Nuclear receptor DAX-1 in human common epithelial ovarian carcinoma: An independent prognostic factor of clinical outcome
Manar Abd-Elaziz,1 Jun-ichi Akahira,2 Takuya Moriya,1 Takashi Suzuki,1 Nobuo Yaegashi2 and Hironobu Sasano1
Departments of 1Pathology and 2Obstetrics and Gynecology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575
(Received August 5, 2003/Revised September 4, 2003/Accepted September 5, 2003)
DAX-1 is a member of the nuclear receptor superfamily and is thought to be involved in the regulation of steroidogenesis. Its expression has been detected primarily in endocrine neoplasms such as adrenocortical as well as pituitary tumors in human, but its biological roles have not been examined well in sex steroid- dependent neoplasms. The aim of this study is to detect the ex- pression of DAX-1 in common epithelial ovarian carcinomas in or- der to evaluate its possible biological significance. DAX-1 immunoreactivity was examined using immunohistochemistry. The correlation between the status of DAX-1 immunoreactivity and clinicopathological parameters and disease-free survival of the patients in a series of 60 cases of common epithelial ovarian carcinoma was examined. The status of DAX-1 immunoreactivity was evaluated using H score. DAX-1 immunoreactivity was widely detected in the nuclei of common epithelial ovarian carci- noma cells. There was a significant positive correlation between DAX-1 immunoreactivity and clinical staging (P=0.0241), tumor grade (P=0.0115), the residual size of the tumor (P=0.0014) and Ki-67 labeling index (P =< 0.0001). In univariate survival analysis, a significant association was detected between DAX-1 immunore- activity and shortened patient survival (P=0.0157). Other signifi- cant prognostic parameters were clinical stage, residual size of tumor and Ki-67. In multivariate analysis, DAX-1 immunoreactiv- ity, clinical stage, residual size of tumor and Ki-67 all turned out to be independent prognostic factors for shortened survival. In conclusion, DAX-1 immunoreactivity is considered to be a new in- dependent marker of poor prognosis or adverse clinical outcome in patients with epithelial ovarian carcinoma, possibly through al- tering in situ steroids production. (Cancer Sci 2003; 94: 980-985)
C ommon epithelial ovarian carcinomas are characterized by a broad spectrum of biological behavior ranging from tu- mors associated with excellent prognosis and response to ther- apy to those with poor prognosis and a high mortality rate. This marked variety is related to the fact that many patients are diag- nosed at an advanced clinical stage.
A number of prognostic factors have been proposed for com- mon epithelial ovarian carcinoma, and are expected to be useful in deciding which patients need additional postoperative treat- ment and whether they may get benefit from treatment in terms of prolongation of disease-free and overall survival. The most important prognostic factors are clinical staging, residual size of the tumor, tumor grade and the patient’s age.1,2) In addition to these relatively well established parameters, other prognostic parameters that have been proposed include estrogen and progesterone receptors (ER and PR), Ki-67 labeling index (LI),3-5) and the preoperative serum marker CA125.6) The iden- tification of new prognostic parameters should contribute to further improvement of treatment and the clinical outcome of the patients.
DAX-1 (Dosage-sensitive sex reversal Adrenal hypoplasia congenita critical region on the X chromosome, gene 1) is a
member of the nuclear receptor superfamily.7) Its expression is largely restricted to steroidogenic tissues such as adrenal cor- tex, ovary, Leydig cells and other endocrine cells such as testic- ular Sertoli cells, pituitary gonadotropes, ventromedial hypothalamic nucleus cells and others.8, 9) DAX-1 acts as a neg- ative regulator of steroid production by repressing the expres- sion of steroidogenic acute regulatory protein (StAR), 10-12) which is essential for the first and rate-limiting step in steroid biosynthesis and the transfer of cholesterol to the inner mito- chondrial membrane,10, 13, 14) where side-chain cleavage P450 (P450scc) converts cholesterol into pregnenolone. Preg- nenolone is further transformed into progesterone by 3-hy- droxysteroid dehydrogenase (3ß-HSD).15) In addition, DAX-1 has also been demonstrated to negatively regulate the transcrip- tion activity of steroidogenic factor-1 (SF-1),16) which is an- other member of the nuclear receptor superfamily that positively regulates the expression of multiple cytochrome P450 steroid hydroxylases and StAR.17)
Among human neoplasms, the status of DAX-1 has been ex- tensively examined in adrenal gland and adrenocortical tumors,16, 18) but its roles in sex steroid-dependent neoplasms, in which in situ steroid production and metabolism are considered to play important roles, have not been examined well. In our present study, we examined the status of DAX-1 in common epithelial ovarian carcinoma and correlated the findings with prognostic factors, such as tumor stage, the size of the residual tumor, tumor grade, the age of the patients, the preoperative se- rum marker CA125 and the LI of Ki-67, ER and PR, in order to evaluate its possible clinical and biological significance in pa- tients with common epithelial ovarian carcinoma.
