Circulating tumor cells in pancreatic cancer patients: Methods of detection and clinical...

Circulating tumor cells in pancreatic cancer patients: methods of

detection and clinical implications

Kjersti Tjensvoll, Oddmund Nordgård and Rune Smaaland

Department of Haematology and Oncology, Stavanger University Hospital, N-4068 Stavanger,

Norway.

Short title: Implication of CTCs in pancreatic cancer patients.

Article category: Mini review

Corresponding author: Kjersti Tjensvoll, Department of Haematology and Oncology,

Laboratory for Molecular Biology, Stavanger University Hospital, N-4068 Stavanger,

Norway. Phone: +47 47809206; E-mail: [email protected], [email protected]

Keywords

Circulating tumor cells; pancreatic cancer; pancreatic adenocarcinoma; prognosis, survival.

International Journal of Cancer

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ijc.28134

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Abstract

The poor prognosis of pancreatic cancer patients is associated with the frequent and early

dissemination of the disease, as well as late detection due to unspecific and late symptoms

from the primary tumor. Pancreatic cancers frequently spread to the liver, lung, and skeletal

system, suggesting that pancreatic tumor cells must be able to intravasate and travel through

the circulation to distant organs. Circulating tumor cells (CTCs) are tumor cells that have

acquired the ability to enter the circulatory system; this cell population is ultimately

responsible for the development of metastases in distant organs. Clinical studies have revealed

that the presence of CTCs in blood is correlated with disease progression for other cancers,

such as breast, colorectal and prostate cancer. However, as CTCs are extremely rare, both

enrichment and sensitive methods of detection are required for their enumeration. This review

highlights various enrichment procedures and methods for the detection of CTCs.

Furthermore, we systematically review previously reported studies of the clinical relevance of

CTC detection in pancreatic cancer patients. There is evidence that the presence of CTCs also

correlates with an unfavorable outcome in pancreatic cancer patients. However,

technical/methodological issues may explain why some studies only show a trend toward an

association between CTC detection and disease progression. Larger studies, as well as

characterization of the CTC population, are required to achieve further insight into the clinical

implications of CTC detection in pancreatic cancer patients.

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John Wiley & Sons, Inc.


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Introduction

Pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer-related death in Western countries,

with a patient survival rate that is among the worst of any solid cancer. The most prominent

risk factors are a family history of pancreatic cancer and cigarette smoking 1, 2.

Computed tomography (CT) is the most frequently used method for diagnosis and

clinical disease staging of pancreatic cancer, although histological or cytological evidence is

required for verification of the adenocarcinoma diagnosis. Clinical staging classifies patients

into the categories of resectable, borderline resectable, locally advanced, and metastatic

disease. Tumor staging according to the TNM system stratifies the patients according to

tumor size, regional lymph node involvement, and distant metastases 3. CT imaging, with

increasing use of magnetic resonance imaging and positron emission tomography-CT, is the

method used to monitor treatment response. Presently, measurements of carbohydrate antigen

19-9 (CA19-9) and carcinoembryonic antigen (CEA), although not highly specific, are the

only blood-based biomarkers for diagnosis, monitoring of treatment, and prediction of

survival in pancreatic cancer patients 4-6.

Gemcitabine is still generally recommended as the standard first-line treatment for

locally advanced and/or metastatic pancreatic cancer 7. Although gemcitabine administration

leads to a statistically significant, clinically beneficial response by patients, the magnitude of

the objective, radiographically measured response is rather small. Thus, the overall 5-year

survival rate among patients with pancreatic cancer is less than 5% 8, 9. Approximately 90% of

the patients who present with advanced pancreatic cancer survive less than one year; 80-85%

of the patients with resectable disease also experience recurrence, and die within five years of

diagnosis 8. For metastatic patients treated with chemotherapy, the median survival time is

approximately 5-6 months. This high death rate is not only caused by de novo and subsequent

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development of drug resistance but is also a consequence of diagnosis at a late stage due to

lack of early symptoms and extensive metastasis already at the time of diagnosis 8.

Accordingly, the vast majority of pancreatic cancer patients are primarily treated with a

palliative intent to reduce symptoms, as well as to prolong life for some patients 10. However,

promising results from two clinical trials published last year give hope for more effective

treatment of these patients in the immediate future 11, 12

.

Although pancreatic cancer has been highly characterized at the molecular level

(reviewed in 2, 8), the link between genetic alterations and the aggressiveness of this disease is

poorly understood. Pancreatic cancer has the highest frequency (>85%) of KRAS mutations

among all human cancers 13. The KRAS mutations seem to occur at an early stage of

pancreatic cancer development, suggesting that KRAS activation may be one of the most

crucial early genetic events leading to tumorigenic transformation 14, 15

. Nonetheless, a

prognostic value for KRAS mutations has not been detected in pancreatic cancer patients 16, 17

.

Circulating tumor cells (CTCs)

CTCs are cells that have detached from the primary tumor and entered the blood circulation.

Accordingly, CTCs can be transported to distant sites to form metastases 18. CTCs may enter

the blood circulation in two ways: by passive tumor cell shedding from the primary tumor or

by an active mechanism involving the epithelial-to-mesenchymal transition. Passive shedding

of tumor cells from the primary tumor into the circulation may occur in large numbers, and in

early stages of tumor formation 19, 20

. The epithelial-to-mesenchymal transition refers to a

complex molecular and cellular program by which epithelial cells lose their differentiated

epithelial characteristics, instead acquiring mesenchymal features including motility,

invasiveness, and a resistance to apoptosis 21. Although still controversial, the epithelial-to-

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mesenchymal transition is presumed to be required for invasion and metastatic dissemination

of carcinoma cells 21.

Striking disparities among CTCs, tumor cells comprising the primary tumor, and

tumor cells comprising overt metastases have been reported for several solid cancers 22, 23

.

Furthermore, considerable heterogeneity within the CTC population has also been

demonstrated 23, 24

. The two modes of CTC dissemination described above may contribute to

this large heterogeneity, reflecting heterogeneity within the primary tumor itself. It also seems

likely that tumor cells shed from distant metastases contribute to a heterogeneous CTC

population 25, 26

. Most of the CTCs will die, with only as few as 0.01% giving rise to overt

metastases 27, 28

. These tumor-initiating cells, which are called cancer stem cells 14, 29, seem to

persist in an inactive non-proliferative dormant state for years that may confer resistance to

current chemotherapeutics 30-32

.

