Immunohystochemical profile of cytokeratins (5, 7, 14, 18, 20) in retrospective cases of breast fibroadenomas

Author(s): Marcia Bernardo*, Andreia Fabiana do Vale Franco*, Gil Facina and Angela Flavia Logullo Waitzberg Introduction: Fibroadenomas (FAs) are the most common benign tumors in the breast. Described as a biphasic stromal and epithelial lesion, it is usually stable and clinically well managed. Although the cytokeratins (CKs) distribution among ductal cells is well described in carcinomas and normal breast tissue, in FAs the differentiation and distribution of epithelium CK ... Abstract View Full Article View


Introduction
The human mammary gland epithelia were generally classifi ed, until recently, as a tissue containing two morphologically distinct cell populations: luminal and basal glandular myoepithelial cells. Currently, there is a more 036 https://www.peertechz.com/journals/annals-of-cytology-and-pathology Citation: Bernardo  in situ carcinoma and lobular carcinomas [2][3][4]. To identify these cells or their different stages of maturation, distinct keratin expression may be used. Cytokeratins are keratin proteins that make up intermediate fi laments found in the intracytoplasmic cytoskeleton of epithelial tissue, arranged and expressed in organ specifi c [5,6]. In epithelial cells they function is important to the cellular structuring and maintenance of the cellular shape, besides infl uencing the maintenance of polarity, migration and healing. When cells lose adhesion and gain motility CK expression are transient and defi nitive changes in their systems that interfere with polarity and cell adhesion occur [7]. Alterations of cytokeratin profi le may be a subtle and early modifi cation of epithelial cells in metaplasia, dysplasia and neoplasia [3].
However, in addition to the epithelial population, the mammary gland contains an important contingent of stromal cells, which indirectly infl uence the epithelial population.
One of the indirect effects of stromal cells is to control the proliferation and differentiation of the epithelial component [8].
In this context of the interrelationship of ductal and stromal cells, FAs are fi broepithelial lesions where there is a progressive increase of both mammary components, stromal and epithelial and the mesenchymal cells arise proportionally with a potential paracrine effect on the oppressed epithelial cells. They are formed by the proliferation of specialized stroma with secondary distortion of the preexisting glands, which contains a single layer of cuboidal cells and myoepithelial cells [9]. The stroma is usually myxoid and compresses the epithelial cells in a concentric arrangement progressively.
Clinically FA comprehends the most common benign tumors in breast, especially in younger women, are usually stable and clinically well managed. FAs may be single or multiple, and the same patient may have one or more lesions throughout life [9,10]. They are devoid of atypical and mitosis, and are called cellular FA when their diagnosis is more cellular [11].
The malignant transformation of FA, albeit an extremely rare event, is associated with the Phyllodes tumor (PT).
Literature data suggest that this entity would be the malignant variant of FA [12,13].
Although these two entities are biphasic, that is, they contain epithelial and stromal components, molecular studies comparing the two types of tumors are rare [14,15]. Noguchi and colleagues recorded the fi rst study on clonality of these entities by amplifi cation and PCR, showing that FAs are polyclonal in both stroma and epithelium, while PT is polyclonal in the epithelium, but monoclonal in the stroma [16]. More recently specifi c mutations in MED-12 gene, similar to those found in uterine leyomiomas, were largely reported in FAs and up to 60% of cases [17]; and also in Phyllodes tumors, albeit more frequently in benign and border line lesions. [15,17].
The evidence of common genetic mutational alterations in both lesions has risen the idea of a possible continuum and association, which was evidenciated by pareja, et al. in PT lesions bearing fi broadenoma-like areas harboring the same mutations of those of classic PT microdissected areas [18].
Although the CKs distribution among ductal cells is well described in carcinomas and normal breast tissue, in FAs the differentiation and distribution of epithelium is scarcely known. In the literature there are several studies reporting changes in epithelial population profi le according to the different expression of cytokeratin in subpopulations in various breast diseases; however, such evaluation of ductal epithelium in FA is scarce. Previous studies in fi broadenomas reporter epithelial profi le with similar proliferation index that normal breast tissue.
The distribution of different ductal cell subtypes at different stages and maturation could imply on a potential switch favoring were subset in detriment of others or even infl uence on differentiation or proliferation process. Since epithelial cells are prone to mesenchymal cells infl uence, early and subtle phenotypic alterations would be refl ected in cytokeratin structure and profi le and could rise a alert of FA lesions with potential risk of clonality.

