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Clinical Cancer Research Vol. 11, 6528-6535, September 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Identification of Mammaglobin as a Novel Serum Marker for Breast Cancer

Jonine L. Bernstein1, James H. Godbold1, George Raptis2, Mark A. Watson4, Brooke Levinson1, Stuart A. Aaronson3 and Timothy P. Fleming5

Authors' Affiliations: Departments of 1 Community and Preventive Medicine, 2 Medicine, and 3 Oncological Sciences, Mount Sinai School of Medicine, New York, New York and Departments of 4 Pathology and Immunology and 5 Surgery, Washington University School of Medicine, St. Louis, Missouri

Requests for reprints: Jonine L. Bernstein, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, 307 E. 63rd Street, 3rd Floor, New York, NY 10021. Phone: 646-735-8155; Fax: 646-735-0012; E-mail: bernstej{at}mskcc.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Early detection of breast cancer has implications for the management and treatment of patients with this disease. Currently, there exist no highly sensitive and specific serologic biomarkers for detection of breast cancer. Mammaglobin is predicted to be a secreted protein, and expression of this gene seems to be highly specific in breast cancer. The present studies were undertaken to develop the mammaglobin protein as a serum biomarker for detection of breast cancer.

Experimental Design: We characterized the mammaglobin protein as a secreted, 14- to 21-kDa species, which is likely post-translationally processed based on its predicted 7-kDa size. Immunostaining for mammaglobin was conducted. An ELISA was developed for the detection of the mammaglobin protein in serum, and levels were compared between women with and without breast cancer. A receiver operating characteristic curve was used to show sensitivity and specificity for cut points on the continuous mammaglobin scale.

Results: The protein was detectable by immunostaining in 72% of breast tumors and not in other tumor types. The ELISA was highly sensitive and specific for detection of mammaglobin protein in tissue culture fluids of breast cancer cells and sera of breast cancer patients. The ELISA differentiated healthy women from those with breast cancer with accurate, repeatable results across time and under varying storage conditions.

Conclusion: Our results indicate that mammaglobin, as measured by the ELISA, holds significant promise for breast cancer screening with the realistic potential to impact management of this disease.

Currently, there exist no sensitive and specific surrogate serologic biomarkers that are reliable enough to detect breast cancer early or to monitor minimal disease recurrence. For example, elevated carcinoembryonic antigen (CEA) and CA27-29 are commonly used in clinical practice as serum breast cancer markers and as predictors of high risk of recurrence; however, these markers are elevated in <50% of patients with metastatic disease and rarely at a significant interval before a clinically observable recurrence (1). An ideal breast tumor serologic marker would serve a diagnostic as well as a prognostic function; it would be used as a screening tool for early detection of the disease among general or high-risk populations and would correlate with tumor burden, recurrence, and overall response to treatment. The presence of such a marker in serum would allow for the development of noninvasive serologic tests to diagnose and monitor the progression of disease among breast cancer patients.

The mammaglobin gene was initially identified using the differential display PCR technique as a gene transcriptionally up-regulated in breast carcinoma (2, 3). Subsequent studies have indicated that mammaglobin expression is specific for breast tissues compared with other tissues, leading to its application in the detection of lymph node metastasis of breast tumors (4–6). The 93–amino acid polypeptide encoded by the mammaglobin gene predicts a secreted protein with a classic hydrophobic leader sequence (2). Moreover, the predicted protein is homologous to a family of proteins that includes rat steroid-binding protein (prostatein) subunit C3 (rPSC3), rabbit uteroglobin (rUg), The Clara cell 10-kDa protein, human mammaglobin B protein (also designated as lacryglobin or lipophilin C), and lipophilin B (also known as BU101) and cat major allergan. Uteroglobin family members form homodimers and heterodimers, and mammaglobin heterodimerizes with lipophilin B in an antiparallel manner that allows for formation of three disulfide bridges between the two molecules (7, 8). These genes are localized and clustered at 11q13 and this family of proteins has recently been designated secretoglobins to emphasize their secreted nature and globin-like structural features (9). The present studies were initiated to identify the secreted mammaglobin protein, to compare its expression in breast and other tumor types using immunostaining, and to establish a reliable ELISA for the detection and analysis of mammaglobin in the sera of breast cancer patients.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mammaglobin antibodies
Rabbit anti-Mamm is a polyclonal antiserum generated against a 16-residue peptide (EVFMQLIYDSSLCDLF) corresponding to the COOH terminus of mammaglobin (3). Rabbit monoclonal antibodies to native mammaglobin were provided by the Corixa Corp. (Seattle, WA) and were produced as recombinant proteins in Chinese hamster ovary cells.

