Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer

Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P= 0.005 Spearman's rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary. © 2001 Cancer Research Campaign http://www.bjcancer.com

We have previously shown that several cytochrome P450 enzymes (CYP1A, CYP1B1, CYP2C and CYP3A) are overexpressed in a variety of tumours (Murray et al, , 1994(Murray et al, , 1997McKay et al, 1993;Murray 2000). In particular CYP1B1 appears to be a tumour-related form which is over-expressed in a number of primary tumours (Murray et al, 1997 but is undetectable in normal tissues especially liver. We have shown strong CYP1B1 immunoreactivity in a small number of primary serous cystadenocarcinomas the most common histological subtype of ovarian cancer (Murray et al, 1997). However, its presence in metastatic tumours has not previously been investigated.
We have recently shown that CYP1B1 inactivates docetaxel and our in vitro studies have shown that the presence of CYP1B1 in cells exposed to docetaxel increases the resistance of those cells to the cytotoxic effects of this anti-cancer drug (McFadyen et al, 2001;Rochat et al, 2001). The findings that CYP1B1 may inactivate docetaxel, a drug currently used as second line treatment of ovarian cancer, and which is currently being evaluated for licensing as first line treatment of ovarian cancer, also necessitated a clearer understanding of the presence of CYP1B1 in the different histological subtypes of both primary and metastatic ovarian cancer. This study investigated the presence of CYP1B1 in both primary and metastatic ovarian cancer.

Tissue
Samples of ovarian cancer and corresponding normal ovary submitted to the Department of Pathology, University of Aberdeen for diagnosis, over a 5-year period (1993)(1994)(1995)(1996)(1997)(1998), were used in this study. Samples of primary ovarian cancer were available from 167 patients. Of these, 43 patients had corresponding synchronous omental metastases, (i.e. both primary and metastatic tumour available for study). In addition a further 5 cases from patients with metastatic tumours were available which had no corresponding primary tumour (i.e. a total of 172 cases 167 + 5 = 172). In 49 out of the 167 patients with primary ovarian cancer contralateral normal ovarian tissue was also available for study. All the tissue samples had been fixed in 10% neutral buffered formalin for 24 hours and then routinely processed to paraffin wax.
The diagnosis of ovarian cancer was performed with haematoxylin and eosin stained sections using standard histopathological criteria. All tumours were reviewed by a specialist gynaecological pathologist (IDM). The tumours were classified according to criteria described by FIGO (Federation Internationale de Gynecologie et d'Obstetrique). The median age of patients in this study was 63 years with an age range from 30-89 years (Table 1). Of the 172 patients used in this study information on treatment was available for 170 and details are listed in Table 2. The following anti-cancer drugs (cisplatin, carboplatin, cyclophosphamide, paclitaxel, and docetaxel) were used to treat the patients. Most patients received either cisplatin or carboplatin. The other 3 drugs were usually given in combination in a platinum-based regime.
Following diagnosis, the disease status of patients was assessed by 2 gynaecological oncologists as follows, at 3 monthly intervals for the first year, at 6 monthly intervals for the following 2 years, and thereafter. If patients had stable disease or were disease-free, they were seen at yearly intervals. Following surgical debulking only 19% of patients remained disease-free over the period of follow-up, 42% had recurrent disease with a median disease-free interval of 23 months and an overall disease-free range of 1 to + 70 months, 39% of the patients had incomplete tumour debulking at surgery and showed no disease-free interval. The median overall survival of the patients was 17 months with an overall survival range of 1 to at least +70 months.

Immunohistochemistry
In this study a monoclonal antibody (MAb) raised against a 15 amino acid peptide located in the C-terminal third of the human CYP1B1 protein (McFadyen et al, 1999) was used to identify sites of CYP1B1 immunoreactivity. Immunohistochemical detection of CYP1B1 was performed using a peroxidase-based tyramine signal amplification method as described previously (McFadyen et al, 1999).
Sections of normal ovary and ovarian cancer were dewaxed, rehydrated and washed in cold water, prior to heat-based antigen retrieval in 0.01 M citrate buffer, pH 6 for 20 minutes. The sections were then immunostained with the anti-CYP1B1 monoclonal antibody. Sites of CYP1B1 immunoreactivity were identified using a fluorescein tyramide technique (NEN, Hounslow, Middlesex, UK) with diaminobenzidine as the enzyme substrate. Sections of breast cancer which had previously been shown to contain CYP1B1 by immunohistochemistry (McFadyen et al, 1999) were used as the positive control. The negative control used TBS in place of the primary antibody.
To establish the presence or absence of CYP1B1 and its distribution, intensity and cellular localization, the sections were counterstained with haemotoxylin and examined using bright field light microscopy by 2 independent observers (MCEM, GIM). CYP1B1 immunoreactivity in the tumours was assessed as either strong (3), moderate (2), weak (1) or negative (0). Tumours exhibiting CYP1B1 immunoreactivity in more than 5% of the cells were considered as positive. A tumour was classified as negative if less than 5% of tumour cells showed positive immunoreactivity (McFadyen et al, 1999).

Statistics
Statistical analysis including survival analysis was performed using SPSS version 7.5 for Windows 95™. The comparison between CYP1B1 expression in primary and metastatic ovarian cancer was performed using the Spearman's rank correlation test. Comparison between survival, CYP1B1 status and the different chemotherapeutic treatments was performed with the Kaplan-Meier log rank test.

