Non-tumor Tissue Samples Research Articles

Mitotic spindle positioning protein serves as prognostic biomarker in patients with colorectal cancer

Background Colorectal cancer (CRC) ranks among the most aggressive types of cancer globally. Currently, clinical tumor prognostic biomarkers still lack accuracy. Mitotic spindle positioning (MISP) protein connects microtubules to the actin cytoskeleton and adhesive plaques, playing a critical role in spindle positioning, orientation, and the process of cell division. MISP can regulate the malignant biological functions of pancreatic cancer and intrahepatic cholangiocarcinoma and it acts as biomarker for prognosis, but its role in CRC remains unclear. Methods This study has collected 37 CRC tissue samples and 37 corresponding adjacent nontumor tissue samples, and 57 additional CRC tissues samples. Clinical data were obtained from the patients with CRC. MISP mRNA and protein expression levels were analyzed in normal control and CRC tissues using the GEPIA and Human Protein Atlas website. MISP protein levels in the collected tissues were analyzed using immunohistochemistry. Results MISP mRNA and protein expression levels were significantly increased in CRC tissues compared to adjacent nontumor tissues. Higher MISP protein levels were associated with distant metastasis, recurrence, and lower survival rates. Kaplan–Meier analysis showed that high expression levels of MISP protein were associated with recurrence and death in CRC patients. In addition, a high expression level of MISP protein, lymph node metastasis, and distance metastasis were risk factors for recurrence and a poor prognosis in patients with CRC. Conclusion Elevated MISP protein correlated with tumor metastasis, recurrence, and lower survival rates in patients with CRC, and thus, MISP has the potential to become a prognostic marker for CRC.

Abstract C094: Differential expression analysis with transcriptome data in FFPE samples from Colombian patients with colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is a significant public health issue in Colombia, It occupies third place in incidence and ranking as the fifth leading cause of cancer-related deaths. Understanding the sociodemographic, clinicopathological, and molecular characteristics of CRC patients is crucial for prognosis, effective management, and treatment. Several molecular alterations and clinicopathological variables have been associated with the prognosis of the disease in different populations; However, we do not know if there are differentially expressed genes specific to Colombian population that are associated with CRC prognosis. Objective: Perform differential expression analysis using RNA-seq data obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples from Colombian patients with CRC. Methodology: For this preliminary analysis were included 5 patients diagnosed with CRC between 2010-2014 at Instituto Nacional de Cancerologia from Colombia. From each patient, formalin-fixed and paraffin-embedded (FFPE) samples of tumor tissue and non-tumor tissue from the surgical remnant were obtained. Extracted RNA was used to perform whole-genome RNA sequencing (RNA-seq) at a depth of 100X. Bioinformatic analysis was performed using Fastqc, Cutadapt, Ribodetector, and STAR on a Linux server. The DESeq2 package in RStudio was used to perform the differential expression analysis between tumor samples vs. non-tumorous. A significant differential expression was considered using an FDR-adjusted p ≤0.05 and a log fold change of 2. Results and discussion: A list of 18 differentially expressed genes was obtained which 5 underexpressed stand out: B3GNT6 (LFC:-10.66; padj=0.0093), ITLN1 (LFC:-6.23; padj=0.0231), NR3C2 (LFC:-3.34; padj=0.0231), NLGN1 (LFC:- 0.46; padj=0.0301), CEACAM7 (LFC:-5.91; padj= 0.0301); and one overexpressed gene: RPL9 (LFC:5.44; padj=0.0267). These genes have been previously characterized and associated with the progression and prognosis of this disease in other populations. Conclusions: 6 differentially expressed genes were found using RNA-seq in FFPE samples. These genes have been associated with CRC progression and prognosis in previous studies, but have not been evaluated in the Colombian population. From this perspective, we propose to continue with the analysis by increasing the number of samples and evaluating their association with the different clinicopathological variables and their impact on the prognosis of the disease. Citation Format: Wendy J. Montero Ovalle, Silvia J Serrano Gómez, Diego F Ballen Lozano. Differential expression analysis with transcriptome data in FFPE samples from Colombian patients with colorectal cancer [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 C094.

