Mammaglobin mRNA Research Articles

Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples.

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX. Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients' blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses. Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression. Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes.

<p>Detection of human mammaglobin mRNA in breast cancer cells among Vietnamese women</p>

BackgroundDetecting circulating tumor cells (CTCs) is a promising approach for evaluating the progression of occult metastases as well as the efficacy of treatment therapies among patients with breast cancer. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) technique has been proposed for detecting CTCs due to its high sensitivity. In this study, we aimed to validate the RT-PCR technique for human mammaglobin (hMAM) mRNA detection among Vietnamese women with breast cancer.Patients and methodsPeripheral blood samples and breast cancer tissues from 43 patients suffering from breast cancer and 21 patients with fibroids were obtained. Real-time RT-PCR and gene sequencing techniques were employed to detect hMAM gene in CTCs of breast cancer cell lines and cancer tissues.ResultshMAM mRNA transcription was detected in 36 out of 43 (83.7%) breast cancer tissues and in blood of 23 out of 43 (53.5%) breast cancer patients, while it was detected in only 9.5% out of tissues and 0% of the blood of fibrosis patients. hMAM mRNA in the peripheral blood of breast cancer patients increased with tumor size, stage of cancer and distant metastasis (P<0.05). No difference in the expression of hMAM mRNA was found in breast cancer tissue regarding age, distant metastasis, lymph node, stages of cancer and histopathology (P>0.05).ConclusionThe study highlighted the expression of hMAM mRNA in breast cancer cells and tissues. This reveals the overall picture of the replication of hMAM mRNA in breast cancer, suggesting the role of hMAM mRNA in breast cancer molecular pathology.

M-RNA mammaglobin expression in metastatic breast cancer patient at Medan city, Indonesia

Breast cancer is the most common causes of women’s death in the world. Metastatic spread presents a major clinical problem in about 30% of the patients. The study aims to investigate the clinical reliability of mammaglobin mRNA as a marker of circulating cancer cells in breast cancer patients. The positivity of blood was analyzed in relation to clinical and pathological characteristics. This study was on 29 breast cancer patients (13 metastatic, 16 non- metastatic patients), where28 were invasive intraductal carcinoma type and 1 was invasive lobular carcinoma type. Breast cancer patients were according to the histologic grade into grade I (7 patients),grade II (6 patients) and grade III (15 patients). All individuals included in this study were subjected to detection of mammaglobin m-RNA of circulating tumor cells in peripheral blood using RT-PCR technique. Positivity for mammaglobin in blood samples was in 38% of patients with metastatic but not in the non-metastatic patients. The presence of mammaglobin correlated with metastatic tumor (P = 0.011). Mammaglobin overexpression in breast tissue was significantly positive in low-grade tumors (I and II).

Abstract P2-01-22: The long term outcomes of Metasin RTqPCR intra-operative sentinel node analysis in early breast cancer

Abstract Background: Axillary lymph node involvement is a prognostic factor in breast cancer and it is used to guide adjuvant therapy. Axillary clearance remains the standard of care in lymph node positive disease in most parts of the world. Usually this is performed as second procedure but immediate intra-operative node analysis allows clearance to be performed as part of the initial procedure where necessary. The Metasin assay targets the breast epithelial cell markers CK19 and mammaglobin mRNA and detects the presence of breast tissue (metastatic disease) in the sentinel nodes. Evidence shows the Metasin assay to be fast (average assay time 41.2min) and accurate with a discordance rate below 4% compared with histology. The cost effectiveness of the assay has been reported in our previous studies. Aim: The aim of this study is to assess the risk of axillary recurrence following the use of the Metasin assay to guide axillary management. Method: This is a single centre retrospective study which included all patients presenting to a district general hospital with early clinically node negative breast cancer undergoing sentinel node biopsy between Oct 2011 and Dec 2014. Alternate 2 mm slices of the node were examined intraoperatively using the Metasin assay and the remainder sent for histological examination. The results of the Metasin assay and histology were compared. The risk of axillary recurrence using the Metasin test to select patients for immediate axillary clearance was assessed. Results: 1073 sentinel nodes from 545 patients were analysed during this three-year period. 2 patients were lost to follow up. 94 patients were node positive and underwent axillary clearance as part of their primary surgery. 449 patients had sentinel node biopsy with no further axillary procedure. There were 36 nodes (3.34%) with discordant results. Median follow up was 32 months (range 18 to 55 months). Fourteen patients presented with recurrences (2.56%). The mean event free interval was 15 months. Of the 14 patients, 5 patients tested with Metasin had macrometastases and underwent immediate axillary clearance during the primary surgery. 7 patients were node negative and 2 patients were shown to have micrometastases. 11 patients recurred with distant metastases, 3 patients with local recurrence and 2 patients (0.36%) with axillary recurrences. The two axillary recurrences occurred at 3 and 5 months after primary surgery. Both patients underwent Metasin intraoperative analyses of sentinel nodes which were negative. Subsequent histological examination confirmed no metastatic node involvement in one patient but micro-metastases in the other. Both of these patients had aggressive disease with local, axillary and distant metastasis and subsequently died of their disease. Conclusions: The Metasin assay is a reliable intraoperative test for sentinel node involvement and when used to guide surgical axillary management is associated with very low axillary recurrences (0.36% with a median follow up of 32 months). Citation Format: Udayasankar S, Ashley E, Jenkins KS, Huws A, Sharaiha Y, Sai-Giridhar P, Thomas D, Munir A, Holt S, Khawaja S. The long term outcomes of Metasin RTqPCR intra-operative sentinel node analysis in early breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-01-22.

