ation of hormonal receptors in breast carcinoma is useful as a predictor of ... cells was assessed as positive or negative, with moderate to strong nuclear staining ...
Anatomic Pathology / HORMONAL RECEPTORS IN MAMMARY PHYLLODES TUMOR
Hormonal Receptors Expression in Epithelial Cells of Mammary Phyllodes Tumors Correlates With Pathologic Grade of the Tumor A Multicenter Study of 143 Cases Gary M.K. Tse, FRCPC,1 C. Soon Lee, FRCPA,5 Fred Y.L. Kung,1 Richard A. Scolyer, FRCPA,5 Bonita K.B. Law, FRCS,2 Tai-shing Lau, PhD,3 and Thomas C. Putti, Dip Am Bd (Path)4 Key Words: Phyllodes tumor; Estrogen receptor; Progesterone receptor; Malignancy; Immunohistochemistry
Abstract We used immunohistochemical analysis to detect the presence of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) protein expression in the epithelial and stromal cells of 143 phyllodes tumors (PTs). Expression of epithelial ER and PR proteins was common, occurring in 43% to 84% of PTs. Expression of epithelial AR protein and stromal ER, PR, and AR proteins was low (5% or less) in all tumors. An inverse relationship of epithelial ER and PR protein expression with degree of malignancy in PT was found (P < .05), and ER expression also correlated with mitotic count (P < .05). When considering PT with the expression of ER or PR proteins and the coexpression of both, the inverse relationship with tumor grade also was significant (P < .05). As the hormonal receptor protein expression shows a consistent decrease with increasing malignancy, we infer that the epithelium has a crucial role in the pathogenesis or progression of PT.
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Mammary phyllodes tumor (PT) is an uncommon stromal-epithelial neoplasm with a reported incidence of 0.3% to 0.5% of female breast tumors.1-5 The median and mean ages of patients are 45 years, and the average tumor size is 4 to 5 cm. Rarely these lesions can occur in young or elderly women or in men. Pathologically, PTs are divided into benign, borderline, and malignant categories based on assessment of a combination of histologic features, namely mitotic count, cellularity and nuclear pleomorphism of the stromal cells, stromal overgrowth, and circumscription of the tumor border.2,6 While borderline and malignant PTs can rarely metastasize, all PTs can recur locally. This propensity to recur makes proper and adequate treatment imperative, even in benign cases. Currently the mainstay of treatment of mammary PT remains surgical, and this also applies to recurrences and metastases. The expression of estrogen receptor (ER) and progesterone receptor (PR) in mammary neoplasms is well documented, particularly in carcinoma of the breast. The evaluation of hormonal receptors in breast carcinoma is useful as a predictor of response to endocrine manipulation therapy and as a prognostic index of the course of the disease.7 While the usefulness of hormone receptor status of mammary PT is unknown, potentially this may offer similar therapeutic and prognostic information. We evaluated the expression of hormonal receptor proteins (ER, PR, and androgen receptor [AR]) in mammary PTs by immunohistochemical analysis with the aim of documenting the hormone receptor status of a large cohort of tumors from 3 centers in the Oceania region in an attempt to gain further insight into the pathobiology of these uncommon tumors. © American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
Materials and Methods
Results
The histopathology files of 3 participating departments from the Asia-Pacific region and Australia were searched for PTs during a 14-year period from 1987 to 2000, yielding a total of 143 cases. The paraffin blocks were retrieved, and 4-µm slides were prepared routinely and stained with H&E. All slides were reviewed for the following histologic parameters: (1) stromal cellularity, (2) nuclear pleomorphism, (3) stromal overgrowth, (4) mitotic count, and (5) margin of the tumor, whether infiltrative or rounded. Parameters 1 and 2 were graded as low/mild, moderate, or severe; stromal overgrowth was graded as mild, moderate (scanty epithelial elements within a low-power field), or severe (absence of epithelial element within a low-power field [×40; Nikon Labophot, Nikon, Tokyo, Japan; field area, 1.9 mm 2 ]); and the mitotic count was given as the number of mitotic figures per 10 high-power fields (×400; Nikon Labophot; field area, 0.19 mm 2 ). A diagnosis of benign PT was made when there was low cellularity, no stromal overgrowth, mild pleomorphism, a rounded margin, and a mitotic count of 2 or less per 10 high-power fields. Malignant PT was diagnosed when the mitotic count was 5 or more per 10 high-power fields together with stromal overgrowth and an infiltrative margin. Borderline PT was diagnosed when the criteria for malignant PT were not totally fulfilled. For hormonal receptor staining, a most representative slide was taken from each case and stained for ER, PR, and AR using the standard avidin-biotin method (for ER [DAKO, Glostrup, Denmark], 1:100 dilution; for PR [Zymed, San Francisco, CA], 1:100 dilution; for AR [Novocastra, Newcastle upon Tyne, England], 1:30 dilution with microwave antigen retrieval). The staining of cells was assessed as positive or negative, with moderate to strong nuclear staining of at least 20% of cell nuclei considered positive. Staining was evaluated in the epithelial cells and in the stromal cells. Analysis of variance was used to study the differences in age, the 5 histologic parameters, and tumor size among the benign, borderline, and the malignant groups. Logistic regression was used to study the effect of hormonal receptor proteins and other histologic factors on diagnosis. The Pearson correlation coefficient was calculated to study the linear association among the hormonal receptors and the histologic factors. The Tukey procedure was used for multiple comparisons. We also applied the trend test 8 to show the decreasing trend of ER or PR expression and the coexpression of ER and PR in the 3 diagnostic groups. Statistical significance was established at P less than .05.
