Mammary Hamartomas: An Immunohistochemical Study of Ten Cases

PATHOLOGY RESEARCH AND PRAGICE

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Mammary Hamartomas: An Immunohistochemical Study of Ten Cases Rosistella Chiacchio'. Luigi Panico'. Antonio D'Antonio'. Paolo Delrio'. Delfina Bifano 1, Maurizio Avallone 1 and Guido Pettinato 1 'Dipartimento di Scienze Biomorfologiche e Funzionali, Settore di Anatomia Patologica, University of Medicine and Surgery, Federico II, Naples, Italy; 2Department of Surgery, University of Medicine and Surgery, Federico II, Naples, Italy

Summary

Introduction

Ten cases of breast hamartomas were reviewed; the patient s' age ranged from 31 to 55 (mean 40.4, median 39). All cases presented with a palpable, sometimes tender, lump. The typic al mammographic feature was a well defined, round to lens shaped, variable den se mass, occasionally surrounded by a thin radiolucent zone. All hamartomas were unilateral (4 in the right and 6 in the left breast, respectively) and no recurrence occurred after local exci sion . The tumor size ranged from 5 to 150 mm (mean 54 mm). Histologically all hamartomas were composed of a typical fibrous, adipose and glandular tissue combination. Immunohistochemically there was a strong positivity for cytokeratin and epithelial membrane antigen in the epithelial cells, a positive finding for vimentin and muscle-specific actin in stromal and myoepithelial cells , and for S-l 00 protein in myoepithelial cells. Vessels endothelial cells were immunoreactive for Factor VIII. Immunohistochemical analy sis of hormone receptors completed on formaldehyde-fixed paraffin-embedded specimens, showed estrogen and progesterone receptors positi vity in 9 case s and estrogen positive progesterone negati ve receptors in one case. In all cases the receptorial positivity was limited to the epithelial elements. These data revealed that 1) brea st hamartoma is a benign, tumor-like lesion, histologically dissimilar from other lesions such as fibroadenoma and pseudoangiomatous hyperplasia; and 2) hamartoma tissue is influenced by hormones like the surrounding normal breast parenchyma.

Mammary hamartoma is a benign tumor-like lesion of the female breast. A diagno sis is achieved by combining clinical, radiological and pathologic results. By definition, hamartomas are focal malformations consisting of an abnormal mixture of the tissues normally existing in a specific organ. The term mammary ham artoma has been used to encompass a heterogeneous group of lesions like the cartil age- and smooth musclecontaining variants [ 3, 4, 5, 16]. The etiology of hamartoma is not known though a connection of hamartoma with pregnancy and lactation [11] has been suggested a hypothesis that has never been confirmed by subsequent reports. Arrigoni et al. [2] and Linell et al. [12] did recognize that pregnancy and lactat ion could increase cellular growth rate while Hessler et al. [9] thought that the development of hamartomas was independent from pregnancy and lactation hormonal influences. Therefore, it seems feasible that breast hamartomas are affected, not caused, by hormonal factors [13 , 16, 18]. We have evaluated serie s of ten breast hamartomas in the clinical, histological and immunohistochemical aspect s to further characterize the benign nature of this lesion and its correlation with estrogen and progesterone receptors.

Key words: Mammary hamartomas - Immunohistochemistry - Steroid receptors Pathol. Res. Pract. 195: 231-236 (1999)

Address for correspondence: Prof. Guido Pettinato, Institute of Pathology University of Medicine and Surgery, Federico II, v, S. Pansini 5, 80131 Naple s, Italy. Tel.: ++39/0 81/7 46 34 33, Fax: ++39/0 81/7 46 34 75 0344-0338/99/195/4-231 $12.00/0

232 . R. Chiacchio et al.

