Breast cancer is the most common cancer in women and
a leading cause of cancer death worldwide. Management
of breast cancer relies on the availability of robust clinical
and pathological prognostic and predictive factors to
guide patient decision making and the selection of
treatment options. In early-stage breast cancer, where the
use of systemic therapy has to be determined for every
patient, the three main prognostic determinants used in
routine practice are lymph node (LN) status, tumor size,
and histological grade. Th e Nottingham (Elston-Ellis)
modi fi cation of the Scarff -Bloom-Richardson grading
system, also known as the Nottingham Grading System
(NGS) , is the grading system recommended by
various professional bodies internationally (World Health
Organi zation [WHO], American Joint Committee on
Cancer [AJCC], European Union [EU], and the Royal
College of Pathologists (UK RCPath) [2,3]).
Th e prognostic relevance of NGS in breast cancer was
initially demonstrated in 1991  and has been validated
subsequently in multiple independent studies [4-14].
Since NGS has independent but equally powerful prog-
nostic value, it has been combined with LN stage and
tumor size to form prognostic indices: the Nottingham
Prognostic Index (NPI) , which includes NGS and LN
stage with equal weighting, and the Kalmar Prognostic
Index , in which grade is given a higher-weighted
value. Owing to the prognostic information provided by
NGS, it has also been incorporated in algorithms (for
example, Adjuvant! Online ) and guidelines (for
example, the St. Gallen guidelines ) to determine the
use of adjuvant chemotherapy. NGS provides a simple,
inexpensive, and routinely applicable overview of the
intrinsic biological characteristics and clinical behavior
of tumors, adding important informa tion to other
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 profi ling 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 profi ling studies have generated clinically useful data that
have signifi cantly 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 off ered
by current parameters. Here, we present an analysis of the validity of histological grade as a prognostic factor and
a consensus view on the signifi cance of histological grade and its role in breast cancer classifi cation and staging
systems in this era of emerging clinical use of molecular classifi ers.
© 2010 BioMed Central Ltd
Breast cancer prognostic classifi cation in
the molecular era: the role of histological grade
Emad A Rakha1, Jorge S Reis-Filho2, Frederick Baehner3, David J Dabbs4, Thomas Decker5, Vincenzo Eusebi6,
Stephen B Fox7, Shu Ichihara8, Jocelyne Jacquemier9, Sunil R Lakhani10, José Palacios11, Andrea L Richardson12,
Stuart J Schnitt13, Fernando C Schmitt14, Puay-Hoon Tan15, Gary M Tse16, Sunil Badve17 and Ian O Ellis*1
1Department of Histopathology, Nottingham University Hospitals NHS Trust,
Nottingham City Hospital Campus, Hucknall Road, Nottingham, NG5 1PB, UK
Full list of author information is available at the end of the article
Rakha et al. Breast Cancer Research 2010, 12:207
© 2010 BioMed Central Ltd
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 patho-
logical assessment of the morphological features such as
tumor grade has increased these concerns. Th e intro-
duction 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 .
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 . 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 sub-
jec tivity associated with histological grading and called
into question the utility of histological grade as a prog-
nostic 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
Molecular methods undoubtedly provide prognostic
and predictive information and may help identify new
therapeutic targets, and the interest in molecular classi-
fi ers and their potential application is perfectly under-
standable. 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 expen-
sive new technologies may not be possible but that
eff ective cost-effi cient methods, such as routine histo-
patho logical evaluation, are available for all . 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 alter-
na tive molecular prognostic tests. We present in a prag-
matic way a comparison between NGS and recent
molecular prognostic tests, taking into account evidence-
based clinical and biological signifi cance, cost-eff ective-
ness, practicality of application in diff erent parts of the
world, and the impact of this on future plans for improve-
ment 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 signifi -
cance are relatively uncommon. As a consequence, the role
of histological typing in clinical management decision
making is currently limited .
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 morpho-
logical characteristics and is a relatively simple and low-
cost 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) mito-
tic count (Figure 1). For details, see [1,2] and Supple-
mentary Information .
