Breast cancer prognostic classification in the molecular era: the role of histological grade

Breast cancer is a heterogeneous disease with varied morphological appearances, molecular features, behavior, and response to therapy. Current routine clinical management of breast cancer relies on the availability of robust clinical and pathological prognostic and predictive factors to support clinical and patient decision making in which potentially suitable treatment options are increasingly available. One of the best-established prognostic factors in breast cancer is histological grade, which represents the morphological assessment of tumor biological characteristics and has been shown to be able to generate important information related to the clinical behavior of breast cancers. Genome-wide microarray-based expression profiling studies have unraveled several characteristics of breast cancer biology and have provided further evidence that the biological features captured by histological grade are important in determining tumor behavior. Also, expression profiling studies have generated clinically useful data that have significantly improved our understanding of the biology of breast cancer, and these studies are undergoing evaluation as improved prognostic and predictive tools in clinical practice. Clinical acceptance of these molecular assays will require them to be more than expensive surrogates of established traditional factors such as histological grade. It is essential that they provide additional prognostic or predictive information above and beyond that offered by current parameters. Here, we present an analysis of the validity of histological grade as a prognostic factor and a consensus view on the significance of histological grade and its role in breast cancer classification and staging systems in this era of emerging clinical use of molecular classifiers.

signi fi cant and at least partly time-dependent prognostic factors, such as tumor size and LN status.
Although the current well-established clinical and histological factors and some well-defi ned biological factors (that is, hormone receptors and HER2 expression) show strong association with prognosis and outcome, there are increasing concerns that these variables are limited in their ability to capture the diversity of clinical behaviors of breast cancer and that they would not be suffi cient to tailor the therapy to individual patients. In addition, the perceived subjective nature of histo pathological assessment of the morphological features such as tumor grade has increased these concerns. Th e introduction of high-throughput technologies that survey thousands of genes and their products in a single assay, coupled with powerful analytical tools, has opened up new avenues for classifying breast cancer into biologically and clinically distinct groups based on gene expression patterns [18,19] and DNA copy number alterations [20]. However, these expression profi ling studies have suggested that molecular tests could perform better than the traditional histopathology and may replace it as the 'gold standard' for prognostication and prediction of response to therapy [21]. Recent studies leading to the development of the 21-gene recurrence score (trade name Oncotype DX; Genomic Health, Inc., Redwood City, CA, USA [22,23]) have highlighted the issue of subjec tivity associated with histological grading and called into question the utility of histological grade as a prognostic tool. Regrettably, these results have been perceived as direct evidence that molecular tests provide an objective and reproducible assessment of prognostic features of estrogen receptor (ER)-positive breast cancers but that histopathological analyses are subjec tive and not reproducible.
Molecular methods undoubtedly provide prognostic and predictive information and may help identify new therapeutic targets, and the interest in molecular classifi ers and their potential application is perfectly understandable. However, it is important to understand their limitations and critically evaluate their role in improving breast cancer prognostication above and beyond the traditional variables in a practical and cost-eff ective way [24,25]. Th e role of NGS as a simple and cost-eff ective method of assessment of tumor biology should not be neglected. It is also important to recognize that in countries with limited health resources, access to expensive new technologies may not be possible but that eff ective cost-effi cient methods, such as routine histopatho logical evaluation, are available for all [26]. In fact, there are numerous lines of evidence to suggest that these molecular tests complement rather than replace the traditional pathological variables, such as NGS, to defi ne the optimal therapy for patients with breast cancer.
Here, we present an overview of the current evidence of the signifi cance of breast cancer grading in view of the availability of an increasing number of potentially alterna tive molecular prognostic tests. We present in a pragmatic way a comparison between NGS and recent molecular prognostic tests, taking into account evidencebased clinical and biological signifi cance, cost-eff ectiveness, practicality of application in diff erent parts of the world, and the impact of this on future plans for improvement in breast cancer prognostication and management.

