Comprehensive proteogenomic characterization of rare kidney tumors.

Non-clear cell renal cell carcinomas (non-ccRCCs) encompass diverse malignant and benign tumors. Refinement of differential diagnosis biomarkers, markers for early prognosis of aggressive disease, and therapeutic targets to complement immunotherapy are current clinical needs. Multi-omics analyses of 48 non-ccRCCs compared with 103 ccRCCs reveal proteogenomic, phosphorylation, glycosylation, and metabolic aberrations in RCC subtypes. RCCs with high genome instability display overexpression of IGF2BP3 and PYCR1. Integration of single-cell and bulk transcriptome data predicts diverse cell-of-origin and clarifies RCC subtype-specific proteogenomic signatures. Expression of biomarkers MAPRE3, ADGRF5, and GPNMB differentiates renal oncocytoma from chromophobe RCC, and PIGR and SOSTDC1 distinguish papillary RCC from MTSCC. This study expands our knowledge of proteogenomic signatures, biomarkers, and potential therapeutic targets in non-ccRCC.

Frozen tissue coring and layered histological analysis improves cell type-specific proteogenomic characterization of pancreatic adenocarcinoma.

BACKGROUND: Omics characterization of pancreatic adenocarcinoma tissue is complicated by the highly heterogeneous and mixed populations of cells. We evaluate the feasibility and potential benefit of using a coring method to enrich specific regions from bulk tissue and then perform proteogenomic analyses. METHODS: We used the Biopsy Trifecta Extraction (BioTExt) technique to isolate cores of epithelial-enriched and stroma-enriched tissue from pancreatic tumor and adjacent tissue blocks. Histology was assessed at multiple depths throughout each core. DNA sequencing, RNA sequencing, and proteomics were performed on the cored and bulk tissue samples. Supervised and unsupervised analyses were performed based on integrated molecular and histology data. RESULTS: Tissue cores had mixed cell composition at varying depths throughout. Average cell type percentages assessed by histology throughout the core were better associated with KRAS variant allele frequencies than standard histology assessment of the cut surface. Clustering based on serial histology data separated the cores into three groups with enrichment of neoplastic epithelium, stroma, and acinar cells, respectively. Using this classification, tumor overexpressed proteins identified in bulk tissue analysis were assigned into epithelial- or stroma-specific categories, which revealed novel epithelial-specific tumor overexpressed proteins. CONCLUSIONS: Our study demonstrates the feasibility of multi-omics data generation from tissue cores, the necessity of interval H&E stains in serial histology sections, and the utility of coring to improve analysis over bulk tissue data.

Epigenetic and transcriptomic characterization reveals progression markers and essential pathways in clear cell renal cell carcinoma.

Identifying tumor-cell-specific markers and elucidating their epigenetic regulation and spatial heterogeneity provides mechanistic insights into cancer etiology. Here, we perform snRNA-seq and snATAC-seq in 34 and 28 human clear cell renal cell carcinoma (ccRCC) specimens, respectively, with matched bulk proteogenomics data. By identifying 20 tumor-specific markers through a multi-omics tiered approach, we reveal an association between higher ceruloplasmin (CP) expression and reduced survival. CP knockdown, combined with spatial transcriptomics, suggests a role for CP in regulating hyalinized stroma and tumor-stroma interactions in ccRCC. Intratumoral heterogeneity analysis portrays tumor cell-intrinsic inflammation and epithelial-mesenchymal transition (EMT) as two distinguishing features of tumor subpopulations. Finally, BAP1 mutations are associated with widespread reduction of chromatin accessibility, while PBRM1 mutations generally increase accessibility, with the former affecting five times more accessible peaks than the latter. These integrated analyses reveal the cellular architecture of ccRCC, providing insights into key markers and pathways in ccRCC tumorigenesis.

Histopathologic and proteogenomic heterogeneity reveals features of clear cell renal cell carcinoma aggressiveness.

Clear cell renal cell carcinomas (ccRCCs) represent ∼75% of RCC cases and account for most RCC-associated deaths. Inter- and intratumoral heterogeneity (ITH) results in varying prognosis and treatment outcomes. To obtain the most comprehensive profile of ccRCC, we perform integrative histopathologic, proteogenomic, and metabolomic analyses on 305 ccRCC tumor segments and 166 paired adjacent normal tissues from 213 cases. Combining histologic and molecular profiles reveals ITH in 90% of ccRCCs, with 50% demonstrating immune signature heterogeneity. High tumor grade, along with BAP1 mutation, genome instability, increased hypermethylation, and a specific protein glycosylation signature define a high-risk disease subset, where UCHL1 expression displays prognostic value. Single-nuclei RNA sequencing of the adverse sarcomatoid and rhabdoid phenotypes uncover gene signatures and potential insights into tumor evolution. In vitro cell line studies confirm the potential of inhibiting identified phosphoproteome targets. This study molecularly stratifies aggressive histopathologic subtypes that may inform more effective treatment strategies.

