Cell-free Circulating Tumor DNA Research Articles

Abstract A004: Clinical test design affects tumor tissue and ctDNA FGFR gene status in metastatic urothelial cancer: a prospective study

Abstract Fibroblast growth factor receptor (FGFR) gene alterations are common in urothelial cancer (UC) and targetable with erdafitinib. The co-approved companion diagnostic is an amplicon-based tumor tissue test that detects a limited number of recurrent FGFR alterations. However, FGFR status may change during disease progression, and rare FGFR alterations not typically covered by existing tests may be of clinical importance. Therefore, in a prospective pan-Canadian study (NCT06129084), we compared standard archival tissue testing to broad targeted sequencing of cell-free circulating tumor DNA (ctDNA) to detect FGFR alterations in UC. Eligible patients had progressing metastatic UC and were undergoing tissue FGFR testing. Blood was drawn at study enrollment and plasma cell-free DNA and matched white blood cell DNA underwent deep-targeted sequencing with a hybridization capture panel including the introns and exons of FGFR1-4 and the common breakpoint region of TACC3. Archival tissue selection and testing was initiated by the treating physician and typically used the Oncomine Focus assay. 210 patients were enrolled across 12 sites, and 140 patients have tissue and ctDNA results available for concordance assessment. 43/140 (31%) of cell-free DNA samples had low tumor fraction (<0.5%) and were considered inconclusive. For the remaining 97 same-patient tissue and ctDNA pairs, FGFR status was identical in 88 (91%). In 7 of 9 discordant cases, the tissue was negative, but ctDNA was positive for FGFR alterations: these were enriched for FGFR3-TACC3 fusions (4/7). Two discordant fusions had canonical breakpoints that can be identified by current amplicon-based companion diagnostics, indicating potential spatiotemporal heterogeneity in somatic FGFR status. The other two discordant (ctDNA-only) fusions had non-canonical breakpoints that are not amenable to detection by current amplicon-based companion diagnostics. In two mutation discordant cases (S249C positive in ctDNA only), lack of signal in tumor tissue could be explained by the presence of additional FGFR3 variants within 25bp of S249 and in cis - disrupting primer binding sites. There were only two cases of tissue-only FGFR alterations among the 97 evaluable pairs (FGFR3-TACC3 fusion, S249C). ctDNA testing of metastatic UC with a broad capture-based approach covering all exons and introns of FGFR genes can identify additional alterations not detected with current amplicon-based companion diagnostics applied to archival tissue. Our results support ctDNA testing as a valuable adjunct to tissue testing, but not as a replacement due to the high frequency of inconclusive ctDNA results, secondary to tumor fraction below the threshold. Assay design, including target enrichment technique, is a significant source of variability in tissue and ctDNA FGFR status and should be carefully considered in future clinical biomarker testing strategies. Citation Format: David C. Müller, Gillian Vandekerkhove, Andrew J. Murtha, Jack V.W. Bacon, Carlos Vasquez-Rios, Maria Stephenson, Kimia Rostin, Elena Schönlau, Connor Wells, Sunil Parimi, Krista Noonan, Naveen S. Basappa, Jenny J. Ko, Daygen Finch, Nimira Alimohamed, Tarek A. Bismar, Gang Wang, Andreas Papadakis, Lucia Nappi, Matti Annala, Cecily Q. Bernales, Alan Spatz, Kim N. Chi, Alexander W. Wyatt, Bernhard J. Eigl. Clinical test design affects tumor tissue and ctDNA FGFR gene status in metastatic urothelial cancer: a prospective study [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A004.

Abstract PO2-13-02: Detection of SPEN mutations in advanced breast cancer by circulating tumor cell-free DNA

Abstract Background: Alterations in the SPEN gene are rare in primary breast cancer (~3%, ref: TCGA). SPEN is a hormone-inducible transcriptional repressor with known functions in orchestrating X-inactivation in females. Additionally, SPEN has been implicated as a potential estrogen receptor co-repressor and molecular partner of NCOR2 as well as epigenetic modifiers such as KMT2D and HDACs via C-terminal interactions. SPEN loss-of-function is associated with tamoxifen resistance in preclinical models. However, the landscape of SPEN alterations in advanced breast cancer remains poorly described. The primary objective of this study was to evaluate the frequency and different types of SPEN alterations in metastatic breast cancer (MBC). Methods: A query was performed on all patients at an academic institution with available genomic testing from Guardant360, a next-generation sequencing cell-free DNA (cfDNA) assay, evaluating 500 genes in total. Patients with MBC were identified, and specific alterations in SPEN as well as co-alterations were extracted. Silent and germline mutations were excluded from analysis. Retrospective review was conducted to determine clinicopathologic characteristics such as age, hormone and HER2 receptor status, menopausal status, and therapy at time of the detected mutation. Results: Among 366 patients with MBC and available cfDNA, 8.5% (n=31) had an alteration detected in the SPEN gene. Median age of patients with SPEN mutations was 66 years (range 33-92). A majority of patients (90%) had HR+ disease (n=28/31), with HR-/HER2+ disease and TNBC additionally representing 3% (n=1/31) and 6% (n=2/31) of cases, respectively. SNVs were most frequent, comprising 89% (n=42/47) of all alterations. Indels were additionally identified in 11% (n=5/47), and no CNVs or fusions were noted. Nonsense and frameshift mutations represented 8/47 (17%) and 2/47 (4%) of all alterations respectively. No recurrent mutational hotspots were detected. The most common co-alterations in descending order were PIK3CA, TP53, ERBB2, ARID1A, MDM4, PPM1D, NF1, DNMT3A, ATM, and ESR1. Association between SPEN mutations and clinical outcomes will be presented at the meeting. Conclusions: SPEN mutations were detected mostly in patients with HR+/HER2- MBC and at a higher frequency than reported in primary disease via TCGA. SNVs were common, though it remains unclear whether these comprise driver versus passenger mutations. While no mutational hotspots were identified, several novel loss-of-function truncating mutations were identified, which warrant further validation of functional and clinical significance. SPEN alterations in metastatic breast cancer Alterations detected by cell-free DNA sequencing in patients with metastatic breast cancer and annotated by type of alteration and receptor subtype. Citation Format: Charles Dai, Haley Barnes, Arielle Medford, Annika Putur, Jennifer Keenan, Beverly Moy, Seth Wander, Ryan Corcoran, Aditya Bardia. Detection of SPEN mutations in advanced breast cancer by circulating tumor cell-free DNA [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-02.

