Comprehensive Expression Analysis Research Articles

Evaluation of the Immunohistochemical Scoring System of CDX2 Expression as a Prognostic Biomarker in Colon Cancer.

Encoded by the CDX2 homeobox gene, the CDX2 protein assumes the role of a pivotal transcription factor localized within the nucleus of intestinal epithelial cells, orchestrating the delicate equilibrium of intestinal physiology while intricately guiding the precise development and differentiation of epithelial tissue. Emerging research has unveiled that positive immunohistochemical expression of this protein shows that the CDX2 gene exerts a potent suppressive impact on tumor advancement in colorectal cancer, impeding the proliferation and distant dissemination of tumor cells, while the inhibition or suppression of CDX2 frequently correlates with aggressive behavior in colorectal cancer. In this study, we conducted an immunohistochemical assessment of CDX2 expression on a cohort of 43 intraoperatively obtained tumor specimens from patients diagnosed with colon cancer at Colțea Clinical Hospital in Bucharest, between April 2019 and December 2023. Additionally, we shed light on the morphological diversity within colon tumors, uncovering varying differentiation grades within the same tumor, reflecting the variations in CDX2 expression as well as the genetic complexity underlying these tumors. Based on the findings, we developed an innovative immunohistochemical scoring system that addresses the heterogeneous nature of colon tumors. Comprehensive statistical analysis of CDX2 immunohistochemical expression unveiled significant correlations with known histopathological parameters such as tumor differentiation grades (p-value = 0.011) and tumor budding score (p-value = 0.002), providing intriguing insights into the complex involvement of the CDX2 gene in orchestrating tumor progression through modulation of differentiation processes, and highlighting its role in metastatic predisposition. The compelling correlation identified between CDX2 expression and conventional histopathological parameters emphasizes the prognostic significance of the CDX2 biomarker in colon cancer. Moreover, our novel immunohistochemical scoring system reveals a distinct subset of colon tumors exhibiting reserved prognostic outcomes, distinguished by their "mosaic" CDX2 expression pattern.

Comprehensive analysis of differential long non-coding RNA and messenger RNA expression in cholelithiasis using high-throughput sequencing and bioinformatics.

The etiology of gallstone disease (GSD) has not been fully elucidated. Consequently, the primary objective of this study was to scrutinize and provisionally authenticate the distinctive expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in GSD. RiboNucleic Acid (RNA) sequencing was used on four paired human gallbladder samples for the purpose of this study. Differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were identified and subjected to analysis of their biological functions. The Pearson's correlation coefficients between DElncRNAs and DEmRNAs were computed to construct a co-expression network delineating their associations. Furthermore, both cis- and trans-regulatory networks of selected lncRNAs were established and visualized. Additionally, a competing endogenous RNA (ceRNA) regulatory network was constructed. To validate the RNA-sequencing data, we performed a Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) on 10 paired human gallbladder samples, assessing the expressions of the top 4 DEmRNAs and DElncRNAs in gallstone and control samples. A total of 934 DEmRNAs and 304DElncRNAs were successfully identified. Functional enrichment analysis indicated a predominant involvement in metabolic-related biological functions. Correlation analysis revealed a strong association between the expressions of 597 DEmRNAs and 194 DElncRNAs. Subsequently, both a cis-lncRNA-mRNA and a trans-lncRNA-Transcription Factor (TF)-mRNA regulatory network were meticulously constructed. Additionally, a ceRNA network, comprising of 24 DElncRNAs, 201 DEmRNAs, and 120 predicted miRNAs, was established. Furthermore, using RT-qPCR, we observed significant upregulation of AC004692.4, HECW1-IT1, SFRP4, and COMP, while LINC01564, SLC26A3, RP1-27K12.2, and GSTA2 exhibited marked downregulation in gallstone samples. Importantly, these findings were consistent with the sequencing. We conducted a screening process to identify DElncRNAs and DEmRNAs in GSD. This approach contributes to a deeper understanding of the genetic factors involved in the etiology of gallstones.

