Plasma DNA Concentrations Research Articles

Quantification of cerebrospinal fluid tumor DNA in lung cancer patients with suspected leptomeningeal carcinomatosis

Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman’s ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

Exertional heat stress promotes the presence of bacterial DNA in plasma: A counterbalanced randomised controlled trial

ObjectivesThe primary aim was to explore the impact of exertional-heat stress (EHS) promoted exercise-associated bacteraemia. A secondary aim was to examine if an amino acid beverage (AAB) intervention may mitigate exercise-associated bacteraemia. DesignCounterbalanced randomised control trial. MethodsTwenty endurance trained male participants completed two randomised EHS trials. On one occasion, participants consumed a 237 mL AAB twice daily for 7 days prior, immediately before and every 20 min during EHS (2 h running at 60 % V̇O2max in 35 °C). On the other occasion, a water volume control (CON) equivalent was consumed. Whole blood samples were collected pre- and immediately post-EHS, and were analysed for plasma DNA concentration by fluorometer quantification after microbial extraction, and bacterial relative abundance by next generation 16s rRNA gene sequencing. ResultsIncreased concentration of microbial DNA in plasma pre- to post-EHS was observed on CON (pre-EHS 0.014 ng/μL, post-EHS 0.039 ng/μL) (p < 0.001) and AAB (pre-EHS 0.015 ng/μL, post-EHS 0.031 ng/μL) (p < 0.001). The magnitude of change from pre- to post-exercise on AAB was 40 % lower, but no significant difference was observed versus CON (p = 0.455). Predominant bacterial groups identified included: phyla-Proteobacteria (88.0 %), family-Burkholderiaceae (59.1 %), and genus-Curvibacter (58.6 %). No significant variation in absolute and relative change in α-diversity and relative abundance for phyla, family, and genus bacterial groups was observed in AAB versus CON. ConclusionsThe increased presence of microbial-bacterial DNA in systemic circulation in response to EHS appears positive in all participants. An amino acid beverage supplementation period prior to and consumption during EHS did not provide significant attenuation of EHS-associated bacteraemia.

Abstract 2416: Personalized minimal residual disease detection using tumor-derived structural variants in cell-free DNA

Abstract We demonstrate development and technical validation of a novel approach for analysis of plasma circulating-tumor DNA (ctDNA) for residual disease detection and monitoring that offers improvements over existing technologies. ctDNA in blood can be leveraged to monitor treatment response, detect cancer recurrence, and inform treatment decisions for cancer patients. However, accurate detection and quantification of ctDNA is technically challenging due to low concentration of plasma DNA, especially in the adjuvant setting. Most current assays rely on interrogating multiple patient-specific point mutations to achieve exquisite limits of detection but struggle with polymerase errors, sequencing artifacts and limited conversion of input DNA. To overcome these challenges, we developed an approach called Structural Variant Enrichment and Normalization (SVEN) that targets patient-specific genomic rearrangements to achieve sensitive detection and quantification of plasma ctDNA. This method leverages intrinsic features of tumor-derived structural variants (SVs) to improve enrichment and confidence of detection at low tumor fraction through multiplexed PCR and amplicon-based next generation sequencing. To evaluate the assay, we performed whole genome sequencing of DNA from two breast cancer cell lines, as well as matched FFPE tumor and buffy coat samples from 21 early-stage breast cancer patients. We identified consensus SVs, and designed multiplexed SV-specific primers for each subject. We then conducted PCR amplification of cell-free DNA followed by sequencing, amplicon identification, and quantification. Analytical validation was conducted using 284 replicates of cell line dilutions from 1% to 0.003%, and 5 to 40 ng of input. We developed multiplexed assays targeting 25 and 32 SVs across two cell lines, and 3 - 43 SVs across breast cancer patients (median 23 per patient). 91% and 42% of PCR targets were successfully validated for cell lines and FFPE samples respectively. Cell line dilutions demonstrated a sensitivity of 89% at 0.003% tumor fraction with 10 ng input or more. Tumor fractions measured using SVEN agreed with known values (R2 = 0.989). Application of validated panels to patient plasma samples and tumor/germline dilutions is ongoing. SVEN is a novel approach for tumor-guided ctDNA detection and quantification with potential relevance for residual disease detection. On-going studies are investigating the performance of this approach for ctDNA detection in patients with early and locally advanced breast cancer treated with neoadjuvant therapy. Citation Format: Bradon R. McDonald, Kirsten L. Dennison, Amanda L. Schussman, Stephanie M. McGregor, Barbara A. Pockaj, Muhammed Murtaza. Personalized minimal residual disease detection using tumor-derived structural variants in cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2416.

