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Liquid Biopsies: Miracle Diagnostic or Next New Fad?

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Article

Liquid Biopsies: Miracle Diagnostic or Next New Fad?

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Angelo DePalma, PhD

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GATCLIQUID is the world's first service line for comprehensive liquid biopsy analysis. The plasma-based tests offer sensitive and cost-efficient detection of mutations in circulating tumour DNA. The services advance cancer research and diagnostics through detailed assessment of the tumour exome, of selected cancer drivers or by monitoring of single mutations.

" Highly sensitive methods are essential, to detect cancer specific SNPs in ctDNA with high confidence. Therefore, we offer unique services, which can be used to get a deeper understanding of cancer and its dynamics."
Dr Tobias Paprotka – Director Research & Development at GATC Biotech

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Droplet Digital™PCR (ddPCR™ technology has emerged as the clearest path for liquid biopsy to the clinic. Its superior precision, sensitivity, and rapid time to results in detecting and quantifying low abundance DNA targets have allowed laboratories to develop tests for monitoring residual disease, tracking treatment efficacy, and identifying disease recurrence earlier and more confidently than ever before.

" Bio-Rad is advancing liquid biopsy analysis through our comprehensive offering of ddPCR mutation detection assays and reagents. We are also committed to achieving regulatory clearance for our ddPCR systems and solutions."
Viresh Patel, Marketing Director, Digital Biology Group, Bio-Rad Laboratories

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QIAGEN’s liquid biopsy solutions empower you to sensitively, specifically and rapidly reveal meaningful insights from your samples with an efficient, streamlined and complete workflow for analyzing circulating cell-free DNA, exosomes and CTCs for PCR and NGS applications, giving you the first step towards uncovering valuable biomarkers in your samples.

" Our mission is to provide you with the best-in-class solutions from Sample to Insight in the emerging field of Liquid Biopsy that has the potential to transform biomarker research and healthcare."
Michael Kazinski, Senior Director, Molecular Preanalytic Technology, QIAGEN

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Diagnosing, monitoring, and managing cancer through blood tests has been a decades-long goal of medical diagnostics. Complete blood counts, protein biomarkers, and cancer antigen tests (e.g. for prostate-specific antigen) provide clues regarding disease status, but do not always render definitive diagnoses.

Liquid biopsies (LBs) are an emerging, disparate group of technologies that seek to broaden the scope and sensitivity of blood-based cancer diagnosis. Since they usually require only a blood sample (vs. solid tissue) LBs are primarily non-invasive. And thanks to the development of highly specific gene-amplification and sequencing technologies LBs access more biomarkers relevant to more cancers than ever before.

Traditional tissue biopsies range in cost from $15,000 to $60,000 U.S. dollars, depending on whether invasive surgery is required, the level of sample preparation, pathology services, and follow-on genetic tests. Because so many hands are in play, traditional biopsies have a failure rate of approximately 25%. Additionally, biopsy tissue from tumors is simply not available for monitoring patient response over the course of treatment, when liquid biopsies become the only feasible option.

LBs seek out two major targets: circulating cancer cells (CTCs) and cell-free tumor DNA (cfDNA). CTC analysis focuses on isolating and expanding populations of rare cells for downstream analysis. cfDNA LBs identify multiple circulating tumor gene mutants at multiple time points.

LBs have generated great attention for their ease of use and applications across many different diseases and health conditions, such as noninvasive prenatal testing, transplant medicine, and oncology – far and away the most commercially lucrative market.

It’s in the Genes

“Liquid biopsies will provide clinicians with faster, cheaper, broader, less invasive ways to assess cancer patients’ clinical status, and help to deliver the right treatment for the right target without delay,” says Dr. Chen-Hsiung Yeh, Chief Scientific Officer at Circulogene Theranostics (Birmingham, Alabama, USA). Yeh believes DNA-based liquid biopsies for multiple mutation profiles could become the gold standard for next-generation cancer management and precision medicine. “Clinical tests employing cfDNA are inherently specific, sensitive, and capture both intra- and inter-tumor heterogeneity in real time,” he adds.

Table 1: Comparison of liquid biopsy and tissue biopsy. Note that these benefits apply to liquid biopsies based on CTCs.

Key CharacteristicsLiquid BiopsyTissue Biopsy
Invasiveness No Yes
Suitability throughout the disease process Yes No
Ex vivo sample stability Yes (less so for CTCs) Stable when processed
Provides longitudinal disease monitoring Yes No
Cost Low High
Processing Time Short Long (involvement of tissue sectioning, staining and pathologists)
Rejection/Failure rates Low High due to insufficient sample or unidentified tumor
Starting material for multiple testing Ample Scarce

Source: Circulogene Theranostics

Circulating cfDNA is highly fragmented and exists at very low concentrations, making its isolation from blood challenging. Isolation efficiency using silica membrane/bead techniques is extremely low due to the unavoidable losses during gene binding, washing, and elution. Circulogene’s cfDNA enrichment technology enables nearly full-recovery of cfDNA from both necrotic and apoptotic cells.

