Haploinsufficiency underlies the neurodevelopmental consequences of SLC6A1 variants.

Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.

Bacterial genome sequences of uncharacterized Chitinophaga species isolated from the International Space Station.

We report four Chitinophaga sp. strains isolated from wastewater collected onboard the International Space Station. Here, we present three finished and one draft genome. Taxonomic ranks established by genome-based analysis indicate that these Chitinophaga sp. strains represent candidates for a new species.

Fibre-based fluorescence-lifetime imaging microscopy: a real-time biopsy guidance tool for suspected lung cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.

Síndrome de Aarskog: um relato de caso / Aarskog syndrome: a case report

Introdução: A Síndrome de Aarskog-Scott (AAS) é uma rara displasia faciogenital ligada ao gene FGD1, afetando principalmente meninos. Relato de caso: Descreve-se um caso de um menino de 4 anos com AAS, destacando sua importância científica devido à raridade, escassez de descrições e morbidade associada. Ele apresentou fenda sacral, criptorquidia bilateral, atrasos no crescimento e histórico familiar semelhante. A AAS é caracterizada por estatura baixa, anomalias faciais e diversos comprometimentos. Este caso ressalta a importância do acompanhamento médico especializado. Considerações finais: A escassez de estudos comparáveis destaca a relevância dos relatos de casos para aprofundar a compreensão de condições clínicas singulares.

Introduction: Aarskog-Scott Syndrome (AAS) is a rare faciogenital dysplasia linked to the FGD1 gene, primarily affecting boys. Case report: We describe a case of a 4-year-old boy with AAS, highlighting its scientific importance due to its rarity, scarcity of descriptions, and associated morbidity. He presented with sacral cleft, bilateral cryptorchidism, growth delays, and similar family history. AAS is characterized by short stature, facial anomalies, and various impairments. Final considerations: This case underscores the importance of specialized medical care, and the scarcity of comparable studies highlights the relevance of case reports in deepening the understanding of unique clinical conditions.

Virtual histological staining of unlabeled autopsy tissue.

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.

Pulmonary lesions in early response assessment in pediatric Hodgkin lymphoma: prevalence and possible implications for initial staging.

BACKGROUND: Disseminated pulmonary involvement in pediatric Hodgkin lymphoma (pHL) is indicative of Ann Arbor stage IV disease. During staging, it is necessary to assess for coexistence of non-malignant lung lesions due to infection representing background noise to avoid erroneously upstaging with therapy intensification. OBJECTIVE: This study attempts to describe new lung lesions detected on interim staging computed tomography (CT) scans after two cycles of vincristine, etoposide, prednisolone, doxorubicin in a prospective clinical trial. Based on the hypothesis that these new lung lesions are not part of the underlying malignancy but are epiphenomena, the aim is to analyze their size, number, and pattern to help distinguish true lung metastases from benign lung lesions on initial staging. MATERIALS AND METHODS: A retrospective analysis of the EuroNet-PHL-C1 trial re-evaluated the staging and interim lung CT scans of 1,300 pediatric patients with HL. Newly developed lung lesions during chemotherapy were classified according to the current Fleischner glossary of terms for thoracic imaging. Patients with new lung lesions found at early response assessment (ERA) were additionally assessed and compared to response seen in hilar and mediastinal lymph nodes. RESULTS: Of 1,300 patients at ERA, 119 (9.2%) had new pulmonary lesions not originally detectable at diagnosis. The phenomenon occurred regardless of initial lung involvement or whether a patient relapsed. In the latter group, new lung lesions on ERA regressed by the time of relapse staging. New lung lesions on ERA in patients without relapse were detected in 102 (7.8%) patients. Pulmonary nodules were recorded in 72 (5.5%) patients, the majority (97%) being<10 mm. Consolidations, ground-glass opacities, and parenchymal bands were less common. CONCLUSION: New nodules on interim staging are common, mostly measure less than 10 mm in diameter and usually require no further action because they are most likely non-malignant. Since it must be assumed that benign and malignant lung lesions coexist on initial staging, this benign background noise needs to be distinguished from lung metastases to avoid upstaging to stage IV disease. Raising the cut-off size for lung nodules to ≥ 10 mm might achieve the reduction of overtreatment but needs to be further evaluated with survival data. In contrast to the staging criteria of EuroNet-PHL-C1 and C2, our data suggest that the number of lesions present at initial staging may be less important.

Radiomics Correlation to CD68+ Tumor-Associated Macrophages in Clear Cell Renal Cell Carcinoma.

