Review

Metastatic Breast Cancer: Cytology Diagnosis with Implications

for Treatment
Alaa Hrizat and Elena Brachtel *

Department of Pathology, Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA

* Correspondence: [email protected]

Abstract: Breast cancer is among the most frequent malignancies in women worldwide. While
early detection and effective treatment provide many women with a cure and prevent their cancer
from spreading, metastases to distant sites still occur in around 20% of women suffering from
breast cancer. These relapses occur in many forms and locations and are as varied as the primary
breast tumors. Metastatic spread makes a cancer incurable and potentially lethal, but new, targeted
treatments can offer control of the cancer cells if the features of new targets are unlocked by advanced
diagnostic testing. The article offers an overview of the pathomechanisms of metastatic progression
and describes the types of metastases, such as hormone-receptor-positive and -negative breast cancers,
and HER2-overexpressing or triple-negative types. Once distant metastatic spread occurs, cytology
allows a precise diagnosis to confirm the breast origin. Other molecular targets include ESR1 and
PIK3CA mutations, MSI, NTRK fusion, PD-L1 expression and others, which can be obtained also
from cytology material and used to determine eligibility for emerging targeted therapeutic options.
We outline the diagnostic features of metastatic breast cancer in cytology samples, together with
validated and emergent biomarkers that may provide new, targeted treatment options.

Keywords: breast cancer; metastatic; diagnosis; molecular; cytology

1. Introduction and Overview

Citation: Hrizat, A.; Brachtel, E. More than 2.3 million patients with breast cancer are newly diagnosed globally, and
Metastatic Breast Cancer: Cytology an estimated 685,000 women died of the disease in 2020 [1,2]. Approximately 3.5 million
Diagnosis with Implications for women in the United States are in treatment or living with a history of breast cancer. While
Treatment. J. Mol. Pathol. 2023, 4, the overall survival rate is favorable with early detection and advanced treatment options,
1–14. https://doi.org/10.3390/ 15–25% of women eventually develop distant metastatic disease, and around 40,000 women
jmp4010001 die from breast cancer each year in the U.S. [1,2].
These overall numbers of breast cancer incidence, obtained through public databases,
Academic Editors: Fernando Schmitt
and Gary M. K. Tse
include a portion of around 25% that are ductal carcinoma in situ, without demonstrating
stromal invasion and (theoretically) with no possibility of metastasis. The term “breast
Received: 9 November 2022 cancer” in this article generally refers to invasive breast cancer [3,4].
Revised: 19 December 2022 With increased awareness and breast cancer screening programs in place for decades,
Accepted: 19 December 2022 only approximately 5% of western women present with breast cancer at an advanced
Published: 24 December 2022
stage—that is, with distant metastatic disease (or stage IV cancer) [5]. Regional lymph
nodes are usually the first site of cancer spread outside the breast; these lymph node
metastases, typically in the axillary lymph nodes, are considered a local disease for staging
Copyright: © 2022 by the authors.
and treatment purposes. In this article, emphasis is placed on distant metastatic spread to
Licensee MDPI, Basel, Switzerland. other organs away from the breast.
This article is an open access article Globally, there are significant regional differences in morbidity and mortality. While
distributed under the terms and the rate among women in low-resource countries might be lower than in the western world,
conditions of the Creative Commons their likelihood of dying from this disease is also higher [2]. Estimates prognosticate a
Attribution (CC BY) license (https:// steep increase in both frequency and risk of death in the next 20 years. With this worrisome
creativecommons.org/licenses/by/ perspective of a higher disease burden on a global scale, effective methods for diagnosis in
4.0/). every layer of the medical resource spectrum are necessary [1,2,6].

