The complete sequence of a human Y chromosome.

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4,5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY, DAZ and RBMY; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

DECIPHER: Improving Genetic Diagnosis Through Dynamic Integration of Genomic and Clinical Data.

DECIPHER (Database of Genomic Variation and Phenotype in Humans Using Ensembl Resources) shares candidate diagnostic variants and phenotypic data from patients with genetic disorders to facilitate research and improve the diagnosis, management, and therapy of rare diseases. The platform sits at the boundary between genomic research and the clinical community. DECIPHER aims to ensure that the most up-to-date data are made rapidly available within its interpretation interfaces to improve clinical care. Newly integrated cardiac case-control data that provide evidence of gene-disease associations and inform variant interpretation exemplify this mission. New research resources are presented in a format optimized for use by a broad range of professionals supporting the delivery of genomic medicine. The interfaces within DECIPHER integrate and contextualize variant and phenotypic data, helping to determine a robust clinico-molecular diagnosis for rare-disease patients, which combines both variant classification and clinical fit. DECIPHER supports discovery research, connecting individuals within the rare-disease community to pursue hypothesis-driven research.

Storage trends, usage and disposition outcomes following egg freezing.

RESEARCH QUESTION: What happens to eggs after egg freezing? DESIGN: A retrospective cohort study was performed spanning 2012-2022. Data were obtained from seven assisted reproductive technology clinics in Victoria, Australia. Aggregated, de-identified data were collected on cycles that resulted in egg freezing and the following outcomes, including treatment involving thawed eggs and disposition outcomes of surplus eggs. RESULTS: The number of patients with eggs in storage grew rapidly from 144 in 2012 to 2015 in 2022. In 2022, 73% of patients had stored their eggs for <5 years, 25% for 5-10 years, and 2% for ≥10 years. Most thaw cycles (600/645, 93%) involved eggs that had been frozen for <5 years, of which 47% had been frozen for <6 months. Overall, the live birth rate per initiated thaw cycle was 12%. Across the study period, 2800 eggs from 286 patients were either discarded, donated or exported. Of the 128 patients who discarded their eggs, 32% had stored their eggs for <5 years, 32% for 5-10 years and 36% for >10 years. Of the 23 patients who donated their eggs to someone else, all but four had stored their eggs for <5 years. No eggs were donated to research over the study period. CONCLUSIONS: This study shows that very few patients have made the decision to use or relinquish their eggs. Strategies may be needed to address the prolonged storage of surplus eggs, and ensure that patients are supported to make decisions regarding the fate of their eggs which align with their preferences and values.

Strong and reliable synaptic communication between pyramidal neurons in adult human cerebral cortex.

Synaptic transmission constitutes the primary mode of communication between neurons. It is extensively studied in rodent but not human neocortex. We characterized synaptic transmission between pyramidal neurons in layers 2 and 3 using neurosurgically resected human middle temporal gyrus (MTG, Brodmann area 21), which is part of the distributed language circuitry. We find that local connectivity is comparable with mouse layer 2/3 connections in the anatomical homologue (temporal association area), but synaptic connections in human are 3-fold stronger and more reliable (0% vs 25% failure rates, respectively). We developed a theoretical approach to quantify properties of spinous synapses showing that synaptic conductance and voltage change in human dendritic spines are 3-4-folds larger compared with mouse, leading to significant NMDA receptor activation in human unitary connections. This model prediction was validated experimentally by showing that NMDA receptor activation increases the amplitude and prolongs decay of unitary excitatory postsynaptic potentials in human but not in mouse connections. Since NMDA-dependent recurrent excitation facilitates persistent activity (supporting working memory), our data uncovers cortical microcircuit properties in human that may contribute to language processing in MTG.

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

The European Variation Archive: a FAIR resource of genomic variation for all species.