CtDNA improves prognostic prediction for patients with relapsed/refractory MM receiving ixazomib, lenalidomide, and dexamethasone

AbstractIt remains elusive how driver mutations, including those detected in circulating tumor DNA (ctDNA), affect prognosis in relapsed/refractory multiple myeloma (RRMM). Here, we performed targeted-capture sequencing using bone marrow plasma cells (BMPCs) and ctDNA of 261 RRMM cases uniformly treated with ixazomib, lenalidomide, and dexamethasone in a multicenter, prospective, observational study. We detected 24 and 47 recurrently mutated genes in BMPC and ctDNA, respectively. In addition to clonal hematopoiesis–associated mutations, varying proportion of driver mutations, particularly TP53 mutations (59.2% of mutated cases), were present in only ctDNA, suggesting their subclonal origin. In univariable analyses, ctDNA mutations of KRAS, TP53, DIS3, BRAF, NRAS, and ATM were associated with worse progression-free survival (PFS). BMPC mutations of TP53 and KRAS were associated with inferior PFS, whereas KRAS mutations were prognostically relevant only when detected in both BMPC and ctDNA. A total number of ctDNA mutations in the 6 relevant genes was a strong prognostic predictor (2-year PFS rates: 57.3%, 22.7%, and 0% for 0, 1, and ≥2 mutations, respectively) and independent of clinical factors and plasma DNA concentration. Using the number of ctDNA mutations, plasma DNA concentration, and clinical factors, we developed a prognostic index, classifying patients into 3 categories with 2-year PFS rates of 57.9%, 28.6%, and 0%. Serial analysis of ctDNA mutations in 94 cases revealed that TP53 and KRAS mutations frequently emerge after therapy. Thus, we clarify the genetic characteristics and clonal architecture of ctDNA mutations and demonstrate their superiority over BMPC mutations for prognostic prediction in RRMM. This study is a part of the C16042 study, which is registered at www.clinicaltrials.gov as #NCT03433001.

Evaluation of sonobiopsy feasibility and safety in a mouse model of diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of pediatric brain cancer mortality, emphasizing the need for precise molecular diagnosis to advance therapeutic development. The tumor's location in the brainstem poses challenges for invasive biopsy, prompting the exploration of liquid biopsies. However, both blood-based and cerebrospinal fluid (CSF)-based liquid biopsies exhibit limited sensitivity in diagnosing DIPG. Sonobiopsy, an innovative technique utilizing focused ultrasound and microbubbles, aims to enhance the sensitivity of liquid biopsies. This study assessed the feasibility and safety of sonobiopsy in a DIPG mouse model. The model was established by intracranial injection of enhanced green-fluorescent-protein (eGFP) transduced DIPG tumor cells. Mice were divided into sonobiopsy and conventional liquid biopsy groups. Sonobiopsy, employing MRI-guided focused ultrasound and microbubbles, demonstrated increases in plasma and CSF eGFP DNA, RNA, and protein concentrations compared to conventional liquid biopsy. The enrichment effect varied based on the biomarker type. No observed hemorrhage or tissue damage attributed to sonication suggests the safety of this approach. These results affirm the feasibility and safety of sonobiopsy in enriching DIPG-specific biomarkers in both plasma and CSF, suggesting sonobiopsy as a promising noninvasive molecular diagnostic tool for DIPG.

B-269 Plasmodium Detection: Sera, Blood, Do They All Work?

