We promote whole-genome pooled sequencing data as a persistent, reusable resource to improve management and utilization of heterogeneous germplasm collections. Using 14.9 Tbp of DNA sequence data from 4987 individuals in the sugar beet ( Beta vulgaris L. ssp. vulgaris ) primary gene pool as a test case, we demonstrate appropriate analytical procedures to reveal population structure, assemble optimized subsets, perform allele mining, and contribute to gene discovery. Table, sugar, fodder, and leaf beets were found to be genetically distinct, with an affinity shown between wild and leaf beets. Differing genetic trajectories were inferred for germplasm releases from four regional U.S. Department of Agriculture-Agricultural Research Service sugar beet breeding programs. Using a germplasm panel developed to represent broad-sense variation in B. vulgaris L. ssp. maritima , we show that the wild relative is variable, divergent, and remains underexploited despite an established, successful history of wild introgressions. We discover that novel Rz2 -type rhizomania disease resistance alleles are common in table beets and the wild relative but are uncommon in US sugar beet germplasm. Phenotypic characterization data held in gene banks can be used with pooled sequencing data for association analyses—a whole-genome signature of selection scan identified BvWIP2 as a candidate gene for monogerm seed development, a valuable trait in beets, consistent with a recent association study using single individuals. Mass production of whole-genome pooled sequencing datasets linked to gene bank collections would minimize the need to re-sequence individuals, in some cases eliminating the wet lab component of genetic studies, shifting the emphasis of gene discovery to phenotyping and bioinformatics.

Bottlebrush squirreltail (Elymus elymoides) and big squirreltail (Elymus multisetus) are high-priority species for restoration of millions of hectares of rangeland in the western United States that have been degraded by accelerated wildfire and introduced annual grasses. Previous research has compared potential germination and seedling performance of these species in a common environment and noted significant genetic differentiation in characteristics that are associated with their environments of origin. In this experiment, we used wet-thermal germination models and long-term simulations of seedbed microclimate to conduct a virtual reciprocal-garden analysis of the potential germination response of these species. We confirmed significant species differentiation in germination rate that appears to confer site-specific advantages for initial seedling establishment in their respective habitats of origin. Our results specifically highlight the relative importance of late-fall seeding for the more rapidly germinating E. multisetus in order to avoid early-fall germination and post-germination seedling mortality from freezing conditions in the winter. In contrast, the slower-germinating E. elymoides ssp. brevifolius A is less likely to germinate in the fall and likely avoids inherently harsher winter temperatures in the seedbed. Virtual simulations of this type might lead to identification of complex-trait genetic markers that are associated with intraspecific and interspecific adaptations to specific environments. Identification of these traits could also inform management of plant communities that are under threat from invasive weeds and climate change.

213 Background: Radiation therapy (RT) can improve survival in oligometastatic prostate cancer (OMPC) patients (pts). Reports implicate immune system contribution to both local tumor control and systemic regression of metastases. Understanding molecular and immune drivers of clinical responses in irradiated OMPC pts may elucidate mechanisms of RT-driven antitumor immune activity. Methods: Prospective samples from consented pts with OMPC (de novo/oligorecurrent) receiving RT included baseline (pre-RT) and 3 post-RT blood samples (RT end, 3-, 6-months). Peripheral blood mononuclear cells (PMBC) were obtained at each timepoint. PBMC sample barcoding and antibody labeling was performed prior to analysis by mass cytometry by time of flight (CyTOF). Data were debarcoded/analyzed using standard deconvolution/gating/clustering algorithms. Wilcoxon signed-rank tests identified longitudinal differences in comparison to baseline. P <0.05 denotes significance. Results: Between 2020-2022, 14 OMPC pts (56 samples) met inclusion criteria (2 de novo, 12 oligorecurrent). All received androgen deprivation therapy. De novo OMPC pts received simultaneous RT to the prostate, pelvis, and OM lesions. 6/12 oligorecurrent pts received simultaneous RT to a pelvic field along with OM lesions, while the remainder received RT to the OM lesions alone. Median follow-up was 17 months (r, 3-29). Four pts (28.6%) experienced recurrence (defined by PSA progression). Median time to recurrence after RT was 10.5 months. Significant differences in immune cell subsets after RT compared to pre-RT levels were identified by CyTOF analysis. CD4+ naïve cells peaked 6 months post-RT, while CD4+ effector memory (EM) cells peaked immediately post-RT (p=0.0012) and decreased to baseline levels at 3 months. CD4+ effector cells expressing TIGIT, OX40, ICOS, and CD69 were significantly more abundant immediately post-RT (p<0.05) compared to baseline. CD8+ cells expressing ICOS increased post-RT and were significantly increased at 6 months (p=0.03). In marked contrast to recurrent pts, non-recurrent pts exhibited increasing CD4+ T regulatory cells that peaked 6 months post-RT (p=0.04) and CD4+ EM cells that peaked post-RT (p=0.02). CD4+ effector cells expressing ICOS (p=0.01) and PD-1 (p=0.01) were both increased immediately post-RT, while cells expressing 4-1BB continued to increase and peaked 6 months post-RT (p=0.04). These changes were not observed or not significant in the recurrent population. Conclusions: Longitudinal immune cell subset changes can be observed after RT for OMPC. These data support immune activation, particularly immediately post-RT in non-recurrent pts, with some longitudinal changes lasting up to 6 months. Further analyses include TCR sequencing, inclusion of serum proteomics, and baseline genomic markers that may predict immune system activation and clinical outcomes.

