Download Comparison of assay methods for detection of circulating tumor cells

IJC
International Journal of Cancer
Comparison of assay methods for detection of circulating
tumor cells in metastatic breast cancer: AdnaGen AdnaTest
BreastCancer Select/DetectTM versus Veridex CellSearchTM
system
E. Andreopoulou1, L.-Y. Yang2, K.M. Rangel2, J.M. Reuben3, L. Hsu1, S. Krishnamurthy4, V. Valero1,
H.A. Fritsche2 and M. Cristofanilli5
1
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX
Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX
3
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX
4
Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX
5
Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA
Early Detection and Diagnosis
2
The detection of CTCs prior to and during therapy is an independent and strong prognostic marker, and it is predictive of poor
treatment outcome. A major challenge is that different technologies are available for isolation and characterization of CTCs in
peripheral blood (PB). We compare the CellSearch system and AdnaTest BreastCancer Select/Detect, to evaluate the extent
that these assays differ in their ability to detect CTCs in the PB of MBC patients. CTCs in 7.5 ml of PB were isolated and
enumerated using the CellSearch, before new treatment. Two cutoff values of 2 and 5 CTCs/7.5 ml were used. AdnaTest
requires 5 ml of PB to detect gene transcripts of tumor markers (GA733-2, MUC-1, and HER2) by RT-PCR. AdnaTest was scored
positive if 1 of the transcript PCR products for the 3 markers were detected at a concentration 0.15 ng/ll. A total of 55
MBC patients were enrolled. 26 (47%) patients were positive for CTCs by the CellSearch (2 cutoff), while 20 (36%) were
positive (5 cutoff). AdnaTest was positive in 29 (53%) with the individual markers being positive in 18% (GA733-2), 44%
(MUC-1), and 35% (HER2). Overall positive agreement was 73% for CTC2 and 69% for CTC5. These preliminary data
suggest that the AdnaTest has equivalent sensitivity to that of the CellSearch system in detecting 2 or more CTCs. While there
is concordance between these 2 methods, the AdnaTest complements the CellSearch system by improving the overall CTC
detection rate and permitting the assessment of genomic markers in CTCs.
Metastatic breast cancer (MBC) is an incurable disease that is
treated with palliative intent. The clinical significance of circulating tumor cells (CTCs) for the prognosis and prediction
of survival outcomes in patients with MBC has been well
documented.1–7 The detection of CTCs prior to and during
therapy is an independent and strong prognostic marker, and
it is predictive of poor treatment outcome. The prognostic
implications of detecting CTCs prior to or during therapy for
breast cancer raise important questions about the biological
Key words: circulating tumor cells, evolving technology, metastatic
breast cancer, Prognostic markers, predictive markers
Grant sponsor: Oncovista and AdnaGen
DOI: 10.1002/ijc.26111
History: Received 15 Jul 2010; Accepted 3 Feb 2011; Online 5 Apr
2011
Correspondence to: Dr. E. Andreopoulou, Department of Breast
Medical Oncology, Unit 1354, The University of Texas MD
Anderson Cancer Center, 1515 Holcombe Blvd., Houston TX 77030,
USA, Tel: þ1-713-792-2817; Fax: þ1-713-794-4385,
E-mail: [email protected]
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Int. J. Cancer: 130, 1590–1597 (2012) V
nature of these cells. A comprehensive analysis of CTCs may
provide new insights into the biology of breast cancer that
could have a potential impact on the clinical management of
breast cancer patients.
