Changes In cfDNA Research Articles

Distinct methylome profile of cfDNA in AMI patients reveals significant alteration in cAMP signaling pathway genes regulating cardiac muscle contraction

BackgroundThe role of epigenetics in cardiovascular diseases has paved the way for innovative therapeutic approaches. Investigating epigenetic changes using cell-free DNA (cfDNA) holds substantial promise beyond mere diagnostics, especially for heart-related conditions like acute myocardial infarction (AMI), where obtaining tissue samples is a challenge. This study explores the methylation patterns of cfDNA in AMI patients and compares them with genomic DNA (gDNA) from the same individuals, aiming to evaluate the effectiveness of cfDNA as a valuable resource for studying heart-related diseases.MethodologyWe generated global methylome profiles of cfDNA and gDNA from 25 AMI patients using EM-Seq. Tissue deconvolution analysis was performed to estimate tissue specificity based on the methylation patterns. Differentially methylated loci were identified and explored to understand AMI pathophysiology.ResultsComparative analysis of cfDNA and gDNA methylation patterns in AMI patients reveals cfDNA holds more significance than gDNA. Principal component analysis revealed distinct clusters for cfDNA and gDNA, indicating distinct methylome profiles. cfDNA originated from multiple sources, predominantly from neutrophils (~ 75%) and about 10% from the left atrium, highlighting cardiac-specific changes. In contrast, immune cells are the major source of gDNA, indicative of inflammatory responses. Gene set enrichment analysis (GSEA) associates cfDNA methylation patterns with pathways related to cardiac muscle contraction, inflammation, hypoxia, and lipid metabolism. The affected genes include G protein-coupled receptors (GHSR, FFAR2, HTR1A, and VIPR2) that are part of the cAMP signaling pathway.ConclusionEpigenetic changes in cfDNA are more specific to cardiac tissue compared to those in gDNA, providing better insights into the molecular mechanisms involved in AMI. Genes that are differentially methylated in cfDNA and regulate core pathways, such as cAMP signaling, could be targeted for clinical applications, including the development of effective biomarkers and therapeutic targets.

Quantitative Detection and Integrality Analysis of Plasma Circulating Cell-Free DNA in Multiple Myeloma

To investigate the role of plasma circulating cell-free DNA (cf-DNA) in the screening and diagnosis of patients with newly diagnosed multiple myeloma (MM) and explore the changes of cf-DNA in terms of content and integrality in the assessment of disease in patients treated with chemotherapy. Peripheral blood specimens were collected from 35 newly diagnosed MM patients and 18 healthy volunteers, and 13 of the 35 patients who had finished 3 courses of standard induction chemotherapy were selected as follow-up group. The ALU247 and ALU115 fragments were used as the target genes, and the cf-DNA content in the plasma of patients and healthy controls was measured by quantitative polymerase chain reaction (qPCR). The integrality of cf-DNA was calculated by the ratio of ALU247 to ALU115. Both the concentration of ALU247 and ALU115 fragments and the integrality of cf-DNA in patients were significantly higher than those in healthy controls (all P < 0.05). Patients who had underwent 3 courses of induction chemotherapy had significantly decreased concentration of ALU247 and ALU115 fragments and integrality of cf-DNA after treatment (all P < 0.05), and strong positive correlations were manifested between cf-DNA integrality and serum M protein content, as well as proportion of abnormal plasma cells in bone marrow before and after treatment (r =0.703, 0.705). Cf-DNA has certain positive significance for the screening and diagnosis of MM. Furthermore, cf-DNA may be a synergism or alternative to serum M-protein content and proportion of abnormal plasma cells in bone marrow in assessing the condition, curative effect and prognosis of patients.

A mathematical description of nonself for biallelic genetic systems in pregnancy, transfusion, and transplantation.

A central issue in immunology is the immunological response against nonself. The prerequisite for a specific immunological response is the exposure to the immune system of a nonself antigen. Mathematical equations are presented, which define the fraction of all outcomes with a nonself allele in biallelic systems at the population level in pregnancy and transfusion/transplantation medicine. When designing assays, the mathematical descriptions can be used for estimating the number of genetic markers necessary to obtain a predetermined probability level in detecting nonself alleles of a given frequency. For instance, the equations can be helpful in the design of assays, where the nonself allele can be detected by analysis of cfDNA in plasma from pregnant women, to estimate fetal fraction or to monitor changes in cfDNA in plasma of transplantation patients. The equations give exact, quantitative descriptions of all nonself situations in pregnancy and transfusion/transplantation.

Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Radiation Biological Toximetry Using Circulating Cell-Free DNA (cfDNA) for Rapid Radiation/Nuclear Triage.

Optimal triage biodosimetry would include risk stratification within minutes, and it would provide useful triage despite heterogeneous dosimetry, cytokine therapy, mixed radiation quality, race, and age. For regulatory approval, the U.S. Food and Drug Administration (FDA) Biodosimetry Guidance requires suitability for purpose and a validated species-independent mechanism. Circulating cell-free DNA (cfDNA) concentration assays may provide such triage information. To test this hypothesis, cfDNA concentrations were measured in unprocessed monkey plasma using a branched DNA (bDNA) technique with a laboratory developed test. The cfDNA levels, along with hematopoietic parameters, were measured over a 7-day period in Rhesus macaques receiving total body radiation doses ranging from 1 to 6.5 Gy. Low-dose irradiation (0-2 Gy) was easily distinguished from high-dose whole-body exposures (5.5 and 6.5 Gy). Fold changes in cfDNA in the monkey model were comparable to those measured in a bone marrow transplant patient receiving a supralethal radiation dose, suggesting that the lethal threshold of cfDNA concentrations may be similar across species. Average cfDNA levels were 50 ± 40 ng/mL [±1 standard deviation (SD)] pre-irradiation, 120 ± 13 ng/mL at 1 Gy; 242 ± 71 ng/mL at 2 Gy; 607 ± 54 at 5.5 Gy; and 1585 ± 351 at 6.5 Gy (±1 SD). There was an exponential increase in cfDNA concentration with radiation dose. Comparison of the monkey model with the mouse model and the Guskova model, developed using Chernobyl responder data, further demonstrated correlation across species, supporting a similar mechanism of action. The test is available commercially in a Clinical Laboratory Improvement Amendments (CLIA) ready form in the U.S. and the European Union. The remaining challenges include developing methods for further simplification of specimen processing and assay evaluation, as well as more accurate calibration of the triage category with cfDNA concentration cutoffs.

Abstract 981: Robustness of fragmentation-based cell-free DNA approaches to clonal hematopoiesis

Abstract INTRODUCTION: Clonal hematopoiesis (CH) is the result of the clonal expansion of hematopoietic stem cells which acquire and pass on somatically gained mutations, including SNVs as well as arm level losses and gains, defined here as mosaic deletions and amplifications (mDAs). The proportion of blood cells in circulation containing CH alterations increases with age and environmental exposures such as smoking, leading to a higher prevalence in many high-risk cancer screening populations. CH remains a major confounder for mutation-based liquid biopsy tests evaluating circulating cell-free DNA (cfDNA), since these assays are unable to discriminate between mutations originating from the tumor as opposed to CH. While the impact of CH mutations on fragmentation-based methods of detecting ctDNA appears to be limited, the effect of mDAs has not yet been explored. METHODS: To assess the impact of mDAs on screening efforts using whole genome sequencing (WGS) of cfDNA, we performed copy number analyses on cfDNA from plasma collected from 895 individuals consisting of high-risk individuals meeting the USPSTF guidelines for annual lung cancer screening using low-dose computed tomography. From a subset of 203 available donors (157 without cancer and 46 with lung cancer), matched genomic DNA from white blood cells (WBCs) were extracted from the isolated buffy coat and WGS was performed to evaluate whether ploidy in cfDNA plasma is derived from WBCs. Genome-wide fragmentation features (short to long ratios and fragment size densities), coverage-based statistics, and copy number (CN) statistics adjusting for mDAs were calculated (CNadj). Machine learning models to calculate the probability of cancer based on these features were then derived. RESULTS: 3.8% (95% CI 0.9%-6.8%) of individuals without cancer had an mDA in their WBCs that were also observed in the plasma (p &amp;lt; 0.01, permutation test). Fragmentation features were not different in regions with mDAs in individuals without cancer (p = 0.619; permutation test) but were altered in regions of tumor-derived chromosomal changes in patients with cancer (p &amp;lt; 0.01; permutation test). Coverage-based statistics were impacted by mDAs (p &amp;lt; 0.01; Wilcoxon test). Machine learning models fit with CNadj showed improved performance over CN. However, machine learning models fit with fragmentation features compensated for this difference overall showing robustness to mDAs (p = 0.45; bootstrap) demonstrating the robustness of cfDNA fragmentomes for detecting lung cancer. CONCLUSIONS: While coverage-based statistics of WGS analyses of cfDNA may be affected by mDAs, fragmentation-based approaches relying on cfDNA fragment length are robust to effects of CH. CNadj is an improved statistic for detecting tumor-derived chromosomal copy number changes in cfDNA, providing increased specificity by conditioning on fragment size distributions. Citation Format: Stephen Cristiano, Bahar Alipanahi, Jamie E. Medina, Lorenzo Rinaldi, Nicholas Dracopoli, Robert B. Scharpf, Alessandro Leal, Victor E. Velculescu, Jennifer Tom. Robustness of fragmentation-based cell-free DNA approaches to clonal hematopoiesis [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 981.

Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection.

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

Diagnostic Utility of Next-Generation Sequencing in Circulating Free DNA and a Comparison With Matched Tissue in Gallbladder Carcinoma

Mutation detection for therapy monitoring in cell-free DNA (cfDNA) is used clinically for some malignancies. Gallbladder carcinoma (GBC) presents a diagnostic challenge and has limited late-stage treatment options. To our knowledge, this novel study examines, for the first time, genomic alterations in cfDNA from GBC to assess diagnostic accuracy and therapeutic options. The concordance of somatic genomic changes in cfDNA and DNA from paired tumor tissue was analyzed. Paired serum and tissue samples from 40 histologically proven GBC, 20 cholecystitis, and 4 normal (noninflamed gallbladder) controls were included. Targeted next-generation sequencing with a 22-gene panel (Colon and Lung Cancer Research Panel v2, Thermo Scientific) in cfDNA and tumor tissue with high depth and uniform coverage on ION Personal Genome Machine (ION, PGM) was performed. A spectrum of 223 mutations in cfDNA and 225 mutations in formalin-fixed paraffin-embedded tissue DNA were identified in 22 genes. Mutations ranged from 1 to 17 per case. In cfDNA frequent alterations were in TP53 (85.0%), EGFR (52.5%), MET (35%) CTNNB1, SMAD4, BRAF (32.5%), PTEN (30%), FGFR3 and PIK3CA (27.5%), NOTCH1 (25.0%), and FBXW7 and ERBB4 (22.5%). At least one clinically actionable mutation was identified in all cfDNA samples. Paired samples shared 149 of 225 genetic abnormalities (66.2%). Individual gene mutation concordance ranged from 44.44% to 82.0% and was highest for EGFR (82.0%), BRAF and NOTCH1 (80.0%), TP53 (73.08%), MET (72.22%), and ERBB4 (71.42%) with a significant level of correlation (Spearman r = 0.91, P ≤ .0001). The sensitivity and specificity of the TP53 gene at the gene level was the highest (94.44% and 100.0%, respectively). Overall survival was higher for ERBB4 and ERBB2 mutant tumors. The adenocarcinoma subtype revealed specific genetic changes in ERBB4, SMAD4, ERBB2, PTEN, KRAS, and NRAS. NGS-based cfDNA mutation profiling can be used to diagnose GBC before surgery to guide treatment decisions. Targeted therapy identified in GBC included SMAD4, ERBB2, ERBB4, EGFR, KRAS, BRAF, PIK3CA, MET, and NRAS.

Multi-Cancer Early Detection Test Is Sensitive and Accurate in Detecting Shared DNA Methylation Signal in a Variety of Lymphoid and Plasma Cell Neoplasms

