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Importance: In a randomized clinical trial, treatment guided by tumor-informed circulating tumor (ct)DNA testing reduced adjuvant chemotherapy use without compromising recurrence-free survival in patients with stage II colon cancer. The potential effects of adopting ctDNA testing into routine patient care is unknown. Objective: To compare the total cost of patient care scenarios with and without the adoption of ctDNA testing. Design, Setting, and Participants: This budget impact analysis was conducted from the perspectives of US commercial health and Medicare Advantage payers. A decision-analytical model was populated with age-specific incidence of colon cancer, use of adjuvant chemotherapy, and use of single-agent or multiagent regimens. Total cost was estimated with the costs of ctDNA testing, drug acquisition, administration, surveillance, and adverse events. The analysis was conducted from September 2023 to January 2024. Exposures: The adoption of ctDNA testing. Main Outcomes and Measures: The incremental cost in the first year following the adoption of ctDNA testing, where testing will affect patient treatment and costs. Results: In hypothetical plans with 1 million individuals covered, 35 commercial health plan members and 102 Medicare Advantage members aged 75 years and younger were eligible for ctDNA testing. In the base case with a 50% adoption rate, total cost savings were $221â¯684 (equivalent to $0.02 per member per month [PMPM]) for a commercial payer and $116â¯720 (equivalent to $0.01 PMPM) for a Medicare Advantage payer. Cost savings were robust to variations in assumptions of all parameters in the commercial population but sensitive to variations in assumptions of adjuvant chemotherapy use rates in the Medicare Advantage population. The number needed to test to avoid 1 patient receiving adjuvant chemotherapy was 4 in the commercial population and 10 in the Medicare Advantage population. The budget-neutral cost for ctDNA testing was $16â¯202 for a commercial payer and $5793 for a Medicare Advantage payer. Conclusions and Relevance: Use of tumor-informed ctDNA testing to guide adjuvant chemotherapy in postsurgery patients with stage II colon cancer was projected to result in cost savings for both commercial and Medicare Advantage payers. Adoption of ctDNA testing is therefore advantageous from a budgetary perspective.
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DNA Tumoral Circulante , Neoplasias do Colo , Medicare Part C , Humanos , Neoplasias do Colo/economia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/sangue , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Estados Unidos , Medicare Part C/economia , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Idoso , Feminino , Masculino , Orçamentos , Pessoa de Meia-Idade , Análise Custo-BenefícioRESUMO
OBJECTIVES: The aim of this pilot study was to evaluate the presence of somatic mutations in matched tumor and circulating DNA (ctDNA) samples from patients with primary head and neck squamous cell carcinoma (HNSCC) and assess the association of changes in ctDNA levels with survival. MATERIALS AND METHODS: Our study included 62 patients with stage I-IVB HNSCC treated with surgery or radical chemoradiotherapy with curative intent. Plasma samples were obtained at baseline, at the end of treatment (EOT), and at disease progression. Tumor DNA was extracted from plasma (ctDNA) and tumor tissue (tDNA). The Safe Sequencing System was used assess the presence of pathogenic variants in four genes (TP53, CDKN2A, HRAS and PI3KCA) in both ctDNA and tDNA. RESULTS: Forty-five patients had available tissue and plasma samples. Concordance of genotyping results between tDNA and ctDNA at baseline was 53.3%. TP53 mutations were most commonly identified at baseline in both ctDNA (32.6%) and tDNA (40%). The presence of mutations in this restricted set of 4 genes in tissue samples at baseline was associated with decreased overall survival (OS) [median 58.3 months for patients with mutations vs. 89 months for patients without mutations, p < 0.013]. Similarly, patients presenting with mutations in ctDNA had shorter OS [median 53.8 vs. 78.6 months, p < 0.037]. CtDNA clearance at EOT did not show any association with PFS or OS. CONCLUSIONS: Liquid biopsy enables real-time molecular characterization of HNSCC and might predict survival. Larger studies are needed to validate the utility of ctDNA as a biomarker in HNSCC.
