Received: 19 October 2019 | Accepted: 21 January 2020

DOI: 10.1111/pin.12910

REVIEW ARTICLE

Invasive breast cancer: Current perspectives and

emerging views

Eric Ka Ho Shea1,2 | Valerie Cui Yun Koh2 | Puay Hoon Tan3

1
Department of Clinical Pathology, Tuen Mun Hospital, Tuen Mun, Hong Kong
2
Department of Anatomical Pathology, Singapore General Hospital, Singapore, Singapore
3
Division of Pathology, Singapore General Hospital, Singapore, Singapore

Abbreviations:
ASCO, American Society of Clinical Oncology;
CAP, College of American Pathologists; DCIS,
ductal carcinoma in‐situ; EGFR, epidermal
growth factor receptor; ER, estrogen receptor;
H&E, hematoxylin and eosin; HER2, human
epidermal growth factor receptor 2; NST, no
special type; PR, progesterone receptor; TIL,
tumor infiltrating lymphocyte; WHO, World
Health Organization
Correspondence
Puay Hoon Tan, MBBS, FRCPA, FAMS, MD,
FRCPath, Division of Pathology, Singapore
General Hospital, 20 College Road, Academia,
Diagnostics Tower, Level 7, Singapore 169856,
Singapore.
Email: tan.puay.hoon@singhealth.com.sg
Invasive breast cancer constitutes a heterogeneous group of tumors. They comprise
various histological types that differ in clinical presentation, imaging features, histo-
pathological characteristics, biomarker profiles, prognostic and predictive parameters.
The current classification of invasive breast cancer is based primarily on histopatho-
logical features. Invasive carcinoma of no special type accounts for the majority, with
some rare entities also being described. With recent research and advances, there are
emerging concepts, including new genetic insights of invasive breast cancer and the
role of the stromal microenvironment. With greater understanding of the pathogenesis
of invasive breast cancer, changes based on the correlation of histologic and genetic
findings have been incorporated in the latest World Health Organization classification
of breast tumors. Medullary carcinomas are subsumed as invasive carcinoma of no
special type with basal‐like and medullary features, regarded as part of the spectrum
of tumor infiltrating lymphocyte‐rich breast cancers. Tall cell carcinoma with reversed
polarity is proposed as a distinct entity in recognition of unique IDH2 mutations. This
article reviews conventional prognostic parameters, new histological entities, and
updates on breast cancer classification, with inclusion of some genetic insights into
breast cancer and the role of tumor infiltrating lymphocytes.
KEYWORDS
breast neoplasms, diagnostic molecular pathology, prognostic factors, tumor infiltrating
lymphocytes

INTRODUCTION largest category.1 Invasive breast cancer types have clinical

Invasive breast cancer is a malignant epithelial neoplasm
of the breast with malignant cells breaching the myoepithelial
and basement membrane layers to extend into the
surrounding stroma. It comprises a heterogeneous group of
tumors. Current classification of invasive breast cancer is
based primarily on histopathological features, with invasive
carcinoma of no special type (NST) accounting for the
importance in terms of prognostic implications. The 2019
World Health Organization (WHO) Classification of Tumors
of the Breast2 recognizes different histological types of
invasive breast carcinoma with distinct morphologic features
(Table 1).
Invasive carcinoma of no special type, as its name implies,
is composed of tumors without characteristic features of any
specific histological type.

