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Circulating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines. Short stature homeobox 2 ( ) and septin 9 ( ) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance. In the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category ( < 0.001). Initially increased methylation levels were associated with a higher risk of death [ : hazard ratio (HR) = 5.27, = 0.001; : HR = 2.32, = 0.024]. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival ( : HR = 2.78, = 0.022; : HR = 2.50, = 0.026), and correlation with tumor and nodal category ( <0.001) were successfully validated. Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes.

γ-Secretase inhibitors (GSIs) are being investigated for their potential to modify the progression of Alzheimer disease based on their ability to regulate amyloid-β (Aβ) accumulation. BMS-708163 (avagacestat) is an oral GSI designed for selective inhibition of Aβ synthesis currently in development for the treatment of mild to moderate and predementia AD. In addition to the desired effect on Aβ synthesis, GSIs affect Notch processing, which is thought to mediate some toxic adverse effects reported with this drug class. Avagacestat produced up to 190-fold greater selectivity for Aβ synthesis than Notch processing in preclinical studies and may therefore produce less toxic adverse events than other less selective compounds. Presented here are the results of the first in-human study for this new GSI compound. The goal of this study was to assess the tolerability profile, pharmacokinetic properties, and effects on pharmacodynamic markers (Aβ, trefoil factor family 3 protein, dual specificity phosphatase 6, and hairy and enhancer of split-1) of single, oral doses of avagacestat in healthy, young, male volunteers. This was a multicenter, randomized, double-blind, placebo-controlled, single-ascending dose study in 8 healthy young men (age, 18–45 years) per dosing panel. Each study participant was randomized to receive a single dose of placebo (n = 2) or avagacestat (n = 6 for each dose) as an oral solution in 1 of 9 sequential dose panels (0.3, 1.5, 5, 15, 50, 100, 200, 400, and 800 mg). For determination of avagacestat, blood samples were obtained before dosing and for up to 144 hours after dosing. For participants in the 800-mg avagacestat dose panel, additional samples were obtained at 216, 312, and 648 hours. For 40–amino acid isoform of Aβ (Aβ1–40) assessment, plasma samples were collected before avagacestat administration and up to 72 hours after dosing. Avagacestat concentrations peaked quickly after oral administration and then had a biphasic decrease in concentrations with a prolonged terminal phase. Exposures were proportional with doses up to 200 mg. Avagacestat was well tolerated at single oral doses up to 800 mg, with a biphasic effect on plasma Aβ1–40. Adverse events were predominately mild to moderate in severity with no evidence of dose dependence up to 200 mg. Results from this single-ascending dose study suggest that avagacestat was tolerated at a single-dose range of 0.3 to 800 mg and suitable for further clinical development. ClinicalTrials.gov identifier: NCT01454115.

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis-free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.

•A multiplex methylation SNaPshot assay was replicated in blood samples.•Postmortem changes can alter the methylation status among specific loci in blood.•Differences in the methylation levels occur between different populations.•The multiplex methylation SNaPshot is a useful method for age estimation. Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1–94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28–86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Retinoblastoma (RB) is the most common intraocular malignancy of childhood, yet molecular assessment of disease dissemination and minimal residual disease (MRD) remains challenging due to the contraindication of intraocular biopsy. Here, we evaluate the feasibility of cell-free RNA (cfRNA)- and circulating tumor cell RNA (ctcRNA)-based liquid biopsy for the sensitive detection of disseminated retinoblastoma using digital PCR (dPCR) targeting the retina-specific markers CRX and RBP3. We analyzed 433 bone marrow (BM), peripheral blood (PB) and cerebrospinal fluid (CSF) samples collected longitudinally from 50 patients with RB. dPCR assays demonstrated high analytical sensitivity. cfRNA detection showed complete sensitivity and negative predictive value in bone marrow compared with myelogram analysis, frequently identifying molecular positivity in cytologically negative samples. In cerebrospinal fluid, cfRNA detection was highly specific but less sensitive, reflecting compartment-specific biological constraints. Longitudinal analysis revealed that changes in CRX and RBP3 ctcRNA levels closely tracked treatment response, preceded cytological evidence of bone marrow involvement in several cases, and identified molecular persistence or re-emergence during follow-up, including after hematopoietic stem cell transplantation. Together, these findings demonstrate that cfRNA- and ctcRNA-based liquid biopsy using CRX and RBP3 enables sensitive and dynamic detection of disseminated retinoblastoma, particularly in bone marrow, and supports its potential utility for MRD monitoring. Longitudinal patient analyses will be required to define prognostic thresholds and establish the clinical role of this approach in risk stratification and long-term surveillance.

