JOURNAL/nrgr/04.03/01300535-202512000-00025/figure1/v/2025-01-31T122243Z/r/image-tiff In clinical specialties focusing on neurological disorders, there is a need for comprehensive and integrated non-invasive, sensitive, and specific testing methods. Both Parkinson's disease and multiple system atrophy are classified as α-synucleinopathies, characterized by abnormal accumulation of α-synuclein protein, which provides a shared pathological background for their comparative study. In addition, both Parkinson's disease and multiple system atrophy involve neuronal death, a process that may release circulating cell-free DNA (cfDNA) into the bloodstream, leading to specific alterations. This premise formed the basis for investigating cell-free DNA as a potential biomarker. Cell-free DNA has garnered attention for its potential pathological significance, yet its characteristics in the context of Parkinson's disease and multiple system atrophy are not fully understood. This study investigated the total concentration, nonapoptotic level, integrity, and cell-free DNA relative telomere length of cell-free DNA in the peripheral blood of 171 participants, comprising 76 normal controls, 62 patients with Parkinson's disease, and 33 patients with multiple system atrophy. In our cohort, 75.8% of patients with Parkinson's disease (stage 1-2 of Hoehn & Yahr) and 60.6% of patients with multiple system atrophy (disease duration less than 3 years) were in the early stages. The diagnostic potential of the cell-free DNA parameters was evaluated using receiver operating characteristic (ROC) analysis, and their association with disease prevalence was examined through logistic regression models, adjusting for confounders such as age, sex, body mass index, and education level. The results showed that cell-free DNA integrity was significantly elevated in both Parkinson's disease and multiple system atrophy patients compared with normal controls ( P < 0.001 for both groups), whereas cell-free DNA relative telomere length was markedly shorter ( P = 0.003 for Parkinson's disease and P = 0.010 for multiple system atrophy). Receiver operating characteristic analysis indicated that both cell-free DNA integrity and cell-free DNA relative telomere length possessed good diagnostic accuracy for differentiating Parkinson's disease and multiple system atrophy from normal controls. Specifically, higher cell-free DNA integrity was associated with increased risk of Parkinson's disease (odds ratio [OR]: 5.72; 95% confidence interval [CI]: 1.54-24.19) and multiple system atrophy (OR: 10.10; 95% CI: 1.55-122.98). Conversely, longer cell-free DNA relative telomere length was linked to reduced risk of Parkinson's disease (OR: 0.16; 95% CI: 0.04-0.54) and multiple system atrophy (OR: 0.10; 95% CI: 0.01-0.57). These findings suggest that cell-free DNA integrity and cell-free DNA relative telomere length may serve as promising biomarkers for the early diagnosis of Parkinson's disease and multiple system atrophy, potentially reflecting specific underlying pathophysiological processes of these neurodegenerative disorders.

The development of electrochemiluminescence (ECL) sensors for the sensitive detection of circulating tumor DNA (ctDNA) associated with non-small cell lung cancer (NSCLC) offers a promising approach for early diagnosis; however, the lack of robust and efficient luminophore remains a key limitation to the analytical performance of ECL sensors. Herein, a ECL biosensor is developed using a novel His@ZIF-8/Fe-TCPP (HZTCP) luminophore for the sensitive detection of NSCLC-related ctDNA. The HZTCP luminophore, synthesized using a histidine imidazole framework (His@ZIF-8) as the precursor and tetra-(4-carboxyphenyl) porphyrin ferric chloride (Fe-TCPP) as the luminescent ligand, integrates the photoelectrochemical activity of porphyrin with the porous structure of the MOF, achieving excellent ECL performance. In addition, polyethyleneimine and gold nanoparticle-functionalized covalent organic frameworks (P-COF-AuNPs) serve as interfacial materials to significantly enhance the effective area and conductivity of the sensing interface, increase the solid-state loading of the capture probe, and improve the biosensor's sensitivity. The ECL biosensor achieves a wide detection range of 1 fM to 100 nM with a limit of detection of 0.35 fM, enabling the differentiation of NSCLC patients from healthy individuals through ctDNA detection. This work provides a straightforward approach for designing efficient ECL luminophores and presents a promising method for the rapid detection of non-small cell lung cancer-related ctDNA.

Introduction : Elevated cell-free DNA (cfDNA) was observed in patients receiving venoarterial (VA) extracorporeal membrane oxygenation (ECMO), but the clinical relevance of cfDNA is still not clear. We aimed to establish a predictive model based on the cfDNA to predict the prognosis for patients on ECMO, and reveal the values of cfDNA for complications of limb ischemia and bleeding/thromboembolic events. Methods : Single-center, retrospective evaluation of patients with ECMO support from 2018 through 2023. The derivation cohort included 133 adults diagnosed with cardiogenic shock who received VA-ECMO for circulatory support. We developed three independent features and combined them with a logistic model to predict mortality. Predictive performance was assessed through Bootstrap analysis and validated by another cohort of 27 patients. The values of cfDNA for complications were analyzed by restricted cubic spline analysis, receiver-operating characteristic curves and multivariate regression analyses. Results : A total of 133 adults who underwent VA-ECMO for refractory cardiogenic shock were entered into the derivation cohort. The logistic model, consisted of cfDNA, the worst mean arterial pressure (MAP) before ECMO and the worst lactate within 24 h of VA-ECMO implantation was predictive and performed similarly for validation cohorts (area under the receiver operating characteristic curve: 0.768 vs. 0.747). Restricted cubic spline analysis revealed a positive linear relationship for the risk of limb ischemia (linear, P = 0.006; area under the receiver operating characteristic curve of 0.75 [95% CI, 0.656-0.848]), a U-shaped trend for bleeding events (nonlinear, P = 0.214), and a negative trend for thrombotic events (linear, P = 0.552). Conclusions: In addition to MAP and lactate levels, elevated cfDNA levels within 48 h of ECMO support were highly associated with mortality for patients. Additionally, cfDNA is predictive of limb ischemia.

