Noninvasive Early Identification of Therapeutic Benefit from Immune Checkpoint Inhibition.

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.

Bridging biological cfDNA features and machine learning approaches.

Liquid biopsies (LBs), particularly using circulating tumor DNA (ctDNA), are expected to revolutionize precision oncology and blood-based cancer screening. Recent technological improvements, in combination with the ever-growing understanding of cell-free DNA (cfDNA) biology, are enabling the detection of tumor-specific changes with extremely high resolution and new analysis concepts beyond genetic alterations, including methylomics, fragmentomics, and nucleosomics. The interrogation of a large number of markers and the high complexity of data render traditional correlation methods insufficient. In this regard, machine learning (ML) algorithms are increasingly being used to decipher disease- and tissue-specific signals from cfDNA. Here, we review recent insights into biological ctDNA features and how these are incorporated into sophisticated ML applications.

Integrating genomic features for non-invasive early lung cancer detection.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.

Circulating Tumor DNA Analysis Guiding Adjuvant Therapy in Stage II Colon Cancer.

BACKGROUND: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. METHODS: We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. RESULTS: Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. CONCLUSIONS: A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).

Bioreaction-Compatible Bivariate Lanthanide MOF Sensor Enables Stimulus-Multiresponsive Platform for ctDNA On-Site Detection.

Detection of circulating tumor DNA (ctDNA) in liquid biopsy is of great importance for tumor diagnosis but difficult due to its low amount in bodily fluids. Herein, a novel ctDNA detection platform is established by quantifying DNA amplification by-product pyrophosphate (PPi) using a newly designed bivariable lanthanide metal-organic framework (Ln-MOF), namely, Ce/Eu-DPA MOF (CE-24, DPA = pyridine-2,6-dicarboxylic acid). CE-24 MOF exhibits ultrafast dual-response (fluorescence enhancement and enzyme-activity inhibition) to PPi stimuli by virtue of host-guest interaction. The platform is applied to detecting colon carcinoma-related ctDNA (KARS G12D mutation) combined with the isothermal nucleic acid exponential amplification reaction (EXPAR). ctDNA triggers the generation of a large amount of PPi, and the ctDNA quantification is achieved through the ratio fluorescence/colorimetric dual-mode assay of PPi. The combination of the EXPAR and the dual-mode PPi sensing allows the ctDNA assay method to be low-cost, convenient, bioreaction-compatible (freedom from the interference of bioreaction systems), sensitive (limit of detection down to 101 fM), and suitable for on-site detection. To the best of our knowledge, this work is the first application of Ln-MOF for ctDNA detection, and it provides a novel universal strategy for the rapid detection of nucleic acid biomarkers in point-of-care scenarios.

Liquid biopsy-based circulating tumour (ct)DNA analysis of a spectrum of myeloid and lymphoid malignancies yields clinically actionable results.

AIMS: Liquid biopsy (LBx)-based next-generation sequencing (NGS) of circulating tumour DNA (ctDNA) can facilitate molecular profiling of haematopoietic neoplasms (HNs), particularly when tissue-based NGS is infeasible. METHODS AND RESULTS: We studied HN LBx samples tested with FoundationOne Liquid CDx, FoundationOne Liquid, or FoundationACT between July 2016 and March 2022. We identified 271 samples: 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytoses, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukaemia (AML). Among 73.4% with detectable pathogenic alterations, median maximum somatic allele frequency (MSAF) was 16.6%, with AML (36.2%), MDS (19.7%), and MPN (44.5%) having higher MSAFs than DLBCL (3.9%), NHL (8.4%), HL (1.5%), PCN (2.8%), and histiocytoses (1.8%) (P = 0.001). LBx detected characteristic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML. Among 24 samples, the positive percent agreement by LBx was 75.7% for variants present in paired buffy coat, marrow, or tissues. Also, 75.0% of pairs exhibited alterations only present on LBx. These were predominantly subclonal (clonal fraction of 3.8%), reflecting the analytical sensitivity of LBx. CONCLUSION: These data demonstrate that LBx can detect relevant genomic alterations across HNs, including at low clonal fractions, suggesting a potential clinical utility for identifying residual or emerging therapy-resistant clones that may be undetectable in site-specific tissue biopsies.

Comparing a fecal immunochemical test and circulating tumor DNA blood test for colorectal cancer screening adherence.