What is histological grade?
Invasive carcinomas are morphologically subdivided accord ing to their growth patterns and degree of diff erentiation, the latter of which refl ects how closely they resemble normal breast epithelial cells. Th is subdivision is achieved by assessing histological type and histological grade, respectively. Although tumor type provides useful prognostic information, the majority (60% to 75%) of breast cancers have no special type of characteristics (that is, invasive ductal carcinoma of no special type, or NST); those special types that show distinct prognostic significance are relatively uncommon. As a consequence, the role of histological typing in clinical management decision making is currently limited [27].
Histological tumor grade is based on the degree of diff erentiation of the tumor tissue. In breast cancer, it refers to the semi-quantitative evaluation of morphological characteristics and is a relatively simple and lowcost method, requiring only adequately prepared hematoxylin-eosin-stained tumor tissue sections to be assessed by an appropriately trained pathologist using a standard protocol. NGS is based on the evaluation of three morphological features: (a) degree of tubule or gland formation, (b) nuclear pleomorphism, and (c) mitotic count ( Figure 1). For details, see [1,2] and Supplementary Information [28].

Histological grade and prognosis
Multiple independent studies have shown that NGS has prognostic value that is equivalent to that of LN status [29] and greater than that of tumor size [4,15]. In a large study, Henson and colleagues [14], who assessed survival rates of 22,616 cases of breast cancer, demonstrated that patients with histological grade 1, stage II disease had the same survival as those with grade 3, stage I disease. Th e authors also found that patients with grade 1 tumors of less than 2 cm in size had an excellent prognosis, with 99% 5-year survival, even when they presented with positive LN. Th ese results are supported by a recent study from the Nottingham group [11], which included 2,219 operable breast cancer cases with long-term follow-up. Th is study has demonstrated that grade is an important determinant of breast cancer outcome and complemen tary to LN stage through the ability to infl uence the outcome of patients in diff erent LN stage categories. Th ese results provide evidence that histological grade, when used in conjunction with LN stage, can improve the prediction of outcome for individual patients and support its inclusion and use in multifactorial indices such as the NPI and Adjuvant! Online. Similar long-term validation has been demonstrated in screen-detected breast cancer in the Swedish Two-County Trial, which demonstrated that tumor grade, LN status, and tumor size at the time of diagnosis have a lasting infl uence on subsequent survival [10].
Th ere is compelling evidence to suggest that histo logical grade can accurately predict tumor behavior, particularly in earlier small tumors (tumor, node, meta stasis [TNM] stage pT1), more than other 'time-dependent' prognostic factors such as tumor size (pT1a, pT1b, and pT1c) [4,9,11,15]. Studies have also demonstrated that grade is an independent prognostic factor in specifi c subgroups of breast cancer, including ER-positive breast cancer patients who have not [30] or who have received neoadjuvant endocrine therapy [31] and patients with LN-negative [5,11,13,32] or -positive [7,11] breast cancer regardless of ER expression. Recently, Desmedt and colleagues [33] demonstrated that in the ER-positive/ HER2-negative tumors (n = 628), only histological grade and the proliferation module retained their association with relapse-free survival (RFS) in the multivariate analysis (hazard ratio [HR] = 2.00, 95% confi dence interval [CI] 1.18 to 3.37; P = 0.01). In the Nottingham series [11,34], histological grade was an independent predictor of RFS in the ER-positive/HER2-negative tumors (n = 1,077) (HR = 2.13, 95% CI 1.79 to 2.53; P <0.0001). Similar associations between grade and survival were found in (a) the LN-negative subgroup (n = 797), who received only adjuvant hormone therapy (HR = 1.85, 95% CI 1.46 to 2.34; P <0.0001, with rates of 10-year risk of relapse of 7% for grade 1, 14% for grade 2, and 31% for grade 3), and in (b) ER-positive tumors with small-volume LN metastasis (pN1; one to three LNs positive) (n = 316) (HR = 2.07, 95% CI 1.51 to 2.86; P <0.0001, with rates of 10-year risk of relapse of 5% for grade 1, 24% for grade 2, and 43% for grade 3) [11,34]. Th erefore, histological grade can provide important prognostic information for clinically relevant subgroups in which the benefi t of chemotherapy is less certain (for example, LN-negative/ER-positive or in patients with low-volume LN metastatic disease).
We have noted, consistently with the biological and clinical roles of histological grade on breast cancer behavior, an important association between histological grade and pattern of survival. Akin to high-grade lymphoma, high-grade breast cancers tend to recur and metastasize early following diagnosis, typically within the fi rst 8 years; thereafter, breast cancer-related deaths decrease in frequency. Low-grade tumors tend to show a very good outcome, and few (if any) events occur; those that do occur, do so relatively late in the lifetime of the patients. Grade 2 tumors show an intermediate outcome during the early years of follow-up; however, on longterm follow-up, they show an obvious trend for continued recurrence and impaired outcome in the long term [11,35] (Figure 2). In contrast, LN stage, which can provide information on the likelihood of death or survival after breast cancer, shows limited value in predicting the timescale of these events (Rakha EA, Ellis IO, unpublished data). Th is important observation provides further insight into the appropriate management strategies of patients with breast cancer. High-grade tumors, with their risk of early recurrence and death, require consideration for prompt use of adjuvant chemotherapy, whereas patients with grade 1 tumors, which are almost invariably ER-positive, could be off ered a long-term follow-up with or without a potentially less toxic systemic therapy (that is, endocrine therapy).

