Performed Gene Set Enrichment Analysis Research Articles

SNAP25 as a prognostic marker in transcriptome analysis of meningioma.

Meningiomas are the most common intracranial tumors and their diagnosis relies mostly on neuroimaging and histology. However, the histology grades cannot predict the outcome exactly and some meningiomas tend to recur after resection of even benign tumors. Therefore, it is necessary to explore prognostic and diagnostic molecular targets. Differential expression analysis between meningiomas and meninges was performed based on the merged data of GSE43290 and GSE84263. Next, we performed gene set enrichment analysis (GSEA), immune cell infiltration analysis, protein-protein interaction analysis, and survival analysis using public data. The expression level of Synaptosome-associated-protein-25kDa (SNAP25) was verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting in meningioma tissues. There were 263 upregulated and 592 downregulated genes identified in meningiomas by differential expression analysis. GSEA results revealed that meningiomas were negatively related to the pathway of soluble N-ethylmaleimide sensitive factor attachment protein receptor interactions in vascular transport and chemokine signaling. SNAP25 was characterized as a hub gene and downregulated in meningiomas. The Kaplan-Meier plot indicated that high expression of SNAP25 is a favorable factor. SNAP25 was downregulated and identified as a potential prognostic marker in meningioma.

Construction of a Myeloid Gene Signature Revealing Novel Therapeutic Targets for Myeloid Malignancies

FH, SL and BG equal contributors Myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML) exhibit shared hallmarks of hyperinflammation and aberrant clonal expansion. Chronic MPNs exhibit a propensity for transformation to secondary AML (sAML), which imparts a dismal prognosis with limited treatment options. However, the pathogenesis of leukemia transition for MPN and the bidirectional relationship between MPN and AML remain incompletely understood. In this study, we investigated commonly-featured genes and pathways of these two diseases to explore novel therapeutic strategies. To dissect the causal association between MPN and AML, we first performed two-sample bidirectional mendelian randomization (MR) analysis which revealed that MPN predicts the risk of AML across multiple statistical methods with consistent directionality and significance. Heterogeneity, pleiotropy, or outlier effects were ruled out by sensitivity analyses with no reverse causality from AML to MPN. To identify a potential myeloid signature, we estimated causal effects of 2,940 plasma proteins on MPN via a two-sample MR framework and identified 55 candidates significantly associated with increased risk of MPN (P < 0.05, OR > 1). Utilizing data from the BEAT-AML cohort, we further screened the prognostic value of these candidates using Cox and log-rank test survival analyses and identified a 4-gene myeloid signature comprising MPO, CDCP1, CRISP3 and DXCR, with each conferring prognostic significance individually. Our scRNA-seq results confirmed enrichment of myeloid signature genes in myeloid progenitor cells, with further elevation in MPN. Of note, expression of myeloid signature genes was induced by Jak2V617F and MPLW515L in Ba/F3 cells. We then investigated the combined prognostic significance of these four genes by constructing a risk score via LASSO regression modeling and divided patients into high- and low-risk groups accordingly. Cox analysis validated the risk score as an independent prognostic factor (HR: 3.01 (1.2 - 7.6), P = 0.019) and Kaplan-Meier survival analysis demonstrated that low-risk AML patients had a significantly better survival than high-risk AML patients with median survival of 21.9 months vs. 11.9 months (P < 0.0001). The prediction accuracy of the myeloid signature was successfully validated in the TCGA-LAML cohort. The risk score significantly correlated with several key clinical parameters, including age, platelet count, ELN2017 staging, monocyte percentage and bone marrow cellularity. To interrogate the underlying mechanism of the myeloid signature, we performed gene set enrichment analysis (GSEA) and observed enrichment of several inflammatory pathways in high-risk AML patients. A similar enrichment of inflammatory pathways in MPN samples compared to healthy donors observed in our previous scRNA-seq results reinforced hyperinflammation as a shared etiology of myeloid malignancies. To explore potential therapeutic strategies, we performed in silico drug sensitivity screening which predicted specific vulnerability of high-risk AML to compounds targeting PI3K/AKT/mTOR pathway signaling. Activation of PI3K/AKT/mTOR signaling in both MPN and AML samples observed across multiple single cell and bulk RNA-seq datasets further prompted us to evaluate targeted therapies against this pathway in myeloid malignancies. MPN and AML cells exhibited sensitivity to mTOR inhibitors as indicated by suppression of cellular proliferation, metabolism, colony formation, and inflammatory cytokine secretion in conjunction with induction of apoptosis. We further evaluated the therapeutic effects of mTOR inhibition in vivo via administration of omipalisib, a potent dual PI3K/mTOR inhibitor, to JAK2V617F knock-in mice. Omipalisib treatment significantly ameliorated features of myeloid malignancies including splenomegaly and leukocytosis which were both significantly reduced following 4 weeks of treatment, without affecting body weight. In summary, our MR analyses reveal that MPN predicts the risk of AML and enabled the construction of a novel myeloid signature which risk stratified AML patients across two different cohorts. We further demonstrate shared activation of PI3K/AKT/mTOR pathway across myeloid malignancies, with in vitro and in vivo data providing a rationale for therapeutic targeting of PI3K/AKT/mTOR pathway in these diseases.

