Fraction Of cfDNA Research Articles

Donor Fractions of Cell-Free DNA Are Elevated During CLAD But Not During Infectious Complications After Lung Transplantation.

During the last few years, cell-free DNA (cfDNA) has emerged as a possible non-invasive biomarker for prediction of complications after lung transplantation. We previously published a proof-of-concept study using a digital droplet polymerase chain reaction (ddPCR)-based method for detection of cfDNA. In the current study, we aimed to further evaluate the potential clinical usefulness of detecting chronic lung allograft dysfunction (CLAD) using three different ddPCR applications measuring and calculating the donor fraction (DF) of cfDNA as well as one method using the absolute amount of donor-derived cfDNA. We analyzed 246 serum samples collected from 26 lung transplant recipients. Nine of the patients had ongoing CLAD at some point during follow-up. All four methods showed statistically significant elevation of the measured variable in the CLAD samples compared to the non-CLAD samples. The results support the use of ddPCR-detected cfDNA as a potential biomarker for prediction of CLAD. These findings need to be validated in a subsequent prospective study.

Diagnosis of pediatric central nervous system tumors using methylation profiling of cfDNA from cerebrospinal fluid

Pediatric central nervous system tumors remain challenging to diagnose. Imaging approaches do not provide sufficient detail to discriminate between different tumor types, while the histopathological examination of tumor tissue shows high inter-observer variability. Recent studies have demonstrated the accurate classification of central nervous system tumors based on the DNA methylation profile of a tumor biopsy. However, a brain biopsy holds significant risk of bleeding and damaging the surrounding tissues. Liquid biopsy approaches analyzing circulating tumor DNA show high potential as an alternative and less invasive tool to study the DNA methylation pattern of tumors. Here, we explore the potential of classifying pediatric brain tumors based on methylation profiling of the circulating cell-free DNA (cfDNA) in cerebrospinal fluid (CSF). For this proof-of-concept study, we collected cerebrospinal fluid samples from 19 pediatric brain cancer patients via a ventricular drain placed for reasons of increased intracranial pressure. Analyses on the cfDNA showed high variability of cfDNA quantities across patients ranging from levels below the limit of quantification to 40 ng cfDNA per milliliter of CSF. Classification based on methylation profiling of cfDNA from CSF was correct for 7 out of 20 samples in our cohort. Accurate results were mostly observed in samples of high quality, more specifically those with limited high molecular weight DNA contamination. Interestingly, we show that centrifugation of the CSF prior to processing increases the fraction of fragmented cfDNA to high molecular weight DNA. In addition, classification was mostly correct for samples with high tumoral cfDNA fraction as estimated by computational deconvolution (> 40%). In summary, analysis of cfDNA in the CSF shows potential as a tool for diagnosing pediatric nervous system tumors especially in patients with high levels of tumoral cfDNA in the CSF. Further optimization of the collection procedure, experimental workflow and bioinformatic approach is required to also allow classification for patients with low tumoral fractions in the CSF.

PATH-04. OPTIMIZING CELL-FREE DNA ASSAYS FOR LOW INPUT LIQUID BIOPSY SAMPLES FROM PEDIATRIC BRAIN TUMOR PATIENTS

Abstract BACKGROUND Genetic and epigenetic profiling have transformed biological understanding, diagnostics, and therapeutic approaches for pediatric central nervous system (CNS) tumors. Obtaining tumor tissue may be associated with neurosurgical risk, may not be feasible in some cases, and is not routinely performed during follow-up or at relapse. Liquid biopsies (LBs) have emerged as a minimally invasive, serially collectable source of tumor-derived DNA. Here, we collected cerebrospinal fluid (CSF) and serum LBs from a large, unselected, cross-entity pediatric CNS tumor cohort (n=55 patients) for method optimization. METHODS Cell-free DNA (cfDNA) isolated from CSF and serum samples was analyzed 1) by low-coverage whole genome sequencing for copy number variation (CNV)-based tumor detection and 2) by enzymatic conversion (EC)-based DNA methylation profiling using the EPIC DNA methylation array and the Heidelberg Brain Tumor Classifier. All library protocols were optimized for LB samples with low cfDNA amounts in the picogram range. Matched tumor samples were used as reference. RESULTS Our lcWGS-based cfDNA profiling protocol had a high technical success rate across the whole cohort (>95%), despite very low cfDNA amounts for most CSF samples. The majority of CSF LBs captured tumor-derived CNVs and displayed a high fraction of tumor-derived cfDNA (>5%, range 0-78%). For serum LBs, only few samples showed detectable CNVs. Proof-of-concept methylation analyses leveraging a new EC-based workflow allowed molecular classification using CSF, particularly for embryonal brain tumors (11/15 samples). Clinical workup of selected cases illustrates the potential of LBs to aid as diagnostic tool, monitor disease, and characterize tumor evolution. CONCLUSIONS Tackling a major roadblock of clinical translation of LBs, this study delineates new methods tailored to low input cfDNA samples. Results highlight the clinical utility of LBs for the management of brain tumor patients, providing a strong rationale for prospective validation within pediatric neuro-oncology trials.

The clinical utility of plasma cell-free DNA (cfDNA) in NET-02: Randomised, phase II trial of liposomal irinotecan (nal-IRI)/5-fluorouracil (5-FU)/folinic acid or docetaxel as second-line (2L) therapy in patients (pts) with progressive poorly differentiated extra-pulmonary neuroendocrine carcinoma (PD-EP-NEC).

