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PubMed2026

Site-Specific Measurement of Meiotic Crossing-Over Rate with Droplet Digital PCR.

Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.

Journal Article↗

PubMed2026

A droplet digital PCR assay targeting 16 human papillomavirus genotypes.

Background. Cervical screening with high-precision assays such as human papillomavirus (HPV) DNA testing is essential for the detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective. To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes, with comparison to a commercial test and validation on clinical samples. Methods. Primers and probes were designed to target the E6 region of 16 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards and clinical screening samples (n=303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results. Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit or genome equivalent per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7 of 234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion. Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.

PCR↗

PubMed2026

Detection of antibiotic heteroresistance in clinical microbiology: current and emerging methodologies.

BACKGROUND: Antibiotic heteroresistance (HR) is characterised by the coexistence of susceptible and resistant subpopulations within an apparently isogenic bacterial isolate. Because routine antimicrobial susceptibility testing (AST) primarily assesses the dominant population, HR may escape detection, potentially leading to discrepancies between laboratory susceptibility categorisation and the underlying bacterial population structure. OBJECTIVES: To provide a critical and practice-oriented evaluation of current and emerging methodologies for HR detection and to discuss their strengths, limitations, and potential for clinical implementation. SOURCES: Narrative review based on PubMed searches, complemented by screening of key reference lists and relevant EUCAST and CLSI documents. Peer-reviewed literature was prioritised. CONTENT: Phenotypic approaches, particularly population analysis profiling, remain the reference method for HR definition, but their labour-intensive workflows, long turnaround times, and limited standardisation restrict routine implementation. Alternative strategies, including modified AST assays, metabolic assays, and single-cell platforms, offer gains in speed or throughput but require broader validation. Molecular approaches such as quantitative PCR, droplet digital PCR, targeted deep sequencing, and whole-genome sequencing improve detection of minority resistance determinants. Emerging computational frameworks, including machine learning models integrating phenotypic and genomic data, represent a promising frontier for scalable HR prediction. IMPLICATIONS: Available evidence supports the clinical relevance of HR, although its association with adverse outcomes varies across bacterial species and antibiotic classes. Harmonised methodologies and clinically validated interpretive criteria are needed to support integration of HR assessment into routine diagnostics. Prospective multicentre studies and further standardisation, including engagement with EUCAST and CLSI, will be important to advance clinical implementation.

Antimicrobial resistance↗

PubMed2026

Single-cell vector copy number analysis of phenotypically defined long-term hematopoietic stem cells for gene therapy safety assessment.

Hematopoietic stem cell (HSC)-based gene therapy has emerged as a transformative approach for the treatment of genetic diseases; however, accurate evaluation of vector copy number (VCN) remains critical for ensuring safety. Conventional bulk VCN assays, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR), do not resolve clonal heterogeneity and cannot identify rare high-VCN cells that may contribute disproportionately to insertional mutagenesis risk. Here, we developed an accessible single-cell VCN profiling method by combining fluorescence-activated cell sorting (FACS) of phenotypically defined long-term HSCs (Lineage- CD34+ CD38- CD90+ CD45RA- cells) with whole-genome amplification followed by conventional qPCR. This approach enabled resolution of VCN distributions at single-cell level using standard laboratory techniques. Notably, single-cell analysis revealed a high VCN tail that bulk VCN analysis could not resolve. Furthermore, in a humanized mouse transplantation model, single-cell VCN profiling demonstrated that overall VCN distributions could be analyzed after engraftment, although inter-donor and inter-mouse variability was observed. Collectively, this method provides a rapid, cost-effective, and phenotypically resolved strategy for assessing VCN heterogeneity in gene-modified HSCs. Single-cell VCN profiling offers complementary insights beyond conventional bulk assays and may enhance preclinical safety evaluation of gene and cell therapy products.

lentiviral vector↗

PubMed2026

Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.

BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.

Humans↗

PubMed2025

Hierarchical integration of mNGS, PCR, and other conventional methods for precision TB diagnostics.

