Prevalence of c-myc expression in breast lesions associated with microcalcifications detected by routine mammography

ABSTRACT CONTEXT AND OBJECTIVE: Genetic abnormalities in cell proliferation-regulating genes have been described in premalignant lesions. The aims here were to evaluate c-myc protein expression in non-palpable breast lesions associated with microcalcifications, detected by screening mammography, and to compare these results with histopathological, clinical and epidemiological variables. DESIGN AND SETTING: Analytical cross-sectional study, with retrospective data collection, in a university hospital in São Paulo. METHODS: Seventy-nine female patients who underwent routine mammography between 1998 and 2004 were studied. Lesions classified by the Breast Imaging Reporting and Data System (BI-RADS) as 4 or 5 underwent percutaneous biopsy using a large-core needle. Ninety-eight lesions were studied anatomopathologically. Paraffin blocks properly representing the lesions were selected for immunohistochemical analyses using the streptavidin-biotin-peroxidase technique with monoclonal mouse c-myc antibodies. RESULTS: Among the 98 lesions, 29 (29.6%) contained malignant neoplasia; 40 (40.8%) had a positive immunohistochemical reaction for c-myc. When the groups were divided between lesions without atypias versus atypical lesions plus malignant lesions, 31.03% of the 58 lesions without atypias were positive for c-myc and 55% of the 40 malignant and atypical lesions (P = 0.018). Comparing the atypical lesions with ductal carcinoma in situ versus the benign lesions without atypias, c-myc was present in 51.61% of the 31 atypical lesions and 31.03% of the benign lesions without atypias (P = 0.057). CONCLUSION: C-myc protein was more frequently expressed in atypical and malignant lesions than in benign lesions without atypias. C-myc expression correlated with the presence of atypias (P = 0.018).


INTRODUCTION
At present, the best screening method for breast cancer is routine annual mammography examinations for women over 40 years of age.This has been correlated with a reduction of up to 30% in the mortality rates due to breast cancer, around the world.Screening using mammography is an extremely sensitive method for detecting both malignant and benign breast lesions, and the great majority of breast lesions seen on mammographs are non-palpable.The high sensitivity of the method is largely due to findings of microcalcifications associated with the lesions.Areas of microcalcifications often need to be investigated by means of biopsies.2][3][4] Precursor lesions or even minimally invasive lesions associated with these microcalcifications provide greater accuracy in identifying lesions at early stages.][7] Today, there are several methodological approaches for performing breast biopsies.The gold standard is still surgical removal of the lesions by means of open surgery. 4However, less invasive methods consisting of percutaneous biopsy with a large-core needle, by means of both automatic propulsion (core biopsy) and vacuum assistance ("mammotomy"), are increasingly used. 8The disadvantage of these less invasive types of approach is that they sometimes do not remove the lesions, which leads to the need for follow-up regarding the patient's remaining microcalcifications. 9 Genetic abnormalities in cell proliferation-regulating genes have been described in premalignant lesions. 10Among the oncogenes connected with controlling the cell cycle, the role of the oncogene c-myc stands out.Since its discovery by Bishop, in the 1970s, its central role in the proliferation and malignant transformation of animal tissue has been widely demonstrated. 11ne important carcinogenesis route that has been studied in relation to breast cancer is the Wnt pathway.The first Wnt gene was cloned from the rat genome in 1982.This family produces an enormous quantity of proteins that are related to various normal cell functions, from differentiation and proliferation to cell survival capacity. 12The best known and most studied are Wnt itself (described in Wilms tumors), adenomatous polyposis coli (APC) (responsible for hereditary colon polyposis syndrome), catenin (a protein for cell support and connection with the basal membrane) and T-cell transcription factor (TCF).There is growing evidence that the proteins in the Wnt signaling pathway and/or its components have an extensive relationship with human breast cancer. 13ecause this is a complex pathway for activating and producing cell proteins, it acts in reactions that include several oncogenes.One of the target genes that have been described is c-myc. 14For this reason, c-myc protein expression is evaluated as an indirect marker for this pathway in breast carcinogenesis.
The c-myc protein acts as a transcription factor, i.e. a protein that is capable of binding to specific deoxyribonucleic acid (DNA) sites with the purpose of modulating the gene transcription.Expression of the c-myc gene is unregulated in several types of human neoplasia.Contrary to other oncogenes such as those of the ras family, activation of c-myc expression does not require mutations or changes in amino acid sequences.
The mechanisms through which the functions of the c-myc gene can be altered include action by viral particles; amplification of the locus on the chromosome, such as in neuroblastomas (N-myc) and pulmonary neoplasia (L-myc); chromosome translocations, such as in Burkitt's lymphomas; or overexpression of the protein due to gene or transcription factor mutations.A gene regulation pathway for the c-myc gene through the expression line of the Wnt gene has been described.By means of the intermembrane receptor, the Wnt gene acts on the protein APC and the latter binds to catenin to form a complex in the cytoplasm.When this complex is not formed, which could be through changes in the action of the Wnt gene, APC or catenin, the latter actively moves to the nucleus and acts as a transcription factor for the c-myc gene. 14nder physiological conditions, the main role of c-myc is to promote cell replication, in response to extracellular signals that arouse the cell cycle from rest. 157][18][19][20] Carcinogenesis relating to c-myc occurs when this is expressed aberrantly through amplification or genetic alteration, thus leading to events that go from promotion of excessive proliferation of altered cells to inhibition of apoptosis. 21,22Amplification and/ or overexpression of this gene have been described in almost all types of malignant neoplasia in human beings. 23mplification of the c-myc oncogene has already been described in benign breast lesions such as fibroadenoma. 11We do not know of any reports on abnormalities in premalignant lesions associated with microcalcifications that were found by means of screening mammography.

