Peran Sel pada Sistem Imun dalam Jaringan Payudara dan Imunoterapi

sebagai Pilihan Terapi pada Kanker Payudara

(The Role of Immune Cells in Breast Tissue and Immunotherapy for the Treatment of Breast Cancer)

Disusun untuk memenuhi tugas dan melengkapi persyaratan dalam menempuh Program
Pendidikan Magister Biomedik

Widoasti Putri Utami

22010120410005

PROGRAM PENDIDIKAN MAGISTER BIOMEDIK

FAKULTAS KEDOKTERAN
UNIVERSITAS DIPONEGORO
SEMARANG
2020
 Review

The Role of Immune Cells in Breast Tissue and

Immunotherapy for the Treatment of Breast
Cancer
Stephanie L. Goff, David N. Danforth
Abstract
Immune cells are present in normal breast tissue and in breast carcinoma. The nature and distribution of the immune
cell subtypes in these tissues are reviewed to promote a better understanding of their important role in breast cancer
prevention and treatment. We conducted a review of the literature to define the type, location, distribution, and role of
immune cells in normal breast tissue and in in situ and invasive breast cancer. Immune cells in normal breast tissue are
located predominantly within the epithelial component in breast ductal lobules. Immune cell subtypes representing
innate immunity (NK, CD68þ, and CD11cþ cells) and adaptive immunity (most commonly CD8þ, but CD4þ and CD20þ
as well) are present; CD8þ cells are the most common subtype and are primarily effector memory cells. Immune cells
may recognize neoantigens and endogenous and exogenous ligands and may serve in chronic inflammation and
immunosurveillance. Progression to breast cancer is characterized by increased immune cell infiltrates in tumor pa-
renchyma and stroma, including CD4þ and CD8þ granzyme Bþ cytotoxic T cells, B cells, macrophages and dendritic
cells. Tumor-infiltrating lymphocytes in breast cancer may serve as prognostic indicators for response to chemo-
therapy and for survival. Experimental strategies of adoptive transfer of breast tumor-infiltrating lymphocyte may allow
regression of metastatic breast cancer and encourage development of innovative T-cell strategies for the immuno-
therapy of breast cancer. In conclusion, immune cells in breast tissues play an important role throughout breast
carcinogenesis. An understanding of these roles has important implications for the prevention and the treatment of
breast cancer.

Clinical Breast Cancer, Vol. -,

No. -, --- Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Immunology, Breast ductal cells, Intraepithelial lymphocytes, Tumor immunology, Tumor-infiltrating lymphocytes

Introduction factors, cytokines, chemokines, and prostaglandins. Among the

Breast cancer is the most common malignancy in women, with
over 316,000 cases annually in the United States.1 Most breast
cancers arise in the breast milk ducts, either as ductal carcinoma-in-
situ (DCIS) or as invasive ductal carcinoma. Breast cancer develops
through the accumulation of mutational changes, most commonly
in the ductal epithelium. The development and progression of these
changes can be influenced by multiple elements in the ductal
microenvironment, both cellular such as immune cells, adipocytes,
fibroblasts, and the microbiome, and soluble including growth
Surgery Branch, Center for Cancer Research, National Cancer Institute, National In-
stitutes of Health, Bethesda, MD
Submitted: Apr 3, 2020; Revised: Jun 10, 2020; Accepted: Jun 29, 2020
Address for correspondence: David N. Danforth, MD, Surgery Branch, CCR, NCI,
NIH, Bethesda, MD 20892
E-mail contact: david_danforth@nih.gov
most important of these elements are the immune cells, which are
considered to play a critical role throughout the course of breast
carcinogenesis, beginning in normal breast tissue with immuno-
surveillance and continuing through primary and metastatic breast
cancer. The ductal cellular layer in the normal breast contains a
significant immune cell population consisting of CD8þ and CD4þ
T cells, B cells, dendritic cells, macrophages, NK (natural killer)
cells, and other immune cell subtypes.2-4 Together, these immune
cells provide important innate and adaptive immunity to the
epithelial layer for both protection against exogenous and endoge-
nous agents, and elimination of transformed cells. An understanding
of the immune cellular population in normal breast tissue may thus
have important implications for breast cancer prevention, improved
methods of risk assessment, and the regulation of breast
carcinogenesis.
Progression through the carcinogenic pathway from normal
breast tissue to breast cancer is accompanied by quantitative and

-1
1526-8209/Published by Elsevier Inc. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
https://doi.org/10.1016/j.clbc.2020.06.011 Clinical Breast Cancer Month 2020
 Role of Immune Cells
qualitative changes in the nature and location of the immune cell
population, including an increase in immune cell content in both
the parenchymal and stromal compartments. The immune cell
infiltration in breast cancer consists of multiple cellular subtypes,
including CD3þ (CD4þ and CD8þ) cells, B cells, monocytes/
macrophages, dendritic cells, and NK cells.4-6 The presence of
multiple immune cell subtypes in both the parenchyma and stroma
places these cells in close proximity to tumor cells and other cells in
the microenvironment, and allows these cells to influence tumor
growth in multiple ways, either directly through CD4þ and CD8þ
cell-mediated cytotoxicity, or indirectly through immunosuppres-
sive or immunostimulatory actions from secreted cytokines, growth
factors and other agents. Their distribution and characteristics may
also vary according to the subtype of breast cancer, their estrogen
responsiveness, their mutational load, and the formation of tertiary
lymphoid structures. Importantly, detailed analysis of tumor-
infiltrating lymphocytes (TILs) has demonstrated prognostic prop-
erties for these cells,7-10 and collecting and administering TILs to
patients has resulted in durable complete regression of solid tumors
as well providing knowledge to allow development of cellular
therapy with gene-modified T-cell receptors (TCRs) for treatment
of breast and other cancers.11 Together these findings indicate an
important role for immune cells in the breast throughout breast
carcinogenesis, and significant changes in these cells during the
course of these events. To clarify these significant properties, we
have conducted a review of the literature of the nature and char-
acteristics of ductal immune cells in normal breast tissue, of TILs in
breast cancer, and the efforts to translate the latter findings into
both valuable prognostic indicators and successful immunothera-
peutic treatment options for breast cancer, including ongoing and
innovative trials at our institution.
Materials and Methods
A literature search was conducted through PubMed and cross-
references to identify publications describing the nature and distri-
bution of immune cells in normal breast tissue and in breast cancer.
Normal breast tissue may include tissue from women at normal risk
(such as from reduction mammoplasty), as well as from the
contralateral breast or from tissue adjacent to a breast cancer in
women with a breast cancer. For studies of breast cancer, emphasis
was placed on immune cells in the primary tumor.
Results and Discussion
Immune Cells in the Normal Breast
Breast Ductal Anatomy. The ductal anatomy and its relationship to
the distribution of immune cells in the normal breast is illustrated in
Figure 1. The principle unit in the breast ductal system is the acini,
which drains into an intralobular and then the extralobular duct;
together, these structures constitute the terminal ductal lobular unit
(TDLU). The intralobular terminal ducts are lined by cuboidal
epithelium, and the extralobular terminal duct and major ducts are
lined by pseudostratified columnar epithelium or a double layer of
cuboidal epithelium. The TDLU is considered to be the predominant
site of the origin of breast cancer.12 The vast majority of breast cancers
are carcinomas that arise in the epithelium and include in-situ and
invasive ductal and invasive lobular carcinoma. Gene expression
2 - Clinical Breast Cancer Month 2020
studies have indicated that hormone-related pathways are highly
enriched in the TDLU, including various hormone-related genes that
are associated with breast carcinogenesis. It has been proposed that the
imbalanced hormone-reactions may result in the early onset of
neoplastic transformation that occurs mostly in breast TDLUs.12 The
breast ducts are surrounded by stroma that consists of extracellular
matrix (ECM), fibroblasts, adipocytes, immune cells, microbiome,
and blood vessels. The breast ducts and stroma together comprise the
breast microenvironment.
Distribution of Immune Cells within Normal Breast Tissue. Normal
breast tissue contains immune cells of both myeloid (monocytes,
macrophages, dendritic cells) and lymphoid (T lymphocytes and B
lymphocytes) lineage. Immune cells in normal breast tissue are
predominantly localized to the lobules rather than the stroma and
fat (Figure 1),2 comprise cells of both the innate and adaptive im-
mune systems, and thus may potentially provide protection against
bacterial and other pathogens as well as immune surveillance and
elimination of epithelial cells with mutational changes. Immune
cells identified in early studies were predominantly lymphocytes,13
including CD8þ and CD4þ T lymphocytes,14,15 and macro-
phages.13-15 Five recent studies have further characterized this
intraepithelial immune cell population in normal breast tissue.2-
4,6,16
Overall, immune CD45þ cells are prominent among the
ductal epithelial cells. CD8þ T cells are the most common cells in
all series, and in the study by Degnim et al,2 CD8þ T cells and
CD11cþ (dendritic) cells were present in virtually all lobules and
among the most numerous across lobules, while CD68þ cells
(macrophages/monocytes) were also numerous across lobules;
CD4þ and CD20þ cells were less frequent. They also found both
dendritic cells and CD8þ cells were consistently observed in inti-
mate association with the epithelium of lobular acini and were
primarily located at the basal aspect of the epithelium. The
anatomical distribution of immune cells in normal breast tissue and
their relationship to epithelial cells is depicted in Figure 1. The
presence of the immune cells in the in TDLU places them at the
predominant site of the origin of breast cancer - TDLU epithelial12;
this close proximity provides an important opportunity for the
immune cells to influence the behavior of epithelial cells. Zumwalde
et al,3 using flow cytometry of breast tissue organoids from reduc-
tion mammoplasty specimens, reported that CD8þ cells represented
75% of CD3þ cells; essentially all of the CD8aþ cells expressed
CD8þb, indicating they were distinct from intestinal intraepithelial
cells, which are CD8þaa.6 Ruffell et al6 examined normal breast
tissue either adjacent to breast cancer or from prophylactic mas-
tectomy specimens and found CD3þ (CD4þ and CD8þ) cells were
the most common, while myeloid-lineage cells including macro-
phages, dendritic cells and neutrophils were also prominent. The
distribution of immune cell types thus appears to be quite consistent
across multiple types of normal breast tissue. Interestingly, in the
study of normal breast organoids by Zumwalde et al, the authors
identified an important subtype of CD3þ cells, gd T cells. The
TCRs of these cells consists of g and d chains rather than the
conventional a and b chains, and the receptor is considered to act as
a pattern recognition receptor and a bridge between innate and
adaptive immune responses.17 These cells possess cytotoxic activity,
 Stephanie L. Goff, David N. Danforth
Figure 1 Nature and Distribution of Immune Cells in Normal Breast Tissue. A Variety of Immune Cells are Present in Normal Breast
Tissue, including Both Lymphocytes and Myeloid-Derived Cells. Immune Cells are Located Primarily Within the Ductal
Epithelium (Intraepithelial) but May be Present in the Stroma as Well (Not Depicted). The Stromal Extracellular Matrix (ECM)
Also Contains a Variety of Cell Types and Structures that May Interact With Immune Cells and the Epithelium. Adapted From
Degnim et al2

