Volume 46, Issue 4 - Special Issue: Liquid Profiling – Circulating Nucleic Acid Diagnostics; Issue Editors: Stefan Holdenrieder, Hanns-Georg Klein and Christof Winter

Genomes of diverse origins are continuously shed into human body fluids in the form of fragmented cell-free DNA (cfDNA). These molecules maintain the genetic and epigenetic codes of their originating source, and often carry additional layers of unique information in newly discovered physico-chemical features. Characterization of cfDNA thus presents the opportunity to non-invasively reconstruct major parts of the host- and metagenome in silico . Data from a single specimen can be leveraged to detect a broad range of disease-specific signatures and has already enabled the development of many pioneering diagnostic tests. Moreover, data from serial sampling may allow unparalleled mapping of the scantily explored landscape of temporal genomic changes as it relates to various changes in different physiological and pathological states of individuals. In this review, we explore how this vast dimension of biological information accessible through cfDNA analysis is being tapped towards the development of increasingly powerful molecular assays and how it is shaping emerging technologies. We also discuss how this departure from traditional paradigms of snapshot genetic testing may pave the way for an onrush of new and exciting discoveries in human biology.

Circulating tumor DNA (ctDNA), accurately described by the term liquid profiling (LP), enables real-time assessment of the tumor mutational profile as a minimally invasive test and has therefore rapidly gained traction, particular for the management of cancer patients. By LP, tumor-specific genetic alterations can be determined as part of companion diagnostics to guide selection of appropriate targeted therapeutics. Because LP facilitates longitudinal monitoring of cancer patients, it can be used to detect acquired resistant mechanisms or as a personalized biomarker for earlier detection of disease recurrence, among other applications. However, LP is not yet integrated into routine care to the extent that might be expected. This is due to the lack of harmonization and standardization of preanalytical and analytical workflows, the lack of proper quality controls, limited evidence of its clinical utility, heterogenous study results, the uncertainty of clinicians regarding the value and appropriate indications for LP and its interpretation, and finally, the lack of reimbursement for most LP tests. In this review, the value proposition of LP for cancer patient management and treatment optimization, the current status of implementation in standard care, and the main challenges that need to be overcome are discussed in detail.

The liquid profiling approach is currently at the threshold of translation from research application in various clinical trials to introduction into the management of cancer patients in the context of clinical care. Routine application has focused primarily on the analysis of single blood-based biomarkers for companion diagnostics. However, liquid profiling promises much broader diagnostic potential, which is discussed and illustrated in this manuscript through several case reports. These clinical cases range from identification of druggable targets to the detection of subclonal resistance mechanisms. In addition, liquid profiling can be used in clinical practice to identify complex molecular patterns or as a personalized tumor marker for patient-specific monitoring of response to treatment. These examples highlight both the value and limitations of liquid profiling in various clinical settings, which could be overcome by an integrative diagnostic approach with imaging techniques. The complementary advantages of both diagnostics approaches will allow combining high sensitivity with genetic tumor profiling and topologic assignment. Therefore, we advocate interdisciplinary integrated collaboration between laboratory and imaging experts to unravel the potentials of precision diagnostics in cancer care.

Early detection is crucial for optimal treatment and prognosis of cancer. New approaches for pan-cancer screening comprise the comprehensive characterization of circulating tumor DNA (ctDNA) in plasma by next generation sequencing and molecular profiling of mutations and methylation patterns, as well as fragmentation analysis. These promise the accurate detection and localization of multiple cancers in early disease stages. However, studies with real screening populations have to show their clinical utility and practicability.

The heterogeneity of each individual oncologic disease can be mirrored by molecular analysis of a simple blood draw in real time. Liquid biopsy testing has been shown useable for cancer detection, proof of minimal residual disease, therapy decision making and monitoring. However, an individual blood analyte does not present a comprehensive picture of the disease. It was recently shown that multi-modal/multi-parametric/multi-analyte liquid biopsy testing has the advantage of generating a high-resolution snapshot of the disease complexity. The different blood analytes such as circulating tumor cells, circulating immune cells, tumor-educated platelets, extracellular vesicles, cell-free DNA, cell-free RNA and circulating proteins complement each other and have additive value for clinical cancer management. We, here, like to review the studies leading to these promising conclusions and like to, at the end, mention that many challenges lie ahead before the translation into the clinic can be accomplished, including issues concerning clinical utility, method standardization, cost reimbursement and data management.

