Napsin A as a marker of clear cell ovarian carcinoma

Epithelial ovarian cancer (EOC) is the main cause of death among women with gynecologic malignancies [1]. At present, post-surgical therapy is mainly dependent upon tumor stage and grade rather than histological subtype [2]. On the basis of a series of morphologic and molecular genetic studies various types of ovarian cancer can be classified into two groups designated type I and type II [3, 4]. Clear cell carcinoma (CCC), which constitutes 5-6% of ovarian malignancies exhibit morphologic, molecular, and clinical features that do not entirely resemble either Type I or Type II tumors and unlike, other Type I tumors, clear cell carcinoma CCC is high grade at presentation [4, 5].

Consequently, most authorities in the field recommend that ovarian clear cell carcinomas should be automatically classified as grade 3 [2, 3]. This is also in agreement with recent findings from Zannoni et al. [4], where they show that clear cell ovarian carcinoma should be studied separately, but still in comparison with the groups of Type I and Type II tumors. In a study from Chan et al. [6] it was concluded, that women with clear cell ovarian carcinoma are likely to be younger at diagnosis. Clear cell carcinoma usually presents as a pelvic mass with higher frequency of capsule rupture than other subtypes and it is known since before that this subtype is associated with endometriosis [7, 8]. Clear cell ovarian tumors were furthermore, in a large clinical trial, found to present mostly as FIGO-stages I/II at diagnosis [9].

Many studies have found that clear cell carcinoma has a distinct aggressive biologic behaviour with poor response to platinum-based therapy compared to other subtypes of epithelial ovarian cancer [6]. Ovarian clear cell carcinoma may be more closely related to other clear cell tumors than other subtypes of ovarian cancer [10]. Cisplatin act mainly by inducing apoptosis in cancer cells and it has been shown in mouse studies as well as in the clinics that intact wild-type p53 is required for efficient execution of apoptosis. In clear cell carcinoma wild-type p53 is mostly present and mutations are uncommon [10, 11].

Recently, it was shown in genome-wide screening for genes involved in chemo-resistance, that NAPA consistently was over-expressed in cisplatin-resistant cells [12]. The gene NAPA has been detected on chromosome 19q 13.33 and the corresponding protein Napsin A is an aspartic peptidase [13]. It was found to represent an anti-apoptotic protein that promotes resistance to cisplatin by degradation of the tumor suppressor p53, which is regulator of the cell cycle and co-works with cyclin kinase inhibitors as p21 and p27 [14‐16]. The p21 gene is a primary mediator of p53-induced cell cycle arrest [17]. Napsin A is known to be present in primary lung adenocarcinomas as well as renal cell carcinoma (papillary and clear cell subtypes). As Napsin A is usually absent in the neoplastic cells in squamous carcinoma (pulmonary and non-pulmonary) it has been used as a diagnostic tool to distinguish between these two types of tumors [18].

In the present study it was confirmed that ovarian clear cell carcinomas exhibit clinico-pathological and immuno-histochemical features that is different from classical Type I and Type II tumors. The immunohistochemical profil based on staining for Napsin A and the apoptosis regulators p21, p53 and concomitant p21 and p53 were unique in the group of clear cell carcinomas compared to other histological subtypes of tumors (Type I and Type II).

Our findings related to the clinico-pathological differences are supported by earlier studies [4, 7, 10] but results about the immunohistochemical profile for Napsin A is new.

Clear cell carcinomas resembles Type I tumors based on relative genetic stability and frequent presentation in stage I, but on the other hand, clear cell carcinoma is high grade at diagnosis. Furthermore, wild-type p53 is mostly present and mutations are uncommon in clear cell tumors contrary to Type II tumors, which are genetically unstable and have a high frequency of p53 mutations [10, 11]. Ovarian clear cell carcinomas constitute a heterogeneous disease at the genomic level despite having similar histological features. Previous data from Tan et al. [11] has suggested that the pattern of genome-wide copy number aberrations may predict clinical outcome.

Based on our results positivity for Napsin A seems to be more frequently detected in clear cell carcinomas than in other histological subtypes. The results are novel but in agreement with results from Kandalaft et al. [26] who has shown that Napsin A is highly expressed in 12 out of 12 (100%) ovarian clear cell tumors. We show further that concomitant positivity for p21 and negativity for p53 was strongly associated to positive staining for Napsin A and clear cell tumors, respectively in the present study. Some difficulties may arise in the morphological diagnosis between clear cell carcinomas and high grade serous or endometrioid ovarian carcinomas [10]. Therefore, concomitant positivity for p21 and negativity for p53 of a Napsin A positive ovarian tumor might strengthen the pathological diagnosis of clear cell ovarian carcinoma. Thus, our findings may facilitate the morphological diagnosis of clear cell ovarian carcinoma when there are difficulties in the pathological distinction between clear cell carcinomas and high grade serous or endometrioid ovarian carcinomas [10].

