Paired Tissue Samples Research Articles

Eutopic and Ectopic Endometrial Interleukin-17 and Interleukin-17 Receptor Expression at the Endometrial-Myometrial Interface in Women with Adenomyosis: Possible Pathophysiology Implications.

Adenomyosis involves the infiltration of endometrial glands and stroma deep into the uterine tissue, causing disruption to the endometrial-myometrial interface (EMI). The role of interleukin-17 (IL-17) has been extensively studied in endometriosis, but its involvement in adenomyosis remains unclear. This study aimed to investigate the expression of IL-17 in eutopic and ectopic endometrium (adenomyosis) of individuals with adenomyosis at the level of EMI. Paired tissues of eutopic endometrium and adenomyoma were collected from 16 premenopausal women undergoing hysterectomy due to adenomyosis. The IL-17 system was demonstrated in paired tissue samples at the level of EMI by the immunochemistry study. Gene expression levels of IL-17A and IL-17 receptor (IL-17R) were assessed through quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Comparative gene transcript amounts were calculated using the delta-delta Ct method. By immunohistochemical staining, CD4, IL-17A, and IL-17R proteins were detected in both eutopic endometrium and adenomyosis at the level of EMI. IL-17A and IL-17R were expressed mainly in the glandular cells, and the expression of both IL-17A and IL-17R was found to be stronger in adenomyosis than in endometrium. 3-Diaminobenzidine (DAB) staining revealed greater IL-17A expression in adenomyosis compared to eutopic endometrium. Quantitative RT-PCR showed 7.28-fold change of IL-17A and 1.99-fold change of IL-17R, and the fold change level of both IL-17A and IL-17R is significantly higher in adenomyosis (IL-17A: p = 0.047, IL-17R: p = 0.027) versus eutopic endometrium. We found significantly higher IL-17 levels in adenomyosis compared to eutopic endometrium at the level of EMI. The results showed that the IL-17 system may play a role in adenomyosis.

RNA-Seq analysis for breast cancer detection: a study on paired tissue samples using hybrid optimization and deep learning techniques

ProblemBreast cancer is a leading global health issue, contributing to high mortality rates among women. The challenge of early detection is exacerbated by the high dimensionality and complexity of gene expression data, which complicates the classification process.AimThis study aims to develop an advanced deep learning model that can accurately detect breast cancer using RNA-Seq gene expression data, while effectively addressing the challenges posed by the data’s high dimensionality and complexity.MethodsWe introduce a novel hybrid gene selection approach that combines the Harris Hawk Optimization (HHO) and Whale Optimization (WO) algorithms with deep learning to improve feature selection and classification accuracy. The model’s performance was compared to five conventional optimization algorithms integrated with deep learning: Genetic Algorithm (GA), Artificial Bee Colony (ABC), Cuckoo Search (CS), and Particle Swarm Optimization (PSO). RNA-Seq data was collected from 66 paired samples of normal and cancerous tissues from breast cancer patients at the Jawaharlal Nehru Cancer Hospital & Research Centre, Bhopal, India. Sequencing was performed by Biokart Genomics Lab, Bengaluru, India.ResultsThe proposed model achieved a mean classification accuracy of 99.0%, consistently outperforming the GA, ABC, CS, and PSO methods. The dataset comprised 55 female breast cancer patients, including both early and advanced stages, along with age-matched healthy controls.ConclusionOur findings demonstrate that the hybrid gene selection approach using HHO and WO, combined with deep learning, is a powerful and accurate tool for breast cancer detection. This approach shows promise for early detection and could facilitate personalized treatment strategies, ultimately improving patient outcomes.

Reconstructing oral cavity tumor evolution through brush biopsy

Oral potentially malignant disorders (OPMDs) with genomic alterations have a heightened risk of evolving into oral squamous cell carcinoma (OSCC). Currently, genomic data are typically obtained through invasive tissue biopsy. However, brush biopsy is a non-invasive method that has been utilized for identifying dysplastic cells in OPMD but its effectiveness in reflecting the genomic landscape of OPMDs remains uncertain. This pilot study investigates the potential of brush biopsy samples in accurately reconstructing the genomic profile and tumor evolution in a patient with both OPMD and OSCC. We analyzed single nucleotide variants (SNVs), copy number aberrations (CNAs), and subclonal architectures in paired tissue and brush biopsy samples. The results showed that brush biopsy effectively captured 90% of SNVs and had similar CNA profiles as those seen in its paired tissue biopsies in all lesions. It was specific, as normal buccal mucosa did not share these genomic alterations. Interestingly, brush biopsy revealed shared SNVs and CNAs between the distinct OPMD and OSCC lesions from the same patient, indicating a common ancestral origin. Subclonal reconstruction confirmed this shared ancestry, followed by divergent evolution of the lesions. These findings highlight the potential of brush biopsies in accurately representing the genomic profile of OPL and OSCC, proving insight into reconstructing tumor evolution.