Comprehensive microRNA expression analysis of pediatric gonadal germ cell tumors: unveiling novel biomarkers and signatures.

microRNAs (miRNAs) are small endogenous noncoding RNAs, and alterations in their expression may contribute to oncogenesis. Discovering a unique miRNA pattern holds the potential for early detection and novel treatment possibilities in cancer. This study aimed to evaluate miRNA expression in pediatric patients with gonadal germ cell tumors (GCTs), focusing on characterizing the miRNA profiles of each histological subtype and identifying a distinct histological miRNA signature for a total of 42 samples of pediatric gonadal GCTs. The analysis revealed distinct miRNA expression profiles for all histological types, regardless of the primary site. We identified specific miRNA expression signatures for each histological type, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma, compared to healthy controls. Furthermore, we identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, and miR-367-3p) showing significant overexpression. Notably, miR-302b-3p exhibited a significant association with all the evaluated clinical features. Our findings suggest that miRNAs have the potential to aid in the diagnosis, prognosis, and management of patients with malignant GCTs.

Comprehensive Analysis of CXCR4, JUNB, and PD-L1 Expression in Circulating Tumor Cells (CTCs) from Prostate Cancer Patients.

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance.

Abstract PO4-24-02: Comprehensive Characterization of BCL2 Family Genes in Metaplastic Triple Negative Breast Cancer

Abstract Background: Metaplastic Breast Cancer (MBC) is rare (0.2-5%), aggressive form of BC characterized by chemotherapy resistance and has worse outcomes in comparison to other BC subtypes. Resistance to chemotherapeutic agents is related to defects in intact intrinsic apoptosis pathway and the BCL2 family of proteins are the central regulators of this pathway. Majority of MBC have triple-negative receptor status and have no standard therapeutic approach and validated prognostic markers. Here, we examine the association of BCL2 family pro-apoptotic and anti-apoptotic genes expression with metaplastic TNBC (MTNBC) patient survival. Methods: 13,036 BC samples (MTNBC, n=102; metaplastic non-TNBC, n=20) were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. MTNBC with PMAIP1-high(H) and -low(L) expression was classified by top and bottom quartile, respectively. Pathway enrichment was determined by GSEA (Broad Inst). Real world overall survival (OS) was extracted from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: MTNBC had enrichment of apoptosis (NES: 1.4, FDR: 0.05), glycolysis (NES: 1.48, FDR: 0.03), PI3K/AKT/mTOR signaling (NES: 1.43, FDR: 0.05), P53 (NES: 1.51, FDR: 0.06), NOTCH signaling (NES: 1.39, FDR: 0.05), TGFβ signaling (NES: 1.41, FDR: 0.05), WNTβ catenin signaling (NES: 1.41, FDR: 0.05) and IL2/STAT5 signaling (NES: 1.33, FDR: 0.09) pathways compared to metaplastic non-TNBC. MTNBC had higher expression of BCL2 pro-apoptotic genes BAX (Fold Change (FC): 1.6), BAK1 (FC: 1.3), BID (FC: 2), BAD (FC: 1.6), BCL2L11 (FC: 2.5) and BBC3 (FC: 1.5), and anti-apoptotic genes BCL2L1 (FC: 1.6) and BCL2L2 (FC: 1.3) (all p < 0.05) compared to metaplastic non-TNBC. Higher PMAIP1 gene expression was associated with worse MTNBC patient survival (mOS: 14.3 month; HR: 0.37; 95% CI 0.19-1.41; p=0.002) (Table 1), but not in metaplastic non-TNBC (mOS: Inf; HR: 1.96; 95% CI 0.64-6.02; p=0.23). PMAIP1-H MTNBC had higher expression of immune checkpoint genes CD274 (FC: 2.8), PDCD1 (FC: 2.2), TIM3 (FC: 1.8), LAG3 (FC: 2.1) and IDO1 (FC: 5.7) (all p < 0.05), compared to PMAIP1-L MTNBC. PMAIP1-H MTNBC had higher frequency of IHC-PD-L1 positivity (68.4% vs 14.3%, p < 0.05). PMAIP1-H MTNBC had higher expression of stem cell related genes CD44 (FC: 1.7), ALDH1A2 (FC: 2.2), ALDH1A3 (FC: 2.6), SOX2 (FC: 5.21) and NANOG (FC: 2.13) (all p < 0.05) compared to PMAIP1-L MTNBC. Conclusion: This is the first comprehensive analysis of expression and prognostic role of BCL2 family proteins in MBC. Our data suggest a strong association of higher expression of PMAIP1 with worse MTNBC patient survival, potentially attributed to higher immune checkpoint, stem cell-related genes expression, and higher frequency of PD-L1 positivity in PMAIP1-H tumors. These findings indicate PMAIP1 as a potential prognostic biomarker candidate in MTNBC but needs further validation in large prospective studies Table 1. Overall survival of metaplastic TNBC patient based on BCL2 family gene expression. Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Milan Radovich, George Sledge Jr, Dario Trapani, Evanthia Roussos Torres, Stephanie Graff, Asher Chanan-Khan. Comprehensive Characterization of BCL2 Family Genes in Metaplastic Triple Negative Breast Cancer [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 PO4-24-02.