The European Variation Archive (EVA; https://www.ebi.ac.uk/eva/) is a resource for sharing all types of genetic variation data (SNPs, indels, and structural variants) for all species. The EVA was created in 2014 to provide FAIR access to genetic variation data and has since grown to be a primary resource for genomic variants hosting >3 billion records. The EVA and dbSNP have established a compatible global system to assign unique identifiers to all submitted genetic variants. The EVA is active within the Global Alliance of Genomics and Health (GA4GH), maintaining, contributing and implementing standards such as VCF, Refget and Variant Representation Specification (VRS). In this article, we describe the submission and permanent accessioning services along with the different ways the data can be retrieved by the scientific community.

The Ensembl COVID-19 resource: ongoing integration of public SARS-CoV-2 data.

The COVID-19 pandemic has seen unprecedented use of SARS-CoV-2 genome sequencing for epidemiological tracking and identification of emerging variants. Understanding the potential impact of these variants on the infectivity of the virus and the efficacy of emerging therapeutics and vaccines has become a cornerstone of the fight against the disease. To support the maximal use of genomic information for SARS-CoV-2 research, we launched the Ensembl COVID-19 browser; the first virus to be encompassed within the Ensembl platform. This resource incorporates a new Ensembl gene set, multiple variant sets, and annotation from several relevant resources aligned to the reference SARS-CoV-2 assembly. Since the first release in May 2020, the content has been regularly updated using our new rapid release workflow, and tools such as the Ensembl Variant Effect Predictor have been integrated. The Ensembl COVID-19 browser is freely available at https://covid-19.ensembl.org.

Can serum progesterone concentration direct a fresh or freeze-all transfer strategy in the first in vitro fertilisation cycle?

PURPOSE: To examine the interaction between serum progesterone concentration on the trigger day and choice of freeze-all and fresh transfer strategies on live birth in an unselected population as well as in patients over 35 years old. METHODS: We performed a retrospective cohort study of 26,661 patients commencing their first IVF cycle in a large fertility centre between 2015 and 2019, including 4687 patients over 35 years old. We performed a multivariable fractional polynomial interaction analysis within a logistic regression model to investigate the interaction between serum progesterone concentration and the choice of freeze-all or fresh transfer strategy following the first transfer. RESULTS: 15,539 patients underwent a fresh embryo transfer and 11,122 underwent a freeze-all strategy in their first IVF cycle. The freeze-all group had a higher live birth rate compared to the fresh group (43.9% vs 40.3%). After adjusting for confounding factors, there was a positive interaction between serum progesterone concentrations and the choice of a freeze-all versus fresh embryo transfer on live birth (p for interaction 0.0001), with a larger magnitude of effect when progesterone concentration was higher. Such an interaction was also observed in patients over 35 years old (p for interaction 0.01), but the treatment effect curve over progesterone concentrations was almost flat. CONCLUSIONS: In an unselected population, frozen transfer is associated with greater chances of live birth, especially in patients with higher serum progesterone concentration. In patients over 35 years old, the benefit of a freeze-all policy appears small across all serum progesterone concentrations.

SUsPECT: a pipeline for variant effect prediction based on custom long-read transcriptomes for improved clinical variant annotation.

Our incomplete knowledge of the human transcriptome impairs the detection of disease-causing variants, in particular if they affect transcripts only expressed under certain conditions. These transcripts are often lacking from reference transcript sets, such as Ensembl/GENCODE and RefSeq, and could be relevant for establishing genetic diagnoses. We present SUsPECT (Solving Unsolved Patient Exomes/gEnomes using Custom Transcriptomes), a pipeline based on the Ensembl Variant Effect Predictor (VEP) to predict variant impact on custom transcript sets, such as those generated by long-read RNA-sequencing, for downstream prioritization. Our pipeline predicts the functional consequence and likely deleteriousness scores for missense variants in the context of novel open reading frames predicted from any transcriptome. We demonstrate the utility of SUsPECT by uncovering potential mutational mechanisms of pathogenic variants in ClinVar that are not predicted to be pathogenic using the reference transcript annotation. In further support of SUsPECT's utility, we identified an enrichment of immune-related variants predicted to have a more severe molecular consequence when annotating with a newly generated transcriptome from stimulated immune cells instead of the reference transcriptome. Our pipeline outputs crucial information for further prioritization of potentially disease-causing variants for any disease and will become increasingly useful as more long-read RNA sequencing datasets become available.

