The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring.

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.

Unexpected Slow Relaxation Dynamics in Pure Ring Polymers Arise from Intermolecular Interactions.

Ring polymers have fascinated scientists for decades, but experimental progress has been challenging due to the presence of linear chain contaminants that fundamentally alter dynamics. In this work, we report the unexpected slow stress relaxation behavior of concentrated ring polymers that arises due to ring-ring interactions and ring packing structure. Topologically pure, high molecular weight ring polymers are prepared without linear chain contaminants using cyclic poly(phthalaldehyde) (cPPA), a metastable polymer chemistry that rapidly depolymerizes from free ends at ambient temperatures. Linear viscoelastic measurements of highly concentrated cPPA show slow, non-power-law stress relaxation dynamics despite the lack of linear chain contaminants. Experiments are complemented by molecular dynamics (MD) simulations of unprecedentedly high molecular weight rings, which clearly show non-power-law stress relaxation in good agreement with experiments. MD simulations reveal substantial ring-ring interpenetrations upon increasing ring molecular weight or local backbone stiffness, despite the global collapsed nature of single ring conformation. A recently proposed microscopic theory for unconcatenated rings provides a qualitative physical mechanism associated with the emergence of strong inter-ring caging which slows down center-of-mass diffusion and long wavelength intramolecular relaxation modes originating from ring-ring interpenetrations, governed by the onset variable N/ND, where the crossover degree of polymerization ND is qualitatively predicted by theory. Our work overcomes challenges in achieving ring polymer purity and by characterizing dynamics for high molecular weight ring polymers. Overall, these results provide a new understanding of ring polymer physics.

Variability in Severe Acute Respiratory Syndrome Coronavirus 2 IgG Antibody Affinity to Omicron and Delta Variants in Convalescent and Community mRNA-Vaccinated Individuals.

The emergence of the omicron and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters, and whether existing mRNA vaccines for the original SARS-CoV-2 strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1-specific salivary Abs to the omicron and delta variants using a cohort of mRNA-vaccinated (n = 109) and convalescent (n = 19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5, 26.7, and 14.7% in measurable signal to the omicron strain and 13.4, 2.4, and -6.4% mean reduction to the delta variant for convalescent, Pfizer-, and Moderna-vaccinated groups, respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves.

Self-assembled microtubular electrodes for on-chip low-voltage electrophoretic manipulation of charged particles and macromolecules.

On-chip manipulation of charged particles using electrophoresis or electroosmosis is widely used for many applications, including optofluidic sensing, bioanalysis and macromolecular data storage. We hereby demonstrate a technique for the capture, localization, and release of charged particles and DNA molecules in an aqueous solution using tubular structures enabled by a strain-induced self-rolled-up nanomembrane (S-RuM) platform. Cuffed-in 3D electrodes that are embedded in cylindrical S-RuM structures and biased by a constant DC voltage are used to provide a uniform electrical field inside the microtubular devices. Efficient charged-particle manipulation is achieved at a bias voltage of <2-4 V, which is ~3 orders of magnitude lower than the required potential in traditional DC electrophoretic devices. Furthermore, Poisson-Boltzmann multiphysics simulation validates the feasibility and advantage of our microtubular charge manipulation devices over planar and other 3D variations of microfluidic devices. This work lays the foundation for on-chip DNA manipulation for data storage applications.

Variability in SARS-Cov-2 IgG Antibody Affinity To Omicron and Delta Variants in Convalescent and Community mRNA Vaccinated Individuals.

The emergence of Omicron and Delta variants of SARS-CoV-2 has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters and whether existing mRNA vaccines for the wildtype strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1 specific salivary antibodies to the Omicron and Delta variants using a cohort of mRNA vaccinated (n=109) and convalescent (n=19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5%, 26.7%, and 14.7% in measurable signal to the Omicron strain and 13.4%, 2.4%, and âˆ'6.4% percent mean reduction to the Delta Variant for convalescent, Pfizer, and Moderna vaccinated groups respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves. Key Points: AMPERIAL assay developed to quantify salivary SARS-CoV-2 S1 IgG antibodies to Omicron and Delta variantsThere was a reduction in affinity to both Delta and Omicron VariantsThe reduction in affinity was more pronounced to Omicron than for Delta VariantsThere was a significant difference between IgG affinities in Individuals vaccinated with Pfizer versus Moderna Vaccines.

The Kinetics of COVID-19 Vaccine Response in a Community-Vaccinated Population.

