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Perioperative tumor cell dissemination in patients with
ARTICLE IN PRESS
Available online at www.sciencedirect.com
EJSO xx (2009) 1e5
www.ejso.com
Perioperative tumor cell dissemination in patients with primary
or metastatic colorectal cancer
J.G. Tralh~ao a,b,*, E. Hoti c, M. Serôdio a, P. Laranjeiro d, A. Paiva d, A.M. Abrantes b,
M.L. Pais d, M.F. Botelho b, F. Castro Sousa a
b
a
Department of Surgery, Surgery 3, Faculty of Medicine, Coimbra University Hospital, Coimbra, Portugal
Biophysics and Biomathematics Institute, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
c
Liver Transplant Unit, Saint Vincent’s University Hospital, Elm Park, Dublin 4, Ireland
d
Center of Histocompatibility of Center, Coimbra, Portugal
Accepted 2 July 2009
Abstract
Introduction: Although there is general correlation between the TNM stage of colorectal cancer (CRC) and its prognosis, there is often
significant variability of tumor behaviour and individual patient outcome, which is unaccounted for by pathologic factors alone. Our
aim was to estimate perioperative tumor cell dissemination in patients with primary or CRC liver metastases as a possible factor influencing
the outcome.
Methods: Forty patients were prospectively enrolled in the study from the year 2007 to 2008. Eighteen patients had histologically proven
CRC (50% rectal, 44% colonic, 6% colonic and rectal). Sixteen patients (47%) had CRC liver metastases only. The remaining six patients
who underwent colon or liver resection for benign conditions, acted as the control group. All patients with malignant pathologies had R0
resections. Blood samples were taken before the surgical incision (T0), immediately after tumor resection (T1) and at the end of the surgical
intervention (T2). Data acquisition was performed using a dual-laser FACSCalibur flow cytometer. Circulating malignant cells were identified as being CD45/cytokeratinþ.
Results: The analysis of patients overall (CRC resection subgroup and hepatectomy subgroup) revealed that there was no statistically significant difference of the tumoral cell count in the blood per million of hematopoietic cells at T0, T1 and T2.
Conclusions: This study demonstrates no differences in the detected circulating numbers of tumor cells at different stages of surgical
intervention.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Colorectal cancer; Circulating tumor cells; Flow cytometry detection
Introduction
Colorectal cancer (CRC) is a common and often lifelimiting disease. Approximately 20e45% of patients with
CRC undergoing curative resection subsequently develop
local recurrence or metastatic disease in lymph nodes, liver,
lung and peritoneum.1,2
Although there is general correlation between the TNM
stage and prognosis, there is often significant variability of
* Corresponding author. Department of Surgery, Surgery 3, Hospitais da
Universidade de Coimbra, Praceta Prof. Mota Pinto, 3000-075 Coimbra,
Portugal. Tel.: þ351 239 400 417; fax: þ351 239 480 258.
E-mail address: [email protected] (J.G. Tralh~ao).
tumor behaviour and individual patient outcome, which is
unaccounted for by pathologic factors alone. The detection
of lymph node (LN) metastases constitutes the most important prognostic factor in CRC and as the primary indicator
of disease spread, LN status determines the choice of postoperative adjuvant chemotherapy. However, the limitations
of TNM staging are emphasised by the considerable prognostic heterogeneity of patients within a given tumor stage
(not all patients with LN-negative tumors are cured and not
all patients with LN-positive tumors die from their disease).
This resulted in a number of efforts to develop more accurate staging protocols.3e6
Tumor progression after curative resection of CRC is
caused by tumor cell dissemination, currently undetected
0748-7983/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ejso.2009.07.003
Please cite this article in press as: Tralh~ao JG et al., Perioperative tumor cell dissemination in patients with primary or metastatic colorectal cancer, Eur J
Surg Oncol (2009), doi:10.1016/j.ejso.2009.07.003
ARTICLE IN PRESS
J.G. Tralh~ao et al. / EJSO xx (2009) 1e5
2
by standard clinical staging techniques. The detection of
disseminated tumor cells could help to identify a subgroup
of patients at risk for disease relapse that could benefit from
adjuvant therapy.6,7 In this context, flow cytometry is one of
the methods used to identify subgroups at risk for disease
relapse. Flow cytometry allows an accurate quantification
of cells and at the same time a multi-parameter characterization of each cell present in the blood samples. This technique is widely used in the detection of rare events, such as
minimal residual disease in hematological malignancies.
Disseminated CRC cells have immunophenotypic characteristics distinct from those of hematopoietic cells, which
allow their identification and quantification in peripheral
blood by flow cytometry.
