cfDNA Yield Research Articles

BIOM-64. EXPLOITING CELL-FREE DNA TO DISSECT MECHANISMS OF RESISTANCE TO TEMOZOLOMIDE IN GLIOBLASTOMA CELLS

Abstract Temozolomide (TMZ) is a cytotoxic alkylating agent that serves as a cornerstone in glioblastoma (GBM) treatment, yet is notorious for eliciting hypermutation in recurrent tumors that have developed drug resistance by downregulating mismatch repair (MMR). We reasoned that cell-free DNA (cfDNA), a non-invasive measure for detecting tumor-derived biomarkers, could be used to temporally examine the mechanism of resistance to TMZ. We therefore designed an in-vitro experiment using the patient-derived GBM43 cell line. Over five days, batches of cells were treated with either TMZ or vehicle. Cell counts and cfDNA were collected every 24 hours. We observed an associated increase in both dead cell counts and cfDNA yield in treated cells compared to untreated cells. In TMZ-treated samples, cfDNA showed a periodic fragmentation pattern consistent with internucleosomal cleavage and TMZ-induced apoptosis. Intriguingly, higher numbers of variants were found in the cfDNA of untreated samples when compared to treated cfDNA, possibly reflecting TMZ-induced selection of resistant subclones in treated samples. This was further supported by the finding that the average variant allele frequency (VAF) increased over time in TMZ-treated samples, consistent with purifying selection. Conversely, cfDNA from vehicle-treated samples demonstrated a decrease in the average VAF over time, consistent with clonal diversification. Although we did not observe an enrichment of the TMZ-associated mutational signature SBS11, we did find higher levels of defective MMR mutation signature SBS44 in treated samples, potentially indicating the selection of subclones with defective MMR mechanisms. Overall, our preliminary findings show that cfDNA can be used to resolve mechanisms of TMZ resistance over time. We are addressing open questions by performing longer treatment times, testing other patient-derived cell lines, and performing murine xenotransplantation experiments to test TMZ-associated hypermutation in vivo. Our work will contribute to our understanding of TMZ resistance, and importantly push the development of cfDNA-based biomarkers for measuring TMZ-sensitivity in patients.

COMPARISON OF YIELD AND QUALITY OF CIRCULATING CELL-FREE DNA FROM K2EDTA AND K3EDTA COLLECTIONS IN HEALTHY SUBJECTS

Background: Circulating cell-free DNA (cfDNA) is a biomarker for various clinical applications, including detecting and monitoring cancer. However, blood collection tubes can affect the yield and quality of cfDNA. Since specific cfDNA collection tubes are costly, K2EDTA and K3EDTA anticoagulant tubes are alternatives in routine clinical laboratories. Objectives: This study aimed to compare the efficiency of cfDNA extraction from plasma collected in K2EDTA and K3EDTA tubes and evaluate implementation for molecular diagnostics. Methods: Blood samples from 38 healthy subjects were collected in K2EDTA and K3EDTA tubes that were processed within 2 hours. The extracted cfDNA was measured and performed using SYBR Green-based qPCR for three endogenous reference genes (GAPDH, HPRT1, TFRC). The cfDNA yield and the amplification efficiency of these genes were compared between K2EDTA and K3EDTA tubes using the Mann-Whitney U test. Results: There were no significant differences in cfDNA concentration between K2EDTA and K3EDTA tubes (p=0.051). However, qPCR analysis revealed significantly higher copy numbers of TFRC and HPRT1 in K2EDTA tubes than in K3EDTA tubes (p<0.05). No significant difference was found for GAPDH. Conclusion: The results indicate that K2EDTA and K3EDTA tubes are an alternative option for cfDNA analysis if samples are processed quickly after a blood draw, which offers flexibility and cost savings in resource-limited areas.

132 Optimizing Success Rates of CSF Liquid Biopsy Examined With Next Generation Sequencing in Pediatric Central Nervous System Tumors

INTRODUCTION: Cerebrospinal fluid (CSF) liquid biopsy presents an attractive option for minimally invasive diagnosis and follow up for pediatric brain tumors. Best practice for obtaining appropriate cfDNA for next generation sequencing (NGS) is not currently known. METHODS: CSF was collected from patients under a prospective study to evaluate diagnostic utility of NGS. Samples were collected from control (non-tumor) and CNS tumor patients. CSF was collected at the dura opening, immediate ventricular catheter (IVC) from CSF divergent procedures, or ventricular shunt taps. Data was gathered on demographics, pathological diagnosis, tumor location, volume of CSF extraction, and DNA yield. RESULTS: CSF was extracted from 72 CNS tumor patients and 20 controls (48 female, 44 male). Of the CNS tumors, there were forty low-grade tumors (40/72) and thirty-two high-grade tumors (32/72). CSF collection location included dura opening (57), ventricular catheter (23), and shunt taps (12). cfDNA yield (ng) for CNS tumors displayed a mean of 135.5 ng (±353.4) and controls displayed a mean of 95.6 ng (±241.0). CNS tumor patients generated a significantly higher (p < 0.0001) DNA concentration (ng/cc) with a mean of 18.9 ng/cc (±82.7) compared to 0.5 ng/cc (±0.7) mean for controls. High grade tumors demonstrated a significantly greater amount of cfDNA concentration with a mean of 33.6 ng/cc (±116.6) whereas low grade tumors had a mean of 4.2 ng/cc (±5.0). CSF acquisition was 90.2% successful for NGS with a mean volume of 4.9 cc. In addition, infratentorial tumors revealed a 96% success rate with a mean volume of 5 cc. CONCLUSIONS: Extracting 4.9 cc of CSF had an 90.2% success rate for NGS. CSF may be acquired from either ventricular catheter or dura opening. High grade tumors had significantly higher cfDNA yield and concentration in CSF compared to low grade.

