Since becoming commercially available over the last 10–15 years, liquid biopsies have been enthusiastically embraced, particularly for noninvasive prenatal screening and increasingly for oncology diagnostics. Liquid biopsies also are emerging as helpful tools for monitoring transplant patients, and a range of other conditions and diseases are following. Strengthening every link in the process — from sample to insight — is key to expanding clinical use of liquid biopsies.
Delivering patient benefits today
Unquestionably, liquid biopsies — still a relatively young science — have been on the fast track in basic and translational research and are emerging as a powerful diagnostic tool. Importantly, they also are gaining ground in companion diagnostics for personalized medicine, helping doctors to stratify patients for suited therapies and to understand a patient’s response to those therapies. Their ability to derive information from throughout the body, rather than a single site tissue sample, is making them increasingly critical diagnostic aids to discover residual and even reoccurring disease. For this reason, they hold significant potential for broadscale screening tests for cancer and other diseases.
From a patient’s perspective, they are a winner. The collection of simple blood, urine, or even saliva samples are far less invasive, easier to collect, and less risky than more invasive, traditional tissue samples, particularly where lesions are difficult to access or present risk of hemorrhage or other complications. To be clear, it is unlikely that extracted tumor tissue or tissue biopsies will be unseated from their primacy in molecular and other pathology tests and technologies such as staining and immunohistochemistry for diagnostic and prognostic purposes. Nevertheless, liquid biopsies have become a strong complementary or alternate tool in patient care.
For example, tissue samples of a tumor remain important to grade and stage a tumor, but prior to and after both surgery and other treatment, liquid biopsies can deliver important information about remaining undetected metastasis, the tumor’s response to therapy, and whether additional or alternate treatments are necessary. Liquid biopsies also offer clear utility in post-surgical patient surveillance for discovering tumor relapses, finding minimal residual disease (MRD), and earlier recognition of new metastases.
The method gives treating oncologists (and their patients) a proven method to understand treatment decisions in their battle with many forms of cancer.
Expanded use relies on sensitivity, specificity
Understanding, treating, and monitoring other diseases and conditions is a rapidly developing area for liquid biopsies. Much like any other evolving technology for diagnostics, liquid biopsies require more development to realize their full potential in additional applications. Sensitivity, specificity, and accuracy, among other test characteristics are paramount. Today, the race is on to achieve these goals and enable expanded adoption in clinical practice, particularly in patient stratification required for personalized medicine, early cancer screening, and the screening and detection of other diseases.
Liquid biopsies derive improved sensitivity from a specified, verified, and validated diagnostic workflow — from sample collection, to the dedicated analytical diagnostic test, to treatment insight. Liquid biopsy-based diagnostics, therefore, rely on the integrity and precision of the processes around them. Hence, it is crucial to choose the right state-of-the-art platform, including instruments, chemistries, software, bioinformatics, and all workflow components for the application targeted.
Another driver in the broad effort to improve sensitivity is the potential offered by the combining of tests for the different components of a liquid biopsy sample. Such multimodal approaches would offer a much sharper picture of the patient’s system including circulating cell-free DNA (ccfDNA), including methylated DNA, RNA in extracellular vesicles (such as exosomes), and molecular profiles of circulating tumor cells. Combining liquid biopsy insights from different specimens from the same patient, such as parallel analysis from blood and urine, can further broaden this approach. The multimodal capability offers tremendous insight for the patient profile and a roadmap for future application.
Sensitivity relies on the absolute integrity of the entire workflow
An entire diagnostic workflow takes place at different locations. It starts with patient preparation at home for professional specimen collection. It continues at a hospital or doctor’s office with the specimen collection itself, including analytes stabilization for preventing post-collection changes. Next comes specimen storage and transport to the medical laboratory where the carrier’s workflow is critical (e.g., temperature, duration). The next stages of the workflow are within the purview of the laboratory where the isolation of the required analytes, such as ccfDNA, occurs. The analytical test itself is conducted along with bioinformatic analysis, and then the post-analytical steps such as the creation of the test report for the physician and patient are completed.
The validity of that report is only as strong as the weakest link in the workflow chain — be it an unsuited specimen collection device, unsterile catch or blood draw, mislabeling of a sample, out-of-specification temperature variance during sample transport, a nucleic acid isolation that causes bias in the targeted analyte profile, varying analytical test technology and so on.
It cannot be overstated that liquid biopsies highly depend on the strength and integrity of the entire workflow. During the development of a diagnostic test, it is critical to assess risk and specify, verify, and validate all workflow steps.
