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SOX17 Promoter Methylation in
Circulating Tumor Cells and Matched
Cell-Free DNA Isolated from Plasma of
Patients with Breast Cancer
M. Chimonidou, A. Strati, N. Malamos,
V. Georgoulias, and E.S. Lianidou
January 2013
www.clinchem.org/content/59/1/270.full
© Copyright 2013 by the American Association for Clinical Chemistry
© Copyright 2009 by the American Association for Clinical Chemistry
Introduction
Circulating Tumor Cells (CTCs)
•CTCs play a critical role in the metastatic spread of carcinomas and their
detection is associated with prognosis in many human cancers, while their
enumeration has been cleared by the FDA for follow up of breast, colon,
and prostate cancer patients with verified metastasis.
•CTCs represent a promising new diagnostic tool, especially for
advanced-stage cancer patients where they can be used as a “liquid
biopsy,” allowing physicians to follow cancer changes over time and tailor
treatment accordingly.
•However, it is quite clear now that simple enumeration of CTCs is not
enough.
•CTC molecular characterization is very important since it can play a
crucial role in understanding the biology of metastasis and in selecting
patients for targeted therapy.
© Copyright 2009 by the American Association for Clinical Chemistry
Introduction
Cell free DNA (cfDNA)
•cfDNA circulates in plasma of patients with cancer at increased
concentrations.
•Many teams have focused on the development of assays that allow the
specific detection of small amounts of tumor specific cfDNA in the
peripheral blood of patients with cancer.
•The detection of tumor specific DNA alterations such as mutations and
methylation in cfDNA provides a less invasive, more easily accessible
source of DNA for genetic analysis than tumor biopsies.
•Several studies have described methylation of tumor suppressor genes in
serum or plasma samples and in the corresponding primary breast tumors,
while DNA methylation was not detected in plasma or serum of healthy
donors.
© Copyright 2009 by the American Association for Clinical Chemistry
Introduction
SOX17
•Is a member of the SOX (Sry-related high mobility group box) family of
transcription factors.
•Plays a critical role in the regulation of development and stem/precursor
cell function partly through repression of the canonical Wnt/beta-catenin
signaling pathway.
•Global analysis of CpG island hypermethylation and gene expression in
cancer cell lines revealed that SOX17 gene silencing is associated with
DNA hypermethylation of a CpG island located in the promoter region
•The SOX17 promoter is methylated in CTCs isolated from peripheral
blood of patients with breast cancer.
© Copyright 2009 by the American Association for Clinical Chemistry
Question:
Is there a direct connection between the
presence of CTCs and cell free DNA in patients
with operable breast cancer where the primary
tumor has already been resected?
To address this question, the authors chose to use the same marker and
the same methodology in matched clinical samples.
The authors evaluated whether SOX17 promoter methylation in CTCs
was associated with the methylation pattern of this gene in matched
cfDNA isolated from plasma of patients with breast cancer.
© Copyright 2009 by the American Association for Clinical Chemistry
Early breast cancer
(N=55)
Figure 1.
Schematic diagram of the
workflow of the study.
Advanced breast cancer
(verified metastasis)
(N=59)
Healthy Individuals
(N=43)
21 ml peripheral
blood in EDTA
20 mL:
Isolation of PBMC’s by density gradient
centrifugation
(ficol)
1 mL:
plasma separation
Positive selection of CTC by
immunomagnetic separation
(EpCAM)
Isolation
of cfDNA from plasma
(200μL)
CTC fraction
Isolation of total RNA
(Trizol)
Isolation of mRNA
(oligo-dTbeads)
cDNA synthesis
RTq-PCR for CK-19
Isolation of genomic DNA
(Trizol)
SB Conversion of genomic
DNA
Real time MSP for SOX17
© Copyright 2009 by the American Association for Clinical Chemistry
20 mL
Peripheral
blood
Methodology
PBMCs
Ficoll
gradient
Cell count
CTCs isolation
CellFreeDNA
isolation
Plasma
DNA
extraction
From
CTCs
Positive
selection
(EpCAM)
Apply magnet
Figure 2. Outline of the extraction of cell free DNA and CTCs.
