ctDNA Detection Research Articles

Personalized circulating tumor DNA analysis used for ctDNA detection and response monitoring among patients with advanced or recurrent endometrial cancer

Personalized circulating tumor DNA analysis used for ctDNA detection and response monitoring among patients with advanced or recurrent endometrial cancer

Circulating DNA in Rectal Cancer to Unravel the Prognostic Potential for Radiation Oncologist: A Meta-analysis.

Liquid biopsy, with its noninvasive nature and ability to detect tumor-specific genetic alterations, emerges as an ideal biomarker for monitoring recurrences for locally advanced rectal cancer (LARC). Completed studies have small sample sizes and different experimental methods. To consolidate and assess the collective evidence regarding the prognostic role of circulating DNA (ctDNA) detection in LARC patients undergoing neoadjuvant chemoradiotherapy (nCRT). Computerized bibliographic searches of MEDLINE and CANCERLIT (2000 to 2023) were supplemented with hand searches of reference lists. Study selection: studies evaluating oncological outcomes of patients with LARC treated with a nCRT comparing patients with positive and negative liquid biopsy at baseline and after nCRT. Data extraction: data on population, intervention, and outcomes were extracted from each study, in accordance with the intention to treat method, by 2 independent observers, and combined using the DerSimonian method and Laird method. Nine studies follow inclusion criteria including 678 patients treated with nCRT. The pooled RD rate of ctDNA negative between measure at baseline and after nCRT is statistically significant 61% (95% CI: 53-70, P=0.0002). The hazard ratio (HR) of progression-free survival between ct-DNA negative and positive is significant 7.41 (95% CI: 4.87-11.289, P<0.00001). ctDNA can identify patients with different recurrence risks following nCRT and assess prognosis in patients with LARC. Further prospective study is necessary to determine the utility of ctDNA in personalised therapy for patients with LARC.

Longitudinal monitoring of circulating tumor DNA to detect relapse early and predict outcome in early breast cancer.

Detection of molecular residual disease (MRD) allows for the identification of breast cancer patients at high-risk of recurrence, with the potential that early initiation of treatment at early stages of relapse could improve patient outcomes. The Invitae Personalized Cancer Monitoring™ assay (PCM) is a newly developed next-generation sequencing approach that utilizes up to 50 patient-specific, tumor-informed DNA variants, to detect circulating tumor DNA (ctDNA). The ability of the PCM assay to detect MRD before clinical relapse was evaluated. The cohort included 61 female patients with high-risk breast cancer who underwent neoadjuvant chemotherapy. Plasma samples were collected before and during neoadjuvant therapy, after surgery and during monitoring. PCM was used to detect ctDNA at each time point. The sensitivity to detect ctDNA in plasma from patients who relapsed during the monitoring phase was 76.9% (10/13). Specificity and positive predictive values were both 100% with all (10/61, 16%) of the patients who had ctDNA detected during the monitoring phase subsequently relapsing. Detection of ctDNA during monitoring was associated with a high-risk of future relapse (HR 37.2, 95% CI 10.5-131.9, p < 0.0001), with a median lead-time from ctDNA detection to clinical relapse of 11.7months. PCM detected ctDNA in patients who relapsed with a long lead-time over clinical relapse, shows strong association with relapse-free survival and may be used to identify patients at high-risk for relapse, allowing for earlier intervention.

MBene Nanosheets with DNA Adsorbability for Circulating Tumor DNA Assay via Fluorescence Biosensing and Paper‐Based Microfluidic POCT

AbstractThe detection of circulating tumor DNA (ctDNA) in blood is significant for non‐invasive cancer diagnosis and treatment monitoring. Herein, MBene nanosheets is synthesized and compared with MXene via Density Functional Theory (DFT) calculations and fluorescence kinetic evaluations, for first time, revealing MBene's exceptional DNA adsorbability and discrimination to single‐stranded DNA (ssDNA) and double‐stranded DNA (dsDNA). Then, a sensitive fluorescence biosensor for ctDNA detection is developed, demonstrating impressive performance. To facilitate point‐of‐care testing (POCT) ctDNA, a paper‐based microfluidic chip incorporated a delay area and a mixing channel is designed. The constricted‐expanded structure channel optimization is guided by numerical simulations and experiments. A WeChat mini program named “ctDNA Detection” is designed for readout assay. Furthermore, cell and mice serum samples are analyzed, with Magnetic Bead@Graphene Oxide (MB@GO) and clutch probes for magnetic pre‐enrichment. The results accuracy is confirmed by its consistency with standard qPCR results (AUC = 1). The successful detection of ctDNA in post‐surgery mouse models underscored the biosensor's potential for cancer treatment monitoring. Thus, this research not only advanced the understanding of the MBene‐DNA interaction in biosensing, but also can pave the way for novel applications in bioimaging and nanopore‐based nucleic acid sequencing, leveraging the digital transformation of DNA base‐MBene adsorption differences.

