Field Of Liquid Biopsy Research Articles

Abstract A039: A sensitive tool for the detection of RNA modifications in blood samples

Abstract The epitranscriptome is the set of chemical modifications applied naturally to RNA to regulate seemingly all aspects of its function, including splicing, translation initiation and fidelity, trafficking, RNA folding and stability, and transcript lifetime. Detecting RNA in blood reliably continues to be a challenge due to its labile nature, and detecting RNA modifications is an even greater challenge. For this reason, current liquid biopsy approaches concentrate on determining the mutational burden in cell-free DNA and do not harness the potential of cfRNA. In contrast to cfDNA, cfRNA is a biomarker for dynamic events that are known to occur at early stages of carcinogenesis, such as alterations to transcriptional profiles and other aspects of the regulatory state of cells. In many RNA species, the modification state is linked to the RNA lifecycle, thus providing important insights into cell state. For example, pre-miRNA is modified before miRNA maturation, tRNA cleavage is initiated by changes to the modification pattern and mRNA is decapped for storage in stress granules. AlidaBio is introducing the EpiPlex platform, an assay platform with an integrated bioinformatics solution that is designed for ultra-low RNA input and has been tested with blood samples. In addition to its sensitivity, the EpiPlex platform has the advantage of detecting multiple RNA modifications in the same reaction, enabling users to elucidate how RNA modifications act in concert to fine-tune RNA function. The approach uses targeted, multiplexed barcoding to reveal co-localization of modifications on the same molecule and to provide semi-quantitative data on mod abundance. This sensitive, multiplexed target detection has the potential to advance the field of RNA liquid biopsy with new biomarker discovery. Citation Format: Andrew Price, Zachary Miles, Byron Purse, Gudrun Stengel. A sensitive tool for the detection of RNA modifications in blood samples [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A039.

Liquid biopsy: the analysis of circulating tumour DNA

Circulating tumour DNA (ctDNA) fragments found in blood represent a critical source of information about the tumour. However, the analysis of ctDNA is challenging due to the overwhelming presence of DNA from non-malignant cells and requires the development of highly sensitive laboratory techniques. This review discusses the major categories of ctDNA biomarkers, their detection methodologies, and explores the potential of emerging technologies within the liquid biopsy field.

Hidden messages in fluids: A review of clinical and fundamental perspectives on post-lymph node dissection drains.

In recent years, there has been a growing interest in liquid biopsy due to its non-invasive diagnostic value. Postoperative drainage fluid (PDF) is the fluid exudate from the wound site following lymph node dissection. PDF is regarded as a medical waste with no specific clinical significance. Nevertheless, the liquid biopsy of PDF may enable the reuse of this fluid. PDF contains a variety of body fluids, including blood and lymph. PDF contains a variety of biological components, including cytokines, extracellular vesicles (EVs), proteins, nucleic acids, cells and bacteria. These components are indicative of the postoperative inflammatory response, the immune response and the therapeutic response. In this review, we examine the current state of research in the field of liquid biopsy in PDF, elucidating how the analysis of its components can assess the prognosis of patients after lymph node dissection, monitor real-time changes in patient status, and identify new biomarkers and potential therapeutic targets.

Predicting somatic mutation origins in cell-free DNA by semi-supervised GAN models

MotivationDistinguishing between pathogenic cancer-associated mutations and other somatic variants present in cell-free DNA (cfDNA) is one of the challenges in the field of liquid biopsy. This distinction is critical, since the misclassification of mutations stemming from clonal hematopoiesis (CH) as tumor-derived and vice versa could result in inaccurate diagnoses and inappropriate therapeutic interventions for patients. ResultsWe addressed this by developing a specialized machine learning technique to differentiate tumor- or CH-related mutations in cfDNA. We established a comprehensive in-house reference catalog, comprising approximately 25,000 single nucleotide variants (SNVs), each linked to either tumor or CH origin. This reference serves as a foundation for training a deep learning model, which is structured on the semi-supervised generative adversarial network (SSGAN) architecture. By analyzing genomic coordinates and nucleotide composition of cfDNA variants, our model attains 95 % area under the curve (AUC) in classifying uncharacterized variants as CH or tumor-derived. In conclusion, our research emphasizes the potential of genomic feature prediction, using cfDNA data, to stand as a robust alternative to conventional multi-analyte sequencing methods. This approach not only enhances the accuracy of distinguishing CH from tumor mutations in liquid biopsy data, but also highlights the potential of advanced data analysis techniques and machine learning in genomics and personalized medicine. Availability: https://github.com/FPalizban/SSGAN.