4130 Background: In NET-02, nal-IRI/5-FU, but not docetaxel, met the primary endpoint of 6-month (mo) progression-free survival (PFS) rate in pts with progressive PD-EP-NEC (1). The prognostic potential of cfDNA in progressive PD-EP-NEC has not been explored in a prospective randomised trial. Methods: NET-02 was a multi-centre, randomised (1:1), phase II trial of IV nal-IRI/5-FU/folinic acid, Q14 days (ARM A), or IV docetaxel, Q21 days (ARM B), as 2L therapy in pts with progressive PD-EP-NEC. Plasma samples were taken at baseline (T0), following 6 weeks of treatment (T1) and at progression (T2). Samples were analysed using a multi-omic next-generation sequencing (NGS) approach (T7-MBD-seq) (measures genome-wide methylation and low pass whole genome copy number alterations (CNA)). The tumour fraction (TF) of cfDNA was determined using ichorCNA and correlated with clinical outcomes using Cox proportional hazards model and Logistic regression. Results: Of 56 pts evaluable for efficacy, there were 54 (96%) T0 samples (48 passed quality control), 31 (55%) at T1 and 20 (36%) at T2. At T0, 36/48 (75%) samples had detectable TF by copy number (using 3% TF cut-off). For the entire cohort, T0 TF was negatively prognostic for overall survival (OS) (Hazard Ratio (HR) 1.17, 95% Confidence interval (CI) 1.00-1.37, P=.044), but not for 6 mo PFS rate (Odds ratio (OR) 1.25, 95% CI 0.91-1.73, P=.17), response rate (RR) (OR 1.11, 95% CI 0.67-1.76, P=.65) or PFS (HR 1.04, 95%CI 0.91-1.20, P=.53). In the entire cohort, median OS split by TF (N=16 each) was 10.2 mo (95% CI 4.1-not available (NA)) for low (TF≤8.6%), 6.9 mo (95% CI 3.4-14.8) for medium (8.6<TF≤24.3%) and 3.7 mo (95% CI 2.8-NA) for high (TF>24.3%). T0 TF was also negatively prognostic for 6 mo PFS rate and OS in ARM A (P=.02, P=.03), but not ARM B (P=.61, P=.48), but was not prognostic for RR or PFS (Table). TF correlated with the presence of liver metastases (Wilcoxon Rank Sum P=.009), but not with ECOG PS (P=.14), sex (P=.10), Ki-67 (P=.39) or age (Spearman rank correlation P=.45). Longitudinal copy number and methylation cfDNA analysis is on-going. Conclusions: These results suggest that it may be possible to stratify prognosis based on amount of baseline TF in patients with progressive PD-EP-NEC, and may also identify patients who would benefit most from the nal-IRI combination. 1. McNamara 2023, EClin Med. Clinical trial information: 03837977. [Table: see text]

Diagnosis and genetic characterization of CNS metastases in lung cancer via shallow whole-genome sequencing of CSF cell-free DNA.

e14013 Background: The genetic characterization of CNS metastases in lung cancer is hindered by the limited sampling opportunities and genetic heterogeneity between intracranial and extracranial lesions. To address these challenges, this study uses cerebrospinal fluid (CSF) and blood samples, to detect and characterize CNS metastasis variants through shallow whole-genome sequencing (sWGS). Methods: From Dec. 2021 to Aug. 2023, 57 lung cancer patients were recruited, comprising 29 with leptomeningeal metastases (LMs) and 28 with only parenchymal brain metastases (PMs). A total of 66 blood and 112 CSF samples were collected for cfDNA sWGS to analyze chromosomal arm CNVs, focal amplifications (CN > 5) and SNV/InDels. Cytology was also conducted on 95 of these CSF samples. Variant differences between PMs and extracranial metastases (EMs) were analyzed using the MSK-MET database. Results: CSF cfDNA concentration was significantly lower than that in plasma (p < 0.0001), with 41.7% of samples unquantifiable, yet all samples successfully completed sWGS. The cfDNA tumor fraction in CSF was significantly higher than in paired plasma (p < 0.0001), and most arm CNVs and gene amplifications exclusively detected in CSF. Cytology, as the gold standard for LM diagnosis, had a sensitivity of 0.76. The sensitivity for LM diagnosis based on aneuploidy and genomic amplification fraction (GAF) were 0.51 and 0.80, respectively, without compromising specificity. Gains in 1p, 7p, 11q, 16p, and 17q, as well as losses in 9p and 9q, were significantly more frequent in PMs compared to EMs and further increased in the LMs (p < 0.05). Notably, losses in 1p, 4p, 5q, and 11p, significantly more frequent in PMs than in extracranial metastases, but showed a significant increase in gain frequency in LMs (p < 0.05). In the LM group, the frequency of EGFR L858R and amplification of nearly the entire 7p where the EGFR located, were significantly increased, whereas KRAS G12D frequency significantly decreased (p < 0.05). Conclusions: The characteristics of extremely low concentration but high tumor fraction of CSF cfDNA make it suitable for sWGS, enhancing LM diagnostic sensitivity without compromising specificity, and identifying molecular characteristics of CNS metastasis. These molecular features require further validation in larger prospective clinical cohorts, offering potential for personalized diagnosis and treatment of CNS metastases.