Material and methods
The study was performed in a consecutive series of 104 patients who were evaluated surgically treated and diagnosed at the Perola Byington Hospital, and who underwent a nodulectomy from January to March 2006. Only patients diagnosed with FA without preview entities, and cases with paraffi n blocks and Hematoxylin-Eosin (HE) stained slides available and reviewed by the pathologist were included. Patients diagnosed with FA associated with others pathologies or inappropriate samples (without slides or paraffi n blocks) were excluded. Clinical data regarding variables such as age, laterality, size tumor and recurrence were collected and organized from the clinical fi les.
Representative areas containing both components of the benign fi broepithelial lesions were marked for Tissue Microarray (TMA). And arranged in two distinct TMAs from the 104 cases elected for the study, as previously described in the literature [19,20]. Briefl y, from the original cut stained by HE, the areas of interest were marked with a sharpie pen; then the equivalent area in the original paraffi n block was marked. From this point, the selected areas were transferred to the receiving block through the use of Tissue Microarrayer TMA Master (3D HISTECH). The TMA block was made with 1.5mm cylinders, with samples arranged in 13 rows and 8 columns [20]. From this block, 20 serial histological sections were prepared on silanized slides by a 4μm mechanical microtome previously treated with special adhesive fi lm (Instrumedics Inc. Hackensack, NJ USA). All reactions were accompanied by a positive control, known to be positive for each antibody tested, and a negative control of the reaction (removal of the primary antibody and removal of the secondary complex).

Immunohistochemistry
The slides were then submitted to the immunohistochemical After antigenic retrieval, the histological slides were cooled at room temperature (15 minutes) and washed with running water (5 minutes) [21]. The slides were placed in 3% hydrogen peroxide (3 times, 5 minutes each) for blocking the endogenous peroxidase and then washed in running water (5 minutes).
The cuts were subjected to blocking of non-specifi c proteins with casein (Protein Block Serum-Free-DakoCytomation, Carpinteria, USA) at room temperature (20 minutes).
The primary antibody was incubated in the sections at room temperature (2 hours); the dilution of the antibodies followed the recommendations of each manufacturer after the adaptation to the laboratory of the Hospital A.C. Camargo Cancer Center (Table 1). Then, for signal detection slides were washed with saline-phosphate buffer (PBS) (3 times, 5 minutes each). After this, they were incubated with a polymer (horse radish peroxidase).  [16].
To analyze the statistical signifi cance of the patient's age, size tumor, and proliferative index on tumoral recurrence parametric statistical tests with normal distribution (test t-student) were used and nonparametric tests (Fisher's exact test) in the case of normality has not been identifi ed. In order to test the mean differences in the cytokeratin's expression in the epithelium was used the McNemar test. The level of statistical signifi cance considered was 5%.

Demographic, clinical and epidemiological data
The epidemiological data collected in the patient's medical records reveal that this series is comparable to others in the literature, refl ecting the normal distribution of patients who present FAs as the main complaint without association with other comorbidities and the recurrence pattern average value. Table 2 shows the epidemiological variables distribution.
Our series presented a mean age of 27 years (median of 25 years). As expected, the distribution shows a predominance of

Relationship between immunohistochemistry expression of different CKs
In general luminal and basal CKs are co-expressed.
Analyzing the difference in the expression of CKs in the tumor epithelium, it was possible to observe that there was no disagreement between CK5 and CK18, that is, these CKs present  [26]. Unlike, women with single FAs often have a family history of theses tumors [27]. An important observation in our work is that majority of our samples presented as relapses may actually be multiple FAs.
Analyzing the age of the patients regarding tumor recurrence, no difference was observed between the cases that presented tumor relapses and those that did not (p=0.0662).
A possible connection between the presence of multiple FAs and oral contraceptives has been proposed, but this connection is not yet well established in the literature [28]. Apparently cases with tumor relapses or multiple FAs did not focus on any specifi c age group.
The epidemiological data collected in the patient's medical records reveal that this series is comparable to others in the  it was observed that the tumor size did not infl uence this correlation either. The size of FAs acquires importance in the reports in which they appear as giant FAs, which are diffi cult to excise, have worse post-operative esthetic results, but not necessarily relapse [29].

Relationship between cytokeratin immunoexpression and clinical variables
In the literature, there are few contributions to the evaluation of proliferation in FAs. In a line developed at UNIFESP, the infl uence of tamoxifen on cell proliferation in FAs was evaluated [24]. This study had a prospective randomized approach where they observed that proliferation rates were lower in epithelial cells when tamoxifen dose was 20 mg/day (data measured by PCNA expression). In this study, proliferation was 32.4% in epithelial cells [30].
A few years later, a study evaluated 33 patients, and noted that proliferation data were not associated with oral contraceptives [31]. In another publication, with 75 patients evaluated, they found mean Ki67 results of 27.88 and 37.88 positive cells per thousand evaluated in the follicular and luteal phases, respectively. As in this study, proliferative activity in FAs did not differ between cases and did not suffer signifi cant hormonal infl uence. The values were not associated with age, according to this study [32]. Although