BACKGROUND AND AIM: Colorectal cancer (CRC) screening programs are most effective at reducing disease incidence and mortality through sustained screening participation. A novel blood test modality is being explored for CRC screening, but it is unclear whether it will provide sustained screening participation. This study aimed to investigate whether a circulating tumor DNA (ctDNA) blood test improved CRC screening re-participation when compared with a fecal immunochemical test (FIT) and to define the predictors of sustained CRC screening in an Australian population. METHODS: South Australians who initially participated in CRC screening using a ctDNA blood test (n = 36) or FIT (n = 547) were offered the same CRC screening test approximately 2 years later through an extended phase of a randomized controlled trial. Surveys collected demographic, psychosocial, and clinical information. Predictors of CRC screening re-participation were explored using chi-square, Wilcoxon tests, and logistic regression. RESULTS: Participants offered a second ctDNA blood test were equally likely to re-participate in CRC screening as those who completed a FIT in the first round and who were offered the same test (61% vs 66% re-participation respectively, P = 0.6). CRC fatalism, health activation, and self-efficacy were associated with repeated screening participation. Test awareness was predictive of repeated FIT-based CRC screening. CONCLUSIONS: Targeted interventions to improve CRC screening awareness and increase patient health activation may improve CRC screening adherence. A ctDNA blood test may be a suitable CRC screening option to maintain CRC screening adherence in people who do not participate in screening with FIT.

The Uncharted Territories of Esophageal Cancer with Cardiac and Skeletal Muscle Metastasis: A Case Report and Literature Review.

Background: Esophageal cancer (EC) comprises 1% of all diagnosed cancers in the USA. It is more common in other parts of the world. If there is distant metastasis, the relative survival rate is 6%. There are no standardized screening methods for EC. Case Presentation: We reported a four-year case of esophageal cancer, a P53-positive mutation with atypical distant metastasis to the cardiac and skeletal muscles. The patient was managed with multimodal therapy, including immunotherapy, which could have been a factor in prolonged survival. Conclusions: Distant metastases are typically seen postmortem, and with prolonged survival, we are able to find such unique metastases antemortem. Despite a history of negative scans, the patient's ctDNA (circulating tumor DNA) remained positive, which was a better predictor of recurrence in this case. Future research is required to establish cost-effective screening methods and standardized treatments.

Early detection and stratification of lung cancer aided by a cost-effective assay targeting circulating tumor DNA (ctDNA) methylation.

BACKGROUND: Detection of lung cancer at earlier stage can greatly improve patient survival. We aim to develop, validate, and implement a cost-effective ctDNA-methylation-based plasma test to aid lung cancer early detection. METHODS: Case-control studies were designed to select the most relevant markers to lung cancer. Patients with lung cancer or benign lung disease and healthy individuals were recruited from different clinical centers. A multi-locus qPCR assay, LunaCAM, was developed for lung cancer alertness by ctDNA methylation. Two LunaCAM models were built for screening (-S) or diagnostic aid (-D) to favor sensitivity or specificity, respectively. The performance of the models was validated for different intended uses in clinics. RESULTS: Profiling DNA methylation on 429 plasma samples including 209 lung cancer, 123 benign diseases and 97 healthy participants identified the top markers that detected lung cancer from benign diseases and healthy with an AUC of 0.85 and 0.95, respectively. The most effective methylation markers were verified individually in 40 tissues and 169 plasma samples to develop LunaCAM assay. Two models corresponding to different intended uses were trained with 513 plasma samples, and validated with an independent collection of 172 plasma samples. In validation, LunaCAM-S model achieved an AUC of 0.90 (95% CI: 0.88-0.94) between lung cancer and healthy individuals, whereas LunaCAM-D model stratified lung cancer from benign pulmonary diseases with an AUC of 0.81 (95% CI: 0.78-0.86). When implemented sequentially in the validation set, LunaCAM-S enables to identify 58 patients of lung cancer (90.6% sensitivity), followed by LunaCAM-D to remove 20 patients with no evidence of cancer (83.3% specificity). LunaCAM-D significantly outperformed the blood test of carcinoembryonic antigen (CEA), and the combined model can further improve the predictive power for lung cancer to an overall AUC of 0.86. CONCLUSIONS: We developed two different models by ctDNA methylation assay to sensitively detect early-stage lung cancer or specifically classify lung benign diseases. Implemented at different clinical settings, LunaCAM models has a potential to provide a facile and inexpensive avenue for early screening and diagnostic aids for lung cancer.