Abstract 7030: Reference materials for analysis of DNA methylation in cell-free circulating tumor DNA

Abstract Aberrant DNA methylation is associated with many cancers including breast, liver, bone, and colon and has been identified as a biomarker for early cancer screening. Liquid biopsies are beginning to screen for DNA methylation in cancer-derived DNA, but accurate assessment of methylation is not trivial, and methods like bisulfite sequencing can result in biased data. Reference materials for establishing the analytical validity of measurements of CpG methylation are not widely available. In order to prepare such reference materials, genomic DNA (gDNA) was extracted from the well characterized human reference cell line GM24385 and fragmented to a circulating tumor DNA (ctDNA)-like size distribution. This DNA was processed further to prepare CpG-methylated and unmethylated versions. The size distribution was determined by using the Bioanalyzer High Sensitivity DNA Kit (Agilent, Santa Clara, CA). Methylation status in the reference materials was confirmed by both digital PCR (dPCR) and NGS based methods. A digital PCR method was developed to analyze heterozygous single nucleotide polymorphisms (SNPs) using the Bio-Rad QX200™ Droplet Digital PCR (ddPCR) System. To determine methylation levels by NGS, the NEBNext™ Enzymatic Methyl-seq Kit was used (EM-seq). Paired-end sequencing of the libraries was performed on a NextSeq™ 2000 (Illumina, San Diego, CA). Data alignment to hg19 was done with the bwa-meth (Pedersen, BS., et al.) aligner, and assessment of methylation in the aligned data was done with MethylDackel (Ryan, D.). Each reference material had an average peak size of ~160 bp in length, similar to patient cfDNA. By dPCR, the unmethylated reference material had an average of 0.73% DNA methylation, while the methylated reference material had an average of 100.10% DNA methylation. Values can be above 100% by dPCR due to unequal representation of both alleles. By NGS, the unmethylated reference material had an average of 0.93 % CpG methylation, which was similar to the background of 1.13 % and 1.31 % for CHG and CHH methylation, respectively. The methylated reference material had an average of 93.95 % CpG methylation, with a background of 0.69 % and 0.74 % for CHG and CHH methylation, respectively. Because EM-seq relies on enzymatic protection and conversion steps, it may be that these reactions did not allow for full protection and full conversion, which led to values above 0 % for unmethylated and below 100 % for methylated materials. We have developed reference materials for the assessment of DNA methylation in ctDNA. One unmethylated and one methylated reference material was created. These reference materials can be mixed together to the level of methylation required to help aid in assay design, optimization, and validation. Citation Format: Alysha Higgs, Angelique Ryan, Jayanthi Ramprakash, Dana Ruminski Lowe, Yves Konigshofer, Catherine Huang, Andrew Anfora, Russell Garlick, Bharathi Anekella. Reference materials for analysis of DNA methylation in cell-free circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7030.

Abstract 3672: Development of liquid biopsy HPV (Human Papilloma Virus) cell free DNA (cfDNA) based approach for prognosis of cervical cancer

Abstract Introduction: Recent progress in the analysis of blood samples for circulating tumor cells or cell-free circulating tumor DNA (ctDNA) has shown that liquid biopsies may have potential applications in the detection and monitoring of cancer. Human papillomavirus (HPV) circulating cell-free (ccf) DNA may act as a unique marker for the prognosis of high-risk HPV-related cancers. However, detection of circulating markers for cervical cancer (CC) requires extremely sensitive techniques that can measure circulating human papillomavirus DNA. Here, we used the sensitive droplet digital PCR (dd PCR) based approach for the detection and quantification of circulating human papillomavirus DNA in plasma from patients diagnosed with cervical cancer at baseline and subsequent follow up with intention of validating its utility as a prognostic marker in CC. Method: Blood sera form 34 Patients diagnosed with cervical cancer (Stage 1-1V) at AIIMS Delhi was collected at baseline and after 3 months of the treatment. 10 healthy controls were also recruited in the study after due approval from AIIMS IEC. Plasma samples were stored at -80°C. ccf DNA was isolated from 1 ml of plasma and further processed to detect the presence of circulating high risk Human papilloma virus (HPV) i.e., HPV-16 and HPV18 cf DNA using dd PCR. Result: The median cf DNA concentration was 9.3 ng/uL (range 3-26.6) for all patients while median cf DNA concentration was 7.2 ng/uL (Range 3-57.3) for healthy controls (n=10). Furthermore, the levels of cfDNA decreases post 3 months of treatment with average concentration of 8.07 ng/uL (Range 4.6-19.3). We further processed our samples for dd PCR based screening to detect low copy no HPV cf DNA and ddPCR detection yielded a positive rate of 51% (18/35) for circulating HPV18 DNA while a positivity of 88.5% (31/35) was seen for the circulating HPV16 DNA in the sera of CC patients. By comparing HPV16 and HPV18 genotypes, we observed detection of circulating HPV 16 DNA more frequently than circulating HPV18 DNA in CC patients coming at our center. Furthermore, we found that copies of circulating viral DNA (HPV18 and HPV16) decreased post induction therapy in CC patients indicating progression ability of thid marker. Conclusion: In the current studies we provide evidence that cell free DNA concentration in CC patient can predict tumor burden and detection of low copy no of circulating HPV DNA could be a promising indicator for prognosis in cervical cancer patients and can predict recurrence of the disease. Citation Format: Gunjan Dagar, Ashna Gupta, Ravi Chauhan, Abhishek Shankar, Daya Nand Sharma, Muzafar A. Macha, Ajaz A. Bhat, Rajeev Goyal, Vaishali Suri, Mayank Singh. Development of liquid biopsy HPV (Human Papilloma Virus) cell free DNA (cfDNA) based approach for prognosis of cervical cancer [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 3672.

Circulating Tumor DNA Dynamics Fail to Predict Efficacy of Poly(ADP-ribose) Polymerase/VEGFR Inhibition in Patients With Heavily Pretreated Advanced Solid Tumors.