The clinical utility of plasma circulating tumor DNA in the diagnosis and disease surveillance in non-diffuse large B-cell non-Hodgkin lymphomas.

Aim: Advances in circulating tumor DNA (ctDNA) analysis for diffuse large B-cell lymphoma (DLBCL) have prompted the evaluation of its utility in other non-Hodgkin lymphomas (NHLs), leading to significant insights into its potential applications.Methods: We retrospectively studied paired plasma and tissue/bone marrow biopsies of 203 non-DLBCL NHLs [87 follicular lymphomas (FL), 64 mantle cell lymphomas (MCL), 30 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL) and 22 marginal zone lymphomas (MZL)]. Genomic profiling was performed using a targeted next generation sequencing panel (Hemasalus™). Longitudinal analyses were performed to explore plasma ctDNA utility in disease monitoring.Results: High plasma ctDNA detection rates were observed across NHL subtypes (FL: 88.5%, MCL: 90.6%, CLL/SLL: 100%, MZL: 68.2%), with high concordance of actionable mutations (FL: 87.4%, MCL: 93.8%, CLL/SLL: 93.3%, MZL: 81.8%) and multiple genetic aberrations exclusively identified in plasma. Particularly, IGH-BCL2 and IGH-CCND1 fusions were concordant between plasma and tumor biopsies in FLs (91.1%) and MCLs (91.3%), respectively. Longitudinal data demonstrated that ctDNA clearance correlated with complete response but ctDNA increases preceded radiological relapses.Conclusion: ctDNA exhibited high concordance with tumor biopsy in detecting genetic aberrations and demonstrated potential as a promising noninvasive approach to disease surveillance in non-DLBCL NHLs.

Prospective assessment of circulating tumor DNA in patients with metastatic uveal melanoma treated with tebentafusp

Tebentafusp, a bispecific immune therapy, is the only drug that demonstrated an overall survival benefit in patients with metastatic uveal melanoma (MUM). Circulating tumor DNA (ctDNA) has emerged as a potential prognostic and predictive marker in the phase 3 IMCgp100-202 trial using multiplex PCR-based next-generation sequencing (NGS). In this study (NCT02866149), ctDNA dynamics were assessed using droplet digital PCR (ddPCR) in 69 MUM patients undergoing tebentafusp treatment. Notably, 61% of patients exhibited detectable ctDNA before treatment initiation, which was associated with shorter overall survival (median 12.9 months versus 40.5 months for patients with undetectable ctDNA; p < 0.001). Patients manifesting a 90% or greater reduction in ctDNA levels at 12 weeks demonstrated markedly prolonged overall survival (median 21.2 months versus 12.9 months; p = 0.02). Our findings highlight the potential of ddPCR-based ctDNA monitoring as an economical, pragmatic and informative approach in MUM management, offering valuable insights into treatment response and prognosis.

Circulating tumour DNA and risk of recurrence in patients with asymptomatic versus symptomatic colorectal cancer.

