February 1, 2018 (Vol. 38, No. 3)

New Advances Suggest That the Warning Attached to Many Biofluid-Based Diagnostic Tests May Be Temporary

Cancer metastasis causes 90% of all cancer-related deaths. Scientists agree that the metastases occur when cancer cells are shed from the primary tumor, carried by the peripheral blood, and deposited at a distal site to create a new colony. An ability to etect circulating tumor cells (CTCs) or cell-free cancer DNA (cfDNA) in blood or saliva opens possibilities to suspect cancer even before it is visible on radiological images.

Minimally invasive bodily fluid testing—dubbed liquid biopsies—allows for frequent testing for residual disease or cancer recurrence, and could be uniquely beneficial when cancer develops in sites inaccessible to surgical biopsies. But what is the best technology to capture and detect cancer material in liquid biopsies? Multiple creative approaches are currently being tested, jostling to reach the clinical diagnostic market.

Cancer Cells as Gutter Balls

The research team led by Steven Soper, Ph.D., University of Kansas, focuses on the oncology-related aspects of liquid biopsies. “Our goal is to develop a universal technology capable of isolating all three oncological markers: cell-free DNA, exosomes, and CTCs,” says Dr. Soper. “But more importantly, the isolated material has to be fully compatible with downstream genetic analysis, which requires high purity isolates.”

As a part of the liquid biopsy initiative, the laboratory created an efficient CTC-capture microfluidic device. The microfluidic chip consists of 50 monoclonal-antibody laden sinusoidal channels.

“The sinusoidal architecture is optimized for capturing rare CTCs. As tumor cells roll along the channel surface, centrifugal forces push the cells to the microchannel walls,” explains Dr. Soper. “The chip’s geometry results in a prolonged rolling path and an extended interaction time to capture cells with low expression levels of the surface antigens.”

This is of particular importance for capturing cells from clinical samples, which contain cells with a wide expression range of target surface antigens. Recent data support the claim of ~90% purity, superior to other existing capture technologies.

The assay was tested in a proof-of-concept study to detect minimal residual disease (MRD) in patients with acute myelogenous leukemia (AML). Antibodies to three separate antigens (CD33, CD34, and CD117) were immobilized on the chips. Only 3 mL of whole unfractionated blood is required, underscoring the vital importance of liquid biopsies in the diagnostic workflow.

As compared with the current diagnostic process, which includes bone marrow biopsy followed by flow cytometry, PCR, or cell staining, microfluidic surveillance presents a simplified, high-yield approach. In the published study, the team was able to perform 39 microfluidic tests as opposed to only 8 when using bone marrow biopsy–based tests.

“Signs of relapse were identified faster,” emphasizes Dr. Soper. “Circulating tumor cells appear variably and unpredictably, such that MRD in AML can be detected at a treatable stage only by frequent sampling.” Dr. Soper’s newly formed company, BioFluidica, is dedicated to commercialization of the microfluidic chips for all three oncological liquid biopsy biomarkers (Figure 1). 

Figure 1. The droplet biopsy chip: selective capture of circulating tumor cells (CTCs) based on density gradients on nanodevices. [bradyurology.blogspot.com]

Chip-Based DNA Capture and Detection

Epidermal growth factor (EGFR) mutations are commonly acquired genetic alterations in lung cancer. Molecular analysis for EGFR mutations is performed on tumor cells obtained via bronchoscopy, limiting its utility in diagnosis and prognosis.

A team of researchers led by David Wong, D.M.D., D.M.Sc., Fang Wei, Ph.D., Wei Liao, Ph.D., and Michael Tu, Ph.D., zeroed on the EGFR exon 19 deletion and exon 21 point mutations. “We explore clinical usefulness of saliva as a noninvasively sampled body fluid for detection of EGFR gene mutations,” says Dr. Wong. “Our goal is to perfect the detection technique that would work without fractionation and processing, [would use] a small sample volume, and [could deliver] results in under 30 min.”

Electric field-induced release and measurement (EFIRM) utilizes an electrochemical chip in a 96-well format of bare gold electrode surfaces (EZLife Bio USA). Several pulses of electric field lead to polymerization of the capture probes onto the gold electrodes, which are hybridized only to mutated DNA. The detector probes, conjugated with horseradish peroxidase, are optimized to hybridize both wild-type and mutation DNA.

Figure 2. Point-of-care EFIRM liquid biopsy. Current clinical practice for lung biopsies is monitoring of indeterminate pulmonary nodules or bronchoscopy and biopsy genotyping. Point-of-care EFIRM liquid biopsy allows detection of ctDNAs that are pathopneumonic to lung cancer in a drop of plasma or saliva with performance that exceeded that of droplet digital PCR and next-generation sequencing (NGS). UCLA

Hybridization occurs when the targets are brought in the proximity of both probes during the positive phase of the electric wave. Unbound probes are washed away with the change of polarity. The pulses are just milliseconds long, and underpin the precise control of specificity and sensitivity of the EFIRM technology (Figures 2 and 3).

“Many believe that liquid biopsy is not quite ready for clinical practice,” agrees Dr. Wong. “EFIRM is poised to narrow the gap.”

The technology was tested in two prospective blinded studies of clinical saliva and plasma samples. The assays demonstrated high concordance with biopsy-based genotyping (96%). Moreover, the cancer-specific molecular fingerprints were clearly identified in pre-surgery samples, even though it is not yet understood how circulating DNA ends up in saliva.

