|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
Journal of Clinical Oncology, Vol 22, No 4 (February 15), 2004: pp. 691-698 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.01.072 Mammaglobin Is Associated With Low-Grade, Steroid Receptor-Positive Breast Tumors From Postmenopausal Patients, and Has Independent Prognostic Value for Relapse-Free Survival TimeFrom the Departments of Chemical Endocrinology and Medical Oncology, University Medical Center Nijmegen, Nijmegen; Division of Endocrine Oncology, Department of Medical Oncology, Erasmus Medical Center—Daniel den Hoed, Rotterdam, the Netherlands; and Division of Laboratory Medicine, Washington University School of Medicine, St Louis, MO Address reprint requests to P.N. Span, PhD, 530 Department of Chemical Endocrinology, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, the Netherlands; e-mail: p.span{at}ace.umcn.nl
PURPOSE: The tumor mRNA expression levels of mammaglobin, a novel breast-specific and breast cancer-associated marker, were correlated with disease outcome in 280 patients with primary breast cancer. PATIENTS AND METHODS: Mammaglobin expression levels were assessed by quantitative reverse transcriptase polymerase chain reaction in frozen tumor tissue from breast cancer patients with a median age of 60 years (range, 30 to 88 years) and a median follow-up of 85 months (range, 2 to 169 months). RESULTS: High expression levels were associated with low-grade tumors (P = .018), with positive estrogen and progesterone receptor status (P < .001), and postmenopausal status (P = .010). In the analysis of all patients, low tumor mammaglobin expression levels predicted an early relapse both in Cox univariate (hazard ratio [HR], 0.52; 95% CI, 0.34 to 0.79; P = .002) and multivariate regression analyses corrected for the traditional prognostic factors (HR, 0.55; 95% CI, 0.35 to 0.88; P = .012). The association of mammaglobin expression with the rate of relapse was particularly favorable in patients who received adjuvant tamoxifen treatment (HR, 0.35; 95% CI, 0.17 to 0.71; P = .004). CONCLUSION: These results demonstrate that the assessment of the tumor mRNA expression level of the breast-specific protein mammaglobin could be useful to stratify patients for individual adjuvant treatment strategies.
Mammaglobin has recently been identified as a novel breast-specific and breast cancer-associated marker [1]. This 93-amino acid protein with a 19-amino acid hydrophobic peptide signal sequence and a predicted molecular mass of 10.5 kd is a member of a family of epithelial secretory proteins, uteroglobins, located on chromosome 11q12.2 [2]. Given that mammaglobin expression is restricted to normal breast epithelium and is frequently upregulated in breast cancer [1,3], several studies have explored the merit of mammaglobin as a marker of metastases in blood [4-8], lymph nodes [9-14], and bone marrow [15,16]. A significant proportion of breast cancer cell lines or tumor samples, however, do not express mammaglobin [1,3,9,14,15,17-19]. This counteracts the use of mammaglobin as a marker for circulating tumor cells because tumor cells that are shed from mammaglobin-negative tumors will not be detected using mammaglobin-specific reverse transcriptase polymerase chain reaction (RT-PCR). To date, studies on the promoter sequence of mammaglobin were unsuccessful in elucidating the factors that regulate mammaglobin expression [18]. We reasoned that it is important to establish the limits of mammaglobin as a marker of metastases by identifying the subset of breast tumors with low mammaglobin expression levels that are more likely to escape detection by mammaglobin PCR. We set out to assess the variation in mammaglobin expression in a large number of unilateral sporadic breast cancer samples by quantitative RT-PCR and investigated the associations with clinicopathologic parameters and prognosis. This is the first report on the prognostic value of tumor mammaglobin expression in primary breast cancer.
