Plasma ctDNA Samples Research Articles

Detection of molecular residual disease in stage II and III melanoma utilizing circulating tumor DNA.

e21564 Background: Overall survival for patients with melanoma has significantly improved with advancements in immunotherapy, especially with immune checkpoint inhibitors and BRAF/MEK inhibitors becoming standard of care for advanced melanoma. However, the decision to proceed with adjuvant therapy should be considered against the risk of adverse side effects, particularly in early-stage melanoma patients. Accurate biomarkers are needed to risk stratify patients, determine who will most likely benefit from adjuvant therapy, and identify when the ideal time is to initiate treatment. This study evaluates the prognostic value of personalized, tumor-informed circulating tumor DNA (ctDNA) testing in patients with stage II and III melanoma. Methods: In this real-world study, plasma samples (n =429) were analyzed from 90 stage IIA-IIID cutaneous melanoma patients treated at Rush University Medical Center between 03/30/2021 and 01/30/2024. A clinically-validated, personalized, tumor-informed 16-plex PCR assay (SignateraTM, Natera, Inc.) was used for detection and quantification of ctDNA in plasma samples. Results: Personalized, tumor-informed ctDNA assays were created using tissue samples from either initial biopsy or surgical excision specimen. Assay design was successful for 100% (90/90) of patients with sufficient tissue; test requests for 4 patients could not proceed due to insufficient tissue. ctDNA was detectable in 28% (25/90) of patients at one or more time points. There was a higher ctDNA-positivity rate in stage III patients (20/50, 40%) compared to stage II patients (5/40, 13%). Of the 25 patients with identifiable ctDNA during surveillance, 13 tested positive on the initial test (2 stage II and 11 stage III). Eighteen of the 25 ctDNA-positive patients received immunotherapy; eight patients achieved sustained ctDNA clearance, 2 had transient clearance, 2 have remained positive on ctDNA testing, and 6 continue to receive adjuvant IO and follow-up ctDNA testing. Clinical or radiographic recurrence occurred in 11/90 patients, 10 of whom tested positive for ctDNA. Conclusions: Personalized, tumor-informed ctDNA offers an adjunct to conventional monitoring for melanoma patients to detect molecular residual disease and was found to be prognostic in our patient cohort. Prospective studies are needed to evaluate the role of ctDNA in clinical decision making for surveillance imaging intervals and appropriate initiation, intensification, or discontinuation of adjuvant therapy.

Longitudinal circulating tumor DNA kinetics and correlation with patient outcomes in metastatic melanoma.

e21538 Background: Previous studies have shown circulating tumor DNA (ctDNA) to be an accurate noninvasive method for detecting minimal residual disease and predicting therapeutic response. This study aims to investigate if positive ctDNA and subsequent clearance kinetics may be used as a predictive and prognostic tool in patients with stage IV metastatic melanoma. Non-clearance of ctDNA may be associated with poorer outcomes for progression-free survival (PFS) and overall survival (OS). Methods: In this retrospective analysis of preexisting data from a cohort of 27 patients with stage IV melanoma treated at Rush University, longitudinal ctDNA plasma samples collected between 05/01/2020 and 01/01/2024 were analyzed. A tumor-informed ctDNA assay (SignateraTM, reported in mean tumor molecules per mL (MTM/ml), low limit of detection of 0.01% variant allele frequency) was used for detection and quantification of ctDNA in plasma samples. Progression was determined based off of radiographic studies, overall survival was determined by time from stage IV diagnosis to patient death. Results: A total of 192 ctDNA timepoints were analyzed. The median number of ctDNA timepoints per patient was 5 (range 1-26). 8 patients were identified whose initial ctDNA result was negative and remained negative throughout their treatment course; 5/8 of these patients had baseline negative values available prior to the initiation of treatment. 19 patients had at least 1 positive ctDNA result at any given time; 1/19 of these patients was ctDNA-negative at baseline, 7/19 were positive at baseline, and 11/19 did not have baseline values available. The PFS rate was higher for ctDNA-negative patients compared to ctDNA-positive patients (62.5% vs. 57.9%). The OS rate was also higher for ctDNA-negative patients compared to ctDNA-positive patients (75% vs. 52.6%). Of the ctDNA-positive patients, those who achieved persistent clearance or had decreasing values of ctDNA had higher PFS rates when compared to ctDNA-positive patients who neither downtrended nor achieved clearance (71.4% and 66.7% vs. 22.9%). Patients who achieved persistent clearance or had decreasing values of ctDNA also had higher OS rates when compared to ctDNA-positive patients who neither downtrended nor achieved clearance (71.4% and 100% vs. 33.3%). Conclusions: Within the limitations of a small sample size, our data suggests that for patients with stage IV melanoma, a positive ctDNA result is associated with poorer outcomes for PFS and OS, when compared to patients with negative ctDNA results. Additionally, ctDNA-positive patients who achieved persistent clearance or downtrending ctDNA values showed more favorable outcomes for PFS and OS than ctDNA-positive patients who did not. [Table: see text]

Molecular characterization and biomarker identification in pediatric B-cell acute lymphoblastic leukemia.

6524 Background: B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent hematologic malignancy in children and a leading cause of mortality. Despite high cure rates, the management of B-ALL remains challenging due to its high heterogeneity and propensity for relapse. This study aimed to delineate molecular features of pediatric B-ALL and explore the clinical utility of circulating tumor DNA (ctDNA) as a biomarker in B-ALL. Methods: The study analyzed 146 primary pediatric B-ALL patients who were diagnosed from August 2020 to April 2023 and received systemic chemotherapy in Wuhan Children's Hospital. Baseline bone marrow (BM) and plasma samples were collected for next-generation sequencing (NGS) to profile the mutational landscape. BM samples collected on day 19 of the treatment were utilized for minimal residual disease (MRD) testing to assess treatment efficacy. Transcriptomic profiling of baseline BM samples was performed via bulk RNA sequencing. Fisher's exact test and the Wilcoxon rank sum test were utilized to compare categorical and continuous variables between groups, respectively. A two‐tailed P-value<0.05 was considered statistically significant unless indicated otherwise. Results: Transcriptomic analysis revealed that 86.3% of patients (126 out of 146) could be categorized into 13 distinct molecular subtypes, with hyperdiploidy emerging as the predominant subtype. Baseline BM NGS identified gene fusions in 61% of patients, including 37 novel fusions previously unreported in B-ALL. Specifically, the KMT2A-TRIM29 novel fusion was detected and validated in a male child diagnosed with the KMT2A-rearranged subtype. Despite initially responding well to therapy, the patient experienced disease progression after a year, indicating a poor prognosis. We also found that elevated mutant counts and maximum-variant-allele-frequency (maxVAF) in baseline BM were associated with significantly poorer response to chemotherapy ( P=0.0012 and 0.028, respectively). Additionally, MRD-negative patients exhibited upregulated expression of immune-related pathways ( P<0.01) and increased CD8+ T cell infiltration ( P=0.047), indicating a more active immune microenvironment in better responding tumors. Baseline plasma ctDNA, which exhibited high mutational concordance with paired baseline BM samples, also demonstrated significant associations with chemotherapy efficacy, highlighting its potential as a non-invasive tool for disease monitoring and treatment outcome prediction. Conclusions: This study elucidated novel gene fusions and potential biomarkers for treatment response in pediatric B-ALL. Importantly, both baseline plasma ctDNA and BM samples offer promising prognostic insights into chemotherapy responses, paving the way for non-invasive monitoring strategies in the management of pediatric B-ALL.