The role of pyroptosis-related genes in breast cancer progression

Breast cancer (BC) is one of the most common malignant cancers affecting females worldwide. Pyroptosis, a form of programmed cell death associated with inflammation and triggered by pro-inflammatory signals, is not well understood in the context of BC. This study aimed to investigate the role of pyroptosis in BC patients. Paired tumor and adjacent normal tissue samples were obtained from The Cancer Genome Atlas. Using a least absolute shrinkage and selection operator Cox analysis, we identified 15 prognostic genes associated with BC. Among these, fibrinogen C domain-containing 1, calcium voltage-gated channel subunit alpha1 H, heat shock protein family B (small) member 8, and peroxidasin-like (PXDNL) were classified as high-risk genes in BC. In contrast, the remaining 11 genes, such as proteasome activator subunit 2 and DIRAS (DIRAS family GTPase 3), were low-risk genes. Kyoto Encyclopedia of Genes and Genomes and Gene ontology analyses revealed that immune-related genes were enriched but showed reduced immunological status in the high-risk group, indicating that pro-inflammatory factors and immune antigens were produced during cell death. Consequently, targeting immune antigens with new immunosuppressants may offer a novel approach to treating BC. Moreover, the expression levels of the prognostic genes PXDNL, armadillo-like helical domain-containing 1 (ARMH1), APOBEC3D, and APOBEC3F in BC cell lines were assessed using quantitative polymerase chain reaction and western blotting. PXDNL and ARMH1 exhibited high levels of expression in BC, suggesting they could be potential therapeutic targets.

Abstract C156: Examining the role of T cell responses in colorectal cancer sex disparities

Abstract Purpose: Persistent sex disparities in colorectal cancer (CRC) incidence and mortality exist and vary by race and ethnicity. The overarching goal of this proposal is to quantify differences in the CRC tumor immune microenvironment by sex in the Latino Colorectal Cancer Consortium (LC3). Methods: The LC3 is a consortium of pooled individual-level data from Latino participants diagnosed with CRC from California, Florida, and Puerto Rico. Biospecimens, epidemiologic data, and clinical outcomes were collected from each contributing study. DNA was extracted from tumor tissue and paired germline or normal tissue samples. Whole exome sequencing was performed using established protocols. Global genetic ancestry was estimated from whole exome sequence data. High-throughput T cell receptor (TCR) sequencing was used to estimate features of the T cell receptor (TCR) repertoire (e.g. abundance, clonality), and tumor-infiltrating lymphocyte (TIL) counts were obtained through pathology review. We evaluated differences in TCR abundance and TILs by sex using linear models for continuous measurements and generalized linear models for binary or count measurements. Primary analyses were unadjusted, and secondary analyses adjusted for age at diagnosis, stage at diagnosis, tumor location (right colon vs. left colorectum), and microsatellite instability (MSI) status. To examine how genetic ancestry modified the relationship between sex and T cell responses, we fit separate models with an interaction term between each genetic ancestry proportion variable (i.e. African, European, Native American) and sex, and assessed effect modification using a likelihood ratio test. Results: To date, 240 participants had TCR repertoire profiling and whole exome sequencing data generated. 52% were male, with a mean age of 58. 17% of participants had rectal tumors. With respect to Hispanic ethnic heritage or country of origin, 49% of participants were of Mexican descent, followed by 13% of Central or South American descent, and 10% were of Puerto Rican descent. Mean T cell abundance was 0.18 (standard deviation (SD): 0.16) and mean clonality was 0.05 (SD: 0.05). Further analysis is underway. Conclusions: This project leverages within-Latino genetic diversity to disentangle the contributions of sex and genetic ancestry to variability in immune function and to understand how this may further sex disparities in CRC. We anticipate that results from this analysis will provide insight on the relationship between the tumor immune microenvironment and sex disparities in CRC. Citation Format: Nicole C. Loroña, Ya-Yu Tsai, Daniel Sobieski, Eric Cockman, Stephanie L. Schmit, Jane C. Figueiredo. Examining the role of T cell responses in colorectal cancer sex disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C156.

