Oestrogen and Progesterone Receptors

Oestrogen and progesterone are steroid hormones that are produced mainly in the ovaries and are essential for sexual and reproductive development in women, help regulate the menstrual cycle and play a significant role in pregnancy. The oestrogen receptors (ERs) exist in two forms, namely the ERα and ERβ and similarly, the progesterone receptors (PgRs) also exist in two forms, namely PRA and PRB. ^{Reference Dai, Xiang, Li and Bai3,Reference Duffy, Harbeck and Nap5,Reference Platet, Cathiard, Gleizes and Garcia8} The validation of the clinical role of the two ERs has currently only been established for the ERα and current immunohistochemistry (IHC) measurements are capable of detecting only the ERα, although IHC can detect both the PRA and PRB. ^{Reference Dai, Xiang, Li and Bai3,Reference Duffy, Harbeck and Nap5,Reference Platet, Cathiard, Gleizes and Garcia8} Ulaner et al. ^{Reference Ulaner, Riedl, Dickler, Jhaveri, Pandit-Taskar and Weber9} and Dai et al. ^{Reference Dai, Xiang, Li and Bai3} reported that ERs are found in approximately 70–75% of primary breast cancers and ER-positive breast cancers use oestradiol as the main growth stimulus, thus, ER status in tumours is a critical index of sensitivity to endocrine therapies. It has also been reported that the expression of ER and PgR are strongly related with each other with PgR expression being dependent on ER signalling and therefore, the most common breast cancer phenotype expresses both ER and PgR and these accounts for over 50% of all breast cancers. ^{Reference Dai, Xiang, Li and Bai3,Reference Ulaner, Riedl, Dickler, Jhaveri, Pandit-Taskar and Weber9–Reference Burstein, Lacchetti and Anderson11} According to Chan et al. ^{Reference Chan, Chang and Gonzalez10} invasive breast cancers expressing only one hormone receptor are less common and account for approximately 15–20% of all breast cancers and are predominantly ER-positive:PgR-negative (expressing ER, but not PgR) tumours, a phenotype associated with greater growth factor signalling and lower endocrine sensitivity. Furthermore, ER-negative:PgR-positive (expressing PgR, but not ER) tumours are uncommon and account for about 2–8% of breast cancers with less known about their characteristics and responsiveness to treatment. ^{Reference Ulaner, Riedl, Dickler, Jhaveri, Pandit-Taskar and Weber9,Reference Chan, Chang and Gonzalez10}

Duffy et al. ^{Reference Duffy, Harbeck and Nap5} reported that both ER and PgR are predictive markers for endocrine therapy and their expressions are used in the neoadjuvant, adjuvant and advanced disease settings. Furthermore, the utilisation of ER-positive versus ER-negative for characterising breast cancer could potentially allow the delineation of diseases that may appear quite similar but represent two different types of diseases that behave biologically different, have different treatment and prognostic implications, and thus may also possibly have different risk factor patterns. ^{Reference Dai, Xiang, Li and Bai3,Reference Duffy, Harbeck and Nap5} Colleoni and Montagna ^{Reference Colleoni and Montagna12} also reported that tumour subtypes that are associated with different responses to neoadjuvant treatments can be identified through the evaluation of pathological features such as tumour grade and ER and PgR expression levels. Furthermore, neoadjuvant endocrine therapy should be considered in place of cytotoxic neoadjuvant therapy for postmenopausal patients with tumours of low grade or proliferation and high expression levels of ER and PgR. Studies ^{Reference Duffy, Harbeck and Nap5,Reference Ulaner, Riedl, Dickler, Jhaveri, Pandit-Taskar and Weber9,Reference Burstein, Lacchetti and Anderson11,Reference Bardou, Arpino and Elledge13,Reference Dowsett, Houghton and Iden14} have reported that patients with ER-positive tumours derive significant benefit from 5 years of tamoxifen in reducing the odds of recurrence and mortality, whereas those with ER-negative tumours do not, and the derived benefit is directly proportional to the expression level of ER, such that patients with higher tumour ER levels derive the highest benefit from the therapy. Ulaner et al. ^{Reference Ulaner, Riedl, Dickler, Jhaveri, Pandit-Taskar and Weber9} also reported that breast cancer patients with high expression levels of PgR have better outcomes, but there is the less predictive value of PgR expression level for hormonal treatment. According to Duffy et al., ^{Reference Duffy, Harbeck and Nap5} both ERs and PgRs should be measured on all newly diagnosed invasive breast cancers for the selection of patients likely to respond to endocrine therapy and where feasible, the measurement of ER and PgR should be performed on recurrent lesions and the primary invasive tumour.

Several studies ^{Reference Chan, Chang and Gonzalez10,Reference Bardou, Arpino and Elledge13–Reference Goss, Ingle and Pritchard19} have investigated the clinical applications of ERs and PgRs in the selection of patients with invasive breast cancer for treatment with endocrine therapy, such as the administration of selective ER modulators (tamoxifen), third-generation aromatase inhibitors (anastrozole, letrozole or exemestane), Luteinizing hormone-releasing hormone (LHRH) agonists (leuprolide, goserelin), pure ER down-regulators (fulvestrant), oophorectomy and other endocrine therapies. Bardou et al. ^{Reference Bardou, Arpino and Elledge13} evaluated the clinical outcome of patients who received adjuvant hormonal therapy to determine whether PgR status could independently identify patients with a greater likelihood of benefiting from adjuvant hormonal therapy. They observed in a multivariate analysis, including lymph node involvement, tumour size and patient age that PgR status is independently associated with disease-free and overall survival among patients who received endocrine treatment. Furthermore, they reported a reduction in relative risk of recurrence of 25% for ER-positive:PgR-negative patients and 53% for ER-positive:PgR-positive patients when compared with ER-negative:PgR-negative patients. They concluded that accurate measurement of PgR status is an independent predictive marker for the benefit from adjuvant endocrine therapy. Therefore, PgR status should be taken into account when discussing relative risk reductions expected from endocrine treatment with individual patients. Chan et al. ^{Reference Chan, Chang and Gonzalez10} retrospectively reviewed a database of 815 patients diagnosed with early-stage invasive breast carcinoma to evaluate the clinicopathological features, natural history and outcomes of ER-negative:PgR-positive breast tumours compared to ER-positive and ER-negative:PgR-negative tumours. They reported that ER-negative:PgR-positive phenotype is associated with higher grade, with overexpression of human epidermal growth factor receptor 2 (HER2) and are observed more commonly in younger women. Furthermore, they identified that the risk of relapse and mortality are more closely related to ER-positive than ER-negative:PgR-negative tumours suggesting that this phenotype represents a group of patients with more aggressive hormone receptor-positive tumours.

Davies et al. ^{Reference Davies, Pan and Godwin20} investigated the increased benefit of continuing tamoxifen treatment to 10 years as opposed to stopping at 5 years for patients with ER-positive early breast cancer. They reported that continuing tamoxifen to 10 years instead of stopping at 5 years further reduces the risk of breast cancer recurrence, breast cancer-specific mortality and overall mortality for patients with ER-positive disease. They concluded that 10 years of tamoxifen treatment can reduce breast cancer mortality by half during the second decade after diagnosis. Dowsett et al. ^{Reference Dowsett, Houghton and Iden14} conducted a study of 813 breast cancer patients who had been randomised to receive 2 years tamoxifen or no adjuvant therapy in two randomised clinical trials and whose ER, PgR, HER2 and epidermal growth factor receptor (EGFR) status were determined. They observed that ER-positive patients benefited from tamoxifen treatment, and although ER-negative patients also showed a strong trend to benefit from tamoxifen it was largely confined to the PgR-positive group. They concluded that the measurement of PgR status in ER-negative patients will define a group of patients who would benefit from tamoxifen, but may be excluded from tamoxifen therapy on the basis of ER status alone. Purdie et al. ^{Reference Purdie, Quinlan and Jordan17} re-evaluated PgR expression as a potential therapeutic guide in 1,074 early breast cancer, ER-positive, lymph node-negative cancer patients. They reported an overall survival and breast cancer-specific survival of 71·0 and 83·0% respectively at a median follow-up of 8·34 years. Furthermore, PgR negativity expression was significantly associated with poor prognosis for overall survival, breast cancer-specific survival and disease-free survival and was not influenced by endocrine therapy. They demonstrated that PgR negativity is a strong, independent prognostic marker in-operable, primary breast cancer patients receiving endocrine therapy. They suggested that PgR-negative expression should be re-evaluated as a biomarker for poor prognosis in ER-positive breast cancer patients and those patients should be considered for additional systemic therapy.

