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Annals of Clinical & Laboratory Science 39:17-24 (2009)
© 2009 Association of Clinical Scientists

Different Panels of Markers Should Be Used to Predict Mammary Paget’s Disease Associated with In Situ or Invasive Ductal Carcinoma of the Breast

Todd Lester1, Jianmin Wang2, Patricia Bourne1, Qi Yang1, Li Fu1 and Ping Tang1
1 Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York; 2 RTI Health Solution, Research Triangle Park, North Carolina, and Cancer Hospital of Tianjin Medical University, Tianjin, China

Address correspondence to Ping Tang, M.D., Ph.D., Department of Pathology and Laboratory Medicine, Box 626, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; tel 585 275 6640; fax 585 273 3637; e-mail ping_tang{at}urmc.rochester.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammary Paget’s disease (MPD) is a rare manifestation of breast carcinoma involving the nipple. Our objective was to identify molecular markers and molecular subtypes that may predict patients at high risk of developing MPD. Immunohistochemical (IHC) analyses were performed with antibodies to estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), HER2, epidermal growth factor receptor (EGFR), and several cytokeratins (CK5/6, CK14, CK17, CK8, CK18) on representative sections of 121 cases of ductal carcinoma of the breast, including 28 cases with MPD, 81 cases with neither MPD nor nipple involvement, and 12 cases of non-MPD with nipple involvement. The rates of receptor expression and subtype distributions of 3 IHC-based molecular classifications were compared among these groups. The results showed that: (1) MPD is more likely to be associated with ER- and PR-negative ductal carcinoma in situ (DCIS), but not invasive ductal carcinoma (IDC); (2) MPD is more likely to be associated with HER2-over expression subtype DCIS, but not IDC; and (3) carcinomas with non-MPD nipple involvement differ from those with MPD, since they are more likely to be ER- and PR-positive, HER2-negative, and luminal A subtype. In summary, different panels of markers should be used to predict MPD associated with different underlying lesions; for DCIS, the ER-negative, PR-negative, and HER2-subtype and not basal-like subtype is most predictive of MPD; for IDC, the luminal B-subtype is most predictive of MPD.

Keywords: mammary Paget’s disease, ER, PR, AR, HER2, EGFR, cytokeratin markers


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sir James Paget of England is credited with the first description, in 1874, of a disease of the mammary nipple/areola that is known eponymically as mammary Paget’s disease (MPD) [1]. MPD is an adenocarcinoma localized to the epidermis of the nipple and/or areola, and may be asymptomatic or associated with focal reddening to classical eczematous appearance. Estimated rates for MPD range from 1 to 4.3% of all breast carcinomas [2]. MPD usually occurs in conjunction with an underlying in situ ductal (DCIS) or invasive ductal carcinoma (IDC) of the breast. The prognosis of patients with MPD largely depends on the underlying disease. Chen et al recently reported that the incidence of MPD is decreasing, likely due to the advent of mammography and other methods for early detection of breast carcinomas [3]. Since MPD generally co-exists with DCIS or IDC, and shares similar phenotypic and immunologic patterns with its underlying lesion, an epidermotropic spread from the underlying carcinoma is the mechanism currently accepted by many [4–11]. However, MPD does rarely occur without associated breast carcinoma [12]. It is believed that such MPD arises from so-called Toker cells present within the epidermis of the nipple/areolar region [13,14]. Recent reports suggest that Toker cells may be the true precursors for MPD, while the underlying tumors are coincidental neoplastic lesions [15,16].

Numerous studies have shown that MPD is often associated with high grade ductal carcinomas, which are often ER and PR negative and HER2 positive [8–10,17]. Recent immunohistochemical (IHC) investigations indicate that breast carcinomas can be classified in two subtypes, ie, basal and non-basal, and further divided into 5 different subtypes: luminal A, luminal B, HER2 over-expression, basal-like, and triple-negative (TN) non-basal [18,19]. These categories are similar to the molecular classification derived from gene expression profiling [20–22], and they also show differences in clinical outcome. The basal and HER2 over-expression subtypes are associated with poor prognosis [18].

Currently, three immunohistochemical (IHC)-based classifications are frequently used in the literature: The cytokeratin (CK) classification defines a basal subtype having positive stains for basal CK markers (CK5/6, CK14, and CK17) and associated with poor prognosis [23,24]. The triple-negative (TN) classification includes tumors that are negative for ER, PR, and HER2 as a basal subtype. This subtype has been shown to be the sole prognostic marker in node-negative tumors [25]. The third classification (CK/TN), a combination of the CK and TN systems, defines the basal-like subtype as ER, PR, HER2 negative, and CK5/6 and/or EGFR positive [18,19].

