**2. Histopathological alterations of the mammary gland during carcinogenesis in rats induced by DMBA-estrogen combination**

The mammary gland is a hormone-dependent organ that is influenced by steroid hormones secreted by the ovary. It was previously known that DMBA induces mammary cancer under hormonal conditions in the presence of adequate levels of estrogen. Estrogen stimulates ductal growth, epithelial hyperplasia, and the development of connective tissue-enclosed ducts and lobules, increasing the number of progestogen receptors in mammary cells, and promoting lobular acinar development has been shown in numerous studies [8].

Histopathological analysis plays an important role in tumor diagnosis and research. Although the histopathological features of the human breast cancer are well known, details about the mechanisms involved in breast cancer and the associated histomorphology alterations are still unclear. The morphological features of rat mammary carcinoma are similar to those of human breast carcinoma. In this study, histopathological observation of a rat mammary cancer model induced by the DMBAestrogen combination was performed using the Hematoxylin–Eosin (HE) staining method (**Figure 1**).

7,12-Dimethylbenz(α)anthracene (DMBA), a toxic tumor-inducing agent that has been proven in animal models, has also been reported to induce human breast cancer [14]. It is mainly reported to induce the formation of terminal ductal lesions, and induce ductal epithelial cell hyperplasia, atypical hyperplasia, and subsequent ductal changes [8].

Our study also confirmed that many types of mammary gland lesions that occurred in rats induced by DMBA and estrogen combination are similar to lesions in human breast cancer. As a result of the induction of DMBA, in the treatment group, alveolar epithelial cells have dysplasia, and their shape and structure have changed compared with control group. The alveolar cells are no longer round, their arrangement is irregular, and there is a change in the shape of the epithelial cells. The normal alveolar epithelial cells are cuboid in layers (**Figure 1A**). The dysplasia found in the alveoli indicates that there has been a neoplastic or cancerous process in the breast. In the treatment group, hyper-chromatin is also found in the cell nucleus so that it looks darker (**Figure 1B**). The mammary duct is normally covered by a layer of epithelial cells and the ductal lumen is empty (**Figure 1C**). The treatment group showed a hyperplasia or proliferation of epithelial cells from the surface of the duct to the lumen of the duct (**Figure 1D**). According to Young et al., tissue in the mammary or stroma is in the form of connective tissue and fatty tissue that surrounds the lobes (**Figure 1E**). In treatment group, new cancer cells form new epithelial cell clusters that had spread to the surrounding tissue (**Figure 1F**).

**Figure 1.**

*Histopathological alterations in rat mammary cancer model induced by DMBA-estrogen combination. Mammary alveolar on control group shows a normal morphology (a). The formation of neoplastic cell on mammary alveolar is shown with dysplasia of alveolar epithelial cell (B). The mammary duct in control group is normally covered by a layer of epithelial cells and the ductal lumen is empty (C). Hyperplasia of ductal epithelial cell to basement membrane (D). Stroma in the form of connective tissue and fatty tissue that surrounds the lobes (E). Invasion of cancer cell to mammary stroma (F) (HE, original magnification ×400).*

According to Feng et al. [8], the carcinogen DMBA and hormone intervention can cause the presence of pre-malignant lesions such as usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) (**Figure 2**) [15]. The UDH sample showed proliferative cells that were arranged in a mixed and disorderly pattern (**Figure 2A**). In the ADH samples, the ductal epithelial cells were bigger than

*Molecular Histopathology of Mammary Carcinogenesis as Approach to Cancer Prediction… DOI: http://dx.doi.org/10.5772/intechopen.110840*

**Figure 2.** *Histopathological changes in breast tissue in the SD rat model. UDH tissue showed proliferative cells in a mixed and disorderly pattern (a). ADH tissue showed that the ductal epithelial cells were larger than normal cells and had an increased cytoplasm-nucleus ratio (B). DCIS tissue showed that the lumen of the duct was filled with atypical proliferative cells (C). IDC tissue showed that the proliferative cells (D) (HE, original magnification ×400). Adapted from Feng et al. [8].*

normal cells and had an increased cytoplasm-nucleus ratio (**Figure 2B**). In the DCIS sample, the lumen of the duct was filled with atypical proliferative cells that formed a cribriform (**Figure 2C**). Further, proliferative tumor cells were observed to damage the basement membrane and then infiltrate into the fibrous connective tissue, namely IDC (**Figure 2D**).

#### **3. Histopathological classification of mammary cancer**

The animal mammary cancer classification is adapted from the breast cancer classification system. Breast cancer classification is based on pathology and molecular biology [16, 17]. To study the breast/mammary cancer morphology, it is necessary to understand whether the tumor is confined to the epithelial component of the breast, invades the surrounding stroma, or develops in the ducts or lobes of the mammary gland [3]. In the practice of histopathology, cell type features, number of cells, type and location of secretion, immunohistochemical profile, and structural features, in addition to their subclassification, are used to determine whether the tumors are ductal or lobular.