From breast cancer and colorectal cancer there is considerable evidence that detection

of CTCs identifies a patient population at high risk for disease recurrence 33-38

, but this

hypothesis has not been extensively explored for pancreatic cancer patients. This review

therefore focuses on studies describing the detection of CTCs in pancreatic cancer patients, as

well as the clinical implications of CTCs.

Methods for detecting CTCs

Enrichment of CTCs

Due to the very low frequency of CTCs in blood (∼1 CTC per billion blood cells), it is

technically challenging to both identify and distinguish them from normal epithelial cells;

detection of CTCs is therefore usually preceded by an enrichment procedure (Figure 1).

Traditionally, enrichment of tumor cells has been performed using density gradient

centrifugation (e.g. 39, 40

). Mononuclear cells, including tumor cells, are then enriched by

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centrifugation in an isoosmotic medium (LymphoPrepTM

(Axis-Shield PoC AS), Ficoll-

HyPaqueTM (Sigma-Aldrich), or Oncoquick

R (Greiner bio-one)) that separates the cells based

on their buoyant density. The RosetteSep™ Human Circulating Epithelial Tumor Cell

Cocktail (StemCell Technology) is also based on density centrifugation, but utilizes a

tetrameric antibody complex that couples white blood cells to red cells to increase their

density. Enrichment procedures via membrane filtration according to cell size are also

available. The size of the filter pores causes the CTCs to be retained on the filter, as they are

generally larger than peripheral blood leukocytes. The most acknowledged membrane

microfilter devices are the isolation by size of epithelial tumour cells (ISET) device 41 and a

micro electro-mechanical system-based device (MEMS) 42. However, other devices have also

become available in recent years 43-47

.

To achieve more specific enrichment of CTCs, immunological capture techniques

have been developed. These approaches use antibodies that bind target proteins present at the

cell surface. Both positive and negative selection strategies have been implemented. In the

absence of strictly tumor-specific antigens, epithelial-specific proteins have been employed

for positive selection of CTCs, with epithelial cell adhesion molecule (EpCAM) serving as the

most frequently used antigen for this application 48. In contrast, negative selection strategies

deplete leukocytes from the blood sample via antibodies against leukocyte-specific surface

antigens such as CD45. In this approach, the antibodies are immobilized on a solid support,

either magnetic beads or the insides of flow cells. When using magnetic beads, cells bound to

the beads are separated from those that are unbound by exposing the container (tube or

column) to a strong permanent magnet 49. Several immunomagnetic bead separation systems

are commercially available including the magnetic-activated cell sorting system (Miltenyi

Biotec GmbH), EasySep cell separation (StemCell Technologies), cell isolation by Dynabeads

(Invitrogen), and the Cell Search system (Veridex).

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For flow cells, cells exhibiting the antigen of interest are retained inside the container

by binding to antibodies that coat the flow cell; other cells are removed by buffer flow 50. The

CTC-chip is perhaps the most acknowledged enrichment flow cell 50. However, due to limited

interactions of target cells with the EpCAM-coated micro posts, and the fact that the complex

micro post structure proved challenging to scale up for high-throughput production and

larger-scale clinical applications, the herringbone-chip or “HB-Chip was developed. The HB-

Chip applies passive mixing of cells through the generation of micro vortices caused by

herringbone-shaped ribs within the flow cell, thus significantly increasing the number of

interactions between target CTCs and the EpCAM-coated chip surface. Several other flow cell

systems have also been developed in recent years, including the MagSweeper 51,

dielectrophoretic field-flow fractionation (depFFF) 52, geometrically enhanced differential

immunocapture (GEDI) 53, and the aptamer-mediated, micropillar-based microfluidic device

54.

Many of the immunological enrichment approaches utilize antibodies against the

EPCAM cell surface antigen. However, several technical challenges are associated with this

strategy, such as heterogeneous expression and down-regulation of EpCAM in some CTCs 55,

as well as tumor-like antigen expression on the surface of normal blood cells.

Immunocytological detection of CTCs

The two main approaches presently employed for the detection of CTCs are

immunocytochemistry and the polymerase chain reaction (PCR) (Figure 1). In

immunocytochemistry, the cells to be analyzed are attached to a solid support to allow easy

handling. After fixation, the cells are stained with one or several antibodies. Antibodies

against epithelial-specific proteins, typically cytokeratins (CKs), indicate whether and how

many CTCs are present. In addition, leukocyte-specific antibodies can be used to avoid false

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positives. Staining may be performed via primary antibodies with detectable tags (such as a

fluorescent molecule or gold particles), or via tagged secondary antibodies that bind the

primary antibody. The cells are visualized through enzymatic color reactions or by

fluorescence. The CTCs are detected and counted with a light or fluorescence microscope.

Other methods available for the detection of immunofluorescence-labeled CTCs on glass

slides include ultra-speed automated digital microscopy using fiber-optic array scanning

technology (FAST) 56, 57

and Cytotrack (CytoTrack ApS).

The Cell Search system (Veridex) allows automated immunomagnetic enrichment of

CTCs expressing EpCAM followed by immunocytochemical identification of the tumor cells.

The captured cells are labeled with fluorescent antibodies specific for leukocytes (CD45) and

epithelial cells (CK 8, 18, and 19). The CTCs are identified based on CK+/CD45- staining

and counted. So far, this method has been approved by the United States Food and Drug

Administration for the detection of CTCs in patients with metastatic breast, colon, or prostate

cancer 58. However, the system has also been used for CTC detection in several studies of

pancreatic cancer 59-61

.

Epithelial immunospotting (EPISPOT) is another immunological approach focusing

on the secretion or active release of specific marker proteins from viable tumor cells. In

EPISPOT the cells are cultured on plates coated with specific antibodies, and secreted

proteins are directly immunocaptured by antibodies immobilized on the bottoms of the wells.

The presence of the released protein is revealed by the addition of a fluorochrome-conjugated

secondary antibody. EPISPOT has been used primarily for the detection of CTCs in blood

from breast cancer patients, where MUC-1 and CK19 were used as marker proteins 62.

