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Molecular Oncology, Markers, Clinical Correlates |
Departments of Cancer Biology [S-K. K., R. S., L. G., G-H. H., L. H. F., L. B. C.] and Adult Oncology [P. R., A. E.], Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7/106 mononuclear cells in positive samples). To improve
the specificity of the rare event detection, a double-labeling protocol
combining intracellular cytokeratin with epithelial cell adhesion
molecule (Ep-CAM) (breast, ovarian, colon, and lung carcinoma
antigen) or disialo-ganglioside (GD2) antigen (small cell lung
carcinoma, neuroblastoma, melanoma antigen) was developed. Examples of
doubly labeled cultured cells and cancer cells from breast and small
cell lung cancer patients are shown. Using the double-labeling
protocol, no "positive" cells were seen in samples of healthy blood
donors. Automated rare event detection (cytokeratin single-staining)
was applied to 355 PB, BM, and stem cell (SC) samples from breast
cancer patients before autologous BM transplantation.
Cytokeratin-positive cells were found in 52% of BM, 35% of PB, and
27% of SC samples at frequencies of 1–1020 positive
cells/106 mononuclear cells, thereby establishing the
efficacy of the technique in the detection of rare cancer cells in
hematopoietic tissue samples of cancer patients. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The detection of residual tumor cells in autologous grafts from both BM and PB SCs after high-dose chemotherapy has been reported by many investigators (11) and was ascribed prognostic significance in both advanced and locally recurring breast cancer (12 , 13) . The presence of cancer cells in blood or BM is therefore being evaluated as an indicator for metastatic disease in patients with solid tumors, e.g., carcinomas of the breast, lung, colorectum, and prostate (14 , 15) , and their quantitation is important in the evaluation of BM or peripheral SC preparations that serve as autologous transplants after high-dose chemotherapy. In addition, repeated determinations during a treatment period may help to monitor the degree of response to therapy, and the cancer cells in these readily accessible tissues may be used to further characterize the disease (e.g., presence of markers associated with specific phenotypes).
The detection of MRT cells relies on differential expression of normal or abnormal genes and the monitoring of their transcripts or products. Different methods have been used to do so (15, 16, 17) , and they fall into three main categories: clonogenic, where the tumor cells are grown in culture and characterized; molecular, where the transcript levels of a marker gene are determined; and immunological, which monitors marker gene products with antibodies. Clonogenic assays are quite specific, but the sensitivity of the clonogenic approach is insufficient to score MRT contamination in hematopoietic samples (13 , 18) . The reverse transcription-PCR method to determine the presence of marker transcripts offers theoretically unparalleled sensitivity (up to 1 tumor cell in 107 or better), but this method is marred by high false-positive determinations and should be complemented with other methods for the confirmation of the positive hits, yet it does not allow direct visualization of the rare positive cells (19, 20, 21, 22) .
Immunological methods are therefore the procedures of choice, whereby antibodies directed to characteristic cellular constituents are used to stain the cells of interest. One can distinguish approaches based on flow cytometry (23 , 24) and on image cytometry (25, 26, 27, 28) . Flow cytometry allows the analysis of a large number of cells in a few minutes. Combined with an immunomagnetic separation technique, frequencies below one epithelial cell/ml blood can be detected (23) . However, confirmation of the "positive events" by visual inspection of tumor cell morphology or other cell characteristics remains necessary. Rare cell enumeration can be performed manually under the microscope, but this is a laborious task and moreover, some positive events can easily be missed, especially when present at low frequencies. Therefore, attempts have been made to automate the microscopic detection and quantification of rare cells (23, 24, 25, 26, 27) , and although still slower than flow cytometry, the performance of microscopic detection systems is improving. These methods offer the advantage that an image of each detected event can be stored in computer memory for later visual evaluation; the detected cells can also be relocated on the microscope slide at any time, if necessary, because the coordinates of all detected objects are known (26 , 29) .