Exploring cancer heterogeneity is crucial for both understanding cancer and developing prognostic tools to monitor cancer progression during treatment through the liquid biopsy concept. Herein, a nanoparticle-based "dispersible electrodes" biosensor was used to detect ultra-low concentrations of microRNA-155 (miRNA-155) from a single breast cancer spheroid for the first time. The results from the sensor were comparable to the standard real-time polymerase chain reaction analysis, but in a much shorter detection time and without any sample purification or amplification. Owing to the unique ability of the sensor to measure biomarker expression from unaltered and undiluted cancer liquid biopsy from a single cancer spheroid, we then tracked dynamic changes in miRNA-155 expression in a single spheroid treated with the anti-cancer drug doxorubicin. The ability to track dynamic biomarker changes in a single cancer spheroid opens the door to understanding key biological processes such as response to treatment on the cellular and molecular levels, paving the way for adapting liquid biopsy insights to guide oncologists and more personalised treatment strategies.

Small-cell lung cancer (SCLC) remains one of the deadliest cancers worldwide. Patients' survival remains poor due to its rapid growth, high metastatic rate and limited possibilities of treatment. For many years, SCLC management has been based mostly on chemo and radiotherapy. However, new therapeutic approaches have been proposed in the past few years, including immunotherapy, which is currently implemented in clinical practice. Unfortunately, in many cases, response to therapy, especially chemotherapy, remains poor, or the patient becomes resistant to initially effective treatment. One of the crucial problems during SCLC patient care is a lack of appropriate predictive biomarkers for various therapeutic approaches. Another critical issue is the scarcity of collected tissue during biopsy, which may be insufficient or of too poor quality for analysis. A liquid biopsy might be the key to solving both of those problems as it is collected in a non-invasive way and enables the measurement of various biomarkers, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). In this review, we discuss various approaches to potentially incorporating liquid biopsy into clinical application - as a companion to imaging during SCLC diagnostics, a new approach to molecular subtyping, and a material enabling predictive or prognostic biomarkers assessment. We also summarize ongoing clinical trials encompassing SCLC patients in which liquid biopsy is collected and examined.

Genetic mutation detection for colorectal cancer (CRC) is crucial for precision diagnosis and treatment, yet current methods often suffer from challenges such as low sensitivity, time consumption, and high costs. In our preliminary bioinformatic analysis of 751 CRC cases from The Cancer Genome Atlas and 131 Chinese patient samples, APC, TP53, and KRAS were identified as the most frequently mutated genes. Among them, KRAS missense mutations emerged as key diagnostic biomarkers. In this study, we applied a fluorescence-based long block displacement amplification (LBDA) sensing method for the rapid, high-throughput, and cost-effective detection of KRAS genetic mutations. In the LBDA system, SYBR Green dye binds to the amplified double-stranded DNA, generating a fluorescence signal that directly reflects the abundance of mutant types (MTs). This real-time signal output enables the enrichment and sensitive detection of MTs, establishing LBDA as an efficient biosensing platform for KRAS genotyping. Using this technique, a detection limit of 0.08% variant allele frequency was achieved with 20 ng of synthetic DNA input. To evaluate clinical performance, the LBDA method was applied to 118 tissue samples from 59 CRC patients, including tumor and matched peritumoral tissues. For 59 CRC tumor samples, LBDA successfully identified KRAS mutations in 37.29% of cases, closely matching results (42.37%) obtained by next-generation sequencing and achieving 88% sensitivity and 100% specificity. In conclusion, this study presents a rapid and cost-effective mutation detection method based on optical biosensing, offering strong potential for advancing personalized CRC diagnosis and treatment.

To construct a prognostic model for predicting cancer-specific survival in lymph node-positive colorectal cancer patients treated with adjuvant chemotherapy after surgery.

Data were collected from the 2010-2015 SEER database and from CRC patients at the Second Affiliated Hospital of Bengbu Medical University (2017-2023). Lasso regression and random survival forest methods were used to screen ten clinicopathologic features. Cox regression analysis identified independent prognostic factors for CRC. Nomogram plot model was used to predict 1-, 3-, and 5-year survival rates, with its accuracy verified through ROC curves, calibration curves, and decision curve analysis (DCA). The X-tile software differentiated between high and low-risk groups and illustrated survival differences using Kaplan-Meier curves.

Age, histologic grade, stage, CEA, nerve invasion, and LNR were independent prognostic risk factors for colorectal cancer (P < 0.001); and LNR were the five variables used to construct the Nomogram. The area under the curve (AUC) was 0.83, 0.85, and 0.84 at 1, 3, and 5 years for the training cohort; 0.83, 0.85, and 0.84 at 1, 3, and 5 years for the internal validation cohort; and 0.83, 0.85, and 0.84 at 1, 3, and 5 years for the external validation cohort, respectively. calibration curves, C-indexes, and DCA curves validated the accuracy of the model, respectively. The survival prognosis of the high-risk group was lower than that of the low-risk group in all three data sets. (HR = 6.37, CI:6.05-6.71, P < 0.05; HR = 7.05, CI:6.52-7.64, P < 0.05; HR = 2.69, CI:1.66-4.37, P < 0.05).

LNR represents a new independent prognostic factor for lymph node-positive CRC. The optimal threshold determined by the Nomogram method effectively categorizes subgroups of lymph node-positive CRC cases after surgical chemotherapy, crucial for guiding clinical treatment strategy selection.

