P63 Expression in P63 Expression in Breast CancerBreast Cancer

A Highly Sensitive and Specific Marker of MA Highly Sensitive and Specific Marker of Metaplastic Carcinomaetaplastic Carcinoma

Meryem M. Koker, MD and Celina G. Kleer, MDMeryem M. Koker, MD and Celina G. Kleer, MD (Am J Surg Pathol 2004(Am J Surg Pathol 2004 ；； 2828：： 1506 – 1512)1506 – 1512)

AbstractAbstract• p63, a member of the p53 gene family, is involved in celp63, a member of the p53 gene family, is involved in cel

lular differentiation and is expressed in the nuclei of mlular differentiation and is expressed in the nuclei of myoepithelial cells of normal breast.yoepithelial cells of normal breast.

• In this study, we determined p63 expression in a large In this study, we determined p63 expression in a large number of breast carcinoma, including metaplastic carnumber of breast carcinoma, including metaplastic carcinoma, and in Phyllodes tumors and sarcoma.cinoma, and in Phyllodes tumors and sarcoma.

• 189 invasive breast carcinomas, including 15 metaplastic 189 invasive breast carcinomas, including 15 metaplastic carcinomas, as well as 10 Phyllodes tumors, and 5 pure scarcinomas, as well as 10 Phyllodes tumors, and 5 pure sarcoma of the breast for pattern and intensity of p63 stainarcoma of the breast for pattern and intensity of p63 staining using an anti-p63 antibody (clone 4A4, Neomarkers)ing using an anti-p63 antibody (clone 4A4, Neomarkers)

• p63 is strongly expressed in 13 of 15 metaplastic carcinop63 is strongly expressed in 13 of 15 metaplastic carcinoma (86.7%).ma (86.7%).

• p63 is positive in all the metaplastic carcinoma with spindp63 is positive in all the metaplastic carcinoma with spindle cell and/or squamous differentiation (12 od 12). le cell and/or squamous differentiation (12 od 12).

• Only 1 of 174 (0.6%) nonmetaplastic invasive carcinomOnly 1 of 174 (0.6%) nonmetaplastic invasive carcinomas was positive for p63.as was positive for p63.

• All Phyllodes tumors and sarcomas were consistently All Phyllodes tumors and sarcomas were consistently negative for p63 expression.negative for p63 expression.

• The sensitivity and specificity of p63 as a diagnostic mThe sensitivity and specificity of p63 as a diagnostic marker for metaplastic carcinoma was 86.7% and 99.4%arker for metaplastic carcinoma was 86.7% and 99.4%

• p63 as part of the diagnostic workup of challenging spip63 as part of the diagnostic workup of challenging spindle cell tumors of the breast as a highly specific markndle cell tumors of the breast as a highly specific marker for metaplastic carcinomas. er for metaplastic carcinomas.

前言：前言：• Metaplastic carcinoma of the breast (MCB) are a rare anMetaplastic carcinoma of the breast (MCB) are a rare an

d heterogeneous group of tumors defined by a cellular d heterogeneous group of tumors defined by a cellular component with an appearance other then epithelial ancomponent with an appearance other then epithelial and glandular.d glandular.

• These tumors may be biphasic and contain glandular elThese tumors may be biphasic and contain glandular elements mixed with a nonglandular component.ements mixed with a nonglandular component.

• The majority of metaplastic carcinoma exhibit spindle cThe majority of metaplastic carcinoma exhibit spindle cell with or without squamous differentiation.ell with or without squamous differentiation.

• The spindle cell component of metaplsic carcinoma froThe spindle cell component of metaplsic carcinoma from low-grade “fibromatosis-like” lesions to high grade sm low-grade “fibromatosis-like” lesions to high grade spindle cell malignancies resembling fibrosarcoma or mpindle cell malignancies resembling fibrosarcoma or malignant fibrous histiocytoma.alignant fibrous histiocytoma.

• The diagnosis of metaplastic carcinoma is straightforwThe diagnosis of metaplastic carcinoma is straightforward when a direct transition from glandular epithelial to ard when a direct transition from glandular epithelial to metaplastic elements is evident, but it becomes a diagnmetaplastic elements is evident, but it becomes a diagnostic challenge when the neoplasm is entirely composeostic challenge when the neoplasm is entirely composed of spindle cells.d of spindle cells.

