Understanding Triple Negative Breast Cancer

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1 Article ID: WMC ISSN Understanding Triple Negative Breast Cancer Peer review status: No Corresponding Author: Dr. Sanjay Katiyar, Associate Professor, Department of Zoology, University of Delhi - India Submitting Author: Ms. Sneha Balani, Student, Department of Zoology, University of Delhi - India Article ID: WMC Article Type: Review articles Submitted on:04-jun-2015, 10:29:15 PM GMT Article URL: Subject Categories:CANCER Keywords:Breast, Cancer, Causes, Risks, Mechanisms Published on: 08-Jun-2015, 03:54:04 PM GMT How to cite the article:balani S, Katiyar S. Understanding Triple Negative Breast Cancer. WebmedCentral CANCER 2015;6(6):WMC Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source(s) of Funding: Not applicable Competing Interests: There is no conflict of interest associated with the publication of this article. WebmedCentral > Review articles Page 1 of 16

2 Understanding Triple Negative Breast Cancer Author(s): Balani S, Katiyar S Abstract Triple Negative Breast Cancers (TNBCs) are distinct breast malignancies that are characterized by the lack of clinically relevant expression of estrogen-, progesterone- receptors (ER/PR) and HER2/neu proteins. With a molecular profile similar to the basal-like carcinomas, the aggressive behavior of TNBCs is orchestrated with a display of distinct metastasis patterns. In absence of targeted therapies they rapidly transform into the most dreaded cancers affecting various parts of the body, with usually worst prognosis. Majority of BRCA1 associated breast cancers are triple negative, but the extent of BRCA1 s involvement is still under investigation. Epidemiologically, TNBCs are prevalent among young African-American women, who display a unique risk factor profile, are characterized by distinct recurrence patterns and a redilection for brain metastasis. Clinical trials for chemicals agents targeting epidermal growth factor receptor (EGFR) and inhibitors of poly ADP-ribose polymerase (PARP) hold the promise in the treatment of TNBC. Triple Negative Breast Cancer: Introduction Breast carcinomas represent a heterogeneous group of tumors of distinct biologic subtype that are also diverse in terms of response to the therapy and prognostic outcomes. Various molecular subtypes of these carcinomas have been identified through gene expression profiling; these are luminal, HER2 positive/ ER negative, and basal-like breast cancers (BLBC) (1). Critical determinants of these subtypes are the steroid hormone receptors such as estrogen receptor (ER) and progesterone receptor (PR) together with the oncogene, human epidermal growth factor receptor 2 (HER2). The luminal subtypes are mostly ER positive whereas the basal-like subtype is characterized by low expression of ER and HER2 related genes. The BLBCs are clinically (usually but not always), ER/PR negative and when they exhibit this characteristic combination with the lack of HER2 expression they constitute the triple negative phenotype (2). This means such a tumor has overcome its dependence on hormones like estrogen, progesterone or the EGF protein. TNBC constitute approximately 10% to 15% of all breast carcinomas. Multiple studies have shown that their intrinsic subtypes display variable prognosis, with inferior outcomes demonstrated among the two hormone receptor negative subgroups when compared to the luminal subtypes (3, 4). There is an increased likelihood of distant recurrence and death in patients with TNBC compared to those with other types of breast cancers. This difference also persists after controlling the established prognostic factors. Recent studies have shown a high incidence of visceral metastasis and brain metastasis in patients with TNBC (5). This clinically challenging scenario is therefore an area of active research. Targeted agents specifically aimed at TNBC are not yet available, unlike the other subtypes. There is therefore an intense need and interest in advancing novel therapeutic strategies beyond chemotherapy for this subset of breast cancer. Molecular Basis Evidence from molecular profiling studies has suggested that 70% of TNBCs display basal-like subtype, while the remainder consist of a variety of molecular subtypes that are biologically different (6). To understand the TNBC phenotype, an extensive review of the normal mammary gland parenchymal cells including their immunophenotype is essential. Classically, the central luminal cells express low molecular weight cytokeratins including CK-7, -8, -18, and -19 and MUC1α-6 integrin; BCL1, ER & PR receptors; and trans-acting T-cell specific transcription factor GATA 3. Moving outward towards the basement membrane, myoepithelial cells comprising the basal cell layer express high molecular weight cytokeratins including CK-5, -14, and -17 along with smooth muscle-specific markers, calcium binding proteins calponin and caldesmon, p63 tumor protein, β4 integrin, laminin, maspin tumor suppressor, neutral endopeptidase CD10, P-cadherin (placental), caveolin-1 (CAV-1), nerve growth factor receptor (NGFR), and S100 proteins (7-12). TNBCs are associated with a number of pathological features (Table 1). In addition to lack of ER, PR, and HER2 expression, they frequently express basal cytokeratins such as CK-5, -14, and -17 and the WebmedCentral > Review articles Page 2 of 16