Imlunestrant, an oral selective estrogen receptor degrader (SERD), in combination with human epidermal growth factor receptor 2 (HER2) directed therapy, with or without abemaciclib, in estrogen receptor (ER) positive, HER2 positive advanced breast cancer (aBC): EMBER phase 1a/1b study.

1027 Background: Imlunestrant is a next-generation, oral SERD designed to deliver continuous ER-target inhibition. In the first-in-human Phase 1a/b EMBER study (NCT04188548), imlunestrant demonstrated favourable safety, PK, and clinical benefit when administered as monotherapy or with targeted therapy in ER+, HER2- aBC. Here we present clinical data of imlunestrant with HER2 targeted therapy, in patients (pts) with ER+, HER2+ aBC enrolled in EMBER. Methods: Pts were randomized to imlunestrant (400mg po daily) + trastuzumab (H), with or without (±) abemaciclib (Part C) or received maintenance treatment with imlunestrant + H + pertuzumab (P) (Part E), at standard doses in 21-day cycles. Key eligibility (Part C): ≥ 2 prior HER2 directed regimens, no prior CDK4/6i or fulvestrant; (Part E): received 1st line induction taxane chemotherapy (any duration) + H + P, without disease progression and ≤1 prior therapy for aBC. Key endpoints included safety, PK, ORR, CBR and PFS. Results: Overall, 45 pts with ER+/HER2+ aBC received the following combinations: imlunestrant + H (n=18), imlunestrant + H + abemaciclib (n=21; 1 pt received 800 mg imlunestrant), and imlunestrant + H + P (n=6). Baseline characteristics, safety, and preliminary efficacy are presented below (Table). Most TEAEs were grade 1-2. Common TEAEs with imlunestrant + H were fatigue (33%) and nausea (28%); with imlunestrant + H + abemaciclib were diarrhea (100%), neutropenia (57%), fatigue (52%) and nausea (48%); and with imlunestrant + H + P were diarrhea (50%) and arthralgia (50%). Imlunestrant PK was consistent with those previously reported with no drug-drug interactions observed with the combinations. From Part C, sequencing of baseline plasma ctDNA samples (n=37) identified prevalent mutations in TP53 (49%), PIK3CA (30%), ESR1 (24%), and GATA3 (14%). Clinical activity of imlunestrant in this ER+/HER2+ population, while based on small numbers, is promising. Conclusions: Imlunestrant in combination with trastuzumab ± abemaciclib or pertuzumab was well tolerated, showed no drug-drug interactions, and demonstrated preliminary antitumor activity in pts with ER+/HER2+ aBC. Clinical trial information: NCT04188548 . [Table: see text]

CtDNA monitoring for breast cancer at high risk of recurrence: Interim analysis of JCOG1204A1.

518 Background: Monitoring ctDNA after definitive surgery has emerged as an accurate predictor of breast cancer (BC) recurrence. However, further data is needed to determine the proportion and dynamics of ctDNA positivity in different BC subtypes. Here we present ctDNA status and dynamics in patients from an ancillary study (A1) of the prospective randomized phase III JCOG1204 trial (UMIN000012429; Registration: November 2013 - January 2020, N = 1034) in which intensive post-operative follow-up is compared to standard follow-up in BC patients at high risk of recurrence. Methods: Eligible patients enrolled in JCOG1204 were required to have either (1) ≥4 lymph node metastases for estrogen receptor (ER)+ BC without neoadjuvant therapy, (2) ≥1 lymph node metastases for ER- without neoadjuvant therapy, or (3) ≥1 lymph node metastases after neoadjuvant therapy. Additional inclusion criteria for ancillary study JCOG1204A1 include no more than 4 years after surgery and relapse-free at the enrollment. A personalized, tumor-informed assay (Signatera, Natera Inc.) was used for ctDNA surveillance every 3 - 6 months for 2 years for ER- or 5 years for ER+ BC. ctDNA status, time of ctDNA detection and association with subtype were examined. The JCOG DSMC approved the publication of this interim analysis. Results: A total of 248 patients were enrolled (July 2020-October 2023) in JCOG1204A1. Personalized assays were successfully designed for 88% patients, the main reason for failure was insufficient tissue (83%; 25/30). A total of 1440 plasma ctDNA samples were collected from 211 patients. The median age at the time of surgery was 50 years. The most prevalent subtype was ER+ HER2- (Luminal; n = 130), followed by ER- HER2- (TN; n = 38), ER- HER2+ (HER2; n = 24), and ER+ HER2+ (Luminal-HER2; n = 18). 25 patients (11.8%) had at least one positive ctDNA result, with overall sample-level positivity proportion of 4.5% (65 / 1440); 76% (19/25) became detectable with longitudinal testing. Surveillance in TN [the first time point median 31.5 months (12.6-55.8) from surgery] did not result in ctDNA detection. Of the 130 patients with Luminal subtype [the first time point median 36.6 months (12-57) from surgery], 21 (16.2%) had ctDNA first detected at a median 43.5 months (18-72). Additionally, 5 patients (2.4%) had ctDNA detection for the first time after 5 years of surgery. The proportion of ctDNA positivity increased by ~2% annually in Luminal subtype; varied rates of ctDNA level increase have been observed. Conclusions: Monitoring ctDNA in high-risk BC reveals a detection proportion of 11.8% at the patient level (TNBC: 0%; Luminal: 16.2%) Longitudinal testing revealed ctDNA detection in patients with luminal subtype beyond 5 years from surgery, suggesting a correlation with late recurrence. Subsequent reports will focus on investigating the association of ctDNA status with relapse and overall survival.

