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Annals of Surgical Oncology 8:667-674 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Lysozyme Expression by Breast Carcinomas, Correlation With Clinicopathologic Parameters, and Prognostic Significance

Francisco Vizoso, PhD, Elena Plaza, MD, Julio Vázquez, PhD, Carlos Serra, PhD, María L. Lamelas, PhD, Luis O. González, MD, Antonio M. Merino, PhD and Jaime Méndez, PhD

From the Servicios de Cirugía General (FV), Anestesia (EP), Ginecología (JV), and Anatomía Patológica (LOG) del Hospital de Jove de Gijón, Spain; Servicio de Cirugía General (CS) del Hospital Virgen de los Lirios de Alcoy, Alicante, Spain; Servicio de Anatomía Patológica (AMM) del Hospital de Cabueñes de Gijón, Spain; and Servicio de Cirugía General B (JM) del Hospital de Basurto, Bilbao, Spain.

Correspondence: Address correspondence and reprint requests to: Dr. Francisco Vizoso, Servicio de Cirugía General, Hospital de Jove, Avda. Eduardo Castro s/n, 33290 Gijón, Spain; Fax: 34-985-315710; E-mail: vazquezj{at}sego.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Here we evaluate the expression and prognostic value of lysozyme, a milk protein that is also synthesized by a significant percentage of breast carcinomas, in women with breast cancer.

Methods: Lysozyme expression was examined by immunohistochemical methods in a series of 177 breast cancer tissue sections. Staining was quantified by using the HSCORE system, which considers both the intensity and the percentage of cells staining at each intensity. The prognostic value of lysozyme was retrospectively evaluated by multivariate analysis that took into account conventional prognostic factors.

Results: A total of 126 of 177 carcinomas (69.4%) stained positive for this protein, but there were clear differences among them with regard to the intensity and percentage of stained cells. Lysozyme values were higher in well-differentiated and moderately differentiated tumors than in poorly differentiated tumors (P < .05). Similarly, lysozyme levels were higher in small and node-negative tumors than in large and node-positive tumors (P < .05). Moreover, results indicated that low lysozyme content predicted shorter relapse-free survival and overall survival (P < .005). Separate Cox multivariate analysis in subgroups of patients as defined by node status showed that lysozyme expression was an independent prognostic factor able to predict both relapse-free survival and overall survival in node-negative patients (P < .05).

Conclusions: Tumoral expression of lysozyme is associated with lesions of favorable evolution in breast cancer. This milk protein may be a new prognostic factor in patients with breast cancer.

Key Words: Lysozyme • Breast cancer • Prognosis • Prolactin receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysozyme, also termed muramidase, is a ubiquitous enzyme discovered by Fleming1 in 1922. It acts on ß1 to ß4 glucosidic bonds in peptidoglycans of the bacterial wall. It is believed to play a role in the primitive nonspecific defense mechanism associated with the monocytic-macrophagic system.2,3 There is a clear relationship between lysozyme and breast physiology, because it is a protein component of milk from lactating women. In addition, it has been reported that nearly 98% of healthy nonlactating women show detectable lysozyme in their breast secretions.4

We have previously demonstrated that breast secretions from nonlactating women may be classified into two types, according to their major protein components. Type I secretions, characterized by containing Zn-{alpha}2-glycoprotein, apolipoprotein D, and gross cystic disease fluid protein-15, are found in most women without breast pathology or with benign breast diseases. Type II secretions are characterized by the presence of lactoferrin, lysozyme, and {alpha}-lactalbumin as major protein components.5 This type II protein pattern is found in the majority of women who have given birth in the last 4 years and in a high proportion of oral contraceptives users, and also in a significant percentage (47%) of breast cancer patients, but in only 7% of healthy women after excluding these groups.6 In addition, it has also been demonstrated that this type II polypeptide pattern is associated with a peak of prolactin secretion after the thyroid-releasing hormone stimulation test in premenopausal nonlactating women.7