Cell-free circulating tumor DNA (ctDNA) has shown its potential as a quantitative biomarker for longitudinal monitoring of response to anticancer therapies. However, ctDNA dynamics have not been studied in patients with heavily pretreated, advanced solid tumors, for whom therapeutic responses can be weak. We investigated whether changes in ctDNA could predict clinical outcomes in such a cohort treated with combined poly(ADP-ribose) polymerase/vascular endothelial growth factor receptor inhibitor therapy. Patients with metastatic pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), small-cell lung cancer (SCLC), or non-small-cell lung cancer (NSCLC) received up to 7 days of cediranib 30 mg orally once daily monotherapy lead-in followed by addition of olaparib 200 mg orally twice daily. Patients had progressed on a median of three previous lines of therapy. Plasma samples were collected before and after cediranib monotherapy lead-in and on combination therapy at 7 days, 28 days, and every 28 days thereafter. ctDNA was quantified from plasma samples using a multigene mutation-based assay. Radiographic assessment was performed every 8 weeks. ctDNA measurements were evaluable in 63 patients. The median baseline ctDNA variant allele fractions (VAFs) were 20%, 28%, 27%, and 34% for PDAC, TNBC, SCLC, and NSCLC, respectively. No association was observed between baseline VAF and radiographic response, progression-free survival, or overall survival (OS). Similarly, no association was found between ctDNA decline and radiographic response or survival. However, an increase in ctDNA at 56 days of combination therapy was associated with disease progression and inferior OS in a landmark analysis. ctDNA levels or dynamics did not correlate with radiographic response or survival outcomes in patients with advanced metastatic malignancies treated with olaparib and cediranib.

Paired Circulating Tumor DNA in Cerebrospinal Fluid and Peripheral Blood: Reliable Technology for Diagnosing and Monitoring Primary and Secondary Central Nervous Lymphoma

Objective: This research delineates the temporal alterations of cell-free circulating tumor DNA (ctDNA) in the plasma and cerebrospinal fluid (CSF) of patients harboring primary and secondary central nervous system (CNS) lymphoma. The aim is to establish that CSF serves as a superior reservoir of ctDNA compared to plasma, thereby aiding in diagnostic processes and monitoring therapeutic efficacy. Concurrently, this study underscores the enhanced response and survival rates in patients administered Bruton's tyrosine kinase (BTK) inhibitors over an extended period exceeding three months. Methods: Data including cranial-enhanced magnetic resonance imaging (MRI) scans, leukocyte and protein concentrations in the CSF, exfoliated CSF cytology, flow cytometry, interleukin-10 to interleukin-6 ratios, ctDNA in plasma and CSF, were gathered from patients presenting primary and secondary CNS lymphoma（7 PCNSL,9 SCNSL）. The temporal ctDNA modifications in plasma and CSF during the patient's disease progression-initial onset, remission, and relapse-were scrutinized. Additionally, the implications of BTK inhibitor usage on patient response rates and survival rates were assessed. Results: Within the cohort of primary CNS lymphoma patients, ctDNA was detected in the CSF and plasma of 2 and 5 patients, respectively. Conversely, in patients with secondary CNS lymphoma, CSF and plasma ctDNA were discovered in 8 and 4 cases, respectively. The detection rate was contingent upon the central lesion's magnitude and position and the patient's surgical history related to the brain. Four patients, including two from each group, underwent cranial-enhanced MRI to appraise tumor burden at various disease stages, simultaneously with the monitoring of ctDNA in plasma and CSF. It was revealed that ctDNA levels in the CSF were in synchrony with the tumor burden, whereas plasma ctDNA levels remained relatively static. This indicates that CSF ctDNA may serve as a viable tool to assess therapeutic responses in CNS lymphoma patients. In two patients where surgical biopsy was unfeasible and both CSF exfoliation cytology and flow cytometry returned negative results, CSF ctDNA was positive, thereby underscoring its auxiliary role in diagnosing CNS lymphoma. Furthermore, the study indicated improved response and survival rates in patients subjected to BTK inhibitors for over three months. Conclusion: CSF ctDNA demonstrates potential in detecting CNS lymphoma and in the dynamic monitoring of therapeutic responses. Compared to cranial-enhanced MRI, exfoliated CSF cytology, and flow cytometry, CSF ctDNA testing offers superior sensitivity. For patients for whom brain surgical biopsy is not an option, CSF ctDNA testing emerges as a valuable supplementary diagnostic tool. CSF ctDNA allows for dynamic monitoring of therapeutic responses in CNS lymphoma patients, enabling the detection of residual lesions, and thereby guiding the formulation and duration of the treatment regimen. Patients administered oral BTK inhibitors for more than three months demonstrate higher treatment response and survival rates.

Prospective, Cell-Free Circulating Tumor DNA (ctDNA) Profiling in Locally Advanced Lung Cancer Treated with Chemoradiation

Liquid biopsy for cell-free circulating tumor DNA (ctDNA) allows for non-invasive, comprehensive genomic profiling. We explored the utility prospective liquid biopsy for (ctDNA) in among non-small cell lung cancer (NSCLC) patients treated with definitive chemoradiation. This prospective clinical cohort consists of unresectable, locally advanced NSCLC patients who had liquid biopsy testing prior to initiation of cancer therapy. Liquid biopsy testing was performed using a next-generation sequencing assay (MSK-ACCESS) which includes 129 genes and paired white blood cell sequencing. An FDA-recognized database (OncoKB) was utilized to classify alterations associated with radiation resistance (including KEAP1, NFE2L2, STK11, and PIK3CA) and radiation sensitivity (including ATM, ATR, BRCA1/2, ARID1A, MLH1 and other DNA Damage Repair Pathway alterations). We evaluated progression-free survival (PFS) from the completion of chemoradiation using the Log-rank test. Among 25 patients with prospective ctDNA testing prior to therapy initiation, 17 patients had stage III disease (68%), 8 patients had stage II disease (32%), 18 patients had adenocarcinoma (72%), 7 patients had squamous cell carcinoma (28%), and 23 (92%) were former or current smokers. The median radiation dose was 60 Gy in 30 fractions (range: 55 to 66 Gy in 20 to 33 fractions). 76% of patients (n = 18) had one or more alterations detected (median: 3 alterations, range: 1 - 8) including genomic markers of radiation response in 2 patients in BRCA1/2 (n = 2) and radiation resistance in 1 patient in KEAP1. The most common driver alteration detected was KRAS in 24% of the cohort (n = 6). Among patients with baseline detectable ctDNA, the median PFS was 21.3 months and was not reached among patients without baseline ctDNA detection (HR 4.54, p = 0.04). Using an institutional assay, the presence of baseline cell-free ctDNA appeared prognostic in patients with unresectable, locally advanced NSCLC treated with definitive chemoradiation. We also detected driver alterations and potential markers of radiation resistance and response using ctDNA testing. Prospective cell-free ctDNA profiling may offer pathways to therapy personalization among patients with locally advanced unresectable lung cancer.