Patients A series of 280 patients with unilateral, operable breast cancer who underwent resection of their primary tumor between November 1987 and December 1997 were selected by the availability of frozen tissue in the tumor bank of the Department of Chemical Endocrinology of the University Medical Center Nijmegen (Nijmegen, the Netherlands). This bank contains frozen tumor tissue of patients with breast cancer from five different hospitals of the Comprehensive Cancer Center East in the Netherlands. The measurement of estrogen receptor (ER) and progesterone receptor (PgR) levels was centrally performed in our hospital for these participating hospitals. The clinical data were retrospectively collected. Patients had no previous diagnosis of carcinoma, no distant metastases at time of diagnosis, and no evidence of disease within 1 month after primary surgery. Furthermore, patients receiving neoadjuvant therapy or with carcinoma-in-situ only were excluded. The median age was 60 years (range, 30 to 88 years). Patients underwent modified radical mastectomy (n = 204) or a breast-conserving lumpectomy (n = 76). Postoperative radiotherapy, to the breast after an incomplete resection or after breast-conserving treatment, or parasternal when the tumor was medially localized, was administered to 203 patients. Lymph node involvement was found in 146 patients. Subsequent systemic adjuvant therapy was given on the basis of established clinical evaluation at that time. Patients treated with endocrine therapy (n = 91) received tamoxifen 40 mg twice daily for at least 2 years. In total, 30 patients received adjuvant chemotherapy. Adjuvant chemotherapy consisted of the classic schedule: cyclophosphamide 100 mg/m2 days 1 to 14, methotrexate 40 mg/m2 days 1 to 8, and fluorouracil 600 mg/m2 days 1 to 8 for six cycles, every 4 weeks. Some patients received adjuvant chemotherapy with nine cycles of fluorouracil, epirubicin plus doxorubicin, and cyclophosphamide. Eighteen patients received both endocrine therapy and chemotherapy. The median follow-up time of patients was 85 months (range, 2 to 169 months). Patients were seen (including history, physical examination, and routine laboratory investigations) once every 3 months during the first 2 years, once every 6 months for 5 years, and once a year thereafter. Once-yearly x-ray mammography was performed, and if findings were suggestive of recurrence, a mamma-magnetic resonance imaging scan also was performed. During follow-up, 103 patients had a recurrence (24 patients had locoregional recurrences, 77 patients had distant metastases, and two patients had both) and 80 patients died (62 deaths were confirmed to be related to breast cancer; two deaths were related to other malignancy, and the causes of 16 deaths were unknown). Contralateral breast cancer or second malignancies were not considered as recurrent disease.
Tissue Processing
RT-PCR
Immunohistochemistry
Statistical Analyses
Mammaglobin mRNA expression in primary breast tumors was undetectable after 40 rounds of amplification in 19 of 280 patients (6.8%). Tumors from the other patients exhibited a wide, log-normal range of expression (ie, from 0.0002 to 229; median, 0.11) after normalization for ß-actin expression (Fig 1).
Immunohistochemical staining of mammaglobin protein in breast carcinomas also yielded heterogeneous results (Fig 2), with tissues expressing abundant (Fig 2A), marginal (Fig 2B), or undetectable (Fig 2C) levels of mammaglobin. Staining is largely confined to the cytoplasm of invasive tumor cells. In tumors with marginal mammaglobin expression (Fig 1B), intense staining is seen in a subpopulation of invasive tumor cells rather than global, weak staining in all tumor cells.
No correlation could be ascertained between the relative amount of tumor tissue and mammaglobin levels (Spearman correlation coefficient [Rs] = -0.160; P = .230) in 58 breast tumor tissues, of which the relative amount of tumor cell nuclei (range, 35% to 90%; median, 70%) was recorded. This strongly suggests that the presence of normal components does not have an appreciable impact on the results presented here. Median mammaglobin expression levels were calculated for patient groups divided on the basis of several clinicopathologic factors, as shown in Table 1. The highest mammaglobin levels were found in tumors that were ER-positive (P < .001) or PgR-positive (P < .001; Table 1; Figs 3A and 3B). A significant (Spearman) correlation could also be found between mammaglobin and continuous ER (Rs = 0.274; P < .001) or PgR (Rs = 0.270; P < .001) levels. Older (P = .015) and postmenopausal patients (P = .010) showed significantly higher tumor mammaglobin mRNA expression levels (Table 1; Fig 3C). Furthermore, low- and intermediate-grade (I and II) tumors expressed significantly (P = .005) higher mammaglobin levels than high-grade (III) tumors.