Abstract 3662: Cell-free DNA dynamics following neuroblastoma resection

Abstract Introduction: Prior studies have demonstrated the utility of circulating tumor DNA (ctDNA) as a biomarker in the postoperative period for oncologic risk stratification. However, its utility may be limited in the early postoperative period by surgical trauma causing an increase in total cell-free DNA (cfDNA) due to tissue destruction, inflammation, and wound healing. No studies have identified the optimal timing of postoperative sample collection in pediatric patients undergoing resection of high-risk neuroblastoma. The purpose of this study is to assess the postoperative dynamics of cfDNA following neuroblastoma resection to determine optimal timing for ctDNA investigations. Methods: An institutional database of banked cell-free DNA samples was retrospectively queried for patients with a diagnosis of neuroblastoma who had blood samples collected prior to resection and postoperative samples collected within 2 months of the operation. Plasma was collected from whole blood samples. cfDNA was extracted from plasma samples using the QIAGEN QIAsymphony SP system, and samples were quantified using the Advanced Analytical Fragment Analyzer Automated CE System with the High Sensitivity Genomic DNA Analysis Kit. Results: Seventeen patients were identified undergoing 18 total operations and 37 total blood draws yielding cfDNA. There were 12 male and 5 female patients. Median age at operation was 5.5 years (range 1.4y to 20.5y). Two patients were INRG stage L2, and 15 patients were stage M. Operative indications were primary tumor resection in 10 patients and resection of recurrence/progression in 8 patients. Three postoperative samples were drawn within 10 days of the operation, and 16 samples were collected between postoperative weeks 4 and 6. There was a statistically significant difference (p=0.029) in cfDNA yield between each timepoint sampled: preoperative (median 6ng/mL, range 2-30ng/mL), 0-10 days postop (median 34ng/mL, range 15 - 85 ng/mL), and 4-6 weeks postop (median 6ng/mL, range 2 - 17ng/mL). Pairing the pre- and post-operative samples for each patient, those drawn within 10 days had a median increased yield of 180% (range +67% to +652%) while the samples collected at 4-6 weeks had a median decreased yield of 23% (range -55% to +491%; p = 0.047). Of note, there were three outliers in the 4-6 week group who had increases in cfDNA yield of >200%. On further review of these charts, each patient was being treated for obstructive uropathy at the time the postoperative sample was collected. Conclusion: Plasma cfDNA yield increases in the early postoperative period before dropping by postoperative week 6. Tumor resection may be associated with an overall decrease in total cfDNA. Postoperative renal pathology may cause a persistent elevation in cfDNA. Further studies are necessary to clarify the role of ctDNA in the management of patients with neuroblastoma. Citation Format: Joshua N. Honeyman, Andrew Chi, Shakeel Modak, Fiorella Iglesias Cardenas, Michael P. La Quaglia, Neerav N. Shukla, J. Ted Gerstle. Cell-free DNA dynamics following neuroblastoma resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3662.

Abstract 5017: Analytical performance of contrived samples for validation of liquid biopsy assays

Abstract Introduction: Cell-free DNA (cfDNA) extracted from plasma is critical for the analytical validation of liquid biopsy assays. cfDNA is comprised of genetic material from tumor and non-tumor cells, which can be highly variable across tumor types and disease stage. Relative to tumor tissue, cfDNA yield is lower and may not always capture all clinically relevant variants (e.g. gene fusions). Supplementing analytical validation studies with contrived samples prepared from cell line derived DNA to mimic the features of cfDNA allows for more robust testing of rare variants in clinical samples. To determine the commutability of contrived sample DNA to clinical plasma sample cfDNA, a retrospective analysis of sensitivity studies using two methods of contrived sample preparation was performed. Methods: The PGDx elio Plasma Focus assay was used to compare sample base size post library preparation and limit of detection (LoD) of clinically relevant variants. Both methods used fragmented cell line DNA with a variant of interest and for method #1, cell line DNA was spiked into noncancerous donor plasma, which was then extracted resulting in a cfDNA and cell line DNA mixture. This contrived sample was diluted using wild-type (WT) DNA extracted from noncancerous donor plasma to obtain a target variant allele frequency (VAF). Method #2 used fragmented cell line DNA diluted with fragmented WT cell line DNA to obtain a target VAF. This retrospective data analysis will include a minimum of 28 samples run in replicate (3-20) across multiple studies. Results: Data obtained from a DNA fragment analyzer post library preparation show similarities between fragmented cell line DNA spiked into plasma, fragmented cell line DNA, and clinical plasma cfDNA, with fragment peaks ranging from 290-320 base pair (bp). The median LoDs across the three sample types (n=28) were also similar for single nucleotide variants (SNVs) at VAFs of 0.70%, 0.76%, 0.72%, indels at VAFs of 0.70%, 1.0%, 0.78%, amplifications at folds of 1.4, 1.3, 1.5, and translocations at fusion read fractions of 0.5%, 0.61%, 0.55%. When comparing the same variants in a contrived sample functional characterization study, the call rates of various dilution levels were similar. A Fisher’s Exact Test of the Hit Rate showed no significant difference for KRAS G12V SNV, ERBB2 amplification, and RET translocation between cell line DNA and clinical cfDNA. Conclusions: This data demonstrates that contrived samples perform with general equivalence to and may be an appropriate substitute for clinical plasma samples, though certain DNA sources may influence overall performance. Overall, fragmented cell line DNA spiked into plasma and fragmented cell line DNA diluted with WT fragmented cell line DNA performed similarly to clinical cfDNA samples representative of the intended use population. Citation Format: Amanda E. Harvey, Cindy K. Maddox, Sonomi O. Dillon, Omar T. Khawar, Ellen L. Verner, Amy Greer, Kenneth C. Valkenburg, Brian J. Caveney, Shakti Ramkissoon. Analytical performance of contrived samples for validation of liquid biopsy assays [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 5017.