3 epidermal growth factor HER1. These features are associated with a poor prognosis in breast cancer (13-17). TNBCs, when compared to other tumor types are more likely to express myoepithelial markers such as CAV-1 and CAV-2 (Caveolins), the high expression of which makes the cells more aggressive and metastatic (15). They also display a higher expression of the stem cell factor, c-kit, which is a proto-oncogene and P-cadherin (14). TNBCs are less likely to express epithelial markers such as E-cadherin (epithelial) and have high expression of genes associated with proliferation, such as Ki67 and TOP2A (13). High levels of cyclin E and low levels of cyclin D1 are also observed (18). Among the characteristic markers of TNBCs, several are potentially targetable notably, HER1/EGFR and c-kit. EGFR and c-kit are expressed in approximately 60% and 31% of basal-like breast tumors respectively (19). EGFR is now used as a candidate for targeted therapy as described below. TNBCs display an aberrant expression of several molecules that are integrally involved in DNA repair, and this may have implications in chemotherapy and drug sensitivity. High p53- immunopositivity or p53 gene mutations are common in BLBCs. Furthermore, one study illustrated that 82% of BLBCs expressed a p53 mutations compared with only 13% in the luminal (3). By immunostaining and mutational analysis 50% of TNBCs display aberrant p53 expression or expression of its homolog, p63. Many of these features are also associated with a poor prognosis in breast cancer (13-20). Other targetable molecular pathways implicated in the pathogenesis of BLBC include the mitogen-activated protein kinase (MAPK) pathway and the poly-adp-ribose polymerase (PARP) pathway, which are addressed in detail in context of therapeutics (below). Relationship between BLBC and TNBC It was demonstrated in several studies that basal-like tumors are not necessarily triple-negative. ER and HER2 are expressed in 15-45% and 14% of BLBCs respectively, indicating that not all BLBCs are triple-negative regardless of the classification method (3, 21). Conversely, 16-44% of TNBCs are negative for all basal markers namely, CK-5/6, -14, and EGFR, 7.3% of non-tnbcs also express these (14, 15). 71% of TNBCs were reported to be positive for at least one basal marker such as CK-5/6, -17, -14, or EGFR as shown in later studies (22). BLBC subtype expresses genes normally expressed in cells of the normal, non-luminal basal, myoepithelial layer of the breast duct and lobular system including CK-5, -14, and -17, smooth muscle actin, EGFR, P-cadherin, CAV-1 and CAV % of all invasive breast cancers have all these basal-like features (1, 3, 16, 23). BLBCs and TNBCs share a number of common features that are represented in Table 1. Typically, they are grade 3 i.e. poorly differentiated, ductal carcinomas with an elevated mitotic count and apoptotic rate. They also show geographic or central tumor necrosis or fibrosis, a pushing margin of invasion and a stromal lymphocytic response (23-25). The terms TNBC and BLBC are interchangeably used. TNBCs are identified by the lack of detectable expression of only three genes ER, PR, and HER2 ascertained by gene expression assays and immunohistochemistry (IHC). On the other hand, the detection and definition of BLBC is based on assessment of mrna expression profiling of ~ 500 genes (26). The investigations involving gene expression profiling have shown that 10-35% of TNBCs are not basal-like and only 40-80% of TNBCs express basal protein markers (14-16, 26). In one study, Pam50 based prediction analysis of microarray gene expression profiling was used to examine the molecular subtypes of 142 TNBCs from patients participating in two clinical trials. It was revealed that ~70% were BLBCs and the remainder was a variety of identifiable non-blbc tumors (6). These findings suggest that the TNBCs and BLBCs are distinct category of tumors with some overlap in their profiles. The use of TNBC in place of BLBC will therefore result in their misclassification (Figure 1). The BRCA1 pathway and p53 association The BRCA1 tumor suppressor gene was originally identified in 1994 by positional cloning on chromosome 17q21. It is a multifocal protein, involved in many normal cellular processes such as DNA repair, transcriptional regulation, cell cycle control checkpoint and ubiquitination (27). Genomic instability ensues in absence of BRCA1 and so is its predisposition in the development of the malignant disease. Previous studies have shown that most breast tumors in women carrying germline mutations in BRCA1 tumor suppressor gene are triple negative (28) and show similarities to BLBCs which also suggests that BRCA1 dysfunction may play a pivotal role in BLBCs. As WebmedCentral > Review articles Page 3 of 16

