| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Oncology, Markers, Clinical Correlates |
Department of Laboratory Medicine and Pathology [T. E. W., T. K., P. C. R.], Division of Medical Oncology [J. N. I.], Department of Surgery [C. G., J. D.], Section of Biostatistics [V. J. S.], Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, and ChromaVision Medical Systems, Inc., San Juan Capistrano, California 92690 [B. B., D. H., J. T-B., K. D. B.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experimental Design: Blood specimens from 34 normal donors (negative controls), 15 samples to which carcinoma cells were added (positive controls), and 84 breast cancer patients [27 node-negative (N-), 29 node-positive (N+), and 28 metastatic] were studied. RBCs were lysed with ammonium chloride and the resulting cell suspension incubated with anti-EpCAM-conjugated immunomagnetic beads for carcinoma cell enrichment. Immunomagnetically selected cells were placed on slides; stained for CKs 8, 18, and 19; and evaluated with an automated digital microscopy system that rapidly scanned the slide and collected images of cells meeting predefined staining and cytomorphological criteria. A montage of the CK+ cells was reviewed to confirm tumor cell morphology.
Results: Eighteen specimens (9 normal, 2 N-, 4 N+, and 3 metastatic) were excluded because of poor cytomorphology or staining artifact. All 15 of the positive controls [95% confidence interval (CI), 78–100%] and none of the 25 negative controls (95% CI, 0–14%) demonstrated CK+ cells. Twenty-one of the 75 (28%; 95% CI, 18–40%) samples from breast cancer patients demonstrated CK+ cells including 76% of patients with metastatic disease (95% CI, 55–91%), 8% with N+ disease (95% CI, 1–26%), and none of those with N- disease (95% CI, 0–14). The mean number of CK+ cells detected in the 21 CK+ patients was 18.4 (range, 1–120).
Conclusions: Breast carcinoma cells can be detected in the blood from a significant fraction of metastatic breast cancer patients using immunomagnetic cell enrichment and digital microscopy. The incidence of CK+ cells was low in those with resected N+ disease (at most 26%) and those with resected N- breast cancer (at most 14%). This technique could be used in large prospective studies of patients with breast cancer to learn whether the detection of rare carcinoma cells is a useful predictive or prognostic factor.
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Searching for rare tumor cells in blood or bone marrow with light microscopy is tedious and not amenable to the routine diagnostic laboratory. Technological advances such as immunomagnetic enrichment (9) , digital microscopy (10) , and molecular methods such as RT-PCR (11, 12, 13, 14) have improved the assays and have made them more applicable to clinical research. Enrichment of the sample for carcinoma cells is especially useful when blood samples are being evaluated. By use of these immunomagnetic enrichment methodologies, rare (approximately 10-7–10-8) tumor cells may be detected in the peripheral circulation of patients with breast cancer (11) . The detection of rare tumor cells in the blood has been additionally improved with the use of a novel automated digital microscope (ACIS; ChromaVision Medical Systems, Inc.). Key features of this technology are automated (unattended) scanning of up to 100 slide-based specimens followed by the active participation of the laboratory professional (pathologist) in reviewing interpreting collected images, thereby minimizing risk for false positive determinations. A recent report (10) suggests that this technology demonstrates superior sensitivity for rare tumor cell detection relative to manual microscopy with excellent reproducibility.
Flow cytometry (9 , 15) and molecular techniques also have been shown to offer a sensitive means for rare circulating tumor cell identification. Mammaglobin is a glycoprotein, which is expressed preferentially in adult breast tissue, in 80–95% of malignant breast tumors, and lymph node metastases from breast cancer patients (13) . RT-PCR using primers to mammaglobin or to CKs has been used to detect metastatic tumor cells (14 , 16) . Although RT-PCR and flow cytometry are sensitive techniques, they do not permit visual confirmation of malignant cells, and false positive results on normal controls have been reported (11 , 12 , 15) . Nonetheless, these early studies suggest that CK+ cells can be detected in the blood and bone marrow with multiple methods and that this may be an important new addition to the staging of breast cancer and the predicting of prognosis.