We used a noninvasive electrochemical quantitative assay for IgG Abs to SARS-CoV-2 S1 Ag in saliva to investigate the kinetics of Ab response in a community-based population that had received either the Pfizer or Moderna mRNA-based vaccine. Samples were received from a total of 97 individuals, including a subset of 42 individuals who collected samples twice weekly for 3 mo or longer. In all, >840 samples were collected and analyzed. In all individuals, salivary SARS-CoV-2 S1 IgG Ab levels rose sharply in the 2-wk period after their second vaccination, with peak Ab levels seen at 10-20 d after vaccination. We observed that 20%, 10%, and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop, respectively, from peak levels during the duration of monitoring, and in two patients, Abs fell to prevaccination levels (5%). The use of noninvasive quantitative salivary Ab measurement can allow widespread, cost-effective monitoring of vaccine response.

The Kinetics of COVID-19 Vaccine Response in a Community Vaccinated Population.

We used a noninvasive electrochemical quantitative assay for IgG antibodies to SARS-CoV-2 S1 in saliva to investigate the kinetics of antibody response in a community-based population who had received either the Pfizer or Moderna mRNA-based vaccines. Samples were received from a total of 97 individuals including a subset of 42 individuals who collected samples twice-weekly for 3 months or longer. In all, 840 samples were collected and analyzed. In all individuals, salivary antibody levels rose sharply in the 2-week period following their second vaccination, with peak antibody levels being at 10-20 days post-vaccination. We observed that 20%, 10% and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop respectively from peak levels during the duration of monitoring and two patients fell to pre-vaccination levels (5%). The use of non-invasive quantitative salivary antibody measurement can allow widespread, cost-effective monitoring of vaccine response. ARTICLE SUMMARY LINE: COVID-19 antibodies were measured in saliva and 20% of vaccinated subjects experienced a 90% drop in peak antibody levels over the course of monitoring.

Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva.

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

Longitudinal Circulating Tumor DNA Analysis in Blood and Saliva for Prediction of Response to Osimertinib and Disease Progression in EGFR-Mutant Lung Adenocarcinoma.

Background: We assessed whether serial ctDNA monitoring of plasma and saliva predicts response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma. Three ctDNA technologies-blood-based droplet-digital PCR (ddPCR), next-generation sequencing (NGS), and saliva-based EFIRM liquid biopsy (eLB)-were employed to investigate their complementary roles. Methods: Plasma and saliva samples were collected from patients enrolled in a prospective clinical trial of osimertinib and local ablative therapy upon progression (NCT02759835). Plasma was analyzed by ddPCR and NGS. Saliva was analyzed by eLB. Results: A total of 25 patients were included. We analyzed 534 samples by ddPCR (n = 25), 256 samples by NGS (n = 24) and 371 samples by eLB (n = 22). Among 20 patients who progressed, ctDNA progression predated RECIST 1.1 progression by a median of 118 days (range: 61-272 days) in 11 (55%) patients. Of nine patients without ctDNA progression by ddPCR, two patients had an increase in mutant EGFR by eLB and two patients were found to have ctDNA progression by NGS. Levels of ctDNA measured by ddPCR and NGS at early time points, but not volumetric tumor burden, were associated with PFS. EGFR/ERBB2/MET/KRAS amplifications, EGFR C797S, PIK3CA E545K, PTEN V9del, and CTNNB1 S45P were key resistance mechanisms identified by NGS. Conclusion: Serial assessment of ctDNA in plasma and saliva predicts response and resistance to osimertinib, with each assay having supplementary roles.

Development and validation of a highly sensitive and specific electrochemical assay to quantify anti-SARS-CoV-2 IgG antibodies to facilitate pandemic surveillance and monitoring of vaccine response.

Amperial™ is a novel assay platform that uses immobilized antigen in a conductive polymer gel followed by an electrochemical detection. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARSCoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

Ultra-Short Circulating Tumor DNA (usctDNA) in Plasma and Saliva of Non-Small Cell Lung Cancer (NSCLC) Patients.