Abstract 5020: Pre-analytical characterization of cell-free DNA to enable liquid biopsy for solid tumors

Abstract Background: The integration of liquid biopsy in oncology has transformed clinical management of patients with cancer. Cell-free DNA (cfDNA) represents extracellular nucleic acid fragments shed during cellular apoptosis, necrosis, or secretion, and can be extracted from whole blood. PGDx elio plasma focus analyzes cfDNA to enable non-invasive genomic profiling with next-generation sequencing. In this study, we characterized the cfDNA fraction (%) and yield (ng) across multiple tumor types and stages. Methods: cfDNA fraction was obtained using automated electrophoresis (Agilent 4200 TapeStation System) to assess a region of 50-700 bp across 293 clinical plasma samples (tumor stages I - IV). The fraction of cfDNA to total DNA present was then calculated to determine overall cfDNA percentage. The overall DNA yield (ng) was quantified via Qubit fluorometer immediately following extraction from plasma and normalized to volume of plasma (ng of cfDNA/mL of plasma). In addition, data was evaluated from 100 clinical plasma cases with strict extraction conditions allowing for yield normalization to plasma volume. Sequencing metrics were evaluated for all clinical cases with a recommended input of 25 ng DNA using PGDx elio plasma focus. Results: Average cfDNA fraction for individual tumor types ranged from 78.0% - 91.4%, with a mean (n=293) of 84.8%. When examining the yield normalized cohort (n=206), the average yield was 23.4 ng of cfDNA/mL of plasma. cfDNA yield and fraction varied across tumor types and increased with cancer stage. Despite these differences, the majority of cases (99.7%) yielded sufficient cfDNA to proceed with PGDx elio plasma focus testing. No statistical differences in sequencing performance (variants reported and coverage for each tumor type) were observed and the PGDx elio plasma focus assay success rate was 97%. There was a slightly higher failure rate (17.6% (3/17)) for samples below 60% cfDNA, indicating that high amounts of contaminating genomic DNA from white blood cells may obscure the number of available cfDNA fragments for analysis. Conclusions: These data characterize pre-analytical factors that impact performance of PGDx elio plasma focus using cfDNA isolated from plasma of patients with solid tumors. We showed cfDNA yield and fraction varied across tumor types and increased with cancer stage. The overall success rate for testing was 97% while failed samples were associated with higher levels of genomic DNA contamination. Citation Format: Robert J. Summersgill, Jesse M. Fox, Jennifer S. Dickey, Liam T. Cox, Cal D. Palumbo, Kenneth C. Valkenburg, Brian J. Caveney, Shakti Ramkissoon, Jennifer B. Jackson. Pre-analytical characterization of cell-free DNA to enable liquid biopsy for solid tumors [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 5020.

Abstract 3670: Reducing the patient burden for ctDNA biomarkers: Advancing small volume home-based collection technologies

Abstract There is broad consensus from regulatory agencies, clinicians and patients for the need to move to decentralized trials while focusing on patient-centric approaches with the COVID-19 pandemic being the catalyst. This requires the development of new sample collection capabilities and workflows to ensure that the necessary biomarkers can be measured. Circulating tumor DNA (ctDNA) has become an increasingly important biomarker for cancer detection and genetics, and monitoring patients during treatment. Typical measurements of ctDNA involve venous draws of >10mls of whole blood that require patients to be in clinic. Here, we test the feasibility of using a new collection device, Tasso+, to collect capillary blood for ctDNA analysis using the Guardant360 panel. A pilot of whole blood microsamples (< 1 mL) with a cfDNA yield of 0.7-1.4 ng/ml spiked to 0.5% MAF with Seraseq ctDNA reference material. This resulted in detection of ~20% of the total expected variants across variant classes showing the feasibility of using the G360 panel to detect low volume/low MAF samples. Initial testing of Tasso+ collected samples revealed a 5-10x increase in high MW gDNA compared to traditional venipuncture. SPRI bead-based size selection was used in the extraction protocol to remove >95% of the high MW DNA, resulting in a 35% loss of target cfDNA fraction. Traditional venipuncture samples and Tasso+ samples were collected from 5 cancer patients and run using the G360 platform. Microsamples detected 75% of the variants across all variant classes found in venipuncture samples. Tasso+ lost sensitivity of variants when the MAF was <0.5%. To expand usage of Tasso+ to at-home collection of blood for ctDNA analysis, we sought to determine if lyophilization of the Streck DNA BCT RUO tube reagent could still provide stability to samples and a potential framework for at-home collections. Venipuncture and Tasso+ samples were collected from control patients, pooled and spiked with Seraseq cfDNA reference material. Samples were aliquoted into standard liquid Streck tubes or tubes with lyophilized reagent at the concentration per manufacturer’s directions and evaluated for stability over time. The Tasso+ samples with a target MAF between 0.5%-2.0% detected ~26% of all variants compared to the standard samples with a similar MAF. Lyophilization of the Streck reagent had minimal effect (<20% reduction in variants detected) on the reagent’s performance as there were approximately an equal number of total variants detected when comparing liquid and lyophilized samples for both the standard tubes and Tasso+ samples. Overall, these results provide support and a framework for using a home-based patient-centric sampling approach for monitoring ctDNA from cancer patients in clinical development. Citation Format: Brad R. Evans, Robert J. Foley, Steven M. Townson, Emily J. Welch, Kevin P. Bateman. Reducing the patient burden for ctDNA biomarkers: Advancing small volume home-based collection technologies [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 3670.

Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients’ care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35–10380 and 500–10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

463 Fetal fraction of cfDNA from noninvasive prenatal testing as a predictor for Placenta Accreta Spectrum

463 Fetal fraction of cfDNA from noninvasive prenatal testing as a predictor for Placenta Accreta Spectrum

Comprehensive tissue deconvolution of cell-free DNA by deep learning for disease diagnosis and monitoring

Plasma cell-free DNA (cfDNA) is a noninvasive biomarker for cell death of all organs. Deciphering the tissue origin of cfDNA can reveal abnormal cell death because of diseases, which has great clinical potential in disease detection and monitoring. Despite the great promise, the sensitive and accurate quantification of tissue-derived cfDNA remains challenging to existing methods due to the limited characterization of tissue methylation and the reliance on unsupervised methods. To fully exploit the clinical potential of tissue-derived cfDNA, here we present one of the largest comprehensive and high-resolution methylation atlas based on 521 noncancer tissue samples spanning 29 major types of human tissues. We systematically identified fragment-level tissue-specific methylation patterns and extensively validated them in orthogonal datasets. Based on the rich tissue methylation atlas, we develop the first supervised tissue deconvolution approach, a deep-learning-powered model, cfSort, for sensitive and accurate tissue deconvolution in cfDNA. On the benchmarking data, cfSort showed superior sensitivity and accuracy compared to the existing methods. We further demonstrated the clinical utilities of cfSort with two potential applications: aiding disease diagnosis and monitoring treatment side effects. The tissue-derived cfDNA fraction estimated from cfSort reflected the clinical outcomes of the patients. In summary, the tissue methylation atlas and cfSort enhanced the performance of tissue deconvolution in cfDNA, thus facilitating cfDNA-based disease detection and longitudinal treatment monitoring.

Abstract 235: Establishing clinical utility for genomic profiling with plasma cell free DNA in urothelial carcinoma

Abstract Purpose: Genomic profiling in urothelial carcinoma (UC) conventionally uses tissue DNA. However, tissue biopsy is so invasive that it can be difficult in some cases. Cell-free DNA (cfDNA) assay is a minimally invasive tool obtaining a comprehensive genomic information reflecting a tumor heterogeneity. Recently, the investigation of cfDNA assay in UC is widely conducted, but its clinical utility remains largely unknown. We aimed to establish plasma cfDNA assay and clarify its clinical role in UC. Method: Blood and matched tissue were obtained from 48 cases with advanced UC before 1st line systemic therapy or radical surgery. For genomic profiling of all samples, we applied a customed targeted DNA-sequencing strategy capturing 54 UC-relevant genes. Somatic mutations were required to be supported by a minimum variant allele frequency (VAF) of 0.5% in plasma cfDNA and 5% in tissue DNA. All mutation calls were filtered against matched leukocyte DNA. The result of mutational analysis was validated with digital PCR. The proportion of tumor-derived cfDNA (circulating tumor DNA (ctDNA) fraction) was estimated. We investigated the association between genomic profile and molecular subtyping with RNA sequencing and immunohistochemistry (IHC). Result: Fifty-five somatic mutations were detected in 22 cfDNA samples (22/48, 45.8%). Twenty-six of all somatic mutations identified in cfDNA (26/55, 47.3%) were concurrently present in tumor tissues. In 14 mutations randomly selected from all of those detected in cfDNA, VAF of digital PCR was statistically consisted with the result of NGS (p&amp;lt;0.001, R2=0.95). Furthermore, digital PCR showed that mutations detected in cfDNA alone were cleared via radical surgery. And those were found in another tissue sample from the same patient. Of the 22 cases, 6 had a ctDNA fraction greater than 2% of total cfDNA. High ctDNA fraction was associated with advanced clinical tumor stage, high PD-L1 expression in tumor tissue by IHC and basal molecular subtype (p = 0.04, 0.02, &amp;lt;0.01, respectively). Conclusions: We established cfDNA assay for genome profiling in advanced UC. The result of validation using digital PCR suggested that cfDNA assay can identify the mutation which cannot be detected in tissue sample as a tumor heterogeneity. Additionally, cfDNA assay generate the information for classifying UC cases by molecular subtypes and predicting the expression of immune checkpoint molecules. Further investigation is needed as a promising minimally invasive assay to predict an immunotherapy responsiveness against metastatic UC. Citation Format: Yuki Kita, Takayuki Sumiyoshi, Takeshi Sano, Akihiro Hamada, Toru Sakatani, Kenji Nakamura, Hideaki Takada, Ryousuke Ikeuchi, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishing clinical utility for genomic profiling with plasma cell free DNA in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 235.

