Enzyme-Linked Immunosorbent Assay and Immunohistochemical Localisation

Abstract:

To evaluate the significance of including CEA in serum and tissues in the management protocol of patients with ovarian malignancies. This study was conducted on 68 patients; 21 with ovarian malignancies; 3 with borderline ovarian tumour; 8 with benign ovarian tumour and 36 women without any gynecological disorder as a control group.

Patients and Methods: The study included 68 patients were divided into 3 groups as follows:

• Group [A]: Included 21 patients with malignant ovarian tumours.
• Group [B]: Included 3 patients with borderline ovarian tumours and 8 patients with benign ovarian tumours 
• Group [C]: Included 36 women without any apparent gynaecologic disorder (control group)

Serum level of CEA was measured in all patients in group A and B prior to treatment and at least 12 weeks following therapy. Formalin - fixed and paraffin - embedded tissue blocks taken from 2 different sites of the studied lesions were prepared. Immunohistochemical staining for CEA was performed for the studied tissues.

Results: All the benign and borderline ovarian tumours had negative pre- and post-treatment serum levels of CEA (< 5 ng/ml). while 52.38% of malignant ovarian tumours had positive pre-treatment serum values. After treatment all the malignant ovarian tumours were sero negative for CEA.

The mean pre-treatment serum CEA in malignant ovarian tumours (7.32 ng/ml) was significantly higher than that of the other groups, whereas the mean post-treatment serum values and the mean difference in serum levels showed no significant differences between the 3 groups. 

The mean difference between pre- and post-treatment serum CEA was significant only in malignant ovarian tumours. 12.5% of the benign ovarian tumours, and 42.86% of the malignant ones had a positive reaction for CEA tissue stain. 

The mean values of serum CEA before treatment were significantly higher in positively stained malignant ovarian tumours. The mean difference in serum CEA was significantly higher in positively stained malignant ovarian tumours. The mean pre-treatment serum CEA and also the mean difference in serum levels showed significant progressive increase with the increase in degree of tissue stain of ovarian carcinomas.

Conclusion: This study indicates that immunohistochemical identification of CEA in tumour tissue is prerequisite to the use of that marker in the serum to monitor disease status.

Complete Article

Introduction

Ovarian masses fall into two broad categories, benign and malignant. The former are a nuisance but rarely dangerous, the latter are the most lethal of the common gynaecological malignancies. The surgical management of ovarian cancer is complex and often involves gastrointestinal surgery. Differentiating between benign and malignant masses is of paramount importance^{(1) (2)}. More than ever, we need a reliable cost-effective method for detecting ovarian cancer early. Most patients are diagnosed with advanced-stage disease when the prognosis is poor, despite radical surgery and combined chemotherapy⁽²⁾. In the US, ovarian cancer is the fifth leading cause of cancer death from gynaecologic malignancies. Ovarian cancer can be cured if detected early. However more than 70% of cases are diagnosed at an advanced stage, where 5 years survival approaches only 20%⁽³⁾. Unfortunately, despite advances in surgical technique and novel chemotherapeutic agents, survival rates have not improved significantly over past 25 years⁽⁴⁾.

The oncofoetal antigens comprise one particular group of markers produced by human neoplasms, these antigens have been detected in the sera of patients with gynaecological cancer. The practical use of such markers in the diagnosis and follow-up has been limited by the sensitivity and specificity of their tests ⁽⁵⁾. Carcinoembryonic antigen (CEA) is one of the first known tumour markers. Since then, many more have been described, but CEA, determined alone or in combination with others, is still one of the most used. CEA is not organ specific and abnormal values may be found in a wide range of carcinomas ⁽⁶⁾. In this work, we try through the study of CEA in the serum and tissues to evaluate the significance of, including this tumour marker, in the management protocol of patients with ovarian malignancies.

