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REVIEW
Triple-negative breast cancer and basal-like subtype :Pathology and targeted therapy
Yoshimi Bando1, Tomoko Kobayashi1, Yuko Miyakami1, Satoshi Sumida1, Takumi Kakimoto1, Yasuyo Saijo2,and Hisanori Uehara1
Division of Pathology, Tokushima University Hospital, Tokushima, Japan
Department of Molecular Pathology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan

1
2

Abstract : Triple-negative breast cancer (TNBC) is a heterogenous disease. For personalized medicine, it is essential to identify and classify tumor subtypes to develop effective therapeutic strategies. Although gene expression
profiling has identified several TNBC subtypes, classification of these tumors remains complex. Most TNBCs exhibit an aggressive phenotype, but some rare types have a favorable clinical course. In this review, we summarize
the classification and characteristics related to the various TNBC subtypes, including the rare types. Therapeutic methods that are suitable for each subtype are also discussed. Of the intrinsic breast cancer subtypes identified by gene expression analysis, the basal-like subtype specifically displayed decreased expression of an estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) cluster. We also present results thatcharacterize the TNBC and basal-like phenotypes. TNBC may be categorized into four major classes : basal-like,immune-enriched, mesenchymal, and luminal androgen receptor. Therapeutic strategies for each subtype havebeen proposed along with newly approved targeted therapies for TNBC, such as immune checkpoint inhibitors.
Understanding the classification of TNBC based on gene expression profiling in association with clinicopathological factors will facilitate accurate pathological diagnosis and effective treatment selection. J. Med. Invest. 68: 213-219, August, 2021
Keywords : triple-negative breast cancer, basal-like subtype, intrinsic subtype, targeted therapy

INTRODUCTION
The biological indicators of breast cancer include tumor invasion diameter, number of metastatic lymph nodes, diseasestage, histological grade, nuclear grade, histological type, andlymphatic / vascular invasion (1, 2). The estrogen receptor (ER),progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), and Ki-67 have been evaluated in pathological
specimens using immunohistochemistry (IHC) and fluorescencein situ hybridization. Significant attention has been focused on
the classification of breast cancer based on gene expression
profiling ; however, IHC has the advantage of cost and timecompared with microarray analysis and can also reveal local
expression including in situ lesions and invasive lesions. For routine clinical practice, the immunohistochemical classification ofbreast cancer is used as a surrogate for subtype and a guide fortreatment selection (3, 4).
Triple-negative breast cancer (TNBC) is devoid of hormone
receptor (ER and PgR) expression, as well as overexpression or
amplification of HER2. Both hormone and anti-HER2 therapies
are ineffective for TNBC ; therefore, chemotherapy is required.
TNBC represents a variety of tumors, and ongoing research is
aimed at developing targeted treatments based on its biologicalcharacteristics (5). Cancer genomic panels using formalin-fixed,
paraffin-embedded tumor blocks have enabled the examinationof many important genes and biomarkers that may indicate if atargeted therapy or a clinical trial is warranted for a patient (6).
Received for publication December 7, 2020 ; accepted May 24, 2021.
Address correspondence and reprint requests to Yoshimi Bando,
MD, PhD, Division of Pathology, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503, Japan andFax : +81-88-633-9568.

TNBCs can be subtyped by various molecular classification
schemes. The basal-like subtype is a type of invasive breast
cancer originally defined by gene expression profiling studies.
Protein expression patterns characteristic of the basal-like
subtype include high expression of basal keratins and epidermal
growth factor receptor (EGFR) (7). The basal-like subtype exhibits pathological features including high histological grade, highproliferative rate, and prominent lymphocyte infiltration (8, 9)
and is enriched in patients harboring tumor suppressor protein
53 (TP53) and BRCA1 mutations (10, 11). Most basal-like subtype
breast cancers exhibit poor prognosis (12). However, some rare
TNBC types have exceptionally good prognosis. Basal-like
subtype breast cancer and TNBC share common biological andpathological features with a 70%–80% overlap (13).
This review focuses on the classification of TNBC subtypes
compared with the histology of the basal-like subtype and otherrare subtypes. We also discuss targeted therapies that are available for each TNBC subtype and introduce some of our study
results, including characteristics of TNBC and the basal-likephenotype.

