Invasive Breast Carcinoma of No Special Type with Medullary Pattern: Morphological and Immunohistochemical Features
Mykola LУNDІN1, Nataliia HYRIAVENKO1, Vladyslav SIKORA1, Yuliia LУNDІNA1, Yuliia SOROKA2, Anatolii ROMANIUK1
1Department of Pathology, Medical Institute, Sumy State University, SUMY, UKRAINE
2Department of Fifth-Year Student, Medical Institute, Sumy State University, SUMY, UKRAINE
Keywords: Breast carcinoma, Triple negative breast cancer, Medullary carcinoma, Tumor microenvironment, Tumor infiltrating lymphocyte
Our study investigated the morphological and immunohistochemical characteristics of invasive breast carcinoma of no special type
(IBC-NST) with medullary pattern to explore the inconsistencies between the structural and clinical traits of this category of tumor.
Material and Method: The breast carcinoma samples (n = 26) with medullary pattern (defined according to established criteria) were subjected
to immunohistochemical assays of the following receptors: ER, PR, HER2/neu, Ki-67, p53, Bcl-2, VEGF, MMP1, E-cadherin, EGFR, Hsp70,
Hsp90, CD20, CD3, CD4, CD8, CD68, CD163, CD56, CD138, MPO, S100, IgG, IgM, and PD-L1.
Results: IBC-NST with medullary pattern was found to have negative expression of ER, PR, and HER2/neu; strong positive expression of
Kі-67, mutant р53, Bcl-2, E-cadherin, EGFR, and PD-L1; moderate positive expression of Hsp70 and Hsp90; and low or negative expression of
VEGF and MMP1. Furthermore, there was pronounced variability in the qualitative composition of tumor immune infiltrates with regards to
T-lymphocytes, B-lymphocytes, macrophages, plasmocytes, and granulocytes.
Conclusion: IBC-NST with medullary pattern has many unfavourable morphological and immunohistochemical prognostic characteristics, which
are balanced against the pronounced protective properties of the tumor cells and the qualitative characteristics of the tumor microenvironment.
These can lead to a favourable disease course despite the relatively adverse features of the carcinoma cells.
Breast carcinoma (ВС) is the most common malignancy
worldwide, accounting for 11.7% of all cancer cases,
with a prevalence of up to 24.5% among women. Despite
detection being possible at increasingly earlier stages, the
BC mortality rate remains relatively high (accounting
for 6.9% of all oncological deaths among both genders)
. The course of the disease, treatment sensitivity, and
prognosis depend on the tumor histological type, grade,
stage, vascular invasion, immunophenotype of neoplastic
cells, and tumor microenvironment 2
. Until recently,
medullary carcinoma was considered the most favourable
variant of BC 3
. In the latest World Health Organisation
(WHO) classification of breast tumors, published in 2018,
this type of carcinoma was included in the category of
invasive BC of no special type (IBC-NST). Now it has name
IBC-NST with medullary pattern or tumor -infiltrating
lymphocytes (TILs) rich IBC-NST 2,4
IBC-NST is well-defined and has a syncytial growth pattern
with no glandular structures of high histological grade
as well as prominent TILs 2,5. Cases are mainly triplenegative
without expression of the еstrogen receptor (ER),
progesterone receptor (PR), and human epidermal growth
factor receptor 2 (HER2/neu). Pronounced proliferative
activity of tumor cells and expression of tumor SUPPRESOR
protein р53 are commonly observed 5. However, despite
the potentially unfavourable histological features, IBCNST
with medullary pattern has a relatively good prognosis
2,6,7; notably, this has been associated with the qualitative
features of the tumor microenvironment (TILs) and
pronounced tumor cell adhesion (involving E-cadherin
expression) and absence of matrix metalloproteinase-1
The favourable course of this high-grade carcinoma has
not been explained by the presence or absence of the
abovementioned proteins. Further studies are required to
explain the inconsistencies between the morphological and
clinical features of IBC-NST with medullary pattern.
|Patients and Samples
Cases of IBC-NST with medullary pattern (n = 26) were
identified among patients who underwent mastectomy
and sectoral resection of the breast from 2010 to 2020 at
the Surgical Department of the Sumy Regional Oncology
Centre in Ukraine. Samples were included in the study
group if they met the following criteria: 1) well-defined
tumor contours with a peripheral pseudocapsule; 2)
syncytia formation in >75% of the tumor area; 3) absence
of tubular and glandular tumor features; 4) pronounced
lymphoplasmacytic stromal infiltration; 5) high histological
grade; 6) absence of metastases; and 7) absence of ER, PR,
and HER2/neu expression (2,5). Written informed consent
for tissue investigation was obtained from all patients. The
Bioethics Commission of the Medical Institute of Sumy
State University approved the experimental protocol (no.
5/2 from 12.02.2017).
