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Oncogenes and Male Breast Carcinoma: c-erbB-2 and p53 Coexpression Predicts a Poor Survival

From the Department of Biomedical Sciences and Human Oncology, Section of Pathology, University of Turin; and the Division of Pathology, S. Giovanni Hospital, Turin, Italy.

Address reprint requests to Achille Pich, MD, Dipartimento di Scienze Biomediche e Oncologia Umana, Università di Torino, Via Santena 7, I-10126 Torino, Italy; email pich{at}molinette.unito.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To investigate the prognostic value of biomarkers in male breast carcinoma (MBC).

PATIENTS AND METHODS: Fifty patients (mean age, 62.2 years) with invasive ductal carcinoma were retrospectively studied. All patients received surgery; 35 had adjuvant postoperative therapy. The median follow-up was 59 months (range, 1 to 230 months). c-myc, c-erbB-2, p53, and bcl-2 proteins were immunohistochemically detected on sections from formalin-fixed, paraffin-embedded tissues using 9E11, CB11, DO7, and bcl-2 124 monoclonal antibodies (mAbs). Estrogen, progesterone, and androgen receptors were detected using specific mAbs. Cell proliferation was assessed by MIB-1 mAb.

RESULTS: In univariate analysis, c-myc, c-erbB-2, and p53 protein overexpression was significantly correlated with prognosis. The median survival was 107 months for c-myc–negative and 52 months for c-myc–positive patients (P = .01), 96 months for c-erbB-2–negative and 39 months for c-erbB-2–positive patients (P = .02), and 100 months for p53-negative and 33 months for p53-positive patients (P = .0008). Tumor histologic grade (P = .01), tumor size (P = .02), patient age at diagnosis (P = .03), and MIB-1 scores (P = .0004) also had prognostic value. In multivariate analysis, only c-erbB-2 and p53 immunoreactivity retained independent prognostic significance. All nine patients who did not express c-erbB-2 and p53 proteins were alive after 58 months, whereas none of the 14 patients expressing both proteins survived at 61 months follow-up (P = .0002).

CONCLUSION: Overexpression of c-myc, c-erbB-2, and p53 proteins may be regarded as an additional prognostic factor in MBC. The combination of c-erbB-2 and p53 immunoreactivity can stratify patients into different risk groups.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
ABNORMALITIES IN the structure or activity of proto-oncogenes may contribute to the development or progression of the malignant phenotype.¹ Oncogene alterations have been extensively investigated in female breast cancer (FBC) and proposed as markers of potential clinical utility.² Few studies exist on the role of oncogenes in male breast carcinoma (MBC).

The c-myc oncogene encodes a nuclear DNA binding protein with several functional domains, which are commonly found in transcription factors.³ c-myc amplification has been found in 4% to 41% of FBCs³ and is correlated with a poor prognosis.^4-9 Overexpression of c-myc protein has been detected by immunohistochemistry in 95% to 100% of FBCs.^10-13 To our knowledge, no investigation on c-myc in MBC has been reported so far.

The c-erbB-2 gene, also known as HER-2/neu, encodes a transmembrane 185-kd protein that shows homology to the epidermal growth factor receptor.¹⁴ c-erbB-2 gene amplification correlates with immunohistochemical assessment of the gene product.¹⁵ Overexpression of c-erbB-2 protein has been detected in 9% to 45% of FBCs.^12,16-20 It was found to be associated with histologic grade of differentiation,^16-18 tumor size,^15,18 lymph node metastasis,^12,18 hormone receptors negativity,^18,21 high proliferative activity,^18,21 and poor survival.^17-20 Patients with strong c-erbB-2 expression seem to have a poorer response to hormonal agents such as tamoxifen as well as nonanthracycline chemotherapy.²² Thus c-erbB-2 may be an important predictive marker in guiding therapy decisions. c-erbB-2 protein expression has also been investigated in MBC; positive immunostaining was detected in 0% to 95% of the specimens.^23-32 No association with prognosis was found.^26-28,32 In only one study, c-erbB-2 immunopositivity appeared as a predictor of adverse survival.²⁹