Circulating Tumor DNA in Gastric Adenocarcinoma: Future Clinical Applications and Perspectives.

Gastric cancer (GC) is still one of the most aggressive cancers with a few targetable alterations and a dismal prognosis. A liquid biopsy allows for identifying and analyzing the DNA released from tumor cells into the bloodstream. Compared to tissue-based biopsy, liquid biopsy is less invasive, requires fewer samples, and can be repeated over time in order to longitudinally monitor tumor burden and molecular changes. Circulating tumor DNA (ctDNA) has been recognized to have a prognostic role in all the disease stages of GC. The aim of this article is to review the current and future applications of ctDNA in gastric adenocarcinoma, in particular, with respect to early diagnosis, the detection of minimal residual disease (MRD) following curative surgery, and in the advanced disease setting for treatment decision choice and therapeutic monitoring. Although liquid biopsies have shown potentiality, pre-analytical and analytical steps must be standardized and validated to ensure the reproducibility and standardization of the procedures and data analysis methods. Further research is needed to allow the use of liquid biopsy in everyday clinical practice.

Circulating Tumor DNA Is a Variant of Liquid Biopsy with Predictive and Prognostic Clinical Value in Breast Cancer Patients.

This paper introduces the reader to the field of liquid biopsies and cell-free nucleic acids, focusing on circulating tumor DNA (ctDNA) in breast cancer (BC). BC is the most common type of cancer in women, and progress with regard to treatment has been made in recent years. Despite this, there remain a number of unresolved issues in the treatment of BC; in particular, early detection and diagnosis, reliable markers of response to treatment and for the prediction of recurrence and metastasis, especially for unfavorable subtypes, are needed. It is also important to identify biomarkers for the assessment of drug resistance and for disease monitoring. Our work is devoted to ctDNA, which may be such a marker. Here, we describe its main characteristics and potential applications in clinical oncology. This review considers the results of studies devoted to the analysis of the prognostic and predictive roles of various methods for the determination of ctDNA in BC patients. Currently known epigenetic changes in ctDNA with clinical significance are reviewed. The possibility of using ctDNA as a predictive and prognostic marker for monitoring BC and predicting the recurrence and metastasis of cancer is also discussed, which may become an important part of a precision approach to the treatment of BC.

Integrating circulating-free DNA (cfDNA) analysis into clinical practice: opportunities and challenges.

In the current era of precision medicine, the identification of genomic alterations has revolutionised the management of patients with solid tumours. Recent advances in the detection and characterisation of circulating tumour DNA (ctDNA) have enabled the integration of liquid biopsy into clinical practice for molecular profiling. ctDNA has also emerged as a promising biomarker for prognostication, monitoring disease response, detection of minimal residual disease and early diagnosis. In this Review, we discuss current and future clinical applications of ctDNA primarily in non-small cell lung cancer in addition to other solid tumours.

Error Characterization and Statistical Modeling Improves Circulating Tumor DNA Detection by Droplet Digital PCR.

BACKGROUND: Droplet digital PCR (ddPCR) is a widely used and sensitive application for circulating tumor DNA (ctDNA) detection. As ctDNA is often found in low abundance, methods to separate low-signal readouts from noise are necessary. We aimed to characterize the ddPCR-generated noise and, informed by this, create a sensitive and specific ctDNA caller. METHODS: We built 2 novel complimentary ctDNA calling methods: dynamic limit of blank and concentration and assay-specific tumor load estimator (CASTLE). Both methods are informed by empirically established assay-specific noise profiles. Here, we characterized noise for 70 mutation-detecting ddPCR assays by applying each assay to 95 nonmutated samples. Using these profiles, the performance of the 2 new methods was assessed in a total of 9447 negative/positive reference samples and in 1311 real-life plasma samples from colorectal cancer patients. Lastly, performances were compared to 7 literature-established calling methods. RESULTS: For many assays, noise increased proportionally with the DNA input amount. Assays targeting transition base changes were more error-prone than transversion-targeting assays. Both our calling methods successfully accounted for the additional noise in transition assays and showed consistently high performance regardless of DNA input amount. Calling methods that were not noise-informed performed less well than noise-informed methods. CASTLE was the only calling method providing a statistical estimate of the noise-corrected mutation level and call certainty. CONCLUSIONS: Accurate error modeling is necessary for sensitive and specific ctDNA detection by ddPCR. Accounting for DNA input amounts ensures specific detection regardless of the sample-specific DNA concentration. Our results demonstrate CASTLE as a powerful tool for ctDNA calling using ddPCR.

