Epithelial antigens in carcinomas of the ovaries. Relation to histological classification

A P M I S 102. 533 537. I994 Pr int id in Denniiirk . All ri,?ht\ rr.wrvi~rl

ISSN OY(13-4641

Epithelial antigens in carcinomas of the ovaries. Relation to histological classification

ANNEMETTE J0RGENSEN and KARSTEN NIELSEN

Institute of Pathology. Aalborg Hospital, Aalborg, Denmark

Jnrgensen, A. & Nielsen, K. Epithelial antigens in carcinomas of the ovaries. Relation to histological classification. APMIS 102: 533-537, 1994.

In an attempt to assess and improve the histological classification of ovarian tumors the value of immunohistochemical techniques has been examined in 50 ovarian tumors. A panel of six immunohistochemical markers (two cytokeratins, EP4, EMA, CEA, and vimentin) seems to have no additional value in differential diagnosis and typing of ovarian tumors.

Key words: Ovary; epithelial tumors; immunohistochemistry.

A. Jnrgensen, Institute of Pathology, Aalborg Hospital, DK-9000 Aalborg, Denmark.

The incidence rates of ovarian cancer vary widely throughout the world, being highest in the Western countries, where ovarian cancer is the most frequent cause of death from gynecological malignancies. The treatment and prognosis for ovarian tumors depends on the spread, differen- tiation and exact histopathological diagnosis. About 90'1/0 of ovarian tumors are of epithelial origin. In an attempt to assess and improve the histological classification we have investigated the value of immunohistochemical techniques in determining epithelial antigens of neoplastic cells of the ovaries.

MATERIALS AND METHODS

During the period from January 1991 to June 1992 specimens from 50 women with ovarian tumors were obtained at surgery. The mean age of the women at the time of the operation was 56 years (range 25- 82 years). At the Institute of Pathology, Aalborg Hospital, Denmark, the 50 ovarian tumors were diag-

Received March 7, 1994. Accepted May I I , 1994.

nosed and classified histologically according to WHO criteria as shown in Table 1. Formalin-fixed paraffin- embedded tissue blocks from the ovarian tumors were investigated retrospectively. One representative tissue block from each tumor was selected for immunohistochemical investigation after reviewing the rou- tine hematoxylin-eosin-stained slides.

A biotin-streptavidin method (DAKOPATTS AIS. Denmark) was used. The tissue sections were pretreated either with pronase (0.15'%1 pronase at 37'C for 5 min or in a microwave oven (700 W for 10 min) with the sections placed in 0.01 M sodiumcitrate (2.94 g i l adjusted to pH 6 with NAOH). (see Table 2 ) . They were then incubated for 18 h at 4 C with a primary anti-human antibody (see Table 2), and after rinsing procedures for 15 min with AEC (3-amino-9- ethylcarbazole in acetone diluted with acetate buffer). Microscopically, the cellular immunoreactivity was classified as present or absent, and when present as located diffusely (D) in the cytoplasm of the neoplastic cells or luminally (L) in the neoplastic cells.

Immunoreactivity was graded as strong (+ + +) when the reaction was seen in up to all neoplastic cells, moderate (+ +) when seen in up to approxi- mately two thirds, and weak (+ ) when seen in up to about one third. Absence of immunoreactivity was graded -. The intensity of the immunoreaction was also scored. A strong staining reaction was graded + + +, a moderate staining reaction + +, and a weak staining reaction +.

533

JBRGENSEN & NIELSEN

TABLE 1 . Histological classijkation of 50 ovarian tumors accordinK to WHO criteria

Histological diagnosis Number Serous cystadenocarcinoma 21 Mucinous cystadenocarcinoma 5 Endometrioid adenocarcinoma 2 Serous borderline tumor 10 Mucinous borderline tumor 8 Brenner tumor 1 Unclassified tumor 3

RESULTS

The results of immunohistochemical staining in the different types of ovarian tumors and the location of the immunoreactivity of the neoplastic cells are shown in Table 3.

The results of scoring of immunoreactivity both according to grade of positivity and intensity are shown in Table 4.

In all but two cases, reactions for high-molecular-weight cytokeratin (HMW) were negative. Positivity was found diffusely in one serous cystadenocarcinoma, but only in a few cells and only with moderate intensity. The reactivity in the Brenner tumor was found only in a small focus of planocellular metaplasia (disregarded in Table 4).

In all the ovarian epithelial tumors and the unclassified tumors the neoplastic cells demon- strated diffuse reactivity for low-molecular- weight cytokeratin (LMW) mostly of strong-to- moderate intensity.