We have previously described the role of p53 and bcl-2 oncogenes in a smaller series of MBC patients^33,34 and found that p53 protein expression was a significant prognostic factor. In the present study, we have retrospectively analyzed the expression of c-myc and c-erbB-2 protein in 50 primary MBCs at diagnosis, using immunohistochemistry on sections from formalin-fixed, paraffin-embedded tissues. Our aim was to assess whether the expression of c-myc and c-erbB-2 proteins was associated with tumor clinicopathologic features, sex hormone receptors, p53 and bcl-2 protein expression, cell proliferative activity, and patient survival.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients and Tumors
From 1967 to 1995, 56 patients with MBC were diagnosed in the pathology units of the Department of Biomedical Sciences and Human Oncology of the Turin University and S. Giovanni Hospital (Turin, Italy). Blocks were accessible for all but six cases and were used for analysis. The mean age of these 50 patients at diagnosis was 62.2 years (range, 27 to 86 years). All patients underwent surgery; 45 had radical or modified radical mastectomies, and five had simple mastectomies. Adjuvant postoperative therapy was administered to 35 patients. Adjuvant radiation, hormone therapy, or chemotherapy alone was given to 13, four, and three patients, respectively. Two patients received radiation and hormone therapy, four received radiation and chemotherapy, four received chemotherapy and hormone therapy, and five received chemotherapy, hormone therapy, and radiation therapy. Patients were traced using hospital, pathology, and/or radiotherapy records, as well as direct interviews with patients or their doctors. Causes of death were collected from death certificates. A minimum follow-up of 3 years for censored (surviving) patients or until death was available for all the cases. The follow-up was complete on December 31, 1998. The median follow-up time was 59 months (range, 1 to 230 months) for the whole series and 105 months (range, 38 to 160 months) for censored patients. Carcinomas were classified according to the World Health Organization³⁵ and staged pathologically according to the International Union Against Cancer.³⁶ All carcinomas were cases of invasive ductal carcinoma; 15 were pT1, 17 were pT2, and 18 were pT4. Lymph node status was available in 35 cases; 14 were node negative, and 21 node positive. Histologic grade was assessed according to Elston and Ellis³⁷; nine tumors were grade 1, 29 were grade 2, and 12 were grade 3. Multiple samples from each case were always fixed in 10% formalin for an average of 24 to 36 hours and embedded in paraffin. Serial sections from the same tissue blocks were cut for histology, sex hormone receptors, c-myc, c-erbB-2, bcl-2, p53, and MIB-1 immunostaining.

c-myc and c-erbB-2 Staining and Scoring
Sections 4-µm thick on poly-L-lysine–coated slides were dewaxed, placed in a glass box filled with 10 mmol/L citrate buffer, pH 6, and subjected to microwave irradiation at 750 W for two periods of 5 minutes each. Sections were stained with the specific monoclonal antibodies (mAbs) anti–c-myc oncoprotein (clone 9E11), raised against a synthetic peptide representing residues 408 to 420 on the human c-myc protein, and anti–c-erbB-2 (clone CB11), raised against a synthetic peptide corresponding to a site of the internal domain of the c-erbB-2 oncoprotein (Medac, Hamburg, Germany), at 1:250 and 1:40 dilution, respectively, in phosphate-buffered saline for 2 hours at room temperature in a humidified atmosphere, using the labeled streptavidin biotin method (Dakopatts, Glostrup, Denmark) and diaminobenzidine as chromogen with light hematoxylin counterstaining. Normal mouse serum was substituted for primary antibodies as a negative control. Sections from FBC immunoreactive for c-myc or c-erbB-2 were used as a positive control in each staining run. Scoring of c-myc and c-erbB-2 immunostaining was independently performed by two pathologists (L.C. and A.P.), who had no knowledge of the tumor clinicopathologic data and patient survival, using a standard light microscope equipped with an ocular reticle (magnification x 15) and a x 40 objective. In each case, 2,000 tumor cells were counted from 10 randomly selected areas, ensuring that the whole section was scanned. All c-myc immunoreactive cells were considered positive, regardless of the staining intensity, and the fraction of positive cells was recorded. A case was considered positive if more than 5% cells were stained. For c-erbB-2 immunostaining, cases that showed distinct membrane staining, either focal (only affecting some tumor cells) or diffuse (affecting all or most tumor cells), were classed as positive according to Somerville et al.³⁸ When the scoring discrepancy was greater than 10%, a consensus interpretation was reached after re-examination of the slides with a double-headed microscope.