Molecular profiling using next generation sequencing with high-purity enrichment of circulating tumor cells.

e15054 Background: In the field of liquid biopsy, coupled with next generation sequencing (NGS), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can serve as an alternative substrate to tumor tissue for mutation detection and companion diagnostic purpose. Emerging evidence suggests that complementary sequential CTCs and ctDNA profiling offers promising benefits for prognosis and treatment. In contrast to well-established ctDNA extraction methods, the process of isolation of intact CTCs for NGS analysis has not yet been fully elucidated. Here we show that isolation and harvesting of viable CTCs using CytoGen's Smart Biopsy™ System achieves high CTC purity for NGS analysis in breast cancer and non-small cell lung cancer (NSCLC). Methods: Targeted panel sequencing for CTC was developed to detect representative mutated genes using the Oncomine™ Comprehensive Assay Plus (501 genes) and the Oncomine™ Pan-Cancer Cell-Free Assay (52 genes). Analytical validation was performed with breast cancer cells MDA-MB-231 (50 to 5 cells) and NSCLC cells H358 (10 and 5 cells) which were spiked into 5 mL of healthy blood to mimic CTCs. CytoGen's Smart Biopsy™ System was applied to isolate the CTCs. Subsequently, CD45 depletion and whole genome amplification were performed. Sequencing was performed on Ion S5™ XL Systems using an Ion 550™ chip kit. The sequencing data were then analyzed using the Ion Torrent software with the default configurations. Results: As a result of targeted panel sequencing with Oncomine™ Comprehensive Assay Plus, representative breast cancer mutation genes (BRAF, KRAS, TP53, NF2 and NOTCH3) were detected in at least 20 MDA-MB-231 cells. In the results using Oncomine™ Pan-Cancer Cell-Free Assay, mutated genes (BRAF, KRAS and TP53) were successfully detected in as few as 5 MDA-MB-231 cells and, in addition, KRAS mutation was detected even with only 5 H358 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to detect representative mutated genes in clinical breast cancer and NSCLC patient samples with detection technology that require high sensitivity and specificity. Conclusions: Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity for NGS analysis. In this study, we developed the process for NGS analysis on very small number of high-purity CTCs using CytoGen's Smart Biopsy™ System. Taken together with the results from our ongoing clinical trials, this process could provide information for the diagnosis and appropriate treatment of breast cancer and NSCLC patients.

Circulating Tumor Cell Phenotype Detection and Epithelial-Mesenchymal Transition Tracking Based on Dual Biomarker Co-Recognition in an Integrated PDMS Chip.

Circulating tumor cells (CTCs) are widely considered as a reliable and promising class of markers in the field of liquid biopsy. As CTCs undergo epithelial-mesenchymal transition (EMT), phenotype detection of heterogeneous CTCs based on EMT markers is of great significance. In this report, an integrated analytical strategy that can simultaneously capture and differentially detect epithelial- and mesenchymal-expressed CTCs in bloods of non-small cell lung cancer (NSCLS) patients is proposed. First, a commercial biomimetic polycarbonate (PCTE) microfiltration membrane is employed as the capture interface for heterogenous CTCs. Meanwhile, differential detection of the captured CTCs is realized by preparing two distinct CdTe quantum dots (QDs) with red and green emissions, attached with EpCAM and Vimentin aptamers, respectively. For combined analysis, a polydimethylsiloxane (PDMS) chip with simple structure is designed, which integrates the membrane capture and QDs-based phenotype detection of CTCs. This chip not only implements the analysis of the number of CTCs down to 2 cellsmL-1, but enables EMT process tracking according to the specific signals of the two QDs. Finally, this method is successfully applied to inspect the correlations of numbers or proportions of heterogenous CTCs in 94 NSCLS patients with disease stage and whether there is distant metastasis.

