Prognostic significance of apoptotic activity and its relationship with morphological characteristics in clear cell ovarian adenocarcinoma.

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1 J Med Dent Sci 2007; 54: Original Article Prognostic significance of apoptotic activity and its relationship with morphological characteristics in clear cell ovarian adenocarcinoma. Mikiko Tsugata Human Pathology, Graduate school, Tokyo Medical and Dental University Objective: Clear cell adenocarcinoma of the ovary is more resistant to chemotherapy and has a worse prognosis than other ovarian carcinomas. In order to clarify the factors that contribute to the poor prognosis of clear cell adenocarcinomas, we examined the expression of proliferation- and apoptosis-related factors. In addition, we attempted to discover correlations between the structure of clear cell adenocarcinomas and prognosis. Material and Methods: We evaluated clinicopathological data on 39 patients with ovarian clear cell adenocarcinoma who underwent resection at the Tokyo Medical and Dental University Hospital. The tumors were examined for the expression of proliferation-related factors (mitotic index, Ki-67 labeling index) and apoptosis-related factors (apoptotic index, bcl-2 and p53). In addition, we investigated correlations between the expression of these factors and the cell types and structure of the tumors. Results: High bcl-2 expression and low apoptotic index were associated with a reduced survival rate. The survival rate of patients with tumors of the hobnail cell type was shorter than that of patients with clear cell type, and expression of bcl-2 was significantly associated with the hobnail cell type. Tumors with a papillary structure showed lower apoptotic index and the patients had a lower survival rate than those with a tubulocystic structure. Conclusion: The results of the current study suggest that a low apoptotic activity controlled by bcl-2 correlated with shorter survival in patients with ovarian clear cell adenocarcinoma. Corresponding Author: Mikiko Tsugata Human Pathology, Graduate school, Tokyo Medical and Dental University, , Yushima, Bunkyo-ku, Tokyo , Japan. Received January 29; Accepted August 1, 2007 The cases with poorer prognosis, associated with higher expression of bcl-2 and lower expression of apoptosis-related factors, may be discerned by morphological characteristics of routinely sampled specimens. Key words: clear cell adenocarcinoma, ovary, apoptosis Introduction Ovarian cancer is said to affect 12 women per 100,000 female population in Japan, and the incidence has been increasing annually. The principal histological type is adenocarcinoma. While clear cell adenocarcinoma accounts for only a small percentage of ovarian cancers in Western countries, it accounts for approximately 24% of all ovarian cancers in Japan, evidently being more common in this country 1. It has been reported that clear cell adenocarcinoma is resistant to chemotherapy and has a poor prognosis 2-5. A lower proliferative activity compared to that of other ovarian adenocarcinomas is considered to be one of the important reasons for the chemotherapy resistance of clear cell adenocarcinoma 5. Apoptotic activity and the expression of apoptosis-related molecules, such as bcl-2, bcl-xl, and p53, have been reported to be involved in the chemotherapy resistance and prognosis of ovarian carcinomas Yamasaki et al reported that a high apoptotic index was significantly related to a shorter survival 6. Torre et al reported that a high apoptotic index and low bcl-2 expression level in the ovarian carcinomas correlated with a shorter survival and shorter disease-free period 7. However, no consensus has been reached in relation to the chemotherapy resistance and