Materials and Methods
Patients’ population. The study materials were 4 normal ova- ries and 60 primary epithelial ovarian carcinomas, which were divided as follows: 33 cases of serous carcinoma, 11 cases of endometrioid carcinoma and 16 cases of clear cell carcinoma. All these archival specimens were retrieved from surgical pa- thology files at Tohoku University Hospital, Sendai. Patients’ ages ranged from 36 to 74 years (median 50 years). Clinico- pathological characteristics of these carcinoma cases are sum- marized in Table 1. Tumor stage was assessed according to the International Federation of Gynecology and Obstetrics. Tumors were graded according to the histological grading system pro- posed by Silverberg.19) Histological types were determined ac- cording to the criteria of the World Health Organization (WHO). Both grading and histological subtypes were evaluated by three different observers (MA, TM, JA). Preoperative serum CA125 levels were obtained from the review of medical records of epithelial ovarian carcinoma patients included in this
E-mail: manarrh@patholo2.med.tohoku.ac.jp
study. CA125 was determined by immunoradiometric assay (Centocor, Malvern, PA). The research protocol was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai.
Primary antibodies. Rabbit polyclonal antibody for DAX-1 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibodies for PR (PR-AB, PR-A: hPRa7 and PR-B: hPRa2) were purchased from NeoMarkers (Fremont, CA). ERa and Ki-67 monoclonal antibodies were purchased from Immunotech. (Marseilles, France). The charac- teristics of the primary antibodies used in immunohistochemis- try are summarized in Table 2.
Immunohistochemistry. All specimens were fixed for 24 h in 10% formalin and embedded in paraffin wax. Sections of 3 um were cut and placed on clean glass slides (Matsunami, Tokyo). After deparaffinization, the slides for DAX-1, PR-AB, PR-A, PR-B and Ki-67 were placed in citric acid buffer (2 mmol/liter citric acid and 9 mmol/liter trisodium citrate dehydrate, pH 6.0) and autoclaved for 5 min at 121℃ and those for ERa were placed in boiling citric acid buffer and heated in microwave oven for 15 min. The slides were subsequently allowed to cool at room temperature, and washed in 0.01 M phosphate-buffered saline (PBS). The slides were then reacted with 1% normal rab- bit or goat serum for 30 min at room temperature. The dilutions of primary antibodies used in our study were as follows: DAX- 1, 1/500; Ki-67, 1/100; PR-AB, 1/50; PR-A, 1/100; PR-B, 1/
| Total number | 60 |
|---|---|
| Patients age (years)1) | 50.9±1.2 |
| Histology | |
| Serous | 33 (55%) |
| Endometriod | 11 (18.3%) |
| Clear cell | 16 (26.7%) |
| FIGO stage | |
| I | 14 (23.3%) |
| II | 24 (40%) |
| III | 6 (10%) |
| IV | 16 (26.7%) |
| Silverberg grade | |
| G1 | 19 (31.7%) |
| G2 | 18 (30%) |
| G3 | 23 (38.3%) |
| Residual size of tumor (mm)1) | 1.8±1.97 |
1) Values represent mean±SD.
Other values represent number of cases and percentages.
| Antibodies | Source | Optimal dilution |
|---|---|---|
| DAX-1 (polyclonal) | Cruz Biotechnology, Inc. (Santa Cruz, CA). | 1/500 |
| PR-AB | NeoMarkers | 1/50 |
| (monoclonal) | (Fremont, CA) | |
| PR-A: hPRa7 | NeoMarkers | 1/100 |
| (monoclonal) | (Fremont, CA) | |
| PR-B: hPRa2 | NeoMarkers | 1/100 |
| (monoclonal) | (Fremont, CA) | |
| ERa | Immunotech. | 1/50 |
| (monoclonal) | (Marseilles, France) | |
| Ki-67: MIB-1 | Immunotech. | 1/100 |
| (monoclonal) | (Marseilles, France) |
100 and ERa, 1/50. They were incubated with tissue sections at 4℃ for 18 h.
