Med Oncol (2014) 31:834

DOI 10.1007/s12032-013-0834-y

REVIEW ARTICLE

Triple-negative breast cancer: future prospects in diagnosis

and management
Shereef Elsamany Sakher Abdullah
Received: 25 October 2013/Accepted: 27 December 2013/Published online: 5 January 2014
Springer Science+Business Media New York 2014

Abstract

Triple-negative breast cancer (TNBC) is an

aggressive subtype comprising about 1020 % of breast

cancer patients with an overall poor prognosis. Recently, it
was found to be a heterogeneous disease that has been
classied into six subtypes based on molecular signature.
In preclinical trials, these subtypes have different active
signaling pathways with variable response to chemotherapy. To improve treatment outcome of TNBC, therapy
should be tailored according to the active driving signaling
aberration. Molecular testing represents the optimal way to
stratify patients, but it has some difculties to be implemented in routine clinical practice. This article provides an
assumption for stepped diagnostic algorithm of TNBC
based on immunohistochemistry markers in addition to a
suggested tailored therapeutic strategy for advanced TNBC
based on the driving aberrations. Furthermore, most TNBC
patients develop early relapse despite adjuvant chemotherapy. We provide a design for future adjuvant therapy
for the disease. This design is based on targeting proposed
active pathways in breast cancer stem cells responsible for
regenerating the tumor and disease relapse. Finally, we
provide a proposed design for future clinical trials in
TNBC to allow for investigation of different medications in
this heterogeneous disease based on upfront patient stratication and then allocation to the suitable treatment arms.

S. Elsamany (&) S. Abdullah

Medical Oncology Department, Oncology Centre, King
Abdullah Medical City, 2677 Al-Mashaeer District,
Makkah 57657, Saudi Arabia
e-mail: samanyshereef@yahoo.com

S. Elsamany
Medical Oncology, Mansoura University, Mansoura, Egypt

Keywords Triple negative Breast Future Diagnosis

Management

Introduction

Triple-negative breast cancer (TNBC) constitutes approximately 1020 % of breast cancer patients and represents an
aggressive subtype with poor overall prognosis [1]. TNBC
patients have a higher rate of early recurrence and distant
metastasistobrainandlungscomparedtootherbreastcancer
subtypes [1]. Chemotherapy is the only systemic therapy
currently available for TNBC; however, most patients with
TNBC relapse within 12 years and\30 % of patients survive 5 years despite adjuvant chemotherapy [2].
Despite initial high response rate to chemotherapy in the
metastatic setting, TNBC patients develop rapid disease
progression resulting in a shorter overall survival compared
to ER? breast cancer [3].
The treatment options for TNBC after chemotherapy
failure are limited. Overall, treatment of patients with
TNBC has been challenging due to the heterogeneity of the
disease and the absence of well-dened molecular targets
[4]. Although several small molecule inhibitors and
monoclonal antibodies have been tested in clinical trials in
unselected TNBC patients, none has entered clinical
practice due to limited efcacy [4]. Given so, improving
therapeutic outcome of TNBC patients requires better
understanding of its molecular basis to identify molecular
drivers that can be therapeutically targeted [5].
In this article, we will review recent data about the
molecular basis of TNBC, with a particular emphasis on
potential targets of novel therapies. In addition, we will
provide assumptions of future diagnostic and therapeutic
approaches of this disease.

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Med Oncol (2014) 31:834

Table 1 Subtypes of TNBC based on gene expression proling

Subtype
Gene expression prole

Basal-like 1

High expression of genes involved in cell cycle

progression, cell division and DNA damage
response pathways

Basal-like 2

chemotherapy
[5].response
However,
from
ER-positive
breast to
have enhanced
todata
PI3K
inhibitors
compared
cancer revealed that single-agent PI3K inhibition leads to
feedback activation of ER pathway, while combined inhibition of both PI3K and ER may be more effective than
inhibition of either pathway alone [9]. This displays the
value of combining PI3K inhibitors with other agents to
optimize therapeutic efcacy.

High expression of genes involved in cell cycle

progression, cell division and growth factor

Promising synergism of PARP inhibitors and PI3K

inhibitors

signaling
Immunomodulatory

High expression of genes involved in immune

processes

Mesenchymal

High expression of genes involved in motility

and extracellular matrix

Mesenchymal stem

High expression of genes involved in motility,

Molecular classication of TNBC

like

extracellular matrix and growth factor

signaling
TNBC has been recently
classied into six subtypes on the
Luminal
androgen
High
expression
genes involved
in have
basis of gene analysis (Table
1) [5].ofThese
subtypes
prognostic signicance with the basal-like subtypes having
receptor
hormonally regulated pathways
the worst clinical outcome [4].