The most common special types of breast cancer (**Figure 3**) according to Nascimento and Otoni [3] include the following:

#### **Figure 3.**

*The morphological classification of the main subtypes of invasive breast carcinomas. (A) Medullary carcinoma; (B) metaplastic carcinoma; (C) apocrine carcinoma; (D) mucinous carcinoma; (E) cribriform carcinoma; (F) tubular carcinoma; (G) neuroendocrine carcinoma; (H) classic lobular carcinoma; and (I) pleomorphic lobular carcinoma (adapted from Nascimento and Otoni, [3]).*

#### **3.1 Invasive ductal carcinoma non-specific type (IDC-NST)**

The most common histological subtype in about 40–75% all invasive breast carcinoma. The morphology of tumor cells is pleomorphic, with protruding nucleoli and numerous mitoses.

#### **3.2 Medullary carcinoma**

Special subtype of invasive breast carcinoma, responsible for approximately 5% of all cases, is associated with mutations in BRCA-1 germline. The microscopic morphology is marked with well-circumscribed carcinoma, composed of large and pleomorphic tumor cells, frequent mitotic figures, and prominent lymphoplasmacytic infiltrate (**Figure 3A**). Other commonly seen features are spindle cell metaplasia and giant tumor cells.

#### **3.3 Metaplastic carcinoma**

This subtype is characterized by the dominant component of metaplastic differentiation, representing approximately 1% of all cases. Morphologically marked with a poorly differentiated heterogeneous tumor that contains ductal carcinoma cells mixed with other histological elements, such as squamous cells, spindle cells or other mesenchymal differentiation (chondroid cells, bone cells, and myoepithelial cells) (**Figure 3B**).

*Molecular Histopathology of Mammary Carcinogenesis as Approach to Cancer Prediction… DOI: http://dx.doi.org/10.5772/intechopen.110840*

#### **3.4 Apocrine carcinoma**

This subtype constitutes about 1–4% of all cases, with prominent apocrine differentiation comprising at least 90% of tumor cells, microscopically marked with large tumor cells with an abundant granular eosinophilic cytoplasm and prominent nucleoli. In addition, bizarre tumor cells with multi-lobulated nuclei can also be observed (**Figure 3C**).

#### **3.5 Mucinous carcinoma**

This is a special subtype of breast cancer, also known as colloid, gelatinous, mucous, and mucoid carcinoma, responsible for 2% of all newly diagnosed cases. Morphologically, these tumors have abundant amounts of extracellular mucin, surrounding small clusters of tumor cells with different growth patterns and mild nuclear atypia (**Figure 3D**).

#### **3.6 Cribriform carcinoma**

Special subtype of breast cancer constitutes about 1–3.5% of all breast cancer cases. Microscopically marked with islands of uniform tumor cells, with low grade atypia, cribriform appearance in 90% of the tumor is often associated with DCIS without well-defined stromal invasion (**Figure 3E**).

#### **3.7 Tubular carcinoma**

This subtype is characterized by the proliferation of prominent tubules, which can be angled, oval, or elongated, with a disorganized disposition and open lumen covered by a single layer of epithelium, without presentation of necrosis and mitosis (**Figure 3F**).

#### **3.8 Neuroendocrine carcinoma**

It accounts for about 0.5–5% of all breast cancer cases. Morphological appearance shows an infiltrative growth pattern with solid aggregates of tumor cells arranged in an alveolar, trabecular, or rosette-like pattern, and a peripheral palisade may also be observed. Tumor cells vary in size and generally have an eosinophilic granular cytoplasm (**Figure 3G**).

#### **3.9 Invasive lobular carcinoma**

This subtype is the second largest biologically distinct carcinoma, accounting for approximately 5–15% of all newly breast cancer cases. The classic morphology of ILC is characterized by the presence of small tumor cells with mild atypia, evenly distributed over the stroma in a concentric pattern (**Figure 3H**).

Histology types of induced breast cancer using DMBA are generally papillary carcinoma and cribriform carcinoma [18, 19].

#### **4. The mechanism of molecular carcinogenesis**

Studies of neoplastic disease development, and cellular and molecular mechanisms have identified many individual molecules and signaling pathways, especially in

humans and experimental animals. Despite the widely accepted notion that cancer results from defective genes (mainly oncogenes and tumor suppressor genes), new principles, pathways, and molecules are emerging that are responsible for the neoplastic transformation of cells. It remains to be found as an essential factor in metastatic malignant progression and fatal outcomes. Certain functional groups of molecules are involved in different stages of tumor progression and metastasis, including mediators of apoptosis and DNA repair, oncogenes and tumor suppressors, adhesion molecules, mediators of angiogenesis, and markers of circulating tumor cells [20].