UNLABELLED: This study systematically compared the diagnostic accuracy of seven assays for detecting the Mycobacterium tuberculosis complex, including metagenomic next-generation sequencing (mNGS), droplet digital polymerase chain reaction, real-time quantitative polymerase chain reaction, EasyNAT MTC, GeneXpert MTB/RIF, interferon-gamma release assay (IGRA), and acid‒fast staining (AFS). We try to select appropriate combinations of tuberculosis (TB) detection methods for regions with varying levels of medical resources, based on sensitivity, cost-effectiveness, and operational feasibility. A retrospective analysis was conducted on 141 samples collected from patients with suspected active TB at The First Affiliated Hospital of Sun Yat-sen University between April 2022 and April 2024. Among these samples, there were 100 cases assigned to the case group and 41 cases to the control group, based on the tuberculosis diagnostic criteria. Historical data for Xpert, IGRA, and AFS were collected, and parallel experiments using mNGS, droplet digital PCR (ddPCR), real-time quantitative polymerase chain reaction (RT-qPCR), and EasyNAT were conducted on all samples. Diagnostic performance was evaluated by comparing it with the final clinical diagnoses. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operating characteristic (ROC) curve analysis were conducted, along with DeLong tests for statistical comparison. Compared with the final clinical diagnosis, mNGS demonstrated the highest sensitivity (100%), followed by IGRA (79.2%), EasyNAT (79.1%), RT-qPCR (78.0%), ddPCR (75.8%), Xpert (75.3%), and AFS (16.7%). The specificity was 100% for both Xpert and AFS, followed by ddPCR (97.6%), RT-qPCR (95.1%), EasyNAT (92.7%), IGRA (72.7%), and mNGS (75.6%). ROC analysis revealed a significantly greater area under the ROC curve for mNGS (0.878) than for ddPCR (0.817, P = 0.031). DeLong tests revealed statistically significant differences in diagnostic performance between mNGS and ddPCR (P < 0.05) and between IGRA and AFS (P < 0.01). mNGS uniquely identified the pathogens involved in co-infection and quantified pathogen-specific sequencing reads. Through a comprehensive evaluation of the diagnostic efficacy, cost-effectiveness, and timeliness of tuberculosis detection methods, we propose corresponding combinations of TB testing approaches for regions with different healthcare resources. For undeveloped regions with limited resources, a combination of AFS +EasyNAT + chest X-ray is recommended. Primary care facilities may additionally employ IGRA + RT-qPCR. Intermediate-level hospitals can incorporate Xpert MTB/RIF for drug resistance testing, while tertiary hospitals or specialized centers should, on the basis of these fundamental tests, utilize mNGS for diagnosis and ddPCR for therapeutic monitoring in patients with complex mixed infections. IMPORTANCE: This study is the first to comprehensively evaluate the diagnostic efficacy, cost-effectiveness, and timeliness of seven TB detection methods in a single-center cohort. Our findings provide actionable solutions for optimizing TB diagnostics in diverse healthcare ecosystems, aligning with the WHO's End TB Strategy to ensure equitable access to rapid diagnostics.

Humans↗

PubMed2026

An RPA-assisted homogeneous electrochemical DNA sensor for on-site eDNA detection toward early warning of crown-of-thorns starfish outbreaks.

Crown-of-thorns starfish (COTS) outbreaks seriously threaten coral reef ecosystems, while conventional monitoring approaches are time-consuming and often lack sufficient sensitivity for early warning. Existing electrochemical DNA sensors usually require complex electrode-surface immobilization procedures, which can lead to uneven probe distribution, significant steric hindrance, and poor stability. Meanwhile, the low concentration of environmental DNA (eDNA) in marine environments further complicates detection. To overcome these challenges, this study developed a homogeneous electrochemical DNA sensor assisted by recombinase polymerase amplification (RPA) for COTS eDNA detection. Target DNA was first amplified by RPA, and the amplification products were then hybridized in solution with capture probe (CP)-modified magnetic beads (MB) and biotin-labeled signal probe (SP) to form sandwich-structured MB complexes. These complexes were subsequently magnetically enriched and immobilized on the electrode surface for electrochemical signal readout. Under optimized conditions, the sensor displayed a linear response to COTS genomic DNA from 3.77 fg/μL to 1 ng/μL, with an LOD of 2.02 fg/μL and an LOQ of 3.77 fg/μL. The sensor was applied to Xisha Islands samples, and the results agreed with droplet digital PCR (ddPCR) (P > 0.05), demonstrating its potential for sensitive and reliable on-site COTS eDNA detection.

