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Molecular Oncology, Markers, Clinical Correlates |
v Integrin Expression Is a Novel Marker of Poor Prognosis in Advanced-stage Ovarian Carcinoma
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; Department of Pathology, The Norwegian Radium Hospital [B. D., A. B., J. M. N.] and Department of Oral Biology [M. B.], University of Oslo, Norway, Montebello N-0310 Oslo, Norway; and Department of Pharmacology, Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel [R. R.]
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ABSTRACT |
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v and ß1 integrin chains and survival in advanced-stage ovarian carcinomas, studying two patient groups with extremely different disease outcome.
Experimental design: Sections from 56 primary ovarian carcinomas and metastatic lesions from 34 patients diagnosed with advanced-stage ovarian carcinoma (Fédération Internationale des Gynaecologistes et Obstetristes stages III-IV), divided into long-term (16) and short-term (18) survivors, were evaluated for expression of v and ß1 integrin chains using mRNA in situ hybridization. Protein expression was additionally studied in 52 specimens using immunohistochemistry.
Results: The mean values for disease-free survival and overall survival were 115 and 132 months for long-term survivors, as compared with 4 and 23 months for short-term survivors, respectively. Expression of v integrin mRNA was observed in carcinoma (18 of 56; 32%) and stromal (17 of 56; 30%) cells. ß1 integrin mRNA was similarly detected in carcinoma (25 of 56; 47%) and stromal (19 of 56; 34%) cells. No significant differences were observed when primary and metastatic lesions were compared (P > 0.05). v integrin mRNA was present more often in carcinoma cells in tumors of short-term survivors (P = 0.017 for carcinoma cells). In univariate survival analysis for all cases, v integrin mRNA expression in tumor cells correlated with poor survival (P = 0.012). This finding retained its predictive power in a multivariate survival analysis, in which all of the molecules studied previously in this patient cohort were included (P = 0.031). Immunohistochemistry confirmed the differences in v integrin expression in tumor cells of short-term as compared with long-term survivors, whereas ß1 integrin protein expression was comparable in the two groups.
Conclusions: To our best knowledge, this is the first evidence associating integrin expression with poor survival in ovarian carcinoma. v integrin is, thus, a novel prognostic marker in advanced-stage ovarian carcinoma.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß subunits (1)
. Integrins participate in both cell-cell adhesion and in binding to a large number of basement membrane and ECM ligands, including laminin, fibronectin, collagen, vitronectin, entactin, tenascin, and fibrinogen (1)
. Seventeen and eight ß subunits are known to date, forming 22 different combinations (2)
. Most subunits associate with a single ß subunit, the largest family being the very late activating ß1 integrin family. This family includes the 5ß1 fibronectin receptor, the vß1 fibronectin receptor, and the 6ß1 laminin receptor (3)
. Several subunits, including 4, 6, and V, can in turn associate with more than one ß subunit. Thus, v integrin is part of the receptors for fibronectin, vitronectin, fibrinogen, and several other proteins (3)
. The roles of integrins extend practically to all main aspects of cellular function. They form a structural link between the ECM and the cellular cytoskeleton through the formation of focal adhesions, where both structural proteins and protein kinases localize. Integrin-mediated signals are involved in cell proliferation, apoptosis, migration, invasion, cellular transformation, angiogenesis, and tumor progression (Refs. 2, 3, 4, 5, 6 and references therein). These effects are closely linked to the activation of metastasis-associated molecules, such as MMPs (7) . Altered expression of integrins, in the form of down- or up-regulated expression, have been detected in the majority of malignant tumors but vary considerably according to the origin of the neoplasm (3) .
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 (8) . 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 (8) . 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 the patients being diagnosed with stage III or IV disease (9) .
