ctDNA Mutation Detection Research Articles

Abstract 3750: Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR

Abstract Introduction: Recent advances in technology support the use of liquid biopsy as a noninvasive method for cancer monitoring and diagnosis. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are both regarded as key liquid biopsy biomarkers, offering significant disease-related molecular insights. Despite their potential, dual isolation and profiling have been challenged in a clinical setting by the lack of established platforms and optimized protocols that support this approach. The Genesis System with Celselect SlidesTM captures CTCs based on size and deformability, then the enriched cells can be recovered for subsequent downstream analysis. Concurrently, Droplet Digital PCR (ddPCRTM) can be utilized to interrogate cell-free DNA (cfDNA) to determine the presence of a range of DNA aberrations present in ctDNA. This study, using a model lung CTC-ctDNA sample, illustrates the feasibility of this dual-biomarker approach to detect cancer-associated nucleic acid mutations simultaneously with CTC enumeration, using commercially available kits and instruments. Methods: To model lung cancer samples, blood samples were prepared by spiking in two model circulating markers, cultured cancer cells (model CTCs, human ㅣlung cancer cell line A549) and fragmented genomic DNA containing EGFR mutations (model ctDNA, multiplexed 23 ctDNA [~145 bp band > 95%]), into whole blood from a healthy donor prior to enrichment/extraction. Plasma isolation from the whole blood model samples was performed using either two-step centrifugation or Ficoll-Paque PLUS solution for plasma/buffy coat separation. The extracted plasma layer was used for ctDNA separation, while the remaining cell layer was used for CTC isolation on the Genesis System with a modified protocol. Results: The Genesis System isolated 78-79% of spiked cancer cells in whole blood at a concentration of 40 cells/ml. Despite additional plasma separation steps and liquid handling, the system isolated CTCs spiked in with minimal cell loss (<6%). With an optimized ctDNA extraction, more than 70% of spiked ctDNA in blood samples in the size range of 120-220 bp was extracted. Using the Bio-Rad QX200 ddPCR System and ddPCR EGFR T790M Mutation Detection Assay, blood samples with the model ctDNA spiked in showed positive droplets (17 copies) for EGFR T790M while control blood samples without model ctDNA spiked in showed no positive droplets. Discussion and Conclusions: This work illustrates an optimized protocol for the dual isolation and profiling of CTCs and ctDNA from a single blood sample by using an automated CTC isolation platform and high-sensitivity ddPCR based ctDNA mutation detection. This protocol enables comprehensive liquid biopsy studies using a single blood sample from cancer patients with readily available instruments and kits, which can be applied to many other cancers. Citation Format: Yoon-Tae Kang, Abiodun Bodunrin, Errile Pusod, Kris Simonyi, Adam Corner, David Coe, Michelle Racey, Elizabeth J. Dreskin. Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR [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 3750.

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.

CRISPincette, and detection of low frequency cancer mutations in ctDNA.

61 Background: Liquid biopsy has emerged as a non-invasive and widely applicable approach in clinical practice for cancer detection and monitoring. Detecting low-frequency mutations in circulating tumor DNA (ctDNA) is crucial for the success of liquid biopsy; however, technical limitations have hindered the detection of low variant allele frequency (VAF) mutations. To address this issue, we developed CRISPincette, a novel method which combines next-generation sequencing with an advanced CRISPR system, GeneCker-Cas9. Methods: CRISPincette was designed to efficiently detect mutant alleles by eliminating abundant wild-type alleles from normal cell-induced cell-free DNA (cfDNA) using CRISPR/Cas9. An engineered CRISPR/Cas9 variant, GeneCker-Cas9, was developed to maintain single-base precision outside the PAM region, enabling efficient discrimination of single-base mutations at all 20 positions within a single guide RNA target sequence. This advanced fidelity significantly improved the accuracy compared to high-specificity SpCas9 variants (SpCas9-HF and eSpCas9). To validate the sensitivity and accuracy of ctDNA detection using CRISPincette, we employed NGS standard materials (Horizon HD776, 778, 779) and assessed the improvement in ctDNA detection sensitivity for EGFR gene mutations (EGFR 19del, EGFR L858R, EGFR T790M). Subsequently, we analyzed the clinical applicability of CRISPincette using plasma samples from 7 non-small cell lung cancer (NSCLC) patients (6 patients with EGFR mutations and 1 patient without EGFR mutations). Results: Our analysis showed an approximately 10 to 30-fold enhancement in the detection sensitivity of EGFR gene mutations. Concordant detection of EGFR mutations was observed in the 6 patients with EGFR mutations using real-time PCR tests in each tissue biopsy, and no EGFR mutation was detected in the patient without the EGFR mutation. This confirmed CRISPincette's high accuracy and sensitivity in detecting low-frequency mutations in ctDNA. Conclusions: The implementation of CRISPincette has significantly improved the accuracy and sensitivity of liquid biopsy, making it a reliable and non-invasive alternative for confirming molecular pathological information in cancer patients. Furthermore, the high accuracy of CRISPincette will be a powerful tool for monitoring treatment and prognosis through the timing of anticancer drug administration. Overall, CRISPincette represents a substantial advancement in liquid biopsy technology, with potential implications for cancer diagnosis, treatment allocation, and monitoring the emergence of resistance.