Histological grade: contentious issues
Despite the utility of histological grade as a prognostic factor for ER-positive disease, there are numerous issues that ought to be considered for the correct use of histological grade in the management of patients with breast cancer [36][37][38][39]. Th ese issues are detailed in the following sections.

Grade and size
Th e latest (7th edition) AJCC TNM staging system endorsed NGS, but grade was not included in calculating stage [40]. Th e decision to exclude grade as an element in the TNM staging system, as stated previously [36], is based mainly on the possible interaction between tumor size and histological grade and, in particular, the lack of clear evidence for the role of grade in small tumors (pT1 and pT2). It should be noted that two of the basic principles of breast cancer screening are that outcome of patients with small invasive cancers is good and adverse events are rare. Th e eff ect of all known prognostic factors will therefore be limited in such a patient group. However, despite this constraint, there are several lines of evidence that demonstrate the prognostic signifi cance of NGS in small tumors. Studies that examine the prognostic signifi cance of grade in small tumors quoted in the AJCC article by Singletary and colleagues [36] show marked variations in outcome, follow-up times, and number of patients. Th ere was also variation in the grading method used, and information on histological grade was obtained from systematic pathology review, whereas in others, information about grade was abstracted from pathology reports, medical records, or tumor registry databases. Th ese diff erences in grading systems and study design are expected to lead to diff erent results regarding the prognostic signifi cance not only of grade but also of other variables should they have been assessed. To conclude, although extracting consistent data on the prognostic signifi cance of grade from the diff erent studies cited in the AJCC review [36] is challenging, studies in which modern methods for histological grading were employed have shown that its utility is retained in small tumors [1,5,6,9,11,27,32,[41][42][43]. With the shift that mammographic screening causes in stage distribution, this issue has become increasingly impor tant, with a high proportion of tumors being T1N0M0 at diagnosis, thereby limiting the relevance of TNM staging in routine practice.
In the Nottingham series, development of recurrent disease following diagnosis of grade 1 breast cancer was infrequent, and when observed, the recurrent lesions were either higher-grade tumors or second primaries. Th e number of patients with grade 1 tumors who developed distant metastasis or died without developing a second event of higher-grade tumor was limited (4%) [44]. Th is observation implies that grade 1 breast cancer studies that do not include pathology review and evaluation of the second event are likely to overestimate the risk of adverse outcome.
high-grade and nearly always carry a recommendation for adjuvant chemotherapy, irrespectively of tumor grade. Although a higher proportion of larger tumors are grade 3 [8,11], some forms of lower-grade breast cancer such as hormone receptor-positive, low-grade invasive lobular cancers frequently present as large mammographi cally occult tumors and are responsive to hormone therapy. Furthermore, if tumor size/stage alone largely dictates an oncologist's choice of treatment, an argument could be made, at least in many centers, for the irrelevance of other biological variables, including gene expression signatures such as Oncotype DX, 70-gene signature, 76-gene signature, and genomic grade index (GGI). Likewise, although NGS might have limited prognostic value in HER2-positive and triple-negative cancers as most of these tumors are of high grade (grade 3) [30,33], these tumors also typically exhibit poor-prognosis gene signatures [45]. In addition, molecular classifi ers such as Oncotype DX and GGI [33] and the MammaPrint (Agendia, Amsterdam, Th e Netherlands), the last of which is recommended to all patients, have negligible discriminatory power in ER-negative disease [30,33].
Th erefore, we believe that treatment decisions based on TNM staging system, which measures the anatomic extent of the tumor, can be improved by the addition of histological grade, which measures the intrinsic biological features of the tumor and refl ects the potential of a carcinoma to metastasize or cause death. Integration of histological grade into the relevant TNM staging system has been accepted for other common epithelial tumors such as adenocarcinoma of the prostate. As the prognostic value of NGS has been proven in operable breast cancer (stages I and II), in which decisions about systemic therapy usage and its regimen need to be made, histological grade could be incorporated in the TNM system to improve its ability to stratify cases into risk-associated subcategories corresponding to grade so that chemotherapy can be potentially be avoided in low-risk groups and considered a high priority for patients in a high-risk category. Th e maximum benefi t of grade assessment would be in the subgroup of patients with ER-positive, LN stage N0 or N1 disease. Th e current evidence indicates that grading has limited value in advanced or metastatic breast cancer (stages III and VI) and grading is not expected to change treatment decisions and therefore need not be considered in these cases.

