Moderators: Dr. Allen M. Gown and Dr. Anthony S.Y. Leong
Section 5 -
Major Diagnostic Problems in Breast Pathology Addressed by Immunohistochemistry
Carlos E. Bacchi
Director and Chief Pathologist, Pathology Reference Laboratory
Botucatu, SP, Brazil
The majority of diagnoses in breast pathology are rendered successfully based on the evaluation of
hematoxylin and eosin slides alone. However, the histologic complexity, varied morphology, and
overlapping features of many benign and neoplastic lesions often lead to problems in interpretation.
Epithelial proliferations are the most common source of diagnostic difficulty, and they provided fertile
ground for exploration of the potential benefits of immunohistochemistry. In this section, recent
advances in the use of immunohistochemistry in diagnostic (non prognostic/predictive) breast pathology
are presented in the following context:
- To assess the presence or absence of
myopeithelial cells in order to help in the differential diagnosis of the following diagnostic situation:
- Sclerosing adenosis/radial scar vs
- Intraductal papilloma/sclerosing papillomatosis vs papillary
- DCIS vs invasive ductal carcinoma
- Differentiation of usual ductal hyperplasia from ductal carcinoma in situ
- To evaluate problematic in situ lesions (lobular vs ductal CIS)
The Utility of Myoepithelial Markers in the Distinction Between Invasive and Non-invasive Breast Lesions
The myoepithelial cell layer
It is now widely accepted that the outer myoepithelial layer remains intact in benign proliferative
lesions and in situ (ductal and lobular) carcinomas
with the possible exception of
However, except in pregnancy, lactation and some forms of
sclerosing adenosis/myoepithelial hyperplasia, the myoepithelial layer is difficult, if not impossible,
to identify on H&E stained sections. In invasive carcinomas, in contrast, a myoepithelial layer does
not exist. Thus, in contrast to other immunohistochemical studies in surgical pathology, determination
of stromal invasion in breast carcinoma is based on the absence of a marker,
in this case a marker identifying the myoepithelial cells.
Summary of the Most Important Myoepithelial-associated Proteins
The 'partial' smooth muscle phenotype of myoepithelial cells has resulted in the use of a number of
antibodies to smooth muscle-restricted proteins as putative myoepithelial markers. Perhaps the most
popular breast myoepithelial cell markers currently employed are antibodies to smooth muscle actins
(clone 1A4 )
and muscle actins (clone HFF35
. However, these markers also
identify myofibroblasts in the desmoplastic stroma of both infiltrating carcinoma and high grade DCIS,
which can significantly compromise their utility. Not infrequently, growing infiltrating carcinoma nests
induce a marked myofibroblast response in adjacent stromal tissue, and many of the myofibroblasts can be
in such close proximity to the tumor cells as to create an appearance of 'pseudo-myoepithelial cells'
around the invasive nests. The more exuberant the desmoplastic response, the more difficult will be the
interpretation of the results of anti-actin immunohistochemical studies
|Smooth muscle myosin heavy chain and calponin|
|Others: CD10, maspin, etc.|
Calponin and Smooth Muscle Myosin Heavy Chain
Calponin is a smooth muscle cell specific 34-kd peptide which interacts with actin, tropomyosin and
Antibodies to smooth muscle myosin heavy chain have proved more specific
in identifying myoepithelium (i.e., compared with myofibroblasts) than antibodies to smooth muscle
actins., as demonstrated in our published study . In the latter, muscle actin-positive
myofibroblasts were noted within the stroma of all infiltrating carcinomas, calponin expression was noted
in only a minor subset of myofibroblasts and SMM-HC was expressed only on rare myofibroblasts in 7% of
infiltrating carcinomas [Wang, 1997 #2768].
The nuclear protein, p63, is a member of the p53 gene family, is perhaps the most sensitive and
specific myoepithelial markers to date [Barbareschi, 2001 #3789]. Note that p63 demonstrated contextual specificity in the breast, as this marker identifies, in other tissues,
a limited range of cell types, including transitional epithelium in the bladder and trophoblasts in the
placenta . Unlike the smooth muscle-associated proteins, there is no expression in any
vascular smooth muscle cells or myofibroblasts. However, the major disadvantage of antibodies to p63 is
their nuclear, rather than cytoplasmic, localization. In certain settings, where the myoepithelial cells
are "splayed apart", the nuclear signals can appear interrupted and even lost. Scattered p63 expression
can also be found in the carcinoma cell population, although this is qualitatively distinguished from
the uniform strong p63 expression found in the myoepithelium . P63, in our experience, is
best employed as a companion marker with a cytoplasmic myoepithelial protein such as SMM-HC or
Benign lesions mimicking invasive carcinoma and vice versa
The following represent the more common histologic settings in which definitive diagnosis can be
difficult to establish. In each of these settings, the use of myoepithelial markers may be appropriate
and even necessary to help confirm the diagnosis.