Comprehensive analysis of the differential expression of mRNAs, lncRNAs, and miRNAs in Zi goose testis with high and low sperm mobility

Comprehensive analysis of the differential expression of mRNAs, lncRNAs, and miRNAs in Zi goose testis with high and low sperm mobility

Dysregulation of phosphoenolpyruvate carboxykinase in cancers: A comprehensive analysis

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis, glycolysis, and the tricarboxylic acid cycle by converting oxaloacetate into phosphoenolpyruvate. Two distinct isoforms of PEPCK, specifically cytosolic PCK1 and mitochondrial PCK2, have been identified. Nevertheless, the comprehensive understanding of their dysregulation in pan-cancer and their potential mechanism contributing to signaling transduction pathways remains elusive. MethodsWe conducted comprehensive analyses of PEPCK gene expression across 33 diverse cancer types using data from The Cancer Genome Atlas (TCGA). Multiple public databases such as HPA, TIMER 2.0, GEPIA2, cBioPortal, UALCAN, CancerSEA, and String were used to investigate protein levels, prognostic significance, clinical associations, genetic mutations, immune cell infiltration, single-cell sequencing, and functional enrichment analysis in patients with pan-cancer. PEPCK expression was analyzed about different clinical and genetic factors of patients using data from TCGA, GEO, and CGGA databases. Furthermore, the role of PCK2 in Glioma was examined using both in vitro and in vivo experiments. ResultsThe analysis we conducted revealed that the expression of PEPCK is involved in both clinical outcomes and immune cell infiltration. Initially, we verified the high expression of PCK2 in GBM cells and its role in metabolic reprogramming and proliferation in GBM. ConclusionOur study showed a correlation between PEPCK (PCK1 and PCK2) expression with clinical prognosis, gene mutation, and immune infiltrates. These findings identified two possible predictive biomarkers across different cancer types, as well as a comprehensive analysis of PCK2 expression in various tumors, with a focus on GBM.

Comprehensive genomic characterization and expression analysis of calreticulin gene family in tomato.

