Elevated Levels of Versican but Not Decorin Predict Disease Progression in Early-Stage Prostate Cancer1

Vol. 4, 963-97i, April i998 Clinical Cancer Research 963

Elevated Levels of Versican but not Decorin Predict Disease Progression in Early-Stage Prostate Cancer1

Carmela Ricciardelli, Keiko Mayne, sican (Kaplan-Meier plot, 89% versus 27% PSA progres- Pamela J. Sykes, Wendy A. Raymond, sion-free at 5 years, respectively; P = 0.0001). We conclude that the measurement of prostatic concentrations of versi- Kieran McCaul, Villis R. Marshall, and can, a molecule with reported anticellular adhesive proper- David J. Horsfall2 ties, may be a useful marker of disease progression in pa- Departments of Surgery [C. R., K. M.. V. R. M., D. J. H.], tients with early-stage prostate cancer and that further Haematology [P. J. S.], and Anatomical Pathology [W. A. R.], study of versican in other patient cohorts is warranted. Hinders University School of Medicine and Hinders Cancer Centre, Hinders Medical Centre. Bedford Park, South Australia 5042, and Epidemiology Branch, Public and Environmental Health Service, INTRODUCTION South Australian Health Commission, Adelaide, South Australia 5000 Patients with organ-confined prostate cancer who would [K. M.], Australia benefit from aggressive and potentially curative treatment are not easily identified by current investigative procedures. More ABSTRACT accurate biomarkers of tumor progression are needed to improve Patients with clinically localized prostate cancer who patient selection and hence overall clinical outcome. We re- might be cured by aggressive management are not easily cently reported that CS3 concentration in peritumoral stromal identified using current clinical information. Additional, tissue of the prostate is a potentially useful biomarker of PSA more accurate, biomarkers of tumor behavior need to be progression in patients who have received definitive treatment identified to improve clinical outcome. Our previous studies for early-stage prostate cancer (1). Patients with low CS con- indicated that the concentration of the glycosaminoglycan centration had a significantly better progression-free survival chondroitin sulfate in prostatic stroma might be a useful rate following radical prostatectomy than patients with high biomarker of disease progression in early-stage prostate levels of CS, and CS was a predictor of disease outcome in cancer. In this study, two chondroitin sulfate proteoglycans, patients with moderately differentiated tumors. That study also versican and decorin, were investigated. Versican and indicated that CS and serum PSA concentrations at diagnosis decorin were immunolocalized to the periacinar and peritu- were independent predictors of PSA progression. We concluded moral fibromuscular stroma in sections of nonmalignant that measurement of stromal concentrations of CS-proteogly- and malignant human prostate tissues. Video image meas- cans at diagnosis of early-stage prostate cancer might facilitate urements indicated that the concentrations of both proteo- the selection of patients for aggressive management. glycans were increased in the prostatic tissue of men with CS-proteoglycans influence cell growth and differentiation early-stage prostate cancer compared with tissue from men of numerous tissues and are reportedly elevated in several without cancer (P 0.0006). Cox’s univariate analysis in- malignancies (2-6). The CS side chains of proteoglycans mod- dicated that increases in versican concentration but not in ulate cell attachment to and migration through the ECM (7-12) that of decorin were associated with increased risk of pros- and appear to be important in the malignant progression of at tate-specific antigen (PSA) progression. Versican concentra- least prostate cancer (1). Information regarding the identity of tion was compared with other clinical or biological features CS-proteoglycans in the prostate gland or other tissues of the of prognosis in two-variable regression analyses. Versican urinary system is limited (13, 14). This study is confined to an and serum PSA concentrations were independent predictors examination of the CS-proteoglycans versican and decorin in of PSA progression. Versican was a stronger prognostic the human prostate and whether they are associated with disease factor than tumor grade, and it could predict outcome for progression in prostate cancer. patients with moderately differentiated tumors. Patients Versican, a large proteoglycan (900 kDa) with 15-20 CS with low versican concentration had significantly better pro- side chains, belongs to a family of extracellular, aggregating gression-free survival than patients with high levels of ver- proteoglycans that bind hyaluronan and are involved in matrix assembly (15, 16). This family of proteoglycans also contain

lectin-like and epidermal growth factor-like sequences ( I 7). Four isoforms of human versican have been identified (13, 18).

Received 4/22/97; revised 1/5/98; accepted 1/16/98. Isoforms VO, Vi, and V2 result from alternative splicing of two 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.