Advances in technology have produced various methods
of detecting CTCs in the blood of cancer patients. These
technologies vary with regard to sensitivity, specificity, reproducibility, for isolation and characterization of CTCs in peripheral blood (PB). Current techniques used to detect CTCs
in cancer patients rely on a cell-enrichment step followed by
a detection step. Most enrichment steps utilize antibodybased magnetic capture with magnetically bound antibodies
directed against the epithelial cell adhesion molecule-1
(EpCAM).8–14 CTCs can be detected after immunomagnetic
capture using either direct, antibody-based methods such as
immunocytochemistry (ICC), immunofluorescence (IF), and
flow cytometry (FACS), or by indirect, nucleic acid-based
methods which measure mRNA transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR).8–14
The CellSearch system (Veridex, LLC, Warren, NJ, USA),
which uses immunomagnetic separation to isolate tumor cells
with subsequent visualization by microscopy and counting of
Andreopoulou et al.
cells that express cytokeratin by IF, has been cleared by the
U.S. Food and Drug Administration for use in patient
care.15,16
The novel PCR-based assay developed by AdnaGen AG
(Langenhagen, Germany) is performed on standard technology platforms and makes use of RT-PCR to identify putative
transcripts of genes in EpCAM-positive cells that are isolated
by a magnetic separation method.14,17,18 Because of the combination of different selection and tumor markers, both the
heterogeneity of the tumor cells and possible individual or
therapy-induced deviations in the expression patterns are
taken into account.
The primary purpose of this study was to determine if the
AdnaTest BreastCancer Select/Detect correlated with the CellSearch system at the defined cutoff values of 2 and 5 CTCs
per 7.5 ml of blood. We also examined the individual gene
transcript markers used in the AdnaTest to determine if
HER2 gene amplification of the primary tumor correlated
with HER2 protein expression in CTCs.
cytokeratins (CK 8, 18, 19-phycoerythrin) are used to distinguish epithelial cells from leukocytes. The identification and
enumeration of CTCs were performed using the CellSpotter
Analyzer, a semiautomated fluorescence-based microscopy
system that permits computer-generated reconstruction of
cellular images. CTCs were defined as nucleated cells that
lacked CD45 and that expressed cytokeratin. Technical details
of the CellSearch and CellSpotter systems, including accuracy,
precision, linearity, and reproducibility, have been described
elsewhere.19
Based on previous studies, we used 2 cutoff values to
determine CTC positivity in samples: 2 or more CTCs per
7.5 ml of blood20 and 5 or more CTCs per 7.5 ml of blood.1
Healthy subjects and patients with nonmalignant diseases
have less than 2 CTCs per 7.5 ml of blood. The median progression-free survival among patients reached a plateau at
approximately 5 CTCs per 7.5 ml of blood and this value
was chosen to distinguish patients with an unfavorable prognosis from patients with a favorable prognosis.
Patients and Methods
Detection of CTCs by AdnaTest BreastCancer Select/Detect
We conducted a prospective trial at The University of Texas
MD Anderson Cancer Center to evaluate the CellSearch system and the AdnaTest for CTC detection. The CellSearch
System, is an automated, standardized system for the immunocytochemical detection and quantification of CTC in blood
and has been regulatory-approved. The AdnaTest Breast
Cancer Select/Detect essentially involves the detection of tumor-associated transcripts by RT-PCR after an immunomagnetically enrichment of tumor cells. All patients with progressive, measurable MBC about to start a new treatment were
included. All patients had Eastern Cooperative Oncology
Group scores performance status of 0 to 2. Prior adjuvant
treatment, treatment of metastatic disease, or both were permitted. The Institutional Review Board at MD Anderson
approved the study protocol, and all patients provided written informed consent.
The AdnaTest can be performed on standard technology
platforms. AdnaGen’s two-step ‘‘Combination-of-Combinations Principle" is illustrated in Figure 1. It initially involves
cell isolation, whereby tumor cells are enriched by an antibody-mix linked to magnetic particles and mRNA is isolated
from the selected tumor cells, and subsequent molecular biological detection and analysis, whereby the isolated mRNA is
transcribed into cDNA and a multiplex PCR is carried out
for the analysis of tumor-associated gene expression.14,17,18
Isolation and Enumeration of CTCs by CellSearch
CTC isolation and enumeration were performed using the
CellSearch system as previously described.1,15,16 Briefly, blood
samples were drawn into 10-ml CellSave Vacutainer tubes
(Becton Dickinson), which contained EDTA and a cell fixative. Samples were maintained at room temperature and
processed within 72 h after collection. All evaluations were
performed without knowledge of the clinical status of the
patients.