Histological grade and prognosis
Multiple independent studies have shown that NGS has
prognostic value that is equivalent to that of LN status 
and greater than that of tumor size [4,15]. In a large study,
Henson and colleagues , 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 , 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
Rakha et al. Breast Cancer Research 2010, 12:207
Page 2 of 12
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
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  or who have received
neoadjuvant endocrine therapy  and patients with
LN-negative [5,11,13,32] or -positive [7,11] breast cancer
regardless of ER expression. Recently, Desmedt and
colleagues  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 long-
term 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 strate-
gies 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
Figure 1. Histological grade of breast cancer as assessed by the Nottingham Grading System. (a) A well-diff erentiated tumor (grade 1) that
demonstrates high homology to the normal breast terminal duct lobular unit, tubule formation (>75%), a mild degree of nuclear pleomorphism,
and low mitotic count. (b) A moderately diff erentiated tumor (grade 2). (c) A poorly diff erentiated (grade 3) tumor with a marked degree of cellular
pleomorphism and frequent mitoses and no tubule formation (<10%).
Rakha et al. Breast Cancer Research 2010, 12:207
Page 3 of 12
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 [36-39]. Th ese issues are detailed in the
Grade and size
Th e latest (7th edition) AJCC TNM staging system
endorsed NGS, but grade was not included in calculating
stage . Th e decision to exclude grade as an element in
the TNM staging system, as stated previously , 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 prog-
nostic signifi cance of grade in small tumors quoted in the
AJCC article by Singletary and colleagues  show
marked variations in outcome, follow-up times, and
number of patients. Th ere was also variation in the grad-
ing 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  is challenging, studies in
which modern methods for histological grading were
employed have shown that its utility is retained in small
tumors [1,5,6,9,11,27,32,41-43]. 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
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%)
. 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.
Further justifi cation for the exclusion of grade from
TNM  is that large tumors (pT3 and pT4) tend to be
Figure 2. Relationship between histological grade and breast
cancer-specifi c survival. (a) In the old Nottingham series (1977 to
1989), no systemic therapy was off ered to the patients. Of the 1,816
patients, 404 (17.7%) had grade 1 tumors (gray curve), 621 (36.2%)
had grade 2 (blue curve), and 791 (46.1%) had grade 3 (black curve)
(χ2 = 97.5, P <0.0001). (b) In the recent Nottingham series (1990 to
2002), systemic therapy was off ered to the patients according to
Nottingham Prognostic Index and estrogen receptor expression
as described previously . Of the 3,579 patients, 677 (18.9%) had
grade 1 tumors (gray curve), 1,383 (38.6%) had grade 2 (blue curve),
and 1,519 (42.4%) had grade 3 (black curve) (χ2 = 195.5, P <0.0001).
Analysis of grade 1 and 2 only showed statistical survival diff erence
(χ2 = 20.7, P <0.0001). Both series are consecutive and included
estrogen receptor-positive and -negative and lymph node-negative
and -positive cases [11,75,76].
Rakha et al. Breast Cancer Research 2010, 12:207
Page 4 of 12
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 mammo-
graphi 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 prog-
nostic 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-prog-
nosis gene signatures . In addition, molecular
classifi ers such as Oncotype DX and GGI  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
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 bio-
logical 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 prog-
nostic 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, histo-
logical grade could be incorporated in the TNM system
to improve its ability to stratify cases into risk-associated
subcategories corresponding to grade so that chemo-
therapy 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 docu-
mented in most tumor types, including invasive lobular
carcinomas . 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 . 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 . 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 carci-
nomas, 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
Grading of needle core biopsy specimens
Current evidence suggests that histological grading can
be assessed relatively reliably whereas other well-estab-
lished prognostic factors, such as vascular invasion and
tumor size, cannot [47,48]. However, some cases may be
upgraded when the excision specimen is analyzed follow-
ing 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 non-
operative diagnostic tumor samples. Amat and colleagues
 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 [1,27,50-59]
(Table 1). Th e variation in the proportion of each grade
reported in the diff erent studies can be explained by the
Rakha et al. Breast Cancer Research 2010, 12:207
Page 5 of 12
variation in the grading system used and the diff erence in
the patient cohorts, including
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]) . Th e use of rigorously opti-
mized and standardized methods in Nottingham has
provided a high NGS reproducibility between grading of
a recent series  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 histo-
logical 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) . 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 lympho-
vascular invasion and immunohistochemical determina-
tion 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
Table 1. Inter-observer and intra-observer agreement of breast cancer histological grade.