I Supported by the National Health and Medical Research Council of 3 The abbreviations used are: CS, chondroitin sulfate; BPH, benign Australia, Australian Kidney Foundation, Anti-Cancer Foundation of prostatic hyperplasia; DAB, diaminobenzidine tetrahydrochloride; South Australia, and Hinders Medical Centre Foundation. ECM, extracellular matrix; GAG, glycosaminoglycan; MIOD, mean 2 To whom requests for reprints should be addressed. Fax: 618-8374- integrated absorbance; PSA, prostate-specific antigen; TURP, transure- 0832 or 618-8204-5899. thral resection of prostate.

large exons each encoding a GAG attachment domain. VO, the ination, transrectal ultrasound-guided core biopsy, and bone largest isoform, contains both the GAG-a and the GAG-3 scans. Additionally, serum PSA level was measured at initial domains, whereas Vl lacks GAG-a and V2 lacks GAG-3. diagnosis. PSA levels were determined routinely by a solid Isoform V3 lacks both GAG attachment domains and appears to phase, two-site immunoenzymatic assay (Tandem-E PSA; Hy- be devoid of GAG side chains. Presently, only versican VO and britech, Inc., San Diego, CA). Assessment of disease progres- VI isoforms have been identified at the protein level; the exist- sion by PSA failure and clinical treatment according to stage of ence of the V2 and V3 isoforms is based on reverse transcrip- disease, age, and medical fitness of the individual patient, were tion-PCR (13, 19). The possibility has been raised, however, as described previously (1). None of the patients in this cohort that glial hyaluronan-binding protein, which lacks CS side received adjuvant hormonal therapy at any stage of treatment, chains and was considered to be a proteolytic fragment of prior to or after surgery. Median follow-up for this early-stage versican, might be the V3 isoform (19, 20). Decorin, a small prostate cancer cohort was 54 months (range, 19-90). proteoglycan (130 kDa) with a single CS or dermatan sulfate Tissue Processing and Immunohistochemical Staining side chain, belongs to a family of ECM proteoglycans, which for Proteoglycans. Paraffin-embedded prostatic tissues were are characterized by several leucine-rich repeat sequences in the processed by standard laboratory protocols and sectioned as core protein. Decorin is primarily associated with collagen described previously (1). Immunohistochemical staining was fibrils of the ECM, but it can also bind fibronectin and throm- performed essentially as described previously (1). Rabbit anti- bospondin. Additionally, decorin binds and regulates the bio- body (diluted 1 : 1000) to human versican fusion protein, purified logical activity of transforming growth factor 3 (21, 22). on an immunoadsorbent of 26-mer versican peptide correspond- The peritumoral stroma, a specialized tissue primarily as- ing to amino acid residues 383-408 (exon 8 GAG-3 domain) of sociated with adenocarcinomas, has been proposed as a regula- the Vi isoform (15) was a gift of Dr. R. LeBaron and Prof. E. tor of neoplastic behavior. Tumor cells have been considered to Ruoslahti, La Jolla Cancer Research Foundation (La Jolla, CA). modify their microenvironment by triggering changes in the This antibody recognizes the VO and Vi isoforms but not the V2 tumor stroma toward a permissive milieu that facilitates inva- and V3 isoforms of versican, which lack the GAG-3 domain. sion (6, 23). Considering the reported anticellular adhesive Rabbit antibody (diluted 1 :500) to deglycosylated human fibro- property of CS, promotion of disease progression by matrix blast decorin (26, 27) was a gift of Prof. H. Kresse, Department proteoglycans is likely to be facilitated by alterations in the of Physiological Chemistry and Pathobiochemistry, University matrix concentration of the proteoglycan or the number or of Munster (Munster, Germany). Nonimmune rabbit serum was length of side chains on the proteoglycan. Consequently, we used as control. Epitope binding by specific antibody was visu- propose that altered expression of stromal proteoglycans may alized by a standard streptavidin immunoperoxidase reaction play an important role in the progression of prostate cancer. This using biotinylated secondary antibody (Vector Laboratories, study investigates whether increased concentrations of versican Burlingame, CA) and DAB to yield an insoluble brown deposit. and/or decorin in the pentumoral stroma of early-stage prostate The area and absorbance of DAB deposition were meas- cancers are related to the rate of PSA progression. ured using an automated image analysis system (VideoPro 32; Leading Edge Pit, Marion, South Australia, Australia) as re-