Plasma ctDNA biomarker study in patients with non-small cell lung cancer with EGFR exon 20 insertion mutation treated with sunvozertinib.

8563 Background: There are limited reports on biomarker studies of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutation (exon20ins). Sunvozertinib (DZD9008), an oral, potent, irreversible and selective EGFR tyrosine kinase inhibitor (TKI), has been granted conditional approval in China for the treatment of NSCLC with EGFR exon20ins in the ≥ second-line setting. Here we report biomarker analysis of EGFR exon20ins in large sample size, by pooling data from WU-KONG series of studies. Methods: Serial plasma ctDNA samples were collected from baseline until progressive disease (PD). Next generation sequencing was used to assess EGFR exon20ins in baseline plasma ctDNA, and to analyze genetic alterations in baseline and PD plasma ctDNA to identify potential resistance mechanisms to sunvozertinib. Droplet digital PCR was used to dynamically monitor the quantity of EGFR exon20ins during treatment. In addition, association between baseline EGFR exon20ins and disease characteristics as well as antitumor activity of sunvozertinib was analyzed. Furthermore, in vitro and in vivo experiments were performed to explore potential approach which may overcome the resistance to sunvozertinib. Results: A total of 121 patients were included in this biomarker analysis, among them 81 out of 121 (66.9%) had detectable EGFR exon20ins in baseline plasma ctDNA. There was a positive correlation between detectable EGFR exon20ins and higher number of metastasis sites. In addition, higher EGFR exon20ins abundance was positively correlated with more metastasis sites and brain metastasis. Patients with EGFR exon20ins negative in plasma ctDNA achieved a higher objective response rate (ORR) (68.0% vs. 45.8%) and longer median progression free survival (PFS) (7.4 months vs. 5.5 months) with sunvozertinib treatment. In patients with baseline detectable EGFR exon20ins, the mutant allele decreased over time with treatment in majority of patients (12/14, 85.7%), and the earliest clearance of EGFR exon20ins occurred after one week of treatment. In patients who developed disease progression, 12 of 18 (66.7%) maintained EGFR exon20ins at PD, and among them, 3 concurrently acquired EGFR C797S, all in cis with exon20ins. In addition, EGFR G724S and other genetic aberrations in EGFR downstream signaling pathway, were also detected. Exploratory work on overcoming resistance to sunvozertinib suggests that combination of golidocitinib, a selective JAK1 inhibitor, with chemotherapy could be a potential approach. Conclusions: This study showed that positive EGFR exon20ins in plasma ctDNA is associated with advanced disease characteristics. Sunvozertinib can effectively clear EGFR exon20ins in plasma ctDNA, confirming its direct effect on EGFR pathway. The resistance to sunvozertinib can be through EGFR-dependent and EGFR-independent mechanisms.

Abstract PO5-13-12: Personalized circulating tumor DNA testing for recurrence detection and treatment response monitoring in patients with metastatic invasive lobular carcinoma

Abstract Introduction Metastatic invasive lobular carcinoma (mILC) presents unique challenges during treatment response monitoring. A higher frequency of metastases to the bone, gastrointestinal tract, uterus, meninges, ovary, and diffuse serosal involvement is observed in mILC. The challenge with mILC is that they can be difficult to detect clinically and radiographically, and consequently clinical progression can be difficult to determine with conventional imaging modalities like CT Scans, thus precluding many patients with mILC from enrolling into clinical trials. Therefore more accurate biomarkers are needed to better assess response to treatment in real time. In this study, we demonstrate the feasibility of longitudinal ctDNA testing for treatment response monitoring in patients with mILC. Methods In this real-world study, longitudinal plasma samples (n=219) from 60 patients with mILC, treated across 52 independent clinics between 7/22/21 and 6/15/23, were analyzed. A personalized, tumor-informed ctDNA assay (SignateraTM bespoke, mPCR-NGS assay) was used for detection and quantification of ctDNA in plasma samples. Results In this cohort, the median patient age at diagnosis of metastatic disease (baseline) was 62.9 years (range: 32.2-79.7). Of the 60 patients, 41 (68%) had ER+PR+HER2-, 7 (12%) ER+PR-HER2-, 4 (7%) ER+PR+HER2+, 2 (3%) ER+PR-HER2+, 1 (2%) triple negative breast cancer; receptor status was not available for 5 (8%) patients. CDH1 (75%) and PIK3CA (42%) were observed to be the top two genes with somatic, nonsynonymous mutations. Mutations in TP53 and TBX3 were observed in 13% of patients, mutations in AKAP9, IGFN1, RBM26, and RFC3 in 10% of patients, and mutations in ESR1 in 6.7% of patients. At variant level, PIK3CA p.H1047R (22%) and RBM26 p.Q701Tfs*23 (10%) were the top two somatic variants identified. The most common CDH1 variant, p.Q23*, was found in 5% of the cases. Of the 60 patients, 37 (61.7%) were ctDNA-positive at all time points while on treatment (chemotherapy/endocrine therapy/targeted therapy) with the first time point collected at a median of 6.7 months (range: 2.5-2307 months) from baseline. Of these 37, imaging/biopsy results after baseline were available for 32 patients, 86% (27/32) of whom progressed. Thirty-two of 37 persistently ctDNA-positive patients had serial ctDNA measurements available, of which 14 (43%) had significant increase in ctDNA levels (average: 47-fold increase, standard deviation: 98) from first to last time points. At some point during their care ctDNA was detectable in all (n=51) patients with evidence of clinical progression. Of the 60 patients, 14 (23.3%) were ctDNA-negative at all time points tested. None of the 14 persistently ctDNA-negative patients had evidence of disease recurrence or progression at the time of initial testing or throughout ctDNA monitoring. The first ctDNA time point was collected at a median of 33.1 months (range: 0.68-92 months) from baseline for these patients,a median of 3 time points was tested per patient (1-12). Of the remaining 9/60 patients, 3 (33%) had sustained ctDNA clearance for a median of 46 days (range: 0-224 days) in response to treatment, and 2 (22%) were initially ctDNA-negative but turned positive 105 and 156 days later, indicating potential resistance to treatment and molecular progression. Additional 4/9 (44%) had ctDNA levels consistently low levels (average: 0.42 mean tumor molecules/mL) while on treatment for a median of 260 days (range: 217-442) and showed no evidence of progression. Conclusions ctDNA status and dynamics correlate well with clinical status of patients with mILC, as determined by conventional monitoring tools such as imaging and biopsy. Our results indicate that personalized, tumor-informed, longitudinal ctDNA testing may serve as a useful tool for detection of progression and monitoring treatment response in mILC patients. Citation Format: Steffi Oesterreich, Antony Tin, Samuel Rivero-Hinojosa, Janie Fielder, Jenifer Ferguson, Ekaterina Kalashnikova, Angel Rodriguez, Minetta Liu, Adrian Lee. Personalized circulating tumor DNA testing for recurrence detection and treatment response monitoring in patients with metastatic invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-12.