PCR-based detection of CTCs

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PCR-based detection of CTCs usually exploits mRNAs that are expressed at high levels in the

tumor cells as surrogate markers for CTCs. In the absence of truly tumor-specific mRNAs,

epithelial-specific mRNAs is frequently used for CTC detection. These transcripts are also

expressed in normal blood cells, although at low levels, thus requiring determination of the

highest normal blood level as a lower threshold for CTC detection. Consequently, this

approach is indirect, as it detects elevated marker levels in cell lysates caused by the presence

of CTCs. Quantitative reverse transcription-PCR (RT-qPCR) enables the quantitative

detection of mRNAs, which is required for this strategy. It also allows multiple markers to be

analyzed simultaneously in multimarker assays (e.g. 63). Compared to immunocytochemistry,

the PCR-based method is less subjective, and automation is easily achieved. Several

pancreatic cancer studies have utilized RT-qPCR for the detection of CTCs 64-66

.

RT-qPCR detection has been combined with various enrichment procedures to

enhance the detection of CTCs. AdnaTest (AdnaGen) is an example of a commercially

available kit in which the tumor cells are first enriched from peripheral blood via antibody-

linked magnetic particles, followed by CTC detection by RT-qPCR. Several mRNA markers

are measured to account for the heterogeneity of the CTC population. To date the AdnaTest is

only available for the enrichment and detection of CTCs from patients with breast, colon, or

prostate cancer.

Evidence for the presence of CTCs in pancreatic cancer patients

Although the relevant studies are few, there is some evidence that CTCs can be detected, both

in operable and inoperable pancreatic cancer patients. The current section describes studies

without clinical follow-up data; an overview of these studies is presented in Table 1. The next

section on the other hand, focuses on the pancreatic cancer studies presenting survival

analyses.

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Chausovsky and colleagues (1999) were the first to investigate whether CK20 mRNA

could be used as a surrogate marker for CTC detection in peripheral blood from pancreatic

cancer patients. Of the 116 patients analyzed in this study, which also included patients with

colon and stomach cancer, 28 patients presented with pancreatic cancer. Nested RT-PCR

resulted in CK20 amplification in 22/28 (79%) pancreatic cancer patients, indicating that

CK20 could be a potential biomarker for CTC detection 67. Zhou et al. (2011) also identified

CTCs by conventional RT-PCR, but after immunomagnetic enrichment. They analyzed blood

samples from 25 pancreatic cancer patients via amplification of human telomerase reverse

transcriptase, C-MET, CK20, and CEA 40; the mRNA expression rates of these molecules

were 100% (25/25), 80% (20/25), 84% (21/25), and 80% (20/25), respectively. Also, by

combining the markers into a multimarker approach, all 25 patients exhibited positive CTC

mRNA status 40.

Allard et al. (2004) used the CellSearch system for CTC detection in peripheral blood

from patients with 12 different types of metastatic cancers 59. The CTC prevalence in this

study was determined in 199 patients with non-malignant disease, 964 patients with metastatic

carcinomas, and 145 healthy individuals. Among the 16 patients with metastatic pancreatic

cancer, six patients (37.5%) were CTC-positive; four patients had ≥2 CTCs, one patient had

≥5 CTCs, and one patient had ≥10 CTCs detected per 7.5 mL of blood. Pancreatic cancer

patients were reported to show one of the lowest CTC levels among the included cancer types

59.

Nagrath and colleagues reported the first use of the CTC-chip for enrichment and

detection of CTCs 50. They tested the capacity of the CTC-chip on blood samples obtained

from patients with different metastatic cancer types, including 15 pancreatic cancer patients.

CTCs were identified in 15/15 (100%) pancreatic cancer patients, and the number of detected

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CTCs ranged from 9 to 831 in these patient samples. Moreover, none of the 20 healthy

individuals had any detectable CTCs 50.

In a recent study by Ren et al. (2011), blood samples from 41 stage III pancreatic

cancer patients, obtained before adjuvant therapy and one week after the first cycle of 5-

fluorouracil adjuvant chemotherapy, were evaluated by immunocytochemistry. CTCs were

detected in 33/41 (80.5%) patients before any therapy, but this number decreased to 12/41

(29.3%) patients after adjuvant therapy 68. Marrinucci et al. (2012), on the other hand,

established a fluid phase biopsy approach for identification of CTCs, and applied this

approach to a small cohort of metastatic prostate, breast, and pancreatic cancer patients. CTCs

were identified in 9/18 (50%) pancreatic cancer patients, of whom two had more than 50

CTCs per mL blood 69.

To summarize, several studies reported the presence of CTCs in peripheral blood from

pancreatic cancer patients, but did not evaluate the clinical implications of their findings. The

next section describes several larger studies with evidence for the presence of CTCs in

pancreatic cancer patients; these studies also included clinical follow-up data.

Clinical implications of CTC detection in pancreatic cancer

patients

The clinical relevance of CTCs in pancreatic cancer has not been as extensively investigated

as for other epithelial cancers, such as breast cancer 36, 70

. However, there are some relevant

published studies available for pancreatic cancer, which will now be presented briefly. An

overview of these studies appears in Table 2. This subject has also been discussed by Cen et

al. (2012) 29.

Soeth and colleagues prospectively evaluated the prognostic significance of CK20 RT-

PCR for detection of disseminated tumor cells and/or CTCs in pre-operative bone marrow and

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blood samples from 172 stage I-IV pancreatic cancer patients 39. CK20-positive cells were

detected in 52/154 (33.8%) blood samples. Univariate survival analyses revealed a significant

association between overall survival and the detection of disseminated tumor cells and/or

CTCs. Furthermore, the mean overall survival time of CK20-positive bone marrow and/or

blood samples was 17.9 months as compared to 26.1 months for CK20-negative patients

(P=0.05) 39.