Patients and Methods

The present study was conducted on patients treated at National Cancer Institute, Cairo university and the department of Obstetrics and Gynaecology, Al Hussain Hospital, Al Azhar university. The study included 68 patients. 21 patients with ovarian malignancies, 3 patient with borderline ovarian tumour, 8 patients with benign ovarian tumours and 36 patients without any apparent gynaecologic disorder as control group. The patient were divided into the following groups:

• Group [A]: Including 21 patient with malignant ovarian tumours. The following histological types were recognised among them : mucinous cystadenocarcinoma (9 cases) ; serous cystadenocarcinoma (7 cases), undifferentiated carcinoma (3 cases) and squamous cell carcinoma(2 cases).
• Group [B]: Which consists of 3 patients with borderline ovarian tumours; 2 of them were borderline mucinous tumour and the third was of the serous type. Eight patients with benign ovarian tumours, 4 of them were mucinous cystadenomas and the other 4 were serous cystadenomas.
• Group [C]: Included 36 women without any apparent gynaecologic disorder - serum samples taken from them were subjected to CEA measurement and considered as control.

All the cases in group A and B, were subjected to the following:

• One - Careful history, clinical examination and investigations
• Two - Clinical staging for malignant lesions in group A according to the International Federation of Gynaecology and Obstetrics (FIGO) staging systems ⁽⁷⁾.
• Three - Serum samples were collected from the patients in groups A and B prior to treatment and at least 12 weeks following surgery or completion of radio or chemotherapy. All the patients were clinically free at the time of the post-treatment sample.

The level of CEA was measured in all the serum samples. A monoclonal antibody based immunoassay kit which provides a quantitative measurement of CEA in human serum was used. It is a solid phase enzyme-linked immunosorbent assay based on sandwich principle. A positive result for CEA in serum was taken as 5 ng/ml or more.

The patients with malignant ovarian tumours were treated with surgery alone or combined with radiation therapy or chemotherapy depending upon primary type, histologic differentiation and stage of disease. Those with non malignant lesions were treated only surgically.

Formalin - fixed and paraffin - embedded tissue blocks taken from 2 different sites of the studied lesions were prepared. The tissues were examined by the conventional histological method (Haematoxylin and eosin) for determination of the histological type of the tumour and the degree of tumour differentiation. 

Immunohistochemical staining for CEA was performed for the studied tissues. According to Charpin et al (1982) ⁽⁸⁾. - a grading system was utilized quantify the staining positivity as follows :

• (0) Denoting negative reaction i.e. showing no difference from the control sections
• (+1) Means that up to 25% of the cells were positive
• (+2) Means that >25 - 50% of the cells were positive
• (+3) Means that >50 - 75% of the cells were positive
• (+4) Means that >75% of the cells were positive

Statistical Analysis

Statistical analysis was carried out using an IBM - AT computer and SAS program (SAS, 1988). One way analysis of various (procedure GLM of SAS) followed by Duncan’s multiple range test were used to test the significance between the different variables studied. Paired t-test (procedure Means of SAS) was run to test the significance of the difference in serum CEA levels in relation to the variables studied in the current investigations, while student’s t - test ( procedure test of SAS) was employed to test the significance of change in serum CEA levels between negatively and positively stained lesions in relation to the different variables investigated. Cross tabulation and chi - square test (procedure frequency of SAS) were used to obtain and compare the percentage distribution of the studied cases according to their serum CEA levels and reactions to CEA immunostaining in relation to the studied variables. The probability level 0.05 (p = 0.05) was used to test the significance of the previous tests.

One of the important problems in the clinical management of cancer is the early detection of its occurrence. The demonstration of immunoreactive substances which are specific or associated with cancer and measurable in serum, provides an interesting and promising approach to cancer detection (Khoo and Mackay, 1974). The measurement of tumour-associated antigens as tumour markers in the serum is useful for early diagnosis, differential diagnosis, and the determination of remission after therapy in cases of malignancy. However, it is important to be aware that each tumour-associated antigen reveals only one biologic characteristic of the tumour cells, and that a tumour mass consists of various types of cells. It must also be understood that a tumour -associated antigen is not always present in all the cells forming the tumour and may also be detected in other tumours or normal organs. (Yabushita et al, 1988).

The two best characterized oncofoetal markers are a-foetoprotein (AFP) and carcinoembryonic antigen (CEA). CEA which was described as a gastrointestinal system-specific tumour antigen is still the most widely used and studied member of this group. Anti-CEA monoclonal antibodies have been used in attempts to define cancer-specific epitopes of CEA, in evaluating CEA by imunohistology and as agents for radioimmunoimaging. Similarly anti-AFP monoclonal antibodies have been used to develop sensitive radio - immunoassay, to explore radioimmunoimaging / immunotherapy and to study the control of AFP gene expression (Daa and Lennox, 1987).