Abstract PO2-15-06: ctDNA-based DNADX in hormone receptor-positive and HER2-negative (HR+/HER2-) advanced breast cancer following endocrine therapy and CDK4/6 inhibition: a correlative analysis from the randomized phase 2 PARSIFAL trial

Abstract ctDNA-based DNADX in hormone receptor-positive and HER2-negative (HR+/HER2-) advanced breast cancer following endocrine therapy and CDK4/6 inhibition: a correlative analysis from the randomized phase 2 PARSIFAL trial Background: DNADX, a novel machine learning-based approach, utilizes DNA from tumor tissue or plasma ctDNA to identify clinically relevant phenotypic tumor features and classify breast cancer into 4 subtypes (Nat Comm 2023). Here, we evaluated DNADX's ability to predict prognosis and treatment benefit in HR+/HER2- advanced breast cancer following endocrine therapy and a CDK4/6 inhibitor. Methods: DNADX was evaluated centrally in available baseline plasma ctDNA samples from PARSIFAL trial (NCT02491983) which randomized 486 patients (pts) with HR+/HER2- advanced breast cancer to receive (1:1 ratio) first line palbociclib with either fulvestrant or letrozole. Shallow whole genome sequencing was performed on ctDNA, and the 4 DNA-based subtypes (Clusters-1, -2, -3, and -4) were identified if the ctDNA tumor fraction (TF)≥3%. The main objective was to evaluate the association of DNADX subtypes with progression-free survival (PFS) and overall survival (OS). Secondary objective was to identify the subgroup of pts who benefit more from each endocrine treatment. Uni- and multi-variable Cox regression models were used after adjusting for TF, menopausal status, ECOG status, de novo metastasis (vs. recurrence), visceral disease and number of metastatic sites. Results: DNADX was evaluated in plasma ctDNA samples from 122 pts (25.1%). Clinical variables and median PFS (27.6 months) were similar as the overall PARSIFAL population. DNADX identified 56.6% pts with TF of < 3%, 14.8% with Cluster-1, 19.7% with Cluster-2, 5.7% with Cluster-3 and 3.3% with Cluster-4. In terms of PFS, pts classified with TF< 3% had a lower risk of progression compared to Cluster-1, Cluster-2, Cluster-3, Cluster-4 subtypes (pairwise PFS hazard ratios [HRs] of 1.88, 2.02, 3.15, and 5.62, respectively, with a global log-rank test of p=0.010). Similar results were obtained after adjusting for other clinical-pathologic variables. A numerical benefit of fulvestrant in comparison with letrozole was observed in pts classified in Cluster-1 and Cluster-4 (HR=0.42, 95% CI 0.14-1.24) in contrast to the other groups (HR=1.21, 95% CI 0.68-2.18), and the interaction test was statistically significant after adjusting for clinical-pathologic variables (anova p-value=0.037). In terms of OS, pts classified with TF< 3% had a lower risk of death compared to Cluster-1, Cluster-2, Cluster-3, and Cluster-4 subtypes (pairwise OS HRs of 1.90, 4.23, 11.13, and 6.80, respectively, with a global log-rank test of p=0.003). In the multivariable analysis, results were consistent after adjusting for clinical-pathologic variables and TF. Conclusions: Liquid biopsy-based DNADX subtypes predict outcomes in pts with HR+/HER2- advanced breast cancer on endocrine therapy and CDK4/6 inhibitors, potentially identifying the most optimal endocrine treatment for each pt. Citation Format: Fara Brasó-Maristany, Javier Cortés, José Manuel Pérez-García, Rosario Vega-León, Laia Paré, Guillermo Villacampa, Judit Matito, Francisco Pardo, Marina Gomez-Rey, Mario Mancino, Elena Martínez-García, Carmen Mora Gallardo, Leonardo Mina, Florence Dalenc, Meritxell Bellet- Ezquerra, Manuel Ruíz - Borrego, Miguel Gil-Gil, Peter Schmid, Charles M. Perou, Joel S. Parker, Patricia Villagrasa, Ana Vivancos, Aleix Prat, Antonio Llombart-Cussac. ctDNA-based DNADX in hormone receptor-positive and HER2-negative (HR+/HER2-) advanced breast cancer following endocrine therapy and CDK4/6 inhibition: a correlative analysis from the randomized phase 2 PARSIFAL trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-06.

Abstract PO5-14-05: Circulating tumor DNA fraction correlates with residual cancer burden post-neoadjuvant chemotherapy in triple negative breast cancer patients