347.1: Unraveling the Stage-Shift of Acute Rejection in Renal Allografts

Introduction: The lack of accuracy in current allograft monitoring methodologies for allograft monitoring underscores a critical unmet need for more accurate immunosurveillance to uncover the flux in alloimmunity. QSant – a non-invasive, urine based multi-analyte diagnostic test – was clinically validated to prognosticate injury, risk of evolution, and resolution of acute rejection. QScore—the composite score across measurements of DNA markers: the amount of cell-free DNA, fraction of methylated cfDNA, protein and metabolic biomarkers in the QSant assay—enables this risk prognostication. This study now explores the clinical utility of QSant across the alloimmunity gradient of 32–100 for the early diagnosis of allograft injury and rejection. Methods: Utilizing a large real-world dataset of contemporaneous kidney allograft biopsy and QSant data from 11 US kidney transplant centers, the QScore offers a dynamic view into the changing state of allograft injury and rejection in response to alterations in the patients’ immunosuppression regimen. First analysis included Q-Score distributions in the biopsy-paired validation (n = 162) and prediction (n = 91) data from the Yang cohort, which were collected between 2010 and 2018 [10]. Second was a new analysis of a real-world data (RWD) cohort, comprising of prospectively collected urine samples (n = 235). Results: The RWD cohort (n = 235) of both adult and pediatric renal transplant patients demonstrated a spectrum across the Q-Score distribution. Specifically, 37.9% of the samples were enriched for immune quiescence (Q-Score < 32); the remaining 62.1% were enriched for the injury/rejection spectrum (Q-Score ≥ 32). The QScore <32 classifies stable allograft recipients; QScore between 32-55 exposes a state of alloimmunity flux; and the score between 55-100 provides advanced alloimmunity. Serial monitoring by QSant across a subset of 32 patients (RWD cohort,) over a period of two consecutive quarters (timepoints 1 and 2), captures the essence of the alloimmunity flux (p-value: 0.086 by non-parametric Wilcoxon test). Patients in the immunoquiescent domain (31% [10/32]) by and large continue to be stable (with Q-Scores that track at <32) 22% [7/32]). However, 9.4% (3/32) of patients show Q-Score drifts above 32 with evidence of early rejection. Patients in the alloimmune injury domain follow three trajectories: i) a planar gradient in QScore across time, showing persistence of alloimmunity; ii) an increasing gradient, delineating patients moving into the mature acute rejection domain; iii) a decreasing gradient, delineating patients moving into the immunoquiescent domain, putatively in response to immunomodulation following assessment of active rejection (Figure 1). Conclusion: The observed alloimmunity flux suggested precision monitoring with QSant has the potential to differentiate higher-grade acute rejection episodes from sub-clinical rejection, thereby improving a transplant recipient’s’ quality of life.

232.11: Longitudinal Surveillance of a Kidney Transplant Recipient During Pregnancy Using Quantification of Fetal and Donorderived Cell-Free DNA

Introduction: Following kidney transplantation, women of childbearing age often experience rapid restoration of fertility as the feedback mechanisms of the hypothalamic-pituitary-gonadal axis normalize. Although rates of rejection during pregnancy are comparable to the non-pregnant transplant population, subtle indications of allograft injury such as increases in serum creatinine or proteinuria can be masked by pregnancy related changes. Here, we describe a novel approach of allograft surveillance using quantification of fetal and donor-derived cell-free DNA (dd-cfDNA) throughout gestation and postpartum. Methods: The patient was monitored longitudinally with dd-cfDNA (AlloSure, CareDx) pre-pregnancy, during gestation and postpartum. For this analysis, specific SNPs were selected that are heterozygous in kidney donor and homozygous (same allele) in recipient and fetal cells. An average minor allele frequency (MAF) was calculated from these selected SNPs. This average represents half of the expected donor cfDNA% due to Hardy-Weinberg principle based on Mendelian genetics. Hence, calculated average MAF was multiplied by factor of 2 to calculate the kidney donor percentage. Results: A 39 year old female with a living related kidney transplant (transplanted September 2018) conceived in February 2021. The patient had a baseline creatinine of 1.4 mg/dl and median dd-cfDNA level of 0.44%. dd-cfDNA levels remained stable throughout pregnancy (median 0.42%, IQR 0.31-0.44%) and were consistent with pre-pregnancy baseline, in keeping with absence of allograft injury. The percentage of fetal cfDNA saw an exponential rise in the first trimester, plateaued in the second trimester and showed a rapid decay post-delivery. Allograft function remained stable throughout pregnancy until development of pre-eclampsia with an acute rise in serum creatine to 2.1 mg/dl, prompting delivery at 31 weeks (Figure 1). Conclusions: The estimated percent of fetal fraction of cfDNA increased with gestational age and rapidly declined post-delivery. Throughout pregnancy, dd-cfDNA levels remained consistent with pre-pregnancy baseline in the absence of graft dysfunction, rejection, or clinical events. This case study suggests that longitudinal monitoring of fetal and dd-cfDNA contributions during pregnancy in kidney transplant recipients is a feasible, novel approach for allograft surveillance in this high-risk patient population.