We developed such an automated microscopic system (Rare Event Imaging System) for the detection and analysis of cancer cells in PB or BM preparations. Slides are automatically scanned at low magnification for the detection of tumor cells (positive events), which is based on cytokeratin/rhodamine labeling, and the total cell count, which is based on nuclear DAPI labeling. Cytokeratin, a cytoskeletal component of epithelial and carcinoma-derived cells, is the most widely used and best characterized marker of cancer cell contamination (8 , 30, 31, 32, 33) . After the automated analysis, the user can review all positive events on the computer screen and manually confirm them using higher magnification. In addition, cells can be viewed with different fluorescence filters for multiple-marker analysis: for increased detection specificity and phenotype characterization of residual tumor cells, we established a double-labeling protocol that combines the labeling of cytokeratin with that of surface antigens reported to be expressed in specific types of cancers, e.g., the Ep-CAM for breast, ovarian, colon, and lung carcinomas (34 , 35) and the disialoganglioside GD2 for small cell lung carcinoma, neuroblastoma, glioma, and melanoma (36, 37, 38) . In this paper, we demonstrate the methodological power of the new automated rare event analyzer using model systems, and first results analyzing samples of patients with breast and small cell lung cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
The breast carcinoma cell line MCF-7 and the small cell lung cancer
cell line SW2 were purchased from American Type Culture Collection and
used to evaluate the staining protocol and to determine the sensitivity
of the Rare Event Imaging System. Cell lines were maintained in DMEM
(MCF-7) or RPMI 1640 (SW2) containing 10% FCS, 100 units/ml
penicillin, and 0.1 mg/ml streptomycin.
Sample Preparation for Microscopic Analysis.
Blood or BM samples were mixed with two volumes of 0.17 M
ammonium chloride, incubated at room temperature for 40 min, and
centrifuged at 800 x g for 10 min at room temperature.
The cell pellet was then washed, resuspended in PBS, and the total
number of living PBMCs or nucleated BM cells was counted using trypan
blue dye exclusion. The cells were attached to adhesive slides (Paul
Marienfeld GmbH & Co., KG, Bad Mergentheim, Germany) at 37°C
for 40 min, and the slides were then blocked with cell culture medium
at 37°C for 20 min. The total number of cells applied per slide was
1.5 x 106, and the adhesive area on these
slides consists of three separate circles totaling 530
mm2.
For the single labeling of cytokeratin, cells were fixed in ice-cold methanol for 5 min, rinsed in PBS, and incubated with a rabbit anticytokeratin antiserum directed against class I and II cytokeratins (Biomedical Technologies, Stoughton, MA) at 37°C for 1 h. Subsequently, slides were washed in PBS, incubated with rhodamine-conjugated antirabbit antibody (Jackson Immuno Research, West Grove, PA) at 37°C for 30 min, counterstained with 0.5 µg/ml DAPI (Molecular Probes, Eugene, OR) in PBS at room temperature for 10 min, and mounted in glycerol-gelatin (Sigma, St. Louis, MO). Processed slides were stored at room temperature and analyzed microscopically within a month.
For the double labeling of cytokeratin and the cell surface antigens Ep-CaM or GD2, the cells were fixed in 1% paraformaldehyde in PBS (pH 7.4) at room temperature for 5 min, washed in PBS, and blocked with 20% human AB-serum (Nabi Diagnostics, Boca Raton, FL) in PBS at 37°C for 20 min. Subsequently, primary antibodies directed against the surface antigens Ep-CAM (monoclonal mouse KS1/4 antibody) or GD2 (monoclonal mouse 1418 antibody) were applied at 37°C for 1 h (both antibodies were kindly provided by Dr. Kin-Ming Lo, Lexigen Pharmaceuticals, Lexington, MA). Cells were then washed, fixed in ice-cold methanol for 5 min, blocked with 20% human AB-serum, and incubated with anticytokeratin antiserum at 37°C for 1 h. Secondary antibodies (FITC-conjugated antimouse and rhodamine-conjugated antirabbit antibodies; Jackson Immuno Research) were mixed and applied at 37°C for 30 min, followed by counterstaining of the nuclei with 0.5 µg/ml DAPI in PBS. Doubly labeled cells were mounted in Gel/Mount (Biomeda, Foster City, CA). Slides were stored at 4°C and analyzed microscopically within a week.
