Hydrogen sulfide (H2S) alleviates diabetic myocardial fibrosis by suppressing pyroptosis via inhibiting DNMT3a-mediated Sestrin2 CpG promoter hypermethylation.

BackgroundGlioblastoma (GBM) is the most common and aggressive primary brain tumor, with limited survival despite multimodal treatment strategies. O6-Methylguanine-DNA Methyltransferase (MGMT) promoter methylation is a well-established predictive biomarker for response to temozolomide (TMZ) therapy. However, determining an optimal quantitative methylation-specific PCR (qMSP) cut-off value remains a challenge in clinical practice.ObjectiveThis study aimed to establish an optimal qMSP cut-off value for MGMT promoter methylation and validate its prognostic significance in GBM patients. The impact of MGMT methylation status on survival outcomes was analyzed concerning surgical extent, tumor localization, and white matter tract involvement.MethodsA retrospective analysis of 101 GBM patients (IDH-wildtype) diagnosed between 2008 and 2022 was performed. All patients underwent surgical resection (total/partial excision or stereotactic biopsy) followed by standard chemoradiotherapy. MGMT promoter methylation status was assessed using real-time qMSP. The optimal cut-off value was determined via receiver operating characteristic curve analysis. Kaplan-Meier survival analysis and Cox regression models evaluated the association between MGMT methylation levels, clinical characteristics, and overall survival (OS).ResultsAmong 101 patients with IDH-wildtype glioblastoma, a qMSP cut-off value of 0.242% demonstrated strong diagnostic performance for MGMT methylation status (AUC = 0.875), with 78% sensitivity and 86% specificity. Patients with high methylation levels (≥ 0.242%) showed significantly longer median overall survival compared to those with low methylation (24 vs. 12 months; p = 0.006). This prognostic relevance persisted across surgical and anatomical subgroups. Multivariable Cox regression identified high qMSP methylation (HR ≈ 0.45, p < 0.001) and extent of resection ≥ 90% (HR ≈ 0.30, p = 0.002) as independent predictors of improved survival, whereas TERT promoter mutation (HR ≈ 1.9, p = 0.017) was associated with worse survival. Stratified analysis revealed that TERTp-mutant tumors with low methylation had the worst outcomes. Additionally, excisional surgery and neocortical tumor involvement were associated with significantly better survival (p = 0.0010 and p = 0.0218, respectively). These findings validate within our institutional setting the clinical utility of the 0.242% qMSP threshold for prognostic stratification in glioblastoma, although external multicenter validation is warranted before generalization to routine clinical practice.ConclusionThe identified qMSP cut-off value (0.242) based on the procedure described in this study provides a robust prognostic stratification tool for GBM patients. High MGMT methylation correlates with improved survival, supporting its integration into clinical decision-making. Further multi-center validation studies are warranted to establish standardized MGMT assessment methodologies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15225-2.

The use of vaginal self-sampling for cervical cancer screening is promising and increasing. However, triage cytology cannot be performed on vaginal self-sampling material after a high-risk human papilloma virus (hrHPV)-positive result. In our recent discovery study, we identified a three-marker panel with high sensitivity (82%) and specificity (74%) for CIN3 or worse (CIN3+). In the present study, we performed the clinical validation of this three-marker panel using real-world hrHPV-positive vaginal self-samples obtained through the Dutch screening programme. The markers LHX8, EPB41L3 and ANKRD18CP were analysed using quantitative methylation-specific PCR (QMSP) on a consecutive cohort of hrHPV-positive vaginal self-samples (n = 2482: 408 CIN3+ and 2074 <CIN3). Diagnostic performance was assessed using sensitivity and specificity derived from receiver operating characteristics (ROC) analysis. The three-marker panel showed 73% (298/408) sensitivity and 79% (1640/2071) specificity to detect CIN3+, and identified 96% (21/22) of cervical cancer cases. A scenario analysis was performed on a virtual population of 100 000 hrHPV-positive women using vaginal self-sampling, comparing our methylation triage test with current cytology triage testing. This analysis revealed that more cancers (864 vs. 684 or 770 for 80 or 90% uptake) would be detected with our methylation panel, while referral rates (29% vs. 31% for both 80 and 90% uptake) and detection of CIN3 (72% vs. 68 or 77% for 80 or 90% uptake) would be similar for methylation and cytology. Compared to cytology triage testing, DNA methylation triage analysis using our three-marker panel offers an appropriate alternative to detect CIN3+ in hrHPV-positive vaginal self-samples. Implementation of the DNA methylation triage test would not only increase cancer detection, but would also eliminate the need for physician visits for cytological triage testing. In addition, it would accelerate referral decisions, ultimately reducing uncertainty and ensuring timely screening completion for all women.

