In: Cancer Biomarkers
Editor: Helen C. Kristoff, pp.
ISBN: 978-1-61761-302-9
© 2010 Nova Science Publishers, Inc.
Chapter VI
Cytokeratin 20 by Reverse
Transcriptase-Polymerase Chain
Reaction as a Marker of Tumor Cells in
Peripheral Venous and Mesenteric
Blood of Colorectal Cancer Patients
Josip Lukac1, Dujo Kovacevic2, Ivan Samija1,
Iva Kirac2, and Zvonko Kusic1
1
Department of Oncology and Nuclear Medicine,
Sisters of Charity University Hospital,
Vinogradska cesta 29, Zagreb 10000, Croatia
2
Department of Surgery, Sisters of Charity University Hospital,
Vinogradska cesta 29, Zagreb 10000, Croatia
Abstract
Purpose: To determine the usefulness of reverse transcriptase-polymerase chain
reaction (RT-PCR) recognition of cytokeratin 20-encoding mRNA in assessing the extent
of preoperative and intraoperative hematogenic tumor cell dissemination in patients with
colorectal cancer undergoing primary tumor resection. Methods: Circulating tumor cells
(CTC) were determined using conventional RT-PCR in preoperative peripheral venous
blood of 65, intraoperative peripheral venous blood of 45, and mesenteric venous blood
of 36 patients with colorectal cancer, and in peripheral venous blood of 21 healthy
volunteers. Results: Positive findings were obtained in preoperative blood of 17% of
AJCC stage 0-III vs. 56% stage IV pts (p=0.007), in mesenteric blood of 18% stage 0-III
vs. 89% stage IV pts (p<0.001), and in none of the healthy volunteers. Among patients
with liver metastases, positive results were found in 37.5% peripheral blood and in 100%
mesenteric blood samples (p=0.063). 17% patients with negative preoperative findings
became intraoperatively positive (p=0.031). Conclusion: Positive CTC findings in
preoperative peripheral venous blood correlate to the clinical stage of the disease in
colorectal cancer patients. CTC determination in mesenteric venous blood seems to be
2
Josip Lukac, Dujo Kovacevic, Ivan Samija et al.
more indicative for liver metastases than their detection in preoperative peripheral venous
blood. Surgical manipulation facilitates the dissemination of tumor cells by circulation.
Introduction
Approximately 20–40% of colorectal cancer (CRC) patients ultimately develop local
recurrence or distant metastases following curative surgical resection [1,2]. Distant metastases
develop by the shading of tumor cells from the primary carcinoma prior to or during
operation, which cannot be detected with standard clinical tools. As circulating tumor cells
(CTC) could be the cause of disease relapse, their presence or absence has been considered by
some investigators to be an important prognostic factor pre- or intraoperatively and/or
postoperatively, and an indicator for the decision concerning adjuvant treatment and followup [3,4]. However, the reliability of using CTC has been debated from a methodological
perspective, as their number in the circulation can be very low [5]. To increase the sensitivity
of detection of these early metastases, the detection of CTC at the molecular level, i.e., by
polymerase chain reaction (PCR), was introduced. The major disadvantage of circulating
DNA material is that it can be released by dying tumor cells and remains in the circulation
due to its relatively high stability in comparison to RNA. Due to its stability, the presence of
circulating DNA cannot be used to definitively confirm the presence of viable cancer cells,
and so researchers have turned to detecting mRNA, which has a shorter half-life once
released from tumor cells [6], using reverse transcriptase-polymerase chain reaction (RTPCR). Conventional RT-PCR has the disadvantage of not being able to quantify marker
expression, allowing simply for positive/negative discrimination. To overcome this
disadvantage, real-time PCR was introduced. This allows the quantification of low-level
background transcription and the definition of cutoff values for marker expression, hence
improving sensitivity [7]. Although they are extremely powerful and reliable techniques,
PCR-based techniques still have the disadvantage of potential oversensitivity, i.e., they are
highly sensitive but may be insufficiently specific [8].