Ma terial and Methods A retrospective study of ten breast hamartomas has been attained reviewing the 1989-1994 files of the Institute of Pathology, University of Naples "Federico II." The clinical data of the 10 patients are summarized in Table 1. The patients' age ranged from 31 to 55 years (mean 40.4, median 39); seven patients were premenopausal and three postmenopausal. Four lesions were found in the right breast and six in the left. The site was the upper outer quadrant (UOQ) in three case s, subareolar or central quadrant (CQ) and upper quadrants (UQ) in two cases, respectively, and in the remaining quadrants for three cases. Mammograms dem on strated nodular densities in all cases (well defined, round to oval shaped nodules in 8 patients; not completely well circumscribed nodules in 2 cases). The lump size ranged from 5 mm to 150 mm (mean 54 mm ). Fat tissue was radiographically detectable within the nodule; only once were microcalcifications never detected. Clinically, 6 cases were classified as fibro adenomas and 4 as a "well circumscribed soft mas s." All patients were treated with local excision. The specimens were fixed in formaldehyde and embedded in paraffin. Five-micron thick sections were stained with hematoxylin and eosin (H&E) for the histological diagnosis. Avidin -biotin peroxidase complex (ABC) technique was used in the immunohistochemical study. Subsequently, antibodies against cytokeratin AEI -AE3 (CK; Dakop atts-Denmark, 1:200), epithelial membrane antigen (EMA; Dakopatts-Denmark 1:100), factor VIII-related antig en (FVIII ; DakopattsDenmark, 1:100), vimentin (VIM; Dakopatts-Denmark, I:50), muscle-specific actin (MSA; Dakopatts-Denmark 1:50) and S-100 protein (S- 100; Dakopatts-Denmark, 1:200 ) were utilized. Enzymatic predi gestion with tryp sin was completed before incubation with CK and FVIII. We adopted the ABC technique in all cases (LSAB plus kit Dakopatts-Denmark) following the manufacturer's instructions. The receptorial status was evaluated after incubation with antibodies to the estrogen-receptor protein (ER ; Dakopatts-Denmark, 1:250) and the progesterone-receptor (PR ; EPOQ, I :250). Before incubation with the primary antibody rehydrated sections were pretreated in a microwave oven at 750 W in citrate buffer 0.01 M (pH 6.0) . The ABC techn ique (Vectastain ABC Elite , Vector Laboratorie s, Burlingame, CA, USA) was also utilized in the

hormonal receptor stud y, in conformity with the producer's recommendations. The antibody-antigen react ion was developed with diaminobenzidine; the nuclei were counterstained with hematoxylin. Tissue samples known to express the teste d antigens were used as positive controls, and a non immune serum was used as the negative control. All controls stained appropriately. The antibodies were purposely chosen with the intent of demonstrating the different elements - epithelial, stromal, myoepithelial, vascular- and their hormone receptor statu s within the tumor-like lesion.

Results Pathologic find ings

The size of the lesions varied from 5 to 150 mm. All lesions presented as circumscribed soft-tissue mass; only 5 out of 10 appeared to be encapsulated (Fig. 1).

Fig . I . Gros s examination shows a well circumscribed, encap sulated mas s.

Table 1. Clinicopathologic features Case

Age

Hormonal state

Site

Clin ical presentation

Size (mm)

1 2 3 4 5 6 7 8 9 10

31 46 50 31 47 33 35 43 33 55

premenopausal postmenopausal postmenopausal premenopausal premenopausal premenopausal premenopausal premenopausal premenopausal postmenopausal

RC LUI LUO LUI-UO RC LLO RLI-LO LUO LUI-UO RUO

well circumscribed nodul e soft mass well circumscribed mass fibrou s nodul e subareolar nodule soft mass fibroadenoma-like nodule superficial fibrou s nodule fibroadenoma-like nodule soft mass

30 75 150 30 5 65 25 11 20 130

RC = Right Cen tral, LUI = Left Upper Inner, LUO = Left Upper Outer, LUI-UO = Left Upper Inner-Upper Outer, LLO = Left Lower Outer, RLI-LO =Right Lower Inner-Lower Outer, RUO =Right Upper Outer