Human Epidermal Growth Factor Receptor 2 (HER2)

The HER (ERBB) family of receptor tyrosine kinases include the HER1 (or ERBB1), HER2 (or ERBB2), HER3 (or ERBB3) and HER4 (or ERBB4) and these receptors consist of a ligand-binding extracellular domain and an intracellular tyrosine kinase domain. This family of receptors is involved in the regulation of cell growth and survival, participate in cellular proliferation and differentiation through multiple signal transduction pathways and play a central role in the pathogenesis of several human cancers. ^{Reference Villalobos and Wistuba21–Reference Ross and Fletcher30} The HER2 gene is a proto-oncogene located on the long arm of chromosome 17 at position 12 (17q12) and encodes a tyrosine kinase receptor member of the HER receptor family. ^{Reference Villalobos and Wistuba21,Reference Ross and Fletcher30, Reference Fountzilas, Christodoulou and Bobos31} Amplification of the HER2 gene and/or overexpression at the protein level has been reported to occur in about 13–25% of patients with early-stage breast cancer and about 55% of these cases are ER-negative:PR-negative. ^{Reference Dai, Xiang, Li and Bai3,Reference Dawood, Broglio and Buzdar24–Reference Piccart-Gebhart, Procter and Leyland-Jones28,Reference Ross and Fletcher30–Reference Slamon, Leyland-Jones and Shak37} Furthermore, population-based studies and retrospective analyses have shown that overexpression of HER2 is associated with a number of adverse prognostic factors such as advanced stage, poorly differentiated high-grade tumours, hormone receptor negativity, increased rates of cell proliferation and lymph-node involvement, early systemic metastasis, shortened disease free and overall survivor, poor prognosis, increased mortality, are commonly presented as more aggressive disease, and relative resistance to certain types of chemotherapy. ^{Reference Dai, Xiang, Li and Bai3,Reference Duffy, Harbeck and Nap5,Reference Dawood, Broglio and Buzdar24,Reference Mitri, Constantine and O’Regan25,Reference Burstein27,Reference Piccart-Gebhart, Procter and Leyland-Jones28,Reference Ross and Fletcher30,Reference Colomer, Aranda-López and Albanell32,Reference Slamon, Eiermann and Robert33} According to Slamon et al., ^{Reference Slamon, Eiermann and Robert33} these adverse clinical effects stem from the fundamental changes in the biological features of breast-cancer cells containing such alterations as suppression of apoptosis, increased motility and proliferation, greater invasive and metastatic potential, accelerated angiogenesis and steroid hormone independence. The clinical implications of HER2 amplification have been recognised since 1987 and the prognostic value of HER2 positivity has been reported to be higher in node-positive patients. ^{Reference Dai, Xiang, Li and Bai3} Dai et al. ^{Reference Dai, Xiang, Li and Bai3} reported that the predictive value of HER2 positivity on treatment outcome for patients receiving anthracycline-based chemotherapy is associated with favourable treatment response and that HER2 positivity is predictive of a better response to higher doses of anthracycline-based and taxane-based regimens. Furthermore, they reported that both preclinical and clinical data have shown that HER2 overexpression confers intrinsic resistance to hormonal therapy in ER-positive or PgR-positive tumours, suggesting that either ER-positive or PgR-positive and HER2-positive patients will have less benefit from single-agent hormone therapy. ^{Reference Dai, Xiang, Li and Bai3} Konecny et al. ^{Reference Konecny, Pauletti and Pegram34} observed that HER2 expression is inversely correlated with ER expression, and reported that the relative resistance of tumours with either ER-positive or PgR-positive and HER2-positive to hormone therapy when compared with tumours with either ER-positive or PR-positive and HER2-negative subtype is primarily due to the reduced ER or PgR expression or high proliferation rates instead of HER2 positivity. ^{Reference Konecny, Pauletti and Pegram34} Hu et al. ^{Reference Hu, Fan and Oh35} also reported that ER-positive or PgR-positive and HER2-positive breast cancer may have improved benefit from anti-HER2 therapy combined with chemotherapy. According to Fountzilas et al., ^{Reference Fountzilas, Christodoulou and Bobos31} trastuzumab (a recombinant humanised monoclonal antibody against the HER2 protein) has been found to prolong progression-free and overall survival of patients with metastatic breast cancer and HER2 gene amplification or HER2 protein overexpression. Furthermore, there have also been discoveries of new monoclonal antibodies and other targeted agents aimed at overcoming trastuzumab resistance and thereby improving survival in patients diagnosed with HER2-positive breast cancers. ^{Reference Mitri, Constantine and O’Regan25,Reference Burstein27,Reference Ross and Fletcher30} Pritchard et al. ^{Reference Pritchard, Shepherd and O’Malley29} suggested that the amplification of HER2 gene and/or overexpression of its proteins in breast cancer has the potential to predict the responsiveness to trastuzumab and also can identify patients whose cancers will be unresponsive to certain treatments and therefore could benefit from high-dose chemotherapy or anthracycline-based chemotherapy. Duffy et al. ^{Reference Duffy, Harbeck and Nap5} proposed that HER2 expression or gene copy number determination should be mandatory for selecting patients who are likely to response to new targeted anti-HER2 treatment such as trastuzumab, pertuzumab, lapatinib or ado-trastuzumab emtansine (KADCYLA, Genentech Inc.) which are approved by the US Food and Drug Administration (FDA).

The USA-FDA approval of trastuzumab as first-line treatment in combination with paclitaxel for patients with metastatic HER2-positive breast cancer has led to the addition of trastuzumab to the standard of care chemotherapy in several clinical trials. ^{Reference Dawood, Broglio and Buzdar24} Consequently, several investigators ^{Reference Dawood, Broglio and Buzdar24,Reference Mitri, Constantine and O’Regan25,Reference Piccart-Gebhart, Procter and Leyland-Jones28,Reference Pritchard, Shepherd and O’Malley29,Reference Slamon, Eiermann and Robert33,Reference Romond, Perez and Bryant36,Reference Slamon, Leyland-Jones and Shak37} have conducted various studies to refine and optimise the utilisation of trastuzumab and lapatinib in patients with HER2-positive early-stage breast cancer, determined the potential value of HER2 expression for predicting therapeutic response of breast cancer, and to develop new treatment approaches using monoclonal antibodies and small-molecule tyrosine kinase inhibitors targeting HER2 or other HER family members. Furthermore, these investigations also studied the efficacy of trastuzumab in the adjuvant settings, established the evidence that HER2 gene and protein abnormalities in breast cancer can predict resistance to tamoxifen treatment and the relative sensitivity to chemotherapy regimens. These studies have demonstrated the effectiveness of trastuzumab in combination with chemotherapy to significantly improve (as much as 50%) disease-free survival and overall survival of patients with early-stage HER2-positive breast cancer as well as metastatic HER2-positive breast cancer. ^{Reference Dawood, Broglio and Buzdar24, Reference Mitri, Constantine and O’Regan25} Dawood et al. ^{Reference Dawood, Broglio and Buzdar24} conducted a retrospective study in 2,019 women comparing the prognosis of patients with metastatic HER2-positive breast cancer with and without the addition of trastuzumab with the prognosis of patients with HER2-negative disease. They reported 1-year survival rates of 75·1, 86·6 and 70·2% among HER2-negative cancer patients, HER2-positive cancer patients treated with trastuzumab, and HER2-positive cancer patients who did not receive trastuzumab treatment respectively. Furthermore, in a multivariable model, HER2-positive cancer patients who received trastuzumab had a 44% reduction in the risk of mortality compared with patients with HER2-negative disease. They concluded that HER2-positive cancer patients who received trastuzumab had improved prognosis compared with HER2-negative cancer patients. Piccart-Gebhart et al. ^{Reference Piccart-Gebhart, Procter and Leyland-Jones28} reported on an international, multicenter, randomised trial with 5,081 HER2-positive and either node-negative or node-positive breast cancer patients who had completed locoregional therapy and at least four cycles of neoadjuvant or adjuvant chemotherapy. They randomly assigned patients to different regimens including 1,694 patients assigned to 2 years of trastuzumab treatment, 1,694 patients assigned to 1 year of trastuzumab treatment and 1,693 patients assigned to observation. They reported that 1 year of trastuzumab treatment after adjuvant chemotherapy significantly improves disease-free survival among patients with HER2-positive breast cancer. Pritchard et al. ^{Reference Pritchard, Shepherd and O’Malley29} conducted a randomised, controlled trial using 639 formalin-fixed paraffin-embedded specimens obtained from 710 premenopausal HER2-positive patients with node-positive breast cancer who had received either cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) or cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) as adjuvant chemotherapy. They reported that HER2 amplification was associated with poor prognosis irrespective of the treatment regimen, although, in patients with HER2 amplification, treatment with CEF was superior to CMF treatment when they assessed on the basis of relapse-free survival and overall survival. They concluded that HER2 amplification in breast cancer is associated with clinical responsiveness to anthracycline-based chemotherapy. Romond et al. ^{Reference Romond, Perez and Bryant36} evaluated two clinical trials comparing adjuvant chemotherapy with or without concurrent trastuzumab in patients with surgically removed HER2-positive breast cancer and reported that trastuzumab treatment was associated with a 33% reduction in risk of mortality and that trastuzumab combined with paclitaxel after doxorubicin and cyclophosphamide improved the outcomes among patients with surgically removed HER2-positive breast cancer. Slamon et al. ^{Reference Slamon, Leyland-Jones and Shak37} evaluated the efficacy and safety of trastuzumab, in patients with HER2 overexpressed metastatic breast cancer who were randomly assigned to standard chemotherapy (234 patients) or standard chemotherapy plus trastuzumab (235 patients). They reported that the addition of trastuzumab to standard chemotherapy was associated with a longer time to disease progression, a higher rate of objective response, a longer duration of response, a lower rate of mortality at 1 year, longer survival, and a 20% reduction in the risk of mortality. Thus trastuzumab improves the clinical benefit of first-line chemotherapy in metastatic breast cancer that overexpressed HER2. In another study, Slamon et al. ^{Reference Slamon, Eiermann and Robert33} evaluated the efficacy and safety of non-anthracycline regimen with trastuzumab in 3,222 patients with HER2-positive early-stage breast cancer and reported that the addition of 1 year of adjuvant trastuzumab significantly improved disease-free and overall survival among patients with HER2-positive breast cancer.

