Sensitive detection of tumor cells in
peripheral blood of carcinoma patients
by a reverse transcription PCR method
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Occurrence of tumor cells in the
peripheral blood of individuals suffering
from cancer may serve as an
early indication that the primary
tumor has dispersed from its tissue
of origin. Despite defence mechanisms
of the organism, individual
cancer cells may attach in distant
regions and form colonies as an
initial step of metastasis formation.
Hence, early detection of metastatic
potential can be estimated by
detecting tumor cells in the blood
circulation. Sensitivity and specificity
are the main objectives of any
method applied for this purpose.
Most highly sensitive techniques,
encounter an increasing problem
of specificity due to background
signaling from illegitimate transcription.
AdnaGen established a
method of tumor cell selection,
their specific identification and
analysis with a lower limit of
detection of at least 2 tumor cells in
5 mL blood (1010 cells). To achieve
this we combined the enrichment
of tumor cells using a specifically
designed antibody mixture with
RT-multiplex PCR techniques for
the detection of mRNAs encoding for tumor associated markers.
Preliminary results of case studies
showed the occurrence of tumor
cells in blood of carcinoma patients
indicating a potential tumor
relapse, in some cases several
months prior to an elevation of
serum tumor markers. AdnaGen
provides a sensitive and specific
method to detect disseminated
tumor cells in peripheral blood of
testicular, breast and colorectal
carcinoma patients. This innovative
method is an option for clinicians
as a predictive tool with respect to
metastasis formation and may
result in an appropriate selection
of patients for adjuvant therapy.
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Metastases are the major cause of
death in cancer patients. Metastases
occur as a result of the interaction
between the tumor cells
and the host, and this interaction
adds to the extreme complexity of
the events associated with tumor
dissemination. Spreading of tumor
cells into the blood circulation
from either primary tumors or
subsequently from the lymphatic origin may be a result of dissemination
of the primary tumor or a secondary
event during tumor progression.
Many of these cells reaching
the circulation may be killed by an
individual response of the immune
system but the metastatic potential
of remaining tumor cells cannot be
ruled out. The presence of disseminated
tumor cells in systemic circulation
is an indication for tumor
relapse. Their early detection followed
by an immediate and possibly
even individual therapy may prevent
formation of metastases1. Specificity
and maximal sensitivity are the main
objectives for any method in this
field. A very high sensitivity is possible
by means of expression markers that
are detected and amplified specifically
on transcription level. However, due
to background from illegitimate transcription
in nucleated blood cells,
most techniques described encounter
the problem of specificity. This can
be overcome by specific enrichment
of non-blood cells2-6. This Application
Note describes a sensitive and specific
method to detect testicular,
breast and colorectal carcinoma cells
from blood. The tumor cell selection and analyses are developed and
provided by AdnaGen, using an
Agilent 2100 bioanalyzer for
detection.
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Material and methods
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AdnaGen developed a diagnostic
system which is based on a twostep
procedure:
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AdnaTest CancerSelect
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A combined method based on
immunomagnetic enrichment of
tumor cells directly from whole
EDTA-blood with an antibody
mixture, which was especially
designed to isolate breast, testicular
and colorectal carcinoma cells.
mRNA was extracted with magnetic
oligo-(dT) beads (DYNAL)
followed by a high sensitive
reverse transcription (QIAGEN).
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AdnaTest CancerDetect
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Expression analysis of tumor cells
with multiplex PCR (up to five
markers per assay) for the detection
of mRNAs encoding for tumor
markers. Multiplex PCR reactions
were prepared using the cDNA
from above as template, the
primer mixture provided by
AdnaGen and the HotStar PCR-kit
(QIAGEN). PCR samples were
analyzed using the Agilent 2100
bioanalyzer and the DNA 500
LabChip® kit.
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Inoculation system
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Cell spiking experiments with
different carcinoma cell lines for
each kind of investigated cancer
were used to test the potential
lower detection limit of this technique.