The CellSearch system consists of a semiautomated system
(CellPrep) for the preparation of the sample, and it is used
with the CellSearch Epithelial Cell Kit. The CellPrep system
enriches the sample for cells expressing EpCAM with antibody-coated ferrous particles, and it labels the cell nucleus
with the fluorescent nucleic acid dye 4,2-diamidino-2-phenylindole dihydrochloride. Fluorescently labeled monoclonal
antibodies specific for leukocytes (CD45-allophycocyan) and
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Int. J. Cancer: 130, 1590–1597 (2012) V
The AdnaTest has a reported analytical sensitivity of 2
CTCs per 5 ml of blood21
For the AdnaTest, blood samples (5 ml) were collected in
EDTA tubes (AdnaCollect, AdnaGen), then immunomagnetic
separation was performed using the AdnaTest BreastCancer
Select. Briefly, epithelial cells are isolated by antibody-linked
Dynalbeads directed against EpCAM (also known as GA733-2)
and MUC-1. The immunomagnetic-captured cells were lysed.
The mRNA was isolated from the cell lysates using the Dynabeads mRNA DIRECT Microkit (Dynal Biotech Gmbh, Hamburg, Germany) included in the AdnaTest BreastCancer Detect
kit. The mRNA was then transcribed into cDNA which was
then used as a template in a 2-step quantitative real-time PCR
for selected markers including MUC-1, HER2 and GA733-2
(Fig. 2). The AdnaTest was considered to be positive if at least 1
or more of the 3 markers shown the expression signal intensity
was equal to or greater than 0.15 ng/ll.
Estrogen receptor/progesterone receptor analysis
Immunostaining was performed on 5-lm-thick tissue sections of the primary tumor using primary antibodies directed
against estrogen receptor (ER) (clone 6F11; Novocastra) and
progesterone receptor (PR) (clone PgR1294; Dako Cytomation) using a Bond Max autostainer (Vision Biosystems) with
Early Detection and Diagnosis
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Early Detection and Diagnosis
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Comparison of assay methods for detection
Figure 1. The ‘‘Combination-of -Combinations’’ Principle. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
epitope retrieval (citrate buffer at pH 6.0). Diaminobenzidine
tetrahydrochloride was used for signal recognition of the
antigen. Distinct nuclear staining in the tumor cells was
regarded as positive for the marker. The number of tumor
cells showing nuclear staining was recorded and expressed as
a percentage. Expression of both markers in 1–9% of the tumor cells was regarded as low positive and in 10% or more
as positive.
Fluorescence in situ hybridization analysis
The HER2/neu status of the primary tumor was determined
by immunohistochemical staining of 4-lm-thick sections cut
from a representative paraffin block of the invasive tumor.
The slides were incubated with the anti-HER2/neu monoclonal antibody e2-4001 (1:100 dilution) on a Dako autostainer
(Dako, Corp., Carpinteria, CA) with the LSAB2 peroxidase kit
(Dako, Corp.) using 3,30 -diaminobenzidine. The percentage of
cells displaying complete membranous staining and the inten-
sity of staining were evaluated on a semiquantitative scale
from 0 to 3þ. In all cases exhibiting any positivity (1þ, 2þ,
3þ), we confirmed the protein overexpression with fluorescence in situ hybridization of HER2/neu gene amplification.
We used the PathVysion HER2/neu kit (Abbott Laboratories,
Chicago, IL), which uses 2 directly labeled fluorescent DNA
probes, one specific for the HER2/neu gene locus and one for
the alpha satellite DNA sequence at the centromeric region of
chromosome 17. Signals were counted for 50 nuclei of the tumor cells by using an epifluorescence microscope and the ratio of HER2/neu to chromosome 17 signals was calculated. A
ratio greater than 2.2 was considered to represent HER2/neu
gene amplification, which was regarded as a positive result.
Statistical methods
We used the Pearson’s v2 test to examine the correlation
between the results obtained using the AdnaTest and the
CellSearch system. We also calculated the sensitivity and
specificity of each method, as well as the overall agreement
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Andreopoulou et al.
