Detection of Micrometastatic Disease and Monitoring of Perioperative Tumor Cell Dissemination in Primary Operable Breast Cancer Patients Using Real-Time Quantitative Reverse Transcription-PCR

Abstract

Purpose: We previously found a statistically significant number of cytokeratin 19 (CK19)؉ cells in peripheral blood (PB) of stage IV breast cancer (BC) patients compared with those of healthy volunteers, using a quantitative real-time reverse transcription-PCR. We aimed to apply the technique on bone marrow (BM) of primary operable BC patients. Pre-and postoperative PB samples of these patients were further analyzed to investigate possible shedding of CK19؉ cells during the operation.Experimental Design: In 54 primary operable BC patients, we analyzed 50 BM samples taken preoperatively and 297 PB samples. PB samples were collected before surgery; immediately after surgery; on the first, second, and fifth day postoperatively; and one month postoperatively.Results: In BM of controls and BC patients, we detected a median of 28 and 568 CK19؉ cells/5 ؋ 10 6 leukocytes, respectively (P < 0.001). In preoperative blood (B-1) samples, we measured a median of 109 CK19؉ cells. Using the upper limit of 95% confidence interval of controls as cutoff, 74% and 52% of BM and (B-1), respectively were considered CK19؉. There was no significant correlation between CK19؉ cells in BM and (B-1) and classical prognostic factors. We found no significant difference between blood samples at different time points with respect to the average CK19؉ cells. Conclusions:In primary BC patients, we detected high numbers of CK19؉ cells in BM and PB (B-1) samples compared with controls. However, no significant correlation between the presence of CK19؉ cells in BM and PB and classical prognostic factors was found. We detected no statistically significant influence of surgical manipulation on CK19؉ cells.

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).…”

“…Indeed, Schoenfeld et al (1997) have reported that the concordance between blood and bone marrow samples for occult tumour cell detection was only 6% by immunocytochemistry and 27% by RT-PCR. The lack of a complete overlap between the presence of CK-19 mRNA-positive CTCs and DTCs either before (Ismail et al, 2004;Pierga et al, 2004;Muller et al, 2005;Wiedswang et al, 2006) or after chemotherapy (Slade et al, 2009) could be related to the fact that occult tumour cells are rare events and therefore their evaluation is greatly influenced by sampling variability. In addition, it has been suggested that blood represents a temporary compartment for disseminated cells (Muller et al, 2005), whereas only a subpopulation of CTCs can settle in distant organs such as the bone marrow (Muller et al, 2005).…”

“…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.…”

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

“…This is in agreement with several other studies (Schoenfeld et al, 1997;Slade et al, 1999;Berois et al, 2000;Stathopoulou et al, 2002;Ismail et al, 2004;Pierga et al, 2004), all of whom demonstrate that BM is more likely to be positive than PB for the presence of DEC (Table 4). Cancer cells may be intermittently shed into the bloodstream, which could result in a sampling error if a single-point sampling is evaluated.…”

Dissemination profile of perioperative tumor cells in peripheral blood of colorectal cancer patients detected by multiple marker genes

“…Moreover, tumor cell dissemination is non-linear, and marker gene expression may vary between a primary tumor and its metastasis [19,20] . Therefore, multiple marker genes have been recently applied to improve the detection sensitivity in this cancer patients [21][22][23] , and single gene quantification at multiple time points has also been proposed for exploring the micrometastatic characteristic [24,25] . However, in colorectal cancer patients this method has not been used due to lack of effective marker genes combination applicable in PB.…”