Abstract Background: Pathologic response after preoperative/neoadjuvant chemotherapy is a continuum that can range from no residual cancer to extensive disease. Viable cancers can shed tumor DNA into the blood and we hypothesized that plasma ctDNA levels can reflect pathologic response as continuum. Patients and methods: Post-neoadjuvant plasma samples were obtained after completion of chemotherapy but before surgery from patients with stage I to III TNBC who received treatment with durvalumab concurrent with weekly nab-paclitaxel followed by dose dense doxorubicin/cyclophosphamide in a single arm neoadjuvant clinical trial (NCT02489448). Up to 3 mL of blood was collected into EDTA- and heparin- containing vacutainer tubes, and plasma cfDNA was extracted and twice purified in the lab at Predicine Inc. Pathologic response was quantified on a continuous scale using the Residual Cancer Burden (RCB) score, and also categorized as RCB-0 (complete pathologic response, n=21), -I (minimal residual cancer, n=7), -II (moderate residual cancer, n=13), and -III (extensive residual cancer, n=3). Median follow-up was 35 months (range 25-70 months). Tumor whole exome sequencing (WES) data was previously generated using HiSeq 4000 at the Yale Center for Genome Analysis (dbGaP: PRJNA558949). A personalized MRD panel of up to 60 personalized variants per patient was designed based on the tumor WES data, and ultra-deep next-generation sequencing (100,000X) of plasma cfDNA was performed using the PredicineBEACON assay for MRD detection by Predicine. CtDNA positivity (MRD) was defined as a weighted score equivalent to 2 or more tumor variants detected in the plasma. cfDNA tumor fraction was measured based on mutant allele fraction of observed variants. Adjusted tumor fraction could be confidently measured for 37 out of 44 patients that had enough confidently called personalized ctDNA variants. We compared tumor fraction versus RCB score as continuous variables, and also compared tumor fraction between RCB categories as ordinal variables. Invasive disease-free survival was examined by Kaplan-Meier analysis. Bioinformatic data analysis was performed using Graphpad Prism and R. Results: 44 of 48 plasma samples yielded successful cfDNA generation and sequencing. Six patients (13.6%) were MRD positive. Among these 6 cases, 3 had RCB-0 response (14.3% of RCB 0 cases), 1 had RCB II response (7.7% of RCB II cases), and 2 had RCB III response (66.7% of RCB III cases). The median cfDNA tumor fraction of patients with pathological complete response to treatment (pCR) was significantly lower than patients with residual disease (RD) (0.06% vs. 0.3%, p=0.02). There was a positive correlation between RCB scores and tumor fraction (r= 0.45, Spearman). Tumor fraction was more strongly correlated with later recurrence than a binary ctDNA positivity call. Tumor fraction was significantly higher in patients that later experienced local or metastatic recurrence (n=8) compared to those with no evidence of disease (n=29) (Wilcoxon test, p=0.02). Both ctDNA positive RCB III cases had metastatic recurrence while the remaining 4 ctDNA positive cases currently remain recurrence-free. Mutational genomic landscape of both tumor WES and plasma ctDNA samples identified hotspot mutations in 19 cancer-associated genes. Novel hot-spot mutations were observed in the plasma of two patients following treatment. TP53 mutations were highly prevalent in tissue and plasma, however no significant association of mutation of any gene with treatment response was detected. Conclusions: PredicineBeacon MRD assay was successfully performed on 3 mL plasma samples collected using from EDTA and heparin-containing tubes. Tumor fraction correlated with residual disease, RCB score and disease recurrence. Citation Format: Naing Lin Shan, Billie Gould, Kim Blenman, Julia Foldi, Pan Du, Frank Zhang, Myles Walsh, Lajos Pusztai. Circulating tumor DNA fraction correlates with residual cancer burden post-neoadjuvant chemotherapy in triple negative breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-05.

Personalized, tumor-informed, circulating tumor DNA assay for detecting minimal residual disease in non-small cell lung cancer patients receiving curative treatments.

Circulating tumor DNA (ctDNA) has emerged as a prognostic and predictive biomarker for detection of minimal residual disease (MRD), monitoring treatment response, and early detection of recurrence in cancer patients. In this study, we explored the utility of ctDNA-based MRD detection to predict recurrence in a real-world cohort of primarily early-stage non-small cell lung cancer (NSCLC) patients treated with curative intent. Longitudinal plasma samples were collected post curative-intent treatment from 36 patients with stage I-IV NSCLC. A personalized, tumor-informed assay was used to detect and quantify ctDNA in plasma samples. Of the 24 patients with plasma samples available during the MRD window (within 6 months of curative surgery and before adjuvant therapy), ctDNA was detectable in two patients. Patients with ctDNA-positivity during the MRD window were 15 times more likely to recur compared to ctDNA-negative patients (HR: 15.0, 95% CI: 1.0-253.0, p = 0.010). At any time post-curative intent treatment, ctDNA-positivity was associated with significantly poorer recurrence-free survival compared to persistently ctDNA-negative patients (p < 0.0001). Our real-world data indicate that longitudinal, personalized, tumor-informed ctDNA monitoring is a valuable tool in patients with NSCLC receiving curative treatment to identify patients at high risk for recurrence.

Prognostic and predictive impact of molecular tumor burden index in non-small cell lung cancer patients.

The biomarkers of immune checkpoint inhibitors in the treatment of non-small cell lung cancer (NSCLC) patients have limited predictive performance. In this study we aimed to investigate the feasibility of molecular tumor burden index (mTBI) in circulating tumor DNA (ctDNA) as a predictor for immunotherapy in patients with NSCLC. From February 2017 to November 2020, pretreatment and on-treatment (3~6 weeks after first cycle of immunotherapy) dynamic plasma ctDNA samples from NSCLC patients receiving immune monotherapy or combination therapy were analyzed by targeted capture sequencing of 1021 genes. PyClone was used to infer the mTBI. The impact of pretreatment mTBI on survival outcomes was verified in the POPLAR/OAK trials. We found that patients without detectable baseline ctDNA had better survival outcomes (median overall survival [OS]: not reached vs. 12.8 months; hazard ratio [HR], 0.15; p = 0.035]). RB1 and SMARCA4 mutations were remarkably associated with worse survival outcomes. Furthermore, lower pretreatment mTBI was associated with superior OS (median: not reached vs. 8.1 months; HR, 0.22; p = 0.024) and PFS (median: 32.9 vs. 5.4 months; HR, 0.35; p = 0.045), but not objective response, which was validated in the POPLAR/OAK cohort, suggesting that baseline mTBI was a prognostic factor for NSCLC immunotherapy. Early dynamic changes of mTBI (ΔmTBI) significantly distinguished responsive patients, and patients with mTBI decrease to more than 68% at the final tumor evaluation had longer OS (median: 38.2 vs. 4.0 months; HR, 0.18; p = 0.017) and PFS (median: not reached vs. 2.3 months; HR, 0.24; p = 0.030). ΔmTBI had a good sensitivity to identify potential beneficial patients based on the best effect CT scans, demonstrating that mTBI dynamics were predictive of benefit from immune checkpoint blockade.

Concordance of ctDNA and tissue mutations in NSCLC: A meta-analysis.