and serve to control the integrity of epithelium.18 The subtype
Vg2þ gd T cells was found to be consistently present in prepara-
tions of mammary ductal epithelial organoids.3
Immunogenicity of Immune Cells in Normal Breast Tissue. An
important question is the immunogenicity of the immune cells in
normal breast tissue. It is noted that the immune cells in the breast
ducts are in proximity to draining lymph nodes in the ipsilateral
axilla and mediastinum, and thus all of the principle cellular and
lymphatic components necessary for adaptive cellular immune
response are present within the ductal system. In the study by
Zumwalde et al3 further analysis of the CD8þ T cells indicated they
were almost exclusively CD45ROþ/CD27 cells, and thus were
effector memory T cells (TEM). The presence of TEM in the
intraepithelial lymphocyte population of the normal breast indicates
these cells have been antigen-activated, as well as indicating the
presence of a dynamic immune network between ductal epithelium
and regional lymph nodes. Importantly, TEM may persist for
considerable periods of time,19 and Hussein and Hassan5 have
shown that the CD3þ population of cells in normal breast tissue are
granzyme Bþ cells and thus have cytotoxic functions, suggesting an
important protective role for these intraepithelial immune cells.
There are several potential sources of antigens for activation of these
CD8þ T cells, both exogenous (eg, viral, bacterial) and endogenous
(nuclear and cytosolic proteins, DNA, extracellular proteins).
Neoantigens, for example, could arise through mutational changes
in ductal epithelium from exposure to estrogens and other carcin-
ogens.20 A recent review has demonstrated that normal breast tissue

Clinical Breast Cancer Month 2020 -3

 Role of Immune Cells
at normal risk for breast cancer (such as from a reduction mam-
moplasty) as well as normal breast tissue at increased risk for breast
cancer (such as normal breast tissue adjacent to breast cancer)
contain widespread genomic changes including loss of heterozy-
gosity, small segmental deletions, DNA methylation, and telomere
shortening.21,22 This could provide an excellent source of neo-
antigens through associated frameshift or other mutational changes.
In addition, the release of damage associatede or pathogen-
associated molecular patterns from injured or dying epithelial or
microbial cells may be recognized by macrophages (also present
among ductal intraepithelial immune cells), eliciting release of
cytokines and chemokines, production of chronic inflammation,
and production of reactive oxygen species and reactive nitrogen
intermediates, which may then cause mutations in neighboring
epithelial cells.23-25 As noted by Degnim et al,2 the consistent
presence of CD8þ cells and dendritic cells, interspersed within the
breast epithelium, strongly suggests a role for antigen presentation
and immune effector function, as well as stress response and
maintenance of epithelial integrity. In a subsequent study by Deg-
nim et al,16 they compared the distribution of immune cells in the
lobules of women with benign breast disease to breast samples from
women with no known breast abnormalities and found benign
breast disease lobules showed greater densities of CD8þ T cells,
CD11cþ dendritic cells, CD20þ B cells, and CD68þ macrophages
compared to the normal controls.16 The authors concluded that
elevated infiltration of both innate and adaptive immune effectors in
benign breast disease tissues suggests an immunogenic microenvi-
ronment. Finally, it has also been shown that many of the muta-
tional changes observed in normal high risk breast tissue are also
present in the adjacent breast cancer, thus providing a potentially
comparable immunogenic environment for immune cells.26 Azizi
et al,4 in a study of normal breast tissue from prophylactic mas-
tectomy tissue, observed a large number of normal breast tissue
resident immune cell states, including 13 myeloid and 19 T-cell
clusters that were not observed in circulation or in the secondary
lymphoid tissue. The set of clusters found in normal breast tissue
cells represented a subset of those observed in the breast cancers,
supporting the widespread immunogenic environment of these
tissues.
Intraepithelial Immune Cells and the Microenvironment. The
immune cells within the intraepithelial layer are also surrounded by,
and may be influenced by, major components of the microenvi-
ronment including the ECM and the interstitial matrix (Figure 1).
The ECM consists of the ductal and endothelial basement mem-
branes, and the interstitial matrix is comprised of connective tissue
and cellular components (fibroblast, adipocyte, endothelial, in-
flammatory).27 A dynamic interaction is thought to exist between
the multiple components of the microenvironment, which serves to
maintain and promote the contribution of each component. The
components of the microenvironment are also considered to play a
significant role during breast carcinogenesis. With progression of the
epithelial cells in the carcinogenic pathway, there is increasing in-
fluence of these components, including the ECM, fibroblasts, and
adipocytes on transformed cells. As Ghajar and Bissell28 have
pointed out, simple changes in ECM composition can alter
4 - Clinical Breast Cancer Month 2020
diffusion and permeability through the ECM, and diffusion
restrictions could foster tumorigenesis over short- and long-term
scales by restricting clearance of factors secreted by enveloped
cells. Fibroblasts, a major component of the ECM, can be reprog-
rammed into cancer-associated fibroblasts by cytokines and growth
factors secreted by transformed epithelial cells.29 Cancer-associated
fibroblasts can be activated to promote tumor initiation, remodel-
ing of the ECM, and modulation of immune cells.29 Adipocytes are
also important components of the ECM and are in close proximity
to epithelial cells, tumor and immune cells. Breast cancer cells
induce production of endocrine and paracrine enzymes and bioac-
tive lipids by adipocytes, which in turn drive increased growth and
invasion of tumor cells.30 Leptin is also an important secretory
product of adipocytes, which can stimulate production of several
proinflammatory factors including interleukin (IL)-1, IL-6, and
tumor necrosis factor (TNF) a.31 Obesity is an important risk factor
for breast cancer in postmenopausal women and may promote
procarcinogenic processes in adipocytes. Several factors, including
leptin, TNFa, IL-6, and resistin, are secreted by normal adipocytes,
and are increased with obesity and have procarcinogenic effects.32
These in turn can modify the immune system and promote tu-
mor growth.33 Together, these findings indicate a diverse popula-
tion of cells within the ductal epithelial layer and microenvironment
of normal breast tissue that interact to play a major role in early
breast carcinogenesis.
Estrogenic Effects on Normal Breast Immune Cells. Estrogen is the
predominant hormone in women during both premenopausal and
postmenopausal time periods. Estrogens play an important role
throughout breast carcinogenesis, from influencing risk, to the
development of contralateral breast cancer, to the progression of
primary breast and metastatic breast cancer. These actions are
achieved primarily through estrogenic actions on breast epithelial
cells, however there is important evidence that immune cells contain
estrogen receptors (ERs) and are regulated by estrogens as well. This
would provide an important opportunity for estrogens to influence
immunosurveillance, as well as the development and progression of
breast cancer. Estrogenic actions result from binding to cytoplasmic
receptors ERa and ERb and activation of genomic pathways,
although estrogens may also activate growth factor receptor activity
with signaling through nongenomic pathways by ligand-
independent ERa signaling involving binding of E2 to a
membrane-anchored receptor, the G proteinecoupled estrogen re-
ceptor 1 (GPER1), with subsequent activation of G proteins.34
Immune cells of both the innate (dendritic cells, macrophages,
neutrophils) and adaptive (CD4þ, CD8þ, B cells) cell types, and in
both normal tissue and in breast cancer, have been shown to possess
ERs and to be responsive to estrogens.35-37 The response of immune
cells to the estrogenic effects may include altered immune cell
proliferation, secretion of cytokines, chemokines, and growth
factors.35,38,39
In breast cancer the distribution of immune cell types may vary
according to tumor subtype as well as between stroma and tumor
parenchyma (see below). The distribution of immune cells between
stroma and parenchyma/ductal epithelium provides an opportunity
for these cells, after estrogen stimulation, to influence multiple