Basal-like subtypes

Several PARP inhibitors are being tested in clinical trials

such as olaparib (AZD2281), which has been shown to be
safe and effective in BRCA-related cancers. Meanwhile, the
benet of iniparib in phase II trial was not conrmed in the
subsequent phase III trial [10].
activity
when preclinical
PARP inhibitors
combined with
PI3K
Emerging
data are
demonstrates
synergistic
inhibitors in cell lines with active PI3K pathway [11].
PARP enzymatic activity is necessary for the repair of
single-strand breaks (SSBs) through the base excision
repair (BER) pathway, while BRCA1 and BRCA2 proteins
are essential components for repair of the double-strand
breaks (DSBs) through homologous recombination (HR)
[12]. When PARP is inhibited, unrepaired SSBs can be
transformed to DSBs that, in BRCA-decient cells, can no
longer be repaired by HR, resulting in lethal DNA damage
[13].

Basal-like 1 (BL-1) and basal-like 2 (BL-2) are the most

frequent TNBC subtypes (47 %) [5]. They are characterized by high proliferation rate with an average Ki-67
staining of 70 % in addition to high expression of genes
involved in cell cycle and cell division [5]. They have high
sensitivity to chemotherapy, especially the antimitotic
agents as demonstrated by high rate of pathological complete response (pCR) with neoadjuvant taxan-based chemotherapy (63 % in BL-1 and BL-2 subtypes compared to
1431 % in other TNBC subtypes) [6].

PI3K pathway is required to maintain and stabilize

BRCA1/2 protein levels. In this way, PI3K inhibition
impairs BRCA1/2 protein levels, which sensitizes BRCAprocient TNBC to PARP inhibition as demonstrated in
cell lines and animal models [11]. In addition, in an animal
model of BRCA1-decient breast cancer, combined therapy of the PARP inhibitor olaparib with the PI3K inhibitor
NVP-BKM120 caused a 14-fold delay in tumor growth
compared with therapy with either agent alone [14].

Interestingly, the majority of studies evaluating neoadjuvant chemotherapy have identied high Ki-67 level as a
predictive factor of pCR [7]. In the study of Fasching et al.
[7] involving TNBC patients, pCR was 57 % in patients
with Ki-67 [35 % compared to 4 % in those with Ki-67
\35 %. Interestingly, Adamo and Anders [8] suggested
that TNBC patients can be divided into two subgroups with
differential response and prognosis after preoperative
chemotherapy based on Ki-67 level.

Recent view of androgen receptor-positive TNBC

Mesenchymal and mesenchymal stem-like subtypes

Mesenchymal (M) and mesenchymal stem-like (MSL)
subtypes have low level of proliferation, but they are
enriched with phosphatidylinositol-3-kinase (PI3K) and
Wnt pathways activity. In preclinical trials, these subtypes

The luminal androgen receptor (LAR) subtype was identied in 11 % of TNBC by gene analysis as reported by
Lehmann et al [5]; however, androgen receptor (AR) was
found to be expressed in one-third of TNBC via immunohistochemistry [15]. This subtype showed enhanced
steroid hormone signaling and androgen receptor mRNA
was detected at an average of ninefold higher than other
TNBC subtypes [16]. Interestingly, LAR subtype belongs
to either luminal A or luminal B intrinsic subtype despite
being negative for ER expression [16].
This subtype showed high response rate to anti-androgens in preclinical trials [5]. Furthermore, the anti-

Med Oncol (2014) 31:834

Fig. 1 Wnt/b-catenin signaling in TNBC. a In the absence of Wnt

binding to LRP5/6 and FZD, APC binds to axin and GSK3. This
complex promotes phosphorylation of b-catenin via CK1. Phosphorylated b-catenin is then degraded by proteasome system. b Wnt
proteins bind to LRP5/6 and FZD forming a complex that prevents

androgen bicalutamide is currently being evaluated in a

phase II study in AR-positive ER-/PR-negative metastatic
breast cancer patients [15].
Meanwhile, PI3K pathway activation is involved in the
resistance to anti-androgens as demonstrated in animal
models of AR-positive TNBC treated with bicalutamide
[17]. In addition, synergistic activity between PI3K inhibitors and anti-androgens has been shown in cell lines and
experimental animals [17]. These preclinical data provide
the rationale for future clinical trials, evaluating the combination of AR antagonists with PI3K inhibitors in ARpositive TNBC patients.