Development and validation of a TRIM27-based nomogram for predicting metachronous liver metastasis and prognosis in postoperative colorectal cancer patients

BackgroundColorectal cancer ranks among the most prevalent malignancies globally. Accurate prediction of metachronous liver metastasis is crucial for optimizing postoperative management. Tripartite motif-containing protein 27 (TRIM27), an E3 ubiquitin ligase, is implicated in diverse cellular functions and tumorigenesis.MethodsThis study aimed to develop and validate a TRIM27-based nomogram for prognostication in colorectal cancer patients. Transcriptome sequencing of five paired tumor and normal tissue samples identified TRIM27 as a potential prognostic biomarker. Immunohistochemistry was employed to assess TRIM27 expression in colorectal cancer cohorts from two institutions.ResultsTRIM27 expression correlated significantly with both the prognosis of colorectal cancer patients and the occurrence of metachronous liver metastasis. A nomogram incorporating TRIM27 and clinical factors was constructed and demonstrated robust predictive accuracy in an independent validation cohort.ConclusionThe TRIM27-based nomogram is a valuable prognostic tool for predicting prognosis and metachronous liver metastasis in colorectal cancer patients, aiding in personalized treatment decisions.

The Genetic Determinants and Genomic Consequences of Non-Leukemogenic Somatic Point Mutations.

Clonal hematopoiesis (CH) is defined by the expansion of a lineage of genetically identical cells in blood. Genetic lesions that confer a fitness advantage, such as point mutations or mosaic chromosomal alterations (mCAs) in genes associated with hematologic malignancy, are frequent mediators of CH. However, recent analyses of both single cell-derived colonies of hematopoietic cells and population sequencing cohorts have revealed CH frequently occurs in the absence of known driver genetic lesions. To characterize CH without known driver genetic lesions, we used 51,399 deeply sequenced whole genomes from the NHLBI TOPMed sequencing initiative to perform simultaneous germline and somatic mutation analyses among individuals without leukemogenic point mutations (LPM), which we term CH-LPMneg. We quantified CH by estimating the total mutation burden. Because estimating somatic mutation burden without a paired-tissue sample is challenging, we developed a novel statistical method, the Genomic and Epigenomic informed Mutation (GEM) rate, that uses external genomic and epigenomic data sources to distinguish artifactual signals from true somatic mutations. We performed a genome-wide association study of GEM to discover the germline determinants of CH-LPMneg. After fine-mapping and variant-to-gene analyses, we identified seven genes associated with CH-LPMneg (TCL1A, TERT, SMC4, NRIP1, PRDM16, MSRA, SCARB1), and one locus associated with a sex-associated mutation pathway (SRGAP2C). We performed a secondary analysis excluding individuals with mCAs, finding that the genetic architecture was largely unaffected by their inclusion. Functional analyses of SMC4 and NRIP1 implicated altered HSC self-renewal and proliferation as the primary mediator of mutation burden in blood. We then performed comprehensive multi-tissue transcriptomic analyses, finding that the expression levels of 404 genes are associated with GEM. Finally, we performed phenotypic association meta-analyses across four cohorts, finding that GEM is associated with increased white blood cell count and increased risk for incident peripheral artery disease, but is not significantly associated with incident stroke or coronary disease events. Overall, we develop GEM for quantifying mutation burden from WGS without a paired-tissue sample and use GEM to discover the genetic, genomic, and phenotypic correlates of CH-LPMneg.

Association of XRCC2 with breast cancer, a multi-omics analysis at genomic, transcriptomic, and epigenomic level.

One of the main causes of cancer-related mortality for women worldwide is breast cancer (BC). The XRCC2 gene, essential for DNA repair, has been implicated in cancer susceptibility. This study aims to evaluate the association between XRCC2 and BC risk. The study was conducted at Zheen International Hospital in Erbil, Iraq, between 2021 and 2024 with a total of 88 samples, including 44 paired normal and cancer tissue samples. Mutation analysis was performed using Next-Generation Sequencing, coupled with in silico tools for variant impact prediction. Expression levels were assessed through RT-PCR, and methylation status was determined using methylation-sensitive restriction enzyme digestion PCR. The study identified seven inherited germline variants in the XRCC2 gene, with five of these mutations being Uncertain Significance, one being Likely Pathogenic, and one being Likely benign. RNA purity was found high with mean A260/280 ratios of 1.986 ± 0.097 in normal (N) and 1.963 ± 0.092 in tumor (T) samples. Tumor samples exhibited a higher RNA concentration (78.56 ± 40.87 ng/µL) than normal samples (71.44 ± 40.79 ng/µL). XRCC2 gene expression was significantly upregulated in tumor tissue, with marked increases in patients aged 40-55 and >56 years and in higher cancer grades (II and III) and invasive ductal carcinoma (p-values ranging from <0.0001 to 0.0392). DNA methylation rates in tumor tissues were low (7%), suggesting limited regulation by methylation. The study suggests that XRCC2 can be classified as an oncogene and that its structural investigation by targeted NGS and expression evaluation can be used as a potential biomarker in BC.