Abstract Background When diagnosing malaria, testing employs blood samples using either microscopy, the current gold standard or molecular diagnostic tests (more sensitive but significantly more expensive). In Plasmodium spp. screening programs in at-risk populations where there are not many resources available, dried blood on filter paper is used to ease transport, storage and preservation of the sample. Although Plasmodium detection studies using blood serum as a sample are available, it isn´t always as sensitive as using whole blood-based sampling, as the concentration of DNA in plasma is proportional to the level of parasitaemia. This study aims to confirm the differences in sensitivity using both samples, demonstrating the importance of choosing the right type of sample to use for diagnosis. Methods The study was carried out using the VIASURE Malaria Real Time PCR Detection Kit with sera and blood samples from 94 inhabitants from Nueva Esperanza community in the Peruvian Amazon collected between 2007 and 2020, establishing a comparative analysis between matrixes. Nucleic acid extraction was performed using the QIAamp DNA Mini Kit from QIAGEN and the VIASURE assay was performed using QuantStudio®6 Flex Real-Time PCR System thermocycler (Thermo Fisher Scientific) and the BioRad CFX96TM Real-Time PCR Detection System for analysing peripheral blood samples and the BioRad CFX96TM Real-Time PCR Detection System for analysing sera. Results From the total of samples, only 5 sera (5.31%) showed amplification of Plasmodium spp., whereas from blood samples, 30 resulted to be Plasmodium spp-positive. Therefore, 26.59% of detection is lost when serum is used as a sample, as opposed to blood. Conclusion After this analysis it could be concluded that malaria detection sensitivity is appreciably higher for blood samples than for sera samples. Thus, serum samples aren´t the best choice for Plasmodium spp. testing and whole blood-based matrixes are preferable for this diagnosis. These results reinforce the idea of the importance of the sample type when diagnosing different pathogens, as well as the relevance of a confirming comparative analysis when similar sample types are involved.

Epstein-Barr virus (Herpesviridae: Lymphocryptovirus) types 1 and 2 and other viral markers in patients with nasopharyngeal carcinoma in two geographically and ethnically distinct regions of Russia

The discovery of two types of Epstein-Barr virus (EBV) (EBV-1 and EBV-2) that have different biological properties stimulated the search for neoplasms associated with each type of the virus. The aim of the work is to study the nature of the association of nasopharyngeal cancer (NPC) with EBV-1 and EBV-2, serological activity for each viral type and the concentration of EBV DNA in the blood plasma of two gender, age and ethnic groups of NPC patients that represent geographically and climatically different regions of Russia,. In the blood plasma of patients with NPC and other non- EBV associated tumors of oral cavity (OTOCEBV-) from the North Caucasian (NCFD) and Central (CFD) Federal Districts of Russia, the types of EBV and the concentration of viral DNA were determined using respectively «nested» and real time PCR; titers of IgG and IgA antibodies to viral capsid antigen (VCA) were measured in indirect immunofluorescence assay. The blood plasma samples testing showed that NPC and OTOCEBV- patients were infected with both types of EBV in approximately equal proportions. In two groups of NPC patients infected with one of the virus types only, EBV-1 or EBV-2, respectively, no statistically significant differences were found between the geometric mean values of IgG and IgA anti-EBV antibody titers and viral DNA concentrations in blood plasma. The distribution of virus types was not affected by either patient gender or ethnogeographic origin. The difference was found only between age groups: EBV-2 dominated in NPC patients up to 60 years, and EBV-1 was prevalent in patients over 60 years. The lack of the predominance of one of EBV types in NPC patients that are the representatives of different ethnic groups from geographically and climatically different regions, suggests that none of these factors play an important role in the NPC carcinogenesis. Evidently, each type of EBV, EBV-1 or EBV-2, if the necessary conditions arise, are able to exhibit its oncogenic potential to initiate tumor development.

Selected Vegetables and Spices Improve DNA Quality and Histopathological Abnormalities in Roof Rats (Rattus rattus) Exposed to Cement Dust

Highlights: 1. This study identified feasible and affordable alternatives to ineffective conventional cement production pollution control strategies.2.M. oleifera, H. sabdariffa, T. occidentalis, and Z. officinale are effective as personal interventions to ameliorate the effects of cement pollution. Abstract Conventional pollution control strategies in the cement industry have proven ineffective. As a result, effective and targeted complementary interventions are necessary. This study used roof rats (Rattus rattus) inhabiting the premises of a cement plant in Sagamu, Ogun State, Nigeria, to evaluate the ameliorative effects of moringa (Moringa oleifera), roselle (Hibiscus sabdariffa), fluted pumpkin (Telfairia occidentalis), and ginger (Zingiber officinale) on cement dust exposure. A total of 42 rats were divided into seven groups, with each group consisting of six rats. Group 1 served as the negative control group and was not exposed to any substances, while group 2 served as the positive control group and received standard feed throughout the experiment. Meanwhile, groups 3, 4, 5, 6, and 7 served as the experimental group. Rats in these groups were fed with 400 mg/kg of ethanolic extracts of Z. officinale, M. oleifera, T. occidentalis, H. sabdariffa, and a mixture of the four extracts with a composition of 1:1:1:1, respectively, for 90 days. The plasma DNA concentrations, DNA purity, and lungs of the rats were examined before and after the experiment. Prior to the experiment, the exposed rats had higher plasma DNA concentrations and lower DNA purity, as well as severe fibrosis and congested alveoli in their lungs, compared to the unexposed rats. At the end of the experiment, the experimental groups showed a significant increase in DNA purity (p≤0.05) and a decline in plasma DNA concentrations compared to the positive control group. In addition, the experimental groups showed fewer histopathological abnormalities than the positive control group. The mixture of the extracts yielded the most favorable results, followed by the extracts of Z. officinale, M. oleifera, T. occidentalis, and H. sabdariffa, respectively. These findings suggested that the selected vegetables and spices have the properties to ameliorate the effects of cement dust exposure. Therefore, individuals residing in close proximity to cement plants are encouraged to consume these vegetables and spices.