Grade and tumor type
Th e prognostic value of histological grade has been documented in most tumor types, including invasive lobular carcinomas [46]. Medullary carcinoma might appear to be one subtype in which grading is less signifi cant. By defi nition, these tumors are of high histological grade (grade 3) but may have a more favorable prognosis than their histological grade would imply [38]. However, a recent study shows that medullary carcinomas account for less than 1% of breast cancers as a result of the strict criteria required for its recognition and that they do not have a prognosis signifi cantly diff erent than that of other forms of grade 3 ductal carcinoma with prominent infl am mation [38]. Importantly, a recent study suggested that the 70-gene prognostic signature may also fail to provide prognostic stratifi cation of patients with some special types of breast cancer. Given that NGS has been shown to provide prognostically relevant information for invasive ductal carcinomas of NST and lobular carcinomas, which together account for greater than 80% of all breast cancers, the systematic inclusion of histological type in breast cancer routine synoptic reports is also advo cated.

Grading of needle core biopsy specimens
Current evidence suggests that histological grading can be assessed relatively reliably whereas other well-established prognostic factors, such as vascular invasion and tumor size, cannot [47,48]. However, some cases may be upgraded when the excision specimen is analyzed following grading of core biopsies (that is, grade I in the core biopsy and grade II in the excision specimen; 30% to 40%). On the other hand, a diagnosis of NGS grade III in a core biopsy is not commonly changed when the excision specimen is graded (5% to 8%). Importantly, changes from grade I in the core to grade III in the excision specimen and vice versa are very rare (0% to 1%) [47,48].
Selection of patients for neoadjuvant therapy requires that prognostic information be available from nonoperative diagnostic tumor samples. Amat and colleagues [49] reported that assessment of grade on needle core biopsy (NCB) is a strong predictive factor of response to induction chemotherapy in breast cancer, independently of the type of regimen used. Th erefore, despite the limitations associated with the accuracy of grading core biopsies related to tumor sampling issues and visibility of mitotic fi gures [47,48], assessment of histological grade on NCB can provide information to support preoperative treatment decision making.

