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J. Obstet. Gynaecol. Xes. Vol. 27, No. 6: 313-318 2001

Clinical Factors and Biomarkers Which Affect a New Universal Grading System for Ovarian Epithelial Carcinoma

Shin-ichi Ishioka, Satoru Sagae, Masaki Sugimura, Yoshihiro Nishioka, Kanji Kobayashi, and Ryuichi Kudo Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, Sapporo, Japan

Abstract

Objective: To detect clinical factors and biomarkers which affect a new grading system for ovarian epithelial cancer that was proposed by Shimizu et al. and to analyze the impact of those factors on malignant behaviors of the tumor.

Methods: Review and scoring of specimens of paraffin embedded tissues of epithelial ovarian cancer were carried out according to the new universal grading system in 110 women who underwent initial treatments including the primary operation in our university hospital between January 1990 and June 1999. As biomarkers, expression of Bcl-2, Bax, Bcl-X, and the accumulation of P53 protein were also studied immunohistochemically.

This grading system functioned as a prognostic indicator. Five-year survivals of the patients was 77.6, 36.3, and 17.4%, for Grade 1, Grade 2, and Grade 3, respectively. The grade was also correlated with clinical stages, histologic subtypes, operative completeness, para-aortic lymph nodes metastasis, and the expression of Bax protein, with univariate analysis. Multivariate analysis revealed histological subtypes and para-aortic lymph nodes metastasis to be important factors which affected the grading system.

Conclusion: These results demonstrate that the new universal grading system is useful, and that this grading system might reflect potential of metastasis or dissemination of the ovarian cancer.

Key words: histologic grade, ovarian cancer, P53, Bax

Results:

Introduction

Histologic grade is known to be an important prognostic factor in ovarian cancer.') Until now, various grading systems such as the International Federation of Gynecology and Obstetrics (FIGO) grading system:) the World Health Organization (WHO) grading system?) and the Gynecologic Oncology Group (GOG) grading system4) have

been used in different institutes. However, those grading systems have difficulties in terms of reproducibility and subjectivity. Recently, Shimizu et al . reported a new universal grading system for epithelial ovarian cancer which was based on the architectural grade, nuclear grade, and mitotic ~ o u n t . ~ , ~ ) They concluded that their grading system provided useful prognostic information for all histologic types of ovarian cancer, and that it

Received: Feb. 14,2000 Accepted: Sep. 14,2001 Reprint request to: Dr. Satoru Sagae, Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, Minami 1- jo, Nishi 16-chome, Chuou-ku, Sappopro, Hokkaido 0604061, Japan

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might reflect the biological behavior of tumor cells.6)

In the present study, we reviewed all the paraffin embedded tissues of epithelial ovarian cancer obtained from 110 women who underwent initial treatments including primary surgery in our hospital between January 1990 and June 1999, and scored the specimens according to the universal grading system. Then we assessed the usefulness of this grading system by studying relationships between this grading system and clinical factors. Furthermore, expression of various biomarkers such as P53, and bcl-2 family genes products has recently been reported to be related to the prognosis of patients with ovarian cancer.’) Therefore, we also focused on the relationships between this grading system and the expression of such biomarkers in ovarian cancer in this study.

Materials and Methods

Patients Patients with available histopathologic slides

who were initially diagnosed at Sapporo Medical University as having malignant epithelial ovarian carcinoma between January 1990 and June 1999, were included in the study when they received initial treatments, including primary surgery, in our university hospital. Those who received primary surgery in other hospitals were excluded. Patients with borderline malignant tumors, non-epithelial tumors, metastatic ovarian tumors, and tumors of unknown origin, were also excluded.

Histologic analysis utilized hematoxylin and eosin-stained sections cut from 2 to 24 blocks per case.

All the slides were screened by three indepen- dent observers (S.1, S.S, K.K) without knowledge of the clinical course of the patients. When a con- sensus was not reached, S.S. decided the final grade.

Diagnosis of Histologic Type and the New Grading System

The classification of histologic subtypes was performed according to the World Health Orga- nization classification of the surface epithelial- stromal category tumor. The grade for an indi- vidual tumor was determined according to the method by Shimizu et al. Briefly, architectual grading was scored as follows; score 1 for glan- dular growth pattern, score 2 for papillary growth pattern, and score 3 for solid growth pat-

tern. Nuclear pleomorphism was scored as follows; score 1 for relatively uniform vesicular nu- clei (variation in diameter < 2 : l), a low nuclear: cytoplasmic ratio, no chromatin clumping or prominent nucleoli, score 2 for intermediate variation in nuclear size (between 2 : 1 to 4 : 1) and shape, nucleoli recognizable but small, some chromatin clumping, no bizarre cells, and score 3 for marked variation in nuclear size (> 4 : 1) and shape, a high nuclear: cytoplasmic ratio, prominent chromatin clumping, thick nuclear mem- branes, large eosinophilic nucleoli, and any bizarre cells which may exist. Mitotic count was scored as follows; score 1 for 0-9 mitotic cells/lO HPF (1OX wide field eyepiece, 40X objective), score 2 for 10-24/10 HPF, score 3 for 25 I110 HPF.