Background: Recent technological advances in molecular diagnostics have facilitated development of blood-based multi-cancer early detection (MCED) tests intended to complement standard of care single-cancer screening. Implementing MCED at population scale has the potential to improve survival for patients with lymphoid neoplasms (LyN) and plasma cell neoplasms (PCN). The Galleri test, launched in 2021, is the first commercially available liquid biopsy MCED test in the United States that has been validated.Galleri is a genome-wide targeted methylation sequencing test that detects cancer-related DNA methylation changes in cfDNA that are shared by many cancers. By targeting 100K fragments in the genome and ~1 million CpGs, processed by a machine learning classification algorithm, Galleri is able to not only detect the presence of a cancer signal, but also to identify the most likely origin of the cancer signal (CSO) in a tissue or organ. Here we report specific performance of Galleri in LyN and PCN, reported as either lymphoid or plasma cell CSO from two large clinical studies and real-world implementation (RWE) among 100,000 individuals. Methods: The case-control (CCGA3) Circulating Cell-free Genome Atlas study (NCT02889978) included 2,823 cancer participants (cases, pre-treatment) and 1,254 non-cancer participants (controls), the prospective interventional PATHFINDER study (NCT04241796), included 6,578 asymptomatic individuals aged ≥50 and received an earlier version of MCED to assess the feasibility of implementing MCED testing. With more than 100,000 results returned to providers across the US to date, we also report early data from systematic collection of outcomes for cases with a Cancer Signal Detected (CSD) result via a controlled quality assurance (QA) program to monitor MCED testing in a real-world population. Results: In CCGA3, the overall sensitivity of Galleri in LyN was 52.9% (119/225; 95% CI 46.4%, 59.3%); the overall sensitivity in PCN was 72.3% (34/47; 95% CI 58.2%, 83.1%) ( Table 1). Galleri can detect a shared cancer signal from a variety of lymphoid and plasmacytic neoplasms, including Hodgkin lymphomas (HL) and non-Hodgkin lymphomas; B- and T-cell neoplasms; from mostly indolent follicular lymphoma (FL) to aggressive Diffuse Large B-Cell Lymphomas (DLBCL) and Multiple Myeloma ( Table 1). Sensitivity was higher in more aggressive lymphomas, like DLBCL (70.5%). Galleri demonstrated excellent performance in hematologic cancers predicting lymphoid CSO with 99% accuracy and plasma cell CSO with 100% accuracy in true cancer cases. Galleri is intended to detect bona fide cancers and not preneoplastic conditions (ex. MGUS, MBL, CHIP). The sensitivity increased from stage I to stage IV, likely reflecting higher tumor burden and tumor fraction in cfDNA ( Table 2). The sensitivity for early combined stage I and II cancers, which are more responsive to treatments, was 45.7%. In PATHFINDER, using an updated commercial version of the test, there were 11 cases with CSD result and a top CSO prediction consistent with a hematologic malignancy: 7 lymphoid and 4 plasma cell neoplasia. A total of 7/11 (63.6%) participants had hematologic malignancies diagnosed: 6 lymphomas (stages I-IV) and 1 PCN. Galleri demonstrated a high PPV of 54.5% for cases with predicted CSO either LyN or PCN, and 71.4% for cases with predicted CSO Lymphoma. Early clinical outcomes data from a controlled QA quality assurance (QA) program collected from an initial limited subset of CSD cases with follow up information showed that, similar to our clinical trial data, this test is able to detect a shared cancer signal in a variety of lymphomas (chronic lymphocytic leukemia [CLL], DLBCL, FL, PTCL, HL), some in early stages, such as HL stage I. More detail on the MCED real-world experience will be presented at the meeting. Conclusions: Findings from two large validation studies and RWE demonstrate that Galleri promises to be a state of the art molecular tool that detects a shared cancer signal and is able to identify a large variety of histopathologic entities of lymphoid and plasmacytic origin, which currently have no other standard of care screening regimen. This tool may lead to better treatment outcomes and decreased mortality saving lives through the early detection of these previously unscreened cancers.

Changes in cell-free DNA after short-term palbociclib and fulvestrant treatment for advanced or metastatic hormone receptor-positive and human epidermal growth factor 2-negative breast cancer.