Reproducibility of histological grade
One of the reasons cited in the past for the reluctance to use grading in patient management decisions has been the perceived lack of reproducibility of the method. Th is may be highlighted by the relatively wide variation in the proportion of each grade in published series. However, a substantial number of studies have reported better levels of inter-and intra-observer concordance [1,27,[50][51][52][53][54][55][56][57][58][59] ( Table 1). Th e variation in the proportion of each grade reported in the diff erent studies can be explained by the variation in the grading system used and the diff erence in the patient cohorts, including age distribution, symptomatic versus screening popula tion, early versus advanced breast cancer groups, and details of tissue fi xation. Grading is dependent on a high quality of tissue preservation. Suboptimal levels of tissue fi xation lead to disruption and loss of visibility of mitotic fi gures, one of the three variables assessed in NGS. Assessment of grade in poorly fi xed tissue will therefore introduce a bias leading to a reduction in the proportion of cases classifi ed as grade 3 [1,2,60].
Another important point to improve the inter-observer agreement rates is the introduction of guidelines for standardization of pre-analytical parameters, including tissue handling, fi xation, and preparation, and of the methods for tumor grading. Diff erences between centers can be attributed in many cases to diff erences in the quality of tissue preparation [2,61]. Critical evaluation of these issues and recommendations for good practice have been provided by professional organizations (that is, WHO, EU, UK RCPath, and the International Union Against Cancer [UICC]) [2]. Th e use of rigorously optimized and standardized methods in Nottingham has provided a high NGS reproducibility between grading of a recent series [11] and that of an old series published more than two decades ago from the same institution with a similar percentage of cases in each grade ( Table 2). Signifi cant improvements in the consistency of histological grading have been observed on a national basis in the UK through the publication of guidelines with linked educational activity and associated external quality assurance (EQA) [51]. Th ese guidelines provide not only information on histological grading methodology but also recommendations on the application of these methods and guidance on tissue handling. Adherence to these guidelines and participation in EQA are also expected to improve assessment of other important prognostic factors in breast cancer, such as lymphovascular invasion and immunohistochemical determination of other biomarkers. In addition, the current use of NGS is expected to provide consistency among diff erent studies in the future as evidenced from multiple studies from the Nottingham group and other institutions that endorse NGS [2,39,60,62]. However, despite the objective improvements that have been made to breast cancer grading methods, any assessment of morphological characteristics inevitably retains a subjective element and is heavily dependent on the pre-analytical parameters.
It should be noted that the degree of scrutiny of the inter-observer reproducibility histological grade has never been applied to molecular tests in current clinical use. A more detailed reproducibility study of the performance of gene expression studies has not been conducted as of yet. In fact, issues of reproducibility are well recognized in all forms of medical laboratory testing. Despite the undeniable need to improve the inter-observer agree ment for histological grade, the criticisms directed against NGS should be tempered by the fact that other parameters used to determine the therapy of patients with breast cancer also suff er from inter-observer variability, including the assessment of small-volume nodal metastases (LN stage), HER2 immunohisto chemical and in situ hybridization scoring, ER scoring, assessment of vascular invasion, and even the assessment of tumor size.

Signifi cance of grade 2 tumors
Mis-assignments of grade I to grade III or vice versa are rarely reported, but grade II tumors usually show the lowest degree of concordance. Th is is an expected phenomenon of scoring of a biological variable where scores in the overlap regions are usually most diffi cult to be categorised. Th e similar example of the problem of repro ducibility of classifi cation of a continuous biological variable was noted in the microarray-based gene expression profi ling studies [63,64]. For example, in the studies by Sorlie and colleagues [18] and Chang and colleagues [65], only a propor tion of cases could be accurately classifi ed into the molecular subtypes, 9% to 15% of tumors could not be assigned as grade 1 and grade 3 by GGI [66], and 19% to 24% of cases showed discordance among diff erent gene expression signatures applied to the same set of tumors [45].
In the latest meeting of the St. Gallen International Expert Consensus on the Primary Th erapy of Early Breast Cancer (2009) [17], it was recommended that grade 1 and grade 3 be taken into consideration for the assessment of indications of adjuvant chemotherapy. Grade 2 was regarded as being similar to other parameters of intermediate-risk signifi cance, such as tumor size of between 2 and 5 cm, low numbers (one to three) of involved LNs, and intermediate scores on multigene assays, and it was inferred that they do not provide a defi nitive indication of risk with respect to the clinical decision of whether to give or withhold chemo therapy. However, it was also noted that the presence of these intermediate-risk criteria usually tips the balance toward the use of chemotherapy [17].
Th e advantage of applications of molecular tests as complements to grade is particularly evident with respect to grade 2 tumors. Several attempts have been made to improve biological and clinical signifi cance of histological grading by classifying grade 2 tumors into two distinct subclasses: a grade 1-like subgroup, which has an excellent outcome and may not require adjuvant chemotherapy, and a grade 3-like subgroup, which comprises tumors that behave in a way similar to high-grade cancers and need a more aggressive systemic treatment. Examples of these studies include the application of GGI to subclassify histological grade 2 into two molecular subclasses (GGI1 and GGI3) [66] or the use of proliferation biomarkers such as MIB1 (Ki67) expression (Rakha EA, Ellis IO, unpublished data). However, the clinical useful ness and the cost-benefi t ratios of these studies need to be further evaluated if they are to be translated into routine practice worldwide, particularly in countries with limited resources.