OBJECTIVE
Our objective here was to evaluate the prevalence of c-myc protein expression in non-palpable breast lesions associated with microcalcifications that underwent biopsy following detection by means of screening mammography, and to compare these results with histopathological variables.

Patients and design
This study was granted prior approval by the Research Ethics Committee of our university hospital.Ninety-eight lesions among 79 female patients who were admitted to our service between 1998 and 2004 were studied.All these patients had undergone routine mammography, were found to present microcalcifications and were classified as 4 or 5 using the 2003 version of Breast Imaging Reporting and Data System (BI-RADS 2003). 24The patients who were included underwent percutaneous biopsies with a large-core needle, using the Mammoguide ® system (Philips MammoDiagnost MD 4000 mammograph, Philips Medical Systems BV, Netherlands) as the guide for image and biopsy site identification.The techniques used were stereotaxis-guided mammotomy and core biopsy with a large-core needle of caliber 12 G, adapted to an automatic propulsion system with a 2.2 cm cursor (Biopty, Bard Urological, Covington, Georgia, United States).

Histological analysis
The samples were fixed in 10% formalin, labeled with the patient's name and sent to the pathology laboratory of the Universidade Federal de São Paulo -Escola Paulista de Medicina (Unifesp-EPM).After processing and embedding in paraffin, five-micron sections were mounted on slides and stained with hematoxylin and eosin using the recommended technique.
All the cases included in our study were reviewed from an anatomopathological point of view, by two independent pathologists.The diagnoses were classified by consensus as benign entities without atypias, premalignant or atypical entities (atypical hyperplasia) or malignant entities.

Immunohistochemical analysis
For the immunohistochemical analysis, new five-micron copies from the blocks studied were mounted on blank silanized slides.The faithfulness of all the reactions was tested by comparing each of them with positive and negative controls that had previously been established, which was done concomitantly.All the steps were preceded by two fiveminute baths in phosphate-buffered solution (PBS) at pH 7.4 (except for the secondary antibody).
The c-myc expression was analyzed according to its distribution and positivity pattern.It was analyzed in the epithelial component of the tu-mors.When c-myc protein was found to be present in predominantly nuclear locations in more than 10% of the neoplastic cells, this was taken to be a positive finding.
The results obtained were subjected to statistical analysis using descriptive and analytical techniques.The chi-squared univariance test was performed (in 2 x 2 tables), and when the frequency was less than five, the Fisher test was used.P-values of less than or equal to 0.05 were taken to be significant.