Evaluating the impact of delayed centrifugation on protein profiles analyzed by LC/MS in serum and plasma samples

The pre-analytical steps in blood-based liquid biopsy, involving sample collection techniques and storage conditions, play a critical role in ensuring the integrity and reliability of collected samples. These steps have a directly impact on the accuracy and reliability of test results and are therefore of utmost importance. Mass spectrometry (MS)-based proteomics is an exceptionally powerful tool in the field of liquid biopsy. It enables the comprehensive analysis of the protein content within biological specimens, providing valuable insights into the underlying mechanisms and pathogenesis of diseases. In this study, we aim to explore the variations in the protein landscape between serum and plasma specimens and evaluate the impact of delayed centrifugation on LC/MS-analyzed protein profiles. We seek to provide recommendations on optimal pre-analytical protocols for MS-based proteomics studies. This will enhance the accuracy and reliability of liquid biopsy for precision medicine, ultimately leading to improved patient outcomes.

Charge-Based Isolation of Extracellular Vesicles from Human Plasma.

Extracellular vesicles (EVs) have garnered significant attention due to their potential applications in disease diagnostics and management. However, the process of isolating EVs, primarily from blood samples, is still suboptimal. This is mainly attributed to the abundant nature of soluble proteins and lipoproteins, which are often separated together with EVs in the end products of conventional isolation methods. As such, we devise a single-step charge-based EV isolation method by utilizing positively charged beads to selectively remove negatively charged major impurities from human plasma via electrostatic interaction. By carefully controlling the buffer pH, we successfully collected EVs from undesired plasma components with superior purity and yield compared to conventional EV collection methods. Moreover, the developed process is rapid, taking only about 20 min for overall EV isolation. The charge-based isolation can ultimately benefit the EV-based liquid biopsy field for the early diagnosis of various diseases.

Abstract 3677: Hybrid capture based sequencing from urinary cell free DNA from colorectal cancer patients

Abstract Background: The study of body fluids other than blood is gaining increasing attention in the field of liquid biopsy. Since urine offers a truly non-invasive sampling method, it is a particularly interesting specimen. Evidence suggests that urine cell-free DNA (ucfDNA) harbors information about renal and bladder cancer. However, not much is known about ucfDNA in colorectal cancer (CRC) patients. Therefore, we aimed to test the feasibility of a hybrid capture based NGS approach with and without preanalytical stabilization of urine samples. Methods: Urine samples were collected from 20 patients with metastatic CRC (mCRC), aliquoted and stored with or without a stabilizing agents (PAXgene Urine Liquid Biopsy Set and Streck urine Preserve) at room temperature. Urine cfDNA was isolated at the day of donation and after 3 days. Matched blood samples were additionally collected in PAXgene Blood ccfDNA Tubes (PreAnalytiX). cfDNA from blood and urine was isolated using the QIAsymphony platform (QIAGEN). cfDNA samples were analyzed using the AVENIO ctDNA Analysis Kit (Roche), a hybrid-capture based approach enriching for 17 clinically relevant genes. Results: While all stabilized ucfDNA yielded high quality libraries, library preparation failed in 66.7% of unstabilized urine samples, demonstrating that without stabilization ucfDNA rapidly degrades after urine donation. In native samples, in which sequencing data could be obtained, sequencing depth was significantly decreased compared to stabilized samples. For stabilized samples, sequence analysis revealed full concordance between urine and plasma for putative germline variants. However, when using less than 50ng of input material of ucfDNA, a low signal-to-noise ratio with a high number of false positive low level variants was observed. We therefore adjusted the limit of detection for ucfDNA to 0.5%, at which CRC-related mutations were detected in ucfDNA in one patient. Although these variants, were not observed in plasma cfDNA, they could be detected in all stabilized urine samples, which hints to true variants. Conclusion: Our data demonstrate that a hybrid-capture based analysis approach is feasible for ucfDNA form CRC patients and that urine may provide complementary information about a patient's tumor that may be missed in plasma. Yet, due to degradation and lower concentrations, immediate stabilization of urine after donation is required. Moreover, the limit of detection needs to be adjusted if low amounts of input DNA is available only. Citation Format: Anna Eberhard, Tina Moser, Leandra Ziegler, Georgios Vlachos, Isaac Lazzeri, Martina Loibner, Armin Gerger, Ellen Heitzer. Hybrid capture based sequencing from urinary cell free DNA from colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3677.