components of the microenvironment including other immune
cells, epithelial cells, fibroblasts, adipose cells and endothelium.
Ultimately, the response of immune cells will also be influenced by
cell density, estrogen levels, ECM composition, blood supply, and
the specific ER stimulated.38,40 Although estrogens are probably the
major steroid that influences cell behavior, it is recognized that these
cells may also contain receptors for other steroids, including pro-
gesterone, glucocorticoids, and androgens,38 and respond and be
influenced by these hormones as well. Together, the cellular
responsiveness of immune cells to estrogens (and other hormones)
further clarifies events regulating progression in carcinogenesis, as
well as suggest an additional potentially important role for anties-
trogen therapy.
Immune Cells in Ductal carcinoma in situ (DCIS)
DCIS is an important precursor lesion for invasive ductal carci-
noma and accounts for 16% of all breast cancers annually in the
United States.1 DCIS contains, TILs and multiple studies have
examined TIL in DCIS both according to cell density, according to
the presence of aggressive features, and according to cell type. DCIS
is characterized by an increase in the density and extent of immune
cell infiltration compared to normal breast tissue.5,41-43 The im-
mune cell infiltrate in DCIS is also increased in more aggressive
lesions, being greater in high-grade tumors,41-46 ER-negative tu-
mors,41,44 and human epidermal growth factor receptor 2 (HER2)-
positive or triple-negative breast cancer (TNBC),43,44,46,47 and also
correlates with tumors with necrosis.5,43,44 High-grade DCIS has
significantly higher percentages of FOXP3þ cells, CD68þ and
CD68þPCNAþ macrophages, human leukocyte antigen (HLA)-
DRþ cells, CD4þ T cells, CD20þ B cells, and total TILs compared
to nonehigh-grade DCIS.5,48 Hussein and Hassan5 noted a marked
increase in the density of mononuclear inflammatory cell infiltrates,
including CD20þ, CD68þ, CD3þ and granzyme Bþ cytotoxic T
cells from normal breast tissue to DCIS to invasive breast cancer.
On average, ER DCIS has been found to contain higher numbers
of all TIL subsets than ERþ DCIS, and ERþ DCIS was more likely
to have to have a high CD8/FOXP3 ratio (> 4) than ER DCIS.41
Thompson et al41 observed that CD3þ T cells predominated across
all DCIS subtypes at all ages, with slightly more CD4þ T cells than
CD8þT cells on average. CD20þ B cells were the next most
common TILs, followed by FOXP3þ T regulatory cells (Tregs).
Others have observed B cells in DCIS and in invasive carcinoma,
which were typically found in perivascular locales clustering in ag-
gregates with T cells, forming ectopic follicles,49,50 and indicating
that the presence of B cells in neoplastic mammary tissue is the
result of chronic activation rather than nonspecific chemo-
attraction.49 There is evidence also that TILs in DCIS are immu-
nosuppressive. Programmed death ligand 1 (PD-L1) is an
immunosuppressive surface ligand on lymphocytes; Thompson
et al41 demonstrated that 81% of DCIS lesions contained PD-L1þ
TILs. They considered this to suggest an active immune response
within DCIS and supported TIL expression of PD-L1 as a marker
of downregulation of the body’s immune response within DCIS.
FOXP3þ Treg cells are another important immunosuppressive cell
that are increased in DCIS,41,44,48,51,52 and are associated with high
nuclear grade, comedo-type necrosis, hormone receptor (HR)
Stephanie L. Goff, David N. Danforth
negativity, high Ki-67 proliferation index, and p53 over-
expression.44 Kim et al44 found the high infiltration of FOXP3þ
TIL and the presence of PD-L1þ immune cells were associated with
tumor recurrence in patients with pure DCIS. Pure DCIS associated
with higher numbers of B lymphocytes has also been shown to be
associated with a shorter recurrence-free interval (P ¼ .04).53 Bates
et al52 observed high numbers of FOXP3þ regulatory T cells
identified in patients with DCIS at increased risk of relapse (P ¼
.04). CD8þ HLA-DRþ T cells, CD8þ HLA-DR T cells, and
CD115þ cell populations have also shown to be associated with risk
of recurrence.48 Together, these findings indicate a prominent
presence of TIL, which may play an important immunosuppressive
role in DCIS.
Immune Cells in Invasive Carcinoma of the Breast
The immune cell content of breast tissue increases progressively
from normal breast tissue to breast cancer.4-6,54,55 This is best
illustrated by studies comparing the immune cell distribution in
breast cancer and related normal breast tissue. In a study of mas-
tectomy specimens utilizing immunohistochemistry (IHC) and flow
cytometry, Ruffell et al6 observed that breast cancer tissues con-
tained infiltrates dominated by CD8þ and CD4þ lymphocytes,
with minor populations of NK cells and B lymphocytes, whereas in
the normal breast tissue myeloid-lineage cells including macro-
phages, mast cells, and neutrophils were more evident. A similar
immune profile was observed in breast tissues obtained in prophy-
lactic mastectomy tissue. Activated T lymphocytes also predomi-
nated in tumor tissue, with both CD4þ and CD8þ T cells
displaying increased expression of activation markers CD69 and
HLA-DR. They concluded these findings indicated a shift within
tumors toward a Th2-type response in breast cancer characterized
by the increased presence of B cells and CD4þ T cells, in com-
parison with normal breast tissue.6 At the transcriptional level, Azizi
et al4 compared by singe cell sequencing the immune cell distri-
bution in breast cancer with that of normal adjacent breast tissue.
They found a significant increase in the diversity of cell states, re-
flected in an increase in variance of gene expression, among T cells,
monocytes, and NK cells in tumor compared to normal tissue. This
suggested that the increased heterogeneity of cell states and marked
phenotypic expansions found within the tumor were likely due to
more diverse local microenvironments within the tumor.4 Two
small studies utilizing high-throughput sequencing of the TCR beta
chain to characterize diversity of the T-cell infiltrate have been
performed. Beausang et al56 demonstrated that there are unique
compartments of enriched clonotypes in tumor and tumor-adjacent
normal breast tissue, as well as identifying sequences shared among
patients and unlikely to be involved in specific tumor recognition.
Park et al57 studied tumors before and after neoadjuvant therapy,
and demonstrated a clonal expansion and decrease in diversity in
those patients with pathologic complete response (pCR) to therapy.
The complexity of the composition of immune cells in breast
cancer reflects a cross-talk between components of the innate im-
mune response as it regulates the tumor microenvironment and the
polarity of the adaptive immune response within that tumor.49
Lymphocytes of the myeloid lineage, ie, tumor-associated macro-
phages, myeloid-derived suppressor cells, and dendritic cells, can
Clinical Breast Cancer Month 2020 -5
 Role of Immune Cells
create immunostimulatory or immunosuppressive milieus that affect
the fate of T cells capable of homing to the tumor. In turn,
macrophage polarization can be driven to M1 or M2 phenotypes by
the cytokines expressed by cytotoxic or regulatory T cells.49,58
Within breast cancer, the distribution of immune cells may also
differ between the tumor parenchyma and stroma. In a study of
breast cancer mastectomy specimens, immune cells were delineated
in the tumors and surrounding stroma by IHC staining to identify
CD3 (T cells), CD20 (B cells), CD68 (macrophages), and gran-
zyme B (cytotoxic subset of CD3þ cells).5 The breast samples
ranged the spectrum of breast disease: normal, benign proliferative
disease (usual ductal hyperplasia), DCIS, and invasive ductal car-
cinoma. Although there was a progressive increase in all cell types in
both the parenchyma and stroma moving from normal mammary
tissue to ductal carcinoma, the most striking difference from benign
proliferative disease to DCIS to invasive cancer was the influx of
CD3þ cells in the stroma of the latter two tissue types: average 4.2
cells/mm2 versus 46.6 cells/mm2 versus 77.0 cells/mm2, respectively
(Figure 2). Interestingly, increases in the cytotoxic subset (granzyme
B) were restricted to immune cells in the parenchyma: 16.3 versus
0.7 cells/mm2 in the parenchyma versus stroma of invasive cancer.5
Solinas et al,59 in their review, similarly found that the incidence of
stromal TIL (sTIL) ranged from 15% to 25%, whereas intratumoral
TIL ranged from 5% to 10%. As will be discussed below, sTILs also
have important prognostic value.
Immune Cell Distribution According to Breast Cancer Subtype. The
distribution of TIL varies quantitively and qualitatively according to
subtype in breast cancer. This has been demonstrated in several
series. Stanton et al,54 in a review of 13,914 patients, found a
median of 20% of patients with TNBC demonstrated lymphocyte-
predominant breast cancer (LPBC;  50%-60% lymphocytic
infiltrate) at the time of diagnosis compared to 16% of HER2þ
tumors and 6% of HRþ cancers. A median of 60% of TNBC
samples had infiltrating CD8þ T cells in contrast to only 43% of
HRþ tumors. TNBC tumors were also more likely to have
FOXP3þ infiltrates than the HRþ subtype. Their findings indicated
that HR-positive disease may be the least immunogenic of the
common breast cancer subtypes. Liu et al,60 in another study, re-
ported a high density infiltration of Treg and FOXP3PþTumor, but
not CD8þTumor, correlated significantly with HER2 overexpression.
Increased infiltration of Tregs and cytotoxic lymphocytes was
significantly more common in those tumors with unfavorable his-
tologic features, including high histologic grade and negative ER
and progesterone receptor status. They proposed that further studies
to explore the functional status and action modes of cytotoxic
lymphocytes and Tregs in different tissue locations and in different
breast cancer subtypes will lead to a better understanding of the
nature of breast carcinoma immunity. Denkert et al61 observed the
percentage of tumors with high TILs was higher in TNBC (30%)
and HER2-positive breast cancer (19%) than in lumi-
naleHER2enegative tumors (13%) (P < .0001). Their data sup-
ported the hypothesis that breast cancer is immunogenic and might
be targetable by immune-modulating therapies. Mohamed et al62
reported HER2/neu-positive breast cancer had a significantly
higher CD8þ content (26.1%) compared to luminal A (13.5%),
6 - Clinical Breast Cancer Month 2020
luminal B (17.0%), or TNBC (14.5%). This, however, did not
apply to CD3þ or CD45ROþ type cells, and there was no signif-
icant correlation between CD45ROþ TILs and clinicopathologic
parameters. Their study was conducted in the Sudan, and their
finding that a higher CD8þ content was observed in HER2/neu-
positive breast cancer is in agreement with the other studies, and
suggests that the biological factors regulating TIL distribution ac-
cording to subtype in North Africa may be similar to those in the
United States.
Mutational Load of Breast Cancer Cells. The finding that immune
cell types in breast cancer varied according to breast cancer subtype
suggested the mutational load of breast cancer subtypes may also
vary. When this was studied it was found there are also striking
differences in the mutational load of breast cancer between sub-
types. The genomic characteristics of each subtype has recently been
analyzed and presented in detail in an analysis of cancer-adjacent
breast tissue from The Cancer Genome Atlas Network.22 The
findings for different subtypes can be summarized as follows: the
overall mutation rate was lowest in luminal A subtype and highest in
the basal like and HER2 enriched (HER2E) subtypes. The luminal
A subtype harbored the most significantly mutated genes, with the
most frequent being PIK3CA (45%), followed by MAP3K1,
GATA3, TP53, CDH1 and MAP2K4. Twelve percent of luminal A
tumors contained likely inactivating mutations in MAP3K1 and
MAP2K4. TP53 and PIK3CA mutations were the most common in
Luminal B cancer (29% each); this contrasted with basal-like tu-
mors where TP53 mutations occurred in 80% but PIK3CA muta-
tions were absent or rare. Ten percent of sporadic breast cancer may
have a strong germline contribution. Luminal tumors were mostly
diploid whereas luminal B tumors were mostly aneuploid.
Regarding HER2þ tumors, there are at least two types of clinically
defined HER2 tumors, luminal messenger RNA (mRNA)-subtype/
HER2þ tumors, and HER2E-mRNA subtype (HER2 enriched). A
comparison identified 302 differentially expressed genes. The
HER2E mRNA subtype typically showed high aneuploidy, the
highest somatic mutation rate, and DNA amplification of other
potential therapeutic targets including fibroblast growth factor re-
ceptors, epidermal growth factor receptor, CDK4, and cyclin D1.
TP53 mutations were significantly enriched in HER2E or ER
tumors whereas GATA3 mutations were only observed in luminal
subtypes or ERþ tumors. The basal-like subtype included TNBC
(75%) as well as other mRNA subtypes (25%), and showed basal-
like tumors with a high frequency of TP53 mutations (80%).
PIK3CA was mutated in 9% of cases; however, inferred PI(3)K
pathway activity, whether from gene, protein, or high PI(3)K/AKT
pathway activities was highest in basal-like cancers. Expression
features showed high expression of genes associated with cell pro-
liferation. Although chromosome 8q24 is amplified across all sub-
types, high MYC activation seems to be a basal-like characteristic.
Other major genomic changes include ATM mutations (3%),
BRCA1 (30%) and BRCA2 (6%) inactivation, RB1 loss (20%), and
cyclin E1 amplification (9%). Together, these findings indicate a
significant mutational burden across breast cancer subtypes. This
may have important clinical, therapeutic, and biological implica-
tions. A high mutational burden would be associated with increased
 Stephanie L. Goff, David N. Danforth
Figure 2 Immune Cells in Breast Proliferative Disease and Breast Cancer. Immune Cells, including CD68D (Macrophages/Monocytes),
CD3D (CD4D, CD8D), and Cytotoxic Lymphocytes (CTL) are Present in Breast Proliferative Disease and Increase in the
Parenchyma and Stroma With Progression to Breast Cancer. Relative Increases for Each Cell Type, With Benign Proliferation
as a Baseline, are Shown on a Logarithmic Scale in the Graphs Below the Figure. (Adapted from Data from within Hussein
and Hassan5)

