Clinical and Translational Oncology

https://doi.org/10.1007/s12094-024-03541-1

REVIEW ARTICLE

Biomarkers in breast cancer 2024: an updated consensus statement

by the Spanish Society of Medical Oncology and the Spanish Society
of Pathology
Ramon Colomer1 · Blanca González‑Farré2 · Ana Isabel Ballesteros3 · Vicente Peg4 · Begoña Bermejo5 ·
Belén Pérez‑Mies6 · Susana de la Cruz7 · Federico Rojo8 · Sonia Pernas9 · José Palacios10

Received: 23 May 2024 / Accepted: 25 May 2024

© The Author(s) 2024

Abstract
This revised consensus statement of the Spanish Society of Medical Oncology (SEOM) and the Spanish Society of Patho-
logical Anatomy (SEAP) updates the recommendations for biomarkers use in the diagnosis and treatment of breast cancer
that we first published in 2018. The expert group recommends determining in early breast cancer the estrogen receptor (ER),
progesterone receptor (PR), Ki-67, and Human Epidermal growth factor Receptor 2 (HER2), as well as BReast CAncer
(BRCA​) genes in high-risk HER2-negative breast cancer, to assist prognosis and help in indicating the therapeutic options,
including hormone therapy, chemotherapy, anti-HER2 therapy, and other targeted therapies. One of the four available genetic
prognostic platforms (Oncotype ­DX®, ­MammaPrint®, ­Prosigna®, or ­EndoPredict®) may be used in ER-positive patients
with early breast cancer to establish a prognostic category and help decide with the patient whether adjuvant treatment may
be limited to hormonal therapy. In second-line advanced breast cancer, in addition, phosphatidylinositol-4,5-bisphosphate
3-kinase catalytic subunit alpha (PIK3CA) and estrogen receptor 1 (ESR1) should be tested in hormone-sensitive cases, BRCA​
gene mutations in HER2-negative cancers, and in triple-negative breast cancer (TNBC), programmed cell death-1 ligand
(PD-L1). Newer biomarkers and technologies, including tumor-infiltrating lymphocytes (TILs), homologous recombination
deficiency (HRD) testing, serine/threonine kinase (AKT) pathway activation, and next-generation sequencing (NGS), are
at this point investigational.

Keywords Breast neoplasm · Diagnostic · Gene expression profiling · Prognostic · Therapy predictive

Introduction years have also seen progress in the understanding of single

molecular abnormalities in breast cancer related to specific
Biomarker analysis in cancer provides information that com- molecular therapies, such as BReast CAncer (BRCA​) gene,
plements classical clinical factors, and also enables certain programmed cell death-1 ligand (PD-L1), phosphatidylino-
treatments in patients to be selected [1]. In breast cancer, sitol 3-kinase (PI3K), or estrogen receptor 1 (ESR1). The
biomarker analysis began with testing for hormone recep- clinical potential for monitoring disease using new technolo-
tor expression to guide tamoxifen therapy. The subsequent gies grouped under the term liquid biopsy is currently being
inclusion of targeted treatments against human epidermal studied.
growth factor receptor 2 (HER2) revolutionized the bio- The purpose of these revised consensus guidelines from
marker field. It also highlighted that biomarker test methods the Spanish Society of Medical Oncology (SEOM) and the
need to be standardized and harmonized. The intervening Spanish Society of Pathological Anatomy (SEAP) is to rec-
ommend which biomarkers should routinely be tested in
Ramon Colomer, Ana Isabel Ballesteros, Begoña Bermejo, Susana patients with breast cancer; including conventional markers,
de la Cruz, and Sonia Pernas are experts from the Spanish Society genetic platforms, and newer technologies, as well as those
of Medical Oncology (SEOM); and Blanca González-Farré, Vicente that remain investigational. Recommendations are presented
Peg, Belén Pérez-Mies, Federico Rojo, and José Palacios are experts
in a stratified fashion, depending on whether the breast can-
from the Spanish Society of Pathological Anatomy (SEAP).
cer is in an early or advanced stage.
Extended author information available on the last page of the article

Vol.:(0123456789)
 Clinical and Translational Oncology

Early‑stage breast cancer Hormone receptors

Histological type and grade Estrogen receptor (ER)-alpha and progesterone receptor
(PR) status must be determined in all newly diagnosed breast
Histological typing should be performed according to carcinomas, as well as in metastatic or recurrent tumors [4].
World Health Organization (WHO) criteria. Among lumi- ERs are expressed in about 70% of invasive breast carci-
nal carcinomas, pure tubular, cribriform and mucinous car- noma cases. ER is a strongly predictive factor of a response
cinomas have better prognosis than invasive carcinomas of to hormone therapy as well as a favorable prognostic factor
no special type (IC-NST). It is controversial whether inva- [5]. In the 2020 ER and PR guidelines from the American
sive lobular carcinoma has a different prognosis than IC- Society of Clinical Oncology and the College of American
NST, but an accurate diagnosis of this tumor type, based Pathologists (ASCO-CAP), the cut-off that indicates patients
on both morphological features and E-cadherin expres- who will benefit from endocrine therapy remains at 1% of
sion pattern, is recommended, especially considering the cancer nuclei stained for ER, irrespective of staining inten-
specific clinicopathological and molecular features of this sity. About 2–3% of breast carcinomas will have 1–10% cell
histological type. Among triple-negative tumors (TNBC), staining for ER. This group represents a clinical challenge,
histological types of good prognosis include adenoid not only for the low reproducibility of the results between
cystic carcinoma, secretory carcinoma, and other salivary laboratories, but also for the real benefit of antiestrogenic
gland-type tumors. In addition, fibromatosis-like and low- therapy for these patients (these tumors seem more related
grade adenosquamous carcinomas are two types of meta- at the molecular level to ER-negative tumors). In this sense,
plastic carcinomas with good prognosis. In contrast, high- the ASCO-CAP guidelines divide the ER-positive result
grade metaplastic carcinomas (spindle cell, squamous, and into positive >10% and low positive 1–10%, deciding the
matrix-producing carcinomas) have a worse prognosis and best treatment on the complete information about an indi-
less response to chemotherapy than other TNBC. vidual case [4]. Currently, immunohistochemistry (IHC) is
Histological grade has independent prognostic value at recommended as primary screening. Only nuclear positiv-
all stages of breast cancer. Therefore, all invasive breast ity in tumoral cells is scored, avoiding normal breast tissue
carcinomas, irrespective of their histological type, should be from being mixed with the tumor. Both staining intensity
graded following a protocolized method [1]. The WHO clas- and the percentage of positive cells are recorded. Alterna-
sification recommends using the Nottingham (Elston-Ellis) tively, a score can be reported, like the one described by
modification of the Patey–Scarff and Bloom–Richardson Allred, et al., combining the estimated nuclear positivity
grading system. Grading is evaluated by a numerical scoring rate in cancer cells (a score of 0–5, based on the percentage)
system of 1–3 per category (tubular formation, nuclear pleo- with staining intensity (intensity 0–3) [6]. It is useful to test
morphism, and mitotic count). Only clear central lumina for ER-alpha in ductal carcinoma in situ because hormone
enclosed by polarized cells should be counted for tubular/ suppression treatment can reduce the recurrence risk by 50%
gland formation. Nuclear pleomorphism is scored in the in ER-positive patients [7].
least differentiated tumoral area. Mitotic count is performed PR is expressed in about 60% of cases of invasive ductal
in the most proliferative area, typically at the periphery of carcinoma of the breast. In general, correlation between
the tumor. This parameter has been reported as the most ER-alpha and PR expression is good, although 10% of
important constituent of grade, so only clear mitosis should cases may prove to be ER-alpha-positive and PR-nega-
be counted. The final score should be adapted to the high- tive. These patients have a higher risk of recurrence than
power field size of the microscope used [2]. When these ER-alpha-positive, PR-positive cases [8, 9]. Fewer than
recommendations are strictly followed the inter-observer 5% of patients may prove to be PR-positive, ER-negative.
agreement level is high, and they can be applied to tissue The methodology and quantification used are the same as
obtained by core-needle biopsy [1]. In the future, artificial for ER-alpha, with cut-off in 1% of stained nuclei. Recent
intelligence algorithms may be a helpful tool for improving studies suggest that PR expression <20% might have
reproducibility or even automatically grading breast carci- adverse prognostic implications [9]. PR determination in
nomas [3]. in situ carcinoma is optional [4].
Two-thirds of breast cancers express androgen receptors
(ARs). Their role in carcinogenesis and as a novel therapeu-
tic target has been explored using antiandrogens either alone
or in combination, with some promising yet limited clinical
results [10, 11]. Current guidelines do not include AR deter-
mination as a routine biomarker for clinical practice [12].
Clinical and Translational Oncology