Tumor Cell Dilutions for Determination of Sensitivity.
To determine the sensitivity of our method regarding the detection of
CK+ cells, MCF-7 breast cancer cells were serially diluted in PBMC of a
healthy blood donor. The dilutions tested were
1:103, 1:104,
1:105, 1:2 x 105,
1:5 x 105, and 1:106.
Solutions were attached to adhesive slides and processed for
cytokeratin labeling as described above. Up to eight adhesive slides
were prepared and scanned per dilution. Samples were analyzed for the
number of tumor cells per slide and related to the total cell count.
Automated Microscopic Detection of Tumor Cells and Total Cell
Count.
Slides were automatically scanned using a Rare Event Imaging System,
developed by Georgia Instruments, Inc. (Roswell, GA). The system
employs proprietary image processing algorithms to detect rare
fluorescent events and determine the total number of cells analyzed. It
is comprised of an advanced computer-controlled microscope (Nikon
Microphot-FXA, Nikon, Japan) with autofocus, motorized X-,
Y-, and Z-axis control, motorized filter
selection, and electronic shuttering. Images are taken by an
integrating, cooled CCD detector and processed in a 60-MHz Pentium
imaging workstation.
In the first step, the slide is automatically scanned for the detection of positive events (e.g., CK+ cells) using the rhodamine filter set. The identification of positive events is based on fluorescence intensity and area. The (X,Y) coordinates of each positive event are stored into computer memory, and the image is archived. In the second step, the slide is scanned for the total number of DAPI-labeled nuclei per slide, representing the total cell count. The total scanned area per slide is 448 mm2 (84% of the adhesive area) to avoid edge effects. At the end of the two scans, the number of positive events and the total cell count are given, and a gallery of images containing all positive events is displayed. The user can review the images and recall any of the events for further examination using the stored coordinates attached to each image. The field of interest can then be visualized using higher magnification and additional filter sets (e.g., fluorescein, or UV filter). Images of different fluorescent colors can be electronically overlaid for positive confirmation of the event and for phenotypic evaluation (multiple labeling). The total scanning time (two scans) for one slide is about 1 h. The two scans can be run independently, thereby offering the option of just screening for positive events and thus shortening the scanning time to 30 min/slide.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows a high cell recovery (89%) for
the PB of healthy blood donors, but a somewhat higher cell loss in
samples from cancer patients (64, 58, and 73% recovery for PB, BM, and
SC samples, respectively; P < 0.05 for PB and BM
versus normal PB by t test).
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. The sensitivity of
detection of CK+ cells was tested with increasing tumor cell dilutions
(MCF-7/PBMC) as described in "Materials and Methods." Cancer cells
in expected quantities could be detected up to the most diluted samples
tested, 1 MCF-7 cell/106 PBMCs (Table 2
; expected and observed curves not
statistically different; 
2 test).
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shows SW2 cells labeled with the anti-GD2 antibody and
the anticytokeratin antiserum. The sequential fixation preserved the
antigenic sites of both proteins with regard to their cellular
localization, as demonstrated in the optical sections taken with a
confocal laser scanning microscope (Fig. 1B)
. The cells
clearly showed cytokeratin in the cytoplasm (red) and GD2 at the cell
surface (green). The expression levels of both proteins was quite
heterogeneous within the cell population. A similar result was obtained
when MCF-7 cells were doubly labeled with the anti-Ep-CAM antibody and
the anticytokeratin antiserum (data not shown). Control experiments in
which one of the primary antibodies was omitted but both secondary
antibodies were applied did not reveal any cross-reactivity between the
two detection systems (data not shown).
