Real-time RT–PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

Abstract

This study assessed the ability of real-time reverse transcription -polymerase chain reaction (RT -PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n ¼ 116/78%) and metastatic (stage M þ , n ¼ 32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT -PCR detection method. The mean follow-up time was 786 days ( þ /À 487). Kaplan -Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P ¼ 0.0045 (CK-19) and P ¼ 0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P ¼ 0.551 (CK-19) and P ¼ 0.329 (MAM)). Separate analyses of the M0 and M þ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M þ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.

Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer

“…Nevertheless, the high concordance observed between CTC and DTC detection in our study favours the hypothesis that there is a continuous circulation and exchange of epithelial cells between bone marrow and peripheral blood. The high detection rate of occult tumour cells in both peripheral blood and bone marrow observed in the current study is in overt contrast with previous studies reporting low detection rates using immunocytochemical assays (Ismail et al, 2004;Pierga et al, 2004;Muller et al, 2005;Wiedswang et al, 2006;Benoy et al, 2006) and should be attributed to the high sensitivity and specificity of the quantitative RT-PCR assay used for the detection of minimal residual disease . Moreover, it is interesting to note that only 10% of 1767 patients with early disease had more than one CTC per 23 ml of peripheral blood when tested with the new automated Cell Search System (Rack et al, 2007(Rack et al, , 2008).…”

“…Previous reports evaluating the prognostic significance of disseminated tumour cells in blood and bone marrow concluded that the detection of CTCs is prognostically inferior to the detection of DTCs (Pierga et al, 2004;Benoy et al, 2006;Wiedswang et al, 2006). In this study, the presence of CK-19 mRNA-positive CTCs and DTCs before the initiation of adjuvant chemotherapy was predictive for decreased OS.…”

“…Conversely, the use of blood for detecting CTCs is more convenient and could be easily acceptable by patients, thus representing a valuable alternative solution. Although some previous studies (Ismail et al, 2004;Pierga et al, 2004;Muller et al, 2005;Benoy et al, 2006;Wiedswang et al, 2006) have shown a correlation between the detection of DTCs and CTCs, this fact has not been unanimously accepted. This could be due to the fact that in most studies, the comparison of the detection of CTCs and DTCs was carried out using immunocytochemical assays, which, in general, have a lower sensitivity compared with molecular assays.…”

Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer

“…The MGB1 detection could be improved by using an epithelial cell enrichment step with immunomagnetic beads as has been reported by other investigators (18). MGB1 detection rates in early breast cancer have varied considerably in published studies from 2% to 22% (23,24,38,39). This variation can be attributed to the different detection techniques used with different sensitivities and specificities, the different patient populations studied, and possibly the lack of correlation data about MGB1 expression in the primary tumor.…”

Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression

“…In many types of cancer, isolated (circulating or disseminated) tumour cells (ITC) in the blood and bone marrow are present (1)(2)(3)(4)(5). The detection frequency of these cells, in the blood and bone marrow, varies substantially not only in different types, but in the same type of cancer (6).…”