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Page 10 of 18 Huang et al. J Cancer Metastasis Treat 2019;5:34 I http://dx.doi.org/10.20517/2394-4722.2018.94

(stem cell markers) . Mostert et al. performed a CTC isolation assay and mRNA expression profiling using
[70]
[69]
the CellSearch technique in 142 metastatic colorectal cancer (mCRC) patients. They measured 95 mRNAs by
RT-qPCR and found that 34 CTC-specific mRNAs were higher in patients with ≥ 3 CTCs compared with
healthy donors. This CTC-specific gene panel for mCRC patients, such as KRT19, KRT20 and AGR2, may
aid in characterizing how CTCs with different expression profiles contribute to malignancy, thereby
furthering the realization of individualized cancer treatment. Campton et al. developed a comprehensive
[71]
and sensitive platform, named as AccuCyte®-CyteFinder® system, for identification and characterization
of individual CTCs. Using the whole genome amplification (WGA) product, they confirmed that the TP53
gene, which is known to contain the R175H mutation in SKBR3, enables personalized, molecularly-guided
cancer treatment.

Ampli1 TM (Menarini Silicon Biosystem), a product developed for single-cell WGA, can be used to amplify
DNA for downstream genotyping analysis, including comparative genomic hybridization (CGH) and
next-generation sequencing (NGS). Upon its development in 1992, CGH technology opened a new avenue
in genomic investigation and, more particularly, in cancer gene analysis. In the past, CGH was applied
for analysis of tumor tissues, but many studies have suggested that data from primary tumors alone is
insufficient. Array CGH, which exploits ordered arrays of genomic DNA sequences, is widely used for
analysis of CTCs, including identification of genomic alterations which include insertion/deletion, single-
nucleotide variations, copy number variations (CNVs) ; identification of candidate oncogenes or tumor
[72]
suppressors; identification of novel biomarkers involved in metastasis, cancer progression and therapy
response; and identification of subgroups of CTCs . High-throughput NGS is another strong technology
[73]
to analyze heterogeneity of CTCs and reveal the mechanisms of metastasis, which might be the Achilles’
heel in disease progression. Bertucci et al. utilized aCGH and NGS to compare DNA copy number and
[74]
mutational profiles of 365 cancer-related genes between primary tumors and metastases and discovered a
degree of divergence for actionable driver genes that might be extremely relevant with cancer metastasis.
Profiting from whole genome amplification technology, the limited amount of single-cell genomic DNA
sample can be amplified indistinguishable for sequencing.

Single-cell RNA sequencing was used to detect the heterogeneity of CTCs, unveiling the mechanism of
drug resistance of androgen receptor (AR) inhibitors in prostate cancer . The technique was also used to
[61]
identify conduct a transcriptomic analysis in pancreatic CTCs, finding increased expression of stromal-
derived extracellular matrix (ECM) proteins, which facilitate cell migration and invasiveness, in CTCs from
mice and humans with pancreatic cancer [Figure 3C].
[75]
Single-cell exome sequencing of isolated CTCs from cancer patient, revealed insertion/deletion and single-
nucleotide variation in CTCs after whole genome amplification. The results showed cancer-type specific
CNVs that are reproducible within cells of the same patient, or even between patients with the same type of
cancer .
[76]

CLINICAL IMPLICATIONS
Prognostic and diagnostic value of CTCs
Several studies have highlighted the correlation between CTC burden and treatment effect, indicating
the prognostic value for patients receiving chemotherapy or surgery and the potential of surveillance of
disease recurrence or metastasis [Table 1]. Several years after chemotherapy, patients with high-risk breast
cancer with elevated CTC counts in their peripheral blood were reported to have worst survival prospect .
[77]
Similarly, patients with colorectal cancer with elevated CTC counts were more likely to have recurrence
after 3 years of curative resection, showing the relation between post-operative CTCs and poor prognosis .
[78]
The prognostic value of CTCs was demonstrated by Nicola et al. [Figure 3D] in 60 patients with extensive
[79]
SCLC. After assessment with the CellSearch system, the group isolated and analyzed CTCs in 90% (54/60)