Prognostic value of tissue-type plasminogen activator (tpa) and its complex with the type-1 inhibitor (PAI-1) in breast cancer

Transcription

1 British Journal of Caner (999) 80(/2), Caner Researh Campaign Artile no. bjo Prognosti value of tissue-type plasminogen ativator (tpa) and its omplex with the type- inhibitor (PAI-) in breast aner JH de Witte, CGJ Sweep, JGM Klijn 2, N Grebenshikov, HA Peters 2, MP Look 2, ThH van Tienoven, JJTM Heuvel, J Bolt-De Vries 2, ThJ Benraad and JA Foekens 2 Department of Chemial Endorinology, University Hospital Nijmegen, Geert Grooteplein 8, PO Box 90, 6500 HB Nijmegen, The Netherlands; 2 Division of Endorine Onology (Department of Medial Onology), Rotterdam Caner Institute (Dr Daniel den Hoed Kliniek)/Aademi Hospital Rotterdam, Rotterdam, The Netherlands Summary The prognosti value of tissue-type plasminogen ativator (tpa) measured in samples derived from 865 patients with primary breast aner using a reently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Sine the assay ould easily be adapted to the assessment of the omplex of tpa with its type- inhibitor (PAI-), it was investigated whether the tpa:pai- omplex also provides prognosti information. To this end, ytosoli extrats and orresponding detergent extrats of g pellets obtained after ultraentrifugation when preparing the ytosoli frations for routine steroid hormone reeptor determination were assayed. Statistially signifiant orrelations were found between the ytosoli levels and those determined in the pellet extrats (Spearman orrelation oeffiient r s =, P < for tpa and r =, P < for tpa:pai- omplex). In both Cox univariate and multivariate analysis elevated levels of (total) tpa determined in the pellet extrats, but not in ytosols, were assoiated with prolonged relapse-free (RFS) and overall survival (OS). In ontrast, high levels of the tpa:pai- omplex measured in ytosols, but not in the pellet extrats, were assoiated with a poor RFS and OS. The prognosti information provided by the ytosoli tpa:pai- omplex was omparable to that provided by ytosoli (total) PAI-. Furthermore, the estimated levels of free, unomplexed tpa and PAI-, in ytosols and in pellet extrats, were related to patient prognosis in a similar way as the (total) levels of tpa and PAI- respetively. Determination of speifi forms of omponents of the plasminogen ativation system, i.e. tpa:pai- omplex and free, unomplexed tpa and/or PAI-, may be onsidered a useful adjunt to the analyses of the separate omponents (tpa and/or PAI-) and provide valuable additional prognosti information with respet to survival of breast aner patients. Keywords: tissue-type plasminogen ativator; tpa:pai- omplex; ELISA; ytosol; breast aner; prognosti impat The plasminogen ativation system represents a omplex enzymati asade whih plays a key role in fibrinolysis, tissue remodelling and invasive proesses, inluding aner (Danø et al, 985; Rijken, 995). Central to this system is the onversion of the inative pro-enzyme plasminogen to plasmin, whih is able to dissolve fibrin lots, degrade extraellular matrix proteins and ativate latent pro-metalloproteases (Danø et al, 985; Conese and Blasi, 995; Rijken, 995). Two enzymes, i.e. urokinase-type plasminogen ativator (upa) and tissue-type plasminogen ativator (tpa), are both effiient ativators of plasminogen. Unlike upa, tpa has a strong affinity for fibrin, the presene of whih strongly enhanes tpa ativity (Mignatti and Rifkin, 993). Thus, both plasminogen ativators have different physiologial roles, upa mediating tissue remodelling proesses in a variety of normal and pathologial onditions, whereas tpa is primarily involved in thrombolysis (Danø et al, 985; Mignatti and Rifkin, 993; Rijken, 995). The ativity of upa and tpa is ontrolled in part by two main speifi inhibitors, plasminogen ativator inhibitor type- (PAI-) and type-2 (PAI-2), whih form inative omplexes of : Reeived 3 July 998 Revised 2 November 998 Aepted November 998 Correspondene to: JH de Witte stoihiometry with the plasminogen ativators (Conese and Blasi, 995; Rijken, 995). Both upa and PAI- are established prognosti fators in several types of aner, a high tissue level of both omponents generally being assoiated with tumour progression and with poor disease-free and/or overall survival (reviewed by Duffy, 996; Shmitt et al, 997). Furthermore, several investigators have reported on the potential diagnosti and prognosti relevane of tpa analysed either in plasma or in tumour tissues from patients, inluding those afflited with breast or olon aner (Layer et al, 987; De Bruin et al, 988; Duffy et al, 988; Needham et al, 988; Grøndahl-Hansen et al, 990; Jänike et al, 990, 99; Rella, et al, 993; Yamashita et al, 993, 995; Duggan et al, 997). In addition, some studies even have