ported previously ( 1). Color images were collected at a magni- MATERIALS AND METHODS fication of X 100. A menu-based program driving a motorized Prostatic Tissue. Formalin-fixed tissue from the transi- microscope stage was used to capture 20 contiguous fields for tional zone of prostates from men with no known clinical each prostate sample, beginning adjacent to a randomly chosen

urinary symptoms (n = 1 1 ; median age, 65 years; range, 55- 83) acinus or cancer focus. Captured fields included both glandular were supplied by pathologists of the South Australian and and stromal tissue areas. Due to the exclusive stromal localiza- Victorian Institutes of Forensic Pathology. The pathology of the tion of versican and decorin, video image measurements were tissues in this autopsy cohort has been reported previously (1). confined to the stroma of both malignant and nonmalignant Clinically derived prostatic tissues used in this study were those prostate tissues, with luminal and epithelial areas, including

reported previously ( 1 ). Benign tissues were obtained at TURP cancer cell foci, edited from the images. The integrated absorb- from patients with poor urinary stream. Tissues from 1 1 patients ance of DAB deposited in the stroma for each image was with BPH (median age, 72 years; range, 54-88) were selected determined, and specific antibody staining was expressed as the from archives at random. Malignant tissue was available from MIOD per unit area of stromal tissue of the 20 images. MIOD patients who underwent TURP and/or radical prostatectomy. was recorded in arbitary density units per pixel. Because of the Tissues used in this study were confirmed as BPH or prostate heterogeneous nature of prostatic carcinoma, variability in im- cancer by one pathologist (W. A. R.). Prostate cancers were age measurements were kept to a minimum by the following graded on the surgical specimen, according to the method of procedures. Accuracy and reproducibility in MIOD measure- Bocking et a!. (24). ments were achieved through strict observance of random sam- Early-Stage Prostate Cancer Patients. Forty-three pling procedures (28) and stringent use of inter- and intrarun prostate cancer patients (median age, 67 years; range, 46-85) control tissue sections with known levels of immunostaining. In were accrued retrospectively as consecutive patients with early- addition, the number of fields to be analyzed was derived by the stage prostate cancer visiting academic urologists at two centers cumulative quotient principle to achieve variance in MIOD

between 1987 and 1991 . Disease stage was determined clini- measurements below 5% in most instances (28). Other controls cally using the Union Internationale Contre le Cancer system performed examined whether surgical technique (paired radical (25). Clinical disease staging was based on digital rectal exam- prostatectomy and TURP specimens from six patients), delay in

Fig. 1 Immunoperoxidase staining of prostate cancer tissue see- tions using monoclonal antibodies to the proteoglycans versican and decorin. Proteoglycan staining was restricted to the pentumoral stroma. A, versican, stage I2 patient (Table 1, patient 12) with well-differentiated prostate cancer. B, versican, stage 12 patient (Table 1, patient 33) with poorly differentiated prostate cancer. C, decorin, stage T2 patient (Table 1, __i1’’__. - -,. .. patient 12) with well-differenti- :; ‘: ‘,‘ ated prostate cancer. D, decorin, .. . ..‘., stage T2 patient (Table 1, patient 33) with poorly differentiated i:” I ‘ prostate cancer. Tech-Pan (Kodak) black and white film was used with a green filter to enhance the contrast of immunostaining. A and C, X260; B and D, X190.

