The prognosis and survival rate of breast cancer varies depending on the extent of the disease, performance status of patients and the type of tumour including the status of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Expression of ER and PR generally indicates better prognosis than the overexpression of HER2 and triple negative breast cancer generally indicates more aggressive cancers with a high growth rate \[1\].
Preoperative or neoadjuvant therapy which is also known as primary systemic therapy followed by surgery and adjuvant radiation therapy is recommended for patients with locally advanced breast cancer \[2\]. Studies using primary systemic therapy have demonstrated useful rates of clinical response and pathological complete response(pCR) rates in the breast alone and pathological complete response rates in the axillary notes. The response rates vary considerably, however response rates to cytotoxic chemotherapy have been uniformally higher in ER negative tumors. There is additional improvement in the pathological complete response rates of about 10 % with the addition of a taxane \[4\]. For operable breast cancer, primary systemic therapy can be considered as an alternative to adjuvant systemic therapy for patients who require a mastectomy but who desire breast conservation surgery. In patients with large tumours who can technically have a lumpectomy, primary systemic therapy may permit less extensive surgery and may result in a better cosmetic result. Primary systemic therapy may also be advisable in patients who have medical contraindications to surgery or where delayed surgery is required.
Nanoparticle albumin bound paclitaxel is reported to achieve a higher intracellular tumour paclitaxel concentration via the albumin mediated transendothelial transport system \[5\]. Better tolerability and efficacy has been demonstrated when compared to paclitaxel or docetaxel in the treatment of metastatic breast cancer \[6, 7\]. Nab paclitaxel is being evaluated in the adjuvant treatment of patients with breast cancer \[8, 9\] and in the neoadjuvant setting \[10.\] The preoperative setting provides an opportunity to study the early molecular changes that may occur in response to treatment. Alteration of biomarkers between pre and post chemotherapy including hormone receptors, the Human Epidermal growth factor receptor (HER2)and Ki67 \[11, 12\] as well as gene pathways \[13\] are areas of possible exploration in neoadjuvant studies whereby tissue is available for analysis before and after the chemotherapy treatment. Particular patterns of reduction in tumour size on MRI can be predictive of successful response, detecting residual tumour not apparent on mammogram or U/S and in accurate evaluation of tumour volume \[14.\] Functional imaging biomarkers of response also have potential utility in assessing treatment response \[15.\] A recent study reported that 4 cycles of adjuvant therapy with the combination of nab Paclitaxel and cyclophosphamide, with or without trastuzumab, is feasible and well tolerated in patients with early stage breast cancer. Another small study demonstrated feasibility of nab Paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide (FEC) \[16.\] An anthracycline containing regimen followed by conventional paclitaxel is amongst the most commonly prescribed adjuvant chemotherapy regime for early breast cancer. Cyclophosphamide is given in combination with doxorubicin or its epimer epirubicin. Epirubicin achieves similar efficacy results to doxorubicin but causes less cardiotoxicity \[17 19\].
Although standards of care are varied, adjuvant chemotherapy in 2011 is generally recommended in women with triple negative breast cancer (TNBC) and HER amplified tumors. In women with hormone receptor positive tumors without HER2 amplification, chemotherapy is reserved for tumors that are large or with extensive nodal involvement and/ or high risk biology. The latter includes young age, presence of lymphovascular space invasion, a high proliferative index (Ki67 expression), lower ER/PR expression, higher Oncotype Dx score and luminal B tumors\[20\]. Thus there are evolving trends to tailor therapy based on the tumor characteristics indicating perceived risk, patient factors, particularly comorbid illness and patient preferences as well as prediction of response.
In breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become a widely used method for comparing proliferation between tumour samples\[21\]. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy\[21, 22\]. Ki67 labeling Index has been incorporated as one of the means of identifying tumor subtypes by the 2011 St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer\[20\]. Analysis of gene expression arrays has resulted in the recognition of several fundamentally different subtypes of breast cancer\[23\]. Using gene expression profile distinction can be made between luminal A and luminal B tumors. While both subtypes could be ER positive but luminal A tumours are unlikely to benefit from cytotoxic chemotherapy. Because it is not always feasible to obtain gene expression array information, a simplified classification, closely following that proposed by Cheang et al whereby a cutpoint for Ki67 labeling index of \<15% was established by comparison with PAM50 intrinsic subtyping to differentiate between luminal A and luminal B tumours\[24\]. Local quality control of Ki67 staining is important\[20\].
The Oncotype Recurrence Score is a validated a 21-gene assay that is now offered as a commercial reference laboratory test (Oncotype DX, Genomic Health Inc. Redwood City, CA). The 21-gene panel includes genes involved in tumour cell proliferation and hormonal response, characteristics that have been reported to be associated with chemotherapy response in general. Oncotype DX not only quantifies the likelihood of breast cancer recurrence in women with node-negative, oestrogen receptor-positive breast cancer, but also predicts the magnitude of chemotherapy benefit \[21, 26\]. Likelihood of chemotherapy benefit is reported as low (Recurrence Score \<18, intermediate RS 18-30 and high \>30).\[26\] The main utility of Oncotype DX is in the adjuvant setting where it could be a powerful tool to guide decision regarding the role of cytotoxic chemotherapy in hormone positive tumours. In this neoadjuvant study we will employ Oncotype DX to guide decision about use of chemotherapy in patients with tumours that have high Ki67 (\>15) but low ER expression and also in the other groups that may have low Ki67 (\<15) but high ER expression by IHC.
