- Open Access
Cathepsin B: a potential prognostic marker for inflammatory breast cancer
- Mohamed A Nouh†1,
- Mona M Mohamed†2Email author,
- Mohamed El-Shinawi3,
- Mohamed A Shaalan4,
- Dora Cavallo-Medved5, 6,
- Hussein M Khaled7 and
- Bonnie F Sloane5, 8
© Nouh et al; licensee BioMed Central Ltd. 2011
- Received: 21 August 2010
- Accepted: 3 January 2011
- Published: 3 January 2011
Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB) is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC.
In this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IBC versus non-IBC patient tissues. Previously, we found that CTSB is localized to caveolar membrane microdomains in cancer cell lines including IBC, and therefore, we also examined the expression of caveolin-1 (cav-1), a structural protein of caveolae in IBC versus non-IBC tissues. In addition, we tested the correlation between the expression of CTSB and cav-1 and the number of positive metastatic lymph nodes in both patient groups.
Our results revealed that CTSB and cav-1 were overexpressed in IBC as compared to non-IBC tissues. Moreover, there was a significant positive correlation between the expression of CTSB and the number of positive metastatic lymph nodes in IBC.
CTSB may initiate proteolytic pathways crucial for IBC invasion. Thus, our data demonstrate that CTSB may be a potential prognostic marker for lymph node metastasis in IBC.
- Inflammatory Breast Cancer
- Tumor Embolus
- Positive Lymph Node Metastasis
- Cancer Cell Motility
- Inflammatory Breast Cancer Patient
Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer, with a 3-year survival rate of 40% as compared to 85% for non-IBC . IBC is defined by distinct clinical features including a rapid onset, erythema, edema of the breast and a "peau d'orange" appearance of the skin. High metastatic behavior (for review see ), rapid invasion into blood and lymphatic vessels and formation of tumor emboli within these vessels  are also major characteristics of IBC. Obstruction of lymphatic flow by tumor emboli within the dermal lymphatics causes swelling of the breast tissue and underlies the inflammatory nature of the disease.
Positive axillary lymph node metastasis is a characteristic of IBC at the time of diagnosis and most IBC patients present with extensive lymph node metastasis [3, 4]. Indeed, the number of positive metastatic lymph nodes contributes to poor survival outcome with each positive lymph node increasing risk of breast cancer mortality by approximately 6% . Although IBC is characterized by the extensive presentation of metastatic lymph nodes, the molecular pathways that direct IBC lymph node invasion are not well defined. Recent studies conducted by Ellsworth and colleagues, using laser capture microdissection and gene expression analysis of primary breast tumors and corresponding metastatic lymph nodes, indicate that overexpression of genes involved in degradation of the extracellular matrix (ECM) in primary breast cancer cells induces them to disseminate to nearby lymph nodes .
The invasive properties of IBC are consistent with a crucial role for proteolytic enzymes in the degradation of ECM, cell motility and metastasis . Cathepsin B (CTSB), a lysosomal cysteine protease, has been shown to be a contributor to the progression and invasion of various types of cancer . Specifically, CTSB is involved in proteolytic pathways that lead to the degradation of ECM proteins thereby promoting cancer cell motility and invasion [8, 9]. In cancer cells, CTSB is shuttled to the plasma membrane where it can activate receptor-bound pro-urokinase-type plasminogen activator (pro-uPA). uPA activate plasminogen a serine protease that can digest ECM proteins and activate MMPs, a family of proteolytic enzymes that are also major participants in ECM degradation and cancer cell motility and invasion . CTSB is associated with cell surface caveolae, specialized membrane microdomains that are involved in signaling pathways, endocytosis and proteolysis (for review see [11, 12]). The role of caveolin-1 (cav-1), the main structural protein of caveolae, in cancer progression and invasion is contradictory and appears to depend upon the cancer type and stage of progression. In IBC patient tissues and cell lines, cav-1 is overexpressed , a phenotype observed in other aggressive breast carcinomas that show high metaplastic properties . Overexpression of cav-1 has been shown to be associated with ECM degradation and formation of invadopodia, which contain membrane-type-1-MMP (MT1-MMP) and mediate breast cancer cell motility and invasion . In previous in vitro studies, we have shown that interaction of IBC cells with human monocytes augments invasion of IBC cells through increased ECM degradation, events correlated with an increase in CTSB expression, secretion and activity and an increase in cav-1 expression in the IBC cells . More recently, we have co-localized active CTSB and uPA with cav-1 in caveolar fractions of SUM149 IBC cells (unpublished data).