Five of these six major protein components from breast secretions, Zn-{alpha}2-glycoprotein,8,9 apolipoprotein D,10 gross cystic disease fluid protein-15,11 lactoferrin,12,13 and {alpha}-lactalbumin,14 are expressed by significant but variable percentages of breast carcinomas. In addition, the expression of apolipoprotein D by these tumors has been associated with a good prognosis of patients.10 However, at present, no study has addressed the expression of lysozyme and its clinical significance in breast carcinomas. The aim of this work was to evaluate the expression of lysozyme by breast carcinomas and assess the potential relationship with clinicopathologic parameters of tumors and its prognostic significance in patients with breast cancer.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
This study was performed on a group of 177 women (mean age, 59.3 years; range, 26–89 years) with histologically verified invasive ductal breast cancer diagnosed and treated at Hospital de Jove (Gijón, Spain) between 1983 and 1995. All of them were previously untreated and without other malignant tumors at the time of diagnosis. Patient characteristics with respect to age, menopausal status, and clinical staging of tumors are listed in Table 1. Histological grade of tumors was determined according to criteria reported by Bloom and Richardson,15 whereas nodal status was assessed histopathologically. Estrogen receptor (ER) content was measured in cytosol extracts by using a commercially available kit from Abbot Laboratories (North Chicago, IL). Breast cancers were considered ER positive if they contained >10 fmol/mg total protein.


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  		TABLE 1. Lysozyme HSCORE values classified according to different characteristics of patients and tumors
 
Radical or modified radical mastectomy with radical axillary dissection was performed in all patients included in the study. This was because the modified radical mastectomy was the standard surgical treatment for breast cancer in our department during the majority of the study period. Evaluation of relapse-free survival (RFS) and overall survival (OS) was performed on the group of 166 patients without evidence of metastasis and with a minimum follow-up period of 1 year. In this group, postoperative locoregional radiotherapy was given to 60 of 166 patients (35.7%) with central or medial tumors or positive axillary nodes. Adjuvant systemic therapy with cyclophosphamide, methotrexate, and 5-fluoruracil (CMF) was given to 62 of 166 patients (36.9%), and adjuvant tamoxifen was given to 59 of 166 patients (35.1%). Tamoxifen was not given to all patients with ER-positive tumors because this therapeutic criterion was not established during the first years of the study. All patients were followed for disease recurrence and survival status by clinical, radiological, and biological studies every 3 months for the first 2 years and then yearly. Mean follow-up period for patients with node-positive carcinomas was 58.6 months and 45.7 months for those with node-negative tumors. Sixty-seven of 166 patients developed tumor recurrence, and 52 of them died from recurrence. In addition, five women died from causes unrelated to breast cancer.

Lysozyme Purification and Antiserum Production
Lysozyme was purified from milk of lactating women according to the high-performance liquid chromatography procedure. The purity of the obtained antigen was confirmed by automatic Edman degradation after treatment of the protein with pyroglutamate aminopeptidase. Antiserum against the purified protein was raised in New Zealand White rabbits by following the method described by Vaitukaitis.16 The immunized rabbits were bled 6 weeks after protein injection, and the obtained serum was dialyzed for 24 hours at 4°C against 20 mM phosphate buffer, pH 7.2. Then the dialyzed material was chromatographed in a column of diethylaminoethyl cellulose equilibrated and eluted in the same phosphate buffer. Finally, the immunoglobulin G–containing fractions were collected and stored at -20°C until used.