2 138 M. TSUGATA J Med Dent Sci poor prognosis of clear cell adenocarcinomas. Vang et al reported that expression of bcl-2 could be detected in 11/11 cases of ovarian clear cell carcinoma, but they could not detect any relationship between the expression level of bcl-2 and the prognosis because of the limited number of cases 10. Therefore, in order to identify the factors that contribute to the poor prognosis of clear cell adenocarcinomas, we examined proliferation, apoptosis and expression of proliferation and apoptosis-related molecules in clear cell adenocarcinoma. Clear cell adenocarcinoma exhibits various cellular and structural patterns that can be classified morphologically by ordinary hematoxylin and eosin staining. Several reports have suggested a relationship between the structural pattern and the prognosis In order to verify the utility of the morphological subclassification of clear cell adenocarcinomas we tried to identify the potential correlations between the morphological patterns and prognosis and the expression of the proliferation- and apoptosis-related factors. Table 1. Patient characteristics of the clear cell adenocarcinomas. Materials and Methods We investigated 39 clear cell adenocarcinomas of the ovary that had been surgically resected at the Tokyo Medical and Dental University Hospital between 1996 and For comparison, 19 serous adenocarcinomas were also investigated. Informed consent to use the surgical materials for the investigation was obtained from all the patients. The clinicopathological staging of the clear cell adenocarcinomas was conducted according to the criteria of the International Federation of Gynecology and Obstetrics (FIGO) 14. Thirty (76.9%) patients were classified as stage I (stage Ia; 4, Ic; 26), 4 patients (10.3%) as stage II (IIa; 1, IIc; 3), 4 patients (10.3%) as stage III (IIIc; 4), and 1 patient (2.5%) as stage IV. Patients in early stages (stages I and II) were predominant (34/39, 87.1%) (Table 1). Thirty-five patients underwent total hysterectomy, bilateral salpingo-oophorectomy and omentectomy with pelvic and para-aortic lymphadectomy. Three patients underwent total hysterectomy, bilateral salpingo-oophorectomy and omentectomy, and 1 patient underwent salpingo-oophorectomy. Thirty-seven patients received from 3 to 14 cycles of platinum or paclitaxel-based chemotherapy after the operation. The median follow-up period was months (range, months); 82.0% of the patients were followed up for more than one year. The materials for the study were obtained during the primary cytoreductive surgery; none of the patients had received neo-adjuvant chemotherapy. Survival time was the primary endpoint of the study and was measured from the date of diagnosis to date of death or last contact. Survival curves were generated using the method of Kaplan-Meier. Statistical significance was calculated using the log-rank test (Stat View survival tool version 4.5). We also investigated the correlations between the expression of the proliferation- and apoptosis-related factors in the tumors and the cell types and structural patterns of the tumors. We divided the tumors into two cell types the clear cell and hobnail cell types and into three patterns based on the structural pattern: Solid, papillary, and tubulocystic patterns according to the criteria of the World Health Organization (WHO) 15 (Figure 1). Tumors containing a mixture of the cell types or structural patterns were labeled according to the predominant component accounting for 50% of the tumor. Formalin-fixed, paraffin-embedded sections (4 Òmthickness) were used; before processing, the sections were pretreated by autoclaving at 121 C for 20 minutes in 10 mm sodium citrate buffer (ph 6.0). Endogenous peroxidase activity was blocked by incubation with 1%

3 PROGNOSTIC SGNIFICANCE OF CLEAN CELL OVAVIAN ADENOCARCINOMA 139 Fig. 1. Cell types (A, B) and structural patterns (C-E) of the clear cell adenocarcinoma A: clear cell type, B: hobnail cell type (scale bar: 100Òm) C: solid pattern, D: papillary pattern, E: tubulocystic pattern (scale bar: 500Òm) The clear cells have abundant clear cytoplasm (A). The hobnail cells have scant cytoplasm and the nuclei protrude into the lumens (B). Sheets of polyhedral cells with abundant clear cell cytoplasm separated by fibrovascular or hyalinized stroma are characteristic of the solid pattern (C). The papillary pattern is characterized by thick or thin papillae containing fibrous tissue or abundant hyaline material (D). The tubulocystic pattern is characterized by varying-sized tubules and cysts (E). H 2 O 2 in methanol for 30 minutes, and the slides were washed in PBS. Following protein block, the slides were incubated with the primary antibody overnight at room temperature. The clonal origin, dilution and source of purchase of the primary antibodies were as follows: Ki- 67 (clone MIB1-M7240; dilution 1:1600; DAKO); p53 (clone NCL-p53-DO7; dilution 1:800; NOVO CAS- TRA); bcl-2 (clone M0887; 1:200; DAKO); HNF-1 beta (polyclonal sc-7411; dilution 1:200; Santa Cruz). After rinsing in PBS, an avidin-biotin detection was performed (Vector Laboratories). Color development was performed using diaminobenzidine (DAB) (Nichirei, Japan), and then slides were stained by Mayer s hematoxylin. More than 1,000 tumor cells were examined in at least 3 fields, and the labeling index for each of the antibodies was determined by the percentage of positive cells. The expressions of Ki-67, p53 and HNF-1 beta were identified by dark brown staining of the nuclei. Staining of bcl-2 was identified in the cytoplasm. The mitotic index was examined in hematoxylin-eosin (HE)- stained specimens in 50 high-power (400 ) fields (Figure 2). The terminal deoxynucleotidyl transferase-mediated

