Katerina Kypraiou
Detection, Characterization and Clinical
Validity of CTCs in Melanoma
Research Laboratory of Photobiology & Skin Cancer
Department of Dermatology, University of Athens
Andreas Sygros Hospital
Athens, Greece
Cutaneous Malignant Melanoma
 One of the most aggressive forms of cancer
*curable by surgical excision in early stages
 High tendency to metastasize
 High resistance to chemotherapy
 Thickness of the primary tumor
 Regional lymph node status
Very poor overall survival
Most important clinicopathological factors associated with
metastasis
 Imaging diagnostic methods can only inform us of metastasis in an advanced stage
Detection of CTCs in the peripheral blood could screen out patients with a higher risk of relapse at
the time of diagnosis
METASTASIS
Methods used for CMCs isolation and enrichment
marker independent
• Density Gradient Centrifugation (DGS) (de Vries et al., 2000, Reinhold et al., 2001)
- established method for the collection of CMCs using Sucrose or CsCl
- Ficoll-Hypaque is commonly used for this purpose.
- relies on the physical characteristics of density and size to separate mononuclear cells
and CMCs.
Major disadvantage is low efficiency of cell recovery which may result in loss of
melanoma cells due to shearing or formation of cell aggregates at the bottom of the
tube.
Methods used for CMCs isolation and enrichment
marker independent
• Red Blood Cell Lysis Buffer
- The depletion of erythrocytes from the sample is another common and costefficient enrichment procedure. It involves erythrocyte lyses through the use
of an NH4Cl buffer, followed by centrifugation and discarding of the
supernatant (Liu et al., 2011, Kitago et al., 2009).
This technique offers slightly improvement over DGC but is much easier and
maintains uniformity between samples (Mitropapas et al., 2006, Nezos et al.,
2011).
Methods used for CMCs isolation and enrichment
marker independent
OncoQuick
Methods used for CMCs isolation and enrichment
marker independent
ISET
Methods used for CMCs isolation and enrichment
marker dependent
Positive Immunomagnetic enrichment:
Ags that characterize melanoma and its
surface antigens (Liu et al, 2011).
Immunomagnetic
enrichment:
-Commonly used Abs
are:
Commonly
usedChondroitin
devices: MACs
microbeads and
- Melanoma
Sulfate
separator
(Miltenyi
(StemCell
proteoglycan
alsoBiotech),
known as EasySep
High Molecular
Weight Melanoma-Associated
Ag
Technologies)
and Dynamag Separator
(Invitrogen).
(MCSP/HMW-MAA) (Kitago et al., 2009).
-MCAM/MUC18/CD146
target
with
A key
advantage CMC integrity
and cell
morphological
higherare
metastatic
potential, associated with
features
left intact
aggressive
phenotypes
and
disease
protection (Shih et al., 1994, Xie et al., 1997,
Major
disadvantage
Bardin
et al., 1996).for CMCs is that heterogeneity
requires
multiple
markers
tocassette
positivesubfamily
select CMCs.
- Markers
for ATP
binding
B member 5 (ABCB5), nestin (NES), CD133,
CD271 (Schatton et al., 2008,Fusi et al., 2011,
Boiko et al., 2010).
Multimarker immunomagnetic isolation allows
the enrichment of more CMCs than the use
of individual Abs, supporting the notion that
there is heterogeneity in CMCs.
Methods used for CMCs isolation and enrichment
marker dependent
Negative Immunomagnetic enrichment
- removes all leukocytes so that the
remaining CMC population can be
isolated
•Six studies have used negative enrichment
through CD45 depletion (leukocytes) and/or
CD34 depletion (endothelial cells) (Rao et al.,
2011, Khoja et al., 2013, Liu et al., 2011,
Georgieva et al., 2002, Fusi et al., 2011,
Sakaizawa et al., 2012).
• Georgieva et al., found negative enrichment
to be superior to positive , along with being
time efficient and more practical than other
isolation procedures usually combined with
RT-PCR.
• Sakaizawa et al., stated that a consequence
of CD45 depletion is a potentially low yield as
a result of loosing CMCs with the leukocytes.