Circulating tumour DNA concentration and genetic classification improve risk stratification in newly diagnosed patients with Diffuse Large B‐Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with poor outcomes for the 40% of patients who relapse or are refractory to upfront therapy, and current prognostic tools are unable to identify many of those patients. Recently, novel prognostic biomarkers like genetic classification and circulating tumor DNA (ctDNA) have been developed. Here we explore the value these biomarkers to improve prognostic stratification in DLBCL. Methods: DNA was extracted from 2 to 5 ml of plasma and formalin-fixed paraffin-embedded (FFPE) tumor tissue obtained at diagnosis from patients with DLBCL treated with R-CHOP-like regimes. Samples were sequenced in an Illumia NovaSeq using a panel of 112 genes that are recurrently mutated in lymphoid neoplasms, and variants were called using a pipeline that follows GATK best practices. Genetic subtype (GS) was determined using LymphGen tool, excluding A53, as copy number variation data was unavailable. ST2, EZB MYC negative and BN2 subtypes were considered favorable, while N1, MCD, EZB MYC positive and other were considered unfavorable. ctDNA levels were reported as haploid genome equivalents per mL of plasma and expressed as a base 10 logarithm (log hGE/mL) Results: We included 46 patients with median age of 64.8 years, 58% advanced stage (III-IV) and 65% germinal center DLBCL by Hans’s algorithm. Somatic mutations were detected in FFPE in all patients and in ctDNA in 40 (87%) patients. Most frequently mutated genes were KMT2D, CREBBP, TP53, ARID1A, MYD88 and CARD11. The sensitivity of ctDNA to detect mutations present in paired FFPE samples was 66% (for all variants) and 74.5% (for variants with >5% allelic frequency in FFPE samples). 67.4% of patients were successfully classified by LymphGen, with a concordance between ctDNA and FFPE of 84.7%. Patients without detectable ctDNA presented localized disease (Ann Arbor stage I) and low DNA concentration in plasma (<15 ng/ml). In our cohort, 24 patients presented a poor R-IPI score with a 3-year progression-free survival (PFS) of 61.5% (Figure 1A). These patients were further stratified by ctDNA levels and GS, and those unfavorable GS or ctDNA concentration >4 log hGE/ml presented worse PFS and OS than without risk factors a 3-year PFS of 34.6% and 90.9%, respectively (Figure 1B). Patients were classified into three groups according IPI score modified by ctDNA levels (Figure 1C), with a 3-year PFS of 94.7%, 81.2% and 18.8%, and a 3-year overall survival (OS) of 100%, 87.5% and 37.5%, respectively (Figure 1D–E). Keywords: Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

Short-length DNA fragments in plasma to predict clinical outcome in unresected glioblastoma patients.

