Immunocytochemical staining

Several different assays have been developed to detect DTC in breast cancer and other types of carcinomas. One major approach to identify DTC is immunocytochemical staining with monoclonal antibodies against epithelial or tumor-associated antigens.4-7 To date, cytokera-tins have become the most widely accepted protein markers for the detection of epithelial tumor cells in mesenchymal tissues such as BM, blood or lymph nodes.8-10 However, different staining techniques can result in specificity variations.11,12 Several international organizations have therefore recognized the need for standardization of the immunocytochemical assay and for its evaluation in prospective studies (see www.dismal-project.eu).13,14

Immunocytochemical analysis is usually used in combination with density gradient centrifugation, immunomagnetic procedures or size filtration methods to enrich tumor cells prior to their detection.15-18 One way to improve current detection assays for single tumor cells is to develop better tumor cell enrichment procedures using improved density gradients19 and antibody-coupled magnetic particles.20-22 At present, it is unclear whether these new enrichment techniques provide more clinically relevant information than the standard density gradient procedure used to isolate the mononuclear cell fraction (Figure 7.1).

The use of new automated devices for microscopic screening of immunostained slides may help slides to be read more rapidly and increase reproducibility of the read-out (Figure 7.1).20,23-27 Among the commercially available automated systems, the CellSearch™ system has gained considerable attention because it allows an automated immunomag-netic enrichment and cytokeratin staining of blood samples.28 A recent validation study demonstrated the reproducibility indicating

BM FicoII

FicoII gradient

BM aspirates taken from the upper iliac crest

Immunocytochemistry: CK staining with monoclonal antibodies

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Figure 7.1 Immunocytochemical detection of disseminated tumor cells (DTC) in the bone marrow (BM) of patients with epithelial tumors. The detection process begins with Ficoll density gradient centrifugation to isolate mononuclear cells (MNC) and uses cytokeratin (CK) antibodies. The detection of the stained DTC can be performed automatically and suspect cells are displayed in an image gallery.

that multicenter studies with shipment of samples are possible.29

Polymerase chain reaction approach for the detection of disseminated tumor cells

A widely used alternative to immunocyto-chemical assays for the detection of DTC became molecular detection procedures. In principle, the nucleic acid in a sample can be amplified by polymerase chain reaction (PCR) so that very small numbers of tumor cells can be detected in a heterogeneous population of cells. However, the tumor cells must have changes in DNA or mRNA expression patterns which distinguish them from the surrounding hematopoetic cells. At the DNA level, breast carcinomas are genetically quite heterogeneous, so that there is no universally applicable DNA marker available. Therefore, the main approach to develop molecular diagnostic assays for breast carcinomas has focused on RNA markers. A multi-marker approach with a panel oftumor-specific mRNA markers may improve the sensitivity for the detection of DTC over single marker

30 31

assays.30'31

Up to date, many transcripts have been evaluated as "tumor-specific" markers such as cytokeratins (CK) CK18, CK19 and CK20, mucin-1 (MUC1), and carcinoembryonic anti-gen.32 However, many of these transcripts can also be identified by reverse transcription (RT)-PCR in normal BM, blood, and lymph node tissue.33-35 Preanalytical depletion of the interfering normal cell fraction (e.g. granulo-cytes which express CK20) and/or quantitative RT-PCR determinations with well-defined cut-off values might solve this problem. In addition, expression of the mRNA marker might be downregulated, which argues in favor of the use of a multimarker RT-PCR approach.36

Enzyme-linked immunospot technology

A drawback of both immunocytochemistry and RT-PCR is the fact that these technologies are usually unable to distinguish between viable and apoptotic cells. Recently, a new technique which allows this important

Coating anti-MUC1 Abs

MUC1-releasing breast tumor cells

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Immunocapt^d MUC1 Elimination of cells rmm i

Fluorescent anti-MUC1 Abs

Revelation step: addition of conjugated anti-MUC1 Abs imrni

Immunospots are the MUC1 fingerprints left only by viable releasing tumor cells discrimination was introduced for DTC/CTC analyses.37 This technique was designated epithelial immunospot (EPISPOT) and is based on the secretion or active release of specific marker proteins using an adaptation of the enzyme-linked immunospot (ELISPOT) technology (Figure 7.2). The EPISPOT assay offers the advantage that only viable tumor cells will be detected and that protein secretion can be detected at an individual cell level.38 For the detection of breast cancer-derived DTC/CTC, MUC1 and CK19 were used as marker proteins.39 MUCl-secreting CTC were detected in all metastatic breast cancer patients analyzed, whereas such cells were not observed in healthy controls. Moreover, the enumeration of both MUC1- and CK19-secreting cells allowed the detection of viable DTC in BM of 90% and 54% of breast cancer patients, with and without overt distant metastasis, respectively.39

These data demonstrate the high specificity and sensitivity of the new EPISPOT technology, which reveals a unique fingerprint of single viable tumor cells and therefore opens a new avenue in the understanding of the biology of early metastatic spread.

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