Section 3 -

Are Immunostains Useful Diagnostic Tools in Breast Pathology? Giuseppe Viale European Institute of Oncology and University of Milan Milan , Italy

Immunostains are useful in selected instances, being not routinely performed to aid in the differential diagnosis of breast diseases -at least in the Departments dealing with a high number of breast biopsies or surgical samples- because the peculiar morphological features of benign and malignant lesions have been precisely identified since a long time and thoroughly described in several papers and comprehensive textbooks. The unfortunately high prevalence of breast cancer has facilitated spreading of knowledge and expertise among pathologists in most Institutions. Experts or specialists in breast pathology are commonly encountered in large Pathology Departments, and breast pathology is one of the key issues in the post-graduate or residency programmes for younger pathologists. Still, there are some specific fields of breast pathology where immunohistochemical data are sought even by expert pathologists to facilitate -or at least to confirm- the histopathological diagnosis. This is especially true in the setting of the differential diagnosis between well-differentiated ductal carcinoma (either invasive or in situ) and benign proliferative lesions, and between pure intraductal carcinoma and (micro)-invasive cancer. Also, immunostains may be an useful adiunct in the correct identification of special types of breast carcinomas. [1] Distinguishing well-differentiated invasive ductal carcinoma from benign lesions. As with most human malignancies, a specific positive marker of breast carcinoma is not available for immunohistochemical purposes, with the possible exception of HER2/neu over-expression. This oncogene, however, is over-expressed only in a fraction of breast carcinomas, and most commonly in high-grade carcinomas, where issues of differential diagnosis with benign lesions are unlikely to occur. Therefore, the correct diagnosis of breast cancer must rely upon a constellation of data, which include gross and microscopic features and only a limited number of immunohistochemical stains. Well differentiated invasive ductal carcinomas, including tubular and cribriform variants, may be difficult to be distinguished from some benign proliferative diseases (e.g., tubular adenosis, radial scar) on purely morphological grounds. In these circumstances, highlighting of the myoepithelial cell component, which is invariably present in the benign proliferative lesions (with the possible exception of microglandular adenosis) and absent in invasive breast cancer, may be the diagnostic key. Luckily, there are several sensitive and specific immunohistochemical markers for mammary myoepithelial cells, which are suitable for staining routinely fixed and embedded tissues, and cytological preparations as well. These include, among others, p63, smooth muscle myosin heavy chain, calponin, and caldesmon [2, 3]. I would strongly recommend not to use smooth muscle actin any longer for the identification of myoepithelial cells, because this protein is also expressed by myofibroblasts which may surround the glands or nests of invasive breast carcinomas and lead to an underdiagnosis of malignancy. S-100 protein, on the other hand, may be expressed also by normal and neoplastic epithelial cells of the breast, again leading to a false positive identification of myoepithelial cells. Among the more consistent and effective markers of myoepithelial cells, I most often rely on p63, because it has a nuclear location -which facilitates labelling of myoepithelial cells even in case of extreme clearing of their cytoplasm- and it may be also safely used to decorate the naked nuclei of myoepithelial cells in cytological specimens. [4] Lack of a myoepithelial cell component is a most reliable diagnostic feature for distinguishing well-differentiated invasive duct carcinoma from non malignant breast proliferations. It should be noted, however, that to rely on negative immunohistochemical findings is always risky, because of the possible appearance of false-negative results due to technical artefacts. For this reason it is highly recommended that the sections to be immunostained always include benign breast tissue surrounding the suspicious proliferation, that may function as an internal positive control. Also, it should be considered that some special types of invasive breast cancer have a myoepithelial component (e.g., adenoid-cystic carcinomas, low-grade adenosquamous carcinomas), or show a myoepithelial differentiation (e.g., so-called basal-type carcinomas), and that some metaplastic carcinomas exhibit a variable proportion of neoplastic cells with definite nuclear immunoreactivity for p63. [5, 6] Differentiating ductal carcinoma in situ (DCIS) from florid epithelial hyperplasia. Here again we have to complain of the lack of a positive marker for breast cancer. Indeed, the immunophenotypic differences between DCIS and ductal hyperplasia consist mainly in the different expression of basal-type cytokeratins (especially cytokeratins 5 and 14). These are consistently co-expressed by a variable fraction of epithelial cells in case of florid epithelial hyperplasia, but not by DCIS cells, which only express "luminal" type cytokeratins (mainly cytokeratins 8,18 and19) [7, 8, 9]. It should be noted that some high-grade DCIS