genomic instability and neoantigen development, increased cell
injury and death, and development of chronic inflammation. At the
same time altered secretion of secretory products could influence the
activity, distribution, and interaction of associated immune and
other cell types.
Nature of Immune Cells in Tertiary Lymph Structures in Breast
Cancer. Tertiary lymph structures (TLS) (aka tertiary lymph organs)
are ectopic lymphoid organs that develop at sites of chronic
inflammation including autoimmune diseases, infection, and tu-
mors. TLS have a defined structure, are comprised of multiple cell
types including both innate (dendritic, macrophages, neutrophils)
and adaptive (B cells, T cells) as well as plasma cells and high
endothelial venules (HEV).63,64 TLS are believed to be the site of
immune response activation against tumor by recruiting and acti-
vating TIL.65 The TLS may be within the tumor or in a peritumoral
location. TLS are generally associated with tumors with a more
aggressive phenotype such as high-grade tumors, TNBC, and
HER2þ tumors.62,65,66 Liu et al65 reported TLS were associated
with higher tumor grade, apocrine phenotype, necrosis, extensive
in-situ component, lymphovascular invasion, high TIL, HR nega-
tivity, HER2 positivity, and c-kit expression. A favorable impact of
TLS density on overall survival (OS) and disease-free survival (DFS)
of patients has been observed.62,63,66-71 An important component of
TLS are HEV. These are specialized postcapillary venules found in
lymphoid tissues that support high levels of lymphocyte extravasa-
tion from the blood.72 High densities of tumor HEVs correlated
with increased naive, central memory and activated effector memory
T-cell infiltration and upregulation of genes related to T-helper 1
adaptive immunity and T-cell cytotoxicity.67 In the study by
Martinet et al,67 high densities of tumor HEVs independently
conferred a lower risk of relapse and significantly correlated this
activity with longer metastasis-free, disease-free, and OS rates.
Together, the presence and characteristics of TLS have important
clinical implications: their presence in poor-prognosis tumors may
be important both for selection of therapy and entry into clinical
trials. The identification of an organized immune cell collection
with effector memory cells in and adjacent to breast cancer and
capable of antitumor activity may encourage efforts to promote its
development and expansion in these tumors. The presence in TLS
of HEVs that can enhance movement of lymphocytes into these
tumors also encourages efforts to identify agents that can promote
the activity of these vessels.
Immune Cells as a Prognostic Biomarker. With renewed interest in
immune cells, investigators have interrogated large prospectively
collected patient samples associated with seminal clinical trials to
evaluate the role of immune cells as prognostic markers. The first of
these studies was reported by Denkert et al10 after analysis of the
GeparDuo and GeparTrio trial cohorts. Pretreatment core biopsy
samples of tumor and stroma were analyzed for lymphocytic infil-
trate evaluated by routine hematoxylin and eosin staining. Two
categories of immune infiltrate were defined: iTu-Ly (intratumoral
lymphocytes) in direct contact with tumor cells or within tumor cell
nests and str-Ly (stromal lymphocytes) without direct contact with
tumor cells. LPBC was defined as tumors with > 60% of either
iTu-Ly or str-Ly and represented 11% of the study population.
LPBC demonstrated an increased incidence of pCR (10/24, 41.7%)