Background The median survival for patients with newly diagnosed (ND) higher risk myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) remains poor. The potential curability of MDS and CMML with allogeneic hematopoietic stem cell transplantation supports the concept of anti-tumor immunity and has led to interest in evaluating immune-based therapeutic approaches in myeloid neoplasms. The immune checkpoint molecule TIM-3 (encoded by the gene HAVCR2) is a target of interest in myeloid neoplasms given its expression on leukemic stem cells as well as several subsets of immune cells including T cells, monocytes, dendritic cells and NK cells. Sabatolimab (MBG453) is an investigational IgG4 anti-TIM-3 antibody currently under evaluation for myeloid neoplasms. In this exploratory study, we sought to characterize the effects of sabatolimab combined with the hypomethylating agent (HMA), azacitidine, on the immune landscape using single cell sequencing of samples from subjects with MDS and CMML treated with the combination from a previously reported phase 1b study (NCT03066648; Brunner et al 2022). Methods Following IRB approval, single-cell RNA sequencing (scRNA-seq) and associated proteomic cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) was performed on both blood (BLD) and bone-marrow (BM) derived samples from subjects with: 1) MDS (n=3) or CMML (n=2) treated with HMA therapy alone as part of standard care; 2) ND MDS (n=5) or CMML (n=3) treated with azacitidine combined with sabatolimab; and 3) relapsed/refractory MDS (n=3) treated with sabatolimab alone. Paired BLD and BM samples as well as samples from serial treatment time points were selected whenever feasible (Fig 1). A total of 206,183 cells from BM and 172,421 cells from BLD post-quality control filtering were analyzed. Further, scRNAseq data from normal BLD(200,000 cells) and BM(240,650 cells) generated as part of the human cell atlas (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79) were integrated with this dataset to assist with immune cell subset characterization, enable downstream comparisons to healthy hematopoietic cells, and to help define cell subsets associated with disease states. Results In baseline samples from subjects with MDS treated with azacitidine-sabatolimab, we found that an increased baseline abundance of interferon-responsive CD8 T cells in both the BM and BLD (FDR<0.1) was associated with response to therapy. An increased baseline abundance of plasmacytoid dendritic cells and a granulocyte population also suggested an association with response to therapy. Differential gene expression analysis of baseline samples from responding versus non-responding subjects with MDS treated with azacitidine-sabatolimab showed a higher baseline expression of TNF and IFNG in BM CD8 T cells and up-regulated expression of MHC-II machinery ( HLA-DRB1, HLA-DPA1, CD74) in myeloid cell subsets in responding subjects (FDR <0.1), suggesting that pre-treatment ability of these cells to be involved in antigen-presentation may play a role in response. In subjects with CMML treated with azacitidine-sabatolimab, we observed dynamic changes in cellular abundances and gene expression when comparing baseline to post-therapy timepoints. Specifically, BM CD8 T cell subsets showed an up-regulation of cytotoxicity genes ( GZMA), interferon response genes ( IFIT2, IFITM1, IFITM2) and IL32 cytokine post-therapy (FDR <0.1). When evaluating myelomonocytic cell populations, we observed an increase in CD16 monocytes post-treatment. Additionally, dendritic cells and monocytes showed an up-regulation of interferon response genes ( IFIT1, OAS1, IFI27). Conversely, we observed a down-regulation of metallothionein genes ( MT1E, MT1G, MT2A) as well as a collection of transcription factors ( NR4A1, FOSL2, JUN, CEBPB, CEBPD), NF-κB inhibitors ( NFKBIZ, NFKBIA) and CXCL8 in the post-treatment samples (FDR <0.1). Conclusions Our study provides one of the most comprehensive evaluations of the cellular dynamics of anti-TIM3 immunotherapy in patients to date, allowing for the nomination of novel putative predictive biomarkers of response and identification of potential immunomodulatory mechanisms induced by the combination of sabatolimab with azacitidine in MDS and CMML for further future analysis.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Valuable genetic variation lies unused in gene banks due to the difficulty of exploiting heterogeneous germplasm accessions. Advances in molecular breeding, including transgenics and genome editing, present the opportunity to exploit hidden sequence variation directly. Here we describe the pan-genome data structure induced by whole-genome sequencing of pooled individuals from wild populations of Patellifolia spp., a source of disease resistance genes for the related crop species sugar beet (Beta vulgaris). We represent the pan-genome as a map of reads from pooled sequencing of a heterogeneous population sample to a reference genome, plus a BLAST data base of the mapped reads. We show that this basic data structure can be queried by reference genome position or homology to identify sequence variants present in the wild relative, at genes of agronomic interest in the crop, a process known as allele or variant mining. Further we demonstrate the possibility of cataloging variants in all Patellifolia genomic regions that have corresponding single copy orthologous regions in sugar beet. The data structure, termed a “pooled read archive,” can be produced, altered, and queried using standard tools to facilitate discovery of agronomically-important sequence variation.