Hepatitis B virus (HBV) infection can cause liver damage through oxidative stress (OS) and immune-inflammatory responses. This study aims to explore the clinical significance of fibroblast growth factor 21 (FGF21) in the development and progression of chronic hepatitis B (CHB). A total of 336 participants were recruited, including 320 CHB patients and 16 healthy controls. The expression of FGF21, immune cytokines, and OS-related molecules in peripheral blood mononuclear cells (PBMCs) was detected using real-time quantitative polymerase chain reaction. The methylation level of the FGF21 gene promoter in PBMCs was detected using TaqMan probe-based quantitative methylation-specific PCR. The expression level of FGF21 in the peripheral blood of CHB patients was higher than that of HC, but the methylation level of the FGF21 promoter was lower than that of HC, especially in patients during the immune activation phase. The mRNA expression levels of CXCR3 and CCL5 in PBMCs of CHB patients during the immune activation and reactivation phases were higher than those in other clinical stages. Single-cell analysis revealed that CXCR3 and CCL5 expression in the immune tolerance and immune activation phases with high HBsAg expression was closely related to T lymphocytes (T cells) and natural killer cells (NK cells) and was highly expressed in CD4 and CD8 T cells and NK cells. In addition, the mRNA expression levels of Nrf2 and GPX4 in the reactivation phase were higher than those in other clinical stages. The mRNA expression level and methylation level of FGF21 in PBMCs of CHB patients were correlated with the viral load, immune inflammation, and OS levels during the antiviral treatment course of CHB. The methylation level of the FGF21 promoter has the potential to become a non-invasive biomarker for monitoring the progress of antiviral treatment in CHB.IMPORTANCEThis study conducted an in-depth exploration of the application of methylation detection technology, analyzing its value and driving mechanism in the oxidative stress and immune-inflammatory balance during the course of chronic hepatitis B. The study analyzed the methylation patterns of the FGF21 promoter and the expression levels of its receptor FGFR1, as well as the expression levels of chemokines CXCR3, CCL5, and oxidative stress factors GPX4 and Nrf2 in the immune tolerance period, immune clearance period, immune control period, and reactivation period of chronic hepatitis B. It clarified the association between these molecules and the FGF21/FGFR1 axis and revealed the synergistic or antagonistic mechanisms of these molecules in the oxidative stress and inflammatory vicious cycle. At the same time, this study also explored the value of FGF21 promoter methylation in disease diagnosis and prognosis, providing a theoretical basis for evaluating the antiviral treatment effect and disease progression of chronic hepatitis B.

DNA methylation profiling has emerged as a promising tool for improving pathological diagnosis. This study investigates the diagnostic efficacy and subgroup heterogeneity of SHOX2, RASSF1A, Septin9, and HOXA9 methylation in pleural effusion lymphoma, aiming to enhance diagnostic accuracy and identify high-risk molecular subgroups. A cohort of 109 patients(73 lymphoma, 36 benign pleural effusions)was analyzed using quantitative methylation-specific PCR for the four biomarkers. Receiver operating characteristic (ROC) curves were constructed to determine optimal cutoff values for each marker, and their diagnostic performance was evaluated in different lymphoma subgroups. The cycle threshold values for the internal control β-actin predominantly ranged from18 to 23, suggesting consistent DNA input for methylation analysis. ROC curve revealed that the area under the curve (AUC) values were 0.871 (SHOX2), 0.779 (HOXA9), 0.611 (Septin9), and 0.548 (RASSF1A). When combined, the four markers achieved an AUC of 0.896, with an overall diagnostic sensitivity of 83.6% and a specificity of 94.4%. SHOX2 and HOXA9 demonstrated higher sensitivity within diffuse large B-cell lymphoma (DLBCL) subgroups, with SHOX2 achieving 100% sensitivity in the non-GCB subgroup and HOXA9 achieving 90.9% sensitivity in the GCB subgroup. Septin9 methylation was significantly associated with the presence of B symptoms (P = 0.041). Importantly, RASSF1A methylation was more strongly associated with TP53 deletion rather than with Bcl-2/IGH, Bcl-6, or c-MYC rearrangements. Integratingmethylation markers SHOX2, HOXA9, Septin9, and RASSF1A exhibits significant diagnostic potential in pleural effusion lymphoma, especially within DLBCL subgroups. These markers could prove valuable in identifying high-risk subgroups and guiding clinical decision-making.