Nucleic acid probes, which are short sequences of nucleic acids designed to complement specific DNA or RNA targets, have broad applications in biosensing, genetic studies, and various other fields. In tumor diagnosis and treatment, nucleic acid probes offer a precise and accessible approach that is essential for improving patient care and quality of life. Despite substantial research on nucleic acid probes over the past three decades, few comprehensive reviews have retrospectively examined the field.

This study extracted 30 years of nucleic acid probe-related research articles from the Web of Science Core Collection database. We used CiteSpace, VOSviewer, and R tools to systematically analyze the field's current status and developmental trends, with an emphasis on applications in oncology.

Our findings indicate a continuous growth trend in nucleic acid probe research, with the United States and China, along with their leading institutions and authors, making the most significant contributions. In oncology specifically, nucleic acid probe research has focused primarily on signal amplification, liquid biopsy, and drug delivery. The emergence of novel biomarkers and assay techniques has been a pivotal factor driving advancements in this field.

Nucleic acid probes show strong potential for applications in tumor precise diagnosis and treatment. Continued innovation and closer interdisciplinary collaboration will be vital for further advancements, while large-scale clinical studies are needed to validate their clinical utility.

Diabetic retinopathy (DR), a complication of type 2 diabetes mellitus (T2DM), is typically asymptomatic in its early stages. Diagnosis typically relies on routine fundoscopy for the clinical detection of microvascular abnormalities. However, permanent retinal damage may occur well before clinical signs are appreciable. In the early stages of DR, the retina undergoes distinct epigenetic changes, including DNA methylation and histone modifications. Recent evidence supports unique epigenetic 'signatures' in patients with DR compared to non-diabetic controls. These DNA 'signature' sequences may be specific to the retina and may circulate in peripheral blood in the form of cell-free DNA (cfDNA). In this review, we explore the literature and clinical application of cfDNA sampling as an early, non-invasive, accessible assessment tool for early DR detection. First, we summarize the known epigenetic signatures of DR. Next, we review current sequencing technologies used for cfDNA detection, such as magnetic bead-based enrichment, next-generation sequencing, and bisulfite sequencing. Finally, we outline the current research limitations and emerging areas of study which aim to improve the clinical utility of cfDNA for DR evaluation.

The use of liquid biopsy of total cell-free DNA (cfDNA) to identify otherwise undetectable cancers has attracted interest; however, its efficacy remains unknown. We explored whether analysis using total cfDNA is efficacious for Japanese patients with oral squamous cell carcinoma (OSCC).

We collected total cfDNA from nine patients with OSCC preoperatively, 1 month postoperatively, and every 3 months thereafter to analyze this association. We used a target DNA sequence for genetic mutation analysis of tumor tissues collected from 33 patients, including the aforementioned nine patients.

Patients with good disease control showed negligible changes in preoperative and postoperative total cfDNA concentrations. A rapid increase in total cfDNA concentration was observed in patients who developed systemic metastases. Patients whose tumor tissue DNA showed genetic mutations had the same mutations in preoperative circulating tumor DNA.

Our data suggested that analyzing total cfDNA is efficacious for patients with OSCC.

Patient stratification remains a challenge for optimal treatment of prostate cancer (PCa). This clinical heterogeneity implies intra-tumoural heterogeneity, with different prostate epithelial cell subtypes not all targeted by current treatments. We reported that such cell subtypes are traceable in liquid biopsies through representative transcripts. Expanding on this concept, we included 57 genes representing cell subtypes, drug targets and relevant to resistance as non-invasive biomarkers for stratification. This panel was tested by RT-qPCR (quantitative reverse transcription polymerase chain reaction) in blood of controls and different categories of PCa patients. Overall, circulating transcripts showed predictive value throughout the disease. Those with aggressive pathological features such as intra-ductal carcinoma at diagnosis showed more genes over-expressed. In metastatic patients, signatures of subtypes or resistance were associated with treatments, progression-free survival and overall survival. Altogether, testing markers of cell diversity, an intrinsic feature of tumours, and drug targets via liquid biopsies represents a valuable means to stratify patients and predict responses to current or new therapeutic modalities. Over-expressed drug target genes suggest potential benefit from targeted treatments, justifying new clinical trials to offer patient-tailored strategies to eventually impact on PCa mortality.

DNA nanostructure, as polymeric material with remarkable molecular recognition property, has been widely used in bioassay. However, it still faces some challenges to overcome complexity of signal-amplified strategies and to realize efficient separation of reaction products. Herein, we present an innovative signal-amplified approach by integrating the toehold-mediated strand displacement reaction with DNA tile self-assembly technology to construct superparamagnetism-functionalized DNA polymeric materials, establishing a new signal-amplified analytical strategy for nucleic acids. This strategy enables highly sensitive, rapid, and efficient nucleic acid detection, making it a promising candidate for point-of-care testing (POCT). The analytical performance of this strategy was validated using target DNA (tDNA) and PIWI-interacting RNA-36026 (piRNA-36026), achieving limits of detection (LOD) of 2.4 × 10-10 M and 2.7 × 10-10 M. Moreover, it successfully detected single-base mutations and demonstrated stability over seven days. Comparative experiments confirmed the superior signal-amplified efficiency of DNA arrays. Recovery experiments yielded recoveries of 88.53 %-101.89 % for tDNA and 87.58 %-108.61 % for piRNA-36026. Ultimately, the feasibility of this strategy for real-world applications was validated through detecting piRNA-36026 in cell lysates. In conclusion, this work introduces an innovative and efficient signal-amplified method, while expanding the application prospects of multifunctional DNA polymeric materials in biomedical diagnostics.

