Expression of Maspin in Non-Muscle Invasive Bladder Carcinoma: Correlation withtumor Angiogenesis and Prognosis

Transcription

1 European Urology European Urology 45 (2004) Expression of Maspin in Non-Muscle Invasive Bladder Carcinoma: Correlation withtumor Angiogenesis and Prognosis Martin G. Friedrich a,*, Marieta I. Toma a, Susan Petri b, Jonathan C. Cheng c, Peter Hammerer a, Andreas Erbersdobler b, Hartwig Huland a a Department of Urology, University of Hamburg, University Hospital Hamburg-Eppendorf, Martinistr. 52, D Hamburg, Germany b Institute of Pathology, University of Hamburg, Hamburg, Germany c Department of Molecular Biology and Biochemistry, University of Southern California, Norris Comprehensive Cancer Center, Los Angeles, CA, USA Accepted 5 December 2003 Available online 30 December 2003 Abstract Objectives: Maspin is a member of the serpin (serine protease inhibitor) family and has been shown to be a suppressor of tumor growth and an inhibitor of angiogenesis as well as metastasis in several types of tumors. We studied expression patterns of Maspin in pta/pt1 urothelial carcinoma of the bladder and compared them with microvessel density (MVD) for two vascular markers (CD34 and CD105) and correlated the findings with clinical outcome. Material and Methods: We investigated tumor samples of 110 patients undergoing transurethral resection for pta/pt1 bladder carcinoma (pta, n ¼ 84; pt1, n ¼ 26; grade 1, n ¼ 22; grade 2, n ¼ 81; grade 3, n ¼ 7). Immunohistochemical studies were performed using the monoclonal antibodies, anti-human Maspin (NCL Maspin), anti-cd34 Class II and anti-cd105. Maspin expression level was classified according to the staining intensity ( to þþþ). The blood vessels (CD34) and specifically proliferating blood vessels (CD105) were counted as vessels per field (microvessel density, MVD). Results: Of the 110 tumors, 27 showed a negative immunostaining for Maspin, 46 tumors stained þ, 29 stained þþ, and 8 stained þþþ. Maspin expression correlated inversely with CD34 reactivity. In tumors with loss of or only weak Maspin expression, the MVD for CD34 was 21.7 vessels per field, and 4.2 vessels per field for proliferating vessels (CD105), whereas Maspin-positive tumors had an MVD of 17.7 vessels per field (CD34), and of 6.0 vessels per field (CD105). Complete follow-up data are available in 92 patients. After a median follow-up of 25 months, 18 of the 92 patients (19.6%) had tumor recurrences. Tumors with decreased Maspin expression ( /þ) had a shorter disease-free interval (23 months) than patients with stronger Maspin (þþ/þþþ) expression (29 months), whereas a Kaplan Meier analysis and the log-rank test showed no significant difference in disease-free survival between the patients. Conclusion: The clinical importance of Maspin has been mainly investigated regarding tumor progression or metastasis. We found a decreased Maspin expression in a large portion of pta/pt1 bladder tumors. Even if patients with decreased Maspin expression have a slightly shorter disease-free survival Maspin does not appear to be a promising prognostic marker. # 2003 Elsevier B.V. All rights reserved. Keywords: Bladder cancer; Maspin; Angiogenesis; Recurrence * Corresponding author. Tel. þ ; Fax: þ address: friedric@uke.uni-hamburg.de (M.G. Friedrich) /$ see front matter # 2003 Elsevier B.V. All rights reserved. doi: /j.eururo

2 738 M.G. Friedrich et al. / European Urology 45 (2004) Introduction With more than 50,000 newly diagnosed cases bladder cancer is among the most common in the US. Approximately 75 85% of the tumors are confined to the mucosa or the submucosa. Although these tumors can be removed completely by transurethral resection more than 50% will recur [1]. Depending on various risk factors including invasion of the submucosa, tumor focality and tumor size, the progression rate varies from 7.1% for patients with a low-risk tumor up to 40% for patients with a high-risk bladder carcinoma. Depending on initial staging and grading there is a risk of 4.3% for patients with low-risk tumors and of up to 30% for patients with high-risk tumors to die from initial non-invasive disease [2]. There is a clear need for development of prognostic markers to improve the management of bladder cancer. There is increasing evidence in clinical importance of Angionesis-regulating molecules [3]. Several angiogenic markers, including Thrombospondin-1 [4] and vascular endothelial growth factor (VEGF) [5], are currently discussed to be of clinical importance for bladder cancer. The angiogenesis inhibitor Maspin (Serpin B5) is a member of the serpin (serine protease inhibitor) family and has recently been described as a clinically relevant factor in several types of tumors. Machtens et al. found it to be a clinical relevant inhibitor of tumor progression in prostate cancer [6]. A decreased expression of Maspin was correlated with the progression of breast cancer [7], and was suggested to be a predictor for metastatic disease in squamous cell carcinoma of the tongue [8]. Maspin is expressed in normal but not in tumor-derived epithelial cells [9]. However the underlying mechanism is still controversial it has been shown that Maspin acts as an inhibitor of angiogenesis and suppresses tumor growth and metastasis in vivo [10]. Maspin has a sequence homology with many serpins, including plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2). The encoding gene was found to be located at chromosome 18q (18q21.3). Recent findings indicate that Maspin inhibits fibrinogen-associated tissue type plasminogen activator (tpa) [11] and urokinase type plasminogen activator (upa) [12]. Due to its antiangiogenic effect, it has been described to act as a tumor suppressor protein. In addition, there is increasing evidence that in addition to the antiangiogenic effect, Maspin can induce apoptosis of tumor cells [13]. The role of Maspin in bladder carcinoma is rarely studied. The aim of the study was to investigate the expression patterns of the Maspin protein in specimens of non-muscle invasive urothelial carcinoma of the bladder and to compare it with angiogenic markers, such as CD34 and CD105 to investigate the effects of Maspin in vascular formation as well as in clinical outcome. 2. Material and methods 2.1. Patients We included 110 patients undergoing transurethral resection of the bladder due to primary urothelial carcinoma of the bladder. The age of patients ranged between 33 years and 86 years with a median age of 68 years. Of the patients, 25 (22.7%) were female and 85 (77.3%) were male. Pathologic diagnosis was established using H&E sections and the tumors were classified according to the 5th edition of the UICC and the WHO [14]. Histopathologic examination revealed pta tumors in 84 patients and pt1 tumors in 26 patients, with 22 tumors classified as grade 1, 81 tumors as grade 2, and 7 tumors as grade Adjuvant therapy Post-operative adjuvant intravesical therapy with chemotherapy (Mitomycin C) or immunotherapy (Bacillus Calmette Guerin (BCG) was administered in all patients except small singularly pta, grade I tumors. Overall 91/110 patients (82.7%) received adjuvant intravesical chemo- or immunotherapy Clinical follow-up Follow-up investigations were performed by office urologists according to the guidelines of the European Association of Urology, which also included a cystoscopy every 3 months. All tumor recurrences were histologically confirmed Immunohistochemistry For immunohistochemical investigations, formalin-fixed, paraffin-embedded 4 mm sections were used. After deparaffinating and rehydrating, the endogenous peroxidase was blocked with 3% H 2 O Immunohistochemistry for Maspin We used a monoclonal antibody against Maspin (NCL-MAS- PIN, Novocastra, mouse anti-human monoclonal, clone EAW24) and an ABC detection system (IgG mouse Kit-Vector Laboratories). The slides were micro waved for 5 minutes in citrate buffer (ph 6). Negative controls were performed by replacing the first antibody by normal mouse serum for each tumor. Non-malignant prostate samples were used as positive controls. Non-specific reactions were blocked by incubating the slides with normal serum for 30 minutes at room temperature. The primary antibody (diluted 1:45) was applied and incubated one hour at room temperature. After washing with TBS buffer, the slides were incubated with the secondary antibody for 30 minutes at room temperature. After incubation with avidin-biotin and detection with DAB, the slides were counterstained with hematoxylin. The slides were dehydrated, clarified in xylol and mounted. Examination of the slides and quantitation of the staining were done at 20-fold and 40-fold magnification. The staining intensity was classified by the number of stained cells as the following: negative ( ), faint (þ: 0 4% positive cells), positive (þþ: 5 49% positive cells), or strongly positive (þþþ: >50% positive cells).