Paraffin-block samples stored for histological diagnosis
were used in the immunohistochemical assay following
the method detailed in our previous publication 8. The
antibody panels (purchased from Thermo Scientific, Master
Diagnóstica, and Abcam) shown in Table I were employed
to establish the parenchymal and stromal components
of the immunophenotype of IBC-NST with medullary
Evaluation of Staining
Two pathologists independently analysed the immunohistochemical
staining evaluations and reached a consensus
decision when necessary. The reaction was considered positive
if the cells had cytoplasmic and/or nuclear expression
of heat-shock protein 70 (Hsp70) and 90 (Hsp90); cytoplasmic
and/or membranous expression of CD20, CD3,
CD4, CD8, CD163, CD56, and CD138; exclusively nuclear expression of ER, PR, Ki-67, and tumor SUPPRESOR protein
p53; exclusively cytoplasmic expression of Bcl-2, VEGF,
MMP1, CD68, MPO, S100, IgG, and IgM; and exclusively
membranous expression of HER2/neu, EGFR, and E-cadherin.
To determine the pattern of receptor expression, we
used a simplified 3-tiered scoring system: “-” – negative
expression (0% immunoreactive cells); “+” – low expression
(5–25% immunoreactive cells); “++” – moderate
expression (26–50% immunoreactive cells); “+++” – high
expression (>50% immunoreactive cells). Only the p53
nuclear expression pattern was considered as positive or
negative staining. Ki-67, Hsp70 and Hsp90 expression were
scored in percentage. The percentage of receptor-positive
cells among the total number of tumor cells was scored.
The intensity of the stained cells was considered separately.
PD-L1 expression was considered positive in the presence
of more than 1% of carcinoma cells showing membranous
staining of any intensity. This scoring sheet was discussed
with and approved by two pathologists prior to the start
of the study. No morphologic features of the cancer were
taken into account in this scoring. Data processing was
carried out using the GraphPad Prism 9 statistics software.
Detection and evaluation of the links among indicators
were carried out using the nonparametric Spearman’s rank
correlation coefficient (r). A p-value of 0.05 (95% level of
confidence) was considered statistically significant.
The results of the immunohistochemical study showed
that all cases of IBC-NST with medullary pattern were ER-,
PR- and HER2-negative. At the same time, pronounced
proliferative activity (nuclear Kі-67 expression in 78.4±4.6%
of tumor cells), mutant р53 expression (nuclear expression
in 92% of cases), and hyperproduction of anti-apoptotic
Bcl-2 protein (cytoplasmic expression) were observed in all cases (Figure 1
). Besides the neoplastic cells, some cells
of the immune infiltrate of the tumor microenvironment
were Kі-67- and Bcl-2-positive.
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|Figure 1: IBC-NST with medullary pattern: a pronounced expression of Ki-67, p53, and bcl-2 in tumoral cells (A); immune
microenvironment cells (Ki-67- and bcl-2-positive expression) (B). Immunohistochemical study of Ki-67, p53, and bcl-2; x200.
All cases of IBC-NST with medullary pattern had strong
(immunoexpression in more than 50% of tumor cells)
membranous expression of E-cadherin, EGFR, and PD-L1
in the cancer cells (Figure 2). Moderate positive nuclear and
cytoplasmic expression of Hsp70 and Hsp90 was observed
in 74±6.3% and 68±5.7% of the tumor cells, respectively.
Simultaneously, the majority of the tumor cells were either
negative or had low (weak expression in less than 25% of
tumor cells) VEGF and MMP1 cytoplasmic expression
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|Figure 2: IBC-NST with medullary pattern: a pronounced expression of E-cadherin, EGFR, and PD-L1; moderate expression of
hsp70 and hsp90; week expression of MMP1 and VEGF; negative expression of MPO in tumoral cells, positive in scattered cells of
microenvironment. Immunohistochemical study of E-cadherin, EGFR, hsp70, hsp90, VEGF, MMP1, PD-L1, and MPO ; x400.
We focused on studying the qualitative composition of
the tumor microenvironment of IBC-NST with medullary
pattern. Immunohistochemical analysis of the expression
of CD20 (B-lymphocytes), CD3 (T-lymphocytes), CD4
(T-helper cells), CD8 (T-killer cells), CD56 (natural killer
cells), CD138 (plasmocytes), CD68, S100, and CD163
(macrophages), MPO (granulocytes), IgG, and IgM
revealed the specific features of the immune infiltrate
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|Figure 3: IBC-NST with medullary pattern: Immunohistochemical study of CD20, CD3, CD4, CD8, CD56, CD138, IgG, and IgM ; x400.
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|Figure 4: IBC-NST with medullary pattern: Immunohistochemical study of CD68, S100, and CD163; x400.
The tumor immune-infiltrate microenvironment mainly
comprised T-lymphocytes (44% of immune cells) with a
relatively low number of B-lymphocytes, macrophages,
plasmocytes, and granulocytes (13%, 22%, 9%, and 3% of
immune cells, respectively). T-helper cells (CD4+ cells),
with a relatively small number of T-killer cells (CD8+ cells)
and NK cells (CD56+ cells), were observed among the
T-lymphocytes (Figure 3). The tumor microenvironment
contained single and grouped plasmocytes, which reached
a prevalence of about 8% of the immune-cell infiltrate in
some cases. Most B-lymphocytes and plasmocytes showed
IgG expression, and some showed IgM expression.
