Cf DNA Research Articles

Quantification of Tumor-Associated Cell-Free HPV DNA in Surgical Drain Fluid Equates to Molecular Residual Disease and Correlates with High-Risk Pathology

<h3>Purpose/Objective(s)</h3> Multi-institutional efforts aimed at safely de-escalating treatment in human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPC) patients rely on subjective pathology for risk stratification. While plasma HPV assays are emerging for post-treatment disease surveillance, they have poor sensitivity to detect postoperative molecular residual disease (MRD). Here we present a liquid biopsy assay of the serosanguinous fluid removed from surgical drains, proximal to the surgical site, to sensitively measure postoperative MRD. We hypothesize that HPV quantified in surgical drain fluid (SDF), 1) represents post-operative regional MRD, and 2) could improve patient risk-stratification to guide precision radiotherapy. <h3>Materials/Methods</h3> We collected SDF at 24-48 hours post-surgery for cell-free (cf) DNA extraction. Our 87-patient head and neck cancer cohort included 74 HPV(+) and 8 HPV(-) OPCs, and 5 benign tumors. Paired plasma and tumor samples were collected from approximately half of all patients. We used the HPV16 E6T2aE7 plasmid to compare our Taqman quantitative PCR (qPCR) to digital droplet PCR (ddPCR). We validated HPV(+) OPC PCR results using next-generation HPV sequencing (NGS) in 9 patients. Then we correlated SDF HPV copies to extranodal extension (ENE), AJCC 8^th edition pathological stage, smoking pack-years, recurrence-free survival (RFS), and composite risk scores derived from OPC de-escalation clinical trials. Statistical methods included Fisher's Exact test, Mann-Whitney (MW) test, Kruskal-Wallis (KW) test, and Kaplan-Meier (KM) analysis. <h3>Results</h3> Our HPV qPCR assay's limit of detection was 9.7 copies, similar to what we saw with ddPCR. HPV genotyping was concordant between NGS and PCR, and all SDF cfDNA samples with nondetectable (ND) HPV via PCR were ND via NGS. HPV was detected in 63 of 74 (85%) SDF samples compared to only 7 of 48 (15%) paired plasma samples. Median HPV copies in ENE+ patients' SDF was 15-fold higher than SDF from ENE- patients (P = 0.002). HPV copies also increased with increasing nodal (P = 0.01) but not primary tumor stage (P = 0.4), suggesting post-operative SDF HPV load reflects invasive nodal disease and is a proxy for regional MRD. When we stratified our cohort according to high-risk and low-risk scores drawn from four clinical trials (RTOG 9501, EORTC 22931, ECOG 3311, MC1273), SDF HPV load was consistently higher in patients with trial-defined high-risk pathology (mean AUC = 0.73). Lastly, KM RFS analysis of HPV-high and HPV-low patients, defined by an RTOG 9501-derived SDF classifier, showed HPV-high patients trended toward worse RFS (HR = 3.1; P = 0.1). <h3>Conclusion</h3> Liquid biopsy assessment of SDF, the biofluid most proximal to the nodal environment, enables postoperative regional MRD detection in HPV(+) OPC, and correlates with high-risk nodal pathology and worse RFS. If confirmed, SDF cfDNA analysis could aid traditional pathology and imaging to personalize adjuvant radiotherapy for surgically treated HPV(+) OPC.

Molecular and immunological changes in blood of rats exposed to various doses of asbestos dust.