INTRODUCTION: Renal cell carcinoma (RCC) is the ninth most common cancer worldwide, with clear cell RCC (ccRCC) being the most frequent histological subtype. The tumor immune microenvironment (TIME) of ccRCC is an important factor to guide treatment, but current assessments are tissue-based, which can be time-consuming and resource-intensive. In this study, we used radiomics extracted from clinically performed computed tomography (CT) as a noninvasive surrogate for CD68 tumor-associated macrophages (TAMs), a significant component of ccRCC TIME. METHODS: TAM population was measured by CD68+/PanCK+ ratio and tumor-TAM clustering was measured by normalized K function calculated from multiplex immunofluorescence (mIF). A total of 1,076 regions on mIF slides from 78 patients were included. Radiomic features were extracted from multiphase CT of the ccRCC tumor. Statistical machine learning models, including random forest, Adaptive Boosting, and ElasticNet, were used to predict TAM population and tumor-TAM clustering. RESULTS: The best models achieved an area under the ROC curve of 0.81 (95% CI: [0.69, 0.92]) for TAM population and 0.77 (95% CI: [0.66, 0.88]) for tumor-TAM clustering, respectively. CONCLUSION: Our study demonstrates the potential of using CT radiomics-derived imaging markers as a surrogate for assessment of TAM in ccRCC for real-time treatment response monitoring and patient selection for targeted therapies and immunotherapies.

Neurotropism of SARS-CoV-2: A Pathological Examination of Neurosurgical Specimens.

BACKGROUND AND OBJECTIVES: Neurological manifestations may occur in more than 80% of patients hospitalized with COVID-19 infection, including severe disruptions of the central nervous system (CNS), such as strokes, encephalitis, or seizures. Although the primary pathophysiological mechanism for the effects of COVID-19 in CNS remains unknown, evidence exists for both direct injury from neuroinvasion and indirect effects from disruptions in systemic inflammatory and coagulation pathways. In this study, we analyzed CNS tissue from living patients to better understand these processes. METHODS: With institutional review board approval and patient consent, samples that would be otherwise discarded from patients with active or recent (within 6 days of surgery) COVID-19 infection undergoing neurosurgical intervention were collected and tested for the presence of SARS-CoV-2 using immunohistochemistry, in situ hybridization, electron microscopy, and reverse transcription polymerase chain reaction. RESULTS: Five patients with perioperative mild-to-moderate COVID-19 infection met inclusion criteria (2 male, 3 female; mean age 38.8 ± 13.5 years). Neurosurgical diagnoses included a glioblastoma, a ruptured arteriovenous malformation, a ruptured posterior inferior cerebellar artery aneurysm, a middle cerebral artery occlusion, and a hemorrhagic pontine cavernous malformation. Samples analyzed included the frontal lobe cortex, olfactory nerve, arteriovenous malformation/temporal lobe parenchyma, middle cerebral artery, cerebellum, and cavernous malformation/brainstem parenchyma. Testing for the presence of SARS-CoV-2 was negative in all samples. CONCLUSION: The CNS is likely not a significant viral reservoir during mild-to-moderate COVID-19 infection, although direct neuroinvasion is not definitively excluded. Additional testing to help elucidate the relative contributions of direct and indirect pathways for CNS injury from COVID is warranted.

Improved Sample Preparation Method for Protein and Peptide Identification from Human Hair.

A fast and sensitive direct extraction (DE) method developed in our group can efficiently extract proteins in 30 min from a 5 cm-long hair strand. Previously, we coupled DE to downstream analysis using gel electrophoresis followed by in-gel digestion, which can be time-consuming. In searching for a better alternative, we found that a combination of DE with a bead-based method (SP3) can lead to significant improvements in protein discovery in human hair. Since SP3 is designed for general applications, we optimized it to process hair proteins following DE and compared it to several other in-solution digestion methods. Of particular concern are genetically variant peptides (GVPs), which can be used for human identification in forensic analysis. Here, we demonstrated improved GVP discovery with the DE and SP3 workflow, which was 3 times faster than the previous in-gel digestion method and required significantly less instrument time depending on the number of gel slices processed. Additionally, it led to an increased number of identified proteins and GVPs. Among the tested in-solution digestion methods, DE combined with SP3 showed the highest sequence coverage, with higher abundances of the identified peptides. This provides a significantly enhanced means for identifying proteins and GVPs in human hair.

Higher Prevalence of Nonsense Pathogenic DMD Variants in a Single-Center Cohort from Brazil: A Genetic Profile Study That May Guide the Choice of Disease-Modifying Treatments.

