1Division of Medical Oncology, Department of Medicine and 2Unit of Clinical Pharmacology and New Drug Development, and Divisions of 3Pathology and Laboratory Medicine, 4Senology and 5Epidemiology and Biostatistics, European Institute of Oncology, Milan; 6University of Bari, School of Medicine; 7University of Milan, School of Medicine, Milan, Italy
Received 4 September 2001; revised 27 November 2001; accepted 9 January 2002.
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Abstract |
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Male breast cancer (MBC) is an uncommon disease, and most of our current knowledge of its biology, natural history and treatment has been extrapolated from data on the disease in women. Information is still needed on the molecular biological properties of male breast tumors and their predictive relevance. Kinase inhibitor proteins (KIPs) p27Kip1 and p21Waf1 negatively regulate cell cycle progression by preventing the passage of cycling cells from G1 to S phase through G1 cyclin-dependent kinase activation. No studies exist on the role of these factors in male breast carcinoma.
Patients and methods
We have retrospectively analyzed the immunohistochemical expression of p21Waf1 and p27Kip1 protein in 27 primary MBC and in 101 female breast cancers (FBC) treated at the European Institute of Oncology between 1997 and 2000. We also assessed sex hormone receptors status, p53, bcl-2 and c-erb-B2 protein expression, and Ki-67 labeling index.
Results
We observed a statistically significant difference in the immunostaining of KIPs p27Kip1 and p21Waf1 in male patients compared with females. Expression of p21Waf1 was observed in 19 of the 27 (70.3%) primary MBCs versus 29 of 101 FBC (29%). Fourteen of 22 negative c-erbB-2 MBCs cases expressed immunostaining for p21Waf1 (P = 0.05). p27Kip1 immunoreactivity was been detected in 26 of 27 (96.2%) male breast patients versus 39 of 101 FBC (39.3%) (P = 0.000). Highly positive staining for p27Kip1 was found in 21 of 25 androgen receptor-expressing samples. Higher levels of p27Kip1 were expressed in bcl-2-positive samples (17 of 20). Eighteen of 22 c-erbB-2-negative cases were strongly immunoreactive for p27Kip1.
Conclusions
p27Kip1 and p21Waf1 immunoreactivity is higher in MBCs compared with FBCs. The findings of higher p27Kip1 and p21Waf1 immunostaining may be an additional predictive factor in MBC. These biological features could be possible indicators for different biological pathways in the tumorigenesis of MBCs.
Key words: male breast cancer, p21, p27
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Introduction |
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Human breast cancer is a biologically heterogeneous disorder, and its clinical course may vary from indolent and slowly progressive to rapidly metastatic disease. Oncogene and tumor suppressor gene abnormalities have been extensively investigated in female breast cancer (FBC) and some have been proposed as markers with potential clinical utility. Among those, genes involved in the apoptotic pathway, p53, p21Waf1, p27Kip1 and bcl-2, are likely to be of major relevance.
p53 is a nuclear protein that detects genetic damage and triggers genetic repair mechanisms. In case of irreparable DNA damage, p53 leads to initiation of apoptosis, thus preventing replication of damaged DNA [1]. Mutant p53 has a prolonged half-life compared with the wild-type protein, and therefore accumulates in the cell nucleus, thus becoming easily detectable by immunohistochemistry. Increased staining for p53 has been observed in approximately 25% of breast cancers [2, 3].
p21Waf1 is a downstream effector of p53. It is transcriptionally upregulated by p53 and inhibits cyclin-dependent kinases (cdks), and thus cell cycle progression, by preventing the passage of cycling cells from G1 to S phase [4]. p21Waf1 is therefore an essential part of the p53-mediated growth arrest pathway elicited by DNA damage [5]. It is well recognized that p21Waf1 can also be regulated by a p53-independent mechanism. Higher levels of p21Waf1 might indicate a more indolent type of breast cancer. A recent study in primary breast carcinoma showed a prolonged disease-free interval for patients with a highly p21Waf1-expressing primary tumor, and low p21Waf1 expression was associated with p53 overexpression, high tumor grade and shorter relapse-free and overall survival [6]. However, data on the prognostic relevance of p21 are conflicting.