BackgroundOral squamous cell carcinoma (OSCC) remains a major global health burden, often diagnosed at advanced stages, with limited survival improvement. Epigenetic dysregulation, particularly promoter hypermethylation, plays a significant role in OSCC pathogenesis. Previous study by Demokan et al. identified Gamma-Aminobutyric Acid Receptor Beta-2 (GABRB2) as a candidate gene subject to methylation-dependent transcriptional silencing. This study aimed to evaluate the methylation and expression profiles of GABRB2 in OSCC and to explore its potential as a diagnostic biomarker.MethodsTissue and serum samples from 48 OSCC patients and 15 healthy controls were analyzed for GABRB2 promoter methylation via quantitative methylation-specific PCR and for gene expression levels via quantitative real-time PCR. Relationships between methylation, expression levels, clinicopathological parameters, and patient outcomes were statistically assessed.ResultsGABRB2 promoter hypermethylation was detected in 14.6% of tumor samples, predominantly in floor of the mouth and buccal tumors. Decreased GABRB2 expression was observed in 52.1% of tumor tissues compared to matched-normal tissues, while 22.9% of tumors exhibited increased expression. Methylation-dependent expression loss was confirmed in 71.4% of methylated tumors. Notably, decreased expression and hypermethylation of GABRB2 were correlated with poor prognosis parameters (p < 0.05). ROC analysis showed moderate discrimination for GABRB2 expression in distinguishing tumor from normal tissues (AUC = 0.635, p = 0.022), while serum-based analysis demonstrated poor diagnostic performance. Survival analysis revealed no statistically significant prognostic impact of GABRB2 expression levels (p > 0.05). However, alcohol consumption (p = 0.006) and recurrence developed (p = 0.030) as independent predictors of poor prognosis in multivariate analysis.ConclusionOur study suggests that there was association between methylation-based expression loss of GABRB2 with OSCC’ subgroups. To our knowledge, this is the first study to investigate GABRB2 gene methylation and expression profiling in both invasive/non-invasive samples from OSCC patients. Hypermethylation in the newly identified candidate GABRB2 gene may play a role in the development of tumors originating from the mouth floor and buccal anatomical regions of the oral cavity. However, since the loss of expression seen in the majority of our patients, we thought that only methylation may not the inhibition mechanism for GABRB2 gene decreased expression. The new identified candidate gene may be specific for the OSCC’s subgroups.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12903-025-06899-y.

This study investigated the expression, methylation patterns, and clinicopathological implications of ARHGAP40 in renal cell carcinoma (RCC), the most common urinary malignancy. A total of 60 clear cell renal cell carcinomas (ccRCC), 30 papillary renal cell carcinomas (pRCC), 30 chromophobe renal cell carcinomas (chRCC), and 13 other RCC subtypes were enrolled. ARHGAP40 expression was analyzed in both RCC tissues and matched paracancerous normal tissues using immunohistochemistry (IHC). The methylation status of the ARHGAP40 promoter region was assessed in both normal and tumor samples by bisulfite sequencing PCR (BSP). Circulating tumor DNA (ctDNA) extracted from peripheral blood samples of RCC patients (20), patients with benign renal tumors (1), and healthy controls (14) was quantitatively analyzed for methylation using quantitative methylation-specific PCR (qMSP). ARHGAP40 expression was significantly downregulated in RCC compared to matched normal tissues (P < 0.001). This reduced expression correlated with tumor necrosis (P = 0.009) but showed no significant association with age, gender, tumor location, tumor diameter, TNM stage, or vascular invasion. In the ccRCC subtype, ARHGAP40 expression exhibited a progressive decrease with larger tumor diameter (P = 0.045), advancing histological grade (P = 0.032), and tumor necrosis (P = 0.011). The methylation status of ARHGAP40 was consistent with its expression level in both tumor and adjacent normal tissues. Methylated ARHGAP40 DNA was detectable only in RCC patient ctDNA samples. ARHGAP40 is epigenetically silenced in RCC through methylation-mediated downregulation, which correlates with tumor necrosis and grade. The detection of methylated ARHGAP40 in ctDNA holds promise as a potential biomarker for early RCC diagnosis.