A Novel Gene Signature Based on Immune Cell Infiltration Landscape Predicts Prognosis in Lung Adenocarcinoma Patients.

The tumor microenvironment (TME) is created by the tumor and dominated by tumor-induced interactions. Long-term survival of lung adenocarcinoma (LUAD) patients is strongly influenced by immune cell infiltration in TME. The current article intends to construct a gene signature from LUAD ICI for predicting patient outcomes. For the initial phase of the study, the TCGA-LUAD dataset was chosen as the training group for dataset selection. We found two datasets named GSE72094 and GSE68465 in the Gene Expression Omnibus (GEO) database for model validation. Unsupervised clustering was performed on the training cohort patients using the ICI profiles. We employed Kaplan-Meier estimators and univariate Cox proportional-hazard models to identify prognostic differentially expressed genes in immune cell infiltration (ICI) clusters. These prognostic genes are then used to develop a LASSO Cox model that generates a prognostic gene signature. Validation was performed using Kaplan-Meier estimation, Cox, and ROC analysis. Our signature and vital immune-relevant signatures were analyzed. Finally, we performed gene set enrichment analysis (GSEA) and immune infiltration analysis on our finding gene signature to further examine the functional mechanisms and immune cellular interactions. Our study found a sixteen-gene signature (EREG, HPGDS, TSPAN32, ACSM5, SFTPD, SCN7A, CCR2, S100P, KLK12, MS4A1, INHA, HOXB9, CYP4B1, SPOCK1, STAP1, and ACAP1) to be prognostic based on data from the training cohort. This prognostic signature was certified by Kaplan-Meier, Cox proportional-hazards, and ROC curves. 11/15 immune-relevant signatures were related to our signature. The GSEA results indicated our gene signature strongly correlates with immune-related pathways. Based on the immune infiltration analysis findings, it can be deduced that a significant portion of the prognostic significance of the signature can be attributed to resting mast cells. We used bioinformatics to determine a new, robust sixteen-gene signature. We also found that this signature's prognostic ability was closely related to the resting mast cell infiltration of LUAD patients.

PRMT5 maintains tumor stem cells to promote pediatric high-grade glioma tumorigenesis.

Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors with poor outcomes, for which no standard-of-care drug therapy currently exists. Through a knockdown screen for epigenetic regulators, we identified PRMT5 as essential for PHGG cell growth. We hypothesized that, similar to its effect in normal cells, PRMT5 promotes self-renewal of stem-like PHGG tumor initiating cells (TICs) essential for tumor growth. We conducted in vitro analyses, including limiting dilution studies of self-renewal, to determine the phenotypic effects of PRMT5 KD. We performed ChIP-Seq to identify PRMT5-mediated epigenetic changes and performed gene set enrichment analysis to identify pathways that PRMT5 regulates. Using an orthotopic xenograft model of PHGG, we tracked survival and histological characteristics resulting from PRMT5 KD or administration of a PRMT5 inhibitor ± radiation therapy (RT). In vitro, PRMT5 KD slowed cell cycle progression, tumor growth and self-renewal, and altered chromatin occupancy at genes associated with differentiation, tumor formation and growth. In vivo, PRMT5 KD increased survival and reduced tumor aggressiveness; however, pharmacological inhibition of PRMT5 with or without RT did not improve survival. PRMT5 KD epigenetically reduced TIC self-renewal, leading to increased survival in preclinical models. Pharmacological inhibition of PRMT5 enzymatic activity may have failed in vivo due to insufficient reduction of PRMT5 activity by chemical inhibition, or this failure may suggest that non-enzymatic activities of PRMT5 are more relevant. Implications: PRMT5 maintains and promotes the growth of stemlike cells that initiate and drive tumorigenesis in pediatric high grade glioma.

Proteomics and Incident Kidney Failure in Individuals With CKD: The African American Study of Kidney Disease and Hypertension and the Boston Kidney Biopsy Cohort