Asbestos-related diseases are a group of diseases resulting from the inhalation of asbestos fibres and their subsequent deposition in the lung parenchyma, which causes the development of inflammatory and fibrotic processes in the respiratory system. Cases of the disease often occur in the practice of doctors. The purpose of the study was to examine the level of circulating-free mitochondrial DNA (cf mtDNA), pro-inflammatory cytokines, immunological status and structural changes in the lung of rats exposed to various doses of asbestos dust. Immune monitoring was performed using the peripheral blood samples of 40 male Wistar rats exposed and unexposed to asbestos dust. cf mtDNA copy numbers were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and cytokines were determined using a rat Enzyme Linked Immuno Sorbent Assay (ELISA) kit. A comprehensive assessment of the histopathological study performed under exposure to asbestos at a dose of 25mg and 50mg showed the presence of pronounced structural defects in the lung tissue of laboratory rats. The level of cf mtDNA in plasma of rats exposed to asbestos at a dose of 25mg was reliably higher than that of control rats, and animals exposed to asbestos at a dose of 50mg. The highest levels of pro-inflammatory cytokines IL-6 and TNF-a were also observed after asbestos dusting at a dose of 25mg. According to the results of the immunological status obtained, the decrease in the levels of pro-inflammatory cytokines in the blood plasma at 50mg is due to the immunosuppressive effect in the rat immune system at this dose. A positive correlation was found between TNF-a level and copy numbers of cf mtDNA at a dose of 25mg.

The Overview of Perspectives of Clinical Application of Liquid Biopsy in Non-Small-Cell Lung Cancer.

The standard diagnostics procedure for non-small-cell lung cancer (NSCLC) requires a pathological evaluation of tissue samples obtained by surgery or biopsy, which are considered invasive sampling procedures. Due to this fact, re-sampling of the primary tumor at the moment of progression is limited and depends on the patient’s condition, even if it could reveal a mechanism of resistance to applied therapy. Recently, many studies have indicated that liquid biopsy could be provided for the noninvasive management of NSCLC patients who receive molecularly targeted therapies or immunotherapy. The liquid biopsy of neoplastic patients harbors small fragments of circulating-free DNA (cfDNA) and cell-free RNA (cfRNA) secreted to the circulation from normal cells, as well as a subset of tumor-derived circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA). In NSCLC patients, a longitudinal assessment of genetic alterations in “druggable” genes in liquid biopsy might improve the follow-up of treatment efficacy and allow for the detection of an early progression before it is detectable in computed tomography or a clinical image. However, a liquid biopsy may be used to determine a variety of relevant molecular or genetic information for understanding tumor biology and its evolutionary trajectories. Thus, liquid biopsy is currently associated with greater hope for common diagnostic and clinical applications. In this review, we would like to highlight diagnostic challenges in the application of liquid biopsy into the clinical routine and indicate its implications on the metastatic spread of NSCLC or monitoring of personalized treatment regimens.

Targeted DNA methylation from cell-free DNA using hybridizationprobe capture.

Cell-free (cf)DNA signatures are quickly becoming the target of choice for non-invasive screening, diagnosis, treatmentand monitoring of human tumors. DNA methylation changes occur early in tumorigenesis and are widespread, making cfDNA methylation an attractive cancer biomarker. Already a proven technology for targeted genome sequencing, hybridization probe capture is emerging as a method for high-throughput targeted methylation profiling suitable to liquid biopsy samples. However, to date there are no reports describing the performance of this approach in terms of reproducibility, scalability, and accuracy. In the current study we performed hybridization probe capture using the myBaits® Custom Methyl-seq kit on 172 plasma samples and standards to evaluate its performance on cfDNA methylation analysis. The myBaits® assay showed high target recovery (>90%), demonstrated excellent reproducibility between captures (R 2=0.92 on average), and was unaffected by increasing the number of targets in a capture. Finally, myBaits® accurately replicated 'gold standard' beta values from WGBS (average R 2=0.79). The results of this study show that custom targeted methylation sequencing with myBaits® offers a cost-effective, reliable platform to profile DNA methylation at a set of discrete custom regions, with potential applicability to liquid biopsies for cancer monitoring.

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

P14.04.A TrackingTERTpromoter andIDH1mutations in liquid biopsies - suitable biomarkers for disease monitoring in glioma patients?