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Carcinoma Pulmonar de Células não Pequenas , DNA Tumoral Circulante , Neoplasias de Cabeça e Pescoço , Neoplasias Pulmonares , Humanos , DNA Tumoral Circulante/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Neoplasias Pulmonares/genética , Projetos Piloto , Mutação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/terapia , Biomarcadores Tumorais/genéticaRESUMO
Background: Although the standard of care is to perform surgery of primary breast cancer (BC) after neoadjuvant chemotherapy (NAC), for certain patients achieving clinical complete response (cCR) and pathologic complete response (pCR), omission of surgical treatment may be an option. Levels of circulating tumor DNA (ctDNA) during and after therapy could identify patients achieving minimal residual disease. In this study, we evaluated whether ctDNA clearance during NAC could be a correlate to effective response in human epidermal growth factor receptor 2 positive (HER2+) and triple-negative (TN) BC patients. Methods: A prospective study was conducted to identify patient-specific PIK3CA and TP53 mutations in tissue using next-generation sequencing, which could then be used to track the presence/absence of mutations prior to, during, and following NAC using Sysmex SafeSEQ technology. All patients underwent a surgical excision after NAC, and pCR was assessed. Results: A total of 29 TN and HER2+ BC patients were examined and 20 that carried mutations in the PIK3CA and/or TP53 genes were recruited. Overall, 19 of these 20 patients harbored at least one tumor-specific mutation in their plasma at baseline. After NAC, 15 patients (75.0%) achieved pCR according to the histopathologic evaluation of the surgical specimen, and 15 patients (75.0%) had a cCR; 18 of 20 patients (90.0%) had concordant pCR and cCR. The status of 'no mutation detected' (NMD) following NAC in cCR patients correctly identified the pCR in 14 of 15 patients (93.33%), as well as correctly ruled out pCR in three patients, with an accuracy of 89.47%. During the 12-month follow-up after surgery, 40 plasma samples collected from 15 patients all showed no detectable ctDNA (NMD), and no patient recurred. Conclusion: These findings prompt further research of the value of ctDNA for non-invasive prediction of clinical/pathological response, raising the possibility of sparing surgery following NAC in selected BC patients.
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Molecular testing using blood-based liquid biopsy approaches has not been widely investigated in patients with glioma. A prospective single-center study enrolled patients with gliomas ranging from grade II to IV. Peripheral blood (PB) was drawn at different timepoints for circulating tumour DNA (ctDNA) monitoring. Next-generation sequencing (NGS) was used for the study of isocitrate dehydrogenase 1 (IDH1) mutations in the primary tumor. Beads, Emulsion, Amplification and Magnetics (BEAMing) was used for the study of IDH1 mutations in plasma and correlated with the NGS results in the tumor. Between February 2017 and July 2018, ten patients were enrolled, six with IDH1-mutant and four with IDH1 wild-type gliomas. Among the six IDH-mutant gliomas, three had the same IDH1 mutation detected in plasma (50%), and the IDH1-positive ctDNA result was obtained in patients either at diagnosis (no treatment) or during progressive disease. While the false-negative rate reached 86% (18/21), 15 out of the 18 (83%) plasma-negative results were from PB collected from the six IDH-mutant patients at times at which there was no accompanying evidence of tumor progression, as assessed by MRI. There were no false-positive cases in plasma collected from patients with IDH1 wild-type tumors. BEAMing detected IDH1 mutations in the plasma of patients with gliomas, with a modest clinical sensitivity (true positivity rate) but with 100% clinical specificity, with complete agreement between the mutant loci detected in tumor and plasma. Larger prospective studies should be conducted to expand on these findings, and further explore the clearance of mutations in PB from IDH1-positive patients in response to therapy.
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BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) has a favorable prognosis which has led to efforts to de-intensify treatment. Response-adaptive de-escalated treatment is promising, however improved biomarkers are needed. Quantitative cell-free HPV-DNA (cfHPV-DNA) in plasma represents an attractive non-invasive biomarker for grading treatment response and post-treatment surveillance. This prospective study evaluates dynamic changes in cfHPV-DNA during induction therapy, definitive (chemo)radiotherapy, and post-treatment surveillance in the context of risk and response-adaptive treatment for HPV + OPC. METHODS: Patients with locoregional HPV + OPC are stratified into two cohorts: High risk (HR) (T4, N3, [Formula: see text] 20 pack-year smoking history (PYH), or non-HPV16 subtype); Low risk (LR) (all other patients). All patients receive induction chemotherapy with three cycles of carboplatin and paclitaxel. LR with ≥ 50% response receive treatment on the single-modality arm (minimally-invasive surgery or radiation alone to 50 Gy). HR with ≥ 50% response or LR with ≥ 30% and < 50% response receive treatment on the intermediate de-escalation arm (chemoradiation to 50 Gy with cisplatin). All other patients receive treatment on the regular dose arm with chemoradiation to 70 Gy with concurrent cisplatin. Plasma cfHPV-DNA is assessed during induction, (chemo)radiation, and post-treatment surveillance. The primary endpoint is correlation of quantitative cfHPV-DNA with radiographic response. DISCUSSION: A de-escalation treatment paradigm that reduces toxicity without compromising survival outcomes is urgently needed for HPV + OPC. Response to induction chemotherapy is predictive and prognostic and can select candidates for de-escalated definitive therapy. Assessment of quantitative cfHPV-DNA in the context of response-adaptive treatment of represents a promising reliable and convenient biomarker-driven strategy to guide personalized treatment in HPV + OPC. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov on October 1st, 2020 with Identifier: NCT04572100 .