Ki-67 Antigen

The Ki-67 nuclear antigen (or MKI67) was originally identified by Scholzen and Gerdes in the early 1980s is located on the long arm of chromosome 10 at position 26·6 (10q26·2). ^{Reference Scholzen and Gerdes38–Reference Soliman and Yussif42} The Ki-67 antigen encodes a nuclear protein Ki-67 (pKi67) and is expressed in all active phases of the cell cycle (S, G1, G2 and M), but absent in resting cells (G0). ^{Reference Ingolf, Russalina and Simona40,Reference Inwald, Klinkhammer-Schalke and Hofstädter41} According to Li et al., ^{Reference Li, Jiang, Chen and Zheng39} pKi67 is an established prognostic and predictive marker for the assessment of biopsies from cancer patients and has been shown to be associated with metastasis and clinical stage of tumours in the clinical settings. Furthermore, pKi67 positivity is reported to be associated with proliferative activities of intrinsic cell populations in malignant tumours thereby making it a marker of tumour aggressiveness and can also potentially identify subpopulations of patients who are most likely to respond to a specific treatment. ^{Reference Li, Jiang, Chen and Zheng39} In addition, Ki-67 antigen expression is also reported to be strongly associated with tumour cell proliferation and growth, is significantly higher in malignant tissues with poorly differentiated tumour cells, tends to increase with decreasing tissue differentiation, correlates with the presence of occult metastasis and is used in the routine pathological investigation as a proliferation marker. ^{Reference Li, Jiang, Chen and Zheng39–Reference Inwald, Klinkhammer-Schalke and Hofstädter41} It has also been demonstrated that Ki-67 positivity is also associated with a higher risk of breast cancer recurrence, the worse survival rate in patients with early-stage breast cancer and elevated levels of Ki-67 antigen expression are associated with worse prognosis. ^{Reference Li, Jiang, Chen and Zheng39–Reference Soliman and Yussif42} Viale et al. ^{Reference Viale, Giobbie-Hurder and Regan43} reported that higher Ki-67 expression levels are predictive of responsiveness to pre-operative chemotherapy and a decrease in Ki-67 expression during pre-operative endocrine therapy is associated with pathologic tumour response, however, persistent higher Ki-67 expression after short-term pre-operative endocrine therapy is predictive of shorter disease-free survival. According to Soliman and Yussif, ^{Reference Soliman and Yussif42} the Ki-67 antigen has several potential usages in breast cancers including the estimation of residual risk in patients on standard therapy, as a dynamic biomarker of treatment efficacy in samples obtained before, during and after neoadjuvant endocrine therapy and the determination of prognosis of relative responsiveness and resistance to chemotherapy or endocrine therapy.

Several studies ^{Reference Ingolf, Russalina and Simona40–Reference Wang, Wu and Zhang45} have investigated the diagnostic, prognostic and predictive value of Ki-67 in patients with invasive breast cancer. Fasching et al. ^{Reference Fasching, Heusinger and Haeberle44} investigated both the predictive and prognostic importance of Ki-67 in 552 patients with invasive breast carcinoma who received neoadjuvant treatment and they found Ki-67 to be an independent predictor for pathological complete response, overall survival and distant disease-free survival. They concluded that Ki-67 has both predictive and prognostic value, has the potential as a marker for clinical practice and can provide additional and independent predictive information on the response of patients to chemotherapy and the prognosis of patients receiving neoadjuvant breast cancer treatment. Ingolf et al. ^{Reference Ingolf, Russalina and Simona40} analysed the potential value of Ki-67 to predict the response of breast cancer patients to neoadjuvant chemotherapy and they demonstrated that Ki-67 expression predicts the response to neoadjuvant chemotherapy in breast cancer patients as a function of the molecular subtype. Inwald et al. ^{Reference Inwald, Klinkhammer-Schalke and Hofstädter41} also evaluated the routine use and the importance of Ki-67 as a prognostic marker in breast cancer as well as analysed the associations between Ki-67 and common histopathological parameters in routine clinical settings of 3,658 patients with invasive breast cancer. They reported that Ki-67 expression is associated with common histopathological parameters such as tumour grade and size, lymph node invasion, nodal status, vascular invasion, and ER, PgR and HER2 expressions. Furthermore, they demonstrated that Ki-67 is an additional independent prognostic parameter for disease-free survival and overall survival in breast cancer patients. Similarly, Soliman and Yussif ^{Reference Soliman and Yussif42} analysed the relationship between the Ki-67 index and clinicopathological factors and prognosis (disease-free survival and overall survival) in 107 breast carcinoma patients. They reported that patients with the Ki-67 value of less than 15% had better overall survival and exhibited a lower incidence of metastasis and recurrence than patients with Ki-67 value higher than 15%. They concluded that Ki-67 expression level is a valuable biomarker in breast cancer patients and can be used in treatment and follow-up, although there is the need for the standardisation of Ki-67 assessment and specification of its role in treatment decisions. Viale et al. ^{Reference Viale, Giobbie-Hurder and Regan43} examined the value of Ki-67 both as a prognostic and predictive factor for differential efficacy of letrozole versus tamoxifen treatment as initial adjuvant therapy in postmenopausal patients with endocrine responsive breast cancer. They reported that higher values of Ki-67 labelling index were associated with adverse prognostic factors and with worse disease-free survival and the treatment benefit for letrozole versus tamoxifen was greater amid patients with elevated Ki-67 than amid patients with low Ki-67 labelling index. They concluded that high Ki-67 levels could potentially identify a group of patients who would benefit from initial letrozole adjuvant therapy. Wang et al. ^{Reference Wang, Wu and Zhang45} investigated the prognostic role of Ki-67 expression in 363 patients for the classification of triple-negative breast cancer into subtypes with different prognosis and whether high Ki-67 expression level can predict benefit from carboplatin in the adjuvant setting. They reported that high Ki-67 index were associated with shorter disease-free survival and overall survival and also they demonstrated the benefit of carboplatin treatment in patients with high Ki-67 index. They concluded that triple negative breast cancer is a heterogeneous disease with different characteristics and prognosis, and may potentially be further subdivided giving the Ki-67 expression levels.

Circulating Tumour Cells

Circulating Tumour Cells (CTCs) are tumour cells that have been shed from either the primary tumour or from its metastasis and are circulating in the peripheral blood or lymphatic vessels of patients, and they are considered the main mechanism for tumour metastasis. ^{Reference Banys-Paluchowski, Schneck and Blassl46–Reference Yap, Leong and Chua49} The first account of the presence of CTCs in the peripheral blood was reported by Thomas Ashworth in 1869 in which he hypothesised that CTC cells might potentially offer better understanding of the metastatic process in cancer patients. ^{Reference Perakis and Speicher50–Reference Osei, Al-Ani, Al-Asady and Dang53} According to several studies, one major factor that contributes to cancer progression, relapse, poor survival and mortality in cancer patients is the metastatic tumours induced by the entry of CTC cells into the bloodstream or lymphatic vessels either as single cells or in clusters. ^{Reference Banys-Paluchowski, Schneck and Blassl46,Reference Nadal, Lorente, Rosell and Serrano47,Reference Alemar and Schuur54–Reference Usiakova, Mikulova and Pinterova56} The detection of CTC in blood has been reported in about 10–80% of breast cancer patients and it has been demonstrated to be correlated with tumour burden and are therefore more frequent in patients with metastatic breast disease. ^{Reference Banys-Paluchowski, Schneck and Blassl46,Reference Wang, Chang and Yeh48,Reference Bidard, Proudhon and Pierga55–Reference Hartkopf, Banys and Krawczyk57} According to Hartkopf et al. ^{Reference Hartkopf, Banys and Krawczyk57} CTC detection rates in the blood varies among patients and depends on the detection method and the stage of the disease, with about 10–40% detection rate observed in primary breast cancer and a significantly higher rate (40–80 %) in patients with metastatic disease.

Several studies have demonstrated the prognostic value of CTCs’ in advanced breast cancers (especially CTC detection prior to treatment) as an independent predictor of progression-free survival and overall survival. ^{Reference Banys-Paluchowski, Schneck and Blassl46,Reference Yap, Leong and Chua49,Reference Bidard, Proudhon and Pierga55,Reference Usiakova, Mikulova and Pinterova56,Reference Bidard, Michiels and Riethdorf58} Furthermore, it has been reported that a significant decrease in CTC count is an early indication of individual response to treatment and thus CTC screening could potentially provide a simpler alternative method to monitor the success of patients’ treatment. ^{Reference Banys-Paluchowski, Schneck and Blassl46,Reference Usiakova, Mikulova and Pinterova56} According to Nadal et al. ^{Reference Nadal, Lorente, Rosell and Serrano47} the detections of CTCs could be a potential substitute to invasive biopsies as a source of tumor tissue in the metastatic settings and an alternative and/or supplement to bone biopsy to monitor CTCs in the early-stage settings. Usiakova et al. ^{Reference Usiakova, Mikulova and Pinterova56} also reported that the detection of CTCs has a potential application as a marker for the success of therapeutic intervention in the adjuvant and neoadjuvant settings. It has been demonstrated that the detection of CTCs in the peripheral blood of breast cancer patients is correlated with poor prognosis in patients with early-stage breast cancer and those with metastatic breast cancer and that rising CTC levels in patients’ blood are highly predictive of disease progression. ^{Reference Wang, Chang and Yeh48,Reference Yap, Leong and Chua49,Reference Usiakova, Mikulova and Pinterova56} The detection and characterisation of CTCs in the peripheral blood of breast cancer patients offers opportunities to understand breast cancer metastatic processes, for early detection of metastasis, to guide treatment decisions in metastatic breast cancer, to provide improved treatment approaches and independent prognostic information for breast cancer patients, and might also provide additional information that could be useful for individualised patient-specific breast cancer treatment. ^{Reference Banys-Paluchowski, Schneck and Blassl46,Reference Nadal, Lorente, Rosell and Serrano47,Reference Alemar and Schuur54–Reference Bidard, Michiels and Riethdorf58}

