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 tubular carcinoma Intraductal papilloma/sclerosing papillomatosis vs papillary carcinoma 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 [1, 2, 3, 4, 5], with the possible exception of microglandular adenosis [6, 7, 8]. 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 Muscle actins Smooth muscle myosin heavy chain and calponin p63 Others: CD10, maspin, etc. 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 [9]) and muscle actins (clone HFF35 [10, 11]) . 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 [12, 13, 14, 15, 16]. Calponin and Smooth Muscle Myosin Heavy Chain Calponin is a smooth muscle cell specific 34-kd peptide which interacts with actin, tropomyosin and calmodulin [17, 18, 19]. 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 [20]. 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]. p63 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 [21]. 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 [22]. P63, in our experience, is best employed as a companion marker with a cytoplasmic myoepithelial protein such as SMM-HC or cytokeratin 5. 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 [23, 24, 25, 26, 27]. 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 [28, 29, 30, 31] and issues relating to clinical detection of the tumor [32]. 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 [33], along with those of Jacobs et al [34], 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 [35]. 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 [35], 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 [36]. 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