Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: interim results of a prospective cohort study.

BACKGROUND: We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis. METHODS: Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells. RESULTS: At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (mam) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN). CONCLUSION: This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the mam gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.

Molecular detection of breast cancer cells in the peripheral blood of advanced-stage breast cancer patients using multimarker real-time reverse transcription-polymerase chain reaction and a novel porous barrier density gradient centrifugation technology.

PURPOSE: The goal of this study was to develop a molecular diagnostic assay to detect circulating breast cancer cells in the peripheral blood for the purpose of staging breast cancer. Our aim was to make available an assay that was not limited by the low concentration of circulating breast cancer cells and the background gene expression that is typically found in peripheral blood. EXPERIMENTAL DESIGN: In this study, we investigated the ability of two new technologies to significantly enhance the quantification of gene expression in the peripheral blood: enrichment by a novel porous barrier density gradient centrifugation technology; and multimarker real-time reverse transcription-PCR (RT-PCR). RESULTS: Using fluorescence-labeled breast cancer cells and flow cytometry, we show that processing peripheral blood by porous barrier density gradient centrifugation results in a 300-fold enrichment of breast cancer cells. Real-time RT-PCR analysis confirmed a concomitant reduction in background expression of the CK19 and MUC1 genes after enrichment. In a pilot study, porous barrier density gradient centrifugation and multimarker real-time RT-PCR enabled our laboratory to detect breast cancer-associated gene overexpression in 13 of 20 (65%) stage IV breast cancer patients. Nine of these 14 patients overexpressed three or more markers. CONCLUSIONS: These results confirm the promise of such a molecular diagnostic assay and suggest that additional studies are needed to precisely define the clinical relevance.