Background Although numerous treatment modalities exist for Prostate Cancer (PCa) with a complex pathophysiology, a significant worldwide health concern for males, early identification remains the paramount factor influencing disease prognosis. Proposed new biomarkers, including GSTP1, PTEN, and NKX3.1, may provide predictive and prognostic functions, hence enhancing the efficacy of PSA in diagnosing prostate cancer. Epigenetic variables associated with nutrition influence the blood gene expression levels of the new biomarkers GSTP1, PTEN, and NKX3.1. This study aimed to elucidate the correlation between nutrition-related epigenetic variables and the expression of the tumor suppressor genes GSTP1, PTEN, and NKX3.1 in benign prostate and prostate cancer patients. Methods This study was conducted with 41 newly diagnosed prostate cancer and 42 benign prostate patients, all aged 40 years and older. Participants had specific anthropometric measurements taken, and seven-day food records were kept. Serum levels of novel biochemical markers GSTP1, PTEN, and NKX3.1 were measured using an Enzyme-Linked Immunosorbent Assay (ELISA) method. Expression analyses of tumor-protective GSTP1, PTEN, and NKX3.1 genes were performed on prostate tissues obtained from fresh prostate biopsies taken from the groups and were associated with nutritional status. Results A significant relationship was found between the serum levels of GSTP1, PTEN, and NKX3.1, which are important in the formation and treatment of prostate cancer, and Gleason grade, tumor stage, and surgical margin status ( p  < 0.05). However, significant relationships were found between many macro and micronutrients obtained from the dietary pattern and effective in epigenetic pathways and GSTP1, PTEN, and NKX3.1 gene expressions ( p  < 0.05). Conclusions The findings of this investigation corroborated the notion that prospective novel markers, including GSTP1, PTEN, and NKX3.1, may serve predictive and prognostic functions in prostate cancer, hence enhancing the efficacy of PSA in its diagnosis. Dietary trends may influence the optimization of many epigenetic processes. Targeted therapeutic nutritional interventions designed to enhance gene expression will facilitate the formulation of prostate cancer prevention methods.

Background Lung cancer remains the leading cause of cancer deaths across the world. Early detection of lung cancer by low-dose computed tomography (LDCT) can reduce the mortality rate. However, making a definitive preoperative diagnosis of malignant pulmonary nodules (PNs) found by LDCT is a clinical challenge. This study aimed to develop a prediction model based on DNA methylation biomarkers and radiological characteristics for identifying malignant pulmonary nodules from benign PNs. Methods We assessed three DNA methylation biomarkers ( PTGER4 , RASSF1A, and SHOX2 ) and clinically-relevant variables in a training cohort of 110 individuals with PNs. Four machine-learning-based prediction models were established and compared, including the K-nearest neighbors (KNN), random forest (RF), support vector machine (SVM), and logistic regression (LR) algorithms. Variables of the best-performing algorithm (LR) were selected through stepwise use of Akaike’s information criterion (AIC). The constructed prediction model was compared with the methylation biomarkers and the Mayo Clinic model using the non-parametric approach of DeLong et al. with the area under a receiver operator characteristic curve (AUC) analysis. Results A prediction model was finally constructed based on three DNA methylation biomarkers and one radiological characteristic for identifying malignant from benign PNs. The developed prediction model achieved an AUC value of 0.951 in malignant PNs diagnosis, significantly higher than the three DNA methylation biomarkers (0.912, 95% CI:0.843–0.958, p  = 0.013) or Mayo Clinic model (0.823, 95% CI:0.739–0.890, p  = 0.001). Validation of the prediction model in the testing cohort of 100 subjects with PNs confirmed the diagnostic value. Conclusion We have shown that integrating DNA methylation biomarkers and radiological characteristics could more accurately identify lung cancer in subjects with CT-found PNs. The prediction model developed in our study may provide clinical utility in combination with LDCT to improve the over-all diagnosis of lung cancer.

Abiraterone acetate (AA) serves as a medication for managing persistent testosterone production in patients with metastatic castration-resistant prostate cancer (mCRPC). However, its efficacy varies among individuals; thus, the identification of biomarkers to predict and follow treatment response is required. In this pilot study, we explored the potential of circulating microRNAs (c-miRNAs) to stratify patients based on their responsiveness to AA. We conducted an analysis of plasma samples obtained from a cohort of 33 mCRPC patients before and after three, six, and nine months of AA treatment. Using miRNA RT-qPCR panels for candidate discovery and TaqMan RT-qPCR for validation, we identified promising miRNA signatures. Our investigation indicated that a signature based on miR-103a-3p and miR-378a-5p effectively discriminates between non-responder and responder patients, while also following the drug’s efficacy over time. Additionally, through in silico analysis, we identified target genes and transcription factors of the two miRNAs, including PTEN and HOXB13, which are known to play roles in AA resistance in mCRPC. In summary, our study highlights two c-miRNAs as potential companion diagnostics of AA in mCRPC patients, offering novel insights for informed decision-making in the treatment of mCRPC.