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Ácidos Nucleicos Livres/sangue , DNA Viral/sangue , Monitoramento de Medicamentos/métodos , Neoplasias Orofaríngeas/tratamento farmacológico , Papillomaviridae/genética , Infecções por Papillomavirus/sangue , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/sangue , Carboplatina/administração & dosagem , Quimiorradioterapia , Cisplatino/administração & dosagem , Estudos de Viabilidade , Feminino , Humanos , Quimioterapia de Indução , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/sangue , Neoplasias Orofaríngeas/virologia , Paclitaxel/administração & dosagem , Infecções por Papillomavirus/virologia , Prognóstico , Estudos Prospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Warfarin, a commonly prescribed oral anticoagulant, is burdened by a narrow therapeutic index and high inter-individual variability in response, making it the second leading cause of drug-related emergency room visits. Since genetic factors contribute significantly to warfarin sensitivity, a genotype-guided dosing strategy may reduce the occurrence of adverse events. While numerous methods have been demonstrated for warfarin genotyping, the specifications of most assays with respect to turnaround time and cost are not ideal for routine testing. Here, we present a unique method for warfarin genotyping based on multiplex PCR coupled with Hybridization-induced Aggregation (HIA), a bead-based technique for sequence-specific detection. A multiplex allele-specific PCR reaction was used to generate products corresponding to 3 genetic variants associated with warfarin sensitivity [CYP2C9 *2, CYP2C9 *3, and VKORC1 (1173C>T)] and an internal control product. The products were detected simultaneously on a poly(ethylene terephthalate) (PeT) microdevice using HIA, which provided genotyping results in approximately 15 minutes following PCR. The genotyping results of 23 patient DNA samples using this approach were in 100% concordance with the results of a validated test (WARFGENO test, ARUP laboratories). Additionally, the PCR reaction was successfully transferred to a PeT chip, which provided accurate genotyping results from patient DNA samples in under an hour. This work demonstrates a simple, rapid, and affordable approach to warfarin genotyping based on multiplex allele-specific PCR coupled with HIA detection. By demonstrating both chemistries on PeT microdevices, we show the potential for integration on a single device for sample-to-answer genotyping.
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Técnicas de Genotipagem/métodos , Polietilenotereftalatos/química , Varfarina/administração & dosagem , Citocromo P-450 CYP2C9/genética , Sondas de DNA/genética , Genótipo , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Hibridização de Ácido Nucleico , Vitamina K Epóxido Redutases/genéticaRESUMO
This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme-based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared-mediated PCR (IR-PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme-mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube-based) protocol. Initial microdevice IR-PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near-full profiles that suffered from interlocus peak imbalance and poor adenylation (significant -A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE-ready STR amplicons in less than 2 h (<1 h of hands-on time). Using this approach, high-quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands-free, closed-system alternative to traditional forensic methods.
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DNA/análise , Genética Forense/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase/métodos , DNA/genética , Desenho de Equipamento , HumanosRESUMO
KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways.
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Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Testes Genéticos/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Alelos , Linhagem Celular Tumoral , Análise Mutacional de DNA/métodos , Humanos , Mutação , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Proteínas ras/genéticaRESUMO
Rapid, inexpensive and simplistic nucleic acid testing (NAT) is pivotal in delivering biotechnology solutions at the point-of-care (POC). We present a poly(methylmethacrylate) (PMMA) microdevice where on-board infrared-mediated PCR amplification is seamlessly integrated with a particle-based, visual DNA detection for specific detection of bacterial targets in less than 35 minutes. Fluidic control is achieved using a capillary burst valve laser-ablated in a novel manner to confine the PCR reagents to a chamber during thermal cycling, and a manual torque-actuated pressure system to mobilize the fluid from the PCR chamber to the detection reservoir containing oligonucleotide-adducted magnetic particles. Interaction of amplified products specific to the target organism with the beads in a rotating magnetic field allows for near instantaneous (<30 s) detection based on hybridization-induced aggregation (HIA) of the particles and simple optical analysis. The integration of PCR with this rapid, sequence-specific DNA detection method on a single microdevice presents the possibility of creating POC NAT systems that are low cost, easy-to-use, and involve minimal external hardware.
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Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/instrumentação , Salmonella enterica/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Hibridização de Ácido Nucleico , Polimetil Metacrilato/química , Pressão , Salmonella enterica/genética , Integração de Sistemas , TorqueRESUMO
Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis.
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Análise Mutacional de DNA/métodos , Microesferas , Mutação/genética , Hibridização de Ácido Nucleico , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Humanos , Fenômenos Magnéticos , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC-DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC]=2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency.
