International Journal of Gynecological Pathology 34:74–84, Lippincott Williams & Wilkins, Baltimore r 2014 International Society of Gynecological Pathologists
Original Article
Should Endometrial Clear Cell Carcinoma be Classified as Type II Endometrial Carcinoma? Hyo Sook Bae, M.D., Hyesun Kim, Ph.D., Sun Young Kwon, Ph.D., Kyu-Rae Kim, Ph.D., Jae Yun Song, M.D., Ph.D., and Insun Kim, M.D., Ph.D.
Summary: Endometrial clear cell carcinomas (ECCCs) are considered to be Type II endometrial carcinomas, like uterine serous adenocarcinoma (SCA), and therefore aggressive clinical management is indicated. However, according to the limited clinical, immunohistochemical, and molecular data available in the literature, ECCCs show overlapping features of SCA and endometrioid adenocarcinomas. Therefore, questions regarding their designation as the Type II carcinomas have been raised. We performed immunohistochemical staining for hepatocyte nuclear factor-1b and napsin A for the histologic confirmation of clear cell carcinoma (CCC), and analyzed immunohistochemical findings for estrogen receptor, progesterone receptor, HER2/neu, p53, p16, ARID1A, PTEN, DNA mismatch-repair proteins along with other prognostic factors. We performed DNA sequencing for the K-RAS, BRAF, PIK3CA, and PTEN genes for 16 pure CCCs. No patients with pure CCC experienced recurrent disease or died of the disease (0/16, 0%). ECCCs had SCA-like features with rare expression of estrogen receptor/progesterone receptor (18.8%/6.3%) and no K-RAS mutations, intermediate features regarding expressions of p53 (37.5%) and p16 (25%), and endometrioid adenocarcinoma-like features regarding losses of PTEN (81.3%), ARID1A (25%) and mismatch-repair protein (68.8%), expression of microsatellite instability-high (25%), HER2/neu (12.5%), and PIK3CA mutations (18.8%). Pure ECCC should not be regarded as Type II carcinoma, because it shares the immunohistochemical and molecular characteristics of Type I endometrioid adenocarcinoma and Type II SCA. Key Words: Endometrial carcinoma—Clear cell adenocarcinoma—Endometrioid adenocarcinoma—Immunohistochemistry—DNA sequencing.
Endometrial cancer is a major cause of morbidity and mortality for women worldwide, with nearly 200,000 cases diagnosed every year (1). The incidence of endometrial cancer differs between regions, but it is the most common cancer of the female genital tract in North America and Europe (2). Although the incidence of endometrial cancer is lower in developing countries than in developed countries, the mortality-to-incidence ratio is higher in developing countries, 21,000 to 62,000 (0.34) compared with 29,000 to 136,000 (0.21) in developed countries (1). Endometrial cancers are classified into 2 broad subtypes based on clinicopathologic and molecular characteristics (3). Type I endometrioid cancers are
From the Departments of Obstetrics and Gynecology (H.S.B., J.Y.S.), Korea University Anam Hospital; Pathology (I.K.), Korea University College of Medicine; Department of Pathology (H.K.), Cheil General Hospital, Kwandong University College of Medicine; Department of Pathology (S.Y.K.), Keimyung University Hospital; and Department of Pathology (K-R.K.), Asan Medical Center, Seoul, Korea. The authors declare no conflict of interest. Address correspondence and reprint requests to Jae Yun Song, MD, PhD, Department of Obstetrics and Gynecology, Korea University Anam Hospital, Korea University College of Medicine, 126-1 5ga, Anam-dong, Sungbuk-gu, Seoul 136-705, Korea. E-mail: [email protected] and Insun Kim, PhD, Department of Pathology, Korea University Anam Hospital, Korea University College of Medicine, 126-1 5ga, Anam-dong, Sungbuk-gu, Seoul, 136-705, Korea. E-mail: [email protected].