Cell-Free Circulating Tumor DNA and Epstein-Barr Virus DNA for Early Diagnosis of Epstein-Barr Virus-Associated Cancers.

Cell-Free Circulating Tumor DNA and Epstein-Barr Virus DNA for Early Diagnosis of Epstein-Barr Virus-Associated Cancers.

Impact of androgen receptor alterations on cell-free DNA genomic profiling on survival outcomes in metastatic castration-resistant prostate cancer.

Androgen receptor (AR) gene alterations, as detected by circulating tumor cell-free DNA (cfDNA) genomic profiling, have been shown to emerge after a variable duration of androgen signaling inhibition. AR alterations were associated with inferior outcomes on treatment with androgen receptor pathway inhibitors (ARPI) in the first line metastatic castration-resistant prostate cancer (mCRPC) setting in a phase 2 trial. Here in, we assessed the impact of these AR alterations on survival outcomes in a real-world patient population of mCRPC experiencing disease progression on an ARPI. In this IRB-approved retrospective study, consecutively seen patients with a confirmed diagnosis of mCRPC, with disease progression on a treatment with ARPIs in the first line mCRPC setting, with no prior exposure to an ARPI in the castration sensitive setting, and with available cfDNA profiling from a CLIA certified laboratory were included. Patients were categorized based on AR status: wild-type (ARwt ) or alteration-positive (AR+ ). The objective was to correlate overall survival (OS) after disease progression on the first-line ARPI with the presence or absence of AR alterations. Kaplan-Meier and Cox Regression Tests were used as implemented in R-Studio (v.4.2). A total of 137 mCRPC patients were eligible: 69 with ARwt versus 68 with AR+ . The median OS posttreatment with the first ARPI was significantly higher for ARwt than AR+ patients (30.1 vs. 15.2 mos; p < 0.001). Of 108 patients who received a subsequent line of therapy, 63 received an alternate ARPI (AR+ 39 vs. 24 ARwt ), while 20 received a taxane-based therapy (11 AR+ vs. 9 ARwt ). Among patients receiving an alternate ARPI, AR+ had numerically shorter OS (16.8 vs. 30.4 mos, p = 0.1). Among patients receiving taxane-based regimens, the OS was not significantly different between AR+ and ARwt (14.5 vs. 10.1 mos, p = 0.18). In this real-world study, mCRPC patients with AR alterations on cfDNA had inferior OS after disease progression on the first ARPI, compared to those who did not, and may impact outcomes on a subsequent ARPI but not on subsequent taxane-based therapy received. By providing survival estimates for patients with or without AR alterations, our data may aid in patient counseling, prognostication, treatment decision, and for designing future clinical trials in this setting.

Molecular associations of response to the new-generation BTK inhibitor zanubrutinib in marginal zone lymphoma.

Using tissue whole exome sequencing (WES) and circulating tumor cell-free DNA (ctDNA), this Australasian Leukaemia & Lymphoma Group translational study sought to characterize primary and acquired molecular determinants of response and resistance of marginal zone lymphoma (MZL) to zanubrutinib for patients treated in the MAGNOLIA clinical trial. WES was performed on baseline tumor samples obtained from 18 patients. For 7 patients, ctDNA sequence was interrogated using a bespoke hybrid-capture next-generation sequencing assay for 48 targeted genes. Somatic mutations were correlated with objective response data and survival analysis using Fisher exact test and Kaplan-Meier (log-rank) method, respectively. Baseline WES identified mutations in 33 of 48 (69%) prioritized genes. NF-κB, NOTCH, or B-cell receptor (BCR) pathway genes were implicated in samples from 16 of 18 patients (89%). KMT2D mutations (n= 11) were most common, followed by FAT1 (n= 9), NOTCH1, NOTCH2, TNFAIP3 (n= 5), and MYD88 (n= 4) mutations. MYD88 or TNFAIP3 mutations correlated with improved progression-free survival (PFS). KMT2D mutations trended to worse PFS. Acquired resistance mutations PLCG2 (R665W/R742P) and BTK (C481Y/C481F) were detected in 2 patients whose disease progressed. A BTK E41K noncatalytic activating mutation was identified before treatment in 1 patient who was zanubrutinib-refractory. MYD88, TNFAIP3, and KMT2D mutations correlate with PFS in patients with relapsed/refractory MZL treated with zanubrutinib. Detection of acquired BTK and PLCG2 mutations in ctDNA while on therapy is feasible and may herald clinical disease progression. This trial was registered at https://anzctr.org.au/ as #ACTRN12619000024145.

Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

MFAST-SeqS based aneuploidy score in circulating cell-free DNA and role as early response marker in patients with metastatic prostate cancer treated with androgen receptor signaling inhibitor.

