© 2001 Blackwell Science Inc., 1075-122X/01/$15.00/0The Breast Journal, Volume 7, Number 4, 2001 245–248

Apoptosis in Ductal Carcinoma in

Situ of the Breast

Abelardo Moreno, MD,* Agnès Figueras,* Belén Lloveras, MD,*

Agustín Escobedo, MD,

†

Elvira Griera,* Angels Sierra, MD,

‡

and Angels Fabra, MD

‡

*

Department of Pathology, Ciutat Sanitaria de Bellvitge,

†

Department of Oncology, Institut Català d’Oncologia, and

‡

Department of Cancer i Metastasis,Institut de Recerca Oncológica, Barcelona, Spain

Apoptosis in suppressed by the bcl-2 gene. Overex-pression of the bcl-2 gene and its related bcl-x proteinmay act as a potent inhibitor of apoptosis. In infiltratingmammary cancers there are several studies correlatingbcl-2 gene products and prognosis. Bcl-2-related proteinexpression is an indicator of a low index of recurrenceand distant metastasis both in node-negative cancer pa-tients (1) and in lymph node-positive carcinomas (2).On the other hand, bcl-x overexpression has been re-lated to an increase in axillary node metastases, indicat-ing a poor prognosis (3). According to their biologicalproperties, bcl-2-related protein immunohistochemicalexpression is seen in low-grade cancers (4) and bcl-xoverexpression is associated with high-grade tumors (3).Bcl-x is also unrelated to bcl-2, hormonal status, andp53 expression. The breast cancer line T47D, which isassociated with increased levels of bcl-XL, remains via-ble after transfection with a temperature-sensitive mu-tant of p53 (p53ts) (5), suggesting that endogenous bcl-XL protects cancer cells from p53-mediated apoptosis.

This figures suggest that bcl-2 and bcl-x may have adifferent mechanisms of influencing tumor progression.On the other hand, the wild-type p53 is also a regulatorof apoptosis, and immunohistochemically detected p53overexpression is associated with a poor outcome in in-filtrating breast cancer.

The importance of apoptosis dysregulations in ductalcarcinoma in situ (DCIS) and its relationship with p53mutations and expression of bcl-2 has received little at-tention in the literature. The purpose of this work is to

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Abstract:

Programmed cell death (apoptosis) may play arole in tumor development and progression. The importanceof apoptosis dysregulations in ductal carcinoma in situ (DCIS)and its relationships with p53 mutations and expression ofbcl-2 has received little attention in the literature. In a seriesof 58 DCIS patients, we evaluated the number of apoptoticcells by the TUNEL technique in subgroups of DCIS and corre-lated it with immunohistochemical expression of hormone re-ceptors, c-

erb

B-2, p53, bcl-2, and Ki-67 (MIB-1) and DNA con-tent measured by image cytometry. High apoptotic index(greater than 3%) was related to high tumor grade, negativehormone receptors, c-

erb

B-2 overexpression, aneuploidy andlack of bcl-2 immunohistochemical stain. Apoptosis was notrelated to p53 or proliferative index. The findings are similarto those found in infiltrating breast cancer.

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Key Words:

apoptosis, breast cancer, ductal carcinoma insitu, immunohistochemistry, TUNEL technique

P

rogrammed cell death (apoptosis) may play a rolein tumor development and progression. Dysregula-

tions of genes controlling apoptosis may cause mam-mary cells to become immortal and constitute a cancer.

Address correspondence and reprint requests to: Abelardo Moreno,MD, Department of Pathology, Ciutat Sanitaria de Bellvitge, Feixa Llargas/n, 08907 L’Hospitalet Ll, Barcelona, Spain.

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moreno et al.

evaluate the rate of apoptotic cells in subgroups of DCISand correlate the apoptotic rate with grade, hormone re-ceptors, and expression of genes related to prognosisand apoptosis regulation.