Clinical Breast Cancer Month 2020 -7

 Role of Immune Cells
when compared to those tumors with no lymphocytic infiltrate (1/
36, 2.8%) or focal infiltrate (17/158, 10.8%). This was also
demonstrated in gene expression data of the same cohort.10 In an
analysis of the BIG 02-98 multi-institutional randomized phase 3
trial, increasing lymphocyte infiltration was associated with
improved prognosis in the subgroup of node-positive patients with
TNBC. LPBC represented only 10.6% of the TNBC cohort using a
threshold of 50%, but prognosis improved linearly with each 10%
increase in lymphocyte infiltration.8
Tumor samples from patients in the Eastern Cooperative
Oncology Group (ECOG) 1199 and 2197 trials have been analyzed
for the presence of intraepithelial TIL or sTIL lymphocytes.9
Overall, the median intraepithelial TIL score was 0%, and LPBC
was again a minority of cases (4.4%), although over 80% of cancers
had a sTIL score > 10. Higher sTIL scores were associated with
better prognosis; for every 10% increase in sTILs, a 14% reduction
of risk of recurrence or death was noted (P ¼ .02), and an 18%
reduction of risk of distant recurrence (P ¼ .04), and 19% reduc-
tion of risk of death. However, in Liu et al,60 the effects of
CD8þTumor infiltration, and the ratio of CD8þTumor/
FOXP3PþTumor on OS and progression-free survival, were not
significant, whereas patients with high FOXP3þTumor had a
significantly shorter OS (P ¼ .007) and progression-free survival
(P ¼ .003) both in the ERþ and ER groups. Interestingly, recent
studies have indicated that the spatial location and organization of
CD8þ TILs within the tumor may be important in relation to
relapse-free survival.73 Egelston et al73 reported that the presence of
islands of infiltrating CD8þ T cells was more significantly associated
with relapse-free survival than CD8þ T-cell infiltration into either
tumor stroma or total tumor. The integrin CD103, a marker for
tissue resident memory T cells (TRM) appeared to mediate locali-
zation into cancer islands within tumors. The TRM subset has been
implicated in cancer surveillance of melanoma and lung cancer.74,75
Savas et al76 utilized single-cell RNA sequencing techniques to
derive a CD8þ TRM signature from primary TNBC tumors; when
applied to a bulk RNA data, the signature was associated with
improved patient survival.
A review of 15 studies of TIL classification also identified dif-
ferences in lymphocyte infiltration across tumor subtypes.54 LPBC
was more frequently identified in TNBC (20%) and HER2þ (16%)
specimens than in HRþ/HER2 tumors (6%). Efforts have been
made to unify the classification of TILs within breast cancer spec-
imens, with a focus on stromal lymphocytes as the most repro-
ducible and significant prognostic indicator of likelihood of
response.7 In an analysis by Denkert et al61 of pooled data from the
German Breast Cancer Group, classification of tumors by degree of
stromal lymphocyte invasion allowed prediction of response to
neoadjuvant chemotherapy in all molecular subtypes, but differ-
ences in survival outcomes may suggest a different biology of the
immunologic infiltrate in HRþ tumors. Although pCR was attained
in 28% of luminal HER2 tumors with high TILs (> 60%)
compared to 6% and 11% in low ( 10%) and intermediate TILs,
the presence of TILs was associated with shorter OS in that subtype.
TNBC with high TILs demonstrated a higher pCR rate (50% high
vs. 31% intermediate and low) and was associated with longer DFS
and OS. The gains were more modest in HER2þ tumors with a
48% pCR rate in high TILs specimens versus 39% in intermediate
8 - Clinical Breast Cancer Month 2020
and 32% in low samples, but still associated with longer DFS.61
Together, these findings support a prognostic role of TILs in
breast cancer. The prognostic significance may correlate inversely
with tumor HR status, suggesting there may also be a prominent
hormonal influence on these lymphocytes in HRþ tumors.
Lastly, the effect of neoadjuvant chemotherapy on immune cell
distribution has also been examined. In the study of Ruffell et al6
their findings suggested that neoadjuvant chemotherapy further
altered the complexity of the immune microenvironment of the
residual tumors. They found residual tumors treated with
neoadjuvant chemotherapy contained increased percentages of
infiltrating myeloid cells, accompanied by an increased CD8/CD4
T-cell ratio and higher numbers of granzyme Beexpressing cells,
compared to tumors not treated with neoadjuvant chemotherapy.
The effect of chemotherapy has also been examined by García-
Martínez et al,77 who evaluated tumors using IHC to define CD3þ
cells, subsets of CD4þ and CD8þ cells, and the presence of B cells
and monocytes before and after neoadjuvant chemotherapy.77 They
observed a decrease in the number of CD3þ cells after chemo-
therapy and a decrease in the CD4:CD8 ratio. They concluded that
an IHC-based profile of immune cell subpopulations in breast
cancer is able to identify a group of tumors highly sensitive to
neoadjuvant chemotherapy.
Immune-Based Therapies for Breast Cancer. The majority of the
studies of lymphocytes in breast cancer has focused on character-
izing the nature of the lymphocyte infiltrate, with some gene
expression data that may hint at the functional role of these cells in
an anticancer response. Chemokines thought to be responsible for
lymphocyte migration and gene expression signatures associated
with Type I effector responses have been shown to correlate with
pCR.78,79 Programmed death receptor 1 (PD-1) and one of its li-
gands (PD-L1) have also been correlated with higher pCR rate and
improved prognosis in breast cancer.80 These molecules are part of
the immune checkpoint pathway that limits T-cell response. PD-L1
gene expression of immune cells in breast cancer has been shown to
be positively associated with CD8þ and CD4þ memory-activated T
cells, but not with CD4þ memory resting or T-regulatory cells or
other immune cell subpopulations.81 In other histologies, expres-
sion of PD-L1 may correlate with response to checkpoint inhibi-
tion, but there are significant challenges to its use as a predictive
biomarker, highlighted by the KEYNOTE-86 trial of pem-
brolizumab in patients with TNBC in which PD-L1 status was not
the strongest discriminator between those with disease that did and
did not respond to therapy.82 The overall response rate in that trial
was 18.5%. Similarly modest results were seen in an ERþ popula-
tion in KEYNOTE-28 with an overall response rate of 12%.83,84
Checkpoint inhibitors have been combined with chemotherapy
demonstrating improvement in median OS, more pronounced in
those patients with PD-L1þ tumors (Impassion130 trial),84 and an
increase in the pCR rate in women with TNBC (KEYNOTE-
522).85 A comprehensive review of current and proposed combi-
nation strategies identified 13 ongoing randomized phase 3 clinical
trials investigating the strategy in patients with breast cancer.86
Given the presence of an immune infiltrate in stages as early as
DCIS, strategies to boost the immune response to potential tumor
antigens by vaccination have been explored in prevention, adjuvant,