Here, we investigated the potential predictive and elucidating efficacy of cell-free DNA (cfDNA) changes on clinical outcomes and biological effects, respectively, after short-term palbociclib and fulvestrant treatment for patients with hormone receptor (HR)-positive and human epidermal growth factor 2 (HER2)-negative advanced or metastatic breast cancer (ABC). In this secondary analysis of the Japan Breast Cancer Research Group-M07 (FUTURE) trial, blood cfDNA was obtained before palbociclib treatment and on day 15 of cycle one (28-day cycle). Target enrichment was performed using next-generation sequencing; progression-free survival (PFS) was compared based on cfDNA changes between baseline and day 15 of cycle one after combination therapy. Fifty-six patients (112 paired blood samples) were examined. The median follow-up time was 8.9months. PIK3CA (30.4%, 17/56), FOXA1 (30.4%, 17/56), and ESR1 (28.6%, 16/56) were most frequently mutated at baseline. The number of mutated genes was significantly decreased on day 15 compared with that at baseline (paired t test: P value = 0.025). No significant difference was observed in PFS (decrease group, 7.9m vs the others, 9.3m; log-rank P value = 0.75; hazard ratio, 1.13; 95% confidence interval, 0.53-2.41). Among patients without previous aromatase inhibitor treatment (n = 15), three (20%) had ESR1 mutations after progression to fulvestrant. No significant association was observed between changes in mutated genes after short-term palbociclib and fulvestrant treatment and disease progression; a significant reduction in cfDNA mutation level was observed on day 15 of cycle one. Clinical meanings of cfDNA should be investigated in the future trials.

Cell-free DNA as a plausible biomarker of chronic kidney disease.

Circulating cell-free DNA (cf-DNA) is released from dead and/or apoptotic leukocytes and due to neutrophil extracellular traps contributing to an inflammatory response. Previous clinical studies have reported that the peak concentrations and dynamic changes of cf-DNA may be used asanoninvasive biomarker of worsening kidney function as well asa guide to themanagement of kidney allograft rejection. We hypothesized that the pattern and dynamic changes of cf-DNA might be aplausible predictive biomarker for patients at risk of chronic kidney disease (CKD), including individuals with type 2 diabetes mellitus, heart failure, cardiovascular diseaseand established CKD. Along with it, pre- and posthemodialysis levels of serum cf-DNA appearto be aindependent predictor for all-cause mortality in patients with end-stage kidney disease.

Phase 1 Study of Acalabrutinib Plus Danvatirsen (AZD9150) in Relapsed/Refractory Diffuse Large B-Cell Lymphoma Including Circulating Tumor DNA Biomarker Assessment.

Novel targeted and immunotherapies have improved outcomes in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), but toxicities limit widespread use. The selective Bruton tyrosine kinase (BTK) inhibitor acalabrutinib has activity in patients with R/R DLBCL, but durable responses are uncommon. STAT3 inhibition has demonstrated clinical activity in DLBCL. Final results of the Phase 1 study of acalabrutinib plus STAT3 inhibitor (danvatirsen; AZD9150) in patients with R/R DLBCL are reported. Danvatirsen 200 mg intravenous infusion (Days 1, 3, 5 [Cycle 1]; weekly infusions starting Day 8, Cycle 1) was administered in combination with oral acalabrutinib 100 mg twice daily until progressive disease (PD) or unacceptable toxicity. Primary endpoints were safety and tolerability. Secondary endpoints included efficacy, pharmacokinetics, and immunogenicity. Seventeen patients received combination treatment. One dose-limiting toxicity (Grade 3 liver transaminase) occurred in one patient. The most common reason for treatment discontinuation was PD (65%). In evaluable patients (n=17), objective response rate was 24%; median duration of response was 1.9 months. All responders with available DLBCL cell-of-origin data were either activated B-cell or nongerminal center B-cell like subtype. Genetic subtype did not correlate with response. Baseline and longitudinal plasma cell-free DNA (cfDNA) concentrations were mostly higher in nonresponding patients. cfDNA changes were generally concordant with imaging. Pretreatment circulating B-cell levels were higher in responders versus nonresponders. Targeting both STAT3 and BTK in combination is safe and tolerable but efficacy is limited in R/R DLBCL. Results support evaluation of circulating tumor DNA as a biomarker for clinical response.