Defined numbers of 2-100
cells were added to 5-mL aliquots
of whole blood from healthy
donors, an unspiked blood sample
was used as negative control,
cancer cell lines served as positive
controls. The specificity of the selected antibodies was determined
by flow cytometry.
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Results and discussion
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AdnaGen developed a diagnostic
system for early detection of
disseminated cells in peripheral
blood of testicular, breast and
colorectal carcinoma patients.
This diagnostic approach is
designed to optimize enrichment
and analysis of tumor cells. The
specificity of the method is shown
by immunofluorescence staining
(figures 1 and 2) and the lower
detection limit by RT-PCR (figure 3).
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Specificity
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Figure 1 shows the specific recognition
of tumor cells by the selected
antibodies coupled to magnetic
beads (AdnaTest CancerSelect) by
microscopy. The immunofluorescence
assay (figure 2) documents
the specificity of the antibody mixture
(AdnaTest CancerSelect) used
for immunomagnetic enrichment.
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Lower detection limit
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Cell spiking experiments with
different cell lines were performed
to test the potential sensitivity of
the assay. After inoculation and
immunomagnetic enrichment,
two carcinoma cells were detected
in 5 mL blood samples (1010 cells)
using the AdnaTest CancerDetect
kit (figure 3). In addition specificities
of up to 100 % are obtained
for each marker of the multiplex
PCR. We demonstrated this using
blood of healthy donors, patients
with different non-malign diseases
(e.g. gastrointestinal diseases) and
inoculated non-malignant epithelial
cells. No signals were detected in
the PCR analysis (data not shown).
The combination of the two steps
above provides high specificity
and sensitivity for the diagnostic
approach.
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RNA stabilization
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Due to the high instability of cellular
RNA in vitro, preserving the
RNA expression profile is essential
for reliable analysis of gene expression. To achieve this we
developed a reagent, which stabilizes
cellular RNA for up to 2 days
at 5 °C and 45 °C, and up to 8 days
at room temperature (figure 4).
Storage at 45 °C caused a reduction
in expression level especially
of tumor marker 2, but the tumor
marker could still be detected.
Thus we are able to overcome the
critical aspect of degradation of
mRNA during storage and transport
of patient samples.
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Clinical validation
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At present, the clinical validity of
the diagnostic system is being
investigated in several studies for
each tumor entity (colorectal, testicular
and breast cancer). Exemplary
we investigated 50 colorectal
carcinoma patients at the time of
primary diagnosis and in followups
over a period of six months.
In several cases tumor cells
were detected by multiplex PCR
(AdnaTest Colon CancerDetect)
prior to the standard CEA-ELISA
(table 1). These colorectal carcinoma
patients showed negative ELISA
values after surgery and in followups
over a period of six months.
In two cases (patient 1 and 3) the
multiplex PCR showed positive
results after six months and in the
case of patient 2 after three months
and in all following samples. Until
now, all three cases characterize
the efficiency of surgery and
chemotherapy by the detection
of tumor cells: In the case of
patient 2 tumor cells in the blood
could be detected even during
chemotherapy, indicating an
ineffectual drug treatment. In all
three cases tumor cells could be
detected after surgery prior to the
standard CEA-ELISAs, indicating
a potentially tumor relapse.
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Conclusion
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AdnaGen developed a combined
method of specific tumor cell
selection (AdnaTest CancerSelect)
and a high sensitivity tumor cell
detection (AdnaTest CancerDetect).
The Agilent 2100 bioanalyzer
provides the performance to
detect the PCR products with high
sensitivity. The procedure allows
a sensitive and specific method to
detect disseminated tumor cells in
peripheral blood of testicular, colorectal
and breast carcinoma
patients at an early stage of tumor
progression. There is evidence
that this procedure enables us to
monitor patients during follow-ups
with high sensitivity. The relapse
free interval could be analyzed
more exactly than is possible with
current methods. This procedure
offers new possibilities for monitoring
and prognosis, and may
result in an appropriate selection
of patients for adjuvant therapy.
AdnaGen’s solution will set a new
standard in tumor diagnostics.
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