neoadjuvant and metastatic settings.87 Additional efforts explore the use
of tumor ablative strategies (ie, cryoablation, radiofrequency ablation,
stereotactic radiation) for potential release of tumor-associated antigens
in conjunction with checkpoint inhibitor monoclonal antibodies to
increase response rates.88,89 A recent review highlighted the ways that the
field of breast cancer immuno-oncology is building on information
about TIL phenotypes to branch into different promising avenues of
research.90 Few groups have studied the phenotype and function of TILs
cultured from freshly resected breast cancer. In the Surgery Branch of the
National Cancer Institute, TILs derived from fragments of metastatic
breast cancer and cultured in IL-2 were predominantly CD4þ, in
contrast to our experience with metastatic melanoma-derived TILs,
which are predominantly CD8þ cells.91,92 Whole exome sequencing of
the resected tumor identified candidate neoepitopes for further study,
and TIL cultures were capable of identifying neoantigens processed by
autologous antigen-presenting cells in vitro, in both class I and class II
restricted fashion.11,93 In a study of primary breast tumors, another
group used an agonistic antibody against 4-1BB/CD137 during the
initial TIL cultures and were able to generate more robust CD8þ
populations. In two of these patient-derived cultures, the expanded
CD8þ TILs were capable of mutation-specific recognition of class I
predicted neoepitopes.94 Tumor-specific TIL could be utilized to
identify tumor neoantigens that could then be utilized in patient-specific
vaccine strategies or to create cell-based treatments with autologous TIL
or TIL-derived TCR gene-engineered products. We recently demon-
strated proof of principle in a single case report of complete regression of
refractory metastatic breast cancer after adoptive cell transfer with IL-2
and pembrolizumab.11
Conclusion
Immune cells are identified in breast tissue throughout carcino-
genesis, beginning in normal breast tissue in women at normal risk
for breast cancer and continuing through breast cancer primary
tumors and breast cancer metastases. The immune cells in normal
breast tissue may play an important role in immunosurveillance and
clarify our understanding of the prevention (and development) of
early events in breast carcinogenesis. Our understanding of TILs is
increasing dramatically. These cells have prognostic value for breast
cancer outcomes and serve in adoptive transfer and other capacities
to treat disseminated breast cancer. This knowledge is allowing us to
recognize and to modify T cells in multiple ways, including selec-
tion of tumor neoantigen-specific T cells, to the engineering of
TCRs to recognize specific tumor associated neoantigens and to
insert these TCR into lymphocytes (including peripheral blood
lymphocytes), for the development of vaccines that recognize
tumor-specific antigens in breast cancer metastases. Together, these
findings allow immunology and immunotherapy to serve as the
major fourth modality for the management of patients with cancer,
alongside surgery, chemotherapy, and radiotherapy. This should
greatly improve our ability to prevent and treat breast cancer, as well
as many other malignancies.
Acknowledgment
This research was supported by the Intramural Research Pro-
gram, Center for Cancer Research, National Cancer Institute, Na-
tional Institutes of Health, Bethesda, MD.
Stephanie L. Goff, David N. Danforth
Disclosure
The authors have stated that they have no conflict of interest.
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Clinical Breast Cancer Month 2020 - 11

Peran Sel pada Sistem Imun dalam Jaringan Payudara dan Imunoterapi
sebagai Pilihan Terapi pada Kanker Payudara

Pendahuluan
Kanker payudara merupakan salah satu jenis keganasan yang paling umum ditemukan
pada wanita, dan mencapai angka 316.000 kasus setiap tahunnya di Amerika Serikat. Sebagian
besar kanker payudara berlokasi di saluran ASI, baik sebagai karsinoma duktal-in-situ (DCIS)
atau sebagai karsinoma duktal invasif. Perkembangan dan progresivitas kanker payudara dapat
dipengaruhi oleh berbagai elemen dalam lingkungan mikro duktus, baik dalam tingkat seluler
seperti sel-sel yang berperan dalam sistem imun, adiposit, fibroblas, dan mikrobioma, serta pada
elemen terlarut seperti faktor pertumbuhan, sitokin, kemokin, dan prostaglandin. Salah satu
komponen yang memiliki peran penting dari unsur-unsur ini adalah sel-sel yang berperan dalam
sistem imun. Lapisan sel duktal pada payudara normal mengandung populasi sel imun yang
signifikan yang terdiri dari sel CD8+ dan CD4+T, sel B, sel dendritik, makrofag, sel NK dan
subtipe sel-sel yang berperan dalam sistem imun lainnya. Sel-sel yang berperan dalam sistem
imun ini menyusun respon imunitas bawaan dan adaptif terhadap lapisan epitel untuk
perlindungan terhadap agen eksogen dan endogen, serta eliminasi sel transformatif.
Infiltrasi sel-sel yang berperan dalam sistem imun pada kanker payudara terdiri dari
sejumlah subtipe seluler, antara lain sel CD3+ (CD4+ dan CD8+), sel B, monosit/makrofag, sel
dendritik, dan sel NK. Sejumlah subtipe sel-sel yang berperan dalam sistem imun pada parenkim
dan stroma menempatkan sel-sel ini di sekitar lingkungan mikro sel tumor dan sel lainnya,
sehingga memungkinkan sel-sel ini untuk mempengaruhi pertumbuhan tumor dengan berbagai
cara, baik secara langsung melalui sitotoksisitas CD4+ dan CD8+ yang dimediasi sel, atau secara
tidak langsung melalui tindakan imunosupresif atau imunostimulan dari sitokin yang
disekresikan, faktor pertumbuhan dan agen lainnya.
Salah satu tujuan penyusunan artikel tinjauan ini adalah untuk mengevaluasi secara rinci
karakteristik khusus sel-sel yang berperan dalam sistem imun duktal pada jaringan payudara
normal, TIL pada kanker payudara, dan upaya untuk mengaplikasikan temuan penelitian tersebut
sebagai indikator prognostik yang baik serta tatalaksana imunoterapi yang dapat
dipertimbangkan sebagai salah satu pilihan terapi untuk kanker payudara.
Bahan dan Metode
Pencarian literatur dilakukan melalui PubMed dan referensi silang untuk
mengidentifikasi publikasi yang menjelaskan sifat dan distribusi sel-sel yang berperan dalam
sistem imun pada komponen jaringan payudara normal dan kanker payudara. Jaringan payudara
normal didefinisikan sebagai komponen jaringan payudara pada subjek wanita dengan risiko
normal (contohnya pada mammoplasty reduksi), sedangkan komponen jaringan dari payudara
kontralateral atau dari jaringan yang berdekatan dengan kanker payudara pada wanita dengan
kanker payudara. Penekanan pada sel-sel yang berperan dalam sistem imun pada tumor primer
diutamakan pada penelitian yang mengevaluasi kanker payudara pada subjek.

Hasil dan Pembahasan

Sel-Sel Yang Berperan dalam Sistem Imun pada Payudara Normal
Anatomi Duktus Payudara. Unit utama dalam sistem saluran payudara adalah asini, yang
mengalir ke intralobular dan kemudian menuju duktus ekstralobular; dan bersama-sama, struktur
ini membentuk unit lobular duktal terminal (TDLU). TDLU dianggap sebagai lokasi predominan
asal kanker payudara. Sebagian besar kanker payudara merupakan karsinoma yang muncul di
epitel dan memiliki komponen in-situ serta karsinoma duktal invasif dan lobular invasif. Saluran
payudara dikelilingi oleh stroma yang terdiri dari matriks ekstraseluler (ECM), fibroblas,
adiposit, sel-sel yang berperan dalam sistem imun, mikrobioma, dan pembuluh darah. Saluran
payudara dan stroma bersama-sama membentuk lingkungan mikro payudara.