4 stated earlier, these mutants will express a high proportion of basal-like cytokeratins CK-5, -14 and -17, as well as P-cadherin and HER/EGFR (27, 29). Due to the integral role of BRCA1 in many cellular pathways significant effort has been expended to identify whether BLBCs have BRCA1 dysfunction or deficiency. This is also because the presence of BRCA1 abnormalities has therapeutic implications. In a previous study, involving an unselected cohort of 54 patients with TNBC, 11% patients carried at least one BRCA1 mutation. The incidence of BRCA1 mutations was higher (14%) among patients < years of age (30). All BRCA1 associated cases were profiled into the basal-like subtype in gene expression based analyses (31). It was observed that the frequent cytogenetic aberrations (such as deletion of 5q) that are typically found among BRCA1 carriers are also found at a similar frequency in TNBCs. There is also supports the view that sporadic BLBCs (cancer developing in people who do not carry a high risk mutation) have defective DNA repair similar to BRCA1 mutation carriers (32). The reduced BRCA1 protein expression in the sporadic BLBCs is caused by reduced transcription, a consequence of due to loss of heterozygosity (LOH; loss of normal function of one allele while the other allele was already inactivated) or BRCA1 promoter methylation (33). Alternatively, high levels of id4, a negative regulator of BRCA1 may attenuate the expression of BRCA1 protein (34). Apart from aberrant expression of BRCA1, abnormalities of other member of BRCA1 pathway may also result in functional loss of BRCA1 action. About three-quarters of BRCA1 related breast cancers exhibit a basal-like phenotype validated by gene expression or IHC (31), (35). This is particularly true among younger patients and those with a family history of breast cancer who also harbor mutant p53. p53 is a tumor suppressor with diverse biological functions such as cell cycle checkpoint control, response to DNA damage resulting in apoptosis etc. p53 was mutated found to be mutated in as much as 82% of BLBCs (1), which was also evident from higher level of genetic instability, specific cytogenetic changes, and higher loss of heterozygosity frequently exhibited by these cancers. A common occurrence among BLBC is the gain of 6p21-p25 and loss of 5q11 (36) chromosomal regions which carry several DNA repair and suppressor genes. TNBCs share biological (central necrosis, lymphocytic infiltration and genomic instability) and histological features with other breast cancers containing BRCA1 mutation(s). A significant association of BRCA1 mutated cases with typical characteristic features of TNBC such as EGFR, CK5/6 expression, ER/HER2 negativity, and p53 mutations has been reported (37). Hence, an association of BRCA1 is observed in TNBCs which is in part responsible for DNA repair, the exploitation of this pathway holds therapeutic implications for curing TNBC. Clinical characteristics, Epidemiology and Risk factors A number of clinicopathologic features are characteristic of TNBCs, such as onset of tumors at a younger age, high tumor grade (grade 3), high mean tumor size and higher rate of lymph node positivity (38, 39). Consistent with their morphological nature the tumors also show distinct imaging features in ultrasonograms, mammograms and MRIs. Histologically, TNBCs show a marked increase in mitotic count, geographic necrosis, pushing borders of invasion and stromal lymphocytic response as was previously stated for BLBCs (25). Majority of TNBCs are ductal in origin (arising in the milk ducts), but they can also express highly aggressive metaplastic phenotypes where the cells change form into those of other parts of the body; atypical and typical medullary, in which the cells take up the color of the medulla and adenoid cystic (38, 40). The epidemiological risk factors differ significantly between TNBC and non-tnbc (Table 2). Young African-American women display a higher incidence of basal-like disease compared to Caucasian women. In the Carolina Breast Cancer Study ~500 women were evaluated, the African-American women showed an elevated prevalence of 26% versus 16% in other women (38) for likelihood of observing basal-like tumors. African Americans also displayed 2-3 times higher frequency of occurrence of TNBCs, while the prevalence was up to 47% of breast cancers in other racial groups. By comparison, the percentage of TNBCs was 55% in West-African patients, 31% in Korean patients, 18% in Chinese patients, 16% in UK and Taiwanese patients and 8% in Japanese patients. It was further observed that African-American women with TNBCs were also more likely to be present with larger tumors of higher grade, mitotic activity, tumor necrosis, and apoptotic index in comparison to white women patients. Their tumors showed contrasting expression of several cell cycle and apoptosis related proteins such as p16, p53, Cyclin E, BCL2 and Cyclin D (6) and majority of these women were premenopausal (24%) versus postmenopausal (15%). These findings were consistent with several large WebmedCentral > Review articles Page 4 of 16

