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To address the clinical relevance of molecular detection of occult breast cancer in sentinel lymph nodes and nonsentinel axillary lymph nodes (ALN), we initiated the Minimally Invasive Molecular Staging of Breast Cancer (MIMS) trial, a multi-institutional prospective cohort study. This trial represents the first prospective cohort study in which a multimarker, real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was applied to the detection of breast cancer micrometastases in ALN. Sentinel and/or nonsentinel ALN from 501 breast cancer subjects with T1–T3 primary tumors were analyzed by standard histopathology and multimarker, real-time RT-PCR analysis. Seven breast cancer-associated genes (mam, mamB, PIP, CK19, muc1, PSE, and CEA) known to be overexpressed in metastatic breast cancer compared with control lymph nodes were used. Follow-up data were collected for 5 years. Of the 501 breast cancer subjects enrolled, 348 were node negative and completed the 5-year follow-up. Of these patients (n = 94), 27% demonstrated evidence of molecular overexpression. The 5-year relapse-free survival rate was 95.4% (95% confidence interval [95% CI], 92.4–97.2%). No single gene or combination of study genes was predictive of recurrence. The genes in this study panel failed to be predictive of clinical relapse. This may be a function of several factors: the low event rate at 5 years, the particular gene set, the methodology used for detection/analysis or that our original hypothesis was wrong and that the presence of positive marker signal by real-time RT-PCR is not associated with a worsened clinical outcome.

Background Pathologic evaluation may lack the sensitivity required for accurate staging of the axilla in breast cancer patients. We have completed enrollment of a multi-institutional prospective cohort study designed to determine if molecular analyses can improve axillary staging. In subset analyses, we have attempted to address the following questions: (1) Does molecular analysis improve the sensitivity of sentinel lymph node biopsy (SLNB) and (2) is the sentinel lymph node (SLN) hypothesis valid at the molecular level? Methods Four hundred eighty-nine subjects with T1, T2, or T3 breast cancer and no evidence of axillary lymph node (ALN) involvement were enrolled. ALNs were analyzed by routine pathology (hematoxylin-eosin staining), and by multimarker real-time reverse transcriptase-polymerase chain reaction analysis CRT-PCR to detect breast cancer metastases. Pathology and molecular data for both SLNs and nonsentinel ALNs were available for a subset of 207 subjects. Results The sensitivity of pathologic analysis of the SLN to predict the pathologic status of ALNs was 84.1%. The sensitivity of combining pathologic with molecular analysis of the SLN to predict the pathologic status of ALNs was 92.8%, a statistically significant increase in sensitivity (P = .031 by the McNemar test for correlated proportions). Finally, the sensitivity of combining pathologic with molecular analysis of the SLN to predict the pathologic or molecular status of ALNs was 85.4%. Conclusions The combination of pathologic and molecular analysis of SLNs resulted in the highest sensitivity for prediction of the pathologic status of ALNs. The data also provide evidence that the SLN hypothesis remains valid at the molecular level.

There is increasing evidence that molecular detection of micrometastatic breast cancer in the axillary lymph nodes (ALN) of breast cancer patients can improve staging. Molecular analyses of samples obtained from the Minimally Invasive Molecular Staging of Breast Cancer Trial (n = 489 patients) indicate that whereas the majority of molecular markers are informative for the detection of metastatic breast cancer (significant disease burden), only a few are sensitive for the detection of micrometastatic disease (limited disease burden). Frequency distribution and linear regression analyses reveal that relative levels of gene expression are highly correlated with apparent sensitivity for the detection of micrometastic breast cancer (P < 0.05). These data provides statistical validation of the concept that the most informative markers for detection of micrometastatic disease are those that are most highly expressed in metastatic disease. To test this hypothesis, we developed an innovative microarray strategy. RNA from a metastatic breast cancer ALN was diluted into RNA from a normal lymph node and analyzed using Affymetrix microarrays. Expression analysis indicated that only two genes [mammaglobin (mam) and trefoil factor 1 (TFF1)] were significantly overexpressed at a dilution of 1:50. Real-time reverse transcription-PCR analysis of pathology-negative ALN (n = 72) confirm that of all the markers tested, mam and TFF1 have the highest apparent sensitivity for detection of micrometastatic breast cancer. We conclude that a dilutional microarray approach is a simple and reliable method for the identification of informative molecular markers for the detection of micrometastatic cancer.