5058 Background: Several treatment modalities in metastatic castration resistant prostate cancer (mCRPC) are available to prolong progression-free and overall survival. With the arrival of additional treatment modalities, the need for accurate monitoring of treatment response is becoming more eminent and research for biomarkers pivotal. Cell-free circulating tumor DNA (ctDNA) has emerged as a promising prognostic and predictive biomarker. Several techniques have been employed for the detection and characterization of ctDNA within the total pool of cell-free DNA (cfDNA), one of which is the modified Fast Aneuploidy Screening Test-Sequencing System (mFAST-SeqS). This affordable and robust assay enables the quantitation of a sample’s ctDNA using a genome-wide aneuploidy score (GWA). Our aim was to investigate whether estimation of the GWA by mFAST-SeqS can be used to evaluate early response to treatment and predict response to treatment at baseline in patients with mCRPC. Methods: We prospectively enrolled mCRPC patients before start of treatment with androgen receptor signaling inhibitor (ARSI) abiraterone or enzalutamide. Blood samples were collected at baseline before start of treatment (BL, n= 134) and at an early treatment time point during treatment (ET, n= 20). We isolated cfDNA from plasma collected at both time points using the QiaSymphony or Maxwell system, and performed mFAST-SeqS on 1 ng cfDNA by amplifying and sequencing long interspaced nuclear elements (LINE-1) throughout the genome. The GWA-scores were calculated by taking the summed square of individual Z scores per chromosome arm relative to healthy controls. GWA-score was dichotomized in high (GWAhigh) and low (GWAlow), based on the previously described cut-off of 5. Clinical outcome, defined as treatment duration with ARSI, was compared for patients with a GWAhigh and GWAlow score at baseline and at an early time point. In addition, conversion of GWA score from baseline to early time point was explored. Results: Median age at start of treatment was 68.5 years (inter-quartile range (IQR): 63-73). Median time between start treatment and blood draw at BL and ET was 3 (IQR: 0-10 days before treatment) and 28 (IQR: 18-62 days during treatment) days, respectively. Median number of prior therapy lines was 1 (IQR: 0-10). Treatment duration was significantly shorter in the GWAhigh group versus the GWAlow group both at BL and at ET in patients treated with ARSI (BL: p&lt;0.001, log-rank test, median 221 and 82 days; ET: p&lt;0.01, log-rank test, median 123 and 42 days). Conclusions: The genome-wide aneuploidy score, based on mFAST-SeqS of cfDNA, is a useful tool to predict response to ARSI in patients with mCRPC. The current dataset will be extended to 199 samples at baseline and 78 samples at early time point, and also include mCRPC patients treated with taxanes. Clinical trial information: NCT01855477 , NTR732 .

Real-world (RW) outcomes of patients (pts) with advanced breast cancer (aBC) with and without resistance alterations (alts) detected in cell-free circulating tumor DNA (ctDNA) prior to CDK4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) treatment.

1081 Background: CDK4/6i plus ET is standard of care first-line (1L) therapy for hormone receptor positive (HR+), HER2- aBC. While molecular analyses from trials and pts have identified several mechanisms of resistance to CDK4/6i plus ET, currently, no biomarkers (outside of hormone status) are used for therapy selection. We utilized RW data to assess impact of putative baseline CDK4/6i+ET resistance alts (CDK4/6i+ET-R) on clinical outcomes among pts with aBC treated with CDK4/6i. Methods: Pts with aBC were identified via the Guardant INFORM database; those with a ctDNA test within 90 days prior to CDK4/6i start were included in this analysis. Based on previous studies, oncogenic or likely oncogenic alterations (as characterized by OncoKB) in the following genes were considered CDK4/6i+ET-R alts: RB1, PTEN, AKT1, CCNE1, ERBB2, FGFR1, FGFR2, and KRAS. Pts with &gt;1 CDK4/6i+ET-R alt were compared to pts without any CDK4/6i-R alts. Kaplan-Meier plots, log-rank tests (LR) and multivariate Cox proportional hazards models (HR) adjusted (adj) for age, sex, year of ctDNA test and line of therapy were used to assess differences in time to next treatment (TTNT) and overall survival (OS). Results: 1075 pts with aBC treated with CDK4/6i (27% receiving 1L treatment) met inclusion criteria, 293 of whom (27%) had &gt;1 alt in a CDK4/6i+ET-R gene. Pts with CDK4/6+ETi-R alts had significantly shorter TTNT (8.7 mos vs 14.2 mos, LR p=0.001; adj HR 1.36, 95%CI 1.11-1.66, adj p=0.003) and OS (28.3 mos vs 60.9 mos, LR p=&lt;0.0001; adj HR 2.19, 95%CI 1.69-2.82, adj p=&lt;0.0001) compared to those without. This divergence was more pronounced for patients receiving 1L CDK4/6i (TTNT: 10.7 mos vs 24.6 mos, LR p=0.006, adj HR 1.84, 95%CI 1.18-2.87, adj p=0.007; OS: 23.8 mos vs 67.3 mos, LR p&lt;0.0001, adj HR 3.27, 95%CI 1.89-5.62, adj p&lt;0.0001). Individual gene contributions towards less favorable TTNT are shown. Conclusions: We demonstrate via RW data that patients with &gt;1 pre-treatment ctDNA alt in a curated list of CDK4/6i+ET-R genes have significantly worse outcomes on CDK4/6i plus ET treatment, a difference more pronounced among patients receiving 1L therapy. This data is among the first successful efforts to validate a clinically useful multi-gene clinical biomarker that has the potential to predict CDK4/6i plus ET resistance. Further exploration of this signature’s prognostic and predictive utility for CDK4/6i plus ET use is ongoing to better refine personalized treatment selection in HR+/HER2- aBC. [Table: see text]

Using real-world evidence to evaluate broad panel vs. sequential single and narrow panel sequencing in members with non-small cell lung cancer (NSCLC).

