Circulating tumor cells and DNA as liquid biopsies

Abstract

For cancer patients, the current approach to prognosis relies on clinicopathological staging, but usually this provides little information about the individual response to treatment. Therefore, there is a tremendous need for protein and genetic biomarkers with predictive and prognostic information. As biomarkers are identified, the serial monitoring of tumor genotypes, which are instable and prone to changes under selection pressure, is becoming increasingly possible. To this end, circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) shed from primary and metastatic cancers may allow the non-invasive analysis of the evolution of tumor genomes during treatment and disease progression through 'liquid biopsies'. Here we review recent progress in the identification of CTCs among thousands of other cells in the blood and new high-resolution approaches, including recent microfluidic platforms, for dissecting the genomes of CTCs and obtaining functional data. We also discuss new ctDNA-based approaches, which may become a powerful alternative to CTC analysis. Together, these approaches provide novel biological insights into the process of metastasis and may elucidate signaling pathways involved in cell invasiveness and metastatic competence. In medicine these liquid biopsies may emerge to be powerful predictive and prognostic biomarkers and could therefore be instrumental for areas such as precision or personalized medicine.

Figure 1

Monitoring of tumor genomes using CTCs and ctDNA. Cancer cells can disseminate from the primary site through the lymphatic system (not shown) or by hematogenous routes. In addition, tumor cells can release DNA into the circulation (illustrated as small DNA strands). The figure shows a tumor consisting of two clones, center, indicated in dark and light blue. In this example the light blue clone releases CTCs and DNA into the circulation at a given time. Analysis of CTCs and ctDNA can reveal tumor-specific copy number changes (chromosome 8 is included here as an example, and is depicted as an overrepresentation of the long arm) and mutations at the nucleotide level (illustrated as the allele fraction of mutations at the bottom). If the tumor genome is stable, repeated analyses would reveal no additional copy number changes or mutations. However, cells from one clone may decrease (left, the light blue clone) as a result of selection pressures associated with a given treatment, whereas cells from another (dark blue clone) increase so that CTCs and ctDNA from this clone may be preferentially released into the circulation. As the material in the circulation is now from a different clone, copy number changes (here illustrated as a loss of the entire chromosome 8) and the allele frequency of mutations may differ substantially from the previous analysis. Alternatively (right), the light blue clone could acquire a new mutation - for example, with increased resistance to a given therapy (shown as green cells) - and because they evolved directly from the light blue cells, copy numbers and mutations will be very similar to the earlier analysis. However, new mutations may be detected (indicated here as a high level amplification on 8q and a new mutation).

Figure 2

Workflow of CTC analyses. (a) CTCs (light blue cell) are rare cells in the circulation; the vast majority of nucleated cells are normal blood cells (orange). (b) First, separation steps as outlined in the main text are necessary to isolate these rare cells. (c) After cell lysis, DNA is accessible for whole-genome amplification (WGA). The WGA products can be analyzed for copy number changes on an array platform by comparative genomic hybridization (array CGH). Alternatively, libraries can be prepared and subjected to next-generation sequencing (NGS). By NGS both copy number changes and mutations within genes can be detected.

Figure 3

Workflow of ctDNA analyses. (a) ctDNA (light blue DNA fragments) are present in the circulation of cancer patients together with DNA fragments released from non-malignant cells (most frequently from cells of the hematogenous system, orange). The latter are often the majority, and the percentage of ctDNA may vary depending on various parameters, such as the tumor burden. (b) The entire DNA is isolated from plasma and can be subjected directly to an array for copy number analysis, or a library can be prepared for NGS, allowing assessment of both copy number changes and mutations at the nucleotide level. (c) After alignment, DNA fragments (here shown for one chromosome) are counted relative to their position in the genome. In theory, DNA fragments from normal cells should be present in identical numbers across the entire genome, as indicated by the equal number of orange fragments. In contrast, tumor-specific fragments may vary and reflect the status of copy number changes of cells releasing material into the circulation at the time of analysis, illustrated by the variable number of blue fragments. Using bioinformatics tools, the number of different fragments at a given locus is converted to a copy number (blue line). Similarly, the percentage of ctDNA determines the allele fraction for the identification of tumor-specific somatic mutations.

Figure 4

Analysis of ctDNA and CTC from a patient with colon cancer using array-CGH[37,76]. Green indicates overrepresented, red underrepresented and black balanced regions. (a) Plasma DNA ratio profile demonstrates losses on chromosomes 3, 4, 5, 8p, and 18 and gains on chromosomes 7p, 17q, and 20. (b) The CTC had almost identical copy number changes with those observed with the plasma DNA.