Knockdown of SLC16A3 decreases extracellular lactate concentration in hepatocellular carcinoma, alleviates hypoxia and induces ferroptosis

SLC16A3/monocarboxylate transporter 4 (MCT4) regulates intracellular lactate transport and is highly expressed in many tumors, indicating poor prognosis. It may be related to inducing hypoxia, apoptosis and other mechanisms, but the study of MCT4 in HCC is far from complete. In this study, we first analyzed the expression of SLC16A3 in HCC tumor and non-tumor tissue samples based on TCGA data and immunohistochemistry. Subsequently, the effects of SLC16A3 expression on cell proliferation and invasion were analyzed using hepatocellular carcinoma (HCC) lines, and Western blot (WB) analysis was performed to explore the changes in pathway proteins and ferroptosis proteins. Finally, the drug sensitivity was tested by CCK8 kit. We found that SLC16A3 was significantly upregulated in tumor tissues, and was significantly correlated with TNM stage, histological grade, and macrovascular invasion. TCGA data and WB analysis showed that the high expression of SLC16A3 induced hypoxia, and knockdown could reverse hypoxia and inhibit ERK phosphorylation, thus limiting the malignant behavior of HCC cells. Moreover, knockdown of SLC16A3 significantly increased the level of lipid peroxidation and reactive oxygen species (ROS), while the expressions of GPX4, DHODH and SLC7A11 were inhibited. The expression of SLC16A3 affected the sensitivity of HCC cells to chemotherapy and targeted drugs, and RNA sequencing data suggested that the expression level influenced tumor microenvironment and response to immunotherapy. So, we draw a conclude that SLC16A3 is associated with poor prognosis of HCC. Inhibition of SLC16A3 expression is a potential therapeutic target for HCC.

CD19+CD73+ B cells infiltration indicates poor prognosis and unfavorable responses to immunotherapy in gastric cancer

ObjectivesCluster of Differentiation 73 (CD73) is expressed on immune cells and plays a significant role in tumor inhibition by suppressing antitumor immunity. The objectives of this study were to explore the expression and functional mechanisms of CD73 on B cells in patients with gastric cancer (GC). MethodsThe prognostic significance of CD19+CD73+ B cells was evaluated in 390 GC patients through dual immunohistochemistry staining. Flow cytometry was employed to analyze the phenotype of the CD19 subpopulation using fresh tumor and non-tumor tissue samples from 8 GC patients. A bioinformatics analysis of CD19+CD73+ B cells was also performed within the scRNA-seq cohort, and the CD19+ B cell subtype was assessed using multiple immunofluorescence staining. ResultsThe infiltration of CD19+CD73+ B cells was observed to be elevated in gastric cancer (GC) tissue compared to normal tissues. A strong correlation was observed between high CD19+CD73+ B cell infiltration, poor overall survival, and diminished responsiveness to neoadjuvant immunotherapy in GC. These cells emerged as a novel subset of regulatory B cells (Bregs) linked to adenosine metabolism and the exhaustion of CD8+ T cells. The CD19+CD73+ B cells also correlated with the production of immunosuppressive cytokines IL-10 and TGFB1. Further analysis indicated an association between CD19+CD73+ B cells and advanced-stage GC. ConclusionsThe presence of CD19+CD73+ B cells in GC may serve as a prognostic indicator for clinical outcomes and a predictive marker for poor responsiveness to neoadjuvant immunotherapy. The correlation between the presence of CD19+CD73+ B cells and CD8+ T cell exhaustion, along with immunosuppression, highlights the tumor-promoting function of these cells.

Clinicopathological Factors as Predictors for Establishment of Patient Derived Head and Neck Squamous Cell Carcinoma Organoids

