Total Cell-free DNA Research Articles

Abstract 5876: ctDNA as predictor for systemic therapy response in patients with colorectal cancer: A systematic review and meta-analysis

Abstract Background: The response to systemic therapy against metastatic colorectal cancer (mCRC) is currently assessed by radiologic imaging. However, an increasing number of studies have shown that circulating tumor DNA (ctDNA) as liquid biopsy can be used as an alternative method to assess therapy efficacy. We conducted a systematic review with subsequent meta-analysis of primary studies to assess the predictive value of sequential liquid biopsies in patients with colorectal cancer treated with systemic therapy. Methods: Randomized, non-randomized, as well as prospective observational studies, reporting on the change in ctDNA concentration in total cell-free DNA over the course of systemic therapy of patients treated for metastatic colorectal cancer to predict treatment response according to RECIST criteria were included. Results: Forty-one studies involving 4546 evaluable patients with metastatic colorectal cancer were included in the meta-analysis. ctDNA increase during systemic therapy as compared to baseline was significantly associated with progression-free survival (HR: 2.47, 95%CI: 1.97-3.10) and overall survival (HR: 2.18, 95%CI: 1.66-2.86), which were reported in 31 and 22 studies, respectively. Conclusion: Longitudinal plasma-based ctDNA liquid biopsy has prognostic value and may help to identify those patients that benefit of continuing therapy or therapy switch. Although further standardization and randomized controlled clinical trials are required, based on the available data, we recommend longitudinal ctDNA measurements to be included in clinical practice and inclusion in the RECIST criteria. Protocol registration: The protocol for this review was registered on PROSPERO (CRD42023420012). Citation Format: Anja Holz, Bidisha Paul, Antonia Zapf, Klaus Pantel, Simon A. Joosse. ctDNA as predictor for systemic therapy response in patients with colorectal cancer: A systematic review and meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5876.

Abstract 5890: Personalized detection of circulating cell-free tumor DNA in gynecologic cancer

Abstract Endometrial and ovarian cancers are among the most common gynecologic malignancies in the U.S., with 5-year overall survival rates of 81% and 50% respectively. Ovarian cancer often presents late due to asymptomatic early stages and nonspecific symptoms in advanced stages. Few markers exist for early detection, and they lack the sensitivity and specificity needed for high-risk screening. Given the absence of informative protein blood biomarkers, cell-free DNA analysis has become a promising tool for early detection, treatment evaluation, and monitoring. However, previous studies of circulating tumor DNA (ctDNA) in gynecological cancers had limited sensitivity. We adapted MASQ (Multiplex Accurate Sensitive Quantitation), originally developed to measure residual cells in leukemia, to the measurement of ctDNA in solid cancers. MASQ detects patient-specific mutations simultaneously in up to 50 loci with exceptional sensitivity, below one part per million. This study uses MASQ to measure tumor-specific variants in blood (cell-free DNA) from endometrial and ovarian cancer patients, aiming to determine the range of ctDNA signals at presentation and explore correlations with clinicopathological features and patient outcomes. We analyzed samples from 46 gynecologic cancer patients (12 ovarian, 34 endometrial) treated at Northwell Health. Samples were collected in collaboration with gynecologic oncology research coordinators and the Northwell Health Biorepository. Peripheral blood was collected at diagnosis (n=46) and post-surgery (n=14), along with surgical tumor specimens. Whole genome sequencing of tumor and blood identified thousands of somatic tumor variants per patient. For each patient, 30 representative variants were selected for MASQ sequencing. MASQ libraries were generated from cell-free DNA isolated from pre- and post-surgery plasma. Ultrasensitive variant counts across 30 loci were aggregated and analyzed for clinical correlations. ctDNA signal was detected in 35 of 46 cases at presentation, with variant allele frequencies ranging from below 10^-4 to 10^-1. ctDNA signal was higher in ovarian cancer (13/13 detected) than endometrial cancer, and higher in aggressive Type II endometrial cancer (15/16) vs. Type I (5/13). ctDNA levels correlated with tumor size and copy number imbalance, but not with total cell-free DNA. Detection of ctDNA predicted worse overall survival (p=0.023) and was the strongest predictor of survival in multivariate analysis. Post-surgery follow-up in 10 of 14 patients showed significant ctDNA reduction following treatment, highlighting MASQ’s sensitivity in tracking treatment response. This study highlights the potential of sensitive ctDNA detection for early diagnosis and monitoring in gynecologic cancers. However, limited cell-free DNA abundance necessitates even more sensitive methods to enhance clinical utility. Citation Format: Andrea B. Moffitt, Jude Kendall, Joan Alexander, Asya Stepansky, Arisa Kapedani, Zihua Wang, Dan Levy, Kenny Ye, Abba M. Krieger, Marina Frimer, Gary L. Goldberg, Michael Wigler. Personalized detection of circulating cell-free tumor DNA in gynecologic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5890.