RESULTS
Among the 98 lesions studied, nine had a diagnosis of invasive ductal carcinoma (IDC), 20 were ductal carcinoma in situ (DCIS), 11 were atypical ductal hyperplasia (ADH) and 58 were benign lesions without atypias, such as fibrocystic abnormalities, fibrosclerosis, adenosis and lesions with focal hyperplasia without atypias.
The c-myc antigen was more frequently expressed in cases of IDC and ADH (Table 1).Comparing the atypia cases (ADH) with the benign cases, it was seen that negative findings for c-myc were predominant in the benign cases, and that this was statistically significant (P = 0.039; Table 2).The benign cases also seemed to be associated with negative c-myc findings when compared with the IDC cases and IDC plus DCIS (malignant) cases, although without statistical significance (P < 0.061).
Finally, dividing the lesions into two groups, the first containing the lesions with atypias and the neoplastic lesions (IDC and DCIS) and the second containing the lesions without atypias, the c-myc expression rates were 55% and 31.03%,respectively.We observed a greater frequency of c-myc in the lesions with atypias, and this difference was statistically significant (P = 0.018), as shown in Table 3.

DISCUSSION
The hypothetical model for tumor progression in breast carcinogenesis, through a sequential (multistep) chain of events, was put forward by Lakhani in 1999. 25Despite the complexity of studying this model for the breast, due to the morphological heterogeneity of preinvasive lesions, this author based his theory on three main sources: experiments on animals, reviews on human histological material and genetic analyses on precursory breast lesions.
In experiments on animals following exposure to the tumor virus MuMTV (murine mammary tumor virus), the normal epithelium was found to be transformed into hyperplastic alveolar nodules that, when transplanted into normal breast tissue, present a higher chance of malignant transformation.In reviews on human histological material, ADH and DCIS were found in specimens of invasive carcinoma, which also showed that these lesions occasionally present morphological transition and continuity with each other.Furthermore, atypical hyperplasia was five times more common in breasts containing malignity than in normal breasts. 25ther evidence has come from a review of prospective studies by Page et al. 26  Pearson chi-squared (1) = 5.6286; P = 0.018.
Table 3. Expression of the c-myc antigen according to the presence or absence of cell atypias more when atypical.They found 5.3 times more carcinomas with ADH and 11 times more in conjunction with family histories of cancer.These authors also showed that in breasts with DCIS in biopsies, 30% more invasive carcinomas were found after 6.1 years of follow-up.
To conclude his theory, Lakhani 25 also reported the various progressive genetic similarities between DCIS and IDC, between ADH and DCIS and between the usual ductal hyperplasia and atypical ductal hyperplasia.In our study, we were also able to observe complexity of proliferative lesions in the same patients.Thus, we found three cases of ADH with DCIS and two cases of DCIS with IDC.
It is known that the carcinogenic process of tumor progression covers a series of intermediate stages, including activation of oncogenes and inactivation of tumor-suppressing genes. 27,28The stages preceding tumor invasion that are described as DCIS and ADH frequently involve several early genetic changes. 29These lesions may be found in association with infiltrative neoplasia, which suggests that the cumulative procedure of genetic damage does not always follow a single linear sequence of events. 30he activated c-myc gene may perform an important biological role in the tumorigenesis process. 31One of the most important actions involves the proliferation process.Early induction of c-myc may occur through different pathways, involving the transition from the quiescent cellular state (G0) to proliferation (G1).Moreover, c-myc is continually expressed in cells undergoing proliferation. 31The mechanism for action leading to the entry of cells into the cell cycle involves inactivation of cyclin-kinase antagonist proteins and inactivation of the cyclin E antagonist.The presence of c-myc from G1 onwards seems to be essential for the cell cycle sequence. 32On the other hand, in cells that enter the differentiation process, the expression decreases and this is important for terminal differentiation.Since c-myc expression is also associated with the apoptosis process, lack of control over c-myc expression may lead to increased cell death. 31n a meta-analysis, Deming et al. 33 observed that, on average, 15.5% of breast cancer biopsies presented c-myc gene amplifications of more than three times normal values.Studies using the immunohistochemical technique have demonstrated that, in 50 to 100% of the cases, there were increased levels of the c-myc protein. 34Mai showed that there was a greater percentage of cancer cases with aberrant c-myc protein levels than of cases showing gene amplification.This would also imply that changes in the stability of messenger ribonucleic acid (RNA) or its protein could be the mechanism for increased c-myc levels, which would be consistent with reports that overexpression precedes gene amplification, thereby leading to chromosome instability. 35ollowing this line of reasoning, it might be supposed that findings of c-myc protein in benign lesions, as in our study, could precisely represent this initial step of gene overexpression and/or amplification and the initial step in breast carcinogenesis.In fact, when we compared the presence of c-myc in atypical lesions with its presence in benign lesions without atypias, we found a statistically significant difference (P = 0.039).Likewise, dividing the lesions into two groups, such that the first contained the lesions with atypias and the neoplastic lesions (IDC and DCIS) and the second contained the lesions without atypias, the c-myc expression was 55% and 31.03%,respectively.Thus, we observed great-er frequency of c-myc in the lesions containing atypias, and this difference was also statistically significant (P = 0.018).
