Liquid Biopsy Samples Research Articles

Unlocking the promise of liquid biopsies in precision oncology

Liquid biopsies have emerged as a promising and minimally invasive alternative to traditional tissue biopsies for detecting and monitoring cancer. Liquid biopsies offer a comprehensive analysis of cancer genetics and tumor burden by examining circulating cells and cell-derived analytes using a variety of assays, including conventional PCR methods and cutting-edge tools like long-read sequencing and nanotechnology. However, there are still some limitations and challenges that need to be overcome for their implementation in clinical routine, including the need for further research on their sensitivity and specificity, cost-effectiveness, standardization, and regulatory approval. Despite these challenges, liquid biopsies have the potential to become widely used tools in oncology. Here we provide an overview of the current state of liquid biopsies, highlighting recent advancements in the field and their potential benefits in clinical settings for cancer patients. The article further discusses the challenges that need to be addressed in order to facilitate their application worldwide. Prompt resolution of these challenges can be achieved by fostering international research collaborations and establishing standardized guidelines for liquid biopsy sample management and studies.

Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation.

Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.

Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients’ care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35–10380 and 500–10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

Genomic features of lung cancer patients in Indonesia’s national cancer center

IntroductionAdvances in molecular biology bring advantages to lung cancer management. Moreover, high-throughput molecular tests are currently useful for revealing genetic variations among lung cancer patients. We investigated the genomics profile of the lung cancer patients at the National Cancer Centre of Indonesia.MethodsA retrospective study enrolled 627 tissue biopsy samples using real time polymerase chain reaction (RT-PCR) and 80 circulating tumour DNA (ctDNA) liquid biopsy samples using next-generation sequencing (NGS) from lung cancer patients admitted to the Dharmais Cancer Hospital from January 2018 to December 2022. Data were obtained from medical records. Data statistically analysed with p < 0.05 is considered significant.ResultThe EGFR test results revealed by RT-PCR were wild type (51.5%), single variant (38.8%), double variant (8.3%), and triple variant (1.4%), with 18.66% L85R, 18.22% Ex19del, and 11.08% L861Q variant. Liquid biopsy ctDNA using NGS showed only 2.5% EGFR wild type, 62.5% single variant and 35% co-variant, with EGFR/TP53 and EGFR/PIK3CA as the highest.ConclusionEGFR variants are the most found in our centre. Liquid biopsy with ctDNA using NGS examination could detect broad variants and co-variants that will influence the treatment planning.

Prognostic and predictive role of liquid biopsy in lung cancer patients.

Lung cancer (LC) is a leading cause of cancer-related mortality worldwide. Approximately 80% of LC cases are of the non-small cell lung cancer (NSCLC) type, and approximately two-thirds of these cases are diagnosed in advanced stages. Only systemic treatment methods can be applied to patients in the advanced stages when there is no chance of surgical treatment. Identification of mutations that cause LC is of vital importance in determining appropriate treatment methods. New noninvasive methods are needed to repeat and monitor these molecular analyses. In this regard, liquid biopsy (LB) is the most promising method. This study aimed to determine the effectiveness of LB in detecting EGFR executive gene mutations that cause LC. One hundred forty-six patients in stages IIIB and IV diagnosed with non-squamous cell non-small cell LC were included. Liquid biopsy was performed as a routine procedure in cases where no mutation was detected in solid tissue or in cases with progression after targeted therapy. Liquid biopsy samples were also obtained for the second time from 10 patients who showed progression under the applied treatment. Mutation analyses were performed using the Cobas® EGFR Test, a real-time PCR test designed to detect mutations in exons 18, 20, and 21 and changes in exon 19 of the EGFR gene. Mutation positivity in paraffin blocks was 21.9%, whereas it was 32.2% in LB. Solids and LB were compatible in 16 patients. Additionally, while no mutation was found in solid tissue in the evaluation of 27 cases, it was detected in LB. It has been observed that new mutations can be detected not only at the time of diagnosis, but also in LB samples taken during the follow-up period, leading to the determination of targeted therapy. The results showed that "liquid biopsy" is a successful and alternative non-invasive method for detecting cancer-causing executive mutations, given the limitations of conventional biopsies.

Combined DNA Analysis from Stool and Blood Samples Improves Tumor Tracking and Assessment of Clonal Heterogeneity in Localized Rectal Cancer Patients.

Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution.

Microfluidics-based patient-derived disease detection tool for deep learning-assisted precision medicine.

