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Liquid Biopsy: Reading the Signs of Disease From Biofluid

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Liquid biopsy is a novel diagnostic tool, which seeks for signs of disease from various biofluids, such as blood and cerebrospinal fluid. Due to the ease of blood draw and relative ease of cerebrospinal fluid tap compared to tissue biopsies, liquid biopsies have been hailed as a minimally invasive method for diagnosing and monitoring disease progression and assessing response to therapy. Liquid biopsies work by detecting nucleic acids, proteins, cells and extracellular vesicles (packages of cellular content) in blood or cerebrospinal fluid. Liquid biopsies shed insight on disease characteristics by analyzing the nature of these biomolecules. In the context of cancer, for instance, this consists of detecting biomolecules shed by the growing solid tumor into circulation, i.e., blood. This includes circulating tumor-derived cell-free DNA (cfDNA) and tumor-derived circulating tumor cells (CTCs). cfDNA can harbor mutations present in the tumor. If an actionable driver mutation is identified, this can suggest potential therapeutic avenues, such as tyrosine kinase inhibitors against mutations to tyrosine kinases driving tumorigenesis.

A rising star: Liquid biopsies debut in the oncology clinic

A search of PubMed in September, 2022, using the term “liquid biopsy” yielded ~10,550 hits, of which ~7,260 or 69% were published in just the past 5 years. Therefore, it is a rapidly growing technology in a broad spectrum of disciplines. Cancer applications dominate, but active research is being pursued in several other medical fields, including prenatal testing in reproductive medicine, pathogen detection and host-cell interactions in infectious disease, and autoimmune disease, to name just a few. Moreover, liquid biopsy tests for cfDNA in cancer have even made their way into the clinic. Several liquid biopsy diagnostic tests of tumor-derived cfDNA have been cleared by the US Food and Drug Administration (FDA) as companion diagnostics for detecting various cancers and their corresponding actionable mutations. Thus, liquid biopsy is poised to make significant contributions to the clinical care of patients, especially in the oncology domain.

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Cancer liquid biopsy: Advancing approaches

Although liquid biopsies have been extensively leveraged in cancer biology to detect cfDNA, including FDA-cleared tests for breast, ovarian, prostrate and non-small cell lung cancers, research continues to expand the repertoire of amenable tumors and technology.

Over the past decade, Andrew K. Godwin, professor and division director of Genomics Diagnostics, Department of Pathology and Laboratory Medicine, The University of Kansas (KU) Medical Center and deputy director, KU Cancer Center, has been developing liquid biopsy platforms to analyze the various materials shed by tumors to improve patient care. Godwin’s work bridges the “valley of death” between basic science research and the clinical domain. “I became involved in translational medicine before it was even recognized as a research field. A major focus of my career has been to molecularly evaluate patient samples to help guide their therapy, primarily in cancer. I seek methods that can enhance early detection, facilitate diagnosis, and monitor disease burden and response to therapy. Liquid biopsy is an exceptional tool to achieve these goals,” Godwin explained of the motivation behind his research.

“When liquid biopsies first emerged, they detected CTCs, and then evolved to look at other circulating models, such as cfDNA, and now more recently cell-free RNA. There has been an explosion of liquid biopsy tests for cfDNA, which seek tumor mutations when a tissue biopsy cannot be performed,” continued Godwin. In addition to his extensive work on cfDNA, Godwin has gained interest in extracellular vesicles over the past decade. “Undoubtably cfDNA is an excellent biomarker of tumors. However, my philosophy is that we can derive more information from an integrated approach to liquid biopsy by examining additional materials shed by tumors, such as extracellular vesicles.” Extracellular vesicles are membrane bound packages released by cells, which express various surface markers related to their cell of origin and carry cargo ranging from RNAs, proteins and metabolites. “Extracellular vesicles can serve as cancer biomarkers based on their surface and cargo content. For example, they can carry mRNA harboring tumor mutations or biomolecules related to the tumor pathophysiology. We can also leverage them for early cancer detection, diagnosis, identify mechanisms of tumor invasion and drug resistance, and monitor response to therapy,” elaborated Godwin.

Further, the Godwin lab and colleagues are developing microfluidic platforms to isolate circulating cancer biomarkers, including cfDNA or extracellular vesicles. Microfluidic devices are comprised of micro-meter sized channels and chambers that perform analyzes by rapidly manipulating small volumes of liquids. “Microfluidic platforms offer many advantages for isolating biomarkers because they combine the capture or enrichment step, i.e., of cfDNA or extracellular vesicles, with the molecular profiling and/or quantification step, which can be performed ‘on chip’. Thus, they streamline the analytical process,” described Godwin of the technology.

“Another important benefit of microfluidics is the ability to work with small volumes, so that only a tiny amount of patient biofluid sample is needed. We recently illustrated the feasibility of using microfluidic devices to detect exosomes in only 2 micro-liters of plasma from ovarian cancer patients,” Godwin explained of the seminal study. Ovarian cancer is hard to detect early. Known as the cancer that whispers, it manifests with symptoms that women tend to ignore. Unfortunately, it is usually widespread by the time of diagnosis, making it difficult to treat and prognosis poor. Analysis of plasma from 20 ovarian cancer patients versus 10 age-matched controls indicated that extracellular vesicles expressing the folate receptor alpha could potentially serve as a biomarker for early detection and progression.