Animals↗

PubMed2026

Alternative End Joining Dependency Imposed by miR-21-5p Defines Radiation Resistance and a Targetable Vulnerability in Oral Squamous Cell Carcinoma.

PURPOSE: Clinical control of oral squamous cell carcinoma (OSCC) is constrained by heterogeneous radiosensitivity driven by divergent DNA damage response programs. The architecture and functional contribution of alternative end joining (Alt-EJ), an error-prone DNA double-strand break (DSB) repair pathway frequently upregulated in cancer, to radiation resistance remains poorly defined. METHODS AND MATERIALS: We profiled microRNAs in radioresistant OSCC clones and performed multiomic integration across an institutional OSCC cohort, an external OSCC cohort from the Gene Expression Omnibus, The Cancer Genome Atlas pan-cancer tumors, and cell lines characterized by Sanger Genomics of Drug Sensitivity in Cancer to infer DNA damage response characteristics, genomic scar features, drug sensitivity, and radiation therapy outcomes. DSB repair capacity and pathway usage were validated using functional assays, including Alt-EJ reporters and droplet digital PCR quantification of microhomology-mediated repair events. Core Alt-EJ effectors such as PARP1 and POLQ were perturbed genetically and pharmacologically. Therapeutic efficacy of PARP or POLQ inhibition with or without irradiation was tested in a syngeneic OSCC model, followed by bulk tumor transcriptomics to assess pathway engagement. RESULTS: Upregulation of miR-21-5p was not only selectively detected in radioresistant OSCC, but also modulated radiosensitivity in vitro and in vivo, and was associated with inferior postradiation therapy survival. A calibrated miR-21-5p target-gene signature tracked Alt-EJ activity across patient and mouse tumors and cancer cell lines, correlated with microhomology-mediated indels and broader genomic scarring, and predicted sensitivity to clinically available PARP inhibitors. Functionally, enforced miR-21-5p expression increased Alt-EJ usage and accelerated DSB repair, whereas inhibition or depletion of key Alt-EJ effectors reduced repair efficiency and restored radiosensitivity. In vivo, Alt-EJ targeting with PARP or POLQ inhibitor abrogated miR-21-5p-driven radiation resistance; transcriptomic profiling supported suppression of Alt-EJ programs as the operative mechanism. CONCLUSIONS: These findings establish a mechanistic link between miR-21-5p activity and Alt-EJ dependence, provide a clinically deployable signature to identify Alt-EJ-dependent OSCC, and support rational combinations of Alt-EJ targeting agents with radiation therapy to overcome treatment failure and advance precision radiation oncology.

MicroRNAs↗

PubMed2026

HPV circulating tumor DNA as a potential prognostic and predictive biomarker in head and neck squamous cell carcinoma: a systematic review.

