Citations

Abstract Introduction: Single-cell RNA-sequencing (scRNA-seq) pairing with single-cell T-Cell-Receptor-sequencing (scTCR-seq) enables profiling of gene expression and TCR repertoires in individual T cells, elevating our understanding of T-cell-mediated immunity. Here, we present immunopipe, a comprehensive and flexible pipeline (https://github.com/pwwang/immunopipe) for analyzing the paired data. Besides the command-line tool, it offers a user-friendly web interface that allows users with varying programming expertise to configure, initiate, and monitor the pipeline. By combining extensive functionality with ease of use, immunopipe empowers users to unlock valuable insights from the data, which notably advances our understanding of immune response and fosters the development of innovative immunotherapies. Results: Immunopipe is a comprehensive analytical pipeline for scRNA-seq and scTCR-seq data, offering various modules leveraging existing tools and novel methods to ensure data quality and enable downstream analytics. It performs quality control for scRNA-seq data, clusters cells based on gene expression to facilitate the identification of cell populations and provides insights into T-cell heterogeneity and functional diversity through T-cell selection and clustering. Enrichment analysis identifies markers and pathways for T-cell characterization. Our automated T-cell annotation tool annotates cell types accurately and hierarchically. For scTCR-seq data, immunopipe analyzes TCR clones, including clonality, diversity, gene usage, and repertoire overlap. Clone residency analysis examines the presence of clones across samples. Integrative analyses combine scRNA-seq and scTCR-seq data, providing a comprehensive understanding of the cellular landscape. Immunopipe incorporates diverse cell group information to analyze T-cell populations, which enables granular perspectives of the data for comprehensive insights. Immunopipe offers a flexible platform, allowing users to customize the analyses as needed. It supports both core and optional analyses through configuration, where users have control over the adjustable parameters. The pipeline relies on cell grouping information that can be referenced from metadata or modified to generate new variables for analysis and data filtering. Immunopipe can be executed locally or on scheduler platforms, and a docker image is provided for compatibility and simplified installation. It also offers a web interface that facilitates configuration, execution monitoring, and access to results. Conclusion: Immunopipe is a flexible and user-friendly pipeline for analyzing scRNA-seq and scTCR-seq data. It empowers researchers to gain valuable insights that could enhance the understanding of immune responses and facilitate the development of innovative immunotherapies. Citation Format: Panwen Wang, Haidong Dong, Yue Yu, Shuwen Zhang, Zhifu Sun, Jean-Pierre A. Kocher, Junwen Wang, Lin Yi, Ying Li. Immunopipe: A comprehensive and flexible scRNA-seq and scTCR-seq data analysis pipeline [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4959.

Abstract Background The electronic health record (EHR) presents new opportunities for the timely identification of patients at high risk of critical illness and the implementation of preventive strategies. This study aims to externally validate an EHR‐based Elders Risk Assessment (ERA) score to identify older patients at high risk of future critical illness during a primary care visit. Methods This historical cohort study included patients aged ≥65 years who had primary care visits at Mayo Clinic Rochester, MN, between July 2019 and December 2021. The ERA score at the time of the primary care visit was used to predict critical illness, defined as death or ICU admission within 1 year of the visit. Results A total of 12,885 patients were included in the analysis. The median age at the time of the primary care visit was 75 years, with 44.6% being male. 93.7% of participants were White, and 64.2% were married. The median (25th, 75th percentile) ERA score was 4 (0, 9). 11.3% of study participants were admitted to the ICU or died within 1 year of the visit. The ERA score predicted critical illness within 1 year of a primary care visit with an area under the receiver operating characteristic curve of 0.84 (95% CI 0.83–0.85), which indicates good discrimination. An ERA score of 9 was identified as optimal for implementing and testing potential preventive strategies, with the odds ratio of having the primary outcome in patients with ERA score ≥9 being 11.33 (95%CI 9.98–12.87). Conclusions This simple EHR‐based risk assessment model can predict critical illness within 1 year of primary care visits in older patients. The findings of this study can serve as a basis for testing and implementation of preventive strategies to promote the well‐being of older adults at risk of critical illness and its consequences.