High anti-Müllerian hormone (AMH) is associated with increased risks of ectopic pregnancy in women undergoing fresh embryo transfer cycle, a cohort study.

BACKGROUND: Ectopic pregnancy is more common amongst assisted reproduction cycles and is a cause of significant maternal morbidity. Few predictive markers exist to help identify and modify risk of ectopic pregnancy in preparing for embryo transfer. The relationship between serum and AMH and ectopic pregnancy rate is unknown. METHODS: This was a retrospective cohort study investigating women who underwent fresh embryo transfer cycles from January 2017 to December 2019 in Peking University Third Hospital. The primary outcome was ectopic pregnancy. Restricted cubic splines with four knots for AMH concentration (0-3, 3-6, 6-12, 12-max) were used to map out the non-linear relationship between the predicted ectopic pregnancy rate and the serum AMH concentration. Log binomial regression was used to test the crude risk ratio (cRR) and the adjusted risk ratio (aRR) after adjustment for confounders with 95% confidence intervals (CI) to determine the difference across various groups. RESULTS: A total of 13,718 cycles in women undergoing fresh embryo transfer were eligible for analysis. The ectopic pregnancy rate was 1.3% per embryo transfer cycle initiated and 3.3% per pregnancy. Serum AMH concentrations were higher amongst women with ectopic pregnancy than in women with a confirmed intrauterine pregnancy or heterotopic pregnancy or who did not become pregnant (Mean levels: 4.0 ng/ml vs 3.2 ng/ml, 1.7 ng/ml, and 2.8 ng/ml). An AMH concentration of 7 ng/ml represented the best cut-off value to predict ectopic pregnancy. The ectopic pregnancy rate was 3.4% per cycle and 7.5% per pregnancy in women with AMH levels ≥ 7 ng/ml; and 1.2% per cycle and 2.9% per pregnancy in women with AMH levels < 7 ng/ml. Serum AMH concentration ≥ 7 ng/ml was associated with an increased risk of ectopic pregnancy in all fresh embryo transfer cycles started (aRR = 2.35 (1.45, 3.58)) as well in women who became pregnant (aRR = 2.23 (1.49, 3.33). CONCLUSIONS: Baseline AMH concentration ≥ 7 ng/ml is associated with an increased risk of ectopic pregnancy in fresh embryo transfer cycles.

Cellular Substrates of Functional Network Integration and Memory in Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) patients are at risk of memory deficits, which have been linked to functional network disturbances, particularly of integration of the default mode network (DMN). However, the cellular substrates of functional network integration are unknown. We leverage a unique cross-scale dataset of drug-resistant TLE patients (n = 31), who underwent pseudo resting-state functional magnetic resonance imaging (fMRI), resting-state magnetoencephalography (MEG) and/or neuropsychological testing before neurosurgery. fMRI and MEG underwent atlas-based connectivity analyses. Functional network centrality of the lateral middle temporal gyrus, part of the DMN, was used as a measure of local network integration. Subsequently, non-pathological cortical tissue from this region was used for single cell morphological and electrophysiological patch-clamp analysis, assessing integration in terms of total dendritic length and action potential rise speed. As could be hypothesized, greater network centrality related to better memory performance. Moreover, greater network centrality correlated with more integrative properties at the cellular level across patients. We conclude that individual differences in cognitively relevant functional network integration of a DMN region are mirrored by differences in cellular integrative properties of this region in TLE patients. These findings connect previously separate scales of investigation, increasing translational insight into focal pathology and large-scale network disturbances in TLE.

ABCDEFGHI Systematic Approach to wound assessment and management.

