A pan-cancer gamma delta T cell repertoire.

This report presents the largest collection of gamma-delta T cell receptor (γδ TCR) reads in human cancer to date, analyzing about 11,000 patient tumor samples across 33 cancer types using the TRUST4 algorithm. Despite γδ T cells being a small fraction of the T cell population, they play a key role in both innate and adaptive immunity. Our comprehensive analysis reveals their significant presence across all cancer types, specifically highlighting the diverse spectrum and clonality patterns of their γδ receptors. This research highlights the complex roles of γδ T cells in tumor tissues and their potential as prognostic biomarkers. We also demonstrate the utility of T cell receptor gamma (TRG) and delta (TRD) gene expression values from standard RNA-seq data. Ultimately, our work establishes a fundamental resource for future tumor-infiltrating γδ T cell research and may facilitate the development of novel γδ-T-cell-based therapeutic strategies. Together, we demonstrate the strong diversity and prognostic potential of γδ T cells in multiple cancer types.

Risk factors for diabetic retinopathy, diabetic macular edema, and sight-threatening diabetic retinopathy.

OBJECTIVE: To identify the risk factors for the development of diabetic retinopathy (DR), diabetic macular edema (DME), and sight-threatening DR (STDR) based on a city-wide diabetes screening program. RESEARCH DESIGN AND METHODS: Diabetic patients were prospectively recruited between June 2016 and December 2022. All patients underwent dilated fundus photography centered on the disc and macula or macular spectral domain optical coherence tomography (SD-OCT) scan. Complete medical history was documented. Systematic examination, blood analysis, and urinalysis were performed. Multivariate logistic regression analysis adjusting for age and sex was conducted. RESULTS: Out of 7274 diabetic patients, 6840 had gradable images, among which 3054 (42.0%) were graded as DR, 1153 (15.9%) as DME, and 1500 (20.6%) as STDR. The factors associated with DR, DME, and STDR included younger age (odds ratio [OR]: 0.96, 0.97, and 0.96 respectively), lower BMI (OR: 0.97, 0.95, and 0.95 respectively), longer duration of diabetes (OR: 1.07, 1.03, and 1.05 respectively) and positive of urinary albumin (OR: 2.22, 2.56, and 2.88 respectively). Other associated factors included elevated blood urea nitrogen (OR: 1.22, 1.28, and 1.27 respectively), higher LDL-cholesterol, lower blood hemoglobin (OR: 0.98, 0.98, and 0.98), insulin intake, presence of diabetic foot pathologies and diabetic peripheral neuropathy. We also identified novel risk factors, including high serum potassium (OR: 1.37, 1.46, and 1.55 respectively), high-serum sodium (OR: 1.02, 1.02, and 1.04 respectively). Better family income was a protective factor for DR, DME, and STDR. Alcohol consumption once a week was also identified as a protective factor for DR. CONCLUSIONS: Similar risk factors for DR, DME, and STDR were found in this study. Our data also indicates high serum sodium, high serum potassium, low blood hemoglobin, and level of family income as novel associated factors for DR, DME, and STDR, which can help with DR monitoring and management.

Modeling phenotypic heterogeneity towards evolutionarily inspired osteosarcoma therapy.

Osteosarcoma is the most common bone sarcoma in children and young adults. While universally delivered, chemotherapy only benefits roughly half of patients with localized disease. Increasingly, intratumoral heterogeneity is recognized as a source of therapeutic resistance. In this study, we develop and evaluate an in vitro model of osteosarcoma heterogeneity based on phenotype and genotype. Cancer cell populations vary in their environment-specific growth rates and in their sensitivity to chemotherapy. We present the genotypic and phenotypic characterization of an osteosarcoma cell line panel with a focus on co-cultures of the most phenotypically divergent cell lines, 143B and SAOS2. Modest environmental (pH, glutamine) or chemical perturbations dramatically shift the success and composition of cell lines. We demonstrate that in nutrient rich culture conditions 143B outcompetes SAOS2. But, under nutrient deprivation or conventional chemotherapy, SAOS2 growth can be favored in spheroids. Importantly, when the simplest heterogeneity state is evaluated, a two-cell line coculture, perturbations that affect the faster growing cell line have only a modest effect on final spheroid size. Thus the only evaluated therapies to eliminate the spheroids were by switching therapies from a first strike to a second strike. This extensively characterized, widely available system, can be modeled and scaled to allow for improved strategies to anticipate resistance in osteosarcoma due to heterogeneity.

CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies.

Advances in the understanding of the tumor microenvironment have led to development of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.

Automatic Detection of Peripheral Retinal Lesions From Ultrawide-Field Fundus Images Using Deep Learning.

PURPOSE: To establish a multilabel-based deep learning (DL) algorithm for automatic detection and categorization of clinically significant peripheral retinal lesions using ultrawide-field fundus images. METHODS: A total of 5958 ultrawide-field fundus images from 3740 patients were randomly split into a training set, validation set, and test set. A multilabel classifier was developed to detect rhegmatogenous retinal detachment, cystic retinal tuft, lattice degeneration, and retinal breaks. Referral decision was automatically generated based on the results of each disease class. t -distributed stochastic neighbor embedding heatmaps were used to visualize the features extracted by the neural networks. Gradient-weighted class activation mapping and guided backpropagation heatmaps were generated to investigate the image locations for decision-making by the DL models. The performance of the classifier(s) was evaluated by sensitivity, specificity, accuracy, F 1 score, area under receiver operating characteristic curve (AUROC) with 95% CI, and area under the precision-recall curve. RESULTS: In the test set, all categories achieved a sensitivity of 0.836-0.918, a specificity of 0.858-0.989, an accuracy of 0.854-0.977, an F 1 score of 0.400-0.931, an AUROC of 0.9205-0.9882, and an area under the precision-recall curve of 0.6723-0.9745. The referral decisions achieved an AUROC of 0.9758 (95% CI= 0.9648-0.9869). The multilabel classifier had significantly better performance in cystic retinal tuft detection than the binary classifier (AUROC= 0.9781 vs 0.6112, P < 0.001). The model showed comparable performance with human experts. CONCLUSIONS: This new DL model of a multilabel classifier is capable of automatic, accurate, and early detection of clinically significant peripheral retinal lesions with various sample sizes. It can be applied in peripheral retinal screening in clinics.

Diversified Treatment Options of Adult Stem Cells for Optic Neuropathies.

Optic neuropathies refer to a group of ocular disorders with abnormalities or dysfunction of the optic nerve, sharing a common pathophysiology of retinal ganglion cell (RGC) death and axonal loss. RGCs, as the retinal neurons in the central nervous system, show limited capacity in regeneration or recovery upon diseases or after injuries. Critically, there is still no effective clinical treatment to cure most types of optic neuropathies. Recently, stem cell therapy was proposed as a potential treatment strategy for optic neuropathies. Adult stem cells, including mesenchymal stem cells and hematopoietic stem cells, have been applied in clinical trials based on their neuroprotective properties. In this article, the applications of adult stem cells on different types of optic neuropathies and the related mechanisms will be reviewed. Research updates on the strategies to enhance the neuroprotective effects of human adult stem cells will be summarized. This review article aims to enlighten the research scientists on the diversified functions of adult stem cells and consideration of adult stem cells as a potential treatment for optic neuropathies in future clinical practices.

Protocol for the isolation of CD8+ tumor-infiltrating lymphocytes from human tumors and their characterization by single-cell immune profiling and multiome.

Understanding the heterogenicity of tumor-infiltraing lymphocyte (TIL) populations and the immunobiology in human cancer is a key to establish efficient immunotherapies. Here, we have established a protocol for the characterization of CD8+ TILs in tumors by single-cell RNA-seq paired to VDJ profiling and chromatin structure including dissociation of tumor biopsies. We have also provided guidance for subsequent fluorescence-activated cell sorting (FACS), single-cell encapsulation, bioinformatics analysis, and troubleshooting. For complete details on the use and execution of this protocol, please refer to Anadon et al. (2022).

Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells.

BACKGROUND: Accumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of tumor-associated CD8 +T cells has important implications for their therapeutic targeting in the tumor microenvironment. In the present study, we investigated how accumulation of Tregs contributes to T cell dysfunction and clonal constriction of tumor-infiltrating CD8 +T cells. METHODS: Resected melanoma and lung adenocarcinoma tissues from tumor-bearing mice or patients were analyzed. The proportions and phenotype as well as clonal diversity of tumor-associated CD8 +T cells were evaluated by flow cytometry and single-cell T-cell receptor (TCR) sequencing, respectively, at early or advanced tumor stages or under Treg depletion conditions. Furthermore, antigen-specific T cells were evaluated on adoptive transfer into tumor-bearing mice in the presence or absence of anti-CTLA-4 antibody or CTLA-4 Ig. Lastly, tumor-bearing mice were treated with anti-KLRG1 antibody and/or bromodomain inhibitor JQ1 with interleukin (IL)-2 immune complexes to determine therapeutic efficacy. RESULTS: We demonstrate that the emergence of exhaustion-like phenotype and impaired effector functionality in tumor-associated CD8 +T cells is positively correlated with Treg accumulation in the tumor bed and this dysfunctional phenotype becomes reversed on Treg reduction in murine melanoma and lung cancer models. Heightened tumor-associated Treg-expressed CTLA-4 is key to emergence and sustenance of this phenotype. Furthermore, TCR sequencing revealed a clonal shrinkage of tumor-infiltrating CD8 +T cells as tumor progressed, which was associated with reduced survival profile concomitant to increasing Treg proportions. Limited IL-2 availability was a key mechanism contributing to this peripheral repertoire reshaping as Treg depletion improved IL-2 levels, rescued CD8 +T cell viability, and improved their clonal diversity. Finally, targeted reduction of tumor but not peripheral Tregs through JQ1 and/or anti-KLRG1 antibody significantly improved antitumor response in melanoma-bearing mice when supplemented with IL-2 immune complexes. CONCLUSION: Collectively, our study reveals a bimodal program enacted by Tregs to support T cell dysfunction in the tumor bed and highlights a promising therapeutic regimen for localized reprogramming of the tumor microenvironment to curb Treg impairment of antitumor CD8 +T cell response in favor of improved antitumor immunity.

Whole-genome sequencing reveals complex genomic features underlying anti-CD19 CAR T-cell treatment failures in lymphoma.

CD19-directed chimeric antigen receptor (CAR-19) T cells are groundbreaking immunotherapies approved for use against large B-cell lymphomas. Although host inflammatory and tumor microenvironmental markers associate with efficacy and resistance, the tumor-intrinsic alterations underlying these phenomena remain undefined. CD19 mutations associate with resistance but are uncommon, and most patients with relapsed disease retain expression of the wild-type receptor, implicating other genomic mechanisms. We therefore leveraged the comprehensive resolution of whole-genome sequencing to assess 51 tumor samples from 49 patients with CAR-19-treated large B-cell lymphoma. We found that the pretreatment presence of complex structural variants, APOBEC mutational signatures, and genomic damage from reactive oxygen species predict CAR-19 resistance. In addition, the recurrent 3p21.31 chromosomal deletion containing the RHOA tumor suppressor was strongly enriched in patients for whom CAR T-cell therapy failed. Pretreatment reduced expression or monoallelic loss of CD19 did not affect responses, suggesting CAR-19 therapy success and resistance are related to multiple mechanisms. Our study showed that tumor-intrinsic genomic alterations are key among the complex interplay of factors that underlie CAR-19 efficacy and resistance for large B-cell lymphomas.

Monocyte-derived SDF1 supports optic nerve regeneration and alters retinal ganglion cells' response to Pten deletion.

