Poster S11 - Resource-friendly immunodiagnostic detection of breast cancer biomarkers at the point-of-care

Introduction: Breast cancer is the leading cause of cancer deaths in women worldwide, and outcomes are significantly worse in resource-limited settings. A major contributor to this disparity is the limited availability of modern diagnostic pathology infrastructure, which is crucial for effective management because it enables the selection of targeted therapies based on breast tumor biomarker evaluation. We describe a cost-effective immunodiagnostic assay —the “BreastPathD4”— that evaluates key breast cancer biomarkers (ER, PR, and Her2) from tumor biopsy specimens within 90 minutes with minimal infrastructural requirements. The BreastPathD4 evaluates biomarker expression from breast tumor tissues directly from crude tumor lysates, which is possible based on several enabling technologies1,2: (1) a disposable assay cassette that passively automates fluid handling and reagent delivery without needing power; (2) modification of assay surfaces with "non-fouling" thin film coatings that eliminate background noise to increase sensitivity and provide a stable substrate for storing inkjet-printed immunoassay reagents; (3) a portable detector for readout of assay results based on fluorescence signal produced by the immunoassay.

Materials and

Methods: This research is performed under IRB exemption Pro 001100388 and Pro 00108816 for studies involving patient tumor specimens. Fabrication of BreastPathD4 cassettes is accomplished by stacking laser cut adhesive and acrylic sheets in specific configurations to create a microfluidic chip network that utilizes gravity-driven capillary flow. The stacked chip is bonded to a polymerized slide with the printed microspots of detection and capture antibodies for the immunoassay (using Biodot and Scienion printers). In anticipation for in-human studies, feasibility studies thus far have involved solid tumors harvested from nude mice engrafted with patient-derived cell lines (The Jackson Laboratory). Commercially available lysis buffer is added to a solid tumor specimen and then mechanically pulverized with a disposable mortar and pestle. The tumor lysate is briefly pelleted with an low-cost portable mini-centrifuge. The soluble fraction is added directly to the BreastPathD4 cassette at two different concentrations for Her2 (1 mg/mL) and ER/PR (30 mg/mL) detection. Samples are run in triplicate. The sample is incubated for 60 minutes with the printed reagents on the slide. The assay is terminated by a brief wash and drying step. Assay readout is accomplished by quantitating fluorescence intensity of microspots corresponding to each biomarker. Results are normalized against a standardized spiked control to account for inter-assay variation.

Results, Conclusions, and Discussions: The BreastPathD4 can successfully perform multiplex evaluation of Her2, ER and PR with minimal cross reactivity with analytical sensitivity considerably below 1 ng/mL, with detection limits for ER, PR, Her2 estimated at 0.034 ng/mL, 0.218 ng/mL, and 0.063 ng/mL, respectively. Signal normalization yielded consistency in results with acceptable inter-assay variation for ER, PR, Her2. Preliminary evaluation of patient-derived solid tumor xenografts with the BreastPathD4 resulted in an accuracy rate of tumor biomarker positive/negative status for Her2 in 7 of 8 tumors, and ER in 8 of 8 tumors, and PR in 6 of 8 tumors.
The BreastPathD4 platform accomplishes biomarker credentialing of solid tumors for (ER, PR, and Her2) with results available within 2 hours and with minimal infrastructural requirements. This technology is applicable to limited resource settings where infrastructural limitations pose obstacles to timely pathological diagnosis (and hence appropriate treatment). In further studies, we plan to optimize the D4 assay for a set of 60 archived human tumor specimens from patients that we have collected for this study while simultaneously comparing them to the gold standards. This will be followed by testing in a cohort of breast cancer patients at Duke Hospital, followed by deployment of the technology in its intended setting in Tanzania.

Acknowledgements (Optional): References- 1. Joh, D. Y., et al. Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood. Proceedings of the National Academy of Sciences 114.34 (2017).
2. Joh, D.Y., Heggestad, J.T., Zhang, S. et al. Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates. npj Breast Cancer 7, 85 (2021).