Cancer spatial and temporal heterogeneity fuels resistance to therapies. To realize the routine assessment of cancer prognosis and treatment, we demonstrate the development of an Intelligent Disease Detection Tool (IDDT), a microfluidic-based tumor model integrated with deep learning-assisted algorithmic analysis. IDDT was clinically validated with liquid blood biopsy samples (n = 71) from patients with various types of cancers (e.g., breast, gastric, and lung cancer) and healthy donors, requiring low sample volume (∼200 μl) and a high-throughput 3D tumor culturing system (∼300 tumor clusters). To support automated algorithmic analysis, intelligent decision-making, and precise segmentation, we designed and developed an integrative deep neural network, which includes Mask Region-Based Convolutional Neural Network (Mask R-CNN), vision transformer, and Segment Anything Model (SAM). Our approach significantly reduces the manual labeling time by up to 90% with a high mean Intersection Over Union (mIoU) of 0.902 and immediate results (<2 s per image) for clinical cohort classification. The IDDT can accurately stratify healthy donors (n = 12) and cancer patients (n = 55) within their respective treatment cycle and cancer stage, resulting in high precision (∼99.3%) and high sensitivity (∼98%). We envision that our patient-centric IDDT provides an intelligent, label-free, and cost-effective approach to help clinicians make precise medical decisions and tailor treatment strategies for each patient.

The potential of liquid biopsy for detection of the KIAA1549-BRAF fusion in circulating tumor DNA from children with pilocytic astrocytoma.

Low-grade gliomas (LGGs) represent children's most prevalent central nervous system tumor, necessitating molecular profiling to diagnose and determine the most suitable treatment. Developing highly sensitive screening techniques for liquid biopsy samples is particularly beneficial, as it enables the early detection and molecular characterization of tumors with minimally invasive samples. We examined CSF and plasma samples from patients with pilocytic astrocytoma (PA) using custom multiplexed droplet digital polymerase chain reaction (ddPCR) assays based on whole genome sequencing data. These assays included a screening test to analyze BRAF duplication and a targeted assay for the detection of patient-specific KIAA1549::BRAF fusion junction sequences or single nucleotide variants. Our findings revealed that 5 out of 13 individual cerebrospinal fluid (CSF) samples tested positive for circulating tumor DNA (ctDNA). Among these cases, 3 exhibited the KIAA1549::BRAF fusion, which was detected through copy number variation (CNV) analysis (n = 1) or a fusion-specific probe (n = 2), while 1 case each displayed the BRAF V600E mutation and the FGFR1 N577K mutation. Additionally, a quantitative analysis of cell-free DNA (cfDNA) concentrations in PA CSF samples showed that most cases had low cfDNA levels, below the limit of detection of our assay (<1.9ng). While CNV analysis of CSF samples from LGGs still has some limitations, it has the potential to serve as a valuable complementary tool. Furthermore, it can also be multiplexed with other aberrations, for example, to the BRAF V600 test, to provide important insights into the molecular characteristics of LGGs.

Next-generation sequencing-based liquid biopsy may be used for detection of residual disease and cancer recurrence monitoring in dogs.

The purpose of this study was to evaluate the performance of a next-generation sequencing-based liquid biopsy test for cancer monitoring in dogs. Pre- and postoperative blood samples were collected from dogs with confirmed cancer diagnoses originally enrolled in the CANcer Detection in Dogs (CANDiD) study. A subset of dogs also had longitudinal blood samples collected for recurrence monitoring. All cancer-diagnosed patients had a preoperative blood sample in which a cancer signal was detected and had at least 1 postoperative sample collected. Clinical data were used to assign a clinical disease status for each follow-up visit. Following excisional surgery, in the absence of clinical residual disease at the postoperative visit, patients with Cancer Signal Detected results at that visit were 1.94 times as likely (95% CI, 1.21 to 3.12; P = .013) to have clinical recurrence within 6 months compared to patients with Cancer Signal Not Detected results. In the subset of patients with longitudinal liquid biopsy samples that had clinical recurrence documented during the study period, 82% (9/11; 95% CI, 48% to 97%) had Cancer Signal Detected in blood prior to or concomitant with clinical recurrence; in the 6 patients where molecular recurrence was detected prior to clinical recurrence, the median lead time was 168 days (range, 47 to 238). Next-generation sequencing-based liquid biopsy is a noninvasive tool that may offer utility as an adjunct to current standard-of-care clinical assessment for cancer monitoring; further studies are needed to confirm diagnostic accuracy in a larger population.

