Detection and characterization of carcinoma cells in the blood

E Racila

D Euhus

A Weiss

C Rao

J McConnell

L Terstappen

J Uhr

Cancer Immunobiology Center and Departments of ^{Microbiology,}^{Surgery, and}^{Urology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235;}^{Allegheny and Thomas Jefferson Schools of Medicine, Philadelphia, PA 19107; and}^{Immunicon Corporation, Huntingdon Valley, PA 19006}

^{To whom reprint requests should be addressed. e-mail:}ude.demws.tendem@rhuJ.

Contributed by Jonathan W. Uhr

Accepted 1998 Feb 3.

Abstract

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid^{, CD45}^{, and cytokeratin}^{. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.}

Abstract

Evidence is accumulating that primary cancers begin shedding neoplastic cells into the circulation at an early stage (1–4); however, the natural history of these cells, their ability to establish metastases, and their bearing on future relapses are unclear. For instance, circulating tumor cells have been detected by PCR in a variety of patients with a good prognosis who are unlikely to develop metastatic disease (5–8). In addition, immunocytochemistry has detected cancer cells in the bone marrow in a proportion of patients with clinically localized disease (9–11). If tumor cell shedding is, in fact, an early event in tumorigenesis, it may be possible to detect cancer cells in the bloodstream before the primary tumor is large enough to be detected by standard screening examinations.

To explore this possibility, we have developed a cellular assay that is more sensitive than PCR and that allows precise enumeration and characterization of circulating carcinoma cells. In model studies, the sensitivity of the technique is below 1 epithelial cell/ml of blood regardless of the number of leukocytes present and the recovery is between 75 and 100%. The assay was used to study the blood of 30 patients with breast cancer, 3 with prostate cancer, and 13 control individuals. An excess of circulating epithelial cells was found in virtually all of the cancer patients unless they were being treated with chemotherapy. In addition, 8 patients with breast cancer undergoing chemotherapy were followed for 1–10 months to determine whether the level of blood tumor cells correlated with clinical studies. The malignant nature of the cells was demonstrated by their cytology and immunophenotype.

Flow cytometry was used to analyze the positive events obtained from 20 ml of blood from control individuals; from women with breast carcinoma, or from men with prostate cancer. The numbers of epithelial cells in the blood of the controls are statistically different by t test (P ≤ 0.01) and by Kruskal–Wallis nonparametric analysis (P < 0.001) from each of the three groups of the breast cancer patients and the prostate cancer patients. The data in this table were used to establish a preliminary cut-off value for positive samples. This value was determined by averaging the number of circulating epithelial cells in the normal controls (n = 13) and then adding three times the SD. The average (n = 13) was 1.5 and the SD is 1.8. Cut-off: 1.5 + 5.4 = 6.9. There is no statistical difference between male and female controls.

Acknowledgments

We thank Drs. Ellen Vitetta and Louis Picker for advice regarding the manuscript, Drs. Joan Reisch and Barbara Foster for help with the statistical analysis, Mr. Thomas Tucker for assistance in sample collection and data processing, and Ms. Cindy Patterson and Sue Chadwick for excellent secretarial assistance. Dr. Emil Racila was supported by National Institutes of Health Fellowship Awards 5-T32-CA66187-02 and T32-CA09082.

Acknowledgments

ABBREVIATION

EPCAM

epithelial cell adhesion molecule

ABBREVIATION

Footnotes

^{Using only flow cytometry, it was possible to detect one breast carcinoma cell mixed with 10}^{leukocytes (}22). There are also reports of occasionally obtaining a sensitivity of one tumor cell in 10^{leukocytes by PCR or reverse transcription PCR when a particular mutation is available (}23–26).

Footnotes

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