3.1. Breast Cancer Risk Factors
It has long been recognized that ovarian hormones are critical risk factors for breast cancer (Kelsey et al., 1993). Early menarche, late menopause, and postmenopausal hormone replacement therapy all increase breast cancer risk, while early full-term pregnancy has a protective effect (Henderson et al., 1988; Pike et al., 1993). Factors associated with increased estrogen production, such as obesity and increased dietary fat, increase risk for breast cancer. Women who have a history of fibrocystic changes with atypical ductal hyperplasia have 4.5 times higher risk for breast cancer. Women with a family history of breast cancer have a 23% lifetime probability of developing a cancer, and 27% if it is a first degree relative (mother, sister, aunt). Mutations of BRCA1 or BRCA2, two high-penetrance tumor suppressor genes confer a very significant risk for breast cancer (Ford et al., 1998). Despite tremendous efforts in the search of additional high-risk genes, up to date BRCA1 and BRCA2 remain to be the only two identified. It has been hypothesized that the remaining familial clustering of breast cancer may result from common variants of a number of genes; each confers only a small effect on breast cancer risk. Recent findings on CHK2 suggest that it may be one of these low-penetrance breast cancer susceptibility genes (Nature Genet. 2002).

3.2. Clinical features of human breast cancer

The most common clinical manifestations are:
· Palpation of a breast mass, usually stone-hard
· Skin changes, such as fixation, dimpling, edema, redness, and in advanced stages, thickening and ulceration
· Nipple retraction and/or serosanguineous discharge
· Pain
· Axillary or supraclavicular masses.
· Tumors arise more frequently in the upper, outer quadrant.
· Breast Cancer can occur in a preinvasive or In Situ form and clearly invasive.

3.3. Classification of human breast cancer

A. Ductal carcinoma
In situ carcinoma is characterized by the proliferation of malignant epithelial cells within lobules or ducts which are surrounded by an intact basement membrane. In situ breast tumors have been subdivided into ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS), according to whether the lesion involves the ducts in the terminal duct lobular unit (TDLU) (DCIS), or in small ducts and lobules (LCIS).

A1. Ductal Carcinoma In Situ (DCIS)
DCIS is a heterogeneous group of lesions, with several microscopic variants, that originate from the TDLU. With screening mammograms, in situ breast cancer is a frequently diagnosed disease, ranging from 15-33% of all mammographically screened patients. The majority of these lesions correspond to DCIS. A slightly lower average age has been reported for DCIS than for infiltrating ductal carcinoma (IDC), but the mean age of affected patients is 50 years. DCIS is bilateral (in both breasts) in 2.2-10%, and multicentric (multiple tumors throughout the breast)in 12-45.5% of cases. Most DCIS are not visible grossly; however, in large comedocarcinomas the necrotic foci can sometimes be grossly recognized.

Grade Classification

Several microscopic variants of DCIS have been recognized, and frequently more than one can be seen in the same patient. Scott et al have proposed the following classification of DCIS, according to degree of nuclear atypia, architectural patterns and presence of necrosis:

· Low grade: cribriform patterns without nuclear atypia of pleomorphism, mitosis or necrosis.(Cribriform, Clinging)
· Intermediate grade: solid patterns with moderate nuclear atypia. Necrosis and occasional mitosis may be seen.
· High grade: marked nuclear pleomorphism, necrosis, mitoses present. (Comedo)
· Special Variants: micropapillary and apocrine types.

Histologic Types

A1-1. Comedocarcinoma
Comedocarcinoma (Image 1) corresponds to the most common type of high grade DCIS characterized by loosely cohesive cells, high nuclear grade, mitoses and central necrosis. It can affect the lobules in a retrograde fashion and mimic LCIS, but the cells are cytologically more pleomorphic with prominent nucleoli and a lymphocytic infiltrate may surround the lobular units or individual acini.

Comedocarcinoma is the most common type of DCIS to express oncogene products c-erb B2 and p53. Microinvasion should be ruled out especially in large lesions.

Comedocarcinomas show higher recurrence rates than other types of DCIS. Axillary lymph node metastases can occur in up to 1-4% of cases.

A1-2. Cribriform

Cribriform DCIS (Image 2) is one of the more common types of low grade DCIS. It is characterized by ducts filled with uniform appearing cells surrounding small, punched out lumens. The prognosis is favorable if completely excised.

Image 1, Comedo Carcinoma	Image 2, Cribiform DCIS	Image 3, Micropapillary

A1-3. Papillary
Papillary carcinoma in situ (Image 2) is a rare form of low grade type of DCIS composed by true fibrovascular stalks, lined by stratified epithelial cells, sometimes admixed with tufting or cribriform patterns. Nuclei are usually bland appearing, although they can show more atypicality.