The expression and the functional role of integrins have been investigated previously in ovarian carcinoma (10, 11, 12, 13, 14)
, but the prognostic role of these molecules remains largely unknown. In a single study of 19 borderline tumors and 31 carcinomas, no correlation was observed between protein expression of vß3 integrin and disease outcome after a mean follow-up period of 26 months (15)
. We have begun recently a prognostic study of metastasis-related molecules in a cohort of advanced-stage ovarian carcinoma patients, with a follow-up period of 
20 years. We reported previously the role of mRNA expression of MMPs (MMP-2, MMP-9, and MT1-MMP) and their inhibitor TIMP-2 (16)
, as well as that of expression of the carbohydrate antigen Sialyl Lewisx (17)
, the E-cadherin adhesion complex molecule 
-catenin (plakoglobin; Ref. 18
), and the transcription factor Ets-1 (19)
, as predictors of poor survival. Conversely, protein expression of the membrane protein caveolin-1 (20)
and mRNA expression of the angiogenic factors basic fibroblast growth factor, interleukin-8, and vascular endothelial growth factor (21)
did not influence the prognosis of patients in this cohort.
The objective of the present study was to evaluate the prognostic role of v and ß1 integrin mRNA expression in advanced-stage ovarian carcinoma. Protein expression of both chains was studied to consolidate mRNA ISH results.
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MATERIALS AND METHODS |
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Tumors.
Formalin-fixed, paraffin-embedded blocks (56) from the archives of the Department of Pathology at the Sheba Medical Center were included in the study. These consisted of 33 primary ovarian tumors and 23 metastatic lesions from 34 patients with advanced ovarian carcinoma. 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 (I-III, corresponding to well, moderately, and poorly differentiated). Established criteria were used for the microscopic diagnosis and tumor classification (22)
. Tumor staging was established according to Fédération Internationale des Gynaecologistes et Obstetristes criteria (22)
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v and ß1 integrin chains were obtained from Biognostik (Göttingen, Germany). Probe sequences (5'-3') were as follows:
v integrin: TGA CCT TGC CAA TAA AAG CTA CCA GGA CC
ß1 integrin: CCA AGT TTC CCA TCT CCA GCA AAG TGA AAC
A poly d(T)20 oligonucleotide (Biognostik) was used to verify the integrity and lack of degradation of mRNA in each sample.
mRNA ISH.
Tissue sections (4-µm thick) of formalin-fixed, paraffin-embedded specimens were mounted on silane-coated slides. Sectioning was performed in RNase-free water. Slides were dewaxed and rehydrated using xylenes (2 x 10 min) and isopropyl alcohol (5 min). Hybridization was carried out as described previously (23)
. 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.
Strong dark blue staining was interpreted as intense. A minimum of 500 cells, when present, was evaluated. 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% of tumor/stromal cells was scored as focal (1), whereas staining of >20% of cells was interpreted as diffuse (2). Staining intensity was scored as absent (0), weak/moderate (1), or intense (2). Neither the experimenter performing the tests nor the one evaluating the slides were aware of disease outcome or any other clinical parameter concerning the patients.
IHC.
Fifty-two specimens (22 from long-term survivors and 30 from short-term survivors) were evaluated for protein expression of v and ß1 integrin chains. An antibody directed against v integrin was obtained from Chemicon International (Temecula, CA). The antibody directed against ß1 integrin was obtained from Serotec (Kidlington, Oxford, UK). Formalin-fixed, paraffin-embedded sections, 4-µm thick, were mounted onto silane-coated slides. After air drying at 37°C for 24 h, slides were deparaffinized and rehydrated. Staining was performed using the labeled Avidin Biotin method. For both antibodies, pretreatment in the form of protein digestion using Trypsin (10 min at 37°C) was used. Negative controls consisted of sections that underwent a similar staining procedure, with the exclusion of primary antibody application. Positive controls consisted of cases in which immunoreactivity was demonstrated in pilot studies.
Statistical Analysis.