Most T1 gastric cancer (GC) harbor lymph node metastasis (LNM) at a rate of <20%; however, owing to the difficulty in accurately diagnosing LNM preoperatively, many patients with T1 GC undergo unnecessary invasive radical gastrectomy with lymphadenectomy. In the present study, we established an epigenetic liquid biopsy assay for the preoperative diagnosis of LNM in T1 GC. A comprehensive biomarker discovery was performed by analyzing genome-wide DNA methylation profiling. We obtained 277 clinical specimens, including 177 surgical tissues and 100 pre-operative plasmas. DNA methylation biomarkers were trained and validated using quantitative methylation-specific polymerase chain reaction (qMSP) assays. We identified six novel differentially methylated regions, including at least two differentially methylated CpG probes (|Delta-beta| >0.12 and p<0.05) within 100 bp, through genome-wide biomarker discovery. A DNA methylation panel was generated using qMSP assays in clinical tissue specimens, with an area under the curve (AUC) of 0.80. This panel was validated in an independent clinical cohort, and a combined model, which integrated the DNA methylation model with preoperative computed tomography -based findings, was established through multivariate logistic regression analyses (AUC: 0.84). Finally, we translated this model into a liquid biopsy, and this cell-free DNA (cfDNA) methylation model exhibited robust performance for LNM identification in T1 GC (AUC: 0.86) and allowed 44% of patients to avoid unnecessary invasive operations, without missing any LNM-positive patients. We have successfully developed a cfDNA methylation signature-based liquid biopsy diagnostic assay that allows for robust and less-invasive LNM detection in patients with T1 GC.

BackgroundTo assess the status of NEUROG1 methylation in the advanced adenoma and colorectal cancer.MethodsThe NEUROG1 methylation in tissue and stool samples from patients with colorectal cancer (CRC), advanced adenoma (AA), and non-advanced adenoma (NAA) were evaluated using methylation-specific quantitative polymerase chain reaction (PCR).ResultsIn tissue samples, the NEUROG1 methylation detection rates were 36% for CRC, 24% for NAA, and 88% for AA. In stool samples, the NEUROG1 methylation detection had a sensitivity of 63.46% for CRC with a positive predictive value (PPV) of 85.94%. The overall diagnostic specificity of the test for the NAA and the healthy control was 76.32%, with a negative predictive value (NPV) of 40.28%.ConclusionNEUROG1 methylation detection can potentially be used in the CRC and AA screening.