The presence of disseminated tumour cells in the bone marrow of cancer patients is a common phenomenon in solid tumours and can be observed in primary breast cancer patients, however, due to the invasive nature of bone marrow aspiration (which are not well tolerated by most patients), recent investigations have shifted towards the detection of CTCs in patients peripheral blood in the settings of routine clinical practice. ^{Reference Nadal, Lorente, Rosell and Serrano47–Reference Yap, Leong and Chua49,Reference Usiakova, Mikulova and Pinterova56,Reference Bidard, Michiels and Riethdorf58} Bidard et al. ^{Reference Bidard, Michiels and Riethdorf58} conducted a meta-analysis in 2030 non-metastatic breast cancer patients whose CTC counts were determined prior to neoadjuvant chemotherapy and/or before surgery to assess the clinical validity of CTC detection as a prognostic marker. They reported CTCs detections in about 25% of patients prior to neoadjuvant chemotherapy which was associated with tumour size and the number of CTCs detected had a detrimental and decremental impact on overall survival, distant disease-free survival and locoregional relapse-free interval. They concluded that CTCs count in patients’ blood is an independent and quantitative prognostic marker in early-stage breast cancer patients treated with neoadjuvant chemotherapy and also complements current prognostic models based on tumour characteristics and treatment response. Usiakova et al. ^{Reference Usiakova, Mikulova and Pinterova56} assessed CTC-positivity rate in 419 blood samples obtained from 179 patients at different stages of breast cancer who were receiving chemotherapy in the adjuvant and neoadjuvant setting and also they evaluated the potential of CTC analysis to confirm treatment response. Patients with primary tumours were divided into neoadjuvant (n = 38), and adjuvant (n = 100) groups and 41 patients with metastatic breast cancer were evaluated under palliative treatment. They reported CTC detection rates of 35% among patients with early-stage breast cancer who did not show detectable metastases before neoadjuvant treatment, 26% in the adjuvant group and 43% in patients with metastatic breast cancer. However, they observed decreased CTC detection rates to 5% in the neoadjuvant group, to 13% in the adjuvant group and up to 12% in the metastatic breast cancer group after treatment completion. They concluded that post-treatment CTC detection may stratify a subgroup of patients at high risk of developing metastatic disease and CTC status can have a significant impact on early-stage breast cancer management. Wang et al. ^{Reference Wang, Chang and Yeh48} conducted a meta-analysis of four studies comprising 550 patients with stages I–IV breast cancer to evaluate the prognostic value of HER2-positive CTCs detection. They reported that HER2-positive CTCs detection is correlated with worse overall survival and progression-free survival in breast cancer patients without distant metastasis, and patients with HER2-positive CTCs detection might benefit from more aggressive and/or targeted treatments. Yap et al. ^{Reference Yap, Leong and Chua49} investigated the prevalence of CTCs detection in 108 breast cancer patients undergoing neoadjuvant or palliative treatment using a label-free microfluidic technology, and also evaluated its prognostic relevance in metastatic breast cancer. They demonstrated the prognostic significance of CTCs in metastatic breast cancer detected with a label-free size-based enrichment technology and reported that the prognostic relevance of CTCs was most significant in patients with HER2- metastatic breast cancer and patients who had one or more prior treatments.

Tumour-Infiltrating Lymphocytes

Tumour infiltrating lymphocytes (TILs) encompass all lymphocytic cells that have permeated tumour tissues and in breast cancers are comprised primarily of cytotoxic (CD8+) and helper (CD4+) T-cells, and a smaller proportion of B lymphocytes and natural killer cells. ^{Reference Pruneri, Vingiani and Denkert59,Reference Mao, Qu and Chen60} Several studies in breast and other types of cancers support the concept that TILs express an anti-tumour cellular immune response that may control the growth and spread of some tumours and the presence of lymphocytes within tumour cell nests and the degree of infiltration and has been shown to correlate with a better prognosis in several tumour types. ^{Reference Mao, Qu and Chen60–Reference Dushyanthen, Beavis and Savas65,Reference Denkert, von Minckwitz and Brase68} According to Wang et al., ^{Reference Wang, Xu, Zhang and Xue62} the prognostic value of TIL in breast cancer was originally demonstrated in the BIG 02–98 clinical trial and evidence from several studies ^{Reference Wang, Xu, Zhang and Xue62–Reference Denkert, Wienert and Poterie64,Reference Stanton, Adams and Disis66} have shown TILs as a predictive marker for response to neoadjuvant therapy and also it’s potential to predict pathological complete responses in all breast cancer subtypes in patients treated with neoadjuvant chemotherapy. Furthermore TILs prognostic value in response to adjuvant chemotherapy has also been confirmed but with no biologically applicable cut-off point as all the studies demonstrated TIL as a prognostic marker on a continuous scale. ^{Reference Mao, Qu and Chen60,Reference Wang, Xu, Zhang and Xue62–Reference Stanton, Adams and Disis66} Dushyanthen et al. ^{Reference Dushyanthen, Beavis and Savas65} and Stanton et al. ^{Reference Stanton, Adams and Disis66} reported that elevated levels of TILs are associated with higher responses to chemotherapy, independent of oestrogen receptor, progesterone receptor and HER2 status. According to Stanton et al. ^{Reference Stanton, Adams and Disis66} patients with at least 50% lymphocytic infiltrate (i.e., lymphocyte-predominant) breast cancer have exceptional prognosis compared to patients with lower levels of TILs. Furthermore, Stanton and Disis ^{Reference Stanton and Disis67} also reported that triple-negative breast cancers are more likely to be lymphocyte-predominant breast cancers and would gain the greatest survival benefit from each 10% increase in TIL level. According to Dushyanthen et al., ^{Reference Dushyanthen, Beavis and Savas65} the correlation between TILs at diagnosis and disease-free or overall survival is significant in triple-negative and HER2-positive breast cancers patients and that TILs positivity prior to treatment can predict response to therapy and improved prognosis in breast cancer patients. Furthermore, elevated levels of TILs at diagnosis would assure an improved outcome regardless of the attainment of pathological complete responses. On the other hand, patients with low levels of TILs and residual tumour at surgery post-neoadjuvant chemotherapy and anti-HER2 treatment may have worse outcomes, suggesting that this group of patients will require more effective anti-tumour and immune-enhancing strategies. ^{Reference Dushyanthen, Beavis and Savas65–Reference Stanton and Disis67} Dushyanthen et al. ^{Reference Dushyanthen, Beavis and Savas65} further suggested that since TILs levels are highly correlated with response to chemotherapy and trastuzumab, TILs positivity both pre- and post-neoadjuvant chemotherapy should be taken into account during patient treatment decisions.

Several independent studies have shown that tumour-infiltrating lymphocytes are both prognostic and predictive markers in breast cancer with potential relevance for the response to immune-checkpoint inhibitor treatments. ^{Reference Mao, Qu and Chen60–Reference Denkert, Wienert and Poterie64,Reference Stanton, Adams and Disis66,Reference Denkert, von Minckwitz and Brase68} Demaria et al. ^{Reference Demaria, Volm and Shapiro61} evaluated the degree of lymphocytic infiltration and TIL positivity in histological sections from both pre- and post-treatment surgical specimens from 25 breast cancer patients who received neoadjuvant paclitaxel chemotherapy to investigate whether increased apoptosis in tumour promotes an anti-tumour immune response. They observed that anti-tumour T-cell responses were triggered in patients when the chemotherapy-mediated killing of cancer cells in the primary tumour is efficient and concluded that the development of TILs after treatment correlates with clinical response to neoadjuvant paclitaxel therapy. Denkert et al. ^{Reference Denkert, Wienert and Poterie64} reported on a study that was conducted by the international immuno-oncology biomarker working group who investigated the intraclass correlation coefficient for evaluation of TIL by different pathologists. A total of 120 slides were evaluated by a large group of pathologists and they reported that TILs assessment is reproducible in breast cancer and opens the way for standardized reporting of tumour immunological parameters in clinical studies and diagnostic practice. Mao et al. ^{Reference Mao, Qu and Chen60} conducted a meta-analysis of 22,964 patients data to identify the prognostic value of TILs and/or TILs subsets in breast cancer patients stratified by infiltration sites. They reported that TILs are prognostic markers for both disease-free survival and overall survival in triple-negative breast cancer patients. And concluded that TILs and TILs subsets are promising prognostic markers in breast cancer, especially in triple-negative breast cancer patients. Stanton et al. ^{Reference Stanton, Adams and Disis66} also reviewed several published studies that investigated the prognostic value of TILs in patients with breast cancer, to derive estimates of the incidence, magnitude and phenotype of T-cell immunity in the major breast cancer subtypes. They reported that the degree of TIL varies both within and among breast cancer subtypes and the levels of lymphocyte subpopulations could potentially identify breast cancer patients who would be more responsive to immunomodulation. Wang et al. ^{Reference Wang, Xu, Zhang and Xue62} investigated the quantification of the clinical relevance of TILs, the effect of TILs on lymphocyte subpopulations and also assessed their consistency in breast cancer. They reviewed 23 studies comprising 13,100 patients and analysed such parameters as the pathological complete response and survival outcome following chemotherapy in breast cancer patients. They reported that TILs are associated with clinicopathological parameters of biologically aggressive phenotypes such as high tumour grade or ER-negative and PgR-negative, but are not correlated with HER2 expression. Moreover, they expressed that elevated TIL levels are associated with a significantly longer disease-free and overall survival, improved pathological complete response rate compared with a low TIL levels and predicted pathological complete response to chemotherapy and improved survival. Yu et al. ^{Reference Yu, Zhang and Wang63} conducted a meta-analysis of 117 studies consisting of 2,968 patients data to evaluate the total or subtype of TILs as a potential prognostic marker for breast cancer and to determine the relationship between TILs and clinicopathological features. They reported significant correlation between TILs and clinical traits in breast cancer patients and high values of total TILs predicted a favourable outcome for neoadjuvant chemotherapy and was associated with better prognosis. They suggested that TILs should be monitored in breast cancer patients for objective stratification and to adjust treatment strategy.