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

Abstract Background: The integration of liquid biopsy in oncology has transformed clinical management of patients with cancer. Cell-free DNA (cfDNA) represents extracellular nucleic acid fragments shed during cellular apoptosis, necrosis, or secretion, and can be extracted from whole blood. PGDx elio plasma focus analyzes cfDNA to enable non-invasive genomic profiling with next-generation sequencing. In this study, we characterized the cfDNA fraction (%) and yield (ng) across multiple tumor types and stages. Methods: cfDNA fraction was obtained using automated electrophoresis (Agilent 4200 TapeStation System) to assess a region of 50-700 bp across 293 clinical plasma samples (tumor stages I - IV). The fraction of cfDNA to total DNA present was then calculated to determine overall cfDNA percentage. The overall DNA yield (ng) was quantified via Qubit fluorometer immediately following extraction from plasma and normalized to volume of plasma (ng of cfDNA/mL of plasma). In addition, data was evaluated from 100 clinical plasma cases with strict extraction conditions allowing for yield normalization to plasma volume. Sequencing metrics were evaluated for all clinical cases with a recommended input of 25 ng DNA using PGDx elio plasma focus. Results: Average cfDNA fraction for individual tumor types ranged from 78.0% - 91.4%, with a mean (n=293) of 84.8%. When examining the yield normalized cohort (n=206), the average yield was 23.4 ng of cfDNA/mL of plasma. cfDNA yield and fraction varied across tumor types and increased with cancer stage. Despite these differences, the majority of cases (99.7%) yielded sufficient cfDNA to proceed with PGDx elio plasma focus testing. No statistical differences in sequencing performance (variants reported and coverage for each tumor type) were observed and the PGDx elio plasma focus assay success rate was 97%. There was a slightly higher failure rate (17.6% (3/17)) for samples below 60% cfDNA, indicating that high amounts of contaminating genomic DNA from white blood cells may obscure the number of available cfDNA fragments for analysis. Conclusions: These data characterize pre-analytical factors that impact performance of PGDx elio plasma focus using cfDNA isolated from plasma of patients with solid tumors. We showed cfDNA yield and fraction varied across tumor types and increased with cancer stage. The overall success rate for testing was 97% while failed samples were associated with higher levels of genomic DNA contamination. Citation Format: Robert J. Summersgill, Jesse M. Fox, Jennifer S. Dickey, Liam T. Cox, Cal D. Palumbo, Kenneth C. Valkenburg, Brian J. Caveney, Shakti Ramkissoon, Jennifer B. Jackson. Pre-analytical characterization of cell-free DNA to enable liquid biopsy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5020.

Abstract 5042: Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications

Abstract Introduction: The non-invasive collection of urine renders it an attractive and pragmatic substitute for plasma in liquid biopsy applications, fostering the creation of diagnostic methods that are more patient-friendly and less cumbersome for clinicians. This study investigates the feasibility of urine-derived cfDNA as a non-invasive option for acquiring vital diagnostic insights, further enhancing the liquid biopsy field. Methods: Paired male first void urine (FVU) and venous blood samples were collected from healthy donors (n=40) using Colli-Pee UAS devices (Novosanis) and BD K2EDTA Vacutainer tubes, respectively. Out of these, 20 paired urine and blood samples were spiked with 10 ng of cfDNA reference standard containing the KRAS p.G12V mutation. ~35 ml of urinary cell-free supernatant and ~3.5 ml of plasma were obtained after centrifugation of FVU and blood sample, respectively, and used as input material for cell-free nucleic acids extraction using the nRichDX Revolution Max20 cfDNA Isolation Kit. The extracted cfDNAs profile was assessed on 4200 TapeStation System, Agilent, using cfDNA ScreenTape. Extracted nucleic acids from urine samples were subjected to a qPCR assay to quantify endogenous human cfDNA content using 2X iTaq Universal SYBR Mastermix (Bio-Rad). Human cfDNA quantity was assessed using Ct values obtained from the qPCR assay. The actionable cfDNA molecules recovery was determined by qPCR mutation detection assay using TaqMan Genotyping. Results: Male FVU collected in Colli-Pee UAS device showed the presence of 100-250 bp cell-free DNA fragments similar to plasma obtained from whole blood collected in 10 ml vacutainer tubes as demonstrated by the Agilent cfDNA ScreenTape analysis. The cfDNA yield was similar when comparing one tube of blood (~3.5 mL of plasma) to one Colli-Pee of urine (~35 mL) shown by endogenous cfDNA qPCR assay. Additionally, the amplifiability of the KRAS G12V mutation was consistent in both plasma and urine samples, with comparable Ct values. Conclusion: The nRichDX Revolution Sample Prep System demonstrates the ability to extract cfDNA from both plasma and urine samples, with comparable extraction efficiency observed between matched samples from the same donor. This study provides evidence supporting the viability of first void urine samples as a non-invasive alternative for prostate cancer-related liquid biopsy applications. It indicates that cfDNA can be recovered from a sample collected with a single BCT or Colli-Pee UAS urine collection device. Future investigations will be required to assess clinical samples and biomarkers to establish a robust correlation between cfDNA levels and various cancer types and stages. This study reinforces the utility of first void urine samples as a reliable proxy for blood-based liquid biopsy applications. Citation Format: Nafiseh Jafari, Jason Saenz, Lauren Lee, Carlos Hernandez, Andrew Dunnigan, Amit Arora, Rafal Iwasiow, Mayer Saidian. Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications [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 5042.

