As cfDNA markers for cancer detection and monitoring become more accessible, there is a growing need to standardize preanalytical variables to ensure accurate and generalizable results. This talk will review the current recommendations for specimen collection, handling and processing, and will highlight efforts to enhance the sensitivity and specificity of tumor cfDNA detection.

Liquid biopsy next-generation sequencing (NGS) assays help guide treatment selection in cancer patients, particularly when tumor tissue is unavailable or during disease progression. Extensive analytical validation of the targeted 33-gene assay PGDx elio plasma focus Dx assay has shown that detection of cancer-associated variants in circulating tumor DNA (ctDNA) is highly specific, sensitive, reproducible, and accurate. Distribution of this kitted assay, including automated machine-learning software to generate reports, can inform precision oncology decision-making.

I will present our experience testing ctDNA from the plasma of ~4200 patients from NCI-MATCH, the largest precision medicine initiative ever conducted. Most patients had rare or uncommon tumors and were screened for the trial but did not enroll to treatment. Patients enrolled to 3 treatment arms have also had ctDNA tested, including one arm that enrolled patients with mismatch repair deficiency by tumor IHC.   

Friends of Cancer Research (Friends) has an ongoing collaboration to assess associations between change in ctDNA levels and long-term outcomes in aggregate patient-level datasets. The data analyzed to date have focused on aNSCLC with different treatment modalities and the results intend to provide consensus around how ctDNA can be used to inform regulatory decision-making.

High-grade serous ovarian carcinoma (HGSOC) accounts for ~80% of ovarian cancer-associated deaths. Non-invasive, highly specific blood-based tests for pre-symptomatic screening in at-risk women are crucial to reducing the mortality associated with this disease. Since most HGSOCs typically arise from the fallopian tubes (FT), our biomarker search focused on proteins found on the surface of extracellular vesicles released by both FT and HGSOC tissue explants and representative cell lines. Our studies demonstrate that these lineage-associated exo-biomarkers can detect ovarian cancer with high specificity and sensitivity early and potentially while localized to the FT when patient outcomes are more favorable.

Stroke is a leading cause of death and adult disability worldwide, and finding new tools for expediting stroke diagnosis is a critical and unmet need. I am working with my colleagues at the University of Kansas to develop an integrated, innovative technology to permit the measurement of ribonucleic acid expression data in near-real-time to help diagnose ischemic and hemorrhagic stroke, using circulating extracellular vesicles in plasma.

The talk will cover the role of glial EVs in propagating neuroinflammation and senescence to other brain cells in the context of HIV proteins and opiates. Specifically, the role of shuttled micro RNAs in mediating recipient cell reprogramming will be discussed, and how it relates to cognitive impairment in the host. How such an approach can be tapped into for biomarker discovery will also be touched upon.

We have developed a portable instrument consisting of a plastic microfluidic chip possessing micropillars decorated with affinity agents to allow for selecting EVs from plasma. This chip was used to select SARS-CoV-2 virus particles (VPs) and human Respiratory Syncytial Virus (RSV) directly from saliva. In this presentation, we discuss an assay that can accept saliva samples and search for VPs and then count the number of VPs selected using a label-free approach; nano-Coulter Counter chip (nCC). The selection chip affinity targeted viruses using aptamers surface immobilized to pillars and then could count the VPs using resistive pulse sensing.

Serum biomarkers are often insufficiently sensitive or specific to facilitate disease screening or diagnostic testing. In many cancers, biomarkers fail to detect early-stage disease or to substantially impact mortality rates. We developed a perception-based sensing method that captures a biomarker-agnostic ‘disease fingerprint’ of disease from serum. The method uses large data sets of molecular binding interactions, to an array of moderately-selective nanosensors, used to train machine learning algorithms. In an initial study using serum from several hundred patients, we built a prediction model of nanosensor responses that reliably identified high-grade serous ovarian cancer substantially better than CA125.

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Liquid biopsies using circulating tumor DNA (ctDNA) enable early detection and monitoring of cancer. But their sensitivity is limited by the intrinsic scarcity of ctDNA in blood. Despite technical improvements in the accuracy of sequencing technology, the biological limitation of ctDNA scarcity remains a major barrier to clinical use of liquid biopsies. To address this problem, we have developed novel “priming agents” that are administered 1-2 hours before a blood draw and augment the level of ctDNA in a blood draw by attenuating ctDNA clearance from plasma. These priming agents consist of antibodies that directly bind and protect ctDNA from clearance and nanoparticles that inhibit cells responsible for ctDNA clearance. In tumor-bearing mice, our priming agents led to recovery of > 10x more ctDNA and significantly higher sensitivity of ctDNA tests, especially at ultra-low plasma ctDNA levels. In addition, antibody priming agents enabled better representation of fragments normally lost in cfDNA, including those overlapping open chromatin regions. Priming agents could make liquid biopsy tests more effective for patients whose blood samples may otherwise contain little to no tumor DNA. This includes critical points in cancer care, such as when a decision needs to be made regarding adjuvant treatment, for diagnosis of early-stage or indeterminate lesions, and for early detection of recurrence after completing cancer treatment.

Cancer drug development focuses primarily on inhibiting tumor cell growth and rarely targets the causes of metastatic spread.  Our group has invented a microfluidic technology (TetherChip) that secures tumor cells in a non-adherent state that more accurately mimics the free-floating environments encountered by tumor cells during metastasis (bloodstream, lymphatics).  TetherChip analysis can rapidly assess 4 independent metastatic phenotypes in less than 24 hours, as well as drug responses.

The talk will describe advances in the development of biomarkers for cancer cell dissemination that can be obtained from fixed tissue and by MRI in real-time. It will also address the effect of chemotherapy on these biomarkers as well as the observed racial disparity in pro-metastatic tumor microenvironment, potentially related to treatment. Finally, the use of these biomarkers for tailoring neoadjuvant chemotherapy will be explored.

The advancement in DNA methylation-based cancer diagnostics and screening is impeded by the unavailability of cost-effective, easily accessible technologies capable of performing comprehensive assessments of methylation biomarker panels. Current methods also lack the necessary high sensitivity and CpG-site specificity for early detection of tumor-derived cfDNA, crucial in cancer diagnostics. To address these issues, we developed a multiplexed digital high-resolution melt (dHRM) technology. This approach combines the superior analytical performance of digital methylation analysis with methylation-agnostic probes to achieve comprehensive analysis of methylation biomarkers across various cancers, including lung and ovarian. Our dHRM platform offers a high-sensitivity, liquid-biopsy-based method for identifying early-stage cancers at a fraction of the cost associated with current targeted-NGS approaches.

Conventional methylation maker detection methods require extensive manual processing by highly skilled personnel using specialized equipment, making them impractical in resource-limited settings. To overcome this, we developed a compact, portable, multiplexed, and fully automated microfluidic platform capable of conducting droplet magnetofluidic- and methylation-specific qPCR assays. This platform is designed to detect DNA methylation cancer biomarkers, with a particular focus on esophageal cancer—a highly prevalent disease in low- and middle-income countries characterized by dismal survival rates. Our findings demonstrate the potential of our portable microfluidic device as a viable epigenetic diagnostic tool for the early detection of esophageal cancer and possibly other types of cancer, especially in areas with limited resources.