Because high mammaglobin mRNA expression levels correlated with favorable clinicopathologic features, we next assessed whether tumor mammaglobin expression levels might be predictive for the rate of relapse or death. In univariate regression analysis using log-transformed continuous mammaglobin values, higher levels were associated with a favorable RFS (HR, 0.81; 95% CI, 0.72 to 0.92; P = .001) and OS (HR, 0.80; 95% CI, 0.69 to 0.94; P = .006). To allow the analysis of the mammaglobin expression level as a categorized variable, and for visualization in survival curves to classify tumors as high versus low, tumor mammaglobin levels were dichotomized at the median value. In univariate analysis, a high tumor mammaglobin mRNA expression level (  0.11) was strongly associated with a favorable RFS compared with tumors with a low expression level (HR, 0.52; 95% CI, 0.34 to 0.79; P = .002; Table 2; Fig 4A), and the association was somewhat weaker with the length of OS (HR, 0.62; 95% CI, 0.39 to 0.99; P = .044) in the total, unselected group of patients. When in exploratory analyses patients were divided in groups based on the type of adjuvant treatment they received, high tumor mammaglobin expression levels predicted a longer RFS only in the group of 91 patients treated with tamoxifen (HR, 0.35; 95% CI, 0.17 to 0.71; P = .004; Fig 4B). The length of OS, however, was not related to the expression level of mammaglobin (HR, 0.70; 95% CI, 0.26 to 1.87; P = .478) in this patient group. In the 48 patients treated with chemotherapy, the mammaglobin expression level did not significantly predict the length of RFS or OS (HR, 0.86; 95% CI, 0.38 to 2.27; P = .764; and HR, 1.52; 95% CI, 0.51 to 4.53; P = .452, respectively).
In multivariate regression analysis including age, menopausal status, histologic grade, tumor size, involved lymph nodes, ER status, PgR status, and postoperative radiotherapy, mammaglobin expression status significantly added to the model for RFS (HR, 0.55; 95% CI, 0.35 to 0.88; P = .012; Table 2). When adjuvant systemic therapy was added to the model, the HR and 95% CI of mammaglobin did not change (HR, 0.55; 95% CI, 0.34 to 0.89). For OS, mammaglobin did not significantly contribute to the multivariate model (HR, 0.62; 95% CI, 0.37 to 1.03; P = .105). There were no statistically significant interactions between mammaglobin expression status and any of the traditional prognostic factors in the multivariate analyses for RFS or OS. Given that mammaglobin expression was correlated with ER and PgR, and mammaglobin was especially prognostic for RFS in the tamoxifen-treated patients, we also performed exploratory multivariate analyses for RFS for this subgroup. When age, menopausal status, histologic grade, tumor size, involved lymph nodes, ER status, PgR status, and postoperative radiotherapy were included in the multivariate model, only ER (HR, 0.44; 95% CI, 0.21 to 0.95; P = .037) and mammaglobin (HR, 0.34; 95% CI, 0.15 to 0.78; P = .011) remained as factors that contributed significantly to the model of RFS. Because the prognostic value of mammaglobin in this subgroup could be related to steroid receptor levels, and not receptor status, we performed the same analysis with ER and PgR as log-transformed continuous variables. As with ER and PgR as dichotomized variables, ER (HR, 0.77; 95% CI, 0.61 to 0.99; P = .038) and mammaglobin (HR, 0.34; 95% CI, 0.15 to 0.77; P = .010) remained as factors that contributed significantly to the model.
Other investigators previously reported on the detection of mammaglobin in peripheral blood or lymph nodes [4-16]. A high mammaglobin level in these studies was associated with a poor prognosis. This conclusion is based on the fact that mammaglobin is exclusively expressed in breast tissues. Thus, mammaglobin expression in peripheral organs is the result of displaced breast cells or micrometastasis of a breast tumor, which itself signifies a poor perspective. Conversely, our study focused on the mammaglobin levels in the primary tumor and prognosis in primary breast cancer. We found that high levels were associated with a favorable prognosis. This is the first study addressing tumor mammaglobin levels and prognosis in breast cancer. High mammaglobin levels were associated with low-grade, steroid receptor-positive tumors from postmenopausal patients. The tissue mammaglobin levels had prognostic value for the rate of relapse, especially in those patients who received adjuvant tamoxifen treatment. In multivariate regression analysis corrected for the traditional prognostic factors, mammaglobin expression status was a strong independent prognostic factor for the length of RFS. A wide range in mammaglobin mRNA expression and/or protein levels in breast tumor cell lines, and a limited number of patient samples, have been reported previously [1,3,9,14,15,17-19]. Watson and Fleming [1] found that eight of 35 primary breast carcinomas overexpressed mammaglobin relative to normal breast tissue specimens, and Watson et al [18] found that