Individuals with chronic kidney disease (CKD) are at increased risk of morbidity and mortality, particularly as they progress to kidney failure. Identifying circulating proteins that underlie kidney failure development may guide the discovery of new targets for intervention. Prospective cohort. 703 African American Study of Kidney Disease and Hypertension (AASK) and 434 Boston Kidney Biopsy Cohort (BKBC) participants with baseline proteomics data. Circulating proteins measured using SomaScan. Kidney failure, defined as dialysis initiation or kidney transplantation. Using adjusted Cox models, we studied associations of 6,284 circulating proteins with kidney failure risk separately in AASK and BKBC and meta-analyzed results. We then performed gene set enrichment analyses to identify underlying perturbations in biological pathways. In separate data sets with kidney-tissue level gene expression, we ascertained dominant regions of expression and correlated kidney tubular gene expression with fibrosis and estimated glomerular filtration rate (eGFR). Over median follow-up periods of 8.8 and 3.1 years, 210 AASK (mean age: 55 years, 39% female, mean GFR: 46mL/min/1.73m2) and 115 BKBC (mean age: 54 years, 47% female, mean eGFR: 51mL/min/1.73m2) participants developed kidney failure, respectively. We identified 143 proteins that were associated with incident kidney failure, of which only 1 (Testican-2) had a lower risk. Notable proteins included those related to vascular permeability (endothelial cell-selective adhesion molecule), glomerulosclerosis (ephrin-A1), glomerular development (ephrin-B2), intracellular sorting/transport (vesicular integral-membrane protein VIP36), podocyte effacement (pigment epithelium-derived factor), complement activation (complement decay-accelerating factor), and fibrosis (ephrin-A1, ephrin-B2, and pigment epithelium-derived factor). Gene set enrichment analyses detected overrepresented pathways that could be related to CKD progression, such as ephrin signaling, cell-cell junctions, intracellular transport, immune response, cell proliferation, and apoptosis. At the kidney level, glomerular expression predominated for genes corresponding to circulating proteins of interest, and several gene expression levels were correlated with eGFR and/or fibrosis. Possible residual confounding. Multimodal data identified proteins and pathways associated with the development of kidney failure.

Establishment of the Prognostic Signature with Genes Related to G2/M Checkpoint in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality in the world. Prognostic indicators such as clinicopathological characteristics and single molecular signature are far from satisfactory in clinical practice. More and more researches have suggested that polygenic prognostic features could predict the prognosis of cancer more precisely than single genes nowadays. In this study, we performed gene set enrichment analysis (GSEA) to identify the sets of TCGA hallmark genes. Univariate Cox regression analysis was used to select preliminary genes, and then multivariate Cox regression analysis was used to identify genes associated with overall survival (OS). We also used Kaplan-Meier analysis and receiver operating characteristic (ROC) analysis to validate the prognostic gene signature. Lastly, qRT-PCR was used to evaluate the expression of these genes in clinical samples, and immunohistochemical staining was performed to confirm the signature. A 12-gene signature was finally built and the risk score was significantly associated with the survival of the patients. Subsequent validation by qRT-PCR and immunohistochemical staining in clinical specimens confirmed the value of the risk score in predicting the prognosis. We developed a 12-gene signature that could predict the prognosis of HCC patients. This signature is of high precision and would help identifying subgroups of HCC patients with high or low risk of unfavorable survival.

SNAI1: a key modulator of survival in lung squamous cell carcinoma and its association with metastasis

BackgroundSnail family zinc finger 1 (SNAI1) has been implicated in cancer progression and prognosis across various malignancies. This study aims to elucidate the prognostic significance of SNAI1 expression in Lung Squamous Cell Carcinoma (LUSC) using data from The Cancer Genome Atlas (TCGA) database.MethodsSNAI1 expression levels in LUSC patients were stratified using X-tile software to establish optimal cut-off values. Kaplan-Meier survival analysis was performed to assess the impact of SNAI1 expression on overall survival (OS). Univariate and multivariate Cox regression analyses were conducted to evaluate the prognostic value of SNAI1, considering clinical parameters such as age, clinical stage, and TNM classification. Additionally, we explored the interaction between SNAI1 expression and metastatic status, and performed Gene Set Enrichment Analysis (GSEA) to investigate associated cellular pathways. Correlations between SNAI1 and immune checkpoint molecules were also examined.ResultsKaplan-Meier analysis revealed significant differences in OS among high, medium, and low SNAI1 expression groups (p < 0.001), with median survival times of 1.6, 3.0, and 5.8 years, respectively. Dichotomizing patients into high and low SNAI1 expression groups confirmed that high SNAI1 expression was associated with significantly poorer OS (p < 0.001). SNAI1 remained an independent prognostic factor in multivariate analysis. High SNAI1 expression correlated with poorer survival outcomes regardless of metastatic status, and the combination of high SNAI1 expression and metastasis resulted in the poorest survival. GSEA identified significant associations between SNAI1 and inflammatory, immune response pathways. Positive correlations were observed between SNAI1 and key immune checkpoint molecules, suggesting an interplay with immune checkpoint mechanisms.ConclusionsHigh SNAI1 expression is a robust prognostic indicator of poor survival in LUSC, independent of other clinical factors. Its association with immune checkpoint molecules highlights its potential as a therapeutic target. These findings underscore the prognostic and therapeutic relevance of SNAI1 in LUSC and possibly other cancers. Further research is warranted to explore targeted therapies against SNAI1.