Multiple initiatives aim to develop circulating tumour DNA (ctDNA) tests for early cancer detection in asymptomatic individuals. The few studies describing ctDNA-testing in both asymptomatic and symptomatic patients report lower ctDNA detection in the asymptomatic patients. Here, we explore if asymptomatic patients differ from symptomatic patients e.g. by including a 'low-ctDNA-shedding' and 'less-aggressive' subgroup. ctDNA assessment was performed in two independent cohorts of consecutively recruited patients with asymptomatic colorectal cancer (CRC) (Cohort#1: n = 215, Cohort#2: n = 368) and symptomatic CRC (Cohort#1: n = 117, Cohort#2: n = 722). After adjusting for tumour stage and size, the odds of ctDNA detection was significantly lower in asymptomatic patients compared to symptomatic patients (Cohort#1: OR: 0.4, 95%CI: 0.2-0.8, Cohort#2: OR: 0.7, 95%CI: 0.5-0.9). Further, the recurrence risk was lower in asymptomatic patients (Cohort#1: sHR: 0.6, 95%CI: 0.3-1.2, Cohort#2: sHR: 0.6, 95%CI: 0.4-1.0). Notably, ctDNA-negative asymptomatic patients had the lowest recurrence risk compared to the symptomatic patients (Cohort#1: sHR: 0.2, 95%CI: 0.1-0.6, Cohort#2: sHR: 0.3, 95%CI: 0.2-0.6). Our study suggests that asymptomatic patients are enriched for a 'low-ctDNA-shedding-low-recurrence-risk' subgroup. Such insights are needed to guide ctDNA-based early-detection initiatives and should prompt discussions about de-escalation of therapy and follow-up for ctDNA-negative asymptomatic CRC patients.

Feasibility of ctDNA in detecting minimal residual disease and predicting recurrence for colorectal cancer liver metastases.

Approximately 50% of patients diagnosed with colorectal cancer develop colorectal cancer liver metastases (CRLM). Although curative intent liver resection provides 5-year survival of 40-50%, up to 70% of patients develop recurrence of CRLM. Detection of minimal residual disease (MRD) is essential for timely, optimized treatment. This study evaluated the feasibility and utility of using circulating tumor DNA (ctDNA) to identify MRD and predict disease recurrence. Patients with CRLM that underwent liver resection and had known KRAS or PIK3CA mutations were retrospectively identified. Serial blood samples were collected every 3 months following surgery for disease surveillance. ctDNA was isolated from the samples and analyzed with digital PCR (dPCR). KRAS and PIK3CA mutations were identified by dPCR in 29 patients over 115 timepoints. In patients with detectable ctDNA at time of liver resection, 81% (13/16) developed disease recurrence, while 46% (6/13) of the patients with undetectable ctDNA recurred (p=0.064). Presence of ctDNA was detected in 27.6% (8/29) of the initial postoperative samples. Radiologic recurrence was later diagnosed in 100% (8/8) of these patients, while 52% (11/21) who had undetectable ctDNA postoperatively recurred (p=0.026). Detectable ctDNA postoperatively was associated with a shorter disease-free survival (DFS) of 9 months vs 13 months in patients who had undetectable ctDNA (HR 2.95, 95% CI 1.16-7.49; p=0.02). Liquid biopsy using dPCR can identify low levels of ctDNA, enabling early detection of disease recurrence. Additionally, the presence of ctDNA postoperatively was predictive of recurrence. This study corroborates current literature and provides rational for moving toward a clinical trial using ctDNA and dPCR to detect MRD after CRLM resection.

Discerning the impact of ctDNA detection on patient decision-making in early-stage breast cancer

The impact of knowledge of circulating tumor DNA (ctDNA) status on patient decisions in high-risk triple-negative breast cancer (TNBC) weighing benefit and toxicity is unknown. Here, 286 women with a history of non-metastatic breast cancer who had received chemotherapy completed a survey mimicking scenarios of residual TNBC after chemotherapy and unknown, negative, or positive ctDNA status to determine the shift in the decision to receive adjuvant therapy. Participants were then presented scenarios mimicking possible post-neoadjuvant therapies and rated acceptability. A general linear model with repeated measures determined contributions of risk reduction and toxicity. When the hypothetical risk of recurrence mimicked ctDNA negativity, significantly less participants were accepting of adjuvant capecitabine versus no therapy. When presented with ctDNA positivity and increased recurrence risk, the degree of benefit impacted acceptability more than the toxicity profile. As genomic technology advances and ctDNA assays become commercially available, it is imperative to understand the impact on patient decision-making.

