Circulating tumor DNA (ctDNA) provides an opportunity for non-invasive assessment of tumor genotype, and may enable rational use of targeted and/or immune modulating therapies at several clinical milestones. Implementation of ctDNA-based assays across clinical and research settings highlights important assay characteristics and suggests future clinical applications.

We will share advancements in multi-biomarker CTC assays suitable for multi-center trials and introduce the Orion spatial biology platform for highly multiplexed tissue analysis. Orion’s single-day workflow for 21-channel staining and whole-slide imaging will be presented as well as IHC validation data. 

Liquid biopsies refer to blood sampling, used mostly to interrogate circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs). Advantages of liquid versus tissue biopsy include that the former is non-invasive, detects shed DNA from multiple metastatic sites, can be performed serially to detect dynamic changes in the cancer, and may be exploitable for early detection. Disadvantages relate to attenuated sensitivity and mostly smaller genomic panels compared to tissue biopsy.

There is a wide world of circular biomarkers beyond genomics that includes proteins, metabolites, immune complexes, and circulating microparticles. Findings from a systematic search for potential biomarkers relevant to lung cancer applications will be presented.

Circulating tumor (ct) DNA has proven utility in enabling the detection tumor variants that can direct therapeutic decisions, particularly where access to tumor tissue is problematic. New ctDNA approaches are showing promise in the earlier detection of disease, relapse and progression. In this presentation we will explore how this opens up new points of intervention and endpoint opportunities for clinical trial design.

Liquid biopsies provide a valuable tool for clinical management of cancer patients by detecting circulating tumor DNA (ctDNA) in cell-free DNA. Our experience in analytically validating a large 523 gene based ctDNA assay in our laboratory and the technical performance of this assay will be described. Results from a comparison study of variant detection between the plasma and matched tumor specimens will be discussed.

CTCs in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors. An in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of colorectal cancer patients. Thus, the establishment of permanent cell lines from CTCs has become the most challenging task over the past year. We described, for the first time, the establishment of a permanent cell line from CTCs of one colon cancer patient. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. In 2017, we defined the molecular portrait of these metastasis-competent CTCs. Subsequently, we characterized 8 additional CTC lines using blood samples from the same metastatic cancer: a unique biological material collected before and after chemotherapy and targeted therapy, and during cancer progression. Very recently, we also showed that the PI3K/AKT/mTOR signaling pathway plays a key role in the proliferation of the CTC-MCC-41 line. The establishment of CTC lines represents a new opportunity to decipher the metastatic cascade and, hopefully, to find ways to stop cancer dissemination.

Within oncology, significant strides have been made to develop less invasive and more informative assessments of patients with liquid biopsies (ctDNA, ctRNA and CTCs). This presentation will review NeoGenomics’ solid tumor liquid biopsy testing options including circulating tumor nucleic acid testing in clinical trials as well as in routine diagnostics.

Liquid biopsies could enable cancer treatment response monitoring but have limited sensitivity to detect minimal residual disease. Here, I will describe our team’s efforts to increase the sensitivity of liquid biopsies to detect circulating tumor DNA at 1 to 10 parts-per-million. I will present new technologies for mutation enrichment and high-accuracy sequencing and provide proof-of-principle application to small clinical studies.

cfDNA fragmentomic analysis can provide more accurate cancer diagnosis and prognosis. Claret Bioscience’s novel NGS approach called SRSLY – Single Reaction Single-stranded ­Library preparation captures features of cfDNA fragmentation that are lost in traditional library preparation methods. The method captures nicked, single-stranded and shorter (<80 bp) cfDNA fragments leading to improved library complexity. The method retains the native ends of cfDNA facilitating more precise genomic origin determination and cfDNA fragmentomics.