High tissue specificity of lncRNAs maximises the prediction of tissue of origin of circulating DNA

Several studies have made it possible to envision a translational application of plasma DNA sequencing in cancer diagnosis and monitoring. However, the extremely low concentration of circulating tumour DNA (ctDNA) fragments among the total cell-free DNA (cfDNA) remains a formidable challenge to overcome and statistical models have yet to be improved enough to become of practical use. In this study, we set about appraising the predictive value of a variety of binary classification models based on cfDNA sequencing using fragmentation features extracted around transcription start sites (TSSs). We investigated (1) features summarising mapped fragment density around each TSS, (2) long non-coding RNA (lncRNA) genes versus coding genes and (3) selection criteria to generate gene classes to be assigned by the model. Given that, in healthy samples, most of the cfDNA comes from lymphomyeloid lineages, we could identify the model parametrisation with the best accuracy in those lineages using publicly available datasets of healthy patients’ cfDNA. Our results show that (1) the way tissue-specific gene classes are defined matters more than what fragmentation features are included, and (2) in particular, lncRNAs are more tissue specific than coding genes and stand out in terms of both sensitivity and specificity in our results.

A large-scale retrospective study in metastatic breast cancer patients using circulating tumour DNA and machine learning to predict treatment outcome and progression-free survival.

Monitoring levels of circulating tumour-derived DNA (ctDNA) provides both a noninvasive snapshot of tumour burden and also potentially clonal evolution. Here, we describe how applying a novel statistical model to serial ctDNA measurements from shallow whole genome sequencing (sWGS) in metastatic breast cancer patients produces a rapid and inexpensive predictive assessment of treatment response and progression-free survival. A cohort of 149 patients had DNA extracted from serial plasma samples (total 1013, mean samples per patient = 6.80). Plasma DNA was assessed using sWGS and the tumour fraction in total cell-free DNA estimated using ichorCNA. This approach was compared with ctDNA targeted sequencing and serial CA15-3 measurements. We identified a transition point of 7% estimated tumour fraction to stratify patients into different categories of progression risk using ichorCNA estimates and a time-dependent Cox Proportional Hazards model and validated it across different breast cancer subtypes and treatments, outperforming the alternative methods. We used the longitudinal ichorCNA values to develop a Bayesian learning model to predict subsequent treatment response with a sensitivity of 0.75 and a specificity of 0.66. In patients with metastatic breast cancer, a strategy of sWGS of ctDNA with longitudinal tracking of tumour fraction provides real-time information on treatment response. These results encourage a prospective large-scale clinical trial to evaluate the clinical benefit of early treatment changes based on ctDNA levels.

The relationship between clinical-anamnestic data and cell-free fetal DNA level assessed by semiconductor sequencing within non-invasive prenatal testing