Abstract 5039: Preanalytical workflow enabling cfDNA analysis from urine samples

Abstract Introduction: Urine has become an important source of information in the liquid biopsy field. In contrast to blood, specifications for the collection, storage, transport and processing of urine intended for molecular examination are not widely established. Preanalytical specifications were published for urine cell-free DNA (cfDNA) only recently (CEN/TS 17811:2022). In this study, we investigated post-collection changes to cfDNA profiles in urine samples and present the performance of an optimized preanalytical workflow for cfDNA analysis. Methods: Urine from apparently healthy, consented female individuals was spiked with processed cell-free male urine or with a cfDNA standard containing PIK3CA mutations (SensID). The urine was stabilized with a urine collection/stabilization solution under development at PreAnalytiX or was left unstabilized. Urine samples were stored for varying durations at different temperatures in a time course experiment to simulate different urine storage conditions. After storage, urine samples were centrifuged to remove cells and cfDNA was isolated from the supernatant via the QIAsymphony® platform (QIAGEN). Autosomal and male-specific targets were quantified using the Rotor-Gene® Q instrument and the therascreen® PIK3CA qPCR Kit (both QIAGEN) or the QIAcuity® Digital PCR System and a dPCR LNA PIK3CA Mutation Assay (both QIAGEN). Fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). Results: Analysis of cfDNA profiles in unstabilized urine stored for varying durations and temperatures highlighted changes which create artificial cfDNA profiles. The yield of cfDNA drastically decreased over time. Furthermore, the size distribution of cfDNA isolated from unstabilized urine was impacted by release of gDNA as well as DNA degradation. Urine stabilization minimized cfDNA degradation and gDNA release and allowed isolation of cfDNA that was analyzed with qPCR and dPCR even after urine storage. Analysis of isolated cfDNA revealed positive detection of PIK3CA mutations in spiked urine samples. Conclusions: Changes in post-collection cfDNA profile can hinder downstream analysis, resulting in artificial or failed outcomes. Urine stabilization with a collection/stabilization solution under development at PreAnalytiX minimized DNA degradation and gDNA release. It enabled urine storage, allowed analysis of urine cfDNA and target detection by qPCR and dPCR. Citation Format: Daniela Mancarella, Julia Rutsch, Lisa Erkelenz, Moritz Meyer, Laura Sofie Witthaus, Moritz Rath, Thorsten Voss. Preanalytical workflow enabling cfDNA analysis from urine samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5039.