Plasma EGFR mutation ctDNA dynamics in patients with advanced EGFR-mutated NSCLC treated with Icotinib: phase 2 multicenter trial result

Plasma epidermal growth factor receptor mutation (EGFRm) circulating tumor DNA (ctDNA) dynamics exhibit promise in predicting outcomes in patients with EGFRm-advanced non-small cell lung cancer (NSCLC). However, there remains limited trial-level data on integrating ctDNA monitoring into clinical practice. We performed a prospective, multicenter trial to investigate the relationship between EGFRm ctDNA dynamic changes and clinical outcomes in NSCLC patients with EGFRm. Ninety-eight treatment-naive EGFRm-advanced NSCLC patients were recruited and administered icotinib until disease progression. Plasma samples were collected at baseline and four weeks after icotinib administration. ctDNA was analyzed using a droplet-digital polymerase chain reaction. At baseline, 71.4% of patients had detectable EGFRm ctDNA. Among them, 45.9% of patients’ ctDNA became undetectable within four weeks of treatment. These patients demonstrated significantly longer progression-free survival (PFS) and overall survival (OS) than those with detectable ctDNA after treatment (P = 0.004 and < 0.001, respectively) and were comparable to those with undetectable ctDNA at both baseline and four weeks. ctDNA detectable at four weeks emerged as a poor independent risk factor for PFS and OS. Patients whose ctDNA became undetectable after treatment had outcomes similar to those with initially undetectable ctDNA, underscoring the predictive value of ctDNA dynamics in treatment efficacy.Registry and the Registration No. of the study/trial: ChiCTR-DDD-17013131. Date of registration: 2017-10-27.

SY26-2 Development of ctDNA for MRD detection in breast cancer

SY26-2 Development of ctDNA for MRD detection in breast cancer

Blood and cerebrospinal fluid biomarkers in neuro-oncology.

The purpose of this review is to discuss the value of blood and CSF biomarkers in primary CNS tumors. Several analytes can be assessed with liquid biopsy techniques, including circulating tumor cells, circulating cell-free tumor DNA, circulating cell-free RNA, circulating proteins and metabolites, extracellular vesicles and tumor-educated platelets. Among diffuse gliomas of the adult, ctDNA in blood or CSF has represented the most used analyte, with the detection of molecular alterations such as MGMT promoter, PTEN, EGFRVIII, TERT promoter mutation and IDH R132H mutation. In general, CSF is enriched for ctDNA as compared with plasma. The use of MRI-guided focused ultrasounds to disrupt the blood-brain barrier could enhance the level of biomarkers in both blood and CSF. The detection of MYD88 L265P mutation with digital droplet PCR and the detection of ctDNA with next generation sequencing represent the best tools to diagnose and monitoring CNS lymphomas under treatment. In meningiomas, the low concentration of ctDNA is a limiting factor for the detection of driver mutations, such as NF2, AKTs, SMO, KLF4, TRAF7, SMARCB1, SMARCE1, PTEN, and TERT; an alternative approach could be the isolation of ctDNA through circulating extracellular vesicles. Liquid biopsies are being used extensively for diagnosis and surveillance of diffuse midline gliomas, in particular with the detection of the driver mutation H3K27M. Last, specific methylome patterns in CSF may allow the distinction of glioblastomas from CNS lymphomas or meningiomas. This review summarizes the current knowledge and future perspectives of liquid biopsy of blood and CSF for diagnosis and monitoring of primary CNS tumors.

Liquid Biopsy in Cancer Diagnosis-Treatment Methodology and Applications

The comprehensive review of liquid biopsy as a cutting-edge, non-invasive technique for cancer detection, diagnosis, and monitoring may be found in the document “Liquid Biopsy in Cancer Diagnosis – Treatment Methodology and Applications.” Via liquid biopsy, tumor-related indicators present in physiological fluids such as blood, urine, and saliva are examined, including exosomes, circulating tumor cells (CTCs), and circulating tumor DNA (ctDNA). Liquid biopsy enables regular, real-time sampling of tumor dynamics, in contrast to standard tissue biopsies, which are invasive and restricted to particular tumor regions. This renders it a potent instrument for the identification of cancer mutations and the tracking of tumor progression, particularly in cases of metastatic or elusive cancers. Technological developments like digital droplet PCR (ddPCR) and next-generation sequencing (NGS) have greatly improved the sensitivity and precision of ctDNA detection, making it possible to identify unusualexceptionally rare genetic mutations. This enhances the efficacy of tailored cancer treatment and monitoring. The genetic heterogeneity of cancers is captured by liquid biopsies, providing information on several tumor subclones that may be missed by traditional biopsies. The document also describes the use of different body fluids for different forms of cancer, such as head and neck and urological tumors, such as saliva, urine, and blood. Blood is the most often utilized medium for tracking genetic changes due to its ease of accessibility. Saliva and urine are useful in the detection of head and neck malignancies and bladder cancer, respectively. Liquid biopsy has several advantages, but it also has drawbacks, such as the requirement for standardized procedures, high prices, and differences in sensitivity and specificity between platforms.In order to overcome these obstacles and integrate liquid biopsy into standard clinical practice, the paper emphasizes the significance of more research and development. This will improve patient outcomes, early cancer detection, and personalized treatment.

