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Molecular Oncology, Markers, Clinical Correlates |
Department of Pathology, The Norwegian Radium Hospital, Montebello N-0310 Oslo, Norway, affiliated with the University of Oslo, Oslo N-0216 Norway [B. D., A. B., J. M. N.]; Department of Pharmacology, Faculty of Medicine and David R. Bloom Center for Pharmacy, Hebrew University, Jerusalem 91120, Israel [R. R.]; Department of Pathology [I. G., J. K.] and Division of Gynecologic Oncology [W. H. G., G. B-B.], Sheba Medical Center, Tel-Hashomer 52621, Israel, affiliated with Sackler School of Medicine, Tel-Aviv University; and Department of Oral Biology, University of Oslo, Oslo, Norway [M. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ets oncogene (v-ets) was discovered as part of a fusion protein with gag and myb expressed by the E26 avian erythroblastosis virus (3) . The Ets family of transcription factors is divided into subfamilies (Ets-1 and -2, ERG, GABP, PEA3, ELK, ELF, and PU1), based mainly on the sequence and location of the Ets domain, an 84-amino acid sequence present in all members of the family. Ets proteins bind to DNA sequences having the core motif C/AGCAA/T (4) .
Ets transcription factors play a role in a variety of physiological and pathological process, including embryogenesis, wound healing, and tumor progression (4 , 5) . This is largely due to their ability to activate the transcription of several proteases, including urokinase-type plasminogen activator, collagenase I (MMP-12 ), stromelysin I (MMP-3), and gelatinase B (MMP-9; Refs. 6, 7, 8, 9 ) as well as that of integrin ß3 (8) . Several growth factors, including acidic fibroblast growth factor and bFGF, VEGF, and epidermal growth factor, are in turn able to induce the expression of Ets proteins, such as Ets-1 (7) . In addition, Ets-responsive elements are present in the promoter region of the Flt and Kdr type of VEGF receptors. The activation of proteolytic enzyme transcription is central to the metastatic process because of its role in both angiogenesis and tumor invasion.
Ovarian cancer is the sixth most common cancer and the sixth most frequent cause of cancer death in women (4.4% of cases and 4.5% of deaths). It is the leading cause of death from gynecological cancer in women in industrialized countries (10)
. The incidence of ovarian carcinoma appears to be increasing in Western countries, as evidenced by a 30% rise in incidence and an 18% rise in death rate in the United States (10)
. Despite the inclusion of new chemotherapeutic regimens, the mortality rate from ovarian carcinoma has remained largely unchanged. This results from the late clinical presentation of this tumor; two-thirds of patients are diagnosed with stage III or IV disease (11)
. In our studies of metastasis-related genes, a cohort of advanced-stage ovarian carcinoma patients has been followed for a period of up to 20 years. We recently reported the role of mRNA expression of MMPs (MMP-2, MMP-9, and MT1-MMP) and their inhibitor TIMP-2 (12)
as well as the expression of the carbohydrate antigen sialyl Lewisx (13)
and the adhesion molecule 
-catenin (plakoglobin; Ref. 14
) as predictive factors for disease aggressiveness. Conversely, mRNA expression of the angiogenic factors bFGF, interleukin 8, and VEGF did not influence the prognosis of patients in this cohort.3
The expression of Ets-1 in stromal, endothelial, and tumor cells has been reported in several epithelial malignancies (15, 16, 17, 18, 19, 20, 21, 22, 23) . However, no data are available regarding its expression in ovarian carcinoma. The present study evaluates the expression and prognostic value of Ets-1 mRNA in ovarian carcinoma and investigates the potential association between Ets-1 expression and expression of angiogenic genes and MMPs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumors.