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Figure 2. AdnaTest procedure. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Results
A total of 55 MBC patients were enrolled between January
2006 and March 2007. Detection of CTCs was performed in
all patients using both the CellSearch system and AdnaTest.
Patient and tumor characteristics at the time of enrollment
are shown in Table 1. The median age was 49 years (range,
21–85).
Primary tumor hormone receptor status for ER/PR
(detected by IHC) and HER2 overexpression (detected by
IHC and FISH) of primary tumors were positive in 36 (65%)
patients and 12 patients (22%), respectively. Triple-negative
(ER-, PR- and HER2-negative) phenotype was present in 11/
55 (20%) at the time of primary tumor diagnosis. Neither
CTC detection method was found to be associated with ER,
PR, or HER2 status of the primary tumor.
Baseline detection of CTCs by CellSearch
In the CellSearch system, patients were grouped according to
the frequency of positive CTCs using the cutoff values of 2
or more CTCs per 7.5 ml of blood and 5 or more CTCs per
7.5 ml of blood. The CellSearch system reported 26/55
patients (47%) as CTC positive with the cutoff value of 2 or
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Int. J. Cancer: 130, 1590–1597 (2012) V
Table 1. Patient and tumor characteristics
N
All patients
Median age (range)
Hormone receptors
49 (21–85)
36
65%
ER & PR neg
16
29%
3
5%
Positive
12
22%
Negative
28
51%
Equivocal
2
4%
Unknown
13
24%
Triple negative
Metastasis
No. of Met Sites
100%
ER or PR pos
Unknown
HER2/Neu
%
55
11
20%
Visceral
30
55%
Nonvisceral
25
45%
One
28
51%
2 or more
27
49%
more CTCs per 7.5 ml of blood, and 20/55 patients (36%) as
CTC positive with the cutoff value of 5 or more CTCs per
7.5 ml of blood (Table 2).
Detection of CTCs by the AdnaTest
In the AdnaTest, blood samples were regarded as CTC positive
if a PCR fragment of at least 1 of the 3 markers, GA733-2,
Early Detection and Diagnosis
between the AdnaTest and CellSearch results, along with
95% confidence intervals (p < 0.01). All statistical analyses
were performed using the SPSS software (version 17; SPSS,
Chicago, IL).
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Comparison of assay methods for detection
Table 2. Incidence of CTCs by CellSearch
CTC Count
N
%
2
29
53%
2
26
47%
<5
35
64%
5
20
36%
N
MUC-1
HER2
Overall AdnaTest
%
Negative
45
82%
Positive
10
18%
Negative
31
56%
Positive
24
44%
Negative
36
65%
Positive
19
35%
Negative
26
47%
Positive
29
53%
p value
Cell search
GA733-2
<5
5
Negative
33
12
Positive
2
Tests Concordance rate
Incidence of CTCs by AdnaGen Tests
GA 73.3-2
Table 4. Distribution of AdnaTest Results by CellSearch CTCs
(<5 vs. 5)
MUC-1
Negative
25
6
Positive
10
14
Negative
26
Positive
9
Tests concordance rate
<0.01
71%
Tests concordance rate
AdnaTest overall result
8
75%
Tests Concordance rate
HER2
<0.01
10
0.07
10
65%
Negative
22
4
Positive
13
16
<0.01
69%
Comparison between the AdnaTest and CellSearch system
Table 3. Distribution of AdnaTest Results by CellSearch CTCs
(<2 vs. 2)
p value
Cell search
Early Detection and Diagnosis
GA73.3-2
<2
2
Negative
28
17
Positive
1
9
Tests concordance rate
MUC-1
23
Positive
6
Tests concordance rate
HER2
Tests concordance rate
Discussion
8
0
18
75%
Negative
23
13
Positive
6
13
Negative
20
Positive
9
Tests concordance rate
Adna overall result
<0.01
67%
Negative
0.02
65%
6
We found a positive agreement between the AdnaTest and
CellSearch system (with a cutoff value 2 CTCs/7.5 ml of
blood) of 73%, and a positive agreement between the AdnaTest and CellSearch system (with a cutoff value 5 CTCs/7.5
ml of blood) of 69% (Tables 3 and 4, respectively).