Detection of such mutations has potential for monitoring tumor progression before lesions reach a size amenable for diagnostic imaging. PET scans, for example, cannot visualize solid tumors containing fewer than 6 million cells in one location. Analysis beyond a single mutation could also uncover tumor heterogeneity that might influence treatment decision-making. 

“Liquid biopsies are not as spatially limited as tissue biopsies, and display a global spectrum of mutations that occur throughout cancer development,” Yeh says. Nevertheless, the sensitivity of conventional Sanger sequencing is not sufficient for detecting low-frequency variants. Advanced next-generation sequencing bioinformatics, for example developed by Guardant Health, provides a cost-effective, highly sensitive alternative for high-throughput analysis of multiple mutations. Additionally PCR-based amplification techniques from RainDance, Bio-Rad, Qiagen, Thermo Fisher, Transgenomic, Sysmex Inostics and Trovagene, allow single-molecule amplification and/or selective enrichment of tumor-specific cfDNA.

Immersed as he is in this new industry, Yeh believes that larger prospective trials are needed to confirm the clinical utility of cfDNA testing. “Future studies must demonstrate whether cfDNA-derived mutation profiles from blood samples can contribute to improved treatment outcomes in cancer patients.”

Table 2: Advanced technologies employed for circulating cell-free DNA in oncology

 
TechnologiesSensitivityClinical Utility
ARMS-PCR (Amplification Refractory Mutation System) 1 % Treatment decision; Resistance
Suitability throughout the disease process 0.1–1 % Treatment decision; Resistance
Clamping PCR Yes (less so for CTCs) Stable when processed
castPCR (Competitive Allele-Specific TaqMan PCR) 0.1–1 % Treatment decision; Resistance
ICE COLD-PCR (Improved & Complete Enrichment CO-amplification at Lower Denaturation temperature) 0.1-1 % Treatment decision; Resistance
BEAMing Digital PCR (Beads, Emulsions, Amplification and Magnetics) 0.01 % Treatment decision; Resistance
Droplet Digital PCR < 0.1 % Treatment decision; Resistance
NGS (Next-Generation Sequencing) 1-5 % Treatment decision; Metastasis; Resistance; Recurrence
Source: Circulogene Theranostics

“Selective” PCR

Polymerase chain reaction (PCR), the gold standard of targeted gene sequence amplification, is not always directly applicable to diagnostics. “The advertisements state that PCR amplifies specific regions of DNA, but the technique has difficulty picking out subtle base change differences between mutant and wild-type DNA when the PCR product is sequenced,” says Paul Kinnon, CEO of Transgenomic (Omaha, Nebraska, USA). According to Kinnon, PCR amplifies wild-type and mutated genes at approximately the same rate. “If you begin with a million wild-type genes and a thousand mutations you may wind up with 100 million copies of the background DNA and a few million of the target.”  

ICE COLD-PCR™ (Improved and Complete Enrichment CO-amplification at Lower Denaturation temperature PCR), Transgenomic’s signature technology, suppresses wild-type DNA while amplifying mutated genes. The technique harnesses the power of low denaturation temperatures to selectively target amplification of mutated genes, thereby achieving greater sensitivity—down to a level of detection of 0.01% in some cases. Another important aspect of the technology is that it works with any sample--liquid or solid tissue, sputum, urine, blood, or excised tumor samples.

“It is a true needle-in-a-haystack assay,” Kinnon says.

Samples processed via ICE COLD-PCR have the potential to reduce traditional biopsy costs by approximately 75% while providing exquisitely sensitive analysis. A paper released by Transgenomic at the most recent American Society for Cancer Oncology Annual Meeting demonstrated 95% concordance between biopsies based on ICE COLD-PCR technology and high-quality traditional tissue biopsies using standard PCR analysis.

ICE COLD-PCR is not strictly a diagnostic test, and as such is not subject to FDA approval. It is rather a DNA enrichment technique that serves as the front end for all sequencing analysis via droplet digital PCR, Sanger sequencing, next-generation sequencing, etc. The company plans to license its technology worldwide to make it widely available to diagnostic labs, both academic and commercial. “Oncologists are able to send us their samples for analysis in our CLIA lab, but they can also use the technology to conduct the analysis on their own,” Kinnon says.

