• non-palpable breast carcinoma;
  • microcalcification;
  • estrogen receptor;
  • progesterone receptor;
  • c-erbB-2;
  • cell proliferation;
  • apoptosis


  1. Top of page
  2. Abstract

Screening mammography has greatly increased the number of non-palpable breast carcinomas diagnosed in asymptomatic women. Malignant-appearing microcalcifications represent one of the earliest mammographic findings of non-palpable breast carcinomas. Many studies have attempted to correlate radiological and histological features of malignant-appearing microcalcifications. In the present study, we evaluated the association between mammographically detected malignant-appearing microcalcifications and the expression profile of selected biological markers in non-palpable breast carcinomas. Two hundred and eighty patients with non-palpable suspicious breast lesions that were detected during screening mammography were studied. All patients underwent mammographically-guided needle localization-excision breast biopsy. Histological examination showed 74 (26.4%) carcinomas of various subtypes. Immunohistochemistry was carried out in 58/74 carcinomas by using a panel of monoclonal and polyclonal antibodies against estrogen receptor (ER), progesterone receptor (PR), HER-2/neu, Bcl-2, Bax, Fas and DNA fragmentation factor (DFF). Malignant-appearing microcalcifications was the major mammographic finding in 45/58 (77%) patients. Nuclear ER positivity (65.5%) and PR positivity (46.5%) of non-palpable breast carcinomas were statistically correlated with malignant-appearing microcalcifications (p < 0.01 and p < 0.05, respectively). Statistically significant associations were also found between malignant-appearing microcalcifications and HER-2/neu positivity (p < 0.01), Bax positivity (p < 0.01), Fas positivity (p < 0.05) and DFF positivity (p < 0.01), whereas no statistical correlation was found with Bcl-2 positivity (p > 0.05). Malignant-appearing microcalcifications detected during screening mammography represent a diagnostic, prognostic and therapeutic challenge. The mammographic/biological associations and their potential implications in the management of women with non-palpable breast carcinomas are thoroughly discussed. © 2002 Wiley-Liss, Inc.

Breast carcinoma represents a common disease among Greek women and is considered to be one of the main causes of cancer mortality. In the last decade screening programs have been intensified in Greece, based on 8 randomized breast screening trials that have suggested a contribution of mammographic screening to breast cancer mortality,1 although debate continues on this issue.2 The main goal of these programs is the detection of breast carcinomas in an earlier and more curable stage of evolution.3 Noteworthy is the fact that with the introduction of mammographic screening, the incidence of ductal carcinoma in situ (DCIS) in asymptomatic women has increased to 20–25% of all screening-detected breast cancers.4 It has been estimated that mammographic detection of non-palpable breast carcinomas reflects only 20% of the “total lifetime” of breast cancer.5 Mammographically- diagnosed non-palpable breast carcinomas are increasingly encountered and constitute a major clinical entity.

Non-palpable breast carcinomas form a heterogeneous group of lesions with variable findings and different prognosis. Most are small in size and have infrequent nodal and distant metastases. It is well documented that microcalcifications represent one of the earliest mammographically detectable changes associated with in situ and invasive breast carcinomas in asymptomatic women. Microcalcifications are the primary indication for approximately 50% of the breast biopsies carried out for non-palpable mammographic abnormalities, although they do not always represent malignancy. Various investigators have attempted to distinguish mammographically benign vs. malignant microcalcifications.6 Histologic examination of the areas of microcalcifications is not always adequate in terms of clinical decision making.7 New parameters for mammographically detected microcalcifications associated with non-palpable breast carcinomas are desirable.

A wide range of prognostic markers have been proposed for non-palpable breast carcinomas. The clinically available markers such as histological type, size, auxiliary node involvement and cytological grading are not sufficient, considering the biological complexity of this clinical entity.8 Remarkable progress in understanding the molecular foundations of breast carcinogenesis has been made recently, providing new biological factors that can be evaluated easily by means of immunohistochemistry in non-palpable breast carcinomas.9 Estrogen receptor (ER) and progesterone receptor (PR) negativity, c-erbB-2 (HER-2/neu) protein overexpression and dysregulation of apoptosis have all been proposed as markers of more aggressive tumor behavior.

The purpose of our study was to evaluate the association between mammographically detected malignant-appearing microcalcifications and the biological characteristics of non-palpable breast carcinomas, and assess its clinical perspective.


  1. Top of page
  2. Abstract

Patient population

Between 1989–2001, 280 patients with non-palpable suspicious breast lesions that were detected during screening mammography were evaluated in the University Hospital of Patras, Greece. The median age of the patients was 55 years (range, 36–76 years) and none of the patients had previously been screened or shown clinical signs of breast disease.