The RT-PCR technique has been used to detect disseminated tumor cells in the peripheral
blood, bone marrow, and peritoneal lavage of patients with CRC by detecting the mRNA of
various tumor and epithelial markers. Since there is no specific marker for colorectal cancer,
the detection of disseminated colorectal tumor cells is based on epithelial markers, mostly on
the carcinoembryonic antigen (CEA) and cytokeratins (CK) 19 and 20. CEA mRNA
expression in peripheral blood was found to correlate with clinical stage of the disease in
most studies [9-15], although the correlation was not demonstrated in all studies [16,17].
CK19 mRNA expression was also demonstrated to correlate with disease stage by some
authors [17-19] but not by others [12,20]. The findings based on CK20 mRNA expression
gave similar results. Some studies demonstrated the association of CK20 mRNA expression
with the stage of disease [13,14,18,21-23], while others did not [11,12,24-26]. Prognostic
value of CTC detection by RT-PCR was also evaluated in some studies. Dong et al. [26]
concluded that CK20 mRNA expression was predictive for tumor dissemination in CRC
patients, while Friederichs et al. [27] found it indicative of a worse prognosis. Positive CK20
mRNA expression in preoperative peripheral blood was associated with reduced disease-free
[28] and overall [29] survival or both [18,22]. Koch et al. [30] demonstrated that CTC
detected intraoperatively during liver metastases resection by CK20 mRNA expression is an
Cytokeratin 20 by Reverse Transcriptase-Polymerase Chain Reaction…
3
independent prognostic factor for tumor relapse. On the other hand, Wang et al. [31] did not
find CK20 to be an independent prognostic factor for prediction of postoperative metastases.
Topal et al. [32] reported that CK20 mRNA expression in peripheral blood was not related to
cancer recurrence in CRC patients. Schuster et al. [25] concluded that peripheral blood is not
a suitable compartment for the detection of tumor cells in CRC patients.
The liver is the most common site of metastasis in CRC patients and metastatic lesions
emerge from tumor cells that presumably spread from the primary tumor prior to or during
surgery [33]. Two major pathways of tumor cell dissemination have been hypothesized: (i)
tumor cells leave the primary tumor and reach the capillary vessels in the liver via the portal
venous drainage. After establishment of metastasis in the liver, tumor cells may be further
disseminated to reach other distant organs. This theory would explain why the liver is the
preferred site of metastasis in CRC; (ii) alternatively, a large number of tumor cells are
released from the primary tumor and pass through the liver, resulting in a high prevalence of
CTC in venous blood as evidence of systemic hematogenic tumor cell dissemination. In this
case, the predominant occurrence of metastasis in colorectal cancer would be explained by
homing functions likely mediated by specific adhesion molecules on CTC [34].
The determination of CTC in blood samples taken simultaneously from different blood
compartments, i.e., from mesenteric and peripheral venous blood, could help to clarify doubts
regarding the pathways of hematogenic metastasis of CRC and improve the clinical utility of
CTC determination in CRC patients. It has been hypothesized that tumor cells should be
detected more frequently in the local tumor-draining mesenteric vein, before passage through
liver capillary vessels [34]. To our knowledge, five reports have been published on the
determination of CTC in mesenteric blood of CRC patients by CEA and/or CK19/20 mRNA
expression using conventional RT-PCR [34-36] or quantitative/real-time RT-PCR [13,22].
Iinuma et al. [22] were the first to demonstrate that the detection of CTC in tumor drainage
blood was associated with prognosis.
Surgical manipulation of tumors bears the risk of intraoperative tumor cell dissemination.
Turnbull et al. [37] described the “no-touch isolation” technique with initial lymphovascular
ligation for resection of colorectal cancer, which demonstrated an improved survival rate.
This improved prognosis, however, could not be confirmed in other studies [38]. The
presence of circulating tumor cells does not necessarily predict subsequent metastatic disease.