The sections were washed in PBS, and the endogenous per- oxidase activity was blocked by placing the slides in 100% methanol with 0.3% hydrogen peroxide for 30 min at room temperature (22℃). The slides were then incubated with bioti- nylated anti-mouse or anti-rabbit immunoglobulin for 30 min at room temperature, and subsequently incubated with peroxidase- conjugated streptavidin for 30 min at room temperature, using the Histofine Kit (Nichirei, Tokyo). The sections were washed with 0.01 M PBS, and the antigen-antibody complexes were vi- sualized by immersion in DAB solution (1 mmol/liter 3,3’-di- aminobenzidine tetrahydrochloride (DAB), 50 mmol/liter Tris- HCI buffer, pH 7.6 and 0.006% hydrogen peroxide). The sec- tions were counterstained with hematoxylin. Human tissue sec- tions used as positive controls in this study were as follows: adrenal gland for DAX-1, endometrium for PR; PR-A, PR-B and breast cancer for ERa. As a negative control, normal rabbit or mouse IgG was used instead of primary antibodies. No spe- cific immunoreactivity was detected in these sections.
Scoring of immunoreactivity. Semiquantitative analysis of im- munoreactivity of DAX-1 (so-called H score) was performed in this study as reported by McCarty et al.20) with some modifica- tions.21) Briefly, more than 500 tumor cells were counted in each case, and the H score was generated by adding together 2x% for strongly stained nuclei, 1x% for weakly stained nu- clei and 0x% for negative or scattered cells representing less than 10%, giving a possible range of 0-200.21-23)
Ki-67, ERa and PR immunoreactivity were scored in more than 500 tumor cells for each case, and the percentage of im- munoreactivity regardless of immunointensity, i.e. LI, was ob- tained.
Statistical analysis. Statistical analysis was accomplished using SAS software (StatView, version 5.0.1, Cary, NC). The signifi- cance of prognostic factors was assessed using both univariate and multivariate analysis. The statistical significance of the cor- relation between expression of DAX-1 and clinicopathological parameters was performed using regression analysis. For sur- vival analysis, the Kaplan-Meier method and log-rank test were used to generate and to compare the different survival curves. Multivariate progression analysis using the Cox proportional hazards model was performed to test the independent value of each parameter in predicting overall survival of the patients. P values <0.05 were considered significant.
Results
Immunohistochemistry of DAX-1 in common epithelial ovarian carcinomas. DAX-1 immunoreactivity was detected in the nuclei of epithelial cells and stromal cells in 4 cases of normal human ovary (100% of cases) (Fig. 1-A), 30 cases of serous carcinoma (91% of cases) (Fig. 1-B), 11 cases of endometrioid carcinoma (100% of cases) (Fig. 1-C) and 15 cases of clear cell carcinoma of the ovary (94% of cases) (Fig. 1-D). The H score value (mean±SD) for DAX-1 was 156.957±8.05 PR-(A+B), PR-A, PR-B, ERa and Ki-67 LI (mean±SD) values were 41.52%±28.4%, 8%±11.3%, 40%±27.4%, 19.3%±21% and 49.4%±15.7%, respectively.
Correlation between DAX-1 immunoreactivity and clinicopatho- logical parameters. The correlations between DAX-1 immunore- activity and clinicopathological parameters of the cases were first examined by univariate analysis. There was a significant positive correlation between DAX-1 immunoreactivity and FIGO stage (P=0.0241), tumor grade (P=0.0115), the residual size of the tumor (P=0.0014) and Ki-67 LI (P<0.0001), as shown in Fig. 2 and Table 3. However there was no significant correlation between DAX-1 immunoreactivity and age, preop- erative CA125, PR-(A, B, A+B) or ERa of the patients.