|Radial scar vs. infiltrating tubular carcinoma|
|Sclerosing adenosis vs. infiltrating ductal carcinoma|
|DCIS with cancerization of adenosis vs. DCIS with microinvasion|
|Identification of infiltrating cribriform carcinoma|
|Adenoid cystic carcinoma vs. collagenous spherulosis|
|Papillary carcinoma vs. papilloma|
|Nipple adenoma versus infiltrating duct carcinoma|
Recommendations for the use and interpretation of myoepithelial markers
It is generally prudent to perform more than one marker study on two sequential sections of the same
lesion; at the current time, we would recommend antibodies to smooth muscle myosin heavy chain as well as
p63 gene product, in order to maximize the specificity and sensitivity of the studies.
One of the main reasons for problems in the interpretation of myoepithelial marker studies is the
invariable presence of vascular smooth muscle cells and myofibroblasts in close proximity to the
epithelial nests. Therefore, even the better myoepithelial markers such as calponin and SMM-HC will
inevitably mark the smooth muscle cells and pericytes of small blood vessels. When interpreting these
marker studies, sometimes it is difficult to determine if the positive nest represents an epithelial nest
or a small vessel with prominent endothelial layer. In highly vascularized infiltrating carcinomas, the
presence of compressed blood vessels adjacent to invasive nests may lead to "false positive"
interpretations of the presence of a myoepithelial cell layer, leading to an erroneous diagnosis of
DCIS. Markers such as the p63 gene product and the cytokeratin proteins such as CK5 have distinct
advantages in this regard.
Antibodies to cytokeratins can be used to highlight the presence of suspicious cell nests, which may
not have been appreciated by H&E sections alone. These are particularly helpful in situations where
dense inflammatory and desmoplastic response masks the presence of few invasive nests.
Distinction Between Ductal and Lobular Phenotypes: E-cadherin
Distinction between ductal and lobular carcinoma of the breast can usually be made using standard
histologic criteria, but as it is now known that lobular carcinoma can be defined by a "molecular
signature" of mutations and alterations of methylation of the E-cadherin gene or promoter, leading to
loss of expression of this protein
Loss of E-cadherin, a molecule responsible for
homotypic cell binding, accounts for the 'single file' infiltrative pattern characteristic of this
tumor. Identification of the lobular variant of breast cancer is of importance to breast oncologists,
owing to differences in risk of the subsequent contralateral cancers
and issues relating
to clinical detection of the tumor .
Certainly, there are striking differences in the clinical implications of the diagnosis of in situ
lobular vs. in situ ductal carcinoma, with only the latter generally triggering additional treatment.
More recent studies we co-authored ,
along with those of Jacobs et al , have
extended these findings of E-cadherin alterations in in situ disease, and
confirming the role of E-cadherin in classifying in situ lesions with indeterminate features. In both of
these recent studies, virtually all cases of LCIS were found via immunohistochemistry to be E-cadherin
negative, whereas all DCIS cases were found to be E-cadherin positive. In addition, the clinical
significance of demonstrating E-cadherin expression in the context of in situ breast carcinoma has been
demonstrated . In a long follow-up study (average of 21 years), Goldstein et al
demonstrated that LCIS patients, whose tumors show only focal expression of E-cadherin (10% of patients),
more frequently developed subsequent invasive disease (often ductal type) after significantly shorter
time periods than patients with E-cadherin negative tumors , in a manner similar to patients
with low-grade DCIS. This observation, however, may not to apply to the recently described variant of
LCIS, referred to "pleomorphic ductal-lobular" or more commonly known as pleomorphic LCIS. These lesion,
in contrast to the conventional type LCIS with low nuclear grade, show marked nuclear pleomorphism,
sometimes with central necrosis, but they lack E-cadherin . Until sufficient clinical
follow-up data become available, these lesions should not be managed as LCIS of usual type, but rather
Some of the more frequent applications of E-cadherin IHC in breast cancer are (a) the distinction
between DCIS and LCIS and (b) the determination of whether part or the whole invasive tumor has a lobular
phenotype. E-cadherin immunostaining, however, demonstrates that the frequency of true "mixed" tumors,
(e.g., ductal with lobular component and 'tubulolobular') is extremely low. One caveat to interpretation
of E-cadherin immunostaining is the occasional finding of a small subset of lobular carcinomas that
contain very weak, rather than completely absent, signal for E-cadherin immunolocalization
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