TNBC SUBTYPING

Gene expression profiles and classification of the intrinsic TNBC
subtypes
Breast cancer is a heterogenous group of diseases that canbe categorized using gene expression profiling into luminal A,
luminal B, HER2-enriched, basal-like, and normal-like subtypes (14). These so-called intrinsic subtypes show differences inincidence, age at diagnosis, prognosis, and response to treatment(14, 15). Lehmann et al. identified six TNBC subtypes using thegene expression profiling results of 21 breast cancer datasets.
These included the basal-like subtype, which was divided into

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BL1 and BL2, and four other subtypes : immunomodulatory(IM), mesenchymal (M), mesenchymal stem-like (MSL), andluminal androgen receptor (LAR). For BL1, pathways associated with cell division and DNA damage repair (ATR / BRCA)
were enriched, and Ki-67 expression was high, suggesting active
cell proliferation. For BL2, growth factor signaling (EGF, NGF,
MET, Wnt / β-catenin, and IGF1R pathways), glycolysis, andgluconeogenesis-related genes were upregulated. Growth factor
receptors and myoepithelial markers (TP63, CD10) were also
highly expressed (16). A histopathological examination and gene
expression analysis of laser-capture microdissection specimens
revealed that the IM type contained infiltrating lymphocytes
and the MSL type contained many stromal cells in the tumor
tissue. The classification was organized into four subtypes : BL1,
BL2, M, and LAR (TNBC type-4) (17).
The claudin-low subtype was considered to be a new intrinsic
subtype. This subtype was characterized by low or absent expression of luminal differentiation markers and tight junction
proteins, as well as enrichment of epithelial-mesenchymaltransition markers, immune response genes, and cancer stem
cell-like features (18). Most claudin-low subtypes showed M andMSL subtypes as classified by Lehmann et al. (19).
Burstein et al. classified TNBCs into four types using RNA
and DNA profiling : basal-like immune-suppressed (BLIS), basal-like immune-activated (BLIA), mesenchymal (MES), and luminal / androgen receptor (LAR). The expression levels of genes
controlling B cell, T cell, and natural killer cell functions were
low in the BLIS subtype and high in the BLIA subtype. In the
BLIS subtype, the expression of molecules involved in antigen
presentation, immune cell differentiation, and signal communication between immune cells was low ; however, the expressionof SOX family transcription factors was high. The BLIA subtype
exhibited significant STAT transcription factor-mediated pathway activation. Among the four subtypes, BLIA had the best
prognosis, whereas BLIS had the worst (20).
The classification of TNBC is very complex and involves basal
markers, stem-cell markers, mesenchymal traits, androgen receptor (AR) expression, and immune markers (21).

TNBC and basal-like subtype, surrogate subtype classification byimmunohistochemistryHigh molecular weight cytokeratins, such as CK5 / 6, CK14,
and CK17, are expressed in cells near the basement membrane
in many epithelia and are known as basal keratins (22). IHC
analysis of the basal keratins is used to define breast cancer
exhibiting a basal phenotype as a surrogate of gene expression.
Breast cancer with a basal phenotype is common in young
people and is characterized by histologically prominent mitotic
figures and nuclear atypia. It is also accompanied by extensive
necrosis and marked lymphocyte infiltration (8, 9). The majorityof breast cancers that occur in BRCA1 mutation carriers are of
the basal-like subtype (11). Tumors arising in BRCA1 carriers
have many similarities to sporadic basal-like tumors with BRCA1
dysfunction, such as low expression of BRCA1 messenger RNA(23).
In our study, TNBC accounted for 15% of the 513 invasivecancers examined. Among the TNBCs, 61% were positive for
CK5 / 6, CK14, or CK17 by IHC and exhibited a basal phenotype.
Among the TNBCs, those exhibiting a basal phenotype had alarger tumor diameter, higher nuclear grade, and higher EGFR
positivity compared with the non-basal phenotypes. In addition,breast cancer exhibiting a basal phenotype was not significantly
different from the non-basal phenotype within TNBC, although
there was a tendency for early recurrence (24).
The basal-like subtype has much in common with TNBC
histology and clinical behavior, but it is not exactly the same.

