Biomarkers in breast cancer: A consensus statement by the Spanish Society of Medical Oncology and the Spanish Society of Pathology
Biomarkers in breast cancer: A consensus statement by the Spanish Society of Medical Oncology and the Spanish Society of Pathology
R. Colomer 0 1 2 3 4 5 6 7 8 9
I. Aranda-L o´pez 0 1 2 3 4 5 6 7 8 9
J. Albanell 0 1 2 3 4 5 6 7 8 9
T. Garc´ıa-Caballero 0 1 2 3 4 5 6 7 8 9
E. Ciruelos 0 1 2 3 4 5 6 7 8 9
M. A´ . Lo´ pez-Garc´ıa 0 1 2 3 4 5 6 7 8 9
J. Corte´s 0 1 2 3 4 5 6 7 8 9
F. Rojo 0 1 2 3 4 5 6 7 8 9
M. Mart´ın 0 1 2 3 4 5 6 7 8 9
J. Palacios-Calvo 0 1 2 3 4 5 6 7 8 9
0 Pathology Department, General University Hospital of Alicante , Alicante , Spain
1 & J. Palacios-Calvo
2 & R. Colomer
3 Baselga Institute of Oncology (IOB) , Madrid, Barcelona , Spain
4 Vall d'Hebron Institute of Oncology (VHIO) , Barcelona , Spain
5 Medical Oncology Department, Ramo ́n y Cajal University Hospital , Madrid , Spain
6 Pathology Department, Virgen del Rocio University Hospital , CIBERONC, Seville , Spain
7 Medical Oncology Department, Doce de Octubre University Hospital , Madrid , Spain
8 Pathology Department, University Hospital Complex of Santiago , Santiago de Compostela , Spain
9 Medical Oncology Department, Mar University Hospital, Hospital del Mar Medical Research Institute (IMIM), Pompeu Fabra University , CIBERONC, Barcelona , Spain
This consensus statement revises and updates the recommendations for biomarkers use in the diagnosis and treatment of breast cancer, and is a joint initiative of the Spanish Society of Medical Oncology and the Spanish Society of Pathology. This expert group recommends determining in all cases of breast cancer the histologic grade and the alpha-estrogen receptor (ER), progesterone receptor, Ki-67 and HER2 status, in order to assist prognosis and establish therapeutic options, including hormone therapy, chemotherapy and anti-HER2 therapy. One of the four available genetic prognostic platforms (MammaPrint , Oncotype DX , Prosigna or EndoPredict ) may be used in node-negative ER-positive patients to establish a prognostic category and decide with the patient whether adjuvant treatment may be limited to hormonal therapy. Newer technologies including next-generation sequencing, liquid biopsy, tumour-infiltrating lymphocytes or PD-1 determination are at this point investigational.
Breast neoplasm; Diagnostic; Gene expression profiling; Prognostic; Therapy predictive
Departamento de Oncolog´ıa Me´dica, Hospital Universitario
La Princesa, C/Diego de Leo´n, 62, 28006 Madrid, Spain
Biomarker analysis in cancer not only provides
additional information about classical clinical factors, but
also enables patients with a more favourable benefit–risk
balance to receive certain treatments [
]. In breast
cancer, biomarker analysis is routine practice. It originally
began with testing for hormone receptor expression to
guide tamoxifen therapy. The subsequent inclusion of
targeted treatments against human epidermal growth
10 Pathology Department, Fundacio´n Jime´nez D´ıaz University
Hospital, Madrid, Spain
Medical Oncology Department, Gregorio Maran˜ o´n
University Hospital, CIBERONC, GEICAM, Madrid, Spain
12 Pathology Department, Ramo´n y Cajal University Hospital,
CIBERONC, IRYCIS and University of Alcala´, Madrid,
factor receptor 2 (HER2) revolutionised the biomarker
field. It also demonstrated that biomarker test methods
needed to be standardised and harmonised. Recognising
that need, scientific societies in several countries have
written and published consensus guidelines. Among
these were the first guidelines on recommendations for
HER2 testing in breast cancer put forward by the
Spanish Society of Pathology (SEAP) and the Spanish
Society of Medical Oncology (SEOM) in 2009 [
Since then, the main change in the management of
infiltrating breast carcinoma in terms of biomarker
testing has been the inclusion of genetic platforms. These
were initially designed to assist prognosis and to predict
chemotherapy response in patients with tumours that
express hormone receptors, and no lymph-node
metastases. The intervening years have also seen progress in
the understanding of molecular abnormalities in breast
cancer from studies using next-generation sequencing
techniques. The clinical potential for monitoring disease
using new technologies grouped under the term ‘‘liquid
biopsy’’ is currently being studied. Also, as with other
cancers, there is growing interest in knowing what
impact immunotherapy and related biomarker testing
will have on the future management of breast cancer
The purpose of these SEOM–SEAP consensus
guidelines is to recommend which biomarkers should routinely be
tested in patients with breast cancer, including
conventional markers, genetic platforms and newer technologies.