Invasive annual grasses now dominate millions of hectares of rangeland in the Intermountain Western United States. Local annual grass distribution, however, has been shown to follow landscape patterns of slope, aspect, and elevation that are correlated with ecological resilience to stress and disturbance and resistance to annual grass invasion. Although these patterns have previously been linked to soil-climate classes, several mechanistic factors in native-plant seedling establishment are also associated with both topography and seasonal weather patterns in the year following planting. In this study we used the Simultaneous Heat and Water (SHAW) model to estimate long-term weather effects on soil microclimate and hydrothermal-germination models to predict germination response of one fast- and one slow-germinating native grass as a function of planting date, slope, aspect, and elevation in the Boise Foothills in southwestern Idaho. Higher elevation and northerly aspect sites are more likely to defer germination of seeded species until late enough in the fall that they avoid postgermination/preemergence freezing mortality. These sites are also more favorable for survival of emerged seedlings through mid to late summer. Slope, aspect, and elevation effects on modeled restoration outcomes are consistent with previously modeled general patterns of ecological resilience and resistance as a function of soil hydrothermal class, but inclusion of slope and aspect effects may produce finer-scale metrics for mapping these patterns over space. The probabilistic nature of microclimatic variability as a function of elevation may yield useful insights into successful restoration approaches for reestablishment of native plant communities in lower-elevation ecosystems with inherently lower ecological resilience and resistance. The generally arid climate in this region, however, may limit successful restoration outcomes at lower elevation in most years even under conditions of long-term annual grass control.

Several recent studies have hypothesized that postgermination/preemergence soil freezing can have a major negative impact on spring emergence of seeded perennial grasses in the Intermountain western United States. In this experiment, we measured germination and emergence of 4 fall-seeded perennial grasses at 15 sites in southeastern Oregon, north-central Nevada, and southwestern Idaho in 2 different yr. We used hydrothermal germination and seedbed microclimatic models to estimate planting date effects on germination and preemergence mortality and evaluated these simulations relative to measured germination and seedling emergence in the field. There were clear indications that both germination and emergence were responsive to site-year effects, but premature fall germination before winter freezing events was not a factor in emergence rates. In all cases, planting was sufficiently late in the fall to avoid significant postgermination/preemergence freezing injury for the sites and years tested. Emergence was weakly correlated to seedbed favorability in the late winter and spring after the principal period of soil freezing. Our data show that for the 2 yr of this study, we can only account for a relatively small portion of any abiotic effects on spring emergence. General inferences about planting date effects, however, may have been limited by arid conditions during the 2-yr field study relative to long-term climate normals.