• p63 is located on chromosome 3q27. Its gene product is crp63 is located on chromosome 3q27. Its gene product is crucial for maintenance of a stem cell population in several eucial for maintenance of a stem cell population in several epithelial tissue and is necessary for the normal developmepithelial tissue and is necessary for the normal development of the epithelial organs including mammary glands.nt of the epithelial organs including mammary glands.

• p63 is also expressed in the nuclei of myoepithelial cells of p63 is also expressed in the nuclei of myoepithelial cells of normal breast ducts and lobules.normal breast ducts and lobules.

• Recently, p63 and other myoepithelial cell markers have beRecently, p63 and other myoepithelial cell markers have been described in matrix-producing and metaplastic carcinoen described in matrix-producing and metaplastic carcinomas of the breast, suggesting that these tumors share a mmas of the breast, suggesting that these tumors share a myoepithelial cell differentiation.yoepithelial cell differentiation.

• Given the specificity of p63 for myoepithelial cells in the Given the specificity of p63 for myoepithelial cells in the breast and the possible myoepithelial differentiation of sbreast and the possible myoepithelial differentiation of spindle cell metaplastic carcinoma, we postulated that p6pindle cell metaplastic carcinoma, we postulated that p63 would be a good marker to distinguish spindle cell me3 would be a good marker to distinguish spindle cell metaplastic carcinoma from other mesenchymal neoplasm.taplastic carcinoma from other mesenchymal neoplasm.

• Characterize the expression of p63 in a large group of inCharacterize the expression of p63 in a large group of invasive carcinomas of different histologic types, sarcomavasive carcinomas of different histologic types, sarcomas, and Phyllodes tumors of the breast define its diagnosts, and Phyllodes tumors of the breast define its diagnostic utility.ic utility.

Materials and MethodsCase Selection, Materials and MethodsCase Selection, Pathologic Evaluation and ImmunohistochemistryPathologic Evaluation and Immunohistochemistry

• A total of 201 breast tumors were identified by a retrospeA total of 201 breast tumors were identified by a retrospective search through the surgical pathology files at the Uctive search through the surgical pathology files at the University of Michigan, comprising 189 invasive carcinomaniversity of Michigan, comprising 189 invasive carcinomas, including 15 metaplastic carcinomas. Ten Phyllodes tus, including 15 metaplastic carcinomas. Ten Phyllodes tumors and 5 primary breast sarcomas were also retrieved.mors and 5 primary breast sarcomas were also retrieved.

• MCB , the architectural pattern and the presence of epitMCB , the architectural pattern and the presence of epithelial and or heterologous elements were noted.helial and or heterologous elements were noted.

• The MCB were graded on the basis of the sarcomatoid The MCB were graded on the basis of the sarcomatoid component as low-, intermediate-, or high-grade followicomponent as low-, intermediate-, or high-grade following described criteria.ng described criteria.

• In all cases, the diagnosis of MCB was confirmed by poIn all cases, the diagnosis of MCB was confirmed by positive cytokeratin staining including a cytokeratin cocktsitive cytokeratin staining including a cytokeratin cocktail (AE1/AE3, CAM5.2) and/or a high molecular weight cail (AE1/AE3, CAM5.2) and/or a high molecular weight cytokeratin stain (34ßE12)ytokeratin stain (34ßE12)

• A subset of the nonmetaplastic invasive carcioma (160 A subset of the nonmetaplastic invasive carcioma (160 cases) was arrayed in a high-density tissue microarray.cases) was arrayed in a high-density tissue microarray.

• At least three tissue core (0.6 mm diameter) were samplAt least three tissue core (0.6 mm diameter) were sampled from different areas of each tumor to account for tued from different areas of each tumor to account for tumor heterogeneity using an automated arrayer (Beechemor heterogeneity using an automated arrayer (Beecher Instruments).r Instruments).

• Immunohistochemistry using an anti-p63 polyclonal anImmunohistochemistry using an anti-p63 polyclonal antibody was performed on the whole tissue sections and tibody was performed on the whole tissue sections and on the tissue microarray simultaneously using standaron the tissue microarray simultaneously using standard biotin-avidin complex technique.d biotin-avidin complex technique.

• The p63 antibody (clone 4A4, NeoMarkers, Fremont, CThe p63 antibody (clone 4A4, NeoMarkers, Fremont, CA) was used at a 1:100 dilution, incubated for 15 mins A) was used at a 1:100 dilution, incubated for 15 mins with citrate buffer at pH 6, and subjected to microwave with citrate buffer at pH 6, and subjected to microwave antigen-retrieval pretreatment.antigen-retrieval pretreatment.