•Clawson et al., suggests that melanomamacroplhage hybrids are lost.
Methods used for CMCs isolation and enrichment
Cell Search: Semi Automated
Technology
•
•
•
•
immunomagnetic
enrichment
with
EpCAM, MSCP, MCAM.
Ab staining of both CMCs (MCSP) and
contaminating cells (CD45, CD35),
followed by DAPI and visualization
through digital fluorescent microscopy
(Rao et al., 2011, Khoja et al., 2013,
Khoja et al., 2014, Steen et al., 2008).
DAPI positive and at least 4 microns
diameter.
FDA approved.
Automation allows comparison between
studies as it excludes many external
variables.
Further studies need to evaluate Cell Search specificity in CMCs. Rao et al. , 2011;
Khoja et al., 2013 and 2014 have showed that inclusion of more detection markers in
the Veridex melanoma CTC kit will make Cell Search extremely valuable approach.
CMC detection: Direct techniques
Manual Immunocytometry (ICM)
• Can be combined with immnonomagnetic enrichment (Benez et al.,
1999, Siewert et al., 2001).
• Immnostaining is followed by microscopy (Freeman et al., 2012,
Benez et al., 1999, Siewert et al., 2001, Sakaizawa et al., 2012) or
even laser capture microdissection to isolate individual cell for
genotyping (Sakaizawa et al., 2012).
• CMCs remain intact for morphological examination.
• Can use negative CD45 staining to exclude leukocytes .
Disadvantages: Time consuming approach, subjective morphological
interpretation, CMCs can be confused with large leukocytes or circulating
endothelial cells (De Giorgi et al., 2010, Khoja et al., 2014).
CMC detection: Direct techniques
Flow Cytometry: FACS
• Records the fluorescent signal of ab labeled cells as they rapidly pass by
a detector in a stream of individual cells (Liu et al., 2001, Fusi et al.,
2011).
• Allows multi-parameter analysis of individual cells.
• Automatic sorting for further characterization.
Disadvantages: Requires enriched samples or large volume. Non-specific
binding may cause false positives, low sensitivity without enrichment.
Aggregates give false results.
CMC detection: Direct techniques
Photoacoustic Flow Cytometry
- Irradiates cells with pulsed light and then measures ultrasonic high
frequency waves produced by pigmented cells as a result of
thermoelastic expansion (O’Brien et al, 2012, Weight et al, 2009).
- High specificity, no markers needed.
Disadvantages:
Lacks clinical validation
False positive results due to circulating benign nevus cells
False negative results due to amelanotic cells
CMC detection: Indirect techniques
qRT-PCR- quantitative Real Time PCR
-Can quantify the number of mRNA copies in a sample through the use
of DNA probes that generate a measurable fluorescent signal which is
subsequently quantified after each cycle in comparison to control
values.
-Quantitative assay.
- More sensitive than other PCR-based protocol.
-Practical and cost-efficient, independent of additional enrichment
techniques.
The inclusion of both spiked blood samples as positive controls and healthy samples as
negative controls during each round of PCR further ensure assay reliability (Schittek et
al., 1999, Koyanagi et al., 2005, De Vries et al., 1999)
Disadvantage:
Indirect proof, does not determine exact number of CMCs.
Circulating Melanoma Cell Detection
• RT – PCR
Melanocytic Markers
Melanoma Specific
Less Specific for
Melanoma Tumor
Markers
TYR
MART-1/Melan-A
MAGE-A1,-A3,-A6
MITF
Melanotransferrin -P97
PRAME
PAX3
GalNAcT
NY-ESO-1
TRP-1
MIA
S100b
TRP-2/Dct
MUC18/MCAM
YKL-40
gp100
Circulating Melanoma Cell Detection
Some commonly studied markers
TYR
• is a basic enzyme involved in the
conversion of tyrosinase to melanin in
process of melanin biosynthesis.
• is expressed in normal melanocytes,
melanoma cells, brain and colon cancers
and in low levels in testis and normal
colon tissue.