DOI: 10.1097/PGP.0000000000000111
74
ENDOMETRIAL CLEAR CELL CARCINOMA related to unopposed estrogen stimulation, arise in younger women, and have relatively favorable prognoses than compared with Type II cancers which include serous and clear cell carcinoma (CCC). Type II cancers are not overtly associated with hormonal risk factors, arise in older women, and have aggressive clinical courses. Endometrioid cancers are frequently characterized by abnormalities in the PTEN tumor suppressor gene, PIK3CA and ARIDIA, microsatellite instability (MSI) arising from defects in DNA mismatch repair (MMR), and mutations in the b-catenin and K-RAS genes (3–5). In contrast, serous Type II cancers frequently demonstrate alterations in p53, HER2/neu, p16, PPP2R1A, and E-cadherin (5,6). Endometrial CCCs that account for about 5% of endometrial carcinomas and occur in older patients with atrophic endometrium, but not associated with hormonal replacement therapy, are considered to be Type II endometrial cancers, like serous carcinoma. The limited data available regarding clinicopathologic features, immunohistochemistry, and molecular findings of endometrial CCC overlap with those of serous and endometrioid carcinomas (7–10). We conducted the current study to evaluate the clinicopathologic, immunohistochemical, and molecular features of endometrial CCC. We propose a pathogenetic pathway that is unique to this tumor and distinguishes it from Type II serous carcinoma. MATERIALS AND METHODS Patients and Endometrial Clear Cell Carcinoma (ECCC) Tissue Samples Data for patients with endometrial CCC who were diagnosed between 2002 and 2012 were retrieved from the pathology files of Korea University Anam Hospital, Kwandong Cheil Hospital, Keimyung Dongsan Hospital, and Asan Medical Center. Diagnoses were made using hysterectomy specimens in all except one case, in which endometrial curettage was performed. Tumor specimens from 16 patients with endometrial CCC were obtained with informed consent. Histologic findings, including tumor size, invasion depth, involvement of the uterine cervix, lymph node metastasis, and lymphovascular invasion were obtained from pathologic reports, and clinical findings such as clinical stage, recurrence, and follow-up were retrieved from clinical records. The histologic diagnoses were made by gynecologic pathologists at each hospital, and were reviewed by one of the authors (I.K.) in a blind manner.
75
The histologic features of CCC included solid, papillary, tubulocystic pattern of large polygonal cell, cuboidal cell or hobnail cell with clear or eosinophilic cytoplasm, or scanty to moderate cytoplasm. The cores of papillary structures were hyalinized, and the tubulocystic structures contained eosinophilic, secretory material. The nuclei of the large polygonal tumor cells were vesicular and had prominent eosinophilic nucleoli, whereas cuboidal and hobnail cells had hyperchromatic nuclei. All tumors with areas of pure CCC were included, whereas cases with mixed CCC or equivocal histology were excluded. Construction of Tissue Microarray and Immunohistochemical Staining Two representative areas were marked on each hematoxylin and eosin–stained slide. The corresponding formalin-fixed, paraffin-embedded tissue blocks were retrieved and the selected areas were marked. Two cores 2 mm in diameter were punched out from each sample and incorporated into a recipient paraffin block using a precision instrument (UNITMA. Co. Ltd, Seoul, Korea). The tissue microarray block was left in 601C oven for 30 min, embedded in a paraffin block, and cut into sections 4 mm thick. Each section was deparaffinized in xylene and rehydrated through a series of graded ethanol. Antigen retrieval was performed by microwaving each section in Epitope Retrieval Solution (0.01 M citrate buffer, pH 6.0 or 10 mM EDTA, pH 8.0) for 20 min, and endogenous peroxidase was blocked by immersing the section in 0.3% hydrogen peroxide for 10 min. Immunostaining was performed using Dako Autostainer plus Universal Staining System (Dako Cytomation, Carpinteria, CA) with a ChemMate DAKO EnVision Detection kit. The characteristics of primary antibodies against estrogen receptor (ER), progesterone receptor (PR), p53, p16, HER2/neu, MLH1, MSH2, MSH6, PMS2, PTEN, ARIDIA, hepatocyte nuclear factor-1b (HNF-1b) and napsin are summarized in Table 1. Interpretation of Immunostaining The expressions of ER and PR were evaluated using Allred scoring system (11). The proportion of positive stained cells was rated as follows: 0, no cells stained positive; 1, between 0% and 1% positive; 2, between 1% and 10% positive; 3, between 10% and 33% positive; 4, between 33% and 66% positive; and 5, between 66% and 100% positive. In addition to the proportion score, an intensity score was made as follows: 0, negative; 1, weak; 2, intermediate; and 3, Int J Gynecol Pathol Vol. 34, No. 1, January 2015
76
H. S. BAE ET AL. TABLE 1. Characteristics of primary antibodies
Antibodies
Type
Source
Dilution
ER PR P53 P16 HER2 MLH1 MSH2 MSH6 PMS2 PTEN ARIDIA HNF-1b Napsin A
Mouse monoclonal Mouse monoclonal Monoclonal mouse Mouse monoclonal Monoclonal rabbit Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal Rabbit polyclonal Rabbit polyclonal Mouse monoclonal
Dako Dako Novocastra, UK Santa Cruz, USA Thermo Scientific, USA Dako DBS, USA DBA, USA Cell Marque, USA Dako Sigma, USA Proteintech, USA Leica, UK
1:50 1:50 1:50 1:100 1:100 1:50 1:50 1:50 1:50 1:100 1:200 1:50 1:400
ER indicates estrogen receptor; PR, progesterone receptor.
