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p63 is useful in the diagnosis of mammary metaplastic carcinomas
Pathology (February 2006) 38(1), pp. 16–20
ANATOMICAL PATHOLOGY
p63 is useful in the diagnosis of mammary metaplastic carcinomas GARY M. TSE*, PUAY-HOON TAN{, BENJAPORN CHAIWUN{, THOMAS C. PUTTI§, PHILIP C. W. LUId, ALEX K. H. TSANG*, FIONA C. L. WONG* AND ANTHONY W. I. LO*
*Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, {Department of Pathology, Singapore General Hospital, Singapore, {Department of Pathology, Chiang Mai University, Thailand, §Department of Pathology, National University Hospital, Singapore, and dDepartment of Pathology, United Christian Hospital, Hong Kong
Summary Aims: p63 has been recently reported to be expressed in sarcomatoid/metaplastic carcinoma of the breast, in addition to its role as a myoepithelial marker. A large series of 34 metaplastic carcinomas, including cases with pure epithelial component (squamous cell and adenosquamous carcinomas), biphasic tumours with carcinomatous and sarcomatoid components and monophasic tumours with only spindle cell component, were evaluated for p63 expression with respect to the different cellular components. Methods: All of the metaplastic carcinomas were assessed for p63 and conventional epithelial and mesenchymal markers of AE1/3, CAM5.2 and vimentin by immunohistochemistry. Results: All of the different categories of metaplastic carcinomas showed similar clinico-pathological features (patient age, tumour size, nuclear grade, mitotic activity, lymph node status and hormonal receptor status). For metaplastic carcinoma with epithelial component only, p63 was only expressed in the squamous cell component, but not the adenocarcinoma component. Eight of the 10 tumours were positive for p63. For the tumours with sarcomatoid component, either singly or together with carcinomatous component, p63 was positive in 14 of 24 cases. Pure sarcomas and carcinomas were all negative for p63 staining by immunohistochemistry, thus rendering p63 staining highly specific for diagnosing metaplastic carcinoma. Conclusions: Using p63 for diagnosis of metaplastic carcinoma gives a sensitivity of 65%, a specificity of 96%, a positive predictive value of 96%, and a negative predictive value of 66% and an accuracy of 78%. p63 may be used as an adjunct marker in the diagnosis of metaplastic carcinoma. Key words: Breast, metaplastic, p63. Received 8 July, revised 13 August, accepted 15 August 2005
INTRODUCTION One of the p53 homologues and related genes, p63, has been mapped to chromosome 3q27–28.1,2 In rodent models, p63 appears to play a crucial role in the regulation of epithelial proliferation and differentiation. Mice that are p63–/– have no hair follicles, teeth, mammary, lacrimal and salivary glands.3,4 In humans, immunohistochemical
analysis reveals p63 expression in the epithelial cells of stratified epithelium, including skin, oesophagus, exocervix, tonsil, bladder and the basal cells in glandular organs, including breast, bronchi and prostate.5 Furthermore, p63 has been shown to be expressed in the myoepithelial cells in the breast6,7 and as a marker of undifferentiation.8 p63 may be useful in assessing breast lesions as a myoepithelial marker.9–11 There are recent reports6,12–14 suggesting that p63 is also expressed in sarcomatoid/metaplastic carcinoma of the breast and may be a marker for this specific mammary carcinoma subtype. In this study, the authors attempt to evaluate the expression of p63 in malignant lesions with a sarcomatoid/metaplastic phenotype based on morphology and other conventional immunohistochemistry in a series of mammary metaplastic carcinoma, and compare the results with cases of pure adenocarcinoma and sarcoma.