The objective of this study was to determine which molecular markers and molecular subtypes are helpful in predicting patients with ductal carcinoma of the breast who have an increased risk of developing MPD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Twenty-eight cases of MPD (18 cases with DCIS and 10 cases with IDC) were retrieved from the 2,500 cases of mammary carcinoma diagnosed between 1997 and 2007 in the Pathology Department at Strong Memorial Hospital in Rochester, NY. Eighty-one cases of carcinomas with neither MPD nor nipple involvement (non-MPD non-nipple group, 38 DCIS and 43 IDC), along with 12 cases of non-MPD, but with nipple involvement, (non-MPD nipple group) were also retrieved. To simplify the comparison, the DCIS and IDC in each group were graded by nuclear grading based on Holland’s criteria [26]. IHC stains were performed with antibodies against ER, PR, AR, HER2, EGFR, CK5/6, CK 14, CK 17, CK 8, and CK 18 on one representative block of formalin-fixed, paraffin-embedded tissue from each case (Table 1Go). Pretreatments consisted of enzyme digestion or other retrieval methods. Sections were stained using the Dako Autostainer with one of the following reagents: (1) labeled monoclonal polymer, HRP (Envision PlusSystem, Dako Cytomation, Carpentaria, CA); (2) Horse Anti-Mouse IgG-Biotin (Vector Laboratories, Inc., Burlingame, CA), Streptavidin-HRP (Jackson Labs, West Grove, PA), and AEC (Dako Cytomation), and counterstained with hematoxylin. Positive stains were defined as ≥10% of tumor cells with strong cytoplasmic staining for CK markers, 3+ membrane staining for HER2 and EGFR, and ≥10% of tumor cells with nuclear staining for AR, ER, and PR. The slides were examined and evaluated blindly and semiquantitatively by two of the authors (TL and PT). The definitions for each classification are summarized in Table 2AGo, which divides tumors into basal and non-basal subtypes. The CK/TN classification was used to classify the breast carcinomas into luminal A, luminal B, HER2 over-expression, basal-like, and TN non-basal subtypes (Table 2BGo). All p-values were based on Fisher’s exact test.


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  		Table 1. Antibodies used for the immunohistochemical analyses.
 

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  		Table 2. Definitions for molecular classification.
 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MPD and the clinical pathological features of associated cancers (Table 3Go).  Patients with MPD had a mean age of 61 yr; 64% were associated with DCIS; 36% were associated with ID-NOS (not otherwise specified); none were associated with lobular carcinoma. Ninety-three percent of the MPD-associated tumors had high nuclear grade. Of the 81 cases in the non-MPD non-nipple group, 63% were DCIS and 89% had high nuclear grade, comparable to those of the MPD group. Of the 12 cases in the non-MPD nipple group, 83% were associated with invasive carcinoma–two of these being invasive lobular carcinoma–and 2 of the 12 cases were associated with nipple duct involvement by DCIS. Only 58% of the non-MPD nipple group tumors had high nuclear grade.


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  		Table 3. Clinical information.
 
MPD and single marker expression (Table 4Go).  MPD had significantly lower expression rates of PR compared to tumors from the non-MPD non-nipple group. This difference was significant in the MPD-associated DCIS group, but not in the IDC group. For ER expression, there was also a significant difference noted in DCIS but not in IDC. HER2 over-expression was associated with MPD in both the DCIS and IDC groups. The expression of AR and EGFR showed no significant differences between the non-MPD and MPD groups.


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  		Table 4. Single markers and mammary Paget’s disease (MPD).
 
MPD and molecular classifications (Table 5Go).  MPD-associated tumors fell mostly in the non-basal subtype (93% to 96%), compared to 59% to 75% in the non-MPD non-nipple group. One hundred percent of the MPD-associated DCIS fell in the non-basal subtype in all three classifications, and 80–90% of MPD-associated IDC fell into the non-basal subtype. Comparing the five subtypes in the CK/TN classification, we found that MPD-associated DCIS was significantly associated with HER2 subtype (88% vs 29%), but MPD-associated IDC was not (40% vs 37%). Similar observations were seen for basal-like subtype; 0% vs 29% for DCIS and 20% vs 16% for IDC. Vice versa, MPD-associated IDC was significantly more likely to be associated with luminal B subtypes (30% vs 7%), but MPD-associated DCIS was not (6% vs. 5%). Luminal A subtype was not associated with MPD associated with either DCIS or IDC.


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  		Table 5. Molecular classification and MPD.
 