The implantation of circulating tumor cells seems to be very inefficient, and circulating tumor
cells might be destroyed rapidly [39,40]. However, the activation of blood coagulation during
surgery, which may lead to increased entrapment of circulating tumor cells and relative
immune suppression caused by surgical stress, could enhance the metastatic potential of
circulating tumor cells [41]. In addition, the metastatic potential of individual tumor cells
might differ significantly [42]. Intraoperative tumor cell dissemination during resection of
primary colorectal cancer [43] and during liver resection of colorectal metastases has been
reported [30,44].
This study presents the results of determination of CTC by Ck20 mRNA expression
analyzed by conventional RT-PCR in preoperative and intraoperative venous and mesenteric
venous blood of CRC patients. The objectives were to evaluate: (i) the association of
preoperative CTC finding in peripheral blood with tumor stage, (ii) potential differences in
CTC findings between peripheral and mesenteric venous blood, and (iii) potential differences
in CTC findings between preoperative and intraoperative peripheral venous blood.
4
Josip Lukac, Dujo Kovacevic, Ivan Samija et al.
Subjects and Methods
Subjects
Healthy controls. Twenty-one healthy volunteers, aged 35–75 years (median 61) among
them 11 females (52%) and 10 males (48%) served as peripheral venous blood donors for the
extraction of RNA. All blood samples were repeatedly tested for CK20 gene expression.
Patients. The study comprised a group 65 patients with primary colorectal cancer. All of
them were treated at the Department of Surgery and the Department of Oncology and Nuclear
Medicine, Sisters of Charity University Hospital, Zagreb, Croatia.
Preoperative diagnosis was established by standard diagnostic procedures, i.e. endoscopy
and radiography, and when required by ultrasonography and computerized tomography. All
primary tumors were resected and pathohistologically confirmed to be adenocarcinomas.
Tumor staging was carried out by AJCC Cancer Staging system [45]. Details regarding
patient’s age, gender and tumor stage and localization are presented in Table 1. The study was
carried out in compliance with the World Medical Association Declaration of Helsinki [46]
and informed consent was obtained from all patients and healthy controls prior to their
enrollment in the study. Peripheral venous blood was drawn both before and after tumor
resection, and mesenteric blood was drawn just before tumor resection.
Table 1. Patient characteristics
Number of patients:
Gender:
Age, mean (range):
Clinical diagnosis:
PHD:
Tumor localization:
AJCC clinical stage:
Metastases localization
65
male:
29 (44.6%)
female:
36 (55.4%)
65 (42-88) years
colorectal cancer
adenocarcinoma
colon:
21 (32.3%)
rectum:
44 (67.7%)
0:
4 (6.2%)
I:
16 (24.6%)
II:
16 (24.6%)
III:
20 (30.8%)
IV:
9 (13.8%)
liver:
6
lungs + liver:
2
bones:
1
Methods
Blood Samples Collection and Processing. Ten milliliters of blood was collected in tubes
containing ethylenediamine tetraacetic acid (EDTA) as anticoagulant. Blood samples were
processed within two hours after collection. The mononuclear cell fraction of blood was
isolated on Ficoll gradient (1.077 g/cm3; Axis-Shield PoC AS, Oslo, Norway) as described by
Cytokeratin 20 by Reverse Transcriptase-Polymerase Chain Reaction…
5
Boyum [47]. Three milliliters of Ficoll was overlaid with 5 mL of blood diluted with 5 mL of
phosphate-buffer saline (PBS). After centrifugation for 15 minutes at room temperature and
500 g, the layer between the plasma and the Ficoll was transferred into another tube and
washed twice with PBS by centrifugation for 15 minutes at 4 ºC at 800 g. The cell pellet was
used immediately for RNA isolation.