No single marker has been reported to be useful alone to distinguish between high grade serous and clear cell ovarian carcinoma. In a newly presented study where seven different markers for distinction between high grade serous and clear cell ovarian carcinoma did though p53 in combination with p21 (and Cyclin E, ER, HNF-1b, WT1 and Ki-67) correctly classify 84% of tumors with high grade serous compared with clear cell ovarian carcinoma [27]. Furthermore, as p53 missense mutations and consequent overexpression (positivity) is infrequent in clear cell carcinomas, but common in high-grade serous carcinoma, positive staining for p53 could be a potential marker to distinguish between these two tumors [27]. In the present study none of the 16 clear cell carcinoma showed positive p53 staining and our findings are in agreement with others [28, 29].

Differences in the immunohistochemical profile for Napsin A and the apoptosis regulators p21, p53 and concomitant p21 and p53 between the groups of clear cell carcinomas and other histological subtypes of tumors in one group (Type I and Type II) were detected in the present study, whereas differences in the p27 status were limited to comparison between clear cell tumors and Type I tumors.

Findings from ROC curves in the present study showed that the markers Napsin A, p21 + p53- or p21 + p53-Napsin A + all had predictive values which might indicate that they are potential diagnostic markers in the process of differential diagnosis between clear cell ovarian carcinomas and other histological subtypes. However, we found that the p21 + p53- phenotype did not add to the Napsin A phenotype along the process of differential diagnosis between clear cell ovarian tumors and histological subtypes.

In a study on ovarian A2780 cells, it was demonstrated that p27 up-regulation is linked directly to activation of the p21/p53 pathway by a DNA-damaging agent (a cisplatin analogue). Therefore, the p53/p21/p27 axis should become a new focus of attention in checkpoint response to DNA damage [30].

In the clinics it is not always obvious how to handle women with suspected clear cells carcinoma, which most probably is related to its heterogeneity. Ovarian cancer is usually treated by surgery followed by chemotherapy. Though in the group of women diagnosed with clear cells carcinoma defined by commonly used histopathology a platinum resistance often occurs [6]. Based on the results described in this study we propose that Napsin A could not only be used as a diagnostic marker of clear cells carcinoma, but also might Napsin A positivity of clear cell carcinoma predict about platinum sensitivity. Out of there the detection of Napsin A in clear cell carcinoma could help us to understand how it promotes resistance to cisplatin by degradation of the tumor suppressor p53 [12, 14].

Similarities between clear cell carcinomas and Type I tumors, Type II tumors and Type I and Type II tumors in one group, respectively were limited to FIGO-stages and recurrent disease.

Patients with clear cell carcinomas were younger than the Type II tumor patients (55 vs. 60 years; mean age). In SEER data reported by Chan et al. [6], women with clear cell histology were younger than patients with serous cancers. The fraction of patients with BMI > 25 was significantly lower in women with clear cell carcinoma compared to woman with type I tumors. This is in line with previous studies that have reported an elevated risk for epithelial ovarian cancer in woman with higher BMI [31]. This risk seams to be limited to the histological subgroups of low-grade serous and low to intermediate grade endometroid [32].

In our material, clear cell carcinomas showed a significantly higher frequency of capsular rupture compared to type I tumors, Type II tumors and Type I/type II combined. Even these results are in line with earlier findings and likely to contribute to poor prognosis for clear cell carcinomas. In one study improvement was observed in the 5-year disease-free survival for patients in FIGO-stage I without tumor rupture during surgery [33]. Rupture (before and during surgery) was the second most powerful prognostic indicator of disease-free survival (after the degree of differentiation) in a multicenter study including 1545 patients with invasive epithelial ovarian cancer all in FIGO-stage I [34].

In the present study the 5-year-survival was not different for the subgroups of patients with clear cell ovarian carcinoma from survival for patients with Type I tumors, Type II tumors or Type I and II in one group. A number of studies [35] have reported that overall survival of women with clear cell ovarian carcinoma is higher than in high grade serous cancer patients in the low-stages (FIGO I-II), although the survival was worse among women with clear cell ovarian carcinoma in the high stages (FIGO III-IV). However, in one of the largest series to date including 1411 clear cell patients comparing 5-year disease-specific survival rate after adjusting for stage there was a significantly worse survival rate associated with clear cell histology compared with other histological subtypes [6].