A
B
C
D
P value=0.0241
4.5
4
3.5
stage
3
2.5
2
1.5
1
.5
130
135
140
145
150
155
160
165
170
175
DAX-1
P value=0.0115
3.25
3
2.75
2.5
grade
2.25
2
1.75
1.5
1.25
1
.75
130
135
140
145
150
155
160
165
170
175
DAX-1
P value=0.0014
Residual size of tumor
6
5
4
3
2
1
0
-1
130
135
140
145
150
155
160
165
170
175
DAX-1
P value <0.0001
450
400
350
Ki-67
300
250
200
150
100
50
130
135
140
145
150
155
160
165
170
175
DAX-1
DAX-1 immunoreactivity and patient survival. Among 60 pa- tients examined, 35 died of ovarian carcinoma, and 25 were alive. The significance of DAX-1, age, tumor grade, FIGO stage, residual size of tumor, preoperative serum marker CA125, proliferation marker Ki-67 and differentiation markers PR and ER in relation to patients’ survival was evaluated by univariate and multivariate analyses.
When DAX-1 was included with the other covariates (age, tumor stage, tumor grade, residual size of the tumor, preopera- tive CA125, Ki-67, ER and PR) of the patients in the univariate analysis, significant inverse correlations were detected between DAX-1 immunoreactivity (P=0.0157), residual size of the tu- mor (P<0.0001), tumor stage (P=0.0143) and Ki-67 LI (P=0.0261) and the clinical outcome of the patients. Cumula- tive survival curves were calculated according to the Kaplan- Meier method. The log-rank test was used to compare survival curves (Fig. 3, Table 4).
Multivariate survival analysis. The independent prognostic value of DAX-1 immunoractivity, as well as other clinicopatho- logical parameters that were found to be significant in univari- ate analysis, i.e. residual size of tumor, clinical stage and Ki-67 immunoreactivity, was further evaluated by multivariate pro- gression analysis based on the COX proportional hazard model.
| DAX-1 immunoreactivity | ||
|---|---|---|
| P value | r | |
| Patients age (years) | 0.7616 | 0.042 |
| FIGO stage (I, II, III, IV) | 0.0241 | 0.307 |
| Sliverberg grade | 0.0115 | 0.335 |
| Residual size of tumor (mm) | 0.0014 | 0.416 |
| Preoperative CA125 | 0.0706 | 0.261 |
| PR H score | 0.2842 | 0.151 |
| ER H score | 0.9921 | 0.001 |
| Ki-67 LI | <0.0001 | 0.592 |
DAX-1 immunoreactivity (P=0.031), residual size of the tumor (P=0.0014), FIGO stage (P=0.0373) and Ki-67 immunoreac- tivity (P=0.0316) turned out to be independent prognostic fac- tors as evaluated by both univariate and multivariate statistical analyses (Table 4).
Discussion
Information on DAX-1 expression has been broadly restricted to steroidogenic tissues such as human adrenal gland,8, 9, 16, 18, 22, 23 since the DAX-1 gene encodes an orphan nuclear hormone re- ceptor essential for normal fetal development of the adrenal cortex.19) In addition, the mutations in the DAX-1 gene are well known to be responsible for congenital X-linked adrenal hypo- plasia associated with hypogonadotropic hypogonadism.7)
In this study, we examined the immunohistochemical local- ization of DAX-1 in normal human surface epithelium, as well as different carcinomas arising from surface epithelial cells. DAX-1 immunoreactive protein was localized in the nuclei of normal surface epithelium of human ovary, as well as in the stromal cells of all normal ovary specimens examined. The re- sults in our study are in good agreement with those of a recent study by Sato et al.22) They demonstrated the expression pat-
| Variable | Univariate | Multivariate |
|---|---|---|
| P | P | |
| Patients age (years) | 0.2509 | |
| FIGO stage (I, II, III, IV) | 0.0143 | 0.0373 |
| Sliverberg grade | 0.417 | |
| Residual size of tumor (mm) | <0.0001 | 0.0014 |
| Preoperative CA125 | 0.3469 | |
| PR H score | 0.7824 | |
| ER H score | 0.6485 | |
| Ki-67 LI | 0.0261 | 0.0316 |
| DAX-1 H score | 0.0157 | 0.031 |
P value <0.0001
1
Cum. survival
.8
· no residual tumor
.6
residual tumor < 2 mm
.4
.2
residual tumor > 2 mm
0
0
20
40
60
80
100
120
Time
P value=0.0157
1
Cum. survival
.8
- DAX-1 (-)
.6
DAX-1 (+)
.4
.2
0
0
20
40
60
80
100
120
Time
P value=0.0143
1
Cum. survival
.8
· stage I
.6
stage II
.4
stage III
.2
+ stage IV
0
0
20
40
60
80
100
120
Time
terns of DAX-1 in different cell types and follicular stages in normal cycling human ovaries, in which DAX-1 immunoreac- tivity was predominantly detected in granulose cells in ovarian follicle and corpora lutea. In addition, DAX-1 immunoreactiv- ity was relatively high in follicular stages from primordial to non-dominant rather than in preantral and dominant follicular stages. Based on the above results, the authors concluded that DAX-1 may play a role in the modulation of Ad4BP (Adrenal 4 Binding Protein)/SF-1-dependent transcription of steroidogenic enzymes involved in human ovarian steroidogenesis.