Expression of Mammaglobin and Carcinoembryonic Antigen in Peripheral Blood of Patients with Breast Cancer Using Real Time Polymerase Chain Reaction

Background and Objectives: Breast cancer is among the most common causes of cancer related mortality in women worldwide. Early detection and prompt diagnosis of tumor is the first step to prevent cancer-related morbidity and mortality, and a comprehensive understanding of the involved molecular mechanisms can greatly help in this respect. Breast cancer, like many other types of cancer, is caused by a combination of genetic and epigenetic changes such as inactivation of tumor suppressor genes. Materials and Methods: This study was performed on 40 breast cancer patients and 40 healthy controls. Quantitative real time reverse transcription polymerase chain reaction (real time qRT-PCR) was used to assess the expression of carcinoembryonic antigen (CEA) and mammaglobin mRNA in the peripheral blood of patients and healthy controls. The two groups were compared using t-test. Results: The two groups were not significantly different in terms of the mean age. Twenty-nine out of 40 cancer patients were positive for CEA mRNA and its sensitivity was calculated to be 72.5%. Twelve out of 40 healthy controls were positive for CEA mRNA. Twenty-six out of 40 patients were positive for mammaglobin mRNA indicative of 65% sensitivity while only five out of 40 healthy controls were positive for mammaglobin mRNA. Conclusion: Both CEA and mammaglobin mRNA had high sensitivity in cancer patients; thus, they can be used for screening and early detection of breast cancer patients. Further studies with larger sample sizes are required to confirm the current findings.

Relationship between Human Mammaglobin mRNA in Peripheral Blood and Overall Survival in Breast Cancer Patients: A Systematic Review and Meta-Analysis

Background: Breast cancer remains a leading cause of cancer-related death in women. Numerous studies have demonstrated that mammaglobin is one of the most promising markers for diagnosis of breast cancer. However, little is known about the relationship between mammaglobin and overall survival. The small number of published studies has yielded controversial results. Objective: This study was performed to clarify the association between expression of mammaglobin in peripheral blood and prognosis in breast cancer patients. Methods: We systematically reviewed and meta-analyzed the data showing the association between expression of mammaglobin in peripheral blood and prognosis in breast cancer patients. The data were obtained from analysis of disease free survival (DFS)/event free survival (EFS) and overall survival (OS). Results: Our analysis showed that patients expressing mammaglobin in peripheral blood before the removal of the primary tumors or chemotherapy showed shorter DFS/EFS, but no change in OS. The detection of mammaglobin mRNA in peripheral blood was not associated with worse outcome for OS, but the effect size was not small. Conclusion: These data indicate that a meaningful association of mammaglobin might be found if large scale studies are performed. Large prospective studies are necessary to determine the prognostic value of the detection of mammaglobin mRNA in peripheral blood of breast cancer patients.

