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Introduction

Ovarian epithelial carcinoma is classified into four morphologically distinct categories: serous, mucinous, endometrioid and clear-cell. Serous adenocarcinoma accounts for ~50% of all ovarian carcinoma (1). Ovarian serous adenocarcinoma cases (~70%) have progressed into the abdominal cavity at the time of diagnosis, giving them a stage III classification, based on the International Federation of Gynecology and Obstetrics (FIGO) criteria. As it is difficult to completely remove most such tumors by surgery, response to chemotherapy determines their prognosis. Although ovarian serous adenocarcinoma is highly sensitive to chemotherapy, many cases relapse. Corrected actuarial survival rates are 41.0% at 5 years for all stages of ovarian serous adenocarcinoma, 35.1% for stage III, and 19.2% for stage IV (1). Reduction of recurrence for patients with partial or complete response to chemotherapy would greatly improve their prognoses.

Mitotic-arrest deficiency 2 (MAD2) was the first gene of the mammalian mitotic spindle checkpoint pathway to be characterized (2). MAD2 localizes at kinetochores after chromosome condensation and before anaphase (3); it significantly affects the transition from metaphase to anaphase by inhibiting the anaphase-promoting complex/cyclosome (APC/C). This process ensures that the chromosomes are correctly aligned at the metaphase plate prior to daughter-cell segregation (4,5). Therefore, MAD2 is a key component of the mitotic spindle checkpoint pathway, which plays a crucial role in preventing loss or gain of chromosomes within cells (6). A compromised mitotic spindle checkpoint results in an abnormal number of chromosomes, known as chromosomal instability (CIN) (7). CIN is characterized by alterations in chromosome number and is commonly detected as aneuploidy (8,9); it has been reported in most types of human cancer. Although the underlying molecular mechanisms are unclear, MAD2 overexpression in transgenic mice notably results in CIN and initiates carcinogenesis in a wide variety of tumors (10). Some studies suggest that a compromised mitotic spindle checkpoint, through MAD2 overexpression, is a significant step in malignancy progression; in fact, MAD2 overexpression is observed in several cancers (11–15).

We have previously reported that MAD2 overexpression occurs in most cases of mucinous ovarian carcinomas and that it is potentally correlated to mucinous ovarian carcinogenesis (16). However, MAD2 overexpression alone may not be sufficient for mucinous ovarian carcinogenesis. Some studies suggest a correlation between MAD2 overexpression and various clinicopathological characteristics, such as histological grade (differentiation), metastasis and prognosis (11,12,14,15). However, reduced MAD2 expression occurs in some human cancers (4,17) and is associated with in vitro resistance to chemotherapies that use microtubule-targeting agents or DNA-damaging agents (18,19). In this study, we examined the relationship between MAD2 expression and chemotherapy resistance in ovarian serous adenocarcinoma.

Materials and methods

Patients and samples

We initially treated 63 cases of ovarian serous adenocarcinoma at Osaka City University Medical School Hospital (Osaka, Japan), between 2000 and 2007. In this study, we reviewed 41 cases in which maximum debulking surgery and platinum-based anticancer drugs as first-line chemotherapy were used, and from patients who had partial or complete responses from this chemotherapy. Tumor samples were obtained following primary surgery. Written informed consent was obtained from all patients prior to immunohistochemical examination. This study was approved by the Ethics Committee of Osaka City University (IRB no. 2201).

Of the 41 cases, 24 were recurrent (relapsed group), and 17 were disease-free (relapse-free group). Based on the FIGO criteria, 1 case in the relapsed group was classified as stage I, 1 as stage II, 19 as stage III and 3 as stage IV. In the relapse-free group, 3 cases were classified as stage I, 0 as stage II, 14 as stage III and 0 as stage IV (Table I).

Table I Patient characteristics.

Table I

Patient characteristics.

	Relapsed	Relapse-free	P-value
No. of cases	24	17
Age			NS
Mean	56.0	53.2
Range	36–82	26–73
FIGO stage			NS
I	1	3
II	1	0
III	19	14
IV	3	0
Chemotherapy			NS
Taxane + carboplatin	20	15
Platinum only	4	2

[i] FIGO, International Federation of Gynecology and Obstetrics; taxane, paclitaxel or docetaxel; platinum only, cisplatin or carboplatin only; NS, none significance.