PURPOSE: Human papillomavirus circulating tumor DNA (HPVctDNA) has emerged as a promising prognostic biomarker in HPV-related head and neck squamous cell carcinoma (HNSCC). This systematic review aimed to synthesize current evidence on the diagnostic accuracy and prognostic value of HPVctDNA in HNSCC management. MATERIAL/METHODS: We systematically reviewed a PubMed-indexed database of studies published between January 2012 and September 2025. Eligible studies were assessed for design, primary tumor site and stage, treatment modality, HPVctDNA detection method, diagnostic accuracy (sensitivity and specificity), and reported clinical endpoints. Descriptive syntheses were performed; sensitivity and specificity were standardized to proportions and summarized as median values per group. RESULTS: A total of 60 studies, including 8,234 patients were analyzed, of which 41 (68.3%) focused exclusively on oropharyngeal squamous cell carcinoma (OPSCC) and 17 (28.3%) included mixed HPV-related HNSCC subsites and HPV-positive cancers of unknown primary. The median follow-up across the included studies was 23 months. Among the included studies, 19 were retrospective (31.7%) and 33 were prospective (55.0%), with a small proportion of cross-sectional and randomized clinical trials. Overall, 40 (66.7%) evaluated the role of HPVctDNA in a curative setting. Plasma was the most common sample type, analyzed in 55 studies (91.7%), while 5 studies also included saliva. Detection methods varied: 40 employed droplet digital PCR (ddPCR), 16 used quantitative PCR (qPCR) and 4 applied NGS-based assays. Most of these studies (38, 63.3%) evaluated the prognostic utility of HPVctDNA, while only 4 (6.7%) assessed HPVctDNA in a screening or diagnostic setting. Regarding diagnostic accuracy, the median sensitivity across evaluable studies was 91.1%, while the median specificity was 99.4%. In OPSCC-only cohorts, the median sensitivity and specificity were 89.4% and 99.4%, respectively. Dynamic changes in HPVctDNA levels during or after treatment were consistently associated with outcomes: clearance or sustained negativity correlated with higher response rates, improved progression-free survival and overall survival, while persistent positivity or increasing levels predicted disease progression and recurrence. CONCLUSIONS: HPVctDNA demonstrates high diagnostic and prognostic accuracy in HPV-related HNSCC, especially OPSCC, supporting its use for prognosis, treatment monitoring and early detection of recurrence. However, prospective interventional studies are still required to demonstrate that HPVctDNA-guided treatment decisions improve clinical outcomes before routine implementation.

Humans↗

PubMed2026

Deep tissue sequencing improves genetic diagnostic yield in focal cortical dysplasia.

Focal cortical dysplasias (FCDs) are malformations of cortical development associated with drug-resistant focal epilepsy. We analyzed surgical tissue from 25 consecutive cases recruited from adult and pediatric epilepsy surgery programs. We performed high-depth sequencing of lesional tissue, validated somatic variants using droplet digital PCR or amplicon sequencing, and investigated genotype-phenotype correlations. A pathogenic or likely pathogenic variant was detected in 64% (n = 16/25) of cases. Of these, five cases with FCDIIa or FCDIIb had germline variants in NPRL3 (n = 3) or DEPDC5 (n = 2). Somatic variants were identified in 44% (n = 11/25) of cases. The genetic yield for FCDIIb was 77% of cases having a pathogenic mTOR pathway variant detected (n = 10/13), and for FCDIIa 66% (n = 6/9). High depth sequencing approaches allowed detection of somatic variants with very low (down to 0.4%) variant allele fractions (VAFs). No pathogenic variants were detected in 3 cases with FCDI. 62% (n = 15/24) of the cases with ≥12 months follow up experienced a favourable seizure outcome (Engel 1-2) following surgery. Of note, n = 9 patients required repeat surgery to resect residual dysplasia. Determining a genetic diagnosis reveals aetiology and paves the way to precision therapies that may benefit those with FCD who do not respond to current treatments.

Humans↗

PubMed2026

Metabolomics and genomics reveal high diversity and concentrations of cyanopeptides during a Microcystis bloom.

Cyanobacterial blooms are an immense global problem that release complex mixtures of poorly characterized biologically active cyanopeptides into freshwater. In this study, metabolomics and genomics were used to assess the diversity and concentrations of cyanopeptides during a dense Microcystis bloom during the late summer of 2023 in Lake Champlain, a large transboundary lake situated between Canada and the United States. Despite the relatively low genetic diversity of the bloom determined by 16S rRNA metabarcoding, 151 cyanopeptides were detected by non-targeted metabolomics. This represents the most recorded cyanopeptides from a single lake plankton bloom event to date. Fifty-two cyanopeptides were previously reported and 99 represent putative new structures. Standards from the microcystin, cyanopeptolin, microginin, and anabaenopeptin groups were used to either quantify or approximate respective cyanopeptide concentrations over the sampling period. Cyanopeptolins were the most diverse (n = 68) cyanopeptides and the second most abundant, reaching 12,892 μg/L. Microginins were the second most diverse (n = 24) and reached the highest concentrations (18,262 μg/L). Anabaenopeptins were the third most diverse (n = 17) cyanopeptides, reaching 4,818 μg/L. Only 8 microcystins were detected, reaching 4,935 μg/L, where MC-LR was the dominant congener. Target cyanopeptide biosynthesis genes for microcystins (mcyE), cyanopeptolins (mcnC), anabaenopeptins (apnD), microviridins (mdnC), and aeruginosins (aerA) were also quantified using digital droplet PCR (ddPCR). The gene copy numbers for mcyE, mcnC, and apnD were highly correlated with their corresponding cyanopeptide concentrations. Overall, the studied Microcystis bloom produced a very diverse cyanopeptide mixture with high cyanopeptide concentrations including non-microcystin groups.