The final grading was made as follows; total score 3-5 for Grade 1 (well differentiated), total score 6-7 for Grade 2 (moderately differentiated), and 8-9 for Grade 3 (poorly differentiated)?) For the determination of the grade, at least three slides suitable for histologic typing and grading of the tumor were selected. In particular, we tried to find slides that included the periphery of the tumor at which active growth was most likely.

lmmunohistochemical Staining Anti-P53 antibody, anti-Bcl-2 antibody, anti-

Bax antibody, and anti-Bcl-X antibody were pur- chased from DAKO. All samples were fixed in formaldehyde and embedded in paraffin. Sec- tions 6-pm-thick were routinely passed through xylene and a graded alchol series and stained for the antibodies by Envision method (DAKO). Sec- tions were counterstained with hematoxylin for microscopic examination. Whole cancer tissues on each slide were examined, and carcinomas in which 30% or more of the component cells were stained for the antibodies were evaluated as positive (+), whereas tissues containing less than 30% stained cells were counted as negative (-).

Statistical Analyses Survival curves were calculated by the Kaplan-

Meier method. The significance of differences between curves was evaluated with the generalized Wilcoxon test. Multivariate analysis was performed by multiple regression analysis with the Microsoft Excel. Other statistical analyses were carried out by x’ test or Student’s t-test. All results were deemed significant at p < 0.05.

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Results

Characteristics of Patients The relationship of FIGO surgical stage and

histologic subtypes of the patients is shown in Table 1. Majority of patients with serous carcinoma had advanced stage disease at the time of diagnosis, whereas almost half of those with clear cell, endometrioid, or mucinous carcinoma were diagnosed at earlier stages. As to operative treatment modality, total hysterectomy, bilateral sal- pingo-oophorectomy, omentectomy, and pelvic and para-aortic lymphadenectomy were performed as a standard treatment modality for the patients. However, for those who had extremely advanced disease, for those who needed preser- vation of ovarian function, and for those who had severe complication, modified operative proce- dures were performed. As to chemotherapy, 73 patients received platinum-based chemotherapy, 24 patients received paclitaxel-based chemotherapy, 9 received CPT-1 1-based chemotherapy, and 7 received no chemotherapy for severe complication or rejection of further treatments.

Survival of the Patients by FlGO Stage The 3-year and 5-year survivals of the 110 pa-

tients by FIGO stage are described in Table 2. Pa-

tients in advanced stages had poorer prognosis than those in earlier stages. Poorer prognosis of patients with Stage 11, might have been derived from fewer number of patients with Stage 11. FIGO stage was thought to be a significant prognostic factor.

Prognostic Significance of the New Grading System As shown in Table 3, this universal grading

system worked as a significant prognostic indicator in patients with ovarian cancer who were en- rolled in this study. Five-year survivals of patients were 77.6, 36.3, and 17.4%, for Grade l, Grade 2, and Grade 3, respectively. Patients with Grade 1 showed a significantly better prognosis than those with higher grades. Among patients with Stage 1/11 and Stage III/IV, those with Grade 1 also showed better prognoses than those with higher grades, though the differences were not statistically significant.

Table 3 shows the 5-year survivals of patients in the major histologic types in each grade. For serous and mucinous types of cancer, this grading system worked as a significant predictor of survival. For endometrioid and clear cell cancers, patients with Grade 1 showed a better prognosis than those with higher grades, though the differences were not significant.

Table 1. noma

Relationship between histological subtypes and FIGO clinical stages in patients with ovarian carci-

~~ __ Histological subtypes I

FIGO stage

I1 111 IV Total (YO)

Serous 11 1 35 7 54 (49.1) Mucinous 8 1 5 1 15 (13.6) Endometrioid 7 2 5 0 14 (12.7) Clear cell 11 2 9 2 24 (21.8) Others* 1 1 0 1 3 (2.8) Total 38 (34.5%) 7 (6.4%) 54 (49.1%) 11 (10.0%) 110 (100)

* Others include mixed epithelial ovarian cancer and undifferentiated ovarian cancer.