5 scale population based studies indicating that TNBCs are more likely to occur among premenopausal women of African-American descent. Population based studies suggest that TNBCs follow a unique risk factor pattern, that includes high parity, younger age at full-term pregnancy, shorter duration of breast feeding, higher body mass index and higher waist to hip ratio. Also, those who used methods to suppress lactation and have preferentially used oral contraceptives were shown to be at higher risk of developing TNBC (41, 42). Women with abdominal obesity, a risk factor for type II diabetes are also at higher risk. It was shown that the anti-diabetic drug Metformin inhibits proliferation and induces apoptosis of TNBC cells along with inducing other beneficial biological changes in breast cancer of various types (43). Other evidence also indicates an enhanced efficacy of chemotherapy in patients with breast cancer that were using Metformin. Hence, the clinical trials of adjuvant Metformin are currently in progress. Outcome of TNBC Demographic studies have shown lower breast cancer specific survival rates and recurrence among patients with triple negative phenotype compared to those with non-triple negative disease. Patients with TNBC are more likely to relapse during the first three years following therapy and more commonly develop soft tissue and visceral metastasis including metastases of central nervous system (44) Survival A disproportionate number of breast cancer deaths result from TNBCs. In several randomized trials the patients with triple negative or basal-like tumors that were treated with anthracyclins and taxanes had experienced a significantly decreased survival compared to the patients with other tumor types. It is important to note that the prognostic effect of TNBC is independent of poor grade, nodal status, tumor size, and treatment (38). The peak risk of recurrence occurs within the first 3 years after the initial treatment of the disease with the majority of deaths occurring in the first 5 years and a significantly shorter survival rate is observed after the diagnosis of metastatic disease in both basal-like and triple negative breast cancer. These two characteristics further depict the aggressiveness of TNBC. The differences in overall survival between TNBC and non-tnbc wear off at about 10 years during the follow up (39). Patterns of Recurrence A distinct pattern of recurrence is exhibited by TNBCs which is a rapidly increase of disease in the initial years following diagnosis that peaks between second and third year. However, the risk decreases over the next five years slowly diminishing thereafter. Majority of women with TNBCs without an evidence of disease progression after eight years do not show disease recurrence thereafter, unlike women with other types of breast cancers. In addition to a distinct pattern of timing of recurrence, unique patterns of relapse sites have also been recognized in TNBC patients. An increased rate of visceral versus bone metastasis among patients with TNBC compared with non-tnbc has been suggested by several studies (24, 45). The largest study involving 12,858 patients had concluded that these TNBC patients depict an elevated risk for lung and brain metastasis as the primary sites of disease recurrence but a lower risk for bone recurrence (46). In another study, among 344 patients with lymph node negative breast tumors, bone relapse was more likely to occur in luminal subtypes but less likely to occur in the basal-like phenotype. Lung metastasis was frequently observed in the basal-like subtype and less likely in the luminal subtype. Finally, 8 cases of brain metastasis were basal in origin and 2 were luminal in origin of the total 14 cases observed in this series (44). The above observations had indicated a predilection for brain metastasis among patients diagnosed with triple negative disease. Incidence of central nervous system metastases An increased risk of central nervous system metastases (CM) was observed in the patients with TNBC in comparison to the breast cancers of other subtypes. In a study, 3000 patients with brain metastasis arising from breast cancer treated between 1989 and 2006 were examined, and using multivariate analysis had shown that triple negative status was the greatest risk factor for developing cerebral metastasis than HER2 positivity. It was also observed that TNBS patients display a short median interval between primary diagnosis and cerebral relapse (22 months) in comparison to the non-tnbc patients (51 months). This was followed by a trend towards worst survival after the diagnosis of brain metastasis (4 months for triple negative and 8 months for non-triple negative patients) (5). Another study involving 222 patients with brain metastasis treated between 2003 and 2006 showed an inferior median survival for patients with TNBC (3.7 months) compared to HER2 positive patients (9 months) and ER/PR/HER2 positive (15 months) (47). Similarly, a significantly elevated risk of brain metastasis was observed among patients with triple negative and HER2 positive breast cancers compared with other phenotypes in a single institution WebmedCentral > Review articles Page 5 of 16