Mutations identified in the epidermal growth factor receptor (EGFR) predict sensitivity to EGFR-targeted therapy for non-small cell lung carcinoma (NSCLC). We previously reported that Electric Field-Induced Release and Measurement (EFIRM)-based liquid biopsy could detect EGFR ctDNA with >94% concordance with tissue-based genotyping. A side-by-side comparison of concordance of EFIRM and droplet digital PCR (ddPCR) for the detection of the two front-line actionable EFGR mutations was performed with paired plasma and saliva samples from 13 NSCLC patients. Deep sequencing analysis based on single-strand DNA library preparation was employed to determine the size distributions of EGFR L858R ctDNA in plasma and saliva samples. EFIRM detected both EGFR mutations with 100% sensitivity in both plasma and saliva samples, whereas ddPCR detected EGFR mutations with sensitivities of 84.6% and 15.4%, respectively. In saliva samples, the majority of EGFR L858R ctDNA fragments detected were <80 bp. Deep sequencing analysis of ctDNA enriched for the EGFR L858R mutation revealed the significant presence of EGFR L858R ctDNA as ultra-short circulating tumor DNA (usctDNA) with the size of 40-60 bp in patient plasma and saliva. Most of usctDNAs are not amplifiable with the current ddPCR assay. Further examination using cell lines and patient biofluids revealed that the majority of usctDNAs were predominately localized in the exosomal fraction. Our study revealed the abundant existence of EGFR ctDNA in the plasma and saliva of NSCLC patients is usctDNA. usctDNA is a novel type of targets for liquid biopsy that can be efficiently detected by EFIRM technology.

Electric Field-Induced Release and Measurement (EFIRM): Characterization and Technical Validation of a Novel Liquid Biopsy Platform in Plasma and Saliva.

Electric field-induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non-small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma.

Rapid PCR-free meat species mitochondrial DNA identification using Electric Field Induced Release and Measurement (EFIRM®).

This work details the usage of EFIRM® (Electric Field Induced Release and Measurement) for PCR-free rapid electrochemical detection of mitochondrial DNA. EFIRM® was able to perform highly sensitive detection of animal species for meat contamination testing without multistep sample lysis, DNA extraction, or PCR amplification steps, demonstrating the capability to detect the presence of foreign meat species that only constituted 0.1% of the total mass of a food sample (achieving sensitivity equivalent to that of PCR). The EFIRM® strategy utilizes surface immobilized nucleic acid probes that complement to mitochondrial sequence of Ovis Aries, Sus Scrofa, and Bos Taurus and are immobilized in a polypyrrole matrix on a 96-electrode array. Quantification was performed through amperometric measurement of oxidation-reduction reactions on a streptavidin-peroxidase enzyme chain that completes the nucleic acid complex. All electrochemical procedures were performed using a high-throughput potentiostat system that allows parallelized electrochemical measurement and interfacing to the 96-electrode array.

Acoustofluidic Salivary Exosome Isolation: A Liquid Biopsy Compatible Approach for Human Papillomavirus-Associated Oropharyngeal Cancer Detection.

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 variation 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 exosome-based 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.

Clinical validity of saliva and novel technology for cancer detection.

Cancer, a local disease at an early stage, systemically evolves as it progresses by triggering alterations in surrounding microenvironment, disturbing immune surveillance and further disseminating its molecular contents into circulation. This pathogenic characteristic of cancer makes the use of biofluids such as blood/serum/plasma, urine, tear and cerebrospinal fluids credible surrogates harboring tumor tissue-derived molecular alterations for the detection of cancer. Most importantly, a number of recent reports have credentialed the clinical validity of saliva for the detection of systemic diseases including cancers. In this review, we discussed the validity of saliva as credible biofluid and clinical sample type for the detection of cancers. We have presented the molecular constituents of saliva that could mirror the systemic status of our body and recent findings of salivaomics associated with cancers. Recently, liquid biopsy to detect cancer-derived circulating tumor DNA has emerged as a credible cancer-detection tool with potential benefits in screening, diagnosis and also risk management of cancers. We have further presented the clinical validity of saliva for liquid biopsy of cancers and a new technology platform based on electrochemical detection of cancer-derived ctDNA in saliva with superior sensitivity and point-of-care potential. The clinical utilities of saliva for the detection of cancers have been evidenced, but biological underpinning on the existence of molecular signatures of cancer-origin in saliva, such as via exosomal distribution, should be addressed in detail.

Identification of salivary metabolites for oral squamous cell carcinoma and oral epithelial dysplasia screening from persistent suspicious oral mucosal lesions.