Implementation of a Pilot Study to Analyze Circulating Tumor DNA in Early-Stage Lung Cancer.

Liquid biopsies based on plasma circulating tumour deoxyribonucleic acid (ctDNA) have shown promise in monitoring lung cancer evolution. The expression of ctDNA across time, its relationship with clinicopathological parameters and its association with lung cancer progression through imaging allow us to weigh how useful ctDNA could be in monitoring surgically resectable lung cancer. The aim of this study was to assess the impact of ctDNA analysis implementation in early-stage lung cancer. A cohort of 47 patients was sequentially recruited. Only 34 patients with early-stage lung cancer were included. All patients had a tissue specimen and five blood samples drawn: at the preoperative stage, from the pulmonary vein, at surgical discharge, at the first follow-up and at the last follow-up. All blood samples were evaluated for ctDNA expression. On average, the maximum yield of ctDNA was obtained in liquid biopsies at the surgical discharge of patients when compared with PO, PV, and F1 (p < 0.0001, p < 0.0001, p < 0.0001 respectively). No statistically significant differences were found when comparing the last follow-up to surgical discharge ctDNA expression (p = 0.851). The correlation between ctDNA concentration according to five-time points and the four clinicopathological characteristics showed that patients younger than 70 years had a statistically significant reduction of the concentration of ctDNA at the preoperative and surgical discharge time point [β = -16 734 (-27 707; - 5760); p = 0.003; β = -21 785 (-38 447; -5123); p = 0.010], as opposed to an increase of the concentration of ctDNA at the pulmonary vein and last follow-up time points [β = 8369 (0.359; 16 378); p = 0.041; β = 34 402 (12 549; 56 254); p = 0.002] all with a confidence level of 95%. In the cases where actionable mutations were identified in tissue biopsies, the expected mutation was found in five out of six patients plasma samples at the pre-operatory time point and in two out of six patients plasma samples at the pulmonary vein time point. Two out of six patients with actionable mutations had disease progression. The results of this pilot study suggest that the maximum yield of ctDNA is obtained at the surgical discharge of the patients and that the pre-operatory timepoint is the one offering the highest sensitivity for the detection of actionable mutations in ctDNA in early-stage lung cancer.

Circulating Tumor DNA Reflects Histologic and Clinical Characteristics of Various Lymphoma Subtypes.

We designed and evaluated the clinical performance of a plasma circulating tumor DNA (ctDNA) panel of 112 genes in various subtypes of lymphoma. Targeted deep sequencing with an error-corrected algorithm was performed in ctDNA from plasma samples that were collected before treatment in 42 lymphoma patients. Blood buffy coat was utilized as a germline control. We evaluated the targeted gene panel using mutation detection concordance on the plasma samples with matched tissue samples analyzed the mutation profiles of the ctDNA. Next-generation sequencing analysis using matched tissue samples was available for 18 of the 42 patients. At least one mutation was detected in the majority of matched tissue biopsy samples (88.9%) and plasma samples (83.3%). A considerable number of mutations (40.4%) that were detected in the tissue samples were also found in the matched plasma samples. Majority of patients (21/42) were diffuse large B cell lymphoma patients. The overall detection rate of ctDNA in patients was 85.7% (36/42). The frequently mutated genes included PIM1, TET2, BCL2, KMT2D, KLHL6, HIST1H1E, and IRF8. A cutoff concentration (4,506 pg/mL) of ctDNA provided 88.9% sensitivity and 82.1% specificity to predict ctDNA mutation detection. The ctDNA concentration correlated with elevated lactate dehydrogenase level and the disease stage. Our design panel can detect many actionable gene mutations, including those at low frequency. Therefore, liquid biopsy can be applied clinically in the evaluation of lymphoma patients, especially in aggressive lymphoma patients.

