Budget Impact Analysis of Circulating Tumor DNA Testing for Colon Cancer in Commercial Health and Medicare Advantage Plans.

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 $221 684 (equivalent to $0.02 per member per month [PMPM]) for a commercial payer and $116 720 (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 $16 202 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.

Warfarin genotyping with hybridization-induced aggregation on a poly(ethylene terephthalate) microdevice.

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.

Hybridization-Induced Aggregation Technology for Practical Clinical Testing: KRAS Mutation Detection in Lung and Colorectal Tumors.

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.

A simple integrated microfluidic device for the multiplexed fluorescence-free detection of Salmonella enterica.

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.

Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.

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.

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking.

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.

Investigation of the DNA target design parameters for effective hybridization-induced aggregation of particles for the sequence-specific detection of DNA.

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.

Formation and role of exosomes in cancer.

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.

Dual-force aggregation of magnetic particles enhances label-free quantification of DNA at the sub-single cell level.

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 ∼35-fold improvement in time compared to single-plex approach (80 min) and ∼4-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).