Introduction. Currently, non-invasive prenatal testing (NIPT) is widely used to assess a risk of fetal chromosomal anomalies. NIPТ accuracy depends on the cell-free fetal DNA (cffDNA) percentage relative to total cell-free DNA in the pregnant woman's blood (cfDNA fetal fraction, FF). Despite numerous studies, no consensus regarding FF-affecting factors has been reached yet.Aim: to investigate a relationship between FF and clinical-anamnestic parameters of pregnant women, pregnancy characteristics, and outcomes using the developed NIPТ technology.Materials and Methods. A prospective observational study was performed by assessing plasma samples from 5459 women with > 9 week-long singleton pregnancies. NIPТ was performed using semiconductor sequencing followed by bioinformatics data processing, including FF determination, according to a previously developed original algorithm.Results. Median FF was 11.7 [9.5–14.0] %. It was demonstrated that FF depends on blood collection tube type (p < 0.05). FF was found to decrease with woman age and body mass index, and increase with gestational age, elevated early prenatal screening (EPS) biochemical markers – pregnancy-associated plasma protein-A (РАРР-А) and free beta-subunit of human chorionic gonadotropin (β-hCG) levels (p < 0.05). It has been shown that the FF in pregnant women with trisomy 18 is lower than normal (p < 0.05). An increase in FF was observed in pregnant women with fetal congenital anomalies according to ultrasound results (p < 0.05). No association was found between FF and the conception type, first-trimester ultrasound parameters (nuchal translucency, crown-rump length, ultrasound chromosome anomalies markers), fetal trisomy 13 and 21, fetal sex chromosome anomalies, or pregnancy complications – preeclampsia, gestational diabetes, preterm birth, and fetal growth restriction (p > 0.05).Conclusion. The identified patterns are important to take into consideration while using and interpreting NIPТ.

Clinical Utility of Serial Circulating Tumor DNA Analysis as a Minimally Invasive Biomarker in Advanced Urothelial Cancer

PURPOSE Circulating tumor DNA (ctDNA) analysis is an alternative to tissue biopsy for genotyping in various cancers. We aimed to establish a plasma ctDNA sequencing assay, then evaluate its clinical utility in advanced urothelial cancer (UC). MATERIALS AND METHODS This study included 82 patients with muscle-invasive or metastatic UC. A total of 158 blood samples and 73 tumor tissue samples were sequenced using our designed panel covering 53 UC-relevant genes and TERT promoter. We investigated the association between the proportion of ctDNA in total cell-free DNA (ctDNA fraction) and response to pembrolizumab treatment. ctDNA dynamics were explored during systemic therapy. RESULTS In paired tissue-ctDNA samples with estimated tumor fraction, half (50.2%) of the somatic mutations were shared between both sources, while some (17.6%) mutations were found only in ctDNA. In the metastatic setting, on-treatment increase in ctDNA fraction during pembrolizumab treatment was significantly associated with a poor response rate (18.7% v 76.1%; P = .001) and progression-free survival (median 2.8 v 9.8 months; P < .001). A comparison of cancer cell fraction, the fraction of tumor cells carrying somatic mutations, between pembrolizumab initiation and on-treatment showed a strong correlation among patients where ctDNA fraction increased during treatment ( r = 0.73). By contrast, no correlation was observed in patients without ctDNA fraction increase ( r = 0.09). Most mutations newly detected at pembrolizumab resistance have already been identified in tumor tissues at earlier stages. CONCLUSION Our newly established assay is suitable for assessing the genomic profile of ctDNA in patients with advanced UC. Our data may support future analyses of prognostic or predictive biomarkers for UC.

The Efficacy of Liquid Biopsy of Total cfDNA for Predicting Systemic Metastasis in Japanese Patients With Oral Squamous Cell Carcinoma.

The use of liquid biopsy of total cell-free DNA (cfDNA) to identify otherwise undetectable cancers has attracted interest; however, its efficacy remains unknown. We explored whether analysis using total cfDNA is efficacious for Japanese patients with oral squamous cell carcinoma (OSCC). We collected total cfDNA from nine patients with OSCC preoperatively, 1 month postoperatively, and every 3 months thereafter to analyze this association. We used a target DNA sequence for genetic mutation analysis of tumor tissues collected from 33 patients, including the aforementioned nine patients. Patients with good disease control showed negligible changes in preoperative and postoperative total cfDNA concentrations. A rapid increase in total cfDNA concentration was observed in patients who developed systemic metastases. Patients whose tumor tissue DNA showed genetic mutations had the same mutations in preoperative circulating tumor DNA. Our data suggested that analyzing total cfDNA is efficacious for patients with OSCC.

Circulating Levels of Low-Density Granulocytes and Cell-Free DNA as Predictors of Cardiovascular Disease and Bone Deterioration in SLE Patients.