HER2 assessment There is a high concordance (98–99%) between HER2

results in core biopsies and surgical specimens, and a core
HER2 must be analyzed for overexpression or amplification biopsy sample is often the material of choice for HER2
in all breast carcinomas, whether early or advanced. HER2 determination [15], since the availability of pre-treatment
amplification is a predictive factor for anti-HER2 therapies biomarkers status allows clinicians to treat patients with the
and an unfavorable prognostic factor, when not treated. The most appropriate neoadjuvant therapy (NAT) and provide
use of anti-HER2 antibodies in combination with chemo- important prognostic and biological information. Optimal
therapy or hormonal therapy has dramatically improved the tissue handling requirements are of primary importance and
clinical course of HER2-positive breast cancer. A better fixation time should not exceed 72 h. Importantly, in cases
understanding of tumor biology and HER2 signaling has when pre-analytical conditions could not be guaranteed,
led to the development of new strategies to further improve this should be specified in the pathology report. The current
patient outcomes. Current novel HER2-targeted therapies ASCO-CAP recommendations propose that the HER2 test
include dual-HER2 inhibition with monoclonal antibod- be repeated on the excision specimen if there are concerns
ies, such as trastuzumab plus pertuzumab; antibody–drug about discordance between histopathologic findings [14,
conjugates such as trastuzumab emtansine or trastuzumab- 16]. The indications of possible HER2 test discordance are
deruxtecan (T-DXd); and tyrosine kinase inhibitors such as specified in detail in the guidelines [16]. Of note, it is not
lapatinib, tucatinib or neratinib. The measurement and defi- mandatory to retest grade 3 tumors in the absence of other
nition of HER2 amplification or overexpression have also clinical-pathological criteria.
been optimized over the years [13]. HER2 expression should be interpreted according to
HER2 status is routinely assessed using a combination of the 2023 ASCO-CAP guidelines [17], slightly modified in
IHC to evaluate HER2 protein expression levels and in situ Fig. 1, which includes staining intensity, percentage of cells
hybridization (ISH) to assess HER2 gene status. Several and staining localization [16]. However, some specific sce-
HER2 test methods are valid, provided the technology is narios are taken into consideration. For instance, when mod-
standardized according to the manufacturer’s instructions, erate to intense basolateral or lateral membrane IHC staining
and supported by an external quality-control program [14]. is detected (a pattern frequently observed in micropapillary

Fig. 1  Algorithm for HER2 assessment in infiltrating breast carci- IHC: immunohistochemistry. Results framed in dashed lines should
noma with an initial IHC approach. CEP17: chromosome enumera- be accompanied with a comment
tion probe 17; HER2: human epidermal growth factor receptor 2;
 Clinical and Translational Oncology