Testing conventional and non-conventional markers
Histological grade is a parameter that has independent
prognostic value at all stages of breast cancer that adds to
axillary status and tumour size. All invasive breast
carcinomas should therefore be graded [
]. The combined
histological grade simply and efficiently provides
biological information about the tumour, directly related to
proliferation (mitosis), abnormal architecture, nuclear shift,
and the expression of chromosomal instability . The
World Health Organization (WHO) classification and the
College of American Pathologists (CAP) guidelines
recommend using the Nottingham (Elston–Ellis) modification
of the Patey–Scarff and Bloom–Richardson grading system
]. The inter-observer agreement level is very high
when these recommendations are strictly followed. Also,
they can be applied to tissue obtained by core-needle
biopsy (CNB) .
Estrogen receptor and progesterone receptor
Expression of estrogen receptor (ER)-alpha is a favourable
prognostic factor and strongly predictive of a response to
hormone therapy [
]. Approximately 30–40% of patients
with ER-expressing advanced breast cancer will have an
objective response to hormone treatment, and a further
20% of patients will achieve disease stabilisation.
Moreover, the hormone therapy response in patients with early
ER-expressing breast cancer, in terms of overall and
disease-free survival, is well known [
therapy is relatively non-toxic. Its long-lasting clinical activity
justifies its use in any patient with an ER-expressing
The technique used to test for ER can be applied
inexpensively to fixed, paraffin-embedded tissue. It is therefore
readily available in most Pathology Departments.
Examining tissue under the microscope means that positive reactions
can be assessed in tumour cells only, avoiding problems with
low cell density or normal breast tissue included in the
tumour growth. Detailed guidelines addressing methods for
the immunohistochemical analysis of ERs and progesterone
receptors (PRs) are available [
In general, 70–75% of invasive breast carcinomas
express ER-alpha. A positive reaction is seen in the
nucleus. Staining intensity and the percentage of positive
cells can vary. The morphological context should be taken
into account. In apparently negative cases of certain special
histological types, such as tubular, mucinous or lobular
carcinoma, or in histological grade I, confirmation of the
results should be considered. The cut-off point for defining
a positive result is C 1% of nuclei positive, irrespective of
staining intensity. The reported results should include the
antibody clone used. It is advisable to include the
percentage of positive cells. Alternatively, a score can be
reported, like the one described by Allred et al., combining
the estimated nuclear positivity rate in cancer cells (a score
of 0–5, based on the percentage) with staining intensity
(intensity 0–3) [
]. It is also useful to test for ER-alpha in
ductal carcinoma in situ, because hormone suppression
treatment can reduce the recurrence risk by 50% in patients
expressing this receptor.
PRs are regulated by ER-alpha, so expression of PRs
suggests that the oestrogen/ER-alpha pathway is
functional. As with ER-alpha, biochemical methods to test for
PR expression were replaced in the 1990s by
immunohistochemistry, which is the recommended technique [
PRs are expressed in 60–70% of cases of invasive ductal
carcinoma of the breast. In general, correlation between
ER-alpha and PR expression is good, although 10% of
cases may prove to be ER-alpha-positive and PR-negative.
These patients have a higher risk of recurrence than
ERalpha-positive, PR-positive cases. Fewer than 5% of
patients may prove to be PR-positive, ER-alpha-negative.