2025 Background: Early prediction of therapeutic response is an important issue in glioblastoma patients. Cell-free DNA (cfDNA) concentration in plasma shown potential prognostic value. cfDNA release is influenced by non-tumor parameters. Short-length cfDNA (slDNA) may better reflect tumor evolution. The aim was to investigate plasmatic slDNA as prognostic marker in unresected glioblastoma patients. Methods: An ancillary study of a prospective trial was conducted (GLIOPLAK trial). Patients had newly diagnosed and histomolecular confirmed glioblastoma IDH wild-type (WHO 2021 classification). Patients with resected tumor were excluded. All patients underwent radiotherapy/temozolomide (RT/TMZ) schedule after biopsy. Plasmas were collected at three times during first-line treatment: before (pre-), after (post-) RT and at the time of disease progression/relapse. Cell-free DNA (cfDNA) was extracted from plasma using the QIAamp circulating nucleic acid kit (Qiagen). Short-length DNA (slDNA) fragments (equal or lower than 250 bp length) concentration was calculated based on AUC after automated electrophoresis using 4200 TapeStation System. Primary objective was to investigate the impact on survival of slDNA during RT/TMZ phase. Secondary objectives were to explore the association between tumor volume, corticosteroid exposition and slDNA. Results: Thirty-eight patients were included: median age was 63 [interquartile range 55-66], 84.2% (n=32) had Karnofsky index higher than 80% at baseline and median overall survival (OS) was 13.3 months [CI 95% 11.5; 15.5]. slDNA was detected in 69.4% at the pre-RT time (mean 722.3 pg/ml ±sd 1257.7) and in 83.3% at the post-RT-time (686.1 pg/ml ±1217.1). One third of the patients experienced a slDNA decrease between pre- and post-RT time. Presence of slDNA at the pre-RT time was associated with improved OS: median of 11.7 months [9.8-19.6] in the slDNA(+) group (n=25) versus 8.8months [5.5-NA] in the slDNA(-) group (n=11), HR 0.309 [0.133-0.716], log rank p=0.004. Decrease of slDNA concentration was also associated with better outcome compared to stability or increase of slDNA between pre- and post-RT: respectively median OS 14.1 vs 10.1 months, 0.424 [0.197-0.909], p=0.02. Corticosteroid exposition and dose were not correlated to slDNA concentration at pre-RT or slDNA variation (respectively correlation coefficient 0.081 and 0.047). At pre-RT, presence of slDNA was independent of tumor volume: mean 265.0 cm3 ±84.7 in slDNA(+) group vs 237.7 cm3 ±115.7 in slDNA(-) group, p=0.492. Total circulating cfDNA concentration did not influence OS in our cohort. Conclusions: slDNA during the RT/TMZ phase is an independent prognostic marker in unresected glioblastoma patients. The role of slDNA as a biomarker for adaptative treatment after RT as well as non-invasive molecular characterization tool can be explored in dedicated trial. Clinical trial information: NCT02617745 .

Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses.

Biomarkers for osteoarthritis (OA) in horses have been extensively investigated, but translation into clinical use has been limited due to cost, limited sensitivity, and practicality. Identifying novel biomarkers that overcome these limitations could facilitate early diagnosis and therapy. This study aimed to compare the concentrations of synovial fluid (SF) and plasma cell-free DNA (cfDNA) over time in control horses with those with induced carpal OA. Following an established model, unilateral carpal OA was induced in 9 of 17 healthy Thoroughbred fillies, while the remainder were sham-operated controls. Synovial fluid and plasma samples were obtained before induction of OA (Day 0) and weekly thereafter until Day 63, and cfDNA concentrations were determined using fluorometry. The SF cfDNA concentrations were significantly higher for OA joints than for sham-operated joints on Days 28 (median 1430 μg/L and 631 μg/L, respectively, p = 0.017) and 63 (median 1537 μg/L and 606 μg/L, respectively, p = 0.021). There were no significant differences in plasma cfDNA between the OA and the sham groups after induction of carpal OA. Plasma cfDNA measurement is not sufficiently sensitive for diagnostic purposes in this induced model of OA. Synovial fluid cfDNA measurement may be used as a biomarker to monitor early disease progression in horses with OA.

Preoperative cell-free DNA concentration in plasma as a diagnostic and prognostic biomarker of clear cell renal cell carcinoma.

Assessment of renal tumour masses is based on conventional imaging studies (computer tomography or magnetic resonance), which does not allow characterisation of the histopathological type. Moreover, the prediction of prognosis in localised and metastatic renal cell carcinoma requires improvement as well. Analysis of circulating free DNA (cfDNA) in blood is one of the variants of liquid biopsy that may improve diagnostics and prognosis issues of patients with renal tumour masses suspected to be renal cell carcinoma. The aim of the study was to assess the diagnostic and prognostic role of preoperative cfDNA concentration in the plasma samples of clear cell renal cell carcinoma (ccRCC) patients. The preoperative plasma cfDNA concentration was assessed in ccRCC patients (n = 46) and healthy individuals (control group) (n = 17). The circulating free DNA concentration was reflected by the 90 bp DNA fragments determined by real-time polymerase chain reaction. The median cfDNA concentration was significantly higher in ccRCC patients (n = 46) compared to the control g roup (n = 17) (2588 ±2554 copies/ml vs. 960 ±490 copies/ml, p < 0.01). In multivariate analysis, the preoperative plasma cfDNA concentration was the significant factor increasing the probability of ccRCC detection (OR: 1.003; 95% CI: 1.001-1.005). The median cfDNA concentration depended on the stage of ccRCC; it was higher in metastatic ccRCC patients (n = 11) compared to non-metastatic ccRCC patients (n = 35) (3619 ±4059 copies/ml vs. 2473 ±1378 copies/ml, p < 0.03). Kaplan-Meier survival analysis demon-strated that patients with high cfDNA values (above 2913 copies/ml) had significantly worse cancer-specific survival (HR: 4.5; 95% CI: 1.3-16.9, log-rank Mantel-Cox test p = 0.015). Preoperative plasma cfDNA concentration has diagnostic and prognostic potential in ccRCC pa-tients.