carcinomas), or circumferential membrane IHC staining that cancer, it has been observed that relevant HER2 discordance
is intense but within ≤10% of tumor cells (heterogeneous but rates are observed between different metastatic sites and
very limited) is observed, it is preferable to consider those molecular subtypes, therefore highlighting the importance
cases as equivocal (2+) and a new test is desirable. of evaluating potentially therapy-relevant HER2-low dis-
In relation to ISH tests, those laboratories using single- cordance rates between a primary tumor and corresponding
probe ISH assays should be encouraged to include concomi- distant metastases [22]. Regarding the new HER2-low sce-
tant IHC review as part of the interpretation. This concomi- nario, the 2023 ASCO-CAP guidelines do not recommend
tant review should be performed in the same institution to changing reporting terminology for lower levels of HER2
ensure parallel interpretation and quality of the two assays. IHC expression (e.g., HER2-low), while the 2023 ESMO
The current diagnostic approach also includes more rigorous expert consensus statements of HER2-low recommend clas-
interpretation criteria for dual-probe ISH assay and requires sifying all levels of HER2 expression [23]. However, the
concomitant IHC review in certain scenarios to come to the score 0 versus 1+ must be informed in all HER2-negative
most accurate HER2 status designation. Those scenarios cases, to ensure the eligibility criteria for T-DXd therapy.
include an average HER2 copy number <4.0 signals/cell The guidelines also provide best practices for discrimination
with a HER2/CEP17 ratio ≥2.0 or an average HER2 copy of IHC 0 versus 1+.
number ≥6.0 signals/cell or ≥4.0 and <6.0 signals/cell with Neratinib is a kinase inhibitor indicated for the extended
a HER2/CEP17 ratio <2.0. If the concomitant interpretation adjuvant treatment of adult patients with early-stage ER-
of the IHC and ISH techniques in these three scenarios is positive HER2-overexpressed/amplified breast cancer, to
a final negative result, a comment should be added in the follow adjuvant trastuzumab-based therapy, when there is a
final report to clarify the absence of robust evidence sup- high risk of relapse (node-positive disease) [24].
porting the use of anti-HER2 therapies (Fig. 1). Importantly,
if a patient develops a recurrence or metastatic disease, a
new HER2 test should be performed if a tissue sample is Ki‑67
available.
HER2 intratumoral heterogeneity has been described in Ki-67 is a nuclear protein found in all phases of the cell
up to 40% of breast carcinomas, especially in HER2 equivo- cycle except G0. Ki-67 expression is related, although not
cal or HER2 borderline cases, or chromosome 17 polysomy. completely, to the histological grading of breast carcinomas.
The presence of HER2 heterogeneity has been associated Immunohistochemical assessment of Ki-67 is the method
with a worse prognosis and lesser anti-HER2 treatment most widely used to determine the proliferative activity of
response [18]. breast cancer, although the reproducibility of the results
According to current guidelines, classifying breast between laboratories has been disputed [12].
tumors as HER2 IHC-3+ or with HER2 gene amplification Calibrating the method in different laboratories substan-
assessed by ISH is the primary predictor of responsiveness tially increases the concordance between results [25]. An
to HER2-targeted therapies. For some recently developed international Ki-67 working group in breast cancer devel-
anti-HER2 therapies, HER2 expression may be a continuous oped a website (https://​www.​ki67i​nbrea​stcan​cerwg.​org/)
variable in terms of treatment effect. Tumors with IHC-1+ that includes an app for scoring Ki-67 more accurately [26].
or 2+ and with a negative ISH result (until now reported Briefly, to score Ki-67, any cells with any degree/intensity
as HER2-negative) might be re-defined as HER2-low, with of brown nuclear staining are considered positive. The whole
HER2 negativity being limited to IHC-0 (Fig. 1). Based on slide should be evaluated, estimating the percent area with
this definition, up to 55% of breast cancers are HER2-low, negligible, low, medium, or high Ki-67 index. A hundred
comprising a majority of hormone receptor-positive (HR- negative or positive nuclei in each field type should be
positive) tumors (65–83%) with different intrinsic sub- counted and finally a “weighted global score” recorded for
types [13, 19]. Although it has been shown that HER2-low that slide [25].
tumors do not benefit from adjuvant trastuzumab [20], some Cut-off point selection for clinical application remains
HER2-directed antibody–drug conjugates such as T-DXd a controversial matter. Given the lack of standardization,
are effective in HER2-positive disease and also in HER2- both the 2021 St. Gallen consensus [27] and the Ki-67 work-
low tumors with no amplification. In a randomized clinical ing group [28] consider that only very low (<5%) or very
trial performed in 557 HER2-low previously treated meta- high (>30%) values can be reliably categorized as low or
static breast cancers, T-DXd resulted in significantly longer high proliferation by visual scoring of Ki-67 IHC in routine
progression-free survival (PFS) and overall survival (OS) clinical practice. In this way, in ER-positive early breast can-
than the physician’s choice of chemotherapy [21]. Although cer with Ki-67 between 6 and 29%, a multi-parameter gene
primary metastatic breast cancer had a significantly lower expression assay has been recommended to help in guiding
HER2-low discordance rate than secondary metastatic breast adjuvant treatment [27].
Clinical and Translational Oncology

Other potential uses for Ki-67 include prediction of depending on the clinical setting and are not interchange-
responsiveness/resistance to chemotherapy or endocrine able. The Oncotype ­DX® trial was validated with level 1A
therapy, estimation of residual risk in patients on standard evidence for prognosis and predicts the benefit of adjuvant
therapy and dynamic biomarker of treatment efficacy in chemotherapy in node-negative, ER-positive, HER2-nega-
samples taken before, during, and after NAT, particularly tive early breast cancer (post- or pre-menopausal) and in
neoadjuvant endocrine therapy identifying patients who can node-positive post-menopausal cases. ­MammaPrint® has
be spared intensive chemotherapy in the adjuvant setting level 1A prognostic evidence in node-negative, ER-posi-
[29–31]. tive, HER2-negative clinically high-risk breast carcinoma
In advanced breast cancer, Ki-67 has also shown rele- and level 1A evidence for determining prognosis in node-
vance in predicting response to cell cycle inhibitors. PFS in positive disease. Further clinical evidence will clarify the
patients undergoing endocrine therapy plus CDK4/6i was use of multigene testing in the node-positive setting. In
inversely correlated with Ki-67 expression, suggesting that addition, clinicians should be aware of the clinical util-
tumor proliferation has a great impact on cell cycle inhibi- ity and limitations when applying such tests, particularly
tors combined with endocrine therapy [32]. since some authors have suggested that molecular testing to
Ki-67 is a useful prognostic tool that, in combination with deliver personalized chemotherapy risks over- and under-
other clinical factors, has a value comparable to that of more treatment [35].
complex gene expression analyses [33]. The systematic use
of digital imaging analysis will improve its reproducibility Oncotype ­DX®
and value in the coming years [34].
Oncotype ­DX® tests the expression of 21 genes and calcu-
Genetic platforms for prognosis and chemotherapy lates a Recurrence Score (RS). Oncotype ­DX® methodol-
guidance ogy has been optimized for application to formalin-fixed
tissue, and its results have had a proven impact on treatment
Gene expression signatures can be helpful in deciding decisions [42]. The value of Oncotype ­DX® for predicting
whether to use adjuvant chemotherapy in early breast cancer. the benefit provided by chemotherapy and hormone therapy
Several retrospective studies have suggested the clinical util- in these risk groups has been examined in various studies,
ity of genomic signatures, although only Oncotype ­DX® and involving both node-negative and node-positive patients [43,
­MammaPrint® are supported by prospective randomized tri- 44]. The RS defines three prognostic groups. The 10-year
als (Table 1). These signatures provide different information distant recurrence rate in the low RS group is 7%, 14% in the

Table 1  Recommendations for the prognostic and predictive value of different genetic tests in breast cancer
Recommendations Oncotype ­DX® EndoPredict® Prosigna® MammaPrint®

NICE 2018 [36] Prognosis: ER+, HER2−, Prognosis: ER+, HER2−, Prognosis: ER+, HER2−, Not cost-effective
N0/N1 (pre/post-meno- N0/N1 (pre/post-meno- N0/N1 (post-menopausal)
pausal) pausal)
Prediction: ER+, HER2−,
N0
St Gallen 2019 [37] Highly recommended for Generic recommendation for Generic recommendation for Generic recommendation for
T1–T3 N0 stages the use of CT in T1–T3 N0 the use of CT in T1–T3 N0 the use of CT in T1–T3 N0
Generic high recommenda- and TxN1 stages and TxN1 stages and TxN1 stages
tion for the use of CT in
TxN1 stages
ESMO 2019 [38] Evidence: 1A Evidence: 1B Evidence: 1B Evidence: 1A
ER+, HER2−, N0/N1 ER+, HER2−, N0/N1 ER+, HER2−, N0/N1 ER+, HER2−, N0/N1
AJCC 2019 [39] Evidence: 1 Evidence: 2 Evidence: 2 Evidence: 2
ASCO 2019 [40] Evidence: High Evidence: Intermediate Evidence: High Evidence: High
ER/PR+, HER2−, N0 ER/PR+, HER2−, N0 ER/PR+, HER2−, N0 For high-risk patients
NCCN 2021 [41] Prediction:Yes Prediction: No Prediction: No Prediction: No
Prognosis: Yes Prognosis: Yes Prognosis: Yes Prognosis: Yes
Evidence 1 (post-menopau- Evidence: 2A Evidence: 2A Evidence: 1
sal)
Evidence 2A (pre-meno-
pausal)