Cell-Based Technologies

Analysis of CTCs represents the second major LB method. Harvested CTCs provide material for downstream analysis for RNA, DNA, and protein expression, plus viable cells can be cultured and a host of other assays are possible. “You also have the opportunity to work with xenograft models,” Peggy Robinson, U.S. Vice President at ANGLE, a U.K.-based medical technology company. “CTDNA is limited mainly to mutation analysis.” 

ANGLE is commercializing the Parsortix™ cell separation system that harvests rare cells, such as circulating tumor cells (CTCs), for subsequent characterization and analysis. Think of Parsortix as the preparative front end for downstream tests such as FISH (fluorescence in situ hybridization), cell culture, protein staining, DNA/RNA analysis, and other techniques for characterizing CTCs and other rare cells. Parsortix has applications for many cell types of interest outside of oncology, and is currently being used to show equivalence to metastatic solid tumor biopsies.

ctDNA arises from apoptotic or necrotic cells, or those that have otherwise broken apart. But the relationship between ctDNA  and CTCs is not straightforward, however, as concentrations of one may rise while the other falls. So the two techniques are complementary, not mutually exclusive. Together they could provide oncologists with a much broader picture than either approach alone.

CELLSEARCH™ CTC is the only commercial test cleared by the U.S. Food and Drug Administration for enumerating CTCs to assess survival and progression-free survival in cancers of the breast, colon, and prostate. The test was approved under FDA’s 510(k) designation for “substantially equivalent” devices and diagnostics. 

Considered the gold standard for quantifying CTCs, CELLSEARCH uses epithelial cancer cell markers, including epithelial cell adhesion marker (EpCAM) to capture the cells, but not all cancer cells express that protein. According to Robinson, by capturing all rare cells the Parsotix system has the potential to provide a broader picture.

The presence or increase of CTCs in the presence of tumor shrinkage may provide insights into a patient’s immune system and the heterogeneity of the tumor itself. This frequently occurs when cancer is “successfully” removed but cells remain lurking in the body. “Getting to those metastatic sites can be otherwise difficult if not impossible,” Robinson says. It may not be obvious from scans that a patient is in danger, and where solid biopsies are not feasible or imaging is inadequate, studies are starting to demonstrate that CTC levels can more reflect metastatic potential. 

Robinson notes that cell-based liquid biopsies provide insight into tumor heterogeneity and represent a real-time method for monitoring tumor treatment. This offers the potentially to detect tumor recurrence before it can be visualized via standard methods. 

Reality Check

Liquid biopsies are new science based on rapidly-changing technologies. Whether they achieve their potential or fizzle out as so many technologies do is the ultimate question.

Guardant Health prefers to call its proprietary digital sequencing technique “biopsy-free testing” as opposed to liquid biopsy. Its technology overcomes the noise inherent in cancer-gene sequencing, thereby making exclusively blood-based testing a reality. As per this idea the company notes some shortcomings of liquid biopsies: They are not all noninvasive (e.g. collection of cerebrospinal fluid or bone marrow samples), CTCs are rare cells that require expansion before downstream testing, and some tests provide only a very narrow observational window. 

Guardant360, the company’s flagship test platform, overcomes the “window” issue by analyzing 68 clinically relevant genes. It broadly interrogates >150,000 bases – not just ten or so specific mutations.

Guardant’s critique of the state of the industry may be self-serving, but it underscores the harrowing diversity among tests, samples, and analytical platforms.

Surgeon-blogger Dr. David Gorski of the Karmanos Cancer Institute at Wayne State University in Farmington Hills, Michigan, has made a side career of debunking popular medical and scientific trends. Gorski says liquid biopsies “are not ready for prime time.” He notes that contrary to popular belief, early detection of certain cancers (or more so cancer markers) carries no advantage in terms of patient longevity or quality of life. And that “disease progression, including cancer progression, is not always a linear process.” For example prostate cancer lays dormant in many men for decades.

Gorski also cautions against claims by liquid biopsy developers and urges fellow practitioners to take the tests for what they are and no more. Given the heterogeneity mentioned previously, that appears to be sound advice. 

John Witte, Ph.D., co-director of the cancer genetics program in the UCSF Helen Diller Family Comprehensive Cancer Center, works on improving the sensitivity of liquid biopsies to monitor the development and progression of prostate cancers. From his perch as a not-too-disinterested participant, Witte describes the state of liquid biopsies work even more colorfully: “Right now it’s the Wild West.” Liquid biopsies might transform cancer screening and monitoring, but it’s anyone’s guess how the technology will play out. Nevertheless, he argues that “the potential value is enormous and well worth risky ventures.”

Angelo DePalma is a freelance writer living in Newton, New Jersey, USA.
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