MATERIALS AND METHODS The histopathology files of the five involved institutions were searched for metaplastic carcinoma of the breast. All of the cases were formalin fixed and routinely processed. Cases of sarcoma and conventional adenocarcinoma of the breast were also included as control. The histological slides were retrieved and reviewed and the diagnosis confirmed. Each tumour was assessed for three components, namely: (1) adenocarcinoma component, which morphologically demonstrated glandular or tubule formation or the presence of intracellular or extracellular mucin secretion; (2) squamous component, when the cells showed a characteristic polygonal appearance with a moderate amount of eosinophilic cytoplasm, and identifiable intercellular bridges, with or without keratin pearls formation; and (3) sarcomatoid/solid area, with the cells forming poorly cohesive sheets or showing spindle cell morphology. The mammary metaplastic carcinomas were divided into three groups, namely: (1) epithelial, with the tumour expressing both adenocarcinoma and squamous cell carcinoma, or squamous cell carcinoma alone; (2) biphasic, with the tumour expressing carcinoma component (either adenocarcinoma or squamous cell carcinoma) and sarcomatoid/solid component; and (3) monophasic, with the tumour being formed exclusively by the sarcomatoid/solid component. For the sarcoma cases, the diagnosis was confirmed by typical spindle cell morphology of the tumour cells, and the typical negative immunohistochemical profile of cytokeratins (AE1/3, CAM5.2) and positivity for vimentin. Immunohistochemistry was performed on a representative section on all cases for the following antibodies using the avidin–biotin method with microwave antigen retrieval. The antibodies used were: AE1/3 (1:300; Dako, Denmark), CAM5.2 (1:70; Becton Dickinson, USA), vimentin
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020500444625
P63 AND METAPLASTIC CARCINOMA
(1:2000; Dako, Denmark), and p63 (1:300; Dako, Denmark). For AE1/3, CAM5.2 and vimentin the staining was cytoplasmic; moderate to strong staining of more than 30% of the tumour cells was considered strong, and weak if the expression was less. For p63 the staining pattern was nuclear; the tumour was considered positive if moderate to strong staining was observed in 5% or more of the cells.
RESULTS A total of 58 patients were included in this study, including 34 with metaplastic carcinoma, four with sarcoma and 20 with adenocarcinoma (19 [95%] infiltrating ductal carcinoma and 1 [5%] infiltrating lobular carcinoma). Of the 34 metaplastic carcinomas, epithelial component only (group 1) was found in 10 patients; biphasic tumours (group 2) which included epithelial and sarcomatoid/solid component were found in 14 patients, and monophasic tumours (group 3) which included only sarcomatoid/solid component were found in 10 patients. Tables 1–3 show the immunoprofiles of the tumour cells for patients with metaplastic carcinoma. Among the group 1 (epithelial component only) metaplastic carcinomas, eight cases showed squamous cell carcinoma only, and two cases showed mixed squamous cell carcinoma and adenocarcinoma. In these two cases, the
17
tumours were of intermediate to high nuclear grade with high mitotic activity, and were distinctive from the low grade adenosquamous cell carcinoma described in the areolar region.15 All patients were female, and the age range was 42–86 years (mean 56 years). The tumour sizes ranged from 0.5 to 8.0 cm (mean 4.3 cm). Immunohistochemistry results are listed in Table 1. Eight cases were positive, with the staining being localised to the basal layers of the squamous cell component. The adenocarcinoma component, which was present in two cases, was negative for p63 (Table 1; Fig. 1a,b). Of the 14 patients in group 2, representing biphasic (epithelial and sarcomatoid/solid components) tumours, all the patients were female, and the age range was 43–79 years (mean 58 years). The tumour sizes ranged from 1.8 to 11.5 cm (mean 5.1 cm). The immunohistochemistry results are listed in Table 2. The adenocarcinoma epithelial component was negative for p63, and the squamous carcinoma epithelial component was positive for p63. For the sarcomatoid/solid component of these 14 tumours, six were positive and eight were negative for p63 (Table 2; Fig. 2a–d). For group 3 tumours (monophasic), which consisted of sarcomatoid/solid areas only, there were 10 patients, all were female, with an age range of 43–76 years (mean 54
TABLE 1 Tumour immunoprofiles in patients with metaplastic carcinoma, group 1* Adenocarcinoma Patient
AE1/3
1 6 7 8 9 12 16 19 30 32
Strong Strong
CAM5.2 Strong Weak Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma
Squamous cell carcinoma
Vimentin
p63
AE1/3
CAM5.2
Vimentin
p63
Negative Negative component component component component component component component component
Negative Negative
Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong
Strong Weak Strong Negative Strong Strong Weak Weak Strong Strong
Negative Negative Negative Negative Negative Negative Negative Negative Weak Negative
Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive
absent absent absent absent absent absent absent absent
*Epithelial component only.
A Fig. 1
B (A) Carcinoma with squamous cell differentiation (H&E, 6400). (B) p63 staining of the tumour cell nuclei of the squamous cell carcinoma (6200).
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TSE et al.
TABLE 2 Tumour immunoprofiles in patients with metaplastic carcinoma, group 2* Carcinomatous component Patient 2 10 11 13 15 17 18 20 22 24 25 27 28 29
Squamous Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Squamous Adenocarcinoma Adenocarcinoma Squamous Squamous Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma
Sarcomatous/solid component
AE1/3
CAM5.2
Vimentin
p63
AE1/3
CAM5.2
Vimentin
p63
Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong
Strong Strong Weak Strong Weak Strong Strong Strong Strong Strong Weak Strong Strong Weak
Strong Negative Weak Strong Weak Negative Negative Strong Negative Negative Strong Negative Negative Strong
Positive Negative Negative Negative Negative Positive Negative Negative Positive Positive Negative Negative Negative Negative
Strong Negative Strong Weak Negative Weak Weak Negative Weak Weak Strong Weak Strong Negative
Strong Negative Negative Negative Negative Weak Negative Negative Negative Negative Negative Weak Negative Negative
Strong Strong Strong Strong Strong Weak Strong Strong Strong Strong Strong Strong Strong Strong
Positive Negative Negative Positive Positive Negative Positive Negative Negative Positive Negative Negative Positive Negative
*Epithelial component and sarcomatous/solid component.
