Anatomic Pathology / MYOEPITHELIAL CELL STAINING OF PAPILLARY BREAST LESIONS

Myoepithelial Cell Staining Patterns of Papillary Breast Lesions From Intraductal Papillomas to Invasive Papillary Carcinomas Cheryl B. Hill, MD, and I-Tien Yeh, MD Key Words: Papillary breast carcinoma; Myoepithelial cell; Calponin; Smooth muscle myosin heavy chain; p63 DOI: 10.1309/XG7TPQ16DMJAV8P1

Abstract We evaluated 25 intraductal papillomas and 18 papillary carcinomas (invasive, 4; micropapillary ductal carcinoma in situ [DCIS], 5; cases originally classified as intracystic/intraductal papillary carcinoma, 9) by calponin, smooth muscle myosin heavy chain (SMM-HC), and p63 immunostains. Calponin, SMM-HC, and p63 labeled myoepithelial cells (MECs) in all intraductal papillomas and all micropapillary DCIS cases. The invasive papillary carcinoma cases were uniformly negative for all stains. The 9 cases originally diagnosed as intracystic/intraductal papillary carcinoma showed more variable results, with identification of an MEC layer in only 4 cases. Comparison of staining of MECs by these 3 stains showed that calponin was more sensitive and intense than SMM-HC; however, there was cross-reactivity with myofibroblastic cells. Staining with p63 was discontinuous, making interpretation of an intact myoepithelial layer difficult. Of 9 cases originally classified as intraductal papillary carcinoma, 5 showed absence of a basal MEC layer by immunohistochemical analysis. The lack of a basal MEC layer in these cases suggests a spectrum of progression from in situ to invasive disease and might help explain distant metastases from previously reported “intraductal papillary carcinoma.”

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Am J Clin Pathol 2005;123:36-44 DOI: 10.1309/XG7TPQ16DMJAV8P1

Identification of a myoepithelial cell (MEC) layer histologically or by immunohistochemical analysis has become a key feature in distinguishing benign from malignant and in situ from invasive lesions of the breast. Indeed, the medical literature contains numerous studies touting the usefulness of various immunohistochemical stains for this purpose. This exercise is particularly pertinent in the classification of papillary lesions. Histologic criteria for distinguishing benign papillomas from papillary carcinoma have been well defined in several classic, often-cited articles.1-3 However, in atypical lesions, there can be significant interobserver variability based on morphologic criteria alone,4 and the differential diagnosis, particularly on core needle biopsy specimens, can be quite challenging, especially in cases with marked epithelial hyperplasia, sclerosis, and/or atypia. Identification of an MEC layer can help distinguish sclerosing papilloma from tubular carcinoma and benign papilloma from papillary carcinoma. According to Tavassoli,5 the most important feature for distinguishing a papilloma from a papillary carcinoma is the presence of a relatively uniform MEC layer in the proliferating intraluminal component of the lesion, and the absence of the basal MEC layer in the papillary processes almost always indicates a carcinoma. There have been only a few articles published about MEC staining patterns in papillary breast lesions, and half of these were done on cytologic preparations.6-9 Most of these are older studies that used stains that are less specific for MECs and, for the most part, are no longer used. Several newer immunohistochemical stains, including calponin, smooth muscle myosin heavy chain (SMM-HC), and p63, have been used to successfully identify myoepithelium. © American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE

Calponin and SMM-HC, which are both cytoplasmic stains, have been well characterized and are excellent MEC markers. Calponin is slightly more sensitive than SMM-HC.4 However, in addition to MECs, these stains mark vascular smooth muscle cells and myofibroblasts (SMM-HC less so) as well, making interpretation difficult at times if there is a very fibroblastic stroma or if the vessels within the fibrovascular cores are in proximity to the epithelium.8,10 These 2 stains currently are the “gold standard” for identification of MECs in breast lesions.10 The nuclear stain, p63, a member of the p53 gene family, shows no cross-reactivity with myofibroblasts or vascular smooth muscle. It is expressed normally in basal epithelial cells of many organs, including breast, prostate, skin, bladder, and uterine cervix.11 However, Werling et al10 noted that it can show at least focal positivity of luminal epithelial cells in a minority of cases. The terminology used for papillary breast lesions is quite confusing as well. The older term, intracystic papillary carcinoma (IPC), still is used quite frequently. This term, although it generally refers to a localized, in situ lesion arising in a cystically dilated duct, has been used in a variety of contexts, particularly in the clinical literature, where it often is difficult to determine whether the term refers to an in situ or invasive lesion. Some have suggested splitting this entity into 3 groups: IPC alone, IPC plus ductal carcinoma in situ (DCIS), and IPC with invasion (divisions that seem to correlate with prognosis) and reserving the term papillary DCIS for a more diffuse, rather than localized, process that involves multiple ducts.12 Morphologically, these entities can be extremely difficult, if not impossible, to separate, especially in core needle biopsy specimens. Also, given the often marked stromal response surrounding these lesions, the distinction between in situ and invasive papillary carcinoma can be a very difficult distinction to make. There may be areas of sclerosis with entrapment of epithelial elements (as can be seen in benign papillomas). Is it in situ or invasive? Identification of an MEC layer can be key in these situations. We studied the usefulness of newer immunohistochemical stains in differentiating benign from malignant and in situ from invasive papillary breast lesions.