Sixty-six formalin-fixed, paraffin-embedded blocks from the archives of the Department of Pathology at the Sheba Medical Center were included in the study. These consisted of 37 primary ovarian tumors and 29 metastatic lesions from 41 patients with advanced ovarian carcinoma. The material studied included both primary and metastatic lesions from 25 patients, primary tumors from 12 patients, and metastases alone from 4 patients. The distribution of the studied material according to biopsy site is shown in Table 1
. Sections from all tumors were reviewed by two observers (B. D. and J. K.) in consensus sessions to confirm the diagnosis, histological type, and tumor grade (grades I-III corresponded to well, moderately, and poorly differentiated tumors). Established criteria were used for the microscopic diagnosis and tumor classification (24)
. Tumor staging was established according to International Federation of Gynecologists and Obstetricians criteria (24)
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The specificity of the probe was verified using a sense probe. A poly(dT)20 oligonucleotide (Research Genetics) was used to verify the integrity and lack of degradation of mRNA in each sample. The DNA probe was hyperbiotinylated. The stock dilution was diluted with probe diluent (Research Genetics) immediately before use. A working dilution of 1:100 was used. Specific sense oligonucleotides were used for the evaluation of nonspecific activity for each probe.
Colorimetric Nonradioactive mRNA ISH.
Tissue sections (4-µm thick) of formalin-fixed, paraffin-embedded specimens were mounted on ProbeOn Plus slides (Fisher Scientific, Pittsburgh, PA). Sectioning was performed in RNase-free water. ISH was carried out by using the microprobe manual staining system (Fisher Scientific; Refs. 25
and 26
). Hybridization of the probes was carried out as described previously (27)
. A positive enzymatic reaction in this assay stained dark blue. Known positive controls were used in each hybridization reaction. These consisted of two cases for which positive hybridization was reproducible in pilot studies. Controls for endogenous alkaline phosphatase included treatment of the sample in the absence of the probe and use of chromogen alone.
Evaluation of ISH Results.
Staining was scored in carcinoma and stromal cells. Staining extent was scored as 0, 1, or 2, using a cutoff of 20%. Staining of 20% or less of tumor/stromal cells was scored as focal (staining = 1), whereas staining of more than 20% of cells was interpreted as diffuse (staining = 2). Staining intensity was scored as absent (staining = 0), weak/moderate (staining = 1), or intense (staining = 2).
Statistical Analysis.
Staining values in tumor cells and stromal cells were evaluated statistically applying the SPSS personal computer software package (version 9.0; SPSS, Chicago, IL). Probability (P) of <0.05 was considered statistically significant. Analyses of the association between ISH results and biopsy site, patient group, tumor grade, and previous results for MMP-2, MMP-9, MT1-MMP, and TIMP-2 (12)
, as well the angiogenic genes bFGF, VEGF, and interleukin 8, were executed using the two-sided 
2 test. Univariate survival analyses were performed for all specimens, as well as for primary tumors alone. Survival analyses were executed using the Kaplan-Meier method and log-rank test. Both staining extent and intensity were analyzed. Multivariate analyses of survival were performed exclusively for primary tumors using the Cox regression model. The parameters included were protein expression for Slex and -catenin in tumor cells and mRNA expression of MMP-2, MMP-9, MT1-MMP, TIMP-2, and Ets-1 in tumor and stromal cells). Clinical parameters (patient age, tumor type, grade of differentiation, and disease stage) were not included because their prognostic role was nullified by the choice of patients for this study.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The distribution of metastatic sites was comparable for all histological types. Tumor differentiation was as follows: (a) 5 grade I tumors; (b) 3 grade II tumors; and (c) 33 grade III tumors. The fraction of poorly differentiated (grade III) tumors was comparable for both groups (13 of 17 and 20 of 24 tumors).
mRNA ISH.
A positive signal using a poly(dT) probe was detected in all cases (data not shown). Ets-1 mRNA was detected in the nuclei and/or cytoplasm of both tumor (Fig. 1)
and stromal cells. Expression of Ets-1 mRNA was detected in carcinoma cells and stromal cells in 28 of 66 and 22 of 66 lesions, respectively (Tables 3
and 4
). Endothelial expression was seen in some stroma-positive specimens.