Mammary Hamartomas . 233

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The cutting surface showed various appearances. Six lesions were firm and homogeneously gray-white. The remaining 4 nodules presented a fibrofatty appearance, as expressed by the yellow-white intermixed areas, and a consistency similar to the adjacent normal breast tissue. The microscopic features corresponded to the gross appearance. All tumors were clearly circumscribed and easily detached from the adjacent parenchyma, however a true capsule was present in only half of the cases. The firm whitish tumors consisted of abundant densely hyaline and scarcely cellular fibrous connective tissue and only minimal fat. The fibrous tissue was often observed in bundles around lobular ductules (Fig. 2) without any evident compression due to areas of intermingled fat (Fig. 3). Tumors that grossly showed a fatty component contained variable amounts of microscopically mature fat. No correlation was found between the amount of fat tissue present and patient's age. In only two of the 10 cases did the stroma contain a network of anastomosing slit-like spaces that appeared to be lined by flat cells. This pattern was similar to the pseudoangiomatous hyperplasia described by Vuich et al. [17 J and particularly conspicuous in densely fibrous areas (Fig. 4). In all cases, scattered or grouped mast-cells with large nuclei and granular eosinophilic cytoplasm were observed within the stroma. We never observed calcification sand muscular or chondroid areas within the stroma. The epithelial components were represented only by ducts and ductules disposed in disordered fashion without well-formed acinar structures. In all cases these ducts were surrounded by a rim of collagen that separated them from the adipose tissue giving the aspect of adenolipomatous areas (Fig. 3). The ducts were lined by a single layer of epithelial cells surFig. 2. The fibrous connective tissue was densely hyaline and paucicellular between lobular mammary ducts (H&E, 106x). Fig. 3. Areas of fat and fibrous tissue circumscribe the mammary duct (H&E, 400x). Fig. 4. The stroma contained areas of pseudoangiomatous hyperplasia (H&E, 250x).

234 . R. Chiacchio et al. Table 2. Immunohistochemical findings Antibodies

Epithelial Endothelial cells cells

CK EMA FVIII VIM S-100 MSA

++ ++

Table 3. Immunohistochemical analysis of estrogen and progesterone receptors

Stromal Myoepithelial elements cells

++ ++

++

+

+

+ ++ ++

rounded by a myoepithelium that occasionally showed plump elements. Small cysts and apocrine changes were observed in 2 cases, but there was never evidence ofepithelial hyperplasia or atypia.

Case no. and age

Pg receptor

E receptor

1 (31 y. 0.) 2 (46 y. 0.) 3 (50 y. 0.) 4 (31 y. 0.) 5 (47 y. 0.) 6 (33 y. 0.) 7 (35 y. 0.) 8 (43 y. 0.) 9 (33 y. 0.) 10 (55 y. 0.)

++ + +++ + ++ +++ + ++

++ + +++ ++ ++ +++ + + + +

+

- negative; + low positivity; ++ moderate positivity; +++ high positivity Immunohistochemical findings(Tables 2-3)

In all 10 tumors there was a strong, diffuse staining of epithelial ductal cells cytoplasm for CK and EMA and a reactivity for MSA, protein S-100 (Fig. 5), and VIM in the majority of myoepithelial cells. VIM and MSA also highlighted the vessels' endothelial lining. Stromal cells were also reactive for VIM and MSA, while only blood vessels cells were positive for F-VIII. In 9 cases there was a variable number of ductal epithelial cells showing a nuclear staining for both estrogen and progesterone receptors. Only once were estrogen receptors positive alone (Fig. 6). Stromal, endothelial and myoepithelial cells did not show any staining for hormone receptors.

Discussion In 1971 Arrigoni et al. [2J were the first to propose the term "hamartoma" to describe a well circumscribed breast lesion characterized by a mixture of benign mammary elements. Previous studies reported benign breast lesions with histologic features similar to Arrigoni's

Fig. 5. A strong positivity for S-lOO protein in the myoepithelial cells (immunoperoxidase400x). Fig. 6. The ductal cells exhibited estrogen receptors (immunoperoxidase 400x).