Results

All the benign and borderline ovarian tumours had negative pre- and post-treatment serum levels of CEA (< 5 ng/ml). while 52.38% of malignant ovarian tumours had positive pre-treatment serum values (> = 5 ng/ml). After treatment all the malignant ovarian tumours were sero negative for CEA.

The mean pre-treatment serum CEA in malignant ovarian tumours (7.32 ng/ml) was significantly higher than that of the other groups, whereas the mean post-treatment serum values and the mean difference in serum levels showed no significant differences between the 3 types of ovarian tumours (Table I).

The mean difference between pre- and post-treatment serum CEA was significant only in malignant ovarian tumours (Table I).

As shown in (Table II) 12.5% of the benign ovarian tumours, and 42.86% of the malignant ones had a positive reaction for CEA tissue stain, while all the borderline tumours showed a negative reaction. Of all the positively stained ovarian tumours 90% were malignant. The mean values of serum CEA before treatment were significantly higher in positively stained malignant ovarian tumours (13.174 ng/ml) in comparison with the negative ones (2.929 ng/ml). The other 2 groups were not valid for such a comparison as they were almost devoid of positive cases.

The mean serum CEA levels after treatment were decreased in all types of ovarian tumours. This decline was most marked in malignant ovarian tumours but with no significant differences between these cases according to their reaction to stain. The mean difference in serum CEA was significantly higher in positively stained malignant ovarian tumours in comparison with the negative ones.

The mean difference between pre- and post-treatment serum CEA was highly significant in the positively and negatively stained malignant tumours. (Table II).

All the positively stained ovarian tumours (12.5% of this group) belonged to (+1) degree of reaction. Positive staining of malignant ovarian tumours was detected in 42.86% of the studied cases, most of them (33.33%) had a (+1) degree of reaction and only 9.52% showed a (+3) had a (+3) level of positively.

83.33% of the negatively stained ovarian carcinomas had negative pre-treatment serum CEA levels and 16.67% showed positive serum values. Whereas all the positively stained ovarian carcinomas had positive pre-treatment levels of serum CEA. The post-treatment serum CEA was negative for all the studied cases of ovarian carcinomas.

The mean difference between pre- and post-treatment serum CEA was highly significant in negatively stained tumours (degree 0) and in positively stained tumours with (+1) degree of reaction.

The mean pre-treatment serum CEA and also the mean difference in serum levels showed significant progressive increase with the increase in degree of tissue stain of ovarian carcinomas (Table III).

Discussion

This work was planned to deal with carcinoembryonic antigen in the plasma and tumour tissues of patients with ovarian tumours. The aim is to declare the significance of including this tumour marker in the management protocol of such patients and to evaluate the reported differences concerning CEA in other investigations.

All the benign and borderline ovarian tumours in the present study had negative serum CEA levels (< 5 ng/ml) before and after treatment (Table I). The incidence of abnormal CEA values (cut-off level 2.5 ng/ml) in benign pelvic masses studied by Inoue et al. (1992)⁽⁹⁾ , was 2.5%. Tholander et al. (1990)⁽¹⁰⁾, showed an elevated serum CEA (> 5 ng/ml) in 13% of the cases of benign adnexal tumours and in 27% of the cases of borderline tumours. CEA was elevated in 7% of women with benign adnexal masses and in 12% with tumours of low malignant potential and in 33% of women with a frankly invasive epithelial ovarian cancer⁽¹¹⁾. The difference in the technique of serum assay and the small number of borderline lesions in the current investigation may account for the difference in results.

On the other hand, 47.62% of the malignant ovarian tumours had negative serum values of CEA and 52.38% showed positive serum levels (> = 5 ng/ml). After treatment all the malignant group were seronegative for CEA (Table I). Stall and Martin (1981) ⁽¹²⁾, found that the mean incidence of elevated serum levels of CEA in ovarian carcinomas was around 40%. Pre-treatment serum CEA was elevated (>2.5 ng/ml) in 29% of those reported by Tholander et al.(1990) ⁽¹⁰⁾, who used a polycolonal anti-serum for CEA assay and in 21.5% of ovarian carcinomas investigated by Inoue et al.(1992) ⁽⁹⁾, and in 33% of cases with frankly invasive epithelial ovarian carcinoma investigated by Roman et al. 1998⁽¹¹⁾.