EpCAM (epithelial cell adhesion molecule) is a cell surface molecule that is known to be highly expressed in colon and other epithelial carcinomas. EpCAM is involved in cell-to-cell adhesion and has been the target of antibody therapy in several clinical trials. To assess the value of EpCAM as a novel target for breast cancer gene therapy, we performed real-time reverse transcription-PCR to quantify the level of EpCAM mRNA expression in normal breast tissue and primary and metastatic breast cancers. We found that EpCAM is overexpressed 100- to 1000-fold in primary and metastatic breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a 35–80% decrease in the rate of cell proliferation in four different breast cancer cell lines. EpCAM siRNA treatment decreased cell migration by 91.8% and cell invasion by 96.4% in the breast cancer cell line MDA-MB-231 in vitro. EpCAM siRNA treatment was also associated with an increase in the detergent-insoluble protein fraction of E-cadherin, α-catenin, and β-catenin, consistent with the known biology of EpCAM as a regulator of cell adhesion. Our hypothesis is that modulation of EpCAM expression can affect cell migration, invasion, and proliferation by enhancing E-cadherin-mediated cell-to-cell adhesion. These data provide compelling evidence that EpCAM is a potential novel target for breast cancer gene therapy and offer insights into the mechanisms associated with EpCAM gene silencing.

Prostate Specific Ets factor is a recently identified transcriptional activator that is overexpressed in prostate cancer. To determine whether this gene is overexpressed in breast cancer, we performed a virtual Northern blot using data available online at the Cancer Genome Anatomy Project website. Ninety-five SAGE libraries were probed with a unique sequence tag to the Prostate Specific Ets gene. The results indicate that Prostate Specific Ets is expressed in 14 out of 15 breast cancer libraries (93%), nine out of 10 prostate cancer libraries (90%), three out of 40 libraries from other cancers (7.5%), and four out of 30 normal tissue libraries (13%). To determine the possibility that the Prostate Specific Ets gene is a novel marker for detection of metastatic breast cancer in axillary lymph nodes, quantitative real-time RT–PCR analyses were performed. The mean level of Prostate Specific Ets expression in lymph nodes containing metastatic breast cancer (n=22) was 410-fold higher than in normal lymph node (n=51). A receiver operator characteristic curve analysis indicated that Prostate Specific Ets was overexpressed in 18 out of 22 lymph nodes containing metastatic breast cancer (82%). The receiver operator characteristic curve analysis also indicated that the diagnostic accuracy of the Prostate Specific Ets gene for detection of metastatic breast cancer in axillary lymph nodes was 0.949. These results provide evidence that Prostate Specific Ets is a potentially informative novel marker for detection of metastatic breast cancer in axillary lymph nodes, and should be included in any study that involves molecular profiling of breast cancer. British Journal of Cancer (2002) 86, 899–904. DOI: 10.1038/sj/bjc/6600190 www.bjcancer.com © 2002 Cancer Research UK

Real-time RT-PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22- to 2.8 x 10(5)-fold above normal mean expression, whereas PIP was overexpressed from 30- to 2.2 x 10(6)-fold above normal in 13 lymph nodes. Real-time RT-PCR analysis of pathology-negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.