Their prognosis is similar to that of ER-alpha-positive,
PRpositive patients. The methodology and quantification used
are the same as for ER-alpha, with positive cases usually
defined as 1% or more. Some recent studies suggest that
low-level PR expression (\ 20%) might have negative
prognostic implications. Including it as one of the
parameters for distinguishing the Luminal subtype has therefore
been suggested [
Immunohistochemical assessment of Ki-67 is the method
most widely used in clinical practice to determine the
proliferative activity of breast cancer. Ki-67 is particularly
important for distinguishing risk groups in carcinomas
positive for ER-alpha and PR. The available guidelines on
Ki-67 assessment in breast cancer address methodological
issues in the various phases [
]. Calibrating the method in
different laboratories substantially increases the
concordance between results [
]. There is no absolute agreement
regarding cut-off points. It has been recommended that
each pathology department should set its most appropriate
cut-off points [
]. Some guidelines define ‘‘low
proliferative activity’’ as Ki-67 levels below 10%, and ‘‘high
proliferative activity’’ as levels above 30%. However, the
critical point is usually between 10 and 20% [
In combination with PR expression levels, the St Gallen
consensus established four categories based on Ki-67
levels: \ 14, 14–19, 20–25 and [ 25%. A 20% cut-off was
recommended for distinguishing between Luminal A-like
and Luminal B-like tumour types [
]. A recent
metaanalysis concluded that a Ki-67 level of over 25% is
associated with a worse prognosis [
Ki-67 quantification appears to have clinical
applicability in the choice of adjuvant therapy for ER-expressing
tumours. In combination with other clinical factors, its
validity is comparable to that of more complex gene
expression analyses [
]. However, American Society of
Clinical Oncology (ASCO) guidelines on using biomarkers
to guide decisions on adjuvant therapy do not recommend
its use [
]. More international studies of a collaborative
nature are needed, to standardise values of this marker so
that it can be clinically validated [
Along with hormone receptors, HER2 is the most
important prognostic and predictive marker in breast cancer.
Since the early studies by Slamon in 1987, it has been
known that breast cancers that overexpress HER2 represent
a highly aggressive biological subtype [
]. However, the
1998 approval of trastuzumab for therapeutic use changed
the outcome in these patients, whose clinical course
improved very significantly. The introduction of new
targeted anti-HER2 therapies, such as lapatinib, pertuzumab
and trastuzumab emtansine (T-DM1), the last one
administered with no requirement for simultaneous cytostatics,
underlines the importance of identifying patients with
HER2-positive breast cancer.
Any invasive breast carcinoma should be tested for
HER2 overexpression, along with ERs, PRs and Ki-67.
A CNB sample is sufficient, and in most cases the test does
not need to be repeated on material from the surgical
specimen (Fig. 1). Fixation time is much more
standardised for CNBs (normally 6–24 h) than for surgical
specimens, and concordance between the two tests is very high
]. Using CNB material also means that
the information is available for clinicians before making a
decision about possible neoadjuvant therapies. This test is
performed by immunohistochemistry and/or in situ
hybridisation (ISH), fluorescence in situ hybridisation
(FISH) or chromogenic in situ hybridisation (CISH or
Various guidelines conclude that any HER2 test method
is valid, provided the technology is standardised according
to the manufacturer’s instructions, and supported by an
external quality-control programme. This tends to be
routine practice in pathology laboratories across Spain [
In order to ensure high-quality testing, it is very important
for the number of technicians who perform the test, and
especially the number of pathologists who interpret the
results, to be as low as possible . Immunohistochemistry
is the most widely used technique for HER2 expression
status analysis. Not only is it available in all pathology
laboratories, but also it allows the sample to be assessed
cheaply, simply and quickly. In addition, it provides an
overview of the sample, permitting easy identification of
possible small positive foci in heterogeneous cases.