Topoisomerase II Alpha

The deoxyribonucleic acid (DNA) topoisomerases are enzymes that participate in the overwinding or underwinding of DNA and are divided into two types or families (type I and type II) of enzymes. The type I enzymes (topoisomerases I, III and V) change the degree of DNA supercoiling by inducing single-strand breaks and re-ligation, whereas the type II enzymes (topoisomerases II, IV and VI) produce double-strand breaks and the activities of both the type I and type II enzymes are critical during DNA transcription and replication. ^{Reference Champoux69,Reference Dekker, Rybenkov and Duguet70} Heestand et al. ^{Reference Heestand, Schwaederle, Gatalica, Arguello and Kurzrock71} and Ren et al. ^{Reference Ren, Liu, Gou and Xing72} reported that the DNA topoisomerase enzymes could be potential targets for cancer treatment since they regulate DNA function. According to Romero et al., ^{Reference Romero, Martín and Cheang73} the catalytic activity of topoisomerases II in mammalian cells is facilitated by the two isoforms, topoisomerase II alpha (TOP2A) and topoisomerase II beta (TOP2B). The TOP2A gene is located on the long arm of chromosome 17 at position 12·2 (17q12·2) and encodes the TOP2A protein which are highly expressed in proliferating cells, is associated with tumour grade and it is considered a proliferation marker. ^{Reference Fountzilas, Christodoulou and Bobos31,Reference Ren, Liu, Gou and Xing72–Reference Depowski, Rosenthal and Brien76} The TOP2A is an essential nuclear enzyme whose primary function is controlling DNA topological states by inducing transient DNA double-strand breaks and it also plays a vital role in DNA metabolism such as replication, movement and transcription, chromosome formation, enrichment and separation. ^{Reference Heestand, Schwaederle, Gatalica, Arguello and Kurzrock71–Reference An, Xu and Luo74,Reference Depowski, Rosenthal and Brien76} Aberration of TOP2A plays a critical role in chromosome instability and tumorigenesis, and the amplification or deletion of TOP2A gene has been associated with anthracycline sensitivity in advanced breast cancer patients or in high-risk primary breast cancer patients receiving adjuvant chemotherapy and also more closely associated with the benefit of adjuvant anthracycline-based treatment in primary breast cancer. ^{Reference Fountzilas, Christodoulou and Bobos31,Reference O’Malley, Chia and Tu75} Heestand et al. ^{Reference Heestand, Schwaederle, Gatalica, Arguello and Kurzrock71} has also reported that for patients with HER2 amplification receiving anthracyclines, co-amplification of TOP2A is associated with improved outcomes.

According to An et al. ^{Reference An, Xu and Luo74} TOP2A gene expression is elevated in proliferative subtypes of breast cancers such as triple-negative and HER2-enriched diseases compared to luminal type and elevated TOP2A protein expression has been associated with poor prognosis only in luminal breast cancer whereas the prognostic impact varies among different breast cancer subtypes. ^{Reference An, Xu and Luo74,Reference Depowski, Rosenthal and Brien76} Depowski et al. ^{Reference Depowski, Rosenthal and Brien76} has indicated that there is a significant correlation between TOP2A expression with lymph node metastases, high tumour grade, tumour size, distant metastases, negative hormone receptor status, DNA aneuploidy and HER2 onco-protein overexpression in tumours. Furthermore, they reported that most in vitro studies have shown that low levels of TOP2A expression in cancer cells are associated with drug resistance, whereas high levels imply sensitivity. ^{Reference Depowski, Rosenthal and Brien76} Romero et al. ^{Reference Romero, Martín and Cheang73} reported that the sensitivity to topoisomerase II inhibitors such as etoposide, teniposide, doxorubicin, idarubicin, epirubicin and mitoxantrone is dependent on TOP2A expression levels in target cancer cells and that cells with low TOP2A protein levels are less sensitive to Topo II inhibiting drugs compared to cells with high TOP2A protein levels. According to Heestand et al. ^{Reference Heestand, Schwaederle, Gatalica, Arguello and Kurzrock71} etoposide and anthracycline chemotherapies such as doxorubicin, daunorubicin and epirubicin inhibit TOP2A by blocking its ability to repair DNA strands after being cleaved which then results in the interruption of the reproduction of cancer cells.

An et al. ^{Reference An, Xu and Luo74} determined TOP2A protein expression levels via IHC and evaluated its prognostic importance in 434 stages I–II luminal breast cancer patients who received curative surgery. They reported that TOP2A overexpression is associated with higher tumour grade, Ki-67 index and is an independent predictor for poor distant metastasis-free survival, suggesting the potential value of the protein expression as a predictor of late recurrence for this subtype of breast cancer. Furthermore, they reported that patients with elevated TOP2A expression showed a significantly higher rate of distant metastasis and shorter distant metastasis-free survival. Additionally, they also observed that the prognostic value of TOP2A protein expression was more significant 5–8 years after diagnosis, more pronounced in stage II patients, luminal B disease and patients treated with adjuvant endocrine therapy. Depowski et al. ^{Reference Depowski, Rosenthal and Brien76} compared TOP2A expression with tumour stage, grade and size; lymph node metastases; ER and PgR status; disease recurrence; patient survival and HER2 gene status in 184 primary breast cancers. They observed that elevated TOP2A expression is correlated with decreased survival, advanced tumour stage, lymph node metastasis, an aggressive form of breast cancer exhibiting HER2 amplification and predicted disease-related mortality. Fountzilas et al. ^{Reference Fountzilas, Christodoulou and Bobos31} investigated the prognostic role of TOP2A gene amplification and protein expression on the outcome of metastatic breast cancer in 225 patients treated with trastuzumab-based regimens and also evaluated their potential interaction with anthracycline-based treatment. They observed TOP2A gene amplification and deletion in 41 and 16% of the tumours respectively and TOP2A protein expression in 65% of the tumours. The TOP2A amplification was more frequent in ER-negative tumours and its protein expression was associated with ER-positive status, high Ki67 expression, expression of Phosphate and Tensin Homologue Deleted on Chromosome 10 (PTEN) protein and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) mutations. Among the patients with TOP2A gene amplification, those who received anthracycline treatment showed a decreased risk of mortality. They concluded that TOP2A gene amplification is a favourable prognostic marker in HER2-positive metastatic and advanced-stage breast cancer patients treated with trastuzumab and it is a strong predictive factor for improved survival in patients with concurrent or prior exposure to anthracyclines.

O’Malley et al. ^{Reference O’Malley, Chia and Tu75} studied 438 tumours from 710 premenopausal patients with node-positive breast cancer who were treated with cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) or cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) as adjuvant chemotherapy in a randomised clinical trial. They observed that CEF treatment was significantly superior to CMF treatment in terms of relapse-free and overall survival for patients with TOP2A amplifications or deletions, and reported that TOP2A gene amplifications or deletions is correlated with elevated responsiveness to anthracycline-based chemotherapy regimens. They concluded that TOP2A gene alterations negativity patients could potentially be treated with a less toxic regimen such as CMF, whereas TOP2A alterations positivity patients should receive dose-intensive anthracycline-based regimens such as CEF. Ren et al. ^{Reference Ren, Liu, Gou and Xing72} conducted a meta-analysis of 25 different studies to elucidate whether TOP2A could predict the prognosis of breast cancer. They reported that elevated TOP2A protein expression is associated with shorter disease-free survival and TOP2A gene amplification or deletion is significantly related to shorter overall and relapse-free survival and hence, TOP2A is a potential prognostic marker for the prognosis of malignant breast tumours. Romero et al. ^{Reference Romero, Martín and Cheang73} assessed TOP2A status in 61 breast cancer samples via four different techniques: fluorescence in situ hybridisation (FISH), gene expression via DNA microarrays and real-time polymerase chain reaction (PCR), and IHC and evaluated the association and agreement among these detection techniques when used to stratify tumours as either TOP2A positive or negative. Furthermore, they investigated the correlation between TOP2A status and clinical features, such as cell proliferation, hormone receptor status and breast cancer intrinsic subtypes. They observed a significant correlation of TOP2A gene expression between real-time PCR and DNA microarrays and both detection methods predicted the IHC detection. Furthermore, they observed a significant correlation between TOP2A expression with cell proliferation, highly proliferative luminal B, HER2-enriched and Basal-like intrinsic breast cancer subtypes. They concluded that TOP2A expression in breast cancer is associated with high proliferation and aggressive tumour subtypes and these clinical features should be considered when assessing the predictive value of TOP2A for anthracycline-based chemotherapy.