Brief Report: Comprehensive Clinicogenomic Profiling of Small Cell Transformation From EGFR-Mutant NSCLC Informs Potential Therapeutic Targets

IntroductionNSCLC transformation to SCLC has been best characterized with EGFR-mutant NSCLC, with emerging case reports seen in ALK, RET, and KRAS-altered NSCLC. Previous reports revealed transformed SCLC from EGFR-mutant NSCLC portends very poor prognosis and lack effective treatment. Genomic analyses revealed TP53 and RB1 loss of function increase the risk of SCLC transformation. Little has been reported on the detailed clinicogenomic characteristics and potential therapeutic targets for this patient population. MethodsIn this study, we conducted a single-center retrospective analysis of clinical and genomic characteristics of patients with EGFR-mutant NSCLC transformed to SCLC. Demographic data, treatment course, and clinical molecular testing reports were extracted from electronic medical records. Kaplan-Meier analyses were used to estimate survival outcomes. Next generation sequencing-based assays was used to identify EGFR and co-occurring genetic alterations in tissue or plasma before and after SCLC transformation. Single-cell RNA sequencing (scRNA-seq) was performed on a patient-derived-xenograft model generated from a patient with EGFR-NSCLC transformed SCLC tumor. ResultsA total of 34 patients were identified in our study. Median age at initial diagnosis was 58, and median time to SCLC transformation was 24.2 months. 68% were female and 82% were never smokers. 79% of patients were diagnosed as stage IV disease, and over half had brain metastases at baseline. Median overall survival of the entire cohort was 38.3 months from initial diagnoses and 12.4 months from time of SCLC transformation. Most patients harbored EGFR exon19 deletions as opposed to exon21 L858R alteration. Continuing EGFR tyrosine kinase inhibitor post-transformation did not improve overall survival compared with those patients where tyrosine kinase inhibitor was stopped in our cohort. In the 20 paired pretransformed and post-transformed patient samples, statistically significant enrichment was seen with PIK3CA alterations (p = 0.04) post-transformation. Profiling of longitudinal liquid biopsy samples suggest emergence of SCLC genetic alterations before biopsy-proven SCLC, as shown by increasing variant allele frequency of TP53, RB1, PIK3CA alterations. ScRNA-seq revealed potential therapeutic targets including DLL3, CD276 (B7-H3) and PTK7 were widely expressed in transformed SCLC. ConclusionsSCLC transformation is a potential treatment resistance mechanism in driver-mutant NSCLC. In our cohort of 34 EGFR-mutant NSCLC, poor prognosis was observed after SCLC transformation. Clinicogenomic analyses of paired and longitudinal samples identified genomic alterations emerging post-transformation and scRNA-seq reveal potential therapeutic targets in this population. Further studies are needed to rigorously validate biomarkers and therapeutic targets for this patient population.

Droplet Digital PCR as a Molecular Tool for the Detection of the EGFR T790M Mutation in NSCLC Patients with the EGFR Activating Mutations.

Almost 50% of NSCLC patients who initially show a successful response to tyrosine kinase inhibitors targeted therapy (TKI therapy) eventually develop acquired EGFR T790M mutation. The T790M secondary mutation can cause resistance to the targeted therapy and disease relapse. Since this mutation can be present at very low frequencies in liquid biopsy samples, droplet digital PCR (ddPCR), due to its high sensitivity, has opened the possibility for minimally invasive monitoring of the disease during TKI targeted therapy. For this study, a total of 45 plasma samples from NSCLC patients with previously detected EGFR-activating mutations were analyzed. Extracted circulating free DNA was amplified and examined for the presence of T790M mutation using ddPCR technology. For the data analysis, QuantaSoft Software was used. Of 45 tested plasma samples, a total of 14 samples were identified as positive for the T790M mutation. The same samples eventually showed the presence of T790M mutation in FFPE. Droplet digital PCR showed its great advantage in high sensitivity detection of rare allele variants. Our ddPCR assay detected T790M mutant allele in frequencies from 0.1%. The average number of droplets generated by ddPCR was 9571. Monitoring of the T790M mutation has an important role in the examination of the effects of the prescribed TKI therapy. Since monitoring of potential changes during TKI therapy requires repeated sampling, our results showed that ddPCR technology has made it possible to use liquid biopsy as an adequate minimally invasive alternative for single nucleotide polymorphisms (SNP) detection.