CT chemotherapy, ER estrogen receptor, HER2 human epidermal growth factor receptor 2, PR progesterone receptor
 Clinical and Translational Oncology

intermediate RS group, and 30% in high RS patients. Results Prosigna® (PAM50/ROR)

of two prospective trials support recommendations for treat-
ment considering RS and menopausal age/state. TAILORx Prosigna® (NanoString Technologies, Inc.) is a second-gen-
(Trial Assigning Individualized Options for Treatment [Rx]) eration multigene signature, which includes 50 genes based
was a prospective trial designed to determine the progno- on NanoString nCounter technology, approved to estimate
sis of a group of patients who had undergone surgery for the risk of distant relapse in early ER-positive breast can-
ER-positive, HER2-negative, node-negative breast cancer cer with up to 3 positive lymph nodes in post-menopausal
with an RS of 11–25 [45]. At a median follow-up of 9 years, women treated with endocrine therapy alone [50]. PAM50
the hazard ratio (HR) for the endocrine group versus the can be performed on FFPE samples locally and provides a
chemoendocrine group was 1.08 (95% confidence interval proliferation-based measure of gene expression that, com-
[CI], 0.94–1.24), and the distant recurrence rate was 5%, bined with node status and tumor size, defines a risk score
regardless of chemotherapy administration, establishing that called risk of recurrence (ROR). ROR is divided into three
at age < 50 and RS 16–25, benefit can be obtained from the risk groups: low (<10%), intermediate (10–20%) and high
use of chemotherapy, whereas at age > 50 and RS < 25, there (>20%) ROR. It correlates with the probability of distant
is no benefit from chemotherapy. Results from the RS < 11 recurrence at 10 years. ­Prosigna® also identifies molecu-
group reported a 0.7% risk of distant recurrence and a 1.3% lar subtypes. It has been validated as a prognostic tool in
risk of any other recurrence. These results were confirmed the ABCG-8 and TransATAC trials, in patients treated with
in the Surveillance Epidemiology and End Results (SEER) endocrine therapy, with ER-positive and node-negative dis-
database registry [46]. It shows the benefit of chemotherapy ease, although prospective data on its predictive value are
in pre-menopausal women with low RS (≤25) and 1–3 axil- needed, which will be generated in the OPTIMA study in
lary lymph node involvement, with a median follow-up of node-positive early breast cancer [51].
5 years (HR 0.81; 95% CI, 0.67–0.98). Results showed a
46% decrease in invasive disease-free survival and a 53% EndoPredict® (EPclin)
decrease in deaths, leading to an absolute improvement in
OS at 5 years of 1.3%. Post-menopausal women with RS EndoPredict® (Myriad Genetics, Inc.) is a 12-gene prog-
0–25 did not benefit from adjuvant chemotherapy in any nostic test that estimates 10-year relapse risk and provides
subgroup [47]. information on potential long-term (beyond 5 years) hor-
monal therapies. The E ­ ndoPredict® score can be combined
MammaPrint® with tumor size and node status to obtain the more compre-
hensive EPclin risk score. This test can be used to guide the
MammaPrint® is a 70-gene signature test of prognostic value therapy decision for chemotherapy and extended endocrine
that classifies breast cancer patients into high-risk and low- therapy [52]. This multigene test was evaluated in the GEI-
risk groups [48]. Initially, the test required fresh tissue, CAM-9906 trial as an independent prognostic parameter in
although now it is optimized on formalin-fixed paraffin- patients with ER-positive, HER2-negative, and node-posi-
embedded (FFPE) samples. M ­ ammaPrint® was validated tive breast cancer for adjuvant chemotherapy and endocrine
for early luminal breast cancer in node-negative patients in therapy. In the ABCSG6 and ABCSG8 trials, E ­ ndoPredict®
the RASTER trial. Its clinical utility has been demonstrated and EPclin were shown to provide additional information
in the prospective randomized phase III MINDACT trial, on the distant recurrence risk in patients with node-neg-
performed in patients with either negative lymph nodes or ative and node-positive disease, independent of clinico-
1–3 positive lymph nodes. Groups with discrepant genomic pathological parameters. EPclin can also be used to guide
and clinical risks were randomized to receive endocrine ther- decision-making for the use of systemic chemotherapy in
apy versus adjuvant chemotherapy and endocrine therapy, post-menopausal patients with ER-positive, HER2-negative,
revealing that high-risk and low-risk patients had limited and node-negative breast tumors. For node-positive patients,
therapeutic benefit from the use of adjuvant therapy. Over- ­EndoPredict® clinical use is not recommended at this time.
all, ­MammaPrint® has not achieved predictive utility, but
it is the only multigenomic trial with prospective 1A evi- BRCA 1/2 gene mutations and homologous
dence level for evaluating prognosis in high-risk patients recombination deficiency
with node-negative and node-positive early luminal breast
cancer. The WSG-PRIMe study has prospectively demon- Early breast cancer HER2-negative patients with high risk
strated the impact of ­MammaPrint® and BluePrint on treat- of recurrence should be tested for germline BRCA1 and
ment decision [49]. BluePrint is an 80-gene assay that allows BRCA2 mutations. For patients who have had previous
a molecular sub-classification into low-risk luminal tumors, surgery, high risk is defined as a tumor size >2 cm or any
high-risk luminal tumors, HER2 and basal type. involved axillary node in TNBC cases, or ≥4 axillary nodes
Clinical and Translational Oncology