Urokinase-Type Plasminogen Activator and Plasminogen Activator Type-1 Inhibitor

The plasminogen activator system is an enzymatic cascade involved in the control of fibrin degradation, matrix turnover and cell invasion, and plays a major role in embryogenesis, angiogenesis, cell migration, wound healing, inflammatory response and apoptotic cell death and also plays a dominant role in tumour growth, tumour cell invasion, migration and metastasis. ^{Reference Duffy, McGowan and Harbeck77–Reference Meijer-van Gelder, Look and Peters80} The major physiological function of the plasminogen activator system is to convert inactive plasminogen to plasmin and are mediated by two immunologically distinct serine proteases: the tissue-type plasminogen activator (tPA) and the urokinase-type plasminogen activator (uPA). ^{Reference Gouri, Dekaken and Bairi78,Reference Banys-Paluchowski, Witzel and Aktas81} It has been reported by several investigators that the uPA, its receptor (uPAR) and the plasminogen activator inhibitor type-1 (PAI-1) and inhibitor type-2 (PAI-2) promote tumour progression and metastasis and have also been established as prognostic factors in primary breast cancer. ^{Reference Duffy, McGowan and Harbeck77–Reference Meijer-van Gelder, Look and Peters80,Reference Look, van Putten and Duffy82–Reference Harms, Malter and Krämer84} According to Mahmood et al., ^{Reference Mahmood, Mihalcioiu and Rabbani83} the uPA which is a serine protease was originally isolated from human urine in 1947, although it can also be detected in the blood and the extracellular matrix. Banys-Paluchowski et al. ^{Reference Banys-Paluchowski, Witzel and Aktas81} reported that the uPA can be shed from tumour cells into the bloodstream and elevated levels have been observed in patients with malignant diseases than in healthy control, suggesting that, circulating uPA in plasma or serum has the potential as a diagnostic biomarker for early detection of breast cancer. Furthermore, they reported that both uPA and PAI-1 can be measured in breast cancer tissue using enzyme-linked immunosorbent assay (ELISA) and IHC at the protein level and real-time PCR at the mRNA level. Elevated levels of uPA, uPAR and PAI-1 in breast cancer tissues have been reported as independent strong predictors of poor clinical outcome, to correlate with disease aggressiveness and can potentially predict benefit from adjuvant chemotherapy in early breast cancer patients. ^{Reference Duffy, McGowan and Harbeck77–Reference Harbeck, Kates and Schmitt79,Reference Banys-Paluchowski, Witzel and Aktas81} Harbeck et al. ^{Reference Harbeck, Kates and Schmitt79} reported that elevated uPA and/or PAI-1 levels in breast cancer is associated with a high risk of disease recurrence, a decreased overall survival and are also strong prognostic factors for disease-free and overall survival. Meijer-van Gelder et al. ^{Reference Meijer-van Gelder, Look and Peters80} also showed that the uPA and PAI-1 levels in primary breast tumour is predictive for response to adjuvant systemic chemotherapy. According to Gouri et al., ^{Reference Gouri, Dekaken and Bairi78} the use of uPA and PAI-1 as markers hold the promise for personalised breast cancer therapy and could potentially identify patients’ risk for metastatic disease and subsequent relapse, thus could assist clinicians in the decision-making process on when to prescribe adjuvant therapy.

Banys-Paluchowski et al. ^{Reference Banys-Paluchowski, Witzel and Aktas81} evaluated the clinical relevance of uPA levels in serum quantified by ELISA in 252 metastatic breast cancer patients enrolled in a prospective multicentre study and also compared uPA with other blood-based biomarkers. They reported that patients with more than one metastatic site are more likely to exhibit elevated uPA levels, which independently predicted reduced overall survival, progression-free survival and improved prognoses in patients with known CTC status. They also observed correlations between uPA levels and other blood-based biomarkers such as CTC status, serum HER2, RAS p21, carbonic anhydrase IX (CA IX), TIMP metallopeptidase inhibitor 1 (TIMP1) and vascular endothelial growth factor. Duffy et al. ^{Reference Duffy, McGowan and Harbeck77} provided an overview of uPA and PAI-1 as prognostic or predictive biomarkers and discussed the potential therapeutic value of uPA in breast cancer. They reported that lymph node-negative breast cancer patients as identified with uPA and/or PAI-1 measurements had excellent outcome, high levels of uPA and/or PAI-1 could select patients who are more likely to benefit from adjuvant chemotherapy and use of uPA and PAI-1 measurements can lead to personalised treatment for patients diagnosed with lymph node-negative breast cancer. Gouri et al. ^{Reference Gouri, Dekaken and Bairi78} reviewed the biology, roles and the presumed role of uPA and/or PAI-1 in precision medicine in oncology. They reported that the use of uPA and/or PAI-1status as a marker of risk for metastatic relapse could assist in the selection of breast cancer patients who are most likely to benefit from systemic therapy and uPA and/or PAI-1 determination could predict treatment efficacy. Harbeck et al. ^{Reference Harbeck, Kates and Schmitt79} investigated the prognostic value of uPA and PAI-1 in node-negative breast cancer patients in a multicenter prospective randomised clinical trial. They reported that node-negative breast cancer patients with low uPA and PAI-1 levels had favourable prognosis and therefore could be spared the burden of adjuvant chemotherapy. However, patients with elevated uPA and/or PAI-1 levels had increased risk of disease recurrence, greater benefit from adjuvant chemotherapy and the administration of cyclophosphamide, methotrexate, 5-fluorouracil-based chemotherapy in these patients resulted in a substantial reduction in risk of disease recurrence. They concluded that elevated levels of uPA and/or PAI-1 indicate an aggressive phenotype which in the adjuvant settings may be overcome or suppressed by early systemic therapy, but may be too advanced for a response to palliative therapy at a later stage. Meijer-van Gelder et al. ^{Reference Meijer-van Gelder, Look and Peters80} studied the predictive value of uPA, uPAR and PAI-1 by evaluating the association of the expression levels in tumour and the efficacy of tamoxifen therapy in 691 hormone-naive ER-positive breast cancer patients treated for recurrent. They reported that elevated levels of uPA, uPAR and PAI-1 in tumours are associated with lower efficacy of tamoxifen therapy and predicted shorter progression-free survival on tamoxifen. They concluded that uPA, uPAR and PAI-1 are predictive for the efficacy of tamoxifen treatment in patients treated for recurrent breast cancer and determination of their expression levels in tumours might be helpful for personalised therapy.

Circulating Micro-Ribonucleic Acids

MicroRNAs (miRNAs) are small (18–24 nucleotides in length) non-coding regulatory ribonucleic acid (RNA) molecules that are involved in regulating cellular gene expression at the posttranscriptional level by suppressing translation of protein-coding genes, or cleaving target messenger RNAs (mRNAs) to induce their degradation, via imperfect pairing with target mRNAs of protein-coding genes. ^{Reference Si, Sun and Chen85–Reference Ng, Li and Shin89} The miRNAs regulate several biological and pathological processes including stem cell maintenance, embryogenesis, hematopoietic stem cell differentiation, and their expression has been shown to be frequently deregulated in cancers including breast cancer. ^{Reference Si, Sun and Chen85–Reference Fu, Chen and Man86, Reference Ng, Li and Shin89–Reference Kashyap and Kaur90} According to Fu et al., ^{Reference Fu, Chen and Man86} genes encoding miRNAs positioned in chromosome regions that are deleted in tumours behave as tumour suppressors, whereas those amplified in tumours function as oncogenes. Furthermore, several miRNAs have been identified that are aberrantly expressed in breast cancers, suggesting their direct involvement in oncogenesis. ^{Reference Fu, Chen and Man86,Reference Kashyap and Kaur90} Heneghan et al. ^{Reference Heneghan, Miller and Lowery88} reported that the involvement of miRNAs in the initiation and progression of cancers has great potential for the development of new techniques in the diagnostic and therapeutic approaches for the management of breast cancer patients. According to Kashyap and Kaur ^{Reference Kashyap and Kaur90} several studies have reported the detection of cell-free miRNAs in the blood and serum or plasma of patients with breast cancer and have suggested using the miRNAs dysregulated expression as a diagnostic indicator for early breast cancer detection. Fu et al. ^{Reference Fu, Chen and Man86} reported the identification of miR-10b, miR-125b and miR-145 miRNAs that are down-regulated and miR-21, and miR-155 miRNAs being up-regulated in breast cancer. Kashyap and Kaur ^{Reference Kashyap and Kaur90} reported the identification miR-127–3p, miR-148b, miR-409–3p, miR-376a, miR-376c, miR-652 and miR-801 miRNAs being up-regulated in the plasma of breast cancer patients in contrast to healthy controls and also reported over ninety cell-free miRNAs observed in different body fluids and their expression status in breast cancer. Furthermore, they also reported a panel of seven miRNAs (miR-23a, miR-23b, miR-24, miR-25, miR-29a, miR-103 and miR-222) co-regulated in serum and tissue of breast cancer patients that were identified in a genome-wide cell-free miRNA analysis using Sequencing by Oligonucleotide Ligation and Detection (SOLiD) sequencing. The expression of miRNA has been associated with some clinicopathological features such as ER status, PgR status and tumour stage and Fu et al. ^{Reference Fu, Chen and Man86} reported the identification of 15 miRNAs that could be used to differentiate breast cancer from normal tissue. According to Kashyap and Kaur, ^{Reference Kashyap and Kaur90} a group of cell-free miRNAs (i.e., miR-21, miR-23b, miR-200b and miR-200c) show elevated expressions in metastatic condition compared to early breast cancer and reported that miR-21 and miR-200b can potentially differentiate metastatic tumours from early-stage breast cancer and when used in combination with the expression of miR-21, miR-190, miR-200b and miR-200c could further improve the differentiating potential. They have suggested that blood-based miRNAs offer great potential for early-staged, non-invasive, sensitive and specific breast cancer detection and screening.

