5, May 1991. Copyrigt C American Association ofPathologists. Somatostatin Receptors in Differentiated. Ovarian Tumors. J. C.Reubi,* U. Horisberger,* J. G. M. ...
American Journal of Pathology, Vol. 138, No. 5, May 1991 Copyrigt C American Association of Pathologists
Somatostatin Receptors in Differentiated Ovarian Tumors J. C. Reubi,* U. Horisberger,* J. G. M. Klijn,t and J. A. Foekenst
sues not established as SRIF targets also may contain SRIF receptors, for instance lung tumors or breast
From Sandoz Research Institute Berne, * Berne, Switzerland; and the Dr. Daniel den Hoed Cancer Center,t Rotterdam, Holland
tumors.§7
The presence of somatostatin receptors was investigated in 57primary human ovarian tumors using in vitro receptor autoradiography with three different somatostatin radioligands, '25I-[Tyr"]-somatostatin-14, '25I-[Leu8, D-Trp22, Tyr25J-somatostatin28, or '25I-[Tyr31]-SMS 201-995. Three cases all belonging to epithelial tumors were receptor positive; specifically 1 of 42 adenocarcinomas, 1 of 3 borderline malignancies, and 1 of 2 cystadenomas. Four other epithelial tumors (3 fibroadenomas, I Brenner tumor), 4 sex cord-stromal tumors (2fibrothecomas, 2 granulosa cell tumors), and 2 germ cell tumors (1 dysgerminoma 1 teratoma) were receptor negative. In the positive cases the somatostatin receptors were localized on epithelial cells exclusively, were of high affinity (KD = 4.6 nmol/l [nanomolar]), and specificfor somatostatin analogs. These receptors bound somatostatin-14 and somatostatin-28 radioligands with a higher affinity than the octapeptide [Tyr3]SMS 201-995. Healthy ovarian tissue had no somatostatin receptors. A subpopulation of relatively welldifferentiated ovarian tumors, therefore, was identified pathobiochemically on the basis of its somatostatin receptor content. This small group of somatostatin receptor-positive tumors may be a targetfor in vivo diagnostic imaging with somatostatin ligands (Am JPathol 1991, 138:1267-1272)
Somatostatin (SRIF), a neuropeptide produced in the brain, the endocrine pancreas, and the gastrointestinal tract was shown to act in many tissues through specific SRIF receptors.1'2 Not only various healthy animal and human tissues contain SRIF receptors but also several human tumors.2 For instance, most tumors originating from SRIF target tissues have SRIF receptors, including most apudomas and brain tumors.2-4 However it was observed recently that tumors originating from tis-
In the last few years it has been increasingly evident that the identification of SRIF receptors in the various tumors may give important clues in regard to tumor diagnosis,8'9 tumor prognosis,10'11 or prediction of SRIF analog efficacy in tumor therapy.12'13 In the present study, therefore, we evaluated the incidence of SRIF receptors in another tumor type not originating from a SRIF target tissue, the ovarian tumor. Ovarian carcinoma is the second most common gynecologic malignancy and has had an extremely low 5-year patient survival rate when compared to other gynecologic malignancies; one of the reasons for the low survival rate is that most tumors are detected at advanced stages. However ovarian carcinomas probably are composed of several subgroups with extremely different prognosis. This situation gives considerable importance to the search for clinical markers such as receptors for steroid hormones, growth factors, or peptides, which may be of prognostic value or may influence treatment regimens in a favorable manner.
Materials and Methods Tumor Samples Fifty-seven primary human ovarian tumors were taken at surgery and stored frozen at - 700C at the Dr. Daniel den Hoed Cancer Center. A part was taken for steroid hormone receptor and epidermal growth factor (EGF) receptor assay. Another part of the tumor was shipped to Beme and frozen in dry ice for SRIF receptor assay. The tumors were classified as follows: epithelial tumors, including 42 adenocarcinomas, 3 borderline malignancies, 2 cystadenomas, 3 adenofibromas, and 1 Brenner tumor; sex cord-stromal tumors, including 2 fibrothecomas and 2 granulosa cell tumors; and germ cell tumors, including 1 dysgerminoma and 1 teratoma. Among the epithelial tuAccepted for publication January 15, 1991. Address reprint requests to J. C. Reubi, MD, Sandoz Research Institute Berne, P.O. Box, 3001 Beme, Switzerland. 1267
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AJP May 1991, Vol. 138, No. 5
mors, all International Federation of Gynecology and Obstetrics (FIGO) tumor stages (I to IV) were represented equally. Both serous and mucinous types of tumors were included. Furthermore normal human ovarian tissue also was investigated.