The aim of the study was to determine whether resection
of the primary tumor or CRC liver metastases would lead to
an increased dissemination of cancer cells.
Patients and methods
Patients
This prospective study included 40 patients who had undergone surgery in our institution from the year 2007 to
2008. There were 24 male patients with a median age
and 16 female patients with a mean age of 64 10 (range:
41e90).
Eighteen patients had histologically proven colorectal
cancer (50% rectal, 44% colonic, 6% colonic and rectal).
Sixteen patients had colorectal liver metastases only. The
remaining six patients, who acted as the control group
had benign conditions (sigmoid diverticulosis, hepatic adenoma and hemangioma).
All patients were preoperatively staged by biological
and radiological examinations. In addition, patients with
rectal cancer received mandatory magnetic resonance imaging (MRI). Similarly, MRI was performed also for patients with non-conclusive computed tomography imaging
(CT scan) concerning liver metastases originating from either rectal or colonic primary tumors.
Seven patients diagnosed with locally advanced rectal
cancer (T3, T4, LN positive) received preoperative radiochemotherapy.
All patients operated for malignancy had R0 resections,
performed according to international standards with at least
10 cm resection margins for colonic tumors or a distal resection margin of at least 2 cm associated with pericolic,
perirectal and perivascular truncal lymphadenectomy for
rectal cancers. High vascular ligation and no touch technique were used for all resections. Similarly, in the group
of patients with liver metastases, liver resection was performed with at least 1 cm surgical margin.
Liver interventions included: right hepatectomy (n ¼ 4),
extended left hepatectomy (n ¼ 1), left hepatectomy
(n ¼ 1), central hepatectomy (n ¼ 1), left lobectomy
(n ¼ 4), bisegmentectomy (n ¼ 3), segmentectomy (n ¼ 2)
and sub-segmentectomy (n ¼ 4). Of the four patients who
had left lobectomy, two had associated right hemicolectomy.
The colorectal procedures included: total coloproctectomy with ileostomy (n ¼ 2), total colectomy (n ¼ 2), subtotal colectomy (n ¼ 1), right hemicolectomy (n ¼ 1), left
hemicolectomy (n ¼ 3), anterior resection (n ¼ 6), anterior
resection with ileostomy (n ¼ 1) and lastly abdominal perineal resection (n ¼ 2).
Blood sampling and processing
Blood was sampled using central venous catheters before placing the surgical incision (T0), immediately after
tumor resection (T1) and at the end of the surgical intervention (T2) and each time a volume of 12 ml was obtained. As
negative control, venous blood specimens were collected
from the six patients who underwent surgery for benign
conditions before (T0), during (T1) and after surgery (T2).
Peripheral blood samples were centrifuged at 540 g
for 10 min, the buffy coat was collected and placed in
a 50 ml tube. Fixative-free NH4Cl lysing solution was
added to each tube (10 ml of lysing solution per 1 ml of
buffy coat) and the samples were incubated for 45 min at
room temperature (RT) to lyse the remaining red blood
cells.
After the incubation period, the samples were centrifuged at 540 g for 10 min. The supernatant was discarded
and the cell pellet washed with 10 ml of phosphate-buffered
saline (PBS, Gibco, Paisley, Scotland, UK) (540 g for
10 min).
The cell pellet was stained with anti-CD45 (clone 30F11, BDB, San Jose, CA, USA) conjugated with peridinin
chlorophyll protein cyanine 5.5 (PerCP Cy5.5) and anticytokeratin (clone MNF116, DakoCytomation, Glostrup,
Denmark) conjugated with fluorescein isothiocyanate and
incubated in the dark at RT for 30 min. At last, the samples
were washed twice in 10 ml of PBS and the cell pellet resuspended in 1 ml of PBS.
Data analysis was performed in a dual-laser FACSCalibur flow cytometer (BDB, San Jose, CA, USA) using the
CellQuest software (BDB, San Jose, CA, USA). First, a total of 20 000 events, corresponding to all nucleated cells in
the sample, was stored. To increase the sensitivity of the
technique, in a second step of acquisition, only cytokeratinþ cells were acquired using an electronic live
gate.
Data analysis was performed using the Paint-a-Gate Pro
software (BDB, San Jose, CA, USA). Circulating CRC
cells were identified as being CD45/cytokeratinþ. The
monoclonal antibody anti-cytokeratin used recognized an
epitope present in keratin 5, 6, 8 and 17.