Abstract BACKGROUND Glioblastoma (GBM) is a universally deadly disease with dismal prognosis. The O6-methylguanine methyltransferase (MGMT) promoter methylation is both predictive in response to standard chemoradiation, as well as prognosis, and unmethylated in approximately 60% of GBM. In newly diagnosed GBM patients with unmethylated MGMT, the median overall survival (OS) is 12.7 months despite combined treatments with surgical resection, temozolomide (TMZ), and fractionated radiotherapy (RT). Poly (ADP-ribose) polymerase (PARP) mediates DNA damage response in GBM. Niraparib is an investigational PARP1/2-selective inhibitor with evidence of excellent tumor pharmacokinetic and pharmacodynamic performance in human GBM compared to other studied PARP inhibitors and an overall survival (OS) of 21.7 months noted in a phase 0/2 study in newly diagnosed GBM. A global registrational Phase 3 study (Gliofocus) will compare the clinical efficacy of niraparib compared to standard of care treatment with temozolomide. MATERIAL AND METHODS In the Phase 3, open-label, 2-arm study (NCT06388733), 450 adult participants with newly-diagnosed, MGMT-unmethylated GBM are randomized to receive niraparib or temozolomide. Participants must have a biopsied or resected GBM, per 2021 World Health Organization classification. MGMT promoter methylation status is determined locally by validated pyrosequencing or quantitative methylation-specific polymerase chain reaction assays. Other key inclusion/exclusion criteria include: (1) Karnofsky performance status of ≥70, (2) no prior treatment for GBM (including brachytherapy or BCNU wafers), (3) no tumor-treating field therapy, (4) suitability for RT of 60 Gy in 30 fractions using ESTRO-EANO ‘single phase’ targeting approach, and (5) no greater than 6 weeks from surgery to treatment initiation. Following 1: 1 randomization, niraparib (Arm A) or TMZ (Arm B) is administered concomitantly with RT and then adjuvantly as monotherapy until disease progression by Blinded Independent Central Review (BICR) or until completion of 6 cycles of TMZ for those on Arm B. The co-primary endpoints of the study are progression-free survival (PFS) (per RANO 2.0; HR = 0.612, 90% power, 1-sided alpha = 0.001) and OS (HR = 0.698, 90% power, 1-sided alpha = 0.0239). Secondary endpoints include overall response rate, health-related quality of life, neurocognitive function, and the safety and tolerability of niraparib compared to TMZ. The first patient was accrued in June 2024 and an interim futility analysis is planned in 2025 Q2. This study, supported by GSK and sponsored by the Ivy Brain Tumor Center, is expected to enroll in a minimum of 115 clinical sites across 11 countries. RESULTS NA/Trial in Progress. CONCLUSION NA/Trial in Progress.

Aim: As a result of the array analyses obtained within the scope of the TUBITAK-SBAG-114S497 project conducted by Demokan and his colleagues, STK32C gene was determined as a potential epigenetic biomarker candidate. In this study, the methylation and expression levels of STK32C gene were examined in a larger patient group consisting of oral squamous cell carcinoma (OSCC) cases; the biomarker potential of this gene in terms of early diagnosis/prognosis evaluation was investigated. Method: DNA and RNA were isolated from tissue samples of 15 OSCC patients, and the methylation and expression levels of the STK32C gene were examined using Quantitative Methylation Specific PCR and Real-Time PCR methods, respectively. Results and Conclusions: In tumor samples of OSCC patients, 93.3% methylation was observed in the promoter region of the STK32C gene. When expression levels were evaluated, expression loss was detected in 53.3% of the samples in tumor tissues compared to normal tissues, while expression increase was observed in 13.3%. Hypermethylation was detected in all patients with decreased expression levels. It is suggested that the loss of expression observed in the STK32C gene due to hypermethylation may play a role in the molecular characterization of a certain subgroup of OSCC patients and that this gene may be a significant biomarker candidate for early diagnosis and prognosis. However, further studies covering larger patient populations are needed to confirm this.

Aim: Oral cancer constitutes 2-4% of all cancers and is the most common cancer in the head and neck region after laryngeal cancer. Oral squamous cell carcinoma (OSCC) constitutes more than 90% of all oral cavity carcinomas. While the 5-year survival rate in OSCC is 40%, this rate increases to 95% in early diagnosis. The GABRB3 gene was identified as a potential epigenetic biomarker candidate in the TUBITAK-SBAG-114S497 project conducted by Demokan et al., and in this study, the differing methylation and expression levels in OSCC patients were examined and its biomarker potential in early diagnosis and prognosis was investigated. Methods: DNA and RNA were isolated from tissue samples taken from 15 patients diagnosed with OSCC, then the methylation status and gene expression levels of the GABRB3 gene were evaluated by Quantitative Methylation Specific PCR and Real-Time PCR methods, respectively. Results and Conclusions: Methylation was detected in the promoter region of the GABRB3 gene in 60% of tumor samples from OSCC patients. When expression levels were evaluated, loss of expression of the GABRB3 gene was detected in 46.7% of samples compared to normal tissues in tumor tissues, while increased expression was observed as 13.3%. Methylation was detected in all patients with decreased expression levels. While decreased expression was observed in 33.3% of tumor tissues having no methylation, increased expression was observed in 50%. It is thought that GABRB3 gene may be responsible for a special subgroup of OSCC patients via methylation-related loss of expression and can be used as a potential biomarker candidate in early diagnosis and prognosis determination. Further studies in larger patient groups are needed to confirm these findings.