Most targeted anticancer therapies fail due to drug resistance evolution. Here we show that tumor evolution can be reproducibly redirected to engineer therapeutic opportunity, regardless of the exact ensemble of pre-existing genetic heterogeneity. We develop a selection gene drive system that is stably introduced into cancer cells and is composed of two genes, or switches, that couple an inducible fitness advantage with a shared fitness cost. Using stochastic models of evolutionary dynamics, we identify the design criteria for selection gene drives. We then build prototypes that harness the selective pressure of multiple approved tyrosine kinase inhibitors and employ therapeutic mechanisms as diverse as prodrug catalysis and immune activity induction. We show that selection gene drives can eradicate diverse forms of genetic resistance in vitro. Finally, we demonstrate that model-informed switch engagement effectively targets pre-existing resistance in mouse models of solid tumors. These results establish selection gene drives as a powerful framework for evolution-guided anticancer therapy.

The present study aimed to clarify the clinical significance of the cell-free DNA (cfDNA) methylation profile of patients with non-small cell lung cancer (NSCLC) showing the epidermal growth factor receptor (EGFR) gene mutation.

In 103 patients, genome-wide DNA methylation analysis using Infinium Methylation EPIC array was performed using samples of pre-tyrosine kinase inhibitor afatinib-treatment plasma cfDNA (n = 101) and post-afatinib cfDNA (n = 84).

Principal component analysis indicated that the cfDNA methylation profile was altered after afatinib treatment. Hierarchical clustering using the pre-afatinib cfDNA methylation profile revealed that cases with a fatal outcome were accumulated in specific clusters. Moreover, Kaplan-Meier analysis showed that the pre-afatinib cfDNA methylation profile was significantly associated with both progression-free survival (PFS) and overall survival (OS), whereas the post-afatinib profile was not. The genes for which pre-afatinib cfDNA methylation levels were associated with PFS were accumulated in the cadherin, Wnt, and EGFR signaling pathways. Activation of EGFR-related signaling due to DNA methylation alterations might overturn the effect of afatinib. Pre-afatinib levels of CEP170 and CHCHD6 cfDNA methylation were associated with both PFS and OS. Both pre- and post-afatinib cfDNA methylation levels of SLC9A3R2 and INTS1 were associated with bone metastasis. Using the cfDNA methylation levels at two CpG sites, cg12721600 and cg05905155, patients showing an overall response were predicted with a sensitivity of 96% or more.

The non-invasively measurable cfDNA methylation profile may reflect the corresponding profile in cancer cells, and that pre-treatment measurement may provide clinically useful information on EGFR mutation-positive NSCLC.

Circulating tumor DNA (ctDNA) is an emerging biomarker in cervical cancer, with elevated levels typically indicating a higher tumor burden. However, its prognostic value in cervical cancer patients remains debated. This meta-analysis aims to clarify the prognostic significance of ctDNA in this patient population.

We searched the PubMed, Cochrane Library, CNKI, and EMBASE databases for studies published up to September 30, 2024, to investigate the prognostic significance of ctDNA in cervical cancer patients. The outcome measures included overall survival (OS) and progression-free survival (PFS)/disease-free survival (DFS).

This analysis included 10 studies encompassing a total of 706 cervical cancer patients. Findings revealed that patients with detectable baseline ctDNA had significantly poorer OS(HR = 1.64, 95% CI = 1.45-1.86, P < 0.001) as well as worse PFS or DFS (HR = 1.42, 95% CI = 1.07-1.89, P = 0.015). Additionally, ctDNA detectability during treatment was strongly associated with poorer OS (HR = 17.22, 95% CI = 4.43-66.89, P < 0.001) and PFS/DFS (HR = 4.16, 95% CI = 2.57-6.73, P < 0.001).

This meta-analysis demonstrates that elevated ctDNA levels are significantly associated with poorer PFS, DFS, and OS in patients with cervical cancer. However, data regarding the association between ctDNA levels and OS are relatively limited, and the number of included studies remains small, with a potential risk of publication bias. Based on the current evidence, ctDNA shows promise as a valuable tool for pre-treatment assessment and an effective biomarker for monitoring therapeutic response and disease progression. Further large-scale, prospective studies are warranted to validate these findings and establish their reliability and clinical applicability.

inplasy.com, identifier INPLASY2024120083.

Continued development of clustered regularly interspaced short palindromic repeats (CRISPR)-powered biosensing system on the electrochemical interface is vital for accurate and timely diagnosis in clinical practice. Herein, an electrochemical biosensor based on manganese metal-organic frameworks (MOFs)-enhanced CRISPR (MME-CRISPR) is proposed that enables the efficient detection of circulating tumor DNA (ctDNA). In this design, customized enzyme stimulants (Mn2+) are co-assembled with Cas12a/crRNA to form enzyme-MOF composites, which can be released quickly under mild conditions. The MOFs-induced proximity effect can continuously provide adequate Mn2+ to sufficiently interact with Cas12a/crRNA during the release process, enhancing the trans-cleavage activity of complex available for biosensor construction. The MOFs-based enzyme biocomposites also afford efficient protection against various external stimulus. It is demonstrated that the developed biosensor can achieve ultrasensitive detection of epidermal growth factor receptor L858R mutation in ctDNA with a low detection limit of 0.28 fm without pre-amplification. Furthermore, the engineered mismatch crRNA enables the biosensor based on MME-CRISPR to detect single nucleotide variant with a high signal-to-noise ratio. More importantly, it has been successfully used to detect the targets in clinical practice, requiring low-dose samples and a short time. This strategy is believed to shed new light on the applications of cancer diagnosis, treatment, and surveillance.