p27Kip1 is a cdk inhibitor (CDI), which has been shown to inhibit the activity of Cyclin Acdk2, cyclin bcdk2, cyclin D cdk4 and cyclin Ecdk2 by preventing cdk activation and thereby precluding cells from entering S phase. In vivo cyclin D cdk4 or cdk6 complexes require p21 or p27 to help form active complexes [7]. Loss of p27Kip1, therefore, may contribute to oncogenesis and tumor progression [8]. Overexpression of p27Kip1 in human breast cancer cells can promote apoptosis [911]. p27Kip1 expression in breast cancer is associated with both proliferative activity and differentiation. p27Kip1 is highly expressed in those tumors with a low S-phase fraction and high estrogen receptor expression [12].
bcl-2 is a cytoplasmic protein whose transcription is regulated by p53, and whose activity is regulated by phosphorylation on Ser70, accomplished by a Ser/Thr kinase. Bcl-2 blocks apoptosis by inhibiting the release of cytochrome c (Apaf 2) and apoptosis-inducing factor (AIF) from the mitochondria to the cytoplasm, and by preventing the activation of caspase 3 (one of the central proteins of the apoptotic pathway) through inhibition of its activator protein, Apaf 1 [13]. Furthermore, it has been shown that the espression of bcl-2 can prevent programmed cell death induced by most chemotherapeutic drugs [14].
The c-erbB-2 gene, also known as HER-2/neu, encodes a transmembrane 185 kDa protein that shows homology to the epidermal growth factor receptor. HER-2 gene amplification correlates with immunohistochemical assessment of the gene product [15]. Overexpression of c-erbB-2 protein was detected in 945% of FBCs [16]. It was found to be associated with high histological grade, larger tumor size, degree of axillary lymph node involvement, low or absent sex steroid hormone receptor expression, high proliferative activity and poor patient survival. Patients with strong c-erbB-2 expression seem to have a poorer response to hormonal agents such as tamoxifen and chemotherapy not containing anthracyclines [17]. Thus, c-erbB-2 may be an important predictive marker in guiding therapy decisions, but information on this subject is controversial [18]. c-erbB-2 protein expression has also been investigated in male breast cancer (MBC). In a recent report, c-erbB-2 overexpression appeared to be a predictor of adverse survival probability [19].
MBC is an uncommon disease, and most of our knowledge of its biology, natural history and treatment has been extrapolated from data on the disease in women. Information is still needed on the molecular biological properties of male breast tumors and their predictive relevance, in order to devise treatment strategies, including optimal systemic therapy regimens. Few data are available on the role of oncogenes and tumor suppressor genes in MBC, and data on molecular markers in these patients yield controversial information [2026].
In the present study, we retrospectively analyzed the immunoreactivity of p21Waf1, p27Kip1, bcl2, p53 and c-erbB2 proteins in 27 primary MBCs and 101 FBCs. Our aim was to compare the expression of the kinase inhibitor proteins (KIPs) p27Kip1 and p21Waf1 in MBC compared with FBCs in order to investigate biological value of these biomarkers in MBCs.
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Materials and methods |
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Median age at diagnosis was 59 years (range 2686 years). All patients underwent surgery (radical or modified radical mastectomy). Adjuvant post-operative therapy was administered to 21 patients: combined adjuvant radiation, chemotherapy and hormone therapy, six patients; adjuvant radiation and endocrine therapy, seven patients; and adjuvant endocrine therapy alone with tamoxifen (proposed), eight patients. All patients were informed that the pathological material and the clinical data would be used for evaluation and reporting.
Formalin-fixed paraffin-embedded specimens were used for immunohistochemical analyses. The samples were coded, and the names of the patients were not revealed.
Tumors were classified according to the World Health Organization and staged pathologically according to the International Union Against Cancer [25, 26]. All carcinomas were ductal carcinoma; five (18.5%) were pTis, 14 (51.8%) were pT1, four (14.8%) were pT2, and three (11.1%) were pT4. Axillary lymph nodes were evaluated in all cases. Seven patients (25.9%) were classified as having node-negative disease, while 20 (74%) had nodal involvement. No patient had overt metastatic disease elsewhere at diagnosis. All tumors were pathologically graded as being well (G1; n = 7), moderately (G2; n = 14) or poorly differentiated (G3; n = 6), according to Elston and Ellis [27]. Vascular invasion was present in nine patients (33.3%).