Background Chronic pancreatitis (CP) is an inflammatory disease characterized by pain, functional deficits and increased mortality. The clinical course is unpredictable, and there are no classification systems or biomarkers to predict this. Identifying patients with high mortality risk is crucial for guiding clinical management and improving outcomes. This study presents a novel approach to a prognostic prediction model that combines clinical parameters and promoter hypermethylation (ph) of genes. Methods We performed methylation-specific quantitative polymerase chain reaction(qPCR) on a panel of 28 genes, using an accelerated bisulfite treatment protocol. We then developed a prognostic prediction model by backwards stepwise elimination using the methylation status of genes with a ph frequency > 5% and seven clinical factors. Survival was assessed with Kaplan-Meier survival curves and Cox regression. Results Ninety-seven patients with CP were included in the study. The final model included: Age, sex, exocrine insufficiency, diabetes, prior history of acute pancreatitis, and the methylation status of MLH1, HIC1, and RASSF1A. The model had an area under the curve (AUC) of 0.84 (95%CI: 0.76–0.92). A risk score was computed, and patients stratified into high and low-risk groups. The high-risk group had a significantly higher hazard ratio (HR) of death of 14.1 (95% CI; 4.3–46.0, p < 0.01). Conclusions This study serves as proof-of-concept that clinical factors can be combined with gene methylation status to provide additional prognostic information in patients with chronic pancreatitis. This could potentially aid the clinician in estimating which patients require intense follow-up. However, external validation is required.

IntroductionIdiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease (ILD) characterized by progressive accumulation of extracellular matrix in the lung and dysregulated activation of specific signaling pathways. Recent advances in the understanding of the biological bases of IPF identified the silencing of secreted protein acidic and rich in cysteine (SPARC) as a key modulator in the pathogenesis of IPF, although the mechanisms underlying the SPARC aberrant modulation remain to be fully elucidated.MethodsHere we investigated the aberrant methylation at the promoter gene region as a possible mechanism of SPARC deregulation in IPF. Formalin-fixed paraffin-embedded (FFPE) tissues from a cohort of 44 patients with IPF and from a control-group of 23 non-idiopathic pulmonary fibrosis (NIPF) were analyzed. DNA methylation analysis at the SPARC promoter region was assessed by quantitative methylation-specific PCR analysis (QMSP) and a total of 11 CpGs located in the gene promoter island were evaluated.ResultsMethylation levels were found to be significantly higher (p < 0.004, Mann-Whitney test) in 44 IPF samples (methylated using the optimal cut-off 20/44, 45%) compared to NIPF surgical biopsies (methylated using the optimal cut-off 3/23, 13%). At the in vitro level, we observed an inverse correlation between SPARC mRNA levels and hypermethylation under 5-Aza-2′-deoxycytidine (5-Aza-CdR) treatment when a primary fibrotic cell line was treated, whereas any variations were observed treating non-fibrotic cells.DiscussionOur explorative study suggests that promoter methylation of the SPARC gene is linked to IPF but not to NIPF, and could represent a potential molecular marker of disease, thus warranting further investigations on larger cohorts.

Junctional adhesion molecule 3 (JAM3) is frequently epigenetically silenced in various cancers, but its role in serous ovarian carcinoma (SOC) was unclear. This study evaluated JAM3 expression and methylation in SOC using immunohistochemistry (IHC), bisulfite sequencing PCR (BSP), and quantitative methylation-specific PCR (qMSP). Cell proliferation, apoptosis, migration, and invasion were examined using CCK8, flow cytometry, scratch-wound, and transwell assays. Pathways downstream of JAM3 were explored through RNA sequencing (RNA-seq) and Western Blot analysis, with rescue experiments using AKT inhibitor (MK2206) to validate pathway dependency. Findings revealed that JAM3 expression is significantly reduced in SOC, correlating with advanced clinical stages and poor prognosis. Methylation levels of the JAM3 promoter were higher in SOC samples compared to normal tissues and were linked to increased Ki67 expression and clinical stages. Functionally, overexpressing JAM3 in SOC cells triggered apoptosis and hindered proliferation, migration, and invasion, whereas JAM3 knockdown produced opposite effects. Mechanism analysis demonstrated that JAM3 affects SOC cell proliferation through the PI3K/AKT signaling pathway. Conclusively, JAM3 acts as a tumor suppressor in SOC by modulating the PI3K/AKT pathway. These insights present JAM3 as a promising therapeutic target for SOC diagnosis and treatment.