Abstract Background Mutations within the telomerase reverse transcriptase promoter (TERTprom) and isocitrate dehydrogenase (IDH) account for the most common genetic alterations in gliomas. Each of these mutations impact clinicopathologic diagnosis and course of diseases. While TERTprom mutations are frequently detected in glioblastoma, IHD mutations are assigned to astrocytoma of grade 2-4, thus mostly associated with better prognosis. In the era of precision oncology, molecular profiling and continuous monitoring of treatment response or relapse are of increasing importance. Accordingly, this study aims to detect TERTprom and IDH mutations in plasma-derived cell-free (cf)DNA of gliomas. The mutant allele frequencies (MAF) will be compared retrospectively to clinico-pathological parameters including extent of resection and tumor progression. Material and Methods Digital droplet PCR (ddPCR) analyses were performed using the QX200TM Digital Droplet System from BioRad. First, to evaluate probes for ddPCR, genomic DNA of several brain tumor cell models (n=6) and tumor tissue (n=1), as well as cfDNA of plasma (n=3) from samples with known TERTprom and IDH mutation status was investigated. For detection of IDH mutations, the unique assay ID dHSaMDV2010055 (IDH1p.R132H) and for TERTprom mutations the TaqMan dPCR Liquid Biopsy Assays for C228T (Hs000000092) and C250T (Hs000000093) were used. The results of ddPCR were analyzed with QuantaSoftTM software and the MAF was calculated Results To validate the detection method for IDH1R132H, we analyzed the MAF in one tissue and corresponding plasma sample of a confirmed IDH1-mutated astrocytoma. In addition, plasma from one astrocytoma grade 2-3 as well as from an IDH1-mutated glioblastoma was tested. Interestingly, both astrocytoma cases exhibited undetectable or very low MAF ranging from 0.1 to 1% in tissue as well as in plasma samples, while in plasma from the high-grade glioblastoma case, IDH1R132H was detected with a frequency of 1.9%. Due to the high GC content of the TERT promoter region, amplification steps are challenging. Accordingly, we first optimized ddPCR conditions for C228T and C250T probes by adding 7-deaza-2-deoxyguanosine-5-triphosphate (7-ddGTP) in varying concentrations to each ddPCR reaction. When using 4µM of 7-ddGTP per sample, a clear separation between mutant and wild-type droplets was reached, detecting MAF between 36-63% in DNA from cell culture models. Conclusion Within this pilot study we optimized the ddPCR method for the detection of IDH1R132H and TERTprom mutations in plasma and tissue samples. Subsequently, we hypothesize that these mutations are suitable liquid biomarkers correlating with extent of resection and tumor progression in gliomas.

1650P The interplay of peripheral circulating free DNA and platelet-to-lymphocyte ratio in patients with differentiated thyroid cancer with/without type 2 diabetes mellitus undergoing radioiodine therapy

Type 2 diabetes mellitus (T2DM) and differentiated thyroid cancer (DTC) are both endocrine system diseases. It has been found that DTC incidence in women with T2DM is constantly growing. T2DM is classified as a metabolic inflammatory disease with an altered number and function of immune cells, both innate and acquired immunity. Circulating cell-free (cf)DNA corresponds to cell-free circulating DNA fragments released in the bloodstream through inflammation, necrosis, apoptosis, or active secretion by nucleated cells such as lymphocytes.

1094P The clinical potential of circulating-free DNA (cfDNA) for real-time longitudinally monitoring clinical outcomes in a real-world first-line non-small cell lung cancer (NSCLC) prospective study

In this real-word study, we prospectively evaluated longitudinal plasma samples to investigate the potential of cfDNA kinetics as early marker of therapeutic efficacy and predictor of prolonged survival in advanced NSCLC patients undergoing standard first-line treatments.