4 140 M. TSUGATA J Med Dent Sci Fig. 2. The mitotic figures (A), immunohistochemical staining for Ki-67 (B), HNF-1beta (C), p53 (D) of the clear cell adenocarcinoma (scale bar: 200Òm) A, B: Arrow head shows mitotic cells (HE stain). Mitotic figures and the expression of Ki-67 were not frequently seen in the clear cell adenocarcinomas. C: The expression of HNF-1 beta was seen diffusely in the clear cell adenocarcinoma. D: Only a few cancer cells expressed p53 in the clear cell adenocarcinomas. deoxyuridine triphosphate nick-end labeling (TUNEL) method was used for in situ labeling of the apoptotic cells. Formalin-fixed, paraffin-embedded sections were used, and after deparaffinization, endogenous peroxidase was blocked with 1% H 2 O 2 and methanol at room temperature for 30 minutes, and protease K (DAKO Cytomation, proteolytic enzyme solution diluted in 0.05 mol/l Tris-HCl, 0.015mol/L sodium azide, ph7.5) treatment was performed at room temperature for 15 minutes. Then, after the sections were washed three times with PBS, the TUNEL reaction was allowed to proceed for 60 minutes at 37 C. After three further washes with PBS, the peroxidase reaction was allowed to occur for 30 minutes at 37 C, and the slides were rinsed, followed by color development with DAB and Mayer s hematoxylin. Apoptotic nuclei were recognized as brown fragmented masses, sometimes with caps, at the nuclear periphery. Statistical analysis of the differences in expression of the proliferation- and apoptosis-related factors among the various cell types and structural patterns of clear cell adenocarcinoma were conducted using the Chisquare test and Fisher s test (statistics soft y-stat 2003). Results The median survival was months for stage I/II cases and months for stage III/IV cases. Six of the 34 stage I/II patients showed recurrence of the disease. The median survival in the patients with recurrence was months and the median survival in the patients with disease-free survival was months. The mitotic index and the Ki-67 labeling index were lower in the clear cell adenocarcinomas (mitotic index, ; Ki-67 labeling index, ) than in

5 PROGNOSTIC SGNIFICANCE OF CLEAN CELL OVAVIAN ADENOCARCINOMA 141 Table 2. Correlation of apoptotic index with the clinicopathologic features in the clear cell adenocarcinomas. Fig. 3. Overall survival of the patients with clear cell adenocarcinoma categorized according to the apoptotic index (AI) and expression of bcl-2. A; Kaplan-Meier curves estimated survival for the apoptotic index. B; Kaplan-Meier curves estimated survival for the expression of bcl- 2. serous adenocarcinomas (mitotic index, ; Ki- 67 labeling index, ) (p<0.01). The expression level of bcl-2 was higher in clear cell adenocarcinomas ( ) than in the serous adenocarcinomas ( ) (p<0.01). Only a few cancer cells expressed p53 in the clear cell adenocarcinomas ( ). HNF-1 beta was selectively overexpressed in the clear cell adenocarcinomas ( ). In the overall survival analysis, a low apoptotic index (<0.50) was associated with a reduced survival rate (Figure 3-A). While a correlation was observed between the apoptotic index and the bcl-2 expression, p53 expression was not correlated with the apoptotic index. Patients in advanced stages were encountered significantly more frequently in the low apoptotic index group. A low apoptotic index was associated with a high mitotic index and a high Ki-67 labeling index (Table 2). On the other hand, a high bcl-2 expression ( 27.0) was associated with a reduced survival rate (Figure 3- B). Patients in advanced stages were encountered significantly more frequently in the high bcl-2 expression group (Table 3). A high mitotic index, high MIB-1 index and high p53 expression tended to be associated with a reduced survival rate, however, the associations did not reach statistical significance. The level of HNF-1 beta expression was not associated with the survival rate. In regard to the cell type of the tumors, the clear cell type was the predominant type in 21 of the 39 cases, while in 18 of the 39 cases the hobnail cell type predominated. The level of bcl-2 expression was higher in the tumors of the hobnail cell type ( ) than in those of the clear cell type ( ), with a significant difference in the labeling indexes between the two types (P=0.0001) (Figure 4-A, C, Figure 5-A). The apoptotic index was lower in the tumors of the hobnail cell type ( ) than in those of the clear cell type ( ) (P=0.0398) (Figure 4-B, D, Figure 5-B). There was no significant difference in the expression levels of p53, Ki-67 and the mitotic index between the two cell types. The median survival of the patients with