Abstract Advances in diagnostic and treatment technology along with rapid developments in translational research may now allow the realization of precision radiotherapy. Integration of biologically informed multimodality imaging to address the spatial and temporal heterogeneity underlying treatment resistance in glioblastoma is now possible for patient care, with evidence of safety and potential benefit. Beyond their diagnostic utility, several candidate imaging biomarkers have emerged in recent early-phase clinical trials of biologically based radiotherapy, and their definitive assessment in multicenter prospective trials is already in development. In this review, the rationale for clinical implementation of candidate advanced magnetic resonance imaging and positron emission tomography imaging biomarkers to guide personalized radiotherapy, the current landscape, and future directions for integrating imaging biomarkers into radiotherapy for glioblastoma are summarized. Moving forward, response-adaptive radiotherapy using biologically informed imaging biomarkers to address emerging treatment resistance in rational combination with novel systemic therapies may ultimately permit improvements in glioblastoma outcomes and true individualization of patient care.

Objective: Given limited critical care resources and an aging population, early interventions to prevent critical illness are vital. In this work, we measured post-implementation outcomes after introducing a novel electronic scoring system (Elders Risk Assessment—ERA) and a risk-factor checklist, Checklist for Early Recognition and Treatment of Acute Illness (CERTAIN), to detect older patients at high risk of critical illness in a primary care setting. Methods: The study was conducted at a family medicine clinic in Kasson, MN. The ADAPT-ITT framework was used to modify the CERTAIN checklist for primary care during 2 co-design workshops involving interdisciplinary clinicians, held in April 2023. The ERA score and modified CERTAIN checklist were implemented between May and July 2023 and identify and assess all patients age ≥60 years at risk of critical illness during their primary care visits. Implementation outcomes were evaluated at the end of the study via an anonymous survey and EHR data extraction. Results: Fourteen clinicians participated in 2 co-design workshops. A total of 19 clinicians participated in a post-pilot survey. All survey items were rated on a 5-point Likert type scale. Mean acceptability of the ERA score and checklist was rated 3.35 (SD = 0.75) and 3.09 (SD = 0.64), respectively. Appropriateness had a mean rating of 3.38 (SD = 0.82) for the ERA score and 3.19 (SD = 0.59) for the checklist. Mean feasibility was rated 3.38(SD = 0.85) and 2.92 (SD = 0.76) for the ERA score and checklist, respectively. The adoption rate was 50% (19/38) among clinicians, but the reach was low at 17% (49/289) of eligible patients. Conclusions: This pilot study evaluated the implementation of an intervention that introduced the ERA score and CERTAIN checklist into a primary care practice. Results indicate moderate acceptability, appropriateness, and feasibility of the ERA score, and similar ratings for the checklist, with slightly lower feasibility. While checklist adoption was moderate, reach was limited, indicating inconsistent use. Recommendations: We plan to use the open-ended resurvey responses to further modify the CERTAIN-FM checklist and implementation process. The ADAPT-ITT framework is a useful model for adapting the checklist to meet the primary care clinician needs.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43–depleted human iPSC–derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43–depleted human iPSC–derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43–depleted human iPSC–derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Ever since its introduction as a diagnostic imaging tool the potential of magnetic resonance imaging (MRI) in radiation therapy (RT) treatment simulation and planning has been recognized. Recent technical advances have addressed many of the impediments to use of this technology and as a result have resulted in rapid and growing adoption of MRI in RT. The purpose of this article is to provide a broad review of the multiple uses of MR in the RT treatment simulation and planning process, identify several of the most used clinical scenarios in which MR is integral to the simulation and planning process, highlight existing limitations and provide multiple unmet needs thereby highlighting opportunities for the diagnostic MR imaging community to contribute and collaborate with our oncology colleagues.Evidence Level5Technical EfficacyStage 5

