Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

Ring, Alistair; Zabaglo, Lila; Ormerod, M.G.; Smith, Ian; Dowsett, Mitch

doi:10.1038/sj.bjc.6602418

Cited by 162 publications

(105 citation statements)

References 21 publications

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“…This assay has been previously developed in our laboratory for the detection of CK-19 and mammaglobin transcripts in peripheral blood and bone marrow samples of patients with breast cancer (Benoy et al, 2004). CK-19 is expressed in the majority of breast carcinomas (Bartek et al, 1985) and has been extensively used as a marker for CTC (Slade et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2003;Benoy et al, 2004;Ring et al, 2005). The specificity of mammaglobin for the detection of breast cancer cells in haematopoietic products has been evaluated in several studies (Leygue et al, 1999;Zach et al, 1999;Suchy et al, 2000;Corradini et al, 2001;Silva et al, 2002;Lin et al, 2003).…”

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confidence: 99%

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer

Auwera

Peeters

Benoy³

et al. 2009

Br J Cancer

162

107

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BACKGROUND: The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice. METHODS: This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/ Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts. RESULTS: As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT -PCR for CK-19 and 54% using RT -PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT -PCR than using the CellSearch System (P ¼ 0.001) or the AdnaTest (Po0.001). CONCLUSION: We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

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confidence: 99%

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer

Auwera

Peeters

Benoy³

et al. 2009

Br J Cancer

162

107

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“…Mammaglobin gene expression has previously been used to detect circulating BC micometastases with no false-positives in the control population (Zach et al, 1999;Grunewald et al, 2000;Suchy et al, 2000;Leone et al, 2001;Silva et al, 2002;Ring et al, 2005).…”

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confidence: 99%

“…Cytokeratin-19 was chosen as it is expressed in the majority of BCs (Bartek et al, 1986) and has been extensively used in studies in this area (Slade et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2003;Benoy et al, 2004;Ring et al, 2005). Mammaglobin gene expression has previously been used to detect circulating BC micometastases with no false-positives in the control population (Zach et al, 1999;Grunewald et al, 2000;Suchy et al, 2000;Leone et al, 2001;Silva et al, 2002;Ring et al, 2005).…”

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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

et al. 2006

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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.

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“…Indeed, the presence of circulating tumor cells (CTCs) in the bloodstream of cancer patients has been recognized for over a century (9), although a lack of sensitive technology precluded the detailed study of these cells until recently. However, technological advances have now facilitated the identification, enumeration, and characterization of individual disseminated cells in breast cancer patients using methods such as PCR (10)(11)(12), flow cytometry (FCM) (13)(14)(15), image-based immunologic approaches (16)(17)(18)(19), immunomagnetic techniques (20,21), and microchip technology (22). In breast cancer patients with either metastatic or apparently localized disease, there is growing evidence that the presence of individual CTCs in the blood or disseminated tumor cells (DTCs) in the bone marrow (BM) may be an important indicator of the potential for metastatic disease and poor prognosis [reviewed in (23)(24)(25)(26)(27)].…”

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confidence: 99%

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

et al. 2008

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The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen-fluorescein isothiocyanate and CD45-PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting. ' International Society for Advancement of CytometryKey terms breast cancer; metastasis; preclinical animal models; tumor cell dissemination; flow cytometry; laser scanning cytometry BREAST cancer is a leading cause of morbidity and mortality in women (1,2), primarily because of the failure of effective clinical detection and management of metastatic disease in distant sites such as lymph node (LN), bone, lung, liver, and brain (3,4). The metastatic process is comprised of a series of sequential steps, and cancer cells must successfully complete each step in order to give rise to a metastatic tumor. These steps include dissemination of cancer cells from the primary tumor into the bloodstream (intravasation), survival in the circulation, arrest and extravasation into the secondary site, and initiation and maintenance of growth to form clinically detectable metastases (3-7). Breast cancer cells may also disseminate from the primary tumor through the lymphatic system, although the lac...

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Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

Cited by 162 publications

References 21 publications

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer

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

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

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