G2PDeep-v2: a web-based deep-learning framework for phenotype prediction and biomarker discovery using multi-omics data.

The G2PDeep-v2 server is a web-based platform powered by deep learning, for phenotype prediction and markers discovery from multi-omics data in any organisms including humans, plants, animals, and viruses. The server provides multiple services for researchers to create deep-learning models through an interactive interface and train these models using an automated hyperparameter tuning algorithm on high-performance computing resources. Users can visualize the results of phenotype and markers predictions and perform Gene Set Enrichment Analysis for the significant markers to provide insights into the molecular mechanisms underlying complex diseases and other biological processes. The G2PDeep-v2 server is publicly available at https://g2pdeep.org/.

Targeting PERK and GRP78 in colorectal cancer: Genetic insights and novel therapeutic approaches

Colorectal cancer (CRC) ranks among the leading causes of cancer-related deaths worldwide. Enhancing CRC diagnosis and prognosis requires the development of improved biomarkers and therapeutic targets. Emerging evidence suggests that the unfolded protein response (UPR) plays a pivotal role in CRC progression, presenting new opportunities for diagnosis, treatment, and prevention. This study hypothesizes that genetic variants in endoplasmic reticulum (ER) stress response genes influence CRC susceptibility. We examined the frequencies of SNPs in PERK (rs13045) and GRP78/BiP (rs430397) within a South Iranian cohort.We mapped the cellular and molecular features of PERK and GRP78 genes in colorectal cancer, observing their differential expressions in tumor and metastatic tissues. We constructed co-expression and protein-protein interaction networks and performed gene set enrichment analysis, highlighting autophagy as a significant pathway through KEGG. Furthermore, the study included 64 CRC patients and 60 control subjects. DNA extraction and genotyping were conducted using high-resolution melting (HRM) analysis. Significant differences in PERK and GRP78 expressions were observed between CRC tissues and controls. Variations in PERK and GRP78 genotypes were significantly correlated with CRC risk. Utilizing a Multi-Target Directed Ligands approach, a dual PERK/GRP78 inhibitor was designed and subjected to molecular modeling studies. Docking experiments indicated high-affinity binding between the proposed inhibitor and both genes, PERK and GRP78, suggesting a novel therapy for CRC.These findings highlight the importance of understanding genetic backgrounds in different populations to assess CRC risk. Polymorphisms in UPR signaling pathway elements may serve as potential markers for predicting CRC susceptibility, paving the way for personalized therapeutic strategies.

Predictive signature of murine and human host response to typical and atypical pneumonia

BackgroundPneumonia due to typical bacterial, atypical bacterial and viral pathogens can be difficult to clinically differentiate. Host response-based diagnostics are emerging as a complementary diagnostic strategy to pathogen detection.MethodsWe used...

Construction and validation of a TTN mutation associated immune prognostic model for evaluating immune microenvironment and outcomes of gastric cancer: An observational study.

Gastric cancer (GC) is a prevalent form of cancer worldwide, and TTN (titin) mutations are frequently observed in GC. However, the association between TTN mutations and immunotherapy for GC remains unclear, necessitating the development of novel prognostic models. The prognostic value and potential mechanisms of TTN in stomach adenocarcinoma were evaluated by TCGA (The Cancer Genome Atlas)-stomach adenocarcinoma cohort analysis, and an immune prognostic model was constructed based on TTN status. We validated it using the GSE84433 dataset. We performed Gene Set Enrichment Analysis and screened for differentially expressed genes, and used lasso (least absolute shrinkage and selection operator) regression analysis to screen for survival genes to construct a multifactorial survival model. In addition, we evaluated the relative proportions of 22 immune cells using the CIBERSORT algorithm for immunogenicity analysis. Finally, we constructed the nomogram integrating immune prognostic model and other clinical factors. GESA showed enrichment of immune-related phenotypes in patients with TTN mutations. We constructed an immune prognostic model based on 16 genes could identify gastric cancer patients with higher risk of poor prognosis. Immuno-microenvironmental analysis showed increased infiltration of naive B cells, plasma cells, and monocyte in high-risk patients. In addition, Nomo plots predicted the probability of 1-year, 3-year, and 5-year OS (overall survival) in GC patients, showing good predictive performance. In this study, we identified that TTN gene may be a potential clinical biomarker for GC and TTN mutations may be a predictor of immunotherapy in patients. We constructed and validated a new model for prognosis of GC patients based on immune characteristics associated with TTN mutations. This study may provide potential therapeutic strategies for gastric cancer.

