the NUP family are frequently cryptic by conventional karyotyping, their incidence has been underestimated. The COG/NCI TARGET AML initiative has performed comprehensive genome-wide characterization of diagnostic specimens from 200 pediatric AML cases in order to identify novel genetic lesions with prognostic and therapeutic significance. The interrogation of the whole genome and RNA sequencing data generated by this initiative identified numerous fusion transcripts involving the NUP family of genes, including NUP98-NSD1, NUP98-KDM5A, NUP98-HOXA9, NUP98-HMG3, NUP98-HOXD13, NUP98-PHF23, NUP98-BRWD3, CLINT-NUP98 and DEK-NUP214. All computationally identified NUP fusions were verified by orthogonal methodology and high-throughput screening assay was developed for frequency determination. The verified NUP fusions were screened in children treated on COG AAML0531 and AAML03P1 to define their prevalence, clinical characteristics and association with clinical outcome. The impact of NUP fusions was initially evaluated in patients with cytogenetically normal AML (CN-AML). NUP fusions were observed in 14.5% (35 of 242) patients: NUP98-NSD1 (N=21), DEK-NUP214 (N=3), NUP98-HMG3 (N=3), NUP98-HOXD13 (N=2), NUP98-PHF23 (N=2) and NUP98-KDM5A (N=4). The NUP fusions NUP98-BRWD3, NUP98-HOXA9 and CLINT-NUP98 were not found in CN-AML patients. Demographics and disease characteristics of CN-AML patients with and without NUP fusions were compared. Although patients of Asian descent comprised only 7% of the study population, they harbored significantly higher number of NUP fusions (29% vs 5%, P =0.002). Among those of Asian descent with CN-AML, 35% harbored a NUP fusion. We also noted an inverse association between NUP fusions and African-Americans where NUP fusions were not identified in any of African-American patients (P =0.031). NUP fusions were correlated with other common mutations in AML. NPM1 (9% vs 28%, P =0.007) and CEBPA (6% vs 19%, P =0.06) were rare in patients with NUP fusions, whereas FLT3/ITD (62% vs 34%, P =0.002) and WT1 (32% vs 8%, P Given the high co-occurrence of NUP fusions and FLT3-ITD, we investigated the prevalence and clinical correlation of NUP fusions in all FLT3-ITD-positive patients. The prevalence of NUP fusions in FLT3-ITD patients was 26% (43 of 164). The CR rate was lower in patients co-expressing the NUP fusion and FLT3-ITD (40% vs 71%, P In this study we report on the discovery, verification and frequency validation of NUP fusions, a new class of genetic alterations in AML. We demonstrate that NUP fusionsare common in pediatric patients and patients with CN-AML harboring NUP fusions have poor outcome and are more likely to have post-induction MRD than those without thesefusions. Furthermore, there is a high co-occurrence of FLT3-ITD and NUP fusions and patients harboring both genetic lesions have a lower CR rate and high post-induction MRD than those with FLT3-ITD alone. NUP fusions define a new subgroup of pediatric AML patients with an overall poor prognosis. AML harboring NUP fusions likely share similar mechanisms of leukemogenesis and targeting these genetic lesions will likely improve outcome in a significant subset of pediatric AML patients. Disclosures No relevant conflicts of interest to declare.

Activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) are present in ~80% of patients with Chronic Neutrophilic Leukemia (CNL) Despite the high frequency of these mutations in CNL, they are quite rare in adult acute myeloid leukemia (AML), in which only a single CSF3R mutated case was found in the TCGA AML analysis (0.5%). We have previously demonstrated significant variation in genomic variants between pediatric and adult malignancies, thus prevalence of genomic variants identified in adults need be fully characterized in children. As part of COG/NCI TARGET AML initiative we interrogated the genomic makeup of 200 cases of childhood AML (discovery cohort) using whole genome sequencing and performed subsequent frequency determination of the variants in 787 unselected cases from COG AAML0531 (validation cohort). Somatic variants in CSF3R were initially found to be recurrent in the discovery cohort and underwent frequency determination in the validation cohort to establish their prevalence and correlations with clinical characteristics and outcome. Frequency determination of CSF3R mutation in 787 pediatric patients with available CSF3R data from AAML0531 identified 16 distinct CSF3R mutations in 28 patients (3.6%). Somatic mutations in CSF3R identified in pediatric AML included known oncogenic variants mutations such as T618I and T615A, previously identified in adult CNL studies as well as novel truncations of the CSF3R cytoplasmic domain (Q749X, Y767fs, Y787X and P819/820fs), and missense mutations (E149D, A208V, R223Q, E405K, A431V, and Q516K). Interestingly, although CSF3R truncations usually occur along with a T618I or T615A mutation in CNL/aCML, these two mutation categories were mutually exclusive in pediatric AML. Initial correlation of all CSF3R variants with demographic and clinical/laboratory parameters determined that CSF3R variants were less prevalent in younger patients (age 0-2, p=0.039), with significantly higher association with t(8;21) (32% vs. 14%, p=0.012) and CEBPA mutations (35% vs. 5%, p Compared to non-mutated cases, transforming CSF3R variants had a significant association with CEBPA mutations (44% vs. 5%, p CSF3R mutations define a distinct molecular subset of pediatric AML, which could be therapeutically targeted in the future using kinase inhibitors such as ruxolitinib. The oncogenic CSF3R mutations found in pediatric AML are either the same point mutations or similar truncation mutations as seen in CNL, suggesting that other cooperating genomic alterations may be important in directing these distinct diseases. Interestingly, we found that the majority of pediatric AML patients with CSF3R mutation have either a core binding factor alteration (such as t(8;21)) or a mutation in CEBPA. The enrichment of CEBPA mutations with CSF3R mutations is particularly striking, as CEBPA mutations are ~9 fold more frequent in patients with transforming CSF3R mutations than those without. Understanding the role of cooperating genomic alteration in CSF3R-driven myeloid malignancies will be the subject of future work. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures Radich: Novartis: Consultancy, Research Funding; Incyte: Consultancy; Gilliad: Consultancy; Ariad: Consultancy.