Results should be interpreted according to the
recommendations in the ASCO/CAP guidelines [
]. The main
change introduced by the current guidelines was the
additional inclusion of incomplete membrane staining in the
definition of equivocal cases (2 ?). This change has been
criticised for entailing an unnecessary increase in ISH tests
]. The rationale for introducing it was mainly based on
micropapillary carcinomas, which often show
moderate-tointense lateral or basolateral staining and can display
]. The new version of the ASCO/CAP
guidelines currently being prepared is likely to revert to the
previous definition of 2 ?, which required complete
membrane staining [
]. At the moment, heterogeneous
cases that are mostly negative but have a small focus
(B 10% of cells) of complete, intense, positive staining are
also classified as 2 ?. ISH is recommended for cases that
are equivocal (2 ?) or indeterminate (cannot be assessed
for technical reasons), and also in all cases of dubious
interpretation (1 ? versus 2 ? or 3 ? versus 2 ?).
Whether tests should be repeated because of histological
discordance is also debatable. This recommendation was
introduced in the 2013 guidelines. In particular, it seems
unnecessary to retest grade 3, HER2-negative cases
], or to repeat tests on the surgical specimen when
CNB results were negative . In the case of available
metastatic, however, the test may be repeated due to the
possibility of cells becoming positive, because it is very
rare for them to become negative. In biopsies of bone
metastases, the process of decalcifying the material
generally prevents reliable HER2 testing, and can lead to false
negatives. Even so, if a mass is available, the test can safely
be done. The arrival of a new generation of weaker
decalcifiers, better at preserving both antigens and nucleic
acids, might solve this problem in the future.
The technique of ISH complements
immunohistochemistry. It has several advantages: it is little affected by fixation;
results are read objectively by counting signals; and normal
cells and tumour cells provide a positive internal control.
However, it is slower to read than immunohistochemistry
because of the counting procedure required, and small foci of
amplification can more easily be missed. In the current
ASCO/CAP guidelines, the threshold for the HER2/CEN17
ratio was simplified back to the original 2.0, in addition to
taking account of the number of HER2 signals per cell [
Thus, even with a negative ratio, if the number of HER2
signals per cell is 6 or more, the result is positive; and if it is
between 4 and 6, the result is equivocal and the test must be
repeated. This can either be done on the same sample, using
an alternative chromosome 17 probe such as RARA or TP53,
or the test can be repeated on material from the surgical
specimen. If the result is still equivocal after repetition, the
oncologist may consider prescribing an anti-HER2 therapy,
which is normally done to minimise risks [
]. It is important
for future guidelines to avoid these ambiguities so that
pathologists’ reports are conclusive for therapy. Analysis of
the response obtained in large series of patients with
polysomy, following treatment of equivocal cases, might provide
valuable data on this issue. In fact, in the draft of the new
version of the ASCO/CAP guidelines, if the specimen test
result is equivocal both by IHC and FISH, it is recommended
that the sample be considered HER2 negative.
Heterogeneous amplification, although uncommon in
breast carcinomas compared with gastric tumours, often raises
doubts about quantification. If a cohesive amplified clonal
focus is seen, only that clone should be counted, with a
generally positive result. If, in contrast, amplified cells appear
mingled with unamplified cells (‘‘salt and pepper’’) they
should all be counted and reported in terms of the means
obtained, and the percentage of amplified cells should also be
Prognostic genetic platforms: molecular phenotypes and translation to the clinic
In the last few years, clinical practice in Spain has witnessed
the arrival of four genetic platforms for determining the
prognosis of patients with ER-positive, HER2-negative
tumours of favourable prognosis, without lymph nodes
involved. All these platforms are used to evaluate the risk of
recurrence. However, they differ substantially in the
methodology used to quantify gene expression, the genes
tested, the clinical and pathological variables included, risk
group stratification, and whether or not testing takes place in
centralised laboratories. It should therefore come as no
surprise that, although they are all of proven clinical usefulness
and analytically validated, results from the various platforms
can place the same patient into different risk categories.
Cost-effectiveness analyses have suggested that the use of
genetic platforms is cost effective in that it reduces
chemotherapy use, and prevents the occurrence of events
during the clinical course [
], although the current use
of generic chemotherapy drugs poses some doubts about the
actual economic impact. The various European and
American clinical guidelines, and several expert groups, make a
range of recommendations for using genetic platforms in
different clinical contexts in hormone-dependent breast
cancer, either as a prognostic tool or to establish the benefit of
supplementing hormone therapy with chemotherapy
] (Tables 1, 2). A very comprehensive scientific
review, that includes economic implications of the
platforms, has been very recently been published .