strong. The intensity score and the proportion score were added to obtain the total score. Scores >2 were considered to be positive. The expression of p53 was scored as the percentage of cells with positive nuclear staining. HNF-1b is expressed in the nucleus, whereas napsin is expressed in the cytoplasm and/or membrane. The staining was interpreted as positive when at least 5% of the tumor cells expressed the stained antigen. MMR proteins (MLH1, MSH2, MSH6, and PMS2), PTEN and ARID1A, were interpreted as abnormal when there was complete loss of nuclear staining in tumor cells compared with normal lymphocytes and stromal cells. HER2/neu expression was evaluated according to the recommendations of the American Society of Clinical Oncology/College of American Pathologists for breast cancer (12,13). No staining or membranous staining in 10% of the tumor cells was scored as 1+, weak or moderate staining of the entire membrane in >10% of the tumor cells was scored as 2+, and strong staining of the entire membrane in >30% of the tumor cells resulted in a score of 3+. The expressions of p16 were interpreted as >90%, 10% to 90%, and 2.2 or HER2 gene copies per number >6.0, whereas a FISH ratio of
Original Article
Should Endometrial Clear Cell Carcinoma be Classified as Type II Endometrial Carcinoma? Hyo Sook Bae, M.D., Hyesun Kim, Ph.D., Sun Young Kwon, Ph.D., Kyu-Rae Kim, Ph.D., Jae Yun Song, M.D., Ph.D., and Insun Kim, M.D., Ph.D.
Summary: Endometrial clear cell carcinomas (ECCCs) are considered to be Type II endometrial carcinomas, like uterine serous adenocarcinoma (SCA), and therefore aggressive clinical management is indicated. However, according to the limited clinical, immunohistochemical, and molecular data available in the literature, ECCCs show overlapping features of SCA and endometrioid adenocarcinomas. Therefore, questions regarding their designation as the Type II carcinomas have been raised. We performed immunohistochemical staining for hepatocyte nuclear factor-1b and napsin A for the histologic confirmation of clear cell carcinoma (CCC), and analyzed immunohistochemical findings for estrogen receptor, progesterone receptor, HER2/neu, p53, p16, ARID1A, PTEN, DNA mismatch-repair proteins along with other prognostic factors. We performed DNA sequencing for the K-RAS, BRAF, PIK3CA, and PTEN genes for 16 pure CCCs. No patients with pure CCC experienced recurrent disease or died of the disease (0/16, 0%). ECCCs had SCA-like features with rare expression of estrogen receptor/progesterone receptor (18.8%/6.3%) and no K-RAS mutations, intermediate features regarding expressions of p53 (37.5%) and p16 (25%), and endometrioid adenocarcinoma-like features regarding losses of PTEN (81.3%), ARID1A (25%) and mismatch-repair protein (68.8%), expression of microsatellite instability-high (25%), HER2/neu (12.5%), and PIK3CA mutations (18.8%). Pure ECCC should not be regarded as Type II carcinoma, because it shares the immunohistochemical and molecular characteristics of Type I endometrioid adenocarcinoma and Type II SCA. Key Words: Endometrial carcinoma—Clear cell adenocarcinoma—Endometrioid adenocarcinoma—Immunohistochemistry—DNA sequencing.