A
B
C
D
Fig. 2 (A) Carcinoma with glandular and spindle cell components (H&E). (B) AE1/3 staining, with the glandular cells showing stronger and spindle cells showing weaker staining. (C) Positive p63 staining of the spindle cells but negative for glandular cells. (D) Positive vimentin staining of the spindle cells but negative for glandular cells (a–d: 6400).
P63 AND METAPLASTIC CARCINOMA
years). The tumour size ranged from 1.3 to 10 cm (mean 4 cm). Eight of the 10 patients had axillary dissection, with three showing lymph node metastases. Other clinical details are tabulated in Table 3. Among this group, eight of the 10 tumours showed strong and two cases showed weak positivity for AE1/3. Three cases were strongly positive, two were weakly positive and five were negative for CAM5.2; all were strongly positive for vimentin and eight were positive and two were negative for p63 (Table 3). For the four sarcoma cases, all patients were female, with an age range of 44–70 years (mean 54 years), and a mean tumour size of 3.5 cm. All of the tumours were strongly positive for vimentin only, and were negative for AE1/3, CAM5.2 and p63. For the 20 cases of adenocarcinoma, there were 19 cases of infiltrating ductal carcinoma, and one case of infiltrating lobular carcinoma. All patients were female, with an age range of 43–77 years (mean 53 years). The tumour size ranged from 0.6 to 4.3 cm (mean 2.2 cm). Among this group, all of the tumours were strongly positive for AE1/3 and CAM5.2, two were also strongly positive and one weakly positive for vimentin. Only one case was positive for p63. If one considers all the cases of metaplastic carcinoma of the breast used in the conventional sense, including those with squamous differentiation and those with sarcomatoid/ solid component, using positive p63 expression for diagnosis of metaplastic carcinoma will give a sensitivity of 65%, specificity of 96%, positive predictive value of 96%, negative predictive value of 66% and an accuracy of 78%. If one considers only the assessment of spindle cells in the differentiation of metaplastic carcinoma (groups 2 and 3) from sarcoma, then positive staining of p63 gives a sensitivity of 58%, specificity of 100%, and accuracy of 77%. The small number of sarcomas partially contributes to the lower sensitivity.
DISCUSSION p63 has been investigated for its usefulness in aiding the diagnosis of a multitude of histological entities, utilising its role as a myoepithelial marker in the breast to differentiate invasive from non-invasive malignancies,10,11 and in cytological preparations.9 The staining of p63 has been investigated in epithelial breast lesions and was reported to be essentially negative.16 Other investigators have reported
TABLE 3 Tumour immunoprofiles in patients with metaplastic carcinoma, group 3* Patient
AE1/3
CAM5.2
Vimentin
p63
3 4 5 14 21 23 26 31 33 34
Strong Weak Strong Weak Strong Weak Strong Strong Strong Strong
Strong Negative Strong Negative Weak Negative Negative Weak Strong Negative
Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong
Negative Positive Positive Positive Negative Positive Positive Positive Positive Positive
*Sarcomatous/solid component only.