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and 4A
). However, although infrequent, intense staining was seen exclusively in metastatic lesions (P = 0.031 for tumor cells; Table 3A
). Ets-1 mRNA expression was more diffuse in both carcinoma and stromal cells in tumors of short-term survivors (P = 0.038 for carcinoma cells; P > 0.05 for stromal cells; Table 4B
).
We subsequently evaluated the association between Ets-1 expression and expression of the previously studied angiogenic factors. Ets-1 mRNA expression showed an association with mRNA expression of VEGF in both staining extent (P = 0.001 for carcinoma cells; P = 0.004 for stromal cells) and intensity (P = 0.001 for carcinoma cells; P = 0.032 for stromal cells). Furthermore, diffuse (>20% of cells) expression of Ets-1 mRNA was associated with intense expression of bFGF in tumor cells (P = 0.049). Ets-1 expression in stromal cells showed an association with expression of MT1-MMP in tumor cells (P = 0.045). In survival analysis for all cases, Ets-1 expression in both tumor (P = 0.018) and stromal (P = 0.026) cells correlated with poor survival (Table 4B
; Fig. 2, A and B
). These findings were reproduced in an analysis of primary tumors alone (P = 0.039 for tumor cells; P < 0.001 for stromal cells; Fig. 2, C and D
). In multivariate analysis, Ets-1 expression in stromal cells (but not in tumor cells) retained its predictive value (P = 0.007), together with TIMP-2 mRNA expression in stromal cells (P < 0.001) and the expression of MMP-9 (P = 0.001), sialyl Lewisx (P = 0.001), and -catenin (P < 0.001) in tumor cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early studies analyzing various benign and cancerous tissues have localized Ets-1 mRNA to endothelial (17) and stromal (17 , 23) cells. No expression was seen in tumor cells. These findings were reproduced in an additional study of preinvasive and invasive bronchial tumors (22) . However, a larger study of 54 lung carcinomas detected the presence of Ets-1 mRNA in carcinoma cells, although to a lesser extent than that observed in the stromal compartment (21) . Expression of c-ets-1 protein was subsequently reported in oral, gastric, pancreatic, and thyroid carcinomas (15 , 16 , 18 , 19) . The latter four studies did not score expression in stromal cells. We found c-ets-1 expression in both tumor and stromal cells in a roughly similar number of specimens. Furthermore, the extent and intensity of expression did not differ markedly between cells in the two compartments. This finding is in agreement with the cellular localization of MMP-2, MMP-9, and TIMP-2 (12) as well as that of angiogenic genes3 in this patient cohort and provides additional evidence supporting the dual (tumor and stromal cell) origin of metastasis-associated molecules in ovarian carcinomas.
The comparative expression of Ets-1 in primary and metastatic lesions has not been studied to date. Bolon et al. (21) evaluated 11 lymph node metastases among 54 lung carcinomas studied. However, the comparative analysis stratified stage I and II versus stage III and IV tumors and metastases. The authors reported a significantly elevated expression of Ets-1 mRNA in the group that included disseminated tumors and metastases. We found a similar elevation in expression in tumor cells from metastatic lesions when signal intensity was the evaluated parameter. However, this finding reflects the presence of intense signals exclusively in metastatic lesions, which were present in only three cases. Larger studies are therefore necessary to confirm this finding. We are currently studying a large number of serous effusions and solid tumors from ovarian carcinoma patients for this purpose.