MATERIALS AND METHODS

A series of 58 DCIS patients were retrieved from thefiles of the Department of Pathology, Ciutat Sanitaria deBellvitge, Barcelona, Spain. The patients had been diag-nosed between 1980 and 1995 and followed for a mini-mum of 4 years. The clinical and histologic characteris-tics of the series and the methods for classification androutine immunohistochemical staining (for hormone re-ceptors, c-

erb

B-2, p53, bcl-2 product, and Ki-67/MIB-1)have been reported previously (6) and are summarizedin Table 1. All the patients were women between theages of 33 and 86 years (median age 52 years). DCISpresented as microcalcifications detected in mammogra-phy (56%) or tumors. Tumor size was evaluated on his-tologic specimens or by mammography (microcalcifica-tions). Cases were classified as low or high grade,depending on cytologic (nuclear atypia, nucleoli, mitoticfigures) and architectural features.

DNA content was measured by image cytometry.Briefly cell suspensions from paraffin-embedded sec-tions (50

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m thick) were obtained from representativeareas. The sections were deparaffinized in xylene and re-hydrated. Mechanical disruption (scissors, nylon filters)and pepsin digestion (0.5% in 0.9% ClNa, pH 1.5 for 1hour at 37

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C) were performed. Feulgen stain was car-ried out on cytocentrifuge slides. An external control ofa normal breast specimen was included in each batch.Computerized densitometric measurements of nucleiwere done using the Discovery system (Becton-Dickin-son). After automatic selection of nuclei, visual controlhelped in discarding artifacts and nontumoral cells.

Apoptosis was detected by the TUNEL [terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP-biotin neck end labeling] technique (7). In summary, af-

ter antigen retrieval with proteinase K (Boehringer Mann-heim) digestion (12.5 mg/ml for 15 minutes at 37

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C),the slides are incubated with the terminal deoxytrans-ferase and biotinylated dUTP (Boehringer Mannheim)1.5 hours at 37

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C. The reaction is stopped with bufferTB (15 minutes at room temperature). An avidin-biotinkit and diaminobencidine are used to visualize the reac-tion.

The results were independently evaluated by two ob-servers and expressed as follows: Hormone receptors,bcl-2, and c-

erb

B-2 were judged positive when more than10% of the cells/nuclei were stained. p53 was recorded aspositive when 25% of the nuclei were stained. For evalu-ation of the

TUNEL

technique, the percentage of positive(stained) nuclei were counted in five high-power fields(

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400) in areas of viable tumor cells lacking necrosis.The cut value was 3% (in order to compare with resultsin infiltrating cancer obtained by our group) (8).

The distribution of frequencies was compared by chi-squared and Fisher’s exact tests. The level of significancewas 0.05.

RESULTS

Twenty-six tumors were classified as high-grade and32 as low-grade intraductal carcinomas. Tumor sizeranged from 4 to 120 mm, without significant differ-ences between low- and high-grade cancers. Forty caseswere classified as low apoptotic rate (less than 3% posi-tive nuclei) counted in high-power fields and the remaining18 tumors were considered as high apoptotic rate (3% ormore positive nuclei). From the series, 38 cases (65.5%) hadpositive estrogen receptors and 30 cases (51.7%) positiveprogesterone receptors; 18 cases (31%) expressed c-

erb

B-2membrane positivity, 11 (19%) were positive for p53, and37 (63.8%) were positive for bcl-2. DNA content couldbe evaluated in 37 cases and 17 of them (45.9%) demon-strated either aneuploid or tetraploid peaks. Heteroge-neous tumors (more than one morphologic type) de-picted minor variations in the positivity from microscopicfield to field, each lesion was tabulated according to the pre-dominant pattern.

In general, low-grade DCIS was associated with posi-tive hormone receptor, bcl-2 expression, and diploidDNA content, while high-grade DCIS was frequentlyhormone receptor negative, overexpressed c-

erb

B-2 andp53. A higher proliferative index, measured by Ki-67,was detected in high-grade DCIS compared to low-grade cancers (18.24% versus 9.16%, respectively).