Immunostaining of tumor tissues
Mammaglobin immunostaining was done using published methods (3). Briefly, tissue sections were deparaffinized and rehydrated in graded solutions of ethanol and distilled water. Tissue sections were preincubated with normal goat serum (Vector Laboratories, Burlingame, CA) at a 1:100 dilution in 3% bovine serum albumin/PBS and then with rabbit anti-Mamm at a 1:1,000 dilution for 1 hour at room temperature. After several rinses in PBS, sections were incubated in a solution of normal goat serum (1:1,000), 3% bovine serum albumin, and 6 µg/mL biotinylated goat anti-rabbit IgG (Vector Laboratories) in PBS for 1 hour. The secondary antibody solution was rinsed four times in PBS, and tissues were then incubated with a 1:1,000 dilution of streptavidin peroxidase (Boehringer Mannheim, Indianapolis, IN) also in a solution of 3% bovine serum albumin/PBS. After a 30-minute incubation, slides were again rinsed four times in PBS and exposed to chromagen solution containing 1 mg/mL 3,3'-diaminobenzidine tetrahydrochloride (DAKO, Carpinteria, CA) and 0.02% hydrogen peroxide for 3 minutes. Slides were rinsed briefly in deionized water, counterstained with Harris' hematoxylin, and mounted under coverslips. Immunopositivity was scored as follows: 0, no staining; 1, weak and sporadic staining in <50% of tumor cells; 2, weak staining in >50% of tumor cells; 3, strong diffuse cytoplasmic staining in <50% of tumor cells; and 4, strong, diffuse cytoplasmic staining in >50% of tumor cells. Only sections scoring 3 or 4 were considered to be mammaglobin positive. All slides were evaluated blindly.

Immunoblot analyses
Cells were lysed [60 mmol/L Tris (pH 6.8), 2% SDS, 100 mmol/L DTT] and centrifuged for 10 minutes at 10,000 x g to remove debris. onditioned medium (~20 µL) centrifuged for 10 minutes at 1,000 x g to remove cellular debris or cell lysate (25 µg) was loaded onto a 5% to 15% polyacrylamide gradient gel. Immunoblot analysis was done with rabbit anti-Mamm at a 1:1,000 dilution. To assess specificity of the detected bands, the same antibody was preincubated for 1 hour at room temperature with 30 µg of the corresponding peptide before immunoblot analysis.

Mammaglobin immunodetection by ELISA
A two-antibody sandwich technique was used for detection of mammaglobin. The first monoclonal antibody was purified and bound to a solid phase (using a 96-well plate). Native mammaglobin was allowed to bind, and unbound proteins were removed by washing. Next, a biotin-conjugated rabbit monoclonal antibody directed against a different mammaglobin epitope was allowed to bind to the captured mammaglobin protein. After washing, the assay was quantified by measuring the amount of labeled secondary antibody bound using avidin-peroxidase reagent. The sensitivity limit of the assay was ~20 pg/mL of purified native mammaglobin, and detection was linear over a range of 30 pg to 25 ng of the native mammaglobin (see Results).

Ascertainment of human biospecimens
Tumor tissue. The human tumor tissues used in this study were obtained from the Washington University Siteman Cancer Center and the Department of Surgery (St. Louis, MO); The Cooperative Human Tissue network (www.chtn.ims.nci.nih.gov), and Vanderbilt School of Medicine (Nashville, TN). All samples were analyzed without knowledge of age, sex, race, or tumor stage.

