CIRCULATING TUMOR CELLS

THE FUTURE OF CANCER DIAGNOSIS

Dr. Nyan Lin Aung

National Health Laboratory
• Blood-borne metastasis from the primary tumor is directly responsible for
most cancer-related deaths
• Tumor cells that are identified in transit within the blood stream are
referred to as circulating tumor cells (CTCs)
• Patients with metastatic lesions are more likely to have CTCs
• However, these have also been reported in some localized cancers

(Yu et al, 2011)

• (Allard et al, 2004)
• (Cristofanilli et al, 2004)
• (Cohen et al, 2008)
• (Rhim et al, 2012)
• Circulating tumor cells originate from the primary tumor or metastatic
deposits after invading and intravasating through the tumor vasculature
• Most CTCs die in the circulation
• A proportion are preprogrammed with homing receptors (e.g., integrins
and chemokines) that enable them to attach to the vasculature in specific
organs, adhere and extravasate and proliferate to form metastases

(Danilla et al, 2011)

• The range of CTC phenotypes includes
• cells with stem or stem cell–like properties
• those with an Epithelial-to-Mesenchymal Transition (EMT) phenotype
• EMT describes the phenotypic change of epithelial tumor cells to a
fibroblastic cell morphology
• decrease in expression of epithelial adhesion molecules (e.g., E
cadherin)
• increased expression of mesenchymal molecules (e.g., vimentin)
leading to increased mobility and invasiveness
(Danilla et al, 2011)
Circulating tumor cells in the process of metastatic progression
(Danilla et al, 2011)
CTC detection technologies

• The presence of CTCs in an autopsy of a patient with advanced metastatic

cancer was first reported in 1869 (Ashworth, 1869)
• A variety of currently used approaches rely on
• physical properties
• expression of biomarkers
• functional characteristics of CTCs

(Yu et al, 2011)

Nucleic acid–based detection of CTCs

• Free DNA and, to a lesser extent, RNA circulating in plasma from patients
with cancer have been described
• Origin of these nucleic acids may also include direct shedding from
necrotic cells in tumor deposits, tumor-derived exosomes, or cellular
fragments or lysis of CTCs in the bloodstream
• Some approaches have focused on isolating nucleic acids directly from
cell-free plasma
(Yu et al, 2011)
Nucleic acid–based detection of CTCs

• At the RNA level, RT-PCR analyses are applied

• Tumor cell–specific markers include
• Cytokeratins, prostate-specific antigen (PSA), mucin-1 (MUC-1),
HER2, AFP (α-fetoprotein), and the CEA (carcinoembryonic antigen)
gene family

(Yu et al, 2011)

Detection of CTCs based on their physical properties
• Several physical properties distinguish CTCs from most normal blood cells
• Larger size of most epithelial cells
• differences in density, charge, migratory properties
• some properties of specific cell types (e.g., melanocytic granules in
melanoma cells)
• Isolation of CTCs by virtue of their increased size has been applied using
several different filtration-based approaches

(Yu et al, 2011)

Detection of CTCs based on their physical properties
• Although most CTCs are larger than leukocytes, there is a significant
variation in their size range
• Several different pores and filter-based approaches have been developed to
prevent clogging and facilitate the retrieval of CTCs

(Yu et al, 2011)

Images of the surface of a wafer
membrane.
(A) A photo of a single wafer batch with
30 fabricated microfilters, a 160 000
pore design and a 7 μm diameter
pore opening (left).
(B) A scanning electron microscope
image (SEM) showing the
hexagonal array pattern of the
microfilter pores (upper right).
(C) Enlarged SEM shows a single round
uniform pore (lower right)
(Adams et al, 2014)
Capture of CTCs using antibodies against cell surface antigens