Identification of key genes and pathways in duck fatty liver syndrome using gene set enrichment analysis

High-laying ducks are often fed high-energy, nutritious feeds to maintain high productivity, which predisposes them to lipid metabolism disorders and the development of fatty liver syndrome (FLS), which seriously affects production performance and has a substantial economic impact on the poultry industry. Therefore, it is necessary to elucidate the mechanisms underlying the development of fatty liver syndrome. In this study, seven Shan Partridge ducks, each with fatty liver syndrome and normal laying ducks, were selected, and Hematoxylin Eosin staining (HE staining), Masson staining, and transcriptome sequencing were performed on liver tissue. In addition to exploring key genes and pathways using conventional analysis methods, we constructed the first Kyoto Encyclopedia of Genes and Genomes (KEGG) database-based predefined gene set containing 12,764 pathways and 16,836 genes and further performed gene set enrichment analysis (GSEA) on the liver transcriptome data. Finally, key nodes and biological processes were identified via the protein-protein interaction (PPI) network. The results showed that the liver in the FL group exhibited steatosis and fibrosis, and a total of 3,663 genes with upregulated expression versus 2,296 downregulated genes were screened by conventional analysis. GSEA analysis and PPI network analysis revealed that the liver in the FL group exhibited disruption of the mitochondrial electron transport chain, leading to decreased oxidative phosphorylation and the secretion of excessive proinflammatory factors amid the continuous accumulation of lipids. Under continuous chronic inflammation, cell cycle arrest triggers apoptosis, while fibrosis becomes more severe, and procarcinogenic genes are activated, leading to the continuous development and deterioration of the liver. In conclusion, the predefined gene set constructed in this study can be used for GSEA, and the identified hub genes provide useful reference data and a solid foundation for the study of the genetic regulatory mechanism of fatty liver syndrome in ducks.

CDCA gene family promotes progression and prognosis in lung adenocarcinoma.

The cell division cycle-associated (CDCA) family participates in the cell cycle, and the dysregulation of its expression is associated with the development of several types of cancers. However, the roles of CDCAs in lung adenocarcinomas (LUAD) have not been investigated in systematic research. Using data retrieved from The Cancer Genome Atlas (TCGA), the expression of CDCAs in LUAD and normal tissues was compared, and survival analysis was performed using the data. Also, the correlation between clinical characteristics and the expression of CDCAs was assessed. Using data from cBioPortal, we investigated genetic alterations in CDCAs and their prognostic implications. Immunohistochemical analyses were performed to validate our findings from TCGA data. Following this, we created a risk score model to develop a nomogram. We also performed gene set enrichment analyses (GSEA), gene ontology, and KEGG pathway analysis. We used Timer to analyze the correlation between immune cell infiltration, tumor purity, and expression data. Our results indicated that all CDCAs were expressed at high levels in LUAD; this could be associated with poor overall survival, as indicated in TCGA data. Univariate and multivariate Cox analyses revealed that CDCA4/5 could serve as independent risk factors. The results of immunohistochemical analyses confirmed our results. Based on the estimation of expression levels, clinical characteristics, alterations, and immune infiltration, the low-risk group of CDCA4/5 had a better prognosis than the high-risk group. Immune therapy is also a potential treatment option. In conclusion, our findings indicate that CDCAs play important roles in LUAD, and CDCA4/5 can serve as diagnostic and prognostic biomarkers and therapeutic targets in LUAD.

Unveiling HOXB7 as a novel diagnostic and prognostic biomarker through pan-cancer computer screening

We attempted to investigate the role of HOXB7 in tumor progression and evolution by means of an extensive computer screening analysis of various cancer types. We performed univariate Cox regression and Kaplan-Meier survival analyses to assess the impact of HOXB7 on overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in different types of cancer. Furthermore, we examined the relationship between HOXB7 and several clinical features: tumor microenvironment, immune regulatory genes, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI). We performed gene set enrichment analysis to gain deeper insights into the potential molecular mechanisms of HOXB7, and validated our findings through functional assays in cells, including methyl thiazolyl tetrazolium cytotoxicity and Transwell invasion assays. HOXB7 expression was associated with different clinical characteristics in numerous malignancies. Higher HOXB7 expression was associated with worse OS, DSS, and PFI in some cancer types. In particular, HOXB7 expression was favorably associated with immune cell infiltration, immune regulatory genes, immunological checkpoints, TMB, and MSI in malignancies. Furthermore, we identified a strong link between copper death-associated gene expression and HOXB7 expression. According to the findings of this study, HOXB7 might serve as an appealing focus for tumor diagnosis and immunotherapy and a prospective indicator of prognosis.