p53, bcl-2, MIB-1, and Sex Hormone Staining and Scoring
Tumor sections 4-µm thick were placed on poly-L-lysine coated slides and were stained with specific mAbs using the labeled streptavidin biotin method (Dakopatts) and diaminobenzidine as chromogen. For p53, bcl-2, and MIB-1 staining, the sections were microwave pretreated for two periods of 5 minutes each in a glass box filled with 10 mmol/L citrate buffer, pH 6, at 750 W. p53-specific mAb DO7 (Oncogene Science, Uniondale, NY), which reacts with both wild-type and mutant type of p53 protein, at 1:75 dilution, mAb antihuman bcl-2 oncoprotein (Dako-bcl-2, 124) (Dakopatts) at 1:40 dilution, and MIB-1 mAb (Immunotech, Marseille, France) at 1:100 dilution were then applied for 2 hours at room temperature in a humidified chamber. Sections of FBC known to express p53 and bcl-2 protein or MIB-1 antigen were included in each staining run as positive controls. For estrogen (ER) and progesterone (PGR) receptor staining, ER-immuno cytochemical assay (ICA) and PGR-ICA (Abbott Laboratories, North Chicago, IL) were used at kit dilution. Sections from known ER/PGR-positive FBC were used as positive controls. For androgen receptors (ARs), sections cut on the day of immunostaining (freshly cut sections) were stained with anti-AR mAb (clone 2F12) (Novocastra, Newcastle, United Kingdom) at 1:10 dilution for 18 hours at 4°C in a humidified atmosphere, following the procedure described elsewhere.³⁹ Freshly cut sections from paraffin-embedded blocks of human hyperplastic prostate were used as a positive control. For all markers except AR, immunostaining was performed within 3 to 4 days after sections had been cut from blocks. Each staining run included no more than 10 sections. All sections were independently scored by two pathologists (E.M. and A.P. for AR, ER, and PGR; L.C. and A.P. for p53, bcl-2, and MIB-1), following the procedure reported for c-myc and c-erbB-2 staining. Cases were considered positive for bcl-2 if they showed immunoreactivity in more than 30% of the neoplastic cells because this value has given the most significant prognostic information in a large series of FBC patients.⁴⁰ For p53 immunostaining, nuclear staining in more than 10% of neoplastic cells was used as a cutoff because, in sections from paraffin-embedded FBCs, all cases with strong immunopositivity in at least 10% of cells had a detectable mutation in the p53 gene, giving a 100% concordance between p53 immunopositivity and mutations.⁴¹ Ten percent positive cells was chosen as cutoff for sex hormone receptor positivity because this value for ER-ICA and PGR-ICA showed the best correlation with biochemical ER/PGR determination of positivity and clinical response in FBC.⁴² For MIB-1 immunoreactivity, the absolute percentage of the stained cells was recorded, and the median value was used as a cutoff because of the size of our sample and the normal distribution of MIB-1 scores.

Statistical Analysis
Associations between c-myc and/or c-erbB-2 positivity/negativity and tumor clinicopathologic features, sex hormone receptors status, and p53 and bcl-2 expression were assessed by the Yates-corrected ² test. Association between expression of the oncogenes and MIB-1 scores was evaluated by one-way analysis of variance. Univariate survival analyses were based on the Kaplan-Meier product-limit estimates of survival distribution, and differences between survival curves were tested using the generalized Wilcoxon rank sum test. The relative importance of all the variables considered in univariate analysis (age, histologic grade, T stage, lymph node status, ER/PGR/AR status, and c-myc, c-erbB-2, p53, bcl-2, and MIB-1 immunoreactivity) was estimated using the Cox proportional hazards regression model. All data were processed with BMDP (BDMP Statistical Software Inc, Los Angeles, CA) statistical software. As a level of significance, P < .05 was used.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Distribution of c-myc and c-erbB-2 Immunoreactivity in MBCs
Five cases showed no immunoreactivity for c-myc protein; four additional cases showed very faint cytoplasmic immunoreactivity in only 0.4% to 4.2% of cells and were considered negative. Forty-one (82%) of the 50 cases showed perinuclear and/or cytoplasmic immunoreactivity in more than 5% of the neoplastic cells (positive cases). In these cases, the percentage of positive cells ranged from 10.5% to virtually all neoplastic cells (Fig 1). Staining intensity varied from case to case; a degree of staining heterogeneity could also be seen within the neoplastic cells of positive cases. No difference between infiltrative margins and tumor center was evident. The results are summarized in Table 1. No association was seen between c-myc and clinicopathologic variables. However, there were too few cases for definitive conclusions.

View larger version (122K): [in this window] [in a new window]   Fig 1. Immunocytochemical detection of c-myc protein in male breast carcinoma. All neoplastic cells show intense staining (labeled streptavidin biotin immunoperoxidase method with light hematoxylin counterstain: original magnification x400).

View larger version (121K): [in this window] [in a new window]   Fig 2. Male breast carcinoma showing strong membrane staining for c-erbB-2 protein (labeled streptavidin biotin immunoperoxidase method with light hematoxylin counterstain: original magnification x400).