Mataki et al. (2004) evaluated 53 patients with biliary pancreatic cancer, of which 20

patients had a curative-intended operation for pancreatic cancer 71. Blood samples were

obtained every three months after surgery and were enriched for CTCs by density

centrifugation before the CEA mRNA level was determined by nested RT-PCR. Of the 20

pancreatic cancer patients, six (30%) demonstrated positive CEA mRNA status during follow-

up. Of these 5/6 experienced disease recurrence, versus 2/12 patients who remained CEA

mRNA-negative (P=0.007). A similar difference was observed for the overall patient group

(P<0.0001), which included ampullary and biliary duct cancers. The increase in CEA mRNA

was detected before or at the time of recurrence detection by imaging diagnostics, suggesting

that CTC assessments may add to and improve the armamentarium for disease monitoring 71.

Kurihara et al. (2008) used the CellSearch system to examine the level of CTCs in

peripheral blood from 26 (both operable and inoperable) pancreatic cancer patients, to

elucidate whether these levels were predictive of survival 60. Eleven of the 26 (42%) patients

were CTC-positive in their study, and these patients exhibited a significant decrease in

median overall survival time (P<0.001) 60.

Recently, de Albuquerque and colleagues published results from an investigation in

which CTCs were enriched by immunomagnetic separation, followed by RT-qPCR detection

using a multimarker mRNA panel consisting of CK19, MUC1, EpCAM, CEACAM5, and

BIRC5 66. In this study, 16/34 (47.1%) pancreatic cancer patients were positive for at least

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one tumor-associated mRNA marker in blood obtained before systemic treatment. CTC

detection was associated with shorter progression-free survival (66 versus 138 days, P=0.01)

66.

Other studies have failed to demonstrate a significant prognostic relevance for CTC

detection in pancreatic cancer patients, although trends have been detected. Z’graggen et al.

(2001) assessed both blood and bone marrow samples from 105 pancreatic cancer patients

(both operable and inoperable) and 66 healthy individuals by immunocytochemistry 72. CTCs

were detected in blood samples from 3/32 (9%) patients with resectable disease, and in 24/73

(33%) patients with unresectable pancreatic cancer (P=0.023). There was a tendency toward

an association between CTC detection and disease progression, although it was not

statistically significant (P=0.08) 72.

Khoja and colleagues compared CellSearch and ISET with regard to CTC enumeration

and characterization in 54 patients with newly diagnosed, progressive metastatic or inoperable

adenocarcinoma of the pancreas 61. Their hypothesis that ISET would detect a higher number

of CTCs than Cell Search, since ISET is a marker-independent enrichment procedure, was

validated. As they assumed ISET did detect more CTCs than CellSearch (CTCs/7.5 mL of

blood: 9 (range 0-240) versus 0 (range 0-144), respectively). ISET also resulted in more

flexible CTC characterization than CellSearch. Although CTC detection did not correlate with

progression-free- or overall survival, there was a non-significant trend (progression-free

survival, P=0.26; overall survival, P=0.19) toward shorter survival for patients with CTCs

detected by CellSearch 61.

Hoffmann et al. (2007) investigated the prognostic potential of CK19 mRNA detection

in blood, bone marrow, and peritoneal lavage from 53 patients, including 37 primary

pancreatic cancer patients, comparing qualitative nested PCR with RT-qPCR for detection of

CTCs in blood and disseminated tumor cells in bone marrow 64. By RT-qPCR, 24/37 (64%)

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blood samples from pancreatic cancer patients were positive for CK19 mRNA. Moreover, the

CK19 mRNA levels in blood samples obtained one day and 10 days after surgery tended to be

lower than the levels in pre-operative samples. At the end of the study, 14 patients had died;

eight of these patients had at least one CK19-positive sample. However, no significant

survival benefit was observed for patients negative for CK19 mRNA expression, although a

trend toward a survival difference was observed (P=0.15) 64.

Sergeant et al. (2011) established a RT-qPCR assay for EpCAM mRNA detection and

evaluated pre-, peri-, and post-operative peripheral blood samples as well as peritoneal cavity

samples from 48 patients (40 resectable, eight unresectable) with pancreatic ductal

adenocarcinoma 65. EpCAM detection in blood significantly increased immediately after

surgery, before decreasing to levels comparable to pre-operative levels one day after surgery.

Pre-operative blood samples were EpCAM-positive in 10/40 (25%) patients compared to

27/40 (67.5%) patients immediately after surgery (P<0.0001). Six weeks after surgery, 8/34

(23.5%) patients were considered to be positive for EpCAM. However, the study did not

uncover any associations between EpCAM positivity and cancer-specific or disease-free

survival at any of the sampling time points, although some tendencies were observed for the

pre-operative sampling (P=0.17 and 0.28, respectively) 65.

Given the available evidence, the clinical implications of CTC detection in pancreatic

cancer patients are to some extent unclear at the moment. Some investigations demonstrated

significant associations between CTC detection and survival, whereas others did not. Yet,

several studies were rather small, resulting in low statistical power, and different detection

technologies and marker choices further complicate the picture. Several of the studies that

employed RT-qPCR for CTC detection also included patients with chronic pancreatitis or

benign diseases as normal control samples, which may have resulted in higher thresholds for

CTC positivity, increasing the number of false-negative samples. However, as most studies

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lacking significant association between CTC detection and survival did report distinct

tendencies, taking the low patient numbers into account, the evidence seems to support a

prognostic value for CTC detection in pancreatic cancer patients (Table 2). A proper meta-

analysis would be required to verify this interpretation. Nevertheless, larger studies exploring

the clinical impact of CTC analysis in pancreatic cancer are also highly warranted.

Conclusion and future perspectives

Although there are some diverging results, our review of the present literature seems to

support a clinical utility for CTC analysis in pancreatic cancer. However, challenges exist in

this field, as there is growing evidence that the CTC population is heterogeneous; only a small

proportion of tumor cells are able to proliferate extensively and form new tumors. The ability

to identify, isolate, propagate, and molecularly characterize these cancer-initiating cells, also

called cancer stem cells, may reveal new biomarkers and thus expand our understanding of

the biology underlying metastases. The heterogeneity of primary tumors suggests that

multiple tissue biopsies are required for complete molecular characterization of a tumor. In

this respect, CTC analyses can be considered “liquid biopsies” and may provide a potential

alternative to invasive tissue biopsies. Liquid biopsies have broad implications for clinical

cancer management, including detection, diagnosis, and monitoring of cancer. Repeated blood

sample analyses may increase the chances of isolating the most relevant CTCs, while causing

minimal discomfort to the patient. Repeated blood sample analyses would also make it

possible to reveal changes in CTC levels during treatment, adding information to improve

prognosis with regard to therapy selection as well as shifting of drug(s) due to resistance.