The same reactivity was found for epithelial protein antigen (EP4), except that luminal reactivity was seen in one serous cystadenocarcinoma. The results of the immunoreactivity for epithelial membrane antigen (EMA) were nearly the same as for LMW and EP4, but a few more tumors with luminal reactivity were seen: one mucinous cystadenocarcinoma, three

TABLE 2. Characterization of primar): anti-human antibodies Antibodv Cloneicode Dilution Pretreatment High-molecular-weight cytokeratin DAKO

A575-callus 1 :600 Pronase

Low-molecular-weight cytokeratin Becton Dickinson 1 :200 Microwave oven CAM 5.2

Vimentin Boehringer Mannheim 1 : 1000 Microwave oven VIM 3B4

Carcinoembryonal antigen (CEA) DAKO 1:100 Pronase A5B7

Epithelial membrane antigen (EMA) DAKO 1:1200 Pronase E29

Epithelial protein antigen (EP4) DAKO Ber-EP4

1 :200 Pronase

TABLE 3. Results of immunoreactivity for 50 ovarian tumors Histological diagnosis Number HMW LMW EP4 EMA CEA Vim Serous adenocarcinoma 2 1 1 (D) 21 (D) 20(D), 1 (L)18 (D), 3 (L) - -

Mutinous adenocarcinoma 5 -

Endometrioid adenocarcin- 2 -

oma Serous borderline 10 - lO(D) lO(D) lO(D) -

Mucinous borderline 8 8 (D) 8 (D) 8 (D)*** 7 (L) Brenner tumor 1 I * 1 (D) 1 (D) 1 (D) 1 (D) Unclassified tumor 3 HMW = high-molecular-weight cytokeratin. LMW = low-molecular-weight cytokeratin. EP4 = epithelial protein antigen. EMA =epithelial membrane antigen. CEA = carcinoembryonal antigen. Vim = vimentin. - = n o immunoreactivity. D = diffuse. L = luminal. * = immunoreactivity in focus of planocellular metaplasia. ** = one failure in staining reaction. ***=one tumor with additional luminal reaction.

534

4 (D), 1 (L) 4 (L) -

- - 5 (D) 5 (D) 2 (D) 2 (D) 2 (D)

8 (D)

3 (D) 3 (D) 2 (D), 1 (L) - 1 (D)

- -

-

- **

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JEIRGENSEN & NIELSEN

serous cystadenocarcinomas, and one unclassified tumor.

Immunoreactivity for carcinoembryonic antigen (CEA) was absent in serous cystadenocarcinomas, serous cystadenomas of the borderline type, endometrioid adenocarcinomas and unclassified tumors. Strong-to-moderate immunoreactivity for CEA was found only in mucinous cystadenocarcinomas (4/ 5) and in mucinous cystadenomas of the borderline type (7/8); the reactions, which were of moderate intensity, were located luminally. The Brenner tumor showed weak diffuse positivity in a few cells.

The epithelial tumors were all negative for vimentin, except for a few moderate-to-weakly positive cells showing a diffuse reaction in serous cystadenomas of the borderline type (8/ 10) and in one unclassified tumor.

DISCUSSION

Differential diagnosis is a major problem in the histopathology of ovarian tumors and throughout the years immunohistochemical techniques have been used to help solve the difficulties in- volved in differential diagnosis and typing. In an attempt to assess and improve the histological classification we have here investigated the value of immunohistochemical techniques in determining epithelial antigens of neoplastic cells of the ovaries.

In this study we found no reaction for HMW cytokeratins in any type of ovarian neoplasm, except for a moderate reaction in a few cells of one serous cystadenocarcinoma, which is in contrast to the results of other investigators who found positivity for HMW cytokeratins in carcinomas (7, 12). Using LMW cytokeratins we found a reaction in all the epithelial ovarian tumors examined, which is in accordance with the results of others (2,6, 12). LMW cytokeratin together with other epithelial markers (EMA, pKeratin) has been used to demonstrate occult microinvasion in some serous ovarian tumors (8). According to our investigation no improve- ment in histological classification can be achieved using EP4 and EMA because we dem- onstrated immunoreactivity for these two markers in all the epithelial ovarian neoplasms examined. Other investigators have reached similar conclusions ( 1, 6).

536

We found vimentin in serous cystadenomas of the borderline type in 80‘% of cases, but no reaction in other epithelial tumors. Others have found positivity in 50% of endometrioid carcinomas (1 ) and 30% of serous tumors (12). Further studies are needed in order to reach a definite conclusion as to whether vimentin can differentiate between serous cystadenocarcinomas of high and of low malignant potential.

Several workers have looked at the relation- ship between histological type and presence of CEA in ovarian tumors (1, 3, 4, 5, 6, 9, 10, 1 1 , 12, 13, 14, 15, 16). In our study using monoclonal CEA we found positive reactions not only in mucinous tumors of high malignant potential, but also in mucinous tumors of low malignant potential. In addition, the Brenner tumor investigated showed a positive reaction. All other tumors were negative. Our results concerning CEA are in accordance with the reports of many others (4, 5, 10, 1 1 , 13, 14, 15), but the results in the literature vary widely and there are some reports of positivity for CEA in serous tumors (9, 12). The conflicting findings may be due to the use of different primary antibodies against CEA (5). In conclusion, we find that the immunohistochemical reaction for HMW cytokeratins, LMW cytokeratins, EP4, EMA, CEA, and vimentin is of no additional value in the differential diagnosis and typing of ovarian tumors.

The authors would like to thank Nor~yl lands Amts ForskningsrHd for supporting this project.

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Epithelial antigens in carcinomas of the ovaries. Relation to histological classification