Extracellular DNA in blood: an index of &lt;i&gt;in vivo&lt;/i&gt; inflammatory response

Increased concentration of cell-free DNA (cfDNA) in the circulating blood of humans and animals is a sign of inflammatory conditions and a distinctive characteristic of various pathophysiological processes in the body. The aim of the present study was to investigate the possible role of tumor necrosis factor (TNFα) in changes of cfDNA contents in peripheral blood as a response to experimentally induced systemic inflammation.We used 40 female hybrid mice (C57Bl/6xDBA/2) F1 at the age of 6-8 weeks. The concentration of cfDNA and its individual fractions was determined using a PicoGreen fluorescent dye. The dynamics of inflammatory process was evaluated after 4, 8, 11 and 24 hours following LPS injection. A significant increase in the blood plasma cfDNA levels was shown under the action of E. coli lipopolysaccharide (LPS), along with simultaneous decreased levels of cfDNA, associated with cell surface. The ratio of cell surface-bound cfDNA to the total cfDNA contents was reduced in dose-dependent manner as early as 4 hours after LPS injection to the animals, thus allowing us to consider this ratio a characteristic sign of netosis of neutrophilic granulocytes during the development of acute inflammation. The described effects are significantly suppressed with co-injection of recombinant TNFα neutralizing protein along with LPS, whereas increased intake of neutrophils in the tissues is determined by some other factors which are not directly related to the production of this cytokine.Based on the obtained data, we proposed a following hypothesis: induction of netosis by inflammatory stimuli causes an increase in the concentration of cfDNA in blood plasma not only due to de novo emerging extracellular DNA by neutrophil netosis, but also by the release of distinct cfDNA fraction that was previously firmly bound to cell membranes in multiple body tissues under the action of proteases released during netosis.

Abstract 3738: Monitoring chemotherapy response status of colorectal cancer patients using liquid biopsy samples

Abstract Background and aims: The early detection of colorectal cancer (CRC) recurrence and the monitoring of therapeutic response are crucial steps in the determination and modification of treatment strategies. The currently used gold standard tumor markers such as CEA and CA 19-9 and the different imaging techniques have several limitations in many cases. Therefore, we aimed to establish a liquid biopsy-based approach for tracking tumor dynamics in post-operative non-metastatic (n=32) and metastatic (n=23) CRC patients. Methods: Blood samples were collected from each patient before and during chemotherapy, and finally, patients were classified according to disease outcome. Longitudinal investigations of the total amount, global and local (SFRP2 and SDC2 genes) DNA methylation pattern of cell-free DNA (cfDNA) fraction were performed. The plasma concentration of homocysteine was also determined, as it is one of the main components of the DNA methylation process, and its level defines the methylation potential. We examined how the parameters mentioned above were affected depending on the different therapy responses. Results: The average cfDNA amount was significantly higher (p&amp;lt;0.05) in patients with recurrent cancer (30.4±17.6ng) and progressive disease (PD) (44.3±34.5ng) than individuals who achieved remission (REM) (13.2±10.0ng). More than 10% elevation of cfDNA from first to last sample collection was detected in 92% of PD patients, while reduced cfDNA concentration was observed in 67% of the non-metastatic CRC patients with REM. An effective differentiation was detected between patients achieving remission and showing tumor progression based on cfDNA level with 94% sensitivity and 81% specificity. The average global cfDNA methylation was determined by bisulfite pyrosequencing analysis of long interspersed nuclear element-1 (LINE-1), and it was significantly lower (p&amp;lt;0.05) in the PD group compared to people with remission (71.0±6.7% vs. 78.9±2.0%). Methylation level changes between the study beginning and end indicated a decline (75.5±3.4% vs. 68.2±8.4%) in PD; in contrast, we found a reverse change in remission. The mean relative change of homocysteine concentration revealed an opposite trend with the global DNA methylation suggesting a linkage between these parameters, as it showed an increase (+12.5%) in the case of PD and a decrease (-15.8%) in patients with REM. Regarding local DNA methylation, SFRP2 and SDC2 genes revealed higher promoter hypermethylation in the PD set compared to patients with remission. Conclusion: Our study offers the possibility to monitor the therapeutic response during chemotherapy with a minimally-invasive blood-based method that combines the analysis of cfDNA, its global and local DNA methylation pattern, and homocysteine level. Citation Format: Barbara Kinga Bartak, Tamás Fodor, Alexandra Kalmár, Zsófia Brigitta Nagy, Sára Zsigrai, Krisztina Andrea Szigeti, William Kothalawala, Gábor Valcz, Péter Igaz, István Takács, Magdolna Dank, Béla Molnár. Monitoring chemotherapy response status of colorectal cancer patients using liquid biopsy samples [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 3738.

Using nucleosome levels, copy number alterations, and DNA methylation to profile disease state through liquid biopsy.