IntroductionPatient derived organoids (PDOs) are 3D in vitro models and have shown to better reflect patient and tumor heterogeneity than conventional 2D cell lines. To utilize PDOs in clinical settings and trials for biomarker discovery or drug response evaluation, it is valuable to determine the best way to optimize sample selection for maximum PDO establishment. In this study, we assess patient, tumor and tissue sampling factors and correlate them with successful PDO establishment in a well-documented cohort of patients with head and neck squamous cell carcinoma (HNSCC).MethodsTumor and non-tumorous adjacent tissue samples were obtained from HNSCC patients during routine biopsy or resection procedures at the University Medical Center Utrecht. The tissue was subsequently processed to establish PDOs. The sample purity was determined as the presence of epithelial cells in the culture on the day of organoid isolation as visualized microscopically by the researcher. PDO establishment was recorded for all samples. Clinical data was obtained from the medical records and was correlated to PDO establishment and presence of epithelial cells.ResultsOrganoids could be established in 133/250 (53.2%) primary tumor site tissues. HNSCC organoid establishment tended to be more successful if patients were younger than the median age of 68 years (74/123 (60.2%) vs. 59/127 (46.5%), p = 0.03). For a subset of samples, the presence of epithelial cells in the organoid culture on the day of organoid isolation was recorded in 112/149 (75.2%) of these samples. When cultures were selected for presence of epithelial cells, organoid establishment increased to 76.8% (86/112 samples).ConclusionThis study found a trend between age and successful organoid outgrowth in patients with HNSCC younger than 68 years and emphasizes the value of efficient sampling regarding PDO establishment.

Small nucleolar RNA expression profiles: A potential prognostic biomarker for non-viral Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a challenging cancer with high mortality rates, limited predictability, and a lack of effective prognostic indicators. The relationship between small nucleolar RNAs (snoRNAs) and HCC is poorly understood. Based on the literature data, snoRNA studies were primarily focused on viral-related causes of HCC, such as Hepatitis B or C viruses (HBV or HCV). According to these studies, we selected four snoRNAs (snoRA12, snoRA47, snoRA80E, and snoRD126) for exploration in the context of non-viral-related causes, including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver diseases (NAFLD), and alcohol steatohepatitis. The primary goal of this study was to gain a deeper understanding of how snoRNA expression affects patient outcomes and whether it can serve as a prognostic tool for non-viral HCC. We conducted a study on tissue samples from 35 HCC patients who had undergone resection at Pilsen University Hospital. SnoRA12, snoRA47, snoRA80E, and snoRD126 were studied by quantitative real-time PCR (qRT-PCR) in tumor and non-tumor adjacent tissue (NTAT) samples. Kaplan-Meier analysis was performed to assess the association of snoRNAs expression levels with patient outcomes: time to recurrence (TTR), disease-free survival (DFS) and overall survival (OS). In tumor tissues, snoRA12, snoRA47 and snoRA80E were upregulated, while snoRD-126 was downregulated compared to NTAT. Low expression of snoRA47 and snoRD126 in patients was associated with longer TTR and DFS. The individual expression of snoRA12 and snoRA80E did not show associations with TTR and DFS. However, a combination of medium expression of snoRD126 and snoRA80E was associated with longer TTR and DFS, while high and low expressions of the combined snoRA126 and snoRA80E showed no significant association with TTR, DFS, and OS. Conversely, a combination of high expression of snoRA12 and snoRD126 was associated with shorter TTR. In conclusion, the results indicate that snoRA47 and snoRD126 exhibit good prognostic power specifically for non-viral related HCC. Both snoRA47 and snoRD126 showed favorable prognostication in single and combined analysis when assessing patient outcomes. Also, in combination analysis, snoRA80E and snoRA12 showed favorable prognosis, but not alone.

The Nucleolar Protein C1orf131 Is a Novel Gene Involved in the Progression of Lung Adenocarcinoma Cells through the AKT Signalling Pathway.

Lung adenocarcinoma (LUAD) is the most widespread cancer in the world, and its development is associated with complex biological mechanisms that are poorly understood. Here, we revealed a marked upregulation in the mRNA level of C1orf131 in LUAD samples compared to non-tumor tissue samples in The Cancer Genome Atlas (TCGA). Depletion of C1orf131 suppressed cell proliferation and growth, whereas it stimulated apoptosis in LUAD cells. Mechanistic investigations revealed that C1orf131 knockdown induced cell cycle dysregulation via the AKT and p53/p21 signalling pathways. Additionally, C1orf131 knockdown blocked cell migration through the modulation of epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. Notably, we identified the C1orf131 protein nucleolar localization sequence, which included amino acid residues 137-142 (KKRKLT) and 240-245 (KKKRKG). Collectively, C1orf131 has potential as a novel therapeutic marker for patients in the future, as it plays a vital role in the progression of lung adenocarcinoma.