Abstract P1-05-27: Liquid Biopsy for HER2 Status Assessment in Breast Cancer Using Surrogate DNA Methylation Markers

Abstract Background: Emerging HER2-targeted drugs especially antibody–drug conjugates (ADCs) are promising and provide more options for breast cancer management. Current assessment of HER2 status and treatment decisions are mainly dependent on immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on the primary tumor tissues. With the disease progression, the molecular status of the tumor may evolve and become discordant with the primary site. However, longitudinal monitoring of HER2 status is limited by infeasible repeated sampling of the tumor tissues. A non-invasive and accurate approach to obtaining real-time samples for measuring HER2 alterations is thus an unmet need for surveillance and guiding treatment selection in breast cancer. Detecting HER2 aberration in cell-free DNA (cfDNA) can allow repeated sampling and avoid effects from tumor heterogeneity of tissue biopsy. Previous approaches for HER2 status determination using liquid biopsy were mostly dependent on the detection of copy number changes in cfDNA, but the limited signal-to-noise ratio poses a great challenge to the accuracy and robustness of the tests. In this study, we identified a group of DNA methylation markers for determining HER2 status in cfDNA for breast cancer. Methods: Genome-wide DNA methylation sequencing was conducted in tissue (25 HER2-positive and 35 HER2-negative) and plasma (32 HER2-positive and 107 HER2-negative) samples to identify specific methylation markers for HER2 status. HER2-positive samples were defined by IHC 3+ and 2+ with FISH positive, while HER2-negative ones were IHC 0/1+ and 2+ with FISH negative. Candidate markers were verified in another two sets of plasma samples (1. 30 HER2-positive and 40 HER2-negative; 2. 33 HER2-positive and 53 HER2-negative) by using quantitative methylation-specific PCR (qMSP). The performance of the markers was estimated by the Wilcoxon test, receiver operating characteristic curve, and logistic regression modelling. Results: 36 HER2 status-specific markers were discovered from genome-wide DNA methylation sequencing. Based on the qMSP results, 11 markers were verified by the performance analyses. The individual area under the curve (AUC) of these markers was from 0.58 to 0.68. From logistic regression modelling and 2-fold cross-validation, a 7-marker diagnostic model was built and validated on plasma samples, with the highest AUC of 0.812. Conclusion: cfDNA methylation detection inferring HER2 overexpression is a novel and non-invasive option for monitoring HER2 status in breast cancer patients, with a potential application in response prediction of HER2-targeted treatments. Further validation of the test is undergoing in large multi-centre cohorts in China. Citation Format: Xianyu Zhang, Shiyao Lu, Hui Li, Xin Liu, Jun Wang, Liuhong Zeng, Zhipeng Lu, Siyu Liu, Yanling Yin, Marina Bibikova, Zhiwei Chen, Jian-Bing Fan, Da Pang. Liquid Biopsy for HER2 Status Assessment in Breast Cancer Using Surrogate DNA Methylation Markers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-27.

Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia

BackgroundSince the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA.Methods and ResultsNewborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40–45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for β-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group.ConclusionsBoth, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses.

Circulating DNA in the neoadjuvant setting of early stage colon cancer

Background While circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III colon cancer, no study has ever analysed the value of this biomarker in colon cancer patients treated with neoadjuvant chemotherapy. We sought to fill this gap by using prospectively collected plasma samples from 80 stage III colon cancer patients, receiving one cycle of neoadjuvant FOLFOX followed by surgery +/− adjuvant FOLFOX in the PePiTA trial. Material and Methods Samples were collected at baseline, 2 weeks and surgery. NPY and WIF1 were selected as universal methylation markers for ctDNA, and analysed with ddPCR technology. ROC curves were applied for cut-off points, and outcome measures included 5-year disease-free survival (DFS) and 6-year overall survival (OS). Results After a median follow-up of 52.5 months, baseline circulating-free (cf) DNA was an independent prognostic factor for DFS (HR 3.35, 95% CI: 1.15–9.77, p = .03), and a trend towards a similar association was observed for relative cfDNA changes between baseline and surgery (HR 2.57, 95% CI: 0.94–7.05, p = .07). Among 60 ctDNA assessable patients, 25 (42%) had detectable ctDNA at baseline. While detection of ctDNA at any pre-operative timepoint was not associated with outcome, patients with ctDNA increase (change of the worst trending methylation marker ≥11%, or mean ctDNA change of NPY and WIF1 ≥ 0%) between baseline and surgery showed a trend towards worse 5-year DFS (HR 3.66, 95% CI: 0.81–16.44, p = .09). Conclusion This is the first study of ctDNA in the neoadjuvant setting of early-stage colon cancer. Results are hypothesis-generating and should be confirmed in larger series.

