HHE Pathology and diagnostics supplement 2018 | Page 9

pathology and diagnostics
Circulating tumour cells as
liquid biopsy: technical aspects
This article focuses on the current technologies used for the enrichment
and detection of circulating tumour cells and the innovative technologies
that have been developed to improve methods for detection
Laure Cayrefourcq PhD
Catherine Alix-
Panabières PhD
Laboratory of Rare
Human Circulating Cells,
University Medical Center
of Montpellier, EA2415
University of Montpellier,
France
Cancer is one of the leading causes of death
worldwide, and metastasis is responsible for more
than 90% of the mortality of cancer patients.
Metastasis occurs when tumour cells leave the
primary tumour, travel through the bloodstream
as circulating tumour cells (CTCs), and then
colonise secondary tumour sites distant from the
primary tumour. CTCs, which are cells that have
become detached from primary tumours, are the
most representative biomarkers of the biological
functions of metastatic development and their
analysis has the potential to reveal the key
mechanisms of tumour progression.
The analysis of CTCs in the blood of patients
with cancer was termed ‘liquid biopsy’. 1,2 Blood
samples can be obtained and analysed at the time
of diagnosis and during treatment. The analysis of
the liquid biopsy provides important information
on the molecular properties of tumour lesions.
This information contributes to the early
detection of metastatic lesions and aids in the
personalised treatment of cancer patients, such
as prognostic evaluation, stratification of patients
for targeted therapies, real-time monitoring of
treatment efficacy, identification of therapeutic
targets, and resistance mechanisms. Numerous
clinical studies and meta-analyses, including large
cohorts of patients, have shown that the number
of CTCs is an important indicator of the risk of
progression or death in patients with metastatic
solid cancer (for example, breast, prostate,
colon). 3–7
However, in-depth investigation of CTCs
remains technically challenging. CTCs occur
as very low concentrations of one tumour cell
among millions of blood cells. Their identification
and characterisation require extremely sensitive
and specific analytic methods, which are usually
a combination of enrichment and detection
procedures.
This article focuses on current technologies
used for the enrichment and detection of CTCs.
A number of innovative technologies to improve
methods for CTC detection have been developed,
including CTC microchips, filtration devices,
quantitative reverse-transcription polymerase
chain reaction (PCR) assays, automated
microscopy systems and functional assays. Among
the considerable number of promising CTC
detection techniques that have been developed,
the analytical specificity and clinical utility must
be demonstrated for their introduction into
clinical practice.
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Strategies for CTC enrichment
CTC enrichment includes a large panel of
technologies based on the different properties
of CTCs that must differentiate them from the
normal haematopoietic cells: (i) physical
properties (for example, size, density, electric
charges and deformability); and (ii), biological
properties (for example, surface protein
expression and invasion capacity; see Table 1).
Biological properties
Biological properties are mainly used in
immunological procedures with antibodies
against either tumour-associated antigens
(positive selection) or common leucocyte antigen
CD45 (negative selection). Positive enrichment
typically attains high cell purity, which depends
on antibody specificity. Negative enrichment
technologies evade some of the pitfalls of positive
enrichment; for example, CTCs are not tagged
with a difficult-to-remove antibody, they are not
activated or modified via an antibody–protein
interaction and antibody selection does not bias
the subpopulation of CTCs captured. However,
these advantages come at the cost of purity, as
negative enrichment strategies typically have
a much lower purity than positive enrichment 8–10
and require a suitable CTC detection step.