In the present study, we assessed the expression levels of CTSB and cav-1 in IBC versus non-IBC patient breast tissues. Furthermore, we examined the correlation between these proteins and the number of metastatic lymph nodes in IBC versus non-IBC patient tissues. Our results revealed an overexpression of CTSB and cav-1 in IBC tissues and demonstrated a positive correlation between CTSB expression and the number of positive lymph node metastases. We speculate that CTSB expressed by tumor cells and localized in caveolae may promote IBC metastasis to lymph nodes by enhancing ECM degradation and tumor invasion.
Patients and Tissue Specimens
Tissue samples were fixed in 10% neutral buffered formalin and processed into paraffin blocks for routine sectioning and immunohistochemistry (IHC). Pathological data regarding tumor size, tumor grade , and the presence of lymphovascular invasion, dermal tumor emboli and tumor parenchyma emboli [2, 18] were assessed (M.A.N), reviewed (H.I.) and tabulated for statistical analysis. Additional sections were generated from the paraffin tissue blocks and immunostained for estrogen receptor (ER), progesterone receptor (PR) and HER2-neu expression status. IHC staining for CTSB, and cav-1 was performed as described below.
Mouse anti-caveolin-1 was purchased from BD Biosciences (San Diego, CA, USA) and polyclonal rabbit anti-human CTSB antibody was previously prepared in house (B.F.S.) . Antibody diluent with background reducing components and DakoCytomation EnVision+ Dual Link System-HRP (DAB+) kits were purchased from Dako (Carpinteria, CA, USA); and Permount® was from Fisher Scientific (Pittsburgh, PA, USA).
Tissue sections were prepared from paraffin blocks and stained with hematoxylin and eosin to select tissue sections for immunostaining and scoring. IHC staining for each marker was performed in duplicate on 5 μm thick tissue sections. Tissue sections were first deparaffinized and rehydrated followed by antigen retrieval. Tissue sections were incubated for 1 hour at room temperature with the following primary antibodies prepared in Dako Antibody diluent with reduced background components: polyclonal CTSB antibody (1:500) and monoclonal anti-cav-1 (1:150). Detection was carried out by incubating tissue sections with 100 μl of horse radish peroxidase-labeled rabbit or mouse secondary antibody [EnVision+ Dual Link System-HRP (DAB+)] for 45 min. Staining was achieved by adding 100 μl of DAB+ diluted 1:50 in substrate buffer [EnVision+ Dual Link System-HRP (DAB+)] for 15 min. Nuclei were counterstained with hematoxylin and specimens were rinsed in PBS and mounted using Permount® for microscopic examination. Negative control slides were run in parallel in which each primary antibody was replaced with PBS.
Two independent readers (M.A.N. and M.M.M.) assessed immunostaining of CTSB and cav-1 using light microscopy (Olympus, CX41, Japan). Discordant results were resolved by consultation with a third reader (H.I.). The expression of CTSB B and cav-1 was scored according to both the intensity of staining and the proportion of positive staining carcinoma cells within the entire slide: "0", no immunostaining was observed within carcinoma cells; "+", less than 10% of carcinoma cells showed cytoplasmic staining of moderate to marked intensity; "++", 10-50% of carcinoma cells showed cytoplasmic staining of moderate to marked intensity; and "+++", greater than 50% of carcinoma cells show cytoplasmic staining of moderate to marked intensity.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Immunoblotting
Peroxidase-labeled goat anti-rabbit secondary antibody and tetramethyl benzidine (TMB membrane peroxidase substrate were purchased from Kirkegaard and Perry Laboratories Inc (Gaithersburg, MD, USA).