Immunohistochemical Staining
All specimens of biopsy were fixed in 10% neutral buffered formalin and stored after embedding in paraffin at room temperature from 5 months to 8 years before further testing. Immunohistochemical assays were performed on 6-µm sections by using the streptavidin-biotin supersensitive method (Biogenex, San Ramon, CA). Incubation with antiserum against lysozyme (diluted 1/200 in 20 mM phosphate buffer, pH 7.2, and 1% bovine serum albumin) was performed at room temperature for 30 minutes. Then, slides were incubated for 20 minutes with the second biotinylated antibody obtained from Biogenex. Endogenous alkaline phosphatase was blocked with levamisole (diluted 1/50). The streptavidin-alkaline phosphatase complex reagent (Biogenex) was performed for 20 minutes at room temperature, and the reaction was developed with fast red in Tris buffer, pH 7.2, with naphthol-phosphate. Later, the slides were contrasted with Mayer hematoxylin and mounted in Aquatex (Merck, Darmstadt, Germany). Specificity of staining was determined by using controls that involved incubation of tissue sections with buffer alone or with an equal amount of immunoglobulin G from nonimmunized rabbits. In both cases, there was no significant staining. Furthermore, immunostaining was completely abolished by antiserum preincubation with lysozyme purified from maternal milk as described previously. Tissue sections were scored in a semiquantitative fashion according to the method described by McCarty et al.,17 which considers both the intensity and percentage of cells staining at each intensity. Intensities were classified from 0 (no staining) to 3 (very strong staining), whereas 10% groupings were used for the percentage of cells that stained positive. For each slide, a value designated as HSCORE was obtained after the application of the following algorithm: HSCORE = {Sigma} (I + 1) x PC, where I and PC represent the intensity and the percentage of cells that stained at each intensity, respectively. The immunostained sections were evaluated independently by two pathologists without knowledge of patients’ clinical data at the time of review. Reproducibility of the scoring method between two observers was >90%. In the remaining cases, in which discrepancies had been noted, differences were settled by consensus review of corresponding slides.

Statistical Analysis
For analysis of data, patients were subdivided into groups on the basis of different clinical or pathologic criteria. Analysis of differences in lysozyme values between two groups was performed with the Mann-Whitney U-test. Relationships between more than two groups were evaluated by the Kruskal-Wallis test. In the univariate study, curves for RFS and OS rates were calculated with the Kaplan-Meier18 method and compared by use of the log-rank test.19 Cox’s regression model20 was also used to examine several combinations and interactions of prognostic factors in a multivariate analysis. The following variables were included in the analysis: menopausal status, tumor size, histological grade, and nodal status. ER status was not included because of the absence of corresponding data in a significant number of tumors. In addition, separate multivariate analyses were also performed for node-negative and node-positive subgroups. Selection of prognostic variables was performed with Cox’s model by using the stepwise regression option of BMDP (program 2L) software.21 The P value to enter or remove covariates was 5%. The scoring for covariates as menopausal status and nodal status were 1 or 2. However, tumor size was scored at 4 levels and histological grade at 3 levels. Statistical significance was defined as P < .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 126 of 177 (69.4%) carcinomas stained positive for lysozyme, although there was a wide variability in the intensity and percentage of positivity. Immunostaining was localized in the cytoplasm of tumoral cells. Representative examples of positive and negative staining are shown in Fig. 1. Adjacent normal epithelium was present in 36 analyzed tissue sections, of which 15 cases (41.6%) showed a positive immunostaining for lysozyme, which was a diffuse and intracytoplasmic pattern in the epithelial cells. As indicated in Fig. 2, HSCORE values in breast carcinomas varied from 0 to 360, with an average of 104.1 ± 6.9. In the group of 126 lysozyme-positive tumors, a total of 37 positive tumors were weakly stained (HSCORE <100), 59 were moderately stained (HSCORE between 100 and 200), and the remaining 30 were strongly positive (HSCORE >200). However, there was no significant difference in lysozyme staining results among the several groups of tissue samples according to their time of storage (data not shown).



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  		FIG. 1. Immunohistochemical staining of lysozyme in human breast cancer. (A) Positive tumor. (B) Negative tumor (original magnification x200).

 


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  		FIG. 2. Distribution of HSCORE values obtained by immunohistochemical staining of lysozyme in 177 human breast carcinomas.