The standard craniocaudal and lateral views were carried out in all patients. Cone-down magnification views were obtained when necessary. All the mammograms were evaluated by 2 experienced radiologists. Mammographic findings requiring further exploration with breast biopsy were considered the following: 1) microcalcifications; 2) mass with or without microcalcifications; 3) architectural distortion with or without microcalcifications; and 4) asymmetric density with the greater diameter <1 cm with or without microcalcifications.10 Based on published observations, we evaluated malignant-appearing microcalcifications according to their shape (e.g., pleomorphic, irregular, fragmented, casting), density (highly variable), distribution (e.g., clustered) and size (highly variable).

Breast lesion localization

All patients with suspicious non-palpable breast lesions underwent pre-operative mammographically-guided breast lesion localization with a Kopan breast localization needle (19G, 9 cm length).11


Histological examination of the specimens showed 74 (26.4%) carcinomas. Histological subtypes were: 40 ductal invasive carcinomas (54%); 29 ductal in situ carcinomas (39.1%); and 5 lobular invasive carcinomas (6.7%).


Immunohistochemical analyses was carried out in 58/74 carcinomas, on the basis of tissue availability. Tissue sections were obtained from the files of the University Hospital of Patras (Department of Pathology). A panel of monoclonal/polyclonal antibodies were employed for ER (Novocasta 6F11), PR (Novocasta 1A6), c-erbB-2 (Biogenex CB11), Bcl-2 (Dako MO887), Bax (Dako A3533), Fas (Dako M3554) and DNA fragmentation factor (Novocasta NCL-DFFp) immunodetection.

Avidin-streptavidin immunohistochemical method (Biogenex Kit) and microwave antigen retrieval was carried out. Negative controls were processed by substituting the primary antibody with nonimmune mouse serum. Positivity scoring of immunohistochemistry for ER, PR, c-erbB-2, Bcl-2, Bax, Bas and DNA fragmentation factor (DFF) was carried out according to criteria established previously.12, 13, 14, 15

Statistical analysis

The χ2 and Fisher's exact tests were used to evaluate the associations between mammographically detected malignant-appearing microcalcifications and the immunohistochemical profile of each of the molecular factors studied. All statistical tests were 2-sided and were carried out at the time p = 0.05 level.


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  2. Abstract

Mammographic findings

Two hundred and eighty patients with suspicious mammographically-found non-palpable breast lesions who underwent mammographically-guided needle excision biopsy were evaluated. Malignant-appearing microcalcifications as an isolated finding or in combination with a mass, density or distortion were detected in 195 (69.6%) patients. Seventy-four of 280 (26.4%) non-palpable breast lesions proved histologically to be carcinomas (40 [54%] ductal invasive, 29 [39.1%] ductal in situ, 5 [6.7%] lobular invasive). Malignant-appearing microcalcifications was the prominent finding in 57/74 (77%) patients with non-palpable breast carcinoma (Table I). Immunohistochemistry was conducted in 58/74 carcinomas, on the basis of tissue availability. Malignant-appearing microcalcifications was the main mammographic finding in 48/58 (82.7%) of these patients.

Table I. Mammographic Appearance of Histologically Proven Non-palpable Breast Carcinomas
Histology/mammographyInvasive ductal carcinomaIn situ ductal carcinomaInvasive lobular carcinomaTotal
Density, mass or distortion114217
Density, mass or distortion and microcalcifications117220

Relationship between malignant-appearing microcalcifications and hormone receptor status

Thirty-eight of 58 (65.5%) non-palpable breast carcinomas proved to be ER-positive. Malignant-appearing microcalcifications was the major mammographic feature in 28/38 (73.6%) ER-positive carcinomas (p < 0.01). Twenty-seven of 58 (46.5%) breast carcinomas were positive with regard to PR, and the prominent mammographic appearance was malignant-appearing microcalcifications in 18 (66.7%) of them (p < 0.05).

Relationship between malignant-appearing microcalcifications and c-erbB-2 expression

Forty of 58 (68.9%) non-palpable breast carcinomas displayed c-erbB-2 overexpression. Immunostaining for c-erbB-2 was considered positive when at least 10% of the cells exhibited clear-cut membrane staining. In 30 (75%) of them, malignant-appearing microcalcifications was the major mammographic finding (p < 0.01) (Fig. 1b).

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Figure 1. (a,b) Membrane positive immunoreaction for c-erbB-2 protein in an in situ comedo ductal breast carcinoma and the mammographically detected malignant-appearing microcalcifications of the same non-palpable breast carcinoma. (c,d) Strong cytoplasmic Bcl-2 immunostaining in an in situ ductal breast carcinoma and the mammographically detected density of the same lesion. (e,f) Strong granular cytoplasmic Bax immunostaining in a Grade II breast carcinoma and the mammographically detected density and malignant-appearing microcalcifications of the same breast carcinoma. (g) Granular cytoplasmic Fas immunostaining in a Grade I breast carcinoma. (h) Nuclear DFF immunostaining in a Grade I breast carcinoma.