e18892 Background: The clinical benefits of biomarker testing for patients with non-small cell lung cancer (NSCLC) are substantial, as increased use of appropriate targeted therapies is associated with improved survival rates. Next generation sequencing (NGS) testing has become increasingly important as it enables the identification of multiple biomarkers simultaneously and efficiently while minimizing the number of biopsies required. The purpose of this study is to assess the total cost of care (TCOC) of broad panel sequencing (BPS) ( &gt; 5 genes) and narrow panel sequencing (NPS) in NSCLC patients. Methods: This study evaluated members with a NSCLC diagnosis undergoing biomarker testing insured by a large commercial fully-insured and Medicare health plan in the United States from January 1, 2021, to December 31, 2021. Due to the complex and inconsistent practices of billing among lab vendors, claims were analyzed and classified using supervised machine learning models to identify the correct test category from Concert Genetics (CG) test taxonomy. Test categories within CG test taxonomy were then mapped to the higher-level classifications of BPS and NPS. CG’s BPS categories included: Cell-Free Circulating Tumor DNA Lung Cancer Panel Tests, Cell-Free DNA Cancer Profiling Panel Tests (5-50 genes), Cell-Free DNA Cancer Profiling Panel Tests (≥ 51 genes), Lung Cancer Diagnostic Algorithmic Tests, Lung Cancer Panel Tests, Tumor-Type Agnostic Molecular Solid Tumor Profiling Panel Tests (≥ 51 genes), Tumor-Type Agnostic Solid Tumor Molecular Profiling Panel Tests (5-50 genes), Tumor-Type Agnostic Solid Tumor Molecular Profiling Panels Tests with IHC and Cytogenetic Analyses. Claims matched to other categories were considered NPS. No price concessions were allowed for external vendors to account for cost differences when evaluating TCOC. Results: In this study, 356 members were evaluated; members were on average 72.5 years (std = 7.5), predominately male (n = 199; 55.9%) with Medicare (n = 332; 93.3%) coverage. These members underwent BPS testing (n = 151; 42.4%) and were NCCN treatment compliant (n = 221; 62.1%). The most common comorbidities identified for these members were hyperlipidemia (n = 286; 80.3%) and hypertension (n = 281; 78.9%). After adjusting for age, gender, and insurance type, the TCOC for patients undergoing BPS testing was $1,750 more than NPS testing (p = 0.03). Conclusions: There was a difference in TCOC between BPS and NPS testing. Standardized billing processes need to be implemented to evaluate clinical outcomes to adequately compare their clinical utility and identify the influence of explanatory variables on costs.

Genomic profiling of NGS-based ctDNA from Chinese non-small cell lung cancer patients.

Cell-free circulating tumor DNA (ctDNA) in plasma enables rapid and repeat testing of actionable mutations. Next-generation sequencing (NGS) is an attractive platform for multiplex sequencing capabilities compared to traditional methods such as PCR. The purpose of this study is to evaluate the value of the NGS-based ctDNA assay and to identify the genomic alteration profile of ctDNA in real-world Chinese non-small cell lung (NSCLC) patients. In total, 294 Chinese patients with pathological diagnosis of Phase III-IV NSCLC were enrolled. 3-4mL peripheral blood was collected and NGS-based analysis was carried out using a 20-gene panel. The analytical sensitivity and specificity of ctDNA NGS-based assay was validated using droplet digital PCR (ddPCR). We have tested 570 sites from 286 samples using ddPCR, which included 108 positive sites and 462 negative sites from NGS results, and the concordance rate was 99.8% (418/419) for single-nucleotide variants (SNVs) and 96.7% (146/151) for insertions and deletions (InDels). The most frequent genes were TP53 (32%), EGFR (31.97%), KRAS (6.46%), PIK3CA (4.76%), and MET (4.08%). Exon 19 deletion (19del) was the most common alteration in EGFR and G12C was the most common alteration in KRAS. Furthermore, the detection rate of TP53 was higher in the male and patients with squamous cell carcinoma. We also found the prevalence of TP53 in L858R was higher than in 19del (61.29% vs. 40%; p = 0.1115). The results indicate that the results of NGS-based ctDNA assay are highly consistent with ddPCR. In Chinese NSCLC patients, TP53 mutation was more frequently associated with male and squamous cell carcinoma. The prevalence of concomitant mutations in L858R may be different from that in 19del.

Abstract 6689: Whole genome cell-free tumor DNA mutational signatures from blood for early detection of recurrence of low stage lung adenocarcinoma

Abstract Introduction: Lung cancer remains the leading cause of cancer-related deaths. Surgery is the best option for early lung cancer, and the role of adjuvant therapy remains controversial. Liquid biopsy offers a noninvasive approach to monitor cancer burden. Targeted sequencing of circulating cell free tumor DNA (ctDNA) in blood has shown success for diagnosis; however, low tumor burden and dynamic evolution of low stage disease is challenging for targeted panels. Thus, we hypothesized that a whole genome sequencing (WGS)-derived patient specific mutational signature from a matched tumor-normal WGS can provide sensitive and specific approach to detect mutations and copy numbers in ctDNA for monitoring of lung adenocarcinoma patients. Methods: We successfully profiled 50 Stage 1 or 2 lung adenocarcinomas. ctDNA was extracted from 1-2 mL of plasma, tumor DNA was extracted from pathology tissue and normal germline DNA from the white blood cells. WGS using was performed on matched tumor and normal DNA, and ctDNA extracted from plasma. WGS coverage was 40x for matched tumor-normal and 20x for ctDNA. We derived a personalized mutational pattern for each tumor and used an AI-based error suppression model for quantification and ultra-sensitive detection of ctDNA in plasma samples. A patient-specific personalized genome-wide compendium of somatic mutations and copy numbers was established and ctDNA tested at 3 to 18 available time points during the therapy or follow up. A personalized mutational signature for detection ctDNA from WGS was quantified and the ctDNA Tumor Fraction (TF) was compared to the clinical status and time to recurrence. Results: Tumor specific signatures were derived from matched tumor-normal samples with &amp;gt;5% tumor purity and &amp;lt;30% duplications rate. Out of all patients, 33 patients showed no recurrence and 12 recurred. Tumor-specific signatures detected the presence of the tumor signature in plasma with TF as low as 10−5. Based on positive minimal residual disease in plasma, the recurrence prediction sensitivity was 0.75 and specificity 0.82, with positive predictive value of 0.6 and negative predictive value 0.9. WGS ctDNA predicted recurrence with a median lead time of 508 days before clinical/imaging recurrence. In one case we were able to identify the second primary by deconvoluting known and novel ctDNA mutations. ctDNA mutational profiles enabled identification of smoking mutational signature matching clinical history, and APOBEC and ageing signatures as well as tumor mutational burden. Conclusions: Patient-specific WGS tumor signature from plasma derived ctDNA enables specific and ultrasensitive tracking of minimal residual disease in low stage lung adenocarcinoma patients. Molecularly positive status can be used to predict recurrence and identify patients with clinical low stage disease that may benefit from adjuvant therapy. Citation Format: Ivy Tran, Alejandro Vargas, Reid Wilkins, Isabella Pizzillo, Kenneth Tokoro, Danielle Afterman, Tomer Lauterman, Maja Kuzman, Santiago Gonzalez, Dunja Glavas, James Smadbeck, Dillon Maloney, Jurica Levatic, Samuel Phillips, Sunil Deochand, Michael Yahalom, Ryan Ptashkin, Iman Tavassoly, Zohar Donenhirsh, Eric White, Ravi Kandasamy, Ury Alon, Paz Polak, Boris Oklander, Asaf Zviran, Matija Snuderl, Harvey I. Pass. Whole genome cell-free tumor DNA mutational signatures from blood for early detection of recurrence of low stage lung adenocarcinoma. [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 6689.