3 M.G. Friedrich et al. / European Urology 45 (2004) Immunohistochemistry for CD34 Microvessel density (MVD) was assessed by the technique of Weidner et al. [15]. For CD34 immunohistochemistry, we used the monoclonal antibody Class II, Clone QBEnd 10, DAKO, mouse anti-human. Antigen unmasking was performed by micro waving the slides 3 times for 5 minutes in citrate buffer (ph 6.0). After washing in PBS, the primary antibody was applied for one hour at room temperature. For the detection system, we used LSAB2 (DAKO, K0675) with the secondary antibody as biotinylated link (30 minutes) followed by streptavidine-peroxidase (30 minutes). After every step, the slides were washed in PBS (ph 7.4). The reaction was detected by incubating with DAB for 5 minutes. After counterstaining with hematoxylin, the slides were dehydrated, clarified and mounted. The slides were then examined at 40-fold magnification. A positive vessel was defined for the identification of a vessel lumen with at least one positive stained endothelial cell. The stained vessels were counted in 5 consecutive fields from the representative tumor zone. The mean value was considered as microvessel density (MVD) Immunohistochemistry for CD105 Antigen unmasking was performed with trypsin (2%) for 30 minutes at 37 8C. After washing with PBS (ph 7.4), the primary antibody anti-cd105 (GP160, DAKO M3537) (diluted 1:15) was applied for 1 hour at room temperature. For the detection system, we used LSABþ (DAKO, K0690) with the secondary antibody as biotinylated link (30 minutes) followed by streptavidine-peroxidase (30 minutes). The detection was performed with DAB for 5 minutes, and the slides were counterstained with hematoxylin. After dehydrating and clarifying, the slides were mounted in Eukit. MVD was assessed as described before for CD Statistical analysis Statistical analysis was performed with commercially available software (StatView 4.5, Abacus Concepts Inc., Berkeley, CA, USA). The Fisher s exact test was used to test the correlation between immunohistochemical findings with conventional clinical features such as pathologic stage, histological grade and tumor recurrence rate. The Kaplan Meier method was used to derive the recurrence-free survival function, and the log-rank test to compare curves for two or more groups; p values <0.05 were considered to reflect significant differences between groups [16]. for Maspin. Samples with increasing grades of dysplasia showed stronger Maspin reactivity in dysplastic urothelial cells. Of the 110 patients 27 (24.5%) showed a complete negative Maspin staining, whereas an at least faint Maspin immunoreaction was detected in 83 (75.5%) carcinomas. When classifying the tumor samples as retained ( /þ) or non-retained (þþ/þþþ) Maspin expression, 73 tumors showed a negative or only a faint ( /þ) Maspin expression, whereas 37 tumor samples showed a non retained Maspin expression. The correlation of Maspin staining intensity and histopathological staging/grading is shown in Table 1. Concomitant nuclear and cytoplasmic expression of Maspin was found in 11 cases. In contrast to the majority of tumors that showed a homogenous Maspin staining, the neoplastic urothelium of 8 patients displayed an increased Maspin reaction at the basal cell layer (Figs. 1 and 2) CD34 staining The median MVD of all investigated tumors was 21.0 (8.78) vessels per field; however, MVD did not differ between patients with a pta tumor (MVD 20:0 8:49) and patients with a pt1 tumor (MVD 18:8 10:05 vessels per field) (Figs. 3 and 4). Table 1 Maspin expression according to histopathologic staging and grading n ¼ 110 Negative Pos. (þ) Pos. (þþ) Pos. (þþþ) pta (27.3%) 35 (41.6%) 21 (25%) 5 (5.9%) pt (15.4%) 11 (42.3%) 8 (30.8%) 3 (11.5%) Grade (36.4%) 12 (54.5%) 1 (0.5%) 1 (0.5%) Grade (23.5%) 30 (37.0%) 25 (30.9%) 7 (8.6%) Grade (0%) 4 (57%) 3 (43%) 0 (0%) Total 27 (24.5%) 46 (41.8%) 29 (26.4%) 8 (7.2%) 3. Results 3.1. Clinical follow-up Follow-up data from 91 of 110 (82.7%) patients were available for further analysis. The follow-up interval was 4 52 months (median follow-up 26 months). Tumor recurrences were observed in 18 of 91 (19.7%) patients. One recurrent carcinoma progressed to muscle invasive disease. This patient underwent radical cystectomy and reconstruction of an ileal neobladder Immunohistochemistry Maspin expression The cytoplasm of normal urothelium in control samples showed a negative or weakly positive staining Fig. 1. Maspin expression in a pta, G2 tumor, displaying an evenly expression of Maspin in the cytoplasm.