Pathology International. 2020;1–11. wileyonlinelibrary.com/journal/pin © 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd | 1
2 E. K. H. Shea et al.
Table 1 Different histological types of invasive breast carcinoma
Histological diagnosis
Invasive carcinoma of no special type (NST)
Microinvasive carcinoma
Invasive lobular carcinoma
Tubular carcinoma
Cribriform carcinoma
Mucinous carcinoma
• Mucinous cystadenocarcinoma
Invasive micropapillary carcinoma
Carcinoma with apocrine differentiation
Metaplastic carcinoma
Rare and salivary gland type tumors
• Acinic cell carcinoma
• Adenoid cystic carcinoma
• Secretory carcinoma
• Mucoepidermoid carcinoma
• Polymorphous adenocarcinoma
• Tall cell carcinoma with reversed polarity
Pathological prognostic and predictive factors of in-
vasive breast cancer inform survival outcomes and ad-
juvant therapy selection. Conventional prognostic
parameters: tumor type, grade, size and lymph node
status, provide information on the likely course of dis-
ease. Predictive markers, such as estrogen receptor
(ER), progesterone receptor (PR) and human epidermal
growth factor receptor 2 (HER2), allow selection of
therapy with the highest likelihood of efficacy to the in-
dividual patient and can be regarded as a form of per-
sonalized medicine.
There have been some notable changes in the classi-
fication and terminology of breast tumors over the years. In
the 2019 WHO classification, carcinomas with medullary
features are subsumed as a combined morphologic subset
under ‘invasive carcinoma NST with basal‐like and me-
dullary features’, regarded as part of the spectrum of tumor
infiltrating lymphocyte (TIL)‐rich breast cancers. Terminol-
ogies for ‘solid papillary breast carcinoma resembling the
tall cell variant of papillary thyroid carcinoma’ and ‘solid
papillary carcinoma with reverse polarity’, have been
harmonized into ‘tall cell carcinoma with reversed polarity’.
The IDH2 mutation has been shown in up to 75% of these
cases.3
The molecular classification of invasive breast carci-
noma has gained recognition since the publication of the
molecular portraits of breast tumors in 2000.4 There are at
least five intrinsic molecular subtypes (luminal A, luminal
B, HER2 enriched, basal‐like and normal breast‐like)
currently recognized, based on hierarchical cluster anal-
ysis of genes.5,6
© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
CONVENTIONAL PROGNOSTIC PARAMETERS OF
INVASIVE BREAST CANCER
Prognostic factors provide information on the likely course of
disease in an untreated individual. Most patients however,
undergo adjuvant treatment that influences the natural
course of disease. Conventional prognostic factors include
tumor size, histological type, histological grade, lymph node
status, lymphovascular invasion and tumor stage.
Tumor size
Tumor size is one of the most powerful predictors of behavior
in breast cancer. Node‐negative patients with tumors less
than 10 mm in greatest dimension have a 10‐year disease‐
free survival of about 90%.7
The greatest dimension of the invasive component is used
in determining pathological T stage. It is measured grossly to
the nearest mm. For small invasive cancers less than
10 mm, tumor size is measured off the glass slide. For dif-
fuse and poorly defined invasive lesions, an estimate is
made with explanatory comments. When two or more dis-
crete tumors are present, each should be measured and
reported separately.
In measuring tumor size, there are potential pitfalls, in-
cluding asymmetry of tumors, slicing of the specimen and/or
tumor prior to receipt in the laboratory, effect of prior core
biopsy, multifocality of tumor and changes of neoadjuvant
treatment. Correlation with radiological finding in such cases
is of key importance.
Microinvasion is defined as one or more clearly separate
microscopic foci of invasive tumor cells extending beyond
the basement membrane into adjacent tissue, each focus
being not more than 1 mm in size. The biological behavior is
similar to ductal carcinoma in‐situ (DCIS).8–10
Histological grade
Histological grade reflects the degree of tumor differentiation
and represents its intrinsic biological characteristics. Grading
of invasive breast carcinoma has been shown to be a robust
predictor of clinical outcome.11 Invasive breast carcinomas,
including lobular and medullary‐like carcinomas, are rou-
tinely graded based on semi‐quantitative methods, using the
Nottingham grading scheme.11,12 Tubule formation, nuclear
pleomorphism and mitotic count are evaluated using clearly
defined criteria in the invasive tumor component, with each
characteristic scored from 1 to 3. Tubule formation is defined
as tubular structures exhibiting central lumina surrounded by
polarized tumor cells. The cut‐off points of 75% and 10% are
used in allocating score 1 (more than 75% tubules), score 2
 Invasive breast cancer pathology 3