#### **4.1 Tumor growth: proto-oncogenes and oncogenes**

Neoplastic growth of mammary tissue is marked by increased cell proliferation compared to non-neoplastic mammary epithelium. Mammary tumor cells have overexpression of growth-promoting gene products (proto-oncogenes), decreased expression levels, or functional activity of growth-inhibiting gene products (tumor suppressors) involved in promoting growth. Proto-oncogenes are directly involved in cell cycle progression, such as members of cell cycle checkpoints (cyclins, cyclindependent kinases [CDK], retinoblastoma proteins), and indirectly involved in cell cycle progression. Any of the members of the molecular network induces growth factor receptor pathway. Growth factor signaling can have both growth-stimulatory and growth-inhibitory effects on breast tumors. Some of the growth-promoting signaling pathways in breast tumors are epidermal growth factor 2 (ERBB2) and epidermal growth factor 1 (EGFR1). Other than EGFR, metastatic canine mammary carcinomas show decreased expression of several transmembrane growth factor receptors compared to non-metastatic carcinomas or normal glands, such as members of the transforming growth factor beta receptor (TGFBR), fibroblast growth factor receptor (FGFR), and growth hormone receptor (GHR).

Proteomic analyses comparing metastatic and non-metastatic simple breast cancer identified an alteration in expression patterns of several growth-promoting and proliferation-related genes. These genes include the Ran/TC4-binding protein (RANBP1), which is involved in spindle cell assembly, the protein elongation factor 1δ (EEF1D), which is potentially involved in neoplastic transformation and carcinogenesis, and the proliferation nuclear antigen (PCNA), which is a requirement for DNA replication.

#### **4.2 Loss of growth inhibition: tumor suppressors**

Dysregulated cell proliferation in non-neoplastic cells is tightly regulated by multiple molecular pathways, including cell cycle checkpoints and various tumor suppressors. Malignant breast tumors have an increased proportion of cells with active cell cycle progression, as shown by Ki67 and PCNA immunohistochemistry. One of the most characterized tumor suppressors and growth inhibitors is p53. This protein is activated by multiple triggers, including oxidative stress and DNA damage, leading to irreversible cell cycle arrest. Cell cycle arrest by p53 is mediated by increased transcription of p21, an inhibitor of cyclin E/Cdk2, at the G1-to-M cell cycle transition checkpoint. The increased expression of p21 in metastatic breast tumors may be an attempt by p53 induction to inhibit cell cycle progression, which fails in the majority of tumor cells. Like p21, p27 regulates cell cycle progression through interaction with cyclin E/Cdk2 or cyclin D/Cdk4. Moreover, p27 expression is decreased in metastatic carcinoma and its metastases, as well as in adenomas, suggesting that loss of p27 expression occurs early in malignant transformation of mammary epithelium.

*Molecular Histopathology of Mammary Carcinogenesis as Approach to Cancer Prediction… DOI: http://dx.doi.org/10.5772/intechopen.110840*

The phosphatase and tensin homolog (PTEN) appears to be another interesting tumor suppressor with prognostic relevance for mammary tumors. PTEN reduces cell proliferation but is involved in apoptosis induction and cell adhesion.

#### **4.3 Cell proliferation and prognosis**

Increased cell proliferation is a clear feature of malignant mammary tumors. Three molecular markers that have been successfully used to measure proliferative activity through histological methods are PCNA, Ki67, and AgNOR.

PCNA is present in the cell nucleus and acts as a cofactor for the DNA polymerase δ, thus increasing DNA replication. PCNA concentrations peak during the G1 and S phases of the cell cycle but are also found in cells that have recently completed the M phase due to its long half-life of 8–20 hours. Ki67 is a heterodimeric protein with peak expression during the M phase of the cell cycle. A nuclear organizer region (NOR) is a chromosomal segment involved in ribosome biogenesis. They are composed of an argyrophilic protein and thus AgNOR that co-localizes with the nucleolar ribosomal DNA loop. The rearrangement of these loops is directly related to cell proliferative activity.

#### **4.4 Apoptosis: loss of the emergency switch**

In non-neoplastic cells, genetic and metabolic imbalances that can lead to malignant transformation are prevented by multiple molecular mechanisms that ultimately lead to apoptosis or programmed cell death. Severe DNA damage under physiological conditions; imprecise DNA replication; dysregulation of cell cycle progression; hypoxic stress is a typical event that causes the activation of pro-apoptotic pathways or the suppression of anti-apoptotic pathways and molecules.