Abstract 2299: More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA

Abstract Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. While early work in liquid biopsy focused on the identification of actionable somatic variations at specific loci, the past decade has seen an expansion into non-genetic features, notably methylation. 5-methylcytosine (5-mC) profiles of cancer are differential from non-cancer at many more loci and so provide a stronger signal. Moreover, recent research has suggested that 5-hydroxymethylcytosine (5-hmC) profiles in cfDNA can be a marker for early cancer. However, a standard blood draw yields an average of only 10ng of cfDNA, presenting the dilemma of how to use limited sample to obtain maximum information. Existing methods for measuring methylation using next-generation sequencing cannot distinguish between 5-mC and 5-hmC and are limited in their ability to detect mutations, with approaches that measure both 5-mC and 5-hmC requiring two separate workflows. Hence, to measure complete genetics, 5-mC and 5-hmC using existing technologies requires three separate workflows, each of which require separate DNA input. We will present a technology which sequences at single base resolution the complete genetic sequence of input DNA fragments integrated with the modification status (unmodified, 5-mC or 5-hmC) for each CpG from low nanogram input quantities of DNA. A hairpin construct is used to create a copy of the original DNA strand. Enzymatic conversion followed by next-generation sequencing enables coupled decoding of bases across the original and copy strand, uniquely reporting one of A, C, G, T, 5-mC, or 5-hmC for each position in the input DNA fragment. Using this technology, we generated whole genome 6-letter data (measuring A, C, G, T, 5-mC, and 5-hmC at single-base resolution across the genome) on cell-free DNA extracted from plasma of healthy volunteers and patients with colorectal cancer at various stages of progression. We demonstrate how the technology can be used to compare 5-mC and 5-hmC methylomic profiles, genomics, and fragmentomics, across different stages of colorectal cancer. In particular, we show how disease progression can be marked by changes in 5-mC and 5-hmC across several loci. We propose that the ability to measure 5-mC and 5-hmC at high accuracy and single base resolution, alongside genomic and fragmentomic profiles, from a limited quantity of DNA will enable greater insight into the ctDNA in plasma and help advance the field of liquid biopsy towards fulfilling its promise. Citation Format: Tom J. Charlesworth, Fabio Puddu, Robert Crawford, Annelie Johansson, Ermira Lleshi, Aurelie Modat, Jamie Scotcher, Michael Wilson, Nicholas Harding, Jean Teyssandier, Jens Fullgrabe, Walraj Gosal, Paidi Creed. More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2299.

Abstract 5042: Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications

Abstract Introduction: The non-invasive collection of urine renders it an attractive and pragmatic substitute for plasma in liquid biopsy applications, fostering the creation of diagnostic methods that are more patient-friendly and less cumbersome for clinicians. This study investigates the feasibility of urine-derived cfDNA as a non-invasive option for acquiring vital diagnostic insights, further enhancing the liquid biopsy field. Methods: Paired male first void urine (FVU) and venous blood samples were collected from healthy donors (n=40) using Colli-Pee UAS devices (Novosanis) and BD K2EDTA Vacutainer tubes, respectively. Out of these, 20 paired urine and blood samples were spiked with 10 ng of cfDNA reference standard containing the KRAS p.G12V mutation. ~35 ml of urinary cell-free supernatant and ~3.5 ml of plasma were obtained after centrifugation of FVU and blood sample, respectively, and used as input material for cell-free nucleic acids extraction using the nRichDX Revolution Max20 cfDNA Isolation Kit. The extracted cfDNAs profile was assessed on 4200 TapeStation System, Agilent, using cfDNA ScreenTape. Extracted nucleic acids from urine samples were subjected to a qPCR assay to quantify endogenous human cfDNA content using 2X iTaq Universal SYBR Mastermix (Bio-Rad). Human cfDNA quantity was assessed using Ct values obtained from the qPCR assay. The actionable cfDNA molecules recovery was determined by qPCR mutation detection assay using TaqMan Genotyping. Results: Male FVU collected in Colli-Pee UAS device showed the presence of 100-250 bp cell-free DNA fragments similar to plasma obtained from whole blood collected in 10 ml vacutainer tubes as demonstrated by the Agilent cfDNA ScreenTape analysis. The cfDNA yield was similar when comparing one tube of blood (~3.5 mL of plasma) to one Colli-Pee of urine (~35 mL) shown by endogenous cfDNA qPCR assay. Additionally, the amplifiability of the KRAS G12V mutation was consistent in both plasma and urine samples, with comparable Ct values. Conclusion: The nRichDX Revolution Sample Prep System demonstrates the ability to extract cfDNA from both plasma and urine samples, with comparable extraction efficiency observed between matched samples from the same donor. This study provides evidence supporting the viability of first void urine samples as a non-invasive alternative for prostate cancer-related liquid biopsy applications. It indicates that cfDNA can be recovered from a sample collected with a single BCT or Colli-Pee UAS urine collection device. Future investigations will be required to assess clinical samples and biomarkers to establish a robust correlation between cfDNA levels and various cancer types and stages. This study reinforces the utility of first void urine samples as a reliable proxy for blood-based liquid biopsy applications. Citation Format: Nafiseh Jafari, Jason Saenz, Lauren Lee, Carlos Hernandez, Andrew Dunnigan, Amit Arora, Rafal Iwasiow, Mayer Saidian. Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5042.