4 740 M.G. Friedrich et al. / European Urology 45 (2004) Fig. 2. Maspin expression in a pta, G2 urothelial carcinoma displaying a more basal expression. Fig. 5. pt1 tumor with CD105 staining showing one stained microvessel. Fig. 3. pta tumor with CD34 staining showing two microvessels CD105 staining The median MVD of all tumors was 5.1 (4.6) vessels per field. There was no difference in MVD between patients with pta tumors (MVD 4:5 4:6 vessels per field) and patients with pt1 tumors (MVD 5:0 6:2 vessels per field) (Figs. 5 and 6). Fig. 4. pta tumor with CD34 staining showing ten microvessels. Fig. 6. pta tumor with CD105 staining showing one immature vessel at the basal membrane Correlation of Maspin expression with microvessel density The MVD (CD34) was higher in patients with a retained Maspin expression. In contrast to CD34 reactivity, a higher vascularization regarding to CD105 was associated with higher Maspin expression. Statistical analysis did not display a significant correlation or inverse correlation between Maspin expression and the MVD for CD34 or CD105 except for subgroups. (Table 2) Correlation of histopathologic staging and grading, Maspin expression, and MVD with clinical outcome Complete follow-up data are available in 91 patients. After a median follow-up of 25 months, 18 of the 92 patients (19.6%) had tumor recurrences. The recurrence rate was 18.2% in pta tumors and 23.1% in pt1 tumors. The disease-free interval was 26.5 months in patients with pta tumors and 26.0 in patients with pt1 tumors. Regarding tumor grading the recurrence rates

5 M.G. Friedrich et al. / European Urology 45 (2004) Table 2 Microvessel density (vessels per field) according to Maspin expression Maspin CD34 p vs. (negative) p ( /þ) vs. (þþ/þþþ) Negative ( ) Pos. (þ) p ¼ 0:057 Pos.(þþ) Pos. (þþþ) Maspin CD105 Negative ( ) Pos. (þ) p ¼ 0:15 Pos.(þþ) Pos. (þþþ) were 15% in grade 1 tumors, 23.4% in grade 2 tumors, and 0% in grade 3 tumors. The recurrence free intervals were 26.5 months (grade 1 tumors), 25 months (grade 2 tumors), and 35 months (grade 3 tumors). The Kaplan Meier analysis and the log-rank test did not reveal a significant difference in disease-free survival intervals for staging and grading ( p > 0:05). Among 62 patients with a retained Maspin expression ( /þ), 12 (19.3%) experienced a tumor recurrence, among patients with positive or strong Maspin staining (þþ/þþþ), the recurrence rate was 6 of 30 (20%). The disease-free survival interval was 23 months in patients with retained Maspin immunoreactivity and 29 months in patients with positive or strong expression of Maspin. The Kaplan Meier analysis and the log-rank test did not display a significant difference in disease-free survival ( p > 0:05). In contrast, patients with a basal staining of Maspin showed tumor recurrences in 3 of 5 cases. According to CD34 staining, patients with a tumor recurrence displayed an MVD of (9.7) vessels per field, whereas in cases with tumor recurrence an MVD of 20.3 (6.1) vessels per field was observed. For CD105 staining, In contrast to CD34 staining, patients without a tumor recurrence displayed a higher MVD (9:9 5:2 vessels per field) than patients with a tumor recurrence (6:1 1:9 vessels per field) ( p > 0:05, Fishers exact test). For the Kaplan Meier analysis the average MVD for all samples was used as threshold for the grouping variable. Using a threshold of 21.0 vessels per field (CD34) and 5.1 vessels per field (CD105) the Kaplan Meier analysis did not show a significant difference in the disease-free survival ( p > 0:05). 4. Discussion Maspin has been described as an inhibitor of angiogenesis in vitro and in vivo. Zhang et al. have demonstrated that Maspin acts directly on cultured endothelial cells by stopping their migration towards basic fibroblast growth factor and vascular endothelial growth factor and limiting mitogenesis and tube formation. In addition, they showed that Maspin blocks neovascularization in vivo in a rat cornea pocket model [10]. In a xenograft mouse model, they have demonstrated that Maspin blocked tumor growth of LnCAP prostate tumors and decreased microvessel density. Concerning Maspin effects, it has been described that the