ABSTRACT: The ABCDEFGHI approach introduces a systematic approach to wound care. It instructs the clinician to Ask pertinent questions, including those that may identify local and systemic Barriers to wound healing. After obtaining a thorough history, the clinician may proceed to Clean the wound and Do a physical examination, specifically looking for Exposed structures and Factors that will complicate the healing process. Good Healing strategies involving various dressings can then be implemented to promote healing. If necessary, a referral can be made to Involve specialists using various referral pathways. Information used to synthesize this approach was obtained through a review of national and international guidelines and Google Scholar, MEDLINE, and PubMed databases. The ABCDEFGHI approach to wound assessment and management is a simple and easy-to-follow guide that can be easily implemented into practice, thereby improving clinician confidence and competence in wound care.

DECIPHER: Supporting the interpretation and sharing of rare disease phenotype-linked variant data to advance diagnosis and research.

DECIPHER (https://www.deciphergenomics.org) is a free web platform for sharing anonymized phenotype-linked variant data from rare disease patients. Its dynamic interpretation interfaces contextualize genomic and phenotypic data to enable more informed variant interpretation, incorporating international standards for variant classification. DECIPHER supports almost all types of germline and mosaic variation in the nuclear and mitochondrial genome: sequence variants, short tandem repeats, copy-number variants, and large structural variants. Patient phenotypes are deposited using Human Phenotype Ontology (HPO) terms, supplemented by quantitative data, which is aggregated to derive gene-specific phenotypic summaries. It hosts data from >250 projects from ~40 countries, openly sharing >40,000 patient records containing >51,000 variants and >172,000 phenotype terms. The rich phenotype-linked variant data in DECIPHER drives rare disease research and diagnosis by enabling patient matching within DECIPHER and with other resources, and has been cited in >2,600 publications. In this study, we describe the types of data deposited to DECIPHER, the variant interpretation tools, and patient matching interfaces which make DECIPHER an invaluable rare disease resource.

Annotating and prioritizing genomic variants using the Ensembl Variant Effect Predictor-A tutorial.

The Ensembl Variant Effect Predictor (VEP) is a freely available, open-source tool for the annotation and filtering of genomic variants. It predicts variant molecular consequences using the Ensembl/GENCODE or RefSeq gene sets. It also reports phenotype associations from databases such as ClinVar, allele frequencies from studies including gnomAD, and predictions of deleteriousness from tools such as Sorting Intolerant From Tolerant and Combined Annotation Dependent Depletion. Ensembl VEP includes filtering options to customize variant prioritization. It is well supported and updated roughly quarterly to incorporate the latest gene, variant, and phenotype association information. Ensembl VEP analysis can be performed using a highly configurable, extensible command-line tool, a Representational State Transfer application programming interface, and a user-friendly web interface. These access methods are designed to suit different levels of bioinformatics experience and meet different needs in terms of data size, visualization, and flexibility. In this tutorial, we will describe performing variant annotation using the Ensembl VEP web tool, which enables sophisticated analysis through a simple interface.

Ensembl Genomes 2020-enabling non-vertebrate genomic research.

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the context of the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of interfaces to genomic data across the tree of life, including reference genome sequence, gene models, transcriptional data, genetic variation and comparative analysis. Data may be accessed via our website, online tools platform and programmatic interfaces, with updates made four times per year (in synchrony with Ensembl). Here, we provide an overview of Ensembl Genomes, with a focus on recent developments. These include the continued growth, more robust and reproducible sets of orthologues and paralogues, and enriched views of gene expression and gene function in plants. Finally, we report on our continued deeper integration with the Ensembl project, which forms a key part of our future strategy for dealing with the increasing quantity of available genome-scale data across the tree of life.

ABCDEFGHI Systematic Approach to Wound Assessment and Management.

GENERAL PURPOSE: The purpose of this continuing education article is to introduce the ABCDEFGHI approach to wound assessment and management. TARGET AUDIENCE: This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. LEARNING OBJECTIVES/OUTCOMES: After participating in this educational activity, the participant will:1. Distinguish barriers to wound healing.2. Summarize best practices for wound treatment strategies.3. Explain the usefulness of complete blood count results during a wound investigation.4. Identify a major factor to consider when deciding on a referral for wound management.