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

Abstract There is broad consensus from regulatory agencies, clinicians and patients for the need to move to decentralized trials while focusing on patient-centric approaches with the COVID-19 pandemic being the catalyst. This requires the development of new sample collection capabilities and workflows to ensure that the necessary biomarkers can be measured. Circulating tumor DNA (ctDNA) has become an increasingly important biomarker for cancer detection and genetics, and monitoring patients during treatment. Typical measurements of ctDNA involve venous draws of >10mls of whole blood that require patients to be in clinic. Here, we test the feasibility of using a new collection device, Tasso+, to collect capillary blood for ctDNA analysis using the Guardant360 panel. A pilot of whole blood microsamples (< 1 mL) with a cfDNA yield of 0.7-1.4 ng/ml spiked to 0.5% MAF with Seraseq ctDNA reference material. This resulted in detection of ~20% of the total expected variants across variant classes showing the feasibility of using the G360 panel to detect low volume/low MAF samples. Initial testing of Tasso+ collected samples revealed a 5-10x increase in high MW gDNA compared to traditional venipuncture. SPRI bead-based size selection was used in the extraction protocol to remove >95% of the high MW DNA, resulting in a 35% loss of target cfDNA fraction. Traditional venipuncture samples and Tasso+ samples were collected from 5 cancer patients and run using the G360 platform. Microsamples detected 75% of the variants across all variant classes found in venipuncture samples. Tasso+ lost sensitivity of variants when the MAF was <0.5%. To expand usage of Tasso+ to at-home collection of blood for ctDNA analysis, we sought to determine if lyophilization of the Streck DNA BCT RUO tube reagent could still provide stability to samples and a potential framework for at-home collections. Venipuncture and Tasso+ samples were collected from control patients, pooled and spiked with Seraseq cfDNA reference material. Samples were aliquoted into standard liquid Streck tubes or tubes with lyophilized reagent at the concentration per manufacturer’s directions and evaluated for stability over time. The Tasso+ samples with a target MAF between 0.5%-2.0% detected ~26% of all variants compared to the standard samples with a similar MAF. Lyophilization of the Streck reagent had minimal effect (<20% reduction in variants detected) on the reagent’s performance as there were approximately an equal number of total variants detected when comparing liquid and lyophilized samples for both the standard tubes and Tasso+ samples. Overall, these results provide support and a framework for using a home-based patient-centric sampling approach for monitoring ctDNA from cancer patients in clinical development. Citation Format: Brad R. Evans, Robert J. Foley, Steven M. Townson, Emily J. Welch, Kevin P. Bateman. Reducing the patient burden for ctDNA biomarkers: Advancing small volume home-based collection technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3670.

Abstract 5028: Detection of non-small cell lung and bladder cancer signatures in peripheral blood using a novel antibody-free chromatin capture assay

Abstract Immune checkpoint inhibition (ICI) via anti-PD-1/PD-L1 has improved clinical outcomes for multiple cancers, including bladder and non-small cell lung cancer (NSCLC). Yet, the response rates remain low highlighting a significant knowledge gap in the mechanisms driving response and resistance. While the expression of PD-L1 has been established as a predictive biomarker of ICI response, other features, including molecular tumor burden, tumor microenvironment (TME), and the host’s immune mechanisms may contribute to overall response. The complex interactions between a tumor and the immune system underscore the need for a more comprehensive approach to explore novel biological and immunological insights of cancer progression. Circulating tumor DNA assays, while integral to contemporary decision-making in oncology, fall short in delineating the transcriptional programs that dictate cancer phenotypes and their evolution over the disease trajectory. To bridge this gap, we have developed a plasma-based active chromatin capture method that enables the comprehensive epigenetic and transcriptomic interrogation of both tumor and immune components. In this study, we quantified epigenetic profiles from plasma samples (n=100) of patients with bladder cancer (n=20) and NSCLC (n=24) undergoing ICI therapy, along with samples (n=70) from healthy individuals (n=5). As expected, the total cfDNA yield (ng) of lung (1.7x, p=2.3e-08) and bladder (1.98x, p=1.6e-11) cancer samples were higher than those of healthy individuals. More Interestingly, the longer cfDNA fragments derived from regulatory-active chromatin, also showed a significantly higher abundance in plasma samples from both lung (2.0x, p=2.5e-09) and bladder (3.2x, p=3.2e-14) cancer patients. Furthermore, the patterns of epigenetic regulations across cancer and healthy conditions showed distinct and stable clusters, consistent between gene bodies and transcription start sites, highlighting the epigenetic changes that may be driving cancer progression. We also observed a common enrichment of immune epigenetic signatures in both lung and bladder cancer samples, likely attributed to shared mechanisms associated with the modulation of the tumor microenvironment and immune interactions. Understanding these changes at an epigenetic level could provide insights into novel therapeutic approaches that target the tumor microenvironment and the immune interactions. Citation Format: Kevin Lai, Katharine Dilger, Rachael Cunningham, Timothy Barnes, Khiet Truong, Kathy Lam, Rhea Boquiren, Maggie C. Louie, Diana Abdueva. Detection of non-small cell lung and bladder cancer signatures in peripheral blood using a novel antibody-free chromatin capture assay [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 5028.