Mammary Hamartomas . 235 hamartoma but defined with different names: mastomas, adenolipoma [17 J and fibroadenolipoma [11]. We consider all these entities true breast hamartomas. The term "hamartoma" has been used to encompass breast lesions that macro- and microscopically reproduce the characteristic of a typical lobular early involution and sclerosis [12]. Some tumors show abundant fatty replacement of the stroma [4, 17J and seldom present heterologous stromal differentiation (i.e. myoid [3, 5, 6J or cartilagineous [14]). The stroma is sometimes hyalinized [4, 9, 16J, in other cases it contains a network of anastomosing slit-like spaces that appear to be lined by flat cells [9]. The overall appearance matches the description of the "pseudoangiomatous hyperplasia" illustrated by Vuitch et al. in 1986 [19 J, a benign disease of the female breast that can produce a mass lesion. Microscopically it is characterized by stromal pathologic changes due to a myofibroblastic proliferation that progresses from open, slit-shaped, anastomosing empty channels to more proliferative lesions, composed of bundles of plump spindle cells. It is possible that the stromal myofibroblastic proliferation is a focal exageration of the hormone-related physiologic of lobular proliferation [18J. In breast hamartoma the epithelial component consists of disarrayed ducts and duetules with an incomplete lobular formation. The ducts seldom appear cystic [13J with epithelial apocrine metaplasia [6]. A constant feature is the juxtaposition between ductules and adipose tissue (adenolipomatous areas) [12J; their identification helps the microscopic diagnosis because this is not a normal characteristic feature of breast epithelium and intralobular connective tissue. The main differential diagnosis is between hamartoma and fibroadenoma. Both lesions are macroscopically well circumscribed and microscopically composed of glandular element and fibrous stroma. However, the stroma in fibroadenomas tends to be more cellular and to distort the lobules while adipose tissue is an unusual feature [8 J. It is essential that the marked stromal vascularity of hamartoma be differentiated from the true pseudoangiomatous stromal hyperplasia [8J. Anderson et al. [1] reported that pseudoangiomatous hyperplasia should not be included in the diagnostic range of breast hamartomas because: 1) hamartomas do not recur while pseudoangiomatous hyperplasia do, especially when the excision is not complete; and 2) stromal cells in pseudoangiomatous hyperplasia show intense reaction for progesterone receptors while they do not in hamartomas. In fact in our study only epithelial cells stained positively with antibodies for estrogen and progesterone receptors. The etiology of breast hamartoma is not known. Some authors suggested that these tumors be the result of a revival of segregated embryonal elements. The term "hamartoma" better reflects a dysembryogenetic

origin [4, 18J, as the definition of "malformation with a mixture of the organ normal tissues" implies. Breast hamartomas present as circumscribed lumps that, even when very large, compress rather than replace the normal tissue [13]. That is the reason why after their removal the residual breast tissue can expand to the normal size. In our series the clinical and pathologic features showed that breast hamartoma is a non neoplastic lump of sequestered normal or mildly altered breast tissue. In all 10 cases we observed normal clinical and mammographic results. The local excision was the treatment of choice and no relapse was observed. The epithelial components, which stained with CK and EMA, formed a single layer of benign ductal cells. No atypical cells were observed. These cells were surrounded by a layer of occasionally plump myoepithelial cells that were positive, like in the normal breast, for S-lOO, VIM and MSA. Vessel spindle cells, stromal fibroblasts, as well as the areas of pseudoangiomatous hyperplasia were positive for VIM and MSA [15]. However, the factor VIII negativity suggests that these cells have a myofibroblastic differentiation. These immunohistochemical findings are important in differentiating the stromal areas of pseudoangiomatous hyperplasia from true vascular hyperplasia or low grade angiosarcoma. Although pseudoangiomatous stromal hyperplasia is a recurrent microscopic feature in breast hamartoma, as above reported these two terms are not synonyms. The role of hormones in the etiopathogenesis of breast hamartomas has varied in different studies. Hogerman and Ostberg [11] reported three post-lactational cases and postulated a causal relationship between hamartomas and lactation. Arrigoni et al. [2 J accepted that pregnancy and lactation could influence the rate of hamartoma growth while Hessler et al. [10J denied any relationship. In the present series we could not find any association among hormone receptor positivity, patient's age, and menopausal state (Table 1 and 3). In conclusion, the abundance of estrogen and progesterone receptors in ductal cells and the presence of areas of pseudoangiomatous hyperplasia suggest that mammary hamartoma can be influenced and changed by hormonal factors and that this lesion might be the result of a mild abnormal development in the breast tissue. Acknowledgements. We are grateful to Mr. Armando Coppola for histechnical assistance.

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Received: January 1, 1998 Accepted in revised version: February 18, 1999