Circulating cell-free DNA (cfDNA) consists of small fragments of extracellular DNA from mammalian and bacterial cells found in bodily fluids such as blood and saliva, and it has been strongly recognized as a critical biomarker for various disease diagnoses, prognoses, and therapeutic monitoring. In this study, we present a reproducible protocol for efficiently isolating cfDNA from murine saliva using an innovative swabbing method in conjunction with the QIAamp MinElute ccfDNA Mini Kit. The quantification of isolated cfDNA is detected by a Qubit Fluorometer. Moreover, qualification assessment is conducted through BioAnalyzer analysis. This protocol facilitates research on saliva-derived cfDNA in the context of oral and systemic diseases in murine models.

The widespread use of immune checkpoint inhibitors (anti-CTLA4 or PD-1) has opened a new chapter in tumor immunotherapy by providing long-term remission for patients. Unfortunately, however, these agents are not universally available and only a minority of patients respond to them. Therefore, there is an urgent need to develop novel therapeutic strategies targeting other co-inhibitory molecules. However, comprehensive information on the expression and prognostic value of co-inhibitory molecules, including co-inhibitory receptors and their ligands, in different cancers is not yet available.

We investigated the expression, correlation, and prognostic value of co-inhibitory molecules in different cancer types based on TCGA, UCSC Xena, TIMER, CellMiner datasets. We also examined the associations between the expression of these molecules and the extent of immune cell infiltration. Besides, we conducted a more in-depth study of VISTA.

The results of differential expression analysis, correlation analysis, and drug sensitivity analysis suggest that CTLA4, PD-1, TIGIT, LAG3, TIM3, NRP1, VISTA, CD80, CD86, PD-L1, PD-L2, PVR, PVRL2, FGL1, LGALS9, HMGB1, SEMA4A, and VEGFA are associated with tumor prognosis and immune cell infiltration. Therefore, we believe that they are hopefully to serve as prognostic biomarkers for certain cancers. In addition, our analysis indicates that VISTA plays a complex role and its expression is related to TMB, MSI, cancer cell stemness, DNA/RNA methylation, and drug sensitivity.

These co-inhibitory molecules have the potential to serve as prognostic biomarkers and therapeutic targets for a broad spectrum of cancers, given their strong associations with key clinical metrics. Furthermore, the analysis results indicate that VISTA may represent a promising target for cancer therapy.

Endometriosis is currently considered a systemic inflammatory disease and different non-invasive inflammatory markers, such as cell-free DNA (cfDNA), have recently been evaluated. Hormonal treatments are frequently prescribed as first-line treatments to improve symptoms, reduce lesions and improve the quality of life of patients with endometriosis. The most frequently used hormonal treatments are estroprogestins and progestins due to their effectiveness and well-tolerated clinical profile. However, the impact these hormonal treatments may have on these markers has yet to be determined. The aim of this study was to assess whether cfDNA levels are modified under the two main first-line hormonal treatments in patients with deep endometriosis (DE).

Ninety patients diagnosed with DE were analyzed in this prospective, observational study. Forty-five received daily oral treatment with dienogest 2 mg, and 45 with 2 mg dienogest/30 μg ethinylestradiol. Plasma cfDNA levels were evaluated by fluorescent assay prior to initiation of treatment and at 6 and 12 months of treatment.

An increase in cfDNA levels was observed during the follow-up at 6 and 12 months. However, these higher levels were only statistically significant at 12 months of treatment. The increase of cfDNA levels was similar with both treatments.

Higher cfDNA levels were observed in DE patients at 12 months of oral hormonal treatment showing similar results with dienogest or dienogest/ethinylestradiol. This increase could be explained by apoptosis of the endometriosis foci due to the treatment.

Neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), are characterized by the progressive and gradual degeneration of neurons. The prevalence and rates of these disorders rise significantly with age. As life spans continue to increase in many countries, the number of cases is expected to grow in the foreseeable future. Early and precise diagnosis, along with appropriate surveillance, continues to pose a challenge. The high heterogeneity of neurodegenerative diseases calls for more accurate and definitive biomarkers to improve clinical therapy. Cell-free DNA (cfDNA), including fragmented DNA released into bodily fluids via apoptosis, necrosis, or active secretion, has emerged as a promising non-invasive diagnostic tool for various disorders including neurodegenerative diseases. cfDNA can serve as an indicator of ongoing cellular damage and mortality, including neuronal loss, and may provide valuable insights into disease processes, progression, and therapeutic responses. This review will first cover the key aspects of cfDNA and then examine recent advances in its potential use as a biomarker for neurodegenerative disorders.

Plasma cell-free DNA (cfDNA) has been used to monitor gene mutations and diagnose tumors. Discriminating parathyroid carcinoma (PC) from parathyroid adenoma (PA) before surgery is difficult because of the overlap in clinical features between parathyroid neoplasms. We aimed to detect cfDNA mutations in plasma samples from PC and PA patients before surgery to predict the CDC73 status in tumor tissue and help in the differential diagnosis of parathyroid neoplasms.

Eighteen PC patients and 13 PA patients were enrolled. Plasma cfDNA was detected using next-generation sequencing, with DNA from matched peripheral white blood cells used as a control. CDC73 gene mutations were detected via whole-exome sequencing or parafibromin staining via immunohistochemistry of tumor tissues. Logistic regression was used to evaluate the ability of cfDNA mutations to predict the CDC73 status in tumor tissue and for differential diagnosis. CDC73 gene mutation or parafibromin staining loss were defined as CDC73 abnormalities.

