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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 immuno- histochemical 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 wide- ly 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 immuno- histochemical 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 neoplas- tic 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 neoplas- tic cells are shown in Table 3.
The results of scoring of immunoreactivity both according to grade of positivity and inten- sity are shown in Table 4.
In all but two cases, reactions for high-mol- ecular-weight cytokeratin (HMW) were nega- tive. 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 re- activity was seen in one serous cystadenocarci- noma. 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 im- munoreactivity. 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)
- -
-
- **
TAB
LE 4
. Sco
rinp
of
irnr
nuno
reac
tivity
ucc
ordi
na t
o nu
mbe
r of
cel
ls r
eact
inp
and
inte
nsity
of
reac
tion
in 5
0 ov
aria
n tu
tnor
s H
isto
logi
cal
diag
nosi
s H
MW
L
MW
EP
4 E
MA
C
EA
V
im
Gra
de I
nt.
Gra
de
Int.
Gra
de
Int.
Gra
de
Int.
Gra
de
Int.
Gra
de I
nt.
-
-
-
Sero
us
+I
++
I +
++
I7
++
+lo
+
++
I6
++
+I3
+
++
I6
++
+I3
-
-20
-20
++
2
+ +6
+ +5
+
+8
+
+4
+ +
8
+2
+S
+
1 +
++
3
++
+3
+
++
4
++
+2
+
++
2
++
+I
++
+3
+
+4
-
aden
ocar
cino
ma
(2 I )
Mut
inou
s -
-
-
++
3
++I
++
3
++
1
-1
+1
+I
-1
aden
ocar
cino
ma
(5)
+ +2
+
2
++
1
Endo
met
rioi
d -
-
aden
ocar
cino
ma
(2)
++
1
Sero
us b
orde
rlin
e (1
0)
-
-
-
+++I
+
+2
+
++
2
++
2
++
2
++
2
-
++
+lo
+
++
6
++
+7
+
++
I
++
+I0
+
++
4
-
+8
+
+3
-2
+s
+I
-2
-
-
-
+ +4
+
+3
+ $
7 + +
6
-
++
+5
$
++
3
++
+3
+
++
I +
++
I +
++
6
++
+I
++
7
-
-
-
Mut
inou
s bo
rder
line
(8)
++
3
+ +5
+ +4
+
+6
+
+7
+
+2
++
s -1
+
I +
I +
I -
-
Bre
nner
tum
or f
1)
-
-
++
I ++
I +I
+
l +
++
I ++
I +
I +I
+I
+l
-2
-2
-
++
+3
+
++
3
++
+2
va
ryin
g +
++
2
vary
ing
-
-
-
Unc
lass
ified
tum
or (
3)
++I
++
I
0 5 2 >
z
HM
W =
high
-mol
ecul
ar-w
eigh
t cy
toke
ratin
. L
MW
= lo
w-m
olec
ular
wei
ght
cyto
kera
tin.
EP4
= ep
ithel
ial
prot
ein
antig
en.
EM
A =
epith
elia
l m
embr
ane
antig
en. C
EA
= ca
rcin
oem
bryo
nal
antig
en. V
im =
vim
entin
. Gra
de =
num
ber
of c
ells
with
im
mun
orea
ctiv
ity. I
nt.
=int
ensi
ty o
f im
mun
orea
ctiv
ity.
ul
w
ul
JEIRGENSEN & NIELSEN
serous cystadenocarcinomas, and one unclassi- fied tumor.
Immunoreactivity for carcinoembryonic anti- gen (CEA) was absent in serous cystadenocarci- nomas, serous cystadenomas of the borderline type, endometrioid adenocarcinomas and un- classified tumors. Strong-to-moderate immuno- reactivity for CEA was found only in mucinous cystadenocarcinomas (4/ 5) and in mucinous cy- stadenomas 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 se- rous 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 through- out 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 determi- ning 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 carci- nomas (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 carci- nomas (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 cystadenocarcino- mas 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 mono- clonal 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 inves- tigated 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 im- munohistochemical reaction for HMW cytoker- atins, LMW cytokeratins, EP4, EMA, CEA, and vimentin is of no additional value in the differential diagnosis and typing of ovarian tu- mors.
The authors would like to thank Nor~yl lands Amts ForskningsrHd for supporting this project.
REFERENCES
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