ISH results in tumor cells and stromal cells were evaluated statistically, applying the SPSS-PC package (version 9.0; SPSS, Chicago, IL). Probability of <0.05 was considered statistically significant. Analyses of the association between ISH results and biopsy site, patient group, and tumor grade were executed using the two-sided 
2 test. Univariate survival analyses for all specimens studied were executed using the Kaplan-Meier method and Log-rank test. Both staining extent and intensity were analyzed. Multivariate analyses of survival for all specimens studied were performed using Cox regression model. The parameters included were the expression of Sialyl Lewisx and -catenin protein in tumor cells; mRNA expression of MMP-2, MMP-9, MT1-MMP, TIMP-2, and Ets-1 in tumor and stromal cells; and v integrin expression in tumor cells (see also "Results"). Clinical parameters (patient age, tumor type, grade of differentiation, and disease stage) were not included, as their prognostic role was nullified by the choice of patients for this study (see also "Results").
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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: 4 grade I tumors, 2 grade II tumors, and 28 grade III tumors. The fraction of poorly differentiated (grade III) tumors was comparable for both groups (12 of 16 and 16 of 18 tumors).
ISH.
A positive signal using a poly d(T) probe was detected in all cases (Fig. 1-A)
. Negative controls showed only contrast staining by nuclear fast red (Fig. 1-B)
. v and ß1 integrin mRNA was detected in tumor and/or stromal cells (Fig. 1-C–F)
. Expression of v integrin mRNA was detected in carcinoma cells and stromal cells in 18 of 56 (32%) and 17 of 56 (30%) of the lesions, respectively. ß1 integrin mRNA expression was detected in carcinoma cells and stromal cells in 25 of 56 (47%) and 19 of 56 (34%) of the lesions, respectively (Tables 3
and 4
). v integrin expression was more diffuse in both tumor and stromal cells in metastases, whereas intense signal was more often seen in tumor cells of primary tumors (Tables 3a
and 4a
). None of these differences achieved statistical significance (P > 0.05). The expression of ß1 integrin mRNA was comparable in primary and metastatic lesions in terms of both intensity and extent (P > 0.05; Tables 3a
and 4a
). v and ß1 integrin mRNA expression in stromal cells was comparable in tumors of long-term and short-term survivors (P > 0.05; Tables 3b
and 4b
). However, marked differences were observed in tumor cells. Diffuse and intense labeling for v integrin mRNA was detected more often in carcinoma cells in tumors of short-term survivors (16 versus 8% for intensity, 27 versus 0% for extent), significantly so for labeling extent (P = 0.017; Fig. 1-E and 1-F
; Tables 3b
and 4b
). Similarly, intense and diffuse expression of ß1 integrin in carcinoma cells was more frequently seen in tumors of short-term, as compared with long-term, survivors (20 versus 8% for intensity, 37 versus 19% for extent; Tables 3b
and 4b
). However, these findings failed to reach significance (P > 0.05).
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v and ß1 integrin protein expression was detected in both tumor and stromal cells (Fig. 1-G and 1-H)
. Frequent labeling of endothelial cells for ß1 integrin was also seen (Fig. 1-I)
. Protein expression results showed good correlation with those of mRNA expression using ISH. Thus, v integrin was detected in 23 of 30 (77%) specimens obtained from short-term survivors but only in 10 of 22 (45%) tumors of long-term survivors. It was in general more diffuse and intense in the former group. Expression in fibroblasts was practically universal in tumor-positive cases. In contrast, ß1 integrin protein expression was comparable in both patient groups (14 of 30 tumors of short-term survivors, 11 of 21 tumors from long-term survivors; 47 and 52%, respectively). Stromal immunoreactivity for ß1 integrin was less frequent than that observed for tumor cells.
Survival Analysis.
In univariate survival analysis of all cases, diffuse (>20% of cells) v mRNA integrin expression in tumor cells correlated with poor survival (P = 0.012; Fig. 2, -A and -B
). v integrin expression retained its predictive power as a marker of poor survival in a multivariate survival analysis, in which all molecules studied previously in this patient cohort were included (P = 0.031), together with TIMP-2 mRNA expression in stromal cells (P = 0.005) and the expression of MMP-2 (P = 0.004), Sialyl Lewisx (P = 0.007), -catenin (P = 0.003), and Ets-1 (P = 0.043) in tumor cells.