Somatostatin Receptors Somatostatin receptors were measured with autoradiography on 1 0-pum cryostat sections with three different radioligands, 12514Tyr11 ]-SRIF-1 4, 1251-[Leu8, D-Trp22, Tyr25]-SRIF-28, and 1251-[Tyr3]-SMS 201-995. All three ligands were iodinated and purified with highperformance liquid chromatography as described previously.1116 Receptor autoradiography was performed as described previously.3,5,16 Briefly, the tumors were cut on a cryostat (Leitz 1720) in 10 ,um sections, mounted on precleaned microscope slides, and stored at - 200C for at least 3 days to improve adhesion of the tissue to the slide. Sections then were incubated for 2 hours at ambient temperature in 170 mmol/l (millimolar) TRIS-HCI buffer (pH 7.4) containing bovine serum albumin (BSA) 1%, bacitracin (40 ,ug/ml), and MgC2 (5 mmol/ I) to inhibit endogenous proteases, in the presence of iodinated ligand (0.15 - 0.30 x 106 dpmlml, about 80 160 pmol/l [picomolar]). Nonspecific binding was determined by adding the respective unlabeled peptide SRIF-14, SRIF-28, or the octapeptide analog [Tyr3]-SMS 201-995 at a concentration of 1 p.mol/l (micromolar). Incubated sections were washed twice for 5 minutes in cold incubation buffer containing 0.25% BSA. Sections were washed in distilled water and dried quickly, apposed to 3H-ultrofilms (Cambridge Instruments, Nussloch, FRG) and exposed for 1 to 3 weeks in x-ray cassettes. In selected cases, displacement experiments using successive sections of a tumor were performed with increasing concentrations of various biologically active or inactive peptides. The autoradiograms were quantified using a computer-assisted image processing system, as previously described.3 Tissue standards for iodinated compounds (Amersham Laboratories, Little Chalfont, UK) were used for this purpose. A tumor was defined as SRIF receptor positive when the optical density measured over a tumor area in the 'total binding' section was at least twice the optical density in the 'nonspecific binding' section.
Steroid Hormone and EGF Receptors Steroid and EGF receptors were measured in tumor samples adjacent to those used for SRIF receptor assay. Estrogen receptors (ER) and progesterone receptors (PR)
were measured using enzyme immunoassay as described previously.17 For ovarian tumor samples an excellent correlation existed between the amounts of steroid receptors measured by the classical biochemical ligand binding assays and the enzyme immunoassays, ie, with Spearman correlations of Rs = 0.82 for ER and Rs = 0.85 for PR (n = 39, 2P < 0.001; Foekens JA, unpublished observations). Epidermal growth factor receptors were measured in membrane homogenates of the pellet fraction in the same tumor sample, as described earlier.18
Results Three of the fifty-seven ovarian tumors were found to be SRIF receptor positive (Table 1); one was a cystadenoma, one a borderline malignancy, and one a moderately differentiated FIGO stage Ic adenocarcinoma. All three SRIF receptor-positive cases, therefore, are relatively well differentiated; however not all differentiated ovarian tumors tested are SRIF receptor positive. Furthermore all three tumors are EGF receptor positive but estrogen and progesterone receptor negative (Table 2). As seen in Figure 1 in the example of the borderline malignancy, the epithelial cells of these ovarian tumors were strongly SRIF receptor positive, but the stroma was not. Interestingly the SRIF receptors in all three ovarian tumors were labeled with 1251-[Tyr11]-SRIF-14 or 1251. [Leu8, D-Trp22, Tyr25]-SRIF-28, as seen in Figure 2. [Tyr11 ]-SRIF and [Leu8, D-Trp22, Tyr25]-SRIF-28 radioligand labeling was comparable, except that the [Tyr11 ]SRIF-14 radioligand had a significant nonspecific binding. Labeling with the octapeptide 1251-[Tyr3]-SMS 201995 (= 1251-204-090) was, however, found to be negligible (Figure 2). Somatostatin receptors were of high affinity (KD = 4.6 nmol/l) and specific for SRIF analogs because unrelated peptides such as luteinizing hormone-releasing hormone showed no affinity (Figure 3). We observed a lower affinity (more than 30 times lower) for SMS 201-995 compared to SRIF-14 in the tumor A Table 1. Incidence of SRIF Receptors According to the Type of Ovarian Cancer N SRIF Tumor type N tested receptor positive Adenocarcinomas 42 1 Borderline malignancies 3 1 2 Cystadenomas 1 Adenofibromas 3 0 2 Fibrothecomas 0 Granulosa cell tumors 2 0 1 Dysgerminoma 0 Teratoma 1 0 Brenner tumor 1 0 SRIF, somatostatin.