Statistical analysis
Continuous data were presented as mean and standard
deviation (SD). Skewed and non-gaussian continuous data
Please cite this article in press as: Tralh~ao JG et al., Perioperative tumor cell dissemination in patients with primary or metastatic colorectal cancer, Eur J
Surg Oncol (2009), doi:10.1016/j.ejso.2009.07.003
ARTICLE IN PRESS
J.G. Tralh~ao et al. / EJSO xx (2009) 1e5
3
were analyzed using non-parametric tests (ManneWhitney
U test or the KruskaleWallis test, whenever there were two,
three or more samples to compare); Chi-square test was applied for the purpose of comparing proportions. Statistical
analysis was performed using StatisticaÒ, version 7. A
p value of 0.05 was considered as statistically significant.
per million of hematopoietic cells was 9 4 (range
0e14) at T0. Instead at T1 the tumoral cell count in the
blood per million of hematopoietic cells was 7 4 (range
0e13) and lastly at T2 the tumoral cell count in the blood
per million of hematopoietic cells was 6 9 (range 0e26),
which is statistically not significant ( p < 0.088).
Ethics
Discussion
Written informed consent was obtained for all the enrolled patients. The protocol of the study conformed to
the ethical guidelines of the 1975 Declaration of Helsinki
and also with the guidelines of our institution.
In this study we have investigated the hypothesis that curative resection of a primary or secondary colorectal tumor
can lead to shedding of malignant cells into the peripheral
circulation. Our results obtained by using flow cytometry
with the pancytokeratin antibody would suggest that such
procedures are not associated or do not disseminate tumoral
cells in the peripheral blood.
Results
Patients of the control group
In this group of patients (n ¼ 6), none of the studied tumor markers were detected at any of the mentioned surgical
times (T0, T1 and T2).
Patients with colorectal cancer or CRC liver
metastases
We analyzed 102 blood samples from 34 patients with
malignancy and detected circulating cancer cells in 31
blood samples from 34 patients who underwent curative
surgical resection.
The median value of the tumoral cell count in the blood
per million of hematopoietic cells was 7 7 (range 0e26)
at T0 (before the placement of surgical incision). At T1
(immediately after the removal of the specimen) the tumoral cell count in the blood per million of hematopoietic
cells was 4 4 (range 0e13) and lastly at T2 (end of the
surgical intervention) the tumoral cell count in the blood
per million of hematopoietic cells was 4 6 (range
0e26). However, this value was statistically not significant
( p < 0.501).
Patients with colorectal cancer
In this subgroup of patients the median value of the tumoral cell count in the blood per million of hematopoietic
cells was 6 8 (range 0e26) at T0. Instead, at T1 the tumoral cell count in the blood per million of hematopoietic
cells was 2 2 (range 0e6) and lastly at T2 the tumoral
cell count in the blood per million of hematopoietic cells
was 3 4 (range 0e10). Similar to the overall group of patients the value of tumoral cell count at T2 was statistically
non-significant ( p < 0.141).
Prognostic factors and management of colorectal
cancer
Tumor progression can result from disseminated tumor
cells in lymph nodes, blood or bone marrow, sites which
are not detected by current staging methods. The objective
of adjuvant therapy is to eradicate viable disseminated tumor cells, thereby decreasing disease relapse and improving patients’ survival.8 Candidates for post-operative
adjuvant therapy are usually patients at high risk for disease
relapse, as judged by current clinical and pathological staging. In the group of patients without distant metastases,
lymph node metastases are the most important prognostic
factor.9 Consequently, adjuvant chemotherapy is recommended for patients with positive lymph nodes. For patients
with colon cancer stage I or II, adjuvant chemotherapy cannot achieve a survival benefit, and thus, adjuvant therapy is
not recommended for these patients. Although considered
at low risk, 10e20% of patients with colorectal cancer
stage I and II ultimately will develop recurrent disease.10,11
It is in this population that prognostic markers may identify
a subgroup of patients who are at a higher risk for disease
relapse and who may also benefit from adjuvant therapy, especially from antitumoral agents with low systemic toxicity
such as monoclonal antibodies, which have also proven to
be effective against dormant tumor cells.6,7 In this regard,
there are several studies that have demonstrated that tumor
cell detection is clearly related to an early relapse and decreased survival of the respective patients.6,7,12,13 This
new prognostic factor may change the surgical management of patients with colorectal liver metastases and may
help to individualize the treatment of these patients with
systemic or regional chemotherapy.14
Patients with colorectal liver metastases
Flow cytometry in the detection of disseminated
colorectal cancer cells
The analysis in this subgroup of patients revealed that
the median value of the tumoral cell count in the blood
Among the different approaches to screen disseminated
colorectal cancer cells in bone marrow aspirates, peripheral
Please cite this article in press as: Tralh~ao JG et al., Perioperative tumor cell dissemination in patients with primary or metastatic colorectal cancer, Eur J
Surg Oncol (2009), doi:10.1016/j.ejso.2009.07.003