RNA Isolation and Reverse Transcription. Total cellular RNA was isolated from
mononuclear cell fraction using the TriPure Isolation Reagent (Roche, Indianapolis, IN, USA)
following the manufacturer’s instructions. This isolation is based on guanidiniumisothiocyanate-phenol-chloroform method described by Chomczynski and Sacchi [48]. RNA
pellet was dissolved in deionized water and stored at –70 ºC. For reverse transcription (RT), 1
g of RNA was mixed with 0.5 g Oligo d(T)18 primer (New England Biolabs, Beverly,
MA, USA) and incubated for 4 minutes at 70 ºC. Other components were added to this
mixture in the final concentrations of 1x RT buffer (50 mmol/L Tris-HCl, 30 mmol/L KCl, 8
mmol/L MgCl2, 10 mmol/L DTT; New England Biolabs), 0.5 mmol/L of each dNTPs
(Sigma, Saint Louis, MO, USA), 1 U/l RNase inhibitor (Roche, Mannheim, Germany) and
1.25 U/L M-MuLV reverse transcriptase (New England Biolabs) in a total volume of 20

ºC, and stored at –20 ºC.
PCR Reaction. Cytokeratin 20 (CK20) expression was analyzed by nested PCR using two
pairs of primers for each marker (Table 2). For the first round of PCR analysis, 2 L of
complementary DNA (cDNA) was added to the reaction mixture containing final
concentrations of 1 x Taq buffer with Mg2+ (50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L
Tris-HCl; Eppendorf, Hamburg, Germany), 0.2 mmol/L of each dNTPs (Sigma), 0.2 mol/L
sense primer (1.f and 139.f for the first and second round, respectively), 0.2 mol/L antisense
primer (558.r and 429.r for the first and second round, respectively), and 0.02 U/L Taq DNA
polymerase (Eppendorf) in a total volume of 25 L. PCR reaction conditions were as follows:
one cycle of 3 minutes at 96 ºC followed by 35 cycles of 30 seconds at 96 ºC for denaturation,
30 seconds at 60 ºC for primer annealing, and 30 seconds at 72 ºC for polymerase extension,
followed with a final 10-minute extension at 72 ºC. For the second round of PCR analysis, 5
L of the first-round PCR product were used. The composition of PCR reaction mixture and
cycling conditions were the same as for the first-round PCR, with the exception of primer
annealing, being carried out at 72oC. For all samples, a housekeeping gene for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified to determine the
integrity of the RNA (Table 2). The composition of PCR reaction mixture was the same as for
CK20 and the cycling conditions were as follows: one cycle of 3 minutes at 96 ºC followed
by 30 cycles of 30 seconds at 96 ºC for denaturation, 30 seconds at 60 ºC for primer
annealing, and 45 seconds at 72 ºC for polymerase extension, followed with a final 7-minute
extension at 72 ºC. Positive control (RNA isolated from human colon adenocarcinoma grade
II cell line HT29, American Type Culture Collection, Manassas, VA, USA) and negative
control (RNA isolated from healthy volunteer blood) samples were included in each RT-PCR
assay. All PCR products were analyzed by electrophoresis on 2% agarose gel stained with
ethidium bromide and directly visualized under UV light at 302 nm. DNA molecular weight
markers VIII or IX (Roche, Manheim, Germany) were included in all gels. A sample was
considered positive if a band of expected size (290 bp for CK20 and 623 bp for GAPDH) was
present.
6
Josip Lukac, Dujo Kovacevic, Ivan Samija et al.
Table 2. Sequences of oligonucleotide primers used for detection of target genes in
patients with melanoma by reverse transcription-polymerase chain reaction (RT-PCR)
Target
gene*
GAPDH
Ck20
Primer
Sequence
Product size (bp)
GAPDH1 (sense)
GAPDH2
(antisense)
Ck20 (outer sense)
Ck20 (outer
antisense)
Ck20 (inner sense)
Ck20 (inner
antisense)
5'-AAC GGA TTT GGT CGT ATT GGG C-3'
5'-AGG GAT GAT GTT CGT GAG AGC C-3'
623
5'-ATG GAT TTC AGT CGC AGA-3'
5'-ATG TAG GGT TAG GTC ATC AAA G-3'
557
5'-TCC AAC TCC AGA CAC ACG GTG AAC TAT G-3'
5'CAG GAC ACA CCG AGC ATT TTG CAG-3'
290
* GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ck20 – cytokeratin 20.