Abstract PO5-13-10: Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection

Abstract Introduction: Success of therapeutic interventions largely rely on the pathological and biological characteristics of the tumor and varies due to the heterogeneous nature of breast cancers. Signal transduction pathways play an important role and are often used as target for specific therapies like hormone treatment, PI3K inhibitors etc. Immunohistochemistry (IHC) is currently the first assessment to divide tumors for subtyping and treatment and, especially in more advanced disease, mutation analyses are often used. Both IHC and mutation analyses only analyze a specific part of a signal transduction pathway and do not correlate well with outcome. Therefore, there is a need for new approaches to look at signaling pathways in cancer. We have developed a methodology that measures and quantifies signal transduction pathways (STPs) activities to reveal potential tumor-driving STPs to identify new options for targeted therapy in breast cancer. Method: Using the mRNA-based OncoSIGNal pathway activity profiling qPCR test (InnoSIGN), STP activities of 7 pathways (AR, ER, PI3K, MAPK, HH, TGFβ and Notch) were measured and quantified on a scale from 0-100. By using a set of non-tumor epithelial breast tissue samples, a reference range for normal physiological pathway activity was defined. A threshold was set on the 95th percentile of this range. A pathway activity score above this threshold was considered to be high (activated). Using this methodology, 201 ER positive (based on IHC) and 64 triple negative FFPE breast tumor samples obtained from different study sites and commercial biobanks were analyzed, including samples from primary and metastatic lesions, to determine for each individual tumor its pathway activity profile and identify activated tumor-driving pathways. Results: In the 195 primary ER positive tumors, the ER pathway was found to be activated in 78% of the samples, even though all samples were strongly ER immunohistochemistry stain positive. In addition to ER pathway activation, occasionally other pathways were activated: PI3K in 37%, HH in 25%, MAPK in 19%, AR in 20%, TGFβ in 6%, and Notch in 1%, of these samples. Analysis of 27 metastatic samples of ER positive tumors revealed in only 63% an active ER pathway. Additionally, more frequently PI3K pathway was observed in 70% of the samples. Other frequencies of pathway activation were: AR in 7% and MAPK in 4%, No Notch and TGFβ activity was found in this cohort. In 76 of the 78 triple negative tumors (TNBC) the ER pathway was inactive as expected. Remarkably, 2 samples appeared to have an activated ER pathway. An activated MAPK pathway was found in 50%, PI3K in 49%, HH in 18%, AR in 14%, TGFβ in 5% and Notch in 3% of these TNBC tumors. Conclusion: Compared to ER positive primary tumors we observed in metastatic ER positive tumors less frequent activation of the ER and more frequent activation of the PI3K pathway, suggesting that the latter pathway is associated with a more aggressive tumor phenotype. The triple negative subtype is characterized predominantly by activation of the PI3K, MAPK, AR and/or HH pathways, which may create new options for personalized targeted therapy of this subtype. OncoSIGNal can be used to determine the tumor driving signaling pathways in breast cancer patients, guiding selection of personalized targeted therapies. Citation Format: Paul van de Wiel, Yvonne Wesseling-Rozendaal, Anke Pierik, Dianne van Strijp, Saskia Vermeer, Fatema Tashrifwala, Josette Hartnett, Robert Babkowski, Suzanne Rose. Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection [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 PO5-13-10.

Multi-omics characterization of esophageal squamous cell carcinoma identifies molecular subtypes and therapeutic targets.

Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer and is characterized by an unfavorable prognosis. To elucidate the distinct molecular alterations in ESCC and investigate therapeutic targets, we performed a comprehensive analysis of transcriptomics, proteomics, and phosphoproteomics data derived from 60 paired treatment-naive ESCC and adjacent nontumor tissue samples. Additionally, we conducted a correlation analysis to describe the regulatory relationship between transcriptomic and proteomic processes, revealing alterations in key metabolic pathways. Unsupervised clustering analysis of the proteomics data stratified patients with ESCC into 3 subtypes with different molecular characteristics and clinical outcomes. Notably, subtype III exhibited the worst prognosis and enrichment in proteins associated with malignant processes, including glycolysis and DNA repair pathways. Furthermore, translocase of inner mitochondrial membrane domain containing 1 (TIMMDC1) was validated as a potential prognostic molecule for ESCC. Moreover, integrated kinase-substrate network analysis using the phosphoproteome nominated candidate kinases as potential targets. In vitro and in vivo experiments further confirmed casein kinase II subunit α (CSNK2A1) as a potential kinase target for ESCC. These underlying data represent a valuable resource for researchers that may provide better insights into the biology and treatment of ESCC.