Sera from women with and without breast cancer. A hospital-based case series of women with and without breast cancer were included in these studies. All women were recruited through either the Radiology Associates Practice (56 women without breast cancer) or through the integrated Medical Oncology Clinic and Faculty Practice of the Mount Sinai Medical Center in New York City (26 women with metastatic breast cancer). Before participation, all women signed an institutional review board–approved consent form indicating their willingness to participate.

Sera from women without evidence of breast cancer. Women with negative screening mammograms who were between ages 35 and 70 years with no previous history of cancer, benign breast disease, breast biopsies or surgeries, or other severe or debilitating medical illness and who were not currently pregnant using oral contraceptives or using hormone replacement therapies were recruited into the study. All women completed an in-person interview and provided a baseline blood sample. The follow-up protocol varied accordingly: (a) 28 premenopausal women completed a total of five blood draws over 3 months, one weekly for 4 weeks and a final draw 2 months later and (b) 28 postmenopausal women completed a total of three blood draws over the 3 months, the first at the initial visit, the second 2 weeks later, and the last was at month 3.

Sera from breast cancer patients. Patients with histologically confirmed measurable or evaluable breast cancer who were receiving hormonal therapy, chemotherapy, and/or immunotherapy for their disease were recruited into the study. Exclusion criteria included pregnancy, use of oral contraceptives or hormone replacement therapy, history of prior malignancy, or concomitant uncontrolled medical illness. Information from a full comprehensive cancer history and physical exam as well as details regarding each patient's breast cancer history was obtained from a patient interview and a review of the medical chart. A blood sample was collected at time of the interview.

Statistical analyses of serum mammaglobin levels by ELISA
Data for statistical analysis for determining normal mammaglobin levels consisted of three replications collected at each of five time points for the premenopausal women and at each of three time points for the postmenopausal women. To examine the effect of the week of the menstrual cycle on mammaglobin levels among premenopausal women, we used the self-reported cycle length information to calibrate the difference between the date of the blood draw and the date of the beginning of the last menstrual cycle. To determine whether mammaglobin levels were stable over the course of the study period and across the menstrual cycle, a mixed model (implemented by PROC MIXED in SAS) was used. Because the data were positively skewed, each value of the replications was log transformed to satisfy the normality assumption of the mixed model analysis. In this model, subjects were regarded as random effects, and time was considered as a fixed effect.

Descriptive univariate statistics were calculated, and nonparametric Mann-Whitney tests were used to evaluate differences between the means of the mean log-transformed mammaglobin levels. For analyses across time periods, at each time point, we used the mean of the mean log mammaglobin levels of replicate values to determine the time point–specific mammaglobin level. For analyses that combined mammaglobin levels across time points, we calculated the mean of these time point–specific mammaglobin levels (i.e., the mean log mammaglobin levels of the replicate measures called "mammaglobin levels"). Among the metastatic breast cancer cases, Spearman rank correlation coefficients (10) were used to calculate the association of the mean log mammaglobin levels with the log of the CEA and CA27-29 values.

To operationalize a screening test based on a continuous variable, a cut point must be used to categorize individuals as either "not elevated" or "elevated," and obviously, the sensitivity and specificity of the test will be a function of the cut point. The receiver operating characteristic (ROC) curve is a graphical device, plotting the true-positive fraction (sensitivity) versus the false-positive fraction (1 – specificity; ref. 11). This approach shows the tradeoffs in sensitivity and specificity for a range of cut points on the underlying continuous scale (11). An ideal test would generate a ROC curve that increases sharply along the vertical axis to ~100% and then be virtually horizontal, resulting in an area under the curve (AUC) of ~1.0. In contrast, the ROC curve for a test that has no benefit in detecting disease would be a diagonal line from the bottom left corner to the top right corner of the graph, with an AUC of 50% (12). The AUC, in the context of mammaglobin as a potential screening test for breast cancer, represents the probability that, given two women, one with breast cancer and one without breast cancer, the mammaglobin level will be higher in the woman with breast cancer than in the woman without breast cancer. A test with no predictive ability would be expected to produce a curve where the AUC is 50%. ROC curves with AUC >50% indicate potentially useful tests, with greater utility being associated with greater area (12).