242.2: Is There Any Role of Combined Use of Gene Expression Profiling and Donor Derived Cell Free DNA in Worsening Renal Allograft Function

Introduction: There are multiple noninvasive tests available to assess risk of renal allograft rejection. Donor-derived cell-free DNA (dd cf DNA) test is used to assess rejection in the settings of worsening renal allograft function while Gene profiling is used to assess the subclinical rejection in the settings of stable graft function. OmniGraf™ is a combination of Gene profiling and dd cf DNA tests and used for diagnosing possible subclinical rejection in the settings of stable renal allograft function. But not much has been observed in the settings of worsening renal allograft function. Here we are presenting our study aiming to correlate OmniGraf™ result with the biopsy outcomes. Methods: We selected 40 patients who had a biopsy either for cause or for protocol along with OmniGraf™ and serum creatinine checked around the same time of the biopsy. Patients were divided into 2 major groups, 1.protocol, and 2. For-cause biopsy, later subdivided into 4 groups depending on the OmniGraf™ result combinations. TX is considered No Rejection, Non-TX is considered increased risk of subclinical rejection.TRAC is the dd cf DNA test, High TRAC more than 0.7% is considered increased risk of rejection. Result: In the protocol biopsy group 12 out of 19 patients who had TX with normal TRAC had rejction on the biopsy.The normal result of OmniGraf™ TX with normal TRAC was supposed to predict no rejection, so if biopsy was not performed it might have missed 57% of rejection.This suggest that the normal result does not predict absence of rejection. The subgroup NonTX with Normal TRAC had 66% patients showing rejection,showed 100% rejection. This suggests that Non TX, irrespective of TRAC level is good in predicting possible rejection, irrespective of the type of rejection.In the for-cause biopsy group, the subgroup 2 of 2 patients with TX and normal TRAC had rejection on biopsy. Normal result of OmniGraf™ is not reliable to rule out rejection. The subgroup TX with high TRAC is good in capturing rejection for 6 out of 6 patients. This suggest that Tx may not be useful in this group in predicting rejection as dd cf DNA test, alone might have been able to predict the rejection. The subgroup Non TX with normal TRAC is showing 37.5 % patients, positive for rejection on biopsy while NON TX with high TRAC group has 60% of patients with rejection. This suggest that Non-TX part is good in predicting increasing risk of rejection in both groups irrespective of the High TRAC. Conclusion: Checking a single OmniGraf™ test to evaluate the risk without doing biopsy will miss subclinical rejection.It is probably more useful in serial monitoring of OmniGraf™ than one single value to capture subclinical rejection. Our data are limited to suggest clinical or statistical relevance, It would be useful to evaluate these results on a larger scale to understand the usefulness of this test in kidney transplant recipients with worsening renal allograft function.

Multisite assessment of the impact of cell-free DNA-based screening for rare autosomal aneuploidies on pregnancy management and outcomes.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

External Quality Assurance programs for processing methods provide evidence on impact of preanalytical variables

An annual External Quality Assurance (EQA) program has been provided to processing laboratories over the last ten years, allowing them to assess the performance of their processing methods, such as nucleic acid extractions or peripheral blood mononuclear cell (PBMC) isolation and cryopreservation. The objective of this study was to perform a global analysis on almost 1000 EQA scheme/participant data in order to assess (i) the impact of critical preanalytical factors on quantitative or qualitative attributes of different types of specimens and (ii) laboratory performance pattern over time. Statistical analysis was performed within each EQA scheme based on categorized preanalytical data provided by the participants and on centralized measurements of relevant quality attributes of the produced specimens (z-scores): DNA, cell-free (cf)DNA or RNA extraction from blood, DNA or RNA extraction from formalin fixed tissue, DNA or RNA extraction from frozen tissue, DNA extraction from saliva or stool, viable PBMC isolation and cryopreservation. The most critical preanalytical factors in nucleic acid extraction schemes were the nucleic acid extraction method and kit, the elution buffer, the enzymes used during extraction, the input material quantity and the storage temperature. Several indications of laboratory performance improvement over time could be seen. The conclusions are that EQA for processing methods provides unique evidence-based insights into the impact of preanalytical factors and the comparative performance of different processing methods and kits, while supporting laboratories in validating their processing methods.

