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[...]the detection of very low amounts of ctDNA and CTCs in blood samples from early-stage cancer patients, and assessment of the possible clinical significance of the resulting findings, is still a challenge [4,5]. More sensitive technologies or the addition of other cancer-specific mutations, or combinations thereof, might in the future be able to increase the sensitivity of liquid biopsy in this type of setting. [...]quite large plasma volumes could be required for early cancer detection, because the ctDNA concentration in early-stage lung cancer patients can be as low as one genome equivalent in 5 ml blood [6].

Cell-free DNA (cfDNA) derived from tumours is present in the plasma of cancer patients. The majority of currently available studies on the use of this circulating tumour DNA (ctDNA) deal with the detection of mutations. The analysis of cfDNA is often discussed in the context of the noninvasive detection of mutations that lead to resistance mechanisms and therapeutic and disease monitoring in cancer patients. Indeed, substantial advances have been made in this area, with the development of methods that reach high sensitivity and can interrogate a large number of genes. Interestingly, however, cfDNA can also be used to analyse different features of DNA, such as methylation status, size fragment patterns, transcriptomics and viral load, which open new avenues for the analysis of liquid biopsy samples from cancer patients. This review will focus on the new perspectives and challenges of cfDNA analysis from mutation detection in patients with solid malignancies.

The detection and molecular characterization of circulating tumor cells (CTCs) are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker. The aims of research on CTCs include (a) estimation of the risk for metastatic relapse or metastatic progression (prognostic information), (b) stratification and real-time monitoring of therapies, (c) identification of therapeutic targets and resistance mechanisms, and (d) understanding metastasis development in cancer patients. This review focuses on the technologies used for the enrichment and detection of CTCs. We outline and discuss the current technologies that are based on exploiting the physical and biological properties of CTCs. A number of innovative technologies to improve methods for CTC detection have recently been developed, including CTC microchips, filtration devices, quantitative reverse-transcription PCR assays, and automated microscopy systems. Molecular-characterization studies have indicated, however, that CTCs are very heterogeneous, a finding that underscores the need for multiplex approaches to capture all of the relevant CTC subsets. We therefore emphasize the current challenges of increasing the yield and detection of CTCs that have undergone an epithelial-mesenchymal transition. Increasing assay analytical sensitivity may lead, however, to a decrease in analytical specificity (e.g., through the detection of circulating normal epithelial cells). A considerable number of promising CTC-detection techniques have been developed in recent years. The analytical specificity and clinical utility of these methods must be demonstrated in large prospective multicenter studies to reach the high level of evidence required for their introduction into clinical practice.

Different technologies, such as quantitative real-time PCR or microarrays, have been developed to measure microRNA (miRNA) expression levels. Quantification of miRNA transcripts implicates data normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data. In this review article we discuss the reliability of the use of small RNAs, commonly reported in the literature as miRNA expression normalizers, and compare different strategies used for data normalization. A workflow strategy is proposed for normalization of miRNA expression data in an attempt to provide a basis for the establishment of a global standard procedure that will allow comparison across studies.

Over the past decade, the amount of research and the number of publications on associations between circulating small and long non-coding RNAs (ncRNAs) and cancer have grown exponentially. Particular focus has been placed on the development of diagnostic and prognostic biomarkers to enable efficient patient management — from early detection of cancer to monitoring for disease recurrence or progression after treatment. Owing to their high abundance and stability, circulating ncRNAs have potential utility as non-invasive, blood-based biomarkers that can provide information on tumour biology and the effects of treatments, such as targeted therapies and immunotherapies. Increasing evidence highlights the roles of ncRNAs in cell-to-cell communication, with a number of ncRNAs having the capacity to regulate gene expression outside of the cell of origin through extracellular vesicle-mediated transfer to recipient cells, with implications for cancer progression and therapy resistance. Moreover, ‘foreign’ microRNAs (miRNAs) encoded by non-human genomes (so-called xeno-miRNAs), such as viral miRNAs, have been shown to be present in human body fluids and can be used as biomarkers. Herein, we review the latest developments in the use of circulating ncRNAs as diagnostic and prognostic biomarkers and discuss their roles in cell-to-cell communication in the context of cancer. We provide a compendium of miRNAs and long ncRNAs that have been reported in the literature to be present in human body fluids and that have the potential to be used as diagnostic and prognostic cancer biomarkers.