BackgroundPrimary liver cancer (PLC) is a global health concern. The plasma dual-target methylation (PDTM) test, which interrogates the methylation status of GNB4 and Riplet, exhibits a commendable ability to discriminate hepatocellular carcinoma (HCC) from controls. Nevertheless, its performance in detecting PLC in larger populations remains to be validated.ResultsA multicenter, double-blind, cross-sectional study was conducted. Blood samples were collected from all participants for the PDTM test, which is based on a triplex quantitative methylation-specific polymerase chain reaction (qMSP) platform. Additionally, Sanger sequencing was performed to confirm the accuracy of methylation detection by the PDTM test. The study enrolled 430 PLC patients and 752 controls. The PDTM test demonstrated an overall sensitivity of 92.3% (95% confidence interval [CI], 89.4–94.7) for PLC patients and an overall specificity of 93.4% (95% CI, 91.3–95.0) for controls with benign liver disease (BLD) or non-liver primary malignancies (NLPM). Specifically, the sensitivities of the PDTM test for patients with HCC, intrahepatic cholangiocarcinoma (ICC) or early-stage (TNM stages I and II) PLC were 91.9% (95% CI, 87.6–95.0), 93.3% (95% CI, 85.9–97.5) and 88.7% (95% CI, 83.9–92.5), respectively. Furthermore, the kappa values for both GNB4 and Riplet between the PDTM test and Sanger sequencing exceeded 0.99, indicating a high level of consistency.ConclusionsThe PDTM test demonstrates excellent diagnostic performance for PLC, particularly in cases of early-stage PLC. It is a promising early screening or surveillance tool for PLC, and further prospective research is required to ascertain its full utility.

Predictive value of BRCA1/RAD51C methylation in HGSOC - An ancillary study of the PAOLA-1/ENGOT-ov25 phase 3 trial.

The high mortality and recurrence rates of genitourinary (GU) malignancies, which include bladder, prostate, renal, testicular, penile, and adrenal tumors, pose serious clinical problems. Foe better results, early detection is essential, yet conventional diagnostic techniques frequently have little sensitivity and are instructive. Non-invasive methods have become viable substitutes for the identification, tracking, and treatment of malignant tumors, especially urine liquid biopsies. Early bladder cancer (BC) diagnosis may be possible with urinary biomarkers such as cytokeratin’s, FGFR3, NMP22, BTA, and miRNAs (e.g., miR-126, miR-141-3p). While miRNAS and exosomal content provides information on the molecular mechanisms underlying the disease and its response to treatment, methylated DNA markers (such as DAPK and TERT) also aid in the diagnosis of BC. The predictive and diagnostic potential of urine biomarkers are being improved by developments in metabolomics, multi-omics, and epigenetic studies. Though problems like tumor heterogeneity, biomarker standardization, and false positives still exist, methods like electrochemical biosensors, real-time PCR, and quantitative methylation-specific PCR are increasing the biomarkers detection sensitivity and specificity. Exosome analysis combined with cutting-edge bio sensing technologies opens up new possibilities for customized medicine in the diagnosis and treatment of BC. Resolving these issues and improving clinical results for patients with GU cancer require ongoing innovation in biomarker research and diagnostic technologies.

Abstract Our earlier research discovered that C1QL1 was expressed less in breast cancer (BrCa) tissues than in normal breast tissues by analyzing the gene profile of RNA sequences. However, up to now, the biological function of C1QL1 and its molecular mechanism in BrCa remains unknow. Public database analysis, qRT-PCR, western blot, immunohistochemistry, and quantitative methylation specific PCR were used to analyze C1QL1 expression and promoter methylation. The effects of C1QL1 on breast cancer proliferation, cell cycle, apoptosis, metastasis, were assessed using CCK8, flow cytometry analysis, TUNEL assays, transwell in vitro and nude mice experiments in vivo. LC-MS/MS, CoIP and western blot were performed to identify factors that mediate effects of C1QL1. In BrCa, C1QL1 is often silenced due to promoter methylation, and its expression is favorably connected with prognosis. Overexpression of C1QL1 inhibits BrCa cell proliferation, metastasis and promotes cancer cell apoptosis both in vitro and in vivo. Conversely, C1QL1 knockdown increases the proliferation and spread of BrCa cells. Mechanistically, C1QL1 is located at endoplasmic reticulum (ER) and interacts with HSP90α and VCP to facilitate their ubiquitin-mediated degradation. This leads to the caspase-dependent apoptosis that occurs in breast cancer cells as a result of ER stress (ERS)/unfolded protein response (UPR). Our results support that C1QL1 can act as a tumor suppressor of BrCa by modulating C1QL1/HSP90α/VCP-ERS/UPR pathway, implying that the promoter methylation status of C1QL1 or the expression of C1QL1 may represent a potential marker for the diagnosis or prognosis of BrCa. Citation Format: Ningning Zhang, Qing shao, Tingxiu Xiang, Xiaohua Zeng. C1QL1 inhibits breast cancer through HSP90α/VCP-ERS/UPR axis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-02-14.