The ABCDEFGHI approach introduces a systematic approach to wound care. It instructs the clinician to Ask pertinent questions, including those that may identify local and systemic B arriers to wound healing. After obtaining a thorough history, the clinician may proceed to C lean the wound and D o a physical examination, specifically looking for E xposed structures and F actors that will complicate the healing process. G ood H ealing strategies involving various dressings can then be implemented to promote healing. If necessary, a referral can be made to I nvolve specialists using various referral pathways. Information used to synthesize this approach was obtained through a review of national and international guidelines and Google Scholar, MEDLINE, and PubMed databases. The ABCDEFGHI approach to wound assessment and management is a simple and easy-to-follow guide that can be easily implemented into practice, thereby improving clinician confidence and competence in wound care.

GnRH agonist and hCG (dual trigger) versus hCG trigger for follicular maturation: a systematic review and meta-analysis of randomized trials.

BACKGROUND: Traditionally, final follicular maturation is triggered by a single bolus of human chorionic gonadotropin (hCG). This acts as a surrogate to the naturally occurring luteinizing hormone (LH) surge to induce luteinization of the granulosa cells, resumption of meiosis and final oocyte maturation. More recently, a bolus of gonadotropin-releasing hormone (GnRH) agonist in combination with hCG (dual trigger) has been suggested as an alternative regimen to achieve final follicular maturation. METHODS: This study was a systematic review and meta-analysis of randomized trials evaluating the effect of dual trigger versus hCG trigger for follicular maturation on pregnancy outcomes in women undergoing in vitro fertilization (IVF). The primary outcome was the live birth rate (LBR) per started cycle. RESULTS: A total of 1048 participants were included in the analysis, with 519 in the dual trigger group and 529 in the hCG trigger group. Dual trigger treatment was associated with a significantly higher LBR per started cycle compared with the hCG trigger treatment (risk ratio (RR) = 1.37 [1.07, 1.76], I2 = 0%, moderate evidence). There was a trend towards an increase in both ongoing pregnancy rate (RR = 1.34 [0.96, 1.89], I2 = 0%, low evidence) and implantation rate (RR = 1.31 [0.90, 1.91], I2 = 76%, low evidence) with dual trigger treatment compared with hCG trigger treatment. Dual trigger treatment was associated with a significant increase in clinical pregnancy rate (RR = 1.29 [1.10, 1.52], I2 = 13%, low evidence), number of oocytes collected (mean difference (MD) = 1.52 [0.59, 2.46), I2 = 53%, low evidence), number of mature oocytes collected (MD = 1.01 [0.43, 1.58], I2 = 18%, low evidence), number of fertilized oocytes (MD = 0.73 [0.16, 1.30], I2 = 7%, low evidence) and significantly more usable embryos (MD = 0.90 [0.42, 1.38], I2 = 0%, low evidence). CONCLUSION: Dual trigger treatment with GnRH agonist and HCG is associated with an increased live birth rate compared with conventional hCG trigger. TRIAL REGISTRATION: CRD42020204452 .

Somatostatin neurons in the central amygdala mediate anxiety by disinhibition of the central sublenticular extended amygdala.

Fear and anxiety are two defensive emotional states evoked by threats in the environment. Fear can be initiated by either imminent or future threats, but experimentally, it is typically studied as a phasic response initiated by imminent danger that subsides when the threats is removed. In contrast, anxiety is a sustained response, initiated by imagined or potential threats. The central amygdala (CeA) is a key structure active during both fear and anxiety but thought to engage different neural systems. Fear responses are triggered by activation of somatostatin (SOM) expressing neurons in the lateral division of the CeA (CeL), and downstream projections from the medial division. Anxiety responses engage the central extended amygdala that includes the CeA, central sublenticular extended amygdala (SLEAc) and bed nucleus of the stria terminalis, but the nature of connections between these regions is not understood. Here using a combination of tract tracing, electrophysiology, and behavioral analysis in mice, we show that a population of SOM+ neurons in the CeL project to the SLEAc where they inhibit local GABAergic interneurons. Optogenetic activation of this input to the SLEAc has no effect on movement, but is anxiogenic in both open field and elevated plus maze. Our results define the inhibitory connections between CeL and SLEAc and establish a specific CeL to SLEAc projection as a circuit element in mediating anxiety.