tissue collection by autopsy, or differences in fixation times Overall, versican staining was reduced in intensity compared between prostate specimens or between pathology laboratories with decorin staining. were potential sources of loss of epitope (1). These controls Measurement of Versican and Decorin Concentration indicated that there was no significant loss of epitope staining in in Prostatic Stroma. The stromal concentrations (MIOD) of any case. Furthermore, image analysis measurements of immu- versican and decorin were measured by image analysis in tissues nohistochemically detected CS epitopes have been shown in our from asymptomatic men and from patients with BPH or clini- hands to be accurate and reproducible when compared with cally localized prostate cancer (Fig. 2). Versican and decorin biochemical measurements made on the same (guinea pig) pros- levels in prostatic stroma were significantly greater in cancer tatic tissues (29). Image analysis enables independent measure- patients than in asymptomatic men or BPH patients. Poorly ments to be made of cancer or stroma associated immuno- differentiated carcinomas had significantly greater levels of stainingwithin a tissue without the need to fractionate the tissue stromal versican than well and moderately differentiated pros- compartments before measurement (29). tate cancers (Fig. 3A). The histological grade and versican Statistical Analysis. Progression-free survival was cal- measurement of the outlying point in the well-differentiated culated from the date of diagnosis to the date of progression. cohort (Table 1, patient 43) were independently confirmed. Survival times were censored on January 30, 1996. Patients who There was no significant difference in decorin staining accord- died from causes other than prostate cancer were censored on ing to cancer grade (Fig. 3B). To determine whether the increase the date of their death. Progression-free survival was used as the in versican and/or decorin staining was related to the increased end point in Cox’s proportional hazards regression analyses level of CS staining reported previously (1), linear correlation (SPSS package, SPSS Inc., Chicago, IL). Variables in multiva- analyses were performed. The concentrations of versican and nate Cox models were examined for multicollinearity using the CS demonstrated a high degree of concordance (r = 0.742; P < method of Davis et al. (30). Progression-free survival of patient 0.0001 ; Fig. 4A). There was no relationship between decorin and subgroups was compared using the Kaplan-Meier product-limit CS concentrations (r = 0.197; P = 0.103; Fig. 4B). method and the log-rank test. Differences between groups of Correlates of Disease Progression in Early-Stage Pros- continuous variables were analyzed by one-way ANOVA tate Cancer. A summary of the clinical and pathological data (Kruskal-Wallis ANOVA). Data are expressed as median for the early-stage prostate cancer cohort is provided in Table 1. (range). Statistical significance was set at P < 0.05. With the exception of a single patient clinically staged at 13, the patients were stage 11-12. Tumor-positive lymph nodes were RESULTS absent from the 27 patients treated by radical surgery. Lymph Immunohistochemical Localization of Versican and node status of the remaining 16 patients was unknown. Extra- Decorin. Immunoperoxidase staining of clinical BPH tissues capsular extension of disease was found in 12 of 27 patients localized versican to the basement membrane of the glandular treated by radical surgery. Tumor-positive surgical margins acini and both versican and decorin to the penglandular stromal were reported for 5 of the 27 patients, and 4 patients had seminal matrix, i.e. , regions where CS was localized previously (1). vesicle involvement (2 of whom also had tumor extension to the Prominant peritumoral staithng of stromal matrix for versican surgical margins). and decorin was identified in sections ofprostate cancer (Fig. 1). The association of several clinical parameters and tumor

A A 30 30 S 6 Q! 2 a) 20 0 20 a) 0 .C U 0

0 ;C Ill .

H 1o 10 0 I* H -9.. * 0 ;; H 0

-I- 0 0. - - f aged-matched BPH cancer weD moderate poor control B B 40 40

0 U 0 0 30 H U 0 U a) H U C -H- U 20 U -I- I:: 1 U 0 U H U 0 U -0- U 10 10 0 I U a 0 0 H

0 0. 0 , . , I well moderate poor aged-matched BPH cancer Fig. 3 Relationship between proteoglycan immunostaining and tumor control grade in early-stage prostate cancer. Tumor grading of well (n = 15),

Fig. 2 Comparison of proteoglycan immunostaining in normal, hyper- moderately (n = 17), and poorly (n = 1 1) differentiated tumors, ac- plastic. and malignant prostate tissues. Patients were as follows: normal cording to the method of Bocking et al. (24). Stromal concentration (asymptomatic) males ages 55-83 years (median, 65 years: n = 1 1): (MIOD) of proteoglycan was recorded in pixel density units. A, versican patients with clinical BPH ages 54-88 years (median, 72 years; n = (*, significantly different from well-differentiated tumor group by 1 1 ); patients with early-stage prostate cancer ages 46- 85 years (median, Kruskal-Wallis ANOVA), P 0.0025; B, decorin (no significant dif-

67 years: n = 43). Stromal concentration (MIOD) of proteoglycans was ference between groups), P = 0.8786. recorded in pixel density units. *, significantly different from age- matched control and BPH tissue groups by Kruskal-Wallis ANOVA. A,

versican, P = 0.0006; B. decorin, P 0.0078. increase in risk of progression compared with patients with tumors below the cut-point. Several further cut-points of versican concentration were tested around the median value, and all features (age, serum PSA value at diagnosis, CS concentration, were found to be significant (data not shown). The largest tumor grade, and mode of treatment) with progression-free increase in risk was found at a MIOD of 6.3, the mean versican survival has been reported for this cohort of patients (1). In this concentration for the cohort. Stromal decorin concentration was study, versican and decorin concentrations were analyzed for not associated with disease progression, whether examined as a any association with progression-free survival (Table 2). Ele- continuous or dichotomized variable. To ensure adequate statis- vated stromal versican expression was significantly associated tical analysis in this cohort, versican concentration was then with an increased risk of PSA progression in early-stage tumors, compared in two-variable regression analyses with the clinical whether analyzed as a continuous or a dichotomous variable. and tumor features, which showed significant association with Used as a continuous variable, there was a 16% increase in risk disease progression (Table 3). Versican and preoperative PSA for each unit increment in versican concentration. When dichot- values were independent predictors of progression in men with omized using an initial cut-point of the median versican con- early-stage prostate cancer. Versican was a stronger predictor of centration for the cohort (MIOD, 6.0), patients with tumors cancer progression than tumor grade. Bivariate analysis of CS equal to or above the cut-point had an average of 7- 8-fold and versican indicated that CS was a stronger predictor of