Abstract 5152: Mutation-agnostic detection of colorectal cancer-specific cell-free DNA using targeted methylation sequencing

Abstract BACKGROUND: Tumor-specific methylation changes in DNA CpG sites commonly occur in cancer and are believed to drive oncogenesis through gene silencing. Detection of methylation changes in circulating cell-free DNA (cfDNA) can offer a novel approach for cancer diagnostics. METHODS: Plasma samples from healthy controls and from patients with advanced colorectal and non-colorectal cancers were included in the study. Bisulfite conversion of cfDNA extracted from plasma was performed using EZ DNA Methylation Lightning Kit (Zymo Research) and was followed by library preparation using Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) and target enrichment using xGen Hybridization Capture for NGS Kit (IDT). Targeted methylation sequencing was done using NextSeq500 mid-output flow cell (300 cycles) (Illumina). Detection rates of methylation ratios in colorectal cancer samples were compared to non-colorectal cancers and healthy controls. RESULTS: First, we reviewed methylation changes in nearly 9,000 CpG sites in colorectal cancer (through TCGA database) and healthy controls. Subsequently, 32 CpG sites with greater than 50% methylation ratio in colorectal cancer and less than 1% methylation ratio in healthy controls were selected to develop targeted methylation sequencing based cfDNA assay. The assay was performed in 32 plasma samples from 20 individuals with advanced colorectal cancer who had tumor KRAS mutation, 8 individuals with advanced non-colorectal cancer who had tumor KRAS mutation (ovarian, n=2; endometrial, n=2; pancreatic, n=2; and lung cancer, n=2), and 4 healthy controls. Colorectal cancer specific methylation changes in cfDNA were detected in 85% (17/20) of colorectal cancer patients with a specificity of 92%. In colorectal cancer patients with confirmed KRAS mutation in cfDNA, methylation changes were detected in 92% (11/12) in comparison to 75% (6/8) in colorectal cancer patients without KRAS mutation in cfDNA. Median methylation ratio for target CpG sites was higher in colorectal cancer patients compared to patients with non-colorectal cancers and healthy controls (p&amp;lt;0.001). In 17 colorectal cancer patients with plasma samples collected before initiation of systemic cancer therapy, detection of methylation changes in cfDNA was associated with a shorter median progression-free survival compared to no detection (PFS; 8 weeks versus 54 weeks; p=0.027). CONCLUSIONS: Targeted methylation sequencing of cfDNA demonstrated high sensitivity and specificity for detection of colorectal cancer-specific cfDNA. Colorectal cancer patients with methylated cfDNA had shorter PFS while on cancer therapy. Citation Format: Mohamed A. Gouda, Dzifa Y. Duose, Morten Lapin, Stephanie Zalles, Helen J. Huang, Yuanxin Xi, Xiaofeng Zheng, Amira I. Aldesoky, Alshimaa M. Alhanafy, Mohamed A. Shehata, Jing Wang, Scott Kopetz, Funda Meric-Bernstam, Ignacio I. Wistuba, Rajyalakshmi Luthra, Filip Janku. Mutation-agnostic detection of colorectal cancer-specific cell-free DNA using targeted methylation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5152.

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Monitoring early dynamic changes of plasma cell-free DNA and pretreatment pre-albumin to predict chemotherapy effectiveness and survival outcomes in advanced non-small cell lung cancer.

