Metastases in breast cancer may arise through either lymphatic spread or through hematogenous dissemination. While the local treatment of breast cancer is quite successful and modern treatment can adequately deal with local lymph node metastases, a substantial proportion of breast cancer patients ultimately die from metastases to distant organs or tissues. Micrometastatic disease may be cured by adjuvant systemic therapy and the ability to detect it reliably would thus be a significant advance. We have recently shown that the tendency for hematogenous spread can already be shown in small tumours, using messenger-RNA (mRNA) expression profiling (van 't Veer et al, 2002). In addition, we and others have developed immunological and RNA-based methods to detect circulating tumour cells in blood, bone marrow and other nonbreast tissues (Lambrechts et al, 1998). There is evidence that the presence of epithelial cells in the bone marrow of patients with early breast cancer correlates with prognosis (Braun et al, 2000). Antibody-based methods to detect these occult tumour cells have, however, not gained widespread clinical use because of significant numbers of false-positive results (Lambrechts et al, 1998,1999).
RNA-based methods have been used to detect the presence of mRNA species in RNA isolations from peripheral blood mononuclear cells that are characteristic of epithelial cells and that may originate from circulating tumour cells. The main problem of RNA-based assays continues to be the almost universally present background signal (Traweek et al, 1993; Datta et al, 1994; Burchill et al, 1995; Krismann et al, 1995; Fields et al, 1996; Moscinski et al, 1996; Mapara et al, 1997; Zippelius et al, 1997). We have recently developed a method that overcomes these problems by making use of two relatively recent developments. First, a truly quantitative PCR has become available, which is known as ‘real-time PCR’ (Bieche et al, 1999). Second, we have selected mRNA species using SAGE that are not present in peripheral blood RNA isolates or in bone marrow cells of healthy volunteers, but that are highly expressed in most breast cancers (Bosma et al, 2002). A panel of four marker genes (p1B, PS2, CK19 and EGP2) was used to detect circulating tumour cells in the peripheral blood of breast cancer patients; peripheral blood samples of healthy females were used as controls. Using a quadratic discriminant analysis (QDA), a score function based on the mRNA levels of the four genes was derived which, when positive, predicts the presence of circulating tumour cells (Bosma et al, 2002). This assay yields positive results in roughly 30% of the patients receiving treatment for advanced breast cancer. Here we show that a positive discriminant value is more frequent in patients with bone metastases and is furthermore associated with a reduced survival.