Table 1 Summary of clinical and patholog y data for th e early-stage prostate cancer cohort Progression Follow-up Patient Clinical Tumor Surgical Versican Decorin (months post- (months post- no. Age stage PSA” Treatment differentiation ECE5 margins level” levele operation) PSA’ operation)

1 72 I1 NIY Rx Well 1.75 22.44 90A”

2 67 T1 <1.0 RP Well - - 0.22 16.06 87A

3 70 12 3.0 RP Moderate + + 0.78 20.68 65A 4 66 I, 2.7 Rx Well 2.81 24.10 86A 5 73 I1 7.0 Rx Well 5.99 31.24 28A

6 62 12 41 RP Moderate - - 0.19 17.22 81A

7 64 I2 7.9 RP Well - - 0.58 18.16 84A 8 67 T ND None Well 7.55 23.84 43 14.6 65B

9 56 T1 ND RP Moderate - - 5.92 2 1.06 84A

10 62 I 0.7 RP Well - - 0.57 13.68 54A

1 1 67 I2 17.5 RP Moderate + -‘ 2.90 24.49 90 0.8 90A

12 74 12 7.4 RP Well - - 2.03 8.03 62 1.8 78A

13 64 12 ND RP Moderate - - 1.55 20.61 53A

14 58 T2 <1.0 RP Moderate - - 1.28 13.10 76A 15 62 T 1.2 RP Well + + 5.83 26.07 39A

16 68 T, 9.5 RP Well - - 5.94 20.66 51A

17 64 T <1.0 RP Well - - 4.27 26.55 67A

18 46 12 1.4 RP Well + - 1.81 26.39 40A

19 64 T, 1.7 RP Well - - 3.43 15.83 78A 20 7 1 T ND None Well 5.41 24.49 53A

21 65 I, ND RP Poor + - 9.83 14.99 74 4.2 79B

22 68 T, <1.0 RP Moderate + - 2.72 17.35 78A 23 68 T2 ND RP Poor + +‘ 4.68 17.82 36 5.0 54B

24 7 1 12 25.2 RP Poor - - 6.67 30. 12 36A

25 57 1, <1.0 RP Poor - - 13.02 28.34 90 1.1 90B

26 69 I2 <4.00 RP Moderate + - 8.63 22.79 37 0.6 37C 27 69 T2 <1.0 RP Moderate + + 6.46 32.83 27 121 41B

28 62 I1 <1.0 RP Moderate - - 6.17 20.07 68B 29 74 I1 3.70 None Moderate 9.36 25.46 24 6.9 62B 30 65 I2 84.0 Rx Poor 12.47 12.58 II 23.6 7lB

31 68 T1 7.30 RP Moderate + -‘ 3.89 25.87 42A 32 71 12 44.70 RP Moderate + + 5.18 20.48 60 5.0 78B

33 64 12 1.50 RP Poor + +‘ 6.85 20.52 58 13.0 74B 34 74 T2 30.20 Rx Moderate 11.27 16.10 48 13.0 MC 35 79 T ND None Poor 7.11 15.00 65A 36 85 T2 11.2 None Moderate 9.11 30.76 19 14.0 19B 37 60 T1 ND Rx Poor 18.20 26.03 58 25.5 103C 38 60 T2 ND Rx Moderate 11.54 21.79 18 >100 45B 39 75 13 11.0 Rx Poor 8.18 18.00 24 11.9 56B

40 59 I2 5.2 RP Moderate - - 6.61 8.61 7lA 41 63 1, ND Rx Poor 6.56 26.67 37 7.7 91B 42 72 T2 54.0 None Poor 16.22 35.87 10 36.8 SOB 43 77 I, 5.10 Rx Well 19.25 30.69 45 6.4 54B

a PSA level (ng/ml) at diagnosis.

b ECE, extracapsular extension of tumor; + , present; - , absent.