Determination of threshold for detection of CK20 by RT-PCR. Healthy volunteer
peripheral blood samples were spiked with serially diluted HT29 cells. HT29 cells were
cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum.
The cells were collected after trypsinization with 0.25 % trypsin and 0.01 % EDTA, washed
with PBS, counted and serially diluted to 105, 104, 103, 102, 10 and 1 cell/ml PBS. One ml of
each dilution was added to a separate 9 ml-aliquot of the healthy volunteer blood. The number
of peripheral blood leukocytes was determined in each blood sample, to allow the expression
of the threshold for detection of the assay as the number of tumor cells per ml of blood and
per peripheral blood leukocyte number. Blood samples were processed and analyzed by RTPCR as described above. The spiking experiment was repeated three times by using three
independently cultured cells and one blood sample for each dilution each time.
Statistical Analysis
The results are presented as numbers/proportions of positive/negative CK20 findings and
statistical significance of association of the marker (CK20) with other nominal categorical
variables was determined by χ2 test with continuity correction for independent samples and
McNemar’s test for dependent samples, using MedCalc 9.4.2.0 statistical package (MedCalc
Software, Mariakerke, Belgium). P values < 0.05 were considered significant.
Results
Threshold for Detection of CK20 RT-PCR Assay
After two rounds of 35 cycles nested PCR with primers for CK20, a 290-bp PCR product
was detected in healthy volunteer blood samples enriched with HT29 cells up to a dilution of
100 HT29 cells in 10 ml of blood, i.e. in a relative ratio of one HT29 cell in 0.82×106
peripheral blood leukocytes.
Cytokeratin 20 by Reverse Transcriptase-Polymerase Chain Reaction…
7
Specificity of CK20 RT-PCR Assay
Blood samples from 21 healthy volunteers were consistently negative for CK20 gene
expression.
Patients
Positive CK20 gene expression was detected in pre- and intraoperatively drawn
peripheral blood as well as in mesenteric blood of approximately one fifth of colorectal
cancer (CRC) patients (Table 3). The proportions of positive findings in preoperatively drawn
peripheral blood were related to clinical stage of disease (Table 4), with the highest
proportion seen in stage IV patients. Similar relation, but with statistically higher
significance, was observed with mesenteric blood too, with the highest proportion of positive
findings also seen in stage IV patients (Table 4). No significant differences in positive
findings were found between preoperatively drawn peripheral blood and mesenteric blood in
any stage of disease. However, a trend to higher proportion of positive findings in mesenteric
than in preoperatively drawn peripheral blood was demonstrated in patients with liver
metastases, with 3 out of 8 (38%) positive findings in preoperatively drawn peripheral blood
versus 8 out of 8 (100%) positive findings in mesenteric blood, although this difference did
not reach statistical significance (p=0.063) (Table 5). A significant proportion (17%) of
patients with preoperatively negative finding had positive CK20 gene expression in
intraoperatively drawn peripheral blood (Table 6).
Table 3. CK20 in pre- and intra-operatively drawn peripheral and in mesenteric blood
in the whole patient group
Blood sample
Patients, n
65
Positive CK20,
n
14
Positive CK20,
%
21.5
Peripheral blood, preoperative
Peripheral blood , intraoperative
45
10
22.2
Mesenteric blood*
36
10
27.7
* For all of these patients data regarding both pre- and intra-operative testing were also available.
