**3.1.2 Molecular classification**

72 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

tissue, or medulla. It is an invasive breast cancer that forms a distinct boundary between tumor tissue and normal tissue. Metaplastic breast cancer is a form of invasive ductal cancer, meaning that it forms in the milk ducts and then moves into other tissues of the breast. Metaplastic breast carcinomas constitute a heterogeneous group of neoplasms, accounting for less than 1% of all invasive mammary carcinomas (Reis-Filho et al., 2005), such as squamous (skin) or osseous (bone) cells. The other groups of invasive breast cancers are invasive lobular carcinoma, adenoid cystic carcinoma, micropapillary carcinoma, mucinous carcinoma (formed by the mucus-producing cancer cells), etc as shown in Fig. 3.

Fig. 3. Histology of breast carcinoma. Breast carcinoma is classified into Ductal (A), Lobular carcinoma (B) and Inflammatory carcinoma. (C). It can be further classified into noninvasive (A-B) and invasive carcinoma (C-L). Invasive cancer includes Inflammatory (C),

Invasive lobular (D), tubular (E) apocrine (F), medullary, (G) metaplastic (H),

micropapillary, (I) adenoid cystic (J), mucunous carcinoma (K), and paget disease (L).

Breast cancer is a clinically heterogeneous disease. Histologically similar tumors may have different prognosis and may respond to therapy differently. It is believed that these differences in clinical behavior are due to molecular differences between histologically similar tumors. DNA microarray technology, Immuno-histochemistry (IHC), Fluorescent in situ hybridization (FISH), and quantitative reverse transcription polymerase chain reaction (RT-PCR) are ideally suitable techniques to reveal molecular differences among the same or different groups of histopathological specimens. Each of these molecular techniques has the potential for proper prognosis and prediction of human cancers, including breast. IHC was developed more than 30 years back and it is used for classification of breast cancer into ER positive and ER negative tumors. FISH was developed 20 years back and is used to classify breast tumors into *HER-2* amplified or non amplified categories. Breast cancer cells generally overexpress estrogen receptor (ER)/ progesterone receptor (PR), and human epidermal growth factor-2 (HER-2) receptor for breast tumor formation and progression. Thus, breast cancer can be classified into three sub-groups (i) ER/PR positive (ii) ER negative or HER-2 positive and triple negative (ER, PR and HER-2 negative) on the basis of receptor status. The classification of breast cancer on the basis of ER status improves the prognosis and clinical outcome of ER+ tumors as ER+ cancer cells depend on estrogen for their growth, and the treatment of patients with anti-estrogen agents (e.g. tamoxifen) will inhibit the effect of estrogen and thus improves the treatment outcome. Generally, HER-2+ had a worse prognosis, however HER-2+ cancer cells respond to drugs such as the monoclonal antibody, trastuzumab, (in combination with conventional chemotherapy) and this has improved the prognosis and pathological complete response significantly (Chang et al., 2010). Triple-negative breast cancer is a high risk breast cancer that lacks the benefit of specific therapy that targets these proteins. It can be categorized in basal subtypes (Rakha et al., 2007). It is found in 10-20% of breast cancer cases and mostly diagnosed in younger women with BRCA1 and BRCA2 mutations (Dent et al., 2007; Dawood et al., 2009). The rate of recurrence is very high, and it reaches its peak within first 3 years and then declines after that. Patients with triple negative breast cancer are most likely to die within 5 years than

Breast Cancer: Classification Based on Molecular Etiology Influencing Prognosis and Prediction 75

**5-year RFS\*** 

**10-year OS (%)** 

**10-year RFS (%)** 

**(%)** 

**5-year OS+ (%)** 

Luminal A 50-60 85-95 80-90 75-85 75-85

Luminal B 5-10 70-80 65-75 55-65 54-64

Basal 10-20 63-73 60-70 57-67 45-55 ERB-2 10-20 55-65 15-20 45-55 15-30 Normal-like 10-15 84-94 80-90 75-85 72-82

Table 3. Breast cancer outcomes in molecular types of breast cancer.RFS: The percentage of people without any further symptoms of breast cancer during the interval elapsed between the date of breast surgery and the date of diagnosed further episode of breast cancer, whether the breast cancer was classified as a recurrence or second primary, and whatever the histology. OS: The percentage of people survived during the interval elapsed between the date of breast surgery and the date of breast cancer-related or un-related death

Breast cancers can be classified by different schemata. Classification aspects include clinical (age, tumor, node), histopathological (grade, ER and HER-2 status, ductal, lobular, invasive) and molecular (normal-like, luminal, basal, HER-2) values. Every aspect influences treatment response and prognosis as shown in Table 2 and Table 3. The true prognostic or predictive value of the various molecular classes is unknown because there is a strong correlation between molecular class and conventional histopathologic variables (ER status, grade). For example, in one study, all luminal-type cancers were ER-positive and 63% of these were also low or intermediate grade, in contrast to 95% of basal-like cancers that were ER-negative, 91% of which were high grade (Pusztai et al., 2003). These associations partly explain the different clinical outcome observed in different molecular classes. Rouzier et al. studied the pathological outcomes of different molecular subclasses of breast cancer patients. They obtained tumor tissue biopsies from 82 patients with newly diagnosed breast cancer before they were given a commonly used chemotherapy (Taxol/5-fluorouracil, doxorubicin, and cyclophosphamide). Patients with basal-like and erbb-2+ subgroups were found to have the highest rates (45% each) of a pathological complete response (CR), while only 6% of luminal tumors had a complete response. Among the normal-like cancers, no response was seen (Rouzier et al., 2005). None of the 61genes associated with pathologic CR in the basal-like group were associated with pathologic CR in the HER-2+ group, which suggest that the mechanisms of chemotherapy sensitivity may vary across the subtypes. As molecular classification was not independently associated with pathologic CR, the predictive accuracy of the logistic regression models including (a) clinical + pathologic variables, (b) clinical variables + molecular classification, and (c) clinical + pathologic variables + molecular class (Fig. 5) was measured by constructing Receiver Operating