J. Mol. Pathol. 2023, 4, 1–14. https://doi.org/10.3390/jmp4010001 https://www.mdpi.com/journal/jmp

J. Mol. Pathol. 2023, 4 2

In most clinical studies of primary breast cancer treatment, distant metastatic disease
represents an endpoint and treatment failure. Although much progress has been made with
the early detection of breast cancer, advanced hormonal therapy, chemotherapy regimens
and targeted treatments to improve the prognosis of breast cancer patients, only modest
improvements are found once the cancer shows a distant metastatic relapse. These patients
are excluded from primary treatment schemes, and their psychological, social and economic
needs are often not realized [6]. New treatment options are needed to improve the outlook
for patients with metastatic breast cancer [7,8].
With the majority of breast cancers being hormone-receptor-positive, hormonal treat-
ment has played an important role for many years [9,10]. As it became better known that
hormonally responsive breast cancers may show a pattern of late relapses beyond the first
five years after the primary cancer diagnosis and initial treatment, new anti-hormonal
agents were discovered and treatment regimens refined [11,12]. These may include agents
such as aromatase inhibitors, which subsequently may cause resistance through mutations,
or selective estrogen receptor degraders, with which treatment can be continued and also
escalated in the metastatic setting [13–15].
The discovery of human epidermal growth factor receptor 2 (HER2/c-erbB2) gene
amplification and protein overexpression as a predictive factor and treatment target for
humanized monoclonal antibodies was a breakthrough in breast cancer treatment. Cur-
rently, several treatment regimens target HER2-positive tumors in the neoadjuvant and
adjuvant setting and have greatly improved the survival of patients with this type of breast
cancer [16,17].
In addition to established predictive and prognostic markers for primary breast cancer,
new molecular targets are being identified, adding new treatment options and improving
the diagnosis for breast cancer patients with metastatic disease [7,10,18]. One such example
is immune checkpoint or cyclin dependent kinase (CDK 4/6) inhibitors, which may allow
further treatment options with agents such as pembrolizumab; primarily, this applies to the
triple-negative subset of breast cancers that are found to express programmed death ligand
(PD-L1) on immune cells in the tumor [19,20]. A new and promising agent to expand the
treatment options for triple-negative breast cancers (TNBC), especially in BRCA 1/2 gene
mutation carriers, is poly-ADP ribose or PARP inhibitors [21].
A large study that compared genomic profiles between primary and metastatic breast
cancers showed more frequent estrogen receptor (ESR1), phosphatase and tensin homolog
(PTEN), cadherin-1 gene (CDH1) and retinoblastoma (RB1) mutations; mouse double
minute 4 (MDM4) and myelocytomatosis (MYC) gene amplifications; and AT-rich interac-
tion domain 1A (ARID1A) deletions in metastatic breast cancers [22].
There are validated biomarkers with actionable treatment options including phosphati-
dylinositol-4,5-bisphosphate 3-kinase catalytic unit alpha (PI3KCA), estrogen receptor mu-
tations (ESR1), microsatellite instability (MSI) and neurotrophic tyrosine receptor kinase
(NTRK) fusion. Emergent biomarkers include Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2),
protein kinase B (AKT), phosphatase and tensin homolog (PTEN), homologous recombina-
tion repair (HRR), CD274 amplification, retinoblastoma (RB1) and neurofibromin 1 (NF1)
mutations [18,22].
Genomic testing of metastatic lesions is necessary to identify actionable targets, and
tissue sampling is at the core of this diagnostic process to determine the presence of
therapeutic targets [23]. Cytology is often the method of choice to sample metastatic
lesions because of the location, accessibility and choice of a minimally invasive sampling
technique [24–26]. The role of liquid-based biopsy from the peripheral blood or other body
fluids is considered later in this article.

2. How Does Metastatic Spread Occur?

Most of the diagnostic parameters and initial treatments in early breast cancer focus
on the primary tumor. Features that are associated with spread outside of the breast are
lymphatic vessel invasion and lymph node metastases. Involvement of axillary lymph
J. Mol. Pathol. 2023, 4 3

