Volume 35, Issue 4

Liquid biopsies, in particular the profiling of circulating tumor DNA (ctDNA), have long held promise as transformative tools in cancer precision medicine. Despite a prolonged incubation phase, ctDNA profiling has recently experienced a strong wave of development and innovation, indicating its imminent integration into the cancer management toolbox. Various advancements in mutation-based ctDNA analysis methodologies and technologies have greatly improved sensitivity and specificity of ctDNA assays, such as optimized preanalytics, size-based pre-enrichment strategies, targeted sequencing, enhanced library preparation methods, sequencing error suppression, integrated bioinformatics and machine learning. Moreover, research breakthroughs have expanded the scope of ctDNA analysis beyond hotspot mutational profiling of plasma-derived apoptotic, mono-nucleosomal ctDNA fragments. This broader perspective considers alternative genetic features of cancer, genome-wide characterization, classical and newly discovered epigenetic modifications, structural variations, diverse cellular and mechanistic ctDNA origins, and alternative biospecimen types. These developments have maximized the utility of ctDNA, facilitating landmark research, clinical trials, and the commercialization of ctDNA assays, technologies, and products. Consequently, ctDNA tests are increasingly recognized as an important part of patient guidance and are being implemented in clinical practice. Although reimbursement for ctDNA tests by healthcare providers still lags behind, it is gaining greater acceptance. In this work, we provide a comprehensive exploration of the extensive landscape of ctDNA profiling methodologies, considering the multitude of factors that influence its development and evolution. By illuminating the broader aspects of ctDNA profiling, the aim is to provide multiple entry points for understanding and navigating the vast and rapidly evolving landscape of ctDNA methodologies, applications, and technologies.

This narrative review aims to provide a comprehensive overview of the current state of circulating tumor cell (CTC) analysis and its clinical significance in patients with epithelial cancers. The review explores the advancements in CTC detection methods, their clinical applications, and the challenges that lie ahead. By examining the important research findings in this field, this review offers the reader a solid foundation to understand the evolving landscape of CTC analysis and its potential implications for clinical practice. The comprehensive analysis of CTCs provides valuable insights into tumor biology, treatment response, minimal residual disease detection, and prognostic evaluation. Furthermore, the review highlights the potential of CTCs as a non-invasive biomarker for personalized medicine and the monitoring of treatment efficacy. Despite the progress made in CTC research, several challenges such as standardization, validation, and integration into routine clinical practice remain. The review concludes by discussing future directions and the potential impact of CTC analysis on improving patient outcomes and guiding therapeutic decision-making in epithelial cancers.

Body fluids contain cell-derived particles called extracellular vesicles (EVs). EVs are released by cells and are present in all body fluids (i. e. liquid biopsies). EVs contribute to physiology and pathology and offer a plethora of potential clinical applications, ranging from biomarkers to therapeutic applications. In this manuscript we provide an overview of this new and rapidly growing research field, along with its challenges and opportunities.

Genetic predisposition is one of the major measurable cancer risk factors. Affected patients have an enhanced risk for cancer and require life-long surveillance. However, current screening measures are mostly invasive and only available for certain tumor types. Particularly in hereditary cancer syndromes, liquid biopsy, in addition to monitoring therapy response and assessing minimal residual disease, holds great potential for surveillance at the precancerous stage and potentially even diagnostics. Exploring these options and future clinical translation could help reduce cancer risk and mortality in high-risk individuals and enhance patients’ adherence to tailored surveillance protocols.

Monitoring ctDNA by liquid biopsies seems to represent the perfect match for precision oncology and its cornerstone clinical framework: the molecular tumour board (MTB). Detecting and scrutinising the success of targeted therapies or tracking and, for that matter, addressing the therapy with the evolutive nature of a tumour are some of the main advancements one considers to be important for the MTB. One challenge is correlating the estimated allele frequency of each identified genetic alteration determined by analysing the ctDNA sequencing results and matching these with the range of suitable drugs, which may limit the simultaneous treatment of all tumour variations. This limitation arises because a new biopsy would typically be required to evaluate the response to treatment. As a result, evaluating the success of MTB recommendations relies on traditional staging methods, highlighting an existing diagnostic gap. Thus, optimising liquid biopsy technology could enhance the efficacy of MTB treatment recommendations and ensuing tailored therapies. Herein, we discuss the prospect of ctDNA analyses in the molecular tumour board.

Liquid biopsy for minimally invasive diagnosis and monitoring of cancer patients is progressing toward routine clinical practice. With the implementation of highly sensitive next-generation sequencing (NGS) based assays for the analysis of cfDNA, however, consideration of the utility of liquid biopsy for clinical genetic testing is critical. While the focus of liquid biopsy for cancer diagnosis is the detection of circulating tumor DNA (ctDNA) as a fraction of total cell-free DNA (cfDNA), cfDNA analysis reveals both somatic mosaic tumor and germline variants and clonal hematopoiesis. Here we outline advantages and limitations of mosaic and germline variant detection as well as the impact of clonal hematopoiesis on liquid biopsy in cancer diagnosis. We also evaluate the potential of cfDNA analysis for the molecular diagnosis of monogenic mosaic disorders.

It is now well-established that non-invasive prenatal testing (NIPT), originally designed to screen cell-free DNA (cfDNA) in maternal blood for the presence of common fetal trisomies, can lead to incidental detection of occult maternal malignancies. Retrospective evaluations have demonstrated that the detection of multiple copy number alterations in cfDNA is particularly suggestive of an incipient tumor and that cancer detection rates not only depend on tumor biology but also on applied NIPT technologies and downstream diagnostic investigations. Since the identification of a maternal cancer in pregnancy has implications for both woman and the unborn child, prospective studies are needed to provide evidence on best clinical practices and on clinical utility in terms of patient outcomes.