6 study involving 3193 patients. It was further observed that the risk of cerebral metastasis was particularly pronounced among young patients with node-positive disease (nodal-metastasis). The incidence of cerebral metastasis among node positive patients below 50 years was 20% in TNBC compared to 4.8% in HER2 positive patients (48). Diagnosis of cerebral metastasis among patients with TNBC was followed by a shorter median survival of 3 to 5 months compared with the other subtypes showing a median survival of 7 to 12 months. In % of TNBC patients, cerebral metastasis was seen as the first site of distant relapse. But the survival rate of such a relapse was as low as 3 months for those where cerebral metastasis was the first site of relapse to 6 months, where it is the latter site. Finally, an overall survival after diagnosis of cerebral metastasis was 3 months in the presence of systemic therapy and 4 months in the absence of it (5, 48, 49). Efforts focusing on the predilection of patients at highest risk for breast cancer related to the brain metastasis include clinical nomograms (output of a graphical calculating device) and gene expression strategies. Recently, a nomogram was used to calculate the probability of developing cerebral metastasis particularly in patients with TNBC (50), however, its clinical implications remain unclear. Treatment Strategies TNBC and BLBC are characterized by their overall poor prognosis and tendency to relapse with distant metastasis. Thus there exists an urgent need for the development of effective systemic therapies against these breast cancers. Chemotherapy is widely used for these diseases, as therapies directed towards hormone receptors and HER2 are not effective. Though historically chemotherapy has resulted in poor outcomes in TNBC and BLBC, it is still only effective means to treating these cancers. Thus, the improvements in chemotherapy are likely to preferentially benefit this subtype of breast cancer because of rapid proliferation rates and defects in DNA repair (21, 51). Anthracycline/ Taxane Based Chemotherapy High sensitivity to anthracycline or anthracycline/ taxane was shown in studies involving neoadjuvants in chemotherapy for BLBCs compared to luminal subtypes. These involved the administration of therapeutic agents prior to the main treatment. In a study involving 100 patients, the clinical response to doxorubicin (adriamycin)/ cyclophosphamide chemotherapy was compared, where 32% patients tumors depicted basal-like (ER/HER2 negative) phenotype, 10% had HER2 positive/er negative tumors while 58% were luminal (ER positive) tumors. When treated with neoadjuvant AC- doxorubicin (adriamycin)/ cyclophosphamide, the highest response rates of 85% were observed in patients with basal-like tumors, followed by 70% among HER2 positive, and 47% among luminal subtype tumors. Despite initial chemosensitivity, disease-free survival and overall survival remained poorest among those with basal-like and HER2-positive tumors compared to luminal tumors (52). In another study involving more than 1,000 patients, comprising of 23% patients was triple negative tumors, the treatment with a variety of anthracycline/ taxane-based neoadjuvant strategies showed consistent results that were in concurrence with results obtained in previous studies. Higher (22%) pathologic complete response (CR) was observed among triple negative patients in comparison to the ER positive patients (11%). The triple negative status was also associated with decreased 3 year progression free and overall survival rate of 63% in multivariate analysis when compared to the other subtypes which showed survival rate of 76% (45). In spite of the high response rates, relapse rates are usually high in patients who do not achieve a pathologic complete response resulting in the decreased overall survival in the patients with basal-like and triple negative tumor groups than in patients with luminal tumors. TNBCs showed highest response to anti-metabolites like 5-fluorouracil when compared to other breast cancers (52). This observation and other retrospective data suggests that the anti-metabolites such as cyclophosphamide, methotrexate and 5-fluorouracil may be more effective in treating TNBCs than other tumor types (15). Loss or inactivation of BRCA1 function is thought to be associated with particular sensitivity to DNA-damaging chemotherapy. Recent studies have shown that BRCA1 associated breast cancer patients are less sensitive to taxane chemotherapy when compared to other sporadic breast cancers (53, 54). The currently available targeted agents including endocrine therapy and HER2 directed therapies are ineffective whereas, as previously mentioned TNBC is highly responsive to primary anthracycline and anthracycline/ taxane chemotherapy, however if the tumor is not eradicated a high risk of relapse remains (21, 42, 52). A population based study by von Minckwitz et al (55) investigated the expression of nuclear (n) and cytoplasmic (cparp) expression using immunohistochemistry on 638 pretreatment cancer biopsies to ascertain the prognostic significance of these markers after neoadjuvant WebmedCentral > Review articles Page 6 of 16