in HR-positive disease. In patients who have had NAT, high event-free survival. The percentage with pCR was significantly
risk derives from either any residual cancer in TNBC or higher among those who received pembrolizumab plus neoad-
high-grade residual disease in HR-positive disease (defined juvant chemotherapy (64.8%) than among those who received
as a Clinical Pathological State + ER Grade [CPS + EG] placebo plus neoadjuvant chemotherapy (51.2%). The benefits
score ≥3). of pembrolizumab-chemotherapy with respect to pCR were
The OlympiA study was a phase III, double-blind, ran- similar in PD-L1-positive and PD-L1-negative subgroups,
domized trial involving patients with HER2-negative early although only 97 (16%) of the 602 cases in the trial were PDL-
breast cancer with BRCA1 or BRCA2 germline pathogenic 1-negative. In a subsequent analysis, event-free survival was
or likely pathogenic variants and high-risk clinicopathologi- also improved in the combination group [60].
cal factors who had received local treatment and neoadjuvant
or adjuvant chemotherapy. Patients were randomly assigned Tumor‑infiltrating lymphocytes
to 1 year of oral olaparib or placebo. The primary endpoint
was invasive disease-free survival. Olaparib treatment was Breast cancer is an immunogenic tumor and, in the last few
associated with significantly longer survival free of invasive years, morphological evaluation of tumor-infiltrating lym-
or distant disease than placebo [53]. One year of adjuvant phocytes (TILs) in breast cancer has been proposed as a
olaparib is currently indicated either alone or concurrently potentially useful biomarker. It has been reported that every
with endocrine therapy in early breast cancer with BRCA1- 10% increment of stromal lymphocytes is associated with a
or BRCA2-mutated high-risk patients that have received 16% reduction of risk of death in TNBC, and values around
local treatment and neoadjuvant or adjuvant chemotherapy. 30–50% have been proposed for potential de-escalation of
Homologous recombination deficiency (HRD) may allow chemotherapies in this type of breast cancer. In the case of
consideration of using DNA-damaging agents such as PARP HER2-positive tumors, values >20% have been proposed
inhibitors. To analyze the impairment of the homologous for potential de-escalation of trastuzumab [61]. In contrast,
recombination pathway, specific mutations in homologous increased TILs seemed to be an adverse prognostic factor
recombination repair genes other than BRCA1 or BRCA2, for survival in luminal HER2-negative breast cancer, sug-
such as PALB2, ATM, CHEK2 and others, can be examined. gesting a different biology of the immunological infiltrate
The use of genomic scars, mutational signatures [54], or the in this subtype [62].
development of functional tests can also be considered [55]. In 2014, the International TILs Working Group described
Currently, olaparib is not indicated in HRD cases, although a method to quantify TILs on hematoxylin and eosin-stained
HRD testing may be useful in the future. slides using light microscopy [63] (Table 2). Recently, to
maximize inter-observer reproducibility, the International
PD‑L1 TILs working group has created a website (www.​tilsi​nbrea​
stcan​cer.​org) in which free training is available, and refer-
Programmed cell death-1 (PD-1) protein is an immune ence images are provided to allow direct visual comparison
checkpoint inhibitor expressed on the surface of T cells, B [64]. This methodology has been subsequently applied in
cells, natural killer T cells, monocytes, and dendritic cells, lymph nodes and metastatic tissues [61]. We strongly recom-
but not resting T cells. PD-1 binds to two ligands, PD-L1 mend the use of this tool for self-validation before starting
(B7-H1) and PD-L2 (B7-DC). Activation of PD-1 by PD-L1 routine reporting of TILs.
or PD-L2 induces downregulation of T-cell activity, reduced Emerging data suggest that TILs quantification can help
cytokine production, T-cell lysis, and induction of tolerance clinicians to identify breast cancers with better response to
to antigens. In solid tumors, the PD-1/PD-L1 inhibitory PD-1/PD-L1 inhibition and better prognosis especially in
pathway can silence the immune system by increasing the TNBC. In the neoadjuvant setting, TILs are predictive of
expression of PD-L1 on the tumor cell surface [56, 57]. pCR with chemotherapy [65].
Currently, neoadjuvant therapy in patients with early TNBC Although not a standard biomarker, we recommend quan-
would include the combination of chemotherapy with pem- tifying and reporting TILs to add valuable information about
brolizumab, regardless of PD-L1 expression. In this setting, the immune response associated with each tumor.
the combination of chemotherapy with pembrolizumab (KEY-
NOTE-522 trial) has showed evidence of efficacy [58, 59].
The Keynote 522 trial evaluated patients with stages II–III Advanced‑stage breast cancer
TNBC treated with 4 cycles of paclitaxel plus carboplatin with
the addition of 4 cycles of either pembrolizumab (n = 784) or PIK3CA
placebo (n = 390). Patients received postoperative pembroli-
zumab or placebo for up to 9 cycles. The primary endpoints I n a p o p u l a t i o n o f 8 2 4 c a s e s o f H R- p o s i -
of the study were pathological complete response (pCR) and tive, HER2-negative tumors, the prevalence of
 Clinical and Translational Oncology

Table 2  Recommendations of the TILs Working Group for assessing TILs in breast cancer [63]

1. One section (4–5 µm, magnification ×200–400) per patient is considered to be sufficient. Full sections are preferred over biopsies (in pre-
therapeutic neoadjuvant setting, cores can be used); currently, no validated methodology has been developed to score TILs after neoadjuvant
treatment
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, hyalinization 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 tumor area should be used
6. It should be scored as a continuous variable that will allow categorization of different thresholds and more accurate statistical analyses

TILs: tumor-infiltrating lymphocytes. For more information and self-training: www.​tilsi​nbrea​stcan​cer.​org [64]

phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic HER2 non-overexpressing metastatic breast cancer that had
subunit alpha (PIK3CA) mutations was 31.4% [66]. Treat- received ≤2 prior therapies in the advanced setting. PFS, the
ment with alpelisib–fulvestrant has been found to prolong primary endpoint, was significantly prolonged with olaparib
PFS among patients with PIK3CA-mutated, HR-positive, versus standard therapy (7.0 vs. 4.2 months). There were no
HER2-negative, advanced breast cancer who had received differences in OS, either at the interim or the final analysis
endocrine therapy previously in the SOLAR-1 phase III [69].
clinical trial [67]. Currently, alpelisib is indicated in combi- The phase III EMBRACA trial enrolled patients with
nation with fulvestrant for the treatment of post-menopausal gBRCA1/2-mutated HER2-negative advanced breast cancer.
women, and men, with HR-positive, HER2-negative, locally Patients received talazoparib or physician’s choice of chemo-
advanced or metastatic breast cancer with a PIK3CA muta- therapy. Median PFS was significantly longer in the talazo-
tion after disease progression following endocrine therapy parib group than in the standard therapy group (8.6 months
as monotherapy. vs. 5.6 months), but there were no differences in OS both at
Alpelisib monotherapy has also shown efficacy in heavily the interim and the final analysis [70].
pretreated ER-positive breast cancer with PIK3CA mutations Olaparib and, in some countries, talazoparib, are indi-
(30% response rate) [68]. It is interesting that a recent real- cated as single agents for previously treated breast cancer
life data study of 233 patients prospectively registered in the patients who have HER2-negative or HR-positive locally
French alpelisib Early Access Program opened to PIK3CA- advanced or metastatic breast cancer with germline BRCA1/2
mutant, HR-positive, HER2-negative ABC patients treated mutations, or as first-line therapy when patients are not suit-
with alpelisib and fulvestrant, showed that patients had able for standard therapies.
received a median number of 4 prior systemic treatments for BRCA1/2 testing, therefore, can provide a therapeu-
ABC, including CDK4/6 inhibitor (97.4%), chemotherapy tic opportunity in advanced breast cancer with germline
(77.3%), or everolimus (56.2%), respectively. BRCA1/2 mutations and should be performed in this setting,
Furthermore, it has been reported at the 2023 San Anto- if PARP inhibitors are available.
nio Breast Cancer Symposium that inavolisib, another PI3K
inhibitor, was more effective than placebo when combined PD‑L1
with palbociclib and fulvestrant in the first-line treatment of
patients with PIK3CA-mutated, HR-positive, HER2-nega- The randomized controlled trials IMpassion130 [71] and
tive, locally advanced or metastatic breast cancer (phase III Keynote-355 [58] have demonstrated the benefit of anti-
INAVO1209). PD-1/PD-L1 agents atezolizumab and pembrolizumab,
Therefore, patients with advanced hormone-sensitive respectively, plus chemotherapy, in first-line metastatic
breast cancer should have their tumors tested for PIK3CA TNBC, with PFS and OS improved in PD-L1-positive
mutations, considering that at least one-third of cases harbor patients [59, 71, 72]. Therefore, measurement of PD-L1
PIK3CA mutations and may benefit from PI3K inhibitors. levels is a critical component in predicting patient benefit.
The combination of atezolizumab and nab-paclitaxel is
BRCA 1/2 gene mutations recommended in the European Union as first-line treatment
for PD-L1-positive (≥1%) metastatic TNBC, based on the
In advanced breast cancer, the phase III OlympiAD study of results of the phase III IMpassion130 trial.
olaparib compared with physician’s choice of chemother- In the KEYNOTE-355 trial, with a more robust statistical
apy was conducted in patients with BRCA​ mutations and design and results, 847 patients with advanced (unresectable,
Clinical and Translational Oncology