Microcystis↗

PubMed2025

Utility of Plasma Cell-free Chromatin Immunoprecipitation to Detect Cardiac Allograft Rejection.

BACKGROUND: Antibody-mediated rejection (AMR) remains the major risk factor for allograft loss across all solid organ transplantation. Unfortunately, its diagnosis relies on biopsy, an invasive gold standard that often sample unaffected allograft tissue leading to missed diagnosis. Plasma donor-derived cell-free DNA (dd-cfDNA) is noninvasive biomarker that has high sensitivity but low specificity for AMR diagnosis. This proof-of-concept study assessed the utility of cell-free chromatin immunoprecipitation (cfChIP) as a surrogate for gene expression to detect cardiac AMR and the associated pathobiology. METHODS: The discovery GRAfT multicenter cohort of heart transplant patients (NCT02423070) identified AMR, acute cellular rejection (ACR), and stable controls based on biopsy and ddcfDNA results. Plasma cfChIP-sequencing was performed to identify peaks, associated genes and pathobiological pathways. Plasma from an external cohort (GTD, NCT01985412) was also analyzed to verify pathways identified. Digital droplet PCR (ddPCR) assays targeting differential regions were constructed to test the diagnostic performance of cfDNA to detect AMR/ACR from stable controls (rejection-specific assays) or AMR from ACR (AMR-specific assays). RESULTS: The cohort included 21 AMR, 28 ACR, and 45 stable controls from GRAfT and GTD, and 23 healthy controls. cfChIP detected expected active genes, including housekeeping genes and gene targets of transplant immunosuppressive drugs but not inactive genes. Unsupervised clustering of the discovery GRAfT cohort assigned 95% of samples correctly as AMR, ACR or stable control. Differential analysis identified pathobiological pathways of AMR such as neutrophil degranulation and complement activation. The pathways were consistent in GTD samples. Rejection-specific assays detected AMR/ACR from controls with AUC of 0.78 - 0.95. AMR-specific assays detected AMR from ACR with AUC of 0.71 - 0.85, sensitivities of 0.73 - 0.94 and specificities of 0.73 - 0.80. CONCLUSION: This study provides valuable preliminary data supporting the use of cfChIP to detect AMR and the associated pathobiological pathways.

Allograft rejection↗

PubMed2026

Analyzing Meiosis in Maize.

Meiosis is central to sexual reproduction and the main source of genetic diversity in plants. Understanding how meiotic processes are regulated has direct relevance to agriculture. As meiotic recombination is the vehicle of plant breeding, gaining the ability to influence recombination patterns can accelerate crop improvement. Maize is a powerful model for studying plant meiosis, thanks to its large chromosomes, well-developed genetics, and the availability of diverse cytogenetic and molecular tools. Insights gained from maize studies can extend to other species. In this review, we describe a variety of approaches for examining meiosis and meiotic recombination in maize. Cytological techniques, including protein immunolocalization and fluorescence in situ hybridization (FISH), enable visualization of chromosome structure and behavior, as well as crossover (CO) formation. Chromatin immunoprecipitation (ChIP) is used in meiosis research to determine locations of recombination proteins, identify recombination sites, and elucidate chromatin features, such as histone modifications. Quantification of COs at specific genomic sites through pollen typing by droplet digital PCR allows precise high-resolution measurement of recombination rates. Combining cytology, protein localization, and molecular assays provides a multiscale picture of meiosis, linking molecular mechanisms to chromosome behavior and, ultimately, to genetic variation.