Heneghan et al. ^{Reference Heneghan, Miller and Lowery87} investigated the value of cancer-specific circulating miRNAs as novel breast cancer biomarkers and a potential relationship between circulating miRNA levels and clinicopathological features of breast cancer, such as tumour subtype, stage of disease, nodal status or hormone receptor status. They used real-time quantitative PCR (qPCR) analysis to quantify miRNA in tissue and blood specimens of 148 breast cancer patients and 44 age-matched and disease-free control women. They observed that specific circulating miRNAs are associated with certain clinicopathological features, such as nodal and ER status and they reported that cancer-specific miRNAs are detectable and are significantly dysregulated in the circulation of breast cancer patients and that increased systemic miR-195 levels in breast cancer patients are replicated in breast tumours. Furthermore, they observed that circulating miR-195 and let-7a levels decreased in curative post-operative cancer patients to similar levels as control women. They concluded that systemic miRNAs can potentially be used as novel breast cancer biomarkers and may have significant value in clinical management during peri-operative stages. Ng et al. ^{Reference Ng, Li and Shin89} studied the development of a sensitive and specific blood-based diagnostic assay for breast cancer detection using plasma samples from 260 clinically diagnosed breast cancer patients. They observed a significant increase in miR-16, miR-21 and miR-451 levels and reduction in miR-145 levels in the plasma of breast cancer patients. However, a combination of miR-145 and miR-451 levels provided the best biomarker to distinguish breast cancer from healthy controls with a receiver operating characteristic (ROC) curve area of 96%. The optimal sensitivity and specificity were 90 and 92% respectively in differentiating breast cancer from controls. They concluded that circulating miRNAs is a potential cancer-specific biomarker for breast cancer screening. Si et al. ^{Reference Si, Sun and Chen85} also investigated the differential expression of miRNAs and the potential of circulating miRNAs as novel primary breast cancer biomarkers on 48 tissue and 100 serum samples of patients with primary breast cancer and 20 control samples of healthy women. They detected expression levels of ten miRNAs (miR-106b, miR-125b, miR-17, miR-185, miR-21, miR-558, miR-625, miR-665, miR-92a and miR-93) which were measured using real-time quantitative reverse transcription PCR (qRT-PCR). They reported significantly lower levels of miR-92a and elevated levels of miR-21 in tissue and serum samples of breast cancer patients compared to healthy controls, suggesting a significant and independent potential quantification of miR-92a and miR-21 expression levels in serums. They also observed that decreased levels of miR-92a and elevated levels of miR-21 were associated with some clinicopathological data such tumour size and a positive lymph node status of breast cancer patients.

Cyclin D1 and Cyclin E

Cyclins are a family of regulatory proteins that control the progression of the cell cycle and activate cyclin-dependent kinases (CDKs), which control cell cycle processes through phosphorylation (e.g., cyclin E exerts its actions by activating cyclin-dependent kinase 2 (CDK2) whereas cyclin D1 exerts its actions by activating cyclin-dependent kinase 4/6 (CDK4/CDK6)). There are five main types of cyclins found in humans viz the cyclin A (A1), cyclin B (B1 and B2), cyclin C, cyclin D (D1, D2 and D3) and cyclin E (E1 and E2). ^{Reference Aaltonen, Blomqvist and Amini91–Reference Han, Park and Bae96} The Cyclin D1 (CCND1) protein is encoded by the cyclin D1 gene located on the long arm of chromosome 11 at position 13·3 (11q13·3) whereas the cyclin E1 (CCNE1) protein is encoded by the cyclin E1 gene located on the long arm of chromosome 19 at position 12 (19q12). ^{Reference Roy and Thompson94} The cyclin D1 and E are the most extensively investigated cell cycle regulators and mediate positive growth stimuli for cell cycle progression and cell proliferation. Furthermore, they control the cell cycle progression through the restriction point during the late-G1 phase when cells lose their dependency on mitogens and commit to DNA synthesis, which is a critical checkpoint controlling cell entry into mitosis. ^{Reference Aaltonen, Blomqvist and Amini91–Reference Lodén, Stighall and Nielsen95} According to Hui et al. ^{Reference Hui, Finney and Carroll93} deregulated cyclin D1 or cyclin E expressions render the growth of normal cells less dependent on growth factors thereby could accelerate through the G1 phase of the cell cycle. Hui et al. ^{Reference Hui, Finney and Carroll93} reported that overexpression of either cyclin D1 or cyclin E has been shown to lead to mammary carcinoma in transgenic mice, suggesting their roles as oncogenes in mammary epithelium. Studies ^{Reference Aaltonen, Amini and Landberg92, Reference Hui, Finney and Carroll93, Reference Lodén, Stighall and Nielsen95} have reported that both cyclins D1 and E play vital roles in breast carcinogenesis and has been linked to tamoxifen resistance, overexpression of either gene has been associated with poor prognosis in breast cancer and according to Hui et al. ^{Reference Hui, Finney and Carroll93} expression levels of cyclin D1 and cyclin E may influence therapeutic sensitivity to anti-oestrogens tumours.

According to Roy and Thompson, ^{Reference Roy and Thompson94} cyclin D1 is one of the frequently overexpressed proteins and commonly amplified genes in breast cancer. It is overexpressed in about 40–90% of invasive breast cancers, while gene amplification has been observed in 5–20% of tumours ^{Reference Roy and Thompson94} and Aaltomen et al. ^{Reference Aaltonen, Amini and Landberg92} reported that the amplification of cyclin D1 gene and posttranscriptional deregulation would lead to cyclin D1 overexpression. Roy and Thompson ^{Reference Roy and Thompson94} further reported that cyclin D1 expression is strongly associated with ER and PgR status in breast cancer and Aaltomen et al. ^{Reference Aaltonen, Amini and Landberg92} also expressed that cyclin D1 overexpression is associated with non-aggressive features in breast cancers such as ER positivity. According to Aaltonen et al. ^{Reference Aaltonen, Blomqvist and Amini91} lower expression levels of cyclin D1 in breast cancer type-1 (BRCA1) tumours and both lower and higher expression levels in breast cancer type-2 (BRCA2) tumours have been observed in patients with breast cancer. Aaltomen et al. ^{Reference Aaltonen, Amini and Landberg92} reported that oestrogen stimulates the expression of cyclin D1 rendering it an important mediator of oestrogen stimulation of cell cycle progression. Loden et al. ^{Reference Lodén, Stighall and Nielsen95} reported a correlation between cyclin D1 overexpression and improved prognosis and cyclin D1 expression and low tumour grade. And Roy and Thompson ^{Reference Roy and Thompson94} also reported that overexpression of cyclin D1 is a good prognostic factor in invasive breast cancer and it is associated with improved patient overall survival and relapse-free survival, especially for ER-positive patients.

Although amplification of the cyclin E gene in tumours is rare, it has been reported to be overexpressed in about 30% of breast cancer cases ^{Reference Hui, Finney and Carroll93,Reference Lodén, Stighall and Nielsen95} and according to Aaltonen et al. ^{Reference Aaltonen, Blomqvist and Amini91} elevated expression levels of cyclin E have been observed in BRCA1 or BRCA2 tumours. Aaltonen et al. ^{Reference Aaltonen, Blomqvist and Amini91} reported that elevated cyclin E expression is common in hormone receptor-negative and high-grade aggressive breast cancers, causes chromosomal instability, is an adverse prognostic factor and has predicted endocrine therapy failure and is often associated with aggressive disease features (i.e., hormone receptor negativity, high grade and large tumour size) in breast cancer. ^{Reference Aaltonen, Blomqvist and Amini91} Hui et al. ^{Reference Hui, Finney and Carroll93} also reported that patients with overexpressed cyclin E breast cancer have a significantly increased risk of relapse and mortality.

