Protein expression of c-erbB-2 and p53 in normal ducts, ductal carcinoma in situ and invasive carcinoma of the same breast

ABSTRACT CONTEXT AND OBJECTIVE: Breast cancer is thought to derive from progressively aberrant, non-invasive breast lesions, but it is not known exactly how invasive breast cancer develops from these lesions. The aim of this study was to verify the changes in c-erbB-2 and p53 protein expression between non-neoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma found in the same breast. DESIGN AND SETTING: This was a cross-sectional study at Centro de Atenção Integral à Saúde da Mulher, Campinas, Brazil. METHODS: Fifty-six women with invasive ductal carcinoma and ductal carcinoma in situ in the same breast were included. The expression of c-erbB-2 and p53 proteins was assessed in non-neoplastic and neoplastic cells using immunohistochemical techniques. RESULTS: The c-erbB-2 protein was absent in non-neoplastic ducts but was present in 46% and 36% of in situ and invasive carcinoma components, respectively. Only 2% of non-neoplastic ducts, and 18% and 16% of ductal carcinoma in situ and invasive carcinoma components, respectively, were positive for p53 protein. No significant difference in c-erbB-2 and p53 protein expression was observed between in situ and invasive components. The nuclear grade agreement between in situ and invasive carcinoma was very good. CONCLUSIONS: The invasiveness of ductal carcinoma in situ seems to be independent of the Her-2/neu and TP53 genes. The general features of an occurrence of breast carcinoma are formulated at the outset of carcinogenesis, and the Her-2/neu and TP53 genes are involved.


INTRODUCTION
Breast cancer is thought to derive from progressively aberrant, non-invasive breast lesions such as atypical hyperplasia and ductal carcinoma in situ, but it is not known exactly how invasive breast cancer develops from these lesions.Chromosomal imbalances occur, with gain or loss at multiple loci, as hyperplastic lesions progress from ductal carcinoma in situ to invasive breast cancer. 1,2Nevertheless, the presence of shared chromosomal changes in both ductal carcinoma in situ and synchronous, adjacent invasive cancers demonstrates their clonal, evolutionary relationship. 3ER-2/neu and TP53 gene expression in normal breast tissue is different from what is found in invasive breast carcinoma, and this variation can be assessed by immunohistochemistry. 4,5 Many studies have already analyzed c-erbB-2 and p53 protein expression in ductal carcinoma in situ and invasive ductal carcinoma, but most of these studies were carried out in tissue from different women, thereby restricting the usefulness of these data for studying tumor progression. 4,5rogression from ductal carcinoma in situ to invasive carcinoma occurs at specifi c points within the preexisting in situ lesion, and therefore ductal carcinoma in situ and invasive ductal carcinoma are frequently found in the same breast. 6We made the assumption that such cases would be a good model in which to study the relationship between non-neoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma.

OBJECTIVE
The aim of this study was to verify the changes in protein expression between nonneoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma found in the same breast.

MATERIALS AND METHODS
Ninety-eight women diagnosed with invasive ductal carcinoma and ductal carcinoma in situ in the same breast were selected consecutively for this study.The patients were seen in our service (Centro de Atenção Integral à Saúde da Mulher, CAISM) or in a private medical service in Campinas, São Paulo, Brazil, between 1994 and 1999.The paraffin-embedded blocks were sectioned and the slides were stained with hematoxylin-eosin to identify the tissue areas in which non-neoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma were found.Forty-two of these women were excluded from the study because the remaining breast tissue in the paraffi n blocks was insuffi cient for immunohistochemical analysis.The remaining fi fty-six women were enrolled in the study: thirty-eight of them were diagnosed as having invasive ductal carcinoma in clinical stage I and eighteen in stage II.Less prevalent histological types were not included, in order to obtain a homogeneous sample.
The same pathologist performed the pathoanatomical analyses and established the fi nal histological diagnosis.Ducts were defi ned as non-neoplastic when they were normal ducts (one layer of cells) or presented typical ductal hyperplasia (up to four layers of cells without atypia).The nuclear grade of tissue components was also evaluated and classifi ed according to the 1997 Consensus Conference on the Classifi cation of Ductal Carcinoma In Situ. 7he expression of c-erbB-2 and p53 proteins was assessed using immunohistochemistry. Specifi c monoclonal primary antibodies for c-erbB-2 (RxH, Dako, code A0485-1, at 1:300 dilution) and for p53 (MxH, clone D07, Dako, code M7001-1, at 1:100 dilution) were used, and steam heating was used to unmask the antigens.The slides were incubated using Envision CONTEXT AND OBJECTIVE: Breast cancer is thought to derive from progressively aberrant, non-invasive breast lesions, but it is not known exactly how invasive breast cancer develops from these lesions.The aim of this study was to verify the changes in c-erbB-2 and p53 protein expression between non-neoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma found in the same breast.

DESIGN AND SETTING:
This was a crosssectional study at Centro de Atenção Integral à Saúde da Mulher, Campinas, Brazil.

