Patients diagnosed with epithelial ovarian cancer (OC) have a 5-year survival rate of 49%. For early-stage disease, the 5-year survival rate is above 90%. However, advanced-stage disease accounts for most cases as patients with early stages often are asymptomatic or present with unspecific symptoms, highlighting the need for diagnostic tools for early diagnosis. Liquid biopsy is a minimal invasive blood-based approach that utilizes circulating tumor DNA (ctDNA) shed from tumor cells for real-time detection of tumor genetics and epigenetics. Increased DNA methylation of promoter regions is an early event during tumorigenesis, and the methylation can be detected in ctDNA, accentuating the promise of methylated ctDNA as a biomarker for OC diagnosis. Many studies have investigated multiple methylation biomarkers in ctDNA from plasma or serum for discriminating OC patients from patients with benign diseases of the ovaries and/or healthy females. This systematic review summarizes and evaluates the performance of the currently investigated DNA methylation biomarkers in blood-derived ctDNA for early diagnosis of OC. PubMed’s MEDLINE and Elsevier’s Embase were systematically searched, and essential results such as methylation frequency of OC cases and controls, performance measures, as well as preanalytical factors were extracted. Overall, 29 studies met the inclusion criteria for this systematic review. The most common method used for methylation analysis was methylation-specific PCR, with half of the studies using plasma and the other half using serum. RASSF1A, BRCA1, and OPCML were the most investigated gene-specific methylation biomarkers, with OPCML having the best performance measures. Generally, methylation panels performed better than single gene-specific methylation biomarkers, with one methylation panel of 103,456 distinct regions and 1,116,720 CpGs having better performance in both training and validation cohorts. However, the evidence is still limited, and the promising methylation panels, as well as gene-specific methylation biomarkers highlighted in this review, need validation in large, prospective cohorts with early-stage asymptomatic OC patients to assess the true diagnostic value in a clinical setting.

Cell-free DNA (cfDNA) serves as a valuable biomarker for early disease detection and monitoring. However, the use of cfDNA for analysis faces challenges owing to general low but variable abundance and fragmentation. Preanalytical factors, including cfDNA extraction, impact cfDNA quality and quantity. Efficient and robust cfDNA extraction is essential for reliable results in downstream applications, and various commercial extraction methods exist, each with trade-offs. To aid researchers and clinicians in choosing the proper cfDNA extraction method, manual, semiautomated, and automated methods were evaluated, including the QIAamp Circulating Nucleic Acid Kit (manual and QIAcube), QIAamp MinElute ccfDNA Kit (QIAcube), and QIAsymphony DSP Circulating DNA Kit (QIAsymphony). For each extraction method, cfDNA was extracted on two separate days, using samples obtained from 18 healthy donors. This study assessed extraction efficiency, quantity, and quality using droplet digital PCR and TapeStation. The QIAamp Circulating Nucleic Acid Kit, both manual and semiautomated, outperformed the QIAamp MinElute ccfDNA Kit (QIAcube) and QIAsymphony DSP Circulating DNA Kit (QIAsymphony), showing higher recovery rates and cfDNA quantity. All methods were reproducible, with no day-to-day variability and no contamination by high-molecular-weight DNA. The QIAamp Circulating Nucleic Acid Kit offers high yield without compromising quality. Implementation of the method should consider specific study and clinical needs, taking into account each method's advantages and limitations for optimal outcomes.

Homologous recombination deficiency (HRD) can arise from germline or somatic pathogenic variants as well as other genomic damage and epigenetic alterations in the HR repair pathway. Patients with tumors presenting with an HRD phenotype can show sensitivity to Poly (ADP-ribose) polymerase inhibitors (PARPis). Several promising tests to detect HRD have been developed based on different HRD definitions, biomarkers, and algorithms. However, no consensus on a gold standard HRD test has been established. In this systematic review, a comprehensive list of tests for the detection of HRD was identified and compared regarding HRD definition, biomarkers, and algorithms. PubMed’s Medline and Elsevier’s Embase were systematically searched, resulting in 27 eligible articles meeting the inclusion criteria. The primary challenge when comparing HRD tests lies in the lack of a consensus definition of HRD, as the HRD definition influences the proportion of samples being classified as HRD and impacts the classification performance. This systematic review provides an overview of available HRD tests that can inspire other researchers in searching for a gold standard HRD definition and highlights the importance of the factors that should be considered when choosing an HRD definition and tests for future planning of clinical trials and studies.