Systematic evaluation of the consistency between circulating tumor DNA (ctDNA) in plasma and tumor tissue samples in mutations in non-small cell lung cancer (NSCLC) patients. To collect publicly published literature from numerous important international medical databases through computer retrieval. To compare the differences in literature data related to gene mutations between plasma ctDNA and tumor tissue samples, a meta-analysis was performed using Stata 12.0 software while taking into account the inclusion and exclusion criteria. This article includes a total of 15 research data and collected data reports from 15 groups of NSCLC patients. The results are displayed using tissue samples as the gold standard. The Pearson correlation coefficients of sensitivity and specificity were used to calculate rho=0.044, and P=0.893. The Q-test found poor homogeneity and high heterogeneity in sensitivity and specificity among research data from various literature studies (I2>50%, P<0.1). The area under the SROC curve is 0.97 (95% CI: 0.96~0.99). The small sample subgroup has high heterogeneity, and the combined diagnosis effect size is 26[6~111], lower than the large sample subgroup 185320 [0~2.7×1012]. When taking 200 as the cut-off point, the combined effect size of the small sample subgroup is 46 [12~183], still lower than that of the large sample subgroup 429 [52~3574]. From this, it can be concluded that the consistency of small-sample studies is higher than the quality of large-sample studies, and the heterogeneity is relatively low. From the perspective of mutation types, the heterogeneity of literature with EGFR gene mutations alone is higher than that of literature with non-EGFR mutations alone, and the consistency is lower. The consistency of using plasma ctDNA to detect mutations in NSCLC patients with tumor tissue samples is influenced by the type of mutation gene and sample size measured by the patient, and there is a significant bias in related studies.

Comparative analysis of genomic profiles between tissue-based and plasma-based next-generation sequencing in patients with non-small cell lung cancer

ObjectivesGenotype-guided personalized therapy has become an essential part of routine clinical care in non-small cell lung cancer (NSCLC) patients. However, small tissue specimens often yield inadequate molecular testing material. Plasma ctDNA-based liquid biopsy is an increasingly common non-invasive alternative to tissue biopsy. This study examined the similarities and differences in the molecular profiling of tissue and plasma samples to provide insight into sample selection in clinical practice. Materials and MethodsSequencing data from 190 NSCLC patients who underwent concurrent tissue-based next-generation sequencing (tissue-NGS) and plasma-based NGS (plasma-NGS) using a 168-gene panel were analyzed. ResultsTissue-NGS identified genomic alterations in 97.4% (185/190) of the enrolled patients and plasma-NGS identified genomic alterations in 72.1% (137/190) of the enrolled patients. Considering all NSCLC guideline-recommended biomarkers in the entire cohort of 190 cases, 81 patients had positive concordant mutations detected in both tissue and plasma samples, while 69 patients had no predefined alterations detected in either tissue or plasma samples. Additional mutations were found in the tissues of 34 patients and the plasma of six patients. The overall concordance rate between tissue and plasma samples was 78.9% (150/190). The tissue-NGS and plasma-NGS sensitivities were 95.0% and 71.9%, respectively. In the 137 patients with detectable ctDNA in plasma samples, the concordance rate between tissue and plasma samples reached 91.2%, and the sensitivity of plasma-NGS reached 93.5%. ConclusionOur findings indicate that plasma-NGS is less capable of detecting genetic alterations than tissue-NGS, especially for copy number variations and gene fusions. Tissue-NGS remains the preferred method for evaluating the molecular profile of NSCLC patients when tumor tissue is available. We suggest that the concurrent use of liquid biopsy and tissue biopsy is the optimal approach in clinical practice; alternatively, plasma can be used as substitute material when tissue is unavailable.

Ultrasensitive detection and monitoring of central nervous system tumors from plasma using personalized whole-genome ctDNA profiling.

2064 Background: Patients with the central nervous system (CNS) tumors are largely followed up by imaging. Current plasma-based liquid biopsy techniques have limited utility in neuro-oncology due to a low circulating cell-free tumor DNA (ctDNA) burden, blood-brain barrier, and low number of mutations in coding regions. Whole genome sequencing (WGS)-derived patient specific mutational signature from a matched tumor-normal WGS can provide a personalized, highly sensitive and specific approach to detect mutations in ctDNA and provide blood-based monitoring in brain tumor patients. Furthermore, it can be performed on lower amount of peripheral blood since WGS requires less sequencing depth compared to targeted ctDNA panels. Methods: We have profiled a cohort of 28 extra- and intra-axial adult and pediatric brain tumors including adult and pediatric low- and high-grade glioma (9), meningiomas (11), medulloblastomas (5), ependymomas (2), neurocytoma (1). Tumor DNA was extracted from archival pathology tissue, normal DNA from unsorted white blood cells, and ctDNA from 1-2 mL of post-surgery plasma. WGS was performed with 40x coverage for Tumor-Normal DNA and 20x for ctDNA. Using WGS of matched Tumor-Normal and plasma samples, we derived a personalized mutational pattern using SNVs, indels, and copy numbers for quantification and ultra-sensitive detection of ctDNA in plasma samples. An AI-based error suppression model was implemented to filter out the noise in the cell-free DNA (cfDNA) while the personalized mutational signature was used to detect the ctDNA in the cfDNA and to amplify the somatic signal to determine the Tumor Fraction at the time of diagnosis, during the therapy or surveillance period. The ctDNA Tumor Fraction (TF) was compared to the clinical status and MRI-based imaging. Results: All subtypes of brain tumors contained enough mutations to derive personalized mutational signatures. Most mutations were distributed in the noncoding DNA. TF correlated with clinical status and with the disease course on imaging at given time points reaching a 10-4 minimal residual disease detection sensitivity. We were able to detect ctDNA across all WHO grades ranging from WHO 1 meningioma to WHO 4 glioblastoma and medulloblastoma. Furthermore, we were able to detect tumor-specific copy number aberrations such as MYCN amplification in plasma samples and mutational signatures. Conclusions: Here we demonstrate that patient-specific WGS tumor signature in ctDNA from plasma can be used for sensitive monitoring of adults and children with primary low- and high-grade CNS tumors.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with surgically resected stage II/III melanoma.