Endometrial cancer is a major cause of morbidity and mortality for women worldwide, with nearly 200,000 cases diagnosed every year (1). The incidence of endometrial cancer differs between regions, but it is the most common cancer of the female genital tract in North America and Europe (2). Although the incidence of endometrial cancer is lower in developing countries than in developed countries, the mortality-to-incidence ratio is higher in developing countries, 21,000 to 62,000 (0.34) compared with 29,000 to 136,000 (0.21) in developed countries (1). Endometrial cancers are classified into 2 broad subtypes based on clinicopathologic and molecular characteristics (3). Type I endometrioid cancers are
From the Departments of Obstetrics and Gynecology (H.S.B., J.Y.S.), Korea University Anam Hospital; Pathology (I.K.), Korea University College of Medicine; Department of Pathology (H.K.), Cheil General Hospital, Kwandong University College of Medicine; Department of Pathology (S.Y.K.), Keimyung University Hospital; and Department of Pathology (K-R.K.), Asan Medical Center, Seoul, Korea. The authors declare no conflict of interest. Address correspondence and reprint requests to Jae Yun Song, MD, PhD, Department of Obstetrics and Gynecology, Korea University Anam Hospital, Korea University College of Medicine, 126-1 5ga, Anam-dong, Sungbuk-gu, Seoul 136-705, Korea. E-mail: [email protected] and Insun Kim, PhD, Department of Pathology, Korea University Anam Hospital, Korea University College of Medicine, 126-1 5ga, Anam-dong, Sungbuk-gu, Seoul, 136-705, Korea. E-mail: [email protected].
DOI: 10.1097/PGP.0000000000000111
74
ENDOMETRIAL CLEAR CELL CARCINOMA related to unopposed estrogen stimulation, arise in younger women, and have relatively favorable prognoses than compared with Type II cancers which include serous and clear cell carcinoma (CCC). Type II cancers are not overtly associated with hormonal risk factors, arise in older women, and have aggressive clinical courses. Endometrioid cancers are frequently characterized by abnormalities in the PTEN tumor suppressor gene, PIK3CA and ARIDIA, microsatellite instability (MSI) arising from defects in DNA mismatch repair (MMR), and mutations in the b-catenin and K-RAS genes (3–5). In contrast, serous Type II cancers frequently demonstrate alterations in p53, HER2/neu, p16, PPP2R1A, and E-cadherin (5,6). Endometrial CCCs that account for about 5% of endometrial carcinomas and occur in older patients with atrophic endometrium, but not associated with hormonal replacement therapy, are considered to be Type II endometrial cancers, like serous carcinoma. The limited data available regarding clinicopathologic features, immunohistochemistry, and molecular findings of endometrial CCC overlap with those of serous and endometrioid carcinomas (7–10). We conducted the current study to evaluate the clinicopathologic, immunohistochemical, and molecular features of endometrial CCC. We propose a pathogenetic pathway that is unique to this tumor and distinguishes it from Type II serous carcinoma. MATERIALS AND METHODS Patients and Endometrial Clear Cell Carcinoma (ECCC) Tissue Samples Data for patients with endometrial CCC who were diagnosed between 2002 and 2012 were retrieved from the pathology files of Korea University Anam Hospital, Kwandong Cheil Hospital, Keimyung Dongsan Hospital, and Asan Medical Center. Diagnoses were made using hysterectomy specimens in all except one case, in which endometrial curettage was performed. Tumor specimens from 16 patients with endometrial CCC were obtained with informed consent. Histologic findings, including tumor size, invasion depth, involvement of the uterine cervix, lymph node metastasis, and lymphovascular invasion were obtained from pathologic reports, and clinical findings such as clinical stage, recurrence, and follow-up were retrieved from clinical records. The histologic diagnoses were made by gynecologic pathologists at each hospital, and were reviewed by one of the authors (I.K.) in a blind manner.