A total of 143 PTs from 137 patients were reviewed, including the initial excision specimens and single recurrences from 4 patients and 2 recurrences from 1 patient. The patient’s ages ranged from 15 to 77 years (mean, 43 years), and the tumor size ranged from 1 to 22 cm (mean, 5 cm). Of the patients, 62.0% were Chinese, 21.2% were white, and 16.8% were Indo-Chinese or Indian. The tumors occurred on the left side in 51.0% of cases and on the right side in 47.6%; in 2 cases (1.4%), the side was not known. Of all cases, there were 87 benign (60.8%), 37 borderline (25.9%), and 19 malignant (13.3%) PTs. For the 87 benign PTs, the patient age range was 17 to 62 years (mean, 40.8 years), and the range of tumor size was 1 to 22 cm (mean, 4.3 cm). For the 37 borderline PTs, the patient age range was 15 to 77 years (mean, 44.6 years), and the range of tumor size was 1 to 20 cm (mean, 5.8 cm). For the 19 malignant PTs, the patient age range was 30 to 76 years (mean, 49.8 years), and the range of tumor size was 2 to 22 cm (mean, 6.8 cm). The mean age of patients increased with the degree of malignancy of PT. The difference between age of patients in the benign and malignant groups was significant (P = .005), but the difference was not significant between the benign and borderline groups or between the borderline and malignant groups. The size of the PTs also increased with increasing degree of malignancy. A statistically significant difference was established between the benign and malignant groups (P = .0158), but the difference was not significant between the borderline and malignant groups or between the benign and borderline groups. For all PTs, ER was expressed in the epithelium in 83 cases (58.0%) and in the stromal cells in 4 cases (2.8%). PR was expressed in the epithelium in 107 cases (74.8%) and in the stromal cells in 2 cases (1.4%). AR was expressed in the epithelium and the stromal cells in 2 (1.4%) PTs each. The expression of ER, PR, and AR in benign, borderline, and malignant PTs is given in ❚Table 1❚. Most ER and PR expression was found in the epithelium. The expression of ER and PR in the stromal cells and of AR in both epithelium and stromal cells was low, 5% or less. If only the epithelial expression of ER is considered, a small increase with age was found (regression coefficient, 0.0067; P = .067). For PR expression, a gradual decrease was noted with increasing age; however, this was not statistically significant (regression coefficient, –0.004; P = .1762). When the epithelial expression of ER and PR was considered among PTs of differing degrees of malignancy, ER expression was higher in benign PTs than in borderline and malignant PTs. Analysis of variance revealed that the difference was statistically significant (P = .0318). For PR expression, both benign and borderline PTs showed similar
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❚Table 1❚ Expression of Hormone Receptors in Phyllodes Tumors of Benign, Borderline, and Malignant Subgroups* Estrogen Receptor Phyllodes Tumor Subgroup Benign (n = 87) Borderline (n = 37) Malignant (n = 19) *
Progesterone Receptor
Androgen Receptor
Epithelial
Stromal
Epithelial
Stromal
Epithelial
Stromal
58 (67) 16 (43) 9 (47)
3 (3) 0 (0) 1 (5)
67 (77) 31 (84) 9 (47)
1 (1) 0 (0) 1 (5)
1 (1) 1 (3) 0 (0)
1 (1) 0 (0) 1 (5)
Data are given as number (percentage).