may co-express cytokeratins 5 and 14 (so-called basal-type DCIS): the nuclear features of these neoplasms, however, allow easy recognition of their malignant nature [10]. Identifying Microinvasion in DCIS The correct identification of microinvasive foci in DCIS is in my opinion one of the most challenging tasks in breast pathology, and I am afraid that inter-observer disagreement in case of microinvasive breast cancer is exceedingly common. Unfortunately, the over-estimation of microinvasion may have very harmful therapeutic implication, including undue axillary surgery and adjuvant interventions. This is particularly true in case of high-grade tumors, most often lacking any estrogen and progesterone receptors, for whom adjuvant chemotherapy would be recommended, in case of microinvasion being reported. I believe that we should be very accurate in diagnosing microinvasion and use stringent morphological criteria. The attitude to diagnose microinvasion just because "this DCIS is too large and too bad: it must be invasive somewhere" has to be discontinued. It is very unfortunate that immunohistochemistry is only marginally useful in this scenario. Indeed, many pathologists rely on immunostaining for basal membrane proteins and/or myoepithelial cell markers to ascertain or rule out microinvasion, but this may be useless and sometimes very misleading. Invasive breast cancer may indeed express basal membrane components leading to under-estimation of microinvasion. On the other hand, lack of a myoepithelial cell layer is not a marker of invasion, because the myoepithelial cells disappear in many high-grade DCIS, leading to a possible over-diagnosis of invasion. [11, 12] Identifying Special Types of Breast Cancer Immunostainings may facilitate recognition of special types of breast carcinomas. Lobular carcinomas are characterized by the lack of e-cadherin immunoreactivity, apocrine carcinoma express androgen receptors and GCDFP-15, adenoid cystic carcinomas are immunoreactive for myoepithelial cell markers and c-kit [13, 14], myoepithelial carcinomas may be identified using myoepithelial cell markers, and so-called basal-type breast cancer variably express cytokeratins 5,14, and 17, EGFR and p63. [15, 16] References Moriya T, Kasajima A, Ishida K, Kariya Y, Akahira J, Endoh M, Watanabe M, Sasano H. New trends of immunohistochemistry for making differential diagnosis of breast lesions. Med Mol Morphol 2006;39(1):8-13. Barbareschi M, Pecciarini L, Cangi MG, Macri E, Rizzo A, Viale G, Doglioni C. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol. 2001;25(8):1054-60. Werling RW, Hwang H, Yaziji H, Gown AM. Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain. Am J Surg Pathol. 2003;27(1):82-90. Reis-Filho JS, Milanezi F, Amendoeira I, Albergaria A, Schmitt FC. Distribution of p63, a novel myoepithelial marker, in fine-needle aspiration biopsies of the breast: an analysis of 82 samples. Cancer 2003 25;99(3):172-9. Reis-Filho JS, Milanezi F, Steele D, Savage K, Simpson PT, Nesland JM, Pereira EM, Lakhani SR, Schmitt FC. Metaplastic breast carcinomas are basal-like tumours. Histopathology 2006;49(1):10-21. Tse GM, Tan PH, Chaiwun B, Putti TC, Lui PC, Tsang AK, Wong FC, Lo AW. p63 is useful in the diagnosis of mammary metaplastic carcinomas. Pathology 2006;38(1):16-20. Otterbach F, Bankfalvi A, Bergner S, Decker T, Krech R, Boecker W. Cytokeratin 5/6 immunohistochemistry assists the differential diagnosis of atypical proliferations of the breast. Histopathology 2000;37(3):232-40. Boecker W, Moll R, Dervan P, Buerger H, Poremba C, Diallo RI, Herbst H, Schmidt A, Lerch MM, Buchwalow IB. Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J Pathol 2002;198(4):458-67. Rabban JT, Koerner FC, Lerwill MF. Solid papillary ductal carcinoma in situ versus usual ductal hyperplasia in the breast: a potentially difficult distinction resolved by cytokeratin 5/6. Hum Pathol 2006;37(7):787-93. Bryan BB, Schnitt SJ, Collins LC. Ductal carcinoma in situ with basal-like phenotype: a possible precursor to invasive basal-like breast cancer. Mod Pathol 2006;19(5):617-21. Ribeiro-Silva A, Zamzelli Ramalho LN, Garcia SB, Zucoloto S. Is p63 reliable in detecting microinvasion in ductal carcinoma in situ of the breast? Pathol Oncol Res 2003;9(1):20-3. Collins LC, Carlo VP, Hwang H, Barry TS, Gown AM, Schnitt SJ. Intracystic papillary carcinomas of the breast: A reevaluation using a panel of myoepithelial cell markers. Am J Surg Pathol 2006;30(8):1002-7. Mastropasqua MG, Maiorano E, Pruneri G, Orvieto E, Mazzarol G, Vento AR, Viale G. Immunoreactivity for c-kit and p63 as an adjunct in the diagnosis of adenoid cystic carcinoma of the breast. Mod Pathol 2005;18(10):1277-82. Azoulay S, Lae M, Freneaux P, Merle S, Al Ghuzlan A, Chnecker C, Rosty C, Klijanienko J, Sigal-Zafrani B, Salmon R, Fourquet A, Sastre-Garau X, Vincent-Salomon A. KIT is highly expressed in adenoid cystic carcinoma of the breast, a basal-like carcinoma associated with a favorable outcome. Mod Pathol 2005;18(12):1623-31. Rakha EA, Putti TC, Abd El-Rehim DM, Paish C, Green AR, Powe DG, Lee AH, Robertson JF, Ellis IO. Morphological and immunophenotypic analysis of breast carcinomas with basal and myoepithelial differentiation. J Pathol 2006;208(4):495-506. Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, Moore DT, Perou CM. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 2006;19(2):264-71.