Grade and molecular profi ling
Recent profi ling studies of breast cancer have emphasized the relevance of tumor biology in governing breast cancer behavior and hence the importance of histological grade. Tumors of diff erent histological grades show distinct molecular profi les at the genomic, transcriptomic, and immuno histochemical levels. Th ese results suggest that the majority of high-grade tumors are unlikely to stem from the progression of low-grade cancers and that grade 1 and 3 breast tumors are probably diff erent diseases [67].
Gene expression studies have demonstrated that histological grade better refl ects the molecular makeup of breast cancer than LN status and tumor size do [68,69]. Sotiriou and colleagues [66] developed a 97-gene classifi er that can accurately identify cases diagnosed as NGS I or NGS III. Th eir studies have shown an asso ciation between a 'gene signature' developed to recapitulate histological grade (GGI) of breast cancer and patient outcome, indepen dently of LN status or tumor size [66]. Th is assay is currently being commercialized in Europe (MapQuant Dx; Ipsogen, Marseille, France). When the prognostic performance of GGI was compared with the Oncotype DX [22] and 70-and 76-gene signatures, a similar separation in distant metastasis-free survival between low-and high-risk groups by the three signatures was found [45]. Another group has similarly demonstrated that the genetic grade signature (RNAbased) remained signifi cantly associated with disease recurrence in most cases. However, recent meta-analyses of microarray-based expression profi ling studies have demonstrated that the prognostic impact of the signatures investigated stems from the proliferation-related genes [30,33]. In fact, when several of the published signatures were divided into partial signatures composed of proliferation-related genes and genes not related to proliferation, the latter failed to show prognostic signifi cance, whereas the prog nostic power of some signatures even improved by the removal of genes not related to proliferation [30]. Most importantly, in numerous studies using molecular signatures, histological grade remained an independent prognostic factor for ER-positive tumors even after the inclusion of gene signatures in the multivariate models [22,25].
Th ere are several lines of evidence to suggest that the objective contribution of gene signatures above and beyond the current clinicopathological parameters is limited. Dunkler and colleagues [70] demonstrated that the explained variation of prognosis (that is, the proportion of patients whose prognosis is determined solely by a given parameter) by prognostic signatures is limited (for example, 3% for the 70-gene signature) when grade and other clinicopathological variables such as LN stage, patient age, and ER status are included in the survival models [30,70,71]. It is important to mention, however, that there are relatively few head-to-head compari sons of NGS versus molecular signatures and that most of them so far have a competitive tone to them. Studies that combine molecular assays and NGS in a balanced manner would be warranted.