Our investigation details the technical performance of combined immunomagnetic enrichment and digital microscopy for the detection and enumeration of CK+ cells in peripheral blood specimens. Initial results are based on the analysis of biological controls [blood from normal donors (negative controls) and blood samples spiked with BT474 breast cancer cells (positive controls)]. The false positive rate of the assay to detect CK+ cells is next evaluated using clinical specimens from normal donors and the prevalence of CK+ cells in women with breast cancer.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Epithelial Cell Enrichment Procedure.
Peripheral blood specimens were processed for the detection of epithelial cells within 24 h of phlebotomy. In 12 cases, <20 ml was obtained by phlebotomy (all cases 
14 ml). Peripheral blood was first enriched for nucleated cells using RBC lysis then enriched for epithelial cells by positive immunomagnetic selection. All of the enrichment steps and centrifugations were performed at room temperature. Erythrocytes were lysed by adding 4 volumes of 1x RBC lysis buffer [155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.2); Sigma Chemical Co., St. Louis, MO], mixing by inverting, and incubating for 5 min. Lysed blood was then centrifuged at 300 x g for 5 min. The lysis step was repeated and the cell pellet was then resuspended in 7 ml PEB buffer. PEB buffer is 1 x PBS, 0.1 mM EDTA (Sigma Chemical Co.), and 0.5% BSA fraction V (Sigma Chemical Co.); 1 x PBS is 0.144 g/liter KH2PO4, 0.795 g/liter Na2HPO4-7H2O, and 9 g/liter NaCl (pH 7.4; Life Technologies, Inc., Gaithersburg, MD). Samples were centrifuged at 300 x g for 5 min. Finally, the cell pellet was resuspended into 0.9 ml PEB.
Positive Selection Procedure.
HEA125-microbeads (100 µl; Miltenyi Biotec Inc., Bergisch Gladbach, Germany) recognizing EpCAM were added to the 0.9 ml cell suspension. The cell-microbead mixture was incubated for 30 min on a shaker (Labquake; Barnstead/Thermolyne). An LS+ column was assembled on a MidiMACS magnet (Miltenyi Biotec) and washed with 3 ml PEB buffer. The cell-microbead suspension was then loaded on the LS+ column. The flow-through was collected and used for negative selection in 46% (54 of 118) of cases. The column was washed first with 2 ml of PEB and then twice with 2 ml of PBS. The column containing the positively selected cells was then disassembled from the magnet, and the cells were eluted from the column with 3 ml of PBS and collected into a Hettich cytospin chamber assembled with a silanized microscope slide (DAKO, Carpinteria, CA). The column was washed again with 3 ml of PBS, which was gently pushed through with the column plunger. Cytocentrifugation was performed at 500 rpm for 15 min with a Universal 16A cytocentrifuge (Hettich/RevPro, Monroe, CT). The supernatant was finally aspirated and the slide dried at room temperature. The slide was kept a maximum of 4 days in a dry box with desiccant before CK staining.
Negative Selection Procedure.
In 54 cases (9 normal, 18 N-, 25 N+, and 2 metastatic) the flow-through was also processed by negative immunomagnetic selection. An equivalent maximum of 75 x 106 cells from the flow-through was depleted with 150 µl CD45 microbeads (Miltenyi Biotec Inc.) in a 1-ml cell suspension using a LS+ column assembled on a MidiMACS magnet. The flow-through was collected, and this negatively selected fraction was then cytocentrifuged onto a microscope slide with a maximum of 2 x 106 cells/slide. As necessary, samples with more cells were cytocentrifuged onto additional slides with the same maximum cell number/slide.
BT474-spiked Controls.
To evaluate the performance (i.e., tumor cell recovery) of immunomagnetic cell enrichment, control studies were performed using the BT474 breast carcinoma cell line. BT474 cells were maintained in culture medium (RPMI 1640, 10% fetal bovine serum, 1 x penicillin/streptomycin/glutamine; Life Technologies, Inc.). After trypsinization, cells were counted using a hemacytometer to determine the cell titer. A calculated volume corresponding to 
20 cells (in 1 case 10 cells) based on the cell titer was spiked into 20 ml of normal blood and processed as for patient specimens by immunomagnetic enrichment, cytocentrifugation and anti-CK immunocytochemistry, and ACIS digital microscopy analysis.