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some positivity of p63 in high-grade mammary carcinoma, but not in the low-grade tumours.7,17 p63 has also been thought to be useful in the assessment of undifferentiated/poorly differentiated carcinoma. It has been shown previously that p63 is differentially expressed in poorly differentiated squamous cell carcinoma and transitional cell carcinoma in comparison to other adenocarcinomas from various internal organs.8 Metaplastic carcinoma of the breast denotes a heterogeneous group of uncommon malignant entities. Conventionally the term is used to denote tumours with mixed malignant epithelial and malignant mesenchymal components, as well as primary squamous or mixed adenocarcinoma and squamous carcinoma. Those cases with mixed epithelial and mesenchymal components can be classified into monophasic, composed of spindle cells, or biphasic, showing distinct carcinomatous and sarcomatoid components.17 Other authors have systematically grouped the tumours into various diagnostic labels, based on the presence of heterologous element or osteoclastic giant cells, or whether the tumour cells are monophasic, biphasic or squamoid.18–22 In this study, all cases that fulfilled the conventional usage as metaplastic carcinomas were included. Taken as a group, the tumours were mostly of moderate to high nuclear grade, with brisk mitotic activity. This was particularly true for the sarcomatoid/solid component. Many patients also had axillary metastases at the time of surgery, indicating an aggressive behaviour. The three groups of metaplastic carcinomas (epithelial only, biphasic and monophasic) did not show any significant difference in the clinico-pathological parameters, including patient age, tumour size, lymph node status, and hormonal receptor expression. In this study, there were nine cases that showed epithelial component only (group 1); these were technically adenosquamous or pure squamous cell carcinomas, which were negative for mesenchymal markers. p63 staining for the tumour components was positive for the squamous cell carcinoma, but not the adenocarcinoma component. More interesting was the staining pattern of the remaining cases, which could be classified as biphasic (group 2) or monophasic (group 3), with a unifying theme of the presence of spindle cells that expressed both epithelial and mesenchymal phenotypes by immunohistochemistry. Not surprisingly, the staining of these spindle cells showed strong vimentin expression, and variable coexpression of epithelial markers. However, p63 staining was positive in many of these cases, some of which showed only weak and focal epithelial marker(s) expression. Thus, it appears that p63 can be a useful marker in the identification of this group of lesions in the breast, particularly as the pure sarcomas and infiltrating ductal carcinomas were negative for p63. If the broader definition of metaplastic carcinoma is adopted to include carcinoma with squamous cell component, then p63 is able to identify all the tumours with squamous cell component. The underlying mechanism of p63 expression in these spindle cell carcinomas remains uncertain. p63 has been shown to be important in squamous epithelial development, as well as initiation of the stratification program and maintenance of the stratified epithelium.3,4,23 Furthermore, p63 may also play a role in the ectodermal-mesenchymal
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TSE et al.
cross talk.24 Thus, one may postulate that p63 is involved in the early stage of epidermal-mesenchymal differentiation in malignancies, as there is evidence that both the carcinomatous and the sarcomatoid components of metaplastic carcinomas are likely to be derived from the same progenitor, based on loss of heterozygosity pattern,25 and p53 mutation pattern.13 We have demonstrated that p63 is a specific marker for spindle cell carcinoma in mammary metaplastic carcinoma, particularly when staining with conventional markers is equivocal. p63 staining can also serve a second purpose of identifying areas of subtle squamous differentiation, particularly in metaplastic carcinoma of the adenosquamous cell type. p63 staining appears to be specific, as conventional adenocarcinoma and sarcomas are negative. Thus, p63 may be included in the panel of antibodies to be used in the evaluation of malignant lesions with spindle cell morphology, whether it is monophasic or biphasic, being intermixed with other better defined carcinomatous (adenocarcinoma or squamous cell carcinoma) components. Address for correspondence: Dr G. M. Tse, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Ngan Shing Street, Shatin, NT, Hong Kong SAR, China. E-mail: [email protected]
References 1. Yang A, McKeon F. P63 and P73: p53 mimics, menaces and more. Nat Rev Mol Cell Biol 2000; 1: 199–207. 2. Little NA, Jochemsen AG. p63. Int J Biochem Cell Biol 2002; 34: 6–9. 3. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999; 398: 708–13. 4. Yang A, Schweitzer R, Sun D, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999; 398: 714–8. 5. Di Como CJ, Urist MJ, Babayan I, et al. p63 expression profiles in human normal and tumour tissues. Clin Cancer Res 2002; 8: 494–501. 6. Barbareschi M, Pecciarini L, Cangi MG, et al. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol 2001; 25: 1054–60. 7. Ribeiro-Silva A, Zambelli Ramalho LN, Britto Garcia S, Zucoloto S. The relationship between p63 and p53 expression in normal and neoplastic breast tissue. Arch Pathol Lab Med 2003; 127: 336–40.