Grade in the era of molecular profi ling tests
Prognostic molecular tests for patients with breast cancer, including Oncotype DX [22] and MammaPrint [72], have already been approved for clinical use. Undoubtedly, these assays support breast cancer prognos tication and can be used as a complement to the wellestablished variables currently used in routine practice [73], as recently recommended in the St. Gallen guidelines [17].
Th e cost of MammaPrint and Oncotype DX [74] is orders of magnitude higher than that of histological grading. Oncotype DX has undergone health economic evaluation in the US and has been reported to be costbenefi cial through reduction of widespread use and appropriate targeting of use of adjuvant chemotherapy. In countries or centers where chemotherapy is less widely prescribed or where targeting is based on other tests, there may be a reduced benefi t and justifi cation of the test cost [26]. Th ere is a trend in the research community not to consider cost-eff ectiveness when promoting the use of a newly developed molecular test, even though costs typically are taken into consideration when evaluating new interventions [26,74]. Th e costs of these modern assays are likely to remain high, and it should be borne in mind that there are still many parts of the world that do not and will not have ready access to these costly tests. Th erefore, histological grading, when carried out properly on well-fi xed specimens, provides a simple, inexpensive, and highly accurate alternative method for assessing tumor biological characteristics and patient prognosis and identifying patients at high and low risk for adverse outcomes. In addition, the cost and availability are not the only factors limiting the routine applicability of currently approved or recommended molecular prognostic assays as there may also be some skepticism of the scientifi c rigor of industry-sponsored cost-benefi t economic models.
Given that grade has been shown by multiple independent groups to be prognostic and that the levels of interobserver agreement have increased with the adoption of NGS, it is rather surprising that clinical practice has changed with molecular tests that have not been as comprehensively tested but has not changed with NGS. Possibly, this stems from the purported objectivity of molecular tests and the denounced subjectivity of histopathological analysis [25]. However, molecular tests also suff er from subjectivity in terms of the biostatistical approaches employed, the stability of the molecular subgroups identi fi ed by the tests, and the reproducibility of assays performed with cell extracts without careful micro dissection of tumor cells (that is, contamination with normal breast epithelial cells or proliferating stromal cells may change the results of molecular tests based on the assessment of ER-and proliferation-related genes) [25]. Th erefore, it should be recognized that both molecular assays and NGS have their own strengths and weak nesses, which vary in diff erent situations. Both can provide valuable prognostic information and both should complement rather than compete with each other and this should be understood when they are used for patient management decision making. When used in combi nation, molecular tests such as GGI are potentially important in the subclassifi cation of grade 2 breast tumors. However, the application of molecular tests to known grade 1 and grade 3 breast cancers in the treatment decision-making process may need further validation and confi rmation of any additional prognostic value and cost benefi t.

Conclusions
Th ere is an international consensus that NGS should be considered the 'gold standard' for breast cancer grading. Th e adoption of the objective criteria of NGS has been shown to overcome many of the previous problems of reproducibility of grading, problems that resulted from using a variety of approaches. To provide a consistent and uniform way of assessing histological grade and to improve its reproducibility, consensus criteria and guidelines have been published with critical evaluation of these issues and recommendations for good practice [2]. Strict adherence to these criteria is expected to improve consistency and reproducibility of breast cancer grading among diff erent institutions. Histological grading, when adequately carried out, provides a simple, inexpensive, and highly accurate method for assessing tumor biological characteristics and patient prognosis. Th is is of particular importance for breast cancer patients in parts of the world where access to new molecular technology is not currently available. Molecular assays and NGS should complement rather than compete with each other. We conclude that the assessment of histological grade is an important determinant of breast cancer prognostication and should be incorporated in staging systems and in algorithms to defi ne therapy for patients with breast cancer.

Take-home messages
Th e Nottingham Grading System, when adequately carried out, provides a simple, inexpensive, accurate, and validated method for assessing patient prognosis.
Consensus criteria for histological grading and recommendations for good practice have been published [2,51] and should be followed.
Th e Nottingham Grading System is a validated alternative to molecular tests in parts of the world where access to new molecular technology is not currently available or likely to become available in the near future.
Assessment of histological grade is an important determinant of breast cancer prognostication and should be incorporated in algorithms to defi ne therapy for patients with breast cancer.

Search strategy and selection criteria
Literature databases, including PubMed, Medline, and the Cochrane Library, were searched for articles published from 1980 to 2009 in English. Th e keywords used for the search were 'breast cancer' , 'grade' , 'histologic(al) grade' , 'molecular profi le' , and 'reproducibility' in relation to biology, prognosis, prediction, and patient outcome. Articles published before 1980 or in another language were also considered if they were commonly referenced or were highly regarded older publications. Th e search also included the references list for these articles and selected additional articles and webpages that were judged to be relevant. Data from publications submitted as abstracts were excluded.