Strengthening every link in the chain
Several studies have demonstrated that the largest contribution to diagnostic errors comes from the pre-analytical workflow steps. The molecular profile of clinical samples can change drastically during the pre-analytical workflow, which involves all steps from specimen collection to the isolation of the target analytes, such as nucleic acids.
Specifically, transporting, storing, or archiving a specimen, or isolating the molecules carried in the sample, can change analytical target profiles, such as circulating cell-free nucleic acid profiles including circulating cell-free tumor DNA (ctDNA) and circulating tumor cells (CTCs), upon which most liquid biopsies rely. Absent a rigorously guided workflow, the tests bear a high risk that the diagnostic analytical test cannot measure the analyte profile as it was in the patient body. Instead, the test studies artificial levels that were generated post collection during the pre-analytical workflow, thus risking a wrong or unreliable diagnostic result. For most liquid biopsy applications, it is therefore key to preserve the analytical target profiles in liquid biopsies by using specimen collection devices with stabilizers for ensuring that such critical changes, such as those caused by post-collection cellular changes or chemical modifications, do not occur.
Circulating cell-free DNA (ccfDNA) offers a good example. This analyte provides real-time mutational information that can be used to detect and monitor biomarkers of cancer and other diseases in a simple blood test. Circulating ccfDNA is often found in low concentrations with rare mutations or in a fragmented condition that can undergo critical post-collection changes as white blood cells die after removal from their natural environment in the body. In this process, white blood cells release huge amounts of additional DNA that can significantly reduce the analytical test sensitivity, leading to false negative results. Reliable tools offer confidence in the signal detected (or not) from a sample.
If the entire diagnostic workflow including its pre-analytical steps is not specified, verified, and thus standardized for the dedicated diagnostic test by developers and manufacturers and then managed by users according to the diagnostic product’s instructions for use, the research and diagnostic results can be invalid or unreliable. Preserving every possible target analyte profile through a well-managed workflow — and analyzing them with equal rigor — offers the most profound basis for obtaining correct and reliable information for diagnostics and treatment consideration.
Developing verified workflows
The importance of workflow integrity as a prerequisite for the continued use and expanding future of high-quality liquid biopsies, is widely appreciated. As a result, steps in the process are being verified and standardized.
Diagnostic companies and diagnostic laboratories are increasingly focused on specified and verified generic pre-analytical workflows for different specimen types and analytical targets. These verifications can include tools such as the specimen collection devices with analyte stabilizers and nucleic acid isolation kits; links to state-of-the-art, well-designed, specified, and verified analytical test technologies including related instrument platforms (such as NGS [next generation sequencing], qPCR [quantitative polymerase chain reaction], and dPCR [digital polymerase chain reaction]) with verified software; and integrated post-analytical steps such as bioinformatics for data analysis and interpretation. All are increasingly becoming the building blocks and basis for the development of new, safe, and reliable analytical tests.
Automation has become more crucial than ever, especially with increased throughput capabilities. Standardization of target analyte isolation and analysis can be achieved by integrated, automated instruments for sample processing including nucleic acid extraction as well as for analytical, including multimodal testing. The automation requires reliable hardware and software as well as optimized chemistry to ensure maintenance of required sensitivity and specificity.
Developing evidence-based standards for pre-analytical, analytical and post-analytical workflows for routine in-vitro diagnostics is critical work being done in Europe and in the United States. Based on scientific evidence, new standards are mostly developed via the International Organization for Standardization (ISO) Committee “Clinical laboratory testing and in vitro diagnostic test systems” for the global markets or European Committee for Standardization (CEN) Committee “In vitro diagnostic medical devices” for the European markets if global standards are not available. QIAGEN and sample workflow specialist PreAnalytiX were drivers of consortia for the first large pre-analytical standards projects in this field. Guidance documents also are published by other professional institutions such as the U.S. Food and Drug Administration and Clinical & Laboratory Standards Institute (CLSI).
Conclusion
Liquid biopsies offer genuinely exciting potential for understanding disease and treating patients across the entire continuum of care. With stronger specified, verified, validated, and complete diagnostic workflows, liquid biopsy diagnostics will shed any lingering doubt on the quality of results, benefitting researchers who will have more and faster reproducible data to drive the discovery and development of new biomarkers. And new biomarkers will allow care providers to expand the clinical use of minimally invasive biopsies as part of a new generation of patient care.