• Expression of TYR in blood indicates the
presence of CMCs since normal
melanocytes are not present in the
circulation.
• Although TYR has been negative in some
cases has also been very helpful in many
cases with patients in advanced stage
melanomas (III – IV) (Tsao et al, 2001).
Circulating Melanoma Cell Detection
Most studied
PAX3
•
is a transcription factor involved in
melanoblast proliferation, migration, and
differentiation during development.
•
positively regulates other CMC biomarkers
(MITF, TGFb1, MCAM) (Medic et al., 2009,
Medic et al., 2011).
•
In some cases has been a useful marker of
both primary malignant melanoma cells and
CTCs (Blake et al, 2005, Koyanagi et al, 2005,
Takeuchi et al, 2004).
•
Interestingly was found even in melanoma cell
lines that were amelanotic, where other
melanocytic factors had been repressed
(Vachtenheim et al., 1999).
Lin and Fisher, Nature 2007
Circulating Melanoma Cell Detection
Most studied
MITF
• is implicated in melanocyte determination and differentiation
during development and even more in regulation of melanoma
proliferation (Carreira et al, 2005, Garraway et al, 2005, Carreira, et
al, 2005) and invasiveness (Loercher et al, 2005).
• Until now MITF has been proved as a very important marker for
disease progression and overall survival of melanoma patients
(Koyanagi et al, 2006), especially the MITF-M isoform has been
shown to be melanocyte specific (Lin et al, 2004).
• Moreover most melanoma markers TYR, TYRP-1, gp 100 are
dependent on MITF.
Circulating Melanoma Cell Detection
Some commonly studied markers
MAGE A3 (MAGE-A3)
• is commonly expressed by various tumours and not by healthy
tissue, except male germ line and placenta (Miyashiro I et al.,
2001, Takeuchi H et al., 2004, Gkalpakiotis S et al., 2010).
• Using RT-PCR has been proven as the most sensitive progression
marker (Arenberger P. et al, 2008).
Circulating Melanoma Cell Detection
Most studied
MART1
• is specific for the melanocyte lineage, found in normal skin, uvea, retina
and melanocytes but not in other normal tissues (Nezos et al., 2009).
• is detected at AJCC stage I between 5-20% and at stage II between 730%. However the detection level at AJCC stages III and IV is between
30-76% (Keilhoz et al, 2004, Sarantou et al, 1997, Palmieri et al, 2003,
Enk et al, 2004, Reynolds et al, 2003, Koyanagi et al, 2005, Mocellin et
al, 2004, Curry et al, 1999).
• Moreover TYR and MART-1 have been found in the blood of patients
with uveal melanoma and patients with liver cancer from uveal
melanoma (Keilhoz et al, 2004).
• Although a good marker the detection levels vary due to differential
gene expression between patients and even the same patient over time
(Arenberger et al, 2008, Enk et al, 2004).
Khoja L et al., Annals of Oncology 00:1-7,2014
Patients:
• Number of patients: 111-817
• Multiple sampling in most studies
• Median follow up: 19 – 75 months
Methods:
• whole blood or PBMCs
• Used RT-PCR
• All used tyrosinase (plus Melan A/Mart 1, MAGE3, gp-100)
CTC detection:
• Detection rate of CTCs: 13-80%
• A >2 marker assay more sensitive than
tyrosinase alone
• Longer follow up and multiple sampling
increased positivity
Clinical outcome:
• Different outcome measures:DFS,OS,DMFS
• Conflicting results on prognostic value
Khoja L et al., Annals of Oncology 00:1-7,2014
• Hashimoto et al., CTC detection at any point was associated with worse DFS and OS;
Detection of more than 1 markers was an adverse prognostic marker
• Quanglino et al., new metastases in all patients were associated with positive CTC
detection of more than 1 time points; patients with detectable CTCs during follow up had
iferior time to progression and OS
• Khoja et al., showed that detection of more than 2 CTCs / 7.5 ml blood is a an adverse
prognostic factor ; Patients receiving treatment in whom CTC number remained more than
2 CTCs during treatment had shorter median OS than those who maintained less than 2
CTCs