© Copyright 2009 by the American Association for Clinical Chemistry
Methodology
Methylation Specific PCR (MSP)
MSP is a bisulfite conversion based PCR technique for the study of DNA CpG
methylation.
Sodium bisulfite converts all unmethylated, but not methylated, cytosines to uracil.
© Copyright 2009 by the American Association for Clinical Chemistry
How were the methylation specific primers
and probes designed?
© Copyright 2009 by the American Association for Clinical Chemistry
Methodology
Primer design for MSP
For maximal discrimination between
methylated and unmethylated alleles,
primers should contain at least one
CpG site at the most 3’-end.
GATUUTGATTGC
Other than CpG sites at the most 3’
end, more CpG sites in primer
sequences are preferred.
Both MSP primers and probe should
contain T bases derived from modified
unmethylated C regions so as to
discriminate and amplify the converted
from unconverted DNA.
© Copyright 2009 by the American Association for Clinical Chemistry
Specificity of the SOX17 MSP assay
Bisulfite treatment
of genomic DNA
MSP using methylated
specific primers and probe
Real time
Methylation
Specific PCR
100%methylated
control
50%
methylated
1%methylated
0%methyaled
© Copyright 2009 by the American Association for Clinical Chemistry
Results- characteristic graphs
Realtime-MSP
for SOX17
using DNA
isolated from
CTCs fraction
from:
a) healthy donors
b) operable breast cancer
c) verified metastasis
Realtime-MSP
for SOX17
using DNA
isolated from
cfDNA from:
a) healthy donors
b) operable breast cancer
c) 2009
verified
metastasis
© Copyright
by the
American Association for Clinical Chemistry
RESULTS
Heat map of SOX17 promoter methylation in matched samples:
CTCs Fraction and cfDNA of patients with: (a) operable breast cancer (n=55), (b)
verified metastasis (n=59)
In parallel, the expression of the epithelial marker CK-19 is shown in CTCs fraction
Early breast cancer (n=55)
CTCs SOX17
CELL FREE DNA
CK19
Verified metastasisis (n=59)
CTCs SOX17
CELL FREE DNA
CK19
Methylated SOX17
Unmethylated samples
© Copyright 2009 by the American Association for Clinical Chemistry
Operable breast cancer
cell free DNA
(N=55)
meth SOX17
unmeth SOX17
Total
meth SOX17
11
8
19
unmeth SOX17
8
28
36
Total
19
36
55
CTC fraction
Table 1.
Outline of the
extraction of cell
free DNA and
CTCs.
Agreement 39 of 55 (70.9%), P*=0.008
Verified Metastasis
cell free DNA
(n=59)
meth SOX17
unmeth SOX17
Total
meth SOX17
13
14
27
unmeth SOX17
11
21
32
Total
24
35
59
CTC fraction
Agreement2009
34 ofby
59 the
(57.6%),
P*=0.283
© Copyright
American
Association for Clinical Chemistry
Question
 Why is SOX17 promoter methylation in CTCs and in
matched cfDNA highly correlated in primary breast
cancer in contrast to verified metastasis?
© Copyright 2009 by the American Association for Clinical Chemistry
Discussion
• SOX17 promoter methylation in CTCs and in matched cfDNA is highly
correlated in early breast cancer but not in metastasis.
• This finding leads toward a direct connection between the presence
of CTCs and cfDNA in patients with operable breast cancer after
surgical removal of the primary tumor.
• In the group of patients with verified metastasis no such connection
was observed, even if in many cases there was a concordance
between SOX17 methylation in CTCs and cfDNA.
• This could possibly be due to the fact that in this case the metastasis
is already present and cfDNA can be also released from apoptotic
cells escaping from the metastatic site.
© Copyright 2009 by the American Association for Clinical Chemistry
Conclusions
• SOX17 promoter methylation in CTCs and in matched cfDNA is
highly correlated in early breast cancer but not in metastasis.
• This finding leads toward a direct connection between the
presence of CTCs and cfDNA in operable breast cancer
patients after surgical removal of the primary tumor.
• Since this study was prospective, the clinical importance of this
finding must be evaluated further, when the clinical outcome of
these early breast cancer patients is known.
© Copyright 2009 by the American Association for Clinical Chemistry
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© Copyright 2009 by the American Association for Clinical Chemistry