Abstract Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells by conferring survival advantages over normal cells. However, the functional importance of ecDNA replication and maintenance is yet to be evaluated, and the molecular components that contributed to these processes remain largely unknown. Here, using CRISPR-C (circularization of genes and chromosomes by CRISPR) technology and synthetic circular DNA mimicking ecDNA identified in tumors, we generated ecDNA-positive (ecDNA+) cell models. Using these models, we identified the proteins involved in ecDNA replication and maintenance and found DNA damage response (DDR)-related pathways were significantly enriched. We further demonstrated that ecDNA replication led to increased DDR and genome instability and DDR was also essential for ecDNA maintenance, consistent with observations that ecDNA+ tumors exhibited higher DDR scores than ecDNA-negative (ecDNA–) tumors in patients. ATM, MDC1, and CHK2, but not ATR, were activated in ecDNA+ cells compared to ecDNA– cells. Moreover, ecDNA+ cells were more sensitive to ATM and CHK2 inhibition. We further found that type II topoisomerase TOP2B and DNA ligase III (LIG3) were critical regulators for ecDNA replication and maintenance and related DDR. In summary, our findings of the reciprocal interactions between ecDNA and DDR provide new insights into detection and therapy for ecDNA+ tumors.

Genetic variation at the transmembrane protein 106B gene ( TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

Abstract Monoclonal B-cell lymphocytosis (MBL) progresses to chronic lymphocytic leukemia (CLL) requiring therapy at 1% to 5% per year. Improved prediction of progression would greatly benefit individuals with MBL. Patients with CLL separate into 3 distinct epigenetic subtypes (epitypes) with high prognostic significance, and recently the intermediate epitype has been shown to be enriched for high-risk immunoglobulin lambda variable (IGLV) 3-21 rearrangements, impacting outcomes for these patients. Here, we employed this combined strategy to generate the epigenetic and light chain immunoglobulin (ELCLV3-21) signature to classify 219 individuals with MBL. The ELCLV3-21 high-risk signature distinguished MBL individuals with a high probability of progression (39.9% and 71.1% at 5 and 10 years, respectively). ELCLV3-21 improved the accuracy of predicting time to therapy for individuals with MBL compared with other established prognostic indicators, including the CLL international prognostic index (c-statistic, 0.767 vs 0.668, respectively). Comparing ELCLV3-21 risk groups in MBL vs a cohort of 226 patients with CLL revealed ELCLV3-21 high-risk individuals with MBL had significantly shorter time to therapy (P = .003) and reduced overall survival (P = .03) compared with ELCLV3-21 low-risk individuals with CLL. These results highlight the power of the ELCLV3-21 approach to identify individuals with a higher likelihood of adverse clinical outcome and may provide a more accurate approach to classify individuals with small B-cell clones.

Abstract Microglia, the major resident immune cells in the central nervous system, serve as the frontline soldiers against cerebral ischemic injuries, possibly along with metabolic alterations. However, signaling pathways involved in the regulation of microglial immunometabolism in ischemic stroke remain to be further elucidated. In this study, using single‐nuclei RNA sequencing, a microglial subcluster up‐regulated in ischemic brain tissues is identified, with high expression of Igf1 and Trem2, neuroprotective transcriptional signature and enhanced oxidative phosphorylation. Microglial depletion by PLX3397 exacerbates ischemic brain damage, which is reversed by repopulating the microglia with high Igf1 and Trem2 phenotype. Mechanistically, Igf1 serves as one of the major down‐stream molecules of Trem2, and Trem2‐Igf1 signaling axis regulates microglial functional and metabolic profiles, exerting neuroprotective effects on ischemic stroke. Overexpression of Igf1 and supplementation of cyclocreatine restore microglial glucometabolic levels and cellular functions even in the absence of Trem2. These findings suggest that Trem2‐Igf1 signaling axis reprograms microglial immunometabolic profiles and shifts microglia toward a neuroprotective phenotype, which has promising therapeutic potential in treating ischemic stroke.