Dystrophinopathies are muscle diseases caused by pathogenic variants in DMD, the largest gene described in humans, representing a spectrum of diseases ranging from asymptomatic creatine phosphokinase elevation to severe Duchenne muscular dystrophy (DMD). Several therapeutic strategies are currently in use or under development, each targeting different pathogenic variants. However, little is known about the genetic profiles of northeast Brazilian patients with dystrophinopathies. We describe the spectrum of pathogenic DMD variants in a single center in northeast Brazil. This is an observational, cross-sectional study carried out through molecular-genetic analysis of male patients diagnosed with dystrophinopathies using Multiplex Ligation-dependent Probe Amplification (MLPA) followed by Next-Generation Sequencing (NGS)-based strategies. A total of 94 male patients were evaluated. Deletions (43.6%) and duplications (10.6%) were the most recurring patterns of pathogenic variants. However, small variants were present in 47.1% of patients, most of them nonsense variants (27.6%). This is the largest South American single-center case series of dystrophinopathies to date. We found a higher frequency of treatment-amenable nonsense single-nucleotide variants than most previous studies. These findings may have implications for diagnostic strategies in less-known populations, as a higher frequency of nonsense variants may mean a higher possibility of treating patients with disease-modifying drugs.

A review of pulmonary neutrophilia and insights into the key role of neutrophils in particle-induced pathogenesis in the lung from animal studies of lunar dusts and other poorly soluble dust particles.

The mechanisms of particle-induced pathogenesis in the lung remain poorly understood. Neutrophilic inflammation and oxidative stress in the lung are hallmarks of toxicity. Some investigators have postulated that oxidative stress from particle surface reactive oxygen species (psROS) on the dust produces the toxicopathology in the lungs of dust-exposed animals. This postulate was tested concurrently with the studies to elucidate the toxicity of lunar dust (LD), which is believed to contain psROS due to high-speed micrometeoroid bombardment that fractured and pulverized lunar surface regolith. Results from studies of rats intratracheally instilled (ITI) with three LDs (prepared from an Apollo-14 lunar regolith), which differed 14-fold in levels of psROS, and two toxicity reference dusts (TiO2 and quartz) indicated that psROS had no significant contribution to the dusts' toxicity in the lung. Reported here are results of further investigations by the LD toxicity study team on the toxicological role of oxidants in alveolar neutrophils that were harvested from rats in the 5-dust ITI study and from rats that were exposed to airborne LD for 4 weeks. The oxidants per neutrophils and all neutrophils increased with dose, exposure time and dust's cytotoxicity. The results suggest that alveolar neutrophils play a critical role in particle-induced injury and toxicity in the lung of dust-exposed animals. Based on these results, we propose an adverse outcome pathway (AOP) for particle-associated lung disease that centers on the crucial role of alveolar neutrophil-derived oxidant species. A critical review of the toxicology literature on particle exposure and lung disease further supports a neutrophil-centric mechanism in the pathogenesis of lung disease and may explain previously reported animal species differences in responses to poorly soluble particles. Key findings from the toxicology literature indicate that (1) after exposures to the same dust at the same amount, rats have more alveolar neutrophils than hamsters; hamsters clear more particles from their lungs, consequently contributing to fewer neutrophils and less severe lung lesions; (2) rats exposed to nano-sized TiO2 have more neutrophils and more severe lesions in their lungs than rats exposed to the same mass-concentration of micron-sized TiO2; nano-sized dust has a greater number of particles and a larger total particle-cell contact surface area than the same mass of micron-sized dust, which triggers more alveolar epithelial cells (AECs) to synthesize and release more cytokines that recruit a greater number of neutrophils leading to more severe lesions. Thus, we postulate that, during chronic dust exposure, particle-inflicted AECs persistently release cytokines, which recruit neutrophils and activate them to produce oxidants resulting in a prolonged continuous source of endogenous oxidative stress that leads to lung toxicity. This neutrophil-driven lung pathogenesis explains why dust exposure induces more severe lesions in rats than hamsters; why, on a mass-dose basis, nano-sized dusts are more toxic than the micron-sized dusts; why lung lesions progress with time; and why dose-response curves of particle toxicity exhibit a hockey stick like shape with a threshold. The neutrophil centric AOP for particle-induced lung disease has implications for risk assessment of human exposures to dust particles and environmental particulate matter.

Inferring the Nominal Molecular Mass of an Analyte from Its Electron Ionization Mass Spectrum.