nodes, which provide the first site of lymphatic drainage to the breast, is still indicative
of local disease but indicates a higher risk of distant relapse [5]. Regional lymph node
involvement is unfavorably correlated with diagnosis as a staging parameter.
Pathogenetically, several models help to explain how tumor cells disseminate into
other sites of the body: tumor cells may gain the capacity to metastasize within the primary
tumor in a linear progression model to later spread through the genetic evolution of
metastasis-capable founder cells. Another possibility assumes the early dissemination of
tumor cells with the acquisition of new mutations at the new site that would allow this.
In a historical hypothesis formulated by Stephen Paget in 1889, cancer cells (“seed”) may
settle preferentially in the selected microenvironment (“soil”).
Following this model, tumor cells encounter a pre-metastatic niche, which they colo-
nize; after various lengths of dormancy, and mediated by tumor-initiated soluble factors
and by possibly creating an immune-suppressed field, these tumor cells eventually cause a
metastatic outgrowth at the new site. In the reality of metastatic spread, various pathways
might be used by traveling tumor cells to remain dormant or establish new growth sites [27].
Eventually, a metastatic cascade gives rise to innumerable foci of tumor cells that become
resistant to treatment and lead to the patient’s demise. The genetic underpinnings of why,
when and where tumor cells precisely cause distant spread are the subject of ongoing
investigation [28].
As to the preferred sites of breast cancer metastases, the molecular subgroups based
on hormone receptors and HER2 expression are associated with certain metastatic patterns
and tropisms. Estrogen-receptor-positive types (luminal A/B type carcinomas following
the intrinsic subtype model) more frequently cause bone metastases. As a morphological
subtype, lobular carcinomas, which are mostly ER-positive, have different metastatic pat-
terns to breast cancers of a ductal/no special type, with more frequent visceral, serosal and
gynecological metastases (40). Triple-negative (basal-like) and HER2-positive carcinomas
were shown to cause brain metastases more frequently, posing particular challenges to
treatment [22,29,30].

3. Diagnosis of Breast Cancer Metastases

Different types of breast cancer based on morphology, histologic grade, hormone
receptor and HER2 expression show different patterns in relapse and metastatic behavior.
While some changes can occur in morphology and immunohistochemical profile, most
metastatic lesions resemble the primary tumor, and key morphologic features in metastases
often lead to a further diagnostic investigation [31,32]. For the diagnosis of primary breast
cancers and their respective metastases, the current classification for tumor diagnosis is
followed [33].
Morphological comparison with the primary may be most helpful when metastasis
from a known breast primary is suspected. In conjunction with clinical correlation, a
selected immunohistochemical profile of the metastatic focus will help to narrow down the
differential diagnosis. A panel of immunohistochemical markers, including estrogen and
progesterone receptors, cytokeratins and GATA3, may allow us to confirm the primary site
in the breast. Other successfully employed markers are mammaglobin and GCDFP-15, but
now often the nuclear marker GATA3 is used. GATA3 is not specific to the breast but, in the
appropriate clinical context, has proven very useful as a diagnostic marker for metastatic
breast cancer [34]. In combination with SOX10 (SRY-box transcription factor 10), it has
been shown to be helpful to diagnose triple-negative breast cancer [35]. More recently, the
novel marker TRPS-1 (trichorhinophalangeal syndrome type 1) was used to confirm the
breast origin [36]. The hormone receptor and HER2 profile of a known breast primary can
be utilized to confirm the metastatic site. It is important to note at this point is that most
metastatic foci resemble the primary tumor immunomorphologically, but exceptions and
“switches” from hormone-receptor- or HER2-positive to HER2-negative, and vice versa,
may occur in a considerable proportion of cases [22,26].
J. Mol. Pathol. 2023, 4 4

The breast ©tself is not a frequent site of metastasis. Nonetheless, some tumors can
metastasize to the breast, such as disseminated lymphomas, melanomas and small-cell
lung cancers. However, this topic is beyond the scope of this discussion [31].
Estrogen-receptor-positive breast cancers can recur late (after 5, 10 or even 20 years),
often causing bone metastases. Other visceral sites, such as multiple liver or lung metastasis,
are seen as part of the metastatic cascade.
Triple-negative breast cancers tend to be of high histologic grade and typically recur
in the first three to five years after the primary diagnosis. Both triple-negative and HER2-
positive breast cancers appear to cause brain metastases more frequently than hormone-
receptor-positive tumors [29].
With the various organs involved, cytology is often the most accessible means of
tissue sampling for breast cancer diagnosis. Depending on the practice patterns, predictive
markers such as hormone receptors and HER2 studies may be determined on the metastatic
lesions as a standard of care and following international practice guidelines [37,38]. In addi-
tion, molecular profiles are often requested to determine patients eligible for novel targeted
treatments such as checkpoint inhibitors, PIK3CA inhibitors or immunotherapy [22,39].
Individual protocols vary and depend on departmental preferences and practice patterns:
some departments routinely apply molecular tests on liquid cytology remnants, while other
use cell block material or send samples for molecular testing.
The subsequent paragraphs illustrate cytological samples of breast cancer subtypes
from metastatic lesions, including examples of breast cancer of ductal/no special type,
special types such as lobular carcinoma or others and the groups based on hormone receptor
and HER2 expression.