• Normal myoepithelial cells around ducts and lobules seNormal myoepithelial cells around ducts and lobules served as positive internal controls.rved as positive internal controls.

• A tumor was considered positive when A tumor was considered positive when 10% of the ne10% of the neoplastic cells unequivocally expressed p63 in the nucleoplastic cells unequivocally expressed p63 in the nuclei and negative when less than 10% of the malignant celli and negative when less than 10% of the malignant cells stained for p63.s stained for p63.

ResultsResults• Histopathologic featuresHistopathologic features

• Of the 189 invasive carcinomas, 159 were ductal, 10 were lOf the 189 invasive carcinomas, 159 were ductal, 10 were lobular, 5 had ductal and lobular features, and 15 were metaobular, 5 had ductal and lobular features, and 15 were metaplastic.plastic.

• Of the metaplastic carcinomas, 12 tumors had spindle and/Of the metaplastic carcinomas, 12 tumors had spindle and/or squamous areas and 3 had cartilage (Fig 1)or squamous areas and 3 had cartilage (Fig 1)

• Table 1Table 1

• Fig 1 Histologic feature of metaplastic carcinoma. Fig 1 Histologic feature of metaplastic carcinoma. A, Biphasic metaplastic carcinoma with well-demarcated spindle and A, Biphasic metaplastic carcinoma with well-demarcated spindle and

epithelial components. epithelial components. B, Metaplastic carcinoma with spindle and epitheloid cellsB, Metaplastic carcinoma with spindle and epitheloid cells

• Fig 1 C, biphasic metaplastic carcinoma with a squamous area that Fig 1 C, biphasic metaplastic carcinoma with a squamous area that blends imperceptibly with the surrounding spindle cell elements. blends imperceptibly with the surrounding spindle cell elements. D, metaplastic carcinoma with spindle cells and dense collagenized D, metaplastic carcinoma with spindle cells and dense collagenized stroma, referred to as “keloidal type”stroma, referred to as “keloidal type”

• The low- and intermediate- grade spindle cell MCB werThe low- and intermediate- grade spindle cell MCB were characterized by elongated, fusiform cells with minime characterized by elongated, fusiform cells with minimal to moderate cytologic atypia and rare or no mitoses. al to moderate cytologic atypia and rare or no mitoses. In contrast, the high-grade spindle cell carcinomas disIn contrast, the high-grade spindle cell carcinomas displayed marked pleomorphism, hyperchromasia, and nuplayed marked pleomorphism, hyperchromasia, and numerous atypical mitoses.merous atypical mitoses.

• The neoplastic cells in both low- and high- grade spindlThe neoplastic cells in both low- and high- grade spindle cell carcinomas infiltrated adjacent mammary and adie cell carcinomas infiltrated adjacent mammary and adipose tissue and were interrupted by dense collagen bapose tissue and were interrupted by dense collagen bands.nds.

• p63 Expression in Invasive Carcinomas of the Breastp63 Expression in Invasive Carcinomas of the Breast

• p63 was expressed in the nuclei of myoepithelial cells of nop63 was expressed in the nuclei of myoepithelial cells of normal ducts and lobules adjacent to the carcinomas, which alrmal ducts and lobules adjacent to the carcinomas, which also served as internal positive controls in all cases (Fig 2a)so served as internal positive controls in all cases (Fig 2a)

• p63 was not detectably expressed in the luminal cells of norp63 was not detectably expressed in the luminal cells of normal structures in any cases. Stromal fibroblasts and myofibrmal structures in any cases. Stromal fibroblasts and myofibroblasts were consistently negative in all case.oblasts were consistently negative in all case.

• Tab 2, Fig 2Tab 2, Fig 2

• Fig 2 A. p63 expression in normal breast and in metaplastic carcinomFig 2 A. p63 expression in normal breast and in metaplastic carcinomas. A, normal terminal-duct lobular unit with p63 expressing myoepithas. A, normal terminal-duct lobular unit with p63 expressing myoepithelial cells, which served as positive internal control.elial cells, which served as positive internal control.