The performance of three algorithms for predicting nominal molecular mass from an analyte's electron ionization mass spectrum is presented. The Peak Interpretation Method (PIM) attempts to quantify the likelihood that a molecular ion peak is contained in the mass spectrum, whereas the Simple Search Hitlist Method (SS-HM) and iterative Hybrid Search Hitlist Method (iHS-HM) leverage results from mass spectral library searching. These predictions can be employed in combination (recommended) or independently. The methods were tested on two sets of query mass spectra searched against libraries that did not contain the reference mass spectra of the same compounds: 19,074 spectra of various organic molecules searched against the NIST17 mass spectral library and 162 spectra of small molecule drugs searched against SWGDRUG version 3.3. Individually, each molecular mass prediction method had computed precisions (the fraction of positive predictions that were correct) of 91, 89, and 74%, respectively. The methods become more valuable when predictions are taken together. When all three predictions were identical, which occurred in 33% of the test cases, the predicted molecular mass was almost always correct (>99%).

On the challenge of unambiguous identification of fentanyl analogs: Exploring measurement diversity using standard reference mass spectral libraries.

Fentanyl analogs are a class of designer drugs that are particularly challenging to unambiguously identify due to the mass spectral and retention time similarities of unique compounds. In this paper, we use agglomerative hierarchical clustering to explore the measurement diversity of fentanyl analogs and better understand the challenge of unambiguous identifications using analytical techniques traditionally available to drug chemists. We consider four measurements in particular: gas chromatography retention indices, electron ionization mass spectra, electrospray ionization tandem mass spectra, and direct analysis in real time mass spectra. Our analysis demonstrates how simultaneously considering data from multiple measurement techniques increases the observable measurement diversity of fentanyl analogs, which can reduce identification ambiguity. This paper further supports the use of multiple analytical techniques to identify fentanyl analogs (among other substances), as is recommended by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG).

Single-Cell Characterization of Pulmonary Nodules Implicates Suppression of Immunosurveillance across Early Stages of Lung Adenocarcinoma.

A greater understanding of molecular, cellular, and immunological changes during the early stages of lung adenocarcinoma development could improve diagnostic and therapeutic approaches in patients with pulmonary nodules at risk for lung cancer. To elucidate the immunopathogenesis of early lung tumorigenesis, we evaluated surgically resected pulmonary nodules representing the spectrum of early lung adenocarcinoma as well as associated normal lung tissues using single-cell RNA sequencing and validated the results by flow cytometry and multiplex immunofluorescence (MIF). Single-cell transcriptomics revealed a significant decrease in gene expression associated with cytolytic activities of tumor-infiltrating natural killer and natural killer T cells. This was accompanied by a reduction in effector T cells and an increase of CD4+ regulatory T cells (Treg) in subsolid nodules. An independent set of resected pulmonary nodules consisting of both adenocarcinomas and associated premalignant lesions corroborated the early increment of Tregs in premalignant lesions compared with the associated normal lung tissues by MIF. Gene expression analysis indicated that cancer-associated alveolar type 2 cells and fibroblasts may contribute to the deregulation of the extracellular matrix, potentially affecting immune infiltration in subsolid nodules through ligand-receptor interactions. These findings suggest that there is a suppression of immune surveillance across the spectrum of early-stage lung adenocarcinoma. SIGNIFICANCE: Analysis of a spectrum of subsolid pulmonary nodules by single-cell RNA sequencing provides insights into the immune regulation and cell-cell interactions in the tumor microenvironment during early lung tumor development.

Differentiation between rebound thymic hyperplasia and thymic relapse after chemotherapy in pediatric Hodgkin lymphoma.

BACKGROUND: Rebound thymic hyperplasia (RTH) is a common phenomenon caused by stress factors such as chemotherapy (CTX) or radiotherapy, with an incidence between 44% and 67.7% in pediatric lymphoma. Misinterpretation of RTH and thymic lymphoma relapse (LR) may lead to unnecessary diagnostic procedures including invasive biopsies or treatment intensification. The aim of this study was to identify parameters that differentiate between RTH and thymic LR in the anterior mediastinum. METHODS: After completion of CTX, we analyzed computed tomographies (CTs) and magnetic resonance images (MRIs) of 291 patients with classical Hodgkin lymphoma (CHL) and adequate imaging available from the European Network for Pediatric Hodgkin lymphoma C1 trial. In all patients with biopsy-proven LR, an additional fluorodeoxyglucose (FDG)-positron emission tomography (PET)-CT was assessed. Structure and morphologic configuration in addition to calcifications and presence of multiple masses in the thymic region and signs of extrathymic LR were evaluated. RESULTS: After CTX, a significant volume increase of new or growing masses in the thymic space occurred in 133 of 291 patients. Without biopsy, only 98 patients could be identified as RTH or LR. No single finding related to thymic regrowth allowed differentiation between RTH and LR. However, the vast majority of cases with thymic LR presented with additional increasing tumor masses (33/34). All RTH patients (64/64) presented with isolated thymic growth. CONCLUSION: Isolated thymic LR is very uncommon. CHL relapse should be suspected when increasing tumor masses are present in distant sites outside of the thymic area. Conversely, if regrowth of lymphoma in other sites can be excluded, isolated thymic mass after CTX likely represents RTH.