“The clinical reality of the future is such that even plasma sampling may not always be possible due to cultural or political limitations,” points out Dr. Wong. “EFIRM provides a viable path forward for early cancer assessment and therapeutic monitoring applications using circulating DNA in saliva, which is much shorter and not amplifiable by PCR.” 

Figure 3. EFIRM technology captures and monitors key oncogene mutations in biofluids. The core technologies include: an electrochemical chip; nucleic acid probes to specifically amplify electrochemical signals from low number of targets (

Rapid Biomarker Classification by Electrical Signature

“The familiar microarray format has several significant advantages over other methods of capturing and isolating CTCs,” suggests Balaji Panchapakesan, Ph.D., small systems laboratory, Worcester Polytechnic Institute. “The number of capture wells can be expanded nearly infinitely, CTCs can be both captured and analyzed in the same well, and a wide variety of antibodies can be functionalized on the same device. The microarray setup is easily automated with the existing robotic instrumentation.”

In the foundation of the miniature CTC chip is the hydrophobic layer of carbon nanotubes. Nickel and gold electrode layers are plated onto the carbon film, and covered by a protective polymer layer. Dr. Panchapakesan explains that even though the chip contains hundreds of microscopic wells bored into the polymer layer, it’s the hydrophobicity of the carbon surface that enables creation of a perfect droplet of blood. Unexpectedly, Dr. Panchapakesan’s team discovered that CTCs sediment to the bottom of the well faster than leukocytes, further increasing the chip’s sensitivity (Figure 4).

The carbon layer is exceptionally sensitive to changes in electrostatic potential: Multiple random interactions of sample proteins with antibodies produces chaotic electrical signals. On the other hand, specific interaction of surface proteins on CTCs with capture antibodies results in a defined electrical signature. Dynamic time warping mathematical analysis is used to distinguish specific and nonspecific patterns.

“We are working on deriving classifiers for each specific biomarker,” adds Dr. Panchapakesan. “We believe that our technology will cause a quantum leap in early cancer diagnostics.”

Static microarray separation has a unique advantage, in that it is able to isolate cancer cells that downregulated all cancer-specific surface markers, such as triple-negative breast cancer cells. A microarray covered with collagen matrix may be able to capture cells that escaped therapy, and be otherwise “invisible” to antibody-detection methods.

“The chip can also capture single cells, which is invaluable for studies of metastatic initiators,” says Dr. Panchapakesan. “Live viable cells from devices that give a positive electric signal are easily released with the pH change for downstream confirmatory analysis, such as PCR.”

Figure 4. Left: Picture of a circulating tumor cell (CTC) in peripheral blood. Upper middle: Scanning electron micrograph of the sinusoidal CTC microfluidic chip. Upper right: Fluorescence image of several CTCs that were captured using an anti-EpCAM antibody sinusoidal CTC microfluidic chip. The cells were stained for the nucleus (blue), CD45 (green, cells were negative for CD45), and cytokeratines (red, the cells were positive for this marker). Bottom right: DNA sequencing trace of the P53 gene harvested from CTCs. TP53, the gene behind P53, has been labeled the “Guardian of the Genome”: It is mutated in approximately half of all human cancers. [University of Kansas]

Fluidly Tracking ctDNA Signatures

“Four milliliters of plasma contain just a few thousand cell equivalents of cell-free DNA,” says Janet Jin, Ph.D., Roche Sequencing Solutions in Pleasanton, CA. “Only a small fraction of this DNA consists of molecules derived from tumor cells. A successful circulating tumor DNA (ctDNA) detection assay should achieve maximum assay efficiency, reduce background errors, and provide broad coverage to ensure that a wide range of mutations could be detected by the same kit.”

According to the company, extensive optimization of every step in its AVENIO liquid biopsy workflow (for research use only) results in superior recovery rates, as evidenced by nearly 70% target DNA recovery at the recommended sequencing depth of 40 million reads. Significant error reduction is achieved by creative use of molecular barcodes incorporated into the DNA library adapters.

Such barcodes enable the precise tracking of individual molecules, making it possible to distinguish true somatic mutations from artifacts. A complex barcode algorithm in the amplified library allows for precise reconstruction of parental double-stranded DNA duplexes.

The AVENIO family of products includes a targeted panel covering guideline-driven biomarkers, an expanded panel with additional emerging biomarkers, and a surveillance panel, primarily focusing on frequently mutated regions previously identified in lung and colon cancers (Figure 5). “The AVENIO Surveillance Panel provides broad mutation coverage to establish an initial pretreatment baseline, a personalized biomarker signature,” explains Dr. Jin. “This signature could be tracked over time to monitor for re-emerging tumors.”

In a retrospective study, unique molecular signatures were identified in the tumor tissue of 144 subjects diagnosed with stage two or three colorectal cancer. Approximately 10 days post-surgery, plasma was sampled. Presence of ctDNA post-surgery strongly correlated with disease recurrence, and may, in the future, provide evidence basis for adjuvant chemotherapy treatments. Adds Dr. Jin, “Roche provides a comprehensive end-to-end solution for liquid biopsy ctDNA analysis, combining hybrid-capture target enrichment with next-generation sequencing and analysis solution. We are one step closer to reliable diagnostics using ctDNA derived from clinically practical blood-collection volumes.”

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