Comparing Efficiency of micro-RNA and mRNA Biomarker Liberation with Microbubble-Enhanced Ultrasound Exposure

Blood biomarkers are potentially powerful diagnostic tools that are limited clinically by low concentrations, the inability to determine biomarker origin and unknown patient baseline. Recently, ultrasound has been shown to liberate proteins and large mRNA biomarkers, overcoming many of these limitations. We have since demonstrated that adding lipid-stabilized microbubbles elevates mRNA concentration an order of magnitude compared with ultrasound without microbubbles, in vitro. Unfortunately the large size of some mRNA molecules may limit efficiency of release and hinder efficacy as an ultrasound-liberated biomarker. We hypothesize that smaller molecules will be released more efficiently with ultrasound than larger molecules. Although investigation of large libraries of biomarkers should be performed to fully validate this hypothesis, we focus on a small subset of mRNA and micro-RNAs. Specifically, we focus on miR-21 (22 base pairs [bp]), which is upregulated in certain forms of cancer, compared with previously investigated mammaglobin mRNA (502 bp). We also report release of micro-RNA miR-155 (22 bp) and housekeeping rRNA S18 (1869 bp). More than 10 million additional miR-21 copies per 100,000 cells are released with ultrasound-microbubble exposure. The low- molecular-weight miR-21 proved to be liberated 50 times more efficiently than high-molecular-weight mammaglobin mRNA, releasing orders of magnitude more miR-21 than mammaglobin mRNA under comparable conditions.

RNA Biomarker Release with Ultrasound and Phase-Change Nanodroplets

Microbubbles driven by ultrasound are capable of permeabilizing cell membranes and allowing biomarkers or therapeutics to exit from or enter cancer cells, respectively. Unfortunately, the relatively large size of microbubbles prevents extravasation. Lipid-based perfluorobutane microbubbles can be made seven-fold smaller by pressurization, creating 430-nm nanodroplets. The present study compares microbubbles and nanodroplets with respect to their ability to enhance miR-21 and mammaglobin mRNA release from cultured ZR-75-1 cells. Mammaglobin mRNA and miR-21 release increased with escalating concentrations of nanodroplets up to, respectively, 25- and 42-fold with 2% nanodroplets (v/v), compared with pre-ultrasound levels, whereas cell viability decreased to 62.4%. Sonication of ZR-75-1 cells incubated with microbubbles or nanodroplets caused relatively similar levels of cell death and miR-21 release, suggesting that nanodroplets are similar to microbubbles in enhancing cell permeability, but may be more advantageous because of their smaller size, which may allow extravasation through leaky tumor vasculature.

Metasin—An Intra-Operative RT-qPCR Assay to Detect Metastatic Breast Cancer in Sentinel Lymph Nodes

Nodal status is one of the most important prognostic factors in breast cancer. Established tests such as touch imprint cytology and frozen sections currently used in the intra-operative setting show variations in sensitivity and specificity. This limitation has led to the development of molecular alternatives, such as GeneSearch, a commercial intra-operative real-time quantitative Polymerase Chain Reaction (RT-qPCR) assay that allows the surgeon to carry out axillary clearance as a one-step process. Since GeneSearch has been discontinued, we have developed the replacement Metasin assay, which targets the breast epithelial cell markers CK19 and mammaglobin mRNA and identifies metastatic disease in sentinel lymph nodes. The optimised assay can be completed within 32 min (6 min for RNA preparation and 26 min instrument run time), making its use feasible in the intraoperative setting. An analysis by Metasin of 154 archived lymph node homogenates previously analysed by both parallel histology and GeneSearch showed concordance for 148 cases. The sensitivity and specificity of Metasin compared with GeneSearch were 95% (CI 83%–99%) and 97% (CI 91%–99%) respectively; compared with histology they were 95% (CI 83%–99%) and 97% (CI 91%–99%), respectively. The sensitivity and specificity of GeneSearch compared with histology were 90% (CI 77%–96%) and 97% (CI 93%–99%) respectively. The positive predictive value of Metasin was 90% and negative predictive value was 98% for both histology and GeneSearch. The positive predictive value of GeneSearch was 92% and the negative predictive value was 97% compared to histology. The discordance rates of Metasin with both GeneSearch and histology were 3.89%. In comparison, the discordance rate of GeneSearch with histology was 4.5%. Metasin’s robustness was independently evaluated on 193 samples previously analysed by GeneSearch from the Jules Bordet Institute, where Metasin yielded comparable results.