The MammaPrint 70-gene expression platform yields a
signature that divides breast carcinomas into two risk
categories, i.e. high and low [
The platform has been validated in several studies, and
provides prognostic information for distant disease-free
survival independently of the usual clinical and
pathological criteria [
In 2007, the platform was approved by the Food and
Drug Administration (FDA) for determining prognosis in
patients aged 60 years or under with node-negative, stage
I–II tumours measuring B 5 cm. In 2009, it obtained a
second approval for patients over 60 years old. More
recently, MammaPrint has been validated for
paraffinembedded material [
Various studies have indicated its prognostic value for
determining 10-year distant metastasis-free survival in
patients with breast cancer involving 1–3 axillary lymph
nodes, in women at low risk, and for HER2-positive
]. It has also been shown that
MammaPrint is useful for establishing the benefit of
administering chemotherapy [
The MINDACT trial (Microarray in Node 0–3 positive
Disease may Avoid Chemotherapy, EORTC10041/BIG03/
04, NCT00433589) was a multicentre, prospective,
randomised, Phase III study involving over 6000 patients. It
demonstrated that, in 1550 cases of high clinical risk but
low genomic risk, 5-year metastasis-free survival was 94%,
suggesting that approximately 46% of high-risk cases
might not need chemotherapy (level of evidence IA)
The 2017 update of the ASCO Clinical Practice
Guideline of Biomarkers use for the adjuvant therapy of
breast cancer, focused on the use of MammaPrint ,
specified that MammaPrint may be used in patients with
HR?, HER2-negative cases with 1–3 positive nodes AND
a high clinical risk to inform decisions on withholding
adjuvant chemotherapy. The ASCO guideline warns that
these patients should be informed that a benefit of
chemotherapy cannot be excluded, particularly in patients
with C 1 nodes involved. On the other hand,
MammaPrint does not have a use in the low-risk category nor
in patients with HER2? or triple-negative breast cancer,
according to the guideline [
Oncotype DX tests the expression of 21 genes (16
cancer-related genes and 5 reference genes) and calculates a
Recurrence Score (RS) [
Oncotype DX methodology has been optimised for
application to formalin-fixed tissue, and its results have a
proven impact on treatment decisions [
The RS defines three groups: low RS with a value under
18; intermediate RS from 18 to 30; and high RS with
values of 31 or over. Several studies have shown that the
10-year distant recurrence rate is 7% in the low RS group,
14% in the intermediate RS group, and 30% in high RS
St Gallen Prognostic value and predictive of the benefit of adjuvant chemotherapy
ASCO American Society of Clinical Oncology, CT chemotherapy, ESMO European Society for Medical Oncology, NA not available, NCCN
National Comprehensive Cancer Network, RS Recurrence Score, SEOM Spanish Society of Medical Oncology
The value of Oncotype DX for predicting the benefit
provided by chemotherapy and hormone therapy in these
risk groups has been examined in various studies,
involving both node-negative and node-positive patients
], although the 2016 ASCO Guideline recommends
the use of Oncotype to guide decisions about adjuvant
chemotherapy only in cases without lymph node
5 years 10 years
Oncotype DX has been shown to provide information
above and beyond the clinical and pathological features in
postmenopausal patients with hormone-dependent breast
cancer treated with an aromatase inhibitor.
TAILORx (Trial Assigning Individualized Options for
Treatment [Rx]) was a prospective trial designed to
determine the prognosis of a group of patients who had undergone
surgery for ER-positive, HER2-negative, node-negative
breast cancer, with an RS of 11–25 [
]. Recently published
results from the RS \ 11 group reported a distant recurrence
risk of 0.7%, and a 1.3% risk of any other recurrence. These
results were confirmed in the Surveillance, Epidemiology
and End Results (SEER) database registry [
Lastly, the RxPONDER study (Rx for Positive NoDe,
Endocrine Responsive Breast Cancer) will prospectively
report the benefit of chemotherapy in women with low RS
and involvement of 1–3 axillary lymph nodes.