The goal is to use the best combination, sequence and duration of therapy together with predicting and monitoring response with high fidelity in the individual patient. Further studies are needed to optimize treatment regimens so as to increase pathologic response rates and ultimately survival, with a further goal of reducing risk and adverse events.
This study uses a tailored approach to select treatment involving the choice of NAB Paclitaxel and EC based on the individual patient and tumour characteristics.
Breast Cancer Stem Cells
It is now widely accepted that our inability to cure cancer is largely due to the presence of a subset of cells within a cancer that constitutes a reservoir of self sustaining \[25\]. Current radiation and cytotoxic chemotherapies more effectively destroy the proliferating cells that form the bulk of the tumour, but are largely ineffective against the cancer stem cells (CSC)\[26, 27\]. Breast cancer was the first solid malignancy from which CSCs were identified\[28\], via specific cell surface marker proteins CD44 and epithelial cell adhesion molecules (EpCAM)\[29, 30\]. EpCAM and CD44v6 are among best available, clinically relevant breast cancer stem cell markers for the proof of principle work in this project \[30\] .
Aptamers
Aptamers are short, singlestranded RNA or DNA that fold into specific 3D structures and bind to their target molecules with high affinity and specificity\[31\]. Unlike antibodies, aptamers remain structurally stable across a wide range of temperature and storage conditions. They are generally nonimmunogenic, nontoxic and are 20 to 25 times smaller than monoclonal antibodies. Thus aptamers offer several advantages for tissue penetration and have shorter circulation time and faster body clearance resulting in a low background noise during imaging and lower radiation dose. In addition, aptamers can be produced rapidly, relatively inexpensively, and with high homogeneity \[32, 33\].
HDACi
Histone deacetylases (HDACs) play an important role in gene regulation. Inhibitors of HDACs (HDACi) are novel anticancer drugs, which induce histone (hyper) acetylation and counteract aberrant gene repression\[34\]. HDACi also evoke nonhistone protein acetylation, which can alter signalling networks relevant for tumorigenesis and these agents can also promote the degradation of (proto) oncoproteins. Adult stem cells are maintained in a quiescent state but are able to exit quiescence and rapidly expand and differentiate in response to stress. The quiescence of cancer stem cells (CSCs) is highly relevant to cancer therapy since the quiescent CSC is often resistant to both conventional therapy and targeted therapies. The p53 gene plays a critical role in regulating stem cell quiescence \[35\]. CSCs promote chemotherapy and radiation resistance through an increase in DNA repair capacity and in histone H3 deacetylation\[35\]. Recently the role of HDACi in moving latent or quiescent cells to an activated state and sensitizing them to other treatments has become a focus of investigation in both HIV and cancer. The HDAC inhibitors have been studied in many hematologic and solid malignancies but little work has focused on breast cancer and particularly CSC\[36, 37\]. A study of HDACi on quiescent CSCs in breast tumours and their radiation and chemotherapy responses would be of great interest in developing new therapeutic paradigms using this class of agents.
NAD(P)H:quinone oxidoreductase 1 and NQO1\*2 genotype (P187S)
Nicotinamide adenine dinucleotide, (NAD+), is a coenzyme found in all cells. In metabolism, NAD+ is involved in reduction oxidation (redox) reactions, carrying electrons from one reaction to another. The coenzyme is found as NAD+, which is an oxidizing agent and forms NADH. This can then be used as a reducing agent. Electron transfer reactions are the main function of NAD+. However, it is also used in other cellular processes, the most notable one being a substrate of enzymes that add or remove chemical groups from proteins, in posttranslational modifications. There is evidence that genetic variants in oxidative stress related genes predict resistance to chemotherapy in primary breast cancer and that germline polymorphisms can affect chemotherapy sensitivity in patients with breastcancer\[38\].
The status of superoxide dismutases and NAD (P) H quinone oxidoreductases have prognostic significance in breast carcinomas\[39\]. The NQO1 enzyme guards against oxidative stress and carcinogenesis and stabilizes p53 tumor suppressor\[40, 41\]. NQO1 deficient mice show reduced p53 induction and apoptosis. NQO1\*2 is a missense variant (NP\_000894:p.187P4S) that is homozygous in 4-20% of human population\[42\]. Cells with the homozygous NQO1\*2 genotype have no measurable NQO1 activity, reflecting the very low levels of the NQO1 P187S protein, which undergoes rapid turnover via the ubiquitin proteasome pathway\[43\]. Response to epirubicin is impaired in NQO1\*2homozygous breast carcinoma cells in vitro, reflecting both p53linked and p53independentroles of NQO1. A potential defective anthracycline response in NQO1deficient breast tumors may confer increased genomic instability promoted by elevated reactive oxygen species, and suggest that the NQO1 genotype is a prognostic and predictive marker for breast cancer. A homozygous common missense variant (NQO1\*2, rs1800566(T), NM\_000903.2:c.558C4T) that disables NQO1 strongly has been shown to predicts poor survival among two independent series of women with breast cancer, an effect particularly evident after anthracycline based adjuvant chemotherapy with epirubicin\[44\]. As part of this study the NQO1\*2 genotype status of all patients will be assessed. A correlation can be explored between NQO1\*2 genotype status and response rate in this setting. The study will evaluate the feasibility and safety of tailored primary systemic therapy in the study population.
