The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.
Using broad range cell-free DNA sequencing (BRcfDNA-Seq), a nontargeted next-generation sequencing (NGS) methodology, we previously identified a novel class of approximately 50 nt ultrashort single-stranded cell-free DNA (uscfDNA) in plasma that is distinctly different from 167 bp mononucleosomal cell-free DNA (mncfDNA). We hypothesize that uscfDNA possesses characteristics that are useful for disease detection. Using BRcfDNA-Seq, we examined both cfDNA populations in the plasma of 18 noncancer controls and 14 patients with late-stage nonsmall cell lung carcinoma (NSCLC). In comparison to mncfDNA, we assessed whether functional element (FE) peaks, fragmentomics, end-motifs, and G-Quadruplex (G-Quad) signatures could be useful features of uscfDNA for NSCLC determination. In noncancer participants, compared to mncfDNA, uscfDNA fragments showed a 45.2-fold increased tendency to form FE peaks (enriched in promoter, intronic, and exonic regions), demonstrated a distinct end-motif-frequency profile, and presented with a 4.9-fold increase in G-Quad signatures. Within NSCLC participants, only the uscfDNA population had discoverable FE peak candidates. Additionally, uscfDNA showcased different end-motif-frequency candidates distinct from mncfDNA. Although both cfDNA populations showed increased fragmentation in NSCLC, the G-Quad signatures were more discriminatory in uscfDNA. Compilation of cfDNA features using principal component analysis revealed that the first 5 principal components of both cfDNA subtypes had a cumulative explained variance of >80%. These observations indicate that the distinct biological processes of uscfDNA and that FE peaks, fragmentomics, end-motifs, and G-Quad signatures are uscfDNA features with promising biomarker potential. These findings further justify its exploration as a distinct class of biomarker to augment pre-existing liquid biopsy approaches.
Recent advances in circulating cell-free DNA (cfDNA) analysis from biofluids have opened new avenues for liquid biopsy (LB). However, current cfDNA LB assays are limited by the availability of existing information on established genotypes associated with tumor tissues. Certain cancers present with a limited list of established mutated cfDNA biomarkers, and thus, nonmutated cfDNA characteristics along with alternative biofluids are needed to broaden the available cfDNA targets for cancer detection. Saliva is an intriguing and accessible biofluid that has yet to be fully explored for its clinical utility for cancer detection. In this report, we employed a low-coverage single stranded (ss) library NGS pipeline “Broad-Range cell-free DNA-Seq” (BRcfDNA-Seq) using saliva to comprehensively investigate the characteristics of salivary cfDNA (ScfDNA). The identification of cfDNA features has been made possible by applying novel cfDNA processing techniques that permit the incorporation of ultrashort, ss, and jagged DNA fragments. As a proof of concept using 10 gastric cancer (GC) and 10 noncancer samples, we examined whether ScfDNA characteristics, including fragmentomics, end motif profiles, microbial contribution, and human chromosomal mapping, could differentiate between these two groups. Individual and integrative analysis of these ScfDNA features demonstrated significant differences between the two cohorts, suggesting that disease state may affect the ScfDNA population by altering nuclear cleavage or the profile of contributory organism cfDNA to total ScfDNA. We report that principal component analysis integration of several aspects of salivary cell-free DNA fragmentomic profiles, genomic element profiles, end-motif sequence patterns, and distinct oral microbiome populations can differentiate the two populations with a p value of < 0.0001 (PC1). These novel features of ScfDNA characteristics could be clinically useful for improving saliva-based LB detection and the eventual monitoring of local or systemic diseases.
Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies.