In this study, a ROC curve was fitted to the observed data, and the AUC was calculated (11). Maximum likelihood estimates of the ROC variables were obtained using the algorithms LABROC4 and LABROC5 developed by Metz et al.6 (13). These algorithms are implemented in the ROCKIT 0.9 B free software distributed by Metz et al. The area under the fitted ROC curve, along with its 95% confidence limits, was calculated based on the maximum likelihood estimates; the area was independently calculated from the empirical data using the Wilcoxon statistic (14). The empirical curve and the fitted ROC curve were plotted using CHART options in EXCEL.

Results with a P < 0.05 were deemed statistically significant. SAS version 8.2 was used for all statistical analyses.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Immunodetection of mammaglobin as a secreted protein. To characterize mammaglobin protein expression, we used rabbit anti-Mamm. Immunoblot analysis of tissue culture fluids from a human breast cancer cell line, MDA-MB-415, which expresses high levels of the mammaglobin transcript (2), revealed a major 21-kDa protein (Fig. 1A). Cell lysates contained mammaglobin immunoreactive species, including a major 14-kDa species, as well as several higher molecular weight proteins. These results suggest extensive intracellular post-translational processing of the mammaglobin protein, which has a predicted 7-kDa coding sequence. Mammaglobin is one of the rare members of the secretoglobin gene family that is glycosylated, having two consensus N-glycosylation sites. Work in our laboratory has shown that N-linked glycosylation is responsible for the observed 21-kDa band when mammaglobin is detected by Western blot under reducing conditions. As a result, immunodetection of the 14- to 21-kDa proteins in cell lysates and conditioned medium, respectively, was competed with the mammaglobin peptide used for immunization, further indicating the mammaglobin-specific nature of these species. Specifically, the high molecular weight protein (~45 kDa) is generally not observed during Western blotting when using the COOH-terminal peptide antibody described in Materials and Methods. When it is observed, it may represent incompletely reduced native mammaglobin, the mammaglobin/lipophilin B heterodimer. This antibody does detect the nonreduced native mammaglobin complex, albeit poorly, and this native mammaglobin protein complex would be blocked by the specific peptide used to generate the polyclonal antibody. In contrast, proteins of higher molecular weight were not efficiently competed, indicating that they likely represented nonspecific bands. Other breast tumor cell lines and breast tumor tissue lysates analyzed by this approach also revealed similar-sized species (data not shown), further confirming the mammaglobin-specific nature of the 14- to 21-kDa proteins.



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  		Fig. 1. Immunodetection of the mammaglobin protein in human breast cancer cells and tissues. A, conditioned medium (S) and cell lysate (C) from mammaglobin transcript–positive MDA-MB-415 breast tumor cells were resolved on a 5% to 15% polyacrylamide gel. Immunoblot analysis was done with rabbit antiserum directed against the COOH terminus of mammaglobin in the absence (left) or presence (right) of the peptide used for immunization as described in Materials and Methods. B, immunostaining with the same rabbit antiserum to mammaglobin of formalin-fixed normal breast tissue (a) and high-grade invasive breast carcinomas with minimal (b) and high (c) levels of mammaglobin expression as described in detail in Materials and Methods (x10).