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8. Kaufmann O, Fietze E, Mengs J, Dietel M. Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol 2001; 116: 823–30. 9. 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; 99: 172–9. 10. Reis-Filho JS, Milanezi F, Amendoeira I, Albergaria A, Schmitt FC. p63 staining of myoepithelial cells in breast fine needle aspirates: a study of its role in differentiating in situ from invasive ductal carcinomas of the breast. J Clin Pathol 2002; 55: 936–9. 11. 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: 20–3. 12. Reis-Filho JS, Schmitt FC. p63 expression in sarcomatoid/metaplastic carcinomas of the breast. Histopathology 2003; 42: 94–5. 13. Wang X, Mori I, Tang W, et al. Metaplastic carcinoma of the breast: p53 analysis identified the same point mutation in the three histological components. Mod Pathol 2001; 14: 1183–6. 14. Koker MM, Kleer CG. p63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma. Am J Surg Pathol 2004; 28: 1506–12. 15. Denley H, Pinder SE, Tan PH, et al. Metaplastic carcinoma of the breast arising within complex sclerosing lesion: a report of five cases. Histopathology 2000; 36: 203–9. 16. Wang X, Mori I, Tang W, et al. p63 expression in normal, hyperplastic and malignant breast tissues. Breast Cancer 2002; 9: 216–9. 17. Elston CW, Ellis IO. The Breast: Systemic Pathology. Vol 13. 3rd ed. Edinburgh: Churchill Livingstone, 1998; 323–31. 18. Wargotz ES, Norris HJ. Metaplastic carcinomas of the breast. I. Matrix-producing carcinoma. Hum Pathol 1989; 20: 628–35. 19. Wargotz ES, Deos PH, Norris HJ. Metaplastic carcinomas of the breast. II. Spindle cell carcinoma. Hum Pathol 1989; 20: 732–40. 20. Wargotz ES, Norris HJ. Metaplastic carcinomas of the breast. III. Carcinosarcoma. Cancer 1989; 64: 1490–9. 21. Wargotz ES, Norris HJ. Metaplastic carcinomas of the breast. IV. Squamous cell carcinoma of ductal origin. Cancer 1990; 65: 272–6. 22. Wargotz ES, Norris HJ. Metaplastic carcinomas of the breast: V. Metaplastic carcinoma with osteoclastic giant cells. Hum Pathol 1990; 21: 1142–50. 23. Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR. p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 2004; 18: 126–31. 24. Fuchs E, Raghavan S. Getting under the skin of epidermal morphogenesis. Nat Rev Genet 2002; 3: 199–209. 25. Kung FY, Tse GM, Lo KW, Law BK, Chang AR, Chen MH. Metachronous bilateral mammary metaplastic and infiltrating duct carcinomas: a molecular study for clonality. Hum Pathol 2002; 33: 677–9.
ANATOMICAL PATHOLOGY
p63 is useful in the diagnosis of mammary metaplastic carcinomas GARY M. TSE*, PUAY-HOON TAN{, BENJAPORN CHAIWUN{, THOMAS C. PUTTI§, PHILIP C. W. LUId, ALEX K. H. TSANG*, FIONA C. L. WONG* AND ANTHONY W. I. LO*
*Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, {Department of Pathology, Singapore General Hospital, Singapore, {Department of Pathology, Chiang Mai University, Thailand, §Department of Pathology, National University Hospital, Singapore, and dDepartment of Pathology, United Christian Hospital, Hong Kong
Summary Aims: p63 has been recently reported to be expressed in sarcomatoid/metaplastic carcinoma of the breast, in addition to its role as a myoepithelial marker. A large series of 34 metaplastic carcinomas, including cases with pure epithelial component (squamous cell and adenosquamous carcinomas), biphasic tumours with carcinomatous and sarcomatoid components and monophasic tumours with only spindle cell component, were evaluated for p63 expression with respect to the different cellular components. Methods: All of the metaplastic carcinomas were assessed for p63 and conventional epithelial and mesenchymal markers of AE1/3, CAM5.2 and vimentin by immunohistochemistry. Results: All of the different categories of metaplastic carcinomas showed similar clinico-pathological features (patient age, tumour size, nuclear grade, mitotic activity, lymph node status and hormonal receptor status). For metaplastic carcinoma with epithelial component only, p63 was only expressed in the squamous cell component, but not the adenocarcinoma component. Eight of the 10 tumours were positive for p63. For the tumours with sarcomatoid component, either singly or together with carcinomatous component, p63 was positive in 14 of 24 cases. Pure sarcomas and carcinomas were all negative for p63 staining by immunohistochemistry, thus rendering p63 staining highly specific for diagnosing metaplastic carcinoma. Conclusions: Using p63 for diagnosis of metaplastic carcinoma gives a sensitivity of 65%, a specificity of 96%, a positive predictive value of 96%, and a negative predictive value of 66% and an accuracy of 78%. p63 may be used as an adjunct marker in the diagnosis of metaplastic carcinoma. Key words: Breast, metaplastic, p63. Received 8 July, revised 13 August, accepted 15 August 2005
INTRODUCTION One of the p53 homologues and related genes, p63, has been mapped to chromosome 3q27–28.1,2 In rodent models, p63 appears to play a crucial role in the regulation of epithelial proliferation and differentiation. Mice that are p63–/– have no hair follicles, teeth, mammary, lacrimal and salivary glands.3,4 In humans, immunohistochemical
analysis reveals p63 expression in the epithelial cells of stratified epithelium, including skin, oesophagus, exocervix, tonsil, bladder and the basal cells in glandular organs, including breast, bronchi and prostate.5 Furthermore, p63 has been shown to be expressed in the myoepithelial cells in the breast6,7 and as a marker of undifferentiation.8 p63 may be useful in assessing breast lesions as a myoepithelial marker.9–11 There are recent reports6,12–14 suggesting that p63 is also expressed in sarcomatoid/metaplastic carcinoma of the breast and may be a marker for this specific mammary carcinoma subtype. In this study, the authors attempt to evaluate the expression of p63 in malignant lesions with a sarcomatoid/metaplastic phenotype based on morphology and other conventional immunohistochemistry in a series of mammary metaplastic carcinoma, and compare the results with cases of pure adenocarcinoma and sarcoma.