Mechanical characteristics of glioblastoma and peritumoral tumor-free human brain tissue

BackgroundThe diagnosis of brain tumor is a serious event for the affected patient. Surgical resection is a crucial part in the treatment of brain tumors. However, the distinction between tumor and brain tissue can be difficult, even for experienced neurosurgeons. This is especially true in the case of gliomas. In this project we examined whether the biomechanical parameters elasticity and stress relaxation behavior are suitable as additional differentiation criteria between tumorous (glioblastoma multiforme; glioblastoma, IDH-wildtype; GBM) and non-tumorous, peritumoral tissue.MethodsIndentation measurements were used to examine non-tumorous human brain tissue and GBM samples for the biomechanical properties of elasticity and stress-relaxation behavior. The results of these measurements were then used in a classification algorithm (Logistic Regression) to distinguish between tumor and non-tumor.ResultsDifferences could be found in elasticity spread and relaxation behavior between tumorous and non-tumorous tissue. Classification was successful with a sensitivity/recall of 83% (sd = 12%) and a precision of 85% (sd = 9%) for detecting tumorous tissue.ConclusionThe findings imply that the data on mechanical characteristics, with particular attention to stress relaxation behavior, can serve as an extra element in differentiating tumorous brain tissue from non-tumorous brain tissue.

28. IS THE SWAB USEFUL AS AN ALTERNATIVE METHOD OF COLLECTING MICROBIOTA SAMPLES IN PATIENTS WITH COLON CANCER?

Abstract Introduction This study aims to investigate the intestinal microbiota in rectal samples obtained by swab, comparing them with stool samples, tumor, and non-tumor tissue in patients who underwent colorectal cancer surgery at our hospital. Methods Eight patients (3 women and 5 men) who underwent surgery at our center between 2021 and 2022 were included in the study. The anatomopathological stage was evenly distributed, with 50% in stage II and 50% in stage III. Tumor location was also varied, with 37.5% of patients having neoplasms in the left side colon and 62.5% in the right side. During the preoperative period, stool samples were collected from the patients. Additionally, a rectal exudate was obtained with a swab, and samples of tumor and non-tumor tissue were collected during the procedure. The composition of the intestinal microbiota in all samples was analyzed using rRNA sequencing. Results The analysis of all samples identified the phyla Firmicutes, Bacteroidota, Actinobacteriota, Fusobacteriota, and Proteobacteria. No significant differences were observed in terms of alpha and beta diversity when analyzing the different types of samples (feces, exudate, tumor, and non-tumor tissue). Stage II patients showed a greater abundance of Pasteurellacea, Magasphera, Citrobaceter, and Clostridium in the exudate, while Velillonella and Blautia were more prominent in feces. All the genera were different from those of stage III. The composition did not vary depending on the location of the tumor. Conclusion Swabs are a reliable alternative to stool collection for obtaining microbiota samples in patients with colon cancer.

The Diagnostic and Prognostic Value of Plasma Circulating CircNUP98 for Nasopharyngeal Carcinoma.

Our preliminary sequencing analysis revealed increased expression levels of circNUP98 in nasopharyngeal carcinoma (NPC). This study was therefore carried out to explore the role of circNUP98 in NPC. The present study enrolled 56 patients with NPC, 44 patients with cervical lymphadenitis (CL), 50 patients with nose bleeding (NB), 50 patients with chronic sinusitis (CS), 50 patients with lymph node tuberculosis (LNT), and 50 healthy controls (Control). Plasma samples were obtained from all patients and the controls. In addition, NPC and paired non-tumor tissue samples were collected from the 56 NPC patients. The expression of circNUP98 in both tissue and plasma samples was determined by RT-qPCR. The 56 NPC patients were followed up for 5 years to analyze the associations between plasma expression of circNUP98 and the survival of patients. The diagnostic value of circNUP98 for NPC was analyzed through ROC curve analysis. The plasma expression levels of circNUP98 were only increased in NPC, but not in CL, NB, CS and LNT groups compared to that in the Control group. In addition, increased expression levels of circNUP98 were observed in NPC tissues compared to that in non-tumor tissues. Plasma circNUP98 was closely correlated with circNUP98 in NPC tissues, but not circNUP98 in non-tumor tissues. With plasma circNUP98 as a biomarker, NPC patients were separated from CL, NB, CS, LNT, and the Control groups. The Plasma expression of circNUP98 was found to be positively correlated with the poor survival of patients. Moreover, plasma circNUP98 was only closely associated with tumor metastasis, but not tumor size. The expression of circNUP98 in plasma may be used to diagnose NPC and predict patients' survival.