Abstract PO5-24-11: An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma

Abstract Introduction: Although recent studies have shown that invasive lobular carcinoma (ILC) is associated with worse long-term outcomes compared to invasive breast cancer of no special type (NST), they are treated in a similar manner. Therefore, classification approaches to help understand the molecular characteristics associated with the different BC subtypes are needed to personalize therapy. Here, we describe an integrated analytical approach to comprehensively characterize ILC samples. Methods: We collected genomic and transcriptomic data for 16,087 ILC and NST samples, with clinical and pathological annotation, from 59 datasets and used an internally developed algorithm based on clustering performed after gene expression analysis to classify them into 5 intrinsic subtypes: Basal, Luminal A (LumA), Luminal B (LumB), HER2 high, and HER2 low. We performed gene set enrichment analyses and utilized the DESeq2 algorithm to perform differential expression analysis of RNA-seq data. We performed molecular grade subtyping using methods described by Antysheva et al. (Cancer Res., 2022). Samples that were listed as ILC or had an inactivated CDH1 gene by mutation, deletion or low expression (&amp;lt; −2.5 MAD after median scaling) were defined as having an ILC histomolecular subtype. Using methods described by Bagaev et. al. (Cancer Cell, 2021), 29 functional gene expression signatures were selected, and unsupervised dense Louvain clustering was performed to identify TME subtypes. The Mann-Whitney U test was used for statistical analysis. The tertiary lymphoid structures (TLS) gene signature was evaluated using methods described by Sautès-Fridman et al. (Nat. Rev. Cancer, 2019). Results: In the ILC samples (n=1,887), we identified LumA (46%), LumB (40%), Basal (8%,), HER2 high (3%), and HER2 low (3%) subtypes. In contrast, the NST samples were classified as LumB (41%), LumA (25%), Basal (19%), HER2 high (9%), and HER2 low (5%). Molecular grade subtyping revealed that 67% of ILC samples were low grade and 33% were high grade, compared to NST where 55% were high grade and 45% were low grade. Notably, among high-grade ILC, LumB (64%) and Basal (23%) subtypes were predominant. Key differentially expressed genes included downregulated CDH1 and upregulated MMP23B, IGF1, and TFAP2B across LumA and LumB ILC samples. Further analysis revealed downregulation of TACSTD2 and CDH3 in the Basal subtype for ILC samples compared to NST samples. Transcriptomic-based TME subtyping revealed distinct TME distributions across the BC subtypes and molecular grades for ILC samples (Table 1). In high-grade ILC LumB samples, 38% had a Desert (D; predictive of poor immunotherapy response) and 39% had an Immune-Enriched (IE; predictive of favorable immunotherapy response) TME. In low-grade ILC LumB samples we found that while 51% had a D TME, only 14% had an IE TME. We found an overrepresentation of the TLS signature [Solid TLS] in ILC compared to NST samples, and higher matrix remodeling and CAF signatures in LumA compared to the LumB subtype among low-grade ILC samples. Samples with high CD274 expression levels had higher percentages of T cells and M1 macrophages, and LumA samples had significantly higher (p &amp;lt; 0.001) CD274 expression compared to the other BC subtypes. Conclusion: These results provide in-depth molecular and tumor microenvironment characterization of ILC. Further optimization of this analytical approach could lead to the development of more effective therapeutic strategies. Table 1. ILC subtypes classification Citation Format: Jason Mouabbi, Konstantin Chernyshov, Oleg Baranov, Vladimir Kushnarev, Polina Turova, Anna Butusova, Sofia Menshikova, Jessica Brown, Nikita Kotlov, Patrick Clayton, Krystle Nomie, Nathan Fowler, Debu Tripathy. An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-11.

Regulation of TMEM100 expression by epigenetic modification, effects on proliferation and invasion of esophageal squamous carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy with a high morbidity and mortality rate. TMEM100 has been shown to be suppressor gene in a variety of tumors, but there are no reports on the role of TMEM100 in esophageal cancer (EC). To investigate epigenetic regulation of TMEM100 expression in ESCC and the effect of TMEM100 on ESCC proliferation and invasion. Firstly, we found the expression of TMEM100 in EC through The Cancer Genome Atlas database. The correlation between TMEM100 gene expression and the survival of patients with EC was further confirmed through Kaplan-Meier analysis. We then added the demethylating agent 5-AZA to ESCC cell lines to explore the regulation of TMEM100 expression by epigenetic modification. To observe the effect of TMEM100 expression on tumor proliferation and invasion by overexpressing TMEM100. Finally, we performed gene set enrichment analysis using the Kyoto Encyclopaedia of Genes and Genomes Orthology-Based Annotation System database to look for pathways that might be affected by TMEM100 and verified the effect of TMEM100 expression on the mitogen-activated protein kinases (MAPK) pathway. In the present study, by bioinformatic analysis we found that TMEM100 was lowly expressed in EC patients compared to normal subjects. Kaplan-meier survival analysis showed that low expression of TMEM100 was associated with poor prognosis in patients with EC. Then, we found that the demethylating agent 5-AZA resulted in increased expression of TMEM100 in ESCC cells [quantitative real-time PCR (qRT-PCR) and western blotting]. Subsequently, we confirmed that overexpression of TMEM100 leads to its increased expression in ESCC cells (qRT-PCR and western blotting). Overexpression of TMEM100 also inhibited proliferation, invasion and migration of ESCC cells (cell counting kit-8 and clone formation assays). Next, by enrichment analysis, we found that the gene set was significantly enriched in the MAPK signaling pathway. The involvement of TMEM100 in the regulation of MAPK signaling pathway in ESCC cell was subsequently verified by western blotting. TMEM100 is a suppressor gene in ESCC, and its low expression may lead to aberrant activation of the MAPK pathway. Promoter methylation may play a key role in regulating TMEM100 expression.