Journal Article↗

PubMed2026

High-Sensitivity ctDNA Analysis Uncovers Relevant Signals Missed by NGS in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) carries high mortality despite multimodal therapy, and improved biomarkers are needed to guide perioperative care. This study evaluated the prognostic significance of Kirsten rat sarcoma virus (KRAS)-mutant circulating tumor DNA (ctDNA) detected by next-generation sequencing (NGS) and digital droplet PCR (ddPCR) in localized PDAC. EXPERIMENTAL DESIGN: In this prospective cohort study (2020-2024), patients with localized PDAC undergoing neoadjuvant chemotherapy (NAC) were enrolled across multiple sites within Northwestern Medicine. Blood samples for ctDNA were assessed at diagnosis, after NAC, and after resection using tumor-agnostic NGS and ddPCR targeting KRAS G12D/V/R mutations. Overall survival (OS) was assessed using Kaplan-Meier analysis. RESULTS: The cohort included 106 patients. At diagnosis, KRAS ctDNA was detected in 17.2% (17/99) by NGS and 64.9% (63/97) by ddPCR. Detection by both platforms was associated with shorter OS, with the higher-sensitivity ddPCR assay providing greater prognostic discrimination by identifying additional patients with poor outcomes not captured by NGS (NGS median OS 11.2 vs. 30.5 months, P < 0.001; ddPCR median OS 24.7 vs. 70.9 months, P = 0.004). Stratified by detection method, median OS was shortest in patients with ctDNA detected by both NGS and ddPCR (10.9 months), longest in those not detected by either platform (40.7 months), and intermediate in patients detected only by ddPCR (26.9 months; P < 0.001). CONCLUSIONS: In localized PDAC, KRAS-mutant ctDNA detected by NGS or ddPCR was associated with worse survival. ddPCR identified additional patients missed by NGS. Integrating ddPCR with NGS ctDNA measures may improve perioperative risk stratification, although validation is needed before clinical implementation.

Humans↗

PubMed2025

From Diagnosis, Therapy Decision-Making to Genetic Risk Assessment: The Impact of ctDNA Testing on Comprehensive Cancer Management-A Case Report.

Circulating tumor DNA (ctDNA) testing is a minimally invasive alternative to tissue biopsy and is ideal for inaccessible tumors or limited samples. It captures tumor heterogeneity over time and different anatomic locations, unlike the static snapshot provided by a biopsy. In this report, we describe a 68-year-old female with an initial diagnosis of metastatic pancreatic adenocarcinoma (a pancreas head mass with multiple bilateral lung nodules). Mutation profiling of the pancreatic mass biopsy using a comprehensive cancer next-generation sequencing (NGS) panel was unsuccessful due to insufficient tissue. Consequently, ctDNA testing using a pan-cancer NGS panel was performed, and an EGFR p.L858R variant at 2.15% was identified. Interestingly, this activating variant is highly specific to non-small cell lung cancer (NSCLC), which raised the possibility of a synchronous tumor unrelated to the pancreatic mass. Immunohistochemistry showed the EGFR variant in station 7 lymph nodes but not in pancreatic biopsy tissue, supporting the inference that the variant originated from the lung mass. Droplet digital PCR on the limited pancreatic biopsy identified a KRAS p.Q61 variant, which was absent by ctDNA testing, suggesting a pancreatic primary with low ctDNA levels. In addition to diagnosing a primary lung cancer, ctDNA testing guided treatment decisions. With a primary EGFR p.L858R-mutant NSCLC, osimertinib was administered, resulting in a partial response within 10 months. In addition, given the synchronous primary pancreatic adenocarcinoma, germline testing was performed, revealing a CDKN2A p.I49T variant consistent with melanoma-pancreatic cancer syndrome, prompting comprehensive cancer surveillance and familial testing. This case illustrates how ctDNA testing enabled a comprehensive evaluation by clarifying the diagnosis, identifying actionable biomarkers, and facilitating genetic risk assessment, ultimately having a significant impact on the patient's clinical management.

Humans↗

PubMed2025

Analytical and clinical performance validation of HPV-SEQ, a novel NGS-based liquid biopsy platform for detection and quantification of human papilloma virus circulating tumor DNA.