MATERIALS AND METHODS The histopathology files of the five involved institutions were searched for metaplastic carcinoma of the breast. All of the cases were formalin fixed and routinely processed. Cases of sarcoma and conventional adenocarcinoma of the breast were also included as control. The histological slides were retrieved and reviewed and the diagnosis confirmed. Each tumour was assessed for three components, namely: (1) adenocarcinoma component, which morphologically demonstrated glandular or tubule formation or the presence of intracellular or extracellular mucin secretion; (2) squamous component, when the cells showed a characteristic polygonal appearance with a moderate amount of eosinophilic cytoplasm, and identifiable intercellular bridges, with or without keratin pearls formation; and (3) sarcomatoid/solid area, with the cells forming poorly cohesive sheets or showing spindle cell morphology. The mammary metaplastic carcinomas were divided into three groups, namely: (1) epithelial, with the tumour expressing both adenocarcinoma and squamous cell carcinoma, or squamous cell carcinoma alone; (2) biphasic, with the tumour expressing carcinoma component (either adenocarcinoma or squamous cell carcinoma) and sarcomatoid/solid component; and (3) monophasic, with the tumour being formed exclusively by the sarcomatoid/solid component. For the sarcoma cases, the diagnosis was confirmed by typical spindle cell morphology of the tumour cells, and the typical negative immunohistochemical profile of cytokeratins (AE1/3, CAM5.2) and positivity for vimentin. Immunohistochemistry was performed on a representative section on all cases for the following antibodies using the avidin–biotin method with microwave antigen retrieval. The antibodies used were: AE1/3 (1:300; Dako, Denmark), CAM5.2 (1:70; Becton Dickinson, USA), vimentin
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020500444625
P63 AND METAPLASTIC CARCINOMA
(1:2000; Dako, Denmark), and p63 (1:300; Dako, Denmark). For AE1/3, CAM5.2 and vimentin the staining was cytoplasmic; moderate to strong staining of more than 30% of the tumour cells was considered strong, and weak if the expression was less. For p63 the staining pattern was nuclear; the tumour was considered positive if moderate to strong staining was observed in 5% or more of the cells.
RESULTS A total of 58 patients were included in this study, including 34 with metaplastic carcinoma, four with sarcoma and 20 with adenocarcinoma (19 [95%] infiltrating ductal carcinoma and 1 [5%] infiltrating lobular carcinoma). Of the 34 metaplastic carcinomas, epithelial component only (group 1) was found in 10 patients; biphasic tumours (group 2) which included epithelial and sarcomatoid/solid component were found in 14 patients, and monophasic tumours (group 3) which included only sarcomatoid/solid component were found in 10 patients. Tables 1–3 show the immunoprofiles of the tumour cells for patients with metaplastic carcinoma. Among the group 1 (epithelial component only) metaplastic carcinomas, eight cases showed squamous cell carcinoma only, and two cases showed mixed squamous cell carcinoma and adenocarcinoma. In these two cases, the
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tumours were of intermediate to high nuclear grade with high mitotic activity, and were distinctive from the low grade adenosquamous cell carcinoma described in the areolar region.15 All patients were female, and the age range was 42–86 years (mean 56 years). The tumour sizes ranged from 0.5 to 8.0 cm (mean 4.3 cm). Immunohistochemistry results are listed in Table 1. Eight cases were positive, with the staining being localised to the basal layers of the squamous cell component. The adenocarcinoma component, which was present in two cases, was negative for p63 (Table 1; Fig. 1a,b). Of the 14 patients in group 2, representing biphasic (epithelial and sarcomatoid/solid components) tumours, all the patients were female, and the age range was 43–79 years (mean 58 years). The tumour sizes ranged from 1.8 to 11.5 cm (mean 5.1 cm). The immunohistochemistry results are listed in Table 2. The adenocarcinoma epithelial component was negative for p63, and the squamous carcinoma epithelial component was positive for p63. For the sarcomatoid/solid component of these 14 tumours, six were positive and eight were negative for p63 (Table 2; Fig. 2a–d). For group 3 tumours (monophasic), which consisted of sarcomatoid/solid areas only, there were 10 patients, all were female, with an age range of 43–76 years (mean 54
TABLE 1 Tumour immunoprofiles in patients with metaplastic carcinoma, group 1* Adenocarcinoma Patient
AE1/3
1 6 7 8 9 12 16 19 30 32
Strong Strong
CAM5.2 Strong Weak Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma
Squamous cell carcinoma
Vimentin
p63
AE1/3
CAM5.2
Vimentin
p63
Negative Negative component component component component component component component component
Negative Negative
Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong
Strong Weak Strong Negative Strong Strong Weak Weak Strong Strong
Negative Negative Negative Negative Negative Negative Negative Negative Weak Negative
Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive
absent absent absent absent absent absent absent absent
*Epithelial component only.