Even without reaching significance, we could see a certain tendency (P = 0.06) towards c-myc expression when the IDC and DCIS results and the results from the benign lesions were analyzed together.This was also seen when we compared the IDC findings alone with the benign lesions (P = 0.06).These results allow it to be presupposed that the presence of c-myc may signify increased risk of progression of the benign proliferative lesions, or even of the DCIS.
The number of cases studied did not allow separate identification of the individual pattern of c-myc expression in the benign breast lesions, separated according to the relative risk of progressing to invasive carcinoma.Nevertheless, among the adenosis, papilloma and typical ductal hyperplasia cases (lesions that are said to be proliferative and considered to present a low risk of breast cancer), we found six cases that were positive for c-myc, out of the 14 studied.On the other hand, among the non-proliferative lesions, such as fibrocystic abnormalities, only one case out of 14 was positive.
We also observed that the pattern of c-myc presence was variable and that it could coexist in different lesions in the same patient.For example, in some patients in whom malignant lesions and low-risk benign proliferative lesions (such as adenosis and the usual ductal hyperplasia) coexisted, we found that both types of lesion were positive for c-myc (two cases out of six evaluated).This finding reinforces the supposition that the initial lesion (adenosis) had already been amplified by the c-myc gene much before the evolution into an atypical proliferative lesion or even into carcinoma.It corroborates the hypothetical model for tumor progression through a multistep sequential chain of events for breast carcinogenesis. 25chmitt and Reis-Filho 36 reported positive findings of c-myc in breast cancer cases, ranging from 1% to 94.4%.This large difference in positive findings of c-myc has a variety of reasons.Among these, perhaps the main reason is differences in investigation methods, which may make use of immunohistochemical or hybridization techniques, such as fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH).These latter methods are clearly more sensitive, but much more expensive to perform.
Another reason would be the fact that c-myc presence is related to different types of tumor.For example, high nuclear grade tumors have a higher chance of presenting overexpressed proteins than do low grade tumors. 11einforcing this idea of overexpression of c-myc protein in cells that are more differentiated, Le Roy et al. 37 demonstrated that patients with breast tumor receptors positive for estrogens who received treatment with tamoxifen showed decreased levels of c-myc messenger RNA, in comparison with patients who had not received this drug.This result suggests that tamoxifen antagonized the effect of estrogens in relation to c-myc expression.
It is known that percutaneous biopsies from benign lesions such as typical ductal hyperplasia, florid adenosis, papillomatosis and even atypical ductal hyperplasia result in some cases of underestimated diagnosis.It is also known that, during follow-up, some of these lesions end up evolving into carcinoma.We might therefore suggest that tamoxifen should be used in cases of biopsy findings of benign proliferative lesions that nonetheless are positive for c-myc.
The study by Berns et al. 38 also suggested that there is an inverse relationship between c-myc presence and human epidermal growth factor receptor 2 (HER2/neu) presence.This opens up even greater possibilities for future studies on c-myc involving possible drug preparations that would be capable of acting directly on amplified c-myc, in the same way as with c-erb-b2, such as the use of herceptin and HER2/neu.
Studying the expression of the c-myc gene has produced some guidelines regarding carcinomas that may in the future contribute towards more accurately determining tumor behavior, because of the associations of c-myc with cell proliferation and chemoresistance. 39

CONCLUSION
The c-myc protein is more frequently expressed in atypical and malignant lesions than in benign lesions without atypias.The expression of c-myc correlates with the presence of atypias.

Table 1 .
that found 2.5 times more carcinomas in patients whose previous biopsies showed the usual ductal hyperplasia and five times Expression of the c-myc antigen according to the histopathological classification of breast lesions

Table 2 .
Comparison between positive and negative c-myc expression and the types of breast lesion, grouped according to benign and malignant histological results IDC = invasive ductal carcinoma; DCIS = ductal carcinoma in situ; ADH = atypical ductal hyperplasia.p-values obtained using the chi-squared frequency test.* P-value obtained using the Fisher exact test.