Calreticulin (CRT) is a calcium-binding endoplasmic reticulum (ER) protein that has been identified for multiple cellular processes, including protein folding, regulation of gene expression, calcium (Ca2+) storage and signaling, regeneration, and stress responses. However, the lack of information about this protein family in tomato species highlights the importance of functional characterization. In the current study, 21 CRTs were identified in four tomato species using the most recent genomic data and performed comprehensive bioinformatics and SlCRT expression in various tissues and treatments. In the bioinformatics analysis, we described the physiochemical properties, phylogeny, subcellular positions, chromosomal location, promoter analysis, gene structure, motif distribution, protein structure and protein interaction. The phylogenetic analysis classified the CRTs into three groups, consensus with the gene architecture and conserved motif analyses. Protein structure analysis revealed that the calreticulin domain is highly conserved among different tomato species and phylogenetic groups. The cis-acting elements and protein interaction analysis indicate that CRTs are involved in various developmental and stress response mechanisms. The cultivated and wild tomato species exhibited similar gene mapping on chromosomes, and synteny analysis proposed that segmental duplication plays an important role in the evolution of the CRTs family with negative selection pressure. RNA-seq data analysis showed that SlCRTs were differentially expressed in different tissues, signifying the role of calreticulin genes in tomato growth and development. qRT-PCR expression profiling showed that all SlCRTs except SlCRT5 were upregulated under PEG (polyethylene glycol) induced drought stress and abscisic acid (ABA) treatment and SlCRT2 and SlCRT3 were upregulated under salt stress. Overall, the results of the study provide information for further investigation of the functional characterization of the CRT genes in tomato.

Identification and characterization of two O-methyltransferases involved in methylated 2-(2-phenethyl) chromones biosynthesis in agarwood.

The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-methyltransferases (OMTs) are necessary for the methylated PECs biosynthesis, however, there is little information known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PECs content revealed that the AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. The vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl) chromone. The transient overexpression experiment in Qi-Nan revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.

Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencingof gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPRinterference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.

Value of Non-Coding RNA Expression in Biofluids to Identify Patients at Low Risk of Pathologies Associated with Pregnancy.

Pregnancy-related complications (PRC) impact maternal and fetal morbidity and mortality and place a huge burden on healthcare systems. Thus, effective diagnostic screening strategies are crucial. Currently, national and international guidelines define patients at low risk of PRC exclusively based on their history, thus excluding the possibility of identifying patients with de novo risk (patients without a history of disease), which represents most women. In this setting, previous studies have underlined the potential contribution of non-coding RNAs (ncRNAs) to detect patients at risk of PRC. However, placenta biopsies or cord blood samples are required, which are not simple procedures. Our review explores the potential of ncRNAs in biofluids (fluids that are excreted, secreted, or developed because of a physiological or pathological process) as biomarkers for identifying patients with low-risk pregnancies. Beyond the regulatory roles of ncRNAs in placental development and vascular remodeling, we investigated their specific expressions in biofluids to determine favorable pregnancy outcomes as well as the most frequent pathologies of pregnant women. We report distinct ncRNA panels associated with PRC based on omics technologies and subsequently define patients at low risk. We present a comprehensive analysis of ncRNA expression in biofluids, including those using next-generation sequencing, shedding light on their predictive value in clinical practice. In conclusion, this paper underscores the emerging significance of ncRNAs in biofluids as promising biomarkers for risk stratification in PRC. The investigation of ncRNA expression patterns and their potential clinical applications is of diagnostic, prognostic, and theragnostic value and paves the way for innovative approaches to improve prenatal care and maternal and fetal outcomes.

Comprehensive Genome-Wide Natural Variation and Expression Analysis of Tubby-like Proteins Gene Family in Brachypodium distachyon.