<0.01
20
73%
MUC-1, or HER2, was detected at a concentration of 0.15 ng/ll
or greater. The AdnaTest reported 29/55 patients (53%) as CTC
positive, with the positive expression rates of 18% for GA733-2,
44% for MUC-1 and 35% for HER2 (Table 2). Of the 29
patients reported to be CTC positive by the AdnaTest, 19 were
positive for either 2 or all 3 markers and the remaining 10 were
positive for only 1 marker. The AdnaTest detected HER2
expression in CTCs in 19 patients; but HER2 status of the
primary tumor was positive and concordant in only 5 patients,
negative and discordant in 11, and unknown in 3 of these
patients.
Our data indicate that the AdnaTest is at least equivalent to
the CellSearch system for the detection of CTCs in patients
with MBC. The data indicate detection of CTCs in higher
proportion of cases compared to an appropriate agreement,
considering the differences in methodologies between the 2
technologies. We found that 26/55 patients were reported as
CTC positive by the CellSearch system (with a cutoff value of
2 CTCs/7.5 ml of blood), while 29/55 patients were
reported as CTC positive by the AdnaTest, giving a positive
agreement of 73% between the 2 methods. However, the
AdnaTest was positive in 9 cases in which the CellSearch system was unable to detect circulating tumor cells, and the
CellSearch system was positive in 6 cases in which the
AdnaTest was considered negative considering possibility of
complementary value. Based on recently reported data, the
concordance rate of the Cell Search system and the AdnaTest
is up to 88%.22–24
An interesting finding of this study is that among the 19
patients who were demonstrated to have CTC expression of
HER2, 11 had no demonstrated HER2 gene amplification in
the primary tumor. Assessing CTCs for HER2 overexpression
could potentially help identify patients, who would not be
considered otherwise candidates for HER-2 targeted therapy,
because of negative disease status on the primary tumor. Discordance between HER2 amplification in the primary tumor
and in CTCs has been previously reported. For instance,
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cases with altered HER2 status in CTCs associated with a
clinical response was also observed.33 Furthermore, a number
of other studies reported that up to one-third of patients
whose primary tumors do not overexpress HER2 have CTCs
with amplified HER227,33,34,35 In view of that CTCs are likely
to derive from clones in the primary tumors28,29, our and
others’ data showing the discordance in HER2 status between
the primary tumor and CTCs may indicate that tumor cells
are undergoing genetic changes. Together, these important
observations support the argument that detection of CTCs
and determination of their gene amplification or expression
can be used for better tailoring therapies.
Collecting representative tissue from solid tumor metastases usually requires more invasive procedures that increase
the risk of complications and discomfort. Furthermore, these
procedures may not provide an adequate specimen for
detailed analysis and typically cannot be repeated for
dynamic evaluation of the biological changes during treatments. Theoretically, CTC detection would allow specific
genes (e.g., HER-2, epidermal growth factor receptor, and
mammaglobin B) or more global gene expression to be analyzed while using specific targeted treatment for MBC based
on the expression of the CTCs.33,36,37 This information could
then be used to design specific treatments that more appropriately reflect the dynamics and heterogeneity of MBC.
Conclusions
The AdnaTest BreastCancer Select/Detect is a new approach
for the detection of CTCs in patients with MBC. The AdnaTest has equivalent sensitivity to that of the CellSearch system
in detecting 2 or more CTCs. The advantage of the AdnaTest
over the CellSearch system is that it enhances the possibility
of detecting very low numbers of CTCs or occult CTCs based
on their expression of tumor-associated genes. The immunomagnetic cell capture technology, combined with multiplex
RT-PCR in AdnaTest may potentially allow the detection of
a broad range of molecular abnormalities in CTCs that can
more accurately characterize the biological status of metastatic disease and support design of future studies testing
CTCs directed therapies. AdnaTest complements the CellSearch system for CTC detection.
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