Abstract Introduction: With increasingly effective systemic therapies and expanding indications for radiation, the role of axillary lymph node dissection (ALND) in the management of patients with node-positive breast cancer (BC) continues to evolve. However, while clinical trials have progressively demonstrated that ALND offers little to no added protection from local recurrence or death in various clinical scenarios, ALND remains the only method to fully stage the axilla and differentiate pN1 vs &amp;gt;pN1 disease. However, certain decisions for adjuvant therapies, such as CDK4/6 inhibitor therapy in patients with estrogen receptor (ER) positive BC, still rely on the nodal stage. In this study, we used machine learning algorithms to create epigenetic classifiers predictive of nodal stage (pN1 vs &amp;gt;pN1) based on DNA methylation profiling of primary BC tumors in two independent cohorts of patients (UCLA and Duke) with ER-positive, HER2-negative BC. Methodology: Eligibility criteria included women aged 18-80 years with ER+, HER2-negative BC, clinically node-positive, who underwent ALND without NAC. Tumoral tissue was microdissected from 8 µm Formalin-Fixed Paraffin-Embedded (FFPE) slides, previously annotated by a trained pathologist, and DNA was extracted using the Quick-DNA FFPE Miniprep kit (Zymo Research). DNAm profiling was performed using the Illumina Infinium Methylation EPIC BeadChip v1. DNAm data was processed using the R/ChAMP package (v.2.34.0), and the batch effect was corrected using the “champ.runCombat” function. The cohort was split into a training cohort (60%, n = 53) and a validation cohort (40%, n = 34). R/VarSelRF (v.0.7-8) was used to identify the combination of probes with the fewest sites and the highest potential to predict the pathological nodal stage. The best-performing classifiers were tested in the validation cohort. Results: DNAm profiling was performed on 91 primary BC samples, with four samples removed due to low data quality. The UCLA and Duke cohorts presented a significant batch effect, which was successfully corrected to avoid potential bias. We identified 1,653 differentially methylated sites between pN1 and &amp;gt;pN1 tumors in the training cohort, successfully stratifying both subgroups of patients. These sites were used to generate a Random Forest-based classifier with the minimum number of probes. We selected the three combinations of probes with the highest Area Under the Curve (AUC) and the fewest CpG sites. We identified three classifiers, comprising 8 to 12 genomic regions, with an AUC between 0.99 and 1 in the training cohort and between 0.81 and 0.82 in the validation cohort. Discussion: As randomized trial data support the omission of ALND in select clinical scenarios in patients with node-positive BC, clinicians will lose the pathologic nodal data that is provided by surgical dissection that guides adjuvant therapy decision-making. This study generated three classifiers that successfully identified patients with higher nodal stage (&amp;gt;pN1). These classifiers use a small number of genomic regions (8 to 12) and can be optimized for low-throughput techniques such as pyrosequencing or quantitative Methylation-Specific PCR, increasing the availability of this diagnostic tool in the clinical setting. Citation Format: Miquel Ensenyat-Mendez, Sandra Iñiguez-Muñoz, Julie Le, Sookyung Ahn, Isabel Eng, Peggy Sullivan, Pere Llinas-Arias, Jennifer L. Baker, Diego M. Marzese, Maggie L. DiNome. Generation and validation of primary breast cancer epigenetic classifiers of pathologic nodal stage in a multi-center breast cancer cohort [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-01-30.