high levels of expression, and the malignant PTs had a much lower PR expression. The difference between malignant and nonmalignant PTs also was statistically significant (P = .0086). By using a logistic regression model, we found that the epithelial expression of ER correlated with the diagnosis (P = .0199) but the expression of PR did not (P = .1925). Further analysis on the correlation of epithelial ER and PR expression with the histologic parameters showed a strong inverse correlation with mitotic count (for ER, correlation coefficient, –0.20; P = .015; for PR, correlation coefficient, –0.21; P = .0134). The logistic regression model with mitoses removed from the analysis revealed that ER expression was significant in predicting the diagnosis (P = .0211), but PR expression was not, indicating that ER expression contains information overlapped with mitotic count in predicting the diagnosis. When expression of ER and PR is combined, the trend is more obvious. Considering the epithelial expression of either ER or PR, 80 (92%) of 87 benign PTs, 32 (86%) of 37 borderline PTs, and 13 (68%) of 19 malignant PTs were positive for either hormonal receptor. When we considered coexpression of ER and PR in the epithelium, 45 (52%) of 87 benign PTs, 15 (41%) of 37 borderline PTs, and 5 (26%) of 19 malignant PTs were positive for both hormonal receptors. Under both conditions, there is a decreasing trend of ER-PR expression as the degree of malignancy increases. By using the trend test, we found that the decreasing trend for ER or PR was significant (P = .0084). Similarly, the decreasing trend for coexpression of ER and PR also was significant (P < .0005).
Discussion Evaluation of hormonal receptor status is now a standard procedure in the assessment of carcinoma of the breast, as this has a bearing on both the prognosis and the prediction of response to adjuvant endocrine therapy. The evaluation of these receptors in other breast lesions is limited in the literature. PT is a fibroepithelial neoplasm that possesses an inherent ability to recur locally in all 524
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cases and to metastasize in borderline and malignant cases. The relatively high recurrence rate despite surgical resection remains an unresolved management problem. An understanding of the expression of hormonal receptors in such neoplasms is crucial for consideration of the potential role of endocrine therapy. In the few studies of hormonal receptors of PTs in the literature, the results are confusing and inconsistent. One of the earliest studies demonstrated, using a biochemical assay, the presence of ER in 20% of PTs and PR in 100% of PTs. By matching the volumes, it was suggested that PR was located in the stroma, whereas ER was in the epithelium. 9 Another report of 1 case of malignant PT with only the stromal component demonstrated persistence of ER by biochemical assay after heterotransplantation into nude mice, 10 suggesting the presence of ER within the stromal component. Other studies used antibodies and the immunohistochemical technique for evaluating hormonal receptors in PTs.11-13 In these studies, the principal findings included the detection of ER and PR in PTs, but the expression was located mostly in the epithelium. Prominent stromal expression has not been demonstrated. The percentage of ER and PR expression in PTs was reported to be 32% and 96%, respectively, in 1 study of 50 cases,12 and in others, the expression was variable, but with PR expression being more extensive than ER expression. 11,14 Another study of 19 PTs demonstrated a statistically significant age trend: an increasing level of ER expression was noted with increasing patient age.13 The results of our study showed the expression of hormonal receptors was located mainly in the epithelial component of PTs. Expression was much less frequent in the stromal cells. This is in agreement with most authors for ER expression9,11-13 but was in contradistinction to Rao et al,9 who claimed to show localization of PR in the stromal cells. This difference may be due to differing methods, as the latter study used a biochemical assay.9,10 The low stromal level of expression of hormonal receptors is consistent with the postulated stromal origin of PT, as the nonneoplastic counterpart seldom expresses hormonal receptors. © American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
Expression of PR was common, with ER being less common and AR uncommon. This finding was similar to those of other series of PTs11-14 and fibroadenomas.14,15 This observation is parallel to carcinoma of the breast,15 which also shows higher PR expression than ER expression in the tumor cells. A decreasing trend of PR expression was observed in PTs with increasing patient age, and this has not been reported previously. This finding, however, was not statistically significant. Similar to other authors, 13 we showed a slight increase in ER expression with age, the significance of which is unknown. There seems to be a relationship between ER and PR expression and the degree of malignancy of PTs. For ER, the expression was high in benign PTs but low in borderline and malignant PTs. For PR, the expression was