Evaluation of the Prognostic Value of Low-Frequency KRAS Mutation Detection in Circulating Tumor DNA of Patients with Metastatic Colorectal Cancer.

KRAS mutation in tumor tissue is a well-known predictor of resistance to the treatment of anti-EGFR antibodies in metastatic colorectal cancers (mCRC). However, the prognostic value of low-frequency plasma circulating tumor DNA (ctDNA) KRAS mutation in predicting treatment resistance in pretreated mCRC patients remains controversial. This study retrospectively reviewed the clinical course, including response to anti-EGFR and anti-VEGF therapies, and changes in serum tumor marker levels along with image studies in mCRC patients with <1.5% KRAS mutations detected in plasma ctDNA by next-generation sequencing (NGS) at a single center in Taiwan. We identified six pretreated mCRC patients with low-frequency KRAS G12V/G12D/G12S/G13D mutations (variant allele frequency 0.26~1.23%) in plasma ctDNA. Co-occurring low-frequency ctDNA mutations in APC, TP53, MAP2K1, KEAP1, or CTNNB1 were also detected. Although all six patients had treatment adjustments within one month after the ctDNA genetic test, image-evident tumor progression was noted in all patients within a median of 4 months afterwards. Re-challenge therapy with a combination of anti-EGFR, anti-VEGF, and FOLFIRI chemotherapy was found to be ineffective in a patient with 0.38% KRAS G12D mutation in baseline ctDNA. Our study suggests that the detection of low-frequency KRAS mutations in ctDNA could be used as a predictor of treatment response in mCRC patients.

Circulating tumor DNA sequencing of pediatric solid and brain tumor patients: An institutional feasibility study

The potential of circulating tumor DNA (ctDNA) analysis to serve as a real-time “liquid biopsy” for children with central nervous system (CNS) and non-CNS solid tumors remains to be fully elucidated. We conducted a study to investigate the feasibility and potential clinical utility of ctDNA sequencing in pediatric patients enrolled on an institutional clinical genomics trial. A total of 240 patients had tumor DNA profiling performed during the study period. Plasma samples were collected at study enrollment from 217 patients and then longitudinally from a subset of patients. Successful cell-free DNA extraction and quantification occurred in 216 of 217 (99.5%) of these initial samples. Twenty-four patients were identified whose tumors harbored 30 unique variants that were potentially detectable on a commercially-available ctDNA panel. Twenty of these 30 mutations (67%) were successfully detected by next-generation sequencing in the ctDNA from at least one plasma sample. The rate of ctDNA mutation detection was higher in patients with non-CNS solid tumors (7/9, 78%) compared to those with CNS tumors (9/15, 60%). A higher ctDNA mutation detection rate was also observed in patients with metastatic disease (9/10, 90%) compared to non-metastatic disease (7/14, 50%), although tumor-specific variants were detected in a few patients in the absence of radiographic evidence of disease. This study illustrates the feasibility of incorporating longitudinal ctDNA analysis into the management of relapsed or refractory patients with childhood CNS or non-CNS solid tumors.

Abstract 5587: Ultra-deep targeted sequencing of cell-free DNA and patient-matched white blood cells for treatment response evaluation in patients with metastatic colorectal cancer