The hallmark of papillary carcinoma is the absence of myoepithelial cells in the papillary processes. Immunohistochemical stains (e.g. negative staining for smooth muscle actin) can help in confirming the diagnosis. It is frequently associated with other types of DCIS that can be identified in the periphery of the papillary growth. The prognosis of pure papillary DCIS is excellent.

A1-4. Micropapillary
In micropapillary (Image 3) variant epithelial tufts project into the duct lumen. Cells are small and uniform. A myoepithelial cell layer is usually present. Some authors propose that one or more ducts measuring at least 2mm should be affected to qualify as DCIS, cribriform or micropapillary. This type of DCIS tends to be extensive and is present in multiple areas of the breast. For this reason some authors recommend simple mastectomy as the main modality of treatment.

Other variants include: Apocrine DCIS which show cells containing abundant granular eosinophilic cytoplasm similar to apocrine metaplasia, but with complex architectural patterns and necrosis, Clear cell DCIS (cells with optically empty or vacuolated cytoplasm), and Signet-ring cell in situ variants.

Microinvasion In Breast Carcinomas

The lesions are predominantly composed of DCIS, but show microscopic foci of basal membrane disruption and stromal invasion. A tongue-like projection of epithelial cells, usually measuring less than 1mm, or single cells invade the stroma. An arbitrary upper limit of 2mm for the invasive component has been proposed. Frequently there is periductal fibrosis and lymphocytic infiltrates within the stroma. The most common type of DCIS associated with microinvasion is high grade comedocarcinoma.

The treatment depends on the grade, type and extent of DCIS. Surgery, whether simple mastectomy or conservative surgery (lumpectomy) are the recommended modalities of treatment.

It has been recommended that high grade DCIS, such as comedocarcinomas, or extensive micropapillary carcinomas should be treated by simple mastectomy. The election of conservative treatment for DCIS should be accompanied by long-term monitoring of the patient, regular physical examination, and mammographic evaluation every 6 months for the first 2 years.

A2. Infiltrating Ductal Carcinoma (IDC)
Pathology

The majority of malignant breast tumors arise from breast duct epithelium. IDC is the most common type of breast cancer, accounting for 2/3 of infiltrating breast tumors. Breast cancers usually have irregular borders, stellate configuration, are hard, gritty, and show whitish/yellowish, chalky streaks. Adequate recording of size and relationship to surgical margins has important prognostic value. Approximately 1/3 of breast cancers have pushing margins. Multicentricity is defined as two or more foci separated by at least 5cm, i.e., usually in different quadrants. A wide range of multicentricity has been reported for IDC, averaging 5-10%. Bilaterality in IDC is 0.2-2%. IDC, not otherwise specified (NOS) (Image 4) is the most commonly encountered form of IDC. It is composed of solid nests of epithelial cells, cords, tubules, and admixtures of all of these architectural patterns recapitulating the normal breast epithelium. Stroma is variably dense and cellular, and in about 20% of the cases, a dense lymphoplasmocytic infiltrate can be identified.

Histopathologic Parameters with Prognostic Significance

Tumor Grade and lympho-vascular invasion are significant prognostic parameters. The degree of anaplasia is a major factor in prognosis of breast carcinomas. Grading takes into consideration growth pattern (histologic grade) and nuclear features (nuclear grade). Considering three parameters; growth pattern, nuclear pleomorphism, and mitotic counts, Bloom and Richardson proposed a grading system for IDC. Elston and Ellis modified the subjective criteria in order to provide a more precise definition for the assignment of points within each category of feature. Each of the three categories is numerically graded from 1-3, and the sum of the three gives a score between 3 and 9. Low-grade tumors have a final score of 3-5, intermediate grade tumors 6-7, and high grade tumors 8-9. This system was adopted by the WHO in 1968. In general, high grade tumors have large, pleomorphic nuclei, with macronucleoli, irregularities of the nuclear membrane and frequent mitoses, while low grade tumors have small, uniform nuclei, inconspicuous nucleoli and rare mitoses. Histologic and nuclear grade coincide in most IDC. Patients with high grade tumors have more frequent lymph node metastases, recurrences, and die more often from metastatic disease than low grade tumors. Especially in patients without node metastases, tumor differentiation correlates with prognosis. Disease-free survival, as well as overall survival is longer in well differentiated tumors, regardless of clinical stage.

Lymphatic permeations in the breast tissue surrounding a tumor have an adverse effect on overall survival, especially in node negative patients. Lymphatic permeations within the tumor have no prognostic value. Death due to breast carcinoma seems to be also more frequent in cases with blood vessel involvement. Dermal lymphatic invasion is associated with the clinical observation of pear d'orange.

In addition to histopathological parameters, the predicted outcome of breast cancer is determined by many other factors such as proliferation rate, erbB2/HER2/neu expression, and other gene expression profiles (see below).