To further validate the staining protocol, we labeled PBMCs
that had been spiked with MCF-7 or SW-2 cells. The goal was to obtain a
bright fluorescent signal of the cancer cells and a low background
signal from the surrounding PBMCs. The two most important factors to
achieve this goal were found to be the sequential application of the
primary antibodies and two blocking steps (20% human AB-serum in PBS)
before the incubation with the primary antibodies. As shown in Fig. 1C
, the doubly labeled MCF-7 cells could clearly be
distinguished from the surrounding PBMCs. At higher magnification, we
were also able to confirm the intracellular cytokeratin labeling and
the surface staining of Ep-CAM (Fig. 1D)
. Similar results
were obtained with PBMCs spiked with SW-2 cells and doubly labeled for
GD2 and cytokeratin (data not shown).
We also applied the double-labeling protocol to PB and BM samples of
cancer patients. Fig. 3B
shows an example of a GD2/CK+ cell
from the PB of a patient with small cell lung cancer. Fig. 3C
shows an Ep-CAM/CK+ cell from the BM of a breast cancer
patient. In this example, the cancer cell is not only bigger than the
surrounding BM cells, but it also exhibits the distinct localization of
the individual stains: cytokeratin (red) is cytoplasmic, whereas Ep-CAM
(green) is concentrated toward the cell periphery, at the cell
membrane.
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, 16–18% of the PB samples
scored positive for cytokeratin using any of the protocols, with the
number of CK+ cells ranging from 1 to 26 labeled
cells/106 WBCs. In contrast, when the samples
where processed with the double-labeling protocol, positivity was
almost completely eliminated from the samples of healthy subjects (a
single doubly positive cell was observed in a total of 77 PB samples).
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. We also tested for
temporal fluctuations in the occurrence of CK+ cells in PB samples. Two
SC samples from each of 96 patients were taken at consecutive days but
without therapeutic intervention. Paired samples showed a statistically
significant concordance with regard to cytokeratin positivity (Fig. 2B)
.
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. The positive cells showed
clear cytoplasmic labeling, whereas the surrounding blood cells were
not stained. We found CK+ cells in 52% of the BM, 34% of the PB, and
27% of the SC samples (Table 4)
. The
frequency of CK+ cells in the positive samples varied from
1/106 to 1020/106. However,
many PB samples from normal subjects displayed a small number of CK+
cells, and these were found to be false-positive cells based on the
double-labeling experiments (Table 3)
. Therefore, to declare definite
positivity in PB samples from cancer patients, we set a cutoff point at
the mean number of CK+ cells plus two times the SD as observed in the
control samples, i.e., 9/106. Applying
this threshold, we still found a higher degree of cytokeratin
positivity in BM (40%) compared to PB (24%) or SC preparations (12%;
Table 4
). Furthermore, patients with stage IV disease were found to be
cytokeratin positive in a significantly higher percentage than patients
with stages II/III disease in all types of samples analyzed (Table 4)
.
We are presently analyzing clinical samples from breast and small cell
lung cancer patients using the double-labeling protocol; the results
will be published elsewhere.
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DISCUSSION |
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For the automated detection of rare events in PB or BM, it was critical
to use a preparation method with minimal cell loss during sample
processing. Simple lysis of erythrocytes was preferred over
Ficoll-based isolation methods to ensure maximal recovery of rare
cancer cells. Our cell preparation/adhesion procedure yielded a
homogeneous cell monolayer, with a recovery comparable to that obtained
in cytospin preparations performed with specially designed buckets and
high centrifugal forces, where minimal cell loss has been reported
(26)
. In contrast, regular cytospin preparations can
result in a loss of up to 2/3 of the cells (39)
.
Information on cell number is unavailable for most studies using
microscopic rare event detection because these fail to record the total
number of cells actually being analyzed on the slides (32
, 40)
. Rather, those papers relate the number of positive events
to the total number of cells processed, assuming a complete recovery.
This introduces a bias: not only did we find that cells are indeed
inevitably lost during preparation, but the recovery can vary greatly
between samples of a given type (see "Range," Table 1
) as well as
according to the type of sample. It is not clear why cell loss was
higher in PB samples from cancer patients compared to like samples from
normal subjects; there are numerous reports that malignant cells
express abnormal levels of a variety of adhesion molecules (including
cadherins, integrins, selectins, laminins, members of the
immunoglobulin superfamily, variant isoforms of the transmembrane
glycoprotein CD44, and others; see Refs. 41, 42, 43, 44, 45, 46, 47
), and
differences in cell-matrix interactions have been reported specifically
between leukocytes (T lymphocytes and dendritic cells) and tumor cells
(invasive melanoma; Ref. 48
). Although we are presently
optimizing the cell deposition procedure and attempting to minimize
variability, we nonetheless conclude in view of these observations,
that the determination of the total number of cells actually included
in the analysis (i.e., on the slides) is imperative in
microscopic rare event detection.