Liquid biopsy for lung cancer: A cross section on the diagnostic routine experience of a referral Italian institution

BackgroundLung cancer is the leading cause of cancer death worldwide. Over the last decade, molecular testing of a growing number of predictive biomarkers has become mandatory in the management of NSCLC patients. However, a major obstacle in routine clinical practice is the scant quantity of available tissue specimens obtainable from advanced stage of NSCLC patients. Thus, liquid biopsy, mostly involving blood sampling, has now been integrated in routine diagnostic practice. However, although liquid biopsies constitute a versatile, compliant, and dynamic source of nucleic acids, many of the current testing approaches pose few technical challenges. Here, to validate the feasibility of implementing NGS-based liquid biopsy approaches in routine diagnostic practice, we overviewed the NGS molecular data generated by our in-house narrow gene panel on plasma samples from real-world NSCLC patients. MethodsOur Institution received testing request on liquid biopsy samples from peripheral institutions not equipped to internally analyze liquid biopsy samples. Molecular data from NSCLC patients following oncological requested for clinically approved plasma-based biomarkers evaluated in a diagnostic routine setting from January 2020 to September 2022 were retrieved from our institutional archive. A customized NGS panel integrated with an optimized bioinformatic pipeline was adopted. ResultsOverall, a total of n = 185 cases were retrieved. Of note, 103 (55.7 %) and 82 (44.3 %) patients were analyzed at basal setting and after resistance to first line TKI administration, respectively. Moreover, 31 out of 185 (16.7 %) cases reveled EGFR clinically relevant alterations. In particular, 6 out of 31 (19.3 %) and 25 out 31 (80.7 %) EGFR mutated patients were tested in basal setting and after first or second line TKIs progression. In addition, exon 20 p.T790 M mutation was also detected in 12 out of 25 (48.0 %) EGFR concomitant mutated cases. Moreover, KRAS hot spot mutations were found in 24 out of 185 (13.0 %) cases. Among them, exon 2 p.G12C clinically relevant mutations were observed in 8 out 24 cases (33.3 %). ConclusionsThis review highlights the technical suitability of an NGS-based liquid biopsy system for the analysis of clinically relevant mutations in NSCLC patients.

Analytical Performance Evaluation of a 523-Gene Circulating Tumor DNA Assay for Next-Generation Sequencing–Based Comprehensive Tumor Profiling in Liquid Biopsy Samples

Next-generation sequencing (NGS)-based comprehensive tumor profiling from liquid biopsy samples can significantly improve diagnosis and monitoring of tumors when high-quality tissue material is difficult to obtain. In addition, it offers the potential to capture the entire complexity of the tumor, which is particularly important for highly heterogeneous or metastatic tumors. Here, we report the findings of an analytical performance evaluation of the TruSight Oncology 500 circulating tumor DNA (ctDNA) assay, a 523-gene NGS panel developed for ctDNA-based comprehensive genomic profiling of tumors, using reference and patient samples. Using 30 ng cell-free DNA, the assay showed high sensitivity and low variant detection variability for single-nucleotide variants, insertions and deletions, and fusions down to a variant allele frequency (VAF) of 0.5% in the reference samples and VAFs that were highly concordant with previous digital droplet PCR results in the patient samples. At reduced input amounts (20, 15, and 5 ng) and below VAFs of 0.5%, sensitivity was considerably lower and variant detection variability increased. Covering 523 tumor-associated genes, the assay demonstrated a convincing performance comparable to NGS-based ctDNA assays with smaller gene panels, highlighting its value to screen large numbers of different genes.

Serum Insights: Leveraging the Power of miRNA Profiling as an Early Diagnostic Tool for Non-Small Cell Lung Cancer.

Non-small cell lung cancer is the predominant form of lung cancer and is associated with a poor prognosis. MiRNAs implicated in cancer initiation and progression can be easily detected in liquid biopsy samples and have the potential to serve as non-invasive biomarkers. In this study, we employed next-generation sequencing to globally profile miRNAs in serum samples from 71 early-stage NSCLC patients and 47 non-cancerous pulmonary condition patients. Preliminary analysis of differentially expressed miRNAs revealed 28 upregulated miRNAs in NSCLC compared to the control group. Functional enrichment analyses unveiled their involvement in NSCLC signaling pathways. Subsequently, we developed a gradient-boosting decision tree classifier based on 2588 miRNAs, which demonstrated high accuracy (0.837), sensitivity (0.806), and specificity (0.859) in effectively distinguishing NSCLC from non-cancerous individuals. Shapley Additive exPlanations analysis improved the model metrics by identifying the top 15 miRNAs with the strongest discriminatory value, yielding an AUC of 0.96 ± 0.04, accuracy of 0.896, sensitivity of 0.884, and specificity of 0.903. Our study establishes the potential utility of a non-invasive serum miRNA signature as a supportive tool for early detection of NSCLC while also shedding light on dysregulated miRNAs in NSCLC biology. For enhanced credibility and understanding, further validation in an independent cohort of patients is warranted.

