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In: Cancer Biomarkers ISBN: 978-1-61761-302-9Editor: Helen C. Kristoff, pp. © 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

1Department of Oncology and Nuclear Medicine, Sisters of Charity University Hospital,

Vinogradska cesta 29, Zagreb 10000, Croatia2Department of Surgery, Sisters of Charity University Hospital,

Vinogradska cesta 29, Zagreb 10000, Croatia

AbstractPurpose: 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

In: Cancer Biomarkers ISBN: 978-1-61761-302-9Editor: Helen C. Kristoff, pp. © 2010 Nova Science Publishers, Inc.

more indicative for liver metastases than their detection in preoperative peripheral venous blood. Surgical manipulation facilitates the dissemination of tumor cells by circulation.

IntroductionApproximately 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 follow-up [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 (RT-PCR). 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…

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.

3

Josip Lukac, Dujo Kovacevic, Ivan Samija et al.

Subjects and MethodsSubjects

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: 65Gender: male:

female:29 (44.6%)36 (55.4%)

Age, mean (range): 65 (42-88) yearsClinical diagnosis: colorectal cancerPHD: adenocarcinomaTumor localization: colon:

rectum:21 (32.3%)44 (67.7%)

AJCC clinical stage: 0:I:II:III:IV:

4 (6.2%)16 (24.6%)16 (24.6%)20 (30.8%) 9 (13.8%)

Metastases localization liver:lungs + liver:bones:

621

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

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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 guanidinium-isothiocyanate-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 L.The mixture was first incubated for 60 minutes at 42 ºC, then for final 5 minutes at 94 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.

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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*

Primer Sequence Product size (bp)

GAPDH GAPDH1 (sense) 5'-AAC GGA TTT GGT CGT ATT GGG C-3' 623GAPDH2 (antisense)

5'-AGG GAT GAT GTT CGT GAG AGC C-3'

Ck20 Ck20 (outer sense) 5'-ATG GAT TTC AGT CGC AGA-3' 557Ck20 (outer antisense)

5'-ATG TAG GGT TAG GTC ATC AAA G-3'

Ck20 (inner sense) 5'-TCC AAC TCC AGA CAC ACG GTG AAC TAT G-3' 290Ck20 (inner antisense)

5'CAG GAC ACA CCG AGC ATT TTG CAG-3'

* 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 RT-PCR 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.

ResultsThreshold 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 0.82×10 6

peripheral blood leukocytes.

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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 Positive CK20, n

Positive CK20, %

Peripheral blood, preoperative 65 14 21.5

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 Mesenteric bloodCK20, positive/total Positive CK20, % CK20,

positive/totalPositive CK20, %

0 0/4 0.0 0/4 0I 1/16 7.1 0/5 0II 4/16 25.0 0/7 0III 4/20 20.0 2/11 18.20-III 9/56 16,7 2/27 7.4IV 5/9 55,6* 8/9 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.

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Table 5. CK20 in preoperatively drawn peripheral blood versus mesenteric blood of patients with liver metastases

CK20, positive/total

Ck20 positive, % P*

Peripheral blood 3/8 37.5Mesenteric blood 8/8 100.0 0.063

*McNemar’s test

Table 6. CK20 in intraoperatively drawn peripheral blood of patients with preoperatively negative CK20 finding

CK20, positive/total

Positive CK20, %

P*

Patients with negative finding in peripheral blood drawn preoperatively

0/35 0.0

Same patients, peripheral blood drawn intraoperatively

6/35 17.1 0.031

P=0.031, McNemar’s test

DiscussionEarly 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

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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

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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.

ConclusionThis 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.

AcknowledgmentThis 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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