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DISCUSSION |
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2 and ß1 integrins have been shown to mediate adhesion of ovarian carcinoma cells to collagen type I (10)
. An additional report documented the role of vß3 and 5ß1 integrins in binding to fibronectin and that of the ß1 chain in binding to laminin and collagen I (13)
. Protein expression of v and ß3 chains and their ligand vitronectin has been additionally shown in normal ovarian surface cells (11
, 13)
. Analyses of integrin expression focusing on clinical specimens of ovarian carcinoma have been, however, few to date. vß3 integrin protein expression was shown to be lower in tumors of low malignant potential, as compared with invasive carcinomas (15)
, inconsistent with a previous study, in which tumors of the low malignant potential category were grouped together with grade I invasive carcinomas and compared with grade II-III carcinomas (12)
. Expression of 3ß1 integrin has also been shown in 26 of 31 ovarian carcinomas (14)
. None of the above-mentioned studies evaluated v and ß1 mRNA integrin expression in ovarian carcinoma, and none scored integrin expression in peritumoral fibroblasts. Our findings suggest that synthesis of v and ß1 integrin chains takes place in both carcinoma cells and stromal cells, although it is more prominent in tumor cells. Synthesis in fibroblasts may thus play a role in interaction with ECM ligands in much the same way as it does in cancer cells and could lead to activation of the synthesis of metastasis-associated molecules, such as MMPs. Our recent report, in which MMP mRNA was localized to both tumor and stromal cells (16)
, supports this hypothesis.
Studies of integrin expression in ovarian carcinoma have focused mainly on primary tumors to date (12
, 14
, 15)
. However, Moser et al. (10)
and Cannistra et al. (13)
analyzed cultures of cells obtained from ascites. No comparative studies of primary and metastatic tumors are available. Our results disclose no significant differences in the expression of v and ß1 integrins at primary and metastatic sites. On the basis of these findings, integrin synthesis appears to be an early event in tumor progression in ovarian carcinoma, in agreement with the role of these molecules in local invasion. The detection of v integrin reported previously in ovarian surface cells supports the possibility of up-regulation in the synthesis of a molecule constitutively expressed in the benign ovarian epithelium.
The prognosis of ovarian carcinoma remains poor, partially because of its late detection, often associated with widespread i.p. disease. Although a considerable amount of data has been acquired by in vitro studies of ovarian carcinoma cells, the prognostic role of the most important metastasis-associated molecules in this malignancy remains unknown. 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 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. Studying this patient cohort, we have identified recently the prognostic role of mRNA expression of MMPs (MMP-2, MMP-9, and MT1-MMP) and their inhibitor TIMP-2 (16)
. The carbohydrate antigen Sialyl Lewisx (17)
, the adhesion complex molecule -catenin (plakoglobin; Ref. 18
), and the transcription factor Ets-1 (19)
were similarly found to predict poor survival. Conversely, protein expression of the membrane protein caveolin-1 (20)
and mRNA expression of the angiogenic factors basic fibroblast growth factor, interleukin-8, and vascular endothelial growth factor did not predict the prognosis of patients in this cohort (21)
.
In the present study, expression of v integrin mRNA was a predictor of poor survival, a finding that retained its power in a multivariate survival analysis. The larger number of IHC-positive cases in both groups, as compared with mRNA results, may be attributable to the pretreatment of sections or the inherent sensitivity of the antibodies. Nevertheless, the differences between the two prognostic patient groups were similarly observed on protein level, supporting our findings using ISH. The prognostic significance of integrin expression has to date been investigated in a single study (15)
. Liapis et al. (15)
evaluated the prognostic role of vß3 integrin expression in 31 ovarian carcinomas and found no correlation between protein expression and disease outcome after a mean follow-up period of 26 months. However, 13 of 23 immunoreactive tumors proved fatal, as compared with 2 of 8 negative tumors (15)
. The absence of correlation with survival in the latter study may be attributed to the short follow-up period, possibly combined with a small number of cases. The cohort studied by us was followed up for an average period of 71 months, in effect considerably longer for nonlethal cases. It is noteworthy that two studies of v integrin expression in lung carcinoma have led to inconclusive results. Clarke et al. (24)
reported an association (though nonsignificant) between protein expression of v integrin and nodal metastasis in a study of 31 carcinomas, whereas Smyth et al. (25)
reported a significant association between loss of expression of this integrin and nodal metastasis. In an additional study, expression of vß3 integrin in vessels predicted disease relapse in breast carcinoma (26)
. To our best knowledge, our findings present the first evidence linking v integrin with poor survival in human epithelial malignancy. v integrin is thus a novel prognostic marker in ovarian carcinoma.