e15014 Background: Nucleosomes are the repeating unit of chromatin that contain important signals for proper genomic function and transcriptional activity. Upon cell death, chromatin is fragmented, and nucleosomes are released into circulation and thus, detectable in liquid biopsy. Cell death naturally occurs over time and increases in a variety of diseases resulting in elevated levels of circulating nucleosomes and the genetic and epigenetic signatures they carry. These signatures contain important information about the cells from which they were derived, as well as the reason for them being in circulation. Methods: We measured circulating nucleosome levels, using Volition’s Nu.Q H3.1 sandwich ELISA across a healthy cohort spanning 40-85 years of age as well as patients whom had been diagnosed with Non-Hodgkin's Lymphoma (NHL), some of which were untreated and others that had undergone treatment. Additionally, to demonstrate that the correlation between nucleosome (Nu.Q) levels and copy number alterations is specific to cancer, we assessed the genomic integrity of an NHL sample with a Nu.Q level of 744 ng/ml and a sepsis sample with Nu.Q level of 3,281 ng/ml using low coverage Oxford Nanopore whole genome sequencing to identify genomic amplifications and deletions. We further used cell type specific methylation patterns to identify the cell of origin of cfDNA in each sample. Results: Importantly, we found no significant correlation between nucleosome level and age in our healthy cohort (R2= 0.0004; 40-49: N = 10, mean = 39 ng/ml; 50-59: N = 10, mean = 18 ng/ml; 60-69: N = 10, mean = 35 ng/ml; 70-79: N = 15, mean = 34 ng/ml; 80-85: N = 5, mean = 32 ng/ml). We did however find elevated nucleosome levels in untreated NHL patients (N = 4, mean 646 ng/ml) and that nucleosome levels in NHL patients that had undergone treatment were reduced (N = 29, mean = 180 ng/ml). Using copy number changes, we found that the fraction of cfDNA estimated to be derived from the tumor in the NHL sample was 54% and were able to detect amplifications in chromosomes 10, 11 and 18 and deletions in chromosomes 1, 8, 17, 18 and 23, whereas we found no copy number alterations present in the cfDNA sample derived from a sepsis patient and a tumor fraction of 0. Furthermore, using cell type specific methylation patterns to deconvolute the data we found that a large fraction of the cfDNA in the NHL sample was derived from B-cells consistent with the NHL sample being of B-cell origin whereas we determined that the cell of origin of the cfDNA in the sepsis sample was Neutrophils. Conclusions: This data shows that nucleosome levels, as measured by Nu.Q, can be used in conjunction with shallow sequencing and DNA methylation profiles to define tumor fraction and cell of origin in liquid biopsy.

Variation in liquid biopsy cfDNA yield predicted by somatic mutation and clinical phenotypes across primary cancers.

e13553 Background: Liquid biopsy provides a non-invasive alternative to tissue biopsy by profiling the genetic mutations and biomarker characteristics of cell-free DNA (cfDNA) derived from primary and metastatic tumors found in patients’ peripheral blood. Previous studies have demonstrated significant variation in cfDNA and circulating tumor DNA (ctDNA) yield based on patient age, sex, primary tumor type, disease stage, and treatment regimen. Somatic mutations are a primary determinant of tumor phenotype, characterizing therapeutic sensitivity and resistance, as well as growth and metastasis rates. However, there has not been extensive investigation as to how presence of somatic mutations affect downstream cfDNA yield and ctDNA fraction. Methods: We screened 140,000 samples analyzed using Guardant360 liquid biopsy test for patient age, diagnosis, treatment, mutations, and associated cfDNA yield (from assay input of 3-10ml peripheral blood, 1 streck tube). We selected and grouped somatic mutations based on prevalence across the cohort. Estimated ctDNA fractions of cfDNA yields were made using the maximum mutant allele frequency (MAF) from among somatic mutations. Samples were organized by phenotype and cfDNA yield and ctDNA fraction was assessed across groups using the Wilcoxon and Kruskal-Wallis for bi-variate and multivariate comparisons, respectively. To understand impact of cfDNA yield variation on assay performance, group comparisons were further evaluated on samples binned by input cfDNA (&lt; 5ng, 5-15ng, 15-30ng, &gt; 30ng) representing cfDNA input requirement ranges current commercial liquid biopsy tests advertise. Results: We found significant yield variation in sample cfDNA and estimated ctDNA fraction between primary disease types ( Myield diff = 16.35ng, p &lt; 0.0001), therapy histories ( Myield diff = 1.49ng, p &lt; 0.001), and patient age groups ( Myield diff = 2.24ng, p &lt; 0.0001), although this finding did not extend to comparisons within input yield bins. We found significant differences in cfDNA and ctDNA fraction across several common somatic mutations, adjusting for test multiplicity, with the greatest difference between TERT splice + and - patients ( Myield diff = 8.57ng, p &lt; 0.0001). Further, we found significant and strong correlations (𝝆 &gt; 0.8, p &lt; 0.001) between EGFR exon 19 deletions and EGFR L858R MAF and estimated ctDNA fraction, and moderate correlation between MAF and cfDNA (𝝆 &gt; 0.25, p &lt; 0.001). Conclusions: Our results indicate that input cfDNA and ctDNA fraction varies by patient age, diagnosis and treatment, as well as with the presence of several common somatic mutations. The demonstrated correlation between somatic mutation presence and cfDNA yield offers a potential explanation for the broad variation of cfDNA yields within tumor and patient phenotypes, and may help inform blood collection strategies for patients with known somatic variants or diagnoses.

C53 DONOR–DERIVED CELL–FREE DNA (DD–CFDNA)–BASED ASSESSMENT OF CORONARY ALLOGRAFT VASCULOPATHY IN HEART TRANSPLANTED PATIENTS