locally advanced, or metastatic) TNBC were randomly monotherapy is indicated by the Food and Drug Administra-
assigned to receive chemotherapy (nab-paclitaxel, pacli- tion (FDA) and European Medicines Agency (EMA) for the
taxel or gemcitabine plus carboplatin) plus pembrolizumab treatment of post-menopausal women and adult men with
or chemotherapy plus placebo. OS improved in patients who HER2-negative, ER-positive advanced breast cancer, with
received pembrolizumab and had tumors with relatively an ESR1-activating mutation who have disease progression
high levels of PD-L1 protein (PD-L1 CPS of at least 10%): after receiving at least one line of endocrine therapy includ-
23.0 months versus 16.1 months in control patients who ing a CDK 4/6 inhibitor. Elacestrant is becoming increas-
received chemotherapy alone [73]. ingly available, and testing for ESR1 mutations in advanced
Patients with advanced TNBC should have PD-L1 tested breast cancer with either liquid or tissue biopsy is currently
in tumor tissue. According to the PD-L1 test that showed recommended by clinical guidelines [41, 77].
positive, pembrolizumab or atezolizumab should be used.
The KEYNOTE studies used the 22C3 PD-L1 immunohis- AKT pathway activation
tochemistry assay (Agilent, Carpinteria, CA) to calculate a
CPS estimated as the ratio of PD-L1-positive cells (tumor An additional mechanism of endocrine resistance in
cells plus immune cells divided by the total number of viable advanced breast cancer is related to serine/threonine kinase
tumor cells ×100 with a cut-off ≥10 [59, 73]). The IMpas- (AKT). AKT is the key node of the PI3K–AKT–PTEN
sion trials, in contrast, used the SP142 PD-L1 immunohis- signaling pathway. Overactivation of the pathway occurs in
tochemistry assay (Ventana, Tucson, AZ), measuring the approximately half of HR-positive, HER2-negative breast
proportion of tumor area that is occupied by PD-L1 staining cancers by means of activating mutations in PIK3CA and
in IC with cut-off in >1% of staining [58, 71, 72]. AKT1 and inactivating alterations in PTEN.
Most laboratories do not regularly use all possible PD-L1 Capivasertib is an orally bioavailable, small molecule
antibodies, and harmonization studies are needed. Rugo, inhibitor of AKT. In a randomized, double-blind, placebo-
et al. compared PD-L1 status on IC (VENTANA SP142, controlled, phase III trial, 708 patients were assigned to
SP263, Dako 22C3) or as a CPS, and concluded that 22C3 receive either oral capivasertib plus fulvestrant or a matching
and SP263 identified more patients as PD-L1-positive than placebo plus fulvestrant. Capivasertib–fulvestrant therapy
SP142 [74]. resulted in significantly longer PFS than treatment with ful-
vestrant alone among patients with HR-positive advanced
ESR1 mutations breast cancer whose disease had progressed during or
after previous aromatase inhibitor therapy with or without
A key mechanism of endocrine resistance is through mis- a CDK4/6 inhibitor. AKT pathway alterations (PIK3CA,
sense mutations in ESR1, the gene that encodes for ER AKT1, or PTEN) were assessed in tumors. Similar results
alpha. ESR1 mutations are present in about 30% of patients were observed in the overall population and in the AKT
with metastatic breast cancer who have received aromatase pathway altered population. At this point, AKT pathway test-
inhibitors, although only in 5% of breast cancer recurring ing for capivasertib use is still in research [78].
after adjuvant aromatase inhibitors, and 1% of endocrine
therapy-naïve metastatic breast cancer [75]. Ligand binding NTRK
domain mutations of ESR1 make the receptor constitutively
active and thus unaffected by aromatase inhibitor depletion NTRK gene fusions are tumor-agnostic biomarkers that
of estrogen. In contrast, ESR1 mutations do not appear to be predict response to NTRK inhibitors. Secretory breast car-
a main mechanism of resistance to tamoxifen or fulvestrant cinoma is a special histological type of breast carcinoma
[76]. Several selective ER modulators or covalent antago- that carries the NTRK3-ETV6 fusion in about 90% of cases.
nists are being tested specifically against ESR1 mutations. Detection of this fusion by fluorescence ISH (FISH) or NGS
Elacestrant is one of these oral selective ER degraders. The can help in the correct diagnosis of this entity and in predict-
randomized phase III EMERALD trial enrolled patients with ing response to inhibitors in advanced tumors. In contrast,
ER-positive, HER2-negative advanced breast cancer who less than 1% of all breast cancer cases harbor NTRK fusions.
had one or two lines of endocrine therapy, required pre-treat- Currently, NTRK testing is not required in advanced breast
ment with a cyclin-dependent kinase 4/6 inhibitor, and had carcinoma.
one or no lines of chemotherapy. Primary endpoints were
PFS by blinded independent central review in all patients TROP‑2
and patients with detectable ESR1 mutations. Detectable
ESR1 mutations in circulating tumor DNA were detected TROP-2 is a transmembrane calcium protein belonging to
in 47.8% of patients. PFS was prolonged in all patients, the EpCAM family that is expressed by normal human mul-
particularly in patients with ESR1 mutation. Elacestrant tistratified epithelia and trophoblast cells. Overexpression
 Clinical and Translational Oncology