Fresh breast tissue specimen obtained from core biopsy or during modified radical mastectomy were minced into small pieces on ice in RIPA buffer [25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Sigma-Aldrich, St. Louis, MO, USA)]. Protein concentrations of cell lysates were measured using Bradford reagent (Sigma-Aldrich, Germany). Samples were equally loaded (20 μg protein/well), separated by 12% SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes as previously described . Immunoblotting analysis was performed using primary antibodies against CTSB (1:4000) and caveolin-1 (1:5000) and a secondary antibody conjugated with horseradish peroxidase (1:10,000) in Tris-buffered saline wash buffer (20 mM Tris, pH 7.5, 0.5 M NaCl) containing 0.5% Tween 20 and 5% (w/v) non-fat dry milk. After washing, bound antibodies were detected by adding a TMB chromagen/substrate solution. Once a signal was detected reactions were terminated by immersing membranes in water for 20-30 seconds.
The data were analyzed using SPSS software version 16.0. Differences were evaluated by Student's t-test and Fisher's exact test. Immunohistochemical scores of 0 and + were considered negative and scores of ++ and +++ were considered positive. Fisher exact test was performed to analyze differences in CTSB and cav-1 immunostaining (i.e., positive versus negative) between IBC and non-IBC groups. Correlations between categorical variables were assessed using Fisher's exact test as previously described .
Clinical and pathological characterization of IBC versus non-IBC patients
Clinical and pathological characterization of IBC versus non-IBC patients
n= 23 (%)
n= 27 (%)
Mean ± SD
40.9 ± 7.5
49.9 ± 9.1
Mean ± SD
6.5 ± 3.3
4.31 ± 2.30
Axillary Lymph Node Status†
Tumor size measurements revealed that 5 IBC patients (21.7%) presented with no tumor mass that could be detected clinically, mammographically or upon examination of the mastectomy specimen; however, tumor emboli were present in skin and core biopsies. For IBC patients with detectable masses, 5.6% of them exhibited tumor masses less than 2 cm and 94.4% had a tumor mass more than 2 cm with tumor sizes ranging from 4-10 cm (mean size of 6.5 ± 3.3 cm). Non-IBC patients had tumor sizes ranging from 1.8-12 cm (mean size of 4.3 ± 2.3 cm) with 3.7% having tumor sizes less than 2 cm and 96.3% having tumor sizes greater than or equal to 2 cm.
Tumor grading revealed that 65% of IBC patients were tumor grade I or II and 35% were tumor grade III. In non-IBC patients 77.8% were diagnosed as tumor grade I or II, and 22.2% were diagnosed as tumor grade III.
We assessed the number of axillary lymph nodes that were positive for metastases in IBC versus non-IBC patients. All IBC patients who underwent surgery had positive metastatic lymph nodes: 15% had 1-3 positive metastatic lymph nodes, 30% had 4-7 positive metastatic lymph nodes and 55% had greater than or equal to 8 positive metastatic lymph nodes. Among non-IBC patients, 25.9% were node negative, 33.4% had 1-3 metastatic lymph nodes, 22.2% had 4-7 metastatic lymph nodes and 18.5% had greater than or equal to 8 positive metastatic lymph nodes. In addition, the difference between the number of positive metastatic lymph nodes in IBC versus non-IBC patients was determined to be statistically significant (P = 0.037).
Lymphovascular invasion was significantly greater (P = 0.000) in IBC (73.9%) versus non-IBC (11.1%) patients. Tumor emboli, a phenotypic hallmark of IBC and defined as tight tumor cell clusters retracted away from the surrounding endothelial lining [2, 18], were detected in 100% of IBC tissue sections as compared to only 11.1% of non-IBC tissue sections (P = 0.000). Positive staining for ER, PR and HER-2 was detected in 27.3%, 31.8% and 18.2% of the IBC patients, respectively. In non-IBC patients, positive staining for ER, PR and HER-2 was 22.2%, 29.6% and 14.8%, respectively.