Exosomes: emerging biomarkers unveiling cellular mysteries—a narrative review

Exosomes have gained recognition as potential diagnostic and prognostic markers due to their stability and disease-specific cargos. Exosomes, once dismissed as cellular debris, are now acknowledged as crucial mediators of intercellular communications and disease. These nanosized vesicles transport dynamic cargos consisting of bioactive molecules, including proteins, lipids, and nucleic acids, enabling cells to exchange information and influence each other’s behavior. This review delves into the multifaceted roles of exosomes, particularly their emergence as biomarkers for various diseases. To conduct research for this review, a comprehensive literature search strategy was employed across various databases using keywords related to exosomes, biomarkers, cellular communication, disease diagnosis, and more. Selected articles were evaluated based on their relevance, and data were extracted, synthesized, and categorized according to thematic content. Quality assessment was performed on different types of articles, ensuring robust findings. In cancer, exosomes act as liquid biopsy samples, revealing genetic mutations, and protein markers. The misfolded proteins characteristic of neurodegenerative diseases such as Alzheimer disease and Parkinson disease are carried by exosomes. Moreover, exosomes have been studied as biomarkers for cardiovascular, infectious, and autoimmune diseases. In addition to their functions as biomarkers, exosomes influence the tumor microenvironment, immune response, synaptic plasticity, and tissue regeneration. Challenges remain to fill research gaps and standardize exosome analyses. Exosomes are pivotal in intercellular communication and for disease diagnosis, offering insights into cellular processes, immunity, and regeneration. Future research may reshape our understanding of cellular communication and drive therapeutic interventions. Although challenges must be addressed, exosomes hold undeniable potential for use in personalized medicine and biomedical research.

DNA-methylation signature accurately differentiates pancreatic cancer from chronic pancreatitis in tissue and plasma

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Differentiation from chronic pancreatitis (CP) is currently inaccurate in about one-third of cases. Misdiagnoses in both directions, however, have severe consequences for...

Development and Validation of a Novel Dual-Drop-off ddPCR Assay for the Simultaneous Detection of Ten Hotspots PIK3CA Mutations.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately 40% of patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer have activating mutations in the PIK3CA gene. We developed a highly sensitive, specific, cost-effective, and reproducible dual-drop-off droplet digital polymerase chain reaction (PCR) assay for the simultaneous detection of ten hotspots of PIK3CA mutations in plasma cell-free (cf) DNA. We first evaluated the analytical specificity, sensitivity, limit of blank, repeatability, and reproducibility of the assay, which simultaneously detects seven mutations in exon9 and three in exon20. We further applied this assay in 11 gDNA and 18 plasma cfDNA samples from healthy donors and 35 plasma cfDNA samples from metastatic breast cancer patients. The assay is highly sensitive, specific, and applicable for clinical samples containing at least 1-5% mutant DNA. We detected PIK3CA mutations in 9/35(26%) plasma cfDNA samples in exon 9 and in 9/35(26%) in exon 20. Direct comparison of the developed assay with amplification refractory mutation system-based PCR (using plasma samples) and with the Food and Drug Administration-approved cobas PIK3CA mutation assay (using formalin fixed paraffin embedded samples) showed high concordance of our developed assay with the cobas PIK3CA assay. The developed assay is cost-effective and can reliably and simultaneously detect ten hotspot PIK3CA mutations in plasma cfDNA. The clinical performance of the assay will be further evaluated in liquid biopsy samples.

Using cfDNA and ctDNA as Oncologic Markers: A Path to Clinical Validation.