Microbubble-Enhanced Ultrasound Liberation of mRNA Biomarkers In Vitro

Blood-borne biomarkers have great potential in diagnostic medicine, but low concentrations, inability to determine their source and lack of a patient baseline have limited their success in both research and clinical medicine. D'Souza et al. previously demonstrated that ultrasound-induced sonoporation can be used to liberate protein biomarkers from a colorectal cancer into the surrounding serum, overcoming many of the limitations of blood-borne biomarkers. In this study we build on D'Souza's work, extending this technique to nucleic acids, specifically mammaglobin mRNA—a potential diagnostic biomarker for breast cancer metastases. Furthermore, we propose to use ultrasound contrast agents, lipid-stabilized microbubbles, to enhance the effects of sonoporation and further amplify the biomarker levels. We demonstrate that microbubbles can enhance mammaglobin mRNA levels by two to three orders of magnitude greater than background levels and one to two orders of magnitude greater than ultrasound alone.

Density-dependent lineage instability of MDA-MB-435 breast cancer cells

The use of cell lines in cancer research is strongly dependent on the avoidance of contaminations, the correct attribution of a cell line to the initial primary tumor and stability. Previous studies have identified expression of melanocytic molecular markers in the widely used breast cancer cell line, MDA-MB-435. In the present study the three breast cancer cell lines, MCF-7, MDA-MB-231 and MDA-MB-435, were systematically analyzed for mRNA and protein expression of major epithelial (cytokeratin isoforms), mammary (mammaglobin) and melanocytic (melan A and S100-protein) markers. Protein expression was identified by immunocytochemistry and quantitative RT-PCR was used to determine mRNA levels. While MCF-7 and MDA-MB-231 cells unambiguously revealed an epithelial/mammary phenotype, MDA-MB-435 cells were found to exhibit epithelial/mammary and melanocytic features dependent on cell density. Subconfluent cells demonstrated epithelial characteristics only, however, densely growing, confluent cells also expressed melanocytic markers. Consistent with gain of melanocytic features, the expression levels of mammaglobin mRNA decreased in these cells. These results indicate that the three cell lines are primarily of epithelial phenotype, however, MDA-MB-435 cells revealed lineage infidelity in dense cultures with a gain in melanocytic phenotype. These characteristics must be taken into consideration when analyzing cancer-relevant genes and their expression profiles in vitro.

P1.18 Mammaglobin Expression as a Marker for the Early Breast Cancer

ABSTRACT There are no any biological makers for the early breast cancer diagnostics. CEA, CA27.29 and CA15.3 proposed for clinical usage lack appropriate specificity and sensitivity in the early breast cancer. One of the possible candidates for this role is the mammaglobin, marker with maximal expression level in the early breast cancer. Materials and methods We studied correlation of mRNA mammaglobin expression measured by qPCR with tumor morphological characteristics in BC patients; also we studied specificity and sensitivity of mRNA mammaglobin determination in peripheral blood by nested PCR as a marker of the early breast cancer. 210 breast tissue samples (66 samples of BC, 40 of fibroademoma (FA) and 99 samples of surrounding morphologically normal tissue (MNT)) were analized. For blood mammaglobin expression analysis we studied 169 blood samples (95 from women with BC, 22 with FA, 24 healthy controls and 28 women with other tumor localization). Results mammaglobin mRNA expression was considerably higher in BC samples as compared to MNT and FA (p = 0,0054). Besides highest mammaglobin level has been found in T1, grade 1, stage 1 breast cancer (p = 0.0375) and it decreases with disease progression. There was positive correlation between mammaglobin expression, estrogen receptor status (p = 0,034) and c-erbB2 amplification (p = 0,024) in tissue samples. Sensitivity of mammaglobin determination in peripheral blood was 60,6% (40 from 66 BC patients), specificity was 92,3% (false-positive results in 10 of 40 patients with FA). There was no mammaglobin expression in healthy controls and patients with other tumors. Conclusion mRNA mammaglobin expression is significantly increased in the early BC and is progradiently decreased during cancer progression. High sensitivity and specificity of mammaglobin determination in peripheral blood in the early breast cancer patients allows to use this method as a breast cancer screening test.