Overexpression of CTSB in IBC versus non-IBC tissues
Scoring of CTSB and cav-1 expression in breast carcinoma cells in IBC versus non-IBC tissues
Fisher's exact test
P = 0.025*
P = 0.001*
IHC scoring results revealed a statistical significance (P = 0.025) in the level of expression of CTSB in IBC versus non-IBC carcinoma cells. In IBC, 34.8% showed CTSB staining score of ++ and 65.2% showed staining score of +++. In non-IBC, CTSB staining was variable with 3.7% scoring 0, 18.5% scoring +, 25.9% scoring ++ and 51.9% scoring +++ (Table 2).
Overexpression of cav-1 in IBC versus non-IBC tissues
Expression of CTSB correlates with positive metastatic lymph nodes in IBC
Correlation between lymph node metastasis and expression of CTSB and cav-1 in IBC versus non-IBC patients.
Lymph node metastasis
Fisher's exact test
P = 0.0478*
P = 0.0717
Thus, our data reveal that the overexpression of CTSB in IBC versus non-IBC is significantly correlated with the increase in number of positive metastatic lymph nodes, suggesting a potential role for this proteolytic enzyme in promoting the invasion of IBC cells into lymphatic vessels.
Criteria for the TNM staging system for breast cancer indicate that the number of positive metastatic axillary lymph nodes is one of the most important prognostic factors for predicting a low survival rate of breast cancer patients . Despite therapeutic regimes, patients with 10 or more positive lymph nodes have a 70% chance of disease recurrence [23, 24]. Indeed, dissemination of IBC cells to lymph nodes is consistent with the aggressive phenotype of IBC although the molecular and cellular pathways underlining this process are poorly understood. In the present study, we show a significant positive correlation between expression of the cysteine protease CSTB and the number of metastatic lymph nodes in IBC patients. In addition, cav-1 was also shown to be overexpressed in IBC tissue as compared to non-IBC tissue.
Our previous in vitro studies showed that increased ECM degradation and invasion of the SUM149 IBC cell line are associated with an overexpression of CTSB and cav-1 . Cav-1 is the main structural protein of lipid raft caveolae, a site that has been hypothesized to localize cell surface proteases involved in pericellular proteolytic events . Indeed, downregulation of cav-1 in colorectal carcinoma cells decreased trafficking of CTSB to caveolae on the surface of these cells and decreased degradation of ECM proteins and cellular invasion . Although the role of cav-1 in breast cancer is contradictory, overexpression of cav-1 is present in aggressive types of breast cancer such as metaplastic carcinoma  and IBC . Moreover, in IBC cell lines and tissues, overexpression of cav-1 is correlated with increased RhoC expression, a GTPase involved in cell motility and invasion . In the present study, overexpression of cav-1 did not significantly correlate with an increase in expression of CSTB; however, current studies in our laboratory have localized CTSB to caveolae of SUM149 IBC cells (unpublished data). Moreover these cells exhibit extracellular degradation of ECM proteins that was partially blocked by cysteine and serine protease inhibitors (unpublished data). Thus, our data suggest that overexpression of cav-1 in IBC cells contributes to proteolytic events involving CTSB that lead to ECM degradation, tumor invasion and metastasis.
IBC is characterized by extensive involvement of positive metastatic lymph nodes, which are associated with the aggressive phenotype of the disease  and are a determining factor in therapeutic decisions [27–29]. As such, we determined whether there were correlations between CTSB and cav-1 and the number of positive metastatic lymph nodes in IBC versus non-IBC patients. Our results revealed a statistically significant positive correlation only between the level of CTSB expression in IBC carcinoma cells and the number of positive metastatic lymph nodes (P = 0.0478). Such a correlation was not detected in non-IBC patients. A positive correlation between CTSB expression and the metastasis of carcinoma cells to lymph nodes has previously been reported in breast , prostate  and gastric  cancers. Overexpression of CTSB in breast cancer has been shown to enhance tumor growth and invasion . This parallels increased recurrence and shortened disease-free survival . Moreover in an animal mammary cancer model, the number of positive metastatic lymph nodes has also been found to be associated with expression of CTSB . Thus, our data are consistent with a crucial role for CTSB in promoting the highly metastatic behaviour of IBC.