 
Distribution of lysozyme values according to patient and tumor characteristics, which included menopausal status, tumor size, axillary node involvement, and histological grade is listed in Table 1. Statistical analysis showed that lysozyme values were significantly correlated with tumor size, nodal status, and histological grade. Thus, lysozyme levels were higher in the small (T1 = 114.3; T2 = 117.9) and the node-negative tumors (N0 = 120.9) than in the large tumors (T3 and 4 = 74.9) and node-positive tumors (N+ = 86.6; P < .05). In addition, the average content of lysozyme was higher in the well-differentiated (grade I = 116.6) and moderately differentiated tumors (grade II = 109.9) than in the poorly differentiated tumors (grade III = 61.2; P < .05).

These results show a relationship between lysozyme content and a favorable outcome of patients with breast cancer. To examine this hypothesis, the potential association between lysozyme immunostaining and RFS and OS was retrospectively evaluated in 166 women without metastasis included in this study. First, we defined the optimal cutoff value by statistical analysis of the ability of lysozyme values for predicting the RFS of the study population. The results showed a continuous association between 80 and 230 HSCORE values and relapse rate (P < .05 for HSCORE values of 80, 90, 120, and 130 and between 180 and 220, and P < .01 for values of 100, 110, and 230). However, as shown in Fig. 3, this analysis led us to define a HSCORE of 100 as the optimal cutoff ({chi}2 = 11.03; P = .0009) with the ability to identify 55.3% of patients as having low or negative lysozyme values. Considering this cutoff value, relapse was confirmed in 41 of 84 patients (48%) with lysozyme-negative tumors but in only 26 of 82 (31%) with lysozyme-positive tumors. Similarly, there were 34 deaths (41%) caused by the disease in patients with lysozyme-negative tumors during the study period and only 18 deaths (21%) in patients whose tumors showed positive immunostaining. Differences between both RFS and OS curves were significant (P = .0009 and P = .0003, respectively; Fig. 4).



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  		FIG. 3. Determination of the cutoff value of lysozyme able to predict relapse-free survival in breast cancer; {chi}2 values obtained for each cutoff value are plotted against the value itself.

 


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  		FIG. 4. Relapse-free and overall survival as a function of lysozyme values in patients with breast cancer. Mean follow-up period was 52.6 months. Differences in relapse-free and overall survival curves were significant at P < .005.

 
The analysis of other well-established prognostic indicators in breast cancer revealed that, as expected, tumor size, axillary lymph node involvement, and histological grade were also significantly associated with relapse and survival in our study population (Table 2). However, multivariate analysis according to Cox’s model confirmed that lysozyme was significantly associated with RFS and OS (P < .005 for both; Table 3). In addition, tumor size and nodal status were also independent prognostic factors for both RFS and OS (P < .05 and P < .01, respectively).


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  		TABLE 2. Univariate analysis of the association of lysozyme with relapse-free and overall survival
 

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  		TABLE 3. Multivariate analysis of association of lysozyme with relapse-free and overall survival
 
Women included in this study were subdivided into two groups according to node status. The possible relationship between lysozyme levels and clinical outcome of disease was examined in both groups. In the node-positive group, relapse was confirmed in 27 of 55 patients (49%) with lysozyme-negative tumors and in 16 of 32 patients (50%) with lysozyme-positive tumors. However, there were 22 deaths (40%) in the group of patients with lysozyme- negative tumors and 14 deaths (44%) in lysozyme-positive tumors during the study period. These differences were significant for OS (P < .05) but not for RFS in the univariate analysis. In addition, lysozyme expression did not reach statistical signification in the multivariate analysis. Only tumor size was an independent prognostic factor in the group of node-positive patients.

In the group of node-negative patients, relapse was observed in 14 of 39 patients (35.8%) with lysozyme-negative tumors but in only 10 of 50 (20%) with lysozyme-positive carcinomas. However, there were 12 deaths (30.7%) because of recurrence in patients with tumors that stained negative for lysozyme and 4 deaths (8%) in lysozyme-positive tumors. These differences were significant at P < .01 and P < .05 for RFS and OS, respectively (Fig. 5 and Table 2). Multivariate analysis confirmed that lysozyme was significantly associated with RFS and OS in this group of node-negative patients (P < .05 for both; Table 3).