The Prosigna test is a genomic classifier based on a
50-gene signature (PAM50). It was initially designed using
RT-qPCR on paraffin-embedded tissue [
]. This test
can be carried out in decentralised laboratories .
Prosigna provides information on the intrinsic tumour
subtype (Luminal A, Luminal B, HER2-enriched or
basallike). It also determines the 10-year risk of distant
recurrence, as a Risk of Recurrence (ROR) score on a scale of
0–100. Scores are categorised as low (ROR score \ 40,
less than 10% risk), intermediate (ROR score 40–60,
10–20% risk), or high (ROR score [ 60, over 20% risk of
The clinical validity of Prosigna has been tested in
several studies. These studies have demonstrated that the
ROR score provides prognostic information above and
beyond the standard clinical and pathological variables
], with level IB evidence . Moreover, the ROR
score is significantly correlated with distant metastasis-free
survival, and adds medium- and long-term prognostic
information (more than 10 years). It has also been
confirmed that Prosigna provides prognostic information
about recurrence after 10 years of hormone therapy.
The impact of Prosigna on therapeutic
decision-making has also been demonstrated [
]. Prosigna has
obtained the CE mark in Europe, FDA accreditation, and
approval by Health Canada for predicting 10-year distant
recurrence in postmenopausal women with 1–3 axillary
lymph nodes involved.
EndoPredict is another second-generation genomic
classifier, based on testing 12 genes by RT-PCR on
paraffinembedded tissue: 8 cancer genes, 3 reference genes for
standardisation, and one for measuring genomic DNA
]. It is a decentralised test that can be carried out in
any laboratory. The clinical validity of EndoPredict for
predicting distant recurrence independently of the classical
clinical and pathological parameters was confirmed in two
clinical trials evaluating adjuvant hormone treatment
(ABCSG-6 and ABCSG-8). It was also validated in a study
by the GEICAM group, in node-positive women treated
with adjuvant hormone therapy and chemotherapy. It
therefore possesses type IB evidence for prognosis [
EndoPredict provides information on the distant
recurrence risk according to gene expression (genomic EP
score [EP]), and the risk adjusted for tumour size and
number of lymph nodes involved (clinical EP score
[EPclin]). On a scale of 0–15, it defines two categories based
on the 10-year distant recurrence risk: low risk (score
\ 3.4; overall risk of 6–8%) and high risk (score [ 3.4;
overall risk of 15–22%).
EndoPredict has been awarded European certification
for clinical use (CE mark for IVD).
Different next-generation sequencing (NGS) studies
] have demonstrated that the most frequently
mutated genes in breast cancer are PIK3CA (31–41%),
TP53 (30–36%), KTMC2 (7–11%), GATA3 (10–11%),
MAP3K1 (7–10%), and CDH1 (10–11%). Whereas
MAP3K1/TP53, GATA3/TP53, CDH1/TP53, and CDH1/
GATA3 mutations were mutually exclusive, concomitant
mutations of MAP3K1/PIK3CA, CDH1/PIK3CA were
Regarding intrinsic breast cancer subtypes, mutations in
PIK3CA were observed in 43–57% of Luminal A and in
31–35% of Luminal B carcinomas, respectively. The most
important difference between both types of tumours was
the frequency of TP53 mutations, which was 11–12% in
Luminal A, but 24–29% in Luminal B breast carcinomas.
TP53 and PIK3CA mutations have been detected in
approximately 70 and 40% of HER2-enriched breast
carcinomas and in 89 and 16% of basal breast carcinomas,
Regarding NGS studies in specific histological subtypes,
Ciriello et al. profiled 127 invasive lobular carcinomas
(ILC) and compared the distribution of mutations with a
subset of infiltrative ductal carcinomas (IDC), particularly
with Luminal A, given that 83% of ILC are classified as
Luminal A by PAM50 [
]. The most frequent mutations
were detected in CDH1 (65%), PIK3CA (48%), RUNX1
(10%), TBX3 (9%), PTEN (8%), TP53 (8%), FOXA1 (7%),
MAP3K1 (6%), and GATA3 (5%). In addition to CDH1
loss, the molecular hallmark of ILC, ILC and IDC differed
in the FOXA1, GATA3 and TBX3, PTEN loss and AKT
activation. The lower incidence of GATA3 and the higher
incidence of FOXA1 mutations in ILC, and their roles as
regulators of ER activity, suggest that GATA3 and FOXA1
regulate the ER receptor by alternative mechanisms. ILC
has the highest levels of AKT activation comparable to
basal IDC, making selective inhibition of this pathway a
hypothetical therapeutic strategy in these tumours. Finally,
14% of ILC showed PTEN inactivation, compared to 3% of
IDC, either by homozygous deletions or mutations, and
were mutually exclusive with PIK3CA. Similar results have
been subsequently reported by Desmedt et al.