METHODS:
Fifty-six women with invasive ductal carcinoma and ductal carcinoma in situ in the same breast were included.The expression of c-erbB-2 and p53 proteins was assessed in non-neoplastic and neoplastic cells using immunohistochemical techniques.

RESULTS:
The c-erbB-2 protein was absent in non-neoplastic ducts but was present in 46% and 36% of in situ and invasive carcinoma components, respectively.Only 2% of non-neoplastic ducts, and 18% and 16% of ductal carcinoma in situ and invasive carcinoma components, respectively, were positive for p53 protein.No signifi cant difference in c-erbB-2 and p53 protein expression was observed between in situ and invasive components.The nuclear grade agreement between in situ and invasive carcinoma was very good.

CONCLUSIONS:
The invasiveness of ductal carcinoma in situ seems to be independent of the Her-2/neu and TP53 genes.The general features of an occurrence of breast carcinoma are formulated at the outset of carcinogenesis, and the Her-2/neu and TP53 genes are involved.labeled polymer components (Dako, code K1491).Development was carried out using DAB (3-3'-diaminobenzidine, Sigma, code D5637).All immunohistochemical assays were performed using external positive controls: invasive ductal carcinoma for c-erbB-2 and borderline ovarian tumor for p53.
Immunohistochemical analysis was carried out using 40x magnifi cation, by a single pathologist who was blinded to the results from the pathoanatomical analyses.The c-erbB-2 protein expression was considered positive when more than 10% of the cells were stained, and p53 protein expression was considered positive when more than 1% of the nuclei were stained, regardless of the intensity of the staining.
Odds ratios were used to evaluate the strength of the association between pairs of variables, and the 95% confi dence intervals (CI) were calculated.The kappa coeffi cient was used to verify the agreement between the nuclear grade of ductal carcinoma in situ and the nuclear grade of invasive ductal carcinoma in the same breast, and was classifi ed as: poor (< 0.20); fair (0.21-0.40); moderate (0.41-0.60); good (0.61-0.80) or very good (0.81-1.00). 8For statistical purposes, nuclear grades 1 and 2 were analyzed together.

RESULTS
The c-erbB-2 protein was absent from nonneoplastic ducts but was present in 46% of the cases of ductal carcinoma in situ, resulting in an odds ratio of 98.18 (95% CI: 5.78-1667.60).There was no statistical difference in c-erbB-2 expression between in situ and invasive components.Only 2% of the non-   neoplastic ducts and 18% of the ductal carcinoma in situ components showed p53 protein expression, resulting in an odds ratio of 11.96 (95% CI: 1.47-96.92),while the difference in p53 positivity between ductal carcinoma in situ and invasive ductal carcinoma gave a nonsignifi cant result (Table 1).Every case with positive c-erbB-2 expression in the invasive carcinoma component showed also positive expression in the in situ component.Eight out of the nine cases with positive p53 expression in the invasive component also showed positive expression in the in situ component (data not shown).
Positive c-erbB-2 protein expression was associated with nuclear grade 3 in both the in situ and the invasive components of ductal carcinoma.The p53 protein expression showed a similar association (Table 2).There were no cases of invasive ductal carcinoma with a higher nuclear grade than what was found in the ductal carcinoma in situ component, and in 52/56 cases both components were found to have the same nuclear grade.The kappa coeffi cient was 0.88 (0.77-0.99), thus indicating very good agreement (Table 3).