Diagnostic and Therapeutic Potential of Circulating-Free DNA and Cell-Free RNA in Cancer Management.

Over time, molecular biology and genomics techniques have been developed to speed up the early diagnosis and clinical management of cancer. These therapies are often most effective when administered to the subset of malignancies harboring the target identified by molecular testing. Important advances in applying molecular testing involve circulating-free DNA (cfDNA)- and cell-free RNA (cfRNA)-based liquid biopsies for the diagnosis, prognosis, prediction, and treatment of cancer. Both cfDNA and cfRNA are sensitive and specific biomarkers for cancer detection, which have been clinically proven through multiple randomized and prospective trials. These help in cancer management based on the noninvasive evaluation of size, quantity, and point mutations, as well as copy number alterations at the tumor site. Moreover, personalized detection of ctDNA helps in adjuvant therapeutics and predicts the chances of recurrence of cancer and resistance to cancer therapy. Despite the controversial diagnostic values of cfDNA and cfRNA, many clinical trials have been completed, and the Food and Drug Administration has approved many multigene assays to detect genetic alterations in the cfDNA of cancer patients. In this review, we underpin the recent advances in the physiological roles of cfDNA and cfRNA, as well as their roles in cancer detection by highlighting recent clinical trials and their roles as prognostic and predictive markers in cancer management.

Combined Liquid Biopsy Methylation Analysis of CADM1 and MAL in Cervical Cancer Patients.

Simple SummaryIn cervical neoplasia, tissue methylation of CADM1 and MAL has been identified as a promising triage tool for HPV-positive women. Here, we performed an exploratory study on the potential of the methylated genes as combined markers for the analysis of cervical smears and peripheral blood samples in cervical cancer patients. The combination of both markers in liquid biopsy resulted in a sensitivity of 83.3% and specificity of 95.5% for discrimination of cervical cancer from healthy controls. The robustness of the assay was proven by a respectably high sensitivity in stage I-II patients.Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.

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.

OTHR-23. Development of a cerebrospinal fluid liquid biopsy platform in pediatric patients with central nervous system tumors

Abstract Pediatric central nervous system (CNS) tumors carry high morbidity and mortality. The advent of targeted agents is aiming to change that through individualized, less invasive, and real-time molecular profiling. We successfully developed a cerebrospinal fluid (CSF) liquid biopsy (LB) platform combining low-pass whole genome sequencing (WGS) and targeted mutation and gene fusion analysis of cell-free (cf) DNA. WGS libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of input cfDNA and paired-end sequenced (Illumina NextSeq 500) for an average 0.5x coverage across the genome. Copy number (CN) profiles were analyzed using the ichorCNA R package and compared with CN abnormalities of the primary tumors detected with CytoScanHD or Oncoscan arrays. Selected cases were analyzed for mutations or gene fusions using a custom glioma hybrid capture-based panel (Twist Bioscience) sequenced to 500-1000x across targeted regions. Mutations identified in LBs were compared with primary tumor OncoKids NGS panel results. CSF LBs from 44 tumor cases and 15 non-tumor controls were analyzed. Sixteen primary tumors had copy number changes, 10 had cfDNA detected in the LB across various primary tumor diagnoses (AT/RT, diffuse midline glioma, ependymoma, high-grade glioma, medulloblastoma, pilocytic astrocytoma). Four primary tumors had sequence alterations from the custom panel, two with detectable cfDNA (diffuse midline glioma H3K27M variant allele frequency (VAF) 49% and low-grade glioma BRAFV600E VAF 1.7%). Three patients had TP53 variants detected, all ultimately germline. Controls were negative across WGS and hybrid gene capture. Tumor DNA is detectable by both WGS and hybrid gene capture. Various histologic subtypes are detectable including embryonal and glioneuronal. CSF seems more enriched for cfDNA in high grade versus low grade tumors. These data suggest that NGS-based LB assays may be used to monitor pediatric patients with a variety of CNS tumors, from diagnosis through treatment and recurrence.

