Background The chance of surviving non-small cell lung cancer (NSCLC) is low, due to the challenge in replicating it in preclinical models, limiting the discovery of effective treatments. Most preclinical models rely on two-dimensional (2D) cancer cell cultures. However, they do not reflect the effects of the 3D tumor architecture on cell proliferation, gene expression, and epigenomic regulation. 3D cell culture models, such as spheroids, can improve the therapeutic predictiveness of preclinical models. Yet, the time of 3D culture where 2D cells will retrieve 3D cell features to faithfully replicate NSCLC remain unknown. Methods We developed a protocol enabling long-term (> 24 days) 3D spheroid culture of NSCLC cell lines. We investigated transcriptomic and epigenomic profiles over time. We compared those changes to 2D cultures and to single-cell transcriptomes of NSCLC patient samples. We performed drug screens to compare the therapeutic vulnerabilities of long-term 3D spheroids to cells in 2D and analyzed their plasticity upon return to 2D culture, at the single-cell transcriptomic and pharmacological levels. Results NSCLC cells within spheroids revealed significant time-dependent epigenomic and transcriptomic changes. While DNA methylation remained stable, we observed time-dependent changes in the methylation and acetylation of histones H3 and H4 that recapitulated those observed in NSCLC. Accordingly, single cell transcriptomic profiles after 24 days of 3D culture were significantly more correlated with those of tumor cells from patients with NSCLC. Long-term spheroid culture increased the stringency of drug screening, inducing resistance to compounds that showed promise in many preclinical studies but failed to obtain approval in NSCLC clinical trials ( e.g. , histone deacetylase (HDAC) inhibitors). We demonstrated novel pharmacological vulnerabilities and synergistic interactions ( e.g ., between protein arginine methyltransferase and HDAC inhibitors). Strikingly, reverting long-term 3D spheroids to 2D culture reversed transcriptomic, epigenetic, and pharmacological signatures within 7 days, demonstrating how variations in cell culture conditions can impact outcomes in preclinical research. Conclusions Our findings demonstrate the value of implementing long-term 3D culture, as compared to short-term spheroids and 2D culture to improve preclinical studies. This method could be adapted to other cancer cell lines to improve preclinical drug development and maximize clinical trial results.

Multivariable regression analysis for the Moderate-severe anxiety, whole cohort.

PLINK results for the Moderate-severe depression.

OBJECTIVES:Distributed computations facilitate multi-institutional data analysis while avoiding the costs and complexity of data pooling. Existing approaches lack crucial features, such as built-in medical standards and terminologies, no-code data visualizations, explicit disclosure control mechanisms, and support for basic statistical computations, in addition to gradient-based optimization capabilities. MATERIALS AND METHODS:We describe the development of the Collaborative Data Analysis (CODA) platform, and the design choices undertaken to address the key needs identified during our survey of stakeholders. We use a public dataset (MIMIC-IV) to demonstrate end-to-end multi-modal FL using CODA. We assessed the technical feasibility of deploying the CODA platform at 9 hospitals in Canada, describe implementation challenges, and evaluate its scalability on large patient populations. RESULTS:The CODA platform was designed, developed, and deployed between January 2020 and January 2023. Software code, documentation, and technical documents were released under an open-source license. Multi-modal federated averaging is illustrated using the MIMIC-IV and MIMIC-CXR datasets. To date, 8 out of the 9 participating sites have successfully deployed the platform, with a total enrolment of >1M patients. Mapping data from legacy systems to FHIR was the biggest barrier to implementation. DISCUSSION AND CONCLUSION:The CODA platform was developed and successfully deployed in a public healthcare setting in Canada, with heterogeneous information technology systems and capabilities. Ongoing efforts will use the platform to develop and prospectively validate models for risk assessment, proactive monitoring, and resource usage. Further work will also make tools available to facilitate migration from legacy formats to FHIR and DICOM.

Multivariable regression analysis for the Trail making test, female participants.