protease inhibits tumor invasion and metastasis by blocking the pericellular plasminogen activation cascade [11]. Also, it has been found that Maspin can sensitize tumor cells to apoptosis [10]. Mutations, hypermethylation and histone deacetylation may lead to decreased Maspin expression [17 19]. Decreased Maspin expression has been associated with impaired outcome in human breast and prostate cancers. Several authors have described that decreased or loss of Maspin expression is related to more advanced tumors [9], less differentiated tumors [20], or with tumor progression in ductal carcinomas [21]. In regards to prostate cancer, Machtens et al. found an association of reduced immunohistochemical expression of Maspin with tumor recurrence in an univariate analysis but not in a multivariate analysis. Decreased Maspin expression was associated with higher histological stage and grade, such as with increased p53 expression [6]. Our study is the first to investigate the clinical relevance of immunohistochemical Maspin staining pattern in bladder carcinoma. We correlated Maspin staining pattern with microvessel density by using anti- CD34 antibody and anti-cd105 antibody. CD34 detects endothelial cells of neoangiogenic vessels as well as of pre-existing vessels. Anti-CD105 antibody is reported to preferentially bind to endothelial cells of newly formed vessels [22,23]. In early bladder carcinoma, we find a retained Maspin expression in the majority of patients with

6 742 M.G. Friedrich et al. / European Urology 45 (2004) superficial cancer (66.4%). The percentage of tumors with reduced Maspin expression was similar in pta (69%) and pt1 tumors (57.7%). Correlating Maspin staining pattern with microvessel density established by CD34 and CD105 immunohistology, there were inconsistent results. Focusing on vascularization in pre-existing or mature vessels (CD34), we found that tumors with retained Maspin expression had a slightly higher vascularization in the tumor zone (not significant). In contrast, focusing on proliferating vessels (CD105), the microvessel density displayed only slight differences in the tumor vascularization. As Maspin has been described as a tumor suppressor for several types of tumors, we correlated the expression of Maspin with the recurrence rate and the disease-free interval in patients with non-invasive urothelial bladder carcinoma. There was no difference in the recurrence rates between patients with expression of the Maspin protein (20%) and in patients with a retained Maspin expression (19.4%) ( p > 0:05 Fisher s exact test). In addition, patients with a retained Maspin expression did not show asignificantly shorter disease free interval (23 months) than patients with a moderate or strong (þþ/þþþ) Maspin expression (29 months). The Kaplan Meier analysis and the log-rank test did not show a statistically significant difference between both groups. The prognostic relevance of Maspin expression is discussed controversially. Several authors have described Maspin as a prognostic marker for tumor impaired clinical outcome. In breast cancer, several authors find a reduced Maspin expression, which correlated with declined prognosis. In contrast, Umekita et al. showed an association of retained Maspin protein with impaired relapse-free survival and with deteriorating overall survival in breast cancer [24]. In ovarian cancer, Sood et al. considered Maspin overexpression as an independent prognostic parameter [25]. There are no data available concerning clinical relevance of Maspin expression in superficial bladder carcinoma. In contrast to several other types of tumors, we could not prove Maspin staining pattern to be a prognostic marker for tumor recurrence in patients with early bladder carcinoma. Though further investigation with larger series may be necessary to draw definite conclusions on the prognostic impact of Maspin, our data demonstrate that immunohistochemical expression of the Maspin protein does not seem to be a useful prognostic marker for the individual patient. 