Gambar 1. Sifat dan Distribusi Sel-Sel yang Berperan dalam

Sistem Imun pada Komponen Jaringan Payudara Normal. Sel-sel
yang berperan dalam sistem imun dapat ditemukan pada jaringan
payudara normal, termasuk limfosit dan sel turunan myeloid. Sel-
sel yang berperan dalam sistem imun tubuh sebagian besar
ditemukan pada epitel duktal (intraepitel) namun dapat pula
ditemukan pada stroma (tidak digambarkan). Matriks ekstraseluler
stromal (ECM) juga mengandung berbagai jenis dan struktur sel
yang dapat berinteraksi dengan sel-sel yang berperan dalam sistem
imun serta epitel. Diadaptasi dari Degnim dkk.
Distribusi Sel-Sel yang Berperan dalam Sistem Imun pada Komponen Jaringan Payudara
Normal. Jaringan payudara normal mengandung sel-sel yang berperan dalam sistem imun baik
pada jalur derivatif myeloid (monosit, makrofag, sel dendritik) maupun limfoid (limfosit T dan
B. limfosit). Sel-sel yang berperan dalam sistem imun yang diidentifikasi dalam sejumlah
penelitian yakni didominasi oleh limfosit, termasuk limfosit T CD8+ dan CD4+, serta makrofag.
Secara keseluruhan, sel CD45+ memiliki karakteristik paling menonjol di antara sel-sel epitel
duktus lainnya. Sel T CD8+ merupakan sel yang paling umum ditemukan pada semua lokasi,
dan dalam sebuah penelitian oleh Degnim dkk, sel T CD8+ dan CD11c+ (dendritc) sel
ditemukan pada hampir semua lobulus dan memiliki jumlah terbanyak di seluruh lobulus.
Demikian juga halnya pada sel CD68+ (makrofag/monosit) yang banyak ditemukan pada seluruh
lobulus; sementara sel CD4+ dan CD20+ cenderung lebih jarang ditemukan. Ruffell dkk
melakukan penelitian terhadap jaringan payudara normal yang lokasinya berdekatan dengan
kanker payudara atau dari spesimen mastektomi profilaksis dan menemukan bahwa sel CD3+
(CD4+ dan CD8+) merupakan jenis sel yang paling umum ditemukan, dan sel-sel jalur derivatif
myeloid seperti makrofag, sel dendritik dan neutrofil juga cenderung mudah ditemukan.

Imunogenisitas Sel-Sel yang Berperan dalam Sistem Imun pada Jaringan Payudara Normal. Sel-
sel yang berperan dalam sistem imun pada saluran payudara berada di dekat kelenjar getah
bening yang mengalir di area ipsilateral aksila dan mediastinum. Dengan demikian semua
komponen seluler dan limfatik utama yang diperlukan untuk respons imun seluler adaptif
ditemukan dalam sistem duktus. Zumwalde dkk pada salah satu penelitiannya mengemukakan
analisis lebih lanjut dari sel T CD8+, yang menunjukkan bahwa sel-sel tersebut merupakan sel
CD45RO+/CD27, dan dengan demikian merupakan sel T memori efektor (TEM). Populasi
limfosit intraepitel TEM pada payudara normal menandakan bahwa sel-sel ini telah diaktivasi
antigen, sekaligus menunjukkan adanya jaringan imun dinamis antara epitel duktus dan kelenjar
getah bening regional. Sejumlah sumber potensial antigen dapat berperan dalam aktivasi sel T
CD8+ ini, baik secara eksogen (misalnya, virus, bakteri) dan endogen (protein nukleus dan
sitosol, DNA, protein ekstraseluler). Sebuah penelitian lanjutan yang dilakukan oleh Degnim dkk
membandingkan distribusi sel-sel yang berperan dalam sistem imun pada lobulus individu yang
terdiagnosis penyakit payudara jinak dengan sampel jaringan payudara dari wanita tanpa
kelainan payudara dan menemukan lobulus pada penyakit payudara jinak menunjukkan
kepadatan sel T CD8+, sel CD11c+dendritik, Sel CD20B, dan makrofag CD68 yang cenderung
lebih tinggi bila dibandingkan dengan individu kontrol normal. Infiltrasi terkait dari efektor imun
bawaan dan adaptif pada penyakit payudara jinak menunjukkan adanya lingkungan mikro
imunogenik.

Sel-Sel yang Berperan dalam Sistem Imun Intraepitelial dan Lingkungan Mikro. Sel-sel yang
berperan dalam sistem imun pada lapisan intraepitel juga dikelilingi dan dapat dipengaruhi oleh,
komponen utama dari lingkungan mikro seperti ECM dan matriks interstisial. ECM terdiri dari
membran basalis duktal dan endotel, dan matriks interstisial terdiri dari jaringan ikat dan
komponen seluler (fibroblast, adiposit, endothelial, inflammatory). Komponen lingkungan mikro
juga dianggap memiliki peran penting selama proses karsinogenesis payudara. Perubahan
sederhana dalam komposisi ECM dapat mengubah difusi dan permeabilitas melalui ECM, dan
pembatasan difusi dapat mendorong tumorigenesis. Fibroblas, komponen utama ECM, dapat
diprogram ulang menjadi fibroblas terkait kanker oleh sitokin dan faktor pertumbuhan yang
disekresikan oleh sel epitel yang mengalami transformasi. Fibroblas terkait kanker dapat
diaktifkan untuk mendorong inisiasi tumor, pemodelan ulang ECM, dan modulasi sel-sel yang
berperan dalam sistem imun. Selain fibroblas, adiposit juga merupakan salah satu komponen
penting dari ECM dan berada di dekat sel epitel, tumor, dan sel-sel yang berperan dalam sistem
imun. Sel kanker payudara menginduksi produksi enzim endokrin dan parakrin serta lipid
bioaktif oleh adiposit, yang pada gilirannya mendorong peningkatan pertumbuhan dan invasi sel
tumor. Leptin juga merupakan produk sekretori penting dari adiposit, yang dapat merangsang
produksi sejumlah faktor inflamasi termasuk interleukin (IL)-1, IL-6, dan faktor nekrosis tumor
(TNF)α. Obesitas merupakan faktor risiko yang cukup signifikan pengaruhnya terhadap insidensi
kanker payudara pada wanita pascamenopause dan dapat mendorong proses prokarsinogenik
pada adiposit. Sejumlah faktor seperti leptin, TNFα, IL-6, dan resistin yang disekresikan oleh
adiposit normal, akan meningkat secara paralel pada penderita obesitas dan memiliki efek
prokarsinogenik. Proses ini dapat memodifikasi sistem kekebalan tubuh dan meningkatkan
pertumbuhan tumor.

Efek Estrogenik pada Sel-Sel yang Berperan dalam Sistem Imun Jaringan Payudara Normal
Estrogen merupakan salah satu hormon predominan pada wanita selama periode premenopause
dan postmenopause. Estrogen memiliki peran penting dalam proses karsinogenesis payudara. Hal
ini terjadi melalui aktivitas estrogenik pada sel epitel payudara, namun terdapat evidensi bahwa
sel-sel yang berperan dalam sistem imun mengandung reseptor estrogen (ERs) dan pada saat
bersamaan aktivitasnya diatur oleh estrogen. Aktivitas hormonal ini akan memberikan
kesempatan bagi estrogen untuk mempengaruhi immunosurveillance, serta perkembangan dan
progresivitas kanker payudara. Aktivitas estrogenik dihasilkan dari pengikatan reseptor
sitoplasma ERα dan Erβ serta aktivasi jalur genomik, walaupun estrogen juga dapat
mengaktifkan aktivitas reseptor faktor pertumbuhan dengan pensinyalan melalui jalur
nongenomik oleh sinyal ERα independen ligan yang melibatkan pengikatan E2 ke reseptor
terikat membran, G protein-coupled estrogen receptor 1 (GPER1) dengan aktivasi protein G.
Sel-sel yang berperan dalam sistem imun dari kedua jenis sel bawaan (sel dendritik, makrofag,
neutrofil) dan adaptif (CD4+, CD8+, sel B) dalam jaringan payudara normal maupun jaringan
kanker payudara terbukti memiliki ER dan responsif terhadap estrogen.

Sel-Sel yang Berperan dalam Sistem Imun pada Karsinoma Duktal In Situ (DCIS). DCIS
memiliki karakteristik berupa peningkatan kepadatan dan luasnya infiltrasi sel-sel yang berperan
dalam sistem imun dibandingkan dengan jaringan payudara normal. DCIS derajat tinggi
memiliki persentase sel FOXP3+, makrofag CD68 dan CD68+PCNA+, antigen leukosit manusia
(HLA)–DR cells, sel T CD4+, sel CD20+B, dan total TIL yang secara signifikan lebih tinggi bila
dibandingkan dengan DCIS derajat rendah atau moderat. Thompson dkk mengemukakan bahwa
sel T CD3+ cenderung mendominasi di semua subtipe DCIS pada semua usia, dengan jumlah sel
T CD4+ yang lebih sedikit bila dibandingkan rata-rata jumlah sel T CD8+. Sel B CD20+
merupakan TIL yang paling umum ditemukan setelah sel T CD3+, diikuti oleh sel T regulator
FOXP3+ (Treg). Sel Treg FOXP3+ merupakan sel-sel yang berperan dalam sistem
imunosupresif penting lainnya yang cenderung meningkat pada DCIS.