Table 4. CK20 in preoperatively drawn peripheral and mesenteric blood of patients
stratified by AJCC stage
AJCC
stage
Peripheral blood
CK20, positive/total
Positive CK20, %
0
I
II
III
0-III
IV
0/4
1/16
4/16
4/20
9/56
5/9
0.0
7.1
25.0
20.0
16,7
55,6*
Mesenteric blood
CK20,
positive/total
0/4
0/5
0/7
2/11
2/27
8/9
Positive CK20, %
0
0
0
18.2
7.4
88.9**
p=0.047, all stages, peripheral blood; p<0.001, all stages, mesenteric blood; * p = 0,007 vs. peripheral
blood stage 0-III; ** p <0.001 vs. mesenteric blood stage 0-III, χ2 test with continuity correction
factor.
8
Josip Lukac, Dujo Kovacevic, Ivan Samija et al.
Table 5. CK20 in preoperatively drawn peripheral blood versus mesenteric blood of
patients with liver metastases
Peripheral blood
Mesenteric blood
*McNemar’s test
CK20,
positive/total
3/8
8/8
Ck20 positive, %
P*
37.5
100.0
0.063
Table 6. CK20 in intraoperatively drawn peripheral blood of patients with
preoperatively negative CK20 finding
Patients with negative finding in peripheral
blood drawn preoperatively
Same patients, peripheral blood drawn
intraoperatively
P=0.031, McNemar’s test
CK20,
positive/total
0/35
Positive
CK20, %
0.0
P*
6/35
17.1
0.031
Discussion
Early detection of distant metastasis and identification of patients at risk of recurrence are
essential for the management of colorectal cancer. Since cells shed from the primary tumor
might be responsible for the development of distant metastases [11,16,20,44] and for disease
relapse after surgery [39,45], detection of circulating tumor cells (CTC) has been
hypothesized as a useful method to predict of disease recurrence [40] and could be of
prognostic value in patients with colorectal cancer (CRC). Sensitive molecular methods such
as reverse transcriptase-polymerase chain reaction (RT-PCR) for the determination of CTC
have been developed and used for the determination of the presence/absence of CTC in the
peripheral blood [24,26,28,30,49-51], mesenteric venous blood drained from tumors
[13,22,35,36,49], in bone marrow [31,43,50], lymph nodes [43] and in peritoneal washings
[13,52] of CRC patients. The primary targets used are the carcinoembryonic antigen (CEA)
[13,22,53] and cytokeratin gene expression [13,22,34-36G, as there is currently no specific
marker for CRC [22].
The presence of CTC was evaluated in pre- and intraoperatively drawn peripheral venous
blood and in mesenteric venous blood of 65 CRC patients using conventional RT-PCR and
cytokeratin 20 (CK20) as a target gene. As shown in Table 4, CK20 gene expression in
preoperative peripheral venous blood was associated with the clinical stage of disease,
showing significantly more positive findings in stage IV than in stage 0-III patients. This is in
line with most other reports that also demonstrated the association of CTC presence in
preoperative peripheral venous blood with clinical stage of disease [9-15,23] although such an
association was not always demonstrated [16,17]. Circulating tumor cells reflect hematogenic
dissemination of CRC, and as such their detection in preoperative peripheral venous blood
could be of prognostic significance. This has been demonstrated by some authors using CK20
Cytokeratin 20 by Reverse Transcriptase-Polymerase Chain Reaction…
9
as target gene [30,36,50], but not by others [25,31,32,35]. Since the presence of CTC in
preoperative peripheral venous blood of the investigated patient group was significantly
higher in patients with distant metastases (Table 4), this finding would support the usefulness
of CTC determination in preoperative peripheral blood as a potential prognostic tool.