Abstract Background: Treatment response monitoring of patients with metastatic colorectal cancer is currently performed by computed tomography (CT) imaging, which assesses tumor volume. Circulating tumor DNA (ctDNA) testing has the potential to replace or complement CT imaging by assessing the presence and abundance of tumor-specific mutations, allowing for personalized treatment and early adaptation of treatment regimens through risk stratification and the emergence of therapy resistance mutations. However, germline and clonal hematopoiesis-associated alterations can confound the identification of tumor-specific alterations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. Aim: The present study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue-independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC) derived DNA. Methods: In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 metastatic colorectal cancer patients participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay. Results: The combined cfDNA and WBC analysis prevented the reporting of false positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to the EGFR inhibitor panitumumab were identified in 42% of patients. Conclusions: Accurate ctDNA mutation detection by combined cfDNA and WBC genomic DNA analyses in a tumor tissue-independent manner mitigates sample logistics challenges in the clinical setting, and the application of this approach for evaluation of therapeutic response and resistance opens new avenues for early adaptation of treatment regimens. Citation Format: Iris van 't Erve, Jamie E. Medina, Alessandro Leal, Eniko Papp, Jillian Phallen, Vilmos Adleff, Elaine Jiayuee Chiao, Adith S. Arun, Karen Bolhuis, John K. Simmons, Aanavi Karandikar, Kenneth C. Valkenburg, Mark Sausen, Samuel V. Angiuoli, Robert B. Scharpf, Cornelis J. Punt, Gerrit J. Meijer, Victor E. Velculescu, Remond J. Fijneman. Ultra-deep targeted sequencing of cell-free DNA and patient-matched white blood cells for treatment response evaluation in patients with 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 5587.

Preoperative Mutational Analysis of Circulating Tumor Cells (CTCs) and Plasma-cfDNA Provides Complementary Information for Early Prediction of Relapse: A Pilot Study in Early-Stage Non-Small Cell Lung Cancer

We assessed whether preoperativemutational analyses of circulating tumor cells (CTCs) and plasma-cfDNA could be used as minimally invasive biomarkers and as complimentary tools for early prediction of relapse in early-stage non-small -cell lung cancer (NSCLC). Using ddPCR assays, hotspot mutations of BRAF, KRAS, EGFR and PIK3CA were identified in plasma-cfDNA samples and size-based enriched CTCs isolated from the same blood samples of 49 early-stage NSCLC patients before surgery and in a control group of healthy blood donors (n= 22). Direct concordance of the mutational spectrum was further evaluated in 27 patient-matched plasma-cfDNA and CTC-derived DNA in comparison to tissue-derived DNA. The prevalence of detectable mutations of the four tested genes was higher in CTC-derived DNA than in the corresponding plasma-cfDNA (38.8% and 24.5%, respectively).The most commonly mutated gene was PIK3CA, in both CTCs and plasma-cfDNA at baseline and at the time of relapse. Direct comparison of the mutation status of selected drug-responsive genes in CTC-derived DNA, corresponding plasma-cfDNA and paired primary FFPE tissues clearly showed the impact of heterogeneity both within a sample type, as well as between different sample components. The incidence of relapse was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA compared with patients in whom no mutation was detected (p =0.023). Univariate analysis showed a significantly higher risk of progression (HR: 2.716; 95% CI, 1.030-7.165; p =0.043) in patients with detectable mutations in plasma-cfDNA compared with patients with undetectable mutations, whereas the hazard ratio was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA (HR: 3.375; 95% CI, 1.098-10.375; p =0.034). Simultaneous mutational analyses of plasma-cfDNA and CTC-derived DNA provided complementary molecular information from the same blood sample and greater diversity in genomic information for cancer treatment and prognosis. The detection of specific mutations in ctDNA and CTCs in patients with early-stage NSCLC before surgery was independently associated with disease recurrence, which represents an important stratification factor for future trials.

Abstract P5-05-01: Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer

Abstract Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF&amp;lt; 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-01.

The utility of ctDNA in colorectal cancer with peritoneal metastases.

The development of peritoneal metastases (PM) in patients with colorectal cancer (CRC) connotates a poor prognosis. Circulating tumour (ctDNA) is a promising tumour biomarker in the management CRC. This systematic review aimed to summarize the role of ctDNA in patients with CRC and PM. Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, a systematic review of the literature until June 2022 was performed. Studies reporting on the utility of ctDNA in colorectal PM were included. A total of eight eligible studies were identified including a total of 167 patients. The findings from this review suggest an evolving role for ctDNA in CRC with PM. ctDNA can be isolated from both plasma and peritoneal fluid, with peritoneal fluid preferred as the liquid biopsy of choice with higher mutation detection rates. Concordance rates between tissue and plasma/peritoneal ctDNA mutation detection can vary, but is generally high. ctDNA has a potential role in monitoring anti-EGFR treatment response and resistance, as well as in predicting future prognosis and recurrence. The detection of ctDNA in plasma of patients with isolated PM is also possibly suggestive of occult systemic disease, and patients exhibiting such ctDNA positivity may benefit from systemic treatment. Limitations to ctDNA mutation detection may include the size of peritoneal lesions, as well as the fact that PM poorly shed ctDNA. While these findings are promising, further large-scale studies are needed to better evaluate the utility of ctDNA in this subset of patients.