Characterization of the CTC population may contribute to enhanced personalized medicine in

terms of drug development based on CTC characteristics, rather than primary tumor

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characteristics. Hence, focusing on methods to enrich specific CTC populations and

characterize them will most likely improve the clinical value of CTC investigations in the

future.

Acknowledgements

Supported by the Folke Hermansen Foundation

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References

1. Blackford A, Parmigiani G, Kensler TW, Wolfgang C, Jones S, Zhang X, Parsons

DW, Lin JC, Leary RJ, Eshleman JR, Goggins M, Jaffee EM, et al. Genetic mutations

associated with cigarette smoking in pancreatic cancer. Cancer Res 2009;69:3681-88.

2. Vincent A, Herman J, Schulick R, Hruban RH, Goggins M. Pancreatic cancer.

Lancet 2011;378:607-20.

3. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. AJCC cancer

staging manual. 7th ed. New York: Springer, 2010.

4. Halm U, Schumann T, Schiefke I, Witzigmann H, Mossner J, Keim V. Decrease of

CA 19-9 during chemotherapy with gemcitabine predicts survival time in patients with

advanced pancreatic cancer. Br J Cancer 2000;82:1013-6.

5. Katz A, Hanlon A, Lanciano R, Hoffman J, Coia L. Prognostic value of CA 19-9

levels in patients with carcinoma of the pancreas treated with radiotherapy. Int J Radiat Oncol

Biol Phys 1998;41:393-6.

6. Mehta J, Prabhu R, Eshpuniyani P, Kantharia C, Supe A. Evaluating the efficacy of

tumor markers CA 19-9 and CEA to predict operability and survival in pancreatic

malignancies. Tropical Gastroenterol 2010;31:190-4.

7. Burris HA, 3rd, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR,

Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, et al.

Improvements in survival and clinical benefit with gemcitabine as first-line therapy for

patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997;15:2403-13.

8. Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet 2004;363:1049-57.

9. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics,

2008. CA Cancer J Clin 2008;58:71-96.

10. Hidalgo M. Pancreatic cancer. N Engl J Med 2010;362:1605-17.

Page 17 of 34



18

11. Von Hoff DD, Ramanathan RK, Borad MJ, Laheru DA, Smith LS, Wood TE,

Korn RL, Desai N, Trieu V, Iglesias JL, Zhang H, Soon-Shiong P, et al. Gemcitabine Plus

nab-Paclitaxel Is an Active Regimen in Patients With Advanced Pancreatic Cancer: A Phase

I/II Trial. J Clin Oncol 2011;29:4548-54

12. Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y, Adenis

A, Raoul JL, Gourgou-Bourgade S, de la Fouchardiere C, Bennouna J, Bachet JB, et al.

FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med

2011;364:1817-25.

13. Tada M, Omata M, Kawai S, Saisho H, Ohto M, Saiki RK, Sninsky JJ. Detection

of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic

adenocarcinoma. Cancer Res 1993;53:2472-4.

14. Balic A, Dorado J, Alonso-Gomez M, Heeschen C. Stem cells as the root of

pancreatic ductal adenocarcinoma. Exp Cell Res 2011;318:691-704.

15. Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M, Hruban RH, Maitra A,

Kinzler K, Vogelstein B, Goggins M. Presence of somatic mutations in most early-stage

pancreatic intraepithelial neoplasia. Gastroenterology 2012;142:730-33.

16. Hruban RH, van Mansfeld AD, Offerhaus GJ, van Weering DH, Allison DC,

Goodman SN, Kensler TW, Bose KK, Cameron JL, Bos JL. K-ras oncogene activation in

adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of

mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide

hybridization. Am J Pathol 1993;143:545-54.

17. Shibata D, Almoguera C, Forrester K, Dunitz J, Martin SE, Cosgrove MM,

Perucho M, Arnheim N. Detection of c-K-ras mutations in fine needle aspirates from human

pancreatic adenocarcinomas. Cancer Res 1990;50:1279-83.

Page 18 of 34



19

18. Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S. Stem cell and

epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor

cells of metastatic breast cancer patients. Breast Cancer Res 2009;11:R46.

19. Husemann Y, Geigl JB, Schubert F, Musiani P, Meyer M, Burghart E, Forni G,

Eils R, Fehm T, Riethmuller G, Klein CA. Systemic spread is an early step in breast cancer.

Cancer Cell 2008;13:58-68.

20. Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer

2004;4:448-56.

21. Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states:

acquisition of malignant and stem cell traits. Nat Rev Cancer 2009;9:265-73.

22. Stoecklein NH, Klein CA. Genetic disparity between primary tumours,

disseminated tumour cells, and manifest metastasis. Int J Cancer 2010;126:589-98.

23. Klein CA, Blankenstein TJ, Schmidt-Kittler O, Petronio M, Polzer B, Stoecklein

NH, Riethmuller G. Genetic heterogeneity of single disseminated tumour cells in minimal

residual cancer. Lancet 2002;360:683-9.

24. Powell AA, Talasaz AH, Zhang H, Coram MA, Reddy A, Deng G, Telli ML,

Advani RH, Carlson RW, Mollick JA, Sheth S, Kurian AW, et al. Single cell profiling of

circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell

lines. PLoS One 2012;7:e33788.

25. Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH, Norton L, Massague

J. Tumor self-seeding by circulating cancer cells. Cell 2009;139:1315-26.

26. Alix-Panabieres C, Schwarzenbach H, Pantel K. Circulating tumor cells and

circulating tumor DNA. Annu Rev Med 2012;63:199-215.

27. Fidler IJ. Metastasis: guantitative analysis of distribution and fate of tumor

embolilabeled with 125 I-5-iodo-2'-deoxyuridine. J Natl Cancer Inst 1970;45:773-82.

Page 19 of 34



20

28. Gupta PB, Mani S, Yang J, Hartwell K, Weinberg RA. The evolving portrait of

cancer metastasis. Cold Spring Harb Symp Quant Biol. 2005;70:291-7.