Experiments whereby PB samples are mixed with defined numbers of breast
cancer cells show that the detection sensitivity of our Rare Event
Imaging System is at least 1 CK+ cell/1 million PBMCs (Table 2)
, which
should be adequate for clinical applications. The brightly stained CK+
MCF-7 cells are easily distinguished from the dark background of
negative cells. A similar detection sensitivity has been shown for
other automated image analysis systems (26
, 27
, 49)
and
for manual microscopic analysis (50)
. A further increase
in detection sensitivity could be achieved by analyzing more cells
(e.g., 10–20 million/sample). However, although such large
numbers of cells can readily be obtained with regular blood or BM
sampling methods, their microscopic analysis would be very time
consuming.
Speed is therefore a fundamental parameter for the evaluation of automated rare event analysis systems. The system described herein takes about 1 h to scan 1 million cells for positive events (e.g., CK positivity) and for the total cell count. This is similar to the fastest automated microscope-based cell analysis systems described by others (26 , 49) , and it makes the processing of large numbers of cells reasonable. By comparison, the commercially available Laser Scanning Cytometer (CompuCyte Corp., Cambridge, MA) can only scan between 1000–2000 cells/min (Ref. 29 and technical information from CompyCyte Corporation, Cambridge, MA). We are presently developing a much faster system by using a more sensitive CCD camera and faster computer, which will bring down the processing time to a few minutes per million cells, comparable to flow cytometry (23) yet retaining the possibility to observe each positive event at higher magnification or with different optics, for morphological confirmation.
The specificity of the detection of cancer cells in blood or BM
preparations can only be as good as the marker and antibodies used in
the procedure. The most widely used marker is cytokeratin, a
cytoskeletal component of epithelial and carcinoma-derived cells
(8
, 30, 31, 32, 33)
. Although it has been validated as a valuable
marker for breast, prostate, gastric, and colorectal cancer in a large
number of clinical studies, cytokeratin is not a tumor cell-specific
marker and can result in false-positive staining of epidermal cells or
weak cytoplasmic staining of phagocytic cells that contain cytokeratin
debris or dye particles (30)
. In these cases, as mentioned
above, definitive microscopic confirmation of the malignant cytology of
the immunostained cells is crucial. Another source of false-positive
events is cross-reactive staining of the epithelial or cancer cell
marker with blood or BM cells; e.g., mucin-like epithelial
membrane markers are able to cross-react with hematopoietic cells.
Indeed, we found that cytokeratin antibodies can label PBMCs from
healthy blood donors (Table 4)
: 17% of the PB samples from normal
blood donors exhibited cytokeratin positivity, albeit at a low level
(mean, 1.18 CK+/106 cells). It is not clear
whether these CK+ cells in "normal" samples represent benign
epithelial cells, cross-reacting hematopoietic cells, or cancer cells
disseminated from an undiagnosed primary carcinoma.
To improve the specificity of the rare event detection, a
double-labeling protocol was developed for the simultaneous detection
of cytokeratin and the epithelial surface markers, Ep-CAM and GD2. This
procedure dramatically reduced the positivity of "normal" samples,
with only one doubly labeled cell among the 77 samples tested
(compounded of CK/Ep-CAM and CK/GD2; Table 3
), suggesting that the few
CK+ cells detected in normal samples were not of cancer origin. In
addition to the mere detection of cancer cells in blood or BM samples,
efforts have been made to further characterize the phenotype of rare
tumor cells, e.g., with respect to their aggressiveness,
cell cycle stage, or growth behavior (40
, 51, 52, 53)
.