It has been reported that 21% of TNBCs are not basal-like and
31% of breast cancers showing basal-like gene expression are nottriple-negative (13).
There is currently no consensus on the optimal immunohistochemical panel to define the basal-like subtype. At the 2017
St. Gallen Consensus Conference, it was determined that thebasal-like subtype should be defined by gene expression profilingassay only (4) (Figure 1). In the latest WHO classification ofbreast cancer, the basal-like subtype was recognized as one of
the intrinsic breast cancer subtypes based on genetic analysis
like a quantitative RT-PCR-based test with a curated list of 50
genes (the PAM50 gene signature) (25). According to the WHO
classification, breast cancers showing basal-like features aredescribed as carcinomas showing a medullary pattern, which
is one of the special morphological patterns of invasive breast
carcinoma of no special type (26).

Figure 1. Surrogate subtypes with immunohistochemistry andintrinsic subtypes of breast cancer. Modified from Fig.2.88 in WHO
classification of tumours, 5th edition, breast tumours.

HISTOLOGICAL TYPES OF TNBC
Most TNBCs are poorly differentiated, highly malignant invasive ductal carcinomas (8, 9). However, TNBC is a heterogeneous
disease and includes special morphological patterns and raretypes that include carcinoma with medullary pattern, apocrine
carcinoma, adenoid cystic carcinoma (ACC), secretory carcinoma, and metaplastic carcinoma (Figure 2A-H).
Carcinoma with medullary pattern is often associated withsignificant lymphocyte infiltration and has a good prognosis
despite its high-grade histology (27). Carcinoma with medullary
pattern is correlated with the intrinsic IM subtype as classified
by Lehmann et al. (16) and a subgroup of the basal-like subtype(28).
Carcinoma with apocrine differentiation is characterized byincreased androgen signaling and often overlaps with the HER2
group based on their gene expression profiles (29).
ACC is a salivary gland-type breast cancer that is generally
low-grade and shows an indolent clinical behavior (30). Approximately half of ACCs are found in the subareolar area. The presence of a MYB-NFIB fusion gene resulting from a specific t(6 ; 9)chromosomal translocation was found in ACC (31).
Secretory carcinoma is associated with a t(12 ; 15) translocation, which results in an ETV6-NTRK3 fusion gene (32). Most secretory carcinomas exhibit low to moderate grade histology with

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Figure 2. Representative histological appearance of rare triple-negative breast cancer (TNBC). (A) Carcinoma withmedullary pattern. Carcinoma cells show high histological grade and syncytial architecture with no glandular structures. (B,C) Carcinoma with apocrine differentiation. Carcinoma cells exhibit abundant, granular eosinophilic cytoplasm and enlarged
nuclei with prominent nucleoli (B). Immunohistochemistry for androgen receptor showing strong positive staining (C). (D)Adenoid cystic carcinoma. Neoplastic cells are arranged in tubular and cribriform patterns. The pseudolumina are filled withstromal matrix including basement membrane. (E) Secretory carcinoma. The tumor cells are arranged in a microcystic growth
pattern with extracellular secretion, simulating thyroid follicles. (F) Fibromatosis-like metaplastic carcinoma. Spindle cells with
low-grade or no atypia are arranged in wavy fascicles. (G) Matrix-producing carcinoma. The discohesive cells are contained in achondroid matrix. (H) Metaplastic carcinoma, squamous type shows carcinoma cells with abundant eosinophilic cytoplasm andfocal keratinization. Scale bars, 100 µm.

a favorable clinical course (33). Inhibitors that target NTRK3
have recently been developed and show efficacy in patients withsecretory breast carcinoma (34).
Metaplastic carcinomas are a heterogeneous group of tumors
characterized by metaplastic differentiation of the neoplastic
epithelium to squamous and / or mesenchymal cells. Metaplastic
carcinomas show genetic similarities with the MSL subtype
in Lehmann’s classification and the claudin-low subtype (35).
In general, metaplastic carcinoma exhibits a low survival rate
and resistance to chemotherapy (36), but some cases of fibromatosis-like metaplastic carcinoma have been associated with afavorable clinical outcome (37).
Each rare TNBC type has characteristic pathological findings
and a distinct clinical course. Therefore, optimal treatment forthese tumors requires an accurate pathological diagnosis.

CLASSIFICATION OF TNBC AND TARGETED THERAPYTNBC is being refined based on its molecular characteristics
and clinical response to targeted therapies. These molecularsubtypes show distinctive clinical behaviors, including treatment responses (Table 1). According to Lehmann et al., there isoverlap between immune-enriched subtypes and other subtypes.
Regardless of tumor subtype, the IM component for each tumor
was highly correlated with percentage of tumor-infiltrating

lymphocytes. The IM subtype likely reflects the presence of gene
expression contributed by immune infiltrates with the carcinoma cells having the signature of a different subtype. BL1, BL2,
MSL, and LAR-classified tumors had representatives with highcorrelations with the IM subtype. In contrast, M-classified tumors had a very low correlation with the IM group (17) (Figure 3).