7 anthracycline/taxane-based chemotherapy. It was observed that 23.7%, 50.9 and 25.4% breast tumor samples showed high, intermediate and no expression of while cparp. The high cparp expression samples could be were positively correlated with non-lobular histology, undifferentiated grade, positive nodal status, and negative hormone receptor (HR) status but were HER2 positive tumors. TNBC tumors also showed high levels of cparp expression in 35.5% samples and only 24.6% of samples were HER2-positive tumors. It was also noted that the patients whose tumor samples had showed high, intermediate and lack of expression of cparp had 26.5%, 19.1%, and 8.0% respectively pathologic complete response rates after drug therapy. On the contrary no such correlation was for nparp expression. Thus it was concluded that cparp could serve not only serve as a prognostic marker for TNBCs but can also predict the therapy outcome in these patients. Platinum-containing agents The association of TNBC with BRCA1 mutations and dysfunctional DNA repair may indicate an increased sensitivity to DNA-damaging agents such as platinum compounds. It was demonstrated that p53 related transcription factors such as p63 and p73 were over expressed in TNBCs they were sensitive to cisplatin (56). In another study using neoadjuvants in BRCA1 mutation carrier breast cancer patients, 10 out of 12 patients that were treated with cisplatin monotherapy showed a pathologic complete response compared to others treated with some other regimen (57). Another study had indicated that the overall survival in the TNBC patients was worse than other subtypes, but when treated with platinum based chemotherapy, the response rates were as high as 88% compared to 51% in other subtypes. The response rates for patients with advanced disease were also higher in TNBC patients, so was the progression free survival and the overall survival rate (58). In a small prospective study the TNBC patients treated with neoadjuvent cisplatin monotherapy showed a 64% overall response rate and a modest 21% pathologic complete response rate (59). Thus, despite an increasing amount of information obtained from clinical trials confirming the efficacy of platinum agents in treatment of TNBC, it is yet to be determined whether these are more effective than the conventional agents. Also there is an absence of randomized data identifying platinum based chemotherapy as optimal regimen due to safety concerns and toxicity associated with these compounds. Other Chemotherapeutics Trabectedin (a DNA minor groove binding agent), a natural product isolated from sea sponges was the first cytotoxic drug that was evaluated for the treatment of TNBC. A large, nonrandomized phase II trial was carried out for assessing the efficacy of Trabectedin in TNBC patients, who had HER2 positive tumors and those who were BRCA1 mutation carriers had failed to reveal any treatment benefit. Ixabepilone (a cytotoxic drug, binds to β-tubulin causing the microtubule stabilization and mitotic arrest), was later tested on TNBC patients. In a phase III clinical trial, the 443 TNBC patients treated with Ixabepilone in combination with Capecitabine showed the superior response rates and progression free survival. While the monotherapy by Capecitabine alone did not have any treatment benefits over combination therapy (60). Further research can reveal whether TNBC are generally sensitive to cytotoxic drugs or other cytotoxic-specific approaches should be tested to achieve superior responses. Targeted Strategies The current thrust in research on TNBCs is aimed at developing novel strategies that are highly specific and precisely targeted. Several promising agents such as PARP inhibitors, EGFR inhibitors, multi-tyrosine kinase inhibitors and anti-angiogenic agents are being tested for efficacious suppression of these cancers. PARP inhibitors PARPs (poly-adp-ribose-polymerase) are a family of nuclear enzymes involved in the detection and repair of DNA damage (61). These are critical during cell proliferation and are often upregulated in TNBCs and BRCA1-associated cancers. Loss of BRCA1 results in an increased dependence on PARP for DNA repair, so the BRCA1 defective cells are sensitive to PARP inhibitors. These inhibitors enhance the effects of a range of DNA damaging agents including radiation and cytotoxic therapies. A number of clinical trials have been conducted for testing the efficacy of PARP inhibitors (Iniparib and Olaparib), either alone and in combination with platinum based chemotherapy. When used alone or in combination with cytotoxic drugs, Iniparib shows no dose-limiting toxic effects and but displays very promising results when used in combination with Gemcitabine and Carboplatin (62). However, these benefits could not be validated in the phase III trial (63). Olaparib was shown to be safe and effective in BRCA-related cancers, and is currently being tested in clinical trials on TNBCs (64). EGFR Inhibitors EGFR provides a rational for the targeted treatment WebmedCentral > Review articles Page 7 of 16