Foreign body ingestion (FBI) among children is associated with morbidity and mortality. We used the National Electronic Injury Surveillance System to compare FBI trends from 2017–2019 to 2020 during the spread of SARS-CoV-2. The pandemic and associated stay-at-home orders were associated with uptrends in button battery and magnet ingestions but unchanged total FBI trends.

Historically the chemokine receptor CXCR4 and its canonical ligand CXCL12 are associated with the bone marrow niche and hematopoiesis. However, CXCL12 exhibits broad tissue expression including brain, thymus, heart, lung, liver, kidney, spleen, and bone marrow. CXCR4 can be considered as a node which is integrating and transducing inputs from a range of ligand-receptor interactions into a responsive and divergent network of intracellular signaling pathways that impact multiple cellular processes such as proliferation, migration, and stress resistance. Dysregulation of the CXCR4/CXCL12 axis and consequent fundamental cellular processes, are associated with a panoply of disease. This review frames the polyfunctionality of the receptor at a molecular, physiological, and pathophysiological levels. Transitioning our perspective of this axis from a single gene/protein:single function model to a polyfunctional signaling cascade highlights the potential for finer therapeutic intervention and cautions against a reductionist approach.

Plant genebanks provide genetic resources for breeding and research programs worldwide. These programs benefit from having access to high-quality, standardized phenotypic and genotypic data. Technological advances have made it possible to collect phenomic and genomic data for genebank collections, which, with the appropriate analytical tools, can directly inform breeding programs. We discuss the importance of considering genebank accession homogeneity and heterogeneity in data collection and documentation. Citing specific examples, we describe how well-documented genomic and phenomic data have met or could meet the needs of plant genetic resource managers and users. We explore future opportunities that may emerge from improved documentation and data integration among plant genetic resource information systems.

BACKGROUND:Major injury results in an early cascade of immunologic responses that increase susceptibility to infection and multiorgan dysfunction. Detailed immune profiling by mass cytometry has the potential to identify immune signatures that correspond to patient outcomes. Our objective was to determine the prognostic value of immune signatures early after major trauma injury.METHODS:Trauma patients (n = 17) were prospectively enrolled between September 2018 and December 2019. Serial whole blood samples were obtained from trauma patients (mean Injury Severity Score, 26.2; standard error of the mean, 3.7) at Days 1 and 3 after injury, and from age- and sex-matched uninjured controls using a standardized protocol for fixation, storage, and labeling. Computational analyses including K-nearest neighbor automated clustering of immune cells and Spearman's correlation analysis were used to identify correlations between cell populations, clinical measures, and patient outcomes.RESULTS:Analysis revealed nine immune cell clusters that correlated with one or more clinical outcomes. On Days 1 and 3 postinjury, the abundance of immature neutrophil and classical monocytes exhibited a strong positive correlation with increased intensive care unit and hospital length of stay. Conversely, the abundance of CD4 T-cell subsets, namely Th17 cells, is associated with improved patient outcomes including decreased ventilator days (r = -0.76), hospital-acquired pneumonia (r = -0.69), and acute kidney injury (r = -0.73).CONCLUSION:Here, we provide a comprehensive multitime point immunophenotyping analysis of whole blood from patients soon after traumatic injury to determine immune correlates of adverse outcomes. Our findings indicate that alterations in myeloid-origin cell types may contribute to immune dysfunction after injury. Conversely, the presence of effector T cell populations corresponds with decreased hospital length of stay and organ dysfunction. Overall, these data identify novel immune signatures following traumatic injury that support the view that monitoring of immune (sub)-populations may provide clinical decision-making support for at-risk patients early in their hospital course.LEVEL OF EVIDENCE:Prognostic/Epidemiologic, Level IV.