Clinical biomarkers in soft tissue sarcoma A comprehensive review of current soft tissue sarcoma biomarkers.

Soft tissue sarcomas (STS) are a heterogeneous group of tumors that arise from mesenchymal tissue. Investigation at the molecular level has been challenging due to the rarity of STS and the number of histologic subtypes. However, recent research has provided new insight into potential genomic, proteomic, and immunological biomarkers of STS. The identification of biomarkers can improve diagnosis, prognosis, and prediction of recurrence and treatment response. This review provides an understanding of biomarkers, discussing the current status of biomarker research in STS.

Personalized circulating tumor DNA in patients with hepatocellular carcinoma: a pilot study.

BACKGROUND: Mutational analysis of circulating tumor DNA (ctDNA) can potentially be used for early detection of recurrence after resection for hepatocellular carcinoma (HCC). Mutations from tumor may be identified in plasma as an early sign of recurrence. We conducted a pilot study investigating if somatic mutations could be detected in plasma in patients undergoing liver resection for HCC and in patients with advanced non-resectable HCC. METHODS AND RESULTS: We prospectively included patients undergoing curative liver resection for HCC. Tumor tissue was investigated with whole exome sequencing and preoperative blood samples were evaluated for ctDNA using targeted next-generation sequencing (NGS) with TruSight Oncology 500 including 523 cancer-associated genes. Subsequently, the method was evaluated in patients with advanced HCC. We included eight patients curatively resected for HCC, where tumor tissue mutations were identified in seven patients. However, only in one patient tumor specific mutations were found in the preoperative blood sample. In all three patients with advanced HCC, tumor mutations were detected in the blood. CONCLUSIONS: In patients with resectable HCC, ctDNA could not be reliably detected using the applied targeted NGS method. In contrast, ctDNA was detected in all patients with advanced HCC. Small tumors, tumor heterogeneity and limited sequencing coverage may explain the lack of detectable ctDNA.

Making the Rounds: Exploring the Role of Circulating Tumor DNA (ctDNA) in Non-Small Cell Lung Cancer.

Advancements in the clinical practice of non-small cell lung cancer (NSCLC) are shifting treatment paradigms towards increasingly personalized approaches. Liquid biopsies using various circulating analytes provide minimally invasive methods of sampling the molecular content within tumor cells. Plasma-derived circulating tumor DNA (ctDNA), the tumor-derived component of cell-free DNA (cfDNA), is the most extensively studied analyte and has a growing list of applications in the clinical management of NSCLC. As an alternative to tumor genotyping, the assessment of oncogenic driver alterations by ctDNA has become an accepted companion diagnostic via both single-gene polymerase chain reactions (PCR) and next-generation sequencing (NGS) for advanced NSCLC. ctDNA technologies have also shown the ability to detect the emerging mechanisms of acquired resistance that evolve after targeted therapy. Furthermore, the detection of minimal residual disease (MRD) by ctDNA for patients with NSCLC after curative-intent treatment may serve as a prognostic and potentially predictive biomarker for recurrence and response to therapy, respectively. Finally, ctDNA analysis via mutational, methylation, and/or fragmentation multi-omic profiling offers the potential for improving early lung cancer detection. In this review, we discuss the role of ctDNA in each of these capacities, namely, for molecular profiling, treatment response monitoring, MRD detection, and early cancer detection of NSCLC.

Circulating tumour DNA in patients with advanced melanoma treated with dabrafenib or dabrafenib plus trametinib: a clinical validation study.