The Tubby-like proteins (TLPs) gene family is a group of transcription factors found in both animals and plants. In this study, we identified twelve B. distachyon TLPs, divided into six groups based on conserved domains and evolutionary relationships. We predicted cis-regulatory elements involved in light, hormone, and biotic and abiotic stresses. The expression patterns in response to light and hormones revealed that BdTLP3, 4, 7, and 14 are involved in light responses, and BdTLP1 is involved in ABA responses. Furthermore, BdTLP2, 7, 9, and 13 are expressed throughout vegetative and reproductive stages, whereas BdTLP1, 3, 5, and 14 are expressed at germinating grains and early vegetative development, and BdTLP4, 6, 8, and 10 are expressed at the early reproduction stage. The natural variation in the eleven most diverged B. distachyon lines revealed high conservation levels of BdTLP1-6 to high variation in BdTLP7-14 proteins. Based on diversifying selection, we identified amino acids in BdTLP1, 3, 8, and 13, potentially substantially affecting protein functions. This analysis provided valuable information for further functional studies to understand the regulation, pathways involved, and mechanism of BdTLPs.

Abstract 6991: Transcriptomic profiling of human and mouse models highlights YAP pathway hyperactivation as the underlying mechanism of the biological and clinical aggressiveness of Numb-defective bladder cancer

Abstract In this study, we identify a previously uncharacterized role for the tumor suppressor, Numb, in bladder tumorigenesis, defining new predictive biomarkers and potentially actionable targets for the treatment of non-muscle-invasive bladder cancers. We conducted a comprehensive IHC analysis of Numb expression leveraging two retrospective longitudinal cohorts comprising 356 post-cystectomy muscle-invasive bladder cancer (MIBC) patients and 77 high-grade non-muscle-invasive (NMIBC) patients with transurethral resections. We found that Numb expression is frequently deregulated in the tumor compared to the adjacent normal urothelium, with NumbLow status correlating with poorer prognosis both in MIBC, associated with advanced stage at diagnosis, vascular invasion, and worse overall survival, and in NMIBC, where a NumbLow status associates with a higher rate of progression to life-threatening MIBC disease. In a transgenic mouse model with targeted deletion of the Numb gene in the basal layer of the bladder mucosa, we observed that loss of Numb per se is sufficient to drive malignant transformation of the normal urothelium and accelerates tumorigenesis in carcinogen-induced models. Integrative transcriptomic analysis of human and mouse Numb-deficient BC models revealed consistent dysregulation of the YAP1/TAZ1 inhibitory Hippo pathway, identifying YAP1 transcriptional hyperactivation as the most distinguishing trait downstream of NUMB loss. Through genetic and pharmacological functional inhibition studies in relevant mouse and human BC cell line-based models, we provide proof-of-evidence that interfering with YAP pathway activation at multiple upstream levels of the Hippo cascade reverts the aggressive Numb loss-associated tumor phenotypes, in particular growth and invasive/migratory potential, thereby highlighting the YAP pathway as a potential therapeutical vulnerability for the treatment of clinically aggressive Numb-deficient human BC. We also identified a gene signature of early BC depending on loss of Numb that we extensively validated for its potential as a predictor of NMIBC to MIBC progression, arguing for its clinical application as a biomarker for NIMBC patient stratification for targeted anti-YAP signaling treatment to prevent MIBC transition. In summary, our study identifies a previously uncharacterized role of Numb in bladder cancer progression, highlighting its potential as a predictive biomarker of NMIBC to MIBC transition and response to targeted therapies, thereby advancing a framework for precision oncology for NMIBC patients at high risk of progression to life-threatening MIBC disease. Citation Format: Francesco Antonio Tucci, Rosa Pennisi, Maria Grazia Filippone, Roberta Bonfanti, Francesco Romeo, Damiano Rigiracciolo, Stefano Freddi, Francesca Sanguedolce, Giuseppe Renne, Giancarlo Pruneri, Gianluca Vago, Daniela Tosoni, Salvatore Pece. Transcriptomic profiling of human and mouse models highlights YAP pathway hyperactivation as the underlying mechanism of the biological and clinical aggressiveness of Numb-defective 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 6991.