Liquid biopsies are a valuable non-invasive biomarker source for the diagnosis, prognosis and monitoring of cancer patients. The detection of circulating cell-free DNA (cfDNA) derived from tumor cells (ctDNA) has emerged as a promising clinical approach, as their levels are elevated in many cancers and contains tumor-related mutations and specific methylation patterns. ctDNA can be released from tumor cells into the bloodstream, either linked to extracellular vesicles (EV-DNA) or in an EV-free form when associated with nucleosomes and other proteins, or even as a component of macromolecular structures such as neutrophil extracellular traps (NET DNA). These different types of cfDNA can mirror cancer progression and predict patient outcome. This review presents the recent benefits of cfDNA in cancer, distinguishing between EV-DNA and EV-free DNA, and highlights their clinical utility.

The diagnostic potential of exosomes and extracellular vesicles (EVs) for liquid biopsies was first demonstrated over a decade ago, but despite a lot of progress in the scientific field there are still very few applications of EVs that are ready for implementation in clinical laboratories for routine diagnostic use. Despite good options for routine isolation of EVs and a wide analyte target space for assay development (incl. RNA, DNA, proteins and intact EVs) assessable by standard detection technologies, the attrition rate in translating biomarker reports in the academic literature to clinical assays is very high. While there are examples of successful development, the largest obstacle to increased clinical utilization is the lack of good biomarkers that can withstand rigid clinical validation, and which make use of the EVs’ unique capabilities as a biomarker platform.

Programmed cell death, accidental cell degradation and active extrusion constantly lead to the release of DNA fragments into human body fluids from virtually all cell and tissue types. It is widely accepted that these cell-free DNA (cfDNA) molecules retain the cell-type specific genetic and epigenetic features. Particularly, cfDNA in plasma or serum has been utilized for molecular diagnostics. The current clinically implemented liquid biopsy approaches are mostly based on detecting genetic differences in cfDNA molecules from healthy and diseased cells. Their diagnostic potential is limited to pathologies involving genetic alterations, by the low proportion of cfDNA molecules carrying the mutation(s) relative to the total cfDNA pool, and by the detection limit of employed techniques. Recently, research efforts turned to epigenetic features of cfDNA molecules and found that the tissue-of-origin of individual cfDNA molecules can be inferred from epigenetic characteristics. Analysis of, e.g., methylation patterns, nucleosome or transcription factor binding site occupancies, fragment size distribution or fragment end motifs, and histone modifications determined the cell or tissue-of-origin of individual cfDNA molecules. With this tissue-of origin-analysis, it is possible to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies beyond genetics and towards a wide range of pathologies, such as autoimmune disorders, cardiovascular diseases, and inflammation, among many others. In this review, we give an overview on the status of tissue-of-origin approaches and focus on what is needed to exploit the full potential of liquid biopsies towards minimally invasive screening methods with broad clinical applications.

Background Physical activity can have a strong impact on the concentration of several promising candidate biomarkers, including cell-free DNA (cfDNA). Content This narrative review describes the current understanding of how physical strain leads to increases of cfDNA and discusses how this interferes with attempts to standardize cfDNA analysis in clinical laboratory medicine. Summary In general, all cells of the human body can release DNA, whereas neutrophils are described as the major source releasing cfDNA under resting conditions. Event at low physical load, cfDNA is rapidly released by immune cells. We recently, identified neutrophils as the major cell-type contributing to cfDNA increases during acute exercise. Both, endurance and strength training can affect the signal-to-noise ratio of liquid biopsy (LB) analysis, affecting the clinical validity between minutes up to several days. Furthermore, we discuss why physical distress of various kinds in a perioperative cancer setting can improve or compromise signal-to-noise. Therefore, physiological events including, but not limited to, activation of blood cells can provoke pre-analytical challenges for ultra-sensitive detection of cfDNA in LB settings. Outlook We discuss why future attempts to standardize liquid biopsy may therefore profit from a deeper understanding of the physiological release mechanisms of cfDNA.