Multivariable regression analysis for the Moderate-severe anxiety, male participants.

AbstractBackground: An increased risk of neurocognitive deficits, anxiety, and depression has been reported in childhood cancer survivors. Methods: We analyzed associations of neurocognitive deficits, as well as anxiety and depression, with common and rare genetic variants derived from whole-exome sequencing data of acute lymphoblastic leukemia (ALL) survivors from the PETALE cohort. In addition, significant associations were assessed using stratified and multivariable analyses. Next, top-ranking common associations were analyzed in an independent SJLIFE replication cohort of ALL survivors. Results: Significant associations were identified in the entire discovery cohort (N = 229) between the AK8 gene and changes in neurocognitive function, whereas PTPRZ1, MUC16, TNRC6C-AS1 were associated with anxiety. Following stratification according to sex, the ZNF382 gene was linked to a neurocognitive deficit in males, whereas APOL2 and C6orf165 were associated with anxiety and EXO5 with depression. Following stratification according to prognostic risk groups, the modulatory effect of rare variants on depression was additionally found in the CYP2W1 and PCMTD1 genes. In the replication SJLIFE cohort (N = 688), the male-specific association in the ZNF382 gene was not significant; however, a P value<0.05 was observed when the entire SJLIFE cohort was analyzed. ZNF382 was significant in males in the combined cohorts as shown by meta-analyses as well as the depression-associated gene EXO5. Conclusions: Further research is needed to confirm whether the current findings, along with other known risk factors, may be valuable in identifying patients at increased risk of these long-term complications. Impact: Our results suggest that specific genes may be related to increased neuropsychological consequences.

Manhattan plot for the Trail making test

Background: Venous thromboembolism (VTE) is a frequent complication of childhood acute lymphoblastic leukemia (ALL). Objectives: We aimed to identify molecular markers and signatures of the leukemia microenvironment associated with VTE in childhood ALL by the dual-omics approach of gene expression and DNA methylation profiling. Methods: Eligible children aged 1 to 21 years old with newly diagnosed ALL were enrolled in the Dana-Farber Cancer Institute 16-001 trial with available RNA sequencing data from bone marrow at diagnosis. The primary outcome was VTE requiring medical intervention, divided between early events (ETs), within 6 weeks from ALL diagnosis, or late events otherwise. We compared differential gene expression and DNA methylation in children with and without VTE and in the subgroup of children with ETs. The DNA methylation cis-regulation was explored by dual-omics integration. Functional gene set enrichment analyses were performed to assess dysregulated pathways associated with thrombosis. Gene expression profiling-based signature for the thrombosis-free interval was determined using the Kaplan-Meier estimator and log-rank tests. Results: We included 248 patients (median age, 7.5 years; 78% precursor B-cell ALL), of whom 56 (23%) developed VTE. Genes and metabolic pathways involved in coagulation, platelet activation, and neutrophil extracellular trap formation were associated with ETs. Dual-omics analysis indicated that methylation reprogramming might be responsible for the overexpression of genes involved in neutrophil extracellular trap formation and coagulation in patients with ETs. A prothrombotic gene signature, based on VWF, PF4, and CXCL8 expression, predicted a thrombosis-free interval. Conclusion: This suggests that gene markers and epigenetic regulation of the leukemic microenvironment are drivers of VTE, notably ETs in childhood ALL.