(10% to 75% tubules) and score 3 (less than 10% tubules)
respectively. Nuclear pleomorphism is categorized into score
1 (mild), score 2 (moderate) and score 3 (marked) by the
regularity of nuclear size and shape with reference to ad-
jacent normal epithelial cells. The mitotic count per 10 high
power fields is evaluated. To standardize the true area over
which mitoses are enumerated, and to facilitate quantitative
measurements on the digital system, the latest 2019 WHO
classification recommends counting the total number of mi-
tosis per millimeter square, instead of 10 high power fields,
as the areas of high power fields vary in different micro-
scopes. The three scores are added to assign the final grade
as follows: score 3 to 5, grade 1; score 6 to 7, grade 2; score
8 to 9, grade 3.
Histological grade is a powerful prognostic parameter. The
chance of a 10‐year survival in patients with histological
grade 1 carcinomas is 85% compared with less than 45% in
those with histological grade 3 carcinomas.12 Patients with
histological grade 1 and stage II disease have the same
survival rate as those with histological grade 3 and stage I
disease. Patients with histological grade 1 carcinomas of
less than 2 cm in size have 99% 5‐year survival, even with
positive nodal disease.13
The proportion of histological grades varies among dif-
ferent studies, ranging from 19% to 46% for grade 3 tumors.
In general, grades 1, 2 and 3 tumors occur in the proportion
of 2:3:5. There is possible interaction of histological grade
with tumor size. Large tumors of pathological stage T3 and
T4 tend to be high grade, although there are large grade 1
tumors as well. In small tumors, there was a perceived lack
of impact of grade due to inconsistent data used in deriving
this conclusion, though with current grading methods, its
relevance in smaller tumors is maintained.11
The reproducibility of histological grade depends on the
quality of fixation, standardization of pre‐analytical factors
and grading methods. Therefore, the technical factors po-
tentially contributing to inter‐observer variability such as
tissue fixation, preservation and section preparation should
be minimized by the implementation of well‐defined guide-
lines recommended by the College of American Pathologists
(CAP) for tissue handling. In order to avoid pitfalls in histo-
logical grading, established criteria of grading should be
followed. Mitotic counting requires calibration of the micro-
scopic field. The most mitotically active area at the ad-
vancing tumor edge should be selected, and hyperchromatic
nuclei without spiculate extensions should be excluded.
For limited tissue samples such as the core biopsy, tumor
heterogeneity could be a major concern in determining the
grade of invasive breast cancer. There are several studies
evaluating the concordance rates of invasive breast cancer
grading on core biopsy and excision. The concordance rate
between provisional grade on core biopsy and the final ex-
cision grade on excision is 59% to 75%.14–16
Histological type
Histological types of invasive breast cancer show relevant
prognostic implications; hence it is clinically important to
recognize them.17,18 Different histological types show vari-
able prognosis and they are summarized in Table 2.17
Tubular carcinoma is defined as an invasive carcinoma
comprising open tubules with a single layer of tumor cells
enclosing a clear lumen in more than 90% of the tumor.1
Tumors with 50% to 90% tubular growth pattern admixed with
other types should be regarded as mixed type. Tubular

Table 2 Variable prognosis of different histological types

Tumors with excellent Tumors with good Tumors with poor

prognosis (>80%
10‐year survival)
Tubular carcinoma and
cribriform carcinoma
Mucinous carcinoma
Tubulolobular variant of
invasive lobular
carcinoma
prognosis (60–80%
10‐year survival)
Mixed invasive
carcinoma with tubular
and other special types
Mixed carcinoma with
ductal and other
special types (invasive
carcinoma of NST
pattern constitutes
10% to 49% of tumor)
Alveolar variant of
invasive lobular
carcinoma
Tumors with moderate prognosis prognosis (<50%
(50–60% 10‐year survival) 10‐year survival)
Invasive papillary carcinoma Invasive carcinoma
NST
Invasive lobular carcinoma, classic type Solid variant of invasive
lobular carcinoma
Carcinoma with medullary features,
including medullary and atypical
medullary carcinomas*

*Currently regarded as part of the spectrum of invasive carcinomas NST with prominent tumor infiltrating lymphocytes (TILs).

© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
4 E. K. H. Shea et al.
carcinoma constitutes less than 2% of invasive breast cancers,
and 9% to 19% of those detected in screening programmes.19
It occurs more frequently in older patients. It shows smaller
tumor size, less nodal involvement and better prognosis
compared with invasive carcinoma (NST).20–24 Tubular carci-
noma is associated with low‐grade DCIS, lobular neoplasia,
flat epithelial atypia and can be found in radial scars.
Mucinous cystadenocarcinoma, included in the 2019
WHO breast blue book, is a rare lesion resembling that oc-
curring in the pancreas and ovary.25 It is characterized by
cystic structures lined by tall columnar cells with abundant
intracytoplasmic mucin, morphologically similar to pan-
creatobiliary or ovarian mucinous cystadenocarcinoma
(Figs. 1 and 2). It tends to occur in Asian and post‐
menopausal patients, forming well circumscribed cystic
masses with gelatinous contents. There is no myoepithelial
cell layer, and DCIS may be present in surrounding tissue. It
is generally triple negative or occasionally HER2 positive;
CK7 positive, CK20 and CDX2 negative.26,27 The prognosis
is usually good, with no reported distant metastasis.
Lymph node status
Axillary lymph nodes are examined histologically as sentinel
lymph node biopsies or as axillary dissection specimens to
determine nodal staging. Axillary nodal status is an important
predictor of disease‐free survival and overall survival in invasive
breast cancer.28 The size of metastatic focus is also quantified.
Lymph node metastases are classified as macrometastases,
micrometastases and isolated tumor cells. Macrometastases
are tumor deposits larger than 2.0 mm in size. Micro-
metastases are defined as histological metastatic tumor foci
Figure 1 H&E image of mucinous cystadenocarcinoma (low
magnification): Cystically dilated ducts filled with luminal mucin re-
sembling mucocele‐like lesion (Adapted with permission from Tan
PH, Sahin AA. Atlas of Differential Diagnosis in Breast Pathology.
Springer Nature: Springer, 2017).
© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
Figure 2 H&E image of mucinous cystadenocarcinoma (high
magnification): Neoplastic cells containing cytoplasmic mucin and
basally located nuclei (Adapted with permission from Tan PH, Sahin
AA. Atlas of Differential Diagnosis in Breast Pathology. Springer
Nature: Springer, 2017).
larger than 0.2 mm in size and/or more than 200 cells, but not
larger than 2 mm in size. Isolated tumor cells are clusters of
tumor cells not exceeding 0.2 mm in size or 200 cells and they
are regarded as pathological stage N0.1
In assessing nodal status, grossly uninvolved lymph nodes
are entirely submitted for histological evaluation. All macro-
metastases should be identified by thin slicing of lymph nodes
at widths of no more than 2 mm. All slices are embedded and
representative H&E sections from each slice are examined.
Determining nodal metastatic size could be problematic, par-
ticularly in cases with multiple foci of metastatic deposits in the
same lymph node. Currently, the greatest dimension of the
largest contiguous metastatic focus should be used to classify
the node.
Axillary nodal status has prognostic importance. The
disease‐free survival reduces with every positive lymph node.
Patients with four or more positive axillary lymph nodes have
a worse prognosis than those with fewer involved nodes.29–32
Macrometastases are prognostically important while micro-
metastases and isolated tumor cells, depending on studies,
have uncertain prognostic significance.33
Lymphovascular invasion
Lymphovascular invasion is defined as carcinoma within
small vessels outside the main tumor mass, most fre-
quently at the periphery of an invasive carcinoma
(peritumoral lymphovascular invasion) (Fig. 3). It is as-
sociated with lymph node metastasis and is an in-
dependent prognostic factor for local and distant
recurrence.34–36 Lymphovascular invasion in the dermis is
a poor prognostic factor due to frequent local recurrence
and distant metastases.37