MPD comparison to the non-MPD nipple group (Table 6Go).  Besides the 28 cases of MPD, 12 cases with non-MPD nipple involvement were studied. These cases included nipples with ulceration and dermal invasion by invasive carcinoma and nipple duct involvement by DCIS. For the single marker comparison, ER and PR were expressed at significantly higher rates in this group compared to MPD, while HER2 over-expression was significantly lower in this group. There was no significant difference among these groups for the expression of AR and EGFR. For molecular classification, there were significant differences among the groups in all three classifications (p values from 0.0019 to 0.0353). The difference was even greater with the CK/TN classification (p value <0.0001), with 42% of luminal A subtype and 8% of HER2 subtype in the non-MPD nipple involvement group, with none of luminal A subtype and 71% of HER2 subtype in the MPD group.


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  		Table 6. Comparison among all three groups.
 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Breast carcinomas, including MPD, were formerly treated uniformly with mastectomy. With earlier detection and better understanding of tumor biology, increasing numbers of breast carcinomas are being treated with breast conservation therapy. Knowledge of which patients are at higher risk of developing MPD may help oncologists to tailor the treatment choices for each patient. Unfortunately, pre-operative detection of MPD is unreliable clinically because some cases of MPD do not show redness, thickening, or eczematous changes. Chaudary et al reported that only 31% of 35 cases of MPD were associated with a palpable mass [8]. Mammography, the most useful screening modality for breast carcinoma, often fails to detect MPD [27,28]. Recent studies suggest that MRI may be useful in detecting early MPD [29]. Nonetheless, IHC identification of reliably predictive molecular markers can be valuable in the diagnosis and management of MPD.

Fu et al reported that in 14 cases of MPD, only 29% were positive for ER and PR [9], which is comparable with the 11% and 4% positivity rates observed in this study. Fu and Keatings also reported very high–93 to 100%–expression of HER2 [9,30], which is also comparable with our rate of 89%. The over-expression of HER2 due to gene amplification was confirmed by CISH in MPD [31]. Schelfhout et al have reported that heregulin {alpha}, motility factor, and HER2 family ligand secreted by epidermal keratinocytes seem capable of attracting Paget cells within the nipple epidermis by binding to the members of HER2 receptor family highly expressed by Paget cells [32]. In the present study, 1 of 28 cases expressed EGFR, which is comparable with prior data of Llody et al [5]. So far, no data on HER3 and HER4 in MPD are available in the literature. In contrast to ER and PR, AR showed no differential expression between MPD and non-MPD non-nipple groups in the present study, which is consistent with the findings of Liegl et al [33].

The present study provides three unique findings that may help elucidate MPD and its underlying lesions. First, different receptor expression patterns and molecular subtype distributions were observed in two different types of MPD-associated lesions–DCIS and IDC. MPD-associated DCIS was more likely to be ER and PR negative and HER2-subtype tumors, and less likely to be basal-like subtype; while MPD-associated IDC was more likely to be luminal B subtype. These results suggest that different molecular mechanisms may be involved for MPD depending on the types of underlying lesions, and that different panels of markers should be used to predict MPD depending on its underlying lesions. Two consistent factors for both MPD-associated DCIS and IDC are association with HER2 over-expression and non-association with luminal A subtype. Second, HER2 over-expression and HER2 subtype seem to differ between MPD-associated DCIS and MPD-associated IDC. HER2 over-expression is associated with both MPD-associated DCIS and IDC, and HER2 subtype is only associated with MPD-associated DCIS. These observations suggest that HER2 over-expression and HER2 subtype are not interchangeable, and over-expression of HER2 and its relationship with the expression of ER and PR may be important in the pathogenesis of MPD. Third, MPD and non-MPD nipple-involved tumors, although both have nipple involvement, are subgroups with very different expression patterns and subtype distribution, suggesting that distinct mechanisms are involved in their pathologenesis. The non-MPD nipple-involved group seems to be a heterogeneous group, which consists of high-grade and non-high carcinomas, ductal and lobular carcinomas, in situ and invasive carcinoma. The presence of this group poses a challenge in predicting nipple involvement in non-MPD carcinoma.

In summary, the significant differences in ER expression, PR expression, and subtype distribution between MPD associated with two underlying lesions (DCIS and IDC) suggest that different panels of markers should be used to predict MPD in these two lesions. Further studies with a larger number of cases of MPD and prospective follow-up of patients with ductal carcinomas may be useful in elucidating the molecular characteristics that are associated with greatest risk of developing MPD.


Figure 1
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  		Fig. 1. Representative H&E and IHC stains for ER, PR, AR, HER2, CK8, CK5/6, and EGFR of MPD (Original magnification 400X).

 

   Acknowledgment
 
The authors thank Steven I. Hajdu and David Hicks for critical suggestions and Marjorie Goldman for assistance in preparing the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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