Abstract Introduction Cardiac Allograft Vasculopathy (CAV) is a leading cause of long–term graft dysfunction and failure after Heart Transplant (HTx). Early diagnosis is crucial to tailor therapeutic strategies. Despite advancements in cardiac imaging, the standard for CAV diagnosis remains coronary angiography. Donor–derived cell free DNA (dd–cfDNA) is a novel marker of graft injury in solid organ transplants. Elevations in dd–cfDNA levels may reflect CAV development during subclinical inflammation, thus it could represent a non–invasive method of surveillance for CAV rejection in apparently stable post–transplant patients. Given these premises, we decided to evaluate the potential of dd–cfDNA as alternative diagnostic biomarker for CAV. Materials and Methods 31 blood samples from patients who underwent heart transplant at our institution in the last 10 years, were analyzed by Next Generation Sequencing (NGS) according to CareDx Alloseq protocol. The cfDNA was extracted starting from 2mL of plasma (Qiagen). Sequencing was performed on the Illumina Miseq platform. Troponin T (hs–TnT) and NT–proBNP (Brain Natriuretic Peptide) were measured by automated immunoassay analysers. All statistics were performed by STATA software and significance was set at p &amp;lt; 0.05. Results Heart transplant recipients were divided into 2 groups: 16 patients with known CAV status and 15 patients who had not (no–CAV). No significant differences between the two populations were observed, aside from time from HTx (177.28± 84.99 months in CAV vs 93.20 ± 84.39 months in no–CAV; p = 0.033). A significant difference in dd–cfDNA fraction was found between CAV and no–CAV patients (p = 0.03); with mean values of 0,41% e 0,13%, respectively. Neither NT-proBNP (p = 0.08) nor hs–TnT (p = 0.31) concentrations were significantly different between the two groups, but a positive correlation was found between the two biomarkers (r 0.6966; p = 0.0013). A trend of linear relationship emerged between cfDNA and NT-proBNP levels (r 0.4155; p = 0.08). Conclusions With this study, we demonstrated the feasibility of evaluating dd–cfDNA in long–term heart transplanted patients and its possible association with CAV development. Further investigations are warranted to explore a possible association between dd–cfDNA levels other circulating biomarkers and CAV progression. The definition of “homogeneous” subgroups of patients according to a “risk score” will allow to better manage their follow up and improve their prognosis.

Sensitive screening of single nucleotide polymorphisms in cell free DNA for diagnosis of gestational tumours

Tumours expressing human chorionic gonadotropin (hCG), the majority of which are difficult to biopsy due to their vascularity, have disparate prognoses depending on their origin. As optimal management relies on accurate diagnosis, we aimed to develop a sensitive cell free DNA (cfDNA) assay to non-invasively distinguish between cases of gestational and non-gestational origin. Deep error-corrected Illumina sequencing of 195 common single nucleotide polymorphisms (SNPs) in cfDNA and matched genomic DNA from 36 patients with hCG-secreting tumours (serum hCG 5 to 3,042,881 IU/L) and 7 controls with normal hCG levels (≤4 IU/L) was performed. cfDNA from confirmed gestational tumours with hCG levels ranging from 1497 to 700,855 IU/L had multiple (n ≥ 12) ‘non-host’ alleles (i.e. alleles of paternal origin). In such cases the non-host fraction of cfDNA ranged from 0.3 to 40.4% and correlated with serum hCG levels. At lower hCG levels the ability to detect non-host cfDNA was variable, with the detection limit dependent on the type of causative pregnancy. Patients with non-gestational tumours were identifiable by the absence of non-host cfDNA, with copy number alterations detectable in the majority of cases. Following validation in a larger cohort, our sensitive assay will enable clinicians to better inform patients, for whom biopsy is inappropriate, of their prognosis and provide optimum management.

Bronchoalveolar Lavage Fluid (BALF) as a Compartment for Donor-Derived Cell-Free DNA (dd-cfDNA) Assessment in Lung Recipients

<h3>Purpose</h3> Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute lung rejection. It is unknown whether BALF may represent a more sensitive compartment for dd-cfDNA analysis in lung recipients. Additionally, the relative contribution of the recipient or donor to the BALF cfDNA content remains uncertain. We sought to understand the feasibility of distinguishing dd-cfDNA in the BALF and determine the greatest contributor of BALF cfDNA. <h3>Methods</h3> The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with paired BALF and plasma samples (n=320 each). Total cfDNA was isolated from ∼1mL of sample using automated nucleic acid purification. The % cfDNA contributed by the donor vs. recipient was distinguished in plasma and BALF samples using a next generation sequencing (NGS) assay that measures single nucleotide polymorphisms (SNPs) followed by bioinformatics algorithms. Because these algorithms were developed on plasma, for each BALF sample the SNP typing from the paired plasma sample was used to verify the accuracy of the donor vs. recipient cfDNA contribution in the BALF. <h3>Results</h3> Applying standard QC filters, 168 BALF samples "passed" and generated a dd-cfDNA result while 96 "failed" and did not generate a dd-cfDNA result. 45 samples yielded insufficient total cfDNA to support NGS while 11 generated a potentially invalid dd-cfDNA result. To understand reasons underlying inability to estimate BALF dd-cfDNA, we compared the volume of BALF used for total cfDNA isolation and the total cfDNA yield from the BALF in passed vs. failed samples. The BALF volume was similar between these groups, however the total cfDNA yield was notably less among samples that failed (median total cfDNA 0.38ng/uL in failed vs. 1.42ng/uL in passed samples). Considering only passed BALF samples, the median (25^th, 75^th percentile) fraction of cfDNA contributed by the donor was 6.95% (2.40%, 14.00%), with the remainder contributed by the recipient. <h3>Conclusion</h3> Our data point to challenges in consistently generating dd-cfDNA data from the BALF and suggest the dilute nature of BALF may be a contributing factor. Additionally, these results indicate that the recipient, rather than the donor, is the largest contributor to the BALF cfDNA content potentially reflecting intragraft recipient immune cells.