Key extracellular proteins and TF-miRNA co-regulatory network in diabetic foot ulcer: Bioinformatics and experimental insights.

Diabetic foot ulcers (DFUs), a serious complication of diabetes, are associated with abnormal extracellular protein (EP) metabolism. The identification of key EPs and their regulatory networks is crucial for the understanding of DFU formation and development of effective treatments. In this study, a large-scale bioinformatics analysis was conducted to identify potential therapeutic targets and experimental validation was performed to ensure the reliability and biological relevance of the findings. Due to the comprehensive profiling of DFU samples provided by the GSE80178 dataset, we initially selected it to derive differentially expressed genes (DEGs) associated with DFU. Subsequently, utilizing the UniProt database and annotated EP list from the Human Protein Atlas annotation database, we screened for extracellular protein-related differentially expressed genes (EP-DEGs) due to their crucial role in the pathogenesis and healing of DFU. We examined EP-DEG pathway enrichment and protein-protein interaction networks, analyzed paired full-thickness skin tissue samples from 24 patients with DFUs and healthy controls, and performed polymerase chain reaction (PCR) experiments to validate candidate genes. Ultimately, we constructed a transcription factor (TF)-microRNA (miRNA)-hub gene co-regulatory network to explore upstream and downstream regulatory connections based on validated DEGs. Four crucial candidate genes (FMOD, LUM, VCAN, and S100A12) were identified and verified via PCR analysis. The TF-miRNA-hub EP-DEG regulatory network contained the pivotal TFs TRIM28 and STAT3 and the miRNAs hsa-mir-20a-5p, hsa-miR-21, and hsa-miR-203. The findings of this study advance our understanding of the pathology of DFU by defining key roles of specific EPs and elucidating a comprehensive regulatory network. These insights pave the way for novel approaches to improve DFU treatment outcomes.

Per- and polyfluoroalkyl substances in Chinese commercially available red swamp crayfish (Procambarus clarkii): Implications for human exposure and health risk assessment

The extensive utilization of per- and polyfluoroalkyl substances (PFASs) has led to their pervasive presence in the environment, resulting in contamination of aquatic products. Prolonged exposure to PFASs has been linked to direct hepatic and renal damage, along with the induction of oxidative stress, contributing to a spectrum of chronic ailments. Despite the recent surge in popularity of red swamp crayfish as a culinary delicacy in China, studies addressing PFASs' exposure and associated health risks from their consumption remain scarce. To address this gap, our study investigated the PFASs’ content in 85 paired edible tissue samples sourced from the five primary red swamp crayfish breeding provinces in China. The health risks associated with dietary exposure were also assessed. Our findings revealed widespread detection of PFASs in crayfish samples, with short-chain perfluoroalkyl carboxylic acids (PFCAs) exhibiting the highest concentrations. Notably, the total PFAS concentration in the hepatopancreas (median: 160 ng/g) significantly exceeded that in muscle tissue (5.95 ng/g), as did the concentration of every single substance. The hazard quotient of perfluorohexanesulfonic acid (PFHxS) via consuming crayfish during peak season exceeded 1. In this case, a potential total non-cancer health risk of PFASs, which is mainly from the hepatopancreas and associated with PFHxS, is also observed (hazard index>1). Thus, it is recommended to avoid consuming the hepatopancreas of red swamp crayfish. Greater attention should be paid to governance technology innovation and regulatory measure strengthening for short-chain PFASs.

Clonal associations between lymphocyte subsets and functional states in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA.

DNA methylation signatures provide novel diagnostic biomarkers and predict responses of immune therapy for breast cancer.