Diagnostic and prognostic significance of tissue lncRNA HOTAIR and hexokinase 2 mRNA expression in patients with pancreatic ductal adenocarcinoma

BackgroundPancreatic adenocarcinoma, recognized for its aggressive behavior and frequent late-stage diagnosis, imposes significant challenges in early detection and prognosis. This study aimed to evaluate the diagnostic and prognostic potential by measuring the expression levels of long non-coding RNA HOTAIR and the glycolytic enzyme hexokinase 2 (HK2) mRNA in both tumorous and adjacent non-tumorous pancreatic tissue samples (n = 25 each) using RT-qPCR. ResultsBoth lncRNA HOTAIR and HK2 expression levels significantly increased in tumorous pancreatic tissues compared to non-tumorous tissue (P = 0.001). However, their levels in stage T2 and T3 showed no statistically significant difference (P = 0.01). lncRNA HOTAIR and HK2 expression levels positively correlated with each other (P = 0.001; r = 0.95); however, no significant associations were found with serum tumor markers CA19-9 and CEA (P = 0.01; r = 005; p = 0.1, r = 0.2). ROC analysis demonstrated the significant abilities of both lncRNA HOTAIR and HK2 expression levels to discriminate between tumorous and non-tumorous pancreatic tissues (AUC = 0.92 and 0.84, respectively) with 96% and 88% sensitivity, and 72% and 40% specificity, respectively, at optimal cut-off values of 1.12 and 0.84 relative expression units. Patients with elevated lncRNA HOTAIR and HK2 expression had shorter median survival (8 and 7 months, respectively), increasing the risk of adverse outcomes or recurrence 4–4.8 times (HR = 4.08, p = 0.07; HR = 4.8, p = 0.01), thus emphasizing their prognostic potential in pancreatic cancer.ConclusionlncRNA HOTAIR and HK2 expression levels exhibit diagnostic potential in pancreatic tumors. Elevated levels of both markers correlate strongly with adverse outcomes, underscoring their prognostic value.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer.

This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Abstract C046: Rapid autopsy provides unique research opportunity to evaluate KRASG12C inhibitor resistance mechanisms in non-small cell lung cancer

Abstract TThe discovery of small molecule inhibitors that target mutant KRASG12C has revolutionized the treatment paradigm for KRASG12C-mutant non-small cell lung cancer (NSCLC). While the initial response rates to KRASG12C inhibitors (KRASG12Ci) are approximately 40%, the development of heterogenous resistance mechanisms remains a challenge. Understanding the heterogeneity of resistance mechanisms is critical in overcoming this obstacle. Autopsy samples provide valuable insights into therapy resistance and genomic evolution. Moffitt’s Rapid Tissue Donation (RTD) program offers patients with lung malignancies the opportunity to donate primary and metastatic tumor samples, as well as non-tumoral tissue samples and body fluids, for research purposes after their death. 16 patients with KRASG12C-mutant NSCLC enrolled into the RTD, and 15 were treated with KRASG12Ci. Postmortem samples from seven patients were collected to date, including 38 tumor samples from lung, liver, lymph nodes, soft tissue, adrenal, spleen, pancreas, brain and epicardium samples. Radiological measurements were used to evaluate therapy response in each lesion. 26/38 tumor samples were non-responding to treatment whereas the remaining 12 were stable/responding lesions. Among the seven patients, five had both non-responding and stable/responding lesions and the remaining two had only non-responding lesions. Whole exome sequencing (WES) and transcriptomic analyses (RNA-Seq) were performed in 7 tumoral samples and 1 non-tumoral liver sample from one single patient. KRASG12C mutation and PTENR55M mutations were found in all tumor lesions, irrespective of therapy response. A PIK3CAE542K mutation was present in all tumor lesions, except for one subcutaneous tumor that did not respond to treatment. In the non-responding lung tumor, as well as the stable metastatic lymph node, a MAP2K3A203S mutation was identified. Notably, the non-responding liver lesions exhibited an ERBB3A17G mutation, whereas the responding liver lesion did not. Gene expression analyses comparing non-responding and responding liver lesions revealed an upregulation of genes associated with hypoxia, epithelial mesenchymal transition, and inflammation in the non-responding lesions, while a subgroup of genes regulated by MYC was upregulated in the responding liver lesion. Additional genomic analyses of other autopsy samples are underway. Our findings reveal that therapy resistance may be driven by pathway alterations instead of genomic alterations. The collection of rapid autopsy specimens from primary and metastatic tissue sites from patients with KRASG12C-mutant NSCLC is essential to understand the heterogeneous KRASG12Ci mechanisms of resistance and explore biomarkers that may guide clinical decision-making in these patients. Citation Format: Hilal Ozakinci, Gina Nazario, Yaakov Stern, Hitendra S Solanki, Dung-Tsa Chen, Rose Trevor, Matthew Schabath, Paul Stewart, Jamie K Teer, Amer Beg, Theresa A Boyle, Eric Haura, Bruna Pellini. Rapid autopsy provides unique research opportunity to evaluate KRASG12C inhibitor resistance mechanisms in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C046.