Although failing to reach significance, the more frequent expression of ß1 integrin mRNA in carcinoma cells in tumors of short-term survivors merits additional attention, as larger studies may reveal a similar role for this integrin. However, the similar expression on protein level in tumors from the two groups argues against a central role in determining disease outcome in ovarian carcinoma. The more pronounced expression of both v and ß1 integrin chain mRNA, as well as v protein in carcinoma cells rather than in stromal cells, suggests that tumor cell, rather than stromal production of these molecules, is central in the biology of ovarian carcinoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 R. R. is affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University.
3 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; ISH, in situ hybridization; IHC, immunohistochemistry.
Received 3/ 8/01; revised 7/31/01; accepted 8/31/01.
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REFERENCES |
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2ß1 integrin. Int. J. Cancer, 67: 695-701, 1996.[CrossRef][Medline]
vß3 and vitronectin expression by normal ovarian surface epithelial cells: role in cell adhesion and cell proliferation. Gynecol. Oncol., 75: 254-260, 1999.[CrossRef][Medline]
v integrins and their ligand vitronectin in normal ovarian epithelium and in ovarian carcinoma. Gynecol. Oncol., 62: 260-267, 1996.[CrossRef][Medline]
vß3 integrins in ovarian cancer. Gynecol. Oncol., 58: 216-225, 1995.[CrossRef][Medline]
vß3 integrin is less frequent in ovarian epithelial tumors of low malignant potential in contrast to ovarian carcinomas. Hum. Pathol., 28: 443-449, 1997.[CrossRef][Medline]
-v integrin expression and recurrence in node-negative lung carcinoma. Ann. Thorac. Surg., 64: 949-953, 1997.(v)ß3: a new prognostic indicator in breast cancer. Clin. Cancer Res., 4: 2625-2634, 1998.[Abstract]This article has been cited by other articles:
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  Q. Chen, H. J. Millar, F. L. McCabe, C. D. Manning, R. Steeves, K. Lai, B. Kellogg, R. J. Lutz, M. Trikha, M. T. Nakada, et al. {alpha}v Integrin-Targeted Immunoconjugates Regress Established Human Tumors in Xenograft Models Clin. Cancer Res., June 15, 2007; 13(12): 3689 - 3695. [Abstract] [Full Text] [PDF]
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 L. Belvisi, T. Riccioni, M. Marcellini, L. Vesci, I. Chiarucci, D. Efrati, D. Potenza, C. Scolastico, L. Manzoni, K. Lombardo, et al. Biological and molecular properties of a new {alpha}v{beta}3/{alpha}v{beta}5 integrin antagonist Mol. Cancer Ther., November 1, 2005; 4(11): 1670 - 1680. [Abstract] [Full Text] [PDF]
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 M. Maatta, R. Butzow, J. Luostarinen, N. Petajaniemi, T. Pihlajaniemi, S. Salo, K. Miyazaki, H. Autio-Harmainen, and I. Virtanen Differential Expression of Laminin Isoforms in Ovarian Epithelial Carcinomas Suggesting Different Origin and Providing Tools for Differential Diagnosis J. Histochem. Cytochem., October 1, 2005; 53(10): 1293 - 1300. [Abstract] [Full Text] [PDF]
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B. Davidson, I. Goldberg, W. H. Gotlieb, J. Kopolovic, G. Ben-Baruch, and R. Reich PEA3 Is the Second Ets Family Transcription Factor Involved in Tumor Progression in Ovarian Carcinoma Clin. Cancer Res., April 1, 2003; 9(4): 1412 - 1419. [Abstract] [Full Text] [PDF]
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