• Fig 2 B-F, Strong and diffuse expression of p63 in metaplastic carcinomFig 2 B-F, Strong and diffuse expression of p63 in metaplastic carcinomas with spindle and squamous areas. Note that the staining is crisp and as with spindle and squamous areas. Note that the staining is crisp and easily discernible even at low power.easily discernible even at low power.

• The single nonmetaplastic carcinoma that was positive The single nonmetaplastic carcinoma that was positive for p63 was a high-grade invasive ductal carcinoma witfor p63 was a high-grade invasive ductal carcinoma with no special features; however, the percentage of positih no special features; however, the percentage of positive malignant cells was much lower than in the metaplave malignant cells was much lower than in the metaplastic carcinoma (10% vs mean of 70%)stic carcinoma (10% vs mean of 70%)

• p63 was negative in all the other invasive ductal, lobulap63 was negative in all the other invasive ductal, lobular and mixed ductal and lobular carcinoma.r and mixed ductal and lobular carcinoma.

• p63 Expression in Mesenchymal tumors of the Breast p63 Expression in Mesenchymal tumors of the Breast

• p63 was consistently negative in the spindle and epithelip63 was consistently negative in the spindle and epithelial cell components of all benign and malignant Phyllodeal cell components of all benign and malignant Phyllodes tumorss tumors

• Fig 3Fig 3

• The sensitivity and specificity of p63 as a marker for metThe sensitivity and specificity of p63 as a marker for metaplastic carcinoma is 86.7% and 99.4%, respectively, witaplastic carcinoma is 86.7% and 99.4%, respectively, with a 100% specificity for MCB with spindle cell and/or squh a 100% specificity for MCB with spindle cell and/or squamous areas.amous areas.

• Fig 3 A and B P63 is not expressed in Phyllodes tumors and sarcomFig 3 A and B P63 is not expressed in Phyllodes tumors and sarcomas of the breast. A and B. Phyllodes tumoras of the breast. A and B. Phyllodes tumor

• Fig 3.C and D Note that p63 is only expressed in the normal myoepithFig 3.C and D Note that p63 is only expressed in the normal myoepithelial cells associated with the epithelial component. The spindle cells elial cells associated with the epithelial component. The spindle cells are negative. C and D, Primary osteosarcoma of the breast, negative fare negative. C and D, Primary osteosarcoma of the breast, negative for p63or p63

DiscussionDiscussion• Traditional, MCB has been defined as a malignant tumor in Traditional, MCB has been defined as a malignant tumor in

which part of all of the carcinomatous epithelium is transfowhich part of all of the carcinomatous epithelium is transformed into a nonglandular (metaplastic) growth process.rmed into a nonglandular (metaplastic) growth process.

• The most common metaplastic components are spindle celThe most common metaplastic components are spindle cell and squamous, wereas heterologous elements including l and squamous, wereas heterologous elements including osseous, chondroid, and other sarcomas comprise a minorosseous, chondroid, and other sarcomas comprise a minority of tumors.ity of tumors.

• MCB with a prominent spindle cell component display histMCB with a prominent spindle cell component display histologic patterns ranging from those resembling highly pleoologic patterns ranging from those resembling highly pleomorphic and anaplastic sarcomas to bland benign-appearimorphic and anaplastic sarcomas to bland benign-appearing lesions.ng lesions.

• To data, the best mechanism other than morphologic exaTo data, the best mechanism other than morphologic examination for differentiating MCB from these other spindle mination for differentiating MCB from these other spindle cell tumors has been cytokeratin staining. cell tumors has been cytokeratin staining.

• Several studies hae shown that staining with a broad-speSeveral studies hae shown that staining with a broad-spectrum anti-CK antibody or high molecular weight CK (34ßctrum anti-CK antibody or high molecular weight CK (34ßE12) provides the most sensitive marker for MCB. E12) provides the most sensitive marker for MCB.

• Other markers, such as smooth muscle actin, CK5, CK14,Other markers, such as smooth muscle actin, CK5, CK14, CAM5.2, and AE1/AE3, have also been examined for thei CAM5.2, and AE1/AE3, have also been examined for their utility in the diagnosis of MCB, but more than half of thr utility in the diagnosis of MCB, but more than half of the cases were negative for any one of these antibodies.e cases were negative for any one of these antibodies.