PATH-09. Liquid biopsy of cerebrospinal fluid enables detecting and monitoring ofMYC/MYCN amplification in pediatric CNS malignancies

Abstract In the era of precision oncology, rapid molecular profiling as well as continuous monitoring of response to treatment or relapse are of increasing importance. We investigated digital droplet (dd)PCR for cerebrospinal fluid (CSF) derived cell-free (cf)DNA detection as a new method for rapid diagnosing of MYC/MYCN amplification in CNS malignancies - a patient collective with a dismal prognosis. Wet lab validated ddPCR probes for MYC were first investigated in D425 cells, a human medulloblastoma cell line with confirmed MYC amplification. Results of the methylation array of patients with stored CSF/serum samples (-80°C) were screened for patients with MYC/MYCN amplification. Five patients with medulloblastoma (group 3/4) and MYC-amplification and one patient with a diffuse glioma and MYCN-amplification were included in this study. ddPCR was performed on cfDNA isolated from CSF or serum. MYC amplification was detected by ddPCR in 28/33 (85%) CSF samples, with 4/5 negative samples being from one patient, and only one sample showing an unsuccessful analysis. MYC amplification was detectable in all longitudinal samples in 4/5 patients. 1/1 sample from the patient with MYCN amplific ation was positive in ddPCR. We were not able to detect MYC amplification in serum samples. Importantly, a turn-around time of only six hours from sample thawing/acquisition to result generation was easily achievable. Concluding, detection of MYC/MYCN-amplification in CSF by ddPCR is feasible in the clinical setting and allows for a rapid molecular diagnosis. MYC-amplification was constantly detectable in 4/5 patients with longitudinal CSF samples, which is in-line with the tumor burden our patients suffered from. Interestingly, the detectability in the patient’s serum was low, presumably due to the lower percentage of cell-free tumor (ct)DNA in serum when compared to CSF. Our results render ddPCR as a promising tool for bed-side molecular diagnosis and disease monitoring.

Cell-free DNA analysis as a molecular tool to monitor response to immune checkpoint inhibition in endometrial cancer.

5592 Background: Immune checkpoint inhibitors (ICI) targeting PD-1 have meaningful activity in microsatellite instability high (MSI-H), hypermutated or mismatch repair deficient (dMMR) advanced endometrial cancer (EC). We investigated if high-depth circulating cell free (cf)DNA sequencing can be used to assess MSI status and monitor ICI response in EC patients enrolled in a phase II trial (NCT03241745). Methods: Patients with recurrent/persistent MSI-H/dMMR/hypermutated EC with measurable disease and ≥1 prior lines of cytotoxic therapy were treated from 06/2018-01/2022 with nivolumab until progression of disease (PD) or unacceptable toxicity. Radiologic tumor response was assessed every 12 wks by RECIST 1.1 criteria.Pre-treatment ECs and matched normal blood-derived DNA were subjected to whole-exome sequencing (WES); cfDNA from plasma at baseline, 2 wks and every 6 wks thereafter was subjected to high-depth MSK-ACCESS sequencing (129 genes). MSI in WES and cfDNA was calculated by MSIsensor and ADMIE, respectively. Results: Ten patients with ≥10 ng cfDNA at baseline and 2 weeks after nivolumab initiation were included. Most (80%) had grade 3 endometrioid EC; 70% had MLH1 hypermethylated EC. Two patients had partial response (PR), 3 had stable disease (SD) and 5 had PD on nivolumab. A high-depth sequencing assay captured somatic mutations in cfDNA at baseline in all patients (median, 29.5; range, 2-75). Median circulating tumor (ct)DNA fraction in cfDNA was 13.1% at baseline (range, 0.0-86.0%). In 8 cases, ctDNA fraction was sufficient to perform MSI assessment. Liquid biopsy MSI status matched tumor MSI status assessed by WES: in 7 patients with MSI-H disease by tumor WES, cfDNA MSI status was also MSI-H ( &gt; 0; range, 0.21-5.46); in 1 patient with MSI-low but hypermutated tumor, cfDNA MSI was 0.0. In all cases, changes in ctDNA fractions reflected ICI response. Median ctDNA fraction percent change from week 0 to week 8 was -51.6% (IQR, -6.2, -85.1) in patients with PR and SD vs +17.7% (IQR, -6.6, +356.6) in patients with PD (p = 0.08). In patients with PR, ctDNA fraction decreased at week 2 and stayed low at week 8 and weeks 32-55 of nivolumab in concordance with ongoing response. In 3 patients with SD, ctDNA fractions decreased by week 2. In 1 patient with SD at week 8 and PD at week 22, ctDNA fraction increased from week 8 to week 20. In 5 patients with PD, 4 had increased ctDNA fraction by week 8. The last patient had a stable ctDNA fraction but increasing allele frequency of 2 truncating B2M alterations, possibly associated with ICI resistance. Conclusions: In patients with advanced hypermutated ECs, cfDNA sequencing can be used to accurately detect MSI status. Early changes in ctDNA fraction may be associated with durable response to ICI or may anticipate radiological progression. Future studies may use ctDNA to assess mechanisms of ICI resistance and offer opportunities for adaptive therapy intervention. Clinical trial information: NCT03241745.