 
Mammaglobin protein is expressed at high frequency and specifically detected in breast cancers. Prior studies using mammaglobin as a biomarker relied on the expression of mammaglobin transcript in breast cancer cells, with little or no focus on the detection of the mammaglobin protein. Thus, we surveyed primary breast cancers for expression of mammaglobin by immunostaining with the rabbit anti-Mamm and found a high frequency of immunoreactivity for mammaglobin (Table 1). Specificity of the reactivity was shown by competition with the immunizing peptide. Immunohistochemical analysis of mammaglobin staining in primary breast cancer shows that ~80% of all breast cancers, regardless of breast cancer type or stage, overexpress the mammaglobin protein complex when compared with normal breast tissue. The cellular staining pattern of mammaglobin was predominantly diffuse and cytoplasmic (Fig. 1B). When we examined mammaglobin immunoreactivity in normal breast tissue, it was generally more localized and focal than in the tumor samples with positive staining in only 10% of the slides. Table 1 summarizes the results of immunostaining a panel of other cancers, including prostate, lung, ovary, and colon. With the exception of uterine cancers, in which 1 of 31 (3%) stained positive, mammaglobin was expressed only in breast cancers.


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  		Table 1. Breast and nonbreast tumors staining positive for mammaglobin expression using immunohistochemistry

 
Development of an ELISA for mammaglobin. To determine whether mammaglobin could be identified in sera, an ELISA was formatted and evaluated for its ability to detect native mammaglobin. As shown in Fig. 2, the assay quantified mammaglobin levels as low as 30 pg/mL and was linear up to 25 ng/mL (r2 = 0.81). The specificity of the ELISA was further shown by analysis of culture supernatants from mammaglobin-positive breast cancer cell lines, which, when immunoprecipitated with anti-Mamm antibodies, were no longer detectably immunoreactive for mammaglobin (data not shown). We also examined whether normal human serum contained no mammaglobin-binding proteins that could affect the ability to detect mammaglobin by the ELISA. Because the standard curve was done using purified native mammaglobin, we tested the native mammaglobin alone or "spiked" into normal sera (at levels identical to those used to construct the standard curve), and compared the results using the ELISA assay. Following overnight incubation, the sensitivity of mammaglobin detection by the ELISA was not different for the spiked serum compared with mammaglobin diluted in PBS (Fig. 2). These results indicate that human serum does not contain proteins that might mask or interfere with mammaglobin detection by the ELISA.



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  		Fig. 2. Immunodetection of native mammaglobin by an ELISA. Native mammaglobin purified from culture supernatants of MDA-MB-415 breast carcinoma cells was diluted either in PBS or in normal human serum with undetectable mammaglobin and evaluated by ELISA as described in detail in Materials and Methods. Points, mean of triplicate wells.

 
To determine whether different methods of storage of human sera affected the reliability of the assay, we monitored the stability of mammaglobin in serum after multiple freeze/thaw cycles following long-term storage. First, we subjected mammaglobin-positive sera to five overnight freeze/thaw cycles in which sera were frozen at –20°C and thawed at 37°C. We observed no statistically significant change in mammaglobin immunoreactivity as measured by the ELISA under these conditions. Tested sera were also analyzed following a year-long storage period at –20°C using standard curves generated with the same purified mammaglobin preparation. We found no detectable change in mammaglobin immunoreactivity (data not shown). Together, these results indicate that the mammaglobin ELISA accurately quantitates mammaglobin levels in stored frozen serum samples.

Serum mammaglobin levels among women with and without breast cancer. Using the mammaglobin ELISA, we analyzed mammaglobin levels in the sera of disease-free women and breast cancer patients. First, we determined "normal" values of mammaglobin among women with no evidence of breast cancer, including women of different ages, race, and menopausal status. CEA and CA27-29 levels were negative among these women. To determine normal intraperson variability over time, we measured repeated serum mammaglobin levels in the same women at different times. We found no statistically significant differences between any of the measurements, either within menopausal status group or across the time period. The levels were stable across all visits; there were no statistically significant differences among the means at the various time points or replications. Figure 3 presents the mean mammaglobin levels across the study period among the premenopausal women. The data for the postmenopausal women were very similar and are therefore not shown.



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  		Fig. 3. Mean log mammaglobin levels measured at each week of the study among premenopausal women with no evidence of breast cancer. Mammaglobin levels were calculated using the mean of the mean log levels of three replicate measures.