Survival Analysis
At the time of analysis, 40 patients (80%) had died, and 10 (20%) were alive. The median survival for the whole series was 61 months (mean, 67 months; range, 1 to 230 months). The median survival for censored patients was 105 months (mean, 97.5 months; range, 38 to 160 months). Overall 5- and 10-year survival rates were 51% and 20%, respectively.

In univariate analysis, c-myc and c-erbB-2 immunoreactivity was strongly associated with survival. The median survival was 107 months for c-myc–negative cases but only 52 months for c-myc–positive cases (P = .01) (Fig 3). Median survival was 96 months for c-erbB-2–negative cases and 39 months for c-erbB-2–positive cases (P = .02) (Fig 4). p53 immunoreactivity (P = .0008), MIB-1 scores (P = .0004), histologic grade (P = .01), and T stage (P = .02) each also had significant prognostic value. Patients younger than 45 years or older than 70 years had a shorter survival than middle-aged men (P = .03). A trend was found for lymph node status (P = .1). Sex hormone receptors and bcl-2 immunoreactivity had no prognostic value. The results are listed in Table 2.

View larger version (13K): [in this window] [in a new window]   Fig 3. Kaplan-Meier survival curves for MBC patients categorized according to expression of c-myc protein.

View larger version (14K): [in this window] [in a new window]   Fig 4. Kaplan-Meier survival curves for MBC patients categorized according to expression of c-erbB-2 protein.

When patients were grouped by c-erbB-2 and p53 immunoreactivity, all nine patients who did not express c-erbB-2 and p53 proteins were alive after 58 months, whereas none of the 14 patients expressing both proteins survived at 61 months of follow-up (P = .0002).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The main results of our study can be summarized as follows: (1) overexpression of c-myc, c-erbB-2, and p53 proteins is associated with a poor prognosis in MBC, and (2) the simultaneous expression of c-erbB-2 and p53 protein identifies a high-risk patient group.

c-myc expression was associated with a shorter survival in univariate analysis; to our knowledge, this is the first report of this association in MBC. Our results are in line with most reports on FBCs^4-9 but contrast with the findings of other investigators who applied different methods for detecting and evaluating c-myc expression.^43-46 It is well known that the expression of c-myc protein, as determined by immunohistochemistry, shows some discrepancy with gene organization and expression.⁴⁷ Indeed, 82% of our cases were immunopositive for c-myc, a rate far higher than that detected by gene amplification in FBC (4% to 41%).³

The rate of c-erbB-2 immunopositivity in our series (56%) was higher than in most FBCs^16-20 and MBCs^25,28,32 but rather similar to that found by Pavelic et al¹² in FBC and by Weber-Chappuis et al³⁰ in MBC. Immunopositivity for c-erbB-2 protein was strongly correlated with poor patient survival and appeared as the most significant independent prognostic variable in multivariate analysis. Our results are in line with several large studies on FBC^17-20 but contrast with reports indicating that c-erbB-2 staining had no¹⁶ or only limited prognostic value.¹⁵ There is only one report of association between c-erbB-2 overexpression and poor survival in MBC,²⁹ whereas most studies have failed to demonstrate any prognostic value.^26-28,32

The conflicting results may be due in part to different scoring systems and cutoff values. FBCs were considered c-erbB-2 positive if they showed at least one focus of positively stained malignant cells,¹⁹ or strong membrane staining, either focal or diffuse,³⁸ or positive membrane staining of any intensity and in any percentage of cancer cells.²⁰ In some reports, quantitative criteria were applied; only cases exhibiting membrane staining in more than 20% of cells³² or in all or a majority of cells¹² were deemed positive. In other series, both quantitative and qualitative criteria were used, and tumors with moderate or strong staining of more than 20% cells were considered positive.²¹

Cytoplasmic immunoreactivity has also been found to correlate with poor prognosis in FBC.⁴⁸ However, only membrane staining was found to reflect gene amplification¹⁵ and appeared significant independently of the degree of reactivity.¹⁷ Thus, we have considered as c-erbB-2 positive all tumors with distinct immunostaining of cell membrane, either focal (only affecting some tumor cells) or diffuse (affecting all or most tumor cells). Similar criteria have been applied by a number of investigators.^19,20,38

Discrepancies may also depend on the number and selection of patients, unequal follow-up, and differing therapeutic regimes. The number of cases in our study is small compared with most studies on FBCs but larger than other analyses of MBC. Only Rayson et al³² tested c-erbB-2 immunoreactivity in 76 cases; however, in their series, radiation, chemotherapy, or hormone therapy was given to only 31%, 5%, and 16% of patients, respectively, whereas 70% of our patients received adjuvant therapy.

