Detection of minimal disease in breast cancer

We investigated the prognostic significance of circulating breast cancer cells in peripheral blood detected by quantitative RT-PCR of marker genes in patients with advanced breast cancer. Blood samples from 94 breast cancer patients with metastatic disease (M1) were examined for circulating tumour cells by studying the mRNA expression of CK19, p1B, PS2, EGP2, mammaglobin and SBEM by real-time PCR. Using a score function, developed for predicting circulating tumour cells by quadratic discriminant analysis (QDA), four expression levels were combined into a single discriminant value. Tumour cells were present in 24 out of 94 (31%) of the patients, as compared with 0 out of 104 controls. The patients with a positive QDA value did have a progression-free survival at 1 year of 3% and overall survival at 2 years of 17%, against 22% and 36% for patients with a negative QDA value (P = 0.015 and P = 0.0053, respectively).

Breast cancer patients with metastatic disease have a significantly worse progression-free survival and overall survival when circulating tumour cells can be detected in their peripheral blood [1]. This method was used but was not sensitive enough to predict survival on the basis of a positive score in peripheral stem cell preparations (PBSC) of patients treated in a high-dose chemotherapy trial. The number of tumour cells in these preparations is too low.

We therefore sought a method that would specifically select breast tumour cells. We now show that a combination of magnetic beads cell separations and one-step cDNA synthesis of mRNA increases the sensitivity by at least 10-fold. Ten MCF7 cells mixed into 10 ml peripheral blood of a healthy control can be easily identified.

To validate this new method we will analyze blood samples of 200 stage I or stage II breast cancer patients and 50 PBSC of breast cancer patients (and 20 PBSC of patients with lymphoid malignancy as controls). So far, 30 stage I or stage II patients have been evaluated with the marker panel of six genes. A combination of four markers employed in the discriminant score revealed a sensitivity of 10% in detecting epithelial marker genes in peripheral blood.

The cell separation technique allows at least a 10-fold increase in sensitivity to detect tumour cell specific mRNA in mixed cell suspensions. Further analysis of the 50 PBSC patients and the prospective 200-patient stage I and stage II diagnostic study will reveal specificity and sensitivity. Interim results indicate an increase in sensitivity of at least 10%.