 
Table 2 presents mammaglobin levels by characteristics of the 56 women with no evidence of disease. Women with bra cup size of D or greater had a significantly higher mammaglobin level than women with smaller cup sizes. The difference between the mammaglobin level for the 28 premenopausal women (7.90 ± 1.0) and the 28 postmenopausal women (7.52 ± 0.72) approached statistical significance (P = 0.08). There were no other statistically significant differences between subgroups of women defined by the variables of smoking, race, and age, and ever having been pregnant.


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  		Table 2. General characteristics and mammaglobin levels (log scale) among women with no evidence of breast cancer

 
Characteristics of the 26 women with metastatic breast cancer and their mammaglobin levels are shown in Table 3. The Spearman rank correlation coefficients for the association of mammaglobin levels with the log of the baseline CEA and CA27-29 were statistically significant (CEA: rS = 0.56, P = 0.0035; CA27-29: rS = 0.66, P = 0.0003). There were no statistically significant differences in mammaglobin levels associated with age, race, hormone therapy, estrogen receptor/progesterone receptor status, or HER-2/neu in this group of women. The distributions of the mammaglobin levels in the women without breast cancer and in the women with breast cancer are shown in Fig. 4A. Although there was some overlap between the groups, the levels were substantially lower for the majority of the women without breast cancer and higher among the women with breast cancer (P < 0.0001).


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  		Table 3. General characteristics and mammaglobin levels (log scale) among patients with metastatic breast cancer

 


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  		Fig. 4. Ability of the mammaglobin ELISA to discriminate mammaglobin levels among women with and without breast cancer. A, histogram comparing mammaglobin levels (on a log scale) among women with no evidence of breast cancer with those obtained from women with known metastatic disease. B, empirical and fitted ROC curves for mammaglobin as described in Materials and Methods.

 
Sensitivity and specificity of the mammaglobin ELISA for breast cancer detection. Figure 4B shows the empirical curve generated by the mammaglobin data along with the ROC curve fit to these data using the method of Metz et al. (13). This method makes minimal assumptions about the underlying data, viz., only the assumption that the input data follow normal distributions after some unknown monotonic transformation. Each point on the fitted curve is associated with a unique value of mammaglobin used as a cut point for dichotomizing the continuous variable of mammaglobin as either "elevated" or "nonelevated." Furthermore, each point on the fitted curve corresponds to a unique pair of values for sensitivity and specificity. Thus, choosing different potential cut points of mammaglobin (on the log scale), based on the fitted curve, the estimated sensitivity and specificity corresponding to a mammaglobin cut point of 8.8 are 68.8% and 88.8%, respectively. In a similar manner, the cut point of 8.4 results in a sensitivity of 80.2% and a specificity of 82.8% and a cut point of 7.9 yields a sensitivity of 88.7% and a specificity of 75.0%. The AUC for the fitted ROC curve is 0.898, and the 95% asymmetrical confidence interval is 0.812 to 0.950. The AUC for the empirical curve was also calculated using the Wilcoxon statistic, which requires no distributional assumptions. This method resulted in an estimated AUC of 0.892 with a 95% confidence interval of 0.805 to 0.9783.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the potential of the mammaglobin protein to serve as a serum biomarker for breast cancer. Previous studies have shown that mammaglobin mRNA levels were detectable at higher levels in breast tumors compared with normal breast tissue, and mammaglobin was not expressed at the RNA level in several normal tissues (3). Moreover, mammaglobin protein expression in breast tumors was observed at high frequency (3, 15, 16) independent of stage, grade, or histology (2). This evidence of breast tissue specificity has led to the application of mammaglobin as a marker for metastatic spread of breast cancer to lymph nodes (5). In the present series of tumor tissues, we confirmed and extended these findings by showing that mammaglobin protein was expressed in a large majority of breast tumors but was not in detected in tumors of the colon, prostate, lung, or ovary. We did observe that 1 of 31 (3%) uterine tumors expressed mammaglobin. However, it should be noted that we have no other information on this patient, so it is possible that she may also have had a prior or an undetected breast tumor. Although further studies of uterine cancer are needed to determine the frequency of mammaglobin expression in this tumor type, our findings, along with previous studies, strongly indicate that mammaglobin protein expression is highly specific for breast cancer.