One PC patient was not tested for CDC73 abnormalities due to the absence of tumor specimen. CDC73 abnormalities were not detected in all 13 PA patients, whereas 10 PC patients harboured CDC73 abnormalities in tumor specimens (P = 0.001). Among the 10 patients, CDC73 mutations were identified in the cfDNA of 8 patients. In another 20 patients without CDC73 abnormalities in tumors, CDC73 mutation was detected in the cfDNA of 4 patients. Using the CDC73 status in cfDNA, the area under the receiver operating characteristic curve (AUC) for predicting CDC73 abnormalities in tumor tissue was 0.80 (95% CI: 0.622-0.978), and the AUC for predicting malignancy was 0.795 (95% CI 0.632-0.958).

This study is the first attempt to evaluate the gene mutation status of parathyroid neoplasms through the deep sequencing of plasma cfDNA, which could also help to identify PC prior to surgery.

Gallbladder cancer (GBC) frequently mimics gallbladder benign lesions (GBBLs) in radiological images, leading to preoperative misdiagnoses. To address this challenge, we initiated a prospective, multicenter clinical trial (ChicCTR2100049249) and proposed a multimodal, non-invasive diagnostic model to distinguish GBC from GBBLs. A total of 301 patients diagnosed with gallbladder-occupying lesions (GBOLs) from 11 medical centers across 7 provinces in China were enrolled and divided into a discovery cohort and an independent external validation cohort. An artificial intelligence (AI)-based integrated model, GBCseeker, is created using cell-free DNA (cfDNA) genetic signatures, radiomic features, and clinical information. It achieves high accuracy in distinguishing GBC from GBBL patients (93.33% in the discovery cohort and 87.76% in the external validation cohort), reduces surgeons' diagnostic errors by 56.24%, and reclassifies GBOL patients into three categories to guide surgical options. Overall, our study establishes a tool for the preoperative diagnosis of GBC, facilitating surgical decision-making.

Circulating cell-free DNA (cfDNA) is a promising biomarker for physiological stress, including exercise-induced responses. However, the lack of standardization in blood collection tubes (BCTs) for quantification of cfDNA hampers inter-study comparisons. In this study, we assessed the impact of different BCTs on exercise-induced cfDNA dynamics. Eleven participants [25 (SD 2.3) years of age] performed three different treadmill exercise protocols, including an all-out test and combinations of constant and interval load. Blood samples were collected before, 5 min and 30 min post-exercise using EDTA, lithium-heparin (LH) and serum BCTs. Concentrations of cfDNA were quantified using quantitative PCR. The cfDNA increased significantly across all protocols and BCTs. A significant effect of BCT on cfDNA concentrations (P = 0.034) was found, with serum showing higher concentrations than EDTA and LH. Although absolute differences from pre- to post-exercise were comparable across BCTs (P = 0.476), fold changes differed significantly (P = 0.012), with the highest observed in EDTA and the lowest in serum. Bland-Altman analyses demonstrated better agreement between EDTA and LH compared with serum. Significant correlations of cfDNA with energy expenditure and peak oxygen uptake were found. These correlations were stronger in EDTA and LH than in serum. Our findings highlight the crucial influence of BCT choice on cfDNA measurements in exercise settings. Given that EDTA and LH reflected exercise load better, they could be preferred for exercise physiology research. This work underscores the need to account for the choice of BCT to improve data comparability across studies. Additionally, these findings might have broader implications for clinical settings where cfDNA is used as a biomarker.

Difficulties in microbiologically confirming childhood tuberculosis (TB) can result in delayed treatment and increased disease severity.

In this study, we for the first time used whole genome next-generation sequencing (NGS) to detect cell-free DNA (cfDNA) from Mycobacterium tuberculosis (MTB) in plasma from children.

We enrolled 94 children with active TB and 32 children with other respiratory infections. Combining NGS with probe capture enrichment (targeted cfNGS) showed higher coverage and detecting capability than did NGS alone. The targeted cfNGS showed slightly lower sensitivity (31.9% vs. 44.7%, P = 0.072) and specificity (96.9% vs. 100.0%, P = 0.236) to those of sputum tested using Xpert. Agreement between cfNGS-plasma and Xpert-sputum was weak (κ = 0.217). Concordant results were obtained for only 85 children (67.5%; 16 cases positive by both tests and 69 cases negative by both tests). A total of 40 children with MTB culture negative results were tested to have positive cfNGS-plasma or Xpert-sputum outcomes, yielding a significantly increased percentage of children with bacteriological evidence (20.2% [19/94] for MTB culture-positive only vs. 62.8% [59/94] for cfNGS-plasma, Xpert-sputum or culture positive).

These data suggest that cfNGS performed well for diagnosing TB using plasma from children. cfNGS may be a new method for diagnosing patients with paucibacillary TB.

Neglected Tropical Diseases (NTDs) continue to ravage the poorest regions of the world, with over 600 million people being affected in Sub-Saharan Africa. The global burden of NTDs within these regions is staggering, particularly post-COVID-19 pandemic, where the emerging infection intercepted the existing eradication efforts and protocols such as the Mass Drug Administration (MDA). This further complicated the approaches laid down to achieve the endgame program of eliminating the neglect and transmission of NTDs. To compensate for the detriment of COVID-19's interruption, accurate and timely diagnoses play a vital role in attaining the objectives of the WHO's goal of NTD elimination by 2030. To this effect, alternative approaches in diagnostics are urgently needed, particularly with the inadequacy of current diagnostic strategies for NTDs. Cell-free DNA (cfDNA) has shown great promise in detecting NTDs. Several studies have demonstrated its potential for diagnosing diseases such as malaria, leishmaniasis, and schistosomiasis. However, the adoption of cfDNA in NTD research faces several challenges, including the cost of the procedure, standardization, and technical expertise. Proper capacity building and training can mitigate some of these challenges. However, despite these limitations, the affordability of cfDNA detection is improving due to increased awareness of the approach and researchers' integration considerations into current diagnostic routines. In conclusion, while there are challenges to adopting cfDNA in NTD research, it remains a promising alternative strategy to be considered in the fight against NTDs.