Establishment of 5-hydroxymethylcytosine expression model of circulating-free DNA gene in early diagnosis of nasopharyngeal carcinoma.

e18047 Background: Nasopharyngeal carcinoma (NPC) has no obvious symptoms in the early stage of onset, and more than 65% of patients have been diagnosed as intermediate and advanced stage. Studies have shown that early diagnosis and timely treatment of NPC play a key role in the survival of patients.5-hydroxymethylcytosine (5hmC) as an important epigenetic modification of DNA is closely related to gene regulation and NPC pathogenesis. Objective: To clarify the diagnostic value of 5hmC expression in circulating free DNA (cfDNA) gene for NPC. Methods:The expression of 5hmC in cfDNA of 166 NPC and 165 controls was detected with high-throughput technology, and the differentially expressed gene map of 5hmC was drawn. Based on the screened 5hmC Top50 differentially expressed genes, the NPC diagnosis model was established with 70% of the sample size as the training set and 30% as the verification set. The efficacy of 5hmC in distinguishing NPC from healthy controls was evaluated by drawing the receiver operating characteristic curve (ROC) and calculating the sensitivity, specificity, area under the curve (AUC) and accuracy rate (AR). Results: High throughput detection showed that there were significant differences in 5hmC expression of 194 genes between NPC and control cfDNA, of which 185 genes were up-regulated and 9 down regulated in NPC. The results of diagnostic test show that when the Youden index threshold of 0.4749 at the verification set is taken as the cut-off value, the sensitivity, specificity, AUC and AR of the training set are 0.7652, 0.7304, 0.8250 and 0.7478, while in the verification set, they are 0.6863, 0.7400, 0.7388 and 0.7129 respectively. Conclusions: Our findings suggest that cell-free 5hmC signatures are valuable as potential noninvasive diagnostic biomarkers of NPC, which can be transformed into clinical application by further optimizing the diagnostic model.

1. Clinical utility of low-pass whole genome sequencing (LP-WGS) and targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors

We designed a liquid biopsy (LB) platform combining LP-WGS and targeted mutation and gene fusion analysis of cell-free (cf) DNA for diagnosing and monitoring pediatric patients with solid tumors. Sample types included cerebrospinal fluid (CSF) (brain tumors), aqueous humor (retinoblastoma), and blood (sarcomas, germ cell and renal tumors). cfDNA was extracted at diagnosis, during therapy, at remission and/or relapse. WGS libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA and paired-end sequenced (Illumina NextSeq 500) for an average depth of coverage across the genome of 3x.