Table 2 shows the results of the different parameterstested, comparing cases with low and high apoptotic ac-

Table 1. Immunohistochemical Studies

Source Clone Pretreatment Dilution

Estrogen receptor

Dako 1D5 Microwave 1/10

Progesterone receptor

MedacDiagnostika 1A6 Microwave 1/10

c-erbB-2 Biogenex CB-11 Saponin 1/80p53 Biogenex BP53-12-1 Microwave 1/80Bcl-2 Biogenex 100 Microwave 1/20Ki67 Biogenex MIB-1 Microwave 1/10

Apoptosis in Ductal Carcinoma in Situ

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247

tivity. In our series, apoptosis detected by the

TUNEL

technique correlated with tumor grade, hormone (estro-gen and progesterone) receptors, c-

erb

B-2 overexpres-sion, bcl-2 expression, and ploidy. p53 overexpression,detected immunohistochemically, and proliferative in-dex did not correlate with apoptosis.

DISCUSSION

In infiltrating breast carcinomas, the study by Lip-ponen et al. (9) found that high apoptotic index (greaterthan 3/mm

2

) was associated with tumor necrosis, highhistologic grade, dense stromal lymphocytic infiltration,aneuploidy, high mitotic rate, high S-phase fraction, andexpression of p53. In the same series survival was re-lated to apoptotic index in both negative- and positive-node patients, although the statistical significance waslost in multifactorial studies [in node-positive cancerseven the univariate analysis (2) shows no relationshipbetween apoptosis and clinical outcome]. There are alsoseveral reports of apoptosis and DCIS. Bodis et al. (10)found intense

TUNEL

-positive staining associated withhistologic necrosis in 19 cases of DCIS. In contrast, noevidence of staining was found in five grade I DCIS stud-ied. Moreover, apoptosis was independent of immuno-histochemically detected p53 status in our study. On theother hand, Harn et al. (11) found a higher apoptotic la-beling index in DCIS when compared with those in infil-trating ductal carcinomas and suggested that this differ-ence might be regulated by altered p53 expression.

In our series, high apoptotic index is related to hightumor grade, negative hormone receptors, c-

erb

B-2overexpression, aneuploidy, and negative bcl-2 immu-nohistochemical stain, and unrelated to p53 and Ki-67(MIB-1) expression. Only occasional reports regarding

T1 breast cancer (2) have shown no significant associa-tion between apoptosis and bcl-2 expression. In breastcancer, bcl-2 expression is associated with small tumorsize, positive hormone receptors, low grade, low stage,and low MIB-1 expression (8,12). Also a significant re-lationship exists between the presence of bcl-2 proteinand the response to endocrine therapy. Moreover, bcl-2immunostaining is a more accurate predictor of re-sponse than receptor status (13).

In high-grade intraductal cancers, the apoptotic bcl-2-mediated arrest is not an important pathogenic mech-anism. Products other that bcl-2 and p53 (Bax or others)may influence the apoptotic index and prognosis.

Our results of apoptotic incidence in DCIS are con-cordant with the results published in infiltrating breastcancer, indicating that most changes in invasive cancersare already present in breast intraductal tumors, sug-gesting that the same molecular mechanisms are operat-ing in both pathologic entities. The development of sev-eral characteristics of tumor progression, such as invasionand metastasis, may be related to the acquisition of anangiogenic phenotype in a later infiltrating stage.

Acknowledgment

This work was supported by grant 54/95 from LaMarató Tv3. We are grateful to J. Klaustermeier for re-view of the manuscript.

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Table 2. Relationship Between Apoptosis and Prognostic Markers in DCIS

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3%

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3%

p

Low-grade DCIS 29 3High-grade DCIS 11 15

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0.0001ER negative 7 13ER positive 33 5

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0.0001PgR negative 13 15PgR positive 27 3 0.0002c-

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B-2 negative 34 6c-

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B-2 positive 6 12

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0.0001p53 negative 34 13p53 positive 6 5 0.2704Bcl-2 negative 9 12Bcl-2 positive 31 6 0.0012Diploid 18 2Aneuploid 8 9 0.0044Ki67 12.190

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9.928 15.500

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9.107 0.3809

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