Monitoring of Dynamic Changes and Clonal Evolution in Circulating Tumor DNA From Patients With IDH-Mutated Cholangiocarcinoma Treated With Isocitrate Dehydrogenase Inhibitors.

IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.

Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

Novel hybridization- and tag-based error-corrected method for sensitive ctDNA mutation detection using ion semiconductor sequencing

Circulating tumor DNA (ctDNA) analysis has emerged as a clinically useful tool for cancer diagnostics and treatment monitoring. However, ctDNA detection is complicated by low DNA concentrations and technical challenges. Here we describe our newly developed sensitive method for ctDNA detection on the Ion Torrent sequencing platform, which we call HYbridization- and Tag-based Error-Corrected sequencing (HYTEC-seq). This method combines hybridization-based capture with molecular tags, and the novel variant caller PlasmaMutationDetector2 to eliminate background errors. We describe the validation of HYTEC-seq using control samples with known mutations, demonstrating an analytical sensitivity down to 0.1% at > 99.99% specificity. Furthermore, to demonstrate the utility of this method in a clinical setting, we analyzed plasma samples from 44 patients with advanced pancreatic cancer, revealing mutations in 57% of the patients at allele frequencies as low as 0.23%.

Circulating Tumor DNA Mutations in Progressive Gastrointestinal Stromal Tumors Identify Biomarkers of Treatment Resistance and Uncover Potential Therapeutic Strategies.

Liquid biopsy circulating tumor DNA (ctDNA)-based approaches may represent a non-invasive means for molecular interrogation of gastrointestinal stromal tumors (GISTs). We deployed a customized 29-gene Archer® LiquidPlex™ targeted panel on 64 plasma samples from 46 patients. The majority were known to harbor KIT mutations (n = 41, 89.1%), while 3 were PDGFRA exon 18 D842V mutants and the rest (n = 2) were wild type for KIT and PDGFRA. In terms of disease stage, 14 (30.4%) were localized GISTs that had undergone complete surgical resection while the rest (n = 32) were metastatic. Among ten patients, including 7 on tyrosine kinase inhibitors, with evidence of disease progression at study inclusion, mutations in ctDNA were detected in 7 cases (70%). Known somatic mutations in KIT (n = 5) or PDGFRA (n = 1) in ctDNA were identified only among 6 of the 10 patients. These KIT mutants included duplication, indels, and single-nucleotide variants. The median mutant AF in ctDNA was 11.0% (range, 0.38%–45.0%). In patients with metastatic progressive KIT-mutant GIST, tumor burden was higher with detectable KIT ctDNA mutation than in those without (median, 5.97 cm vs. 2.40 cm, p = 0.0195). None of the known tumor mutations were detected in ctDNA for localized cases (n = 14) or metastatic cases without evidence of disease progression (n = 22). In patients with serial samples along progression of disease, secondary acquired mutations, including a potentially actionable PIK3CA exon 9 c.1633G>A mutation, were detected. ctDNA mutations were not detectable when patients responded to a switch in TKI therapy. In conclusion, detection of GIST-related mutations in ctDNA using a customized targeted NGS panel represents an attractive non-invasive means to obtain clinically tractable information at the time of disease progression.

P24.07 An Ultra-Sensitive Protocol for ctDNA Mutation Detection With Application in Lung Cancers