The mean value of pre-treatment serum CEA in patients with benign ovarian tumours was 1.13 ng/ml, that in borderline tumours was 1.929 ng/ml, while in malignant ovarian tumours the value increased to 7.32 ng/ml. In the control group the mean serum level of CEA was 1.024 ng/ml. The mean pre-treatment serum CEA was significantly higher in malignant ovarian tumours in comparison with the other group (Table I). This is in agreement with the finding of Inoue et al.(1992) ⁽⁹⁾. 

After treatment serum CEA was decreased in the 3 groups of tumours (benign, borderline and malignant). The findings in this respect are comparable to those of previously reported studies ^(17)(13). The mean difference between pre- and post-treatment serum CEA was highly significant in malignant ovarian tumours only (Table I). On comparing this mean difference with those of benign and borderline tumours it was found insignificant because of the large standard deviation (S.D) of the malignant group (reflecting wide range of variability) which affects the significance of results.

Reaction of Ovarian Tumours to CEA Immunostain
In the current work, 87.5% of the benign ovarian tumours were negative for CEA tissue stain and 12.5% were positive (Table II). Tohya et al.(1986) ⁽¹⁴⁾, showed positive staining in 25% of the cases of benign ovarian tumours but all their studied cases were of mucinous type. Neunteufel and Britenecker (1989) ⁽¹⁵⁾, reported a lower incidence (16.67%) of positive staining.

The technique of immunohistochemical staining used by these groups of investigators was the same as ours. Using different methods of tissue stain, Motoyama et al.(1990) ⁽¹⁶⁾ found positive staining of benign ovarian tumours in 30.9% of the cases.

On the other hand, all the borderline ovarian tumours showed a negative reaction for CEA tissue staining (Table II). This result is contradictory to the 55% positive staining of Tohya et al.(1986) ⁽¹⁴⁾, 11% of Dietel et al.(1986) ⁽¹⁶⁾, 31.25% of Neunteufel and Britenecker (1989)⁽¹⁵⁾, and 70% of Motoyama et al.(1990)⁽¹⁶⁾. The small number of borderline lesions in the present study did not allow accurate detection of the susceptibility of these tumours to CEA immunostaining.

In the malignant ovarian tumours 57.14% of the studied cases were negative for CEA immunostain and 42.86% were positive. As a whole, 90% of all the positively stained ovarian tumours were malignant (Table II). These results are consistent with the incidence of positive staining of ovarian carcinomas (45.45%) detected by Neunteufel and Breitenecker (1989)⁽¹⁵⁾, using the same tissue staining technique.

Tohya et al. (1986)⁽¹⁴⁾, found positive staining in 100% of the cases of ovarian carcinomas but all the studied cases were of the mucinous type. Motoyama et al. (1990) ⁽¹⁶⁾, reported positive staining of ovarian carcinomas in 37.79% of the cases but they employed methods of immunostaining different from that used in the present investigation.

The mean values of serum CEA before treatment in the positively and negatively stained and borderline tumours can not be compared, all of them were negative except one benign tumour. However, the mean pre-treatment serum CEA in positively stained malignant ovarian tumours (13.174 ng/ml) was highly significant in comparison with the negatively stained ones (2.929 ng/ml) as observed in (Table II). These findings are in agreement with those of Motoyama et al. (1990)⁽¹⁶⁾. After treatment a decrease in serum CEA levels was observed in all types of ovarian tumours with negative and positive tissue stain. This decrease was most marked in malignant ovarian tumours which showed no significant difference between its members according to their tissue staining reaction. (Table II).

The mean difference between pre- and post-treatment serum CEA was significant in the positively and negatively stained malignant ovarian tumours (Table III). Although the mean pre- and post treatment serum CEA in negatively stained carcinomas were within the negative range (< 5 ng/ml). On comparing the two types of tissue reaction the mean difference was significantly higher in positive malignant ovarian tumours (12.25 ng/ml) in comparison with the negative ones (2.302 ng/ml) as seen in (Table III).