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Relationship between malignant-appearing microcalcifications and expression of apoptosis-related factors

Bax immunostaining was cytoplasmic and heterogeneous (Fig. 1e). Cases were scored as Bax-positive if more than 10% of cells exhibited clear-cut nuclear staining. Forty-two of 58 (72.4%) non-palpable breast carcinomas displayed high Bax expression. In 32 (76.1%) of them, malignant-appearing microcalcifications was the dominant mammographic finding (p < 0.01) (Fig. 1f).

Fas (APO-1, CD95) cytoplasmic expression was also evaluated immunohistochemically (Fig. 1g). Thirty-six of 58 (62%) non-palpable breast carcinomas produced positive immunoreaction for Fas protein. In 26 (72.2%) of them, mammography showed malignant-appearing microcalcifications (p < 0.05).

DFF expression was evaluated based on the nuclear immunopositivity it produced (Fig. 1h). Forty-five of 58 (77.5%) non-palpable breast carcinomas exhibited positive immunostaining and 33 (73.3%) of them harbored malignant-appearing microcalcifications as the major mammographic appearance (p < 0.01).

Finally, only 19/58 (32.7%) non-palpable breast carcinomas exhibited cytoplasmic positivity for the Bcl-2 protein, named as cytoplasmic clear-cut positivity of at least 10% of cells (Fig. 1c). Among them, 13 (68.4%) had malignant-appearing microcalcifications as the prominent mammographic finding (p > 0.05) (Fig. 1d).


  1. Top of page
  2. Abstract

The pathobiologic profile of non-palpable breast carcinomas cannot be determined accurately based solely on the mammographic appearance of microcalcifications. Previous studies have attempted to correlate radiological and histological features of malignant microcalcifications with respect to appearance, distribution and size.16 Our aim was to evaluate and interpret the clinical perspective of the relationship between mammographically found malignant-appearing microcalcifications and the expression of selected biological markers, such as ER, PR, c-erbB-2 (HER-2/neu), Bax, Fas, Bcl-2 and DFF in screening detected non-palpable breast carcinomas.

The expanded use of screening mammography has resulted in an increased detection of non-palpable suspicious breast lesions. This diagnostic dilemma has necessitated an increased number of diagnostic breast biopsies. Malignant-appearing microcalcifications found during screening mammography represent a diagnostic, prognostic and therapeutic challenge. Microcalcifications are generally considered as an unfavorable sign in the case of invasive tumors, whereas they seem to be favorable in non-invasive non-palpable breast carcinomas.17

Recent studies failed to show significant differences in pathological and biological features of symptomatic and mammographically detected non-invasive breast carcinomas.18 Many researchers have concluded that the favorable features of screen-detected cancers are due to the effect of length bias on biological factors.19 Other investigators, however, have suggested that breast carcinomas detected by screening programs are small and, although already invasive, have not yet undergone the genetic changes that determine the phenotypic characteristics representing poor prognostic indicators.20 There are also clues that the pathological and biological characteristics of screened breast cancers correspond to a lower degree of malignancy.21

Breast carcinogenesis represents a multistep process via a series of intermediate hyperplastic and preinvasive stages.22 According to a recent thesis, tumor progression occurs late in this preclinical phase, with a proportion of non-palpable carcinomas undergoing de-differentiation with increasing size, thus becoming more invasive and clinically aggressive.23. In our study, DCIS proportion was 39.2% among all screened breast carcinomas, which is in accordance with other published series,24 whereas non-palpable locally invasive carcinomas were found in 60.8% of our patients. There is a considerable need, therefore, to identify new markers associated with progression and tumor aggressiveness in conjunction with breast imaging studies providing more clues that would help breast specialists to make treatment decisions.

Determination of ERs and PRs in our series (65.5% and 46.5%, respectively) produced results similar to those reported in the literature, ranging from 32% to almost 80%.25 The PR ratio of only 46.5% can be attributed to the high incidence of invasive carcinomas. Several studies comparing screened and symptomatic breast carcinomas have found various associations with regard to ER,26 and some have not found any association with regard to PR.20 In our study, a strong association was found between ER positivity and mammographically detected malignant microcalcifications (p < 0.01). We also found a weaker, statistically significant association between PR positivity and malignant microcalcifications (p < 0.05).

Human epithelial breast cell growth is controlled by steroid hormones and growth factors.27 Aberrant transcriptional activity of some oncogenes, which are induced by estrogens augments the production of their protein products resulting in the loss of hormonal control of cell proliferation. Gradual reduction of PR content and ER prevalence in the cell seems to be the first stage of breast cell de-differentiation. ER positivity subsequently decreases as the aggressiveness of the tumor increases. The currently adopted scenario is that the failure of equilibrium between proliferation control mechanisms and programed cell death is responsible for deregulation of cell growth, mutation fixation and ultimately cancer development.