To establish an ultra-high sensitivity protocol for ctDNA mutation detection in blood samples, i.e., PEAC followed by NGS (PEAC+NGS), to achieve the desirable results of targeted panel sequencing and the sensitivity of digital PCR. The PEAC+NGS protocol was proposed to achieve high concordance between the matched blood and tissue samples from patients with early-stage lung cancer. The cfDNA extracted from blood samples was enriched by PEAC technique, and the obtained products were amplified by a second round PCR process to add the adapter sequences used for NGS sequencing. The PEAC+NGS protocol was applied to the blood samples of 46 lung cancer patients, including 36 patients in stage I and II, and 10 patients in stage III and IV. The testing results were used to evaluate the performance of PEAC+NGS in blood ctDNA testing and its consistency with the testing results of matched tissues. The PEAC+NGS protocol can simultaneously detect over 40 variant forms of five genes, i.e., EGFR, KRAS, NRAS, BRAF, and PIK3CA, which are closely associated with lung cancer. With a sequencing depth of over 10,000×, PEAC+NGS can achieve the ability to detect ctDNA variant at 0.01% allele frequencies, and 100% detection specificity. In 36 blood samples from patients with stage I and II lung cancer, 9 cases were detected as positive for mutations and 27 cases were negative by PEAC+NGS. The 9 blood samples with positive ctDNA variants also had positive results in matched tissue samples, while 11 of the 27 negative cases had positive testing results in matched tissues. The concordance between tissue and blood samples of stage I and II patients was 55.6%. Accordingly, for the 10 patients in stage III and IV, two blood samples detected as negative by PEAC+NGS also had negative results in the tissue; while among the remaining 8 patients that were tested positive in tissues, only one was failed to be detected by PEAC+NGS on the blood sample. The concordance between the blood and tissue samples was thus 90% for stage III and IV patients. We established an ultra-sensitive protocol for ctDNA mutation detection in blood samples, PEAC+NGS, which achieved 55.6% concordance in matched blood and tissue samples from patients with stage I and II lung cancer, and 90% concordance in stage III and IV lung cancer patients. These results demonstrate the great value of PEAC+NGS in clinical ctDNA testing.

Circulating tumor DNA mutation as a prognostic marker in melanoma with brain metastasis.

e21560 Background: Prognosis in melanoma with brain metastasis is poor with a median survival of four months and a one-year survival rate of 10–20%. There is an unmet need for surveillance methods that can supplement imaging at regular intervals. Serial analysis of circulating tumor DNA (ctDNA) may aid surveillance and prognostication. A PCR-based, “specimen in/result out” testing device was employed to detect BRAF variants in plasma-derived ctDNA to evaluate the utility of rapid biomarker detection in the management of melanoma with brain metastasis. Methods: Serial blood samples from patients diagnosed with BRAF mutation-positive metastatic melanoma were collected at regular intervals. We employed a real-time PCR-based automated mutation detection system (Idylla; Biocartis, Belgium) to interrogate the plasma samples. The ctDNA mutation detection trend was analyzed relative to disease progression. Results: 39 patients with BRAF mutation positive melanoma were enrolled. 29 patients were treated in the metastatic setting, 10 in the adjuvant setting. 18 of the 29 patients with metastatic disease (62%) had brain metastases. Circulating BRAF mutation was detected in 17 of the 29 (59%) patients with metastatic disease, and was not detected in any patients treated adjuvantly. In the group with metastatic disease, this circulating biomarker changed from undetectable to detectable in eight (28%) and detectable to undetectable in three (10%). No change in circulating mutation status occurred in 18 (62%). In the eight patients who had an initial negative test that later became positive, seven (87%) had brain metastases. In three patients, ctDNA mutation detection occurred before the diagnosis of brain metastases on imaging, with a median lead time of five weeks (range, 3-12 weeks). In one patient with de novo metastatic disease admitted to the ICU, tissue was unavailable for BRAF testing but plasma was found to be positive for ctDNA BRAF detection. BRAF/MEK targeted therapy resulted in a sustained objective response. Five of six (83%) patients that had persistent ctDNA positivity had brain metastases. Among patients with brain metastases, median overall survival (mOS) of patients demonstrating &gt;50% test positivity was numerically longer than those with &lt;50% positivity (mOS 12.3 vs 53.5 months; p = 0.133). Conclusions: Plasma-based, rapid ctDNA testing may be useful as an aid in detecting progression and gauging prognosis in patients with melanoma treated in the metastatic setting. The dynamics of ctDNA test positivity may indicate a need for more urgent imaging, particularly of the brain. Blood-based, semi-automated ctDNA detection may serve as an attractive adjunct to scheduled imaging surveillance in melanoma.

20P Is evaluation of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutational status on circulating tumour DNA (ctDNA) by liquid biopsy ready for prime-time? A systematic review and an individual patient meta-analysis

PIK3CA represents one of the most mutated genes, both in hormone receptor-positive BC and HER2-positive ones. Several clinical trials demonstrated that the detection of PIK3CA mutations in ctDNA could represent a predictive biomarker of PI3K-inhibitor treatment response. BELLE-2, BELLE-3, and SOLAR-1 trials prospectively showed a significant progression-free survival benefit in ctDNA PIK3CA-mutant patients, suggesting PIK3CA mutation detection in ctDNA could represent a PI3K-Is predictive biomarker.