Protocols for multiple marker analysis, combining cytokeratin labeling
with growth factor receptors or proliferation-associated antigens to
analyze breast cancer samples (52)
, or combining
cytokeratin labeling with prostate specific antigen to analyze prostate
carcinoma (53)
have been developed. Also, in gastric
cancer patients, cells that were doubly positive for cytokeratin and
the urokinase plasminogen activator receptor correlated with high
metastatic potential (51)
. A variety of possible
additional (cancer-specific) markers have been described,
e.g., glycoproteins (39)
, gangliosides
(54)
, cell adhesion molecules (55
, 56)
, and
others (57)
. The sensitivity and specificity of the cancer
cell detection method and the quality of tumor characterization we will
be able to make will all improve as new markers become available.
Extensive double-labeling studies of cancer patients’ hematopoietic
samples are under way in our laboratory to explore the biological
properties of MRT cells.
Automated rare event detection using single cytokeratin labeling was
applied to 355 BM, PB, and SC samples from patients with breast cancer
before autologous BM transplantation (Table 4)
. BM showed the highest
percentage of CK+ samples (52%), followed by PB (34%) and SC
preparations (27%). Furthermore, samples from patients with stage IV
disease contained CK+ cells at a higher frequency than patients with
stage II/III disease. Similar gradations have been reported by others
(12
, 32
, 39
, 58)
. These numbers are in general accordance
with previous studies using various rare event detection procedures
(12
, 30
, 39
, 54 , 59)
, although some authors report a
higher proportion of cancer patients with BM or PB contamination than
observed here, possibly due to chemotherapy treatment received by most
of the patients in our cohort shortly before sample collection
(33)
. However, given the large proportion of low-level CK+
samples observed in PB samples from healthy blood donors (Table 3)
, we
set a cutoff in the frequency of CK+ cells required to declare a sample
contaminated (mean + 2 SD of CK+ cell contamination in samples from
healthy subjects, i.e., 9 CK+
cells/106 cells). Using this cutoff, 40%, 24%,
and 12% of the samples were declared contaminated with cancer cells in
BM, PB, and SC samples, respectively. Upcoming double-labeling studies
will not be subject to this false-positive problem or require that a
cutoff background level of contamination be used.
Analyses of paired BM samples of at least 106
cells for spatial sampling heterogeneity showed a good concordance
within pairs (Fig. 2)
. Pantel et al. (30)
had
found that sampling from two sides of the iliac crest is necessary to
obtain reliable results. However, it is not clear whether in that study
the mere doubling of the number of cells analyzed improved the result
or whether true heterogeneity was captured. Our results indicate that
it is sufficient to analyze only one BM sample of at least
106 cells taken from either side of the iliac
crest. Analyses of the temporal sampling heterogeneity revealed that
there was little fluctuation with regard to cytokeratin positivity in
SC preparations within the constraints of our experimental protocol,
indicating a good repeatability of the method.
In summary, an automated microscopic method was developed for the detection of cancer cells in blood or BM samples. It was demonstrated that the system is capable of the detection of rare events (1 per 1 million) in a reasonable time and double-labeling protocols result in a drastic reduction of false-positive determinations. This allows the use of the system in a clinical setting to monitor cancer treatment and to determine recurrences. Furthermore, detected cancer cells can be characterized phenotypically for different markers.
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FOOTNOTES |
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1 Supported by Public Health Service Grant CA13849
from National Cancer Institute, the NIH, Department of Health & Human
Services. 
2 To whom requests for reprints should be
addressed, at Dana-Farber Cancer Institute, Department of Cancer
Biology, Room SM1058, 44 Binney Street, Boston, MA 02115. Phone: (617)
632-3386; Fax: (617) 632-4470; E-mail: drchen{at}shore.net
3 The abbreviations used are: MRT, minimal
residual tumor; BM, bone marrow; CK+, cytokeratin-positive; DAPI,
4',6-diamidino-2-phenylindole; Ep-CAM, epithelial cell adhesion
molecule; PB, peripheral blood; PBMC, PB mononuclear cell; SC, stem
cell.
Received 7/22/99; accepted 10/26/99.
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