Figure 3 Lymphovascular invasion (high magnification): Tumor
embolus in a lymphovascular space (H&E stain).
It is not required to distinguish blood vessels from lym-
phatics. Invasive carcinoma cells may form circumscribed
nests filling the lymphovascular spaces and hence may
mimic DCIS. Necrosis in carcinoma nests and the difficulty in
identifying the endothelial cells lining lymphovascular spaces
could further compound the diagnostic difficulty. On the other
hand, artifactual retraction around tumor nests may produce
an appearance simulating that of lymphovascular invasion.38
Identifying tumor cells in locations where normal lympho-
vascular spaces occur is helpful. Endothelial cell markers,
including CD31, CD34, factor VIII and D2‐40 may be val-
uable in confirming lymphovascular invasion. It is important
to note that D2‐40 not only highlights lymphatic endothelial
cells, but also marks myoepithelial cells; hence, it should be
used with caution in this setting.39–44
EVOLVING TERMINOLOGY AND ENTITIES
Although histopathology remains the basis for current clas-
sification of invasive breast cancer, there have been
changes in terminology and new entities described, recog-
nizing recent genetic insights and better understanding of the
stromal microenvironment.
Classical medullary carcinomas are vanishingly rare in the
breast. To define medullary carcinoma histologically, criteria
of more than 75% syncytial growth pattern, lack of glandular
structures, diffuse lymphoplasmacytic infiltrates, moderate to
marked nuclear pleomorphism and complete histological
circumscription are required.45,46 Atypical medullary carci-
noma has been proposed for tumors with syncytial growth
pattern and two or three other criteria. In the 2012 WHO
classification, medullary carcinoma, atypical medullary car-
cinoma and invasive carcinoma NST with medullary features
are grouped together under ‘carcinomas with medullary
© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
Invasive breast cancer pathology 5
features’, in recognition of their common immune enriched
microenvironment, basal‐like expression and occasional
association with BRCA1 mutations.1 In the 2019 WHO
classification, these tumors are subsumed as a combined
morphologic subset under ‘invasive carcinoma NST with
basal‐like and medullary features’ (Fig. 4), regarded as part
of the spectrum of TIL‐rich breast cancers.
The term ‘tall cell carcinoma with reversed polarity’ (Fig. 5)
has been proposed to replace previous terms ‘solid papillary
breast carcinoma resembling the tall cell variant of papillary
thyroid carcinoma’ and ‘solid papillary carcinoma with re-
verse polarity’. These are rare tumors, typically low grade
and triple negative,47 with favorable prognosis. They harbor
specific morphologic features characterized by cuboidal to
tall tumor cells with reverse nuclear polarity and abundant
eosinophilic cytoplasm. Nuclei are sometimes grooved.48,49
They typically show expression of CK5/6 and calretinin. Ki67
proliferative index is usually low (less than 5%). Despite
previous terms and nuclear features, they are unrelated to
papillary thyroid carcinoma. A molecular analysis described
more than 75% of cases to harbor IDH2 mutations, with
protein immunohistochemistry with a specific antibody IDH1/
2 mutant R132/R172 that can highlight IDH2 mutations.
Transcriptomic analysis showed that the proteoglycan
pathway was significantly enriched.3 Such findings support
the fact that tall cell carcinoma with reversed polarity is a
unique breast tumor that is distinguished from other papillary
breast tumors.
STROMAL MICROENVIRONMENT
The stromal milieu is an important factor in the development
and progression of invasive carcinoma, with interactions
between malignant cells and the stromal microenvironment,
including TILs. It represents an emerging area of active re-
search. In invasive breast carcinoma, the stroma may show
cellular fibroblastic proliferation, scant connective tissue,
marked hyalinization or elastosis. A fibrotic focus is defined
as an area of exaggerated reactive stromal formation larger