Sel-Sel yang Berperan dalam Sistem Imun pada Karsinoma Payudara Invasif. Ruffell dkk
mengamati bahwa jaringan kanker payudara mengandung infiltrat yang didominasi oleh limfosit
CD8+ dan CD4+, dengan sejumlah kecil sel NK dan limfosit B, sedangkan pada jaringan
payudara normal banyak ditemukan sel derivatif myeloid seperti makrofag, sel mast, dan
neutrofil. Limfosit T teraktivasi juga mendominasi jaringan tumor, dengan sel T CD4+ dan
CD8+ menunjukkan peningkatan ekspresi penanda aktivasi CD69 dan HLA-DR. Kompleksitas
komposisi sel-sel yang berperan dalam sistem imun pada kanker payudara mencerminkan
aktivitas silang antara komponen respon imun bawaan karena mengatur lingkungan mikro tumor
dan polaritas respon imun adaptif di dalam tumor tersebut. Walaupun ditemukan peningkatan
progresif pada semua jenis sel baik pada sel parenkim dan stroma yang berbatasan dari jaringan
payudara normal ke karsinoma duktal, perbedaan paling mencolok dari tumor jinak proliferatif,
DCIS dan kanker invasif adalah masuknya sel CD3+ pada stroma. Peningkatan subset sitotoksik
(granzyme B) terbatas pada sel-sel yang berperan dalam sistem imun di parenkim.

Gambar 2. Sel-Sel Yang Berperan Dalam Sistem Imun Pada Penyakit Proliferatif Payudara Dan Kanker Payudara.
Sel-sel yang berperan dalam sistem imun tubuh, termasuk CD68+ (makrofag/monosit), CD3+ (CD4+, CD8+), dan
limfosit sitotoksik (CTL) ditemukan dalam penyakit proliferatif payudara dan mengalami peningkatan pada
parenkim serta stroma pada perkembangan kanker payudara. Peningkatan relatif untuk setiap jenis sel, dengan
proliferasi jinak sebagai baseline, ditunjukkan pada skala logaritmik pada grafik di bawah gambar.

Karakteristik Sel-Sel yang Berperan dalam Sistem Imun pada Struktur Limfe Tersier Jaringan
Kanker Payudara. Struktur limfe tersier (tertiary lymph organ/TLS) merupakan organ limfoid
ektopik yang berkembang pada lokasi peradangan kronis termasuk penyakit autoimun, infeksi,
dan tumor. TLS memiliki struktur yang terdiri dari sejumlah tipe sel imun bawaan (dendritik,
makrofag, neutrofil) dan adaptif (sel B, sel T) serta sel plasma dan venula endothelial (HEV).
TLS merupakan lokasi aktivasi respon imun terhadap tumor yang mengumpulkan dan
mengaktifkan TIL. TLS dapat berlokasi di dalam tumor atau dalam lokasi peritumor. Komponen
penting dari TLS adalah HEV, venula postkapiler khusus yang ditemukan pada jaringan limfoid
dan mendukung ekstravasasi limfosit tingkat tinggi dari darah. Identifikasi kumpulan sel-sel
yang berperan dalam sistem imun yang terorganisir dengan sel memori efektor di dalam dan di
sekitar jaringan kanker payudara serta kemampuan aktivitas antitumor dapat mempengaruhi
perkembangan dan perluasannya pada tumor. Keberadaan TLS dari HEV yang dapat
meningkatkan pergerakan limfosit ke dalam tumor dan juga berkontribusi terhadap upaya
identifikasi agen yang dapat mendorong aktivitas pembuluh darah tersebut.

Sel-Sel yang Berperan dalam Sistem Imun sebagai Biomarker Prognostik. Salah satu penelitian
pertama mengenai topik ini dilakukan oleh Denkert dkk dengan menggunakan biopsi inti tumor
dan stroma sebelum perawatan, untuk dianalisis infiltrat limfositiknya yang dievaluasi dengan
pewarnaan hematoksilin dan eosin rutin. Dua kategori infiltrat imun didefinisikan: iTu-Ly
(limfosit intratumoral) dalam kontak langsung dengan sel tumor atau di dalam kumpulan sel
tumor dan str-Ly (limfosit stroma) tanpa kontak langsung dengan sel tumor. Integrin CD103,
sebuah penanda sel T memori residen jaringan (TRM) memiliki aktivitas mediasi lokasi daerah
kanker di dalam tumor. Sejumlah upaya dilakukan untuk mengklasifikasi TIL dalam spesimen
kanker payudara, dengan fokus pada limfosit stroma sebagai indikator prognostik yang paling
dapat direproduksi dan memiliki kemungkinan respons yang cukup signifikan. Analisis oleh
Denkert dkk dari data yang dikumpulkan dari German Breast Cancer Group mengemukakan
bahwa klasifikasi tumor berdasarkan tingkat invasi limfosit stroma memungkinkan prediksi
respon kemoterapi neoadjuvan pada seluruh subtipe molekuler. Ruffell dkk mengemukakan
bahwa kemoterapi neoadjuvan mengubah kompleksitas lingkungan mikro imun dari tumor-
tumor sekunder. Tumor residual yang diintervensi dengan kemoterapi neoadjuvan menunjukkan
adanya peningkatan persentase dari sel myeloid yang menginfiltrasi, disertai dengan peningkatan
rasio sel CD8/CD4T dan jumlah sel ekspresi granzim yang cenderung lebih tinggi, bila
dibandingkan dengan tumor yang tidak diobati dengan kemoterapi neoadjuvan.

Terapi Berbasis Kekebalan Tubuh untuk Kanker Payudara. Mayoritas penelitian limfosit pada
kanker payudara difokuskan pada karakterisasi sifat infiltrasi limfosit, dengan sejumlah data
ekspresi gen yang mengisyaratkan peran fungsional sel-sel ini dalam respons antikanker.
Kemokin yang dianggap bertanggung jawab terhadap migrasi limfosit dan tanda ekspresi gen
yang terkait dengan respons efektor Tipe I telah terbukti berkorelasi dengan pCR. Reseptor
kematian terprogram 1 (PD-1) dan salah satu li-gandnya (PD-L1) juga menunjukkan korelasi
terhadap tingkat pCR yang lebih tinggi dan prognosis yang lebih baik pada kanker payudara.
Infiltrat imun yang ditemukan pada tahap awal DCIS mendorong pentingnya strategi untuk
meningkatkan respon imun terhadap tumor antigen potensial yakni dengan vaksinasi, yang telah
dieksplorasi baik dalam segi pencegahan, adjuvan, neoadjuvan dan kondisi metastatis. Upaya
tambahan yang dapat dilakukan antara lain mengeksplorasi penggunaan strategi ablatif tumor
(krioablasi, ablasi frekuensi radio, radiasi stereotaktik) sebagai pendorong potensi pelepasan
antigen terkait tumor dengan inhibitor checkpoint antibodi monoklonal untuk meningkatkan
derajat respon. TIL spesifik tumor dapat digunakan untuk mengidentifikasi neoantigen tumor
yang kemudian dapat digunakan dalam strategi vaksin khusus pasien atau untuk membuat
perawatan berbasis sel dengan TIL atau turunan produk rekayasa gen TCR autolog TIL. Peneliti
baru-baru ini menemukan bukti dalam laporan kasus tunggal bahwa terjadi regresi dari kanker
payudara yg bermetastasis pasca transfer sel adoptif dengan IL-2 dan pembrolizumab.

Kesimpulan
Sel-sel yang berperan dalam sistem imun jaringan payudara normal memiliki peran
penting dalam immunosurveillance dan menambah wawasan mengenai pencegahan dan
perkembangan proses karsinogenesis payudara. TIL memiliki nilai prognostik terhadap kanker
payudara dan berfungsi dalam transfer adopsi sebagai tatalaksana persebaran kanker payudara.
Imunologi dan imunoterapi berfungsi sebagai salah satu modalitas utama untuk pengelolaan
pasien dengan kanker, di samping pembedahan, kemoterapi, dan radioterapi.