Clinical Relevance of Human Mammaglobin mRNA in Pleural Effusion from Patients Undergoing Thoracoscopy: A Pilot Study

human mammaglobin (hMAM) expression has been reported in pleural effusions (PE). The aim of this study was to assess the clinical relevance of hMAM mRNA in PE from patients who underwent thoracoscopy. A total of 288 patients with PE were studied, 155 of which were diagnosed with malignant and 133 with non-malignant diseases by thoracoscopy. Cells from PE were analyzed by nested hMAM RT-PCR. Statistical analyses were performed to evaluate the diagnostic performance parameters (DPP), the association between hMAM expression and benign or malignant status and the relative risk of cancer for patients with negative thoracoscopy showing hMAM positivity. hMAM mRNA was found in 68/288 (23.6%) PE samples of which 51 were from the 155 patients diagnosed with malignant diseases and 17 were from the 133 patients diagnosed with non-malignant diseases. A significant correlation between hMAM expression and malignancy was found (OR=3.04) and the DPP were as follows: sensitivity=32.9%, specificity=87.2%, accuracy=58.0%, positive predictive value=75.0% and negative predictive value=52.7%. Among the patients with negative thoracoscopy (n=133), 5/17 (29.4%) hMAM-positive patients had or developed a tumor during the 18-month follow up period, as compared to 10/116 (8.6%) hMAM-negative patients (relative risk of 4.6 for developing a malignancy). These findings suggest a possible application of hMAM RT-PCR detection in PE as to identify a false-negative thoracoscopy in non-specific pleuritis.

Plasma human mammaglobin mRNA associated with poor outcome in patients with breast cancer

Different treatment outcomes and prognoses in patients with breast cancer can be observed with similar clinical predictors; this is because the biology of breast cancer is complex and heterogenous, involving multiple unknown contributing factors. We looked for plasma human mammaglobin (hMAM) mRNA by RT-PCR in 82 Korean patients with breast cancer to determine if there is an association between the presence of plasma hMAM mRNA in these patients and known prognostic factors. The prognostic usefulness of detection of plasma hMAM mRNA expression in these patients was also evaluated by determining overall survival and event-free survival. A significant difference was observed in the rate of positivity of plasma hMAM mRNA between the early stages of cancer (stages I-II, 23.4%) and advanced stages (stages III-IV, 82.9%). The expression rates of estrogen receptor, progesterone receptor, and HER-2/neu in the breast tissue of these patients, by immunohistochemistry, were 69.5, 75.6, and 20.7%, respectively. In the univariate analysis, plasma hMAM expression was significantly correlated with high histological and nuclear grades, nodal metastasis, and negative estrogen receptor and progesterone receptor status. Patients negative for plasma hMAM mRNA had significantly higher rates of event-free survival compared to the patients positive for plasma hMAM mRNA. However, no significant association with overall survival was observed for expression of plasma hMAM mRNA (P = 0.16). Qualitative detection of plasma hMAM mRNA appears to be associated with unfavorable prognostic factors and lower rates of event-free survival in patients with breast cancer.

Expression of human mammaglobin as a marker of bone marrow micrometastasis in breast cancer

The aim of the present study was to detect the expression of human mammaglobin (hMAM) mRNA in the bone marrow (BM) of patients with breast cancer and determine the relationship between micrometastasis and clinicopathological parameters as well as selected molecular markers and breast cancer prognosis. The expression of hMAM mRNA in the BM of patients with breast cancer was determined by RT-PCR. The expression of ER, PR and Cath-D in cancer tissues was detected by immunohistochemistry. A positive expression rate for hMAM of 38.2% in 102 patients with stage I-III breast cancer was found. The expression of hMAM was higher in patients with T(2-3) (>2 cm) tumors than in those with T(1) tumors (≤2 cm) (χ(2)=19.20, P=0.001) and in patients with stage II or III tumors than in patients with stage I tumors (χ(2)=15.101, P=0.001). The expression of hMAM in the BM of breast cancer patients categorized as grade 1 was lower than that in those of grade 2 or 3 (χ(2)=8.522, P=0.014), and hMAM expression was related to the pathological type of tumor (χ(2)=6.892, P=0.032) and the degree of axillary lymph node metastasis (χ(2)=14.050, P=0.001). The expression of hMAM in BM was much higher in patients with ER(-) or ER(+) tumors than in those with ER(++ or +++) (χ(2)=11.800, P=0.003), and those with PR (χ(2)=8.759, P=0.013). hMAM expression in BM was also significantly positively correlated with Cath-D expression (χ(2)=6.623, P=0.036). However, no correlation was found between hMAM expression and patient age (χ(2)=1.056, P=0.304). There was a strong correlation between patients with positive expression of hMAM in the BM and the presence of distant metastases (P=0.009). In conclusion, micrometastasis in the BM correlates with certain clinical pathological parameters and several tumor markers. Patients with positive expression of hMAM in the BM have a greater chance of distant metastasis and poor prognosis. The detection of micrometastasis may be one of the most advantageous markers for predicting the prognosis of breast cancer.