The detection of circulating tumor DNA (ctDNA) in liquid biopsy samples as an oncological marker is being used in clinical trials at every step of clinical management. As ctDNA-based liquid biopsy kits are developed and used in clinics, companies work towards increased convenience, accuracy, and cost over solid biopsies and other oncological markers. The technology used to differentiate ctDNA and cell-free DNA (cfDNA) continues to improve with new tests and methodologies being able to detect down to mutant allele frequencies of 0.001% or 1/100,000 copies. Recognizing this development in technology, the FDA has recently given pre-market approval and breakthrough device designations to multiple companies. The purpose of this review is to look at the utility of measuring total cfDNA, techniques used to differentiate ctDNA from cfDNA, and the utility of different ctDNA-based liquid biopsy kits using relevant articles from PubMed, clinicaltrials.gov, FDA approvals, and company newsletters. Measuring total cfDNA could be a cost-effective, viable prognostic marker, but various factors do not favor it as a monitoring tool during chemotherapy. While there may be a place in the clinic for measuring total cfDNA in the future, the lack of standardization means that it is difficult to move forward with large-scale clinical validation studies currently. While the detection of ctDNA has promising standardized liquid biopsy kits from various companies with large clinical trials ongoing, their applications in screening and minimal residual disease can suffer from lower sensitivity. However, researchers are working towards solutions to these issues with innovations in technology, multi-omics, and sampling. With great promise, further research is needed before liquid biopsies can be recommended for everyday clinical management.

Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer

Methylation patterns in cell-free DNA (cfDNA) have emerged as a promising genomic feature for detecting the presence of cancer and determining its origin. The purpose of this study was to evaluate the diagnostic performance of methylation-sensitive restriction enzyme digestion followed by sequencing (MRE-Seq) using cfDNA, and to investigate the cancer signal origin (CSO) of the cancer using a deep neural network (DNN) analyses for liquid biopsy of colorectal and lung cancer. We developed a selective MRE-Seq method with DNN learning-based prediction model using demethylated-sequence-depth patterns from 63,266 CpG sites using SacII enzyme digestion. A total of 191 patients with stage I–IV cancers (95 lung cancers and 96 colorectal cancers) and 126 noncancer participants were enrolled in this study. Our study showed an area under the receiver operating characteristic curve (AUC) of 0.978 with a sensitivity of 78.1% for colorectal cancer, and an AUC of 0.956 with a sensitivity of 66.3% for lung cancer, both at a specificity of 99.2%. For colorectal cancer, sensitivities for stages I–IV ranged from 76.2 to 83.3% while for lung cancer, sensitivities for stages I–IV ranged from 44.4 to 78.9%, both again at a specificity of 99.2%. The CSO model's true-positive rates were 94.4% and 89.9% for colorectal and lung cancers, respectively. The MRE-Seq was found to be a useful method for detecting global hypomethylation patterns in liquid biopsy samples and accurately diagnosing colorectal and lung cancers, as well as determining CSO of the cancer using DNN analysis.Trial registration: This trial was registered at ClinicalTrials.gov (registration number: NCT 04253509) for lung cancer on 5 February 2020, https://clinicaltrials.gov/ct2/show/NCT04253509. Colorectal cancer samples were retrospectively registered at CRIS (Clinical Research Information Service, registration number: KCT0008037) on 23 December 2022, https://cris.nih.go.kr, https://who.init/ictrp. Healthy control samples were retrospectively registered.

Two-dimensional substrate assisted SERS immunosensor for accurate detection of carcinoembryonic antigen

Accurate and sensitive detection of carcinoembryonic antigen (CEA) is essential for the detection of various diseases in healthcare and the medical field. Currently, due to the high false negative rate of CEA assay in clinical setting and its use as a common indicator for early cancer screening, a novel CEA detection method with high sensitivity, increased specificity and the lower cost has become a clinical challenge. Here, a facile sandwich type immunosensor based on surface-enhanced Raman scattering (SERS) was presented including 4-mercaptobenzonitrile (4MBN) labeled gold core-silver shell nanoparticles (Au@4MBN@Ag NPs) as SERS tag and 4-mercaptophenylboronic acid (4-MPBA) functionalized two-dimensional (2D) silver nanoparticle film (Ag FM) as SERS capture substrate for CEA detection. A linearity of 10-9-10-14M was observed with high sensitivity and excellent selectivity for the detection of CEA. Additionally, the spiking experiment yielded 105.33–127.00% recovery with variation coefficients below 10% demonstrating high assay accuracy and precision. The immunosensor we proposed here is a promising approach to quantify CEA in liquid biopsy samples with high sensitivity, which could be further developed for early cancer screening.