The present work evaluates the predictive value of low-density granulocytes (LDGs) for the development of cardiovascular disease (CVD) and/or bone deterioration (BD) in a 6-year prospective study in systemic lupus erythematosus (SLE). Considering the high SLE-LDG capacity to form neutrophil extracellular traps (NETs), circulating levels of total cell-free DNA (cirDNA) and relative amounts of mitochondrial and nuclear DNA (mtDNA and nDNA, respectively) were tested as LDG-associated biomarkers to identify SLE patients at risk of CVD and BD. The frequency of total blood LDGs, as well as the CD16negCD14neg (nLDG) and CD16posCD14low (pLDG) subsets, was quantified by flow cytometry in 33 controls and 144 SLE patients. Total cirDNA and relative amounts of mitochondrial (mtDNA) and nuclear (nDNA) cell-free DNA were measured by fluorometry or qPCR in plasma from a subgroup of 117 patients and 23 controls at enrolment. Our findings showed increased blood levels of SLE-nLDGs at enrolment associated with prospective CVD development (pCVD) and the presence of BD, thus revealing LDG expansion as a predictor of both comorbidities in SLE progression. The amounts of the different types of circulating DNA analyzed were increased in patients, especially those presenting with traditional CV risk factors or subclinical atheromatosis. Similar to nLDGs, the nDNA concentration could predict the development of pCVD in SLE, supporting the quantification of cirDNA levels as a surrogate marker of LDGs in clinical practice.

Abstract B027: Combining total cell-free DNA (cfDNA) and circulating tumor (ctDNA) to enhance the clinical sensitivity of ddPCR assays to detect minimal residual disease (MRD) in stage IIIB/C/IV melanoma patients on adjuvant immunotherapy in CheckMate 238

Abstract Background: In patients (pts) with resected, stage III/IV melanoma receiving adjuvant immunotherapy, pre-treatment (pre-Tx) and on-treatment (on-Tx) ctDNA measurements are associated with relapse free survival; however, clinical adoption has been hindered by suboptimal assay sensitivity. Methods: We used analytically validated, mutation-specific ddPCR assays to measure ctDNA and cfDNA in pre- and on-Tx plasma samples from pts with resected stage IIIB/C/IV melanoma enrolled in CheckMate 238 (NCT02388906) receiving either adjuvant Nivolumab (NIVO) or Ipilimumab (IPI). Assay choice was personalized based on detection of one of the 7 melanoma hot-spot mutations in pt tumors: BRAF V600E/K, NRAS Q61R/L/K, or TERT promotor C228T or C250T. We evaluated associations between ctDNA detection, or high cfDNA based on cutpoint analysis (>6,000 copies/ml of plasma) and recurrence-free survival (RFS), pre-Tx tumor biomarkers including tumor cell PD-L1 expression, IFNg gene signature, CD8 infiltration, and tumor mutational burden. Survival analysis was performed using univariable and multivariable Kaplan-Meier and Cox regression models. Results: Assay mutations were identified in 87% of resected tumors. In the pre-Tx samples, ctDNA alone was detected in 94/753 (12%) pts. The detection rate increased to 20% when assay positivity was defined as either detectable ctDNA or high cfDNA. The sensitivity of ctDNA detection to predict recurrence ≤6 months(m) from pre-Tx samples increased from 25% (ctDNA alone) to 33% when high cfDNA was added to detectable ctDNA; specificity decreased to 84% from 91% (ctDNA alone). The patient subgroup defined by the combined metric of ctDNA+ or high cfDNA in pre- Tx samples was significantly enriched with higher stage of disease, elevated LDH, tumor cell PD-L1 expression < 5%, and tumor IFNg-RNA signature < median. Pts with detectable pre-Tx ctDNA or high cfDNA was associated with shorter RFS in both NIVO (median RFS 20.5 m [95%CI, 2.88, 59.15]) and IPI (median RFS 7.75 m [95%CI, 2.76, 24.08]) arms compared to those with undetectable ctDNA and low cfDNA (NIVO (median RFS 47.95 m [95%CI, 11.09, 59.89]) and IPI (median RFS 24.15 m [95%CI, 5.78, 58.38])). In a multivariable model that included sex, age, treatment, and IFNg gene signature, ctDNA+ or cfDNA high was an independent predictor of shorter RFS [HR 1.624 (95%CI, 1.275, 2.068), p<0.001]. When combining pre-Tx and on-Tx measurements at week 3 (w3) and w7, the assay performance generally increased compared to pre-Tx alone. For example, pre-Tx assay positivity had a 38% positive predictive value (PPV) and 81% negative predictive value (NPV) to predict recurrence at <6 m. In pts who were undetectable at pre-Tx but became positive at w3 and remained positive at w7, PPV increased to 62% and NPV increased to 90%. Conclusions: Incorporating quantitative measurements of cfDNA with ctDNA detection in the evaluation of pre-Tx and on- Tx plasma samples improves the clinical performance of mutation-specific ddPCR assays to detect MRD in patients with resected stage III and stage IV melanoma. Citation Format: Mahrukh M Syeda, Jennifer M Wiggins, Josephine Alegun, Saim Ali, Soutrik Mandal, Tracy (Hao) Tang, Keyur Desai, Sonia Dolfi, Daniel J Tenney, Paolo A Ascierto, James Larkin, Michele Del Vecchio, Jeffrey S Weber, David Polsky. Combining total cell-free DNA (cfDNA) and circulating tumor (ctDNA) to enhance the clinical sensitivity of ddPCR assays to detect minimal residual disease (MRD) in stage IIIB/C/IV melanoma patients on adjuvant immunotherapy in CheckMate 238 [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B027.