Clinical utility of ctDNA by amplicon based next generation sequencing in first line non small cell lung cancer patients.

The assessment of ctDNA has emerged as a minimally invasive avenue for molecular diagnosis and real-time tracking of tumor progression in NSCLC. However, the evaluation of ctDNA by amplicon-based NGS has been not endorsed by all the healthcare systems and remains to be fully integrated into clinical routine practice. To compare tissue single-gene with plasma multiplexed testing, we retrospectively evaluated 120 plasma samples from 12 consecutive patients with advanced non-squamous NSCLC who were part of a prospective study enrolling treatment-naïve patients and in which tissue samples were evaluated using a single-gene testing approach. While the plasma ctDNA detection of EGFR and BRAF mutations had an acceptable level of concordance with the archival tissue (85%), discordance was seen in all the patients in whom ALK alterations were only detected in tissue samples. Among six responders and six non-responders, early ctDNA mutant allelic frequency (MAF) reduction seemed to predict radiologic responses and longer survival, whereas increasing MAF values with the emergence of co-mutations like BRAFV600E, KRASG12V or TP53M237I seemed to be an early indicator of molecular and radiologic progression. This report using an amplicon-based NGS assay on ctDNA underscores the real-life need for plasma and tissue genotyping as complementary tools in the diagnostic and therapeutic decision-making process.

Self-reinforced D-MOF and autocatalytic AuPd@SnS2 dual-wavelength electrochemiluminescence biosensor for detection of ctDNA and CEA

Trace detection of circulating tumor DNA (ctDNA) and CEA is of great significance for early prevention and diagnosis of cancer. In this work, the well electrochemiluminescence (ECL) of the dual-ligand metal-organic frameworks (D-MOFs) and AuPd@SnS2 QDs nuclear-satellite conjugate was newly explored, and a dual-wavelength biosensing platform based on ECL energy transfer between D-MOFs and AuPd NPs coupled with catalytic effect of AuPd NPs on ECL of SnS2 QDs was developed for detection of trace ctDNA and CEA. Here, thanks to the ordered heterogeneous arrangement and network charge transfer of the luminescent ligand, the prepared D-MOF exhibits highly enhanced ECL without exogenous co-reactants. Target ctDNA was exponentially amplified using an entropy-driven DNA cycle amplification strategy to obtain bandage DNA, which triggered the DNA walker to connect the AuPd@SnS2 core-satellite probe to the electrode. Therefore, the ECL ratio of D-MOF and sensitized SnS2 at 535 nm and 650 nm was used to achieve ultrasensitive detection of trace ctDNA. Moreover, the specific recognition of target CEA to T1 DNA induced changes in dual-wavelength signals to achieve further detection of CEA, and ensure the accuracy of cancer analysis. The work opened up two novel ECL materials and developed a novel ECL biosensor for rapid detection of double tumor markers, providing a new way for early prevention and diagnosis of actual cancer.

Role of cfDNA and ctDNA to improve the risk stratification and the disease follow-up in patients with endometrial cancer: towards the clinical application