Immunohistochemistry
Primary ovarian cancer CYP1B1 immunoreactivity was identified in the majority (92%) of ovarian cancers and was specifically localized to the cytoplasm of   tumour cells (Figure 1). In addition, there was no apparent intratumour heterogeneity. In a high percentage of the ovarian cancers there was moderate (23%) or strong (51%) immunoreactivity for CYP1B1, while in a minority of cases (17%) there was weak immunostaining for CYP1B1 only 8% of ovarian cancers showed no CYP1B1 immunostaining (Table 3). The different stages of ovarian cancer all exhibited a high level of CYP1B1 expression: stage 1, 91%; stage 2, 73%; stage 3, 95% and stage 4, 90%. A high level of expression was also observed for the different histological subtypes, serous 92%; mucinous 87%; endometrioid 94%; mixed malignant Mullerian 100% and clear cell 86%.

Metastatic ovarian cancer
The presence of CYP1B1 was observed in the majority (94%) of metastases with a high proportion showing moderate (46%) or strong (38%) immunoreactivity, weak (10%) immunoreactivity or absence (6%) of immunoreactivity was found in a minority of cases (Table 4). There was no detectable CYP1B1 expression in any of the normal ovarian tissue samples (Figure 1). Similar levels of CYP1B1 expression were exhibited for the different histological subtypes in both primary and metastatic tumour (serous, mucinous endometrioid and clear cell) (Tables 3  and 4). The comparison between CYP1B1 expression in primary ovarian cancer and metastatic deposits showed a significant correlation (P = 0.005 Spearman's rank correlation test) ( Table 5).
In this investigation there was no significant difference in survival of patients on different chemotherapeutic treatments. In addition, the presence of CYP1B1 generally had no effect on the survival of patients on the different treatments. However, in the small group of patients treated with docetaxel either singly or in combination with a platinum-based agent (n = 20), a trend towards a poorer overall survival was observed, in those patients with moderate or strong CYP1B1 immunoreactivity (Kaplan-Meier log rank test P = 0.2716) (Figure 2).

DISCUSSION
This study has investigated the presence of CYP1B1 in a large number of both primary and metastatic ovarian tumours, utilizing a monoclonal antibody to CYP1B1 to demonstrate the localization of CYP1B1 to ovarian tumour cells and lack of expression in normal ovarian tissue. The over-expression of CYP1B1 was observed in all the histological subtypes of epithelial ovarian cancer and at high frequency. This is in agreement with a pilot study which utilized a polyclonal antibody to CYP1B1 (Murray et al, 1997), and showed the over-expression of CYP1B1 in a small number (n = 7) of serous cystadenocarcinomas investigated. This is the most common histological subtype found in patients suffering from this malignancy. We have previously shown the over-expression of CYP1B1 protein in a wide variety of primary malignant tumours of different histogenetic types and the lack of detectable CYP1B1 protein in normal tissue (Murray et al, 1997). However, no information is available on the presence of CYP1B1 in metastatic disease. It is important to determine the phenotype of the metastatic disease as it may not reflect that of the primary tumour.
Due to the lack of available tissue from metastatic deposits, tissue from the primary tumour is often used to predict the response of the metastatic tumour to chemotherapy . This may not be a true reflection of the biological phenotype of the metastatic tumour, indeed differential expression of several proteins has previously been observed between primary and metastatic tumours . These differences in expression are likely to affect the response of metastatic lesions to chemotherapy. Spread of ovarian cancer is through the peritoneal cavity and one of the key findings of this current study is the high frequency of CYP1B1 over-expression observed in the majority of metastatic tumours. Indeed this study is the first to describe the over-expression of CYP1B1 in metastatic tumours. A highly significant correlation of CYP1B1 immunoreactivity was observed  between the primary and metastatic tumours (P = 0.005 Spearman's rank correlation test).
In this study a trend suggesting a poorer survival was shown in those patients with moderate or strong CYP1B1 immunoreactivity who received docetaxel either as a single agent or in combination with a platinum-based agent, compared to those patients with low or negative CYP1B1 immunoreactivity. Although, this finding was not statistically significant (Kaplan-Meier log rank test P = 0.2716), this may be due to the small number of patients treated with docetaxel. However, this finding supports our in vitro studies and the concept that CYP1B1 inactivates docetaxel and increases the resistance of cells expressing CYP1B1 to the cytotoxic effects of this anti-cancer drug (McFadyen et al, 2001;Rochat 2001). Therefore, further investigation with a larger population is required to ascertain whether CYP1B1 does contribute significantly to the response of ovarian cancer to docetaxel.
We have recently shown that CYP1B1 interacts with a number of structurally diverse clinically useful anti-cancer agents in a substrate-dependent manner (Rochat et al, 2001). Therefore, in addition to being a mechanism of docetaxel drug resistance it is likely that CYP1B1 is a mechanism of drug resistance to a variety of anti-cancer drugs. The findings that CYP1B1 immunoreactivity was over-expressed in the majority of primary and metastatic tumours, and the lack of detectable CYP1B1 in normal tissue including the ovary, also highlights CYP1B1 as a target for the development of novel chemotherapeutic agents which could be activated by this P450 (Murray et al, 1997;McFadyen et al, 1999).