Table 2. Three- and 5-year survival of the patients by the clinical stages

Clinical stage ( n = ) 3-year survival (YO) 5-year survival (%)

Iab (15) 100 100 Iab: Ic, N.S

11 (7) 43.0 43.0 IIIab (14) 83.9 47.9 IIIab: IIIc, p < 0.05 IIIc (40) 32.0 25.6 IIIc: IV. p < 0.05

Ic (23) 85.6 70.6 Ic: IIIc, p < 0.01

IV (11) 19.1 0

Five-year survivals were determined by the Kaplan-Meier method. p values are determined by the Generalised Wilcoxon test. N.S means statistically not significant.

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Table 3. Five-year survival of the patients bv the new universal grading svstem

p-value Histology G1 G2 G3 ( n = 1 ( n = ) ( n = 1 ( n = )

All 77.6% 36.1% 17.4% G1 : G2, p < 0.01 (41) (53) (26) G2 : G3, p < 0.01

Histology Serous 100% 45.4% 19.2% GI : G2, p c 0.01 G2 : G3, p < 0.05 G1 : G2, p < 0.05

(24) -

(54) (8) (22) Mucinous 60.0% 20.0%

(15) (10) (5) (0) Endometrioid 100% 66.7% 50.0% N.S

(2) - N.S

(14) (7) (5)

(24) (14) (10) (0)

(45) (27) (14) (0)

(65) (14) (25) (26)

Clear cell 52.9% 46.7Yo

Stage I, I1 84.0% 63.8% - N.S

111, IV 50.8% 22.3% 19.0% N.S

Five-year survivals were determined by the Kaplan-Meier method. p values are determined by the Generalised Wilcoxon test. N.S means statistically not significant.

lmmunohistochemical Staining As shown in Table 4, expression of Bax protein

was significantly correlated with the grade. Among 41 G1 carcinomas, 30 (73.1%) were positive for Bax protein, whereas among 26 G3 carcinomas, only 8 (30.8%) were positive for Bax protein. As to the accumulation of P53 and the grade, the expression of Bcl-2 and Bcl-X protein and the grade, statistically significant relations were not obtained. However, a tendency was found for there to be a connection between negative P53 and G1, and negative Bcl-2 and G1, though it was not significant.

Univariate Analysis of Factors Which May Affect the New Grading System

As clinical factors which may affect the grade, the FIG0 clinical stage, histological subtype, size of the tumor, age of the patient, operative completeness, and existence of para-aortic lymph node (PAN) metastasis, were selected.

Tumor size was divided into groups as follows; less than 10 cm in diameter and 10 cm or larger in diameter. In 3 cases, we were unable to determine the size of the tumor from the operation record. Patients were divided into groups by age as follows: those less than 60 years old and those 60 or more years old. Operative completeness was divided into groups as follows; the size of residual tumor was less than 0.5 cm (optimal operation) and size of the residual tumor was 0.5 cm or larger (suboptimal operation). As to PAN metastasis, 56 of 110 cases received lymphadenec-

Table 4. Grade and the expressions of biomarkers

Grade 1 Grade 2 Grade 3 p value

P53 Positive Negative

Positive Negative

Positive Negative

Positive Negative

Bcl-2

Bax

Bcl-X

9 12 32 31

9 11 32 32

30 21 11 21

15 16 26 27

11 N.S 15

10 NS') 16

8 p =0.001 18

15 N.S 15

a) N.S means statistically not significant.

tomy or sampling of PAN. Those who only received palpation of PAN, and those for whom exploration of PAN was not performed were excluded.

As shown in Table 5, patients with advanced stages, those with the serous subtype, those with residual tumors 0.5 cm or larger, and those who had positive PAN metastasis, had significantly higher grades.

Multivariate Analysis of Factors Which May Affect the New Grading System

The universal grading system provided useful prognostic information even for our epithelial ovarian cancer cases. Univariate analysis revealed that the clinical stage, histological sub-

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Table 5. Univariate analysis of the grade by clinical factors

Index Grade 1 Grade 2 Grade 3 p value

Stage I I1 Ill IV

Histology Serous Mucinous Endometrioid Clear cell Others

Tumor size < 10 cm 2 10 cm

< 60 years old 2 60 years old

Residual tumor < 0.5 cm 2 0.5 cm

PAN meta Negative Positive

Age

23 15 0 4 3 0

13 21 20 1 4 6 < 0.001

8 22 24 10 5 0 7 5 2

14 10 0 2 1 0 < 0.001

15 16 12 24 26 14 N.S

27 30 13 14 13 13 N.S

32 25 6 9 18 20 < 0.001

1 6 12 5 3 1 p=0.004

N.S means, not significant.