five of 10 breast carcinoma cell lines and 13 of 21 metastatic breast tumors exhibited high levels of mammaglobin mRNA. This overexpression did not appear to correlate with histology, tumor grade, tumor stage, or steroid hormone-receptor status. A recent study, however, reported a positive association of mammaglobin with ER status [19]. In another study, 81 of 100 primary human breast tumors were strongly immunopositive for mammaglobin, independent of tumor grade and histologic type [3]. Watson et al [18] mentioned unpublished data suggesting that mammaglobin expression is associated with mammary gland proliferation and terminal differentiation. Our results confirm the earlier-reported correlation between mammaglobin and ER levels [19] in primary breast cancer samples. We also found a correlation between mammaglobin and PgR, suggesting a possible estrogen responsiveness of mammaglobin expression. Remarkably, at the lower serum estradiol levels after menopause, we find high levels of mammaglobin in breast cancer tissue, which would argue against an estrogen-mediated induction of mammaglobin expression. Because other uteroglobin gene family members are regulated by steroid hormones, Watson et al [18] analyzed the mammaglobin gene promoter for steroid-responsive elements. They identified imperfect elements similar to estrogen- and androgen-response elements. However, they did not observe any modulation of mammaglobin transcription by estradiol, progesterone, dexamethasone, or androgens. Similarly, our studies into this issue have been unsuccessful in revealing an estrogen-mediated regulation of mammaglobin expression in the breast tumor cell lines MCF-7, MDA-MB175, and MDA-MB231 (unpublished data). Thus, mammaglobin expression is not influenced by steroid hormones. Possibly, mammaglobin is a differentiation marker of an apocrine epithelial cell type [21], whereas ER/PgR-negative cells are of basal cell origin [22,23]. This explanation would concur with the association of mammaglobin with ER/PgR status and histologic grade. It would also be in line with the IHC data, which show that in tumors with intermediate mammaglobin expression, intense staining is seen in a subpopulation of invasive tumor cells rather than global, weak staining in all tumor cells. On the other hand, we also found ER/PgR-negative tumors that express mammaglobin (Fig 3). This might be due to cellular heterogeneity within the tumor and, therefore, can only be accurately addressed by performing parallel IHC studies with ER and mammaglobin. We report here that the expression levels of mammaglobin have prognostic value in unselected breast cancer tissues, albeit with a limited follow-up of 85 months. The possibility that mammaglobin might also be a predictive factor (ie, could identify patients that benefit most from endocrine therapy) seemed likely given its association with ER and PgR, and its strong independent prognostic value in the tamoxifen-treated subgroup. Adding an interaction variable to the model to assess predictive value in retrospective studies, as reported earlier for urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 in chemotherapy [24], is precluded by the association of mammaglobin with choice of adjuvant treatment through its correlation with ER values. With the present data, we are unable to confirm or refute a possible predictive value of tumor tissue mammaglobin expression for success of tamoxifen therapy. In conclusion, mammaglobin mRNA is expressed in primary breast cancers at a wide range of levels and is associated with low-grade, steroid receptor-positive tumors from postmenopausal patients. Furthermore, tumor mammaglobin expression levels have prognostic value in an unselected cohort and in tamoxifen-treated patients with invasive breast carcinoma. These results suggest that the knowledge of the expression level of mammaglobin mRNA in primary breast tumors might be useful to stratify patients for individual adjuvant treatment strategies.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Owns stock (not including shares held through a public mutual fund): Mark A. Watson, CORIXA. Acted as a consultant within the last 2 years: Mark A. Watson, Pharmacia, Pfizer. Performed contract work within the last 2 years: John A. Foekens, Novartis, Wilex AG. Received more than $2,000 a year from a company for either of the last 2 years: Mark A. Watson, CORIXA.
We thank all of the contributors, especially the surgeons and internists, of the UMC Nijmegen and of the community hospitals in the region: Ziekenhuiscentrum Apeldoorn, Apeldoorn; Rijnstate Ziekenhuis, Arnhem; Maasziekenhuis, Boxmeer; Deventer Ziekenhuis, Deventer; Gelderse Vallei, Ede and Canisius-Wilhelmina Ziekenhuis, Nijmegen, for their assistance in collecting the patients' clinical follow-up data. Doorlène van Tienoven and Anneke Geurts of the Department of Chemical Endocrinology of the University Medical Center Nijmegen are acknowledged for their excellent work in collecting and archiving the breast tumor samples.