Cytokeratin-19 and Mammaglobin Gene Expression in Circulating Tumor Cells from Metastatic Breast Cancer Patients Enrolled in North Central Cancer Treatment Group Trials, N0234/336/436/437

To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β(2)-microglobulin and calibrated to healthy blood using the 2(-ΔΔCq) algorithm; positivity was defined as 2 or more. CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients.

Human Mammaglobin: A Superior Marker for Reverse-Transcriptase PCR in Detecting Circulating Tumor cells In Breast Cancer Patients

Breast cancer is the most frequent cancer in women in the USA and the second most common cause of death in females who develop cancer. Recently, the detection of circulating tumor cells has emerged as a promising tool for monitoring the progression of clinically occult micrometastases in breast cancer patients. Sensitive molecular techniques, primarily based upon the reverse-transcriptase PCR, using various molecules as markers, have been developed to detect circulating tumor cells. Among those molecules, human mammaglobin mRNA has been found to be the most specific marker for the hematogenous spread of breast cancer cells. In this article, we review the current knowledge regarding the use of reverse-transcriptase PCR for detecting human mammaglobin mRNA as a biomarker for circulating tumor cells in breast cancer patients, and evaluate the clinical implications of human mammaglobin since it was first isolated in 1996.

Mammaglobin RT-qPCR for breast cancer metastasis detection in sentinel lymph nodes

(Cancer Sci 2010; 101: 2501) Dear Editor, We have read with great interest a recent paper in Cancer Science by Liu et al.(1) They report on the use of the GeneSearch Breast Lymph Node (BLN) Assay in detecting sentinel lymph node metastases in a substantial cohort of breast cancer patients. This RT-qPCR-based assay detects cytokeratin 19 (an epithelial cell-specific marker) and mammaglobin (a breast cell-specific marker) transcripts. Their results show that the BLN assay is specific and more sensitive than intraoperative imprint cytology. We would like to point out that mammaglobin is not uniformly expressed by all breast cells. In fact, we found that differences in mammaglobin expression can amount to 10 000-fold between different subtypes of breast cancer,(2) and that low-grade, estrogen receptor (ER)-positive tumors express the highest amount of mammaglobin. With the premise that circulating tumor cells retain the characteristics of the primary tumor they stem from, our earlier results suggest that cells from low-grade, ER–positive breast tumors are more likely to be detected in the circulation as these have the highest number of mammaglobin mRNA molecules per cell. In contrast, ER-negative and high-grade tumors are more likely to escape detection as they express lower numbers of mammaglobin mRNA molecules per cell. The enormous variation in mammaglobin levels, coupled with its association with certain tumor characteristics, will lead to a divergence in detection probability for particular tumor types. Indeed, Liu et al.(1) describe that only 19 of the 29 BLN-positive samples were positive for mammaglobin. This is 66% of patients, exactly the proportion of patients that is ER positive in this cohort, and close to the 68% of patients that have tumors with good or mild differentiation according to Table 1 from Liu et al.(1) We would be most interested in learning whether the mammaglobin-positive samples are indeed comprised of mainly low-grade, ER-positive patients. This would be in line with our earlier concerns about using mammaglobin PCR to detect breast cancer circulating cells,(3, 4) and should be kept in mind when clinically evaluating the BLN assay results.