The positive correlation between CTSB and nodal metastatic burden in IBC patients suggests that this proteolytic enzyme may promote nodal metastasis in IBC patients. We hypothesize that the overexpression of cav-1 in IBC increases trafficking of CTSB to the cell surface where it promotes IBC invasion into lymphatic vessels and metastasis to lymph nodes. Further studies to validate CTSB as a prognostic marker in IBC and delineate the mechanisms by which the association of CTSB with cav-1 is involved in lymph node metastasis in IBC patients are in progress.
We acknowledge the contribution of Prof. Hoda Ismail (Department of Pathology, National Cancer Institute, Cairo University, Giza, Egypt) for her assistance in reviewing and scoring of pathology slides. We also thank Ms. A Dhiaa Alraawi and Ms. Marwa Tantawy (Department of Zoology, Cairo University, Giza, Egypt) for their assistance in the statistical analysis and immunoblotting, respectively. The authors were supported by Avon Grant # 02-2007-049 (M.M.M., B.F.S.) and Science and Technology Development Funds (Grant # 343 and 408), Egypt (M.M.M.).
- Lerebours F, Bieche I, Lidereau R: Update on inflammatory breast cancer. Breast Cancer Res. 2005, 7: 52-58. 10.1186/bcr997.PubMed CentralView ArticlePubMedGoogle Scholar
- Gong Y: Pathologic aspects of inflammatory breast cancer: part 2. Biologic insights into its aggressive phenotype. Semin Oncol. 2008, 35: 33-40. 10.1053/j.seminoncol.2007.11.014.View ArticlePubMedGoogle Scholar
- Van Laere SJ, Van den Eynden GG, Van der Auwera I, Vandenberghe M, van Dam P, Van Marck EA, van Golen KL, Vermeulen PB, Dirix LY: Identification of cell-of-origin breast tumor subtypes in inflammatory breast cancer by gene expression profiling. Breast Cancer Res Treat. 2006, 95: 243-255. 10.1007/s10549-005-9015-9.View ArticlePubMedGoogle Scholar
- Alberini JL, Lerebours F, Wartski M, Fourme E, Le Stanc E, Gontier E, Madar O, Cherel P, Pecking AP: 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) imaging in the staging and prognosis of inflammatory breast cancer. Cancer. 2009, 115: 5038-5047. 10.1002/cncr.24534.View ArticlePubMedGoogle Scholar
- Michaelson JS, Chen LL, Silverstein MJ, Cheongsiatmoy JA, Mihm MC, Sober AJ, Tanabe KK, Smith BL, Younger J: Why cancer at the primary site and in the lymph nodes contributes to the risk of cancer death. Cancer. 2009, 115: 5084-5094. 10.1002/cncr.24542.View ArticlePubMedGoogle Scholar
- Ellsworth RE, Seebach J, Field LA, Heckman C, Kane J, Hooke JA, Love B, Shriver CD: A gene expression signature that defines breast cancer metastases. Clin Exp Metastasis. 2009, 26: 205-213. 10.1007/s10585-008-9232-9.View ArticlePubMedGoogle Scholar
- Van den Eynden GG, Van Laere SJ, Van der Auwera I, Merajver SD, Van Marck EA, van Dam P, Vermeulen PB, Dirix LY, van Golen KL: Overexpression of caveolin-1 and -2 in cell lines and in human samples of inflammatory breast cancer. Breast Cancer Res Treat. 2006, 95: 219-228. 10.1007/s10549-005-9002-1.View ArticlePubMedGoogle Scholar
- Mohamed MM, Sloane BF: Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer. 2006, 6: 764-775. 10.1038/nrc1949.View ArticlePubMedGoogle Scholar
- Ren WP, Sloane BF: Cathepsins D and B in breast cancer. Cancer Treat Res. 1996, 83: 325-352.View ArticlePubMedGoogle Scholar