The Correlation between Periodontal Parameters and Cell-Free DNA in the Gingival Crevicular Fluid, Saliva, and Plasma in Chinese Patients: A Cross-Sectional Study.

Purpose: To investigate the correlation between periodontal parameters and cell-free DNA (cfDNA) concentrations in gingival crevicular fluid (GCF), saliva, and plasma. Methods: Full mouth periodontal parameters, including probing depth (PD), bleeding on probing (BOP), and plaque index (PI) were recorded from 25 healthy volunteers, 31 patients with untreated gingivitis, and 25 patients with untreated periodontitis. GCF, saliva, and plasma samples were collected from all subjects. Extraction and quantification assays were undertaken to determine cfDNA concentrations of each sample. Results: GCF and salivary cfDNA levels were increased with aggravation of periodontal inflammation (GCF p < 0.0001; saliva p < 0.001). Plasma cfDNA concentrations in patients with periodontitis were significantly higher than those in healthy volunteers and patients with gingivitis. GCF and salivary cfDNA were positively correlated with mean PD, max PD, BOP, and mean PI (p < 0.0001), whereas plasma cfDNA was not correlated with BOP (p = 0.099). Conclusion: GCF, saliva, and plasma concentrations of cfDNA were significantly elevated in patients with periodontal disease. There were also positive correlations between cfDNA levels in GCF and saliva and periodontal parameters.

Uncovering Novel Prognostic Factors of Sudden Sensorineural Hearing Loss by Whole-Genome Sequencing of Cell-Free DNA.

Sudden sensorineural hearing loss is a common disease with several etiologic hypotheses, such as infection, vascular occlusion, inflammation, oxidative stress, etc. Studies have reported that the concentration of cell-free DNA in plasma will elevate in these situations. Former studies have reported that the whole-genome sequencing of cell-free DNA has high accuracy and sensitivity in inferring gene expressions. In this study, we plan to use the whole-genome sequencing of cell-free DNA to uncover novel prognostic factors of sudden sensorineural hearing loss and provide new insight into the clinical application of cell-free DNA. In this study, 84 sudden sensorineural hearing loss patients (47 in recovery group and 37 in no-recovery group) were enrolled. After whole-genome sequencing of the cell-free DNA, the protein-protein interaction network was constructed using the differentially expressed genes. Multinomial logistics regression analysis was used to analyze the prognostic factors of hearing improvement. In this study, we found distinct patterns of expressed and unexpressed genes in cell-free DNA sequence read depth coverage in sudden sensorineural hearing loss patients. The top centrality hub genes IGF1, NOTCH1, APOE, FAM3C, RPS6KB1, and RELB were identified from the protein-protein interaction network. Multinomial logistics regression analysis demonstrated that the coverage patterns of 3 key differentially expressed genes (NOTCH1, APOE, and RELB) are significantly different in sudden sensorineural hearing loss with and without hearing recovery. The cell-free DNA could have more applications in diverse diseases, and the coverage patterns of 3 differentially expressed genes (NOTCH1, APOE, and RELB) are independent prognostic factors of sudden sensorineural hearing loss. Their expression levels may play a critical role in the hearing improvement of sudden sensorineural hearing loss patients.