Although mammalian retinal ganglion cells (RGCs) normally cannot regenerate axons nor survive after optic nerve injury, this failure is partially reversed by inducing sterile inflammation in the eye. Infiltrative myeloid cells express the axogenic protein oncomodulin (Ocm) but additional, as-yet-unidentified, factors are also required. We show here that infiltrative macrophages express stromal cell­derived factor 1 (SDF1, CXCL12), which plays a central role in this regard. Among many growth factors tested in culture, only SDF1 enhances Ocm activity, an effect mediated through intracellular cyclic AMP (cAMP) elevation and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation. SDF1 deficiency in myeloid cells (CXCL12flx/flxLysM-Cre−/+ mice) or deletion of the SDF1 receptor CXCR4 in RGCs (intraocular AAV2-Cre in CXCR4flx/flx mice) or SDF1 antagonist AMD3100 greatly suppresses inflammation-induced regeneration and decreases RGC survival to baseline levels. Conversely, SDF1 induces optic nerve regeneration and RGC survival, and, when combined with Ocm/cAMP, SDF1 increases axon regeneration to levels similar to those induced by intraocular inflammation. In contrast to deletion of phosphatase and tensin homolog (Pten), which promotes regeneration selectively from αRGCs, SDF1 promotes regeneration from non-αRGCs and enables the latter cells to respond robustly to Pten deletion; however, SDF1 surprisingly diminishes the response of αRGCs to Pten deletion. When combined with inflammation and Pten deletion, SDF1 enables many RGCs to regenerate axons the entire length of the optic nerve. Thus, SDF1 complements the effects of Ocm in mediating inflammation-induced regeneration and enables different RGC subtypes to respond to Pten deletion.

Evaluation of Tumor DNA Sequencing Results in Patients with Gastric and Gastroesophageal Junction Adenocarcinoma Stratified by TP53 Mutation Status.

BACKGROUND: Gastric cancer (GC) and gastroesophageal junction adenocarcinomas (GEJ) are molecularly diverse. TP53 is the most frequently altered gene with approximately 50% of patients harboring mutations. This qualitative study describes the distinct genomic alterations in GCs and GEJs stratified by TP53 mutation status. PATIENTS AND METHODS: Tumor DNA sequencing results of 324 genes from 3741 patients with GC and GEJ were obtained from Foundation Medicine. Association between gene mutation frequency and TP53 mutation status was examined using Fisher's exact test. Functional gene groupings representing molecular pathways suggested to be differentially mutated in TP53 wild-type (TP53WT) and TP53 mutant (TP53MUT) tumors were identified. The association of the frequency of tumors containing a gene mutation in the molecular pathways of interest and TP53 mutation status was assessed using Fisher's exact test with a P-value of <.01 deemed statistically significant for all analyses. RESULTS: TP53 mutations were noted in 61.6% of 2946 GCs and 81.4% of 795 GEJs (P < .001). Forty-nine genes had statistically different mutation frequencies in TP53WT vs. TP53MUT patients. TP53WT tumors more likely had mutations related to DNA mismatch repair, homologous recombination repair, DNA and histone methylation, Wnt/B-catenin, PI3K/Akt/mTOR, and chromatin remodeling complexes. TP53MUT tumors more likely had mutations related to fibroblast growth factor, epidermal growth factor receptor, other receptor tyrosine kinases, and cyclin and cyclin-dependent kinases. CONCLUSION: The mutational profiles of GCs and GEJs varied according to TP53 mutation status. These mutational differences can be used when designing future studies assessing the predictive ability of TP53 mutation status when targeting differentially affected molecular pathways.

Coupling the immunomodulatory properties of the HDAC6 inhibitor ACY241 with Oxaliplatin promotes robust anti-tumor response in non-small cell lung cancer.