Breast cancer (BRCA) is one of the most common malignant tumors affecting women worldwide. DNA methylation modifications can influence oncogenic pathways and provide potential diagnostic and therapeutic targets for precision oncology. In this study, we used non-parametric permutation tests to identify differentially methylated positions (DMPs) between paired tumor and normal BRCA tissue samples from the Cancer Genome Atlas (TCGA) database. Then, we applied non-negative matrix factorization (NMF) to the DMPs to derive eight distinct DNA methylation signatures. Among them, signatures Hyper-S3 and Hypo-S4 signatures were associated with later tumor stages, while Hyper-S1 and Hypo-S3 exhibited higher methylation levels in earlier stages. Signature Hyper-S3 displayed an effect on overall survival. We further validated the four stage-associated signatures using an independent BRCA DNA methylation dataset from peripheral blood samples. Results demonstrated that 24 commonly hypomethylated sites in Hypo-S4 showed lower methylation in BRCA patients compared to healthy individuals, suggesting its potential as an early diagnostic biomarker. Furthermore, we found that methylation of 23 probes from four stage-related signatures exhibited predictive power for immune therapy response. Notably, methylation levels of all three probes from the Hypo-S4 and activity of the Hypo-S4 demonstrated highly positive relevance to PD-L1 gene expression, implying their significant predictive values for immunotherapy outcomes. GO and KEGG pathway enrichment analysis revealed that genes with these 23 immunotherapy-related methylation probes are mainly involved in glycan degradation or protein deglycosylation. These methylation signatures and probes may serve as novel epigenetic biomarkers for predicting tumor immunotherapy response. Our findings provide new insights into precision oncology approaches for BRCA.

A fragment-based algorithm for identifying pan-cancer differential methylation markers from cfDNA.

e22507 Background: Methylation-based sequencing has been developed as a promising approach for Multi-Cancer Early Detection (MCED). Conventionally, cancer-specific methylation markers were identified by comparing paired tumor and adjacent normal tissues using traditional algorithm DSS (Dispersion shrinkage for sequencing data). However, the clinical applicability is limited by the unsatisfactory sensitivity in early-stage disease. Hence, we included blood specific markers derived from paired tissue and blood samples and developed a novel fragment-based algorithm in cfDNA with a better signal to noise ratio (SNR) than DSS to improve the sensitivity of the assay. Methods: A total of 391 plasma samples and 433 tissue samples from 7 cancer types were subjected to whole genome bisulfite sequencing (WGBS) (Table). The proportion of stage I-III and stage IV was 3.5:1 among 7 cancer types. The average sequencing depth was 30X. For each bin, the proportion of fully methylated (M) / un-methylated (U) fragments, defined with methylation level ≥85% / ≤15%, was computed individually. One-sided Wilcoxon rank sum test was employed to test whether cancer cfDNA have significantly (FDR &lt; 0.001) higher proportion of U/M fragments than control cfDNA. Results: To evaluate the performance of fragment-based algorithm in cfDNA, the false positive was assessed by resampling from healthy subjects. The healthy cfDNA were randomly assigned 20 times as pseudo case-control groups of 30 /170. The number of markers among healthy samples was recorded as NFalse Positive (NFP), while the number of markers between cancer and healthy samples was recorded as NPositive (NP). SNR was defined as (NP-NFP)/NFP. We calculated SNR using the fragment-based algorithm and achieved SNR of 135.2 for colorectal cancer, compared to SNR of 2.2 using DSS. The improved SNR was also reproduced in other cancer types. Concordance between plasma and tissue markers was 30.09%-95.92% (median 66.72%) among 7 cancer types, which was related to the proportion of ctDNA for each cancer type. Plasma markers which show weak signals in tissue are defined as blood-specific markers. These markers were enriched in immune-related pathways, suggesting a correlation to the immune response of cancer patients. Conclusions: We developed a novel fragment-based algorithm that achieves much higher SNR than DSS in cfDNA. We also identified blood-specific methylation markers which may improve the sensitivity of early cancer detection in clinical. [Table: see text]

Effect of molecular cartography on recurrence-specific drivers through analysis of matched primary and recurrent rhabdomyosarcoma tumors.