To more accurately estimate the number of carcinoma cells spiked in these experiments, three additional slides were prepared in parallel from each BT 474 cell suspension but without immunomagnetic enrichment. For each of the three slides, a volume of BT474 cells identical to the control sample submitted for immunomagnetic enrichment was added to 1 ml of blood from a normal donor and cytocentrifuged. These three slides were then submitted to anti-CK immunocytochemical staining and ACIS-assisted analysis. The average number of detected CK+ cells was used to calculate the cell recovery for the immunomagnetic enrichment experiments described in Table 3
.
|
1 x 106 WBC and a second slide containing 1 x 106 WBC spiked with cells from a carcinoma cell line (BT474 or HeLa S3) to serve as negative- and positive-staining controls, respectively.
CK+ Cell Detection by Digital Microscopy.
The ACIS (ChromaVision Medical Systems, Inc.) is a computerized microscope, which includes an image processing system that has been optimized for the detection of rare carcinoma cells in hematological or solid tissue (e.g., lymph node) specimens. A complete description of this system can be found in Ref. 10
. Briefly, for rare cell detection, the ACIS makes use of proprietary software allowing for highly sensitive color detection along with the capability for analysis of a variety of morphometric features. The application software available on the instrument involves first scanning a microscope slide at low magnification (x10). The instrument next returns to objects that were originally identified for a second analysis at higher magnification (x40 or x60). In this case, more sophisticated image analysis of color and morphometric characteristics (nuclear size and nuclear shape) is undertaken in an effort to exclude cellular debris, large clumps, and cells with morphological features typical of normal hematological mononuclear cells as opposed to CK+ carcinoma cells. Cellular objects that meet color- and morphometry-based criteria as likely tumor cells are collected and presented as montage images for review and classification by a pathologist or other laboratory professional. Finally, the pathologist reviews all of the collected images to assure that they meet appropriate cytomorphometric criteria for classification as carcinoma cells (e.g., excluding squamous cells, which arise as a result from venipuncture based on nucleus:cytoplasm ratio and chromatin texture). In the data file generated after the analysis of the peripheral blood or bone marrow specimen, the x-y coordinates of each cell are stored. On the basis of saved coordinates, a "revisit" capability allows the user to return to the proper location on the specimen slide for additional review of the cells of interest under manual control of the microscope.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and Fig. 2
) of collected cells were reviewed and classified based on CK staining and cytomorphological characteristics in the absence of knowledge of the specimen source (i.e., normal volunteer or carcinoma patient).
|
|
. A total of 18 specimens were excluded from this study for various reasons. Specimens with poor cytomorphology (poor nuclear morphology and/or cell integrity; 5 of 34 normal and 3 of 84 cancer patient samples) were excluded before their clinical status was revealed. Slides from 6 additional cases (1 of 34 normal and 5 of 84 cancer patient specimens) demonstrated very sparse cellularity. For these cases, although no CK+ cells were identified, poor quality autofocusing also led to their exclusion before clinical status was revealed. Finally, specimens affected by a staining artifact, which appeared to arise from staining errors (3 of 34 normal and 1 of 84 cancer patient samples) were also excluded. In these cases, false positive immunocytochemical staining was observed in WBCs, which could be recognized by their distinctive nuclear morphology.
|
.
|
Studies on Control Blood Samples Spiked with BT474 Carcinoma Cells.
To monitor the quality and the consistency of the CK cell detection method used for the breast cancer patient samples, blood samples were spiked with carcinoma cells and processed in the same manner as clinical specimens at various times over a period of 28 weeks. These control samples consisted of 20 ml of blood from normal donors spiked with a volume corresponding to 1–113 BT474 carcinoma cells (Table 3)
. CK+ cells were detected by ACIS analysis in 100% of the spiked positive control blood samples. The mean CK cell recovery was 86% with a range of 47%–100% (Table 3)
.
Reproducibility of Tumor Cell Detection in Peripheral Blood from Breast Cancer Patients Using ACIS.