Basal-like subtype
For the basal-like subtype, cell cycle and DNA damage response pathways are significantly activated, which results inincreased cell proliferation. Therefore, targeting the DNA damage response pathways is a rational therapeutic approach. This
subtype is highly sensitive to platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors (38, 39). BRCA1 and BRCA2
function as tumor suppressor genes and play major roles in the
DNA repair systems, specifically in repairing double-stranded
breaks by homologous recombination. When homologous recombination is dysfunctional (homologous recombination deficiency,
HRD), which is commonly observed in cases with BRCA1 / 2 mutations, double-stranded breaks result in genomic instability (40).
PARP1 binds to single-stranded breaks during the DNA repair
process. PARP inhibitors can trap PARP1 and induce cell deathby preventing this single-stranded break repair. Double-stranded breaks occur in patients with BRCA mutations that lack afunctional homologous recombination pathway (41).
Masuda et al. have reported a retrospective analysis of TNBC

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Table 1. Triple-negative breast cancer molecular subtype and potential targets for therapy.
SubtypeMolecular Features

Important Markers

Therapeutic Targets / Drugs

BL1

Elevated cell cycle and DNA damage response gene
expression

ATR/BRCA, Ki-67

Cisplatin, PARP inhibitors

BL2

Enriched in growth factor signaling, metabolic
pathways, and myoepithelial marker expression

EGFR, IGF1R, NGF, MET,
Wnt / b-catenin, EPHA2, TP63

Growth factor inhibitors

IM

Genes involved in cytokine and immune signalingtransduction pathways

IL-12, IL-7, NFKB, TNF,
JAK / STAT

PD-1 / PD-L1 inhibitors,
other immune
checkpoint inhibitors

M

Gene expression for EMT, cell motility, anddifferentiation

Wnt, ALK, TGF-β

PI3K / mTOR inhibitors,
growth factor inhibitors,
Src inhibitors

MSL

Increased growth factor signaling compared with (M),low proliferation, enrichment of genes associatedangiogenesis and stem cells, low claudin expression

EGFR, PDGF, ERK1/2,
TGF-β , Wnt /β -catenin

PI3K / mTOR inhibitors,
growth factor inhibitors,
Src inhibitors

LAR

Increased in hormonally regulated pathways, AR
signaling, high rate of PIK3CA activating mutations

AR , FOXA1, KRT18, XBP1

AR antagonists, PI3K
inhibitors, Hsp inhibitors

Modified from Lehmann et al. (16) ; ALK, anaplastic lymphoma kinase ; ATR, ataxia telangiectasia and Rad3- related protein ; EGFR,
epidermal growth factor receptor ; EMT, epithelial-mesenchymal transition ; EPHA2, EPH receptor A2 ; ERK, mitogen-activatedprotein kinase ; FOXA1, forkhead box A1 : Hsp90, heat shock protein 90 ; IGF1R, insulin like growth factor 1 receptor ; IL, interleukin ; JAK, Janus kinase ; MET, hepatocyte growth factor receptor ; mTOR, mechanistic target of rapamycin ; NF-kB, nuclear factor-kappa B ; NGF, nerve growth factor ; PARP, poly(ADP-ribose) polymerase ; PD-1, programmed cell death 1 ; PDGF, platelet-derived
growth factor ; PD-L1, programmed cell death 1-ligand 1 ; PI3K, phosphoinositide 3-kinase ; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ; Src, SRC proto-oncogene ; STAT, signal transducer and activator of transcription ; TGFβ,transforming growth factor beta ; TNF, tumor necrosis factor ; Wnt, Wnt family member ; XBP1, X-box binding protein 1.