Abstract Objective Chemotherapy-induced nausea and vomiting (CINV) is characterized by disabling nausea and emesis that can recur throughout the treatment of cancer and affects approximately 59% of pediatric and young adult patients. CINV can be associated with significant clinical morbidity, frequent hospital admissions, negative effects on health care related quality-of-life and can exhibit downstream effects such as weight loss that can worsen overall outcomes. Despite the vast number and potential combinations of pharmacotherapies and lifestyle modifications available to manage CINV in children, there are currently no clinical action tools offered to manage this condition better at home. We aimed to develop and assess an evidence-based, personalized pictogram-based nausea action plan (NAP) to aid providers, parents, and patients in the management of CINV. Methods The USNAP (Figure 1) facilitates the management of CINV by using a health literacy-informed approach to provide instructions for pharmacotherapies and lifestyle modifications. This study included Part 1 (Pictogram Validation) and Part 2 (Assessment). For Part 1, Pictogram transparency, translucency, and recall were assessed by parent survey with transparency ≥85%, mean translucency score ≥5, recall ≥85% required for validation. For Part 2, the USNAP was assessed by parents, clinical librarians, and clinicians. Patient/caregiver perceptions (n=27) were assessed using the Consumer Information Rating Form (17 questions) to gauge comprehension, design quality and usefulness. Readability was assessed by 5 formulas and a Readability Consensus Score was calculated. Clinical Librarians (n=2) used the Patient Education Materials Assessment Tool to measure the understandability (19 questions) and actionability (7 questions) of the plan and audiovisual educational content (>80% acceptable.) Suitability was assessed by clinicians (n=16) using Doaks' Suitability Assessment of Materials (superior≥70% rating). Results All 15 pictograms demonstrated appropriate transparency, translucency, and recall. Patient/caregiver perceptions reflected appropriate comprehension, design quality, and usefulness. The Readability Composite Score measured at a fourth-grade level. Clinical librarians reported acceptable understandability and actionability. Clinicians reported superior suitability. Conclusion The Uniformed Services Nausea Action Plan (USNAP) is the first clinical action tool designed to assist in managing CINV at home. Although recent clinical practice updates provide guidance on the therapeutic management of CINV in pediatric and young adult patients with cancer, these recommendations do not fully address the needs of the patient at home or adequately inform in instances of low health literacy. The USNAP seeks to mitigate this by making the clinical practice guidance useful to patients and caregivers at home. In addition, the USNAP is poised to identify other urgent clinical developments for patients with cancer that could masquerade as nausea and may serve as an early warning sign in these instances. The USNAP met all criteria for clinical implementation. The USNAP has potential to become an important tool in the care of patients with CINV, improving both quality-of-care and clinical outcomes. Future study of USNAP implementation for treating children with chronic CINV is needed. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Rangeland vegetation in the Great Basin, United States, is frequently disturbed by natural- and human-caused wildfires that facilitate the establishment and dominance of introduced annual weeds such as cheatgrass (Bromus tectorum) and medusahead wildrye (Taeniatherum caput-medusae [L.] Nevski). Ecological resilience and resistance of native and seeded-non-native plant communities in this region, however, appear to follow topographic patterns associated with slope, aspect, and elevation. Currently, resistance and resilience concepts are being used to prioritize rangeland restoration efforts based on soil-climate classification. We hypothesized that probabilistic patterns of shorter-term weather effects on seedbed microclimate might also be correlated with these perceived spatial patterns of resistance and resilience over space. We used a 39-yr gridded weather dataset to estimate seedbed temperature and water potential at seeding depth as a function of slope and aspect using the Simultaneous Heat and Water (SHAW) model. Seedbed temperature and water potential were then used as input to hydrothermal germination response models to generate indices of seedbed favorability for initial germination and emergence and to estimate cumulative germination response as a function of topography and planting date for the very-fast-germinating cheatgrass, fast-germinating bottlebrush squirreltail (Elymus elymoides [Raf] Swezey), and slower-germinating Idaho fescue (Festuca idahoensis Elmer). Topographic mapping of seedbed favorability showed distinct seasonal patterns associated with both slope and aspect. Southern exposures are likely to facilitate both prewinter germination for early-fall-planted seeds and relatively more midwinter germination for seeds planted later in the fall, but these exposures are also less subject to midwinter frost effects. Northern exposures were likely to delay germination into later winter and early spring and thus avoid potential exposure to a higher probability of winter frost mortality. Microclimatic simulations of this type may provide new metrics for improving our understanding of the mechanistic causes of observed patterns of ecological resistance and resilience over space.