Genome-Wide Extrachromosomal Circular DNA Profiling of Paired Hepatocellular Carcinoma and Adjacent Liver Tissues.

Hepatocellular carcinoma (HCC) develops through multiple mechanisms. While recent studies have shown the presence of extrachromosomal circular DNA (eccDNA) in most cancer types, the eccDNA expression pattern and its association with HCC remain obscure. We aimed to investigate this problem. The genome-wide eccDNA profiles of eight paired HCC and adjacent non-tumor tissue samples were comprehensively elucidated based on Circle-seq, and they were further cross-analyzed with the RNA sequencing data to determine the association between eccDNA expression and transcriptome dysregulation. A total of 60,423 unique eccDNA types were identified. Most of the detected eccDNAs were smaller than 1 kb, with a length up to 182,363 bp and a mean sizes of 674 bp (non-tumor) and 813 bp (tumor), showing a greater association with gene-rich rather than with gene-poor regions. Although there was no statistical difference in length and chromosome distribution, the eccDNA patterns between HCC and adjacent non-tumor tissues showed significant differences at both the chromosomal and single gene levels. Five of the eight HCC tissues showed significantly higher amounts of chromosome 22-derived eccDNA expression compared to the non-tumor tissue. Furthermore, two genes, SLC16A3 and BAIAP2L2, with a higher transcription level in tumor tissues, were related to eccDNAs exclusively detected in three HCC samples and were negatively associated with survival rates in HCC cohorts from public databases. These results indicate the existence and massive heterogeneity of eccDNAs in HCC and adjacent liver tissues, and suggest their potential association with dysregulated gene expression.

Assessment of MYC and TERT copy number variations in lung cancer using digital PCR

ObjectiveLung cancer is the second most frequent cancer type and the most common cause of cancer-related deaths worldwide. Alteration of gene copy numbers are associated with lung cancer and the determination of copy number variations (CNV) is appropriate for the discrimination between tumor and non-tumor tissue in lung cancer. As telomerase reverse transcriptase (TERT) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) play a role in lung cancer the aims of this study were the verification of our recent results analyzing MYC CNV in tumor and non-tumor tissue of lung cancer patients using an independent study group and the assessment of TERT CNV as an additional marker.ResultsTERT and MYC status was analyzed using digital PCR (dPCR) in tumor and adjacent non-tumor tissue samples of 114 lung cancer patients. The difference between tumor and non-tumor samples were statistically significant (p < 0.0001) for TERT and MYC. Using a predefined specificity of 99% a sensitivity of 41% and 51% was observed for TERT and MYC, respectively. For the combination of TERT and MYC the overall sensitivity increased to 60% at 99% specificity. We demonstrated that a combination of markers increases the performance in comparison to individual markers. Additionally, the determination of CNV using dPCR might be an appropriate tool in precision medicine.