NAPA was identified as one of nine cisplatin resistance genes in a genome-wide analysis of HeLa cells by using DNA microarrays [12]. The chemotherapeutic agent cisplatin is known to induce apoptosis in actively replicating cells. Mouse studies and clinical evidence suggest that wild p53 is required for efficient apoptosis in tumor cells indicating that intact p53 represents a critical target of chemotherapeutic drugs [36, 37].

Epithelial- mesenchymal transition (EMT) is a multistep process, by which polarized epithelial cells lose epithelial adherence and become capable of free movement through the extracellular matrix. This process is often activated during cancer invasion and metastasis [38]. Recently, it has become clear that mutant p53 proteins after losing their transcriptional function can acquire new functions and drive cell migration, invasion and metastasis [39]. It was reported from one study [40] that wild type p53 can inhibit the focal adhesion kinase (FAK) promoter activity in vitro, but FAK is a critical regulator of adhesion, motility, metastasis and survival signalling. Cells without Napsin A appear susceptible to transition and one of the reasons might be low-level expression of Napsin A. In a further study [38] it was demonstrated in an in vitro EMT model that, Napsin A caused G(0)/(G1) arrest and inhibited the expression of FAK. Recent reports [7] involving large institutional cohorts compared low-stage to high-stage ovarian cancers (I/II vs. III/IV) showed that 57–81% of clear cell carcinoma were diagnosed at stage I/II. One reason of the low frequency of high-stage disease in clear cell ovarian cancer could theoretically be explained by the findings from our study of high frequency of Napsin A positivity and p53 negativity (intact wild type p53) which both might inhibit the process of EMT in clear cell carcinoma.

It has been suggested that Napsin A may have a therapeutic potential as a gene therapy candidate for tumor metastasis as increase in expression of Napsin A and may inhibit the epithelial- mesenchymal transition [41]. In the future the development of high-throughput gene expression and genomic microarray- based analytical platforms may answer many important questions about clear cell ovarian carcinomas [10]. Firstly, if clear cell ovarian carcinomas are more closely related in molecular terms to other clear cell tumors as renal cell carcinomas or endometriod clear cell carcinomas? Does the potential exist for crossover therapeutic targets to be developed for clear cell tumors from a variety of tissue type? Therapy by using the gene NAPA and the protein, Napsin A could be one option of many for treatment of clear cell ovarian tumors from of a variety of tissues in the future.

Some limitations of this work have to be noted. One limitation corresponds to the relative limited number of patients included in the study, which though is conducted in a geographically well defined region in Sweden. However, the frequency of 16 (12.2%) patients with clear cell ovarian carcinoma out 131 consecutive patients all with low-stages epithelial ovarian cancer is in line with the frequency (12.4%) in a previous study in the same region [28]. Furthermore, the non-serous (mucinous, endometrioid and clear cell) subtypes usually are detected in FIGO-stages I-II and therefore represent uncommon diseases which require large scale research trials for searching for subtype specific biomarkers [8]. Another limitation is related to the tissue microarray technology used in this study might also contribute to some limitations of the work. Only two 0.6 mm core biopsies were obtained from each specimen for analysis and there could be a risk of non- representative tissue collected for microarray. As ovarian carcinomas can be very heterogeneous, such specimens may not be representative of the tumor in some cases. Out of there, we used the method of semi-quantitative analysis [22] for the interpretation. Thus, all markers were dichotomized into negative and positive groups [23]. However, in recent years, it has been common practice to perform immunohistochemical studies on tissue microarray, where a large number of antibodies can been screened in a rapid and cost efficient manner [18].

Differences in the immunohistochemical profil for Napsin A and the apoptosis regulators p21, p53 and concomitant p21 and p53 between the groups of clear cell carcinomas and other histological subtypes of tumors in one group (Type I and Type II) were found in the present study, whereas differences in the p27 status were limited to comparison between clear cell tumors and Type I tumors. According to the ROC curves we found that the markers Napsin A, p21 + p53- and p21 + p53-Napsin A + all had some predictive value as diagnostic markers for clear cell ovarian carcinoma. However, the p21 + p53- phenotype did not add to the Napsin A phenotype alone along the process of differential diagnosis between clear cell ovarian tumors and histological subtypes.