than 1 mm within the tumor with or without coagulative ne-
crosis. It is an independent prognostic factor in invasive
breast cancer, with its presence associated with more ag-
gressive behavior.50,51
A number of studies have been performed to determine
the prognostic and predictive significance of TILs in various
solid tumors, including invasive breast cancer.52–57 As im-
mune checkpoint inhibitors and other forms of im-
munotherapy have been successful in diverse solid tumors
of different organs, there is a need for reliable, easily appli-
cable and reproducible assessment of TILs. The assessment
of TILs as a routine clinical biomarker however has not yet
been implemented in invasive breast cancer.
6 E. K. H. Shea et al.
Figure 4 (a) Invasive carcinoma with medullary‐like features is categorized under invasive breast cancer no special type in the 2019 WHO
breast tumor classification. Solid sheets of high‐grade carcinoma cells are accompanied by lymphoplasmacytic infiltrates. (b) Basal‐like
immunophenotype in an invasive breast cancer of no special type, with immunohistochemical expression of the high molecular weight keratin
CK14.
In early stage HER2‐positive and triple negative breast
carcinoma, TILs are recognized in up to 75% of cases, with
up to 20% of dense infiltrates. Conversely, less TILs are
seen in the luminal subtypes.58 Recommendations for the
evaluation of TILs in invasive breast cancer have been made
by an international TILs working group in 2014.59,60 Stand-
ardization of TILs evaluation not only improves reproduci-
bility and consistency, but also helps in assessing the utility
of TILs in the era of immunotherapy. In the evaluation of TILs
score, hematoxylin and eosin (H&E) sections are examined
at the microscopic magnification of 200× (20× objective, 10×
eyepiece) or 400× (40× objective, 10× eyepiece). For prac-
tical purposes, it is considered adequate to examine one
section (4–5 µm) per patient. Full section is preferred to
limited biopsy. Core biopsy can be used in the pre‐
therapeutic neo‐adjuvant setting. TILs should be reported for
Figure 5 Tall cell carcinoma with reversed polarity shows tall
columnar cells with nuclei aligned towards the apical surface (re-
versed nuclear polarity), arranged in papillary and solid papillary
patterns. Courtesy of Dr Wentao Yang.
© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
the stromal compartment and they are scored as a per-
centage of stromal areas alone. Instead of the number of
stromal cells, the area of stromal tissue should be used as a
denominator to determine the percentage of stromal TILs,
that is, area with mononuclear cells is divided by total intra‐
tumoral stromal area. All mononuclear cells, including lym-
phocytes and plasma cells, but not polymorphonuclear leu-
kocytes, in the stromal compartment between areas of
carcinoma are included. Tumor zones with factors precluding
proper assessment, such as necrosis, hyalinization, crush
artifacts and previous biopsy sites should not be included. In
addition, when TILs are noted around DCIS and normal lo-
bules, or outside the boundary of tumor, they are also ex-
cluded. Similarly, peritumoral follicular aggregates and
tertiary lymphoid structures should not be included in the
assessment. If TILs are distributed heterogeneously, an
average rather than a hotspot approach is reported. TILs
should be assessed as a continuous variable, since more
biologically relevant information may be provided and more
accurate statistical analysis can be performed. Rounding up
the percentage to the nearest 5–10% is done by most
pathologists in daily practice. It is recommended to report the
scores in as much detail as they can. Stromal TILs per-
centage is a semi‐quantitative parameter. The international
TILs working group in 2014 has no formal recommendation
for a clinically relevant TIL threshold.60 A valid methodology
was considered more important than determining thresholds
for clinical practice.
MOLECULAR BIOMARKERS
There are three biomarkers, namely ER, PR
and HER2, used routinely in the assessment and clinical
management of invasive breast cancer. They should be