An additional study was performed to investigate the reproducibility of the ACIS for tumor cell detection. Four breast cancer patient specimens, including three CK+ and one CK-, were independently analyzed using three separate ACIS instruments. Each specimen was scanned and analyzed on three separate occasions for each instrument. The results are presented in Table 4
. Analysis of the four specimens revealed identical numbers of cells in all cases.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Other studies have also reported the detection of carcinoma cells in the blood of patients with solid tumors using various detection methods. Racila et al. (15) evaluated the peripheral blood of 30 patients with breast cancer, 3 patients with prostate cancer, and 13 controls using an immunomagnetic enrichment with flow cytometry using antibody to CK. They concluded that CK+ cells were present in 32 of 33 cancer patients. Martin et al. (9) evaluated peripheral blood specimens from patients with advanced carcinoma of the breast, prostate, colon, rectum, and lung, and detected CK+ cells in 21 of 34 patients using immunomagnetic enrichment and flow cytometry. In a study of the blood from 19 patients with breast cancer before and after surgery using immunomagnetic enrichment in a nonautomated slide-based light microscope system, Krag et al. (18) found that 95% (18 of 19) of patients had cells in the blood preoperative, and 30% were positive postoperative. Kraeft et al. (19) reported recently the detection of CK+ cells using an automated fluorescence microscope system. Their initial results showed CK+ cells in 17% of normal blood donors. They subsequently modified their procedure to use double-staining procedures, and this methodological change eliminated "positive" staining in normal donors and resulted in the detection of CK+ cells in 35% of peripheral blood specimens obtained from cancer patients without the requirement for immunomagnetic enrichment.
Interpretation of these results in some studies has been complicated by the finding in some studies of positive (i.e., carcinoma cells) in the peripheral blood of "normal" donors. For example, Racila et al. (15) concluded that 7 of 13 peripheral blood specimens from healthy individuals included CK+ cells after immunomagnetic enrichment and flow cytometric analysis. Other reports using molecular methodologies have documented that false positive determinations can occur, which clearly impact in the interpretation of assay results (11) .
None of the studies of circulating carcinoma cells in blood to date have analyzed the impact of this finding on disease-free survival and overall survival. The main reason for this is that it is important to first develop and rigorously test the performance of the technology for detecting these rare cells before large-scale clinical studies are undertaken. Because carcinoma cells are so rare in the blood, it is impractical to simply review slides with routine light microscopy. Key advantages of digital microscopy-based analysis are the opportunity for automated (unattended) scanning of specimen slides and the opportunity for visual review by the laboratory professional to assure that the collected events, in fact, represent CK+ carcinoma cells (Figs. 1
and 2
).
The combination of immunomagnetic enrichment and digital microscopy as described in this study demonstrate that the technology is now available to be applied to blood samples from large groups of patients. Well-designed clinical studies should now provide the opportunity to learn whether the presence of rare carcinoma cells is useful in the clinical management of patients.
Additional research is required to determine whether this test is of value in metastatic disease. In our study, none of the patients had a known objective status, and it is possible that the presence or absence of circulating cells might correlate with the effects of treatment. This would be a particularly important finding, because many patients do not have easily assessable metastatic disease.
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be addressed, T. Witzig at 628 Stabile Building, Mayo Clinic, Rochester, MN 55905. Phone: (507) 266-9276; Fax: (507) 266-9277; E-mail: witzig{at}mayo.edu 
2 The abbreviations used are: CK, cytokeratin; RT-PCR, reverse transcription-PCR; N-, node-negative; N+, node-positive; ACIS, Automated Cellular Imaging System; CI, confidence interval.