examined the presene of omplexes between tpa and PAI- in plasma from healthy individuals and patients with various diseases (Niwano et al, 992; Hansson et al, 994; Maser et al, 997). In these different studies referred to above, ELISA methods have mostly been employed. Reently, a new ELISA suitable for the assessment of tpa in breast tumour tissue was developed (Grebenshikov et al, 997). This ELISA, based on a ombination of polylonal antibodies, is easily adaptable to the assessment of its omplex with PAI-. In the present study, we applied the ELISA to the determination of tpa and tpa:pai- omplex in breast tumour ytosols and detergent extrats of the orresponding high-speed pellets. The prognosti relevane of the levels of tpa and of the tpa:pai- 286

2 tpa and tpa:pai- omplexes in breast aner 287 Table Relationships of tpa and tpa:pai- omplex levels in 865 ytosols and pellet extrats with patient and tumour harateristis Perentage of tumours above the median value b Charateristis Number of tpa tpa p tpa:pai- tpa:pai- p patients a omplex omplex All patients Age at surgery (years) > P-value d Menopausal status Pre-menopausal Post-menopausal P-value e Tumour size T ( 2 m) T 2 (> 2 5 m) T 3/4 (> 5 m) P-value f < < Nodal status N N N > P-value f 0.23 < Grade Well/moderate Poor P-value e < < ER-positive g No Yes P-value d < < < < PgR-positive g No Yes P-value d < < < 2 a Due to missing values the numbers do not always add up to 865; values for tpa:pai- and tpa:pai- p orrespond to 782 and 822 patients, respetively. b Median antigen levels for tpa, tpa p, tpa:pai- and tpa:pai- p as indiated in the legend to Table II. tpa, tpa p, tpa:pai- and tpa:pai- p denotes tpa and tpa:pai- omplex levels determined with the different ELISAs in ytosols () and pellet extrats (p), respetively. d P-value for Spearman rank orrelation. e P-value for Wiloxon rank-sum test (for grade: well and moderate ombined). f P-value for Kruskal Wallis test, inluding a Wiloxon-type test for trend. g Cut-off point used for ER and PgR: 0 fmol mg protein. omplex in both types of samples was subsequently evaluated. Furthermore, we analysed the prognosti value of the alulated PAI- to tpa ratio and of the free, unomplexed forms of both PAI- and tpa. MATERIALS AND METHODS Patients and tumour harateristis This study was performed on a group of 865 patients with operable primary breast aner in whom tpa antigen levels were measured in both ytosols and pellet extrats. The patients, whose samples were stored in the tumour bank of the Rotterdam Caner Institute (Dr Daniel den Hoed Kliniek), inluded those for whih tumour levels of upa and PAI- were determined previously (De Witte et al, 998). Patients with primary diagnosis of breast aner between 979 and 989, without evidene of distant metastasis at the time of diagnosis, were inluded in the study. Patients with Caner Researh Campaign 999 previous diagnosis of arinoma, exept for basal ell skin aner or ervial aner stage Ia, or with evidene of disease within month after primary surgery were exluded. None of the patients reeived neo-adjuvant treatment before primary surgery. In ase of mastetomy for residual disease after an initial lumpetomy, the mastetomy is onsidered (as part of) primary treatment. The median number of lymph nodes removed surgially was. Median age of the patients at the time of surgery was 56 years (range years). Radiotherapy was given to 82% of the patients: on the breast/thorai wall to 596 patients and/or on the axilla to 250 patients, and/or on one or more lymph node areas other than the axilla to 283 patients. Two of the 434 nodenegative patients reeived adjuvant hemotherapy (CMF-ylophosphamide, methotrexate, 5-fluorourail), while none of these patients reeived adjuvant endorine therapy. Of the 422 nodepositive patients (for nine patients, nodal status missing), adjuvant hemotherapy was given to 38 patients, while 59 patients reeived hormonal therapy, either alone (45 patients) or in British Journal of Caner (999) 80(/2),

3 288 JH de Witte et al ombination with hemotherapy (4 patients). All patients were routinely examined every 3 6 months during the first 5 years of follow-up and one a year thereafter (median follow-up period of patients alive, 00 months; range, 2 67 months). During follow-up, 354 patients (4%) showed relapse and ounted as failures in the analysis for relapse-free survival (RFS). Sixty-two patients (7%) died without evidene of disease and were ensored at last follow-up in the analysis for RFS. Two-hundred and fourteen patients (25%) died after a previous relapse. A total of 276 (62+24) patients (32%) were ounted as failures in the analysis for overall survival (OS). Further harateristis of patients and tumours are listed in Table. Tumour tissue extration Tumour tissues (stored