75
The histologic features of CCC included solid, papillary, tubulocystic pattern of large polygonal cell, cuboidal cell or hobnail cell with clear or eosinophilic cytoplasm, or scanty to moderate cytoplasm. The cores of papillary structures were hyalinized, and the tubulocystic structures contained eosinophilic, secretory material. The nuclei of the large polygonal tumor cells were vesicular and had prominent eosinophilic nucleoli, whereas cuboidal and hobnail cells had hyperchromatic nuclei. All tumors with areas of pure CCC were included, whereas cases with mixed CCC or equivocal histology were excluded. Construction of Tissue Microarray and Immunohistochemical Staining Two representative areas were marked on each hematoxylin and eosin–stained slide. The corresponding formalin-fixed, paraffin-embedded tissue blocks were retrieved and the selected areas were marked. Two cores 2 mm in diameter were punched out from each sample and incorporated into a recipient paraffin block using a precision instrument (UNITMA. Co. Ltd, Seoul, Korea). The tissue microarray block was left in 601C oven for 30 min, embedded in a paraffin block, and cut into sections 4 mm thick. Each section was deparaffinized in xylene and rehydrated through a series of graded ethanol. Antigen retrieval was performed by microwaving each section in Epitope Retrieval Solution (0.01 M citrate buffer, pH 6.0 or 10 mM EDTA, pH 8.0) for 20 min, and endogenous peroxidase was blocked by immersing the section in 0.3% hydrogen peroxide for 10 min. Immunostaining was performed using Dako Autostainer plus Universal Staining System (Dako Cytomation, Carpinteria, CA) with a ChemMate DAKO EnVision Detection kit. The characteristics of primary antibodies against estrogen receptor (ER), progesterone receptor (PR), p53, p16, HER2/neu, MLH1, MSH2, MSH6, PMS2, PTEN, ARIDIA, hepatocyte nuclear factor-1b (HNF-1b) and napsin are summarized in Table 1. Interpretation of Immunostaining The expressions of ER and PR were evaluated using Allred scoring system (11). The proportion of positive stained cells was rated as follows: 0, no cells stained positive; 1, between 0% and 1% positive; 2, between 1% and 10% positive; 3, between 10% and 33% positive; 4, between 33% and 66% positive; and 5, between 66% and 100% positive. In addition to the proportion score, an intensity score was made as follows: 0, negative; 1, weak; 2, intermediate; and 3, Int J Gynecol Pathol Vol. 34, No. 1, January 2015
76
H. S. BAE ET AL. TABLE 1. Characteristics of primary antibodies
Antibodies
Type
Source
Dilution
ER PR P53 P16 HER2 MLH1 MSH2 MSH6 PMS2 PTEN ARIDIA HNF-1b Napsin A
Mouse monoclonal Mouse monoclonal Monoclonal mouse Mouse monoclonal Monoclonal rabbit Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal Rabbit polyclonal Rabbit polyclonal Mouse monoclonal
Dako Dako Novocastra, UK Santa Cruz, USA Thermo Scientific, USA Dako DBS, USA DBA, USA Cell Marque, USA Dako Sigma, USA Proteintech, USA Leica, UK
1:50 1:50 1:50 1:100 1:100 1:50 1:50 1:50 1:50 1:100 1:200 1:50 1:400
ER indicates estrogen receptor; PR, progesterone receptor.
strong. The intensity score and the proportion score were added to obtain the total score. Scores >2 were considered to be positive. The expression of p53 was scored as the percentage of cells with positive nuclear staining. HNF-1b is expressed in the nucleus, whereas napsin is expressed in the cytoplasm and/or membrane. The staining was interpreted as positive when at least 5% of the tumor cells expressed the stained antigen. MMR proteins (MLH1, MSH2, MSH6, and PMS2), PTEN and ARID1A, were interpreted as abnormal when there was complete loss of nuclear staining in tumor cells compared with normal lymphocytes and stromal cells. HER2/neu expression was evaluated according to the recommendations of the American Society of Clinical Oncology/College of American Pathologists for breast cancer (12,13). No staining or membranous staining in 10% of the tumor cells was scored as 1+, weak or moderate staining of the entire membrane in >10% of the tumor cells was scored as 2+, and strong staining of the entire membrane in >30% of the tumor cells resulted in a score of 3+. The expressions of p16 were interpreted as >90%, 10% to 90%, and 2.2 or HER2 gene copies per number >6.0, whereas a FISH ratio of