29. Cen P, Ni X, Yang J, Graham DY, Li M. Circulating tumor cells in the diagnosis

and management of pancreatic cancer. Biochim Biophys Acta 2012;1826:350-6.

30. Paez D, Labonte MJ, Bohanes P, Zhang W, Benhanim L, Ning Y, Wakatsuki T,

Loupakis F, Lenz HJ. Cancer dormancy: a model of early dissemination and late cancer

recurrence. Clin Cancer Res 2012;18:645-53.

31. Uhr JW, Pantel K. Controversies in clinical cancer dormancy. Proc Natl Acad Sci

U S A 2011;108:12396-400.

32. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer

2005;5:275-84.

33. Xenidis N, Ignatiadis M, Apostolaki S, Perraki M, Kalbakis K, Agelaki S,

Stathopoulos EN, Chlouverakis G, Lianidou E, Kakolyris S, Georgoulias V, Mavroudis D.

Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in

patients with early breast cancer. J Clin Oncol 2009;27:2177-84.

34. Ignatiadis M, Xenidis N, Perraki M, Apostolaki S, Politaki E, Kafousi M,

Stathopoulos EN, Stathopoulou A, Lianidou E, Chlouverakis G, Sotiriou C, Georgoulias V, et

al. Different prognostic value of cytokeratin-19 mRNA positive circulating tumor cells

according to estrogen receptor and HER2 status in early-stage breast cancer. J Clin Oncol

2007;25:5194-202.

35. Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A, Reuben JM, Doyle GV,

Matera J, Allard WJ, Miller MC, Fritsche HA, Hortobagyi GN, et al. Circulating tumor cells:

a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol

2005;23:1420-30.

Page 20 of 34



21

36. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM,

Doyle GV, Allard WJ, Terstappen LW, Hayes DF. Circulating tumor cells, disease

progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781-91.

37. Xenidis N, Perraki M, Kafousi M, Apostolaki S, Bolonaki I, Stathopoulou A,

Kalbakis K, Androulakis N, Kouroussis C, Pallis T, Christophylakis C, Argyraki K, et al.

Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells

detected by real-time polymerase chain reaction in node-negative breast cancer patients. J

Clin Oncol 2006;24:3756-62.

38. Iinuma H, Watanabe T, Mimori K, Adachi M, Hayashi N, Tamura J, Matsuda K,

Fukushima R, Okinaga K, Sasako M, Mori M. Clinical significance of circulating tumor cells,

including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients

with Dukes' stage B and C colorectal cancer. J Clin Oncol 2011;29:1547-55.

39. Soeth E, Grigoleit U, Moellmann B, Roder C, Schniewind B, Kremer B, Kalthoff

H, Vogel I. Detection of tumor cell dissemination in pancreatic ductal carcinoma patients by

CK 20 RT-PCR indicates poor survival. J Cancer Res Clin Oncol 2005;131:669-76.

40. Zhou J, Hu L, Yu Z, Zheng J, Yang D, Bouvet M, Hoffman RM. Marker

expression in circulating cancer cells of pancreatic cancer patients. J Surg Res 2011;171:631-

6.

41. Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schutze K, Capron F, Franco D,

Pazzagli M, Vekemans M, Lacour B, Brechot C, et al. Isolation by size of epithelial tumor

cells : a new method for the immunomorphological and molecular characterization of

circulatingtumor cells. Am J Pathol 2000;156:57-63.

42. Zheng S, Lin H, Liu JQ, Balic M, Datar R, Cote RJ, Tai YC. Membrane

microfilter device for selective capture, electrolysis and genomic analysis of human

circulating tumor cells. J Chromatogr A 2007;1162:154-61.

Page 21 of 34



22

43. Zheng S, Lin HK, Lu B, Williams A, Datar R, Cote RJ, Tai YC. 3D microfilter

device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices

2011;13:203-13.

44. Lin HK, Zheng S, Williams AJ, Balic M, Groshen S, Scher HI, Fleisher M, Stadler

W, Datar RH, Tai YC, Cote RJ. Portable filter-based microdevice for detection and

characterization of circulating tumor cells. Clin Cancer Res 2010;16:5011-8.

45. Williams A, Balic M, Datar R, Cote R. Size-Based Enrichment Technologies for

CTC Detection and Characterization. Recent Results Cancer Res 2012;195:87-95.

46. Xu T, Lu B, Tai YC, Goldkorn A. A cancer detection platform which measures

telomerase activity from live circulating tumor cells captured on a microfilter. Cancer Res

2010;70:6420-6.

47. Bhagat AA, Hou HW, Li LD, Lim CT, Han J. Pinched flow coupled shear-

modulated inertial microfluidics for high-throughput rare blood cell separation. Lab Chip

2011;11:1870-8.

48. Parkinson DR, Dracopoli N, Gumbs Petty B, Compton C, Cristofanilli M,

Deisseroth A, Hayes DF, Kapke G, Kumar P, Lee JS, Liu MC, McCormack R, et al.

Considerations in the development of circulating tumor cell technology for clinical use. J

Transl Med 2012;10:138.

49. Neurauter AA, Bonyhadi M, Lien E, Nokleby L, Ruud E, Camacho S, Aarvak T.

Cell isolation and expansion using Dynabeads. Adv Biochem Eng Biotechnol 2007;106:41-

73.

50. Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR,

Kwak EL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, et al. Isolation of rare circulating

tumour cells in cancer patients by microchip technology. Nature 2007;450:1235-9.

Page 22 of 34



23

51. Talasaz AH, Powell AA, Huber DE, Berbee JG, Roh KH, Yu W, Xiao W, Davis

MM, Pease RF, Mindrinos MN, Jeffrey SS, Davis RW. Isolating highly enriched populations

of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device.

Proc Natl Acad Sci U S A 2009;106:3970-5.

52. Gascoyne PR, Noshari J, Anderson TJ, Becker FF. Isolation of rare cells from cell

mixtures by dielectrophoresis. Electrophoresis 2009;30:1388-98.