Immunohistochemical analysis

The expression of MAD2 was investigated in paraffin-embedded sections, using a MAD2 antibody and the avidin-biotin peroxidase complex method. Paraffin sections (4-μm) were de-paraffinized and immersed in 3% hydrogen peroxidase in methanol to block endogenous peroxidase activity. Next, an antigen retrieval procedure was performed by immersing the slides in 10 mM citrate buffer (pH 6.0) and heating in an autoclave at 110°C for 20 min. The sections were then washed in phosphate-buffered saline (PBS). The protocol for the Dako LSAB 2 peroxidase kit (Dako, Kyoto, Japan) was followed. The sections were incubated with the primary antibodies for 2 h at room temperature. The primary antibody used for this study was monoclonal rabbit anti-human MAD2 (1:200; Proteintech Group, Inc., Chicago, IL, USA). Sections were rinsed with PBS for 15 min and incubated for 10 min with secondary antibody (biotinylated goat anti-mouse and rabbit immunoglobulin G; Dako). Sections were then incubated with the streptavidin-peroxidase complex; 3,3′-diaminobenzidine was used as chromogen. Sections were then counterstained with Mayer's hematoxylin. Specificity of immunohistochemical reactions was checked by omitting the primary antibody. Quantitative analysis of MAD2 expression was based on the scoring method of Sinicrope et al (20). Briefly, mean percentages of positive tumor cells were determined in five separate areas (magnification, ×400) and assigned to the following values: 0, <5%; 1, 5–25%; 2, 25–50%; 3, 50–75% or 4, >75%. Immunostaining intensity was scored as: 1+, weak; 2+, moderate or 3+, intense. For each specimen, percentage of positive tumor cells was multiplied by staining intensity to produce a weighted score.

Statistical analysis

Kaplan-Meier and log-rank tests were used for the prognostic analyses. StatView 5.0 (Abacus Concepts, Berkeley, CA, USA) was used for data analysis and P<0.05 was considered significant. Continuous variables were expressed as mean ± standard deviation (SD) or mean ± standard error (SE), as shown in the figures. Weighted scores were compared using the Mann-Whitney U test.

Results

Patient characteristics

The mean age was 56.0 years (range, 36–82) in the relapsed group and 53.2 years (range: 26–73) in the relapse-free group. No significant difference was observed among the two groups (Table I). All patients recieved platinum-based chemotherapy, which was mostly taxane (paclitaxel or docetaxel)/carboplatin.

MAD2 expression

MAD2 was expressed in tumor cell nuclei (Fig. 1). Mean weighted scores were 4.3 in the relapsed and 7.2 in the relapse-free group (Table II), and were thus significantly greater in the relapse-free than in the relapsed group (Fig. 2).

Figure 1 Immunohistochemical staining of MAD2 in ovarian serous adeno-carcinoma. (A) Relapse-free group, score 12; (B) relapsed group, score 8; (C) relapsed group, score 4; (D) relapsed group score 0 (A-D, magnification, ×400). MAD2 was expressed in nuclei of tumor cells. Hematoxylin and eosin stain.

Figure 2 Weighted score for MAD2 in ovarian serous adenocarcinoma. MAD2 expression was significantly greater in the relapse-free group than in the relapsed group. *P=0.023 (Mann-Whitney U test).

Table II Weighted scores in relapsed and relapse-free groups.

Table II

Weighted scores in relapsed and relapse-free groups.

Weighted-score	Relapsed (n)	Relapse-free (n)	Total (n)
0	2	0	2
1	3	3	6
2	3	1	4
Total, n (%)	8 (33.3)	4 (23.5)	12 (29.3)
3	4	0	4
4	2	1	3
6	5	3	8
Total, n (%)	11 (45.8)	4 (23.5)	15 (36.6)
8	4	0	4
9	0	4	4
12	1	5	6
Total, n (%)	5 (20.8)	9 (52.9)	14 (34.1)
Weighted score, mean	4.3	7.2	5.5

Survival

In the relapsed group, mean progression-free survival was 18.8 months (range, 3–49). Mean overall survival was 36.7 months (range, 8–66) for the relapsed group and 73.9 months (range, 46–119) for the relapse-free group.

Grade of MAD2 expression

The 41 cases were re-classified as showing low or high MAD2 expression. The low expression group contained 24 cases with weighted scores from 0–6 and the high expression group contained 17 cases with weighted scores from 8–12. The mean age of the low expression group was 57.1 years (range, 26–82) and that of high expression group was 50.4 years (range, 34–73). Based on the FIGO criteria, 3 low-expression cases were classified as stage I, 1 as stage II, 20 as stage III and 3 as stage IV; among high-expression cases, 1 was classified as stage I, 0 as stage II, 13 as stage III and 0 as stage IV. In the low-expression group, 19 patients relapsed and 17 died; in the high-expression group, 5 patients relapsed and 3 died (Table III). There was no significant difference in the estimated mean progression-free survival between the low-expression group (47.6 months) and the high-expression group (67.1 months) (P=0.0685; Fig. 3). However, overall estimated mean survival was significantly shorter for the low-expression (61.7 months) than for the high-expression group (82.0 months) (P=0.0188; Fig. 4).