Background Whole body ischemia-reperfusion injury (IRI) after cardiopulmonary resuscitation (CPR) induces a generalized inflammatory response which contributes to the development of post-cardiac arrest syndrome (PCAS). Recently, pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and inflammasomes, have been shown to mediate the inflammatory response in IRI. In this study we investigated monocyte PRR signaling and function in PCAS. Methods Blood samples were drawn in the first 12 hours, and at 24 and 48 hours following return of spontaneous circulation in 51 survivors after cardiac arrest. Monocyte mRNA levels of TLR2, TLR4, interleukin-1 receptor-associated kinase (IRAK)3, IRAK4, NLR family pyrin domain containing (NLRP)1, NLRP3, AIM2, PYCARD, CASP1, and IL1B were determined by real-time quantitative PCR. Ex vivo cytokine production in response to stimulation with TLR ligands Pam 3 CSK 4 and lipopolysaccharide (LPS) was assessed in both whole blood and monocyte culture assays. Ex vivo cytokine production of peripheral blood mononuclear cells (PBMCs) from a healthy volunteer in response to stimulation with patients’ sera with or without LPS was assessed. The results were compared to 19 hemodynamically stable patients with coronary artery disease. Results Monocyte TLR2, TLR4, IRAK3, IRAK4, NLRP3, PYCARD and IL1B were initially upregulated in patients following cardiac arrest. The NLRP1 and AIM2 inflammasomes were downregulated in resuscitated patients. There was a significant positive correlation between TLR2, TLR4, IRAK3 and IRAK4 expression and the degree of ischemia as assessed by serum lactate levels and the time until return of spontaneous circulation. Nonsurvivors at 30 days had significantly lower mRNA levels of TLR2, IRAK3, IRAK4, NLRP3 and CASP1 in the late phase following cardiac arrest. We observed reduced proinflammatory cytokine release in response to both TLR2 and TLR4 activation in whole blood and monocyte culture assays in patients after CPR. Sera from resuscitated patients attenuated the inflammatory response in cultured PBMCs after co-stimulation with LPS. Conclusions Successful resuscitation from cardiac arrest results in changes in monocyte pattern recognition receptor signaling pathways, which may contribute to the post-cardiac arrest syndrome. Trial registration The trial was registered in the German Clinical Trials Register ( DRKS00009684 ) on 27/11/2015.

Background It has been reported that microRNA-130a (miR-130a) targets GAX, the growth arrest-specific homeobox, which inhibits proliferation, differentiation, and migration of vascular smooth muscle cells (VSMCs). In the present study, we therefore investigated the effect of miR-130a on proliferation of cultured VSMCs and the potential role of miR-130a in vascular remodeling during hypertension. Methods Proliferation of VSMCs was determined by 5-bromo-2′-deoxyuridine (BrdU) incorporation method. The expression of miR-130a and GAX was analyzed by quantitative reverse transcription-PCR. The protein expression of GAX was analyzed by western blot. The mimic and inhibitor of miR-130a were used in gain-of-function and loss-of-function in vitro studies, respectively. The correlation of miR-130a with vascular remodeling was observed in spontaneously hypertensive rats (SHRs). Results MiR-130a mimic at the concentration of 25 or 50nmol/l significantly promoted proliferation of VSMCs. The expression of miR-130a was upregulated in the remodeled aorta and superior mesenteric artery of SHRs. The expression of GAX was downregulated in VSMCs transfected with miR-130a mimic and in thoracic aorta and superior mesenteric artery of SHRs. Angiotensin II (Ang II) promoted proliferation of VSMCs and upregulated miR-130a expression concomitantly with a decreased GAX expression in a concentration- and time-dependent manner. The proliferative effects of Ang II on VSMCs were suppressed partly by the miR-130a inhibitor. Conclusions These results suggest that miR-130a is a novel regulator of proliferation of VSMCs via inhibiting the expression of GAX, which may contribute to vascular remodeling in hypertension. American Journal of Hypertension advance online publication 14 July 2011; doi:10.1038/ajh.2011.116