While the primary value of steroid hormone receptors is as predictive biomarkers for endocrine therapy, the receptor status of a primary invasive breast cancer can also provide prognostic information. Indeed, several retrospective studies have shown that patients with ER or PRexpressing tumours tend to have a better outcome than those lacking the receptors. 26,37,40,41,42 However, the favourably prognosis associated with ER-positive tumours mostly occurs during the first 5-7 years after initial diagnosis . 40,41,42 Thereafter, the risk of relapse tends to be greater in ER-positive than ER-negative tumours. Steroid hormone receptors alone, thus, cannot be used to identify women whose tumours are more likely to show clinical aggressive behaviour in long-term follow-up. 20 Furthermore, the impact of therapy on the prognostic impact of steroid hormone receptors prognosis is difficult to exclude, as almost all receptor-positive patients are treated with endocrine therapy in either early or advanced disease. Indeed, in some studies, the improved outcome with receptorpositive tumours was only found in patients who underwent endocrine therapy. 41,42 Scoring system. Many scoring systems have been developed to quantify hormonal receptor status in breast cancer samples and many studies have compared their ability to predict treatment response and correlations with outcome. 19 The first scoring system counted the percentage of hormonal receptor-positive cancer cells and ignored staining intensity. 43,44 ASCO/CAP (College of American Pathologists)'s practice guidelines recommend that the cut-off to distinguish "positive" from "negative" cases should be ≥ 1% of ER-/PgRpositive tumour cells, since predictive validity for ER and PgR has been demonstrated with as few as 1% of stained tumour cell nuclei. 26 Therefore, the Panel endorses use of endocrine therapy in patients whose breast tumours show at least 1% of ER-positive cells and discourages such a therapeutic approach if this value is less than 1%. 26 Furthermore, according to the International Breast Cancer Study Group scheme, which constitutes the basis of 2015 St Gallen treatment guidelines 15 , breast cancer is divided into three groups based on the percentage of hormonal receptor-positive cancer cells: responsive (≥10%), response uncertain (1%-9%) and non-responsive (0%). Therefore, among breast tumours defined as hormonal receptor-positive, it is possible to notice different degrees of endocrine responsiveness, which can underlie different therapeutic outcomes in this group of breast cancer patients. 26,15 Another scoring system for ER and PgR is represented by the "Allred score", which combines together both the percentage of positive cells and staining intensity. 45 A total score of 3 characterises the lowest positive result and parallels the St Gallen endocrine response-uncertain category, in which adjuvant hormone treatment can be recommended, but has an uncertain benefit. 15,46 Indeed, this intermediate group comprises tumours whose cells show some features of endocrine responsiveness, but insufficient to be sure that endocrine therapy alone is adequate; patients classified in this category may, therefore, require both endocrine therapy and chemotherapy. 46 Human epidermal growth factor receptor 2The human epidermal growth factor receptor 2 gene ERBB2 (commonly referred to as HER2) is amplified in approximately 18% to 20% of invasive breast cancers, more than a half of which (~55%)are ER-and PgR-negative. 47 ERBB2 is the official name provided by the HUGO Gene NomenclatureCommittee for the v-erb-b2 erythroblastic leukaemia viral oncogene homolog 2 gene that encodes a member of the epidermal growth factor receptor family of receptor tyrosine kinases. 48 Of note, amplification is the primary mechanism of HER2 overexpression and abnormally high levels of a 185kd glycoprotein with tyrosine kinase activity are found in these tumors. 49 There are several possible uses of HER2 status, as both predictive and prognostic biomarker, but the main clinical utility of HER2 measurement is undoubtedly in predicting the response of breast cancer to anti-HER2 therapy in the neoadjuvant, adjuvant and advanced disease settings. 20,48,50 Indeed, even though numerous studies have revealed that HER2 positivity is associated with worse prognosis (i.e., higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy 48,51,52 , the predictive value of this biomarker plays a fundamental role in the choice of administering anti-HER2 targeted therapies and, therefore, in the contest of personalized medicine. 