C tumor present; - , tumor absent. d Concentration in MIOD units.

e Concentration in MIOD units.

I PSA level at documented progression. S ND, not done; RP, radical prostatectomy; Rx, radiotherapy. S A, alive without evidence of progression; B, alive with progression; C, death unrelated to prostate cancer.

I Tumor invasion of seminal vesicles.

progression than versican. There was no evidence of multicol- pared with 27%; P = 0.0001). Progression-free survivals for the linearity in this comparison (condition index, 2.6). whole cohort of patients according to tumor grade were reported Kaplan-Meier product-limit plots comparing progression- previously (1). Progression-free survivals of patients with mod- free survivals for the total patient cohort using the median erately differentiated tumors according to versican concentra- (MIOD, 6.0) cut-point in versican concentration are shown in tion are shown in Fig. 6. Patients with moderately differentiated Fig. 5. Early-stage prostate cancer patients with low stromal tumors expressing low levels of versican had a significant lower concentrations of versican (MIOD, <6.0) had a significantly incidence of progression than similarly grouped patients with lower incidence of PSA progression than patients with higher high versican expression (86% progression-free at 5 years com- versican concentrations (89% progression-free at 5 years compared with 25%; P < 0.0045). Progression-free survivals for the

A Table 2 Univariate Cox regression analysis of progression in early- stage prostate cancers (n = 43) 30 The data for tumor grade, PSA level, treatment, age, and CS

U concentration were previously reported (1).

a Variable Relative risk 95% confidence interval P Q 20 U. #{149} Differentiation I.. 0) U Well 1.00 C U C _ . Moderate 2.56 0.68-9.68 0.1647 #{149}10 #{149}U. U. U Poor 5.21 1.41-19.37 0.0136 U) UU 1.04 C’) U I PSA” 1.01-1.07 0.0051 C.) U Co #{149}UU UU Treatment U Radical 1.00 UU Radiotherapy 2.99 1.10-8.14 0.0325 U 0 No treatment 4.49 1.34-15.06 0.0149 10 20 30 Ageb 1.08 1.01-1.17 0.0301 Versican staining (MIOD) CSC 1.17 1.08-1.27 0.0001 Versican” 1 . 16 1.08-1.25 0.0001 Versicane 7.66 2.53-23.18 0.0003 B Decorin1 1.08 0.93-1.18 0.0784 40 a PSA level at diagnosis (ng/ml) as a continuous variable; there is a 4% increase in risk of progression per unit increment in PSA concentration (n = 32). Q30 b There is an 8% increase in risk of progression per year.

C Concentration (MIOD units) as a continuous variable; there is a

0) U 17% increase in risk of progression for every I .0 MIOD increment in CS C 20 U U U U .co.c UU U concentration. d U Ui lip U Concentration (MIOD units) as a continuous variable; there is a (I) U UU U 16% increase in risk of progression for every 1.0 MIOD increment in c10 U U U versican concentration. (0 U U U UU U e Concentration as a dichotomous variable; median cut point <6.0 U UU versus 6.0. n U 0 10 20 30 4o 1Concentration (MIOD units) as a continuous variable.

Decorin staining (MIOD)

Fig. 4 Relationship between proteoglycan staining and staining for CS (data for CS, epitope 6C3, from Ref. 1). A, versican; linear regression indicated that the CS-proteoglycan versican is a candidate bi- correlation coefficient, r = 0.742; P < 0.0001. B, decorin; r = 0.197; P = 0.103. omarker, whereas another CS-proteoglycan, decorin, is unlikely to be useful in determining progression in prostate cancer. Versican and decorin were both elevated in the peritumoral stroma of prostate cancers compared to nonmalignant hyper- patient subgroup treated by radical prostatectomy according to plastic tissues. Decorin levels showed no evidence of correlation versican concentration are shown in Fig. 7. Patients with versi- with the CS levels previously reported for this cohort of patients can levels lower than the mean versican concentration (MIOD, (1), nor any association with tumor grade or PSA progression. 6.3) for the radical prostatectomy subgroup were statistically Versican levels, in parallel with CS levels, were significantly associated with a lower incidence of progression (89% progres- elevated in poorly differentiated cancers and were strongly sion-free at 5 years compared with 54%; P 0.0221). Use of predictive of PSA progression. Two-variable Cox analysis in- the median cutoff (MIOD, 6.0) in this instance, however, just dicated that versican concentration was independent of serum failed to reach significance (P = 0.0591). PSA values at diagnosis and was a stronger predictor than tumor grade. The fact that the patient with well-differentiated prostate DISCUSSION cancer and elevated versican (Fig. 3A, outlier point; Table 1, Accurate determination of organ-confined disease remains patient 43) was among the relapsing group of patients would a difficult problem in the management of patients with early- support this contention. Kaplan-Meier plots and log rank statis- stage prostate cancer. Lower grade of tumor, small volume of tics indicated that patients with low levels of versican expen- disease at core biopsy, and low serum PSA level (<4 ng/ml) are enced a longer progression-free survival than patients with currently used as guides for determining organ-confined pros- elevated levels of versican. Another important observation of tate cancer. However, the facts that 40-70% of patients treated this study was that versican concentration, again in parallel with by radical prostatectomy for presumed organ-confined disease CS levels, appeared to be capable of discriminating patients with are found to be understaged at pathological examination and that moderately differentiated tumors into those who do and those many of these patients will eventually experience PSA progres- who do not progress after definitive local therapy. An exami- sion indicate that the identification of more accurate biomarkers nation of the subgroup of patients who received radical prosta- of progression must remain a high priority (31, 32). Our previ- tectomy indicated that versican concentration was of borderline ous report (1) indicated that the level of CS in the peritumoral significance in the prediction of progression-free survival in this