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DNA/análise , Fosfolipídeos/química , Polietilenoglicóis/química , Polietilenoimina/química , Espectrometria de Fluorescência , Composição de Bases , DNA/isolamento & purificação , DNA/metabolismo , Dimiristoilfosfatidilcolina/química , Eletroforese Capilar , Corantes Fluorescentes/química , Micelas , Nanogéis , Éteres Fosfolipídicos/química , Reação em Cadeia da Polimerase , TemperaturaRESUMO
In a recent publication, we presented a label-free method for the detection of specific DNA sequences through the hybridization-induced aggregation (HIA) of a pair of oligonucleotide-adducted magnetic particles. Here we show, through the use of modified hardware, that we are able to simultaneously analyze multiple (4) samples, and detect a 26-mer ssDNA sequence at femtomolar concentrations in minutes. As such, this work represents an improvement in throughput and a 100-fold improvement in sensitivity, compared to that reported previously. Here, we also investigate the design parameters of the target sequence, in an effort to maximize the sensitivity of HIA and to use as a guide in future applications of this work. Modifications were made to the original 26-mer oligonucleotide sequence to evaluate the effects of: (1) non-complementary flanking bases, (2) target sequence length, and (3) single base mismatches on aggregation response. The aggregation response decreased as the number of the non-complementary flanking bases increased, with only a five base addition lowering the LOD by four orders of magnitude. Low sensitivity was observed with short sequences of 6 and 10 complementary bases, which were only detectable at micromolar concentrations. Target sequences with 20, 26 or 32 complementary bases provided the greatest sensitivity and were detectable at femtomolar concentrations. Additionally, HIA could effectively differentiate sequences that were fully complementary from those containing 1, 2 or 3 single base mismatches at micromolar concentrations. The robustness of the HIA system to other buffer components was explored with nine potential assay interferents that could affect hybridization (aggregation) or falsely induce aggregation. Of these, purified BSA and lysed whole blood induced a false aggregation. None of the interferents inhibited aggregation when the hybridizing target was added. Having delineated the fundamental parameters affecting HIA-target hybridization, and demonstrating that HIA had the selectivity to detect single base mismatches, this fluor-free end-point detection has the potential to become a powerful tool for microfluidic DNA detection.
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DNA/genética , Hibridização de Ácido Nucleico/métodos , Pareamento Incorreto de Bases , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/análise , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , Desenho de Equipamento , Limite de Detecção , Mutação PuntualRESUMO
Exosomes offer new insight into cancer biology with both diagnostic and therapeutic implications. Because of their cell-to-cell communication, exosomes influence tumor progression, metastasis, and therapeutic efficacy. They can be isolated from blood and other bodily fluids to reveal disease processes occurring within the body, including cancerous growth. In addition to being a reservoir of cancer biomarkers, they can be re-engineered to reinstate tumor immunity. Tumor exosomes interact with various cells of the microenvironment to confer tumor-advantageous changes that are responsible for stromal activation, induction of the angiogenic switch, increased vascular permeability, and immune escape. Exosomes also contribute to metastasis by aiding in the epithelial-to-mesenchymal transition and formation of the pre-metastatic niche. Furthermore, exosomes protect tumor cells from the cytotoxic effects of chemotherapy drugs and transfer chemoresistance properties to nearby cells. Thus, exosomes are essential to many lethal elements of cancer and it is important to understand their biogenesis and role in cancer.
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Exossomos/metabolismo , Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Vacinas Anticâncer/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Metástase Neoplásica , Neoplasias/diagnóstico , Neoplasias/terapia , Microambiente TumoralRESUMO
We recently reported the 'pinwheel effect' as the foundation for a DNA assay based on a DNA concentration-dependent aggregation of silica-coated magnetic beads in a rotating magnetic field (RMF). Using a rotating magnet that generated a 5 cm magnetic field that impinged on a circular array of 5mm microwells, aggregation was found to only be effective in a single well at the center of the field. As a result, when multiple samples needed to be analyzed, the single-plex (single well) analysis was tedious, time-consuming and labor-intensive, as each well needed to be exposed to the center of the RMF in a serial manner for consistent well-to-well aggregation. For more effective multiplexing (simultaneous aggregation in 12 wells), we used a circular array of microwells and incorporated 'agitation' as a second force that worked in concert with the RMF to provide effective multiplexed aggregation-based DNA quantitation. The dual-force aggregation (DFA) approach allows for effective simultaneous aggregation in multiple wells (12 demonstrated) of the multi-well microdevice, allowing for 12 samples to be interrogated for DNA content in 140 s, providing a â¼35-fold improvement in time compared to single-plex approach (80 min) and â¼4-fold improvement over conventional fluorospectrometric methods. Furthermore, the increased interaction between DNA and beads provided by DFA improved the limit of detection to 250 fg µL(-1). The correlation between the DFA results and those from a fluorospectrometer, demonstrate DFA as an inexpensive and rapid alternative to more conventional methods (fluorescent and spectrophotometric).