Page 3 of 7

834

axin, CK1 and GSK3 from inducing b-catenin degradation. Free bcatenin is translocated to the nucleus to bind with TCF transcription
factor. The TCFb-catenin complex activates Wnt target genes. APC
adenomatous polyposis coli, GSK3 glycogen synthase kinase-3, CK1
casein kinase-1

prevents b-catenin degradation, which allows its translocation to the nucleus to enhance cell proliferation [18]
(Fig. 1).
Dysfunction of the Wnt ligands at the cell surface leads
to aberrant activation of Wnt/b-catenin signaling that
drives breast carcinogenesis [19]. Among the 10 FZD
proteins, FZD7, overexpressed in 67 % of TNBC, is likely
the most important one involved in breast cancer tumorigenesis [19]. Furthermore, downregulation of FZD7 or
LRP5/6 suppressed cell growth and tumor transformation
in TNBC cell lines [20].

Porcupine is an acyltransferase enzyme required for the

function and secretion of Wnt ligands. The porcupine
inhibitor LGK974 is a Wnt inhibitor under development for
the treatment of cancers driven by the Wnt pathway in a
Wnt ligand-dependent manner [21]. In preclinical evaluation, LGK974 attenuated tumor growth and induced tumor
Wnt/b-catenin pathway in TNBC
regression as a single agent as well as in combination with
paclitaxel in a human primary breast cancer model [22].
LGK974 is currently evaluated in a phase I trial in patients
Wnt/b-catenin signaling pathway is preferentially activated
advanced
malignancies,
including
malignant
in TNBC [18], especially in BL2 and mesenchymal sub- with
types [5]. Wnt proteins bind to the lipoprotein receptor-

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Page 4 of 7

Med Oncol (2014) 31:834

Fig. 2 Proposed future tailored treatment algorithm of advanced

TNBC. PI3KI, PI3K inhibitor; PARPI, PARP inhibitor. *1 No data
about possible simultaneous Wnt?ve/CAV?ve disease. *2 No data

about possible combinations of Wnt, CAV1 and S6K positivities. *3

Chemotherapy may be considered given that no other options for
those patients

melanoma, pancreatic
icaltrials.gov).

these genes are either targets for dasatinib or substrates for

Src kinases. Interestingly, expression of these genes was
particularlyobservedinTNBCcelllines [26].Finnetal[24]
also reported an association between CAV1 (Caveolin 1)
mRNA expression and sensitivity to dasatinib. Similarly,
Tryfonopoulos et al [25] detected an association between
dasatinib sensitivity and high protein level of CAV1.

cancer

and

TNBC

(www.clin

New insights on Src pathway in TNBC

Src is a non-receptor tyrosine kinase involved in cell

adhesion and motility [23]. Preclinical studies in breast
cancer cell lines displayed that compared to other subtypes,
TNBC is uniquely sensitive to growth inhibition by the Src
inhibitor, dasatinib, and synergistic activity with chemotherapy has been shown in TNBC cell lines [24, 25]. The
mesenchymal-like subtypes, being enriched in cell motility
genes, are the most common subtypes with high Src
activity and were found to be more sensitive to Src
inhibitors in preclinical trials [4].
Src expression is more frequent in TNBC; however, the
level of Src expression is not predictive of dasatinib sensitivity [25]. Huang et al [26] identied a six-gene panel that
could predict sensitivity to dasatinib. These genes included
EPHA2, CAV1, CAV2, ANXA1, PTRF and IGFBP2. All

Despite promising activity in TNBC cell lines, the Src

inhibitor dasatinib had disappointing results in clinical
studies involving unselected TNBC patients. In a phase II
trial including patients with advanced TNBC, dasatinib
displayed modest activity (5 % partial response, 10 %
disease control) [4]. So, proper use of Src inhibitors in
TNBC requires selecting patients who are more likely to
benet from this therapy.

Tailored therapy for advanced TNBC

Evidence from preclinical trials illustrates that there is

differential response to therapeutic agents in TNBC

Page 5 of 7

Med Oncol (2014) 31:834

834

Fig. 3 Proposed future

adjuvant therapy of TNBC.
PI3KI, PI3K inhibitor; PARPI,
PARP inhibitor

Fig. 4 Proposed design for

future clinical trials in TNBC

subtypes depending on the active signaling pathway [5].

Currently, many trials in TNBC are ongoing (clinical trials.gov), but unfortunately, most of these trials involve
unselected patients and are not directed by predictive
biomarkers, which make their success, in a heterogeneous
disease with different driving molecular pathways, extremely doubtful.
In view of this, TNBC can be considered an excellent
example of the value of tailored therapy. A more logic
approach should consider upfront stratication of TNBC
patients based on genetic or surrogate immunohistochemistry (IHC) biomarkers that reect various subtypes and
driving pathways to allow treatment to be personalized
according to the intrinsic tumor signature. Ideally,

stratifying patients based on genetic testing represents the

optimal approach, but the cost, complexity and requirement
of facilities and trained personnel for genetic testing may
make classifying patients based on IHC surrogate markers
a more attractive practical approach.
The following is a suggested stepped testing algorithm
for advanced TNBC patients that may allow for proper
treatment selection.
1. Testing for Ki-67, CAV1 and Wnt proteins expression
via IHC:

Testing for Wnt pathway proteins: FZD7 and LRP

5/6 are selected being the most commonly overexpressed in TNBC.

