Detection and characterization of carcinoma cells in the blood

Abstract

A highly sensitive assay combining immunomagnetic enrichment with multiparameter f low 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 f low 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.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 cell͞ml 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 determin...

Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

“…The detection of such cells could have significant clinical utility in risk stratification in early breast cancer, in early detection of relapse and in monitoring response to treatment. Cytometric techniques based on immunohistochemical analyses and nucleic acid-based approaches to cell detection have been described (Racila et al, 1998;Lambrechts et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2002;Witzig et al, 2002). However, there is considerable variability in the reported sensitivities and specificities of existing techniques with putative carcinoma cells reported to be present in between 0 and 100% of blood samples from patients with metastatic breast cancer (Ring et al, 2004).…”

Detection and enrichment of disseminated renal carcinoma cells from peripheral blood by immunomagnetic cell separation

“…9 In spiking experiments with RCC cell lines, we determined a sensitivity of positive tumor-cell detection of 10 -7 , which is comparable to other reports. 11 In comparison to the detection of disseminated carcinoma cells, we found no false-positive cell-staining artifacts in normal blood cells. Renal cancer cell line Caki-1 had only moderate or weak CK expression detected by several specific anti-CK antibodies (CK 8-18, CK 19) and by pan-anti-CK antibody (MNF 116, AE 1-3) in the periphery of the cytoplasm, which was weaker than in MCF-7 breast-carcinoma cells.…”

Development of a high‐throughput membrane‐array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers

“…25 Additionally, a panel of molecular markers could enhance the sensitivity for CTCs detection, compared with single markers in use. 21 RT-PCR assay is by far regarded widely to be the most sensitive method for detecting tumor-associated molecular markers. [26][27][28][29][30][31][32] However, for multiple gene detection, RT-PCR is too time-consuming and laborious to apply in clinical diagnosis.…”

“…Because of heterogeneity of the expression of tumor-related genes, a multimarker assay is regarded to be more reliable and sensitive than singlemarker assay. [20][21][22] Therefore, a high-throughput assay able to detect simultaneously a panel of informative molecular markers for the presence of CTCs is in urgent need of development, today. The objective of the study was to evaluate the utility of a noninvasive, peripheral blood-based membrane-array assay detecting a panel of tumor-related mRNA markers (hTERT, CK-19, CEA and MUC1) for GC patients.…”