Among neoplasms, DAX-1 expression was detected prima- rily in adrenocortical neoplasm and pituitary adenoma. 16, 18, 24, 25) Many previous studies on DAX-1 expression in adrenocortical and pituitary neoplasms have demonstrated the presence of DAX-1 in non-functioning adrenal and pituitary adenomas. DAX-1 expression was, however, low or even absent in aldos- terone-producing adenomas, adrenocortical carcinomas of the adrenal gland as well as in GH-secreting and PRL-secreting ad- enomas of the pituitary gland. These results indicate that DAX- 1 is one of the factors regulating steroid biosynthesis in these neoplasms.
DAX-1 expression has not been well studied in sex steroid- dependent neoplasms. This is the first study to examine the sta- tus of DAX-1 in malignant epithelial ovarian carcinoma in or- der to evaluate the prognostic impact of this hormonal receptor on these carcinomas. The prognostic impact of DAX-1 was evaluated on the basis of detection of its expression in epithelial ovarian carcinoma in a total of 60 patients, and the results were correlated with other putative prognostic factors of ovarian tu- mors and with the overall survival of the patients.
In the present study, DAX-1 immunoreactivity was detected in the nuclei of epithelial cells of ovarian carcinoma and was directly correlated with tumor grading, residual size of the tu- mor, FIGO stage and Ki-67 labeling index. DAX-1, advanced clinical stage, increasing size of the residual tumor and higher values of Ki-67 LI all turned out to be independent factors for poor prognosis in patients with common epithelial ovarian car- cinoma. It is then important to elucidate the possible mecha- nisms of DAX-1 expression in carcinoma cells in relation to
aggressive biological behaviors commonly seen in epithelial ovarian carcinoma patients.
Previous studies have demonstrated the importance of DAX- 1 in human steroidogenesis and in the regulation of steroid hor- mone production, as described above. Lalli et al.,10) measured the production of pregnenolone and progesterone in Y-1/ hDAX-1 cells (DAX-1-expressing Y-Î cells derived from adrenocortical tumors). They reported that the production of both pregnenolone and progesterone is extremely low in Y-1/ hDAX-1 cells and that in these cells, the first steps of the ste- roidogenic cascade are almost completely suppressed. They further demonstrated that DAX-1 inhibits steroid production and metabolism of Y-1 cells at multiple levels.
Progesterone has been demonstrated to be involved in ovar- ian cancer development and progression.26) In addition, recent studies have demonstrated in situ production of progesterone in human common epithelial ovarian carcinoma.27) Progesterone has also been considered to directly mediate decreased tumor growth28) through inhibition of cell proliferation and induction of apoptosis.29,30) The above findings suggest that DAX-1 may inhibit progesterone hormone production in the tumor tissue through repressing multiple biochemical steps in the ste- roidogenic cascade. This decreased level of progesterone pro- duction may result in increased cellular proliferation and decreased apoptosis of carcinoma cells. The highly significant statistical correlation between DAX-1 immunoreactivity and Ki-67 LI in this study is also consistent with this hypothesis. However, further investigations are required.
This study is the first to demonstrate the significant prognos- tic value of DAX-1 expression in any of the human tumors. In addition, the analysis of DAX-1 in tumor cells of common epi- thelial ovarian carcinoma may be helpful in the management of the patients, possibly representing a surrogate marker of hor- monal or endocrine therapy. In summary, DAX-1 immunoreac- tivity was detected in the nuclei of epithelial ovarian carcinoma cells, and positive DAX-1 immunoreactivity was shown to be an independent marker for poor prognosis in common epithelial ovarian carcinoma.
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