A phenol-chloroform free method for cfDNA isolation from cell conditioned media: development, optimization and comparative analysis

The non-invasive invasive nature of cell-free DNA (cfDNA) as diagnostic, prognostic, and theragnostic biomarkers has gained immense popularity in recent years. The clinical utility of cfDNA biomarkers may depend on understanding their origin and biological significance. Apoptosis, necrosis, and/or active release are possible mechanisms of cellular DNA release into the cell-free milieu. In-vitro cell culture models can provide useful insights into cfDNA biology. The yields and quality of cfDNA in the cell conditioned media (CCM) are largely dependent on the extraction method used. Here, we developed a phenol-chloroform-free cfDNA extraction method from CCM and compared it with three others published cfDNA extraction methods and four commercially available kits. Real-Time PCR (qPCR) targeting two different loci and a fluorescence-based Qubit assay were performed to quantify the extracted cfDNA. The absolute concentration of the extracted cfDNA varies with the target used for the qPCR assay; however, the relative trend remains similar for both qPCR assays. The cfDNA yield from CCM provided by the developed method was found to be either higher or comparable to the other methods used. In conclusion, we developed a safe, rapid and cost-effective cfDNA extraction protocol with minimal hands-on time; with no compromise in cfDNA yields.

Cancer Genomic Profiling and Minimal Residual Disease Monitoring By Cell-Free DNA Sequencing in Pediatric Leukemia

Introduction: Cell-free DNA (cfDNA) sequencing has been reported as a promising non-invasive tool for detecting cancer and monitoring treatment response. We sought to evaluate the potential of a cfDNA-based strategy for detecting diverse classes of somatic mutations relevant to pediatric leukemia and cancers. Methods: Plasma samples collected at diagnosis from 135 acute lymphoblastic leukemia (ALL) and 34 acute myeloid leukemia (AML) patients were evaluated, including 114 B-ALL and 21 T-ALL. We also studied diagnostic plasma samples from 38 solid tumor and 19 brain tumor patients, and plasma collected from 12 patients with B-ALL at days 15, 22 and end of induction chemotherapy (EOI). Plasma cfDNA extraction and library preparation were performed with the NucleoSnap cfDNA kit and ThruPLEX Tag-Seq HV kit, respectively. Next-generation sequencing was performed using a capture panel covering 355 pediatric cancer genes (including noncoding regions covering hotspots of structural variation) and 8,103 heterozygous population polymorphisms. We developed analytic approaches for de novo detection of all types of somatic mutations, including focal copy number alterations, rearrangements, and large chromosomal abnormalities. Whole-genome sequencing (WGS) was also performed for 10 cfDNA samples from leukemia. Data were compared with corresponding whole-genome, exome and transcriptome tumor cell sequencing data at diagnosis. Results: At diagnosis, plasma cfDNA yield was higher for leukemias than for solid and brain tumors (median, 142 ng/ml, 7.16 ng/ml, and 7.07 ng/ml, respectively. The average coverages of leukemia cfDNA sequencing were 790X by capture panel post error suppression and 69X by WGS. We found that 100% cases of leukemia at diagnosis had detectable circulating tumor DNA (ctDNA) in plasma cfDNA, and the median ctDNA purity was 77%, comparable to primary tumor purity from diagnostic sequencing. Overall, our panel covered 86% somatic variants present in the tumor samples. Of those covered events, capture-based cfDNA sequencing detected 98% SNVs/indels, 95% focal copy gains/losses, 100% internal tandem duplications, 95% chromosomal translocations, and 99% aneuploidy. In addition, capture-based sequencing identified cfDNA-specific variants such as NRAS/KRAS SNVs and a IKZF1 deletion that were subclonal in plasma cfDNA. Of those uncovered events, 85% were structural variants with their breakpoints in intronic/noncoding regions, and cfDNA WGS identified all somatic events present in the 10 sequenced tumor samples. To detect minimal residual disease (MRD) in B-ALL patients during induction, we increased cfDNA sequencing depth for low VAF events, and the detection limit was 0.4% for SNV/indel and structural variations. Compared to the MRD results from flow cytometry, somatic mutations were detected in 7/8 MRD-positive and 1/4 MRD-negative patients on day 15, 3/4 MRD-positive patients on day 22, and 0/2 MRD-positive patients at EOI. We also sequenced cfDNA from peripheral blood and bone marrow plasma for 2 B-ALL patients on day 0, day 15, and EOI, with comparable ctDNA purity and dynamics observed between the two compartments. In a patient with concurrent ALL and neuroblastoma (NBL) who experienced NBL relapse four months after the primary diagnosis, cfDNA genomic profiling identified ctDNA derived from ALL (52% of total plasma cfDNA), primary NBL (20%), and relapsed NBL (10%), suggesting that genetic variations of multiple tumors could be detected in a single cfDNA sample. Conclusion: Our study has demonstrated the utility of cfDNA sequencing to identify both sequence and structural somatic mutations in childhood acute leukemia. This approach will be useful to monitor disease, as well as providing a non-invasive diagnostic approach.