Abstract 5627: PPARG-high circulating monocytes exhibit an immunosuppressive phenotype in urothelial cancer patients treated with anti-PD1

Abstract Background: PPARG is expressed in a variety of immune cells such as monocytes, macrophages, and lymphocytes, where it plays a role in their maturation and function. PPARG has been implicated in immune mediated resistance but to date, a comprehensive analysis of PPARG expression in peripheral blood mononuclear cells (PBMCs) in urothelial cancer (UC) patients is lacking. In this study, we identified the immunophenotypes associated with PPARG expression in UC patients treated with anti-PD1 therapy. Methods: We performed single-cell RNA sequencing of PBMCs from 5 UC patients and 3 normal healthy volunteers (NHV). Real-world samples (Discovery Life Sciences) were collected from patients who received prior Pembrolizumab. Time post-treatment to collection ranged from 46 to 307 days. An average of 9,500 cells were recovered per donor reflecting an ~80% QC pass rate. Count matrices were generated using 10x Genomics Cell Ranger pipeline (v7.0.0; GRCh38 reference). Downstream data processing, analysis and visualization were done on Cellenics software. Results: We evaluated 75,725 cells and identified 9 distinct cell populations. The proportion of T-cells was significantly decreased in UC samples compared to NHVs (40% vs 60%, p=0.03). Highest PPARG expression was observed on Classical Monocytes (CMs) cell population. PPARG expression was higher on CMs in UC compared to NHV samples (1.74 vs 0.11 logcounts; padj<0.001). Unsupervised clustering of all monocytes revealed two distinct CM clusters. In cluster A, 95% of the cells originated from UC samples, while most cells in cluster B (81%) originated from NHV samples. PPARG was the top differentially expressed gene upregulated in cluster A (PPARG-high) vs cluster B (PPARG-low) (1.89 vs 0.23 logcounts; padj<0.001). PPARG-high CMs are characterized by a gene signature inclusive of GPRIN3, RGCC, MAFB and IRF2BP, which have been shown to be upregulated in M2 polarized macrophages [1]. Moreover, pathway analysis using GSEA highlighted that PPARG-high CMs are enriched for TREM1 signaling genes (padj<0.001), whose high expression has been associated with immunosuppressive phenotypes in myeloid cells [2] and shorter survival of patients across other cancers [3]. Conclusion: PPARG-high circulating CMs in UC patients exhibit a transcriptomic profile associated with immunosuppression and M2 macrophage polarization. FX-909, a first-in-class PPARG inverse agonist, is being evaluated in the clinic and the study includes an exploratory biomarker approach to assess the potential effect of PPARG inhibition on the CM immune-profile of advanced UC patients enrolled in the Ph1 study [4].

A comprehensive analysis of CD47 expression in various histological subtypes of soft tissue sarcoma: exploring novel opportunities for macrophage-directed treatments

PurposeThe CD47 molecule, often referred to as the “do not eat me” signal, is frequently overexpressed in tumor cells. This signaling pathway limits phagocytosis by macrophages. Our objective was to determine CD47 abundance in various soft tissue sarcomas (STS) to investigate whether it could serve as a potential evasion mechanism for tumor cells. Additionally, we aimed to assess the prognostic value of CD47 expression by examining its association with different clinicopathological factors. This study aimed to elucidate the significance of CD47 in the context of emerging anti-tumor targeting approaches.MethodsIn this retrospective study, formalin-fixed paraffine-embedded (FFPE) tumor tissues of 55 treatment-naïve patients were evaluated by immunohistochemistry for the abundance of CD47 molecule on tumor cells. The categorization of CD47 positivity was as follows: 0 (no staining of tumor cells), 1 + (less than 1/3 of tumor area positive), 2 + (between 1/3 and 2/3 of tumor area positive), and 3 + (more than 2/3 of tumor area positive for CD47). Next, we compared CD47 abundance between different tumor grades (G1–3). We used Kaplan–Meier survival curves with log-rank test to analyze the differences in survival between patients with different CD47 expression. Moreover, we performed Cox proportional hazards regression model to evaluate the clinical significance of CD47.ResultsCD47 is widely prevalent across distinct STS subtypes. More than 80% of high grade undifferentiated pleiomorphic sarcoma (UPS), 70% of myxofibrosarcoma (MFS) and more than 60% of liposarcoma (LPS) samples displayed a pattern of moderate-to-diffuse positivity. This phenomenon remains consistent regardless of the tumor grade. However, there was a tendency for higher CD47 expression levels in the G3 group compared to the combined G1 + G2 groups when all LPS, MFS, and UPS were analyzed together. No significant associations were observed between CD47 abundance, death, and metastatic status. Additionally, high CD47 expression was associated with a statistically significant increase in progression-free survival in the studied cohort of patients.ConclusionThis study highlights the potential of the CD47 molecule as a promising immunotherapeutic target in STS, particularly given its elevated expression levels in diverse sarcoma types. Our data showed a notable trend linking CD47 expression to tumor grade, while also suggesting an interesting correlation between enhanced abundance of CD47 expression and a reduced hazard risk of disease progression. Although these findings shed light on different roles of CD47 in STS, further research is crucial to assess its potential in clinical settings.