Abstract 3358: PLCRC-PROVENC3: assessing the prognostic value of post-surgery liquid biopsy cell-free circulating tumor DNA in stage III colon cancer patients

Abstract Introduction: Surgery followed by adjuvant chemotherapy (ACT) is standard of care in stage III colon cancer. However, 50% of the patients would be cured by surgery alone and are being overtreated, while 30-35% will experience a recurrence despite adjuvant treatment, resulting in only 15-20% of the patients benefitting from ACT. Therefore, there is a need for prognostic biomarkers to better stratify this group of patients for ACT decisions. Recent observational and interventional studies in non-metastatic colon cancer have shown that detection of cell-free circulating tumor DNA (ctDNA) in blood after surgery is highly prognostic for development of recurrence. Hence, ctDNA analysis is a promising approach to guide treatment decisions in stage III colon cancer, but studies with large well-defined patient cohorts are needed to prove clinical utility. Aim: Determine the prognostic value of ctDNA in stage III colon cancer patients treated with ACT to reduce futile treatment. Methods: 241 stage III colon cancer patients treated with ACT were included in the prospective observational study “PROVENC3” (PROgnostic Value of Early Notification by Ctdna in Colon Cancer stage 3), a substudy of the Prospective Dutch Colorectal Cancer cohort (PLCRC). The PLCRC infrastructure accrued patients with colorectal cancer in 23 participating hospitals in the Netherlands. After informed consent, blood was collected pre-surgery, post-surgery, post-ACT and every six months up to 3 years. Tumor-informed detection of ctDNA was performed through integrated whole genome sequencing (WGS) analyses of formalin-fixed paraffin-embedded tumor tissue DNA (80x), germline DNA (40x), and plasma cell-free DNA (30x). Results: Patient accrual was completed in 2021, with a median follow-up of 35.6 months. In total, 1090 blood samples have been collected to date. Analytical studies demonstrated a limit of detection of the test of 0.005% ctDNA utilizing contrived reference models derived from six independent cell lines, with a specificity of 99.6% across 119 noncancerous donor plasma specimens. From the PROVENC3 study, ctDNA analyses are ongoing for pre-surgery (n=68) and post-surgery (n=241 patients) blood samples. Preliminary results demonstrated a ctDNA detection rate of 93.4% pre-surgery and 17.1% post-surgery, which was associated with disease recurrence. Final analysis will enable determination of: 1) the proportion of ctDNA-positive/negative patients after surgery and the corresponding recurrence rates; 2) the prognostic value of post-surgery ctDNA; and 3) the lead time between post-surgery ctDNA detection and recurrence. Future Perspective: Ultimately, the results of this study will be used to model and design a ctDNA-guided interventional trial in stage III colon cancer patients, to reduce futile ACT and its associated side-effects. Citation Format: Carmen Rubio-Alarcon, Steven L. Ketelaars, Ingrid A. Franken, Sietske C. van Nassau, Dave E. van der Kruijssen, Suzanna J. Schraa, Theodora C. Linders, Pien Delis-van Diemen, Maartje Alkemade, Anne Bolijn, Marianne Tijssen, Margriet Lemmens, Miranda van Dongen, Mirthe Lanfermeijer, Annegien Broeks, Lana Meiqari, Linda J. Bosch, Victor E. Velculescu, Amy Greer, Samuel V. Angiuoli, Andrew Georgiadis, David Riley, James R. White, Christopher Greco, Liam Cox, Daan van den Broek, Cornelis J. Punt, Veerle M. Coupé, Miriam Koopman, Jeanine Roodhart, Gerrit A. Meijer, Mark Sausen, Geraldine R. Vink, Remond J. Fijneman. PLCRC-PROVENC3: assessing the prognostic value of post-surgery liquid biopsy cell-free circulating tumor DNA in stage III colon cancer patients [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 3358.

Abstract 6603: BRCA1 promoter methylation in sporadic breast cancer patients detected by liquid biopsy

Abstract Background: BRCA1 promoter methylation (PM) is an early initiating event in cancer, occurring in 3 to 65.2% of all breast tumors, and 30 to 65% of triple negative tumors. BRCA1 PM has been associated with defective homologous recombination repair (HRR), early onset of breast and ovarian cancer, and improved clinical response to adjuvant chemotherapy. Historically, there has been no diagnostic assay that comprehensively evaluates both BRCA1 PM and genomic alterations in cell-free circulating tumor DNA (ctDNA). Here, we describe the novel detection of BRCA1 PM and genomic alterations in a cohort of patients with breast cancer using GuardantINFINITY, a liquid biopsy assay interrogating 800+ genes and genome-wide methylation detection. Method: We assessed for BRCA1 PM in ctDNA from 274 patients with late-stage breast cancer. Genomic sequencing of 800+ genes and PM profiling of 398 genes was performed by GuardantINFINITY. The positive calling threshold for PM was established by comparing cell-free DNA derived from patients with cancer and cancer-free donors. The limit of detection (LoD) was determined through in silico and experimental titrations of ctDNA from clinical samples and cell lines with known gene PM into the plasma of cancer-free donors. Results: Among the 274 patients with advanced breast cancer, 8 (2.9%) had germline pathogenic mutations in BRCA1, BRCA2, or ATM. BRCA1 PM was detected in 11/274 (4.0%) patients at the predefined threshold of &amp;gt;99% specificity. BRCA1 PM detection in this cohort was 8.9% (8/90) when excluding samples with low tumor shedding (&amp;lt;1% epigenomic tumor fraction in cfDNA). Among the 11 patients with BRCA1 PM detected in ctDNA, one had a co-occurring somatic BRCA1 nonsense variant (p.S361*); none of the remaining patients with BRCA1 PM had another HRR-related mutation detected in cfDNA. Among patients without BRCA1 PM detected, pathogenic somatic alterations were detected in BRCA2, ATM, and CHEK2 in 25 (9.4%) patients. In silico simulations using clinical samples with BRCA1 PM indicated an LoD of 0.0408%. BRCA1 PM was not detected in 3210 individual and mixed cancer-free clinical samples, indicating a high specificity for BRCA1 PM calls. Conclusion: GuardantINFINITY, a plasma-based diagnostic assay, detected both BRCA1 PM and genomic alterations in this unspecified advanced breast cancer cohort. The BRCA1 PM detection rates of 4.0-8.9% are consistent with values previously reported in the literature. As BRCA1 PM has important prognostic and therapeutic implications for the management of breast (as well as ovarian) cancers, additional studies are warranted to further describe the PM patterns across breast cancer subtypes and how these patterns both influence and are influenced by disease evolution and therapeutic response. Liquid biopsy thus serves as a suitable method to noninvasively identify and monitor changes in both genomics and epigenomics. Citation Format: Jennifer Yen, Sai Chen, Colby Jenkins, Brooke Overstreet, Yu Fu, Jun Zhao, Tingting Jiang, Leylah Drusbosky, Stephen Pettitt, Michael Dorschner, Lauren Lawrence, Han-Yu Chuang, Andrew Tutt. BRCA1 promoter methylation in sporadic breast cancer patients detected by liquid biopsy [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 6603.