**4. Clinical outcomes of breast cancer in association with clinical,** 

**Molecular types of breast carcinoma** 

**Frequency (%)** 

(documented from hospital records).

Characteristics curve.

**histopathological and molecular classification** 

patients with other breast cancers. All deaths due to breast cancer in patients' with triplenegative cancer occurred within 10 years of diagnosis.

A novel molecular classification of breast cancer based on gene expression profiles segregates breast cancer into four types (i) luminal, (ii) basal, (iii) HER-2 and (iv) normal type (Perou et al., 2000; Sotiriou et al., 2003; Tamimi et al., 2008) (Fig. 4).

Fig. 4. Dendrogram of breast cancer. The tumors were separated into two main groups mainly associated with ER status as analyzed by hierarchical cluster analysis generated by using gene profile data. The dendrogram is further branched into smaller subgroups within the ER+ and ER- classes based on their basal and luminal characteristics: HER-2 subgroup, dark red; basal-like 1 subgroup, pink; luminal-like A subgroup, green; luminal-like B subgroup, yellow; and normal-like breast subgroup, blue.

Luminal express keratin 8/18, ER, GATA binding protein, X-box binding protein 1, annexin XXXI, cytochrome P450 and basal type express keratin 5, keratin 17, integrin β4, matrix metalloprotease 14, laminin α3, basonuclin and mutated *TP53* gene. Luminal type is further classified into luminal A and luminal B. Luminal B expresses HER-2 along with ER where as luminal A doesn't express HER-2. HER-2 subtype express ERB-2/HER-2, growth factor receptor bound protein 7, TNF receptor-associated factor IV, GRB 7. Normal–breast-like group showed the highest expression of many genes known to be expressed by adipose tissue and other non-epithelial cell types. These tumors also showed strong expression of basal epithelial genes and low expression of luminal epithelial genes. It expresses CD36 antigen collagen type I, glycerol 3 phosphate dehydrogenase I, lipoprotein lipase A, alcohol dehydrogenase 2 (Sorlie et al., 2001). The molecular subclasses show difference in clinical outcome as per as overall survival (OS) and relapse free survival (RFS) is concerned as shown in Table 1.3. There was a significant difference in overall survival between the subtypes with basal and HER-2 is as associated with worse outcome and shortest survival time.

patients with other breast cancers. All deaths due to breast cancer in patients' with triple-

A novel molecular classification of breast cancer based on gene expression profiles segregates breast cancer into four types (i) luminal, (ii) basal, (iii) HER-2 and (iv) normal

Fig. 4. Dendrogram of breast cancer. The tumors were separated into two main groups mainly associated with ER status as analyzed by hierarchical cluster analysis generated by using gene profile data. The dendrogram is further branched into smaller subgroups within the ER+ and ER- classes based on their basal and luminal characteristics: HER-2 subgroup, dark red; basal-like 1 subgroup, pink; luminal-like A subgroup, green; luminal-like B

Luminal express keratin 8/18, ER, GATA binding protein, X-box binding protein 1, annexin XXXI, cytochrome P450 and basal type express keratin 5, keratin 17, integrin β4, matrix metalloprotease 14, laminin α3, basonuclin and mutated *TP53* gene. Luminal type is further classified into luminal A and luminal B. Luminal B expresses HER-2 along with ER where as luminal A doesn't express HER-2. HER-2 subtype express ERB-2/HER-2, growth factor receptor bound protein 7, TNF receptor-associated factor IV, GRB 7. Normal–breast-like group showed the highest expression of many genes known to be expressed by adipose tissue and other non-epithelial cell types. These tumors also showed strong expression of basal epithelial genes and low expression of luminal epithelial genes. It expresses CD36 antigen collagen type I, glycerol 3 phosphate dehydrogenase I, lipoprotein lipase A, alcohol dehydrogenase 2 (Sorlie et al., 2001). The molecular subclasses show difference in clinical outcome as per as overall survival (OS) and relapse free survival (RFS) is concerned as shown in Table 1.3. There was a significant difference in overall survival between the subtypes with basal and HER-2 is as associated with worse outcome and shortest survival

subgroup, yellow; and normal-like breast subgroup, blue.

time.

negative cancer occurred within 10 years of diagnosis.

type (Perou et al., 2000; Sotiriou et al., 2003; Tamimi et al., 2008) (Fig. 4).


Table 3. Breast cancer outcomes in molecular types of breast cancer.RFS: The percentage of people without any further symptoms of breast cancer during the interval elapsed between the date of breast surgery and the date of diagnosed further episode of breast cancer, whether the breast cancer was classified as a recurrence or second primary, and whatever the histology. OS: The percentage of people survived during the interval elapsed between the date of breast surgery and the date of breast cancer-related or un-related death (documented from hospital records).