ARTICLE IN PRESS
4
J.G. Tralh~ao et al. / EJSO xx (2009) 1e5
and mesenteric venous blood, immunocytochemistry is the
most widely used method. This method has the advantage
to allow cell morphology characterization, but presents
low sensitivity.6,15,16 RT-PCR based protocols have further
improved the sensitivity and specificity of detection systems for disseminated cancer cells, allowing the identification of approximately one neoplastic cell in 107 normal
peripheral mononuclear blood cells.17
Several studies have proved that the sensitivity of flow
cytometry is similar to PCR’s, ranging from 104 to
105.16,18,19,20 A study based on serial dilutions of breast
cancer cells in normal peripheral blood, showed that, using
the appropriated markers, flow cytometry presented a sensitivity ranging between 106 and 107.21 Despite this, flow
cytometry is not widely used in the detection of disseminated tumor cells in peripheral blood or bone marrow samples, probably because of the limited information in the
literature regarding the phenotype of these cells. Because
of the absence of tumor-specific target antigens, the disseminating tumor cells are identified based on the expression of
epithelium-specific antigens such as cytoskeleton-associated cytokeratins, surface adhesion molecules, or growth
factor receptors, whose quantitative expression obtained
by flow cytometry is not well documented. Moreover, cytokeratin expression might vary along malignant transformations and different tumor stages.21,22 Other reasons that
have limited the use of flow cytometry in this field are
the absence of a consensus for the reagents and methods applied, the need for technical expertise and the long period
required for sample acquisition, in order to obtain a large
number of cells to achieve an acceptable sensitivity.
However, flow cytometry allows an accurate quantification of cells and enables the immunophenotypic characterization of each cell in the sample. Besides the improvement
of flow cytometry technology, the development of new
high-speed flow cytometers allowing the acquisition of
20 000 events per second, has reduced dramatically the acquisition period. Altogether these factors make flow cytometry an attractive method for the quantification of rare
events. This technique has become a method of choice
for the detection of minimal residual disease in hematological malignant neoplasms17,19,23,24 and, more recently, an
important tool in quantifying other rare events such as circulating endothelial cells, circulating progenitor cells,19,25
mesenchymal stem cells25 and disseminated tumor cells
from CRC,26 small and non-small lung cancer,26 prostate
cancer27 and rhabdomyosarcoma.28
In this study, we used an anti-pancytokeratin monoclonal
antibody able to recognize an epitope present in cytokeratin
5, 6, 8 and 17; and anti-CD45 monoclonal antibody. The
analysis of the expression of both pancytokeratin and
CD45 together with the light scatter properties enabled us
to distinguish between non-hematopoietic cells (pancytokeratinþ/CD45) and peripheral blood cells (pancytokeratin/CD45þ). However, before we proceeded to
the quantification of disseminated CRC cells in peripheral
blood, we evaluated the pancytokeratin expression in tumor
cells from tumor biopsies and verified the presence of two
distinct populations: the first with a low pancytokeratin expression and the second with a higher expression. Based on
previous proteomic studies in CCR showing a heightened
cytokeratin 8 expression in tumor tissue compared to normal mucosa from the same individual, we assume that
only those cells with higher pancytokeratin expression
were CRC cells.29,30 Therefore, when peripheral blood
samples were analyzed, we only considered as circulating
CRC cells those with characteristic light scatter properties,
CD45 and with high pancytokeratin expression.
In our results, none of the samples from patients with benign conditions who had surgical resection demonstrated
the evaluated tumor markers. However, these tumor markers
are not limited only to the gastrointestinal epithelium, as
they have been found in a variety of cell types including urothelial, Merkel cells and leucocytes in 1982.31 On the other
hand, several studies have also reported that in terms of molecular screening of circulating blood, the expression of several tumor markers is limited to patients with colorectal
cancer with no expression seen in the controls.32,33
Conclusion
This prospective study using flow cytometry (a very specific and sensitive technique) to detect circulating tumor
cells, demonstrated no differences in the circulating numbers of tumor cells detected at different times of the surgical intervention. These results would lead to the logical
questions: what is the impact of the ‘‘no touch’’ technique
in the oncological outcome of patients with primary colorectal cancer or liver metastases? Do we need to use perioperative adjuvant therapy or to change the surgical
strategies to prevent intraoperative tumor cell shedding?
Our study does not suggest so; however, further studies
should be performed to answer the raised questions.
Conflict of interest
Concerning the present manuscript, the authors declare
that there are no personal, financial or non-financial competing interests or conflicts.
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Surg Oncol (2009), doi:10.1016/j.ejso.2009.07.003
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