BIOM-27. CELL-FREE DNA-BASED LIQUID BIOPSIES FOR THE REAL-TIME TRACKING OF TUMOR BURDEN AND TREATMENT RESPONSE IN GLIOBLASTOMA

Abstract Cell-free DNA (cfDNA) are short fragments of DNA that are released by apoptotic or necrotic cells into bodily fluids. It has emerged as a potential diagnostic tool allowing for the detection and analysis of tumor-derived biomarkers in cancer. Despite the detailed view provided by clinical imaging, its inability to quantify tumor burden and distinguish between pseudo-progression and pseudo-response poses a challenge for patients. In this study, we sought to investigate the effectiveness of cfDNA as a liquid biopsy tool to track tumor burden and disease progression in real time in different glioblastoma models and through different types of biofluids. To examine the feasibility of cfDNA-based tumor tracking we cultivated several patient-derived glioblastoma lines, purified cfDNA and performed sequencing. Further analysis revealed that dead cells were associated with an elevated cfDNA yield and altered fragment size profiles. Genomic sequencing revealed consistent variants shared across serial cfDNA timepoints, strengthening the suggestion that cfDNA can be used as a non-invasive biomarker. In order to demonstrate the clinical implications of our methodology, we collected a preliminary batch of n = 43 longitudinal intraventricular cerebrospinal fluid (CSF) samples from a patient that underwent CAR-T treatment over the span of two years. We observed a marked increase in cfDNA yield post-treatment compared to samples taken prior to treatment administration. Furthermore, cfDNA-based estimates of tumor burden measured as allele fraction specific to the tumor, tracked with clinical imaging-based tumor size estimates. These preliminary findings provide compelling evidence supporting the clinical utility of cfDNA analysis in monitoring the effectiveness and dynamics of CAR-T treatment. By tracking cfDNA levels in CSF, we can identify patterns reflecting treatment response and aid in assessing the therapeutic efficiency of CAR-T treatment in glioma patients. Further investigations with larger patient cohorts and more comprehensive data analysis to validate these findings are actively underway.

Abstract 245: Development of a novel urine cell-free DNA preservation method for molecular profiling in genitourinary tumors

Abstract Introduction: Due to the benefits of its non-invasive testing, liquid biopsy technology has been applied therapeutically as an adjuvant to disease detection and monitoring in areas such as prenatal cell-free DNA (cfDNA) screening and cancer. Although the majority of current liquid biopsy techniques focus on DNA from blood, DNA from urine also conveys important genetic information from the human body. Studies have revealed that urine cfDNA can potentially be utilized for bladder cancer detection and monitoring. Urine samples can be quickly and painlessly collected; however, because urine DNA is readily damaged, maintaining high-quality cfDNA in urine is challenging. In this study, we developed a robust and user-friendly urine preservation method to maintain integrity of cfDNA in urine and to facilitate the transportation of urinary samples to clinical laboratories. Methods: Urine samples from 10 donors were collected with Predicine’s proprietary urine preservation method. Two urine samples were spiked with BL21(DE3) E.coli competent cells to simulate the urinary tract infection. All samples were incubated at 4°C, room temperature, and 37°C to mimic extreme storage or transporting environments. cfDNA was extracted after incubation of 0, 4, and 7 days and measured by Qubit and Fragment analyzer. Extracted DNA was also quantified by ddPCR with two markers: EGFR as an endogenous DNA marker and ARv7 (synthetic oligos containing intron-deleted sequence, post-incubation spike-in) as an inner control for the extraction and detection assays. Whole-genome sequencing was conducted to measure the quality of the cfDNA and bacterial contamination. Other commercial urine preserve buffers are used as control. Results: cfDNA derived from urine stored in Predicine’s proprietary urine preservation buffer remained stable till day 7 at different temperatures. There was no significant change in normalized copies of EGFR in the treated urine samples for up to 7 days. cfDNA yield was considerably higher in the novel preservation buffer than that using commercial buffers. Furthermore, the mapping rates in bacteria-contaminated samples were significantly improved. Conclusion: Predicine’s proprietary urine preservation method demonstrated excellent urine cfDNA preserving capacity at various temperatures for at least 7 days, outperforming commercially available preserve buffers in terms of DNA integrity and yield. The innovative technique offers an easy-to-use solution for obtaining high-quality urine cfDNA for clinical use. Citation Format: jie zhang, fang liu, min wang, cuishan deng, wei mo, seng tao, amy wang, shidong jia, zhixin zhao, pan du, binggang xiang, shujun luo. Development of a novel urine cell-free DNA preservation method for molecular profiling in genitourinary tumors [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 245.

Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology

Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses.

Next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) in advanced pancreatic neuroendocrine neoplasms (PanNENs).