3.1. Metastatic Ductal Breast Cancer (No Special Type)

Invasive ductal carcinomas/carcinoma of no special type constitute approximately
80% of breast carcinomas (Figure 1) and range in histologic grade from well (grade 1) to
moderately (grade 2) to poorly differentiated (grade 3). Usually, metastatic lesions are not
independently graded. Nuclear pleomorphism, necrosis or other features can of course be
used to describe, for example, a poorly differentiated tumor on cytology.

3.2. Metastatic Lobular Carcinoma

Lobular carcinomas are typically low-grade (grade 2 or 1) and represent approxi-
mately 15% of breast carcinomas (Figure 2). Metastatic lobular carcinoma shows more
frequent involvement of visceral sites, the intestinal or gynecological tract or pleural
membranes [40,41]. Characteristically, in effusions, there are medium-sized dyscohesive
tumor cells against a background of reactive mesothelial cells.

3.3. Metastatic Breast Cancer, Other Special Types

Invasive breast cancers occur in several other special types which can be distinguished
by morphological features; only a few can be shown here as examples (33). Some of
these special types are associated with a more favorable prognosis, for example mucinous
carcinomas of the breast (Figure 3), medullary carcinomas or adenoid cystic carcinomas.
On the other hand, matrix producing breast cancers are typically high-grade breast cancers
with an aggressive behavior. Micropapillary carcinomas are characterized by a high rate of
lymph node metastasis and poor prognosis.

3.4. ER-Positive Metastatic Breast Cancer

Most breast carcinomas are positive for estrogen receptors (Figure 4). Hormone
receptors are reported in a semi-quantitative or quantitative way following international
guidelines [37]. While it is feasible to perform immunocytochemical stains for nuclear
receptors on cytology preparations (direct smears as well as liquid-based preparations),
staining protocols need to be separately validated for cytology. Cell block preparations can
J. Mol. Pathol. 2023, 4 5

J. Mol. Pathol. 2023, 4, FOR PEER REVIEW 5

be prepared following several techniques and are feasible for immunohistochemical as well
as for molecular testing [24,25,42].

(a)

(b)

Figure 1. Cont.
J. Mol. Pathol. 2023, 4 6
J. Mol. Pathol. 2023, 4, FOR PEER REVIEW 6

(c)

Figure1.1.(a)(a)
Figure Cytology
Cytology smear
smear of a of a liver
liver fine-needle
fine-needle aspiration
aspiration shows metastatic
shows metastatic ductal carcinoma
ductal carcinoma
(Papanicolaou stain). The smear is very cellular, with three-dimensional clusters
(Papanicolaou stain). The smear is very cellular, with three-dimensional clusters and sheets of tumor and sheets of
cells. (b) Cytology ThinPrep of a pleural effusion with metastatic breast cancer of ductal
tumor cells. (b) Cytology ThinPrep of a pleural effusion with metastatic breast cancer of ductal type. Char-
acteristic are large, three-dimensional groups of tumor cells, so-called “cannonballs” in an effusion
type. Characteristic are large, three-dimensional groups of tumor cells, so-called “cannonballs” in an
specimen. (Papanicolaou stain). (c) Cytology cell block of a pleural effusion with metastatic breast
effusion
cancer specimen.
of ductal type. (Papanicolaou
Groups of tumorstain). (c) Cytology
cells against cell block
a background of a pleural
of reactive effusion
mesothelial cells,with
his- metastatic
breast cancer of ductal type. Groups of tumor cells against a background of reactive
tiocytes and inflammation (hematoxylin and eosin stain). Of note is that the cell block material here mesothelial cells,
ishistiocytes
markedly cellular; the cytology sample appears suitable for diagnostic testing as well as
and inflammation (hematoxylin and eosin stain). Of note is that the cell block materialadvanced
J. Mol. Pathol. 2023, 4, FOR PEER REVIEW
molecular testing. cellular; the cytology sample appears suitable for diagnostic testing 7
here is markedly as well as
advanced molecular testing.
3.2. Metastatic Lobular Carcinoma
Lobular carcinomas are typically low-grade (grade 2 or 1) and represent approxi-
mately 15% of breast carcinomas (Figure 2). Metastatic lobular carcinoma shows more fre-
quent involvement of visceral sites, the intestinal or gynecological tract or pleural mem-
branes [40,41]. Characteristically, in effusions, there are medium-sized dyscohesive tumor
cells against a background of reactive mesothelial cells.