performed on all invasive breast cancers according to the
guidelines published by the American Society of Clinical On-
cology/College of American Pathologists (ASCO/CAP).61,62
Large clinical trials have shown a strong predictive value of
ER to hormonal treatment, such as tamoxifen.63 Invasive
breast carcinomas with phenotype ER‐positive and PR‐
positive are associated with the best response.64 Invasive
breast cancers are considered positive for ER and PR when
at least 1% of tumor cells show nuclear staining.62,65 An ER
assay should be performed on cases of DCIS as well.66
The HER2‐positive invasive breast carcinomas show favor-
able response to therapy targeting specifically the HER2 pro-
tein, such as trastuzumab.67–69 Therefore, accurate and reliable
results are essential. The ER and PR status is determined using
immunohistochemistry. The HER2 status is evaluated with im-
munohistochemistry, fluorescence in‐situ hybridization (Fig. 6)
or other chromogenic methods of in‐situ hybridization.70
There are potential pitfalls in the assessment of ER, PR
and HER2 status. These include pre‐analytic, analytic and
post‐analytic factors.71 Examples of these factors are listed
in Table 3.
The specimen type impacts the fixation. A core biopsy is
generally better fixed than an excisional biopsy for the
same period of fixation. As formalin fixation rate is limited,
surgical specimens must be sliced into sections of 3 to
5 mm thick. Poor tissue fixation is the most common
source of error in hormonal receptor testing. Tissue fix-
ation should be done as soon as possible, preferably
within 20 to 30 min after resection or biopsy. The tissue
should be fixed in 10% neutral‐buffered formalin,
instead of any other unvalidated fixatives as
immunohistochemistry and in‐situ hybridization can be
hampered by non‐formalin fixatives. The duration of
fixation for surgically excised samples should be 6 to 72 h.
Figure 6 HER2 gene amplification demonstrated by fluorescence
in‐situ hybridization assay using a dual‐probe system.
© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
Invasive breast cancer pathology 7
Table 3 Factors that impact the assessment of ER, PR and HER2
status
Pre‐analytic Post‐analytic
factors Analytic factors factors
Type of fixation Assay validation Interpretation criteria
Duration of fixation Equipment Reporting elements
calibration
Method of tissue Laboratory Scoring system
processing procedures
Time to slicing and Staff competence
fixation
Type of antigen
retrieval
Test reagents used
Control materials
Assay conditions
The hormone receptor result should be interpreted with
caution. There are certain clues to questionable hormone
receptor results which are detailed in Table 4.
HER2 testing in invasive breast cancer should be as-
sessed only in the invasive component as HER2 staining can
be seen in up to 50% of DCIS as well.72 The HER2 im-
munohistochemical interpretation should be based on the
ASCO/CAP guidelines and recommendations.61 A positive
HER2 result should be interpreted with caution in cases of
grade 1 invasive carcinoma, special types such as invasive
tubular, cribriform and mucinous carcinomas, classic in-
vasive lobular carcinoma and small invasive cancer amid
extensive high‐grade DCIS.
To maximize successful receptor testing, validation,
standardization and accreditation are essential. The an-
tibody, probe and result should be validated. The guide-
lines and recommendations should be implemented to
ensure optimal quality of testing. The laboratory should
also participate in external quality assurance programs,
such as those offered by the College of American Path-
ologists, the Royal College of Pathologists of Australasia
and the UK National External Quality Assessment
Scheme. Reliable internal and external quality controls
are essential. Regular reviews and discussion in breast
multidisciplinary conferences and tumor boards are also
preferred.
Table 4 Questionable hormone receptor results
Clues to questionable hormone receptor results
Internal and external controls are not working
Grade 1 tumors reported as ER and PR negative
Special types, for example, tubular, cribriform and mucinous
carcinomas reported as ER and PR negative
Medullary carcinoma reported as ER, PR and HER2 positive
8 E. K. H. Shea et al.
CELL PROLIFERATION IN BREAST CANCER: KI67
Ki67 is a labile nonhistone nuclear protein that is tightly linked
to the cell cycle. It is expressed in proliferating cells in mid G1
phase, increasing in level through S and G2 phases, and
peaking in M phase. It is catabolized rapidly at the end of M
phase. It is undetectable in resting (G0 and early G1) cells.28,73
Ki67 is regarded as a marker of cell proliferation. The
percentage of Ki67‐positive tumor cells can stratify patients
with invasive breast cancer into good and poor prognostic
categories. When it is utilized in combination with ER, PR
and HER2 scores to form the ‘IHC4′, Ki67 proliferative index
has a prognostic value in patients with ER‐positive invasive
breast cancer.74 It is a predictor of response to chemo-
therapy.75,76 Ki67 proliferative index is a surrogate marker to
define intrinsic subtypes of invasive breast carcinomas into
luminal A (Ki67 less than 14%) and luminal B (Ki67 high).
This cut‐point for Ki67 labeling index is established by
comparison with PAM50 intrinsic subtyping.77,78 There are
potential pitfalls in the evaluation of Ki67 proliferative index.
The interpretation is limited by lack of standardization of