At present, NGS in breast cancer remains a research
tool. A recent consensus group has suggested that for
selecting breast cancer patients for clinical trials
investigating new drugs, an optimal gene panel should detect
AKT1, PIK3CA, PTEN, ESR1 mutations and FGFR1
amplification, in addition to the study of ER, PR, HER2
and BRCA1/2 [
Liquid biopsy and circulating tumour cells
Liquid biopsies, defined broadly as either circulating
tumour cells (CTCs) of epithelial origin, tumour nucleic
acids (ctDNA, cfmiRNA), or tumour exosomes in the
blood of cancer patients, have received increasing
attention as a new diagnostic tool. To date, diagnosis and
metastasis monitoring is mainly carried out through tissue
biopsy and/or re-biopsy, an invasive procedure limited
only to certain locations and not always feasible in
clinical practice. In order to improve tumour characterisation
and disease monitoring over time, liquid biopsy may
represent a new tool. Technologies for detecting and
isolating CTCs include the FDA-validated CellSearch
system, but other technologies are gaining prominence
CTCs have been proved to be a significant prognostic
factor in both early and metastatic breast cancer [
fact, CTC positivity constitutes an individual risk factor for
breast cancer relapse/death not inferior to the usual
prognostic factors (size, grade, proliferation or node status) that
are currently taken into account for adjuvant treatment
]. However, no definitive evidence supports its
clinical utility at the moment. As opposed to CTCs
enumeration, molecular characterisation of the CTCs might
potentially be helpful as a predictive biomarker for therapy
Emerging data support a potential role of ctDNA in
breast cancer. In a study performed in patients with early
breast cancer treated with neoadjuvant chemotherapy, the
detection of ctDNA post-surgery or during follow-up was
highly predictive of relapse, resistance to therapy, and
prediction of response. Another potential use of ctDNA is
to detect ESR1 mutations, which predict resistance to
aromatase inhibitors (but not fulvestrant) in advanced
ERpositive breast cancer or PI3K mutations, which may
predict the benefit of some PI3K inhibitors; these are under
The use of NGS in liquid biopsy may further improve
our ability to predict relapse, monitor patients, predict drug
activity, or provide early detection of resistance.
In the last few years, morphological evaluation of
tumourinfiltrating lymphocytes (TILs) in breast cancer has been
proposed as a potentially useful biomarker given the
prognostic value observed in triple-negative breast cancer
], and HER2 subtypes [
]. It has been
reported that every 10% increment of stromal lymphocytes
was associated with an 18% reduction of risk of death
However, the majority of panelists of the 2017 St Gallen
Consensus Conference did not recommend using TILs as a
new prognostic factor in TNBC patients, in view of the
absence of standardised guidelines for their evaluation,
data on methodological reproducibility, or clinical
]. The International TILs Working Group
experienced in TILs evaluations recently issued
recommendations for harmonising and improving
consistency in scoring TILs, including detailed guidelines for
annotating the prevalence of lymphocyte infiltration, which
may minimise inter-observer reproducibility [
(Table 3). In order to evaluate the feasibility and utility of
these recommendations in clinical practice, Prunery et al.