- Kobayashi H, Moniwa N, Sugimura M, Shinohara H, Ohi H, Terao T: Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes. Biochim Biophys Acta. 1993, 1178: 55-62. 10.1016/0167-4889(93)90109-3.View ArticlePubMedGoogle Scholar
- Anderson RG: The caveolae membrane system. Annu Rev Biochem. 1998, 67: 199-225. 10.1146/annurev.biochem.67.1.199.View ArticlePubMedGoogle Scholar
- Cavallo-Medved D, Sloane BF: Cell-surface cathepsin B: understanding its functional significance. Curr Top Dev Biol. 2003, 54: 313-341. full_text.View ArticlePubMedGoogle Scholar
- Elsheikh SE, Green AR, Rakha EA, Samaka RM, Ammar AA, Powe D, Reis-Filho JS, Ellis IO: Caveolin 1 and Caveolin 2 are associated with breast cancer basal-like and triple-negative immunophenotype. Br J Cancer. 2008, 99: 327-334. 10.1038/sj.bjc.6604463.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamaguchi H, Takeo Y, Yoshida S, Kouchi Z, Nakamura Y, Fukami K: Lipid rafts and caveolin-1 are required for invadopodia formation and extracellular matrix degradation by human breast cancer cells. Cancer Res. 2009, 69: 8594-8602. 10.1158/0008-5472.CAN-09-2305.View ArticlePubMedGoogle Scholar
- Mohamed MM, Cavallo-Medved D, Sloane BF: Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer. Biol Chem. 2008, 389: 1117-1121. 10.1515/BC.2008.117.PubMed CentralView ArticlePubMedGoogle Scholar
- Dawood S, Merajver SD, Viens P, Vermeulen PB, Swain SM, Buchholz TA, Dirix LY, Levine PH, Lucci A, Krishnamurthy S, Robertson FM, Woodward WA, Yang WT, Ueno NT, Cristofanilli M: International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment. Ann Oncol. 2010,Google Scholar
- Genestie C, Zafrani B, Asselain B, Fourquet A, Rozan S, Validire P, Vincent-Salomon A, Sastre-Garau X: Comparison of the prognostic value of Scarff-Bloom-Richardson and Nottingham histological grades in a series of 825 cases of breast cancer: major importance of the mitotic count as a component of both grading systems. Anticancer Res. 1998, 18: 571-576.PubMedGoogle Scholar
- Bonnier P, Charpin C, Lejeune C, Romain S, Tubiana N, Beedassy B, Martin PM, Serment H, Piana L: Inflammatory carcinomas of the breast: a clinical, pathological, or a clinical and pathological definition?. Int J Cancer. 1995, 62: 382-385. 10.1002/ijc.2910620404.View ArticlePubMedGoogle Scholar
- Moin K, Day NA, Sameni M, Hasnain S, Hirama T, Sloane BF: Human tumour cathepsin B. Comparison with normal liver cathepsin B. Biochem J. 1992, 285 (Pt 2): 427-434.PubMed CentralView ArticlePubMedGoogle Scholar
- Mohamed MM, Cavallo-Medved D, Rudy D, Anbalagan A, Moin K, Sloane BF: Interleukin-6 increases expression and secretion of cathepsin B by breast tumor-associated monocytes. Cell Physiol Biochem. 2010, 25: 315-324. 10.1159/000276564.PubMed CentralView ArticlePubMedGoogle Scholar
- Savage K, Lambros MB, Robertson D, Jones RL, Jones C, Mackay A, James M, Hornick JL, Pereira EM, Milanezi F: Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis. Clin Cancer Res. 2007, 13: 90-101. 10.1158/1078-0432.CCR-06-1371.View ArticlePubMedGoogle Scholar
- Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, Borgen PI, Clark G, Edge SB, Hayes DF: Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol. 2002, 20: 3628-3636. 10.1200/JCO.2002.02.026.View ArticlePubMedGoogle Scholar
- Carter CL, Allen C, Henson DE: Relation of tumor size, lymph node status, and survival in 24,740 breast cancer cases. Cancer. 1989, 63: 181-187. 10.1002/1097-0142(19890101)63:1<181::AID-CNCR2820630129>3.0.CO;2-H.View ArticlePubMedGoogle Scholar