Plasma extracellular DNA and neutrophilic leukocyte activity in patients with rheumatoid arthritis

Neutrophilic leukocytes play a key role for the joint damage in development of rheumatoid arthritis (RA). The specific death mode of these cells (netosis) may be an important reason of increase of cell-free DNA (cfDNA) in peripheral blood of the RA patients. Of great interest would be studies of alleged relationships between the of blood cfDNA contents being able of playing the role of an auto-antigen participating in the initiation of autoimmune reactions, and indices of neutrophil activation in this immunopathological disorder. The aim of the present study was to determine the levels of cfDNA in blood plasma of patients with RA depending on the clinical course of the disease, and to evaluate possible relationships between this index and activation of neutrophilic leukocytes. The study was conducted on 28 conditionally healthy donors and 63 patients with RA from the Rheumatology Department at the Clinic of Immunopathology (Novosibirsk). The level of cfDNA was determined using PicoGreen fluorescent dye. Neutrophils from the peripheral blood of donors and patients with rheumatoid arthritis were isolated in a Ficoll-Urografin density gradient. Neutrophilic leukocytes accounted for more than 98% of the fraction of isolated cells, and their viability was 99%. A portion of freshly isolated neutrophils was stimulated by phorbol myristate acetate. Concentration of myeloperoxidase in blood plasma of donors and patients with RA was determined using the Human MPO ELISA kit. It has been shown that the increased concentration of extracellular DNA in blood plasma of RA patients correlates with an higher degree of disease activity, and this parameter may serve as a relatively independent indicator of the disease intensity. A correlation was found between the level of cfDNA and common biochemical markers used to assess the activity of disease, i.e., DAS-28 and C-reactive protein levels in serum (p 0.05). Decrease of cfDNA concentrations is detected during treatment of the RA patients. This is due to the expected prognosis, i.e., a decreased manifestation of the disease, which also means correct administration of therapy. A relationship was found between the level of cfDNA and blood myeloperoxidase concentration in RA patients. The data obtained during the study suggest a possible connection between increased concentration of extracellular DNA, and activation of neutrophilic leukocytes in rheumatoid arthritis, with increased netosis in the affected joints.

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer.

PurposeThis study aimed to analyze changes in the plasma concentration of EGFR-mutated circulating tumor DNA (ctDNA) occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs).MethodsSerial plasma samples were collected from 30 patients with EGFR-driven non-small cell lung cancer before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 h after the start of the therapy. The content of EGFR alleles (exon 19 deletions or L858R) in ctDNA was measured by ddPCR.ResultsctDNA was detected at base-line in 25/30 (83%) subjects. Twelve (50%) out of 24 informative patients showed > 25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8–12 weeks of therapy. The remaining 12 individuals showed either stable content of EGFR-mutated ctDNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8–12 weeks (p = 0.032, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies at 48 h also correlated with longer progression-free survival (14.7 months vs. 8.5 months, p = 0.013).ConclusionComparison of concentration of EGFR-mutated ctDNA at base-line and at 48 h after the start of therapy is predictive for the duration of TKI efficacy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10147-022-02128-6.

Sex Difference in Plasma Deoxyribonuclease Activity in Rats.

Extracellular DNA (ecDNA) activates immune cells and is involved in the pathogenesis of diseases associated with inflammation such as sepsis, rheumatoid arthritis or metabolic syndrome. DNA can be cleaved by deoxyribonucleases (DNases), some of which are secreted out of cells. The aim of this experiment was to describe plasma DNase activity in relation to extracellular DNA in adult rats, to analyse potential sex differences and to prove whether they are related to endogenous testosterone. Adult Lewis rats (n=28) of both sexes were included in the experiment. Male rats were gonadectomized or sham-operated and compared to intact female rats. Plasma ecDNA and DNase activity were measured using fluorometry and single radial enzyme diffusion assay, respectively. Concentrations of nuclear ecDNA and mitochondrial ecDNA were determined using real-time PCR. Females had 60% higher plasma DNase activity than males ( p=0.03). Gonadectomy did not affect plasma DNase in males. Neither the concentration of total ecDNA, nor nuclear or mitochondrial DNA in plasma differed between the groups. No significant correlations between DNase and ecDNA were found. From previous studies on mice, it was expected, that male rats will have higher DNase activity. In contrast, our study in rats showed the opposite sex difference. This sex difference seems not to be caused by endogenous testosterone. Interestingly, no sex differences were observed in plasma ecDNA suggesting a complex or missing association between plasma ecDNA and DNase. The observed sex difference in plasma DNase should be taken into account in animal models of ecDNA-associated diseases.