5. Conclusion We investigated the impact of the angiogenesis inhibitor Maspin on tumor vascularization and clinical outcome. There was no significant difference in recurrence rates or disease free interval between patients with normal or retained Maspin expression. We therefore conclude that Maspin does not appear to be a promising prognostic marker for patients with pta/pt1 bladder cancer. References [1] Holmang S, Hedelin H, Anderstrom C, Holmberg E, Busch C, Johansson SL. Recurrence and progression in low grade papillary urothelial tumors. J Urol 1999;162(3 Pt 1): [2] Chopin DK, Gattegno B. Superficial bladder tumors. Eur Urol 2002; 42(6): [3] Kausch I, Bohle A. Molecular aspects of bladder cancer III. Prognostic markers of bladder cancer. Eur Urol 2002;41(1): [4] Goddard JC, Sutton CD, Jones JL, O Byrne KJ, Kockelbergh RC. Reduced thrombospondin-1 at presentation predicts disease progression in superficial bladder cancer. Eur Urol 2002;42(5): [5] Crew JP. Vascular endothelial growth factor: an important angiogenic mediator in bladder cancer. Eur Urol 1999;35(1):2 8. [6] Machtens S, Serth J, Bokemeyer C, Bathke W, Minssen A, Kollmannsberger C, et al. Expression of the p53 and Maspin protein in primary prostate cancer: correlation with clinical features. Int J Cancer 2001;95(5): [7] Maass N, Teffner M, Rosel F, Pawaresch R, Jonat W, Nagasaki K, et al. Decline in the expression of the serine proteinase inhibitor Maspin is associated with tumour progression in ductal carcinomas of the breast. J Pathol 2001;195(3): [8] Yasumatsu R, Nakashima T, Hirakawa N, Kumamoto Y, Kuratomi Y, Tomita K, et al. Maspin expression in stage I and II oral tongue squamous cell carcinoma. Head Neck 2001;23(11): [9] Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, Sheng S, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 1994;263(5146): [10] Zhang M, Volpert O, Shi YH, Bouck N. Maspin is an angiogenesis inhibitor. Nat Med 2000;6(2): [11] Sheng S, Truong B, Fredrickson D, Wu R, Pardee AB, Sager R. Tissue-type plasminogen activator is a target of the tumor suppressor gene Maspin. Proc Natl Acad Sci U S A 1998;95(2): [12] Biliran Jr H, Sheng S. Pleiotrophic inhibition of pericellular urokinase-type plasminogen activator system by endogenous tumor suppressive Maspin. Cancer Res 2001;61(24): [13] Jiang N, Meng Y, Zhang S, Mensah-Osman E, Sheng S. Maspin sensitizes breast carcinoma cells to induced apoptosis. Oncogene 2002;21(26): [14] Sobin LH, Wittekind C. TNM Classification of Malignant Tumors, 5th edn. New York: John Wiley & Sons; [15] Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis correlation in invasive breast carcinoma. N Engl J Med 1991;324(1):1 8. [16] Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Statist Assoc 1958;53: [17] Umekita Y, Hiipakka RA, Liao S. Rat and human Maspins: structures, metastatic suppressor activity and mutation in prostate cancer cells. Cancer Lett 1997;113(1 2):87 93.

7 M.G. Friedrich et al. / European Urology 45 (2004) [18] Costello JF, Vertino PM. Methylation matters: a new spin on Maspin. Nat Genet 2002;31(2): [19] Maass N, Biallek M, Rosel F, Schem C, Ohike N, Zhang M, et al. Hypermethylation and histone deacetylation lead to silencing of the Maspin gene in human breast cancer. Biochem Biophys Res Commun 2002;297(1): [20] Hojo T, Akiyama Y, Nagasaki K, Maruyama K, Kikuchi K, Ikeda T, et al. Association of Maspin expression with the malignancy grade and tumor vascularization in breast cancer tissues. Cancer Lett 2001; 171(1): [21] Maass N, Hojo T, Rosel F, Ikeda T, Jonat W, Nagasaki K. Down regulation of the tumor suppressor gene Maspin in breast carcinoma is associated with a higher risk of distant metastasis. Clin Biochem 2001;34(4): [22] Tanaka F, Otake Y, Yanagihara K, Kawano Y, Miyahara R, Li M, et al. Evaluation of angiogenesis in non-small cell lung cancer. Cancer Clin Res 2001;7: [23] Kumar S, Ghellal A, Li C, Byrne G, Haboubi N, Wang JM, et al. Breast carcinoma: vascular density determined using CD105 antibody correlates with tumor prognosis. Cancer Res 1999;59: [24] Umekita Y, Ohi Y, Sagara Y, Yoshida H. Expression of Maspin predicts poor prognosis in breast-cancer patients. Int J Cancer 2002;100(4): [25] Sood AK, Fletcher MS, Gruman LM, Coffin JE, Jabbari S, Khalkhali-Ellis Z, et al. The paradoxical expression of Maspin in ovarian carcinoma. Clin Cancer Res 2002;8(9):