A Fig. 1
B (A) Carcinoma with squamous cell differentiation (H&E, 6400). (B) p63 staining of the tumour cell nuclei of the squamous cell carcinoma (6200).
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TABLE 2 Tumour immunoprofiles in patients with metaplastic carcinoma, group 2* Carcinomatous component Patient 2 10 11 13 15 17 18 20 22 24 25 27 28 29
Squamous Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Squamous Adenocarcinoma Adenocarcinoma Squamous Squamous Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma
Sarcomatous/solid component
AE1/3
CAM5.2
Vimentin
p63
AE1/3
CAM5.2
Vimentin
p63
Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong
Strong Strong Weak Strong Weak Strong Strong Strong Strong Strong Weak Strong Strong Weak
Strong Negative Weak Strong Weak Negative Negative Strong Negative Negative Strong Negative Negative Strong
Positive Negative Negative Negative Negative Positive Negative Negative Positive Positive Negative Negative Negative Negative
Strong Negative Strong Weak Negative Weak Weak Negative Weak Weak Strong Weak Strong Negative
Strong Negative Negative Negative Negative Weak Negative Negative Negative Negative Negative Weak Negative Negative
Strong Strong Strong Strong Strong Weak Strong Strong Strong Strong Strong Strong Strong Strong
Positive Negative Negative Positive Positive Negative Positive Negative Negative Positive Negative Negative Positive Negative
*Epithelial component and sarcomatous/solid component.
A
B
C
D
Fig. 2 (A) Carcinoma with glandular and spindle cell components (H&E). (B) AE1/3 staining, with the glandular cells showing stronger and spindle cells showing weaker staining. (C) Positive p63 staining of the spindle cells but negative for glandular cells. (D) Positive vimentin staining of the spindle cells but negative for glandular cells (a–d: 6400).
P63 AND METAPLASTIC CARCINOMA
years). The tumour size ranged from 1.3 to 10 cm (mean 4 cm). Eight of the 10 patients had axillary dissection, with three showing lymph node metastases. Other clinical details are tabulated in Table 3. Among this group, eight of the 10 tumours showed strong and two cases showed weak positivity for AE1/3. Three cases were strongly positive, two were weakly positive and five were negative for CAM5.2; all were strongly positive for vimentin and eight were positive and two were negative for p63 (Table 3). For the four sarcoma cases, all patients were female, with an age range of 44–70 years (mean 54 years), and a mean tumour size of 3.5 cm. All of the tumours were strongly positive for vimentin only, and were negative for AE1/3, CAM5.2 and p63. For the 20 cases of adenocarcinoma, there were 19 cases of infiltrating ductal carcinoma, and one case of infiltrating lobular carcinoma. All patients were female, with an age range of 43–77 years (mean 53 years). The tumour size ranged from 0.6 to 4.3 cm (mean 2.2 cm). Among this group, all of the tumours were strongly positive for AE1/3 and CAM5.2, two were also strongly positive and one weakly positive for vimentin. Only one case was positive for p63. If one considers all the cases of metaplastic carcinoma of the breast used in the conventional sense, including those with squamous differentiation and those with sarcomatoid/ solid component, using positive p63 expression for diagnosis of metaplastic carcinoma will give a sensitivity of 65%, specificity of 96%, positive predictive value of 96%, negative predictive value of 66% and an accuracy of 78%. If one considers only the assessment of spindle cells in the differentiation of metaplastic carcinoma (groups 2 and 3) from sarcoma, then positive staining of p63 gives a sensitivity of 58%, specificity of 100%, and accuracy of 77%. The small number of sarcomas partially contributes to the lower sensitivity.