BACKGROUND: Melanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes. METHODS: In this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number. FINDINGS: In COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p<0·0001 by univariate analysis), independent of treatment group and baseline lactate dehydrogenase concentrations (1·08 [1·03-1·13], p=0·0020), in COMBI-d. A ctDNA cutoff point of 64 copies per mL of plasma stratified patients enrolled in COMBI-d as high risk or low risk with respect to survival outcomes (HR 1·74 [95% CI 1·37-2·21], p<0·0001 for progression-free survival; 2·23 [1·73-2·87], p<0·0001 for overall survival) and was validated in the COMBI-MB cohort (3·20 [1·39-7·34], p=0·0047 for progression-free survival; 2·94 [1·18-7·32], p=0·016 for overall survival). In COMBI-d, undetectable ctDNA at week 4 was significantly associated with extended progression-free and overall survival, particularly in patients with elevated lactate dehydrogenase concentrations (HR 1·99 [95% CI 1·08-3·64], p=0·027 for progression-free survival; 2·38 [1·24-4·54], p=0·0089 for overall survival). INTERPRETATION: Pretreatment and on-treatment BRAFV600-mutant ctDNA measurements could serve as independent, predictive biomarkers of clinical outcome with targeted therapy. FUNDING: Novartis.

Flap Endonuclease 1-Assisted DNA Walkers for Sensitively and Specifically Sensing ctDNAs.

DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.

Prognostic impact of circulating tumor DNA status post-allogeneic hematopoietic stem cell transplantation in AML and MDS.

This study was performed to assess the utility of tumor-derived fragmentary DNA, or circulating tumor DNA (ctDNA), for identifying high-risk patients for relapse of acute myeloid leukemia and myelodysplastic syndrome (AML/MDS) after undergoing myeloablative allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively collected tumor and available matched serum samples at diagnosis and 1 and 3 months post-alloSCT from 53 patients with AML/MDS. After identifying driver mutations in 51 patients using next-generation sequencing, we designed at least 1 personalized digital polymerase chain reaction assay per case. Diagnostic ctDNA and matched tumor DNA exhibited excellent correlations with variant allele frequencies. Sixteen patients relapsed after a median of 7 months post-alloSCT. Both mutation persistence (MP) in bone marrow (BM) at 1 and 3 months post-alloSCT and corresponding ctDNA persistence (CP) in the matched serum (MP1 and MP3; CP1 and CP3, respectively) were comparably associated with higher 3-year cumulative incidence of relapse (CIR) rates (MP1 vs non-MP1, 72.9% vs 13.8% [P = .0012]; CP1 vs non-CP1, 65.6% vs 9.0% [P = .0002]; MP3 vs non-MP3, 80% vs 11.6% [P = .0002]; CP3 vs non-CP3, 71.4% vs 8.4% [P < .0001]). We subsequently evaluated whether subset analysis of patients with 3 genes associated with clonal hematopoiesis, DNMT3A, TET2, and ASXL1 (DTA), could also be helpful in relapse prediction. As a result, CP based on DTA gene mutations also had the prognostic effect on CIR. These results, for the first time, support the utility of ctDNA as a noninvasive prognostic biomarker in patients with AML/MDS undergoing alloSCT.

Circulating tumor DNA is a sensitive marker for routine monitoring of treatment response in advanced colorectal cancer.

Accurate assessment of chemotherapy response provides the means to terminate ineffective treatment, trial alternative drug regimens or schedules and reduce dose to minimize toxicity. Here, we have compared circulating tumor DNA (ctDNA) with carcinoembryonic antigen (CEA) for the cycle by cycle assessment of chemotherapy response in 30 patients with metastatic colorectal cancer. CtDNA (quantified using individualized digital droplet PCR (ddPCR) assays) and CEA levels were determined immediately prior to each chemotherapy cycle over time periods ranging from 42-548 days (average of 10 time points/patient). Twenty-nine/thirty (97%) patients had detectable ctDNA compared with 83% whose tumors were CEA-positive (>5 ng/ml) during the monitoring course. Over the course of treatment, 20 disease progression events were detected by computed tomography; ctDNA predicted significantly more of these events than CEA (16 (80%) versus 6 (30%), respectively; P-value = 0.004). When progression was detected by both ctDNA and CEA, the rise in ctDNA occurred significantly earlier than CEA (P-value = 0.046). Partial responses to chemotherapy were also detected more frequently by ctDNA, although this was not significant (P-value = 0.07). In addition, another 28 colorectal cancer patients who underwent potentially curative surgery and showed no evidence of residual disease were monitored with ctDNA for up to 2 years. Clinical relapse was observed in 6/28 (21%) patients. Four out of 6 of these patients showed a significant increase in ctDNA at or prior to relapse. Overall, ctDNA analyses were able to be performed in a clinically relevant timeline and were a more sensitive and responsive measure of tumor burden than CEA.