Study Number of cases Number of readers Grade Inter-observer
 613 2 NGS Kappa 0.69
 52 2 NGS Kappa 0.54
 425 2 NGS Complete agreement 76%
 75 6 NGS Kappa 0.43 to 0.74
 12 600 NGS Kappa 0.45 to 0.53 (fi gures after application of guidelines)
 3 NGS Complete agreement 72.3%; kappa 0.57
 24 21 NGS Complete agreement 69%; kappa 0.53
 50 5 NGS Mean polychoric correlation 0.8
 35 13 NGS Kappa 0.5 to 0.7
 93 7 NGS Kappa 0.54
 40 3 NGS Kappa 0.68 to 0.83
 874 2 WHO criteria Complete agreement 78.1%; kappa 0.66
 50 5 NGS Complete agreement 83.3%; kappa 0.73
NGS, Nottingham Grading System; WHO, World Health Organization.
Rakha et al. Breast Cancer Research 2010, 12:207
Page 6 of 12
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 expres-
sion profi ling studies [63,64]. For example, in the studies
by Sorlie and colleagues  and Chang and colleagues
, 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 , and 19% to 24% of cases showed discordance
among diff erent gene expression signatures applied to the
same set of tumors .
In the latest meeting of the St. Gallen International
Expert Consensus on the Primary Th erapy of Early Breast
Cancer (2009) , 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 .
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 chemo-
therapy, 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)  or the use of prolifera-
tion 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 .
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  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 cia-
tion between a ‘gene signature’ developed to recapitulate
Table 2. Proportion of grades among diff erent studies.
Study Number of cases Grade 1 Grade 2 Grade 3
Elston, 1984  625 17% 37% 46%
Davis et al., 1986  1,537 22% 49% 29%
Hopton et al., 1989  874 29% 46% 25%
Le Doussal et al., 1989  1,262 11% 45% 46%
Balslev et al., 1994  9,149 32% 49% 19%
Saimura et al., 1999  741 19 37% 44%
Reed et al., 2000  613 25% 41% 35%
Simpson et al., 2000  368 22% 45% 33%
Lundin et al., 2001  1,554 26% 47% 27%
Frkovic-Grazio and Bracko, 2002  270 38% 38% 24%
Warwick et al., 2004  1,988 23% 37% 40%
Williams et al., 2006  1,058 20% 46% 34%
Rakha et al., 2008  2,219 18% 36% 46%
Thomas et al., 2009  1,650 26% 45% 29%
Blamey et al., 2009  16,944 29% 41% 30%
Rakha et al. Breast Cancer Research 2010, 12:207
Page 7 of 12
histological grade (GGI) of breast cancer and patient
outcome, indepen dently of LN status or tumor size .
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  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 . Another group has similarly
demonstrated that the genetic grade signature (RNA-
based) 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 . 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  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  and MammaPrint
, have already been approved for clinical use.
Undoubtedly, these assays support breast cancer prog-
nos tication and can be used as a complement to the well-
established variables currently used in routine practice
, as recently recommended in the St. Gallen guide-
Th e cost of MammaPrint and Oncotype DX  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 cost-
benefi 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 . 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 availa-
bility 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 indepen-
dent groups to be prognostic and that the levels of inter-
observer 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 . 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)
. 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 na-
tion, molecular tests such as GGI are potentially impor-
tant 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
Rakha et al. Breast Cancer Research 2010, 12:207
Page 8 of 12
decision-making process may need further validation and
confi rmation of any additional prognostic value and cost
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 .