Abstract 4144019: Early total cell-free DNA, but not donor fraction, predicts late events in heart transplantation

Introduction: Donor fraction (DF) cell-free DNA (cfDNA) is an emerging tool for non-invasive rejection surveillance in heart transplantation (HTx). Little is known about the significance of DF and/or total cfDNA (TcfDNA) in the first month post HTx. We explored the relationship between early cfDNA results and later clinical events in adult and pediatric HTx recipients. Aims: 1. Explore the relationship between early cfDNA levels and late events after HTx 2. Describe the decline in DF during the first month post HTx Methods: Retrospective data from the multicenter prospective blinded DTRT study (DNA-based transplant rejection test) was used. DF and TcfDNA results from samples drawn post HTx day 1 (<24h), day 4 (+/- 12h), day 7 (+/- 12h), day 14 (+/- 24h) and day 28 (+/- 7 days) were compared in subjects with or without events >35d to 1 year post HTx. Captured events include cardiac arrest, mechanical circulatory support or death. Exclusions included absent TcfDNA, multiorgan Tx, any PTLD, or other cancer in previous 2 yrs. Cell-free DNA values were compared across event groups and time windows using GEE (generalized estimating equation) with Max Likelihood Estimation. Results: 190 subjects had 566 samples drawn < 35 days post HTx. Median age was 17.8 yrs with a range of 26 days - 73.4 yrs; 51.8% were pediatric. 16 subjects (8.4%) had events bridging or following day 35 post HTx; 23 with events prior to day 35 were included as non-event subjects. TcfDNA was significantly higher in event subjects on days 4, 7 and 28 post HTx. [Fig 1]. There was no difference in DF between event and non-event subjects. Median (IQR) DF at 14 days was 0.26% (0.20, 0.39) and 0.19% (0.13, 0.29) at 28 days. Conclusions: Elevated TcfDNA, even in the setting of normal DF, may be a useful early marker to identify patients at risk for later events in the first year post HTx. The majority of patients reach low DF levels by 28 days post HTx suggesting an opportunity to incorporate cfDNA earlier into rejection surveillance protocols.

Donor-derived cell-free DNA versus left ventricular global longitudinal strain in noninvasive detection of heart allograft injury

