Introduction

Clear cell carcinoma (CCC) accounts for approximately 10% of ovarian and less than 5% of endometrial epithelial carcinomas (1,2). In ovary, the biology and clinical behavior of ovarian clear cell carcinoma (OCCC) is different from other epithelial tumors. Unlike high grade papillary serous carcinomas (HGSC), OCCC affects younger women in association with endometriosis and is frequently diagnosed in early stages (3). When adjusted for stage, OCCC has the worst prognosis in comparison with other epithelial carcinomas (4,5). Efficacy of platinum-based chemotherapy regimens is between 20 and 50% for OCCC compared with 60 to 80% for HGSC (3). In endometrium, clear cell carcinomas involve older patients and are diagnosed in higher stage of disease with worse prognosis compared with endometrioid or type I carcinomas (2,6). Diagnostic histopathological findings are similar in ovary and endometrial clear cell carcinomas (ECCC). In most of the tumors, a mixture of solid, papillary and tubulo-cystic architectural patterns are seen. The lining epithelial cells are hobnail and/or cuboidal with clear and/or eosinophilic cytoplasm. Mitotic activity is variable but is usually low. High nuclear grade is infrequent (7). Although in most of the cases, histopathological findings are distinctive and characteristic, other types of endometrial and ovarian carcinomas can harbor clear cell changes and make diagnostic confusion (8,9). In a study by Han et al., moderate agreement was observed in classification of the endometrial carcinomas with clear cell changes between the reviewers, where only 46% of original ECCCs were accurately diagnosed (10).
In this study we evaluated the diagnostic utility of Napsin-A by immunohistochemistry (IHC) for ovarian and endometrial clear cell carcinomas.

This study was conducted in Imam Khomeini Hospital Complex, Tehran, Iran. Due to scarcity of clear cell carcinoma cases, we designed a case-control study. The study was approved by the Ethics committee of Tehran University of medical Sciences (IR.TUMS.IKHC.REC.1397.304). After reviewing the pathology reports from 2013 to 2018 in 3 general and women’s hospitals in Tehran, ovarian and endometrial clear cell carcinomas were re-evaluated by 2 expert pathologists. Forty-two CCC samples were included as case and 42 non-clear cell carcinomas (non-CCC) in ovary or endometrium were selected as control group. A 4μm thick section from relevant paraffin blocks were taken on charged slides and dried 40 minutes at 60ºC. After deparaffinization and rehydration, heat induced epitope retrieval was performed using Master Diagnóstica EDTA buffer (pH: 6) for 40 minutes at 37ºC. After that, the slides were rinsed with 3-5 changes of distilled water followed by cooling at room temperature for 20 min. Peroxidase solution was used, 10 minutes at room temperature for blocking endogenous peroxidase. After incubation for 60 minutes with primary antibodies for Napsin-A (Master Diagnóstica; Spain), Master Polymer Plus Detection System (HRP) was used for 20 minutes. Finally, the slides were counterstained with haematoxylin and mounted. The IHC scoring was performed based on the intensity of staining (0: negative, 1+: faint cytoplasmic granular, 2+: moderate granular, 3+: coarse cytoplasmic granular positivity) and percentage of positive tumor cells (0: no tumor cell, 1+: 1-25%, 2+: 25-50%, 3+: ≥50%). Tumors with sum score ≥2 were considered positive (Figure 1).

Fig. 1.  (A and B) IHC stained slides (400x) showing strongly positive coarse (1+) cytoplasmic granules (3+), (C) moderate coarse and fine cytoplasmic granules (2+) and (D) fine cytoplasmic granules.

Data analysis was performed using SPSS 22.0 (IBM Inc., Chicago, Illinois, USA). Independent-Sample t-test and Chi-square were used for comparison of continuous variables and nominal and categorical variables respectively. The P-values less than 0.05 were considered statistically significant. Sensitivity, specifity and overall accuracy of Napsin-A were calculated using 2× 2 tables.

Endometrial carcinoma is the most common gynecologic cancer in developed countries (11) and ovarian cancer is the fifth frequent tumor in women (12). Clear cell carcinoma is an uncommon subtype of malignant epithelial tumors in ovary and endometrium. Its biology, molecular features, clinical behavior and response to chemotherapy is different from other kinds of carcinomas, which necessitates its accurate diagnosis. Despite histopathological findings, the diagnosis may be challenging and IHC study is crucial. Similar to serous and endometrioid carcinomas, CCCs express CK7, B72.3 and BerEP4 while CA125 and vimentin are positive in approximately 50% of the cases. ER, PR and WT1 are typically negative in CCCS but expression of p53 and p16, which are intermediate between serous and endometrioid carcinomas, is variable (13). Hepatocyte Nuclear Factor-β (HNF-β) is positive in 90 to 100% of CCCs but its specifity is only 54%. Alpha-methylacyl-Coa Racemase (AMACR) is a specific (specifity 99%) but less sensitive (sensitivity 63%) marker for CCC (14).
Napsin-A is a cytoplasmic aspartic protease, which is predominantly expressed in lung and kidney. In the lung, Napsin-A is expressed in type II alveolar pneumocytes and is involved in the synthesis of surfactant protein. In the kidney, Napsin-A is expressed in proximal tubules and is involved in lysosomal protein catabolism (15). Napsin-A is a well-established new marker for pulmonary adenocarcinoma and is considered to be superior to TTF-1 (16). Its expression in gynecologic CCCs and renal neoplasms has been the subject of some recent studies (14,15,17-24).
In the present study we found 100% specifity and 83% sensitivity for Napsin-A by IHC, for distinction of OCCC and ECCC from non-CCCs. Our results are in concordance with previous studies, Table 2. Since CCCs are uncommon, we conducted a case-control study to participate a higher number of CCC cases. Inability to determine prevalence, positive predictive and negative predictive values is the limitation of our study. But as we know, case-control studies are superior to cross-sectional studies when the prevalence of the target disease is low and the diagnostic test is costly to perform (25). The results of our study were similar to the findings of the study performed by Yamashita et al., which included the largest sample of OCCCs (86 samples) and non-CCC (101 samples) compared with other studies (21).
The role of Napsin-A in OCCC and ECCC is not clear. In lung and kidney, expression of Napsin-A is reduced in high grade tumors with advanced clinical stages, therefore a tumor suppressor role has been proposed for this protein (23). Along with some other previous studies, we could not find a significant statistical difference between pathologic stage, omental involvement and lymph node metastasis in negative and positive samples. Hence larger scale studies are needed for this subject.
Fadare et al. recommended a panel of immuno-histochemical markers including Napsin-A, HNF-1 βand AMACR to obtain the highest sensitivity and specifity when CCC is a diagnostic consideration (26).

Table 2. Review of previous studies evaluating diagnostic accuracy of Napsin-A in ovarian and endometrial clear cell carcinoma.

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Napsin-A is a sensitive and highly specific marker for ovarian and endometrial clear cell carcinoma.

 

The study has been funded and supported by Tehran University of Medical Sciences (TUMS); Grant number 96-02-30-35314.

 

The authors declared no conflict of interest.