DISCUSSION
The expression of c-erbB-2 and p53 proteins showed a large variation between the non-neoplastic ducts and ductal carcinoma in situ components, but most of the cases showed very similar protein expression and good nuclear grade agreement between ductal carcinoma in situ and invasive ductal carcinoma components.
0][11][12][13] A few studies have reported expression of this protein in rare cases of atypical ductal hyperplasia, [14][15][16] and there could be two possible explanations for these fi ndings.First, these rare cases could be genetically different and, second, the interobserver reproducibility of borderline lesions with these diagnostic criteria is poor.Atypical ductal hyperplasia with c-erbB-2 positive expression may have been classifi ed as well-differentiated ductal carcinoma in situ by other observers. 17Few cases of well-differentiated ductal carcinoma in situ show c-erbB-2 protein expression. 3,5 review 18 has shown that overall c-erbB-2 positivity for all types of ductal carcinoma in situ ranges between 21% and 64%, and for comedo ductal carcinoma in situ between 62% and 81%.The positivity is lower in cases of invasive ductal carcinoma, ranging between 16% and 40%, 18 and these data are in agreement with the results from the present study.There is also a clear association between c-erbB-2 positivity and worse nuclear and histological grades, tumor aneuploidy and high rate of proliferation, and this is more frequent in ductal carcinoma in situ than in invasive ductal carcinoma. 5,18,19This profi le for c-erbB-2 protein expression is quite similar to the p53 protein expression profi le. 20,21he morphological features and immunohistochemical profi le of the ductal carcinoma in situ and invasive ductal carcinoma components of the same specimens have been found to be very similar. 5,22Cases of ductal carcinoma in situ have shown a more "malignant picture" than cases of invasive cancer. 5,19n the present study, fi fty-two out of fi fty-six cases showed ductal carcinoma in situ and invasive ductal carcinoma components with the same nuclear grade, while in the other four cases, more malignant features were found in the ductal carcinoma in situ component.This may suggest that undifferentiation and invasiveness of the ductal carcinoma in situ are not necessarily associated.Similar results were obtained by Warnberg et al. 5 when comparing the nuclear grades of ductal carcinoma in situ and invasive ductal carcinoma in the same breast.There was concordance between the nuclear grades of ductal carcinoma in situ and invasive ductal carcinoma in 102 cases out of 259.In 151 cases, the nuclear grade was higher in ductal carcinoma in situ than in invasive ductal carcinoma, and in only six cases was the nuclear grade of invasive ductal carcinoma higher than that of ductal carcinoma in situ.These data suggest that the degree of aggressiveness of the tumor, i.e. its prognosis, is genetically "formulated" at the beginning of carcinogenesis and is maintained throughout the evolution of the disease.
A study of gene expression patterns in ductal carcinoma in situ and invasive ductal carcinoma, carried out using serial analysis of gene expression (SAGE), found that the most dramatic transcriptome change occurs at the time of transition from normal to ductal carcinoma in situ, when there is no clear universal "in situ" or "invasive" molecular signature of the tumor.That study suggested that some genes may be able to defi ne biologically and clinically meaningful subgroups of ductal carcinoma in situ with a high risk of progression to invasive disease. 23nother study using microdissection and DNA microarrays revealed extensive similarities at the transcriptome level among the distinct stages of progression and suggested that gene expression alterations that conferred the potential for invasive growth are already present in the preinvasive stages.In contrast, different tumor grades are associated with distinct gene expression signatures.Furthermore, a subset of genes associated with high tumor grade is quantitatively correlated with the transition from preinvasive to invasive growth. 24typical ductal hyperplasia is an early aberrant breast lesion that may progress to low nuclear grade ductal carcinoma in situ, which would continue accumulating genetic alterations 13 and could lead progressively to intermediate and high nuclear grade ductal carcinoma in situ.
Considering the fi ndings of these studies, the ductal carcinoma in situ cells could acquire the ability to cross the basal membrane and trigger an invasive ductal carcinoma with very similar morphological and immunohistochemical features.Therefore, a low-grade ductal carcinoma in situ could trigger a low-grade invasive ductal carcinoma and the same could occur in cases of intermediate and high-grade tumors.The in situ ductal carcinomas that do not become invasive at an early stage would reach high nuclear grade and would proliferate along the mammary duct, thus accumulating areas of necrosis and calcifi cation.
There are two possible explanations for the few cases in our study in which the ductal carcinoma in situ component showed a higher nuclear grade than did the invasive ductal carcinoma component.First, the cellular clone that becomes invasive may be more differentiated than the other clones present in the ductal carcinoma in situ component.Second, after invasion has occurred, the remaining ductal carcinoma in situ component would continue accumulating genetic alterations, thereby reaching higher nuclear grades than the cellular clone from which the invasive ductal carcinoma originated.
In short, the results from our study suggest that the Her-2/neu and TP53 genes are likely to be involved in the beginning of breast carcinogenesis (induction) and undifferentiation of ductal carcinoma in situ, but not in the progression from ductal carcinoma in situ to invasive carcinoma.We must emphasize that the variation in c-erbB-2 protein expression in the three components of the same specimens is due exclusively to local modifi cations of ductal cells, since there are no genetic differences other than those acquired over the course of the evolution of carcinogenesis.Studies carried out on ductal carcinoma in situ and invasive ductal carcinoma in different women are biased by individual genetic differences.

CONCLUSIONS
The invasiveness of ductal carcinoma in situ seems to be independent of the Her-2/neu and TP53 genes.Our results suggest that the general features of breast carcinoma occurrences are formulated at the outset of carcinogenesis, and that the Her-2/neu and TP53 genes are involved in this.The morphological features and immunohistochemical profi le of the ductal carcinoma in situ and invasive ductal carcinoma components of the same specimens are very similar.

Table 1 .
Association of protein expression of c-erbB-2 and p53 in non-neoplastic ducts, ductal carcinoma in situ and invasive ductal carcinoma of the same breast OR = odds ratio; 95% CI = 95% confi dence interval.* = Both OR included data referring to ductal carcinoma in situ.

Table 3 .
Distribution of cases according to the nuclear grade of ductal carcinoma in situ and invasive ductal carcinoma of the same breast: analysis of nuclear grade agreement Kappa coeffi cient (95% CI): 0.88 (0.77-0.99)NG = nuclear grade; 95% CI = 95% confi dence interval.

Table 2 .
Association of c-erbB-2 and p53 protein expression according to nuclear grade of ductal carcinoma in situ and invasive ductal carcinoma of the same breast