Currently, due to the invasive nature of colonoscopy and the associated pain, people avoid undergoing the procedure, making it difficult to detect the majority of potential early stage colorectal carcinoma/precancerous lesions or advanced adenoma. Advanced colorectal adenoma is the main precursor to the development of colorectal carcinoma. Therefore, improving advanced colorectal adenoma detection rate can significantly decrease the development and morbidity of colorectal carcinoma. Accordingly, a non-invasive method to screen high-risk people for colonoscopy in clinical practice is urgently needed.

With the development of medical technology, screening methods for colorectal carcinoma are emerging rapidly, and diverse non-invasive methods are being developed. Cell-free DNA (cfDNA), commonly referred to as liquid biopsy, has promising application prospects as a minimally invasive strategy for early screening of colorectal cancer. CfDNA has already been applied in the field of prenatal diagnosis, advanced carcinoma, and organ transplantation, and the application cfDNA in advanced colorectal adenoma is at the cutting-edge of current research. Thus, this review summarizes the progress in research on different biological characteristics of cfDNA and its utility in the screening of advanced colorectal adenoma, including sizes of cfDNA molecules, end signature of cfDNA (preferred ends, end motifs, jagged ends), nucleosomal footprints, cfDNA topology, cfDNA methylation, and cfDNA integrity.

We hope that this review will advance this promising research field.

Rapid identification of acute ischemic stroke (AIS) is challenging in both pre-hospital and hospital settings. We aimed to identify the most promising cell-free nucleic acids (cfNAs) as diagnostic biomarkers for IS within 72 h from symptom onset. We searched PubMed, Web of Science, EMBASE, and Cochrane Library for published articles that evaluated blood cfNAs in the early diagnosis of AIS until 10 May 2023. The diagnostic performances of individual cfNAs were pooled by random-effects meta-analysis based on the fold change of biomarkers' level between AIS and non-AIS patients. Of 2955 records, 66 articles reporting 143 different cfNAs met the inclusion criteria. The median sample size was 110, and 21.4% of the studies performed validation. Among selected high-quality studies, miR-106b-5p, miR-124, miR-155, lncRNA H19, and cfDNA showed good diagnostic performance. Data from four studies on cfDNA involving 355 AIS patients and 97 controls were pooled in the meta-analysis, which showed a significant fold change between AIS and controls (pooled ratio 1.48, 95% confidence interval 1.23-1.79, p < 0.001). This review highlights that cfDNA, miR-106b-5p, miR-124, miR-155, and lncRNA H19 are the most promising biomarkers for AIS diagnosis, and further research is needed for verification.

Simultaneous detection of multiple nucleic acid targets from a single sample is a common requirement in molecular diagnosis and basic research. Dividing a bulky polymerase chain reaction (PCR) into many isolated small reaction units through microfluidic technology is commonly used to realize this goal. However, previous microfluidic platforms for multiplex PCR suffer from complex structures and strict operation requirements. In this study, a chip-based universal strategy is constructed to realize multiplex PCR by using wax films for sealing and controllable release of prespotted primers. This microfluidic chip contains twenty-four reaction chambers connected in series by a single channel, and the bottom of each reaction chamber is covered by a wax film generated by solvent volatilization. These wax films can prevent the prespotted primers from being flushed away by the reaction mixture during the injection process. After thermal blocking of the connecting channel to isolate each reaction chamber, the chip is placed on a flat thermal cycler. The wax films melt during the denaturing step of PCR so that the primers are released and mixed with the reaction mixture, a process seamlessly compatible with PCR thermal cycling. After fluid flow simulation and carefully examining its basic performance, this chip was applied to genotype seven deafness-associated hotspot mutations using a competitive allele-specific PCR assay. The genotyping results of clinical samples using this chip were totally concordant with those obtained by Sanger sequencing, demonstrating the practical utility of this universal strategy for multiplex PCR detection.

Tumors constantly shed cancer cells that are considered the mediators of metastasis via the blood stream. Analysis of circulating cells and circulating cell-free DNA (cfDNA) in liquid biopsies, mostly taken from peripheral blood, have emerged as powerful biomarkers in oncology, as they enable the detection of genomic aberrations. Similarly, liquid biopsies taken from pregnant women serve as prenatal screening test for an abnormal number of chromosomes in the fetus, e.g., via the analysis of microchimeric fetal cells and cfDNA circulating in maternal blood. Liquid biopsies are minimally invasive and, consequently, associated with reduced risks for the patients. However, different challenges arise in oncology and pregnancy-acquired liquid biopsies with regard to the analyte concentration and biological (background) noise among other factors. In this review, we highlight the unique biological properties of circulating tumor cells (CTC), summarize the various techniques that have been developed for the enrichment, detection and analysis of CTCs as well as for analysis of genetic and epigenetic aberrations in cfDNA and highlight the range of possible clinical applications. Lastly, the potential, but also the challenges of liquid biopsies in oncology as well as their translational value for the analysis of pregnancy-acquired microchimerism are discussed.