BACKGROUND:Alterations of FLT3 are among the most common driver events in acute leukaemia with important clinical implications, since it allows patient classification into prognostic groups and the possibility of personalising therapy thanks to the availability of FLT3 inhibitors. Most of the knowledge on FLT3 implications comes from the study of acute myeloid leukaemia and so far, few studies have been performed in other leukaemias. METHODS:A comprehensive genomic (DNA-seq in 267 patients) and transcriptomic (RNA-seq in 160 patients) analysis of FLT3 in 342 childhood acute lymphoblastic leukaemia (ALL) patients was performed. Mutations were functionally characterised by in vitro experiments. RESULTS:Point mutations (PM) and internal tandem duplications (ITD) were detected in 4.3% and 2.7% of the patients, respectively. A new activating mutation of the TKD, G846D, conferred oncogenic properties and sorafenib resistance. Moreover, a novel alteration involving the circularisation of read-through transcripts (rt-circRNAs) was observed in 10% of the cases. Patients presenting FLT3 alterations exhibited higher levels of the receptor. In addition, patients with ZNF384- and MLL/KMT2A-rearranged ALL, as well as hyperdiploid subtype, overexpressed FLT3. DISCUSSION:Our results suggest that specific ALL subgroups may also benefit from a deeper understanding of the biology of FLT3 alterations and their clinical implications.

INTRODUCTION:Pregnancy complications, including preeclampsia (PE), preterm birth (PTB), and intra-uterine growth restriction (IUGR) have individually been associated with inflammation but the combined comparative analysis of their placental profiles at the transcriptomic and histological levels is lacking. METHODS:Bulk RNA-sequencing of human placental biopsies from uncomplicated term pregnancies (CTL) and pregnancies complicated with early-onset (EO), and late-onset (LO) PE, as well as PTB and term IUGR were used to characterize individual molecular profiles. We also applied immune-cell-specific cellular deconvolution to address local immune cell compositions and analyzed placental lesions by histology to further characterize these complications. RESULTS:Transcriptome analysis revealed that clinically distinct complications differentiated themselves in unique ways compared to CTLs. Only TMEM136 was commonly modulated. Compared to CTLs, we found that PTB and IUGR were the most distinct, with LOPE being the least distinct. PTB and IUGR revealed differently enhanced inflammatory pathways, where PTB had general inflammatory responses and IUGR had immune cell activation. This inflammation was reflected in the histological profile for PTB only, whereas structural lesions were elevated in all complications. Placental lesions additionally had corresponding enhancement in inflammatory and structural biological processes. We observed that having co-complications, particularly for PTB with or without IUGR, impacted placental transcriptomes. Lastly, cellular deconvolution uncovered shared immune features among the complications. DISCUSSION:Overall, we provide evidence that these pregnancy complications are not only distinct in their clinical manifestations but also in their placental profiles, which could be leveraged to understand their underlying mechanisms and could offer therapeutic targets.

Multivariable regression analysis for the Trail making test, male participants.

Functional description of associations.

Additional analyses from the SJLIFE Cohort.

Childhood B-cell acute lymphoblastic leukemia (B-ALL) is a heterogeneous disease comprising multiple molecular subgroups with subtype-specific expression profiles. Recently, a new type of ncRNA, termed circular RNA (circRNA), has emerged as a promising biomarker in cancer, but little is known about their role in childhood B-ALL. Here, through RNA-seq analysis in 105 childhood B-ALL patients comprising six genetic subtypes and seven B-cell controls from two independent cohorts we demonstrated that circRNAs properly stratified B-ALL subtypes. By differential expression analysis of each subtype vs. controls, 156 overexpressed and 134 underexpressed circRNAs were identified consistently in at least one subtype, most of them with subtype-specific expression. TCF3::PBX1 subtype was the one with the highest number of unique and overexpressed circRNAs, and the circRNA signature could effectively discriminate new patients with TCF3::PBX1 subtype from others. Our results indicated that NUDT21, an RNA-binding protein (RBP) involved in circRNA biogenesis, may contribute to this circRNA enrichment in TCF3::PBX1 ALL. Further functional characterization using the CRISPR-Cas13d system demonstrated that circBARD1, overexpressed in TCF3::PBX1 patients and regulated by NUDT21, might be involved in leukemogenesis through the activation of p38 via hsa-miR-153-5p. Our results suggest that circRNAs could play a role in the pathogenesis of childhood B-ALL.

PLINK results for the Moderate-severe anxiety.

Combined effect model of the APOL2 and C6orf165 genes

Combined effect model of the PTPRZ1, MUC16 and TNRC6C-AS1 genes