BackgroundThere has been a rise in endometrial cancer (EC) incidence leading to increased mortality. To counter this trend, improving the stratification of post-surgery recurrence risk and anticipating disease relapse and treatment resistance is essential. Liquid biopsy analyses offer a promising tool for these clinical challenges, though the best strategy for applying them in EC must be defined. This study was designed to determine the value of cfDNA/ctDNA monitoring in improving the clinical management of patients with localized and recurrent disease.MethodsPlasma samples and uterine aspirates (UA) from 198 EC patients were collected at surgery and over time. The genetic landscape of UAs was characterized using targeted sequencing. Total cfDNA was analyzed for ctDNA presence based on the UA mutational profile.ResultsHigh cfDNA levels and detectable ctDNA at baseline correlated with poor prognosis for DFS (p-value < 0.0001; HR = 9.25) and DSS (p-value < 0.0001; HR = 11.20). This remained clinically significant when stratifying tumors by histopathological risk factors. Of note, cfDNA/ctDNA analyses discriminated patients with early post-surgery relapse and the ctDNA kinetics served to identify patients undergoing relapse before any clinical evidence emerged.ConclusionsThis is the most comprehensive study on cfDNA/ctDNA characterization in EC, demonstrating its value in improving risk stratification and anticipating disease relapse in patients with localized disease. CtDNA kinetics assessment complements current strategies to monitor the disease evolution and the treatment response. Therefore, implementing cfDNA/ctDNA monitoring in clinical routines offers a unique opportunity to improve EC management.Translational relevanceThe study demonstrates that high levels of cfDNA and detectable ctDNA at baseline are strong indicators of poor prognosis. This enables more accurate risk stratification beyond traditional histopathological factors, allowing clinicians to identify high-risk patients who may benefit from more aggressive treatment and closer monitoring. Moreover, longitudinal analysis of cfDNA/ctDNA can detect disease recurrence months before clinical symptoms or imaging evidence appear. This early warning system offers a significant advantage in clinical practice, providing a window of opportunity for early intervention and potentially improving patient outcomes.

Prediction of undetectable circulating tumor DNA by comprehensive genomic profiling assay in metastatic prostate cancer: the SCRUM-Japan MONSTAR SCREEN project.

Undetectable circulating tumor DNA (ctDNA) is an obstacle to performing comprehensive genomic profiling in daily practice to identify genomic alterations. We investigated the associations between clinicopathological factors and undetectable ctDNA using a commercially available comprehensive genomic profiling assay in metastatic prostate cancer. Patients treated with systemic treatment for metastatic prostate cancer were included. ctDNA was analyzed by FoundationOne®Liquid CDx at enrollment. The associations between clinicopathological characteristics and ctDNA detection were analyzed. The number of bone metastasis was associated with ctDNA detection (odds ratio [95% confidence interval], 13.6 [1.71-108], P = 0.014). An algorithm predicting ctDNA detection using clinicopathological parameters was created. If ≥ 4 bone metastases were observed, ctDNA detection was estimated to be 98.9%. Among the patients with < 4 bone metastases, if two or three features among ISUP grade group 5, PSA level ≥ 10ng/ml, and castration resistance were present, the ctDNA detection rate was 96.7% while the ctDNA detection rate was 86.3% if no or only one feature was present. An algorithm created in this study is helpful in determining when to undertake comprehensive genomic profiling assay using blood.

Real-world experience with circulating tumor DNA in cerebrospinal fluid from patients with central nervous system tumors

The characterization of genetic alterations in tumor samples has become standard practice for many human cancers to achieve more precise disease classification and guide the selection of targeted therapies. Cerebrospinal fluid (CSF) can serve as a source of tumor DNA in patients with central nervous system (CNS) cancer. We performed comprehensive profiling of CSF circulating tumor DNA (ctDNA) in 711 patients using an FDA-authorized platform (MSK-IMPACT™) in a hospital laboratory. We identified genetic alterations in 489/922 (53.0%) CSF samples with clinically documented CNS tumors. None of 85 CSF samples from patients without CNS tumors had detectable ctDNA. The distribution of clinically actionable somatic alterations was consistent with tumor-type specific alterations across the AACR GENIE cohort. Repeated CSF ctDNA examinations from the same patients identified clonal evolution and emergence of resistance mechanisms. ctDNA detection was associated with shortened overall survival following CSF collection. Next-generation sequencing of CSF, collected through a minimally invasive lumbar puncture in a routine hospital setting, provides clinically actionable cancer genotype information in a large fraction of patients with CNS tumors.

Abstract A009: Mutant KRAS in Circulating Tumor DNA Assessed by Digit Droplet PCR as a Biomarker in Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy

Abstract Introduction: The detectability and prognostic significance of circulating tumor DNA (ctDNA) in patients with pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant chemotherapy (NAC) is unclear. The objective of this study was to investigate the rate of detection and prognostic value of KRAS mutant ctDNA in PDAC patients after treatment with NAC as assessed by digital droplet polymerase chain reaction (ddPCR). Methods: Patients with newly diagnosed, localized, PDAC were enrolled in a prospective cohort study. Peripheral blood samples were collected at diagnosis, after NAC, and after resection. DNA was extracted from patient plasma samples using the QIAamp Circulating Nucleic Acid Kit #55114 (Qiagen N.V., Venlo Netherlands). Each sample was probed (catalog number 10049550) for KRAS codon 12 exon 2 glycine substitutions for aspartic acid (G12D), valine (G12V), and arginine (G12R). Kaplan-Meier, log-rank, and Cox regression survival analyses were performed. Results: 84 patients were included in the analysis. Patients were most commonly male (53.6%) and had tumors that were radiographically resectable (57.1%). After diagnosis, 79.8% of patients received NAC and 57.1% of patients received curative-intent surgical resection. Samples were collected for analysis from 59 (70.2%), 56 (83.6%), and 45 (93.8%) patients at diagnosis, after NAC, and after resection, respectively. Mutant KRAS ctDNA was detected in 49.3% of patients at diagnosis, 69.6% of patients after NAC, and 54.1% of patients after resection, respectively. The median OS of the cohort was 25.1 months (12.0-not reached [NR]). Detection (hazard ratio [HR] 36.8, 95% confidence interval [CI] 2.9-461.4), copy number (HR 4.0 95% CI 1.6-10.2), and percentage mutation (HR 2.9 95% CI 1.2-6.8) of KRAS G12V ctDNA after resection were all independently associated with shorter OS. Mutant KRAS ctDNA detection was not associated with and OS at other study timepoints. There were 15 (17.9%) patients that cleared mutational ctDNA over the course of treatment. Clearance of ctDNA during NAC was associated with improved overall survival (OS) (18.4 mo. vs NR). Conclusions: In patients with PDAC treated with NAC, KRAS mutations are detectable in ctDNA by ddPCR in the majority of patients over the course of treatment. Detection, copy number, and percentage mutation of KRAS G12V after NAC and resection were each independently associated with shorter OS. Clearance of ctDNA is associated with improved OS. The results demonstrate that KRAS mutant ctDNA predicts prognosis after NAC and resection. Citation Format: Dominic Vitello, Dhavan Shah, Amy Wells, Larissa Masnyk, Madison Cox, Lauren Janczewski, John Abad, Kevin Dawravoo, Arlene D'Souza, Grace Suh, Robert Bayer, Massimo Cristofanilli, David Bentrem, Yingzhe Liu, Hui Zhang, Lucas Santana-Santos, Lawrence Jennings, Qiang Zhang, Akhil Chawla. Mutant KRAS in Circulating Tumor DNA Assessed by Digit Droplet PCR as a Biomarker in Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A009.

Detection of circulating tumor DNA by tumor-informed whole-genome sequencing enables prediction of recurrence in stage III colorectal cancer patients

IntroductionCirculating tumor (ctDNA) can be used to detect residual disease after cancer treatment. Detecting low-level ctDNA is challenging, due to the limited number of recoverable ctDNA fragments at any target loci. In response, we applied tumor-informed whole-genome sequencing (WGS), leveraging thousands of mutations for ctDNA detection. MethodsPerformance was evaluated in serial plasma samples (n = 1283) from 144 stage III colorectal cancer patients. Tumor/normal WGS was used to establish a patient-specific mutational fingerprint, which was searched for in 20x WGS plasma profiles. For reproducibility, paired aliquots of 172 plasma samples were analyzed in two independent laboratories. De novo variant calling was performed for serial plasma samples with a ctDNA level > 10 % (n = 17) to explore genomic evolution. ResultsWGS-based ctDNA detection was prognostic of recurrence: post-operation (Hazard ratio [HR] 6.75, 95 %CI 3.18–14.3, p < 0.001), post-adjuvant chemotherapy (HR 28.9, 95 %CI 10.1–82.8; p < 0.001), and during surveillance (HR 22.8, 95 %CI 13.7–37.9, p < 0.0001). The 3-year cumulative incidence of ctDNA detection in recurrence patients was 95 %. ctDNA was detected a median of 8.7 months before radiological recurrence. The independently analyzed plasma aliquots showed excellent agreement (Cohens Kappa=0.9, r = 0.99). Genomic characterization of serial plasma revealed significant evolution in mutations and copy number alterations, and the timing of mutational processes, such as 5-fluorouracil-induced mutations. ConclusionOur study supports the use of WGS for sensitive ctDNA detection and demonstrates that post-treatment ctDNA detection is highly prognostic of recurrence. Furthermore, plasma WGS can identify genomic differences distinguishing the primary tumor and relapsing metastasis, and monitor treatment-induced genomic changes.