• The most widely used CTC isolation technique

• Rely on antibody-based capture of CTCs, which express epithelial cell
surface markers that are absent in normal leukocytes
• Epithelial cell adhesion molecule (EpCAM) is most commonly used
because its expression is virtually universal in cells of epithelial origin and
it is absent in blood cells
• The CellSearch system (Veridex) - a widely used (Food and Drug
Administration) FDA-approved method
• Conjugation of antibodies against EpCAM to magnetic beads, followed by
purification of captured cells through a magnetic field
• Uses ferrofluids loaded with an EpCAM antibody
• Visualized by staining with a cocktail of antibodies against the cytoplasmic
epithelial cytokeratins or tissue-specific markers
• Staining for the leukocyte-specific marker CD45 is used as a control to
exclude contaminating leukocytes
• CTCs defined as the subset of EpCAM-captured cells that are confirmed as
both positive for cytokeratins and negative for CD45
• dual-positive cells are not well understood, they are excluded from
enumeration
• For enumeration, the entire device is imaged at multiple planes using a
semiautomated imaging system while on-chip lysis allows for DNA and
RNA extraction and molecular analyses
• Captured cells remain viable to a few hours due to the absence of cell
fixation
Summary of CTCs detection by Cell Search Method
Summary of CTCs detection by Cell Search Method
Micrographs of CTCs
Combined fluorescent and reflected light micrographs of a cytokeratin 7/8 (green) stained CTC captured
from breast cancer patient blood and a contaminating CD45-positive (red) white blood cell (A) and
cytokeratin 7/8 (green, B) and PSA (green, C) stained CTCs captured from a prostate cancer patient
(D and E) HER2 (green, D) and cytokeratin 7/8 (green, E) stained CTCs captured from a breast cancer
patient. In all panels, the nuclei are stained with DAPI (4',6-diamidino-2-phenylindole) (blue)
Micrographs of CTCs
(F and G) Individual and merged fluorescent micrographs of CTCs captured from prostate cancer
patients’ blood stained positive for PSA (green) and Ki-67 (red) (F), and PSA (green) and M30 (red,
apoptotic marker) (G)
In all panels, the nuclei are stained with DAPI (4',6-diamidino-2-phenylindole) (blue)
Current and potential applications of CTC technologies
1. CTCs as prognostic markers
• several studies with large cohorts of patients have been performed to
evaluate the clinical utility of CTC enumeration
• these studies have concluded that the presence of detectable CTCs in
the blood serves as an independent prognostic factor in patients with
cancers of the breast, prostate, and colon
Illustration of current and potential
applications of CTC technologies
• Immunostaining for specific markers
and FISH for genomic amplification
and translocation can be applied to
CTCs
• DNA or RNA can be extracted from the
CTCs and subjected to sequencing,
quantitative RT-PCR and potential
expression profile analysis
• Viable cells can be released and
propagated in cell culture
In patients with metastatic breast cancer, CTC counts above five CTCs per 7.5
ml of blood before the start of systemic therapy were associated with a shorter
median progression-free survival and overall survival(Botteri et al, 2010)
Current and potential applications of CTC technologies
2. CTC analyses in monitoring responses to therapy
• The number of CTCs in patients with lung, prostate, and other cancers
declined rapidly after the initiation of effective chemotherapy, hormonal
therapy, or targeted kinase inhibition (Pachmann et al., 2005;
Maheswaran et al., 2008;)
• BaselineCTC numbers among different cancer patients are not well
correlated with standard measures of tumor mass, including tumor
size or serum protein markers, such as PSA levels in prostate cancer
• Number of CTCs in the blood is not simply a measure of tumor
volume, but instead, it may reflect additional biological features,
possibly including tumor vascularity or invasiveness, both of which
may have distinct prognostic contributions
3. Molecular characterization of CTCs (Identification of molecular
aberrations in CTCs)
• Beyond the enumeration of CTCs, their molecular characterization
provides a key to demonstrating their cellular origin from primary and
metastatic tumor deposits, and it may also provide clues to their evolution
during the course of cancer treatment
• FISH analysis performed on CTCs from breast, kidney, prostate, and
colon cancer showed evidence of aneuploidy in some cells, which was
consistent with their malignant origin (Fehm et al., 2002)
• CTCs can be used to test for molecular evolution of tumors during the
course of treatment
• Some HER2-positive CTCs are reported in breast cancer patients
whose primary tumor was HER2 negative (Meng et al., 2004; Wülfing
et al., 2006)
• As well as some HER2-negative CTCs emerging in HER2-positive
breast cancers subjected to anti-HER2 therapy (Hayes et al., 2002).
3. Evaluation of signaling, proliferation, and apoptosis in CTCs