e23546 Background: Despite intensive treatment, the chance of cure for patients with recurrent rhabdomyosarcoma (RMS) is very low, highlighting the need for an improved understanding of recurrent RMS. As most studies to date have focused on diagnosis, our understanding of the molecular and clonal dynamics of pre- and postrecurrence pairs is limited. Methods: We collected critical clinical data from patients with RMS and performed targeted next-generation sequencing on paired tissue and blood samples obtained pre- and postrecurrence. At OrigiMed, which accredited by the College of American Pathologists and certified under the Clinical Laboratory Improvement Amendments, we conducted a thorough examination of a targeted gene panel that included 706 DNA-level and 649 RNA-level cancer-associated genes. Additionally, we executed whole-transcriptome sequencing (WTS) to determine the changes in gene expression and immune infiltration associated with disease recurrence. Cox univariate and multivariate analyses were applied to identify prognostic indicators of progression-free survival (PFS). Results: Paired pre- and post-recurrent tissue and blood samples from15 RMS patients were subjected to targeted gene panel sequencing. Of the 15 patients, 4 underwent WTS. The histological types included nine embryonal RMSs, three alveolar RMSs, and three spindle cell/sclerosing RMSs. Analysis of genome-wide mutation landscapes revealed distinct mutational patterns pre- and postrecurrence. MYOD1 (26.7%), BCOR (20.0%), CDKN2A (20.0%), and TP53 (20.0%) were the most common gene alterations detected at pre-recurrence, whereas TP53 (40.0%), BCOR (20.0%), CDKN2A (20.0%), and COL1A1 (13.3%) were the most common gene alterations observed at post-recurrence. Notably, compared with prerecurrence TP53 mutation was more common in postrecurrent tumors (40.0% vs. 20.0%), which plays an essential role in the pathogenesis of RMS. Pathway analysis between pre- and postrecurrence revealed that the P53 pathway (53.3% vs. 26.7%) was more activated postrecurrence. In particular, after recurrence, alterations in three genes, BCOR (p = 0.024), CDKN2A (p = 0.024), and TP53 (p = 0.036), demonstrated a significant association with the risk groups. These findings imply that these gene alterations may have a substantial connection to recurrence and could act as predictive biomarkers for risk stratification purposes. Cox multivariate analysis revealed that KDM5C (p = 0.015) was an independent poor prognostic factor for PFS. According to the WTS analysis, the expression level of transcripts per million in CAMLG (p = 0.049), GAN (p = 0.049), and HSPA4 (p = 0.049) was higher in postrecurrent tumors than in prerecurrent tumors. Conclusions: Our study reveals multiple potential drivers of recurrent disease in RMS, improving our understanding of tumor evolution and suggesting potential biomarkers. Clinical trial information: NCT05076071 .

Protosappanin B enhances the chemosensitivity of 5-fluorouracil in colon adenocarcinoma by regulating the LINC00612/microRNA-590-3p/Golgi phosphoprotein 3 axis

Background5-fluorouracil (5-FU) is conventionally used in chemotherapy for colon adenocarcinomas. Acquired resistance of 5-FU remains a clinical challenge in colon cancer, and efforts to develop targeted agents to reduce resistance have not yielded success. Protosappanin B (PSB), the main component of Lignum Sappan extract, is known to exhibit anti-tumor effects. However, whether and how PSB could improve 5-FU resistance in colon cancer have not yet been established. In this study, we aimed to explore the effects and underlying mechanisms of PSB in 5-FU-induced chemoresistance in colon adenocarcinoma.MethodsForty-seven paired colon cancer tissue samples from patients who received 5-FU chemotherapy were collected as clinical samples. Two 5-FU resistant colon cancer cell lines were established for in vitro experiments. Reverse transcription-quantitative PCR (RT-qPCR) was performed to determine the mRNA and microRNA (miRNA) expression levels in colon adenocarcinoma tissues and cell lines. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were performed to evaluate cell proliferation and apoptosis, respectively.ResultsLINC00612 was highly expressed in colon adenocarcinoma samples and 5-FU resistant colon cancer cells. LINC00612 knockdown enhances 5-FU chemosensitivity in 5-FU resistant cells. Notably, PSB treatment attenuated LINC00612 expression in 5-FU resistant colon adenocarcinoma cells. Moreover, PSB treatment reversed the increase in LINC00612-induced 5-FU resistance. Mechanistically, LINC00612 specifically bound to miR-590-3p, which promoted 5-FU resistance in colon adenocarcinoma cells and attenuated the inhibitory effect of LINC00612 on GOLPH3 expression.ConclusionPSB attenuates 5-FU chemoresistance in colon adenocarcinoma by regulating the LINC00612/miRNA-590-3p/GOLPH3 axis.Graphical

The m6A reader HNRNPC predicts adverse prognosis and promotes the progression of colorectal cancer.