6 142 M. TSUGATA J Med Dent Sci Table 3. Correlation of the bcl-2 expression level with clinicopathologic features in the clear cell adenocarcinomas. the hobnail cell type was months and that of the patients with the clear cell type was months. The 5-year survival rate of the patients with the hobnail cell type tend to be shorter than that of the patients with the clear cell type (P=0.08) (Figure 6-A). In regard to the structural pattern of the tumors, the tubulocystic pattern was the most frequently encountered (22/39, 56.4%), followed by the papillary pattern (17/39, 43.6%); none of the cases in this series showed a solid pattern. In regard to the relationship between the cell type and structural pattern of the tumors, the clear cell type was significantly associated with the tubulocystic pattern, and the hobnail cell type was significantly associated with the papillary pattern (clear cell type/papillary pattern, 6; clear cell type/tubulocystic pattern, 15; hobnail cell type/papillary pattern, 11; hobnail cell type/tubulocystic pattern, 7) (p=0.041). The level of bcl-2 expression was higher in the tumors of the papillary pattern ( ) than in those of the tubulocystic pattern ( ), with a significant difference in the labeling index between the two types (P<0.0001) (Figure 4-A, C, Figure 5-C). The apoptotic index was lower in the tumors of the papillary pattern than in those of the tubulocystic pattern (papillary pattern; , tubulocystic pattern; ), with a significant difference (p=0.0037) (Figure 4- B, D, Figure 5- D). Analysis of the survival in relation to the structural pattern of the tumors indicated that patients with the papillary pattern showed a significantly shorter survival than those with the tubulocystic pattern (p=0.0298) (Figure 6-B). In this study, among the 39 cases of clear cell adenocarcinoma, the tumor was associated with endometriosis in 21 cases (58.4%). While bcl-2 expression was also found in the epithelium of the endometriotic tissue adjacent to the clear cell adenocarcinoma (16/21, 76.2%), staining for bcl-2 was barely detectable in endometrial cysts and endometriosis of the ovary without an adenocarcinoma (expression of bcl-2; 1/18 cases) (Figure4-E, F). In a Kaplan-Meier curve analysis, no statistically significant difference in the survival rates could be demonstrated between patients with clear cell adenocarcinoma concomitant with or without endometriosis. Accordingly, there was no correlation between the expression of the proliferation- and apoptosis-related factors and the presence of endometriosis. Tsuchiya et al reported that HNF-1 beta, a unique molecular marker of ovarian clear cell adenocarcinoma, may inhibit apoptosis in cancer cell lines 16. In this study, the median survival and Kaplan-Meier curves failed to reveal any statistically significant association with the expression level of HNF-1 beta. There was also no significant difference in the apoptotic index related to the expression level of HNF-1 beta. Discussion Clear cell adenocarcinoma is more resistant to chemotherapy than other ovarian carcinomas, and patients with clear cell adenocarcinoma have a poor prognosis. Several studies have suggested that bcl-2, an apoptosis-related factor, may play an important role in the prognosis of ovarian carcinomas. However, no consensus has been reached in regard to the chemotherapy resistance and poor prognosis of clear cell adenocarcinomas In this study, based on the analysis of an adequate number of clear cell adenocarcinomas, we found that the bcl-2 expression level showed an inverse correlation with the apoptotic

7 PROGNOSTIC SGNIFICANCE OF CLEAN CELL OVAVIAN ADENOCARCINOMA 143 Fig. 4. Immunohistochemical staining for bcl-2(a, C), and apoptotic cells by TUNEL method (B, D) in the clear cell adenocarcinoma and immunohistochemical staining for bcl-2 in the endometriotic tissue E, F). (scale bar: 200Ò) A, B: Clear cell adenocarcinoma with the hobnail cell type and papillary pattern. ; bcl-2 was diffusely expressed (A) and apoptotic cell was not seen (B). C, D: Clear cell adenocarcinoma with the clear cell type and tubulocystic pattern. bcl-2 was not expressed (C) and a few apoptotic cells were seen (arrow head) (D). E; The expression of bcl-2 was also found in the epithelium of the endometriotic tissue adjacent to the clear cell adenocarcinoma. F; bcl-2 was not expressed in endometrial epithelium of the ovary.