Presented in part at the 2002 European Society for Medical Oncology meeting, October 18–22, Nice, France. Authors' disclosures of potential conflicts of interest are found at the end of this article.
1. Watson MA, Fleming TP: Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 56:860–865, 1996
2. Ni J, Kalff-Suske M, Gentz R, et al: All human genes of the uteroglobin family are localized on chromosome 11q12.2 and form a dense cluster. Ann NY Acad Sci 923:25–42, 2000
3. Watson MA, Dintzis S, Darrow CM, et al: Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res 59:3028–3031, 1999
4. Zach O, Kasparu H, Krieger O, et al: Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via a nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA. J Clin Oncol 17:2015–2019, 1999 5. 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 80:1071–1077, 2000[Medline] 6. Suchy B, Austrup F, Driesel G, et al: Detection of mammaglobin expressing cells in blood of breast cancer patients. Cancer Lett 158:171–178, 2000[CrossRef][Medline]
7. Zach O, Kasparu H, Wagner H, et al: Mammaglobin as a marker for the detection of tumor cells in the peripheral blood of breast cancer patients. Ann NY Acad Sci 923:343–345, 2000
8. Silva JM, Dominguez G, Silva J, et al: Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics. Clin Cancer Res 7:2821–2825, 2001 9. Leygue E, Snell L, Dotzlaw H, et al: Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol 189:28–33, 1999[CrossRef][Medline] 10. Kataoka A, Mori M, Sadanaga N, et al: RT-PCR detection of breast cancer cells in sentinel lymph nodes. Int J Oncol 16:1147–1152, 2000[Medline] 11. Ooka M, Sakita I, Fujiwara Y, et al: Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients. Oncol Rep 7:561–566, 2000[Medline] 12. Manzotti M, Dell'Orto P, Maisonneuve P, et al: Reverse transcription-polymerase chain reaction assay for multiple mRNA markers in the detection of breast cancer metastases in sentinel lymph nodes. Int J Cancer 95:307–312, 2001[CrossRef][Medline] 13. Mitas M, Mikhitarian K, Walters C, et al: Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel. Int J Cancer 93:162–171, 2001[CrossRef][Medline]
14. Zehentner BK, Dillon DC, Jiang Y, et al: Application of a multigene reverse transcription-PCR assay for detection of mammaglobin and complementary transcribed genes in breast cancer lymph nodes. Clin Chem 48:1225–1231, 2002 15. Leone F, Perissinotto E, Viale A, et al: Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction. Bone Marrow Transplant 27:517–523, 2001[CrossRef][Medline] 16. Ooka M, Tamaki Y, Sakita I, et al: Bone marrow micrometastases detected by RT-PCR for mammaglobin can be an alternative prognostic factor of breast cancer. Breast Cancer Res Treat 67:169–175, 2001[CrossRef][Medline]
17. 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 58:4581–4584, 1998 18. Watson MA, Darrow C, Zimonjic DB, et al: Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13. Oncogene 16:817–824, 1998[CrossRef][Medline]
19. O'Brien N, Maguire TM, O'Donovan N, et al: Mammaglobin a: A promising marker for breast cancer. Clin Chem 48:1362–1364, 2002 20. EORTC Breast Co-operative Group. Revision of the standards for the assessment of hormone receptors in human breast cancer: Report of the Second EORTC Workshop, held on 16–17 March, 1979, in the Netherlands Cancer Institute. Eur J Cancer 16:1513–1515, 1980
21. Dontu G, Abdallah WM, Foley JM, et al: In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270, 2003 22. Perou CM, Sorlie T, Eisen MB, et al: Molecular portraits of human breast tumours. Nature 406:747–752, 2000[CrossRef][Medline]
23. Gruvberger S, Ringner M, Chen Y, et al: Estrogen receptor status in breast cancer is associated with remarkably distinct gene expression patterns. Cancer Res 61:5979–5984, 2001
24. Harbeck N, Kates RE, Look MP, et al: Enhanced benefit from adjuvant chemotherapy in breast cancer patients classified high-risk according to urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (n = 3424). Cancer Res 62:4617–4622, 2002 Submitted January 13, 2003; accepted December 10, 2003. This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|||||||||||
|
|||||||||||
|
|
Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
|
TOP
ABSTRACT
INTRODUCTION