Moreover, the association of positive CTC findings with the stage of disease was even more
evident in intraoperatively drawn mesenteric venous blood, with a higher degree of statistical
significance between stage 0-III and stage IV patients (Table 4). It has been hypothesized that
tumor cells should be detected more frequently in the local tumor-draining mesenteric vein,
before the passage of released tumor cells through liver capillary vessels [34]. To our
knowledge, there are five reports on results of CTC detection in mesenteric venous blood
using CK20 gene expression by conventional [34-36] or quantitative/real-time RT-PCR
[13,22] in CRC patients. Fujita et al. [36] indicated the prognostic significance of the
presence of CTC in mesenteric venous blood of CRC patients, but questioned the clinical
utility of this assay due to the low recurrence rate in positive patients. However, due to the
small patient group, they did not analyze differences between CTC findings in peripheral and
mesenteric venous blood. Yamaguchi et al. [35] reported that the simultaneous presence of
CEA and CK20 mRNA in mesenteric, but not in peripheral venous blood, is a potent
prognostic factor independent of traditional pathological parameters, but did not analyze two
markers separately. Koch et al. [34] found a significantly higher CTC detection rate in
mesenteric than in peripheral venous blood, which emphasizes the importance of the filtering
function of the liver for circulating tumor cells. Guller [13] reported that the detection of CTC
in peripheral and mesenteric venous blood measured by CEA/CK20 mRNA expression by
quantitative PCR is of potential clinical utility as a prognostic marker, but should be
evaluated in larger clinical studies. However, these authors did not distinguish between CEA
and CK20 or between peripheral and mesenteric venous blood findings, and therefore those
results cannot be compared to those of this study. Iinuma et al. [22] demonstrated that the
combination of CEA/CK20 gene expression in mesenteric venous blood is an independent
prognostic factor for disease-free and overall survival, but also advocate the conductance of
large scale and long-term clinical studies to confirm these findings. They found significantly
more positive CK20 findings in mesenteric than in peripheral venous blood in their patient
group, which is in line with the trend indicated in our patient group (Table 4). They also
found a significant association between the presence of liver mestastases and CEA/CK20
positivity in mesenteric venous blood, but did not present separate results for CEA and CK20,
and as such their results are not comparable. Nevertheless, they reported rate of 72.4% in
mesenteric venous blood of their patients with liver metastases, while this rate in our small
stage IV patient group was 100% (Table 5) which is consistent with Yamaguchi [35] who
also found all seven patients with liver metastases at surgery positive for both CEA and CK20
mRNA in mesenteric blood. The higher detection rate in mesenteric venous blood emphasizes
the importance of the filter function of the liver for circulating tumor cells in portal venous
blood [34].
Regarding surgical manipulation, a significant number of negative findings in peripheral
blood drawn preoperatively turned positive in peripheral blood drawn intraoperatively, i.e.
immediately following tumor resection (Table 6). This might indicate tumor cell shedding
due to surgical manipulation and the possibility of hematogenic tumor cell dissemination.
Although the concept of dissemination of malignant cells during surgical procedures in not
new, the relationship between CTC detection and the development of metastatic disease is
10
Josip Lukac, Dujo Kovacevic, Ivan Samija et al.
still not clear. The results are indicative of tumor cell dissemination during primary tumor
resection, as judged by the determination of CK20 mRNA expression [35,44], though there is
still no strong evidence of the prognostic significance of these findings. However, Koch et al.
[30] demonstrated that the detection CTC by CK20 mRNA expression during hepatic
resection of CRC metastases predicted tumor relapse. This may individualize the therapy for
patients at risk of distant metastases and may aid in developing surgical strategies to prevent
intraoperative hematogenous tumor cell shedding.
Conclusion
This study demonstrated the association of CTC with the stage of disease, the higher
frequency of CTC in mesenteric than in peripheral venous blood, and the higher frequency of
CTC in intraoperatively than in preoperatively drawn venous blood in CRC patients. These
findings indicate that CTC detection by CK20 mRNA expression by RT-PCR in different
peripheral blood compartments and at various times might be helpful in (i) identifying
patients at risk of recurrence, (ii) identifying patients at risk of liver metastases, and (iii) in
developing surgical strategies to prevent intraoperative hematogenous tumor cell
dissemination.
Acknowledgment
This work was supported by Research Grant 0134-1342428-2427 administered by the
Ministry of Science, Education and Sports, Republic of Croatia.
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