9582 Background: Over the past decade, advances in immunotherapy (IO) have dramatically improved overall survival (OS) for patients with metastatic melanoma. Immune checkpoint inhibitors and BRAF/MEK inhibitors also became standard of care for surgically resected stage III melanoma, and most recently pembrolizumab was approved for stage IIB/C patients. Although the phase III trials that led to their respective approvals met their primary endpoints for relapse-free survival, no benefit in OS has been observed. Given that a large proportion of early-stage melanoma patients are cured with surgery alone, the decision to proceed with adjuvant therapy should be weighed against the risk of potential life-altering toxicity. There is a high unmet need to develop biomarkers to accurately select patients who are most likely to benefit from adjuvant IO while sparing a group of low risk patients from unnecessary treatment. In this study, we evaluated the prognostic value of personalized, tumor-informed circulating tumor DNA (ctDNA) testing in patients with resectable stage II/III melanoma. Methods: In this real-world study, longitudinal plasma samples (n = 159) were analyzed in real-time from 45 stage IIA-IIID cutaneous melanoma patients treated at Rush University between 03/30/2021 and 01/12/2023. A personalized, tumor-informed ctDNA assay (Signatera bespoke, mPCR-NGS assay) was used for detection and quantification of ctDNA in plasma samples. Results: Personalized, tumor-informed ctDNA assays were successfully designed for 93% (42/45) of patients using tissue samples from either surgical resection (14/42, 33%) or biopsy (26/42, 67%; 16 shave, 10 excisional, 1 punch, 1 core). ctDNA was detectable at one or more time points in 36% (15/42) of patients. We observed a higher ctDNA-positivity rate in stage III patients (11/20, 55%) compared to stage II patients (4/22, 18%). Twelve of the 15 ctDNA-positive patients received adjuvant IO/treatment escalation after a positive ctDNA result; 6 patients had sustained ctDNA clearance, 2 had transient clearance, and 4 continue to receive adjuvant IO and follow-up ctDNA testing. Radiographic recurrence was observed in 7/42 patients, 6 of whom were ctDNA-positive. Ninety-six percent (26/27) of patients with persistent ctDNA-negativity remain clinically and radiographically progression-free. Conclusions: Our data suggest that molecular residual disease detection using a personalized, tumor-informed ctDNA assay is highly prognostic in melanoma patients undergoing curative resection. Sustained clearance of ctDNA was achieved with adjuvant therapy in a subset of this cohort. Prospective studies evaluating the utility of ctDNA to aid clinical decision-making regarding adjuvant treatment intensification or de-escalation are warranted.

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

Abstract Introduction: Lung cancer remains the leading cause of cancer-related deaths. Surgery is the best option for early lung cancer, and the role of adjuvant therapy remains controversial. Liquid biopsy offers a noninvasive approach to monitor cancer burden. Targeted sequencing of circulating cell free tumor DNA (ctDNA) in blood has shown success for diagnosis; however, low tumor burden and dynamic evolution of low stage disease is challenging for targeted panels. Thus, we hypothesized that a whole genome sequencing (WGS)-derived patient specific mutational signature from a matched tumor-normal WGS can provide sensitive and specific approach to detect mutations and copy numbers in ctDNA for monitoring of lung adenocarcinoma patients. Methods: We successfully profiled 50 Stage 1 or 2 lung adenocarcinomas. ctDNA was extracted from 1-2 mL of plasma, tumor DNA was extracted from pathology tissue and normal germline DNA from the white blood cells. WGS using was performed on matched tumor and normal DNA, and ctDNA extracted from plasma. WGS coverage was 40x for matched tumor-normal and 20x for ctDNA. We derived a personalized mutational pattern for each tumor and used an AI-based error suppression model for quantification and ultra-sensitive detection of ctDNA in plasma samples. A patient-specific personalized genome-wide compendium of somatic mutations and copy numbers was established and ctDNA tested at 3 to 18 available time points during the therapy or follow up. A personalized mutational signature for detection ctDNA from WGS was quantified and the ctDNA Tumor Fraction (TF) was compared to the clinical status and time to recurrence. Results: Tumor specific signatures were derived from matched tumor-normal samples with &amp;gt;5% tumor purity and &amp;lt;30% duplications rate. Out of all patients, 33 patients showed no recurrence and 12 recurred. Tumor-specific signatures detected the presence of the tumor signature in plasma with TF as low as 10−5. Based on positive minimal residual disease in plasma, the recurrence prediction sensitivity was 0.75 and specificity 0.82, with positive predictive value of 0.6 and negative predictive value 0.9. WGS ctDNA predicted recurrence with a median lead time of 508 days before clinical/imaging recurrence. In one case we were able to identify the second primary by deconvoluting known and novel ctDNA mutations. ctDNA mutational profiles enabled identification of smoking mutational signature matching clinical history, and APOBEC and ageing signatures as well as tumor mutational burden. Conclusions: Patient-specific WGS tumor signature from plasma derived ctDNA enables specific and ultrasensitive tracking of minimal residual disease in low stage lung adenocarcinoma patients. Molecularly positive status can be used to predict recurrence and identify patients with clinical low stage disease that may benefit from adjuvant therapy. Citation Format: Ivy Tran, Alejandro Vargas, Reid Wilkins, Isabella Pizzillo, Kenneth Tokoro, Danielle Afterman, Tomer Lauterman, Maja Kuzman, Santiago Gonzalez, Dunja Glavas, James Smadbeck, Dillon Maloney, Jurica Levatic, Samuel Phillips, Sunil Deochand, Michael Yahalom, Ryan Ptashkin, Iman Tavassoly, Zohar Donenhirsh, Eric White, Ravi Kandasamy, Ury Alon, Paz Polak, Boris Oklander, Asaf Zviran, Matija Snuderl, Harvey I. Pass. Whole genome cell-free tumor DNA mutational signatures from blood for early detection of recurrence of low stage lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6689.

Effect of dynamic circulating tumor DNA minimal residual disease detection on recurrence surveillance and second resection rate after curative intent resection of colorectal liver metastasis: A prospective cohort study.