stroma might be a useful biomarker. The present study has group (mean concentration, P = 0.0221 ; median concentration,

Table 3 Two-variable Cox regression analysis of progression in 1.0 - early-stage prostate cancers (n = 43) -+--versican <6.0 (n=9) Relative 95% confidence CS) .8 C Variable risk interval P a) CO Versican and differentiation 0. .6 Versican” 1.17 1.06-1.30 0.0016 0 C Differentiation 0 .4 Well 1.00 0 0. versican>6.0 (n=8) Moderate 2.91 0.74-1 1.53 0.1267 0 Poor 2.05 0.51-8.25 0.3 124 Q_ .2

Versican and PSA (n = 32) Versican I .20 1.09-1 .34 0.0005 nfl PSA” 1.04 1.01-1.07 0.0056 0 20 40 60 80 100 Versican and treatment Progression-free survival (months) Versican 1. 15 1.05-1 .26 0.00 19 Fig. 6 Kaplan-Meier product limit plots of progression-free interval in Treatment patients with moderately differentiated tumors (n = 17). Versican cut- Radical 1.00 point = median concentration (MIOD, 6.0); log-rank statistic, 8.05; P = Radiotherapy 1.63 0.50-5.31 0.4196 0.0045. No treatment 3.86 1 . 1 1-1 3.47 0.0342

Versican and age Versican I . 16 1.07-1 .25 0.0003 Age 1.05 0.98-1.12 0.1488 1.0 1_+ H Versican and CS versican<6.3_(n20) Versican 1.06 0.95-1.19 0.2962 CO .8 f--H ---- - CSC 1.12 1.00-1.25 0.0488 C L 0) a Concentration (MIOD units) used as a continuous variable. CO 0. .6 b Preoperative PSA level (ng/ml) used as a continuous variable. 0 C Concentration (MIOD units) used as a continuous variable. C 0 .4 0 0. 0 0. .2 versican>6.3 (n=7) 1.0

versican <6.0 (n23) An CI) .8 20 30 40 50 60 70 80 90 100 C 0) Progression-free survival (months) CO .6 0. Fig. 7 Kaplan-Meier product limit plots of progression-free interval in 0 C patients with early-stage prostate cancer treated by radical prostatec- 0 t .4 tomy (n = 27). Versican cut-point = mean concentration (MIOD, 6.3): 0 = 0. log-rank statistic, 5.23; P 0.0221. 0 a- .2

_0 20 40 60 80 100 glycan concentrations are associated with the presence of ma- (5, 23, 33-36), few studies have examined the impli- Progression-free survival (months) lignancy cations for disease course or patient treatment. Reduced Fig. 5 Kaplan-Meier product limit plots for progression-free interval in expression of the heparan sulfate proteoglycan syndecan has patients with early-stage prostate cancer (n = 43). Versican cut-point median concentration (MIOD, 6.0); log-rank statistic, 18.86; P = been linked to progression of disease in squamous cell carcino- 0.0001. mas of the head, neck, and uterine cervix (37, 38). We believe, however, that this is the first study to report an association between versican concentration and disease progression in any cancer.