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  		FIG. 5. Relapse-free and overall survival as function of lysozyme values in node-negative breast cancer patients. Differences between relapse-free survival curves were significant at P < .01 and for overall survival at P < .05.

 
Finally, we also analyzed the possible correlation between systemic therapy use and lysozyme expression. Our results did not show significant differences in lysozyme expression between tumors of the patients who received postoperative CMF (n = 62; mean ± SE, 93.9 ± 9.9) and those of patients without this type of systemic therapy (n = 104; 104.4 ± 9.9). Similarly, there was no significant difference in lysozyme content between tumors of the patients with tamoxifen adjuvant therapy (n = 59; 108 ± 11.5) and those of patients without this therapy (107; 95.1 ± 9.2). In addition, lysozyme expression was not a marker of drug sensitivity in our study population of patients with breast cancer (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is, to our knowledge, the first clinical study of the tumoral expression and clinical significance of lysozyme in breast carcinomas. Our results demonstrate that lysozyme is expressed by a significant percentage of breast carcinomas and demonstrate the existence of a significant relationship between this milk protein and clinical and pathologic parameters of tumors, as well as with prognosis of patients.

After demonstrating that lysozyme is one of the major protein components of breast secretions obtained from the nipples of approximately 47% of patients with breast cancer,5,6 we were prompted to examine the potential value of lysozyme in breast carcinomas. Sixty-nine percent of these tumors showed a positive immunostaining for the protein; this is a higher percentage than that previously reported for breast secretion in breast cancer patients. However, there was a wide variability of lysozyme immunostaining values, and this variability seems to be caused by the biological heterogeneity of these tumors. Thus, we first observed that lysozyme values were significantly higher in small or node-negative tumors than in large or node-positive tumors. In addition, the results also showed a significant and independent relationship between levels of this protein and both RFS and OS of patients. Although nodal status was a stronger predictor for outcome than lysozyme expression in the overall group of patients, our results also show that lysozyme was significant and independently associated with both RFS and OS in patients with node-negative breast cancer. Thus, our results suggest that, although nodal status is still the best prognostic indicator, lysozyme may be helpful as a prognostic factor in the important subgroup of node-negative patients.

Several aspects of lysozyme according to cellular differentiation may provide biological support to the clinical data presented here. Although it has been reported that lysozyme is found in 98% of normal breast secretions of nonlactating women,4 this finding used a sensitive method that permits detection of the lowest levels of the protein produced in some part of the breast epithelium. In addition, the finding of this study, that a 41.6% of the normal breast epithelium adjacent to breast carcinomas shows lysozyme immunostaining, is in according with our prior report that lysozyme is a major protein component in breast secretions obtained by nipple in a similar percentage of women with breast cancer (47%).5,6 Thus, considering that lysozyme may be expressed by normal breast epithelium, the finding of lysozyme expression in a subset of breast carcinomas indicates that they possess the required degree of functional differentiation to synthesize the protein. Consequently, a possible explanation of why lysozyme confers a prognostic advantage to breast carcinoma is that its presence may reflect the existence of a complete hormone receptor pathway.

Milk proteins are synthesized by the mammary epithelium in response to a complex hormonal release during pregnancy and lactation. Although different corticosteroid and peptide hormones cooperate in this process, it is widely accepted that prolactin plays a primary role by increasing transcription of milk genes.22 Thus, considering that it has been demonstrated that breast cancer cells may show the ability to synthesize a significant amount of biologically active prolactin,23 we may speculate that prolactin could be responsible of the expression of lysozyme by these functionally well-differentiated tumors. In addition, several authors have described prolactin-receptor (PR) expression in approximately 50% of breast carcinomas24–27 and have claimed that this tumoral receptor expression is more intense in the tumoral tissue than in the adjacent normal breast tissue.28 However, the presence of these hormonal receptors does not necessarily implicate sensitivity to prolactin. Thus, it has been reported that not all PR-positive breast tumors respond to prolactin stimulation by means of an increment of DNA synthesis.29 It has been suggested that the determination of prolactin-inducible proteins, rather than prolactin-receptor content, may represent a more optimal method for evaluating the sensitivity of breast tumors to this hormone.30,31