(a)

Figure 2. Cont.
J. Mol. Pathol. 2023, 4 7

(a)

J. Mol. Pathol. 2023, 4, FOR PEER REVIEW 8

shows metastatic lobular carcinoma that is positive for estrogen receptor protein (immunohisto-
chemical stain).

3.3. Metastatic Breast Cancer, Other Special Types

Invasive breast cancers occur in several other special types which can be distin-
(b)
guished by morphological features; only a few can be shown here as examples (33). Some
ofFigure
these2.2.
Figure special
(a) typesThinPrep
(a)Cytology
Cytology areThinPrep
associated of with a more
a pleural
of a pleural effusion favorable
effusion
aspirate prognosis,
aspirate
shows showsfor
metastatic example
metastatic
lobular mucin- carcinoma
lobular
carcinoma
ous carcinomas
(Papanicolaou
(Papanicolaou of the
stain).
stain). breast
There
There (Figure
are scattered 3), medullary
dyscohesive
are scattered carcinomas
tumor
dyscohesive cells or
that are
tumor adenoid
slightly
cells cystic
that larger
are thancarcino-
slightlythelarger than the
mas. On the mesothelial
surrounding
surrounding other hand,
mesothelial matrix
cells
cells producing
and lymphocytes.
and breast
(b)
lymphocytes. cancers
Cytology
(b) are of
cell block
Cytology typically
a pleural high-grade
cell blockeffusion breast
aspirate
of a pleural effusion aspirate
cancers with an aggressive behavior. Micropapillary carcinomas are characterized by a
shows metastatic lobular carcinoma that is positive for estrogen receptor protein (immunohistochem-
high rate of lymph node metastasis and poor prognosis.
ical stain).

(a)

Figure 3. Cont.
J. Mol. Pathol.
J. Mol. 2023,
Pathol. 4 4, FOR PEER REVIEW
2023, 9 8

(b)

(c)

Figure 3. (a)
Figure 3. (a) Giemsa-stained, air-driedsmear
Giemsa-stained, air-dried smearofofmetaplastic
metaplastic carcinoma
carcinoma (matrix-producing
(matrix-producing carcinoma).
carci-
(b) Papanicolaou-stained,
noma). ethanol-fixed
(b) Papanicolaou-stained, direct
ethanol-fixed smear
direct of of
smear mucinous
mucinousbreast
breast carcinoma with groups
carcinoma with
groups
of tumorof cells
tumorfloating
cells floating in abundant
in abundant extracellular
extracellular mucin.This
mucin. Thistype
typeof
of breast
breast carcinoma
carcinoma isis usually
usually hormone-receptor-positive, with a good prognosis and a relatively low rate of distant me-
hormone-receptor-positive, with a good prognosis and a relatively low rate of distant metastases.
tastases. (c) Cytology liquid-based SurePath preparation of metastatic micropapillary carcinoma,
(c) Cytology liquid-based SurePath preparation of metastatic micropapillary carcinoma, Papanicolaou-
stained. The tumor cells consist of finger-shaped, three-dimensional groups. While cytologically
of intermediate grade, this morphological type of breast cancer shows frequent lymphatic vessel
invasion and has high metastatic potential.
 3.4. ER-Positive Metastatic Breast Cancer
Most breast carcinomas are positive for estrogen receptors (Figure 4). Hormone re-
ceptors are reported in a semi-quantitative or quantitative way following international
guidelines [37]. While it is feasible to perform immunocytochemical stains for nuclear re-
ceptors on cytology preparations (direct smears as well as liquid-based preparations),
J. Mol. Pathol. 2023, 4 staining protocols need to be separately validated for cytology. Cell block preparations 9
can be prepared following several techniques and are feasible for immunohistochemical
as well as for molecular testing [24,25,42].