can be present in several solid tumors, including TNBC. and standardized reporting system that does not incorporate
Approximately 86% of TNBCs are TROP-2-positive [79]. response, but establishes risk of recurrence in patients with
Sacituzumab govitecan is an antibody–drug conjugate residual disease in both the breast and the lymph nodes. The
that combines a humanized monoclonal antibody binding to parameters to be quantified and reported, as well as a calcu-
TROP-2-expressing cancer cells (sacituzumab) with a topoi- lator, are available online [84].
somerase I inhibitor (govitecan). Sacituzumab govitecan as IHC-based biomarkers such as ER, PR, and HER2 may
a single agent is indicated for the treatment of adult patients also be re-assessed if negative prior to treatment, to allow
with unresectable or metastatic TNBC who have received patients to benefit from targeted therapies and to obtain bio-
two or more prior systemic therapies, with at least one for logical explanation for possible causes of intrinsic resistance
advanced disease [80]. to treatments. Although clinical decisions are not still made
Different levels of TROP-2 expression did not have an based on TILs, immune markers are among the most promis-
apparent effect on the efficacy of the treatment in this study. ing biomarkers in the post-NAT setting, in which extensive
Currently, TROP-2 testing is not required in order to use tumor infiltration by lymphocytes indicates a good prognosis
sacituzumab govitecan for this indication. in some breast tumor types, irrespective of residual tumor
size. In TNBC, TILs levels are significantly associated with
Other biomarkers improved recurrence-free survival (RFS) and OS and add
further prognostic information to RCB class, particularly in
Assessment of other biomarkers that are targets of novel RCB class II [85].
therapies, such as CDK amplification, FGFR1 amplification,
or PTEN loss of heterozygosity or mutations, is not currently
recommended. Following neoadjuvant endocrine therapy
in post‑menopausal patients

Biomarker assessment following NAT Ki‑67 after short‑term endocrine treatment

NAT, including chemotherapy, anti-HER2 therapy and hor- Ki-67 expression after 2 weeks of treatment (Ki-672W) may
monal therapy administered before surgery, has become improve the prediction of RFS better than Ki-67 at base-
part of the standard-of-care treatment of patients not only line ­(Ki67B), as observed among patients enrolled in the
with locally advanced breast cancer but also with opera- IMPACT trial [86]. The POETIC trial provided evidence for
ble tumors, particularly in HER2-positive carcinomas and the clinical validity of on-treatment aromatase inhibitor Ki-
TNBC. Besides reducing tumor burden, NAT provides a 672W in addition to Ki-67B to predict those with high resid-
unique opportunity to evaluate the tumor response to dif- ual risk of recurrence in spite of standard-of-care therapy
ferent treatments. The pCR is a well-established surrogate [87]. Patients whose Ki-67B was low had good results, with
marker of improved prognosis in breast cancer. However, 85% of those receiving endocrine therapy alone. Patients
not all patients obtain a pCR, and these patients have a vary- whose tumors had a high baseline Ki-67 and a low Ki-672w,
ing risk of relapse [81]. Importantly, substantial biological had a better prognosis at 5 years than those who continued
differences exist between treatment-naive breast cancer and to have a high Ki-672W.
residual tissue following NAT [82]. Re-assessment of bio-
markers in the residual breast cancer tissue may have both
prognostic and potential therapeutic implications. Post-NAT Preoperative endocrine prognostic index (PEPI score)
pathological stage and biomarker status may help guide
adjuvant treatment decisions. Accordingly, this SEOM- Multivariable testing of post-treatment tumor characteristics
SEAP consensus guideline recommends the following bio- including pathological tumor size, node status, Ki-67 level,
marker determinations, depending on the type of NAT. and ER status were independently associated with RFS and
breast cancer-specific survival. Of note, patients with low
Following neoadjuvant chemotherapy pathological stage at surgery and a favorable biomarker pro-
with or without anti‑HER2 therapy file (preoperative endocrine prognostic index [PEPI] score
0) had such a low rate of recurrence that further adjuvant
Classic histopathological parameters such as ypTNM clas- systemic therapy beyond continuation of endocrine therapy
sification and histological grade provide valuable prognostic seemed unnecessary. In contrast, patients with high patho-
and predictive information when assessed in residual breast logical stage disease at surgery and a poor PEPI score (PEPI
cancer tissue after NAT [83]. Additionally, the Residual group 3) had a significantly higher risk of relapse, and there-
Cancer Burden (RCB) index [81] is a clinically validated fore should be offered appropriate adjuvant treatments [88].
Clinical and Translational Oncology

New technologies of liquid biopsy is ctDNA testing in advanced breast can-

cer. The recent advances in massively parallel sequencing
Next‑generation sequencing (NGS) technologies have empowered liquid biopsies, particularly
ctDNA analysis, to be the new paradigm in personalized
At present, NGS in breast cancer remains a research tool. cancer management.
NGS should not be used as an exploratory tool to prescribe Plasma ctDNA detection may overcome some of the
treatments beyond the indications for which they have current limitations in tumor tissue procurement and serves
been approved, regardless of whether the target genetic as a convenient and non-invasive method to capture tumor
alteration is detected [89]. The SAFIR01 study identi- heterogeneity and genetic evolution along patients’ cancer
fied targetable genomic alterations in 195 cases (46%) journeys. ctDNA can be sequenced for genetic profiling of
in a series of 423 patients with advanced breast cancer, the tumors in selected patients for mutation-directed therapy
although ultimately only 55 received personalized treat- [94–96]. Data from the plasmaMATCH study were recently
ment and just 13 showed any type of response [90]. reported [97], and showed that ctDNA testing for mutations
Therefore, more research is needed into the role of has high sensitivity and accuracy for widespread adoption in
NGS in clinical practice before incorporating it into rou- clinical practice even to screen for rare oncogenic mutations.
tine use, and efforts should be made to educate clinicians It is efficient and rapid for screening and allows evaluation
to increase their knowledge and confidence in such tech- of different acquired mutations throughout the evolution of
nologies [91]. An expert panel has produced a guidance the disease.
document to rank DNA alterations into tiers of evidence Recently, the FDA approved the Therascreen PIK3CA
for clinical utility for selecting breast cancer patients for RGQ polymerase chain reaction assay as a companion diag-
targeted therapies according to ESMO Scale for Clinical nostic assay to detect PIK3CA mutations in breast cancer for
Actionability of molecular Targets (ESCAT) [92]. Among both tissue and liquid biopsies, bringing the role of liquid
40 recurrent driver alterations described in breast cancer, biopsy into breast cancer management. In this context, alpe-
only HER2 amplification, germline BRCA1/2 mutations, lisib, a PI3K inhibitor, was the first agent to be approved by
and PIK3CA mutations were given level 1A evidence as the FDA and EMA [98].
molecular targets whereas NTRK fusions and microsatel- In breast cancer, the current clinical application of ctDNA
lite instability (MSI) were ranked as 1C evidence. includes detection of drug-resistant clones. In this way ESR1
An update of the ESMO recommendations for the mutation detection has already been shown as a predictive
use of NGS in advanced cancer has been published very biomarker, used in clinical practice for metastatic hormone
recently [93]. The authors consider that, since NGS receptor breast cancer and in some cases, early switch of
can substitute germline BRCA​ testing in most patients hormone therapy [99]. ESR1 mutations assessment using
and ESR1 mutations have been reclassified as level 1A, liquid biopsy (by digital-PCR or NGS) [100], is a current
performing NGS in advanced breast cancer (tumor or standard accepted by clinical guidelines [41, 77], as has been
plasma) is recommended in patients with HR-positive/ described earlier in this document.
HER2-negative disease after resistance to endocrine CTC plasma count analysis after curative tumor resection
therapy. surgery may facilitate early detection of minimal residual
disease, aiding in the initiation of adjuvant therapy to pre-
vent recurrence. Furthermore, CTC plasma count can be
Liquid biopsy, circulating tumor cells, used for monitoring disease response, detecting and predict-
and circulating tumor DNA ing risk of progression or relapse [101].
In the field of breast cancer, liquid biopsy has been a
Traditional methods of cancer detection, such as tissue research hot-spot in recent years, playing a key role in mon-
biopsy, can sometimes not be comprehensive enough to itoring breast cancer metastasis, predicting disease recur-
capture the entire genomic landscape of breast tumors. The rence, and assessing clinical drug resistance. Liquid biopsy
role of liquid biopsy in cancer management has been gain- has the advantages of non-invasive, high sensitivity, high
ing increased prominence in the past decade. Various com- specificity, and real-time dynamic monitoring. While clini-
ponents of tumor cells released into the blood circulation cal application is not yet a reality, the research prospects of
can be analyzed in liquid biopsy sampling, some of which CTCs and cfDNA in breast cancer are worth exploring and
include circulating tumor cells (CTCs), circulating tumor discovering.
DNA (ctDNA), cell-free RNA, tumor-educated platelets, Researchers and clinicians are currently working to vali-
and exosomes. These components can be used for different date the clinical utility of ctDNA in diagnostics, prognostics,
purposes. Currently one of the most investigated utilities the surveillance of minimal residual disease, and the moni-
toring of therapeutic response [94–96].
 Clinical and Translational Oncology