Clinical data were obtained from hospital charts and radiotherapy. Two patients (7.4%) presented initially with gynecomastia. Clinical history of other malignancy (prior, concomitant or subsequent: three bladder, one prostate and one colon cancer) was recorded for five patients (18.5%); history of cardiovascular diseases was found in 11 patients (40.7%). Serial sections from the same tissue blocks, selected for the presence of representative tumor tissue, were cut for histological verification and for immunohistochemical assessment of sex hormone receptors, p21Waf1, p27Kip1, bcl2, p53, c-erbB2 and Ki-67 labeling.
Female breast patients
Median age at diagnosis was 48 years (range 2470 years). All patients underwent surgery (71 quadrantectomy, 30 mastectomy). Neoadjuvant chemotherapy was administered to all patients. Patients were informed that the pathological material and the clinical data would be used for evaluation and reporting.
Formalin-fixed paraffin-embedded specimens were used for immunohistochemical analyses. The samples were coded, and the names of the patients were not revealed.
All carcinomas were ductal carcinoma: 45 (45%) were pT1, 43 (43%) were pT2 and seven (7%) were pT3. Axillary lymph nodes were evaluated in all cases. Thirty-three patients (33%) were classified as having node-negative disease, while 63 (64%) had nodal involvement. No patient had overt metastatic disease elsewhere at diagnosis. All tumors were pathologically graded as being well (G1; n = 3), moderately (G2; n = 38) or poorly differentiated (G3; n = 27) [27].
Immunohistochemistry
Immunohistochemical investigations were performed on routinely processed, formalin-fixed, paraffin-embedded tissues. Consecutive 4 µm tissue sections were cut from blocks selected for the presence of representative tumor tissue and mounted on poly-L-lysine-coated slides. Prior to incubation with specific antibodies, the sections were treated with 0.1 mM citrate buffer (pH 6) under microwave irradiation for 10 min at 750 W for antigen retrieval, and with 3% hydrogen peroxide in distilled water for 5 min at room temperature to quench endogenous peroxidase activity. Non-specific binding was blocked with normal horse serum. Immunohistochemistry was performed using an automated immunostainer (TechMate 500; Dako, Glostrup, Denmark) and a peroxidase-based detection system in kit form (ChemMate; Dako) according to the manufacturers instruction.
Primary specific monoclonal antibodies to the following antigens were used at the specified dilutions: estrogen receptors (ER) (clone 1D5; Dako; 1:50); progesterone receptors (PgR) (clone 1A6; Novocastra, Newcastle-upon-Tyne, UK; 1:20); androgen receptors (AR) (clone F39.4.1; Biogenex, S. Ramon, CA, USA; 1:100); Ki-67 (clone MIB-1; Immunotech, Marseille, France; 1:100); c-erbB-2 (clone CB11; Biogenex; 1:80); p27Kip1 (clone AB1; Transduction Laboratories, Lexington, KY, USA; 1:600); p21Waf1 (clone 57; Oncogene Science, Cambridge, MA, USA; 1:100); p53 (clone D0-7; Dako; 1:1000); and bcl-2 (clone 124; Dako; 1:200). After immunostaining, sections were counterstained with 1% modified Harris hematoxylin, dehydrated and mounted. Sections from FBCs and normal prostate with known immunoreactivity for all antigens were used as positive controls. A strong nuclear staining of lymphocytes and stromal cells provided an additional internal positive control for preservation of the p27Kip1 and p21Waf1 immunogenicity in most cases examined. Normal mouse serum was substituted for primary antibodies as a negative control.
Scoring
Immunoreactivity for ERs, PgRs and ARs was scored according to the percentage of neoplastic cells. We chose a relative staining intensity >10% to identify hormone receptor-positive tumors.
p21Waf1 and p27Kip1 immunoreactivity was scored in four groups according to the percentage of neoplastic cells showing definite nuclear staining. Cases were categorized as negative when <1% of cells were immunostained, low when 125%, moderate when 2650%, and intense when >50% were immunostained. Within tumor cells, the staining reaction for p27Kip1 was often localized both in the nucleus and in the cytoplasm, but the nuclear staining was predominant and, in most cases, exclusive.
The immunoreactivity for bcl-2 was dichotomized according to the staining of more or less than 30% neoplastic cells.