NIST Mass Spectrometry Data Center standard reference libraries and software tools: Application to seized drug analysis.

The standard reference libraries and associated custom software provided by the National Institute of Standards and Technology's Mass Spectrometry Data Center (NIST MSDC) are described with a focus on assisting the seized drug analyst with the identification of fentanyl-related substances (FRS). These tools are particularly useful when encountering novel substances when no certified sample is available. The MSDC provides three standard reference mass spectral libraries, as well as six software packages for mass spectral analysis, reference library searching, data interpretation, and measurement uncertainty estimation. Each of these libraries and software packages are described with references to the original publications provided. Examples of fentanyl identification by gas chromatography-mass spectrometry (GC-MS) and by direct analysis in real-time (DART) mass spectrometry are given. A link to online tutorials is provided.

Response-adapted omission of radiotherapy in children and adolescents with early-stage classical Hodgkin lymphoma and an adequate response to vincristine, etoposide, prednisone, and doxorubicin (EuroNet-PHL-C1): a titration study.

BACKGROUND: Children and adolescents with early-stage classical Hodgkin lymphoma have a 5-year event-free survival of 90% or more with vincristine, etoposide, prednisone, and doxorubicin (OEPA) plus radiotherapy, but late complications of treatment affect survival and quality of life. We investigated whether radiotherapy can be omitted in patients with adequate morphological and metabolic responses to OEPA. METHODS: The EuroNet-PHL-C1 trial was designed as a titration study and recruited patients at 186 hospital sites across 16 European countries. Children and adolescents with newly diagnosed stage IA, IB, and IIA classical Hodgkin lymphoma younger than 18 years of age were assigned to treatment group 1 to be treated with two cycles of OEPA (vincristine 1·5 mg/m2 intravenously, capped at 2 mg, on days 1, 8, and 15; etoposide 125 mg/m2 intravenously, on days 1-5; prednisone 60 mg/m2 orally on days 1-15; and doxorubicin 40 mg/m2 intravenously on days 1 and 15). If no adequate response (a partial morphological remission or greater and PET negativity) had been achieved after two cycles of OEPA, involved-field radiotherapy was administered at a total dose of 19·8 Gy (usually in 11 fractions of 1·8 Gy per day). The primary endpoint was event-free survival. The primary objective was maintaining a 5-year event-free survival rate of 90% in patients with an adequate response to OEPA without radiotherapy. We performed intention-to-treat and per-protocol analyses. The trial was registered at ClinicalTrials.gov (NCT00433459) and with EUDRACT, (2006-000995-33) and is completed. FINDINGS: Between Jan 31, 2007, and Jan 30, 2013, 2131 patients were registered and 2102 patients were enrolled onto EuroNet-PHL-C1. Of these 2102 patients, 738 with early-stage disease were allocated to treatment group 1. Median follow-up was 63·3 months (IQR 60·1-69·8). We report on 714 patients assigned to and treated on treatment group 1; the intention-to-treat population comprised 713 patients with 323 (45%) male and 390 (55%) female patients. In 440 of 713 patients in the intention-to-treat group who had an adequate response and did not receive radiotherapy, 5-year event-free survival was 86·5% (95% CI 83·3-89·8), which was less than the 90% target rate. In 273 patients with an inadequate response who received radiotherapy, 5-year event-free survival was 88·6% (95% CI 84·8-92·5), for which the 95% CI included the 90% target rate. The most common grade 3-4 adverse events were neutropenia (in 597 [88%] of 680 patients) and leukopenia (437 [61%] of 712). There were no treatment-related deaths. INTERPRETATION: On the basis of all the evidence, radiotherapy could be omitted in patients with early-stage classical Hodgkin lymphoma and an adequate response to OEPA, but patients with risk factors might need more intensive treatment. FUNDING: Deutsche Krebshilfe, Elternverein für Krebs-und leukämiekranke Kinder, Gießen, Kinderkrebsstiftung Mainz of the Journal Oldtimer Markt, Tour der Hoffnung, Menschen für Kinder, Mitteldeutsche Kinderkrebsforschung, Programme Hospitalier de Recherche Clinique, and Cancer Research UK.