Overexpression of HER-2/neu is considered as an independent poor prognostic factor in premalignant breast lesions, non-invasive and invasive breast carcinomas.28 The reported range of patients found to be positive for c-erbB-2 by immunohistochemistry is 9–44%.29 In our study, the proportion was remarkably high (68.9%), possibly due to the high incidence of invasive carcinomas (60.8%). It is currently suggested, however, that the combination of HER-2/neu status measured by fluorescence in situ hybridization with weakly positive immunohistochemical results, provides more reliable information of c-erbB-2 gene amplification and safer clinical interpretation.30

Overexpression of the HER-2/neu occurs early during breast carcinogenesis.31 More important than its prognostic value is its predictive utility, because c-erbB-2 overexpression has been associated with altered clinical responsiveness to systemic breast cancer treatments (e.g. chemotherapy, antiestrogens), although the experimental and clinical data are still contradictory.32 Moreover, c-erbB-2 is the tumor-specific target for the recently approved antibody trastuzumab that has proved effective in patients with metastatic breast cancer.33 The significant association of c-erbB-2 overexpression and mammographically detected malignant microcalcifications (p < 0.01) found in our study, could be a combined predictive indicator of lower response rates in future therapeutic interventions and poorer overall prognosis.

Apoptosis is regulated by a complex network of cell death inhibitors and enhancers.34 Many apoptosis-triggering molecules have been identified, whereas an increasing number of apoptosis blockers, such as Bcl family proteins (e.g., Bcl-2), also exists.35 The bcl-2 gene product is frequently expressed in breast tumors and is associated with favorable prognosis.36 It has also been postulated that Bcl-2 acts as a modulator of chemotherapy or endocrine therapy.37, 38 The gradual decrease of Bcl-2 expression in premalignant and malignant breast lesions results in increased tumor aggressiveness.38 In this setting, our findings of highly positive c-erbB-2 status (68.9%) and loss of Bcl-2 expression (32.7%), combined with low PR positivity (46.5%) and the non-significant association between mammographically detected malignant microcalcifications and Bcl-2 expression (p > 0.05), could represent a very aggressive tumor profile requiring intensive treatment despite its non-palpable status.

Pro-apoptotic molecules (e.g., Bax) dimerize with Bcl-2 proteins and interfere with many of their functions. Bax expression is commonly induced in tumor cells after successful apoptosis stimulation. In normal breast tissue, cell survival and cell death balance is regulated by complex interplay between Bcl-2 and related proteins (e.g., Bax).39 The high positivity for Bax protein (72.4%) combined with low Bcl-2 expression (32.7%) found in our study might represent apoptosis induction. It is also important to emphasize the significant association found between Bax expression and mammographically detected malignant microcalcifications (p < 0.01).

DNA nucleosomal fragmentation is a characteristic event of late stages of apoptosis resulting from nucleases activation.40 One such nuclease, DFF, is capable of inducing DNA fragmentation after cleavage by caspase-3.41 In our study, an increased DFF positivity was detected (77.5%), along with a strong association with malignant microcalcifications (p < 0.01). These findings indicate the significant association of mammographically detected malignant microcalcifications with apoptosis triggering in the preclinical stage of breast carcinomas.

Bcl-2 does not promote cell proliferation and in the absence of additional genetic alterations Bcl-2-positive tumors tend to be relatively non-aggressive.49 It has been demonstrated, however, that Bcl-2-independent pathways for apoptosis induction also exist.42 In some cell types, apoptosis triggering by Fas and other tumor necrosis factor (TNF) family ‘death’ receptors is not blocked by Bcl-2 overexpression or other anti-apoptotic Bcl family members.43 This implies that Fas and similar TNF family receptors can bypass the Bcl-2-dependent checkpoint that controls cell survival. In our study, there was significant expression of Fas protein (62%) that was statistically associated (p < 0.05) with malignant microcalcifications in non-palpable breast carcinomas.

It is difficult to evaluate the importance of Bcl-2-dependent and independent apoptosis pathways results within the context of clinical response prediction to breast cancer systemic therapy. Given the fact that extensive “cross-talk” between these 2 pathways exists, we pose that a net balance between these proteins and the genetic events controlling their expression and action governs the stage of tumor transformation from the preclinical to the clinical state of the disease. Nevertheless, our study substantiates the hypothesis that tumor progression occurs late in the preclinical phase of the disease, due to an equilibrium failure of proliferation controlling mechanisms and apoptosis pathways.

Further assessment of the role of cell proliferation and apoptosis molecules and their biologic interplay, in conjunction with more accurate digital mammographic evaluation of non-palpable breast malignancies could offer more clues about their prognostic and predictive value in breast carcinogenesis, as well as more efficacious treatment strategies.


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  2. Abstract
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