P = 0.0591). This discrepancy may be due to the small size of How elevated levels of versican or CS in peritumoral the subgroup of patients receiving radical prostatectomy. In stroma might contribute to reduced progression-free survival is addition, the larger number of patients in the radical prostatec- unknown. Nevertheless, mechanisms whereby the highly tomy group with low versican expression most probably reflects charged CS side-chains of versican might promote reduced cell the conservative selection of patients for this treatment, favoring adhesion or increased cell motility have been described recently those with other good prognostic indicators (such as low path- (7, 1 1, 12). Although there are some inconsistencies between ological grade), and thus has produced a selection bias. Exam- these studies (due to species and/or tissue differences in the ination of the predictive ability of versican concentration in a source of ECM components, proteoglycans, and cell lines), the larger cohort of patients receiving radical surgery needs to be general consensus is that versican binds to the RGD (Arg-Gly- undertaken. Asp) motif in fibronectin and laminin and thereby specifically Whereas several studies have shown that elevated proteo- inhibits cell binding. This inhibition appears to be a function of

the intact molecule because treatment of versican-fibronectin- 9. Yamagata, M., and Kimata, K. Repression of a malignant cell- coated substrates with chondroitinase ABC restores cell binding substratum adhesion phenotype by inhibiting the production of the activity to fibronectin, and free CS side-chains are not inhibi- anti-adhesive proteoglycan, PG-Mlversican. J. Cell Sci., 107: 258 1- 2590, 1994. tory. Of particular note is the observation that versican is abun- 10. Ernst, H., Zanin, M. K. B., Everman, D., and Hoffman, S. Receptor- dant in the subcellular interstitium of cultured fibroblasts but is mediated adhesive and anti-adhesive functions of chondroitin sulfate excluded selectively from focal contacts where integrins and proteoglycan preparations from embryonic chicken brain. J. Cell Sci.,

fibronectin colocalize with cytoskeletal components (8). This 108: 3807-3816, 1995. suggests that the role of versican may be to destabilize focal cell 11. Braunewell, K-H., Pesheva, P., McCarthy, J. B., Furcht, L. T., contacts. Schmitz, B., and Schachner, M. Functional involvement of sciatic We conclude from our study that high levels of versican nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and occur in the prostatic stroma of men with early-stage prostate neurite outgrowth. Eur. J Neurosci., 7: 805-814, 1995. cancer in contrast to prostatic tissue from age-matched men with 12. Perris, R., Perissinotto, D., Pettway, Z., Bronner-fraser, M., MOrge- normal or clinically benign hyperplastic prostates. Higher con- lin, M., and Kimata, K. Inhibitory effects of PG-H/aggrecan and P0- centrations of versican appear to be strongly associated with M/versican on avian neural crest cell migration. FASEB J., 10: 293- PSA progression. Consequently, we consider that measurement 301, 1996. of prostatic versican or CS levels at the time of diagnosis of 13. Bode-Lesniewska, B., Dours-Zimmermann, M. T., Odermatt, B. F., early-stage and potentially organ-confined prostate cancer may Briner, J., Heitz, P. U., and Zimmermann, D. R. Distribution of the large be useful in assessing the suitability of patients for surgical aggregating proteoglycan versican in adult human tissues. J. Histochem. Cytochem., 44: 303-312, 1996. treatment. It is important, however, to recognize that cut-points 14. Scholzen, T., Solursh, M., Suzuki, S., Reiter, R., Morgan, J. L., identified in studies such as this require extensive testing in Buchberg, A. M., Siracusa, L. D., and lozzo, R. V. The murine decorin: independent patient cohorts before any newly described marker complete eDNA cloning, genomic organization, chromosomal assign- can be accepted as a useful adjunct to current staging and ment, and expression during organogenesis and tissue differentiation. diagnostic practices (39). We believe that changes in stromal J. Biol. Chem., 269: 28270-28281, 1994. versican concentration are important for, and will lead to a new 15. LeBaron, R. G., Zimmermann, D. R., and Ruoslahti, E. Hyaluronate understanding of, the progression of prostatic cancer. binding properties of versican. J. Biol. Chem., 267: 10003-10010, 1992. 16. Knudson, C. B., and Knudson, W. Hyaluronan-binding proteins in ACKNOWLEDGMENTS development, tissue homeostasis, and disease. FASEB J., 7: 1233-1241, 1993. The authors are very grateful to Graham Sinclair and Peter Suth- 17. Krusius, T., Gehlsen, K. R., and Ruoslahti, E. 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Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 1998 American Association for Cancer Research. Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer.

C Ricciardelli, K Mayne, P J Sykes, et al.

Clin Cancer Res 1998;4:963-971.

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