The Casitas B-Lineage Lymphoma (CBL) gene encodes for an E3 ubiquitin ligase that targets activated receptor tyrosine kinases for degradation. Mutations of the CBL gene have been described in juvenile myelomonocytic leukemia (JMML) but less is known about mutations and variants of CBL in de novo AML. We previously reported that somatic mutations of CBL are rare in pediatric AML. In this report we present a comprehensive evaluation of genomic and transcript variants of CBL including novel deletion events as well as transcript variants which, in combination with somatic mutations, account for over 6% of pediatric AML with extreme association with inv(16) and favorable outcome. Initial assessment of CBL transcript in a cohort of 100 patients identified previously reported deletion of exon 8 (CBL ΔE8, N=2) associated with CBL splice mutations as well as a novel whole exon 8 and 9 deletion variant (CBL ΔE8+9, N=3) without identifiable underlying somatic alterations. Long distance PCR, as well as custom Nanostring CNV array evaluation revealed a genomic deletion underlying this transcript variant. Subsequent whole genome sequencing as part of COG/NCI TARGET AML initiative, identified discrete genomic deletions of 1998, 3588 and 6189 bp across exon 8 and 9, leading to the generation of this novel variant. We evaluated the functional consequence of the novel CBL ΔE8+9 deletion variant by expressing it in IL3-dependent Ba/F3 cell line. Compared to control cells, Ba/F3 cells expressing CBL ΔE8+9 demonstrated cytokine independent growth. A comprehensive profiling of CBL variants was conducted in 796 pediatric de novo AML patients by transcript profiling (transcript variants) or by exome capture sequencing (somatic mutations including point mutations and smaller indels). All patients were treated on Children's Oncology Group studies AAML03P1 (N=167) and AAML0531 (N=629) and presence of CBL variants was correlated with disease characteristics and clinical outcome. Of the 796 patient specimens tested, 50 patients (6.3%) had one of 3 distinct CBL variants; transcript variant (N=28), somatic mutation (N=14), or dual transcript variant and somatic mutation (N=8). All cases of CBL ΔE8+9 were associated with a corresponding genomic deletion. Out of 14 cases of CBL ΔE8 and 1 case of CBL ΔE9, only 4 cases (27%) had a splice site mutation identified as the underlying mechanism of splice variant. Presence of CBL variants was correlated with clinical characteristics and outcome. Those with CBL variants had a significantly higher prevalence of inv(16) compared with CBL wild type (WT) (37% vs. 13%, p Patients with CBL variants had a 100% clinical remission rate by end of induction II compared to 89% for CBL WT patients (p=0.014). Survival from study entry was similar between CBL mutant vs. WT patients (5 year OS 72% vs. 66%, p=0.24; 5 year EFS 61% vs. 50%, p=0.11). Due to the strong association of CBL mutation with core binding factor leukemia, we assessed whether CBL variant was prognostic of outcome within this favorable risk group, but there was no significant difference in outcomes. Variants of the CBL gene in pediatric AML include genetic mutations with and without whole exon deletions. These CBL variants are highly associated with low risk AML but do not provide independent risk prognosis. The cooperating events of CBL variants in core binding factor leukemia deserve greater study. Our initial analysis of the transcript variants in a cell line model suggest that these large exon 8+9 deletions represent important oncogenic events. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures No relevant conflicts of interest to declare.