As the potential of liquid biopsies for prognostic, predictive or early cancer detection applications grows, so does the demand for technical advances to accompany the burgeoning range of applications. A major bottleneck in liquid biopsies is the limited material available for analysis. We present new technical developments that overcome this problem for certain classes of cancer patients and show that cancer biomarkers can be sensitively detected from 100pg or less circulating-DNA. This opens up the possibility of detecting biomarkers from minimal amounts of blood such as that obtained from finger-pricks, thereby enabling minimally invasive testing at multiple time-points following treatment to assess residual cancer. Examples from clinical sample testing will be presented.

This presentation describes the high-sensitivity isolation of rare cells, including circulating tumor cells and fetal cells, using fluorescence activated aliquot sorting. Here, a blood sample is divided into nanoliter volumes or aliquots, then analyzed and sorted based on the presence or absence of a rare cell. By performing two rapid, on the millisecond timescale, back-to-back sorting, we also demonstrate isolation of rare cells with high purity. Finally, we drastically increased the number of nucleated cells sorted by first concentrating peripheral blood mononuclear cells from whole blood, which translated into an equivalent blood volume analyzed by over an order of magnitude, from 8mL to over 100mL per experiment.

An AC electrokinetic microarray device allowed rapid isolation of cancer related biomarkers including exosomes and high molecular weight cell free (hmw) cf-DNA from 50uls of patient plasma sample. In a blinded PDAC cancer patient study of 36 samples, glypican-1 and the hmw cf-DNA levels were elevated in a number of PDAC patient samples compared to those from various IPMN stages, pancreatitis and benign patient samples. Digital PCR analysis for KRAS G12 & G13 codon mutations in the hmw-cf-DNA further confirm which samples were from the PDAC patients.

We are developing a platform which identifies aberrant CACs using a novel 4-color FISH methodology.   It is intended to complement CT scans and lung biopsies by offering additional insights into disease trajectory and management.  This presentation will provide an update on several of our late-stage clinical research programs.

Tumor-derived analytes represent potential liquid biopsy targets to detect and characterize cancer. Extracellular vesicles (EVs) are released by all cells and carry molecular cargo that may provide information about the cell of origin. However, EVs are small, heterogeneous, and difficult to measure. We have developed sensitive and quantitative EV analytics based on vesicle flow cytometry (vFC) that can identify and characterize tumor EVs in complex biofluids for liquid biopsy.

Integration of multiple mutations or fragment features can improve sensitivity of liquid biopsies. Recent studies have shown genome-wide fragmentation patterns in cell-free DNA reflect nucleosome positioning and additional protein-DNA interactions. I will describe results from investigation of fragmentation patterns in plasma and urine cell-free DNA and potential applications for monitoring treatment response and early detection of cancer.

As part of the FDA-led Sequencing Quality Control Phase 2 (SEQC2) project, we conducted a cross-laboratory assessment of five leading liquid biopsy assays. Using the set of known positives and negative positions in the contrived reference samples, sensitivity, reproducibility, and false positive rates were thoroughly examined at different DNA input amounts and variant allele fractions.

ctDNA shows great promise for cancer patient management, but there is a need for standardization in its use. The FNIH Biomarkers Consortium initiated a public-private partnership (PPP) to develop recognized quality control materials (QCM) for widespread use in ctDNA testing. Designed for use across multiple assays, the QCMs will provide confidence in interpretation of ctDNA biomarker assay results, paving the way for more effective clinical research and therapeutic decision-making. This presentation describes the need and acceptable performance characteristics of QCMs and establishment of the PPP and provides an update on current status of the FNIH ctDNA QCM project.

The BloodPAC Data Commons is being developed and is operated by the public-private BloodPAC Consortium to support the liquid biopsy community. We describe the BloodPAC Data Commons, the data it manages, the process that the BloodPAC Consortium used to develop it, and some of the applications that have been developed using its API.

Pre-analytical standardization is a requisite to incorporate Liquid Biopsies in the oncology practice. Our new platform allows automated and standardized Liquid Biopsy preparation at site of blood collection, minimizing sample manipulation and preserving clinical content for highest assay sensitivity and specificity. Our latest data show precise cancer diagnosis and CTC clusters detection in early breast cancer, demonstrating accurate performance in early settings, paving the way for a variety of clinical applications.