Endometrial thickness in the prediction of neonatal adverse outcomes in frozen cycles for singleton pregnancies.

RESEARCH QUESTION: Does endometrial thickness (EMT) predict adverse neonatal outcomes in singleton pregnancies after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) frozen embryo transfer (FET)? DESIGN: This retrospective study involved 13,383 women undergoing IVF/ICSI FET cycles between January 2010 and December 2018 in Women's Hospital of Zhejiang University. The primary outcome was preterm delivery (PTD). The secondary outcomes were small for gestational age (SGA), large for gestational age (LGA) and low birthweight (LBW). RESULTS: A total of 13,383 FET cycles resulting in 5220 singleton live births and 8163 failed cycles were included. Multiple spline regression visualization showed an increasing risk of PTD and LBW for a thin EMT. By comparing multiple cut-off points using area under the curve, a cut-off point of 8 mm was identified, which was used to categorize EMT. A reference point of EMT greater than 8 mm was used; after adjusting for covariates, individuals with EMT less than 8 mm had an adjusted odds ratio of 1.75 (95% CI 1.30 to 2.34) for PTD, 1.57 (95% CI 1.09 to 2.26) for LBW, 0.97 (95% CI 0.63 to 1.50) for SGA and 1.04 (95% CI 0.79 to 1.37) for LGA. Additional analyses showed similar increasing risk with a thin endometrium for both PTD with and without caesarean section, and PTD with low and normal birthweight percentiles. CONCLUSION: A clinical cut-off point of 8 mm has been identified, below which risk of PTD and LBW increases in women undergoing IVF/ICSI.

Identification of prognostic factors in childhood T-cell acute lymphoblastic leukemia: Results from DFCI ALL Consortium Protocols 05-001 and 11-001.

BACKGROUND/OBJECTIVES: While outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) are favorable, there are few widely accepted prognostic factors, limiting the ability to risk stratify therapy. DESIGN/METHODS: Dana-Farber Cancer Institute (DFCI) Protocols 05-001 and 11-001 enrolled pediatric patients with newly diagnosed B- or T-ALL from 2005 to 2011 and from 2012 to 2015, respectively. Protocol therapy was nearly identical for patients with T-ALL (N = 123), who were all initially assigned to the high-risk arm. End-induction minimal residual disease (MRD) was assessed by reverse transcription polymerase chain reaction (RT-PCR) or next-generation sequencing (NGS), but was not used to modify postinduction therapy. Early T-cell precursor (ETP) status was determined by flow cytometry. Cases with sufficient diagnostic DNA were retrospectively evaluated by targeted NGS of known genetic drivers of T-ALL, including Notch, PI3K, and Ras pathway genes. RESULTS: The 5-year event-free survival (EFS) and overall survival (OS) for patients with T-ALL was 81% (95% CI, 73-87%) and 90% (95% CI, 83-94%), respectively. ETP phenotype was associated with failure to achieve complete remission, but not with inferior OS. Low end-induction MRD (<10-4 ) was associated with superior disease-free survival (DFS). Pathogenic mutations of the PI3K pathway were mutually exclusive of ETP phenotype and were associated with inferior 5-year DFS and OS. CONCLUSIONS: Together, our findings demonstrate that ETP phenotype, end-induction MRD, and PI3K pathway mutation status are prognostically relevant in pediatric T-ALL and should be considered for risk classification in future trials. DFCI Protocols 05-001 and 11-001 are registered at www.clinicaltrials.gov as NCT00165087 and NCT01574274, respectively.