Figure 3 Progression-free survival according to level of MAD2 expression. The low expression group (weighted score, 0–6) contained 24 cases and the high expression group (weighted score, 8–12) contained 17 cases. Relapse rates were significantly different between the low expression group, with 19 cases (70.3%) relapsed and the high expression group with 5 cases (35.7%), but these groups showed no significant difference in progression-free survival (P=0.0685; Kaplan-Meier and log-rank tests).

Figure 4 Overall survival according to MAD2 expression levels. The low expression group (weighted score, 0–6) contained 24 cases and the high expression group (weighted score, 8–12) contained 17 cases. In the low-expression group, 17 cases were dead of disease (63.0%) and 3 cases with the high-expression group were dead of disease (21.4%). Death rates differed significantly between the low- and high-expression groups. In addition, there was a significant difference between the low- and high-expression groups in terms of overall survival (P=0.0188; Kaplan-Meier and log-rank tests).

Table III Clinical background of cases with low and high expression of MAD2.

Table III

Clinical background of cases with low and high expression of MAD2.

	Low expression (score: 0, 1, 2, 3, 4, 6)	High expression (score: 8, 9, 12)	P-value
No. of cases	27	14
Age			NS
Mean	57.1	50.4
Range	26–82	34–73
FIGO stage, n			NS
I	3	1
II	1	0
III	20	13
IV	3	0
Relapses, n(%)	19 (70.3)	5 (35.7)	<0.05
Deaths, n(%)	17 (63.0)	3 (21.4)	<0.05

[i] FIGO, International Federation of Gynecology and Obstetrics; NS, not significant.

Discussion

MAD2 is a key component of the mitotic spindle checkpoint pathway, which if compromised, can result in CIN and tumorigenesis. MAD2 overexpression has been shown to promote aneuploidy, tumorigenesis and tumor progression (21). Reportedly, MAD2 overexpression is a critical mediator of CIN, as seen in inactivation of p53 and Rb pathways (22). MAD2 overexpression has also been observed in some human cancers (11–15). A correlation has been implied between MAD2 overexpression and various clinicopathological characteristics, such as histological grade (differentiation), metastasis and prognosis (11,12,14,15). In a study that assessed incidence of metastasis and survival time in 48 cases of human osteosarcoma, MAD2 overexpression was associated with early metastasis and poor prognosis (14). Another study showed MAD2 overexpression to be associated with lung cancer recurrence in mice (23).

In the present study, 39 of 41 samples (95.2%) were stained positively for MAD2. Of the 41 cases, 24 were recurrent and 17 disease-free (Table I). Mean weighted scores for MAD2 expression were significantly less for the relapsed (4.3) than for the relapse-free group (7.2) (Fig 2; Table II). When the 41 cases were classified into 24 low-expression (weighted score, 0–6) and 17 high-expression (weighted score, 8–12) cases (Table III), they showed no significant difference in progression-free survival, but overall survival for the low-expression group was significantly shorter for the high-expression group (Figs. 3 and 4). In this study, 87.8% (36/41) of cases were in advanced stages. However, the above-mentioned study (14) indicated significant association between MAD2 expression and clinical staging, which implies that poor prognoses reflect clinical staging rather than MAD2 expression. Adjuvant chemotherapy in our study could have affected prognoses, implying a relationship between MAD2 expression and chemotherapy resistance.

MAD2 is a key component of spindle assembly checkpoint (SAC) function which meditates attachment of spindle microtubules to kinetochores and accurate chromosomal segregation during mitosis (24,25). One review focused on chemoresistance to taxane treatment in breast cancer, particularly in relation to the SAC and dysfunctional regulation of apoptotic signaling (26). Reportedly, cellular senescence induced by the low MAD2 expression affects paclitaxel sensitivity (27). Low MAD2 expression is also reportedly associated with resistance to paclitaxel in ovarian cancer cells (28) and gastric cancer cells (29), and Bcl-2 may be involved in this process. While failure of SAC function does not fully explain chemotherapy-resistant acquisition, it appears to take an important role. By extension, MAD2 expression level could be a predictor of chemoresistance. Our previous study suggested MAD2 expression to predict efficacy of neoadjuvant chemotherapy for locally advanced uterine cervical cancer (30). In this study, while progression-free survival between groups expressing low and high levels of MAD2 did not significantly differ (P=0.0685; Fig. 3), those expressing lower MAD2 levels had significantly shorter overall survival (P=0.0188; Fig. 4). Therefore, MAD2 expression may be an important predictor of prognosis for ovarian serous adenocarcinoma.

In conclusion, the results of the present study suggest that levels of MAD2 expression can help predict sensitivity to anticancer agents and risk of recurrence for these patients.

Acknowledgements

We thank the gynecologists at Osaka City University Medical School Hospital for their support (Osaka, Japan). This study was supported by the Osaka Medical Research Foundation for Incurable Diseases.

References