SRIF Receptors in Ovarian Tumors 1269 AJP May 1991, Vol. 138, No. 5
Table 2. Receptor Characteristics of the Three SRIF Receptor-positive Ovarian Tumors Nr. A 601 A 635 A 692
Tumor type Adenocarcinoma Borderline malignancy Cystadenoma
SRIF-28
SRIF Receptors SRIF-14
204-090
(fmoles/ mg protein)
+ +
+ +
-
47 15
-
-
-
-
+
+
-
33
-
-
EGF-R
enzyme immunoassay SRIF-28 means binding with the 1251-[Leu8, D-Trp22, Tyr25]-SRIF-28 radioligand, SRIF-14 radioligand, 204-090 means binding with the 1251-[Tyr3]-SMS 201-995 radioligand.
Steroid-R* ER PR
*
________________
means
binding with the 1251-[Tyr"]-SRIF-14
692 in competition experiments using the 1251_ [Tyr11]-SRIF-14 ligand (Figure 3). In normal human ovarian tissue, no specific high>9 ¢~~~~~~~~~~~~~off ' ¢ inihli QQ19= do Loe kn UaeUtec ton^t%+^ %,i;+k aiiiiiity orJIr rn,,r-%terc, Witn aiyi^Uo +kr t1e receUIptors coUIU rollr%
r%
ligands.
Discussion The present report is the first autoradiographic demonstration of SRIF receptors in human ovarian tumors. We measured high affinity, specific SRIF receptors in a small number of cases representing rather differentiated ovar;_ +. "_A~~~~- el.+__S -E_ ian tumors, amounting to 2.4% o0 tne adenocarcinomas, 33% of the borderline malignancies, and 50% of the cystadenomas. The receptors were, in all three cases, strictly localized in the epithelial tumor cells but not on the stroma. Because no SRIF receptors are found in the normal ovary, it is not clear whether the SRIF receptors found in the ovarian tumors are newly expressed due to tumoral transformation or whether they anse from a SRIF-positive cell type present in a too-low density in the ovary to be detected with this method or with a homogenate radioreceptor binding assay.'9 These SRIF receptors in ovarian tumors represent a SRIF receptor subpopulation. They are characterized by a high affinity for SRIF-14 and SRIF-28 but low affinity for the octapeptide analog SMS 201-995 or its [Tyr3] derivative (204-090). These characteristics, ie, a differential affinity of the SS receptor for various SRIF analogs, confirm a recent study by Srkalovic et al19 showing a differential affinity of SRIF receptors in ovarian tumor homogenates for various SRIF analogs. We previously showed that a SRIF receptor subpopulation, characterized by a lower affinitv for SMS 201-995 exists in some hrnin aroaq rh as the substantia nigra, hippocampus, or cortex.16' 21 However a small number of human tumors also seem to possess this type of SRIF receptor subpopulation, in particular insulinomas, carcinoids, or pituitary adenoAt/i
Figure 1. Distribution ofSRIF receptors in an ovarian tunwr(borderline malignancy, A 635). A: Hematoxylin and eosin-stained section. B: Autoradiogram showing total binding of 12ILTSRIF-28 Only ePithelial tumor cells are labeled. C Autoradiogram shoing nonpecific binding of'25IL-SrSPeF28 (in pres10 ence ofl6 olll unlabeled SRIF-28). Bar =1 mm.
mas.
_1_______
312 22 ,
Interestingly the SRIF receptor-positive tumors were all relatively differentiated cases, removed at early stages
1270 Reubi et al AJP May 1991, Vol. 138, No. 5
Figure 2. SRIF receptors in ovarian tumors show a differential affinity for various SRIF analogs. A,B: Autoradiograms showing total (A) and nonspecific (B: in presence of 10-6 mol/I SRIF-14) binding of I[25_7,r"J1-SRIF-14. C,D: Autoradiograms showing total (C) and nonspecific (D: in presence of 10-6 mol/l
SRIF-28) binding of 1251-LTT-SR!F-28. E,F: Autoradiograms showing total (E) and nonspefiic (F: in presence of 10- mol/Il [7r3]SMS 201-995) binding of 1251_[Tyr3]-SMS 201995. Dense labeling of epithelial tumor cells is present in A and C but not in E. Bar = .o
.. ii
c..
