A multiplicity of studies, including retrospective evaluation of randomised clinical trials and meta-analyses, have shown that elevated levels of Ki67 are independently associated with adverse outcome in patients with breast cancer. 85,86,87 In one of the largest studies, Petrelli et al. 86 performed a systematic review of the literature which was followed by a meta-analysis of the individual studies; in total, 41 studies encompassing 64,196 patients were identified. Although different cut-off points over the range 10 to >25% cell staining were investigated, the threshold displaying the strongest prognostic significance for overall survival was found to be >25% cell staining (hazard ratio 2.05; p < 0.00001). 86 The consistent relationship between high Ki67 values and poor outcome in patients with breast cancer has been found despite the reported poor interlaboratory precision for the assays employed, use of different methods to measure Ki67 and use of different cut-off points for differentiating between tumours with low and high Ki67 concentrations. 70 According to Denkert et al. 88 , imprecision is particularly found at borderline or intermediate concentrations of Ki67. Indeed, these investigators suggested that clinical decision-making should not be based on Ki67 levels in the borderline range.However, in contrast to the poor precision at intermediate levels, a multicentre study carried out by the International Ki67 Working Group 89 concluded that good interlaboratory agreement was achievable using centrally stained core needle biopsies when Ki67 scores were higher or lower than intermediate scores (i.e. <10% or >20% cell staining). Furthermore, in 2013, the St Gallen International Expert Consensus Panel, even noticing that standardized cut-offs for Ki-67 had not been establishedand laboratory specific values should be used, voted that a threshold of ≥ 20% was clearly indicative of "high" Ki-67 status. 14 This threshold has also been adopted as cut-off to distinguish, among hormonal receptor-positive HER2-negative breast cancers, those more likely to exhibit an aggressive clinical behaviour and to benefit from the addition of chemotherapy to endocrine therapy (i.e., luminal B-like tumours ─see below). 14 Nonetheless, some authors still rely on the cut-off proposed by Cheang et al. 90 , that is Ki67 ≥ 14%, to characterise luminal B breast cancers.Scoring system. The most commonly used mouse anti-human Ki67 monoclonal antibody, MIB1 clone 91 , has the especially favourable property of detecting an epitope motif unique to Ki67 (ensuring specificity) that is repeated 16 times in the protein (enhancing sensitivity) 92 . Furthermore, given its long and highly validated track record, MIB1 is recommended as the "gold standard" for Ki67 assessment by the International Ki67 in Breast Cancer Working Group. 70 The ki67 score (also named Ki67 index) is defined as the percentage of positively stained cells among the total number of malignant cells scored and, if the staining is homogeneous, the recommendation is to count at least three randomly selected high-power (x40 objective) fields. 70 Of note, Ki67 is a nuclear protein and, while creating a Ki67 index, only nuclear staining (plus mitotic figures which are stained by MIB1) should be incorporated into the score. However, biological heterogeneity of Ki67 staining can occur across specimens, and the location and extent of the area of the cancer that should be scored is controversial. Two types of heterogeneity are prominent: a gradient of increasing staining toward the tumour edge and hot spots. For the former, three fields should be scored at the periphery of the tumour because the invasive edge is widely considered to be the most biologically active part of the tumour and is most likely to drive outcome of the disease. Hot spots, on the other hand, are defined as areas in which Ki67 staining is particularly prevalent and may occur in an otherwise homogeneously stained sample. The approach to scoring hot spots varies across studies; some investigators have focused in particular on the analysis of hot spots, others have included hot spots in a general assessment of Ki67 across the section, and yet others have recommended avoiding them altogether.This issue needs clarification and, in the meantime, when hot spots are present, an approach that assesses the whole section and records the overall average score is recommended. 70 3b. Breast cancer immunohistochemical subtypes and their main clinical implicationsWithin the complex and heterogeneous scenario of breast cancer, the main issue for the clinician is represented by the ability of offering patients the most suitable and effective treatment, according to their prognosis and their potential response to current available therapeutic options. 15 This "challenge" is particularly evident in the adjuvant setting, whereby the choice of the best systemic treatment to be delivered after breast surgery should be tailored on each single patient's risk of recurrence and death, as well as the probability of positive response to a selected therapy. 15 In this contest, conventional clinical factors, such as age, tumour grade and size, lymph node involvement and lympho-vascular invasion, are no longer sufficient as sole prognostic features and biomarkers detected by immunohistochemistry (i.e., ER, PgR, HER2 and Ki67) have been proven to be highly useful in identifying distinct biological breast cancer subtypes with specific predictive and prognostic potential. 93 Indeed, by assessing ER, PgR, HER2 and Ki67 values in breast tumours, it is possible to subdivide breast cancer patients into five subtypes which play a fundamental role in treatment decision-making (Table 2). 14 These subtypes are represented, on one hand, by luminal A, luminal B/HER2-negative and luminal B/HER2-positive breast tumours, which constitute together the "hormone receptor-positive group", and, on the other hand, by HER2-positive (non-luminal) and triple negative breast cancers, which compose the so-called "hormone receptor-negative group".   