Table 6. Multivariate analysis of the grade by some factors

Index Regression Order of coefficient correlation")

Clinical stage 0.059082 5 Histology - 0.33487 1* Operative completeness 0.098251 4 PAN metastasis 0.32338 2, Expression of Bax 0.221427 3

a): * indicates statistically significant correlation by multivariate analysis.

type, operative completeness, PAN metastasis, and the expression of Bax protein significantly affected the universal grading system. Multivariate analysis revealed the histological subtype and para-aortic lymph node metastasis to be two important factors which affected this grading system (Table 6).

Discussion

Although histologic grade has been regarded as an important prognostic factor in patients with ovarian cancer for years, universal agreement on the grading system has not yet been established. FIGO, WHO, GOG, and some other investigators

proposed their own grading systems.24) How- ever, they are complex and lack reproducibility because their systems are based either on architectural features, nuclear grade, a combination of both, or more detailed histopathologic features in addition to both architectural features and nuclear grade.

Shimizu et al. proposed a new universal grading system for epithelial ovarian cancer which was based on the architectural grade, nuclear grade, and mitotic ~ o u n t . ~ , ~ ) In this study, we reviewed all our specimens of epithelial ovarian cancer obtained between January 1990 and June 1999, scored the specimens according to their grading system, and then assessed the usefulness of the system. Furthermore, the relationships between the new universal grading system and some clinical characteristics or biomarkers were investigated in order to understand mechanism by which this grading system works with regard to prognostic factors.

As mentioned above, this universal grading system worked as a prognostic factor. Patients with lower grades had a better prognosis. How- ever, it must be stressed that neither mucinous nor clear cell cancer showed Grade 3 under our scoring. Usually, those 2 histologic types of tumors show less mitosis and less nuclear abnor- mality. For those tumors, it would be hard to score Grade 3, which needs a total of score 8-9. Therefore, it seems that an increase in Grade 2 is likely to occur under this grading system.

In this study, the grade was significantly correlated with the FIGO clinical stage, histological subtype, operative completeness, para-aortic lymph node metastasis, and the expression of Bax protein by univariate analysis. Furthermore, multivariate analysis revealed that both the histological subtype and para-aortic lymph node metastasis were 2 important factors which affected the universal grading system. These results might suggest that for cancers with higher grades it is generally hard to perform an optimal operation probably due to wide dissemination of the tumor at the time of the operation, and that these are likely to metastasize to para-aortic lymph nodes. Or it might be possible to say that serous ovarian cancer with Grade 3 has a high propen- sity for para-aortic lymph nodes metastasis. Shimizu et al. reported that their grading system might reflect the biological behavior of tumor cells6) Our results may support their hypothesis; that is to say, their grading system is likely to reflect the malignant potential of tumors, which is the potential for metastasis or dissemination of

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the tumor. In addition, in this study, we focused on the

accumulation of P53 protein and the expression of bcl-2 family proteins such as Bcl-2, Bax, and Bcl-X, as biomarkers which might affect the new grading system because they might affect the prognosis of patients with ovarian carcinoma through the mechanism of apoptosis. Accumula- tion of P53 protein on immunohistochemical staining, which is thought to indicate the pres- ence of mutant P53, has been regarded as a poor prognostic factor in some cancers, including ovarian carcinoma.') As mutant P53 protein is thought to have lost the ability to induce apoptosis in anticancer drug-treated cells, it can lead to drug resistance.") bcl-2 family proteins are also known to affect chemosensitivity by regulating the apoptotic cascade.') Bcl-2 protein has been reported to suppress apoptosis induced by anticancer drugs, whereas Bax and Bcl-X protein induce apoptosis.'")

In this study, a significant relationship between the expression of Bax protein and lower grades was detected. Positive Bax protein has been reported to be associated with a better prognosis in ovarian carcinoma.") Induction of apoptosis may lead to a good prognosis. Furthermore, a tendency was found for there to be a connection between negative P53 and Grade 1, and negative Bcl-2 and Grade 1 although it was not statistically significant. These results suggest that lower grade tumors have higher apoptotic potential with anti-cancer drugs, which may lead to a better prognosis. As mechanisms by which this grading system works as a prognostic factor, not only the potential of metastasis or dissemination of the tumor, but also the ability to induce apoptosis might be important. Our results showed the significance of the new universal grading system, and its relationship with some

clinical factors and biomarkers. However further studies must be done to improve this system better one.

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