- Fisher B, Bauer M, Wickerham DL, Redmond CK, Fisher ER, Cruz AB, Foster R, Gardner B, Lerner H, Margolese R: Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. An NSABP update. Cancer. 1983, 52: 1551-1557. 10.1002/1097-0142(19831101)52:9<1551::AID-CNCR2820520902>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- Cavallo-Medved D, Mai J, Dosescu J, Sameni M, Sloane BF: Caveolin-1 mediates the expression and localization of cathepsin B, pro-urokinase plasminogen activator and their cell-surface receptors in human colorectal carcinoma cells. J Cell Sci. 2005, 118: 1493-1503. 10.1242/jcs.02278.View ArticlePubMedGoogle Scholar
- Kleer CG, van Golen KL, Merajver SD: Molecular biology of breast cancer metastasis. Inflammatory breast cancer: clinical syndrome and molecular determinants. Breast Cancer Res. 2000, 2: 423-429. 10.1186/bcr89.PubMed CentralView ArticlePubMedGoogle Scholar
- Hudis CA, Seidman AD, Baselga J, Raptis G, Lebwohl D, Gilewski T, Currie V, Moynahan ME, Sklarin N, Fennelly D: Sequential adjuvant therapy with doxorubicin/paclitaxel/cyclophosphamide for resectable breast cancer involving four or more axillary nodes. Semin Oncol. 1995, 22: 18-23.PubMedGoogle Scholar
- Gianni AM, Siena S, Bregni M, Di Nicola M, Orefice S, Cusumano F, Salvadori B, Luini A, Greco M, Zucali R: Efficacy, toxicity, and applicability of high-dose sequential chemotherapy as adjuvant treatment in operable breast cancer with 10 or more involved axillary nodes: five-year results. J Clin Oncol. 1997, 15: 2312-2321.PubMedGoogle Scholar
- Bristol IJ, Woodward WA, Strom EA, Cristofanilli M, Domain D, Singletary SE, Perkins GH, Oh JL, Yu TK, Terrefe W: Locoregional treatment outcomes after multimodality management of inflammatory breast cancer. Int J Radiat Oncol Biol Phys. 2008, 72: 474-484. 10.1016/j.ijrobp.2008.01.039.PubMed CentralView ArticlePubMedGoogle Scholar
- Foekens JA, Kos J, Peters HA, Krasovec M, Look MP, Cimerman N, Meijer-van Gelder ME, Henzen-Logmans SC, van Putten WL, Klijn JG: Prognostic significance of cathepsins B and L in primary human breast cancer. J Clin Oncol. 1998, 16: 1013-1021.PubMedGoogle Scholar
- Sinha AA, Quast BJ, Wilson MJ, Fernandes ET, Reddy PK, Ewing SL, Gleason DF: Prediction of pelvic lymph node metastasis by the ratio of cathepsin B to stefin A in patients with prostate carcinoma. Cancer. 2002, 94: 3141-3149. 10.1002/cncr.10604.View ArticlePubMedGoogle Scholar
- Czyzewska J, Guzinska-Ustymowicz K, Kemona A, Bandurski R: The expression of matrix metalloproteinase 9 and cathepsin B in gastric carcinoma is associated with lymph node metastasis, but not with postoperative survival. Folia Histochem Cytobiol. 2008, 46: 57-64. 10.2478/v10042-008-0007-6.View ArticlePubMedGoogle Scholar
- Vigneswaran N, Wu J, Muller S, Zacharias W, Narendran S, Middleton L: Expression analysis of cystatin C and M in laser-capture microdissectioned human breast cancer cells--a preliminary study. Pathol Res Pract. 2005, 200: 753-762. 10.1016/j.prp.2004.09.005.View ArticlePubMedGoogle Scholar
- Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, Hashim AI, Morse DL, Raghunand N, Gatenby RA, Gillies RJ: Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer Res. 2009, 69: 2260-2268. 10.1158/0008-5472.CAN-07-5575.PubMed CentralView ArticlePubMedGoogle Scholar
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