Unveiling the mitophagy puzzle in non-alcoholic fatty liver disease (NAFLD): Six hub genes for early diagnosis and immune modulatory roles

BackgroundNon-alcoholic fatty liver disease (NAFLD) stands as a predominant chronic liver ailment globally, yet its pathogenesis remains elusive. This study aims to identify Hub mitophagy-related genes (MRGs), and explore the underlying pathological mechanisms through which these hub genes regulate NAFLD. MethodsA total of 3 datasets were acquired from the GEO database and integrated to identify differentially expressed genes (DEGs) in NAFLD and perform Gene Set Enrichment Analysis (GSEA). By intersecting DEGs with MRGs, mitophagy-related differentially expressed genes (MRDEGs) were obtained. Then, hub MRGs with diagnostic biomarker capability for NAFLD were screened and a diagnostic prediction model was constructed and assessed using Nomogram, Decision Curve Analysis (DCA), and ROC curves. Functional enrichment analysis was conducted on the identified hub genes to explore their biological significance. Additionally, regulatory networks were constructed using databases. NAFLD was stratified into high and low-risk groups based on the Riskscore from the diagnostic prediction model. Furthermore, single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT algorithms were employed to analyze immune cell infiltration patterns and the relationship between Hub MRGs and immune cells. ResultsThe integrated dataset comprised 122 NAFLD samples and 31 control samples. After screening, 18 MRDEGs were identified. Subsequently, six hub MRGs (NR4A1, PPP2R2A, P4HA1, TUBB6, DUSP1, NAMPT) with diagnostic potential were selected through WGCNA, logistic regression, SVM, RF, and LASSO models, all significantly downregulated in NAFLD samples compared to the control group. A diagnostic prediction model based on these six genes demonstrated robust predictive performance. Functional enrichment analysis of the six hub genes revealed involvement in processes such as protein phosphorylation or dephosphorylation. Correlation analysis demonstrated a significant association between hub MRGs and infiltrating immune cells. ConclusionWe identified six hub MRGs in NAFLD and constructed a diagnostic prediction model based on these six genes, applicable for early NAFLD diagnosis. These genes may participate in regulating NAFLD progression through the modulation of mitophagy and immune activation. Our findings may contribute to subsequent clinical and basic research on NAFLD.

Abstract 1107: Prospective observational study to evaluate the efficacy of oxygen saturation endoscopic imaging for radiotherapeutic response in head and neck squamous cell carcinoma

Abstract Background: Radiotherapy (RT) has a pivotal role in the management of head and neck squamous cell carcinoma (HNSCC), providing effective tumor control and functional preservation. Hypoxia is a cause of radioresistance in HNSCC. We have developed an oxygen saturation endoscopic imaging (OXEI) method to assess real-time tissue oxygenation levels. However, the clinical significance of OXEI in patients with HNSCC receiving RT remains unclear. We evaluated the association between pretreatment tumor oxygen saturation (StO2) quantified by OXEI and clinical outcomes of patients with HNSCC receiving RT. Methods: We prospectively recruited patients with HNSCC scheduled to receive RT at 2 institutions. The primary endpoint was the association between pretreatment tumor StO2 and complete response (CR) rate 8 weeks after completion of RT. Secondary endpoints evaluated the impact of StO2 on 1-year overall survival (OS) and progression-free survival (PFS) rates after RT initiation. We also evaluated the cumulative incidence of local failure (LF). To explore the relationship between tumor hypoxia assessed by OXEI and gene expression profile, we performed RNA sequencing of 4 tumor samples collected from 2 patients (2 samples/patient) before RT. The hypoxia score for each sample was calculated by gene set variation analysis based on HNSCC mRNA-based hypoxia signatures. We performed gene set enrichment analysis (GSEA) to evaluate whether the biological pathways (Hallmark, Reactome, and KEGG gene sets) showed significant associations with tumor hypoxia. Results: Of the 50 patients enrolled from March 2019 to February 2022, we analyzed 37 who underwent RT and OXEI. Thirty-six (97%) patients received current platinum-based chemotherapy. The median (range) pretreatment tumor StO2 of 60 (45-84)% was lower than that of normal mucosa, 66 (56-84)% (P &amp;lt; 0.01). Patients with tumor StO2 &amp;lt; 60% were classified as the hypoxia group (n =19), and those with tumor StO2 ≥ 60% as the non-hypoxia group (n =18). The CR rate 8 weeks after RT was not significantly different between the hypoxia and non-hypoxia groups (84% vs. 100%, respectively; P = 0.23). After a median (range) follow-up of 33 (9-45) months, the hypoxia group had worse 1-year PFS (74% vs. 94%, P = 0.017) and OS (89% vs. 100%, P = 0.015) and higher LF rates (21% vs. 6%, P = 0.036) compared with the non-hypoxia group. RNA sequencing confirmed that tumor StO2 reflected expression-based hypoxia scores. GSEA showed upregulation of reactive oxygen species (ROS) and KEAP1-NFE2L2 pathways in hypoxic samples (StO2 of 59%) compared with non-hypoxic samples (StO2 of 70%; adjusted P &amp;lt; 0.01), consistent with the fact that hypoxic stress increases ROS production. Conclusion: Our findings suggest that pretreatment tumor StO2 quantified by OXEI correlates with 1-year PFS, OS, and LF rates of patients with HNSCC receiving RT. Citation Format: Kento Tomizawa, Atsushi Motegi, Hidenari Hirata, Hiroki Yamashita, Hironori Sunakawa, Wataru Okano, Tomohiro Enokida, Takeshi Fujisawa, Sadamoto Zenda, Masaki Nakamura, Hidehiro Hojo, Seiichiro Abe, Madoka Sakuramachi, Hiroshi Igaki, Yutaka Saito, Kazuto Matsuura, Makoto Tahara, Tomonori Yano, Tetsuo Akimoto. Prospective observational study to evaluate the efficacy of oxygen saturation endoscopic imaging for radiotherapeutic response in head and neck squamous cell carcinoma [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 1107.

