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The purpose of this study was to determine whether primary breast cancer patients showed evidence of circulating tumour cells (CTCs) during follow-up as an alternative to monitoring disseminated bone marrow tumour cells (DTCs) by immunocytochemistry and reverse transcriptase (RT)-PCR for the detection of micrometastases. We planned to compare CTC and DTC frequency in low-risk and high-risk patients. We identified two cohorts of primary breast cancer patients who were at low (group II, T(1)N(0), n=18) or high (group III, >3 nodes positive (with one exception, a patient with two positive nodes) n=33) risk of relapse who were being followed up after primary treatment. We tested each cohort for CTCs using the CellSearch system on 1-7 occasions and for DTCs by immunocytochemistry and RT-PCR on 1-2 occasions over a period of 2 years. We also examined patients with confirmed metastatic disease (group IV, n=12) and 21 control healthy volunteers for CTCs (group I). All group I samples were negative for CTCs. In contrast, 7 out of 18 (39%) group II primary patients and 23 out of 33 (70%) group III patients were positive for CTCs (P=0.042). If we count only samples with >1 cell as positive: 2 out of 18 (11%) group II patients were positive compared with 10 out of 33 (30%) in group III (P=0.174). In the case of DTCs, 1 out of 13 (8%) group II patients were positive compared with 19 out of 27 (70%) in group III (P<0.001). Only 10 out of 33 (30%) patients in group III showed no evidence of CTCs in all tests over the period of testing, compared with 11 out of 18 (61%) in group II (P=0.033). A significant proportion of poor prognosis primary breast cancer patients (group III) have evidence of CTCs on follow-up. Many also have evidence of DTCs, which are more often found in patients who were lymph node positive. As repeat sampling of peripheral blood is more acceptable to patients, the measurement of CTCs warrants further investigation because it enables blood samples to be taken more frequently, thus possibly enabling clinicians to have prior warning of impending overt metastatic disease.

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19. RT-PCR was done by two 40-cycle rounds using nested primers. In initial experiments, RT-PCR was shown to be capable of detecting one tumour cell in one million normal bone marrow cells, which was at least 10 times more sensitive than immunohistochemistry, while retaining specificity. Five per cent of the peripheral blood and 22% of the bone marrow samples contained K19 positive cells by immunohistochemistry staining. Using RT-PCR, these proportions increased to 25% and 35%, respectively. This represents a significantly greater detection frequency (P < 0.001 and P = 0.03, respectively). RT-PCR for K19 is a more sensitive method for detecting micrometastases in patients with primary breast cancer when compared with immunohistochemistry.