Macrophages (CD68+ cells) were present in significant
numbers both among tumor cells and in the immune
infiltrate (Figure 4). It should be noted that their active
forms (S100+ cells) were mainly localised among the cancer
cells. A significant population of M2 macrophages (CD163+
cells) was identified, and they were diffused throughout all
the components of the neoplastic tissue.
The scores for each cell type in the tumor microenvironment
showed the following ratios: CD3+:CD20+:CD138+:CD56+:
CD68+:MPO = 0.44: 0.13: 0.09: 0.09: 0.22: 0.03.
The malignancy of tumors is determined by various factors,
including morphological and molecular-genetic features of
the affected tissues 2,5,7,8
. The qualitative composition of the tumor microenvironment is of particular interest
. Here we focused on IBC-NST with medullary
pattern, which has unique favourable prognostic features
with simultaneous high-grade tumor growth 2,5-7
Our study investigated the immunohistochemical
features of IBC-NST with medullary pattern to explain
the inconsistency between the morphological and clinical
characteristics of this tumor type. We found that the
cancer cells had pronounced proliferative activity (Kі-67
expression), and blocked both natural and drug-induced
apoptosis (p53 and Bcl-2 expression), as well as the
expression of unfavourable proteins (EGFR, Hsp70, Hsp90,
and PD-L1) that induce progression of the neoplastic
process 8,9,11-13. At the same time, the cells were ER and
PR-negative. In contrast, most of the tumor cells had no HER2/neu expression, low VEGF and MMP1 expression
(weak expression in less than 25% of tumor cells), and high
E-cadherin expression (strong expression in more than
50% of tumor cells), which indicated a favourable course of
the carcinoma 14-17.
Our results showed pronounced variability in the qualitative
composition of the tumor microenvironment of IBC-NST
with medullary pattern. This was represented mainly by
TILs, which were predominantly T-lymphocytes and, in
particular, CD4+ cells. Simultaneously, significant numbers
of CD8+ T-cells, NK cells, В-lymphocytes, and plasmocytes
were found in the immune infiltrate. Furthermore, the
neoplastic tissue was infiltrated with a significant quantity of
macrophages. Notably, most of the activated macrophages
(S100+ cells) were in close contact with cancer cells. At the
same, a significant number of M2 macrophages (CD163+
cells) were found in the tumor microenvironment, which
could have an unfavourable adverse effect on the course of
the neoplastic process 5,9,18,19.
From the abovementioned findings, we concluded that
the unique course of IBC-NST with medullary pattern is
determined by several factors (Figure 5). This tumor type was characterized by a combination of both aggressive
and favourable immunophenotypic features of cancer cells
and the immune microenvironment. On the one hand, ER
and PR were absent in the tumor tissue; there was a high
proliferative index; overexpression of p53, Bcl-2, EGFR,
hsp70, hsp90, and PD-L1; and significant numbers of M2
macrophages. On the other hand, there were insufficient
amounts of pro-metastatic proteins (VEGF and MMP1); an
absence of HER2/neu expression; and the severe adhesive
capacity of tumor cells prevented local and remote tumor
spread. The activation of cellular (macrophages, CD4+
and CD8+ T-cells, and NK cells) and humoral (activated
macrophages, B-lymphocytes, CD4+ T-cells, plasmocytes,
and IgG synthesis) local immune responses limited the
spread of the disease.
Click Here to Zoom
|Figure 5: Interaction of protective and aggressive factors in IBCNST
with medullary pattern.
In IBC-NST with medullary pattern, the favourable factors
(protection indicators) mostly surpassed the adverse
influences. This led to a favourable prognosis for the course
of the malignant process, despite the numerous negative
morphological and immunohistochemical features of the
parenchymal and stromal tumor components.
In summary, IBC-NST with medullary pattern has
many prognostically unfavourable morphological and
immunohistochemical characteristics, which are balanced
by the pronounced protective properties of the tumor cells
and the qualitative traits of the tumor microenvironment.
This leads to a favourable course for this carcinoma despite
the relatively adverse features of the cancer cells.
CONFLICT of INTEREST
The authors declare that they have no competing interests.
This research was supported by the Ministry of Education
and Science of Ukraine Grant № 0121U100472 and research
theme № 0119U100887.
Written informed consent was obtained from patients who
participated in this study.
Concept: ML, YL, YS, AR, Design: ML, NH, AR, Data
collection or processing: ML, NH, VS, YL, YS, Analysis of
Interpretation: ML, NH, VS, YL, YS, AR, Literature search:
ML, NH, VS, YL, YS, Writing: ML, NH, VS, YL, AR,
Approval: ML, VS, AR.
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