Label-free fluorescent sensor for sensitive detection of ctDNA based on water stabilized CsPbBr3 nanosheet

The detection of circulating tumor DNA (ctDNA), as a practical liquid biopsy technique, was of great significance for the study of cancer diagnosis and prognosis. However, reported methods for detection ctDNA still have some limitations, such as tedious process and high cost. In this study, CsPbBr3 nanosheet (CsPbBr3 NS) with high water stability was prepared by etching, and its fluorescence intensity could be stably stored for 1 year. The Ti3C2Tx possessed high quenching efficiency for CsPbBr3 NS and the HOMO-LUMO orbital study revealed that the PET mechanism was responsible for fluorescence quenching. And the Ti3C2Tx showed stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). The probe ssDNA could be adsorbed on the surface of Ti3C2Tx through π-π stacking. After the targets were recognized by probe ssDNA to form dsDNA, its affinity with Ti3C2Tx decreased and the active site of Ti3C2Tx recovered, causing a high quenching efficiency on CsPbBr3 NS. Based on this, a label-free fluorescent biosensor was designed for the sensitive detection of ctDNA (EGFR 19 Dels for non-small cell lung cancer, NSCLC). Under the optimal experimental conditions, this biosensor exhibited a detection limit of 180 fM and a linear range of 50 pM–350 pM with amplification of magnetic beads through strand displacement reaction. In addition, this sensor was applied to the detection of ctDNA in serum samples and cells lysates. This method for ctDNA detection was expected to have great potential for biomarker detection in the field of liquid biopsy.

Recent advances in droplet‐based microfluidics in liquid biopsy for cancer diagnosis

AbstractLiquid biopsy, a noninvasive technique to obtain tumor information from body fluids, is an emerging technology for cancer diagnosis, prognosis, and monitoring, providing crucial support for the realization of precision medicine. The main biomarkers of liquid biopsy include circulating tumor cells, circulating tumor DNA, microRNA, and circulating tumor exosomes. Traditional liquid biopsy detection methods include flow cytometry, immunoassay, polymerase chain reaction (PCR)‐based methods, and next‐generation sequencing (NGS)‐based methods, which are time‐consuming, labor‐intensive, and cannot reflect cell heterogeneity. Droplet‐based microfluidics with high throughput, low contamination, high sensitivity, and single‐cell/single‐molecule/single‐exosome analysis capabilities have shown great potential in the field of liquid biopsy. This review aims to summarize the recent development in droplet‐based microfluidics in liquid biopsy for cancer diagnosis.

Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System.

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.

CRISPR/Cas detection with nanodevices: moving deeper into liquid biopsy.

The emerging field of liquid biopsy has garnered significant interest in precision diagnostics, offering a non-invasive and repetitive method for analyzing bodily fluids to procure real-time diagnostic data. The precision and accuracy offered by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) technology have advanced and broadened the applications of liquid biopsy. Significantly, when combined with swiftly advancing nanotechnology, CRISPR/Cas-mediated nanodevices show vast potential in precise liquid biopsy applications. However, persistent challenges are still associated with off-target effects, and the current platforms also constrain the performance of the assays. In this review, we highlight the merits of CRISPR/Cas systems in liquid biopsy, tracing the development of CRISPR/Cas systems and their current applications in disease diagnosis particularly in liquid biopsies. We also outline ongoing efforts to design nanoscale devices with improved sensing and readout capabilities, aiming to enhance the performance of CRISPR/Cas detectors in liquid biopsy. Finally, we identify the critical obstacles hindering the widespread adoption of CRISPR/Cas liquid biopsy and explore potential solutions. This feature article presents a comprehensive overview of CRISPR/Cas-mediated liquid biopsies, emphasizing the progress in integrating nanodevices to improve specificity and sensitivity. It also sheds light on future research directions in employing nanodevices for CRISPR/Cas-based liquid biopsies in the realm of precision medicine.