While HDAC inhibitors have shown promise in hematologic cancers, their efficacy remains limited in solid cancers. In the present study, we evaluated the immunomodulatory properties of the HDAC6 inhibitor, Citarinostat (ACY241) on lung tumor immune compartment and its therapeutic potential in combination with Oxaliplatin. As a single agent, ACY241 treatment promoted increased infiltration, activation, proliferation, and effector function of T cells in the tumors of lung adenocarcinoma-bearing mice. Furthermore, tumor-associated macrophages exhibited downregulated expression of inhibitory ligands in favor of increased MHC and co-stimulatory molecules in addition to higher expression of CCL4 that favored increased T cell numbers in the tumors. RNA-sequencing of tumor-associated T cells and macrophages after ACY241 treatment revealed significant genomic changes that is consistent with improved T cell viability, reduced inhibitory molecular signature, and enhancement of macrophage capacity for improved T cell priming. Finally, coupling these ACY241-mediated effects with the chemotherapy drug Oxaliplatin led to significantly enhanced tumor-associated T cell effector functionality in lung cancer-bearing mice and in patient-derived tumors. Collectively, our studies highlight the molecular underpinnings of the expansive immunomodulatory activity of ACY241 and supports its suitability as a partner agent in combination with rationally selected chemotherapy agents for therapeutic intervention in NSCLC.

Tumor Expression Quantitative Trait Methylation Screening Reveals Distinct CpG Panels for Deconvolving Cancer Immune Signatures.

DNA methylation signatures in tumors could serve as reliable biomarkers that are accessible in archival tissues for tracking the epigenetic dynamics shaped by both cancer cells and the tumor microenvironment. However, given the ultrahigh dimensionality and noncollapsible nature of the data, it remains challenging to screen all CpG sites to identify the most promising marker panels. In this article, we introduce the concept of tumor-based expression quantitative trait methylation (eQTM) for the prioritization and systematic mining of predictive biomarkers. In melanoma as a disease model, eQTM CpGs and genes represent new and efficient candidate targets to be investigated for both prognostic and immune status monitoring purposes. Three cis-eQTM CpGs (cg07786657, cg12446199, and cg00027570) were strongly associated with and can serve as surrogate biomarkers for the tumor immune cytolytic activity score (CYT). In addition, multiple eQTM genes could be further exploited for predicting immunoregulatory phenotypes. A targeted gene panel analysis identified one eQTM in TCF7 (cg25947408) as a novel candidate biomarker for uncoupling overall T-cell differentiation and exhaustion status in a tumor. The prognostic significance of this eQTM as an independent signature to CYT was validated by both The Cancer Genome Atlas and Moffitt melanoma cohort data. Overall, eQTMs represent a mechanistically distinct class of potential biomarkers that can be used to predict patient prognosis and immune status. SIGNIFICANCE: This study provides a novel and promising approach to identify targeted epigenetic biomarkers in cancer and will spur further analysis in tumor immune phenotyping.

Agonist of growth hormone-releasing hormone enhances retinal ganglion cell protection induced by macrophages after optic nerve injury.

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.

Effects of long-term norepinephrine treatment on normal immortalized ovarian and fallopian tube cells.

Sustained adrenergic stimulation by norepinephrine (NE) contributes to ovarian carcinoma metastasis and impairment of chemotherapy response. Although the effect of sustained NE stimulation in cancer progression is well established, less is known about its role in cancer initiation. To determine the extent to which stress hormones influence ovarian cancer initiation, we conducted a long-term (> 3 months; > 40 population doublings) experiment in which normal immortalized fallopian tube secretory (iFTSEC283) and ovarian surface epithelial (iOSE11) cell lines and their isogenic pairs containing a p53 mutation (iFTSEC283p53R175H; iOSE11p53R175H), were continuously exposed to NE (100 nM, 1 µM, 10 µM). Fallopian tube cells displayed a p53-independent increase in proliferation and colony-forming ability in response to NE, while ovarian surface epithelial cells displayed a p53-independent decrease in both assays. Fallopian tube cells with mutant p53 showed a mild loss of chromosomes and TP53 status was also a defining factor in transcriptional response of fallopian tube cells to long-term NE treatment.

Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3.