Farmers and traders have developed a system of names to refer to different qat (Catha edulis) cultivars, using stem color as the primary trait to differentiate them. In this study, we tested if the named cultivars from Ethiopia, Kenya, and Yemen represent genetic clusters. We also quantified clonal reproduction and tracked the geographic dispersal of cultivated–qat genotypes using microsatellite genotypes of specimens collected from across the major qat growing regions (Ethiopia, Kenya, and Yemen). Specimens were mapped to determine whether names, morphology, genetic clusters, or genotypes (in particular clones) were geographically restricted. Stem color was found to be a plastic trait because specimens of the same genotype have different colored stems. A single cultivar name was often applied to numerous clonal groups and genetic clusters, creating widespread homonymy in the ethnotaxonomy of qat cultivars. The East African Rift (EAR) in central Ethiopia was found to be a barrier to the exchange of both clonal groups and certain cultivar names. In Kenya, both cultivar names and clonal groups were broadly dispersed geographically. Nearly all of these clonal groups originated from the Mt. Kenya/Meru area in central Kenya.

Araucaria (Araucaria angustifolia (Bert.) O. Ktze) is a primarily dioecious species threatened with extinction that plays an important social and economic role especially in the southern region of Brazil. The aim of this work is to investigate the diversity and likely determinants of genetic lineages in this species for conservation management. For this, a collection of 30-year-old Araucaria was used. Accessions collected from 12 sites across the species range were analyzed, with ten individuals per site. The SSR genotyping was conducted with 15 loci and the data were analyzed using several complementary approaches. Descriptive statistics among sampling sites were used and diversity was partitioned non-hierarchically to estimate the size and composition of genetic clusters using a Bayesian assignment method. To explore possible biological implications of differences between Niche Models and habitat suitability, a series of statistical procedures were used, and tests were carried out using the software ENM Tools and Maxent. Populations from the southernmost zone showed higher genetic variation and a lower inbreeding coefficient compared to the northernmost zone, which may correlate with their isolation. A positive relation between genetic differentiation and geographic distance was observed. Two genetic groups (southernmost and northernmost zones) were evident. The Niche modelling showed separate ranges for each genetic lineage suggesting that differences in selection pressure may be playing a role in the apparent differentiation and may be adaptive. Finally, an evident correlation was observed between genetic data and habitat suitability. The two distinct groups observed must be considered as independent units for conservation and hybridization in breeding programs.

e21026 Background: Comprehensive molecular profiling and the use of biomarkers as companion diagnostics have transformed precision medicine for cancer patients. To identify patient-specific tumor microenvironment and biomarker profiles, we assessed the accuracy of our deconvolution algorithm in identifying cellular compositions from whole exome (WES) and whole transcriptome (RNA-seq) sequencing of solid tumors compared with cell populations identified by Mass Cytometry by Time of Flight (CyTOF) in surgically resected tissue from non-small cell lung cancer (NSCLC) patients. Methods: Resected NSCLC tissue was divided for RNA-seq and WES of whole tissue (n = 9) and for generating tissue single cell suspensions through mechanical dissociation and enzymatic digestion (n = 11). Bulk RNA-seq and CyTOF were performed on all cell suspensions. Cellular phenotypes were identified using clustering algorithms in CyTOF and predicted from bulk RNA-seq using our proprietary computational method. Results: Cellular composition reconstructed from RNA-seq correlated with the composition detected by CyTOF (R2= 0.922, n = 7) from cell suspensions. To recover the cell percentage from bulk RNA-seq, a machine learning framework was trained on the cell compendia comprising 7,117 unique cell type RNA-seq profiles. A two-stage hierarchical learning procedure generated a gradient boosting Light GBM model that included training on artificial RNA-seq mixtures of different cell types. With this method, we found that stromal and malignant cells were depleted during single cell suspension preparation, resulting in statistically significant differences in the tumor cell composition reconstructed from solid tissue and single cell suspensions. Immune cell types namely T cells and macrophages were similarly represented in both the bulk tumor tissue and matched single cell suspensions. Transcriptomics revealed a subgroup of patients whose tumors were B-cell-enriched, which was validated in other NSCLC cohorts and was associated with greater CD4+ and CD8+ T cell infiltration and improved clinical outcomes. Conclusions: Since preparation of single cell suspensions leads to the loss of several cellular components, RNA-seq of tumor bulk tissue better describes the molecular and cellular properties of the tumor microenvironment. The combination of RNA-seq and WES of tumor tissue provides a comprehensive profile of cellular composition, suggesting that this combination is ideal for precision medicine applications.