Several studies have revealed the presence of tumor-derived nucleic acids in other body fluids, such as urine, saliva8, and CSF. In patients with lung cancer, ctDNA has also been detected in bronchial washings and pleural fluids. Indeed, the localization of the first tumour and of any metastatic lesions seems to possess a serious effect on the abundance of ctDNA in different body fluids.

At present, quantification of tumor-derived tr­DNA in urine is technically challenging, owing mainly to the low amounts present, although the continual development of DNA-amplification and sequencing technologies will probably facilitate this approach. Moreover, we should be careful of the lik

Several studies have revealed the presence of tumor-derived nucleic acids in other body fluids, such as urine, saliva8, and CSF. In patients with lung cancer, ctDNA has also been detected in bronchial washings and pleural fluids. Indeed, the localization of the first tumour and of any metastatic lesions seems to possess a serious effect on the abundance of ctDNA in different body fluids.

At present, quantification of tumor-derived tr­DNA in urine is technically challenging, owing mainly to the low amounts present, although the continual development of DNA-amplification and sequencing technologies will probably facilitate this approach. Moreover, we should be careful of the likelihood that liquid biopsy of urine might be preferable to the utilization of other body fluids, as this approach may be a truly non­invasive alternative to biopsy sampling and may be performed reception by the patients themselves. Liquid biopsy of tr-DNA offers the fascinating possibility of monitoring minimal residual disease (MRD) after surgery with curative intent.

Technologies to analyse ctDNA
Circulating cfDNA is primarily composed of germ line DNA that originates from normal cells, with
a relatively small and highly variable fraction of ctDNA present in patients with cancer. As such, the sensitivity of traditional approaches to DNA analysis (such as Sanger sequencing) is insufficient for detection of somatic mutations in plasma ctDNA from patients with cancer.

To overcome these limitations, digital-PCR-based technologies with a high level of analytical sensitivity and specificity are developed, enabling high-throughput, targeted amplification of the gene of interest on the background of abundant wild type alleles, reaching limits of detection below 0.0001% — which is mandatory for the detection of rare aberrations in ctDNA.

In addition, non-targeted genome-wide analyses enable the identification of tumor-specific alterations without prior knowledge of the aberrations likely to be present in the tumor therefore, such approaches are often exploited for de novo discovery of genetic changes underlying therapy resistance and therefore the identification of latest actionable targets in patients with cancer. NGS may be a technique that involves immobilization of DNA fragments on a solid support and reading of the sequence as a neighborhood of a DNA-synthesis process.

Using NGS, many ctDNA sequences are often produced during a single reaction, and are subsequently aligned and compared with a reference genome or to the germ line DNA obtained from an equivalent patient (that is, from nonmalignant tissue, typically peripheral blood mononuclear cells), making it possible to spot nucleotide changes (variants or mutations) relative to the reference sequence. Several NGS-based methods have now been devised that enable the detection of not only point mutations and insertions, deletions or rearrangements, but also copy-number alterations and gene fusions.

In the future, digital PCR and NGS will probably both be used complementarily in liquid biopsy analyses. The former approach enables dynamic profiling of individual mutations, but requires a priori knowledge of the mutant allele, whereas the latter technique enables the invention of novel mutated variants, but has higher costs and cannot be readily applied to watch patients longitudinally.

Clinical applications of liquid biopsies

The potential of liquid biopsy assays is way reaching, and their wide-ranging clinical applications are only starting to emerge. Liquid biopsies are often exploited for diagnostic purposes, to spot and track tumor-specific alterations during the course of the disease, and to guide therapeutic decisions107. Clinical implementation will only be achievable, however, if standardized procedures are defined and enormous validation studies are performed.

CTCs as biomarkers and their clinical utility. At the present, the clinical value of CTC analysis remains controversial, although evidence indicates that the abundance of tumor cells within the blood of patients with cancer has prognostic value, which CTC numbers after treatment can be predictive of response to therapy and, thus, treatment outcomes. These findings must be considered with caution, however, because CTC numbers are highly variable between different tumor types, and are subject to biases concerning the variability of CTC-detection methods used. Moreover, the correlation between the number of CTCs detected and patient survival is way
from being defined108, and this limitation is probably going to be overcome only by combining different technologies to improve assay performance.

ctDNA in cancer diagnosis. Quantitative chemical analysis of cfDNA are often wont to assess tumors burden — with diagnostic implications. For instance, the quantity of cfDNA present in plasma is substantially higher in patients with cancer than in healthy individuals or in patients with benign diseases, and seems to increase with tumors stage (and presumably, therefore, tumor volume). Moreover, cfDNA measurements could potentially be wont to determine if a patient is
disease free after curative surgery (and ctDNA analysis are going to be more useful during this regard: see ‘MRD monitoring and early diagnosis of relapse — ctDNA as prognostic biomarker’). In the diagnosis of cancer, the absolute levels of cfDNA in the circulation provide limited; however, when levels of cfDNA are including identification of somatic mutations (that is, that specialize in ctDNA), they supply valuable diagnostic.

Methylation profiles in ctDNA — predicting response to chemotherapy. Promoter hypermethylation at specific CpG sites related to tumors-suppressor genes occurs in many cancers; therefore, methylated ctDNA is a promising biomarker. Several studies have compared
aberrant methylation in tumors tissues and matched ctDNA from blood samples, in settings like lung, gastrointestinal, breast, ovarian, prostate, testicular, and head and neck cancer, and in most cases a good correlation.

Detection of promoter hypermethylation in ctDNA might have higher sensitivity than analyses of instability in microsatellite DNA (at unique GT/CA repeats, either mononucleotide microsatellite sites, like BAT25 and BAT26, or dinucleotide sites, like D2, D5S346, and D17S250), which could potentially be further improved if combined with mutational analysis.