Neoplastic cell enrichment of tumor tissues using coring and laser microdissection for proteomic and genomic analyses of pancreatic ductal adenocarcinoma.

BACKGROUND: The identification of differentially expressed tumor-associated proteins and genomic alterations driving neoplasia is critical in the development of clinical assays to detect cancers and forms the foundation for understanding cancer biology. One of the challenges in the analysis of pancreatic ductal adenocarcinoma (PDAC) is the low neoplastic cellularity and heterogeneous composition of bulk tumors. To enrich neoplastic cells from bulk tumor tissue, coring, and laser microdissection (LMD) sampling techniques have been employed. In this study, we assessed the protein and KRAS mutation changes associated with samples obtained by these enrichment techniques and evaluated the fraction of neoplastic cells in PDAC for proteomic and genomic analyses. METHODS: Three fresh frozen PDAC tumors and their tumor-matched normal adjacent tissues (NATs) were obtained from three sampling techniques using bulk, coring, and LMD; and analyzed by TMT-based quantitative proteomics. The protein profiles and characterizations of differentially expressed proteins in three sampling groups were determined. These three PDACs and samples of five additional PDACs obtained by the same three sampling techniques were also subjected to genomic analysis to characterize KRAS mutations. RESULTS: The neoplastic cellularity of eight PDACs ranged from less than 10% to over 80% based on morphological review. Distinctive proteomic patterns and abundances of certain tumor-associated proteins were revealed when comparing the tumors and NATs by different sampling techniques. Coring and bulk tissues had comparable proteome profiles, while LMD samples had the most distinct proteome composition compared to bulk tissues. Further genomic analysis of bulk, cored, or LMD samples demonstrated that KRAS mutations were significantly enriched in LMD samples while coring was less effective in enriching for KRAS mutations when bulk tissues contained a relatively low neoplastic cellularity. CONCLUSIONS: In addition to bulk tissues, samples from LMD and coring techniques can be used for proteogenomic studies. The greatest enrichment of neoplastic cellularity is obtained with the LMD technique.

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

A proteogenomic portrait of lung squamous cell carcinoma.

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

Proteogenomic Characterization Reveals Therapeutic Vulnerabilities in Lung Adenocarcinoma.

To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.

Effects of prefrontal cortex and hippocampal NMDA NR1-subunit deletion on complex cognitive and social behaviors.

Glutamate N-methyl-D-aspartate receptors (NMDARs) in the medial prefrontal cortex (mPFC) and hippocampus may play an integral role in complex cognitive and social deficits associated with a number of psychiatric illnesses including autism, mood disorders, and schizophrenia. We used localized infusions of adeno-associated virus Cre-recombinase in adult, targeted knock-in mice with loxP sites flanking exons 11-22 of the NR1 gene to investigate the effects of chronic NMDAR dysfunction in the mPFC and CA3 hippocampus on cognitive and social behavior. A 5-choice serial reaction time task (5-CSRTT) was used to monitor aspects of cognitive function that included attention and response inhibition. Social behavior was assessed using Crowley׳s sociability and preference for social novelty protocol. Chronic NMDAR dysfunction localized to the anterior cingulate/prelimbic mPFC or dorsal CA3 hippocampus differentially affected the response inhibition and social interaction. mPFC NR1-deletion increased perseverative responding in the 5-CSRTT and enhanced preference for social novelty, whereas CA3 NR1-deletion increased premature responding in the 5-CSRTT and decreased social approach behavior. These findings suggest that mPFC and CA3 NMDARs play selective roles in regulating compulsive and impulsive behavior, respectively. Furthermore, these findings are consistent with emerging evidence that these behaviors are mediated by distinct, albeit overlapping, neural circuits. Our data also suggest that NMDARs in these regions uniquely contribute to the expression of normal social behavior. In this case, mPFC and CA3 NMDARs appear to inhibit and facilitate aspects of social interaction, respectively. The latter dissociation raises the possibility that distinct circuits contribute to the expression of social intrusiveness and impoverished social interaction.