Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction

Leone, Francesco; Perissinotto, Eliana; Viale, Andrea; Cavalloni, Giuliana; Taraglio, Stefano; Capaldi, Antonio; Piacibello, Wanda; Torchio, Bruno; Aglietta, Massimo

doi:10.1038/sj.bmt.1702815

Cited by 21 publications

(22 citation statements)

References 23 publications

(32 reference statements)

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“…Real-time polymerase chain reaction experiments have shown that high levels of expression are warranted to obtain an effect on prognosis (29). In our data, however, a nonquantitative RT-PCR evaluation of MAM expression on PBPCs also showed a statistically significant correlation with an increased risk of relapse for high-risk breast cancer patients.…”

Section: Discussioncontrasting

confidence: 43%

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Mammaglobin Expression in Leukapheresis Products Is a Predictive Marker of Poor Prognosis in Women with High-Risk Breast Cancer

Ferrucci

Rabascio

Mazzetta

et al. 2004

Clinical Cancer Research

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Purpose: The purpose of this study was to investigate the incidence and prognostic relevance of tumor cell detection in granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cell collections (PBPCCs) using cytokeratin (CK), maspin (MAS), and mammaglobin (MAM) genes as epithelial cell markers. The population on which the study was conducted was drawn from stage III breast cancer patients undergoing high-dose chemotherapy and autologous transplantation with PBPCCs.Experimental Design: One hundred and ninety-four patients were enrolled in the study and analyzed for tumor cell detection on the basis of 481 PBPCCs gathered before administration of chemotherapy. CK, MAS, and MAM gene expressions were investigated by means of the reverse transcription nested polymerase chain reaction, and those samples expressing CK were further hybridized with a radiolabeled internal probe to reduce false-positive results. Sensitivity and specificity were assessed on 37 controls (12 cell lines, 12 healthy donors, and 13 nonepithelial malignancies). Each of the known prognostic variables (age, stage, lymph node status, receptor status, c-ErbB2 status, and Ki67 status) was then analyzed (both individually and together with CK, MAS, and MAM expression on PBPCCs) in relation to patient overall survival (OS) and relapse-free survival (RFS).Results: After a 3-year follow-up, an estimated 83% (95% confidence interval, 77.1-88.8%) of the patients were alive and an estimated 67% (95% confidence interval, 60.1-74.6%) were free of relapse. One hundred and seventy-six of the 194 patients (91%) had contaminated PBPCCs evidenced by at least one positive sample for any of the markers evaluated. The PBPCC frequency of CK, MAS, and MAM positivity (؉) was 71%, 36%, and 16%, respectively. MAM expression on PBPCC was associated with an increased risk of relapse (P ‫؍‬ 0.003), whereas CK and MAS expressions were not associated with changes in either RFS or OS.Conclusions: MAM gene expression on leukapheresis products of high-risk breast cancer patients is an indicator of poor prognosis. The method of evaluation is simple and reproducible and provides new tools for evaluating the role played by tumor cells in apheresis products and their potential in causing metastasis.

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Section: Discussioncontrasting

confidence: 43%

“…MAM seems to be much more specific for breast cancer because it is also detectable in the PB of metastatic breast cancer patients, and it has been shown to be the more affordable marker in detecting epithelial cell contamination in hematologic samples (29).…”

Section: Discussionmentioning

confidence: 99%

Mammaglobin Expression in Leukapheresis Products Is a Predictive Marker of Poor Prognosis in Women with High-Risk Breast Cancer

Ferrucci

Rabascio

Mazzetta

et al. 2004

Clinical Cancer Research

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“…Leone et al (2001) found a high intertumoral variability of mammaglobin expression levels. Zach et al (1999) confirmed that different breast cancer cell lines show different mammaglobin expression levels.…”

Section: Detection Of Dec In Breast Cancer By Rt -Pcr Ih Benoy Et Almentioning

confidence: 99%

“…Reverse transcriptase -polymerase chain reaction according to the Taqman methodology was performed to quantify DEC in leukapheresis products of breast cancer patients by the RGE using a panel of genes related to the breast gland or involved in breast cancer: maspin, mammaglobin and c-ErbB-2 (Leone et al, 2001). Maspin and mammaglobin were not expressed in control samples, and the mammaglobin expression was the most sensitive single marker to detect DEC. An additional reason for false negativity is the inherent mutator phenotype of cancer resulting in heterogeneous gene expression patterns, both topographically and temporally, within a tumour.…”

Section: Detection Of Dec In Breast Cancer By Rt -Pcr Ih Benoy Et Almentioning

confidence: 99%

Real-time RT–PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer

et al. 2004

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Real-time reverse transcriptase -polymerase chain reaction (RT -PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT -PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against b-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT -PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r ¼ 0.7; P ¼ 0.0003), and between hMAM expression and ICC (r ¼ 0.6; P ¼ 0.003). CK-19 expression and ICC (r ¼ 0.9; Po0.0001) showed the strongest correlation. Reverse transcriptase -polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT -PCR, and RT -PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT -PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.

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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%

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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Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction

Cited by 21 publications

References 23 publications

Mammaglobin Expression in Leukapheresis Products Is a Predictive Marker of Poor Prognosis in Women with High-Risk Breast Cancer

Mammaglobin Expression in Leukapheresis Products Is a Predictive Marker of Poor Prognosis in Women with High-Risk Breast Cancer

Real-time RT–PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with 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

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