Plasmatic KRAS Kinetics for the Prediction of Treatment Response and Progression in Patients With KRAS-mutant Lung Adenocarcinoma

IntroductionKRAS is the most common driver mutation in lung cancer. ctDNA-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Monitoring KRAS mutational load in ctDNA may be useful in the management of the patients. MethodsConsecutive patients diagnosed with KRAS mutant lung adenocarcinoma in the tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. KRAS mutations in plasma were quantified using digital PCR and correlated with mutations in tumor and with radiological response and progression. ResultsTwo hundred and forty-five plasma samples from 56 patients were analyzed. The rate of detection of KRAS mutations in plasma in our previously characterized KRAS-mutant cases was 82% overall, reaching 96% in cases with more than 1 metastatic location. The dynamics of KRAS mutational load predicted response in 93% and progression in 63% of cases, 33 and 50 days respectively in advance of radiological evaluation. Progression-free survival for patients in whom ctDNA was not detectable in plasma after treatment initiation was significantly longer than for those in whom ctDNA remained detectable (7.7 versus 3.2 months; HR: 0.44, p=0.004). ConclusionsThe detection of KRAS mutations in ctDNA showed a good correlation with that in tumor biopsy and, in most cases, predicted tumor response and progression to chemotherapy in advance of radiographic evaluation. The liquid biopsies for ctDNA-based molecular analyses are a reliable tool for KRAS testing in clinical practice.

Utility of circulating tumor DNA in the clinical management of patients with BRAFV600E metastatic colorectal cancer.

119 Background: Molecular profiling is critical for oncologists in personalizing treatment decisions for patients (pts) with metastatic colorectal cancer (mCRC). In contrast to archival tumor tissue specimens classically used profiling, sequencing of circulating tumor DNA (ctDNA) is more sensitive at quantifying low mutation allele frequencies and characterize “real time” tumor biology. We assessed the relationship between detection of BRAFV600E mutations in ctDNA and the clinical management of pts with mCRC. Methods: We retrospectively analyzed mCRC patients evaluated at MD Anderson Cancer Center with BRAFV600E mutations on ctDNA. ctDNA was isolated and sequenced for somatic mutations using a 70-gene next-generation sequencing assay (MD Anderson/GuardantHealth LB70 panel). Variant allele frequency (VAF) was characterized as the ratio of mutant reads: total reads for a given gene. BRAFV600E mutations were classified as “clonal” if the relative VAF (rVAF) exceeded 50% of the maximum VAF. “Major” and “minor” subclonal mutations were called for a rVAF of 10-50% and &lt; 10%, respectively. Associations between BRAFV600E clonality and treatment decision were performed using a Fisher’s exact test. Survival outcomes were estimated using the Kaplan-Meier method. Results: 64 patients with mCRC had a BRAFV600E mutation detected in ctDNA. Concordance between tissue and ctDNA for BRAFV600E mutation was occurred in 44/55 (80%) patients with evaluable tumor specimen. There were 9 patients with BRAFV600E mutations identified in the absence of evaluable tumor tissue. Median VAF for BRAFV600E in the ctDNA was 3.6% (interquartile range, 0.50 – 17%). The majority of patients had a clonal BRAFV600E mutation (50/64, 78%). There were 3 (5%) and 11 (17%) patients with major subclonal and minor subclonal BRAFV600E mutations, respectively. Among patients with minor subclonal BRAFV600E mutations, 91% (10/11) had developed resistance to anti-EGFR therapies for management of RASwild-type mCRC. Discordance between tissue and ctDNA BRAFV600E status was associated with minor subclones (odds ratio (OR) 56, p &lt; .0001). Clonal BRAFV600E mutations in the ctDNA were associated with a higher likelihood for treatment with BRAF targeted therapies (OR 5.8, p = .008). Median progression-free survival among 37 evaluable patients was 6.4 months. Conclusions: Reported VAF in the ctDNA served to stratify BRAFV600E according to relative clonality. Lower VAF was linked to acquired resistance to anti-EGFR therapies, whereas higher VAF was associated with receipt of matched targeted therapies for BRAFV600E mCRC. ctDNA technologies for identifying BRAFV600E mutations are feasible and informative for conducting relevant molecular profiling for patients with mCRC.