For Ki-67 labeling, the absolute percentage of the the neoplastic cells showing nuclear immunoreactivity was recorded. The median Ki-67 labeling index was assessed as 20%, and this was used as cut-off point to dicothomize cases with low and high proliferative fraction.
For HER-2 only, the score was assessed as follows. No staining at all, or membrane staining in <10% of the observed tumor cells was considered negative (0). A feint/barely perceptible membrane staining in >10% of tumor cells or staining of part of their membrane was scored as negative (1+). A weak to moderate staining of the entire membrane in >10% of the tumor cells was considered weakly positive (2+). A moderate to strong staining of the entire membrane in >10% of the tumor cells was been scored as strongly positive (3+).
Depending on the percentage of nuclei exhibiting positive staining, tumors were categorized for p53 as follows: negative/patchy (0% to 10% of positive nuclei), positive (>10%).
Scoring of immunostaining results was independently performed by two pathologists (G.R. and G.V.), who had no knowledge of the tumor clinicopathological data and patient survival. In each case, at least 2000 tumor cells were counted from 10 randomly selected areas, ensuring that the whole section was scanned. When the scoring discrepancy between the examiners was >10%, a consensus interpretation was reached after re-examination of the slides with a double-headed microscope.
Statistical analyses
The comparison of the biological markers in men and women was accomplished using Fishers exact test. Log linear models were used to test for differences in the level of the biological markers between men and women adjusting for grade and T stage, using linear interaction tests. The association between tumor biomarker expression and other clinicopathological variables was calculated using contingency table methods and tested for significance using the linear by linear association test [28]. All P values were based on two-sided testing. Exact tests were used because of the small number of patients (StatExact 4, 1998). Testing that the association was the same in men and women was accomplished using a log linear model. This comparison, in particular, has low power as it is a three-way interaction test and there were fewer men than women.
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Results |
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Immunoreactivity for p21Waf1 was observed in 19 of 27 (70.3%) primary MBCs. A mild immunostaining (125% stained cells) was detected in 14 of 19 positive samples. Five samples showed a moderate staining (2650%).
No significant correlation was found between p21Waf1 immunoreactivity and tumor stage or grade. In fact, positive staining was observed in four of five (80%) pTis, 10 of 14 (71%) pT1 and one of two (50%) pT4 tumors (P = 0.3). Similarly, p21Waf1 was found in three of the seven (57.1%) well differentiated (G1), 11 of 14 (78.5%) moderately differentiated (G2) and in five of six (83.3%) poorly differentiated tumors (P = 0.1).
A statistically significant inverse correlation was observed between p21Waf1 immunoreactivity and HER-2 overexpression. Fourteen of 22 (63.6%) negative c-erbB-2 cases showed immunostaining for p21Waf1 (P = 0.05). A borderline statistically significant relationship was ascertained between p21Waf1 and Ki-67 labeling (P = 0.09). Ten of 11 cases (90.9%) with high proliferative fraction (expressing >20% Ki-67) also had p21Waf1 immunoreactivity.
Immunoreactivity for p21Waf1 was observed in 29 of 101 (29%) primary FBCs. A statistically significant difference was when comparing FBCs with MBCs (P <0.01).
p27Kip1 expression was evaluated within tumor cells in all cases. Only cells with a clear nuclear staining were considered positive. The percentage of positive tumor cells within a microscopic field ranged from 099%, and intratumoral heterogeneity of p27Kip1 nuclear immunostaining was common. In all tissue sections there were sufficient normal mammary epithelial cells that could also be evaluated and, in these cases, normal glands also gave immunostaining scores of 13. Normal myoepithelial cells, stromal cells and lymphocytes were also occasionally positive.
p27Kip1 immunoreactivity was detected in 26 of 27 cases (96.2%). Intense (>50% positive cells), moderate (2550%) and low (<25%) p27Kip1 staining were observed in 21 (77.7%), two (7.4%) and three (11.1%) of the primary MBCs, respectively. Intense or moderate p27Kip1 immunoreactivity was found in six of seven (85.7%) well differentiated, 12 of 14 (85.7%) moderately differentiated and five of six (83.3%) poorly differentiated tumors. We emphasize that this set of samples is relatively small. However, a statistically significant correlation was found between AR status and intense staining for p27Kip1 (P = 0.0499). Score 3 staining for p27Kip1 was found 21 of 25 (84%) AR-expressing tumors.