653 Background: PanNENs represent 1-2% of all pancreatic neoplasms. The genomic landscape derived from PanNEN tumor tissue has been described previously. There are little data detailing the frequency of genetic alterations identified in cfDNA in an advanced PanNEN population, the plasma-tissue concordance of detected alterations, and the clinical utility of cfDNA. Methods: Patients (pts) with metastatic PanNENs underwent collection of cfDNA for NGS using the MSK-IMPACT 505 gene assay between March 2017 and April 2020. Matched tissue based NGS with the FDA authorized MSK-IMPACT gene assay was completed when tumor tissue was available. For some pts, plasma and tumor tissue were sequenced at multiple time points. Clinical actionability of sequence variants was annotated by OncoKB. Clinicopathologic characteristics were extracted, and data are herein reported. Results: 25 unique pts with metastatic PanNENs had 32 plasma samples analyzed. The majority had well differentiated (22/25; 88%), intermediate grade disease (13/25; 52%). 6 (24%) pts had well differentiated high grade disease and 3 (12%) had poorly differentiated neuroendocrine carcinomas. After extraction, median cfDNA yield per sample was 23.98ng (range: 3.2 to 500.1). Mutations were detected in 21(66%) of 32 samples (10 pre systemic therapy, 10 at progression, 12 post response to therapy or while stable on therapy). The most frequently mutated genes occurring in >10% of patients were DAXX (28%), TSC2 (24%), MEN1 (24%), ARID1B (20%), ARID1A (12%) and ATRX (12%). 23 (92%) pts underwent tumor tissue sequencing with MSK-IMPACT with a median time of 6.9 (range: 0.5-33.4) months between tissue collection and time of plasma analysis. NGS of cfDNA identified the most common mutations observed in tumor tissue for: DAXX (5/6; 83%), TSC2 (3/6; 50%), MEN1 (5/12; 42%), ARID1A (3/5; 60%) and ATRX (3/6; 50%). In 21/23 (91%) paired samples, additional mutations not seen in tissue were detected in plasma and included TSC2, TP53, EGFR, VHL, and BRCA2. Potentially actionable mutations were identified in sequenced cfDNA in 8/25 (32%) patients including 4 TSC2 mutations (level 3b), 1 ATM mutation (level 3b), 2 ARID1A mutations (level 4) and 1 KRAS mutation (level 4). One patient who was treated with larotrectinib for an ETV6:NTRK3 fusion detected on tumor sequencing ultimately developed resistance with a NRTK3 G623R alteration identified through sequencing of cfDNA at radiographic disease progression. Conclusions: NGS of cfDNA in metastatic PanNENs, across the spectrum of WHO-defined tumor grade/differentiation, revealed tumor-associated genetic alterations in 66% of plasma samples. Clonal evolution, actionable alterations, and resistance mechanisms can be detected through circulating cfDNA genotyping and may serve as a powerful tool to better understand disease biology of a disease that often changes over time and through therapy.

Liquidhope: methylome and genomic profiling from very limited quantities of plasma-derived DNA.

Analysis of the methylome of tumor cell-free deoxyribonucleic acid (DNA; cfDNA) has emerged as a powerful non-invasive technique for cancer subtyping and prognosis. However, its application is frequently hampered by the quality and total cfDNA yield. Here, we demonstrate the feasibility of very low-input cfDNA for whole-methylome and copy-number profiling studies using enzymatic conversion of unmethylated cysteines [enzymatic methyl-seq (EM-seq)] to better preserve DNA integrity. We created a model for predicting genomic subtyping and prognosis with high accuracy. We validated our tool by comparing whole-genome CpG sequencing with in situ cohorts generated with bisulfite conversion and array hybridization, demonstrating that, despite the different techniques and sample origins, information on cfDNA methylation is comparable with in situ cohorts. Our findings support use of liquid biopsy followed by EM-seq to assess methylome of cancer patients, enabling validation in external cohorts. This advance is particularly relevant for rare cancers like neuroblastomas where liquid-biopsy volume is restricted by ethical regulations in pediatric patients.

Abstract B006: Development of a NF1-MPNST-PDX liquid biopsy model using whole-genome sequencing and quantitative PCR of mouse-derived cell-free DNA

Abstract Background: Malignant Peripheral Nerve Sheath Tumors (MPNST) are aggressive, NF1-associated soft tissue sarcomas with a dismal 40% 5-year survival rate, high rates of disease relapse and metastasis, and limited treatment options. Our lab has generated a collection of patient-derived xenograft (PDX) models, which recapitulate the variety of genetic changes that occur in human NF1-MPNST. Longitudinal analysis of PDX tumor burden in preclinical studies is currently limited to imprecise tumor measurement or expensive and time-consuming imaging modalities such as MRI. These methods of analysis fail to allow for the early detection of tumor formation, the assessment of treatment-response dynamics, the formation of resistant subclones, or the detection of molecular residual disease. Liquid biopsies using plasma-derived cell-free DNA (cfDNA) have been successful in addressing these challenges in the context of multiple solid tumor types including MPNST by our group previously (Szymanski et al. 2021). However, liquid biopsy of cfDNA in the context murine model systems is quite limited, primarily due the challenge of collecting sufficient plasma for analysis. We hypothesize that we can detect and longitudinally track tumor burden in response to treatment using human-tumor-specific quantitative PCR (qPCR) and whole-genome sequencing of sequentially collected PDX-bearing murine plasma to develop a NF1-MPNST-PDX liquid biopsy model. Methods: To address this challenge, we sequentially collected ≈50 µL of murine plasma weekly from two independent groups of NF1-MPNST-PDX-bearing mice and corresponding PDX-free controls for up to six weeks. The first group consisted of six PDX-bearing mice who underwent whole-genome sequence of murine-derived plasma cfDNA and paired PDX tissue. The second group consisted of fifteen NF1-MPNST-PDX-bearing mice and five PDX-free controls; plasma cfDNA samples collected from this group underwent human-specific LINE-1 qPCR, which should only detect human DNA derived from implanted PDX and allow for longitudinal tracking of tumor burden. Results: We serially collected ≈50 µL of plasma weekly from NF1-MPNST-PDX mice with no obvious impact on mouse survival or weight. Mean total cfDNA yield from the terminally collected samples was 1.12 ± 1.02 ng per uL plasma (n=21) for PDX-bearing animals with cfDNA fragments canonically appearing between 70 and 450 bps as determined by electropherogram. Whole-genome sequencing of murine plasma cfDNA revealed broad genomic aneuploidy detectable in NF1-MPNST-PDX plasma which corresponded to alterations present in PDX tissue, including MPNST-specific chromosome 8q gain, corresponding with our earlier findings (Dehner et al. 2021). Human-specific LINE-1 qPCR showed specific enrichment of tumor-derived LINE-1 in NF1-MPNST-PDX-bearing mice compared to PDX-free mice which showed no human LINE-1 enrichment. Conclusion: Our data suggest that PDX plasma cfDNA may be an informative biomarker that can be non-invasively collected and used to track tumor burden in NF1-MPNST-PDX models. Citation Format: Paul A. Jones, Wenjia Feng, Xiaochun Zhang, Peter K. Harris, Taylor Sundby, Jeffrey J. Szymanski, Divya Srihari, Faridi Qaium, Jack F. Shern, Aadel A. Chaudhuri, Angela C. Hirbe. Development of a NF1-MPNST-PDX liquid biopsy model using whole-genome sequencing and quantitative PCR of mouse-derived cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B006.