8 approach, since it is over-expressed in approximately 60% of triple-negative breast tumors. EGFR targeting agents such as small molecules and monoclonal antibodies were evaluated in clinical trials in TNBC, along with various studies conducted on Ixabepilone and Cisplatin, with or without Cetuximab. Data from these studies suggests that the EGFR inhibitors have a low efficacy when used alone but a greatly improved efficacy when used in combination with other agents in treating TNBC. In a clinical study where 11 patients with metastatic TNBC were treated with taxane in combination with Cetuximab, 9 patients responded and 3 developed brain metastasis during treatment (65). In another study, patients with metastatic TNBC that were treated with Cetuximab alone revealed a response rate of 6%, however when used in combination with Carboplatin the response rate increased to 18%. Phase II trials using this two-drug combination have reported an increased overall patient benefit and survival (66). Another combination was made of Irinotecan and Carboplatin with or without Cetuximab. When used with Cetuximab a response rate of 49% was seen, whereas without it the response rate was 30%, however incidence of toxicity, diarrhea, vomiting, neutropenia (low number of neutrophils), and thrombocytopenia (low number of platelets) was higher among patients who received Cetuximab (67). Multi-tyrosine kinase inhibitors Tyrosine kinases play a critical role in the development and progression of cancer. Tyrosine kinase receptors such as c-kit are over expressed or mutated in BLBCs. The use of multi-tyrosine kinase inhibitors can be central in treatment of TNBC. Dasatinib, a mutityrosine kinase inhibitor is a small molecule that was approved for the treatment of chronic myeloid leukemia. Phase II study using this molecule in advanced TNBC patients reported a modest activity, with an overall clinical benefit of 9.3%. However, it was observed that discontinuation of the therapy and dose reduction weakened the results of the study (68). Mitogen activated protein kinase (MAPK) pathway regulates various cellular activities such as gene expression, mitosis, differentiation, proliferation, and cell survival. Drugs that can selectively down-regulate this cascade can be valuable therapeutic agents in treating malignant diseases. This has been achieved by the heat-shock protein 90 inhibitor PU-H71 which inhibits the MAPK pathway thus leading to complete response and tumor regression (69). Anti-angiogenic Agents Anti-angiogenic agents are substances that inhibit the growth of new blood cells. Bevacizumab (Avastin), is a monoclonal antibody targeting all forms of vascular endothelial growth factor (VEGF). VEGF is active in a variety of solid tumors including breast cancers. In one clinical study, when Bevacizumab was added to a certain Paclitaxel chemotherapy resulted in an improvement in progression free survival from 6 months (Paclitaxel alone) to 12 months (70). Studies are underway for testing various small-molecule inhibitors of the VEGF pathway that appear to have activity in the subset of pretreated TNBCs (71). The study of Bevacizumab Adjuvant Therapy in Triple Negative Breast Cancer (BEATRICE) was designed to investigate the effect of adding Bevacizumab to adjuvant chemotherapy in TNBC. An oral multi-targeted tyrosine kinase inhibitor Sunitinib malate was found to inhibit the vascular endothelial growth receptor, platelet derived growth factor receptor, c-kit and colony stimulating factor 1 receptor, all of which play important role in the development of mammary gland and carcinogenesis. A phase II study of this inhibitor in patients who were resistant to anthracyclines and taxanes showed an impact in patients with TNBC (72). Other strategies The range of genetic abnormalities that are seen in TNBCs and BLBCs has opened the door to focus on the breast cancer to other therapeutic strategies, many of which are being evaluated in clinical trials including agents targeting kinases, like mtor, androgen receptor, TGF-β, and the trail receptor. Despite the clinical trials it is still too early to examine the clinical efficacy of these strategies in TNBC and BLBC. The status of TNBC in India There is a paucity of data on TNBC from India. As per previous reports ~25% of all breast cancer cases in Indian women may be classified as triple negative (73, 74). In concordance to the western studies where TNBC predominantly affects younger women (39, 52), Indian studies have also revealed similar association at younger age (median age 48 years) (74). On the contrary, Ambroise et al had reported that the occurrence of TNBC in patients who are more than 50 years of age (73). Triple negative breast tumors were commonly grade 3 and showed lymph node positivity similar to those reported in the west (38, 39, 73, 74). Western studies have suggested the association of TNBC with large tumor size (39) however, reports from India have suggested a lack of any such association WebmedCentral > Review articles Page 8 of 16

9 (median tumor was 5 cm in both TNBC and non TNBC groups in a cohort of 636 patients) (74). In the same study the proportion of premenopausal to post-menopausal women was equal (1:1) which was in contradiction to western reports where TNBC patients are mostly premenopausal (38, 74). The molecular markers used to identify triple negative status are ER-, PR-, HER2/neu- negativity along with CK 5/6 and EGFR. In one study, expression of EGFR was seen in 40%, while mutations in EGFR were seen in 50% of the triple-negative samples (75). In another study, of 60 patients studied none showed EGFR positivity (74) whereas in western countries it ranges from 36% to 64% (22). CK 5/6 was expressed in 44.8% of TNBCs which agrees with the findings from western countries (22, 74). Also CK 5/6 expression significantly correlated with younger age, high grade tumor, node positivity, larger tumor size and triple negative status (74). Treatment