• Importantly, CK-positive metaplastic carcinomas, even Importantly, CK-positive metaplastic carcinomas, even those stained with 34ßE12 or broad-spectrum CKs, oftethose stained with 34ßE12 or broad-spectrum CKs, often only show focal or patchy positive, particular in small n only show focal or patchy positive, particular in small core biopsies, leading to potential misdiagnoses.core biopsies, leading to potential misdiagnoses.

• Apart from the CK stains, CD34, smooth muscle actin, Apart from the CK stains, CD34, smooth muscle actin, vimentin, and occasionally Bcl-2 have been proposed avimentin, and occasionally Bcl-2 have been proposed as helpful adjuncts when the differential diagnosis inclus helpful adjuncts when the differential diagnosis includes MCB, sarcoma of the breast, and Phyllodes tumor.des MCB, sarcoma of the breast, and Phyllodes tumor.

• However, borderline and malignant Phyllodes tumors, tHowever, borderline and malignant Phyllodes tumors, the types most likely to cause diagnostic confusion with he types most likely to cause diagnostic confusion with metaplastic carcinomas, are much less likely to be posimetaplastic carcinomas, are much less likely to be positive for any of these markers.tive for any of these markers.

• All the MCB with spindle cell and squamous componenAll the MCB with spindle cell and squamous components stained strongly for p63 in the nuclei, which confirmts stained strongly for p63 in the nuclei, which confirms previous reports.s previous reports.

• Surprisingly, we found that only 1 of the 174 nonmetaplSurprisingly, we found that only 1 of the 174 nonmetaplastic invasive carcinoma (0.6%) was positive for p63 exastic invasive carcinoma (0.6%) was positive for p63 expression, and the percentage of positive cells in this capression, and the percentage of positive cells in this carcinoma was much smaller then in the MCB (10% vs. a rcinoma was much smaller then in the MCB (10% vs. a mean of 70% for MCB)mean of 70% for MCB)

• This tumor was an invasive ductal carcinoma, with high This tumor was an invasive ductal carcinoma, with high histologic grade and no special features.histologic grade and no special features.

• While all the of metaplastic spindle cell carcinomas staiWhile all the of metaplastic spindle cell carcinomas stained strongly with p63, none of the Phyllodes tumors or ned strongly with p63, none of the Phyllodes tumors or primary breast sarcomas showed any p63 expressed.primary breast sarcomas showed any p63 expressed.

• Emerging data suggest that the metaplastic component Emerging data suggest that the metaplastic component and the adenocarcinoma have a clonal origin.and the adenocarcinoma have a clonal origin.

• It has been suggested that these tumors arise from a siIt has been suggested that these tumors arise from a single stem cell, a group of different cells, or a common ngle stem cell, a group of different cells, or a common progenitor cell capable of differentiating into other cell progenitor cell capable of differentiating into other cell types.types.

• Using laser capture microdissection of a tumor with carUsing laser capture microdissection of a tumor with carcinomatous and sarcomatoid component, Wada et al focinomatous and sarcomatoid component, Wada et al found that both areas had identical patterns of X-chromound that both areas had identical patterns of X-chromosome inactivation and the same of p53 mutation, stronsome inactivation and the same of p53 mutation, strongly supporting the hypothesis that this tumor is derivegly supporting the hypothesis that this tumor is derived from a single totipotent stem cell.d from a single totipotent stem cell.

• Furthermore, the concept that the metaplastic spindle eFurthermore, the concept that the metaplastic spindle elements have myoepithelial differentiation is supported lements have myoepithelial differentiation is supported by the present study and by several studies showing thby the present study and by several studies showing that these cells are reactive for myoepithelial markers, inat these cells are reactive for myoepithelial markers, including high molecular weight CK, p63, and maspin.cluding high molecular weight CK, p63, and maspin.

• In summary, p63 is a specific and sensitive marker for In summary, p63 is a specific and sensitive marker for MCB, and particularly specific for metaplastic carcinomMCB, and particularly specific for metaplastic carcinomas with spindle and/or squamous areas as with spindle and/or squamous areas

• It is a highly complementary stain to CK because p63 is It is a highly complementary stain to CK because p63 is a nuclear marker, which has a strong and diffuse a nuclear marker, which has a strong and diffuse staining pattern that is easily discernible, even at low staining pattern that is easily discernible, even at low power. power.

• We propose the inclusion of p63 in the diagnostic We propose the inclusion of p63 in the diagnostic workup of challenging spindle cell tumors of the workup of challenging spindle cell tumors of the breast.breast.