Alterations in genes encoding for proteins that control fucosylation are known to play causative roles in several developmental disorders, such as Dowling-Degos disease 2 and congenital disorder of glycosylation type IIc (CDGIIc). Recent studies have provided evidence that changes in fucosylation can contribute to the development and progression of several different types of cancers. It is therefore important to gain a detailed understanding of how fucosylation is altered in disease states so that interventions may be developed for therapeutic purposes. In this report, we find that fucosylation occurs on many intracellular proteins. This is an interesting finding, as the fucosylation machinery is restricted to the secretory pathway and is thought to predominately affect cell-membrane-bound and secreted proteins. We find that Ribosomal protein S3 (RPS3) is fucosylated in normal tissues and in cancer cells, and that the extent of its fucosylation appears to respond to stress, including MAPK inhibitors, suggesting a new role in posttranslational protein function. Our data identify a new ribosome-independent species of fucosylated RPS3 that interacts with proteins involved in posttranscriptional regulation of RNA, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), as well as with a predominance of non-coding RNAs. These data highlight a novel role for RPS3, which, given previously reported oncogenic roles for RPS3, might represent functions that are perturbed in pathologies such as cancer. Together, our findings suggest a previously unrecognized role for fucosylation in directly influencing intracellular protein functions.

TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.

Association of a novel endometrial cancer biomarker panel with prognostic risk, platinum insensitivity, and targetable therapeutic options.

During the past decade, the age-adjusted mortality rate for endometrial cancer (EC) increased 1.9% annually with TP53 mutant (TP53-mu) EC disproportionally represented in advanced disease and deaths. Therefore, we aimed to assess pivotal molecular parameters that differentiate clinical outcomes in high- and low-risk EC. Using the Cancer Genome Atlas, we analyzed EC specimens with available DNA sequences and quantitative gene-specific RNA expression data. After polymerase ɛ (POLE)-mutant specimens were excluded, differential gene-specific mutations and mRNA expressions were annotated and integrated. Consequent to TP53-mu failure to induce p21, derepression of multiple oncogenes harboring promoter p21 repressive sites was observed, including CCNA2 and FOXM1 (P < .001 compared with TP53 wild type [TP53-wt]). TP53-wt EC with high CCNA2 expression (CCNA2-H) had a targeted transcriptomic profile similar to that of TP53-mu EC, suggesting CCNA2 is a seminal determinant for both TP53-wt and TP53-mu EC. CCNA2 enhances E2F1 function, upregulating FOXM1 and CIP2A, as observed in TP53-mu and CCNA2-H TP53-wt EC (P < .001). CIP2A inhibits protein phosphatase 2A, leading to AKT inactivation of GSK3ß and restricted oncoprotein degradation; PPP2R1A and FBXW7 mutations yield similar results. Upregulation of FOXM1 and failed degradation of FOXM1 is evidenced by marked upregulation of multiple homologous recombination genes (P < .001). Integrating these molecular aberrations generated a molecular biomarker panel with significant prognostic discrimination (P = 5.8×10-7); adjusting for age, histology, grade, myometrial invasion, TP53 status, and stage, only CCNA2-H/E2F1-H (P = .0003), FBXW7-mu/PPP2R1A-mu (P = .0002), and stage (P = .017) were significant. The generated prognostic molecular classification system identifies dissimilar signaling aberrations potentially amenable to targetable therapeutic options.

Managing a Large-Scale Multiomics Project: A Team Science Case Study in Proteogenomics.

Highly collaborative scientists are often called on to extend their expertise to different types of projects and to expand the scope and scale of projects well beyond their previous experience. For a large-scale project involving "big data" to be successful, several different aspects of the research plan need to be developed and tested, which include but are not limited to the experimental design, sample collection, sample preparation, metadata recording, technical capability, data acquisition, approaches for data analysis, methods for integration of different data types, recruitment of additional expertise as needed to guide the project, and strategies for clear communication throughout the project. To capture this process, we describe an example project in proteogenomics that built on our collective expertise and experience. Key steps included definition of hypotheses, identification of an appropriate clinical cohort, pilot projects to assess feasibility, refinement of experimental designs, and extensive discussions involving the research team throughout the process. The goal of this chapter is to provide the reader with a set of guidelines to support development of other large-scale multiomics projects.