Clonal hematopoiesis of indeterminate potential (CHIP) is a hematologic precursor lesion that is defined by the presence of somatic mutations in peripheral blood cells but without evidence for the presence of leukemia or another hematologic neoplasm. CHIP is frequent in elderly individuals and can be detected as incidental finding in liquid profiling of cell-free DNA. While liquid profiling assays aim to reduce the biological noise generated by CHIP and to discriminate solid cancer-associated from CHIP-associated mutation profiles, the finding of CHIP is of potential clinical relevance at its own. Overall, CHIP is associated with a moderate risk of progression to an overt hematologic neoplasm of 1% per year. The risk increases substantially in patients with unexplained blood count abnormalities, multiple mutations, or specific patterns of mutations. In patients with solid cancer, the presence of CHIP increases the risk for development of treatment-related myeloid neoplasms. In addition, CHIP has been associated with a number of non-hematological diseases and represents a previously unrecognized major risk factor for cardiovascular disease. The management of individuals diagnosed with CHIP includes both hematologic and cardiovascular risk assessment in a multidisciplinary setting. Additional evidence from interventional studies is needed to integrate CHIP into a personalized treatment approach for patients with solid cancer.

Since 2012, non-invasive prenatal testing (NIPT) using cell-free DNA from maternal plasma is applied all over the world as highly efficient first-line or contingent screening approach for trisomy 13, 18 and 21. With further technical development the screening has expanded to other genetic conditions such as sex chromosome anomalies (SCAs), rare autosomal trisomies (RATs), microdeletions/microduplications, structural chromosomal aberrations and monogenic diseases. Meanwhile, commercial providers are offering a number of different tests, with variable performance, the application of which needs to be carefully evaluated to apply to the true needs of clinical practice. In our review we present the different NIPT methodologies and discuss the main strengths and limitations in the context of providing a responsible pregnancy management.

Testing for genetic alterations in tumor tissue allows clinicians to identify patients who most likely will benefit from molecular targeted treatment. EXLIQUID – exploiting liquid biopsies to advance cancer precision medicine – investigates the potential of additional non-invasive tools for guiding therapy decisions and monitoring of advanced cancer patients. The term “liquid biopsy” (LB) refers to non-invasive analysis of tumor-derived circulating material such as cell-free DNA in blood samples from cancer patients. Although recent technological advances allow sensitive and specific detection of LB biomarkers, only few LB assays have entered clinical routine to date. EXLIQUID is a German Cancer Consortium (DKTK)-wide joint funding project that aims at establishing LBs as a minimally-invasive tool to analyze molecular changes in circulating tumor DNA (ctDNA). Here, we present the structure, clinical aim, and methodical approach of the new DKTK EXLIQUID consortium. Within EXLIQUID, we will set up a multicenter repository of high-quality LB samples from patients participating in DKTK MASTER and local molecular tumor boards, which use molecular profiles of tumor tissues to guide targeted therapies. We will develop LB assays for monitoring of therapy efficacy by the analysis of tumor mutant variants and tumor-specific DNA methylation patterns in ctDNA from these patients. By bringing together LB experts from all DKTK partner sites and exploiting the diversity of their particular expertise, complementary skills and technologies, the EXLIQUID consortium addresses the challenges of translating LBs into the clinic. The DKTK structure provides EXLIQUID a unique position for the identification of liquid biomarkers even in less common tumor types, thereby extending the group of patients benefitting from non-invasive LB testing. Besides its scientific aims, EXLIQUID is building a valuable precision oncology cohort and LB platform which will be available for future collaborative research studies within the DKTK and beyond.