Over 600,000 people in the US will die from cancer this year. It is estimated that 25% of these deaths could have been prevented by detection in earlier stages. Implementation of minimally-invasive routine screening, such as pap smears for cervical cancer, has proven to be an effective approach for reducing cancer mortality. However, several challenges prevent successful implementation of screening in most cancers, especially ones which are not readily accessible for imaging or tissue biopsy. One promising avenue for cancer diagnostics is through use of circulating DNA from so-called "liquid biopsies" or other accessible sample media circulating throughout the body collecting genetic material from tissues, including tumors. Tumor-specific molecular biomarkers, such as DNA mutations and methylation, can be found in minimally-invasive sample media, such as blood, stool, and urine, but are typically only present in very low copy numbers (<10 copies/mL) and low fractions (<0.1%) among a high background of healthy DNA. Technical limitations as well as practical inaccessibility of currently available tools have precluded research efforts to discover early-stage cancer- specific DNA biomarker panels and subsequent clinical implementation that could improve patient outcomes. To address this, we previously developed a prototype digital microfluidic platform to facilitate highly sensitive, low-cost detection of cancer-specific DNA methylation patterns by highly parallelized single-molecule thermodynamic sequencing. In this Phase 1 SBIR proposal, we will greatly expand upon the capabilities of this platform to increase accessibility and improve analytical performance towards detection of early-stage disease by significantly increasing its digitization power. We will develop a high-degree multiplexing paradigm for detection and methylation profiling of biomarker panels using a multicolor barcoding technique. We will then incorporate this assay into a microfluidic platform that can interrogate hundreds to thousands of single DNA copies by digitizing template molecules into droplets for high-throughput single molecules analysis. The platform will increase accessibility for biomarker research from liquid biopsies by reducing costs to <$25 per sample and turnaround time to 4 hours. The platform will enable single-copy detection even among high background populations (<0.001% sensitivity), which may be necessary for early-stage disease. The proposed work in this Phase 1 project will develop the assay fundamentals for a multiplex, multidimensional analysis of a clinically relevant biomarker panel (Aim 1), incorporate this assay into a highly-parallelized droplet microfluidic platform (Aim 2), and assess its clinical feasibility with plasma samples from a cohort of lung cancer patients and controls (Aim 3). This will lay the groundwork for a subsequent Phase 2 project to design the cartridge and instrumentation for scalable manufacturing and user-friendliness and implement automated, machine- learning image and data analysis pipelines.
Public Health Relevance Statement: Project narrative Detection of cancer biomarkers from minimally-invasive samples such as blood for early diagnostics could save hundreds of thousands of lives each year. Current technologies for research in this space have technical limitations and are inaccessible to most research laboratories around the world. We propose a sensitive, practical platform for detection of molecular biomarkers from minimally-invasive samples to promote discovery of novel biomarkers, enable better understanding of disease progression, and ultimately improve standard of care for patients globally.
Project Terms: clinical relevance; clinically relevant; prototype; tumor; minimally invasive; standard of care; new marker; novel biomarker; novel marker; molecular biomarker; molecular marker; bio-markers; biologic marker; biomarker; Biological Markers; NGS Method; NGS system; next gen sequencing; nextgen sequencing; next generation sequencing; screenings; screening; methylation pattern; cell free circulating DNA; cell free DNA; circulating DNA; methylation marker; methylation biomarker; dimensional analysis; Data Analytics; targeted biomarker; Formulation; biomarker array; marker panel; biomarker panel; cancer markers; cancer biomarkers; parallelization; liquid biopsy; clinical implementation; annual screening; routine screening; imager; data processing pipeline; data analysis pipeline; detection system; detection platform; multiplex assay; manufacture; digital platform; Adoption; Algorithms; Bar Codes; barcode; Biological Assay; Assay; Bioassay; Biologic Assays; Biopsy; Blood; Blood Reticuloendothelial System; malignant breast neoplasm; Breast Cancer; malignant breast tumor; Buffers; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Cause of Death; Malignant neoplasm of cervix uteri; Cervical Cancer; Cervix Cancer; Malignant Cervical Neoplasm; Malignant Cervical Tumor; Malignant Neoplasm of the Cervix; Malignant Tumor of the Cervix; Malignant Tumor of the Cervix Uteri; Malignant Uterine Cervix Neoplasm; Malignant Uterine Cervix Tumor; Uterine Cervix Cancer; Colonoscopy; Color; Cost Analysis; Cost Analyses; assess cost; cost assessment; cost evaluation; evaluate cost; examine cost; Cessation of life; Death; Disease; Disorder; DNA; Deoxyribonucleic Acid; Expenditure; Feces; stool; Fluorescence; Future; Patient Care; Patient Care Delivery; Genes; Genome; Goals; instrumentation; Laboratories; Laboratory Research; Methods; Methylation; mortality; Persons; Optics; optical; Patients; Plasma; Blood Plasma; Plasma Serum; Reticuloendothelial System, Serum, Plasma; Research; Resources; Research Resources; Sensitivity and Specificity; Specificity; Technology; Thermodynamics; Thermodynamic; Time; Tissues; Body Tissues; Urine; Work; Pap smear; Pap Test; Pap screening; Papanicolaou Smear; Papanicolaou Test; Uninsured; improved; Procedures; Image Analyses; image evaluation; image interpretation; Image Analysis; Site; Clinical; Phase; Individual; Malignant Tumor of the Lung; Pulmonary Cancer; Pulmonary malignant Neoplasm; lung cancer; Malignant neoplasm of lung; Disease Progression; tool; Diagnostic; Machine Learning; machine based learning; DNA Methylation; Hour; Dimensions; Frequencies; Complex; Genetic Materials; Reaction; Techniques; System; Early Diagnosis; early detection; Performance; cohort; melting; Sampling; single molecule; Molecular Interaction; Binding; µfluidic; Microfluidics; preventing; prevent; T-Stage; Tumor stage; Address; DNA Sequence Alteration; DNA Alteration; DNA mutation; Genetic mutation; Sequence Alteration; genomic alteration; Detection; Cancer Diagnostics; Resolution; resolutions; Cancer Control; Cancer Control Science; Cancer Detection; Cancer Patient; Clinical Sensitivity; Patient-Focused Outcomes; Patient outcome; Patient-Centered Outcomes; patient oriented outcomes; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Tumor-Derived; Modification; Development; developmental; Colorectal Cancer; Colo-rectal Cancer; Image; imaging; cost; digital; designing; design; cost effective; Population; cancer type