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 bio-
logical 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
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 recom-
mendations 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’, ‘histo-
logic(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.
AJCC, American Joint Committee on Cancer; CI, confi dence interval; EQA,
external quality assurance; ER, estrogen receptor; EU, European Union; GGI,
genomic grade index; HR, hazard ratio; LN, lymph node; NCB, needle core
biopsy; NGS, Nottingham Grading System; NPI, Nottingham Prognostic Index;
NST, no special type; RFS, relapse-free survival; TNM, tumor, node, metastasis;
UK RCPath, Royal College of Pathologists; WHO, World Health Organization.
The authors declare that they have no competing interests.
EAR and JSR-F performed the literature review and helped write the fi rst
draft of the manuscript. SB and IOE helped write the fi rst draft. All authors
contributed to the writing and approval of the fi nal and revised drafts of the
On 16 October 2008, the Susan G Komen for the Cure sponsored a meeting
of pathologists to review and make recommendations regarding the utility of
histological grade in breast cancer staging and prognostication. This report
was conceived in that meeting. JSR-F is funded in part by Breakthrough Breast
Cancer. The funding sources had no role in the literature search or in the
drafting and approval of the manuscript.
1Department of Histopathology, Nottingham University Hospitals NHS Trust,
Nottingham City Hospital Campus, Hucknall Road, Nottingham, NG5 1PB,
UK; 2The Breakthrough Breast Cancer Research Centre, Institute of Cancer
Research, Fulham Road, London, SW3 6JB, UK; 3Department of Anatomic
Pathology, University of California, Divisadero Street, Box 1785, San Francisco,
CA 94143, USA; 4University of Pittsburgh Medical Center, Halket Street,
Pittsburgh, PA 15213, USA; 5Breast-Screening-Pathology, Reference Centre
Münster, Gerhard Domagk-Institute of Pathology, University Hospital Münster,
Domagkstrasse 17, D-48149 Münster, Germany; 6Sezione Anatomia Istologia e
Citologia Patologica ‘M. Malpighi’, Università-ASL Ospedale Bellaria, Via Altura 3,
40139 Bologna, Italia; 7Pathology Department, Peter MacCallum Cancer Centre,
St Andrews Place, East Melbourne, Victoria 3002, Australia; 8Department
of Pathology, Nagoya Medical Center 4-1-1 Sannomaru, Naka ku, Nagoya
460 0001, Japan; 9Unité d’Anatomie et de Cytologie Pathologiques, Institut
Paoli-Calmettes, 13273 Marseille, Cedex 9, France; 10Molecular and Cellular
Pathology, The University of Queensland, Mayne Medical School, Brisbane,
QLD 4006, Australia; 11Servicio de Anatomía Patológica, Hospital Universitario
Virgen del Rocío, Avda. Manuel Siurot, s/n, 41013 Sevilla, Spain; 12Department
of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston,
MA 02115, USA; 13Department of Pathology, Beth Israel Deaconess Medical
Center and Harvard Medical School, 330 Brookline Avenue, #Sl435, Boston, MA
02215, USA; 14Institute of Molecular Pathology and Immunology (IPATIMUP)
and Medical Faculty, University of Porto, R. Roberto Frias, S/N, 4200 - Porto,
Portugal; 15Department of Pathology, Singapore General Hospital, Outram
Road, Singapore 169608; 16Department of Anatomical and Cellular Pathology,
Prince of Wales Hospital, Ngan shing Street, Shatin, Hong Kong; 17Departments
of Pathology and Internal Medicine, Clarian Pathology Lab of Indiana
University, 350 West 11th Street, CPL-4050, Indianapolis, IN 46202, USA.
Published: 24 August 2010
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Cite this article as: Rakha EA, et al.: Breast cancer prognostic classifi cation
in the molecular era: the role of histological grade. Breast Cancer Research
Rakha et al. Breast Cancer Research 2010, 12:207
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