One of the central regulators in the early stages of apoptosis induction is the tumor suppressor p53. In stressed cells or severe DNA damage, activated p53 accumulates within the cell and leads to cell cycle arrest. This either activates the DNA damage response or, in the case of irreparable DNA damage, induces apoptosis *via* the activation of pro-apoptotic proteins (bax). Bax activation ultimately leads to the activation of apoptosis-associated initiators, cysteine peptidases (caspases). Several p53 mutations have been identified in breast tumor cases. Furthermore, p53 mRNA and protein expression data in malignant tumors report differently between normal and elevated expressions compared to benign tumors or normal glands. Proteins involved in downstream signaling of p53 and other triggers of apoptosis have been extensively analyzed. In some studies, breast cancers showed increased or unchanged expression of anti-apoptotic proteins (BCL2, BCLX, SFRP2), but decreased expression of proapoptotic proteins (BAX, caspase-8, caspase-9).

Rapid growth of malignant tumors causes cytotoxic stress such as hypoxia and nutritional deficiencies. As a result, tumor cells accumulate misfolded proteins in the endoplasmic reticulum, ultimately inducing apoptosis and reducing cell viability.

#### **4.5 DNA repair: failure of the DNA quality management**

Carcinogens can initially cause DNA damage, but later stages of cancer progression exhibit chromosomal instability. Aberrant cell proliferation caused by mutations activating proto-oncogenes or inactivation of tumor suppressors can continuously induce DNA replication stress. Chronic hypoxia and/or cycles of hypoxia and reoxygenation contribute to genomic instability. This is not unique to tumor cells, but is

fairly common in non-tumor cells in the body at any time. In consequence of the wide variety of DNA damage forms, mammalian cells possess multiple DNA repair tools, including non-homologous termination; homologous recombination; base excision repair; nucleotide excision repair; mismatch repair; and transregion synthesis. Mutations or epigenetic inactivation of DNA repair genes accumulate during tumor progression, causing alterations in DNA repair mechanisms and accelerating malignant progression by increasing mutation rates.

In human breast cancer, approximately 5–10% are considered hereditary and associated with germline mutations in breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) that play a role in maintaining genomic stability by functioning as a DNA damage sensor and inducer of the DNA repair response. Several single-nucleotide polymorphisms in the BRCA1 and BRCA2 genomic sequences have been identified in breast tumor cases. In immunohistochemical patterns, malignant breast tumors show decreased nuclear expression and increased cytoplasmic expression of the BRCA1 protein. BRCA-mediated DNA repair is accomplished through the interaction and activation of BRCA1 and BRCA2 with DNA repair proteins, such as RAD51 that increases in metastatic malignant tumor, and p53 which is activated after the identification of DNA damage and which induces cell cycle arrest to allow either DNA repair or apoptosis.

## **4.6 Cell adhesion molecules**

The multicellular organisms' development requires a dynamic and well-coordinated intercellular adhesion system. During various physiological and pathological processes such as tissue development, cell growth, differentiation, embryogenesis, immune response, tumor development, and progression, cell junctions are reorganized and regulated by a network of protein complexes. During carcinogenesis, this complex network of cell–cell contacts is altered, leading to disruption of barriers that facilitate cancer progression and metastasis. Alterations in the expression or function of adhesion molecules at tight, adherens, and gap junctions are critical to tumor progression, including detachment of tumor cells from the primary site, vascular invasion, extravasation into distant target organs, and the formation of metastases. Cell adhesion molecules involved in tumorigenesis include cadherins and catenins, sialylated antigens, claudins, connexins, CD44, HEPACAM1, and HEPACAM2.

#### **4.7 Angiogenesis**

Angiogenesis is the formation of new capillaries from pre-existing blood vessels in adults. It is induced in several physiological and pathological (wound healing) non-neoplastic conditions and has profound implications for tumor growth and metastasis. These newly formed blood vessels within the tumor have three main functions; provide nutrients and oxygen to the rapidly growing tumor mass; remove metabolites; and provide an easily accessible exit for metastatic tumor cells entering the systemic circulation.

One of the most important and well-studied stimulators of angiogenesis in malignant tumors is the vascular endothelial growth factor (VEGF) family of proteins. VEGF protein is synthesized and localized in cytoplasmic granules of neoplastic epithelial, endothelial, and stromal cells, suggesting that both autocrine and paracrine signaling induce proliferation of endothelial buds. Besides VEGF, angiopoietins (ANG) 1 and 2 are the only other angiogenic factors analyzed immunohistochemically in malignancies.

*Molecular Histopathology of Mammary Carcinogenesis as Approach to Cancer Prediction… DOI: http://dx.doi.org/10.5772/intechopen.110840*


*(Source: Nascimento and Otoni: Histological and molecular classification of breast cancer: what do we know? Copyright © 2020 Brazilian Society of Mastology. All rights reserved).*

#### **Table 1.**

*Molecular histopathology classification of breast cancer.*