Immune-enriched subtype

Figure 3. Classification of triple-negative breast cancer fromgenomic and transcriptomic data. BL1, basal-like 1 ; BL2, basallike 2 ; BLIA, basal-like immune-activated ; BLIS, basal-like
immune-suppressed ; LAR, luminal androgen receptor ; IM,
immunomodulatory ; MSL, mesenchymal stem-like ; M,mesenchymal ; TNBC, triple-negative breast cancer. Modified from
Lehmann et al. (16, 17), Burstein et al. (20), and Fig.2.88 in WHO
classification of tumours, 5th edition, breast tumours.

subtype based on response rates to neoadjuvant chemotherapy.
The BL1 subtype achieved the highest pathological complete
response rate (52%), while the BL2 subtype was found to havethe lowest response rate (0%) (42). Standard chemotherapeutics,
palatinum salts, and PARP inhibitors may be the most effective
options currently available for patients with the BL1 subtype.
BL2 has unique gene ontologies involving growth factor signaling, such as the EGF, MET, and IGF1R pathways. BL2 subtype
patients may be candidates for EGFR or IGF1R inhibitors.

This subtype includes tumors enriched for genes associatedwith immune cell processes. This phenotype is common in the
IM subtype as classified by Lehmann et al. (16) and the BLIA
subtype as classified by Burstein et al. (20). Immunotherapies, including immune checkpoint inhibitors, have attracted attention
for TNBC treatment.
Programmed death-1 (PD-1) is a T-cell inhibitory receptor that
regulates the immune system by downregulating the T-cell response upon binding with its ligand, PD-L1, which is expressedin cancer cells. While activation of this pathway protects cancer
cells from immune cell-mediated death, inhibition of PD-1 orPD-L1 can restore the antitumor effects of T-cells. In patientswith TNBC, PD-L1 is expressed on tumor-infiltrating immune
cells as well as on tumor cells. When at least 1% or more of
immune cells are immunohistochemically positive for PD-L1
in tumor tissue and at the tumor margin, targeted therapy
with anti-PD-L1 monoclonal antibodies may be considered (43)(Figure 4).

Mesenchymal subtype
The mesenchymal and mesenchymal stem-like subtypes arecharacterized by gene clusters associated with cell motility,
matrix interactions, growth factors, and epithelial-mesenchymal transition. Lehmann et al. proposed that cell models formesenchymal and mesenchymal stem-like TNBC may be sensitive to mTOR inhibitors because these cells exhibit activated
PI3K / AKT / mTOR signaling resulting from the mutation of
PIK3CA or deactivation of PTEN (16).

Luminal AR subtype
The AR belongs to the nuclear steroid hormone receptor family
and regulates cell proliferation, apoptosis, and promotes cell migration and invasion in TNBCs (44). It is expressed in TNBCs,
primarily in the LAR subtype, and is associated with apocrine
histologic features (45). Barton et al. demonstrated that AR suppression enhances sensitivity to chemotherapy by decreasing theresistant cancer stem cell-like population (46). Lehmann et al.

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Figure 4. Histological appearance of triple-negative breast cancer (TNBC) with dense immune cell infiltration. High-gradefeature with large nucleus, prominent nucleoli, and remarkable mitotic activity. H&E (A). Immune cells in the tumor tissue
and at the margin of the tumor tissue exhibit positive immunohistochemical staining for PD-L1 (B) and PD-1 (C). Immune cells
include CD4-positive T lymphocytes (D), CD8-positive T lymphocytes (E), and CD68-positive macrophages (F). Scale bars, 100 µm.

showed that PIK3CA gene mutations frequently occur in AR-positive patients with TNBC. A synergistic effect was observed withthe combination of AR antagonists and PI3K inhibitors in an
AR-positive cell line and xenograft models (47). AR and PI3K
inhibition have been proposed as therapeutic strategies (48).

CONCLUSION AND OUTLOOKThe advent of next-generation sequencing that can be used toanalyze expression changes in many genes has paved the way
for genomic medicine. Nucleic acids contained in formalin-fixedparaffin-embedded tissue blocks used for routine pathologicaldiagnosis may also be used for genomic analysis. A sufficientamount of high-quality nucleic acids is available following the
preparation and examination of paraffin blocks (49). Block selection, indicating the tumor areas on the slide glass for macrodissection, and determination of tumor content have also become
important pathology tasks (50). It is important for clinicians
and pathologists to share data and strive for high-quality pathological and genomic diagnoses. The concept of breast cancer
subtype classification is expected to change with advances andwidespread use of molecular biological tests such as oncogene
and multigene assays. Understanding and accepting the conceptof new subtypes is important, but it is also important to closely
observe the findings of histopathological specimens and provide
accurate and detailed information.

ACKNOWLEDGEMENTS
We thank Edanz Group (https://en-author-services.edanz.
com/ac) for editing a draft of this manuscript.

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All authors declare no conflicts of interests.

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