BACKGROUND: Human papillomavirus (HPV) is the primary causative driver of oropharyngeal squamous cell carcinoma (OPSCC). Accurate detection of HPV-DNA is critical for risk stratification and management of OPSCC. However, assays designed to detect HPV in primary tumors do not allow monitoring of HPV-DNA over time, whereas commercially available droplet digital PCR-based methods for assessment of circulating cell free (cf)HPV-DNA in plasma remain suboptimal, hindering adaptation into clinical practice. We have developed HPV-SEQ, a novel next-generation-sequencing (NGS) based method for detection and quantification of HPV16/18 DNA in plasma of patients with OPSCC. METHODS: The assay uses primers targeting the L1 gene of HPV16 and HPV18 viral genomes and strain specific calibrators at a defined concentration to determine the ratio of native HPV to a known standard, enabling accurate reporting of patient-derived HPV16/18 viral load in a sample. This study was conducted using two different patient populations in addition to healthy donors and contrived material. All experiments were performed to fulfill several applicable analytical, performance and validation guidelines. RESULTS: A thorough analytical characterization and clinical validation of this platform demonstrates that HPV-SEQ detects cfHPV-DNA with exceptional limit of quantification and high precision, providing a foundation for integrating this platform into clinical settings. CONCLUSIONS: This ultra-sensitive HPV profiling method with optimal analytical performance may represent a significant advancement in risk stratification, treatment management, and post-treatment surveillance for patients with OPSCC.

Humans↗

PubMed2025

Methylation-based droplet digital polymerase chain reaction shows high concordance with chronic lymphocytic leukemia IGHV somatic mutation status.

OBJECTIVE: Somatic hypermutation at immunoglobulin heavy chain variable (IGHV) genes, an established prognostic and predictive biomarker for chronic lymphocytic leukemia (CLL), is assessed by gene sequencing. We developed a single methylation-specific droplet digital polymerase chain reaction (methyl-ddPCR) to predict IGHV status in patients with CLL. METHODS: The CLL methylation array and IGHV data from the International Cancer Genome Consortium (ICGC) were used for biomarker discovery. Top-ranked candidate regions were manually screened for PCR primer and probe binding sites. A single methyl-ddPCR was evaluated on an internal cohort of CLLs with mutated (M), unmutated (U), and inconclusive IGHV results originally determined by next-generation sequencing (NGS). RESULTS: Analysis of ICGC data identified array probe cg23844018 as a candidate for the PCR. The corresponding CpG site showed high methylation levels in U-CLL and lower levels in M-CLL. On the internal cohort, a single optimal cutoff correctly classified 104 of 115 U- and M-CLLs (90.4%; area under the curve = 0.96). The PCR data correlated with some prognostic fluorescence in situ hybridization and CLL subset groupings. Limited analysis suggests that the PCR may be able to stratify some patients with CLL who have inconclusive results on IGHV NGS testing. CONCLUSIONS: The methyl-ddPCR showed high concordance with CLL IGHV status in an internal cohort.

Humans↗

PubMed2006

Integrated polymerase chain reaction chips utilizing digital microfluidics.

This study reports an integrated microfluidic chip for polymerase chain reaction (PCR) applications utilizing digital microfluidic chip (DMC) technology. Several crucial procedures including sample transportation, mixing, and DNA amplification were performed on the integrated chip using electro-wetting-on-dielectric (EWOD) effect. An innovative concept of hydrophobic/hydrophilic structure has been successfully demonstrated to integrate the DMC chip with the on-chip PCR device. Sample droplets were generated, transported and mixed by the EWOD-actuation. Then the mixture droplets were transported to a PCR chamber by utilizing the hydrophilic/hydrophobic interface to generate required surface tension gradient. A micro temperature sensor and two micro heaters inside the PCR chamber along with a controller were used to form a micro temperature control module, which could perform precise PCR thermal cycling for DNA amplification. In order to demonstrate the performance of the integrated DMC/PCR chips, a detection gene for Dengue II virus was successfully amplified and detected. The new integrated DMC/PCR chips only required an operation voltage of 12V(RMS) at a frequency of 3 KHz for digital microfluidic actuation and 9V(DC) for thermal cycling. When compared to its large-scale counterparts for DNA amplification, the developed system consumed less sample and reagent and could reduce the detection time. The developed chips successfully demonstrated the feasibility of Lab-On-a-Chip (LOC) by utilizing EWOD-based digital microfluidics.

Dengue Virus↗