BioFluidica (United States)

e13047 Background: HER2 targeted therapies, such as HER2 monoclonal antibodies, HER2 tyrosine kinase inhibitors, and HER2 antibody drug conjugates, are approved for the treatment of HER2-positive breast, gastric and colorectal cancers. However, inter- and intra-tumoral heterogeneity in HER2 status presented a significant challenge in identifying patients that may benefit from HER2-targeted therapies. Detection of ERBB2 amplification in Circulating Tumor Cells (CTCs) may circumvent tissue heterogeneity issues and effectively predict the response of patients to anti-HER2 agents as a non-invasive alternative approach. Methods: A twenty-four patient pilot study was designed to compare HER2 status of traditional needle biopsies to high sensitivity CTC based liquid biopsies. HER2 status was determined using immunohistochemistry (IHC) and Fluorescence in Situ Hybridization (FISH) for tissue samples and FISH analysis for CTCs. Currently 21 patient samples have been compared in this study. Results: CTCs with HER2 amplified signals were found in patients with stages 2, 3, or 4 breast cancers. Based on the biopsies of the primary tumor or metastatic disease, 6 patients (29%) had HER2-overexpressing as defined by either HER2 IHC 3+ or HER2 FISH amplified. Analysis of CTCs showed 100% concordance (HER2 amplification detected in CTCs) in these patients with HER2-overexpressing breast cancer. Furthermore, an additional 4 (19%) patients had HER2 amplification by CTC analysis but not on needle biopsy. Conclusions: Our finding demonstrates that LiquidScan can be used as a rapid analysis tool to evaluate HER2 amplification in CTCs. This approach can potentially help identify additional cohort of patients (HER2 non-amplified tumor / HER2 amplified CTCs) who may benefit from the new generation of HER2 targeted therapies. Studies are ongoing to include an expanded cohort of patients with early stage disease, as well as HER2-low disease, in order to evaluating dynamics of ERBB2 gene amplification, expression, and pathway activation while on active systemic therapy.

Liquid biopsies are becoming popular for managing a variety of diseases due to the minimally invasive nature of their acquisition, thus potentially providing better outcomes for patients. Circulating tumor cells (CTCs) are among the many different biomarkers secured from a liquid biopsy, and a number of efficient platforms for their isolation and enrichment from blood have been reported. However, many of these platforms require manual sample handling, which can generate difficulties when translating CTC assays into the clinic due to potential sample loss, contamination, and the need for highly specialized operators. We report a system modularity chip for the analysis of rare targets (SMART-Chip) composed of three task-specific modules that can fully automate processing of CTCs. The modules were used for affinity selection of the CTCs from peripheral blood with subsequent photorelease, simultaneous counting, and viability determinations of the CTCs and staining/imaging of the CTCs for immunophenotyping. The modules were interconnected to a fluidic motherboard populated with valves, interconnects, pneumatic control channels, and a fluidic network. The SMART-Chip components were made from thermoplastics via microreplication, which lowers the cost of production making it amenable to clinical implementation. The utility of the SMART-Chip was demonstrated by processing blood samples secured from colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) patients. We were able to affinity-select EpCAM expressing CTCs with high purity (0-3 white blood cells/mL of blood), enumerate the selected cells, determine their viability, and immunophenotype the cells. The assay could be completed in <4 h, while manual processing required >8 h.

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

Incorporation of spiral continuous flow (CF) polymerase chain reaction (PCR) devices into a 96-well titer plate format will enable highly parallel analyses of many nucleic acids under steady-state temperature control for the realization of a high throughput CFPCR multi-reactor platform. Prior to realization of the full platform, verifying the feasibility of small footprint CFPCR devices, each device confined to a footprint of 8x8 mm 2 , throughout the manufacturing and amplification processes was essential. Small footprint, CFPCR devices in a CFPCR multi-reactor module were designed and double-side hot embossed on one side of a substrate and structures for thermal isolation on the other to improve thermal management. Successful amplification of 99 bp target DNA fragments from -DNA template was demonstrated in the small footprint, 20-and 25-turn CFPCR devices.

A highly parallel, polymerase chain reaction (PCR) multireactor platform is in high demand to satisfy the high throughput requirements for exploiting the accumulated genetic information from the Human Genome Project. By incorporating continuous flow PCR (CFPCR) devices in a polymer 96-well titer plate format, DNA amplification can be performed with steady-state temperature control and faster reaction speed at lower cost. Prior to the realization of a PCR multi-reactor platform, consisting of a sample delivery chip, a PCR multireactor chip, and a thermal cycler, optimization of the geometry for CFPCR devices in a titer plate-based PCR multi-reactor chip based on manufacturing feasibility is necessary. A prototype PCR multi-reactor chip was designed in a 96-well titer plate format with twelve different CFPCR configurations. High quality metallic, large area mold inserts (LAMIs) were fabricated using an SU-8 based UV-LIGA technique by overplating nickel in SU-8 electroplating templates. Micro molding of polycarbonate (PC) was done using hot embossing, resulting in good replication fidelity over the large surface area. Thermal fusion bonding of the molded PC chips using a custom-made bonding jig yielded acceptable sealing results. The manufacturability investigation throughout the design and the process sequence suggested that the microchannel walls require a minimum width of at least 20 μm and an aspect ratio of 2 for structural rigidity. An optimal CFPCR device for use in a PCR multi-reactor chip can be selected with a series of amplification experiments with the development of a thermal cycler.