Intense immunoreactivity for p27Kip1 was more prevalent in bcl-2-positive samples (17/20, 85%; P = 0.09). Eighteen of 22 (81.8%) negative c-erbB-2 cases expressed score 23 immunostaining for p27Kip1 (P = 0.81).
p27Kip1 immunoreactivity was detected in 67 of 101 FBC cases (67%). Intense (>50% positive cells), moderate (2550%) and low (<25%) p27Kip1 staining were observed in eight (8%), 31 (31%) and 28 (28%) of the primary FBCs, respectively.
Analysis of sex hormone receptor status, Ki-67, p53, bcl-2 and c-erbB2
ER-, PgR- and AR-positive immunostaining was detected in 27 (100%), 24 (88.8%) and 26 (96.2%) of the MBC cases, respectively. No significant relationship was observed between steroid hormone receptor status and all clinicopathological variables. In FBCs we observed 63 ER-positive and 38 PgR-positive samples.
The median labeling for Ki-67 for the whole series was 20% (range 136%). Ki-67 score variations were observed in relation to grading. Ki-67 scores were higher in moderate or poorly than in well differentiated tumors (P = 0.02).
Normal epithelium demonstrated no immunoreactivity for p53. Twenty-six of 27 (96.2%) MBC samples had nuclear staining in <10% positive cells. Eighty-five of 101 (85%) FBC had negative nuclear staining.
bcl-2 immunoreactivity was detected in 20 (74.07%) of the cases, with all cases expressing >30% immunostaining. In the FBC group, 69 (70%) patients showed >30% immunoreactivity for bcl-2.
HER-2 overexpression was observed in four of 27 (14.8%) samples. Weak to moderate and moderate to strong staining of the entire membrane in >10% of tumor cells was found in one and three samples, respectively. HER-2 immunostaining did not display a significant correlation with any parameter analyzed. Out of 101 FBC samples, HER-2 overexpression was observed in nine (9%) patients. Scores of 1+ and 2+ were observed in six and nine patients, respectively.
The comparative analysis of biological parameters investigated in male and female breast cancer is shown in Table 2.
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Discussion |
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In our analysis, upregulation of p27Kip1 and p21Waf1 was more frequently observed in MBC compared with FBC. In fact, expression of p21Waf1 was observed in 19 of 27 (70.3%) primary MBCs. p27 reactivity was detected in 26 of 27 male breast patients (96.2%). The significance of the association between p27Kip1 and p21Waf1 and gender was not influenced by adjustments for grade or T stage.
Reports on FBC showed immunoreactivity of p21Waf1 and p27Kip1 in 32 and 44% of cases, respectively, of large FBC series [6, 12]. In our series we observed immunoreactivity of p21Waf1 and p27Kip1 in 29 and 39% of patients, respectively. In previous studies, p21Waf1 loss and p53 overexpression were associated with shorter disease-free survival [6]. It was also reported that reduced expression of p27Kip1 predicted poor survival in FBC [12]. Catzavelos et al. [29] found, in a study of 168 primary FBC patients, that p27Kip1 expression decreased with increasing tumor grade, and that reduced p27Kip1 expression was a strong predictor for shorter disease-free survival. Porter et al. [30] studied 246 young women and analyzed by immunostaining the levels of expression of both p27Kip1 and cyclin E in their primary breast cancer tissue. Low or absent levels of p27Kip1 were a strong predictor of poor outcome, expecially when associated with upregulation of cyclin E.
We observed a statistically significant inverse correlation between p21Waf1 expression and c-erbB-2. p21Waf1 was more frequently expressed (64%) in c-erbB-2-negative patients. p53-negative samples showed p21Waf1 overexpression in at least 70% of the cases. Similar results were observed for p27Kip1. Higher scores of immunostaining for p27Kip1 were expressed in 82% of the patients without c-erbB-2 overexpression compared with those whose tumors were classified as c-erbB-2 positive. p53-negative samples showed p27Kip1 overexpression in 77% of the cases. In 85% of bcl-2-positive samples we found higher levels of p27Kip1.