8 144 M. TSUGATA J Med Dent Sci Fig. 5. Correlation between expression of bcl-2 or apoptotic index and the cell types (A, B) and structural patterns (C, D) of the tumors. A; Expression of bcl-2 according to cell types of the tumors. The bcl-2 expression in the tumor with hobnail cell type was higher than in the tumor with clear cell type. B;Apoptotic index according to cell types of the tumors. The tumor with hobnail cell type was lower apoptotic index. C;Expression of bcl-2 according to structural patterns of the tumors. The expression of bcl-2 in the tumor with papillary pattern was higher than in the tumor with tubulocystic pattern. D;Apoptotic index according to the structural patterns of the tumors. The tumor with papillary pattern was lower apoptotic index. index. Furthermore, a high bcl-2 expression level and low apoptotic index were associated with a reduced survival rate. Because overexpression of bcl-2 inhibits anticancer drug-induced apoptosis as well as spontaneous apoptosis of the cancer cells, resistance to chemotherapy is one of the reasons why patients with high bcl-2 and low apoptotic index showed a reduced survival rate 17. Clear cell adenocarcinoma of the ovary is frequently associated with endometriosis, and it has been suggested that the tumor may develop from endometriotic tissue 2, The bcl-2 expression found in the epithelium of the endometriotic tissue adjacent to the clear cell adenocarcinoma suggests that the abnormal expression of bcl-2 may be involved early in the multi-step transformation of endometrial epithelium.

9 PROGNOSTIC SGNIFICANCE OF CLEAN CELL OVAVIAN ADENOCARCINOMA 145 Fig. 6. Overall survival of patients with clear cell adenocarcinoma categorized according to the cell types and structural patterns of the tumors. A; Kaplan-Meier curves estimated survival for cell types of the tumors. B; Kaplan-Meier curves estimated survival for structural patterns of the tumors. Survival of patients with the papillary pattern was shorter than with the tubulocystic pattern. Mutated p53 is a well-known anti-apoptotic factor in cancer cells, but expression of p53 was not seen at a high frequency in the clear cell adenocarcinomas in our study 22. No inverse relationship was observed between the apoptotic index and the expression level of p53. Thus, the anti-apoptotic pathway through the overexpression of bcl-2 is more important than abnormality of p53 in clear cell adenocarcinomas. Because many chemotherapeutic agents exert their antitumor effects by inhibiting the cell cycle, the proliferative activity of cancer cells is known to be related to their chemosensitivity. Itamochi et al examined the Ki- 67 labeling index in 41 cases of clear cell adenocarcinoma and 90 cases of serous adenocarcinoma, and reported that the Ki-67 labeling index was significantly lower in the tumors of clear cell adenocarcinoma than in the tumors of serous adenocarcinoma. They also reported that the Ki-67 labeling index in the tumors was significantly higher in the responders than in the nonresponders to chemotherapy 5. In our study, a high mitotic index and high Ki-67 labeling index tended to be associated with a reduced survival rate, however, the differences were not significant. Thus, apoptosisrelated factors seem to be more important in the prognosis of clear cell adenocarcinoma. In relation to the structure of clear cell adenocarcinoma, Okuda et al clearly demonstrated that patients with the papillary tumor structure showed a significantly shorter survival 11,12. Nakano et al also reported that 13 of the 31 clear cell adenocarcinomas in their series exhibited the papillary structure, and that the prognosis of these patients was poor 13. Also in our study, the tumors with the papillary structure were significantly associated with lower apoptosis and shorter survival. In addition, the clear cell adenocarcinomas that were mainly composed of hobnail cells showed significantly higher expression of bcl-2 and tended to be associated with shorter survival. Thus, the cases of clear cell adenocarcinomas with poorer prognosis associated with higher expression of bcl-2 and lower apoptosis may be discerned by the morphological characteristics of routinely sampled specimens. The results of the current study suggest that a low apoptotic activity controlled by bcl-2 correlated with shorter survival in patients with ovarian clear cell adenocarcinoma and that that was one of the causative factors of the poor prognosis. The cases with poorer prognosis associated with higher expression of bcl-2 and lower apoptosis may be discerned by the morphological characteristics of routinely sampled specimens. Acknowledgments I would like to express my gratitude to Dr. Morio Koike, Dr. Touichiro Takizawa, Dr. Yoshinobu Eishi, Dr. Takumi Akashi, Dr. Daisuke Kobayashi, Department of Human Pathology, and Dr. Toshiro Kubota, Department of Comprehensive Reproductive Medicine. I also wish to thank Dr. Katsuiku Hirokawa, Department of Comprehensive Pathology, for his very precious advice, and Mr. Noboru Ando and Mr. Keisuke Uchida, technical specialists, who have lent me a supporting hand throughout the course of this study.

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