Degree of positivity of staining reaction of ovarian tumours
As shown in (Table II), 12.5% of the benign ovarian tumours showed positive staining for CEA, all of them were in the (+1) degree of positively. The positively stained benign ovarian tumours in the study of Charpin et al. (1982) ⁽⁸⁾, showed a (+2) degree of positivity (same grading system of tissue staining reaction) but they used a different method of immunostaining and their cases were of the mucinous type only. Motoyama et al. (1990) ⁽¹⁶⁾, found that positively stained benign ovarian tumours were distributed between 3 levels of positivity of tissue reaction. However, their system of grading of positive reaction was different from that used in our study.

Positive staining in malignant ovarian tumours was found in 42.86% of the studied cases, 33.33% of them had a (+1) degree of reaction, while the remaining 9.52% showed a higher level of positivity (+3) as shown in (Table II). Charpin et al. (1982) ⁽⁸⁾, found that positively stained ovarian carcinomas were distributed between 3 levels of positivity as follows: 13.04% in (+1), 13.04% in (+2) and 6.52% in (+3). Although they had the same system of grading of tissue staining but they used a different technique of immunohistochemical staining. Motoyama et al.(1990) ⁽¹⁶⁾, found different percentage distributions of positively stained ovarian carcinomas among the various grades of positive staining reaction. However, direct comparison with their results is not suitable because they used different grading system and staining techniques.

Among the negatively stained ovarian carcinomas 83.33% of the cases had negative pre-treatment serum CEA levels (< 5 ng/ml), and 16.67% had positive levels (> 5 ng/ml). Whereas, all the positively stained malignant ovarian tumours (+1 and +3 degrees) showed a positive pre-treatment serum CEA. After treatment all the studied ovarian carcinomas were seronegative for CEA. The association between the positivity of tissue stain for CEA and the positive serum levels was also proved by Motoyama et al. (1990) ⁽¹⁶⁾.

A progressive increase in mean pre-treatment serum CEA with increasing degree of tissue stain of ovarian carcinomas for CEA was observed in (Table III). The differences between the 3 groups were statistically significant.

After treatment a marked drop in serum CEA took place in all degree of stain of ovarian carcinomas. Although the mean levels of serum CEA increased with increasing degree of positivity, yet the differences were insignificant (Table III).

The mean difference between pre- and post-treatment serum CEA showed the highest values (23.08 ng/ml) in the (+3) degree, followed by that in (+1) degree (9.157 ng/ml), and the lowest level (2.302 ng/ml) was in the negative group. The differences between these three groups were statistically significant (Table III).

Also the mean difference in serum CEA levels were significant in (0) and (+1) degrees of tissue stain. Although the mean pre- and post-treatment serum CEA were below the cut-off level of the present study in negatively stained ovarian carcinomas (degree “0”), yet the mean difference in serum levels were significant. An important finding which should be considered here is that 16.67% of negatively stained carcinomas had positive pre-treatment serum CEA levels (> 5 ng/ml). In the (+1) degree the result is comparable to those of Motoyama et al. (1990) ⁽¹⁶⁾, and is a logic outcome of the high mean pre-treatment and low mean post-treatment serum levels. As regards the (+3) level of positivity the mean difference in serum CEA levels were insignificant because of the small number of cases in this group.

This study proved the association between positive staining of tissue for CEA and elevated serum CEA level in the majority of the studied cases of malignancy.

In conclusion this study indicate that immunohistochemical identification of CEA in tumour tissue is prerequisite to the use of that marker in the serum to monitor disease status.

Table I : Serum CEA before and after treatment in different types of ovarian tumours

Serum CEA

SD: Standard deviation / dt: Duncan's multiple range t-test / SE: Standard error / T: Paired t-test / N.S: Not significant

Table II : Serum CEA in different types of ovarian tumour's according to their reaction to tissue stain

S.D: Standard deviation / df: Degree of freedom / S.E: Standard error / t: Paired t-test / T: Student's t-test

Table III : Serum CEA in different degree of tissue stain of ovarian carcinomas

+ VE

dt : Duncan's multiple range t- test / T: Paired t-test / Means with the same letters are not significantly different at P = 0.05 / 
SD: Standard deviation / SE: Standard error

References:

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