Abstract Introduction Invasive endomyocardial biopsy (EMB) is still considered the gold standard method for monitoring heart transplant (HTx) rejection. However, it is associated with potentially serious complications, and its histopathologic interpretation has high interobserver variability. Thus, noninvasive methods for surveillance of the transplanted organ remains of great interest. The noninvasive donor-derived cell-free DNA is a marker of graft injury which can indicate acute rejection, even before the histopathological diagnosis of rejection detected by EMB. The assay has a high negative predictive value for acute rejection. Left ventricular global longitudinal strain (LV GLS) is an emerging marker of subclinical myocardial injury which plays a crucial role in the evaluation of several cardiac diseases; however, its role in HTx injury has so far been poorly investigated. Purpose We aimed to investigate the efficacy of a local laboratory-run dd-cfDNA assay-based heart transplant rejection surveillance programme, and the prognostic and discriminatory performance of LV GLS for subsequent heart allograft injury. Methods HTx recipients without a history of allograft rejection requiring intensification of immunosuppressive therapy were transitioned from our EMB-based surveillance protocol to dd-cfDNA-based rejection surveillance. We assessed the percentage of dd-cfDNA to total cell-free DNA. Injury cut-off was defined by a dd-cfDNA level ≥0.20%, while the severe injury threshold was at ≥0.35%. LV ejection fraction was calculated using 2D Simpson’s method. To derive LV GLS, we analyzed apical 4-chamber, 2-chamber, and long-axis images by 2D speckle tracking echocardiography. Results A total of 244 dd-cfDNA samples were analyzed from 46 patients in fifteen runs performed monthly between October 2022 and December 2023. The dd-cfDNA fraction remained below the injury cut-off in most patients (81%). There were no missed rejection episodes. 88% of routine EMBs that would have otherwise been performed were safely avoided. Eighteen for-cause EMBs were performed based on dd-cfDNA levels suggesting injury. One EMB verified moderate cellular rejection (ISHLT grade 2R) that required steroid pulse therapy. Two EMBs proved mild cellular rejection, one EMB confirmed mild antibody-mediated rejection, while one EMB proved mixed rejection. Thirteen EMBs found no rejection. Mean LVEF was preserved (57.6%±7.6%). Mean LV GLS was -15.1%±2.5% in the whole cohort, while -15.6%±4.7% in recipients with HTx rejection on indication EMB. There was no significant correlation between dd-cfDNA and LV GLS (n=159). Conclusion The noninvasive dd-cfDNA assay is effective in ruling out acute rejection and safely decreases the necessity of invasive surveillance EMBs after HTx. LV GLS is less sensitive in comparison with the dd-cfDNA assay for detection of myocardial injury in the early stages of graft rejection in HTx recipients at low risk for rejection.

(1164) - Quantifying Total Cell-Free DNA an a Heart Transplant Patient with Acute Cellular Rejection

(1164) - Quantifying Total Cell-Free DNA an a Heart Transplant Patient with Acute Cellular Rejection

Abstract 3662: Cell-free DNA dynamics following neuroblastoma resection

Abstract Introduction: Prior studies have demonstrated the utility of circulating tumor DNA (ctDNA) as a biomarker in the postoperative period for oncologic risk stratification. However, its utility may be limited in the early postoperative period by surgical trauma causing an increase in total cell-free DNA (cfDNA) due to tissue destruction, inflammation, and wound healing. No studies have identified the optimal timing of postoperative sample collection in pediatric patients undergoing resection of high-risk neuroblastoma. The purpose of this study is to assess the postoperative dynamics of cfDNA following neuroblastoma resection to determine optimal timing for ctDNA investigations. Methods: An institutional database of banked cell-free DNA samples was retrospectively queried for patients with a diagnosis of neuroblastoma who had blood samples collected prior to resection and postoperative samples collected within 2 months of the operation. Plasma was collected from whole blood samples. cfDNA was extracted from plasma samples using the QIAGEN QIAsymphony SP system, and samples were quantified using the Advanced Analytical Fragment Analyzer Automated CE System with the High Sensitivity Genomic DNA Analysis Kit. Results: Seventeen patients were identified undergoing 18 total operations and 37 total blood draws yielding cfDNA. There were 12 male and 5 female patients. Median age at operation was 5.5 years (range 1.4y to 20.5y). Two patients were INRG stage L2, and 15 patients were stage M. Operative indications were primary tumor resection in 10 patients and resection of recurrence/progression in 8 patients. Three postoperative samples were drawn within 10 days of the operation, and 16 samples were collected between postoperative weeks 4 and 6. There was a statistically significant difference (p=0.029) in cfDNA yield between each timepoint sampled: preoperative (median 6ng/mL, range 2-30ng/mL), 0-10 days postop (median 34ng/mL, range 15 - 85 ng/mL), and 4-6 weeks postop (median 6ng/mL, range 2 - 17ng/mL). Pairing the pre- and post-operative samples for each patient, those drawn within 10 days had a median increased yield of 180% (range +67% to +652%) while the samples collected at 4-6 weeks had a median decreased yield of 23% (range -55% to +491%; p = 0.047). Of note, there were three outliers in the 4-6 week group who had increases in cfDNA yield of >200%. On further review of these charts, each patient was being treated for obstructive uropathy at the time the postoperative sample was collected. Conclusion: Plasma cfDNA yield increases in the early postoperative period before dropping by postoperative week 6. Tumor resection may be associated with an overall decrease in total cfDNA. Postoperative renal pathology may cause a persistent elevation in cfDNA. Further studies are necessary to clarify the role of ctDNA in the management of patients with neuroblastoma. Citation Format: Joshua N. Honeyman, Andrew Chi, Shakeel Modak, Fiorella Iglesias Cardenas, Michael P. La Quaglia, Neerav N. Shukla, J. Ted Gerstle. Cell-free DNA dynamics following neuroblastoma resection [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 3662.