Godwin and his research team are also investigating the potential of liquid biopsy for the Ewing sarcoma family of tumors, or ESFT for short. ESFT is a cancer of the bones or soft tissues, molecularly characterized by a chromosomal translocation EWS-ETS. “Although the EWS-ETS translocation is a strong molecular diagnostic of ESFT, unfortunately it requires tissue biopsy of the tumor, which can be painful or inaccessible. Liquid biopsy can offer a potential solution. We can detect EWS-ETS mRNA in extracellular vesicles from a cell model, and, more recently, demonstrated the power of a proteomic signature in extracellular vesicles for diagnosing ESFT,” explained Godwin of the findings.

Godwin continues to drive his research forward, conducting longitudinal studies to assess the utility of liquid biopsy for early detection in at risk populations, such as in BRCA1 and BRCA2 mutation carriers. “I can envision a rapid and sensitive liquid biopsy test for screening of woman at increased risk for ovarian cancer,” he said of the ultimate goal. “Technology drives science and the test may eventually be administered in a point-of-care format, where patients would get their results directly during their visit for their annual exam.”

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Liquid biopsy for organ injury: Paving the way to a generalizable platform

Although cancer biology and the field of oncology have comprised the bulk of liquid biopsy studies, there are research pursuits to expand applications in other areas. “Liquid biopsy is useful for a broad range of diagnostic applications. In addition to cancer, liquid biopsy may be leveraged to monitor organ injury, an area of great interest to us,” elaborated Iwijn De Vlaminck, professor of biomedical engineering at Cornell University, of his research. “Organ injury can occur after transplants and during infectious or immune-related diseases and complications. Liquid biopsy methodology features several advantages to monitor organ injury. First, it is minimally invasive, which causes less discomfort to patients and thus can be employed longitudinally. Second, liquid biopsy can be applied in an untargeted format by leveraging molecular technologies to characterize and quantify cfDNA molecules released by injured organs into biofluid.”

Specifically, De Vlaminck and his team pinpoint what organ cfDNA derives from based on distinct molecular signatures. “Organ injury is a common phenomenon that occurs during various disease processes. Necrotic or apoptotic cells from injured organs spill cfDNA into circulation, which we detect and analyze, generating a well of information on the affected tissues and organs. Organ injury may occur following transplants in patients with terminal organ failure or hematologic cancers. Infection can trigger an inflammatory response, which can cause organ damage.”

In earlier work, De Vlaminck demonstrated that donor-derived cfDNA could be identified in plasma from organ recipients of heart and lung transplants. In both instances, purified cfDNA was sequenced to quantify the fraction attributable to the organ donor, which correlated positively with host rejection by tissue biopsy. Organ recipients are placed on an immunosuppression regimen to lower the risk of transplant rejection, which unfortunately raises patient susceptibility to infection. Indeed, cytomegalovirus, herpesvirus, and adenovirus sequences were present among the nonhuman cfDNA, indicating possible infections. “More recently we reported a comprehensive liquid biopsy platform to assay cfDNA in allogeneic hematopoietic cell transplant recipients for hematologic malignancies. We leveraged cfDNA methylation signatures to trace tissue origin, pinpointing organ-specific damage in recipients, and analyzed fraction of donor-to-recipient cfDNA to evaluate graft rejection. In parallel, we identified mutations in tumor-derived cfDNA to assess cancer relapse and performed cfDNA metagenomic profiling to assess infection,” explained De Vlaminck. Analysis of 170 plasma samples from 27 hematopoietic cell transplant recipients found that elevated solid-organ-derived plasma cfDNA, indicative of generalized organ damage, within one month of transplant correlated with graft-versus-host disease, an occasional immune complication in recipients. Moreover, previous findings were replicated, with higher donor cfDNA fraction predictive of graft rejection, and viral infection and cancer relapse could be detected.

Another focus of the De Vlaminck lab is organ damage as a consequence of infectious disease. “We did a study of urine samples from kidney transplant recipients with and without bacterial or viral urinary tract infection. Of course, we detected the infectious agent using metagenomic profiling of urinary cfDNA, as anticipated. However, in tandem, we also found methylated cfDNA signatures indicative of virus- and bacteria-induced kidney and bladder tissue damage in patients with urinary tract infection,” De Vlaminck concluded of the study.

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De Vlaminck and his team also leveraged their liquid biopsy platform based on cfDNA methylation signatures to monitor organ injury as a surrogate of infection severity in COVID-19 patients. “Very early in the pandemic it was clear that diagnostic tools were needed to assess severity of COVID-19 infection that focused on the patient’s status,” De Vlaminck discussed of his motivation for the study. “So, we investigated cfDNA signatures of organ injury in plasma samples from COVID-19 patients versus those from patients with other viral infections and healthy controls.” cfDNA harboring methylation signatures suggestive of lung, liver and red blood cell progenitor damage were linked to severe COVID-19. Total cfDNA associated with the World Health Organization ordinal scale for disease progression and was significantly elevated in intubated patients. 

De Vlaminck thinks that the information held within cfDNA in liquid biopsy could be very useful in research, and eventually, the clinic. “The goal is to build a generalizable liquid biopsy platform to quantity and pinpoint organ injury. This would be a minimally invasive, rapid, and untargeted diagnostic tool, which could generate a comprehensive picture of organ damage in a patient within a single test.”

Over the past few years, liquid biopsies have rapidly evolved from the research domain to the clinic for cfDNA as a companion diagnostic in cancer. However, research continues to expand the scope of tumor-derived content amenable to liquid biopsy, potentially deriving additional information. Moreover, research pursues the liquid biopsy platform in areas other than cancer to broaden potential applications in medicine.