242 Background: Recurrence rates after curative intent resection of colorectal cancer liver metastases (CRLM) are nearly 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging and CEA measurements. However, recurrence detection often happens too late to resume a second resection. Previous studies have shown that circulating tumor DNA (ctDNA) detection, as a biomarker of minimal residual disease (MRD), is a robust prognostic factor for patients with colorectal cancers. Here, we performed a prospective study to explore the potential use for improving postoperative CRLM surveillance by dynamic ctDNA detection and its impact on the second resection of recurrent patients. Methods: Enrolled patients received curative intent resection of CRLM and were confirmed at the status of no evidence of disease (NED) by CT about one month after surgery. All patients received stand-of-care postoperative surveillance and serial plasma samples were collected, including presurgical and postsurgical samples (4-6 weeks after surgery) and every 3rd month until relapse or up to 24 months after resection. A personalized tumor-informed assay (Signatera/HuaJianwei bespoke multiplex-PCR NGS) was used to quantify ctDNA in plasma samples. Results: A total of 318 plasma samples from 60 patients recruited from July 2020 to July 2021 were included in our analysis. Twenty-five (41.6%) patients received neoadjuvant chemotherapy, and 46 patients (76.7%) received adjuvant chemotherapy after surgery. The median follow-up time was 18.0 months (range, 7.5 to 25.3 months). ctDNA was detectable in 58/60 (96.7%) patients before surgery, in 35/60 (58.3%) patients after surgery, and in 23/58 (39.7%) patients at the end of treatment (EOT). Patients with ctDNA/MRD positive status post-surgery or EOT experienced a significant shorter recurrence-free survival (RFS) than patients with negative ctDNA/MRD [hazard ratio (HR) 4.2; P &lt; 0.001 and HR 9.8; P &lt; 0.001]. The recurrence rate in MRD-positive patients (aka. positive predictive value, PPV) was 91.4% (32/35) at post-surgery and 91.3% (21/23) at EOT, respectively. In 93.0% of postoperative MRD-positive patients, ctDNA detected early relapse with a lead time up to 9.0 months (median 3.4 months) compared with standard radiological imaging. Among 40 patients with recurrence, the total secondary resection rate was 52.5% (21/40) with 40% (21/40) cases reached secondary NED. While among 24 recurrent cases with only liver metastases, the secondary resection rate was 66.7% (16/24) with 58.3% (14/24) cases reached secondary NED. Conclusions: Postoperative ctDNA status is strongly associated with shorter RFS. Serial ctDNA monitoring assists early detection of relapse, and therefore, improves patient outcomes by secondary resection. Clinical trial information: ChiCTR2000035677 .

Loss of Heterozygosity in the Circulating Tumor DNA and CD138+ Bone Marrow Cells in Multiple Myeloma.

Multiple myeloma (MM) is characterized by heterogeneity of tumor cells. The study of tumor cells from blood, bone marrow, plasmacytoma, etc., allows us to identify similarities and differences in tumor lesions of various anatomical localizations. The aim of this study was to compare the loss of heterozygosity (LOH) by tumor cells by assessing STR profiles of different MM lesions. We examined paired samples of plasma circulating tumor DNA (ctDNA) and CD138+ bone marrow cells in MM patients. For patients with plasmacytomas (66% of 38 patients included), the STR profile of plasmacytomas was also studied when biopsy samples were available. Diverse patterns of LOH were found in lesions of different localization for most patients. LOH in plasma ctDNA, bone marrow, and plasmacytoma samples was found for 55%, 71%, and 100% of patients, respectively. One could expect a greater variety of STR profiles in aberrant loci for patients with plasmacytomas. This hypothesis was not confirmed-no difference in the frequency of LOH in MM patients with or without plasmacytomas was found. This indicates the genetic diversity of tumor clones in MM, regardless of the presence of extramedullar lesions. Therefore, we conclude that risk stratification based on molecular tests performed solely on bone marrow samples may not be sufficient for all MM patients, including those without plasmacytomas. Due to genetic heterogeneity of MM tumor cells from various lesions, the high diagnostic value of liquid biopsy approaches becomes obvious.

Application of ddPCR in detection of the status and abundance of EGFR T790M mutation in the plasma samples of non-small cell lung cancer patients.

The third-generation epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitor (TKIs), such as osimertinib, designed for targeting the acquired drug-resistant mutation of EGFR T790M, was approved as the first-line therapy for advanced EGFR-mutated non-small cell lung cancer (NSCLC). Thus, detection of the EGFR T790M mutation for NSCLC is crucial. However, tissue samples are often difficult to obtain, especially in patients at advanced stages. This study assessed the performances of droplet digital polymerase chain reaction (ddPCR) and next-generation sequencing (NGS) in detecting EGFR T790M status and abundance in the plasma ctDNA samples of patients with NSCLC. We also explored the association between T790M status and abundance and the response to third-generation EGFR-TKIs. A total of 201 plasma samples with matched tissues, 821 plasma samples, and 56 patients who received third-generation EGFR-TKIs with response evaluation were included in this study. ddPCR and NGS were used to detect the mutation status and abundance of T790M in the tissues and/or blood samples. The results showed that the sensitivity and the specificity of EGFR T790M mutation status detected by ddPCR in plasma samples were 81.82% and 91.85%, respectively, compared with the tissue samples, with a consistency coefficient of 0.740. Among the 821 plasma samples, the positive rates of EGFR T790M detected by ddPCR and NGS were 34.2% (281/821) and 22.5% (185/821), respectively. With NGS results as the reference, the sensitivity and the specificity of ddPCR were 100% and 84.91%, respectively, and the consistency coefficient of the two methods was 0.717. In addition, we found that a higher EGFR T790M abundance was linked to a higher treatment response rate to the third-generation EGFR-TKIs regardless of the classification of the median value of 0.43% (P = 0.016) or average value of 3.16% (P = 0.010). Taking these data together, this study reveals that ddPCR is an alternatively potent method for the detection of EGFR T790M in the plasma samples of NSCLC patients.

Construction of broom-like Ag@N,O-C sensing interface for electrochemical detection of circulating tumor DNA using entropy-driven DNA walker

In human peripheral blood, circulating tumor DNA (ctDNA) is an excellent tumor biomarker for noninvasive diagnosis, therapy and prognosis of cancer. Herein, an innovative enzyme-free electrochemical sensor sensitized with broom-like Ag@N,O-C hierarchical nanomaterials and entropy-driven DNA walker was designed for PIK3CA E545K ctDNA assay. In this work, N, O-doped carbon layers were used to completely coat Ag nanoparticles, and then were assembled into three-dimensional broom-like nanomaterials with hierarchical structure. The application of Ag@N,O-C broom-like hierarchical nanomaterials (Ag@N,O-C BLHS) as electrode material not only facilitated electron transfer, but also increased functional active sites for a high-density assembly of DNA walking substrates. Via the entropy-driven catalytic reaction of DNA walker, one target ctDNA initiated the dissociation of abundant electroactive tags from the electrode surface, achieving a one-to-multiple amplification effect. By measuring electrochemical responses, PIK3CA E545K ctDNA was quantified with 10 aM detection limit. Additionally, the enzyme-free amplified sensing interface achieved the direct detection of ctDNA in plasma samples from breast cancer patients and healthy human. Overall, the good selectivity, reproducibility and practical application ability confirmed a promising potential of the sensor for disease diagnosis.