As the release of tumor-associated DNA into blood circulation is a common event in patients with cancer, screening of plasma or serum DNA may provide information on genetic and epigenetic profiles associated with breast cancer development, progression, and response to therapy. Quantitative testing of circulating DNA can reflect tumor burden, and molecular characterization of circulating DNA can reveal important tumor characteristics relevant to the choice of targeted therapies in individual patients. Contrary to circulating DNA from blood that presents molecular changes in tumor DNA in real time, tissue biopsies can deliver only a spatially and temporally limited snapshot of the heterogeneous tumor. Analyses of circulating DNA might provide prognostic and predictive information and therefore advance personalized medicine. However, standardization of different technical platforms as well as the control of pre-analytical and analytical factors is mandatory before its introduction into clinical practice. In the present review, we discussed technical aspects and clinical relevance of the analyses of circulating plasma/serum DNA in patients with breast cancer.

Prostate cancer represents the most common non-skin cancer type in men. Unmet needs include understanding prognosis to determine when intervention is needed and what type, prediction to guide the choice of a systemic therapy, and response indicators to determine whether a treatment is working. Over the past decade, the \"liquid biopsy,\" characterized by the analysis of tumor cells and tumor cell products such as cell-free nucleic acids (DNA, microRNA) or extracellular vesicles circulating in the blood of cancer patients, has received considerable attention. Among those biomarkers, circulating tumor cells (CTCs) have been most intensively analyzed in prostate cancer. Here we discuss recent studies on the enumeration and characterization of CTCs in peripheral blood and how this information can be used to develop biomarkers for each of these clinical contexts. We focus on clinical applications in men with metastatic castration-resistant prostate cancer, in whom CTCs are more often detected and at higher numbers, and clinical validation for different contexts of use is most mature. The overall goal of CTC-based liquid biopsy testing is to better inform medical decision-making so that patient outcomes are improved.

Editorial summary Progress in sensitive analytical approaches has opened new avenues for the detection of cells or products such as circulating cell-free DNA released by tumors. These ‘liquid biopsies’ are being explored in clinical trials for early cancer detection, prediction of recurrent disease, and assessment of therapeutic resistance mechanisms.

Because of improvements in the treatment of patients with metastatic breast cancer, the development of brain metastases (BM) has become a major limitation of life expectancy and quality of life for many breast cancer patients. The improvement of management strategies for BM is thus an important clinical challenge, especially among high-risk patients such as human epidermal growth factor receptor 2-positive and triple-negative patients. However, the formation of BM as a multistep process is thus far poorly understood. To grow in the brain, single tumor cells must pass through the tight blood–brain barrier (BBB). The BBB represents an obstacle for circulating tumor cells entering the brain, but it also plays a protective role against immune cell and toxic agents once metastatic cells have colonized the cerebral compartment. Furthermore, animal studies have shown that, after passing the BBB, the tumor cells not only require close contact with endothelial cells but also interact closely with many different brain residential cells. Thus, in addition to a genetic predisposition of the tumor cells, cellular adaptation processes within the new microenvironment may also determine the ability of a tumor cell to metastasize. In this review, we summarize the biology of breast cancer that has spread into the brain and discuss the implications for current and potential future treatment strategies.

Cancer metastasis is the main cause of cancer‐related death, and dissemination of tumor cells through the blood circulation is an important intermediate step that also exemplifies the switch from localized to systemic disease. Early detection and characterization of circulating tumor cells (CTCs) is therefore important as a general strategy to monitor and prevent the development of overt metastatic disease. Furthermore, sequential analysis of CTCs can provide clinically relevant information on the effectiveness and progression of systemic therapies (e.g., chemo‐, hormonal, or targeted therapies with antibodies or small inhibitors). Although many advances have been made regarding the detection and molecular characterization of CTCs, several challenges still exist that limit the current use of this important diagnostic approach. In this review, we discuss the biology of tumor cell dissemination, technical advances, as well as the challenges and potential clinical implications of CTC detection and characterization. Graphical Abstract An overview of the biology of tumor cell dissemination, including an in depth discussion of the current advances and limitations in the detection/isolation of circulating tumor cells and their potential prognostic and diagnostic value.