Aaltomen et al. ^{Reference Aaltonen, Amini and Landberg92} analysed cyclin E and D1 immunohistochemical expression on breast tumour microarrays in a series of 1,348 invasive breast cancers to determine the role of cyclin D1 and E expression in proliferation among ER-positive and ER-negative breast cancers and their correlation with histopathological variables. They reported that elevated cyclin D1 expression was associated with high grade, high cyclin A and Ki67 expression among ER-positive breast cancers, whereas the expression was correlated with low grade and low Ki67 expression among ER-negative breast cancers. Furthermore, they observed cyclin E expression is associated with aggressive breast cancer phenotype and an independent marker of poor metastasis-free survival and both cyclin E and D1 expression correlated positively in ER-positive but showed a negative correlation in ER-negative tumours. They indicated that proliferation in ER-positive and ER-negative breast cancers is driven by different mechanisms and supports the role cyclin D1 plays in tumourigenesis of ER-positive breast cancers. In another study, Aaltonen et al. ^{Reference Aaltonen, Blomqvist and Amini91} assessed cyclin E and cyclin D1 expression in a series of 53 BRCA1-positive, 58 BRCA2-positive, 798 familial non-BRCA1/2 and 439 sporadic breast cancers to determine their role in familial breast cancers. They reported that elevated cyclin E and low cyclin D1 expressions are independent predictors of BRCA2 mutation in comparison with familial non-BRCA1/2 tumours. Furthermore, they observed that lower frequency of high cyclin E expression independently distinguished familial non-BRCA1/2 tumours from sporadic breast cancers. And they concluded that cyclin E and cyclin D1 expression can potentially distinguish familial non-BRCA1/2 tumours from both sporadic and BRCA1- and BRCA2-associated tumours and may reflect the different predisposition and pathogenesis in these groups. Han et al. ^{Reference Han, Park and Bae96} investigated the impact of cyclin E and cyclin D1 expression in tissue arrays of 175 node-negative breast cancer patients treated with cyclophosphamide, methotrexate, 5-fluorouracil (CMF) chemotherapy to the treatment outcome. They observed that Cyclin E expression was significantly elevated in young aged patients and poorly differentiated tumours, was significantly increased in cyclin D1 expressing tumours, was associated with poor prognosis and was significant for the prediction of poor survival in node-negative breast cancer patients after CMF chemotherapy. Hui et al. ^{Reference Hui, Finney and Carroll93} investigated the potential roles of cyclin D1 and cyclin E expression as markers of therapeutic responsiveness to pure steroidal anti-oestrogen ICI 182,780 using T-47D breast cancer cell lines constitutively overexpressing cyclin D1 or cyclin E. They reported that cyclin D1 overexpression decreased sensitivity to anti-oestrogen inhibition whereas cyclin E overexpression produced a less pronounced early cell cycle effect, indicating a partial resistance to a short-term anti-oestrogen inhibition. They concluded that cyclin D1 expression and cyclin E-p27 association play important roles in anti-oestrogen action, and suggest that cyclin D1 or cyclin E overexpression has subtle effects on anti-oestrogen sensitivity. Roy and Thompson ^{Reference Roy and Thompson94} reviewed the roles of cyclin D1 in both normal and abnormal cell-cycle regulation, mammary gland development and carcinogenesis and the relationship to oestrogen in breast tissues. They reported that cyclin D1 is overexpressed in more than half of invasive breast cancers, it interferes with the effect of tamoxifen and ER-positive breast cancers and could potentially account for treatment failure while on tamoxifen treatment.

Autoantibodies

Autoantibodies or natural antibodies are essentially Immunoglobulin M (IgM) and are antibodies that react with self-antigens which may be present in all cell types (e.g., chromatin, centromeres) or be highly specific for a specific cell type in one organ of the body (e.g., thyroglobulin in cells of the thyroid gland). ^{Reference Gómez Lahoz, Liegeois and Zhang97} They may consist of proteins, nucleic acids, carbohydrates, lipids or various combinations of these and provide a first line of defense against infections and contribute to the homeostasis of the immune system. ^{Reference Gómez Lahoz, Liegeois and Zhang97–Reference Lacombe, Mangé and Jarlier101} According to Leslie et al., ^{Reference Elkon and Casali98} autoantibodies can be detected in diseases with a long prodrome and presenting no clinical symptoms, can potentially predict both the likelihood of clinical disease and the disease activity (i.e., rate of progression to disease), it can assist define the nature of the disease and provide markers to stratify the disease for patients with established disease. ^{Reference Elkon and Casali98} Madrid ^{Reference Wang, Figueroa and Wallstrom102} reported that several autoantibodies have been observed in the sera of breast cancer patients and have been identified as either diagnostic or prognostic markers of the disease. According to Blixt et al., ^{Reference Madrid103} autoantibodies to the transmembrane glycoprotein Mucin 1 (MUC1) showed an autoantibody response in early-stage breast cancer patients that is highly correlated to age. Furthermore, autoantibodies to specific glycoforms of MUC1 have also been detected more frequently and at elevated levels in breast cancer patients in comparison with patients with benign breast disease or healthy women. Blixt et al. ^{Reference Madrid103} reported that autoantibody induction to the core3-MUC1 and STn-MUC1 glycoforms is correlated to a decreased incidence of cancer metastases and elevated time to metastasis, which suggests that autoantibodies to cancer-associated glycoforms of MUC1 may play a role in the development and progression of the disease. Other studies have demonstrated that autoantibodies are detectable in the sera of patients and those which are produced against tumour-associated antigens may provide an in vivo amplification of an early carcinogenic cue and therefore may potentially allow early detection of cancer and could be used to guide the clinical management of the disease. ^{Reference Gómez Lahoz, Liegeois and Zhang97–Reference Madrid103}

Zhong et al. ^{Reference Blixt, Bueti and Burford104} interrogated breast cancer T7 complementary DNA (cDNA) phage library to determine tumour-associated proteins, measured autoantibody reactivities to assess phase-expressed proteins in sera of breast cancer patients and control women via immunochemistry and ELISA and evaluated the sensitivity and specificity of single versus combined antibody measurements for predicting the probability of breast cancer. They concluded that serum autoantibody profiling is a promising technique for early detection and diagnosis of breast cancer and that a panel of autoantibodies would potentially achieve superior detection accuracy. Blixt et al. ^{Reference Madrid103} used a microarray platform of 60mer MUC1 glycopeptides to investigate the presence of autoantibodies to cancer-associated glycoforms of MUC1 in early breast cancer patients. They reported elevated levels of autoantibodies in the sera from breast cancer patients in comparison to women with benign breast disease or healthy women and was highly correlated with age. Chapman et al. ^{Reference Leslie, Lipsky and Notkins99} investigated the diagnostic potential for the presence of autoantibodies to tumor protein 53 (TP53), c-MYC, HER2, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), BRCA-1, BRCA-2 and MUC1 antigens via enzyme-linked immunosorbent assay in the sera from 94 healthy women, 97 primary breast cancer patients and 40 patients with ductal carcinoma in situ. They observed reproducible elevated levels of autoantibodies in at least one of the tumour-associated antigens in the sera from 64% of patients with primary breast cancer and 45% of patients with ductal carcinoma in situ at a specificity of 85%. They concluded that autoantibodies against at least one of the tumour-associated antigens is an indication of early-stage breast cancer and autoantibody assays against a panel of antigens could be a useful aid to mammography in the detection and diagnosis of early primary breast cancer, particularly in younger women who may be at increased risk of breast cancer and where mammography is considered to have reduced sensitivity and specificity. In a review by Madrid, ^{Reference Wang, Figueroa and Wallstrom102} the author discussed the rationale for the consideration of autoantibodies as potential biomarkers for early diagnosis of breast cancer, and evaluated reported tumour-associated antigen-antibody systems in breast cancer as well as the implications of autoantibodies detection in sera of breast cancer patients. They reported that the use of genomics, proteomics and high throughput technology has led to the identification of a large group of breast cancer-associated autoantigens recognised by autoantibodies present in the sera of cancer patients and that autoantibodies have the potential as biomarkers and may facilitate the early diagnosis of breast cancer.

Wang et al. ^{Reference Lacombe, Mangé and Jarlier101} evaluated autoantibodies associated with basal-like breast cancer using sera samples from a population-based case-control breast cancer study. They screened 10,000 antigens on protein arrays using 45 basal-like breast cancer patients and 45 controls and they identified 748 potential plasma autoantibodies associated with basal-like breast cancer; however, a further investigation via ELISA assays on the markers of 145 basal-like breast cancer cases and 145 age-matched controls identified 13 autoantibodies capable to distinguish basal-like breast cancer from controls with 33% sensitivity and 98% specificity. They also observed a strong correlation of TP53 autoantibodies with its protein expression in 145 basal-like breast cancer patients and a correlation between meningioma 1 (MN1) and TP53 autoantibodies with worse survival. Yagihashi et al. ^{Reference Zhong, Ge and Zu105} examined the prevalence of anti-survivin and livin antibodies in sera from breast cancer patients via ELISA using full-length recombinant livin protein. They observed that anti-survivin and anti-livin antibodies in sera from breast cancer patients are significantly elevated compared with those in sera from healthy women and concluded that survivin and livin may act as a major cancer antigen in breast cancer patients. Lacombe ^{Reference Chapman, Murray and Chakrabarti100} used a proteomic approach and serological screening of 580 sera to identify autoantibodies associated with early-stage breast cancer and to determine whether a panel of autoantibodies could enhance the detection rate. They identified 67 antigens that produced a specific humoral response in breast cancer and when they selected 5 antigens (GAL3, PAK2, PHB2, RACK1 and RUVBL1) for validation, they observed that GAL3 and RACK1 showed increased reactivity in early-stage breast cancer. They reported that a combination of the five markers significantly discriminated early-stage breast cancer from healthy women, and was elevated in patients with node-negative early-stage primary breast cancer or ductal carcinoma in situ. They concluded that the autoantibody panel could be useful as a diagnostic tool in the screening of early-stage invasive breast cancer and pre-invasive breast cancer and could potentially complement mammography for women with high breast density.