We characterized the mammaglobin protein as a 14- to 21-kDa species, which is likely post-translationally processed from its predicted 7-kDa size. We established its secretory nature by showing its release into tissue culture fluids of mammaglobin transcript–positive breast tumor cells. Further, we showed that the mammaglobin protein could be detected by a sensitive and specific ELISA in the sera of some breast cancer patients. Ideal biomarkers for screening are easy to use, noninvasive, and stable over various conditions. We found mammaglobin to be stable over several cycles of freeze thawing and under conditions of long-term storage. Thus, archived samples in biorepositories should be suitable for analysis in further assessing its utility as a serum biomarker for breast cancer.

Using the ELISA, we determined levels of mammaglobin in the serum of disease-free, nonpregnant, premenopausal and postmenopausal to establish baseline levels of mammaglobin and examined these values according to factors, such as age, body mass index, menopausal status, race, smoking history, or a family history of breast cancer. We found that mammaglobin levels did not fluctuate as a function of menopausal status or any of these characteristics associated with risk of breast cancer. We also investigated the variation of mammaglobin levels over time to examine the effects of ovulatory cycle on baseline levels in serum. We observed no differences in serum mammaglobin levels over the 3 months of measurements, suggesting that the protein is a stable marker across time and also across individuals. However, when the baseline levels of healthy controls were compared with mammaglobin levels in breast cancer patients with metastatic disease, we found that the levels were much higher among the cases. Furthermore, we observed that the serum mammaglobin levels in breast cancer patients were correlated with serum CEA and CA27-29 levels. Combined, these findings suggest the potential utility of this new serum biomarker in confirming the diagnosis of breast cancer in the early detection of recurrence and as an indicator of treatment response. Also of note was our finding that women with bra cup size D had significantly higher mammaglobin levels compared with women with smaller bra cup sizes (P < 0.05). In this series, bra cup size was used as a surrogate measure of breast volume because breast volume measurements were unavailable. As this observation may reflect the possibility that mammaglobin levels are positively correlated with breast volume and perhaps breast density as well, further study of these associations, where all measures are available for both women with and without breast cancer, are warranted.

Our results indicate that there exist cut point levels for mammaglobin that produce values for sensitivity and specificity comparable with those associated with use of prostate-specific antigen as a biomarker for prostate cancer (17). For example, in the case of prostate-specific antigen as a biomarker for prostate cancer, a cut point of 4.0 ng/mL provides 73.2% sensitivity and 85.4% specificity (18). Although the ROC curve cannot be used to determine an appropriate cut point for a continuous variable, our data showed that using a cut point of 8.8 on the log scale results in 68.8% sensitivity and 88.8% specificity. Previous studies have shown that ~70% to 80% of breast tumors are positive for mammaglobin expression and in this study, we observed mammaglobin immunostaining in a similar fraction of breast tumors analyzed. We presume that those tumors, which express little or no mammaglobin by these approaches would be less likely than mammaglobin-positive tumors to release this biomarker into the blood. Therefore, taking into consideration that 20% to 30% of breast tumors lack detectable mammaglobin expression, the high level of sensitivity and specificity of the mammaglobin ELISA is encouraging.

Thus, our studies establish mammaglobin as a novel serum biomarker of breast cancer. As such, this serum marker holds significant promise in improving the survival potential of breast cancer patients through early detection and/or more accurate monitoring of response to therapy and relapse.


   Acknowledgments
 
We thank the women who participated in this study and Rebeca Gogel and Anitha Chetty for their assistance with recruitment and follow-up of women through the Mount Sinai Radiology Associates Practice. We are also grateful to Dr. Roy Jensen for his help in obtaining breast cancer tissue specimens.