As a critical m6A RNA methylation regulator, HNRNPC has been revealed to serve as potential biomarkers in various human cancers. The specific expression and significance of HNRNPC in colorectal cancer remain unknown. This study aimed to confirm HNRNPC expression level and evaluate its function in colorectal cancer progression. 101 paired tissue samples were collected from colorectal cancer patients. HNRNPC levels in colorectal cancer were detected using PCR. CCK8 and transwell assays were conducted to estimate the effect of HNRNPC on cell growth and metastasis with the regulation of HNRNPC by cell transfection. Upregulated HNRNPC was observed in colorectal cancer compared with normal tissues and cells. The higher HNRNPC levels in tumor tissues were associated with the advanced TNM stage and positive lymph node metastasis. Meanwhile, HNRNPC upregulation could indicate adverse outcomes of colorectal cancer patients. In vitro, the knockdown of HNRNPC significantly suppressed the proliferation, migration, and invasion of colorectal cancer cells. Upregulated HNRNPC served as a biomarker for the prognosis and development of colorectal cancer, which provides a novel therapeutic target for colorectal cancer.

Claudin-18 status and its correlation with HER2 and PD-L1 expression in gastric cancer with peritoneal dissemination

BackgroundGastric cancer with peritoneal dissemination (PD) has a dismal prognosis, and current treatments have shown little efficacy. CLDN18.2-targeted therapies have shown promising efficacy against gastric cancers that express high levels of CLDN18. Because of the limited information regarding CLDN18.2 status in PD, we analyzed PD-positive gastric cancers for CLDN18 status in both primary and PD, along with HER2 and PD-L1 combined positive score (CPS).MethodsImmunohistochemical analyses were performed on 84 gastric cancer cases using paired primary and PD tissue samples.ResultsAt 40% cut-off, CLDN18 was positive in 57% (48/84) primary tumors and in 44% (37/84) PDs. At 75% cut-off, 28.6% (24/84) primary tumors and 20.2% (17/84) PDs were CLDN18-positive. The concordance rate between primary tumors and PD was 79.8% at 40% cut-off and 75% at 75% cut-off. When comparing biopsy and surgical specimens, the concordance rates were 87.5% at 40% cut-off and 81.3% at 75% cut-off. Within a tumor, the superficial area tended to have a higher CLDN18-positive rate than the invasive front (P = 0.001). Although HER2 -positivity was only 11.9% in this cohort, CLDN18 positivity in HER2-negative tumors (n = 74) was relatively high: 60.8% at 40% cut-off and 28.4% at 75% cut-off. Among double-negative (HER2 − and PD-L1 CPS < 1) tumors, CLDN18 positivity was 67.6% at 40% cut-off and 26.5% at 75% cut-off.ConclusionsCLDN18 expression is generally maintained in PD and is relatively high even in double-negative tumors, making it a promising therapeutic target for PD-positive gastric cancer.

Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

Chemokine‑ and chemokine receptor-based subtypes predict prognosis, immunotherapy and chemotherapy response in colorectal cancer patients

BackgroundThe clinical significance and comprehensive characteristics of chemokines and chemokine receptors in colorectal cancer (CRC) have not been previously reported. Our study aims to investigate the expression profiles of chemokines and chemokine receptors, as well as establish subtypes in CRC. Methods1009 CRC samples were enrolled in our study. Consensus unsupervised clustering analysis was conducted to establish subtypes, and a risk score model was developed using univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses. 36 pairs of tissue specimens of CRC patients and two CRC cell lines were used to validate the subtypes and risk score in vitro. Quantitative real-time PCR and western blotting were employed to validate mRNA and protein expression levels, respectively. Flow cytometry was utilized for analyzing cell apoptosis, while cell viability assay and EdU assay were conducted to assess cell proliferation ability. ResultsThe Cluster B group shares similarities with the low-risk group in terms of exhibiting a higher level of immune cell infiltration and belonging to hot tumor. Patients CRC in the Cluster B group demonstrate a more favorable prognosis and exhibit better response to immunotherapy and chemotherapy. On the other hand, the Cluster A group resembles the high-risk group as it displays lower levels of immune cell infiltration, indicating a cold tumor phenotype. CRC patients in the Cluster A group have poorer prognoses and show less therapeutic efficacy towards immunotherapy and chemotherapy. Furthermore, we utilized a total of 36 pairs of tissue samples obtained from patients with CRC, along with two CRC cell lines for validation in vitro. This comprehensive approach further enhances the scientific validity and reliability of the identified subtypes and risk score in their ability to predict prognosis, response to immunotherapy, and response to chemotherapy among CRC patients. ConclusionWe first established robust prognostic subtypes based on chemokines and chemokine receptors, which could potentially serve as a novel biomarker for guiding individualized treatment in patients with CRC undergoing immunotherapy and chemotherapy.