In agreement with these considerations, lysozyme could be used to identify a subset of patient candidates to receive an alternative hormonal treatment with PR blocking. It has been demonstrated the inhibition of breast cancer cell growth by using PR antagonists32 and it has been demonstrated that antiprolactin therapy may increase the therapeutic efficiency of antiestrogens in breast cancer patients with metastatic disease.33

In summary, these preliminary findings suggest that lysozyme expression by breast tumors in combination with other well-established prognostic factors may contribute to more accurately identifying subgroups of breast cancer patients with different risks of recurrence or death. In addition, further studies are required to investigate the potential role of lysozyme as a hormonal marker of the existence of a complete hormone receptor pathway in breast cancer.


   Acknowledgments
 
The authors thank Dr. C. López-Otín, from Departamento de Bioquímica, Facultad de Medicina, Universidad de Oviedo, and Dr. MC Diez from Servicio de Cirugra General, Hospital de Jove for support and for helpful comments.

Received for publication December 12, 2000. Accepted for publication May 29, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fleming A. On a remarkable bacteriolitic element found in tissues and secretions. Proc R Soc London 1922; B93: 306–17.
  2. Biggar WD, Sturgess JM. Role of lysozyme in the microbicidal activity of rat alveolar macrophages. Infect Immun 1977; 16: 974–82.[Abstract/Free Full Text]
  3. Klokars ML, Robers P. Stimulation of phagocytosis by human lysozyme. Acta Haematol 1976; 52: 289–95.
  4. Petrakis NL. Physiologic, biochemical and cytologic aspects of the nipple aspirate fluid. Breast Cancer Res Treat 1986; 8: 7–19.[CrossRef][Medline]
  5. Sánchez LM, Vizoso F, Díez-Itza I, López-Otín C. Identification of the major protein components in breast secretions from women with benign and malignant breast diseases. Cancer Res 1992; 52: 95–100.[Abstract/Free Full Text]
  6. Vizoso F, Sánchez LM, Díez-Itza I, Lamelas ML, López-Otín C. Factors affecting protein composition of breast secretions from nonlactating women. Breast Cancer Res Treat 1992; 23: 251–8.[CrossRef][Medline]
  7. Vizoso F, Díez-Itza I, Sánchez LM, Ruibal A, López-Otín C. Relationship between prolactin levels and composition of breast secretions in nonlactating women. J Clin Endocrinol Metab 1994; 79: 525–9.[Abstract]
  8. Bundred NJ, Miller WR, Walker RA. An immunohistochemical study of the tissue distribution of the breast cyst fluid protein Zn-{alpha}2-glycoprotein. Histopathology 1987; 11: 603–10.[Medline]
  9. Díez-Itza I, Sánchez LM, Allende MT, Vizoso F, Ruibal A, López-Otín C. Zn-alpha2-glycoprotein levels in breast cancer cytosols and correlation with clinical, histological and biochemical parameters. Eur J Cancer 1993; 29A: 1256–60.[CrossRef]
  10. Díez-Itza I, Vizoso F, Merino AM, et al. Expression and prognostic significance of apolipoprotein D in breast cancer. Am J Pathol 1994; 144: 310–20.[Abstract]
  11. Mazoujian G, Parish TM, Haagensen DEJr. Immunoperoxidase localization of GCDFP-15 with mouse monoclonal antibodies versus rabbit antiserum. J Histochem Cytochem 1988; 36: 377–82.[Abstract]
  12. Wurster K, Heberling D, Rapp W. Carcinoembryonic antigen (CEA) and lactoferrin (LF) in benign and malignant disease of the breast. A contribution to be immunohistochemical demonstration of marker substances. Geburtshilfe Frauenheilkd 1980; 40: 412–22.[Medline]