high in both benign and borderline PTs but low in malignant PTs. This observation highlights several interesting points. The inverse relationship between hormonal receptor expression and degree of malignancy is a phenomenon similar to that observed in carcinoma of the breast.15 Whether this association is causal or fortuitous remains to be resolved, as this observation has not been reported previously. Second, ER was shown to have an inverse correlation with the mitotic count and overlapped with the mitotic count in predicting the diagnosis. This is particularly interesting, as the expression of ER was in the epithelium, and the mitotic count was determined from the stromal cells. This correlation raises the possibility that the epithelium may have a significant role in triggering the stromal proliferation, and this mechanism may be reflected partly in the histomorphologic features of subepithelial stromal condensation. This type of stromal-epithelial interaction and cell signaling that may lead to epigenetic changes in the stroma has been observed in other tumors, such as prostatic neoplasms.16 Third, as PTs progress from benign to borderline to malignant, the level of ER expression decreases before the level of PR expression does, so in borderline PTs, there is a low level of ER expression but PR expression remains high. It is only when the PTs reach the malignant stage that both ER and PR expression decrease to a low level. This is difficult to reconcile with the current concept that PR expression is ER-dependent. In evaluating the combined expression of ER and PR, we showed that if we considered the expression of either one of the receptors (ER or PR) or of both (ER and PR), both criteria exhibited a decreasing trend of expression with increasing degree of malignancy. An analogy can be drawn from the situation in mammary carcinoma, in which although the same endocrine therapy is given to patients with expression of either one or both hormone receptors, patients with a tumor coexpressing ER and PR have the best survival, ER- and PR-negative patients have the worst survival, and patients with tumors expressing one of the receptors have an © American Society for Clinical Pathology
intermediate prognosis.17 Our findings suggest that the inverse relationship of ER and PR expression patterns and diagnosis potentially would indicate an outcome pattern similar to that in mammary carcinoma. The significance of AR in PT remains largely unknown. In primary carcinoma of breast, AR expression is reported to be present in 35% to 50%, and its presence is considered a favorable prognostic factor. 18-20 Investigators have suggested that loss of AR may be associated with HER-2 expression, indicating a worse prognosis.21 The AR protein has tyrosine kinase receptor properties,22 and it is known that the AR signaling pathway can be activated by a number of ligands for receptor tyrosine kinase.23,24 When there is a low level of androgen, growth factors such as HER-2/neu expressed by stromal cells can activate the AR pathway, even in the absence of androgens.24 Conversely, the low expression or loss of AR in the stroma of PTs may reciprocally up-regulate receptor tyrosine kinase expression, and this can be critical in the multistep evolution of PTs. In our series of PTs, the expression of AR in both epithelial and stromal components in benign, borderline, and malignant categories was low (5% or less), and no difference was demonstrated between PTs of varying degrees of malignancy. AR expression in other breast diseases is variable, including a high level of expression in apocrine lesions, but it has not been evaluated in fibroepithelial or stromal lesions. Further evaluation, particularly at the molecular level, to study the gene expression of these receptors in PTs would be useful to further the understanding of the disease and the possible role, if any, of endocrine therapy.
Conclusions In the present study, we demonstrated that in PTs, there is high epithelial expression of ER and PR, singly or in combination, and the level decreases with increasing degree of malignancy, suggesting a possible role of hormonal effect on tumor progression. The epithelial AR expression, unlike that of some primary carcinomas, is low, and so is the stromal expression of ER, PR, and AR. These findings seem to indicate that the potential role of endocrine therapy is limited, as stromal cells constitute the principal neoplastic cell population in PTs. However, the altered expression of these receptors in the epithelial cells suggests that they may be important in the development of PT. From the Departments of 1Anatomical and Cellular Pathology and 2Surgery, Prince of Wales Hospital, Hong Kong, People’s Republic of China; 3Statistics, Chinese University of Hong Kong, Hong Kong; 4Pathology, National University Hospital, Singapore; and 5Pathology, University of Sydney and Royal Prince Alfred Hospital, Sydney, Australia.
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Address reprint requests to Dr Tse: Senior Medical Officer, Dept of Anatomical and Cellular Pathology, Prince of Wales Hospital, Ngan Shing St, Shatin, NT, HKSAR, China.
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