carried out a retrospective analysis of a series of 897
patients with TNBC [
]. Multivariable analysis
confirmed, in agreement with previous studies, that each 10%
increase in TILs strongly predicted better survival
independent of patients’ age, lymph node status, histological
grade, peritumoural vascular invasion, and Ki-67 labelling
index. Stratified analysis revealed a positive correlation
between TILs and overall survival across all the subgroups
The current recommendation is that the level of TILs
should not be used to withhold chemotherapy or
trastuzumab therapy in TN and HER2-positive breast cancers,
respectively, as the analytical validity and clinical utility of
TILs remains to be firmly established. Whether TILs will
be predictive of response to immunotherapeutic regimens,
in particular T-cell checkpoint inhibition, has yet to be
1. One section (4–5 lm, magnification 2009–4009) per patient is considered to be sufficient. Full sections are preferred over biopsies (in
pretherapeutic neoadjuvant setting, cores can be used); currently, no validated methodology has been developed to score TILs after
2. TILs should be reported for the stromal compartment (% stromal TILs). The denominator used to determine the % stromal TILs is the area
of stromal tissue
3. TILs should be evaluated exclusively within the borders of the invasive tumour, excluding TILs around ductal carcinoma in situ or normal
lobules and zones with artefacts, necrosis, hyalinisation as well as the previous biopsy site
4. All mononuclear cells (including lymphocytes and plasma cells) should be scored, but polymorphonuclear leukocytes are excluded
5. A full assessment of average TILs in the tumour area should be used
6. It should be scored as a continuous variable that will allow categorise different thresholds and more accurate statistical analyses
TILs tumour-infiltrating lymphocytes
Table 4 Summary of biomarkers consensus in breast cancer
ER estrogen receptor, PR progesterone receptor, HER2 human
epidermal growth factor receptor 2, NGS next-generation sequencing,
CTCs circulating tumour cells
Programmed cell death protein (PD-1) is an immune
checkpoint regulator constitutively expressed on the
surface of T cells. Its major ligand, PD-L1, is expressed on the
surface of TILs, antigen-presenting cells, and cancer cells
including breast cancer. When PD-L1 binds to PD-1, a
strong inhibitory signal is transmitted to T cells, which
reduces cytokine production and suppresses T-cell
proliferation. PD-L1 expression in breast cancer has been
associated with poor clinical and pathological features and has
been reported as preferentially expressed by basal and
HER2 breast cancer [
]. Therefore, it might play a
role as a prognostic biomarker in the future.
The presence of tumoural PD-L1-positive TILs
correlates with adverse clinic-pathological features and basal
and HER2 breast cancer, but interestingly also with clinical
response to PD-1 pathway blockade with anti-PD1 or anti
PD-L1 targeted immunotherapy [
]. Given the high
costs and toxicity especially when combined with therapy,
predictive biomarkers are needed. A number of ongoing
trials are trying to elucidate this question.
In order to plan an adequate adjuvant therapy in patients
with primary breast cancer (Table 4), pathology reports
must include in all cases the expression and levels of
ERalpha, PR, HER2 and Ki-67, in addition to histological
grade, to assist prognosis and to establish current
therapeutic options available, including hormone therapy,
chemotherapy and anti-HER2 therapy.
In node-negative ER-positive breast cancer patients, one
of several available genetic prognostic platforms
(MammaPrint , Oncotype DX , Prosigna or EndoPredict )
may be used in order to establish a prognostic category and
to discuss with the patient whether adjuvant treatment may
be limited to hormonal therapy.
Newer technologies including NGS, liquid biopsy,
tumour-infiltrating lymphocytes or PD-1 determination are
still experimental at this point.
Acknowledgements The authors are grateful for the editorial
assistance of Dr. Fernando Sa´nchez-Barbero of HealthCo (Madrid, Spain)
in the production of this manuscript. SEOM and SEAP are grateful for
financial support for this project provided by unrestricted grants from
AstraZeneca, Ferrer Diagnostic, Novartis, Palex, Pfizer, Roche Farma
and NanoString Technologies.
Compliance with ethical standards
Conflict of interest The authors declare that, when writing and
revising the text, they did not know the names of the pharmaceutical
companies that provided financial support for this project, so this
support has not influenced the content of this article.
Ethical statement The study has been performed in accordance with
the ethical standards of the Declaration of Helsinki and its later
amendments. This article does not contain any studies with human
participants or animals performed by any of the authors.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
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