• Dynamic studies of CTCs after therapeutic interventions allow real

time monitoring of treatment responses
• Phospho-PI3K and phospho-Akt in breast cancer (Kallergi et al.,
2008)
• DNA damage marker phosphorylated γ-H2AX in patients treated
with chemotherapeutic agents (Wang et al., 2010)
• Evidence of apoptosis - using analysis of M30-stained cells in
prostate cancer(Larson et al., 2004; Hou et al., 2009; Swennenhuis
et al., 2009)

• Up-regulation of the antiapoptotic BCL2-α transcript in CTCs of

murine prostate cancer model(Helzer et al., 2009).
Future perspectives
• The molecular characterization of CTCs has the potential to revolutionize
understanding of cancer metastasis
• Technological platforms highly sensitive and reliable CTC isolation
• As the technology evolves further, a broad range of potential applications
can be applied
• the noninvasive monitoring of tumor genotypes to the identification
and analysis of metastatic precursor cell subpopulations
• early detection of invasive but localized cancer
These advances are likely to have important implications for
our understanding of cancer metastasis and our treatment of
patients with cancer.
References
• Adams DL, Zhu P, Makarova OV, Martin SS, Charpentier M,
Chumsri S and Tang CM. (2014). The systematic study of circulating
tumor cell isolation using lithographic microfilters. RSC Advances; 9,
4334–4342.
• Danila DC, Pantel K, Fleisher M, and Scher HI. (2011) Circulating
Tumors Cells as Biomarkers: Progress Toward Biomarker
Qualification. Cancer Journal; 17(6), 438–450.
 References
• Hayes DF, Walker TM, Singh B, Vitetta ES, Uhr JW, Gross S, Rao C,
Doyle GV, and Terstappen LW. 2002. Monitoring expression of HER-
2 on circulating epithelial cells in patients with advanced breast
cancer. Int.J. Oncol; 21:1111–1117.
• Maheswaran S, Sequist LV, Nagrath S, Ulkus K, Brannigan B,
Collura CV, Inserra E, Diederichs S, Iafrate AJ, Bell DW. (2008)
Detection of mutations in EGFR in circulating lung-cancer cells. N.
Engl. J. Med. 359:366–377
 References
• Meng, S, Tripathy D, Shete S, Ashfaq R, Haley B, Perkins S, Beitsch
P, Khan A, Euhus D, Osborne C, et al. 2004. HER-2 gene
amplification can be acquired as breast cancer progresses. Proc. Natl.
Acad. Sci; 101:9393–9398
• Pachmann K, Camara O, Kavallaris A, Schneider U, Schünemann S,
and Höffken K. (2005) Quantification of the response of circulating
epithelial cells to neodadjuvant treatment for breast cancer: a new
tool for therapy monitoring. Breast Cancer Res. 7:975–979
References
• Wülfing P, Borchard J, Buerger H, Heidl S, Zänker KS, Kiesel L and
Brandt B. (2006) HER2-positive circulating tumor cells indicate poor
clinical outcome in stage I to III breast cancer patients. Clin. Cancer
Res;12:1715–1720
• Yu M, Stott S, Toner M, Maheswaran S, and Daniel A. (2011)
Circulating tumor cells: approaches to isolation and characterization.
J. Cell boil; 192(3): 373-382.
Thank You!