20 Predictive role. Currently, four anti-HER2 therapies are approved for the treatment of HER2-positive breast cancer 50 : Trastuzumab (an anti-HER2 monoclonal antibody which appears to work by a variety of different mechanisms, including prevention of HER2 cleavage and downstream proliferative signalling, antiangiogenic effects and cellular cytotoxicity 53 ), Lapatinib (an oral small-molecule which acts as a strong and reversible inhibitor of both EGFR and HER2 tyrosine kinase domains 54 ), Pertuzumab (a monoclonal antibody that binds to a different region of the HER2 extracellular domain thanTrastuzumab and can block dimerization of HER2 with other HER family receptors 55 ) and Trastuzumab-Emtansine (also referred to as T-DM1; an antibody-drug conjugate, with Trastuzumab delivering the antimicrotubule agent DM1 [Emtansine] to HER2-positive tumour cells 56 ). Based on the available evidence, HER2 gene amplification/overexpression appears to be necessary but not sufficient for response to all of these anti-HER2 therapies. 50 Several expert panels, including ASCO 57 , recommend that the first-line therapy for patients with HER2positive advanced breast cancer should be Trastuzumab, Pertuzumab and a taxane. For those who progress during or after first-line anti-HER2 treatment, T-DM1 or Lapatinib in combination withCapecitabine should be administered. 57 In the adjuvant setting, Trastuzumab (in combination with chemotherapy) is the standard and still the only approved form of anti-HER2 therapy administered, while, for the neoadjuvant setting, dual treatment with Trastuzumab and Pertuzumab (plus chemotherapy) is approved both in Europe and in the USA. 50,15 It is worth noting that HER2 status is also predictive for systemic therapies other than HER2-targeted ones. 58 In this regard, HER2 positivity appears to be associated with relative, but not absolute, resistance to endocrine therapies in general. 59 Although controversial, preclinical and clinical studies have suggested that this effect may be specific to selective oestrogen receptor modulator therapy, such as Tamoxifen, and perhaps not to oestrogen depletion therapies, such as those with aromatase inhibitors. 60 HER2 status also appears to be predictive for either resistance or sensitivity to different types of chemotherapeutic agents. Indeed, HER2 may be associated with relative, but not absolute, lower benefit from non-anthracycline, non-taxane containing chemotherapy regimens. 61 In contrast, retrospectively obtained results from prospectively conducted randomized clinical trials appear more definitive in suggesting that HER2 positivity is associated with response to anthracycline therapy;although, this effect may be secondary to co-amplification of HER2 with topoisomerase II, which is the direct target of these agents. 62,63 Further data also suggest that HER2 may predict for response and benefit from taxane-containing regimens in either the metastatic or adjuvant settings. 64,65 Scoring system. Although HER2 can be detected using many methods, two are routinely employed:IHC and fluorescence in situ hybridization (FISH). 48 IHC evaluates overexpression of the receptor protein at the surface of the cells, while FISH assesses the status of the HER2 gene in the nucleus. In the majority of HER2-positive cancers, HER2 protein overexpression is the result of gene amplification, thus both methods should be highly correlated. 48 Immunohistochemistry reactions for HER-2 are scored by HercepTest™ (a semi-quantitative immunohistochemical assay for determination of HER2 protein overexpression in breast cancer tissues routinely processed for histological evaluation), whereby 0 and 1+ scores are negative, 2+ is weakly positive and 3+ is clearly positive. A clearly positive HER2 result consists of a uniform and intense membrane staining of more than 30% of tumour cells and further evaluation is unnecessary for invasive cancers that stain definitely positive or negative. On the contrary, weakly positive (i.e., equivocal or 2+) cases should be tested for gene amplification by FISH. A positive result using this method indicates more than 6 HER2 gene copies per tumour cell nucleus or a HER2 gene to chromosome 17 ratio of more than 2.2. 48 Selection of the best treatment, especially if the patient is a candidate for HER2-targeted therapy, depends on accurate laboratory results of the assessment of HER2 status. All aspects of the test are, thus, performed in a highly standardized fashion with good quality control and the quality controls must be continuously monitored. 19 