BackgroundThis study aimed to determine whether plasma cell-free DNA (cfDNA) and pretreatment parameters provide useful therapeutic response and prognostic information for advanced non-small cell lung cancer (NSCLC) patients.MethodsA total of 114 patients with advanced NSCLC who underwent systemic chemotherapy were included in this study. Detection of plasma cfDNA concentration and blood parameters before and at the sixth week after treatment was performed. The prognostic value of cfDNA dynamic changes and laboratory parameters was determined via a receiver operating characteristic (ROC) curve, and then analyzed by comparing with the therapeutic efficacy and progression-free survival (PFS). Based on the ROC curve, it revealed a pretreatment pre-albumin (PA) concentration of 21.7 mg/dL was the cut-off value. The Cox proportional hazards regression model was used to evaluate the predictive factors for treatment response and PFS via univariate and multivariate analyses.ResultsPatients with cfDNA reduction ≥20% at the sixth week after treatment reported a significantly better disease control rate (DCR) and prolonged PFS (median PFS: 10.0 vs. 4.0 months, P<0.001). The median PFS of low PA group (PA <21.7 mg/dL) was 6.0 months, while the median PFS of high PA group (PA ≥21.7 mg/dL) was 8.0 months. The combined assessment of cfDNA and pretreatment pre-albumin was associated with significantly better survival outcomes compared with the remaining population (P<0.001). Multivariate analysis for DCR indicated that cfDNA reduction ≥20% was an independent factor (OR =0.419, P=0.001). In addition, multivariate analysis identified 6 significant factors associated with PFS: cfDNA reduction of ≥20%, age <65 years, Eastern Cooperative Oncology Group (ECOG) score ≥2, driver gene mutation, chemotherapy combined regimen, and treatment response of complete response (CR) and partial response (PR). The nomogram could predict the 2-year PFS probability of advanced NSCLC patients after treatment, and the C-index was 0.817.ConclusionsMonitoring cfDNA changes and pretreatment pre-albumin level in advanced NSCLC patients receiving treatment is an accurate predictor of tumor response and PFS. Combined assessment of cfDNA and pretreatment pre-albumin is helpful for predicting survival outcomes. These findings may assist in identifying high-risk patients and guiding treatment strategies.

CfDNA Changes in Maximal Exercises as a Sport Adaptation Predictor.

Changes of circulating free plasma DNA (cfDNA) are associated with different types of tissue injury, including those induced by intensive aerobic and anaerobic exercises. Observed changes are dependent from induced inflammation, and thus it may be a potential marker for athletic overtraining. We aimed to identify the response of cfDNA to different types of exercise, with association to exercise intensity as a potential marker of exercise load. Fifty volunteers (25 athletes and 25 physically active men) were assigned to the study and performed maximal aerobic (Bruce test) and anaerobic (Wingate Anaerobic Test) test. Blood samples for cfDNA analysis were collected at four time-points: before, 2–5 min after, 30 min after and 60 min after each type of maximal physical activity. The two-way ANOVA revealed a significant effect of group factor on serum cfDNA concentrations (32.15% higher concentration of cfDNA in the athletes). In turn the results of the post hoc test for the interaction of the repeated measures factor and the group showed that while the concentration of cfDNA decreased by 40.10% in the period from 30 min to 60 min after exercise in the control group, the concentration of cfDNA in the group of athletes remained at a similar level. Our analysis presents different responses depending on the intensity and duration of exercise. Our observations imply that formation of cfDNA is associated with response to physical activity but only during maximal effort.

The Potential Use of Dynamics Changes of ctDNA and cfDNA in the Perioperative Period to Predict the Recurrence Risk in Early NSCLC.

BackgroundPostoperative circulation tumor DNA (ctDNA) is a promising method to predict the risk of recurrence. However, the amount of ctDNA in patients with early NSCLC is too small. Cell damages caused during the intraoperative period leads to a significant increase in cell free DNA (cfDNA). Whether cfDNA content is restored to the preoperative level within a short time after surgery may indicate the degree of surgical trauma. In this study, dynamic changes of cfDNA combined with ctDNA in the perioperative period of NSCLC were used to explore the possibility of them as a biomarker to indicate the risk of recurrence.MethodsNSCLC patients who planned to undergo radical resection were investigated. 10ml of peripheral blood was collected before, during and 7 days after surgery. DNA concentration was measured, and a 23-gene NGS panel was performed to detect gene mutations. All the patients would be followed-up for at least 18 months.ResultsA total of 7 patients were sampled. The amount of cfDNA before surgery was 36.6 ± 14.7ng, and increased to 127.2 ± 52.2ng during surgery. 7 days after surgery, it dropped to 45.23 ± 9.41ng in 3 patients and rose to 173.7 ± 80.80ng in the remaining 4. Only 1 patient was ctDNA positive after surgery, with decreasing cfDNA, and he was the only one that relapsed and died within 18 months.ConclusionThe use of ctDNA to predict the risk of postoperative recurrence of NSCLC is a very valuable method, and it may be more reliable if combined with the dynamic changes of cfDNA. The amounts of cfDNA are raised by the operation, but will be polarized after surgery in 7 days. Postoperative NSCLC patients with positive ctDNA and reduced cfDNA have a higher risk of recurrence.