Identification of novel biomarkers with potential for diagnosis and prognosis of gastric cancer: a Bioinformatics Approach

Introduction: Gastric cancer (GC) is the fifth most diagnosed neoplasia and the third leading cause of cancer-related deaths. A substantial number of patients exhibit an advanced GC stage once diagnosed. Therefore, the search for biomarkers contributes to the improvement and development of therapies. Objective: This study aimed to identify potential GC biomarkers making use of in silico tools. Methods: Gastric tissue microarray data available in Gene Expression Omnibus and The Cancer Genome Atlas Program was extracted. We applied statistical tests in the search for differentially expressed genes between tumoral and non-tumoral adjacent tissue samples. The selected genes were submitted to an in-house tool for analyses of functional enrichment, survival rate, histological and molecular classifications, and clinical follow-up data. A decision tree analysis was performed to evaluate the predictive power of the potential biomarkers. Results: In total, 39 differentially expressed genes were found, mostly involved in extracellular structure organization, extracellular matrix organization, and angiogenesis. The genes SLC7A8, LY6E, and SIDT2 showed potential as diagnostic biomarkers considering the differential expression results coupled with the high predictive power of the decision tree models. Moreover, GC samples showed lower SLC7A8 and SIDT2 expression, whereas LY6E was higher. SIDT2 demonstrated a potential prognostic role for the diffuse type of GC, given the higher patient survival rate for lower gene expression. Conclusion: Our study outlines novel biomarkers for GC that may have a key role in tumor progression. Nevertheless, complementary in vitro analyses are still needed to further support their potential.

Evaluation of potential targets to enhance the sensitivity of cholangiocarcinoma cells to anticancer drugs

BackgroundCholangiocarcinoma (CCA) is a highly lethal cancer originated in the biliary tree. Available treatments for CCA are scarcely effective, partly due to mechanisms of chemoresistance, such as aberrant activation of Wnt/β-catenin pathway and dysfunctional p53. AimTo evaluate the impact of enhancing the expression of negative regulators of the Wnt/β-catenin pathway (AXIN1, AXIN2, and GSK3B) and the tumor suppressor gene TP53. MethodsGene expression in paired samples of CCA and adjacent non-tumor liver tissue was determined by RT-qPCR and immunohistochemistry (IHC). Using lentiviral vectors, CCA cells were transduced with genes of interest to assess their impact on the resistome (TLDA), apoptosis (annexin V/propidium iodide), and decreased cell viability (MTT). ResultsIHC revealed marked nuclear localization of β-catenin, consistent with Wnt/β-catenin pathway activation. In silico analysis with data from TCGA showed heterogeneous down-regulation of AXIN1, AXIN2, and GSK3B in CCA. Enhancing the expression of AXIN1, AXIN2, and GSK3B in CCA cells was not enough to block the activity of this signaling pathway or significantly modify resistance to 5-FU, gemcitabine, and platinated drugs. Consistent with impaired p53 function, CDKN1A was down-regulated in CCA. Forced TP53 expression induced p21 up-regulation and reduced cell proliferation. Moreover, the resistome was modified (FAS, BAX, TYMP, and CES2 up-regulation along with DHFR, RRM1, and BIRC5 down-regulation), which was accompanied by enhanced sensitivity to some antitumor drugs, mainly platinated drugs. ConclusionEnhancing TP53 expression, but not that of AXIN1, AXIN2, and GSK3B, in CCA cells may be a useful strategy to sensitize CCA to antitumor drugs.

Determining the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in patients with non-small cell lung cancer

The aim of this study was to determine the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue in patients with non-small cell lung cancer (NSCLC). A relative level of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes was determined by the quantitative methylation-specific PCR in 73 patients with NSCLC. The quantitative methylation-specific reaction was performed both for tumor tissue samples and non-tumor tissue samples of the same patient. For each of the samples, a reaction was set both by the investigated genes (MARCH11, UNCX, HOXA9, and PTGDR) and by the reference beta-actin gene (β-actin). Positive levels of methylation of the HOXA9 gene were established for 83.5 % patients; the MARCH11 gene – for 80.8 % patients; the PTGDR gene – for 68.4 % patients; the UNCX gene – for 84.9 % patients. In the study group of patients with NSCLC, significant differences were found in the relative levels of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue. The data suggest that hypermethylation of MARCH11, HOXA9, PTGDR, and UNCX genes may play a role in NSCLC tumor progression.