DISCUSSION p63 has been investigated for its usefulness in aiding the diagnosis of a multitude of histological entities, utilising its role as a myoepithelial marker in the breast to differentiate invasive from non-invasive malignancies,10,11 and in cytological preparations.9 The staining of p63 has been investigated in epithelial breast lesions and was reported to be essentially negative.16 Other investigators have reported
TABLE 3 Tumour immunoprofiles in patients with metaplastic carcinoma, group 3* Patient
AE1/3
CAM5.2
Vimentin
p63
3 4 5 14 21 23 26 31 33 34
Strong Weak Strong Weak Strong Weak Strong Strong Strong Strong
Strong Negative Strong Negative Weak Negative Negative Weak Strong Negative
Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong
Negative Positive Positive Positive Negative Positive Positive Positive Positive Positive
*Sarcomatous/solid component only.
19
some positivity of p63 in high-grade mammary carcinoma, but not in the low-grade tumours.7,17 p63 has also been thought to be useful in the assessment of undifferentiated/poorly differentiated carcinoma. It has been shown previously that p63 is differentially expressed in poorly differentiated squamous cell carcinoma and transitional cell carcinoma in comparison to other adenocarcinomas from various internal organs.8 Metaplastic carcinoma of the breast denotes a heterogeneous group of uncommon malignant entities. Conventionally the term is used to denote tumours with mixed malignant epithelial and malignant mesenchymal components, as well as primary squamous or mixed adenocarcinoma and squamous carcinoma. Those cases with mixed epithelial and mesenchymal components can be classified into monophasic, composed of spindle cells, or biphasic, showing distinct carcinomatous and sarcomatoid components.17 Other authors have systematically grouped the tumours into various diagnostic labels, based on the presence of heterologous element or osteoclastic giant cells, or whether the tumour cells are monophasic, biphasic or squamoid.18–22 In this study, all cases that fulfilled the conventional usage as metaplastic carcinomas were included. Taken as a group, the tumours were mostly of moderate to high nuclear grade, with brisk mitotic activity. This was particularly true for the sarcomatoid/solid component. Many patients also had axillary metastases at the time of surgery, indicating an aggressive behaviour. The three groups of metaplastic carcinomas (epithelial only, biphasic and monophasic) did not show any significant difference in the clinico-pathological parameters, including patient age, tumour size, lymph node status, and hormonal receptor expression. In this study, there were nine cases that showed epithelial component only (group 1); these were technically adenosquamous or pure squamous cell carcinomas, which were negative for mesenchymal markers. p63 staining for the tumour components was positive for the squamous cell carcinoma, but not the adenocarcinoma component. More interesting was the staining pattern of the remaining cases, which could be classified as biphasic (group 2) or monophasic (group 3), with a unifying theme of the presence of spindle cells that expressed both epithelial and mesenchymal phenotypes by immunohistochemistry. Not surprisingly, the staining of these spindle cells showed strong vimentin expression, and variable coexpression of epithelial markers. However, p63 staining was positive in many of these cases, some of which showed only weak and focal epithelial marker(s) expression. Thus, it appears that p63 can be a useful marker in the identification of this group of lesions in the breast, particularly as the pure sarcomas and infiltrating ductal carcinomas were negative for p63. If the broader definition of metaplastic carcinoma is adopted to include carcinoma with squamous cell component, then p63 is able to identify all the tumours with squamous cell component. The underlying mechanism of p63 expression in these spindle cell carcinomas remains uncertain. p63 has been shown to be important in squamous epithelial development, as well as initiation of the stratification program and maintenance of the stratified epithelium.3,4,23 Furthermore, p63 may also play a role in the ectodermal-mesenchymal
20
TSE et al.
cross talk.24 Thus, one may postulate that p63 is involved in the early stage of epidermal-mesenchymal differentiation in malignancies, as there is evidence that both the carcinomatous and the sarcomatoid components of metaplastic carcinomas are likely to be derived from the same progenitor, based on loss of heterozygosity pattern,25 and p53 mutation pattern.13 We have demonstrated that p63 is a specific marker for spindle cell carcinoma in mammary metaplastic carcinoma, particularly when staining with conventional markers is equivocal. p63 staining can also serve a second purpose of identifying areas of subtle squamous differentiation, particularly in metaplastic carcinoma of the adenosquamous cell type. p63 staining appears to be specific, as conventional adenocarcinoma and sarcomas are negative. Thus, p63 may be included in the panel of antibodies to be used in the evaluation of malignant lesions with spindle cell morphology, whether it is monophasic or biphasic, being intermixed with other better defined carcinomatous (adenocarcinoma or squamous cell carcinoma) components. Address for correspondence: Dr G. M. Tse, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Ngan Shing Street, Shatin, NT, Hong Kong SAR, China. E-mail: [email protected]
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