Abstract PO4-08-09: Concordance analysis of paired breast cancer core needle biopsies and surgical excision samples using the Oncotype DX Breast Recurrence Score® assay

Abstract INTRODUCTION: The Oncotype DX Breast Recurrence Score® (RS) assay is validated to predict risk of distant recurrence at 10 years and the benefit of adjuvant chemotherapy in patients with estrogen receptor (ER)+, HER2- early breast cancer who receive 5 years of endocrine therapy. The feasibility of Oncotype DX® testing with diagnostic core needle biopsies (CNB) was demonstrated through analysis of a large commercial laboratory experience. The neoadjuvant ADAPT study showed the clinical utility of deriving the Recurrence Score® (RS) result from CNB as well. However, data are limited on concordance of RS results from paired CNB and surgical samples. Our primary objective was to determine the degree of concordance of RS results between paired tissue samples from the same primary breast tumor of patients with early stage, ER+, HER2- invasive breast cancer who had a CNB and subsequent surgical excision (SE; lumpectomy or mastectomy) without intervening systemic therapy. METHODS: Patients with a commercial RS result on either CNB or subsequent SE were identified through medical record and clinical database review. A pathologist reviewed archival tissue samples to confirm eligibility and selected the other paired CNB or SE block most representative of the tumor tissue for subsequent study Oncotype DX testing. Descriptive statistics were used to summarize patient and tumor characteristics. Wilcoxon signed rank, McNemar’s and Cochran-Mantel-Haenszel tests were used to compare RS between SE and CNB paired samples. Cohen’s Kappa and 95% confidence intervals (CIs) were used to compare the agreement of categorical RS (0-25 and 26-100) result between SE and CNB paired samples. Lin’s concordance correlation and 95% CIs were used to compare agreement of continuous RS between paired samples. RESULTS: A total of 134 patients were identified with paired CNB and SE samples with a median time of 34 (range 5-103) days between collections. Of the 134 patients, a commercial RS result was available for 25 (18.7%) with CNB and 109 (81.3%) with SE. A study RS result was then generated on the other paired sample. Median age was 62 years (range 33-99; 23 patients [17%] were age ≤50 vs. 111 [83%] age &amp;gt;50). Cases were predominantly node negative (107/134 node negative vs. 26/134 node positive; 1/134 unknown) invasive ductal carcinomas (104/134 ductal vs. 18/134 classic lobular vs. 12/134 other). Mean RS results were 15.6 (range 0-56) and 16.6 (range 0-59) for CNB and SE, respectively (p = 0.003), with no statistically significant differences in RS category defined as low vs. high risk (119/134 CNB vs. 114/134 SE for RS 0-25 and 15/134 CNB vs 20/134 SE for RS 26-100; p = 0.13) or low vs. intermediate vs. high risk (85/134 CNB vs. 78/134 SE for RS 0-17 and 43/134 CNB vs. 48/134 SE for RS 18-30 and 6/134 CNB vs. 8/134 SE for RS 31-100; p = 0.19). No differences were observed based on age relative to 50 years or nodal status (data not shown). Cohen’s Kappa statistic k = 0.64 (95% CI, 0.44-0.83) supports substantial agreement between the CNB and SE samples. Lin’s concordance correlation coefficient (CCC) demonstrates similar agreement for the continuous RS result between paired samples (CCC = 0.86 [0.80-0.90]). Overall percent agreement was 91.8% (87.1-96.4%). CONCLUSIONS: Studies evaluating the level of concordance of ER, progesterone receptor, and HER2 biomarker status between CNB and SE samples have found high concordance rates, albeit varied. We now provide data demonstrating high concordance of RS results between CNB and SE specimens obtained from the same breast primary tumor without intervening systemic therapy. This supports use of either biopsy source for testing with a genomic predictor to identify those patients who will most likely benefit from chemotherapy and which patients may be spared. Citation Format: Aziza Nassar, Jodi Carter, Paige Innis, Satish Seerapu, Christy Russell, Helena Hanna, Abigail Lochala, Minetta Liu. Concordance analysis of paired breast cancer core needle biopsies and surgical excision samples using the Oncotype DX Breast Recurrence Score® assay [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 PO4-08-09.