The utility of liquid biopsy in clinical genetic diagnosis of cancer and monogenic mosaic disorders.

Liquid biopsy for minimally invasive diagnosis and monitoring of cancer patients is progressing toward routine clinical practice. With the implementation of highly sensitive next-generation sequencing (NGS) based assays for the analysis of cfDNA, however, consideration of the utility of liquid biopsy for clinical genetic testing is critical. While the focus of liquid biopsy for cancer diagnosis is the detection of circulating tumor DNA (ctDNA) as a fraction of total cell-free DNA (cfDNA), cfDNA analysis reveals both somatic mosaic tumor and germline variants and clonal hematopoiesis. Here we outline advantages and limitations of mosaic and germline variant detection as well as the impact of clonal hematopoiesis on liquid biopsy in cancer diagnosis. We also evaluate the potential of cfDNA analysis for the molecular diagnosis of monogenic mosaic disorders.

Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis

Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis

Fluorometric Quantification of Total Cell-Free DNA as a Prognostic Biomarker in Non-Small-Cell Lung Cancer Patients Treated with Immune Checkpoint Blockade.

The present study aimed to investigate the potential of basal cell-free fluorometric DNA (cfDNA) quantification as a prognostic biomarker in advanced non-small cell lung cancer (NSCLC) patients treated with an Immune Checkpoint Blockade (ICB). A discovery and validation cohort of 61 and 31 advanced lung cancer patients treated with ICB were included in this study. Quantification of cfDNA concentration was performed before the start of the treatment and patients were followed up for a median of 34 (30-40) months. The prognostic predicted value of cfDNA was evaluated based on ROC, and Cox regression was conducted via univariate and multivariate analyses to estimate the hazard ratio. We observed that a cfDNA cut-off of 0.55 ng/µL before the ICB determines the overall survival of patients with a log rank p-value of 3.3 × 10-4. That represents median survivals of 3.8 vs. 17.5 months. Similar results were obtained in the validation cohort being the log rank p-value 3.8 × 10-2 with median survivals of 5.9 vs. 24.3. The univariate and multivariate analysis revealed that the cut-off of 0.55 ng/µL before ICB treatment was an independent predictive factor and was significantly associated with a better survival outcome. High cfDNA concentrations identify patients with advanced NSCLC who do not benefit from the ICB. The determination of cfDNA is a simple test that could select a group of patients in whom new therapeutic strategies are needed.

HERCULES: A prostate cancer (PC) sequencing panel for parallel analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs).