Efficacy of intraoperative GeneSearch Breast Lymph Node (BLN) Assay for breast cancer metastasis detection in sentinel lymph node in Chinese patients

Although intraoperative assessment of the sentinel lymph node (SLN) is useful, it has not gained popularity in China as it involves a heavy workload for pathologists. We conducted a prospective clinical feasibility study of the GeneSearch Breast Lymph Node (BLN) Assay performed in 158 SLNs from 97 patients by comparison with postoperative permanent section histopathology, to validate its potential usefulness in China. Every SLN was cut into alternating 1.5 to 3.0 mm slabs. The BLN assay processed 50% of the fresh alternating slabs to detect the presence of cytokeratin 19 and mammaglobin mRNA. Assay results were compared with those for permanent section histopathology and intraoperative imprint cytology. Slides for imprint cytology were prepared from the BLN assay node tissue before it was processed. Full axillary lymph node (ALN) dissections were performed on some patients after a SLN biopsy. The BLN assay was successfully performed on 158 SLNs from 97 patients. Overall performance of the BLN assay compared with permanent section histopathology was sensitivity 83.9% (26/31), specificity 95.5% (63/66), positive predictive value 89.7% (26/29), negative predictive value 92.6% (63/68), and overall agreement 91.8% (89/97). The BLN assay detected about 25% more metastases than imprint cytology. Moreover, the BLN assay correctly identified most of the additional non-sentinel ALNs metastases (P = 0.005). Our results from a large series of Chinese patients with breast cancer indicate that the BLN assay may be a viable alternative for the standard intraoperative procedures used for metastases detection, especially in early stage breast cancer patients. Name of the trial register: GeneSearch Breast Lymph Node (BLN) Assay China Registration Study. NCT00869674.

Abstract 1760: Mitotic activity index and bone marrow micrometastsis detection is a strong prognostic combination in lymph node positive and negative breast cancer

Abstract PURPOSE: To investigate the prognostic value of Mitotic Activity Index (MAI) in lymph node negative and lymph node positive operable breast cancer patients in an unselected population and to compare it to bone marrow (BM) micrometastatis status. PATIENTS AND METHODS: Analysis of MAI assessed using a strict protocol with proven strong prognostic value (Baak JP et al. in: JCO 2005, Ann Oncol 2008 and Breast Cancer Res Treat 2009) was compared to classic prognosticators and BM micrometastases detected by a real-time RT-PCR multimarker assay including mammaglobin, cytokeratin 19 and TWIST1 mRNA in 179 consecutive early breast cancer patients (median follow-up: 96 months; range: 0 to 126 months). RESULTS: Systemic relapse occurred in 31 patients (17.3%) and 26 (14.5%) of these patients died of breast cancer-related causes. MAI (&amp;lt;10, &amp;gt;=10) was strongly associated with recurrence both in lymph-node (LN)-negative (HR 3.4, Confidence Interval (CI) 1.11-10.66) but less so in LN-positive patients (HR: 2.2, CI 0.87-5.62). BM-status was highly significant in LN-positive patients (HR: 3.7, CI 1.5-9.22) but not in LN-negative patients (HR: 2.4, CI 0.65-8.81). With multivariate Cox regression, BM micrometasis status and MAI were the strongest variables in LN-positive patients for both recurrence-free survival (HR 3.7, CI 1.5 −9.2 and HR 2.2, CI 0.9-5.7 respectively) and breast cancer specific survival (HR 3.3, CI 1.25-8.49 and HR 3.1, CI 1.1-8.9, respectively). In the LN-negative population ER was the strongest prognosticator (HR 0.24, CI 0.08-0.8) for systemic relapse, whereas MAI was the only significant variable in breast cancer specific survival (HR 7.0, CI 1.7-27.9). A multimarker panel including both BM-status and MAI was explored and was found to be the most significant prognostic marker in both LN-positive (HR 4.4, CI 1.3-15.3 / HR 6.0, CI 1.4 to 26.4) and LN-negative (HR 4.1, CI 1.0-16.5 / HR 7.7, CI 1.2-50) for both systemic recurrence free survival and breast cancer specific survival, respectively, in multivariate Cox regression. CONCLUSION: MAI and BM micrometastatic status are independent prognostic factors and supplementary to each other in identifying patient groups with poor prognosis. Patients with high mitotic activity tend to relapse early, whereas BM micrometastatic disease predicts a constant risk over time. The combination of the proliferation marker MAI and assessment of BM micrometastasis is the strongest prognostic marker in both the LN-positive and LN-negative subgroups of 179 early breast cancer patients. This allows better identification of patients at need for more aggressive treatment and warrants validation in a larger trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1760.