options include surgery, radiotherapy and chemotherapy. Inferior response rates are seen in TNBC patients when given chemotherapy. Also no significant difference has been seen in non-metastatic cases (74). In a study involving 215 cases of breast cancers aged 35 to 70 years triple negativity was significantly seen to influence disease free survival vs. non-triple negative cohort. However triple negativity did not influence overall survival in months, 56 in these vs. 43 in non-triple negative cohort. The mean disease free survival was months (74, 76). Stage of disease, node status, grade and menopausal status did not influence disease free survival and overall survival which suggests that triple negativity can significantly affect progression of breast cancer in Indian breast cancer patients and follow up of 10 years and more is necessary to determine its effects on survival (76). Indian patients show presence of more advanced stages as well as locally advanced breast cancer is seen in 60-65% cases as compared to 10-20% in West (74, 77). This could provide a probable explanation for the disparities observed in Indian and Western studies. As is evident from the conflicting data there is a dire need for more studies elucidate the incidence and characteristics of this clinically and prognostically important subgroup in Indian population and also its influence on survival. Conclusion Triple negative breast cancer represents a distinct subtype of breast carcinoma which exhibits unique molecular and clinical characteristics. It expresses basal-like features and shows morphological similarities to basal-like tumor including elevated mitotic and apoptotic rate with pushing margin of invasion, which led to its misclassification as a basal-like breast cancer. The identification of properties uncharacteristic of the basal-like phenotype in some triple-negative cases along with its association to BRCA1 mutation, provided this aggressive disease an identity. The triple negative phenotype shows inferior prognosis, distinct patterns of recurrence and a predilection for cerebral metastasis. Despite its sensitivity to conventional methods of chemotherapy, it is a challenge to develop targeted agents for this subtype with a poor prognosis and survival rate. PARP inhibitors, EGFR inhibitors, anti-angiogenic agents and identifying unique genetic factors are promising avenues that may help curb this unusual disease. References 1. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature. 2000;406(6797): Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N. Triple-negative breast cancer--current status and future directions. Ann Oncol. 2009;20(12): Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001;98(19): Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003;100(14): Heitz F, Harter P., Traut A, Lueck HJ, Beutel B, A db. Cerebral metastases in breast cancer with focus on triple negative tumors. Journal of Clinical Oncology 2008;26(May suppl; abstract 1010). 6. Carey L, Winer E, Viale G, Cameron D, Gianni L. Triple-negative breast cancer: disease entity or title of convenience? Nat Rev Clin Oncol.7(12): Bocker W, Bier B, Freytag G, Brommelkamp B, Jarasch ED, Edel G, et al. An immunohistochemical study of the breast using antibodies to basal and luminal keratins, alpha-smooth muscle actin, vimentin, collagen IV and laminin. Part II: Epitheliosis and ductal WebmedCentral > Review articles Page 9 of 16

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14 Illustrations Illustration 1 Figure 1: Shared features between triple negative, basal-like and BRCA1 associated breast cancers. Abbreviations: TNBC: Triple Negative Breast Cancers; BLBC: Basal-like Breast Cancers; ER: Estrogen Receptor; PR: Progesterone Receptor; EGFR: Epidermal Growth Factor Receptor WebmedCentral > Review articles Page 14 of 16

15 Illustration 2 Table 1: Pathological features of triple negative breast cancer Pathological Features Frequency in TNBC Frequency in BLBC References Triple Negative 100% (by definition) ~ 80% 3, 21 Grade 3 and ductal histology Mostly grade 3 Incidence more than in TNBC Elevated mitotic count, high apoptotic rate, Most TNBC show these Typical of BLBC central tumor necrosis, pushing margin of invasion, lymphocytic response features Basal cytokeratins (5,14,17) ~ 50% 100% EGFR ~ 50% Increased in BLBC P-cadherin Very high Relatively low 14 C-kit expression High Low 14 Accumulation of p53 mutation ~50% ~ 82% 3, 13 Increased cyclin E and low cyclin D1 Positive correlation between - 18 these two features and TNBC Increased CAV1 and CAV2 More likely in TNBC Less likely in BLBC 15 TNBC: Triple Negative Breast Cancer; BLBC: Basal-like Breast Cancer; EGFR: Epidermal Growth Factor Receptor; CAV1 and CAV2: Caveolin1 and Caveolin 2. WebmedCentral > Review articles Page 15 of 16

16 Illustration 3 Table 2: Risk factors of triple negative breast cancer Features References High parity, young age at the time of first pregnancy, lack of breast feeding, use of oral contraceptives (41, 42) Younger age at diagnosis (<50 years) (38, 39) African-American ethnicity (6, 38) Pre-menopausal (38) Increased body weight (41) Metabolic syndrome, particularly abdominal obesity (43) WebmedCentral > Review articles Page 16 of 16