Matched Molecular Profiling of Cell-Free DNA and Tumor Tissue in Patients With Advanced Clear Cell Renal Cell Carcinoma.

The clinical utility of cell-free DNA (cfDNA) as a biomarker for advanced clear cell renal cell carcinoma (ccRCC) remains unclear. We evaluated the validity of cfDNA-based genomic profiling in a large cohort of patients with ccRCC with matched next-generation sequencing (NGS) from primary tumor tissues. We performed paired NGS of tumor DNA and plasma cfDNA using the MSK-IMPACT platform in 110 patients with metastatic ccRCC. Tissues were profiled for variants and copy number alterations with germline comparison. Manual cross-genotyping between cfDNA and tumor tissue was performed. Deep sequencing with a higher sensitivity platform, MSK-ACCESS, was performed on a subset of cfDNA samples. Clinical data and radiographic tumor volumes were assessed to correlate cfDNA yield with treatment response and disease burden. Tumor tissue MSK-IMPACT testing identified 582 genomic alterations (GAs) across the cohort. Using standard thresholds for de novo variant calling in cfDNA, only 24 GAs were found by MSK-IMPACT in cfDNA in 7 of 110 patients (6%). With manual cross-genotyping, 210 GAs were detectable below thresholds in 74 patients (67%). Intrapatient concordance with tumor tissue was limited, including VHL (31.6%), PBRM1 (24.1%), and TP53 (52.9%). cfDNA profiling did not identify 3p loss because of low tumor fractions. Tumor volume was associated with cfDNA allele frequency, and VHL concordance was superior for patients with greater disease burden. cfDNA-based NGS profiling yielded low detection rates in this metastatic ccRCC cohort. Concordance with tumor profiling was low, even for truncal mutations such as VHL, and some findings in peripheral blood may represent clonal hematopoiesis. Routine cfDNA panel testing is not supported, and its application in biomarker efforts must account for these limitations.

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

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

Next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) in patients (pts) with advanced hepatocellular carcinoma (HCC).

4110 Background: HCC is often diagnosed based on high-quality cross-sectional imaging, and when a biopsy is pursued, acquisition of tissue may be of limited quantity and quality or complicated by underlying medical comorbidities. NGS of tumor derived circulating cfDNA represents an investigational tool for non-invasive molecular profiling, that has the potential to aid in diagnosis, prognosis, and in monitoring disease status. Although prior reports have evaluated such technologies, few studies have included tumor tissues to confirm histology and to explore plasma-tissue gene concordance. Methods: The primary objective of this retrospective cohort study was to define genomic alterations in circulating cfDNA and to explore plasma-tissue genotype concordance in HCC pts. HCC pts underwent collection of cfDNA for NGS using the MSK-ACCESS 129-gene assay between August 2019 and February 2021. Matched tissue-based NGS with the FDA authorized MSK-IMPACT gene assay was completed when tumor tissue was available. Clinical actionability of sequence variants was annotated by OncoKB, an FDA recognized knowledge base. Clinicopathologic characteristics were extracted, and all data were reported with descriptive statistics. Results: 51 unique patients with 53 plasma samples had an HCC histological diagnosis. Pts were male (39, 76%), median age 69 (42-87), viral hepatitis-related (24, 47%), and advanced stage (Stage III:9, 18%; Stage IV:38, 74.5%). Extrahepatic disease and macrovascular involvement were observed in 28 (55%) and 19 (38%) pts, respectively. 22 (43%) pts had AFP ≥400 ng/mL. 49 (92.5%) of 53 plasma samples had detectable genomic alterations. Median cfDNA yield after extraction was 39.43 ng (range: 7.93-287.68). The most frequently mutated genes occurring in > 10% of patients were TERT (57%), TP53 (47%), CTNNB1 (37%), ARID1A (18%) and TSC2 (14%). The most common oncogenic pathways that contained alterations were WNT-β-Catenin (45%) and PIK3-AKT-TOR (25%). 37 (73%) pts underwent tissue sequencing with MSK-IMPACT with a median time of 9.0 months to the time cfDNA testing. MSK-ACCESS identified mutations observed in tumor in most cases: TERT (20/22; 91%), TP53 (16/17; 94%), CTNNB1 (11/12; 92%), ARID1A (6/6; 100%) and TSC2 (6/7; 86%). In 18 (49%) of 37 paired samples, additional mutations in cfDNA not seen in tumor were detected and included KRAS, EGFR, and TP53 alterations. Potentially actionable mutations were identified through cfDNA in 37% of cases including TSC1/2 (18%), BRCA1/ 2 (8%) and PIK3CA (8%). Conclusions: Circulating cfDNA genotyping with MSK-ACCESS identifies previously reported HCC tumor genomic profiles and revealed tumor-associated mutations in 92.5% of plasma samples. Ongoing efforts will explore predictive and prognostic implications of NGS at different HCC stages as well as kinetics of treatment response.