Sex Difference in Plasma Deoxyribonuclease Activity in Rats

Extracellular DNA (ecDNA) activates immune cells and is involved in the pathogenesis of diseases associated with inflammation such as sepsis, rheumatoid arthritis or metabolic syndrome. DNA can be cleaved by deoxyribonucleases (DNases), some of which are secreted out of cells. The aim of this experiment was to describe plasma DNase activity in relation to extracellular DNA in adult rats, to analyse potential sex differences and to prove whether they are related to endogenous testosterone. Adult Lewis rats (n=28) of both sexes were included in the experiment. Male rats were gonadectomized or sham-operated and compared to intact female rats. Plasma ecDNA and DNase activity were measured using fluorometry and single radial enzyme diffusion assay, respectively. Concentrations of nuclear ecDNA and mitochondrial ecDNA were determined using real-time PCR. Females had 60% higher plasma DNase activity than males (p=0.03). Gonadectomy did not affect plasma DNase in males. Neither the concentration of total ecDNA, nor nuclear or mitochondrial DNA in plasma differed between the groups. No significant correlations between DNase and ecDNA were found. From previous studies on mice, it was expected, that male rats will have higher DNase activity. In contrast, our study in rats showed the opposite sex difference. This sex difference seems not to be caused by endogenous testosterone. Interestingly, no sex differences were observed in plasma ecDNA suggesting a complex or missing association between plasma ecDNA and DNase. The observed sex difference in plasma DNase should be taken into account in animal models of ecDNA-associated diseases.

Markers of Epstein–Barr virus in clinical assessment of Russian patients with nasopharyngeal cancer

Introduction. Epstein–Barr virus (EBV) is equally widespread in the endemic and non-endemic world regions for nasopharyngeal cancer (NPC). High incidence of NPC in endemic countries and low in non-endemic countries suggest there are different mechanisms and conditions for tumor occurrence and, possibly, different clinical significance of EBV-associated markers. However, significance of these markers for determining NPC in non-endemic regions is still poorly understood. Objective – to determine clinical significance of titers of IgG/IgA antibodies to EBV capsid antigen and concentrations of the viral DNA in patients’ blood plasma as diagnostic and monitoring markers for NPC in a non-endemic region of Russia. Materials and methods. Titers of EB-specific antibodies were determined by indirect immunofluorescence, and concentration of the viral DNA in plasma was measured using a quantitative polymerase chain reaction in real time. Study group included patients with NPC (n = 96), and control group – blood donors (n = 171) and patients with other head and neck tumors (n = 33).Results. Titers of IgG/IgA antibodies to EBV capsid antigen, being an important diagnostic marker of nasopharyngeal cancer, did not always correlate with patients’ clinical condition. Humoral response to emerging events often delayed due to inertia of the immune system. Concentration of EBV DNA in patients’ blood plasma clearly reflected the dynamics of the pathological process: it decreased to background values in remission and increased while the disease progressed. In contrast to endemic regions, we did not find any correlation between the studied EBV markers and clinical manifestations of the disease, evaluated in accordance with the TNM classification (Tumor, Nodus and Metastasis).Conclusion. In non-endemic countries, such as Russia, serological and molecular markers of EBV can be successfully used for the primary diagnosis of NPC. However, for the disease monitoring, it is preferable to use the value of the concentrations of circulating EBV DNA, which, in contrast to the values of IgG/IgA antibody titers to VCA EBV, more accurately reflect the patient’s clinical condition.

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of anti-EGFR therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer

This study aimed to analyze changes in the plasma concentration of EGFR-mutated DNA occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs). The study included 30 patients with EGFR mutation-driven non-small cell lung cancer (NSCLC). Serial plasma samples were collected before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 hours after the start of the therapy. EGFR-mutated plasma DNA (EGFR+ ctDNA) was detectable at diagnosis in 25 out of 30 study participants. There were different patterns of changes of the amount of circulating tumor DNA, i.e., the consistent decline of ctDNA content, or continuing increase of the number of circulating EGFR mutant copies, or alternating spikes and drops in the ctDNA concentration. Correlation with the disease outcome was observed only for the measurement performed at 48 hours. Twelve (50%) out of 24 informative patients showed &gt;25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8-12 weeks of therapy. The remaining 12 individuals showed either stable content of circulating EGFR+ DNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8-12 weeks (p = 0.014, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies also correlated with longer progression-free survival (PFS; 14.7 months vs. 8.5 months, p = 0.013). Conclusion: Monitoring of plasma EGFR-M+ concentration within the first hours of the TKI therapy may be used as an immediate predictor of tumor response to the treatment.