Histological Variants of IDC

Several histological types of IDC have been described, many of them showing important biological differences with classical IDC, not otherwise specified (NOS).
There are several variants with a favorable prognosis. These include the following:

A2-1. Tubular Carcinoma
Tubular carcinoma (Image 5) comprises 0.7% of all breast carcinomas, although frequencies as high as 8% have been reported. Patients are slightly younger than IDC, NOS. Multicentricity has been reported in up to 28%, and bilaterality in 12-38%.

Pure tubular carcinomas are usually less than 2cm in size. Histologically, this tumor is composed of regular, rounded or angulated tubules, scattered in a fibrous stroma, without any lobular arrangement. They are linked by a single layer of cells with bland-looking nuclei, and rare mitotic figures. Cytoplasmic luminal snouts can be present in about 30% of cases. Pure tubular tumors and mixed tumors (at least 75% of tubular components) have an excellent prognosis.

A2-2. Mucinous (Colloid) Carcinoma
Mucinous carcinoma (Image 6) comprises approximately 2% of breast carcinomas. IDC, NOS usually contains discrete mucinous material in the cells, but in mucinous tumors this amount is considerable in the cells, as well as in the stroma, with cells floating in the PAS (+) material.

Grossly, these tumors are soft, gelatinous, and with well demarcated but pushing borders. At least 50% of the tumor should be mucinous to qualify as colloid. Pure mucinous tumors have a slightly better prognosis than IDC, NOS. The prognosis is less clear for mixed tumors with a minor mucinous component.

Image 4, Infiltrating	Image 5, Tubular Carcinoma	Image 6, Mucinous Carcinoma

A2-3. Medullary Carcinoma
Medullary carcinoma is a rare form of breast cancer, accounting for less than 5% of all breast cancers. It is usually a rounded, well-circumscribed, soft fleshy tumor. Histologically, the hallmark is the presence of solid sheets or nests of relatively poorly differentiated cells, surrounded by a mantle of plasma cells and lymphocytes, sometimes with germinal centers present. Epithelial nuclei are vesicular, with prominent nucleoli, and mitotic figures are frequent.

Medullary carcinomas showing irregular infiltrative margins, only moderate lymphocytic infiltrates, and tubular structures have been called atypical medullary carcinomas. Despite the nuclear features, medullary carcinomas have a more favorable prognosis than IDC, NOS. Nuclear grading is not done in this special type of infiltrating cancer.

A2-4. Papillary Carcinoma
Papillary carcinoma (Image 7) comprises about 1-2% of breast carcinomas. Women are usually slightly older. WHO defines papillary carcinoma as one whose invasive pattern is predominantly in the form of papillary structures. The tumor is grossly well circumscribed, and sometimes cystic.

Histologically, a spectrum of papillary pattern, micropapillae, solid areas, cribriform growth and cysts can be found. Distinction from benign papillomas can be difficult, but immunohistochemistry may assist by demonstrating the presence of myoepithelial cells. Hyperchromatic nuclei, absence of apocrine metaplasia and recognition of frank stromal invasion are features of carcinomas. Nuclear grade is variable. The prognosis is usually more favorable than IDC.

There are several variants with a poor prognosis. These include the following:

A2-5. Metaplastic Carcinoma
Metaplastic carcinoma (Image 8) corresponds to primary breast carcinomas in which the epithelial elements undergo metaplastic changes to a non-glandular pattern. The most frequent metaplastic changes are squamous (3.7% of invasive cancers), and heterologous (0.2%). The extent of metaplastic changes is variable, from few foci in a classical IDC, to almost complete replacement of the tumor by the metaplastic growth pattern. The epithelial elements are usually poorly differentiated carcinomas.

Heterologous elements are usually histologically malignant and composed of bone or cartilage, although rhabdomyoid, adipose, and angiosarcomatous patterns have been reported. Multinucleated giant cells are frequently found in these neoplasms. Heterologous metaplastic tumors have a relatively poor prognosis compared to IDC, NOS. Squamous metaplastic foci in a classical IDC do not seem to have prognostic significance.

Metaplastic carcinoma disseminates via blood vessels with frequent lung metastases. Regional node involvement is rare and therefore axillary dissection is rarely performed.

A2-6. Inflammatory Carcinoma
Inflammatory carcinoma (Image 9) is a clinical, not histopathological designation of a breast cancer that presents clinically with a large, erythematous and painful mass showing purple discoloration of the skin, which is an unfavorable sign.

The histologic correlate of the clinical sign is the presence of lymphatic dermal involvement, although these are sometimes hard to find. Most of the patients die of metastatic disease within two years after the diagnosis.

Chemotherapy is the treatment of choice for these types of malignancies.