Materials and Methods We evaluated histologic sections from 43 breast lesions retrieved from the University Health System files in San Antonio, TX, between March 1994 and February 2003. These cases included 25 benign intraductal papillomas, 9 intraductal papillary carcinomas, 5 micropapillary DCIS cases, and 4 invasive papillary carcinomas. Cases were identified in our computer system by a free text search for the words papillary and breast. Only 4 cases with a diagnosis

of invasive papillary carcinoma were identified during our search period. Intraductal papillomas were more numerous, and a representative group of 25 cases was selected based on the size of the lesion and adequacy of tissue remaining in the block. We were interested in staining larger, more complex lesions, while maintaining a mixture of core needle and excisional biopsy specimens. The 5 micropapillary DCIS cases originally were diagnosed as papillary DCIS; however, on review, we reclassified them as micropapillary DCIS. We decided to keep these cases as part of the study for the sake of completeness. All cases had been fixed routinely in 10% formalin and embedded in paraffin, and sections were stained with H&E using conventional methods. All cases were reviewed by 2 pathologists (C.B.H. and I.Y.) for confirmation of diagnosis and for selection of blocks for immunohistochemical staining. Specimens were from 29 excisions (including excisional biopsy and mastectomy specimens) and 14 core needle biopsies. Immunohistochemical Analysis Deparaffinized 4- to 5-µm sections of 1 block from each case were rehydrated and subjected to heat-induced epitope retrieval procedures optimized for each antibody. All 3 antibodies, calponin (dilution 1:400; DAKO, Carpinteria, CA), SMM-HC (dilution 1:100; DAKO), and p63 (dilution 1:1,500; NeoMarkers, Fremont, CA) were applied to sequential sections from each block. A standard avidinbiotin immunoperoxidase technique was used. Sections were counterstained with 0.1% methyl green. In addition to a separate positive control sample of normal breast tissue that was run with each stain, internal positive control samples were present in every case in the form of nonneoplastic breast tissue. Negative control samples using 10% bovine serum albumin in place of the primary antibody were run with each batch of stains as well. The MEC layer was defined by the presence of immunoreactivity by calponin, SMM-HC, and p63 in the basal layer of the papillary lesions. Immunohistochemical stains were graded based on the percentage of staining of the basal epithelial layer as follows: 0, no staining; 1, less than 10% staining; 2, 10% to 49% staining; 3, 50% to 74% staining; 4, 75% to 89% staining; and 5, 90% to 100% staining. The carcinoma cases then were reevaluated along with the immunohistochemical stains to determine whether a change in diagnosis was warranted. All stains were reviewed by both pathologists, and an interpretation was agreed on.

Results The results of immunohistochemical staining, along with the procedure type, are summarized in ❚Table 1❚. Am J Clin Pathol 2005;123:36-44

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DOI: 10.1309/XG7TPQ16DMJAV8P1

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Hill and Yeh / MYOEPITHELIAL CELL STAINING OF PAPILLARY BREAST LESIONS

❚Table 1❚ Immunohistochemical Staining of Papillary Breast Lesions* Case No.

Procedure Type

Originally diagnosed as Invasive papillary carcinoma 1 Mastectomy 2 Excision 3 Core biopsy 4 Excision Intraductal papillary carcinoma 5 Mastectomy 6 Excision 7 Mastectomy 8 Excision 9 Excision 10 Excision 11 Mastectomy 12 Excision 13 Excision Micropapillary DCIS 14 Core biopsy 15 Excision 16 Core biopsy 17 Excision 18 Excision Intraductal papilloma 19 Excision 20 Core biopsy

Calponin

SMM-HC

p63

0 0 0 0

0 0 0 0

0 0 0 0

5 0 3 5 1 0 0 4 4

4 0 2 4 1 0 0 3 3

4 0 3 4 0 0 0 4 3

5 5 5 4 5

5 5 5 4 5

5 3 5 4 5

5 5

5 5

4 5

Case No.

Procedure Type

Intraductal papilloma 21 Core biopsy 22 Core biopsy 23 Core biopsy 24 Excision 25 Excision 26 Excision 27 Core biopsy 28 Excision 29 Excision 30 Core biopsy 31 Core biopsy 32 Excision 33 Excision 34 Excision 35 Core biopsy 36 Excision 37 Excision 38 Core biopsy 39 Core biopsy 40 Excision 41 Excision 42 Excision 43 Core biopsy

Calponin SMM-HC

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4

4 4 5 5 5 4 5 5 5 4 5 4 5 5 4 5 4 4 4 5 4 4 4

p63

3 3 4 5 5 5 4 5 5 3 4 4 5 5 3 4 3 4 3 4 3 3 4

DCIS, ductal carcinoma in situ; SMM-HC, smooth muscle myosin heavy chain. * Staining was scored as follows: 0, no staining; 1,