5036 Background: In metastatic PC, tracking genomic alterations over time and with treatment can predict disease outcomes, guide treatment, and identify novel therapeutic targets. Analysis of peripheral blood ctDNA offers a non-invasive liquid biopsy for genomic profiling, but ctDNA often comprises a small portion of total cell-free DNA (cfDNA) in the blood, requiring high depth of sequencing which may still miss PC-relevant variants. We and others have shown that single CTCs captured from the same blood sample yield additional PC-relevant genomic data when combined with ctDNA profiling, but currently there is no assay that can be applied uniformly to both analytes. Here, we present a new amplicon-based sequencing panel capable of detecting PC-relevant variants in both cfDNA and single CTCs. Methods: PC-relevant genes were curated by overlapping reviews of cBioPortal for cancer genomics, the catalogue of somatic mutations in cancer (COSMIC), and PC-relevant genomic literature. HERCULES targets 36 genes with 258 amplicons covering PC-associated mutations and copy number variations. AmpliSeq HD targeted sequencing technology was applied for analysis of both cfDNA and CTCs. Analytical validation was performed sequentially, first using AcroMetrix and Seracare DNA control samples, then single and pooled LNCap and PC3 PC cells and cfDNA, then 8 patient peripheral blood samples of matched plasma cfDNA and single CTCs captured using the RareCyte platform from participants in SWOG S1802, an ongoing CTEP/CIRB-approved phase 3 trial for men with metastatic PC. Results: In DNA control samples with allele frequencies of 1%, 0.25%, and 0.1%, HERCULES displayed sensitivity of 100%, 75%, and 50%, respectively. In matched single cells and cfDNA from cell lines, HERCULES detected 100% of database-annotated somatic variants in cfDNA and in pools of 2 or more cells, and it identified >90% of variants in single cells. In 16 matched patient samples of cfDNA and single CTCs, with read coverage depths of >50,000x for 20ng cfDNA and >1,500x for single CTCs, HERCULES detected shared variants (mean N per sample = 6.1) as well as unique somatic variants (mean of 1.1 CTC variants not called in the cfDNA, and 4.1 cfDNA variants not called in the CTCs). Conclusions: HERCULES is, to our knowledge, the first PC-specific targeted sequencing panel capable of parallel genomic profiling of both ctDNA and CTCs. In our preliminary analytical validation, it detected somatic variants with high sensitivity in matched cfDNA and CTC samples. With additional validation, HERCULES has the potential to augment conventional ctDNA profiling by illuminating both cell-free and cellular genomic evolution during PC treatment and progression.

Quantitative analysis of cell-free plasma DNA as a prognostic biomarker in acute ischemic stroke patients

BackgroundStroke has become a leading cause of death and disability worldwide, and despite the introduction of new screening programs, therapies, and monitoring technologies, there is still a need to develop more useful biochemical tests to monitor treatment response and inform clinical decision-making. Cell-free DNA released from damaged neurons in stroke patients may be useful in assessing stroke prognosis. The purpose of this study was to evaluate the role of cell-free DNA and a highly sensitive blood biomarker in acute ischemic stroke. 188 patients with acute ischemic stroke were recruited for the study. The level of cell-free DNA in plasma was estimated using a real-time PCR assay for the β-globin gene (Qiagen-Rotor-Gene Q MDX, Germany). Clinical assessment was performed with the National Institutes of Health Stroke Scale (NIHSS) at the time of admission. After a period of three months from the onset of stroke, (mRS) scores were estimated using the modified Rankin scale.ResultsElevated levels of cell-free DNA were found in patients with higher NIHSS admission scores and mRS 3-month scores (p < 0.05). The regression analysis revealed that the markers are associated with an unfavorable outcome in comparison to other stroke risk factors (R2 = 0.224). Stroke outcome was relatively better in patients with a cell-free DNA level < 10,000 kilogenome equivalents/L (p < 0.05).ConclusionsMeasurement of total cell-free DNA levels is a simpler and less-expensive biomarker suggesting potential clinical application of blood-based test. Cell-free DNA can contribute to the clinical evaluation and optimal management of ischemic stroke patients. This biomarker seems to have the potential to predict the long-term prognosis of acute ischemic stroke.

Total Cell-Free DNA as a Noninvasive Biomarker of a Delayed Graft Function After Kidney Transplantation From Donors After Cardiac Death

Total Cell-Free DNA as a Noninvasive Biomarker of a Delayed Graft Function After Kidney Transplantation From Donors After Cardiac Death

(1210) Total Cell-Free Dna May Reflect Non-Rejection Causes of Lung Allograft Injury

(1210) Total Cell-Free Dna May Reflect Non-Rejection Causes of Lung Allograft Injury