About cytokeratins
Although CKs are traditionally used for differential diagnosis between epithelial lesions of various sites, or for identifying genetic diseases that affect the epidermis, they are also addressed in the literature involved with other clinical aspects.
In mastology, research with CKs has been used to detect circulating bone marrow tumor cells, and this feature correlates with survival [33]. Some CKs, in isolated and specifi c form, possibly participate in the invasion mechanism, as with CK18 and CK14 [34,35], or as prognostic indicators [36].
In adulthood, the mammary epithelium is composed of CK5 is considered a baseline profi le marker, and is usually expressed in myoepithelial and basal cells [37]. When this CK is expressed together with CK14 it may refl ect the presence of mammary progenitor cells capable of giving rise to both glandular and myoepithelial cells.
This CK5 is usually expressed in the mammary ductal epithelium. According to the literature, this expression refl ects the contingent of non-luminal cells or outside the context of the fraction of cells that have already differentiated and will not divide but are generally in a more apical position [37]. Conversely, CK18 refl ects the proportion of cells in differentiation status that usually express hormone receptors.
The relationship between these two markers should not be concomitant, but rather inverse when observed in each cell type.
In fact, it was observed in the study that there was no correlation between these two markers regarding the In this case we would have positive basal cells adjacent to positive luminals cells, however, in our work a more chaotic distribution of this reactivity was observed. In cases with cellular atrophy and progressive stromal hyalinization of FAs, CK5 positivity decreased considerably. One of the most likely explanations for this fi nding is that the three-dimensional spatial distribution of the ducts and their cells, as well as the ordered segmental differentiation of the ductal tree is partially compromised in the FAs. This impairment may be the result of the basic spatial alteration by the progressive strangulation of stromal expansion or, eventually, by the paracrine action of stromal cells next to the other ducts.
Overall, the percentage of CK5 positive cells was also higher in FAs than in normal breast tissue described in the literature. This difference may have a greater signifi cance when we look at it in light of the results of a study with a broad line of research in breast cancer, where we have shown that CK5 positive cells are more present in heterogeneous tumors and constitute a portion of tumor cells that are often resistant to hormonal and radiotherapeutic treatment [39].
Basal cell profi le and proliferation by Ki67 expression was also a different fi nding from normal breast tissue. The larger the number of basal cells, the higher the proliferation rate found. This relationship indicates that cell proliferation is related to an increase in the contingent of CK5 expressing cells, which may mean that the resulting cells are of this profi le, or that proliferation is occurring to rearrange an imbalance of this cell differentiation profi le.
The results of CK7 and CK20 expression in the FA epithelium studied in TMA achieved the expected, that is, strong, homogeneous and continuous positivity in the CK7 epithelium, and diffuse negativity for CK20, which is used for differential diagnosis in lesions of undetermined origin, where the suspicion of mammary origin requires the binomial CK7 + / CK20to maintain this hypothesis. Gastrointestinal origin is indicated by the opposite binomial, ie, CK7 -/CK20 + .
The maintenance of this profi le in FAs suggests that the alterations caused by the epithelial population being contained within an FA are not enough to alter the expression of these CKs and compromise their basic mammary cell profi le. The absence of CK7 expression in stromal cells could suggest that none of the stromal cells sampled underwent epithelium-mesenchyma transformation (EMT), ie, they are not epithelioid cells with mesenchymal morphological characteristics.
CK14 is considered a basal cell marker, as is CK5. It is frequently studied in most studies that try to categorize breast cells according to different stages of differentiation. In contrast to CK5, it has been the subject of study in experimental mammary carcinogenesis where transient recovery of CK14 expression has been recorded, specifi cally in the invasive front of tumors. Recent translational research points out that early and subtle mechanisms such as the phosphorylation of CK segments may infl uence its stability and therefore impair and achieve cell adhesion [40,41]. Mutations in the CK14 gene ie cause Dowling -Meara EBS disease, by forming irregular cytoplasmic non-functioning aggregates [42].
The results of the comparison between CK5 and CK14 expression, as expected, show concomitance in the basal cells.
This contingent of cells refl ects the population of so-called progenitor cells in the breast, which could give rise to both glandular and myoepithelial cells. There are some isolated cases containing CK5 or CK14 expression in luminal position cells in our study, which may refl ect a stage of atrophy. Patients older than 50 years old were shown a higher percentage of CK14-expressing ductal cells. With age, morphologically, myoepithelial cells decline and luminals acquire a profi le more similar to basal ones [43]. As well, it was observed that the immunoexpression of CK14 and CK18 were not always restricted in the basal and luminal layers, respectively. These authors report that this controversial inversion of basal CK expression in luminal cells and / or luminal CK expression in basal cells may be present in type I lobes, ie, less differentiated from breast tissue. We may suggest that this pattern is correlated with lower mammary tissue differentiation in cases of FAs with this characteristic [38,39]. FAs have a contingent of progenitor cells when considered CK5 and CK14 positive, comparable or larger than non-tumoral breast tissue; Ki67 is a nuclear antigen expressed in cells proliferation process. It is used as prognostic indicator, and is considered a luminal B feature when the level of immunoexpression exceeds 14% [16]. Its elevated expression was related to increased mitotic activity, cellular indifferentiation and invasion tendency. Only 1 case did not show positive expression to Ki67.
Cell proliferation in FAs is comparable to normal breast tissue and correlates with CK5 expression.