Exosomal DNA: Role in Reflecting Tumor Genetic Heterogeneity, Diagnosis, and Disease Monitoring.

Extracellular vesicles (EVs), with exosomes at the forefront, are key in transferring cellular information and assorted biological materials, including nucleic acids. While exosomal RNA has been thoroughly examined, exploration into exosomal DNA (exoDNA)-which is stable and promising for cancer diagnostics-lags behind. This hybrid genetic material, combining contributions from both nuclear and mitochondrial DNA (mtDNA), is rooted in the cytoplasm. The enigmatic process concerning its cytoplasmic encapsulation continues to captivate researchers. Covering the entire genetic landscape, exoDNA encases significant oncogenic alterations in genes like TP53, ALK, and IDH1, which is vital for clinical assessment. This review delves into exosomal origins, the ins and outs of DNA encapsulation, and exoDNA's link to tumor biology, underscoring its superiority to circulating tumor DNA in the biomarker arena for both detection and therapy. Amidst scientific progress, there are complexities in the comprehension and practical application of the exoDNA surface. Reflecting on these nuances, we chart the prospective research terrain and potential pitfalls, forging a path for future inquiry. By illuminating both the known and unknown facets of exoDNA, the objective of this review is to provide guidance to the field of liquid biopsy (LB) while minimizing the occurrence of avoidable blind spots and detours.

Method for the extraction of circulating nucleic acids based on MOF reveals cell-free RNA signatures in liver cancer.

Cell-free RNA (cfRNA) allows assessment of health, status, and phenotype of a variety of human organs and is a potential biomarker to non-invasively diagnose numerous diseases. Nevertheless, there is a lack of highly efficient and bias-free cfRNA isolation technologies due to the low abundance and instability of cfRNA. Here, we developed a reproducible and high-efficiency isolation technology for different types of cell-free nucleic acids (containing cfRNA and viral RNA) in serum/plasma based on the inclusion of nucleic acids by metal-organic framework (MOF) materials, which greatly improved the isolation efficiency and was able to preserve RNA integrity compared with the most widely used research kit method. Importantly, the quality of cfRNA extracted by the MOF method is about 10-fold that of the kit method, and the MOF method isolates more than three times as many different RNA types as the kit method. The whole transcriptome mapping characteristics of cfRNA in serum from patients with liver cancer was described and a cfRNA signature with six cfRNAs was identified to diagnose liver cancer with high diagnostic efficiency (area under curve=0.905 in the independent validation cohort) using this MOF method. Thus, this new MOF isolation technique will advance the field of liquid biopsy, with the potential to diagnose liver cancer.

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.

Oxford Nanopore Technology and its Application in Liquid Biopsies.

Advanced medical technologies are transforming the future of healthcare, in particular, the screening and detection of molecular-genetic changes in patients suspected of having a neoplasm. They are based on the assumption that neoplasms release small amounts of various neoplasm-specific molecules, such as tumor DNA, called circulating DNA (cirDNA), into the extracellular space and subsequently into the blood. The detection of tumor-specific molecules and specific molecular changes in body fluids in a noninvasive or minimally invasive approach is known as "liquid biopsy." The aim of this review is to summarize the current knowledge of the application of ONT for analyzing circulating DNA in the field of liquid biopsies among cancer patients. Databases were searched using the keywords "nanopore" and "liquid biopsy" and by applying strict inclusion criteria. This technique can be used for the detection of neoplastic disease, including metastases, guiding precision therapy, and monitoring its effects. There are many challenges, however, for the successful implementation of this technology into the clinical practice. The first one is the low amount of tumor-specific molecules in the body fluids. Secondly, a tumor molecular signature should be discriminated from benign conditions like clonal hematopoiesis of unknown significance. Oxford Nanopore Technology (ONT) is a third-generation sequencing technology that seems particularly promising to complete these tasks. It offers rapid sequencing thanks to its ability to detect changes in the density of the electric current passing through nanopores. Even though ONT still needs validation technology, it is a promising approach for early diagnosis, therapy guidance, and monitoring of different neoplasms based on analyzing the cirDNA.