  13. Charpin C, Lachard A, Poureau-Schneider N. Localization of lactoferrin and nonspecific cross-reacting antigen in human breast carcinomas. Cancer 1985; 55: 2612–7.[CrossRef][Medline]
  14. Simickova M, Lang BA, Rejhar A, Cernoch M, Sakalova J, Pacovsky Z. Immunohistochemical localization of alpha-lactalbumin in human breast cancer tissue. Neoplasma 1989; 36: 11–20.[Medline]
  15. Bloom HJG, Richardson WW. Histological grading and prognosis in breast cancer. r J Cancer 1957; 11: 359–77.
  16. Vaitukaitis JL. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods Enzymol 1981; 73: 46–52.[Medline]
  17. McCarty KS, Szabo E, Flowers JL, et al. Use of a monoclonal anti-estrogen receptor antibody in the inmunohistochemical evaluation of human tumors. Cancer Res 1986; 46: 4244–8.
  18. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457–81.[CrossRef]
  19. Mantel M, Myers M. Problems of convergence of maximum likelihood iterative procedures in multiparameter situations. J Am Stat Assoc 1971; 66: 484–91.[CrossRef]
  20. Cox DR. Regression models and life tables. J R Stat Soc B 1972; 34: 187–220.
  21. Dixon WJ, Brown MB, Engelman L, et al. BMDP statistical software. Berkeley University of California Press: Berkeley, CA, 1985.
  22. Topper YJ, Freeman CS. Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 1989; 60: 1049–106.
  23. Ginsburg E, Vonderhaar BK. Prolactin synthesis and secretion by human breast cancer cells. Cancer Res 1995; 55: 2591–5.[Abstract/Free Full Text]
  24. Holdaway MI, Friesen HG. Hormone binding by human mammary carcinoma. Cancer Res 1977; 37: 1946–52.
  25. Partridge RK, Hahnel R. Prolactin receptors in human breast carcinoma. Cancer 1979; 43: 643–6.[CrossRef][Medline]
  26. Turcot Lemay L, Kelly PA. Prolactin in human breast tumors. J Natl Cancer Inst 1982; 68: 381–3.
  27. Bonneterre J, Peyrat JP, Vandewalle B, Beuscart R, Vie MC, Cappelaere P. Prolactin receptors in human breast cancer. Eur J Cancer Clin Oncol 1982; 18: 1157–62.[CrossRef][Medline]
  28. Touraine F, Martini JF, Zafrani B, et al. Increased expression of prolactin receptor gene assessed by quantitative polymerase chain reaction in human breast tumors versus normal breast tissues. J Clin Endocrinol Metab 1998; 83: 667–74.[Abstract/Free Full Text]
  29. Peyrat JP, Djiane J, Bonneterre J, et al. Stimulation of DNA synthesis by prolactin in human breast tumor explants. Relation to prolactin receptors. Anticancer Res 1984; 4: 257–62.[Medline]
  30. Bonneterre J, Peyrat JP, Beuscart R, Demaille A. Biological and clinical aspects of prolactin receptors (PRL-R) in human breast cancer. J Steroid Biochem Mol Biol 1990; 37: 977–81.[CrossRef][Medline]
  31. Myal Y, Robinson DB, Iwasiow B, Tsuyuki D, Wong P, Shiu RP. The prolactin-inducible protein (PIP/GCDFP) gene: cloning structure and regulation. Mol Cell Endocrinol 1991; 80: 165–75.[CrossRef][Medline]
  32. Fuch G, Wells J. Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines. J Biol Chem 1995; 22: 13133–7.
  33. Bontental M, Foekens JA, Lamberts SW, et al. Feasibility, endocrine and anti-tumour effects of a triple endocrine therapy with tamoxifen, a somatostatin analogue and an antiprolactin in post-menopausal metastatic breast cancer: a randomized study with long-term follow-up. Br J Cancer 1998; 77: 115–22.[Medline]




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