The utility of circulating tumor DNA (ctDNA) analysis has not been well-established for disease detection and monitoring of childhood cancers, especially leukemias. We developed PeCan-Seq, a deep sequencing method targeting diverse somatic genomic variants in cell-free samples in childhood cancer. Plasma samples were collected at diagnosis from 233 children with hematologic, solid and brain tumors. All children with hematologic malignancy (n = 177) had detectable ctDNA at diagnosis. The median ctDNA fraction was 0.77, and 97% of 789 expected tumor variants were identified, including sequence mutations, copy number variations, and structural variations responsible for oncogenic fusions. In contrast, ctDNA was detected in 19 of 38 solid tumor patients and 1 of 18 brain tumor patients. Somatic variants from ctDNA were correlated with minimal residual disease levels as determined by flow cytometry in serial plasma samples from patients with B-cell acute lymphoblastic leukemia (B-ALL). We showcase multi-tumor detection by ctDNA analysis for a patient with concurrent B-ALL and neuroblastoma. In conclusion, PeCan-seq sensitively identified heterogeneous ctDNA alterations from 1 mL plasma for childhood hematologic malignancies and a subset of solid tumors. PeCan-seq provides a robust, non-invasive approach to augment comprehensive genomic profiling at diagnosis and mutation-specific detection during disease monitoring.

Low-dose CT (LDCT) screening effectively reduces lung adenocarcinoma (LUAD) mortality. However, accurately evaluating the malignant potential of indeterminate lung nodules remains a challenge. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6), a potential biomarker for distinguishing benign pulmonary nodules from LUAD, may be leveraged for noninvasive positron emission tomography (PET) imaging to aid LUAD diagnosis.

This study utilized mRNA, protein, and survival datasets of LUAD patients, along with an animal model of malignant pulmonary nodules, to investigate CEACAM6 expression specificity and its correlation with LUAD. Targeting ligands for CEACAM6 were designed using the Rosetta platform, labeled with [68Ga]Ga, and screened through high-throughput PET imaging to identify the optimal tracer.

CEACAM6 was found to be specifically overexpressed in LUAD and was significantly associated with poor prognosis and disease progression. In vivo, [68Ga]Ga-NODA-P3 demonstrated high specificity for delineating CEACAM6-positive A549 xenografts, a LUAD model, via PET imaging, achieving a highest target-to-background ratio of 7.68 ± 0.44. Region of interest (ROI) analysis showed significantly higher tracer uptake in A549 xenografts compared to CEACAM6-negative Huh7 xenografts (a hepatocellular carcinoma model) at 30 min post-injection (1.81 ± 0.10%ID/g vs. 0.54 ± 0.06%ID/g). Pre-treatment with an excess of unlabeled NODA-P3 significantly reduced tumor uptake to 0.52 ± 0.07%ID/g.

These preclinical findings indicate that [68Ga]Ga-NODA-P3 is a candidate radiotracer for the non-invasive visualization of CEACAM6-positive LUAD, demonstrating favorable imaging contrast. Although the current tumor uptake limits its immediate clinical application, ongoing optimization efforts are expected to improve its efficacy, enabling earlier and more accurate diagnosis of malignant pulmonary nodules in LUAD.

Hepatocellular carcinoma (HCC) is a common malignancy and generally develops from liver cirrhosis (LC), which is primarily caused by the chronic hepatitis B (CHB) virus. Reliable liquid biopsy methods for HCC screening in high-risk populations are urgently needed. Here, we establish a porous silicon-assisted laser desorption ionization mass spectrometry (PSALDI-MS) technology to profile metabolite information hidden in human serum in a high throughput manner. Serum metabolites can be captured in the pore channel of APTES-modified porous silicon (pSi) particles and well-preserved during storage or transportation. Furthermore, serum metabolites captured in the APTES-pSi particles can be directly detected on the LDI-MS without the addition of an organic matrix, thus greatly accelerating the acquisition of metabolic fingerprints of serum samples. The PSALDI-MS displays the capability of high throughput (5 min per 96 samples), high reproducibility (coefficient of variation <15%), high sensitivity (LOD ∼ 1 pmol), and high tolerance to background salt and proteins. In a multicenter cohort study, 1433 subjects including healthy controls (HC), CHB, LC, and HCC volunteers were enrolled and nontargeted serum metabolomic analysis was performed on the PSALDI-MS platform. After the selection of feature metabolites, a stepwise diagnostic model for the classification of different liver disease stages was constructed by the machine learning algorithm. In external testing, the accuracy of 91.2% for HC, 71.4% for CHB, 70.0% for LC, and 95.3% for HCC was achieved by chemometrics. Preliminary studies indicated that the diagnostic model constructed from serum metabolic fingerprint also displays good predictive performance in a prospective observation. We believe that the combination of PSALDI-MS technology and machine learning may serve as an efficient tool in clinical practice.

Cell-free DNA (cfDNA) has attracted increasing attention as a promising biomarker in liquid biopsy due to its crucial role in disease diagnosis. However, previous cfDNA detection methods are commonly based on the development of target-specific primers and integrated signal amplification strategies, which may induce false-positive results. This paper presents a sensitive yet accurate method for cfDNA detection that combines phosphorothioated-terminal hairpin creation with a self-priming extension process. This approach initiates a self-priming mediated chain extension-based signal cycle following the trans-cleavage of H0@MBs when the CRISPR-Cas12a complex is activated by target cfDNA, resulting in the production of a substantial quantity of pyrophosphate. A pyrophosphate sensing probe (pp probe) was utilized, facilitating both high-efficiency and stable colorimetric signaling. This innovative technique for colorimetric detection of target cfDNA demonstrated exceptional sensitivity with a low limit of detection of 1.04 fM and greatly enhanced selectivity, with the complete detection process taking around 60 min. In addition, this technique is capable of detecting cfDNA from the culture medium of HEK293 cells, indicating its clinical application potential. Compared with the previous CRISPR-Cas system-based cfDNA method that necessitates an amplification step before detection, Cas12a was directly used to identify a target sequence that can avoid false target amplification. This technique is simple, accurate, and rapid, engineered to identify cancer-associated cfDNA via a highly sensitive colorimetric change, which is expected to be beneficial for applications requiring point-of-care cancer detection.