MSI and MMRd checkpoint inhibitors show the same correlation with TMB
(i.e., atezolizumab) and not all assays have the same thresh-
High microsatellite instability (MSI-H)/Mismatch repair defi- olds. There is a need for harmonization, and care should be
ciency (MMRd) is considered a tumor-agnostic biomarker taken when interpretating TMB for specific treatments [103,
that predicts response to ICI in some advanced cancers [102]. 104].
However, the frequency of MSI-H/MMRd is very low among
breast carcinomas, and currently it is not a recommended
biomarker. While it is controversial whether women with Conclusions
Lynch syndrome have an increased risk of developing breast
carcinomas, about 50% of breast carcinomas in patients with To plan adequate therapy in patients with early breast can-
Lynch syndrome carry MMRd. Immunohistochemistry is cer (Fig. 2), pathology reports should include in all cases
the most frequently used method to detect MMRd, although the expression and levels of ER, PR, HER2, and Ki-67, in
other molecular methods can be used. addition to histological grade (as well as BRCA​ in high-
risk HR-positive, HER2-negative patients, and TNBC) to
Tumor mutational burden assist prognosis and to establish current therapeutic options
available, including hormone therapy, chemotherapy, anti-
Tumor mutational burden (TMB) may be a good biomarker HER2 therapy and PARP inhibitors. In ER-positive HER2-
for the indication of immune checkpoint inhibitors, as it negative early breast cancer patients, one of the several
can reflect a high neoantigen burden, which can lead to an available genetic prognostic platforms (Oncotype D ­ X ®,
® ® ®
increased immune response. Moreover, the F­ oundationOne® ­MammaPrint , ­Prosigna , or E ­ ndoPredict ) may be used to
CDx assay has been approved as a companion diagnostic establish prognosis and to discuss with the patient whether
for tumor-agnostic pembrolizumab in patients with a TMB adjuvant treatment may be limited to hormonal therapy.
of ≥10 mutations per megabase. However, not all immune

Fig. 2  Current routine use, research use, and not-recommended use phatase and tensin homolog; TILs: tumour-infiltrating lymphocytes;
of biomarkers for breast cancer. AKT: serine/threonine kinase; AR: TMB: tumor mutational burden; TROP-2: tumor-associated calcium
androgen receptor; BRCA: breast cancer; CTCs: circulating tumour signal transducer 2. 1Mammaprint®, Oncotype D ­ X®, ­Prosigna® or
cells; ER: estrogen receptor; ESR1: estrogen receptor 1; FGFR1: ­EndoPredict® in early luminal breast cancer with low risk of recur-
fibroblast growth factor receptor 1; HER2: human epidermal growth rence; 2In advanced triple-negative or luminal breast cancer; 3In
factor receptor 2; HRD: homologous recombination deficiency; MSI: advanced triple-negative breast cancer; 4In advanced luminal breast
microsatellite instability; NGS: next-generation sequencing; NTRK: cancer; 5Some studies relate them to responses to neoadjuvant chem-
neurotrophic receptor tyrosine kinase 1; PD-L1: programmed death otherapy, 6Approved in the United States of America as a companion
ligand 1; PIK3CA: phosphatidylinositol 4,5-bisphosphate 3-kinase diagnostic to a PI3K inhibitor
catalytic subunit alpha; PR: progesterone receptor; PTEN: phos-
Clinical and Translational Oncology

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jurisdictional claims in published maps and institutional affiliations.

Authors and Affiliations

Ramon Colomer1 · Blanca González‑Farré2 · Ana Isabel Ballesteros3 · Vicente Peg4 · Begoña Bermejo5 ·
Belén Pérez‑Mies6 · Susana de la Cruz7 · Federico Rojo8 · Sonia Pernas9 · José Palacios10

* Ramon Colomer and Research Institute, C/Diego de León, 62, 28006 Madrid,
[email protected] Spain
2
* José Palacios Pathological Anatomy Service, Clínic Hospital, Barcelona,
[email protected] Spain
3
Blanca González‑Farré Medical Oncology Department, La Princesa University
[email protected] Hospital, Madrid, Spain
4
Ana Isabel Ballesteros Pathological Anatomy Service, Vall d’Hebron University
[email protected] Hospital, Barcelona, Spain
5
Vicente Peg Medical Oncology Department, Biomedical Research
[email protected] Institute INCLIVA, Medicine Department of the University
of Valencia and Clinic University Hospital, Valencia, Spain
Begoña Bermejo
6
[email protected] Pathological Anatomy Service, Ramón y Cajal University
Hospital, Faculty of Medicine, University of Alcalá, IRYCIS
Belén Pérez‑Mies
and CIBERONC, Madrid, Spain
[email protected]
7
Medical Oncology Department, Navarra University Hospital,
Susana de la Cruz
Navarre, Spain
[email protected]
8
Anatomy Service, Fundación Jiménez Díaz University
Federico Rojo
Hospital and CIBERONC, Madrid, Spain
[email protected]
9
Oncology Department, Catalan Institute of Oncology
Sonia Pernas
(ICO)-IDIBELL, L’Hospitalet, Barcelona, Spain
[email protected]
10
Pathological Anatomy Service, Department of Pathology,
1
UAM Personalised Precision Medicine Chair & Medical Ramón y Cajal University Hospital, Faculty of Medicine,
Oncology Department, La Princesa University Hospital University of Alcalá, IRYCIS and CIBERONC,
Ctra. Colmenar Viejo, Km 9,1, 28034 Madrid, Spain