Comprehensive analysis of fibroblast activation protein expression across 23 tumor indications: insights for biomarker development in cancer immunotherapies.

Fibroblast activation protein (FAP) is predominantly upregulated in various tumor microenvironments and scarcely expressed in normal tissues. We analyzed FAP across 1216 tissue samples covering 23 tumor types and 70 subtypes. Elevated FAP levels were notable in breast, pancreatic, esophageal, and lung cancers. Using immunohistochemistry and RNAseq, a correlation between FAP gene and protein expression was found. Evaluating FAP's clinical significance, we assessed 29 cohorts from 12 clinical trials, including both mono and combination therapies with the PD-L1 inhibitor atezolizumab and chemotherapy. A trend links higher FAP expression to poorer prognosis, particularly in RCC, across both treatment arms. However, four cohorts showed improved survival with high FAP, while in four others, FAP had no apparent survival impact. Our results emphasize FAP's multifaceted role in therapy response, suggesting its potential as a cancer immunotherapy biomarker.

Prognostic and immunological implications of paraptosis-related genes in lung adenocarcinoma: Comprehensive analysis and functional verification of hub gene.

Lung adenocarcinoma (LUAD) poses significant clinical challenges due to its inherent heterogeneity and variable response to treatment. Recent research has specifically focused on elucidating the role of Paraptosis-related genes (PRGs) in the progression of cancer and the prognosis of patients. We conducted a comprehensive analysis of the differential expression of PRGs in LUAD. Additionally, univariate Cox regression analysis was utilized to determine the prognostic significance of these genes. Furthermore, consensus clustering was employed to differentiate molecular subtypes within LUAD, while immune heterogeneity was assessed. To evaluate treatment outcomes, the expression of immune checkpoint inhibitors was examined, and the sensitivity of LUAD patients to chemotherapy drugs was assessed. Moreover, machine learning algorithms were employed to construct a Paraptosis-related risk score with prognostic and immunological indicators. Finally, to validate the findings, in vitro experiments were performed to verify the regulatory effect of key PRGs on Paraptosis. Our analysis identified 24 PRGs that exhibited differential expression, with CDKN3, TP53, and PHB emerging as the most prominently upregulated genes in tumor tissues. Among these genes, seven were identified as prognostic markers, with HSPB8 being the sole protective factor. Notably, our analysis also revealed the existence of two distinct molecular subtypes within LUAD, each characterized by unique prognoses and immune responses. Specifically, Subtype B displayed a poorer prognosis but demonstrated increased sensitivity to both chemotherapy and immunotherapy. In addition, our development of a Paraptosis-Associated Risk Score yielded a significant prognostic value in predicting patient outcomes. Furthermore, we found regulatory effect of CDKN3 on Paraptosis in two cell lines. Our study highlights the importance of PRGs in LUAD, particularly in prognosis and treatment response. The identified molecular subtypes and Paraptosis-Associated Risk Score offer valuable insights for personalized treatment strategies.