The role of Ets-1 in the activation of proteolytic enzyme expression has been shown in several studies (7 , 8 , 9 , 20) . Regulation of Ets-1 expression is in turn regulated by angiogenic factors, including VEGF and bFGF (7) . Furthermore, in the absence of Ets-1, angiogenesis is inhibited (29) . These intriguing findings were confirmed in vivo for MMP-1, MMP-3, and urokinase-type plasminogen activator (21 , 23) . However, no data are available regarding the association between Ets-1 expression and expression of angiogenic genes in human tumors. In agreement with the above-mentioned reports, we found a significant association between mRNA expression of VEGF and bFGF and that of Ets-1 in our patient cohort. This finding provides the first in vivo evidence of a relationship between these molecules. Although a similar association between Ets-1 and MMP-9 was reported in cell cultures (8 , 29) , we did not observe it in our study. However, the expression of MT1-MMP mRNA, which is involved in the activation of MMP-2, in tumor cells correlated with Ets-1 mRNA expression in peritumoral stromal cells. In view of the central role of MMP-2 in tumor invasion and angiogenesis (30) and the prognostic role found for both MMP-2 and MT1-MMP in ovarian carcinoma (12) , this finding appears to further support the role of Ets-1 in tumor progression and its use as a marker for disease outcome.
The prognosis of ovarian carcinoma remains poor, primarily due to its late detection, which is often associated with widespread i.p. disease (31)
. To be able to segregate patients with advanced-stage disease into different prognostic groups, one will need to rely on molecular and cellular markers, few of which have an undisputed role in ovarian carcinoma to date. Our study evaluated two groups of patients diagnosed with advanced-stage ovarian carcinoma with a markedly different disease outcome, with a follow-up period of up to 20 years. Established prognostic factors, such as age, stage, grade, and tumor type, were all controlled by patient selection criteria in the design of the study. This selection was meant to facilitate the study of potential prognostic markers. Results recently published using this patient cohort identified MMP-9, MT1-MMP, TIMP-2 (12)
, sialyl Lewisx antigen (13)
, and the adhesion molecule -catenin (14)
as new prognostic markers in ovarian carcinoma and confirmed the findings of earlier reports regarding MMP-2 (12)
.
Studies by other groups had demonstrated the association between expression of Ets-1 and the invasive phenotype of oral, gastric, and lung carcinomas (15 , 16 , 22) . Two of these studies have additionally found an association between Ets expression and the presence of lymph node metastases (15 , 16) . However, the association between Ets-1 and disease outcome has not been demonstrated previously. In the present study, diffuse expression of Ets-1 mRNA in both tumor and stromal cells correlated with rapidly lethal disease. Furthermore, whereas expression in carcinoma cells merely retained its prognostic power in the analysis of primary tumors alone, expression in stromal cells proved to be a considerably more powerful marker of poor survival in this group and retained its power in multivariate survival analysis. These results show that expression of Ets-1 mRNA in both stromal and tumor cells, in either the entire material or primary tumors alone, is a marker of poor survival. Therefore, they all have clinical application in advanced-stage ovarian carcinoma. Prognostic studies, however, are most often performed on primary tumors. This fact, combined with the strong predictive power of Ets-1 expression in stromal cells of primary tumors, makes the analysis of this parameter most useful. This choice is highlighted by the multivariate analysis results. Taken together, our results provide the first evidence of a correlation between Ets-1 mRNA expression and survival in human epithelial malignancy and suggest a role for Ets-1 as a novel prognostic marker in ovarian carcinoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed, at Department of Pathology, The Norwegian Radium Hospital, Montebello N-0310, Oslo, Norway. Phone: 47-22934871; Fax: 47-22508554; E-mail: bend{at}ulrik.uio.no 
2 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane type; ISH, in situ hybridization; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; TIMP, tissue inhibitor of metalloproteinase.
3 B. Davidson, R. Reich, I. Goldberg, W. H. Gotlieb, J. Kopolovic, A. Berner, G. Ben-Baruch, M. Bryne, and J. M. Nesland. Expression of angiogenesis-related genes in ovarian carcinoma—A clinicopathologic study, submitted for publication.
Received 8/31/00; accepted 11/20/00.
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Commentary
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