The percentage of c-erbB-2 overexpressing tumor immunopositivity in our series (15%) was rather similar to that found in most FBC [16], but different from recent data in MBC reporting a 56% incidence [19]. We did not find any association between c-erbB-2 expression and the clinical parameters analyzed. These conflicting results may be due in part to different scoring systems and cut-off values. Our results fitted biologically with a concomitant overexpression of CDIs in this subset of patients.
Only a few MBCs were p53 positive (4%). A 10% cut-off was applied to our series. We observed that MBCs express c-erbB-2 and p53 proteins very infrequently, and found a low simultaneous expression of p53 and c-erbB-2 proteins. A larger number of cases are necessary to test the reliability of this observation. As showed by Pich et al. [19], when 50 male breast patients were grouped according to HER-2 and p53 co-overexpression, all nine patients who did not express c-erbB-2 and p53 proteins were alive at 58 months median follow-up, whereas none of the 14 patients whose tumors expressed both proteins survived after 61 months.
bcl-2 immunoreactivity was detected in 20 (74.07%) of our cases. This finding was similar to the observations of others [19]. There is experimental evidence that estradiol exposure of MCF-7 breast cancer cells significantly increases bcl-2 protein expression and that anti-estrogen tamoxifen induces apoptosis, downregulating bcl-2 [31, 32]. All of these data justify the indication of endocrine therapy in MBC.
Data on treatment results summarized in a recent review on MBCs indicate that, during the last two decades, despite an overall increased use, endocrine therapy and chemotherapy are employed in the adjuvant treatment for MBC less often than for FBC [24]. Furthermore, in this report, the duration of adjuvant endocrine therapy was often <2 years [24]. Studies on FBC with ER-positive disease have conclusively shown that adjuvant treatment with tamoxifen improves overall survival, and that 5 years of adjuvant tamoxifen is more beneficial than 2 years [3335]. It is therefore reasonable to imply that a frequent use of adjuvant tamoxifen, prescribed for 5 years, in MBC may improve the survival of patients with ER-positive disease.
Our data suggest that the immunohistochemical evaluation of p21Waf1 and p27Kip1 expression in male breast carcinomas may be a further useful prognostic marker. The biological features observed could be possible indication for different biological pathways in tumorigenesis of MBC.
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Acknowledgements |
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Footnotes |
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References |
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2. Trock BJ. Molecular epidemiology. In Harris JR, Lippman ME, Morrow M, Hellman S (eds): Diseases of the Breast. Philadelphia, PA: Lippincott-Raven 1996; 213220.
3. Leibermann DA, Hoffman B, Steinman RA. Molecular control of growth arrest and apoptosis: p53-dependent and independent pathways. Oncogene 1995; 11: 199210.[ISI][Medline]
4. Elledge RM, Allred DC. Prognostic and predictive value of p53 and p21 in breast cancer. Breast Cancer Res Treat 1998; 52: 7998.[ISI][Medline]
5. McClelland RA, Gee JM, OSullivan L et al. p21waf1 expression and endocrine response in breast cancer. J Pathol 1999; 188: 126132.[ISI][Medline]
6. Caffo O, Doglioni C, Veronese S et al. Prognostic value of p21 and p53 expression in breast carcinoma: an immunohistochemical study in 261 patients with long term follow up. Clin Cancer Res 1996; 2: 15911599.[Abstract]
7.
Cheng M, Olivier P, Diehl JA et al. The p21(Cip1) and p27(Kip1) CDK inhibitors are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 1999; 18: 15711583.
8. Sgambato A, Cittadini A, Faraglia B, Weinstein IB. Multiple functions of p27(Kip1) and its alterations in tumor cells: a review. J Cell Physiol 2000; 183: 1827.[ISI][Medline]
9. Polyak K, Lee MH, Erdjument-Bromage H et al. Cloning p27Kip1, a cyclin dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 1994; 78: 5966.[ISI][Medline]
10. Alessandrini A, Chiaur DS, Pagano M. Regulation of the cyclin-dependent kinase inhibitor p27 by degradation and phosphorylation. Leukemia 1997; 11: 342345.[ISI][Medline]
11. Katayose Y, Kim M, Rakkar AN et al. Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27. Cancer Res 1997; 52: 54415445.