53. Gleghorn JP, Pratt ED, Denning D, Liu H, Bander NH, Tagawa ST, Nanus DM,

Giannakakou PA, Kirby BJ. Capture of circulating tumor cells from whole blood of prostate

cancer patients using geometrically enhanced differential immunocapture (GEDI) and a

prostate-specific antibody. Lab Chip 2010;10:27-9.

54. Sheng W, Chen T, Kamath R, Xiong X, Tan W, Fan ZH. Aptamer-enabled

efficient isolation of cancer cells from whole blood using a microfluidic device. Anal Chem

2012;84:4199-206.

55. Sieuwerts AM, Kraan J, Bolt J, van der Spoel P, Elstrodt F, Schutte M, Martens

JW, Gratama JW, Sleijfer S, Foekens JA. Anti-epithelial cell adhesion molecule antibodies

and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst

2009;101:61-6.

56. Hsieh HB, Marrinucci D, Bethel K, Curry DN, Humphrey M, Krivacic RT,

Kroener J, Kroener L, Ladanyi A, Lazarus N, Kuhn P, Bruce RH, et al. High speed detection

of circulating tumor cells. Biosens Bioelectron 2006;21:1893-9.

57. Krivacic RT, Ladanyi A, Curry DN, Hsieh HB, Kuhn P, Bergsrud DE, Kepros JF,

Barbera T, Ho MY, Chen LB, Lerner RA, Bruce RH. A rare-cell detector for cancer. Proc

Natl Acad Sci U S A 2004;101:10501-4.

Page 23 of 34



24

58. Miller MC, Doyle GV, Terstappen LW. Significance of Circulating Tumor Cells

Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate

Cancer. J Oncol 2010;2010:617421.

59. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, Tibbe AG,

Uhr JW, Terstappen LW. Tumor cells circulate in the peripheral blood of all major

carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer

Res 2004;10:6897-904.

60. Kurihara T, Itoi T, Sofuni A, Itokawa F, Tsuchiya T, Tsuji S, Ishii K, Ikeuchi N,

Tsuchida A, Kasuya K, Kawai T, Sakai Y, et al. Detection of circulating tumor cells in

patients with pancreatic cancer: a preliminary result. J Hepatobiliary Pancreat Surg

2008;15:189-95.

61. Khoja L, Backen A, Sloane R, Menasce L, Ryder D, Krebs M, Board R, Clack G,

Hughes A, Blackhall F, Valle JW, Dive C. A pilot study to explore circulating tumour cells in

pancreatic cancer as a novel biomarker. Br J Cancer 2011;106:508-16.

62. Alix-Panabieres C, Vendrell JP, Pelle O, Rebillard X, Riethdorf S, Muller V,

Fabbro M, Pantel K. Detection and characterization of putative metastatic precursor cells in

cancer patients. Clin Chem 2007;53:537-9.

63. Tjensvoll K, Oltedal S, Farmen RK, Shammas FV, Heikkila R, Kvaloy JT, Gilje B,

Smaaland R, Nordgard O. Disseminated tumor cells in bone marrow assessed by TWIST1,

cytokeratin 19, and mammaglobin A mRNA predict clinical outcome in operable breast

cancer patients. Clin Breast Cancer 2010;10:378-84.

64. Hoffmann K, Kerner C, Wilfert W, Mueller M, Thiery J, Hauss J, Witzigmann H.

Detection of disseminated pancreatic cells by amplification of cytokeratin-19 with

quantitative RT-PCR in blood, bone marrow and peritoneal lavage of pancreatic carcinoma

patients. World J Gastroenterol 2007;13:257-63.

Page 24 of 34



25

65. Sergeant G, Roskams T, van Pelt J, Houtmeyers F, Aerts R, Topal B. Perioperative

cancer cell dissemination detected with a real-time RT-PCR assay for EpCAM is not

associated with worse prognosis in pancreatic ductal adenocarcinoma. BMC Cancer

2011;11:47.

66. de Albuquerque A, Kubisch I, Breier G, Stamminger G, Fersis N, Eichler A, Kaul

S, Stolzel U. Multimarker Gene Analysis of Circulating Tumor Cells in Pancreatic Cancer

Patients: A Feasibility Study. Oncology 2012;82:3-10.

67. Chausovsky G, Luchansky M, Figer A, Shapira J, Gottfried M, Novis B,

Bogelman G, Zemer R, Zimlichman S, Klein A. Expression of cytokeratin 20 in the blood of

patients with disseminated carcinoma of the pancreas, colon, stomach, and lung. Cancer

1999;86:2398-405.

68. Ren C, Han C, Zhang J, He P, Wang D, Wang B, Zhao P, Zhao X. Detection of

apoptotic circulating tumor cells in advanced pancreatic cancer following 5-fluorouracil

chemotherapy. Cancer Biol Ther 2011;12:700-6.

69. Marrinucci D, Bethel K, Kolatkar A, Luttgen MS, Malchiodi M, Baehring F, Voigt

K, Lazar D, Nieva J, Bazhenova L, Ko AH, Korn WM, et al. Fluid biopsy in patients with

metastatic prostate, pancreatic and breast cancers. Phys Biol 2012;9:016003.

70. Pachmann K, Camara O, Kavallaris A, Krauspe S, Malarski N, Gajda M, Kroll T,

Jorke C, Hammer U, Altendorf-Hofmann A, Rabenstein C, Pachmann U, et al. Monitoring the

response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer

allows detection of patients at risk of early relapse. J Clin Oncol 2008;26:1208-15.

71. Mataki Y, Takao S, Maemura K, Mori S, Shinchi H, Natsugoe S, Aikou T.

Carcinoembryonic antigen messenger RNA expression using nested reverse transcription-

PCR in the peripheral blood during follow-up period of patients who underwent curative

Page 25 of 34



26

surgery for biliary-pancreatic cancer: longitudinal analyses. Clin Cancer Res 2004;10:3807-

14.

72. Z'Graggen K, Centeno BA, Fernandez-del Castillo C, Jimenez RE, Werner J,

Warshaw AL. Biological implications of tumor cells in blood and bone marrow of pancreatic

cancer patients. Surgery 2001;129:537-46.

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27

Tables

Table 1: Studies of CTC detection in pancreatic cancer patients without survival data. Only

studies with 15 or more patients have been included.

Table 2: Studies of the clinical relevance of CTC detection in pancreatic cancer patients.