OBJECTIVE: To identify salivary metabolite biomarkers to differentiate patients with oral squamous cell carcinoma and oral epithelial dysplasia (OSCC/OED) from those with persistent suspicious oral mucosal lesions (PSOML). SUBJECTS AND METHODS: Whole unstimulated saliva samples were collected from age-, sex-, and race-matched patients who had a lesion in the oral cavity and for whom open biopsies were performed. The patients included OSCC (n = 6), OED (n = 10), and PSOML (n = 32). Hydrophilic metabolites in saliva samples were comprehensively analyzed using capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate OSCC/OED from PSOML. RESULTS: Six metabolites were significantly different in OSCC/OED compared with PSOML. From these six metabolites, ornithine, o-hydroxybenzoate, and ribose 5-phosphate (R5P) were used to develop the MLR model, which resulted in a high value for the area under receiver operating characteristic curve (AUC 0.871, 95% confidential interval (CI) 0.760-0.982; p < 0.001) to discriminate OSCC/OED from PSOML. CONCLUSIONS: This is the first study to identify salivary metabolites that discriminate OSCC/OED from PSOML rather than from healthy controls. The profiles of salivary metabolites were significantly different between OSCC/OED and PSOML. The ability to discriminate OSCC/OED from PSOML is important for dentists who are not oral surgery specialists. These salivary metabolites showed potential for non-invasive screening to discriminate OSCC/OED from PSOML. CLINICAL RELEVANCE: Salivary metabolites in this study showed potential for non-invasive screening to discriminate OSCC/OED from PSOML.

Electric Field-Induced Release and Measurement Liquid Biopsy for Noninvasive Early Lung Cancer Assessment.

Previously, we detected circulating tumor DNA that contained two EGFR mutations (p.L858R and exon19 del) in plasma of patients with late-stage non-small-cell lung carcinoma (NSCLC) using the electric field-induced release and measurement (EFIRM) platform. Our aim was to determine whether EFIRM technology can detect these mutations in patients with early-stage NSCLC. Prospectively, 248 patients with radiographically determined pulmonary nodules were recruited. Plasma was collected before biopsy and histologic examination of the nodule. Inclusion criteria were histologic diagnosis of benign nodule (control) and stage I or II adenocarcinoma harboring either p.L858R or exon19 delEGFR mutations. Plasma samples were available from 44 patients: 23 with biopsy-proven benign pulmonary nodules and 21 with stage I or II adenocarcinoma (12 p.L858R and 9 exon19 delEGFR variants). Samples were analyzed for the EGFR mutations using the EFIRM platform. Assay sensitivity was 92% for p.L858R (11 of 12 samples positive) and 77% for exon19 del (7 of 9 samples positive). Specificity was 91% with two false-positive results in 23 patients with EGFR-positive nodules and 95% for the entire 44-patient series. Concordance was 100% with identical mutations discovered in plasma and nodule biopsy. The EFIRM platform is able to noninvasively detect two EGFR mutations in individuals with early-stage NSCLC.

Reduction of emergency department use in people with disabilities.

OBJECTIVES: To examine emergency department (ED) use by individuals with disabilities in safety-net clinics that have adopted the patient-centered medical home (PCMH) model. STUDY DESIGN: This is a retrospective matched cohort study. Prior to matching, we identified 2269 nonelderly Medicaid beneficiaries with disabilities from a Los Angeles Medicaid managed care plan in PCMH clinics and 21,897 in non-PCMH clinics. METHODS: To minimize self-selection bias from clinics and individuals, we created 3 comparison groups through a series of propensity score matching schemes that included matching clinics with similar health service utilization per patient and matching individuals with similar demographic characteristics and underlying health conditions. Rates of having at least 1 ED visit per year and excess ED use (defined as ≥2 ED visits per year) were compared across beneficiaries who received care from PCMH clinics and matched comparisons using logistic regression analyses. RESULTS: After matching on clinic- and individual-level characteristics, the adjusted odds ratio (OR) of excess ED use was 25% to 33% lower (P <.05) in the PCMH group compared with the non-PCMH group. When limiting the study population to patients with at least 1 office visit, the OR of having at least 1 ED visit decreased by 21% (P <.05) for the PCMH group. Similarly, the OR of having excess ED use decreased by 38% (P <.05) for the PCMH group. CONCLUSIONS: Our study highlights that the adoption of the PCMH model in safety-net clinics was associated with reduced ED use in Medicaid beneficiaries with disabilities.

Electric Field-Induced Disruption and Releasing Viable Content from Extracellular Vesicles.

In order to more fully elucidate the biogenesis of exosomes, a type of extracellular vesicles (EVs) and understand the role of EVs in disease processes, it is necessary to develop methods to capture EVs and induce the unloading of viable cargo. Traditionally, ultracentrifugation followed by chemical based EV lysis techniques is used to isolate these extracellular vesicles and release their internal content. Here, we describe a novel technique for capturing and releasing exosomal content through magnetic bead-based EV extraction coupled with electric field induced release and measurement (EFIRM). The usage of low-voltage electric fields allows the EV to be lysed without chemical treatment, and surface immobilized probes can allow for the rapid capture of the content of lysed EVs. EFIRM as an integrated EV lysing and analysis system offers great potential for the investigation of EVs in clinical and basic science contexts.