1 mm.
441-.
*1'" .0 Figure 3. Displacement curve of 125I-[Tyr]-SRIF-14 in tissue sections from a human ovarian cystadenoma (A 692). Tissue sections were incubated with 24,000 cpm/100 _u '251-[Tr]-SRFF-14 and increasing concentrations of unlabeled SRIF-14 (0), SMS 201-995 (U), or 100 nmol/l luteinizing hormone-releasing hormone (IHRH) (A). Optical density of binding was measured as desribed in Materials and Methods. Nonspecific binding (in presence of 100 nmol/I SRIF-14) was subtractedfrom all values. Photographs represent autoradiogramsfrom sections incubated with the corresponding concentration (nmol/l) of unlabeled SRIF-14 (0, 3, 100) or 100 nmol/l LRH (A). Insert represent Scatchard analysis of the SR1F-14 data. KD for SRIF-14 is 4.6 nmol/l.
SRIF Receptors in Ovarian Tumors 1271 AJP May 1991, Vol. 138, No. 5
of tumor development. This feature correlates with previous studies on tumoral SRIF receptors showing their presence preferably in well-differentiated tumors; such an observation was made recently in carcinoids, in glial tumors, in pancreatic tumors, or in neuroblastomas,3'4'1' suggesting that SRIF receptors may be markers for tumor differentiation. When the expression of SRIF receptors is compared to other known receptor markers, such as steroid receptors and EGF receptors, it is interesting to note that the present study of ovarian carcinomas shows a concomitant expression of SRIF receptors with EGF receptors but not with steroid receptors. This is at variance with the results obtained in some other SRIF receptor-positive tumors, including glial, lung, and breast tumors, in which an inverse relationship between incidence of SRIF receptors and EGF receptors was observed.6'1123 Although the presence of EGF receptors may indicate a bad prognosis in several tumor types, this may not be the case for ovarian tumors,24 in which the EGF receptor positivity was associated with a better response to chemotherapy. However, due to the small number of SRIF receptorpositive cases investigated in this study, the above statements regarding correlations with SRIF receptors should be considered preliminary. It should be noted that all three patients with SRIF receptor- and EGF receptor-positive ovarian tumors in this study are still alive 2 years after tumor diagnosis. We know that these particular types of ovarian tumors, ie, cystadenomas, borderline malignancies, or adenocarcinomas stage Ic, exhibit, per se, a good prognosis. The fact, however, that SRIF receptors seem preferentially expressed by those tumors with favorable prognosis may suggest a relationship between these two factors. Therefore it will be of great importance to investigate further whether this subpopulation of SRIF receptor-positive ovarian tumors have such specific characteristics as to permit a more precise pathobiochemical classification and a prognostic evaluation of these tumors. Information about a possible role of SRIF through SRIF receptors in ovanan tumors is completely lacking, either in regard to antiproliferative or to antisecretory function. Therefore, despite the presence of SRIF receptors in certain ovarian tumors suggesting the possibility of a direct tumoral action, it is probably too early to recommend the use of any kind of SRIF analogs as therapeutic agents in these tumors25 before more information on SRIF function in these tissues is available. However diagnostic tests, ie, in vivo visualization of the tumors in the patients using injectable 1231-SRIF analogs, may be a useful and feasible method8'9 for identifying this subpopulation of ovarian tumors. It should, however, be realized that in ovarian tumors SRIF receptors have a higher affinity for SRIF-14 and SRIF-28 than for SMS 201-995. Because we recently
found in insulinoma that this type of SRIF receptor subpopulation cannot be visualized easily with 1231_ [Tyr3]-SMS 201-995 analogs,26 it will be advantageous to develop and use SRIF analogs with the adequate pharmacologic profile.
Acknowledgments The authors thank Dr. C. J. Rodenburg for providing the tumor samples, Dr. S. C. Henzen-Logmans and Professor J. Laissue for histopathologic evaluation of the tissues, and B. Waser and W. Huebener for technical assistance.
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