The importance of structural genetic variants, such as copy number variations (CNVs), in modulating human disease is being increasingly recognized. Several clinical conditions require investigation of human neutrophil antigen (HNA-1), which is encoded by the Fc gamma receptor IIIb gene (FCGR3B), including suspicion of neutropenia, infections, and proactive testing of blood component donors to reduce the potential risk in transfusion. In this study, we compared real-time quantitative polymerase chain reaction (qPCR) with two digital PCR (dPCR) platforms, namely droplet digital PCR and an array-based platform, to determine CNV in FCGR3B. We initially tested 400 anonymous blood donors with qPCR using a commercially available TaqMan probe assay (Applied Biosystems) on a Quant Studio 12 Flex. CNV was determined for all 400 tested individuals with copy numbers ranging from zero to four. Zero copies were detected in 0.2% (1/400), one copy was detected in 3.8% (15/400), two copies were detected in 87.8% (351/400), three copies were detected in 8.0% (32/400), and four copies were detected in 0.2% (1/400) of tested individuals. From this cohort, we selected 32 donors with copy numbers from zero to four for analyses with Digital Real-Time PCR (dPCR) using Lab on an array (LOAA) on an On-Point analyzer from Optolane Technologies Inc. and the Droplet Digital PCR (ddPCR) platform from Bio-Rad Laboratories. We compared the obtained copy numbers of FCGR3B on the three platforms and found full concordance between the copy numbers obtained. We therefore conclude that all three platforms can be used for quantification of copy numbers for FCGR3B, and although dPCR has some advantages over qPCR, it was not necessary for reliably estimating CNV of the FCGR3B gene.

Background: Ovarian cancer remains the most lethal gynecological cancer, primarily due to its asymptomatic nature in early stages and consequent late diagnosis. Early detection improves survival, but current biomarkers lack sensitivity and specificity. Cell-free DNA (cfDNA) released from tumor cells captures tumor-associated epigenetic alterations and represents a promising source for minimally invasive biomarkers. Among these, aberrant DNA methylation occurs early in tumorigenesis and may reflect underlying disease biology. This study aimed to investigate genome-wide cfDNA methylation profiles in patients with ovarian cancer, benign ovarian conditions, and healthy controls to identify cancer-associated methylation patterns that may inform future biomarker development. Results: We performed genome-wide cfDNA methylation profiling using cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) on plasma samples from 40 patients with high-grade serous ovarian carcinoma, 38 patients with benign ovarian conditions, and 38 healthy postmenopausal women. A total of 536 differentially methylated regions (DMRs) were identified between ovarian cancer and controls (n = 76), with 97% showing hypermethylation in ovarian cancer. DMRs were enriched in CpG islands and gene bodies and depleted in repetitive elements, consistent with known cancer-associated methylation patterns. Fifteen genes showed robust hypermethylation across analyses. These genes exhibited methylation across intronic, exonic, and upstream regulatory regions. Separate comparisons of ovarian cancer to each control group (benign and healthy) supported the reproducibility of these findings. Gene Ontology enrichment analysis revealed enrichment in gland development, embryonic morphogenesis, and endocrine regulation, suggesting biological relevance to ovarian tumorigenesis. Conclusions: This study identifies consistent cfDNA hypermethylation patterns in ovarian cancer, affecting genes involved in developmental regulation and hormone-related processes. Our findings underscore the potential of cfMeDIP-seq for detecting tumor-specific methylation signatures in plasma and highlight these 15 hypermethylated genes as biologically relevant targets for future studies on cfDNA methylation in ovarian cancer.

Bone formation is critical to maintain bone integrity. Here, we studied the importance of intact energy metabolism for bone formation in humans. The skeletal impact of impaired oxidative phosphorylation (OXPHOS) was investigated in adult individuals with genetically defective mitochondrial DNA translation (m.3243A>G). Although impaired mitochondrial ATP production in m.3243A>G human bone marrow stromal cells (hBMSC) was compensated by increased glycolytic ATP production (unchanged net ATP production), both in vitro osteoblast differentiation and in vivo ectopic bone formation were decreased. The impaired OXPHOS was associated with mitochondrial stress and disruption of the pro-osteogenic transcriptional program characteristic of hBMSC. Supporting OXPHOS pharmacologically in hBMSC restored mitochondrial ATP production, their transcriptional program and metabolism, leading to upregulation of osteogenic genes and restoration of bone formation capacity. These findings demonstrate a mitochondrial regulation mechanism of the osteogenic capacity of hBMSCs and identify OXPHOS as a potential target for increasing bone formation.