123

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Page 6 of 7

2.

Testing for CAV1 protein level: as a marker of

dasatinib sensitivity.
In Ki-67\35 %: testing for AR and PI3K activity:

AR testing: via IHC.

PI3K testing: through IHC testing for S6 kinase
(S6K), the downstream signaling protein of PI3K
pathway, as a surrogate marker [27].

It is to be noted that Ki-67[35 % is selected as surrogate marker for BL1 and BL2 subtypes based on average
Ki-67 of 70 % in these subtypes [5] and high response rate
to chemotherapy in patients with Ki-67[35 % [7]. Testing
for AR and PI3K activity is suggested to be reserved for
patients with Ki-67\35 % given the low proliferation rate
in LAR and mesenchymal-like subtypes.
Next, based on the above panel of predictive markers,
the following diagram illustrates proposed future tailored
treatment algorithm for advanced TNBC (Fig. 2).

Med Oncol (2014) 31:834

Based on the above, the following therapeutic strategy is

suggested
for (Fig.
early 3).
stage TNBC after nishing adjuvant
chemotherapy
Proposed design of future clinical trials in TNBC

Of course, these proposed approaches of management of

early and advanced TNBC should be evaluated in wellplanned clinical trials. To facilitate patients recruitment
and conduction of trials in this disease with variable subtypes where a plenty of drugs need to be investigated,
upfront testing of patients for predictive markers should be
considered. Multiple treatment arms should be available
and patients can be allocated to the suitable therapy based
on their testing results (Fig. 4).

Conclusion

Treatment outcome of TNBC is still unsatisfactory even in

the early stage of the disease. TNBC should be viewed as a
group of different diseases that have similar phenotype but
Proposed design for future adjuvant therapy
different genotypes with variable response to chemotherapy. Recent data revealed different molecular subtypes of
the disease with different driving molecular pathways and
As stated above, most patients with TNBC relapse within
provided insights about potential therapeutic targets. This
12 years despite adjuvant chemotherapy [2], which concept raises the real need for personalized therapies of
highlights the need for additional therapy in early stage
TNBC to improve the treatment outcome of this disease.
TNBC to prevent rapid relapse. According to cancer stem
Several questions are still to be answered. The proper
cells (CSCs) theory, these cells are intrinsically resistant to
sequence of therapies, possible combinations, duration of
chemotherapy and could later regenerate the tumor therapy and validation of predictive markers are open elds
resulting in relapse [28]. Therefore, drugs that inhibit key
for future research. In addition, possible exclusion of
active pathways in CSCs, such as PI3K and Wnt pathways,
adjuvant chemotherapy needs to be assessed in TNBC
may
suppress
their of
tumor
property,
which may
Acknowledgments
The authors would
like tochemotherapy
thank Dr. Mian Ussubtypes
with low proliferation
where
has
confers
resistance
CSCsinitiating
to radiation
and chemotherapy
man
Farooq
for
his
technical
support
in
the
preparation
of the gures
represent
promising
therapeutic
strategies
in
the
adjuvant
effect and other therapies may be more benecial.
[30]. Noteworthy, Wnt pathway inhibition can control little
of the manuscript.
setting
TNBC
[29]. salinomycin, through induction of
CSCs. of
For
example,
The authors declared that they have no conict
Conict of interest
LRP6
degradation,
was found
to be
a selective breast
CSCs
Constitutive
activation
of the
Wnt/b-catenin
signaling
of interest.
inhibitor
[30].
This
suggests
that
targeting
Wnt/b-catenin
pathway is essential for maintenance, clonogenicity of
pathway
can most
controlimportantly,
breast CSCs Wnt/b-catenin
which may optimize
the
CSCs
and,
signaling
therapy of TNBC [31].
PI3K/Akt/mTOR signaling pathway is critical for CSC
survival, and CSCs were found to be more sensitive to
inhibition of this pathway than normal stem cells [32]. This
has been demonstrated in brain and prostate CSCs exposed
to Akt inhibitors where the drugs affected CSCs, but not
healthy stem cells [33].
Furthermore, adjuvant hormonal therapy improves the
outcome of ER-positive breast cancer patients. This may
justify evaluating the efcacy of anti-androgens in adjuvant
therapy of AR-positive TNBC.

123

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