Received 10/17/01; revised 1/28/02; accepted 2/ 5/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Hiraiwa, H. Takeuchi, H. Hasegawa, Y. Saikawa, K. Suda, T. Ando, K. Kumagai, T. Irino, T. Yoshikawa, S. Matsuda, et al. Clinical Significance of Circulating Tumor Cells in Blood from Patients with Gastrointestinal Cancers Ann. Surg. Oncol., November 1, 2008; 15(11): 3092 - 3100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Alix-Panabieres, S. Riethdorf, and K. Pantel Circulating Tumor Cells and Bone Marrow Micrometastasis Clin. Cancer Res., August 15, 2008; 14(16): 5013 - 5021. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Pantel, C. Alix-Panabieres, and S. Riethdorf Circulating Tumor Cells: Detection and Clinical Relevance Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 603 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Pusztai Current Status of Prognostic Profiling in Breast Cancer Oncologist, April 1, 2008; 13(4): 350 - 360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Riethdorf, H. Fritsche, V. Muller, T. Rau, C. Schindlbeck, B. Rack, W. Janni, C. Coith, K. Beck, F. Janicke, et al. Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System Clin. Cancer Res., February 1, 2007; 13(3): 920 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Quintela-Fandino, J. M. Lopez, R. Hitt, S. Gamarra, A. Jimeno, R. Ayala, J. Hornedo, C. Guzman, F. Gilsanz, and H. Cortes-Funes Breast Cancer-Specific mRNA Transcripts Presence in Peripheral Blood After Adjuvant Chemotherapy Predicts Poor Survival Among High-Risk Breast Cancer Patients Treated With High-Dose Chemotherapy With Peripheral Blood Stem Cell Support J. Clin. Oncol., August 1, 2006; 24(22): 3611 - 3618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Muller, N. Stahmann, S. Riethdorf, T. Rau, T. Zabel, A. Goetz, F. Janicke, and K. Pantel Circulating Tumor Cells in Breast Cancer: Correlation to Bone Marrow Micrometastases, Heterogeneous Response to Systemic Therapy and Low Proliferative Activity Clin. Cancer Res., May 15, 2005; 11(10): 3678 - 3685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Reinholz, A. Nibbe, L. M. Jonart, K. Kitzmann, V. J. Suman, J. N. Ingle, R. Houghton, B. Zehentner, P. C. Roche, and W. L. Lingle Evaluation of a Panel of Tumor Markers for Molecular Detection of Circulating Cancer Cells in Women with Suspected Breast Cancer Clin. Cancer Res., May 15, 2005; 11(10): 3722 - 3732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Meng, D. Tripathy, E. P. Frenkel, S. Shete, E. Z. Naftalis, J. F. Huth, P. D. Beitsch, M. Leitch, S. Hoover, D. Euhus, et al. Circulating Tumor Cells in Patients with Breast Cancer Dormancy Clin. Cancer Res., December 15, 2004; 10(24): 8152 - 8162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. Allard, J. Matera, M. C. Miller, M. Repollet, M. C. Connelly, C. Rao, A. G. J. Tibbe, J. W. Uhr, and L. W. M. M. Terstappen Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases Clin. Cancer Res., October 15, 2004; 10(20): 6897 - 6904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Krivacic, A. Ladanyi, D. N. Curry, H. B. Hsieh, P. Kuhn, D. E. Bergsrud, J. F. Kepros, T. Barbera, M. Y. Ho, L. B. Chen, et al. A rare-cell detector for cancer PNAS, July 20, 2004; 101(29): 10501 - 10504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Seligson, A. J. Pantuck, X. Liu, Y. Huang, S. Horvath, M. H. T. Bui, K.-r. Han, A. J. L. Correa, M. Eeva, S. Tze, et al. Epithelial Cell Adhesion Molecule (KSA) Expression: Pathobiology and Its Role as an Independent Predictor of Survival in Renal Cell Carcinoma Clin. Cancer Res., April 15, 2004; 10(8): 2659 - 2669. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-Y. Pierga, C. Bonneton, A. Vincent-Salomon, P. de Cremoux, C. Nos, N. Blin, P. Pouillart, J.-P. Thiery, and H. Magdelenat Clinical Significance of Immunocytochemical Detection of Tumor Cells Using Digital Microscopy in Peripheral Blood and Bone Marrow of Breast Cancer Patients Clin. Cancer Res., February 15, 2004; 10(4): 1392 - 1400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Pantel, V. Muller, M. Auer, N. Nusser, N. Harbeck, and S. Braun Detection and Clinical Implications of Early Systemic Tumor Cell Dissemination in Breast Cancer Clin. Cancer Res., December 15, 2003; 9(17): 6326 - 6334. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