Lastly, as in several FBC studies, we did not find association between c-erbB-2 expression and tumor histologic grade,^15,21 size,^17,21 lymph node metastasis,^15-17,19 hormone receptor status,^16,19 and cell proliferation.⁴⁹

p53 overexpression was strongly correlated with poor survival and also appeared as an independent variable in multivariate analysis, confirming our previous report on a smaller series of MBCs.³³ Although an abnormal p53 phenotype was found to be associated with a poor clinical outcome in large studies on FBC,^50,51 suggesting both a prognostic and predictive role,⁵² the prognostic significance of p53 in MBC is still unclear. Mutations of gene p53 were only of marginal significance in one study.⁵³ Rayson et al,³² using 20% p53 immunopositive cells as a cutoff, did not find any prognostic value for the marker. However, when the same cutoff was applied to our series, the prognostic significance of p53 overexpression seemed even stronger (P < .0001) than that reported in the present article. We believe that the prognostic relevance of p53 overexpression mainly depends on the association between protein overexpression and cell proliferative activity. Indeed, we found that MIB-1 scores were higher in p53 positive cases (28.08%) than negative cases (19.03%; P < .0001), and cell proliferation, as assessed by MIB-1 scores, was a significant prognostic variable (P = .0004), in line with our previous findings in MBC.^33,39,54

Other biomarkers, such as bcl-2 overexpression and sex hormone status, had no prognostic value, confirming our previous reports in a smaller subset of MBCs.^33,34,39,54 The rate of ER/PGR positivity in the present series was lower than in other studies.^32,55 This may be because of the panel of cases available. In our series, there were only 18% grade 1 carcinomas, and it is known that well-differentiated FBCs are more often receptor-positive⁵⁶; moreover, all low expressor cases have been considered as negative in the present study.

The second main finding of our study is that simultaneous expression of p53 and c-erbB-2 proteins has additive negative impact on survival of MBC patients and allows identification of different risk groups. We are aware that a large number of cases are necessary to achieve reliable results and that, with our relatively small sample size, any conclusion must be taken with caution. However, when our 50 patients were grouped by c-erbB-2 and p53 expression (which were the only two independent prognostic variables in multivariate analysis), all nine patients who did not express c-erbB-2 and p53 proteins were alive at 58 months of follow-up, whereas none of the 14 patients expressing both proteins survived after 61 months (P = .0002). Simultaneous expression of both proteins has also been reported in FBC,^20,57-71 in proportions ranging from 4%⁶¹ to 42%.⁵⁷ Several studies on FBC also showed poorer prognosis for patients whose tumors coexpress c-erbB-2 and p53 proteins.^{20,59,61,62,66,67,69,71}

It is likely that neoplasms with concomitant expression of the p53 and c-erbB-2 genes, both mapped to chromosome 17, might have lost a mechanism for control of cell proliferation and gained an activator of malignant cell potential,⁵⁸ resulting in a very malignant tumor phenotype.

However, other reports on FBC showed no additive impact on patient survival of the coexpression of c-erbB-2 and p53 proteins^60,65 or even a better prognosis for tumors coexpressing both proteins.⁶³ The differing results may depend on the interaction between the two molecular markers and a specific therapeutic regime. Patients with abnormalities of both c-erbB-2 and p53 had a remarkable 10-year overall survival of 90% when treated with high doses of cyclophosphamide, doxorubicin, and fluorouracil.⁷⁰ This implies that the type of treatment should be considered when the prognostic significance of biomarkers is evaluated.

In our series, most patients (70%) received adjuvant therapy. The median survival for the 15 patients who received surgery alone was lower (35 months) than the median survival for the 35 patients also receiving adjuvant therapies (86 months, P = .003). However, it was impossible to assess the precise role of each individual therapy with respect to the various biomarkers because too few patients received radiotherapy, chemotherapy, or hormone therapy alone (13, three, and four, respectively).

In conclusion, immunohistochemical detection of c-myc, c-erbB-2 and p53 proteins provides new additional criteria for assessing prognosis in MBC. Furthermore, with the limitation of the small number of cases, our results suggest that the combination of c-erbB-2 and p53 immunoreactivity may help to stratify patients with MBC into different risk groups.

	ACKNOWLEDGMENTS

 
Supported by grants from the Italian Ministero dell’Università e Ricerca Scientifica e Tecnologica (MURST 60%, Rome, Italy).

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Submitted August 13, 1999; accepted April 4, 2000.

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