Abstract 1254: Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma

Abstract Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. Previous study has shown that homologous recombination genes, BRCA1/2-unmutated HGSC patients possess the potential to be in refractory condition, and their primary response to chemotherapy was heterogenous; thus, there can be subtype-specific determinant factors hatching refractoriness in BRCA1/2-unmutated HGSC patients. To explore the determinant factors, we performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients (p-value &amp;lt; 0.001), and eventually, pathway clustering analysis by variational Bayesian Gaussian mixture model discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1; as a supplement, the propriety of these genetic alterations to develop HGSC models was backed up by genetic profiles of high-grade serous ovarian cancer patients in AACR Project GENIE. After that, we knocked out Nf1 and Pten whose deletions were detected relatively with high frequency in HGSC to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes such as poor survival (p-value &amp;lt; 0.01) and high growth rate (p-value &amp;lt; 0.001). Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents like carboplatin (p-value &amp;lt; 0.001), paclitaxel (p-value &amp;lt; 0.01), and olaparib (p-value &amp;lt; 0.01). To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Citation Format: Tomohiro Tamura, Kenta Masuda, Shimpei Nagai, Keiyo Imaeda, Juntaro Yamasaki, Eiji Sugihara, Hiroyuki Nobusue, Yuji Otsuki, Rui Yamaguchi, Kazuhiro Sakurada, Wataru Yamagami, Hideyuki Saya, Osamu Nagano. Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma [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 1254.

Abstract 7060: Mitochondrial metabolism and racial disparity of bladder cancer

Abstract Bladder cancer mortality is significantly higher in African American patients compared to their European American counterparts. This inferior survival of African American cancer patients is driven at least in part by distinctive intrinsic tumor biology, more specifically altered metabolic activity. However, underlying molecular mechanism for the African American bladder cancer mortality are largely unexplored. Our pioneering metabolomic findings demonstrate alterations in metabolic pathways between African American and European American bladder cancer, specifically those involved in driving energy metabolism. Along these line, we observed increased electron transport chain activity and ATP production in African American bladder cancer could be a result of distinctive rewiring of energy metabolism compared to their European American counterparts. We further performed gene set enrichment analysis by mapping metabolites to genes and observed that oxidative phosphorylation pathway was uniquely enriched in African American tumors compared to European American tumors. In addition, from in vitro assays, mitochondrial complex protein and activity were altered in African American bladder cancer. To determine the role of glutamine mediated increased electron transport chain activity and ATP production in African American bladder cancer, we evaluated the metabolic flux using 13C labeled tracers in both African American and European American bladder cancer cell lines. Metabolic flux analysis revealed that African American cell lines are more glutamine dependent and that producing increased tricarboxylic acid intermediates. Overall, our data indicate that elevated mitochondrial metabolism and oxidative phosphorylation driven by enhanced glutaminolysis may facilitate African American bladder cancer progression. These findings provide the rationale to use mitochondrial-specific inhibitors to target at least a subset of African American bladder cancer patients. Citation Format: Karthik Reddy Kami Reddy, Jun Hyoung Park, Roni J. Bollag, Martha K. Terris, Seth P. Lerner, Minhaj Siddiqui, Yair Lotan, Jianjun Gao, Benny Abraham Kaipparettu, Nagireddy Putluri. Mitochondrial metabolism and racial disparity of bladder cancer [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 7060.