J. Mol. Pathol. 2023, 4, FOR PEER REVIEW 11

(a)

(b)

Figure4.4.(a)(a)
Figure Cytology
Cytology cell block
cell block of a supraclavicular
of a supraclavicular lymph node lymph nodeaspirate
fine-needle fine-needle
shows aspirate
meta- shows
metastatic
static breast breast carcinoma,
carcinoma, present
present in in cords
cords and groupsand groups
of tumor of tumor
cells against cells against aofbackground
a background lym- of
phoid
lymphoidtissuetissue
(hematoxylin and eosin
(hematoxylin and stain). (b) Cell(b)
eosin stain). block
Cellofblock
supraclavicular lymph node
of supraclavicular aspirate
lymph node aspirate
with metastatic breast carcinoma (same case as in Figure 4a) shows strong staining for estrogen
with metastatic breast carcinoma (same case as in Figure 4a) shows strong staining for estrogen
receptor protein by immunohistochemical stain.
receptor protein by immunohistochemical stain.
3.5. HER2-Positive Metastatic Breast Cancer
3.5. HER2-Positive Metastatic Breast Cancer
Human epidermal growth factor receptor (HER2) positive tumors represent around
Human epidermal growth factor receptor (HER2) positive tumors represent around
10–20% of breast cancers (Figure 5). The scoring of tumor tissue is performed by immuno-
10–20% of breast
histochemical cancers
and/or in situ(Figure 5). The techniques,
hybridization scoring of tumor tissue
following is performed
international by immunohis-
guidelines
tochemical and/or in situ hybridization techniques, following international guidelines
[38]. Fluorescent in situ hybridization can also be achieved on cytology preparations with [38].
adapted protocols. Often preferred, however, is HER2 testing on formalin-fixed, paraffin-
embedded cell block and core biopsy specimens, as routine protocols for both immuno-
histochemistry and in situ hybridization procedures can be followed, as per guidelines.
J. Mol. Pathol. 2023, 4 10

Fluorescent in situ hybridization can also be achieved on cytology preparations with adapted
protocols. Often preferred, however, is HER2 testing on formalin-fixed, paraffin-embedded
J. Mol. Pathol. 2023, 4, FOR PEER REVIEW 12
cell block and core biopsy specimens, as routine protocols for both immunohistochemistry
and in situ hybridization procedures can be followed, as per guidelines.

(a)

(b)

Figure (a)Cell
5.(a)
Figure 5. Cellblock
blockshows
shows metastatic
metastatic breast
breast carcinoma
carcinoma withwith strong
strong circumferential
circumferential overexpression
overexpres-
sion (3+)
(3+) of HER2
of HER2 (immunohistochemicalstain).
(immunohistochemical stain). (b)
(b) Cell
Cellblock
blockwith
withHER2-amplified
HER2-amplified breast carci-carcinoma
breast
noma (dual-color fluorescent in situ hybridization, with centromere probe in green
(dual-color fluorescent in situ hybridization, with centromere probe in green (CEP17) and (CEP17) and
HER2 gene
HER2 gene in orange). Usual protocols for HER2 testing apply to cell block and core biopsy samples.
in orange). Usual protocols for HER2 testing apply to cell block and core biopsy samples.
3.6. Triple-Negative Metastatic Breast Cancer
J. Mol. Pathol. 2023, 4 11

3.6. Triple-Negative Metastatic Breast Cancer

Triple-negative breast cancers comprise around 10% of overall breast cancers, which
are often of ductal type (no special type) and poorly differentiated. Some special-type breast
cancers such as matrix-producing breast cancers, spindle cell carcinomas or adenoid cystic
carcinoma (33). As therapeutic targets such as hormonal treatment or HER2 overexpression
do not apply, PD-L1 can be used for possible immune checkpoint inhibitor treatment. PD-L1
testing is performed on primary tumors and is feasible also on cytology samples, to test for
immunotherapy eligibility [26].