protocols. Regarding the optimal scoring methods, there are
uncertainties in defining the cutoffs and thresholds, hotspot
approach versus overall quantification, as well as the
number of tumor cells assessed.
In an analysis to determine the optimal Ki67 threshold for
predicting outcome of invasive breast cancer, quantitative
scoring of Ki67 on tissue microarray sections of 440 invasive
breast cancers was performed using Aperio ePathology
ImmunoHistochemistry Nuclear Image Analysis algorithm,
and tissue microarray slides digitally scanned via a scanning
system. On multivariate analysis, tumors with Ki67 of ≥14%
had an increased likelihood of recurrence and shorter overall
survival. The value of Ki67 associated with luminal B subtype
tumors is found to be ≥17%.79
MOLECULAR CLASSIFICATION
Molecular classification gained recognition in 2000 with the
publication of the molecular portraits of breast tumors by
Perou et al.4 Intrinsic molecular subtypes have been de-
scribed based on hierarchical cluster analysis of genes that
vary more between tumors than between repeated samples
of the same tumor (intrinsic genes). Based on the gene ex-
pression profiling, at least five major molecular subtypes
have been currently identified:4,6,80,81
• Luminal A
• Luminal B
• HER2 enriched
• Basal‐like
• Normal breast‐like
© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
Other molecular subtypes identified in gene expression
profiling also include the molecular apocrine characterized
by activation of gene in the androgen receptor pathway and
the claudin‐low type featuring a high expression of genes
involved in epithelial‐mesenchymal transition and stem cell
features.82–85
Different molecular subtypes are characterized by their gene
expression patterns, clinical features, treatment response and
prognosis. This provides further evidence of the heterogeneity
of invasive breast cancer. The most stable separation has
been identified between basal‐like tumors and other ‘intrinsic
type’ tumors. Tumors of basal‐like group most often show high
histological grade, solid architecture, high mitotic activity,
pushing tumor border, stromal lymphocytic infiltrate and geo-
graphic necrosis. They occur more commonly in African
American women. Although there is currently no international
consensus on the definition of basal‐like tumors, they are
typically characterized by the expression of basal cytokeratins
(such as cytokeratins 5/6, 14 and 17), epidermal growth factor
receptor (EGFR), and/or other basal‐related genes, and they
also tend to be ER, PR and HER2 negative.86–88 Although the
basal‐like tumors often correspond to triple‐negative tumors,
they are not always synonymous with them. On the other
hand, not all triple‐negative breast cancers are basal‐like by
gene expression profiling. Hence, basal‐like and triple‐
negative carcinomas are not synonymous and appropriate
terms ought to be used when describing these tumors. Basal‐
like carcinomas are generally associated with poor prognosis.
The molecular classification is not currently applied in rou-
tine diagnostic practice. Current practice emphasizes the im-
portance in determining between patients who will benefit
from particular therapies and those who will not. Such a dis-
tinction is made by assessing ER, PR and HER2 assays.89
Although it has been suggested that molecular subtypes can
be defined by utilizing a combination of surrogate biomarkers
including ER, PR, HER2, CK5/6, Ki67 and EGFR, the corre-
lation between the result using such surrogate biomarkers
and that defined by gene expression profiling still shows
considerable discordance.90,91 Hence, utilizing surrogate bi-
omarkers in determining the molecular subtypes of invasive
breast cancer on an individual patient may not always be
accurate compared to gene expression profiling. The value of
molecular classification in invasive breast cancer in routine
clinical practice beyond the conventional histopathological
features and biomarker status is yet to be determined.
CONCLUSION
Conventional prognostic parameters of invasive breast can-
cers provide reliable information on disease outcome. Well‐
defined predictive biomarkers assess the likelihood of treat-
ment response and hence allow the identification of patients

who benefit from specific therapies. Histopathological features
remain the basis for current classification of invasive breast
cancer. However, recent molecular and genetic analyses
have gained new insights that refine classification schemes.
Emerging concepts in invasive breast cancer, including the
stromal microenvironment and tumor infiltrating lymphocytes,
in relation to the clinical outcome of invasive breast cancer are
being recognized. There are recommendations on the as-
sessment of tumor infiltrating lymphocytes. New entities and
terminology, such as TIL‐rich breast carcinomas, mucinous
cystadenocarcinoma and tall cell carcinoma with reversed
polarity, have been recognized and proposed.
ACKNOWLEDGMENTS
The contents of this article were presented at the 108th
Annual Meeting of the Japanese Society of Pathology in
Tokyo, May 2019. There is no financial grant and other
funding to this article. The authors would like to thank Dr
Wentao Yang from the Fudan University Shanghai Cancer
Centre for the contribution of Fig. 5.
DISCLOSURE STATEMENT
None declared.
AUTHOR CONTRIBUTIONS
EKHS drafted the manuscript. PHT formulated the scope of
the review. VCYK and PHT edited the manuscript.
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