The primary objective of cancer staging is to be able to classify patients by the extent of disease into groups with similar clinical outcomes and so facilitate patient management. In the setting of breast cancer, one of the most important prognostic indicators is the presence of axillary lymph node (ALN) metastases. Frequently, ALN disease status is the critical parameter for determining whether adjuvant systemic chemo or hormonal therapy is recommended.1–3 As a result, staging for newly diagnosed clinical stage I and II breast cancer patients has traditionally included an ipsilateral ALN dissection (ALND). Unfortunately, standard H&E histopathologic analysis of ALN has limitations. A number of studies have shown that performing additional tissue sections and/or immunohistochemical staining (IHC) of ALN increases metastases detection by up to 25%.4–6 Furthermore, these retrospective studies suggest that the prognosis for patients with occult disease is similar to patients with pathology-positive ALN.4,5,7 These findings imply that the development of more sensitive methods to detect micrometastatic disease in ALN could significantly improve breast cancer staging. The recent identification of genes overexpressed in breast cancer combined with advances in molecular biology provide such an opportunity for improving breast cancer staging.8–16 We and others have shown that the reverse transcription polymerase chain reaction (RT-PCR) is capable of detecting metastatic disease in ALN of breast cancer patients,15,17 with a sensitivity of up to one cancer cell per 107 normal cells.18–20 Ironically, the exquisite sensitivity of RT-PCR has hindered its clinical application because the majority of potential markers have some baseline expression in normal tissues.21,22 Due to the fact that conventional RT-PCR techniques are at best semiquantitative, it has been difficult to differentiate between baseline gene expression in normal tissues and increased gene expression associated with breast cancer.8,21,23–29 As a result, some investigators consider PCR technology to be problematic for clinical application with false positive and/or clinically irrelevant results a concern.8,21,23,25–27,29–31 Real-time RT-PCR solves these limitations through the use of an online fluorescence detection system that precisely quantifies the amount of PCR product. We have previously shown that real-time RT-PCR can differentiate between baseline gene expression in normal tissues and cancer-associated gene overexpression.32,33 For example, CEA, CK19, and muc1 have detectable baseline expression in normal lymph nodes, but expression levels in ALN with metastatic breast cancer is 5-fold to 3500-fold higher.32 Our data indicate that a combination of multimarker analysis and quantitative real-time RT-PCR can be a precise and powerful tool for the detection of breast cancer ALN metastases. Furthermore, the genes mam, PIP, PDEF, CK19, CEA, muc1, and mamB have particular promise for breast cancer detection.15,32,33 Although these results suggest that molecular markers could serve as valid surrogates for metastatic and micrometastatic breast cancer, their clinical relevance is unproven. To address this, the Minimally Invasive Molecular Staging of Breast Cancer (MIMS) trial was initiated. This trial represents the first prospective cohort study in which a multimarker, real-time RT-PCR analysis was applied to the detection of breast cancer micrometastases in ALN. Sentinel and/or nonsentinel ALN from 489 breast cancer subjects with T1–T3 primary tumors were analyzed by standard histopathology and multimarker, real-time RT-PCR analysis. The study was designed with sufficient statistical power to correlate molecular analyses with clinical outcome at 5 years. Although the clinical outcome data are not yet available, we show in this interim report that real-time RT-PCR is able to sensitively detect metastatic breast cancer in ALN and that overexpression of breast cancer–associated genes in subjects with pathology-negative ALN is correlated with traditional indicators of poor prognosis.

Background The rapid evolution of molecular technology and novel markers provides the opportunity to establish a more effective means to detect micrometastatic breast cancer. Given the controversies concerning application and clinical relevance, this review critically evaluates the current status of these molecular staging technologies. Data sources Breast cancer literature addressing (1) molecular detection methodologies (immunohistochemistry, reverse transcriptase polymerase chain reaction, and microarray analysis); (2) specific tissue applications such as lymph nodes, bone marrow aspirate, and peripheral blood; (3) expert commentary concerning the clinical applications and pitfalls of these technologies; and (4) recent data from our molecular diagnostics laboratory. Conclusions Molecular detection technologies such as reverse transcriptase polymerase chain reaction and microarray analyses are being developed that will likely have future application as cancer diagnostics. Further work is needed to establish assays that are validated by prospective clinical studies. Early identification of clinically relevant disease could lead to new treatment or staging approaches for breast cancer.

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.