Image 7, Papillary Carcinoma	Image 8, Metaplastic carcinoma	Image 9, Inflammatory carcinoma

B. Lobular Carcinoma

B1. Lobular Carcinoma In Situ (LCIS)
The average age of lobular carcinoma in situ (Image 10) affected patients is younger, usually premenopausal women 44-46 years in age, which is 15 years younger than the infiltrative variant. The lesion is not clinically apparent, but is frequently multicentric and multifocal (many foci in the same section), as well as bilateral in 35-59% of the cases. The tumors are ER- and PR-positive. Histologically, the neoplastic cells are small uniform and have clear or eosinophilic cytoplasm and round regular nucleus with smooth membrane and inconspicuous nucleolus. Signet ring cells may be present. The cells must fill the acini and expand or distort the entire lobule or at least 1/2 of the acini in the lobular unit. There are no intercellular spaces between the cells and mitosis is rare.

B2. Invasive lobular carcinoma
The frequency of invasive lobular carcinoma (ILC, Image 11) is 6%, although using less strict diagnostic criteria it can be as high as 14% of all breast tumors. The peak age of incidence is 45-56 years. It presents as an ill defined mass, which is in part due to the diffuse tumor growth pattern. Frequently they lack calcifications, a fact that adds more difficulties to their mammographic detection. Bilaterality has been reported from 6-28%, and 9-14% of the patients with lobular carcinoma develop a subsequent breast tumor. Lobular carcinoma has been reported to have a higher rate of metastasis to other sites, such as bone, abdominal viscera, serosa and retroperitoneum.

ILC is usually composed of small, uniform cells with a low mitotic rate. The cells have a characteristic pattern of infiltration, with single-file, linear arrangement ("indian files"), or individual cells embedded in a fibroplastic stroma. The tumor cells have a concentric disposition around ducts and lobules ("targetoid pattern"), and frequent remnants of in situ lobular component expand the affected lobules. The tumors can also show solid, alveolar or tubular patterns with larger cells, with hyperchromatic, pleomorphic nuclei.

C. Paget's Disease of the Nipple
Paget's disease (Image 12) corresponds to 1-5% of breast carcinomas, and is defined by the presence of large cells with abundant pale cytoplasm and large, atypical nuclei in the surface epithelium of the nipple.

The symptoms are redness and eczematoid changes of the nipple. In 95% of the cases an underlying carcinoma (of different types) is recognized. The lesion is interpreted as an adenocarcinoma, arising either independently or from the underlying tumor.

Image 10, LCIS	Image 11, Lobular Carcinoma	Image 12, Paget's Disease

3.4. Molecular Alterations of Human Breast Cancer

¹Lalage Wakefield, ²Wen-Hwa Lee and ²Eva Lee
¹Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD
²Dept of Developmental & Cell Biology and Dept. of Biological Chemistry, University of California, Irvine, CA

Deregulated cell proliferation, apoptosis, and loss of genome stability are central characteristics of cancer cells. Genes involved in these cellular processes have been identified and mutations of these genes have been found in sporadic and hereditary breast cancer. Furthermore, incipient tumor cells recruit collaborating cells from their microenvironment during tumor formation (Hanahan and Weinberg, 2000). There is also abundant evidence of interaction between epithelial cells and their surrounding extracellular matrix (Bissell and Zerb, 2000). Aberrant activities of genes involved in either autonomous cellular function or interaction with cellular environment have been demonstrated in breast cancer.

As discussed earlier, carriers of several tumor suppressor genes, BRCA1, BRCA2, p53, are present at low frequencies. On the other hand, carriers of ataxia telangiectasia mutated (ATM), estimated to represent 0.5-1% of the population, may have significantly increased risk for breast cancer (Scott et al., 2002). Although there is compelling evidence linking mutation of these tumor suppressors to cancer predisposition, the cumulative effects of multiple low-penetrance susceptibility alleles may be more common in the general population (reviewed in Nathanson et al., 2001).

For sporadic breast cancer, a number of genes or pathways have been implicated genetically, through the demonstration of somatic mutations, amplifications or deletions in sporadic breast tumors. Genes falling into this category would include the tumor suppressors p53, Rb and E-cadherin, and the proto-oncogenes HER2/neu, c-myc and cyclin D1. However many more genes and pathways have been shown to have undergone epigenetic alterations in expression or activity in breast tumors and have been strongly implicated in the tumorigenic process by supporting clinical, epidemiological and biological data. Examples in this category would include many growth factor/receptor systems, ras, cyclin-dependent kinase inhibitors such as p27, and various proteases. Genes and gene products that are found to be involved in breast cancer are disscussed in more detail below. These genes are grossly divided into growth factors/receptors, intracellular signalling pathways, regulators of the cell cycle and cell cycle checkpoints, DNA repair genes, survival pathways and pathways for cell adhesion, invasion and migration.

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