Abstract 5714: Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer

Abstract BACKGROUND: Cell-free circulating tumor DNA (ctDNA) assays have been adopted to monitor therapeutic response in both early- and late-stage cancer. However, tests currently available require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the circulation. Recently, we developed a tumor-agnostic approach called DELFI Tumor Fraction (DELFI-TF), a Bayesian probabilistic model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. METHODS: Overall, 692 longitudinal plasma samples collected from 153 patients with initially unresectable colorectal cancer (CRC) liver-only metastases participating in the phase III study CAIRO5 (NCT02162563) were split across a training (RAS/BRAF mutant) and a validation cohort (RAS/BRAF wildtype). For all cell-free DNA (cfDNA) samples of patients within the training cohort (n=312), the tumor burden was quantified as the MAF of the tumor-tissue-proven RAS/BRAF variant measured by digital droplet PCR (ddPCR). Using cfDNA fragment-sequencing statistics from low-coverage WGS data, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints in the training cohort. Changes in DELFI-TF scores during first-line FOLFOX/FOLFIRI and bevacizumab therapy were assessed to predict disease response. A tissue-informed approach was applied to detect minimal residual disease in the subgroup of patients treated with curative intent (complete resection of the primary tumor and liver metastases). RESULTS: In the training cohort, DELFI-TF scores strongly correlated with RAS/BRAF MAF measured by ddPCR (r=0.85, p&amp;lt;0.001). DELFI-TF captured plasma tumor fractions associated with copy number changes when ddPCR failed to detect RAS/BRAF mutations. Before treatment initiation, DELFI-TF correlated with the sum of the largest diameters on CT scans according to RECIST1.1 (r=0.49, p&amp;lt;0.001). Patients with negative and positive changes in DELFI-TF more often exhibited response and progressive disease based on consecutive RECIST assessments, respectively. Median progression-free survival was 9.9 and 12.8 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.007). Overall survival was 28.8 and 58.9 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.001). In patients treated with curative intent, tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after metastasectomy. Most patients presenting molecular relapse were later diagnosed with clinical recurrence by conventional CT imaging. CONCLUSIONS: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with a performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction. Citation Format: Bahar Alipanahi, Iris van 't Erve, Keith Lumbard, Laurel K. Millberg, Zach Skidmore, Lorenzo Rinaldi, Jacob Carey, Jennifer Tom, Cornelis J. A. Punt, Nicholas C. Dracopoli, Gerrit A. Meijer, Robert B. Scharpf, Victor E. Velculescu, Remond Fijneman, Alessandro Leal. Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer. [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 5714.

Abstract 6012: Comparison of pre-analytical methods for DNA methylation analysis of cfDNA

Abstract Liquid biopsies in the form of cell-free circulating tumor DNA (ctDNA) isolated from blood plasma or serum have shown great promise to be used as minimally-invasive markers in cancer patients providing an insight into the composition of both primary tumor and metastases, and an opportunity to assess clonal dynamics throughout the course of disease. CtDNA carries not only tumor- specific changes in its sequence but also distinctive epigenetic marks, namely DNA methylation patterns similar to the patterns in the primary tumor. Deregulation of tissue-specific DNA methylation patterns is an early event in carcinogenesis holding great promise to be exploited for early detection of cancer. However, in contrast to mutation detection, targeted DNA methylation sequencing of cfDNA remains a challenge due to low concentration and heavy fragmentation. The limiting step in detection of any analyte is the recovery of the DNA molecules. The pre-analytical steps in the DNA methylation detection pipeline include extraction of cfDNA molecules and their conversion using sodium bisulfite. Bisulfite conversion is a harsh chemical reaction that induces further fragmentation and degradation of already scarce cfDNA. In this study we compared commercial methods for cfDNA extraction and for bisulfite conversion to evaluate the yield, degradation and procedural loss. We compared five commercial kits for cfDNA extraction from blood plasma, three column-based (Qiagen’s QIAamp Circulating nucleic acids kit, Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification and Macherey-Nagel’s NucleoSpin® Plasma XS) and two based on magnetic beads cleanup (Bioo Scientific’s Next Prep magnetic Cell-Free Circulating DNA isolation kitand Applied Biosystems’ Magmax Cell-Free Circulating DNA isolation kit. The kits used for bisulfite conversion were Zymo EZ DNA Methylation Lightning with column based purification and with magnetic beads purification, Zymo EZ DNA Methylation Direct with column based purification and with magnetic bead purification, Qiagen Epitect Fast DNA Bisulfite and Diagenode Premium Bisulfite kits. Same samples were processed by each of the kits, Sample degradation was determined using Agilent Bioanalyzer dsDNA kit. Yield was evaluated by Picogreen, absolute qPCR for single-copy gene and for contamination of gDNA by a B-cell specific assay, while procedural loss was determined based on the recovery of a synthetic spike-in. Citation Format: Miljana Tanic, Oluwadunni Emiloju, Pawan Dhami, Stephan Beck. Comparison of pre-analytical methods for DNA methylation analysis of cfDNA. [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 6012.