QSOX2 Upregulated in triple-negative breast cancer exacerbates patient prognosis by stabilizing integrin β1

Breast cancer (BC) remains a significant global health threat, with triple-negative breast cancer (TNBC) standing out as a particularly aggressive subtype lacking targeted therapies. Addressing this gap, we propose Quiescin Q6 sulfhydryl oxidase 2 (QSOX2) as a potential therapeutic target, a disulfide bond-forming enzyme implicated in cancer progression. Using publicly available datasets, we conducted a comprehensive analysis of QSOX2 expression in BC tumor and non-tumor tissues, assessing its specificity across different molecular subtypes. We further explored correlations between QSOX2 expression and patient outcomes, utilizing datasets like TCGA and METABRIC. In addition, we performed in vitro experiments to evaluate QSOX2 expression in BC cell lines and investigate the effects of QSOX2 knockdown on various TNBC cellular processes, including cell proliferation, apoptosis resistance, migration, and the epithelial-to-mesenchymal transition (EMT). Our results reveal significantly elevated QSOX2 expression in BC tumor tissues, particularly in TNBC, and establish an association between high QSOX2 expression and increased patient mortality, cancer progression, and recurrence across various BC subtypes. Notably, QSOX2 knockdown in TNBC cell lines reduces cell proliferation, enhances apoptosis, and suppresses migration, potentially mediated through its influence on the EMT process. Furthermore, we identify a significant link between QSOX2 and integrin β1 (ITGB1), suggesting that QSOX2 enhances ITGB1 stability, subsequently exacerbating the malignancy of TNBC. In conclusion, elevated QSOX2 expression emerges as a key factor associated with adverse patient outcomes in BC, particularly in TNBC, contributing to disease progression through various mechanisms, including the modulation of ITGB1 stability. Our findings underscore the potential of targeting QSOX2 as a therapeutic strategy for improving patient prognoses not only in TNBC but also in other BC subtypes.

The m6A reader IGF2BP1 attenuates the stability of RPL36 and cell proliferation to mediate benzene hematotoxicity by recognizing m6A modification

Benzene exposure leads to hematotoxicity, and epigenetic modification is considered to be a potential mechanism of benzene pathogenesis. As a newly discovered post-transcriptional modification, the roles of N6-methyladenosine (m6A) in benzene hematotoxicity are still unclear. m6A can only exert its gene regulatory function after being recognized by m6A reading proteins. In this study, we found that the expression of m6A reader IGF2BP1 decreased in benzene poisoning workers and in 20 μM benzene metabolite 1,4-BQ-treated AHH-1 cells. Further overexpression of IGF2BP1 in mice alleviated 50 ppm benzene-induced hematopoietic damage, suggesting that IGF2BP1 plays a critical role in benzene hematotoxicity. Next, we examined transcriptome-wide m6A methylation in vitro to search for target genes of IGF2BP1. We found that benzene metabolite 1,4-BQ treatment altered the m6A methylation levels of various genes. The comprehensive analysis of mRNA expression and m6A methylation uncovered that the hypomethylated Ribosomal Protein L36 (RPL36) and its consequent reduced expression impaired cell proliferation. Mechanically, m6A modification reduced RNA stability to down-regulate RPL36 expression. Moreover, overexpression of IGF2BP1 relieved RPL36 reduction and cell proliferation inhibition caused by benzene in vitro and in vivo by directly binding with RPL36 mRNA. In conclusion, the m6A reader IGF2BP1 attenuates the stability of RPL36 and cell proliferation to mediate benzene hematotoxicity by recognizing m6A modification. IGF2BP1 and RPL36 may be key molecules and potential therapeutic targets for benzene hematotoxicity.

Regulation of non-canonical proteins from diverse origins through the nonsense-mediated mRNA decay pathway.

Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.