12. Gillett CE, Smith P, Peters G et al. Cyclin-dependent kinase inhibitor p27kip1 expression and interaction with other cell cycle-associated proteins in mammary carcinoma. J Pathol 1999; 187: 200206.[ISI][Medline]
13. Lasorella A, Iavarone A, Usrael MA. Differentiation of neuroblastoma enhances Bcl-2 expression and induces alterations of apoptosis and drug resistance. Cancer Res 1995; 55: 47114716.[Abstract]
14. Bonetti A, Zaninelli M, Leone R et al. Bcl-2 but not p53 expression is associated with resistance to chemotherapy in advanced breast cancer. Clin Cancer Res 1998; 4: 23312336.[Abstract]
15. Coussens L, Yang-Feng TL, Tiao TL et al. Tyrosine kinase receptor with extensive homology to EGF receptors shares chromosomal location with neu oncogene. Science 1985; 230: 11321139.[ISI][Medline]
16. Piccart MJ, Di Leo A, Hamilton A. HER2. A predictive factor ready to use in the daily management of breast cancer patients? Eur J Cancer 2000; 36: 17551761.[ISI][Medline]
17. Wright C, Nicholson S, Angus B et al. Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Br J Cancer 1992; 65: 118121.[ISI][Medline]
18.
Berry D, Muss H, Thor A et al. HER-2/neu and p53 expression versus tamoxifen resistance in estrogen-receptor positive, node-positive breast cancer. J Clin Oncol 2000; 18: 34713479.
19.
Pich A, Margaria E, Chiusa L. Oncogenes and male breast carcinoma: c-erbB-2 and p53 coexpression predicts a poor survival. J Clin Oncol 2000; 18: 29482956.
20. Payne S, Bruce DM, Heys SD et al. Prognostic parameters in male breast cancer. J Pathol 1994; 175 (Suppl): (Abstr 143).
21. Bruce DM, Payne S, Heys SD et al. Male breast cancer: clinico-pathological features, immunocytochemical characteristics and prognosis. Eur J Surg Cancer 1996; 32: 4246.
22. Rayson D, Erlichman C, Suman VJ et al. Molecular markers in male breast carcinoma. Cancer 1998; 83: 19471955.[ISI][Medline]
23. Ravandi-Kashani F, Hayes TG. Male breast cancer: a review of the literature. Eur J Cancer 1998; 34: 13411347.[ISI][Medline]
24. Goss P, Reid C, Pintilie M et al. Male breast carcinoma: a review of 229 patients who presented to the Princess Margaret Hospital during 40 years: 19551996. Cancer 1999; 85: 629639.[ISI][Medline]
25. Clark JL, Nguyen PL, Jaszcz WB et al. Prognostic variables in male breast cancer. Am Surg 2000; 66: 502511.[ISI][Medline]
26. Scarff RW, Torloni H. Histological typing of breast tumors. International histological classification of tumors, No. 2. Geneva: World Health Organization 1968.
27. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow up. Histopathology 1991; 19: 403410.[ISI][Medline]
28. Agresti A. Categorical Data Analyis. New York, NY: Wiley 1990.
29. Catzavelos C, Bhattacharya N, Ung Y et al. Decreased levels of the cell-cycle inhibitor p27 protein: prognostic implications in primary breast cancer. Nat Med 1997; 3: 227230.[ISI][Medline]
30. Porter P, Malone K, Heagerty P et al. Expression of cell-cycle regulators p27 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nature Med 1997; 3: 222225.[ISI][Medline]
31.
Cariou S, Donovan J, Flanagan WM et al. Down regulation of p21 or p27 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl Acad Sci USA 2000; 97: 90429046.
32.
Zhang GJ, Kimijima I, Onda M et al. Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X, without alteration of p53 protein levels. Clin Cancer Res 1999; 5: 29712977.
33. Gupta N, Cohen J, Rosenbaum C. Estrogen receptors in male breast cancer. Cancer 1980; 46: 17811784.[ISI][Medline]
34. Early Breast Cancer Trialist Cooperative Group. Treatment of early breast cancer: worldwide evidence in 19851990. A systematic overview of all avalaible randomized trials in early breast cancer of adjuvant endocrine and cytoxic therapy. Treatment of early breast cancer, Vol. 1. Oxford, UK: Oxford University Press 1990.
35.
Swedish Breast Cancer Cooperative Group. Randomized trial of two versus five years of adjuvant tamoxifen for postmenopausal early stage breast cancer. J Natl Cancer Inst 1996; 88: 15431549.