Only studies with 15 or more patients have been included.

Page 27 of 34



28

Figure legends

Figure 1: An overview of methods used for enrichment and detection of circulating tumor

cells (CTCs). FAST= fiber-optic array scanning technology, EPISPOT= Epithelial

immunospotting, RT-qPCR= Reverse transcription quantitative-PCR.

Page 28 of 34



Table 1: Studies of CTC detection in pancreatic cancer patients without survival data. Only studies with 15 or more patients have been included.

Study Markers Enrichment CTC detection method Number of

patients

Summary

Chausovsky et al., 1999

CK20 mRNA Density centrifugation Nested RT-PCR N=28 79% of the patients were CK20-

positive. No correlation between

CK20 status and CEA and CA19-9

levels.

Allard et al., 2004

CK8, CK18, and

CK19

Immunomagnetic

enrichment with

EpCAM

CellSearch N=16 CTCs were detected in 6/16

pancreatic cancer patients.

Nagrath et al., 2007

CKs Immunomagnetic

enrichment with

EpCAM

CTC-chip N=15 CTCs were detected in all 15

patients; the number of isolated

CTCs ranged from 9 to 831 per mL

blood.

Ren et al., 2011

CA19-9 and CK8/18 Red cell lysis buffer

followed by CD45

depletion

Immunocytochemistry N=41 CTCs were detected in 80.5% of

patients before and in 29.3% of

patients one week after

chemotherapy.

Zhou et al., 2011 h-TERT, C-MET,

CK20, and CEA

mRNAs

Immunomagnetic

enrichment with

EpCAM

Nested RT-PCR N=25 h-TERT-positive CTCs were

detected in 100% (25/25), C-MET

in 80% (20/25), CK20 in 84%

(21/25), and CEA in 80% (20/25)

of the patients.

Marrinucci et al., 2012

CKs No enrichment Immunocytochemistry N=18 CTCs were detected in 50% of the

patients.

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5051525354555657585960

CK = Cytokeratin, PSCA = prostate stem cell antigen, CA19-9 = carbohydrate antigen 19-9, CEA = carcinoembryonic antigen, h-TERT = human telomerase reverse transcriptase.

Page 30 of 34



5051525354555657585960

Table 2: Studies of the clinical relevance of CTC detection in pancreatic cancer patients. Only studies with 15 or more patients have been

included.

Study Markers Enrichment CTC detection method Number of

patients

Summary

Z’graggen et al., 2001

AE1/AE3, CK7,

CK19, CK20, and

glycoproteins

Density centrifugation Immunocytochemistry N=105 CTCs were detected in 3/32 (9%)

patients with resectable disease,

and in 24/73 (33%) patients with

unresectable pancreatic cancer.

There was a trend towards an

association between CTC detection

and disease progression.

Mataki et al., 2004

CEA mRNA Density centrifugation Nested PCR N=20 6/20 (30%) patients had positive

CEA status during follow-up, and

five of these patients experienced

disease recurrence compared to

2/12 CEA mRNA-negative patients

(P=0.007).

Soeth et al., 2005

CK20 mRNA Density centrifugation Nested PCR N=154 CK20-positive cells were detected

in 52/154 (33.8%) patients, and

these patients had significantly

shorter overall survival (P=0.05).

Hoffmann et al., 2007

CK19 mRNA Density centrifugation

after erythrocyte lysis

Compared nested PCR and

RT-qPCR

N=37 24/37 (64%) patients had positive

CK19 mRNA status before surgery

by RT-qPCR. The CK19 level

decreased after surgery, and there

was a trend towards an association

between CK19 mRNA status and

survival.

Kurihara et al., 2008

CK8, CK18, and

CK19

Immunomagnetic

enrichment with

EpCAM

Cell Search N=26 11/26 (42%) patients were CTC-

positive and had significantly

shorter overall survival (P<0.001).

Page 31 of 34



5051525354555657585960

Khoja et al., 2011

CK8, CK18, CK19,

EpCAM, anti-CK,

Vimentin, CK7, and E-

cadherin

ISET and

immunomagnetic

enrichment with

EpCAM

Immunocytochemistry and

Cell Search

N=53 More CTCs were detected by ISET

enrichment. There was a trend

toward decreased survival for

patients with CTCs detected by

CellSearch.

Sergeant et al., 2011 EpCAM mRNA Density centrifugation

after erythrocyte lysis

RT-qPCR N=48 25% of the patients had EpCAM-

positive cells before surgery versus

65% after surgery. There was a

tendency toward an association

between EpCAM mRNA status and

survival in the pre-operative

samples.

De Albuquerque et al., 2012

CK19, MUC1,

EpCAM, CEACAM5,

and BIRC5 mRNAs.

Immunomagnetic

enrichment with

EpCAM and MUC1

RT-qPCR N=34 16/34 (47.1%) patients were

positive for at least one CTC

marker; these patients had

significantly shorter progression-

free survival (P=0.01).

CK = Cytokeratin, MUC1 = Mucin 1, EpCAM = epithelial cell adhesion molecule, CEACAM5 = carcinoembryonic antigen-related cell adhesion molecule 5, BIRC5 = baculoviral inhibitor of apoptosis repeat-

containing 5, CEA = carcinoembryonic antigen.

Page 32 of 34



5051525354555657585960

Peripheral blood sample

Enrichment of CTCs Density gradient centrifugation

Membrane filtration

Immunomagnetic beads

Flow cells

CTC detection

Immunological

PCR-based

Immunocytochemistry FAST Cytotrack

CellSearch

EPISPOT

CTC-chip

Other

RT-qPCR AdnaTest

Figure 1: An overview of methods used for enrichment and detection of circulating tumor cells (CTCs). FAST= fiber-optic array scanning technology, EPISPOT= Epithelial immunospotting, RT-qPCR= Reverse transcription quantitative-PCR.

Page 33 of 34



4445464748495051525354555657585960

Figure 1: An overview of methods used for enrichment and detection of circulating tumor cells (CTCs). FAST= fiber-optic array scanning technology, EPISPOT= Epithelial immunospotting, RT-qPCR= Reverse

transcription quantitative-PCR.

254x190mm (96 x 96 DPI)

Page 34 of 34



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