   Footnotes
 
Grant support: Breast Cancer Research Foundation (S.A. Aaronson), National Cancer Institute Biostatistics Shared Resource grant CA088282 (J.H. Godbold), and NIH grant CA098021 (T.P. Fleming).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: S.A. Aaronson and T.P. Fleming contributed equally to this work. T.P. Fleming and M.A. Watson are named inventors on patents relating to mammaglobin-related technology filed by Washington University School of Medicine and licensed to Corixa Corporation. The remaining authors have no financial or other interest that may lead to conflict of interest.

6 http://www-radiology.uchicago.edu/sections/roc/kitguide/rockit_GUIDE03.html. Back

Received 2/25/05; revised 6/ 1/05; accepted 6/15/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lauro S, Trasatti L, Bordin F, et al. Comparison of CEA, MCA, CA 15-3 and CA 27-29 in follow-up and monitoring therapeutic response in breast cancer patients. Anticancer Res 1999;19:3511–5.[Medline]
  2. Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 1996;56:860–5.[Abstract/Free Full Text]
  3. Watson MA, Dintzis S, Darrow CM, et al. Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res 1999;59:3028–31.[Abstract/Free Full Text]
  4. Leygue E, Snell L, Dotzlaw H, et al. Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol 1999;189:28–33.[CrossRef][Medline]
  5. Min CJ, Tafra L, Verbanac KM. Identification of superior markers for polymerase chain reaction detection of breast cancer metastases in sentinel lymph nodes. Cancer Res 1998;58:4581–4.[Abstract/Free Full Text]
  6. Ouellette RJ, Richard D, Maicas E. RT-PCR for mammaglobin genes, MGB1 and MGB2, identifies breast cancer micrometastases in sentinel lymph nodes. Am J Clin Pathol 2004;121:637–43.[CrossRef][Medline]
  7. Carter D, Douglass JF, Cornellison CD, et al. Purification and characterization of the mammaglobin/lipophilin B complex, a promising diagnostic marker for breast cancer. Biochemistry 2002;41:6714–22.[CrossRef][Medline]
  8. Colpitts TL, Billing-Medel P, Friedman P, et al. Mammaglobin is found in breast tissue as a complex with BU101. Biochemistry 2001;40:11048–59.[CrossRef][Medline]
  9. Klug J, Beier HM, Bernard A, et al. Uteroglobin/Clara cell 10-kDa family of proteins: nomenclature committee report. Ann N Y Acad Sci 2000;923:348–54.[Free Full Text]
  10. Pagano M, Gauvreau K. Principles of biostatistics. Pacific Grove (CA): Duxbury Press; 2000.
  11. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 1982;143:29–36.[Abstract/Free Full Text]
  12. Jekel JF, Elmore JG, Katz DL. Epidemiology, biostatistics, and preventive medicine. Philadelphia (PA): W.B. Saunders Co.; 1996.
  13. Metz CE, Herman BA, Shen JH. Maximum likelihood estimation of receiver operating characteristic (ROC) curves from continuously-distributed data. Stat Med 1998;17:1033–53.[CrossRef][Medline]
  14. Bamber D. The area above the ordinal dominance graph and the area below the receiver operating graph. J Math Psych 1975;12:387–415.[CrossRef]
  15. Span PN, Waanders E, Manders P, et al. Mammaglobin is associated with low-grade, steroid receptor-positive breast tumors from postmenopausal patients, and has independent prognostic value for relapse-free survival time. J Clin Oncol 2004;22:691–8.[Abstract/Free Full Text]
  16. Grunewald K, Haun M, Urbanek M, et al. Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19. Lab Invest 2000;80:1071–7.[Medline]
  17. Catalona WJ, Smith DS, Ratliff TL, et al. Measurement of prostate-specific antigen in serum as a screening test for prostate cancer. N Engl J Med 1991;324:1156–61.[Abstract]
  18. Gann PH, Hennekens CH, Stampfer MJ. A prospective evaluation of plasma prostate-specific antigen for detection of prostatic cancer. JAMA 1995;273:289–94.[Abstract]



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