DOI:https://doi.org/10.1016/j.adaj.2023.05.006
Background
Each day, humans produce approximately 0.5 through 1.5 liters of saliva, a biofluid that is rich in biological omic constituents. Our lack of understanding how omic biomarkers migrate from diseased tissue to the saliva has impeded the clinical translation of saliva testing. Although such biomarkers must be conveyed via the vascular and lymphatic systems to the salivary glands, the molecular mechanisms that underlie this transport remain unclear. Although COVID-19 highlighted the need for rapid and reliable testing for infectious diseases, it represents only one of the many health conditions that potentially can be diagnosed using a saliva sample.
Types of Studies Reviewed
The authors discuss salivaomics, saliva exosomics, and the mechanisms on which saliva diagnostics are based and introduce a novel electrochemical sensing technology that may be exploited for saliva liquid biopsy.
Results
The utility of saliva for screening for lung cancer is under investigation. Saliva testing may be used to stratify patients, monitor treatment response, and detect disease recurrence. The authors also highlight the landscapes of saliva-based SARS-CoV-2 testing and ultrashort cell-free DNA and outline how these fields are likely to evolve in the near future.
Practical Implications
Breakthroughs in the study of saliva research, therefore, will facilitate clinical deployment of saliva-based testing.
doi: 10.3390/ijms241210387
Liquid biopsy is a rapidly emerging field that involves the minimal/non-invasive assessment of signature somatic mutations through the analysis of circulating tumor DNA (ctDNA) shed by tumor cells in bodily fluids. Broadly speaking, the unmet need in liquid biopsy lung cancer detection is the lack of a multiplex platform that can detect a mutation panel of lung cancer genes using a minimum amount of sample, especially for ultra-short ctDNA (usctDNA). Here, we developed a non-PCR and non-NGS-based single-droplet-based multiplexing microsensor technology, “Electric-Field-Induced Released and Measurement (EFIRM) Liquid Biopsy” (m-eLB), for lung cancer-associated usctDNA. The m-eLB provides a multiplexable assessment of usctDNA within a single droplet of biofluid in only one well of micro-electrodes, as each electrode is coated with different probes for the ctDNA. This m-eLB prototype demonstrates accuracy for three tyrosine-kinase-inhibitor-related EGFR target sequences in synthetic nucleotides. The accuracy of the multiplexing assay has an area under the curve (AUC) of 0.98 for L858R, 0.94 for Ex19 deletion, and 0.93 for T790M. In combination, the 3 EGFR assay has an AUC of 0.97 for the multiplexing assay.
https://doi.org/10.1016/j.isci.2022.104554
Plasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mono-nuclesomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mono-nucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggest that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.
J Mol Diagn 2020, 22: 50e59
Previous efforts to evaluate the detection of human papilloma viral (HPV) DNA in whole saliva as a diagnostic measure for HPV-associated oropharyngeal cancer (HPV-OPC) have not shown sufficient clinical performance. We hypothesize that salivary exosomes are packaged with HPV-associated biomarkers, and efficient enrichment of salivary exosomes through isolation can enhance diagnostic and prognostic performance for HPV-OPC. In this study, an acoustofluidic (the fusion of acoustics and microfluidics) platform was developed to perform size-based isolation of salivary exosomes. These data showed that this platform is capable of consistently isolating exosomes from saliva samples, regardless of viscosity vari- ation and collection method. Compared with the current gold standard, differential centrifugation, droplet digital RT-PCR analysis showed that the average yield of salivary exosomal small RNA from the acoustofluidic platform is 15 times higher. With this high-yield exosome isolation platform, we show that HPV16 DNA could be detected in isolated exosomes from the saliva of HPV-associated OPC patients at 80% concordance with tissues/biopsies positive for HPV16. Overall, these data demonstrated that the acoustofluidic platform can achieve high-purity and high-yield salivary exosome isolation for downstream salivary exosomeebased liquid biopsy applications. Additionally, HPV16 DNA sequences in HPV-OPC patients are packaged in salivary exosomes and their isolation will enhance the detection of HPV16 DNA.
Cell. 2019 Apr 4;177(2):463-477.e15. doi: 10.1016/j.cell.2019.02.018.
To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.