3.7. Molecular Profiles in Cytology Samples and Liquid Biopsies

Circulating tumor cells or cell-free DNA (ctDNA) are in various stages of development
to follow treatment responses and for clinical applications [43]. For example, estrogen
receptor mutations (ESR1) following hormonal treatment with aromatase inhibitors or
PIK3CA mutations have been tested by blood samples to follow treatment [10,14,44,45].
Another active area of investigation is liquid biopsies, which are proposed to be used for
staging and molecular characterization in blood and in certain body compartments, such
as cerebrospinal fluid disease (although, for many places, these methods are still beyond
the standard diagnostic repertory) [46–50].

4. Novel Therapeutic Targets in Metastatic Breast Cancer

Multiple novel mutations have been validated and are promising for future person-
alized, targeted treatment. The phosphatidylinositol 3-kinase gene (PI3KCA) is involved
in the cell cycle and cell proliferation, encoding for the class I catalytic isoform p110α. It
is mutated in around 40% of hormone-receptor (HR) positive breast cancer cases [51,52].
Microsatellite instability (MSI) occurs in less than 1% of cases. Neurotrophin receptor
tyrosine kinase (NTRK) translocation is found exclusively in secretory carcinoma [53]. The
activating mutation of the estrogen receptor α (ESR1) gene results in the constitutional
activity of the estrogen receptor independently of the ligand; it is commonly found as a
result of aromatase inhibitor in HR+/HER2− metastatic breast cancer [10,54,55]. Clinical
trials already provide levels of evidence, and molecular testing in tissue and liquid biopsies
is being established.
Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib, ribociclib and abe-
maciclib, are already being used in hormone-receptor-positive metastatic breast cancer [56].
They interrupt cell cycle progression, leading to the inhibition of tumor growth. Adding
them to endocrine therapy has shown improved progression-free survival with minimal
toxicity. The agent pembrolizumab, also an immune checkpoint inhibitor, is being used
for triple-negative breast cancers, for which there are often few treatment options [20]. For
metastatic triple-negative breast cancers in BRCA 1/2 mutation carriers, a patient population
with often aggressive tumors and few treatment options, PARP inhibitors are now used,
with some success [21].
HER2-positive breast cancers are defined by HER2 overexpression and/or amplifica-
tion, which drives the growth of cancer cells. Therapies that target HER2 include human-
ized monoclonal antibodies such as trastuzumab, pertuzumab or other conjugates that are
used in neoadjuvant and adjuvant treatment settings [16,17]. Adding these agents to the
neoadjuvant chemotherapy reduces the tumor size and increases the breast-conserving rate.
Emergent biomarkers include Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), AKT, phos-
phatase and tensin homolog (PTEN), homologous recombination repair (HRR), CD274
amplification, retinoblastoma protein (RB1) and neurofibromin 1 (NF1) mutations [18,22].

5. Conclusions and Outlook

This article describes the current practice in sampling metastatic breast cancer lesions
and determining their molecular profiles to better apply targeted treatments. This ranges
from routine ancillary breast markers such as estrogen receptor proteins to molecular
profiles to assess the response and resistance to novel hormonal treatments. HER2-targeted
J. Mol. Pathol. 2023, 4 12

treatments have expanded from a single anti-HER2 agent to a range of treatment possibili-
ties, also in the metastatic setting. Other actionable treatments include PIK3CA mutations
and immune checkpoint inhibitors for hormone-receptor-positive and triple-negative breast
cancer subtypes. Current oncology guidelines include circulating tumor DNA as a novel
technology to determine the mutational profiles of tumor cells in metastatic breast cancer.

Author Contributions: A.H.: writing and editing. E.B.: conceptualizing, writing and editing. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank Susana Alexandrakis (TJUH), Tonneke van de Beeten (MUMC)
and Diana Cuoco (MGH) for administrative support.
Conflicts of Interest: The authors declare no conflict of interest.

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