in liquid nitrogen) were proessed in the frozen state by pulverization with a mirodismembrator and the resulting tissue powders homogenized in buffer (0 mm K 2 HPO 4, ontaining.5 mm di-potassium hloride EDTA, 3 mm sodium azide, 0 mm monothioglyerol and 0% (v/v) glyerol, ph = 7.4), as reommended by the European Organization for Researh and Treatment of Caner for analysis of steroid hormone reeptors (oestrogen (ER and PgR) (EORTC Breast Caner Cooperative Group, 980). The homogenates were entrifuged for 30 min at g and 4 C to obtain the supernatant frations (ytosols). The g pellets were re-homogenized with an Ultraturrax tissue homogenizer in 20 mm Tris-HCl (ph = 8.5) ontaining 25 mm sodium hloride, after omplete removal of the 50% (v/v) glyerol under whih the pellets were interimly stored at 80 C. After adding Triton X-00 to a final onentration of %, the homogenates (total volume 500 µl) were subsequently inubated for 6 20 h at 4 C under gentle shaking. The supernatant frations obtained by entrifugation at g at 4 C were designated as pellet extrats. Steroid hormone reeptor assays ER and PgR levels were determined by ligand binding assay or enzyme immunoassay in ytosols as desribed earlier (Foekens et al, 989). The ut-off level used as to lassify tumours as ER or PgR positive or negative was 0 fmol mg ytosoli protein. ELISAs ELISA for tpa The antigen levels of tpa in ytosols and pellet extrats were assessed with a newly established ELISA (Grebenshikov et al, 997). Briefly, mirotitreplates were first oated overnight at 4 C with sheep anti-hiken IgY antibodies and then inubated with the speifi hiken antibodies direted against tpa. Bound ligand was deteted by inubation with rabbit anti-tpa antibodies and subsequent inubation with horseradish peroxidase (HRP)- labelled goat anti-rabbit antibodies (Sigma Chemial Co, St Louis, MO, USA) diluted : Reombinant single-hain tpa (Boehringer Ingelheim, Alkmaar, The Netherlands) was used as a standard in the ELISA. The tpa ELISA detets both the free form of tpa, as well as its omplex with PAI- and PAI-2. ELISA for tpa:pai- omplex The tpa:pai- omplex in ytosols and pellet extrats was determined in assay formats based on a ombination of polylonal antibodies employed in the ELISAs for the separate omponents, i.e. tpa and PAI- (Grebenshikov et al, 997). Briefly, the hiken anti-tpa and rabbit anti-pai- antibody (ELISA format A), and/or the hiken anti-pai- and rabbit anti-tpa antibody (ELISA format B) were ombined as athing and tagging antibody in one and the same ELISA frame for assessment of tpa: PAI- omplex. Detetion of omplexes was performed with HRPlabelled goat anti-rabbit antibodies as desribed above. The tpa:pai- omplex standard was a gift from Prof. Andreasen (University of Århus, Århus, Denmark), prepared from the separate omponents as desribed previously (Egelund et al, 997). The tpa:pai- omplex in ytosols and pellet extrats was determined essentially in both ELISA formats, exept for those ases in whih high levels of PAI- or tpa were to be expeted; in these ases only format A ([PAI-] 0*[tPA]) or format B ([tpa] 0*[PAI-]) was hosen, thereby antiipating an underestimation of omplex assessment beause of possible ompetition of (exess) unomplexed PAI- or tpa with tpa:pai- omplex for binding to the athing antibodies. At least two different dilutions of eah patient sample were analysed in ELISA format A and/or format B, and the final onentration was alulated by extrapolation to infinite dilution using linear regression. When applying both ELISA formats, the highest antigen value obtained was onsidered as representing the atual tpa:pai- omplex onentration in the patient s sample. Strong statistially signifiant orrelations were found between the antigen values obtained in ELISA format A and format B (r s = 0.97, P <, for both the ytosols and the pellet extrats). The alulated perentage of tpa being in omplex with PAI-, the antigen levels expressed in molars, varied to a large extent, both in ytosols (median: 44%; inter-quartile range: 8 90%) and in pellet extrats (34%; 2 83%). ELISA reagents were all diluted in phosphate-buffered saline (PBS) with % bovine serum albumin (BSA) and 0.% (v/v) Tween-20. Inubations with standards, samples and ontrols, diluted in PBS-BSA-Tween, were performed overnight at 4 C. All determinations were performed in dupliate. Triton X-00 did not influene the ELISA results up to a onentration of %. The reproduibility of assay performane was ontrolled by analysis of an aliquot of a pooled breast tumour ytosol sample (tpa ELISA) or a pooled plasma sample (tpa:pai- ELISA) in eah assay-run and the between-assay variation was found to be below 0% for both ELISAs. The within-assay variation of samples measured in dupliate was always below 5%. Protein determinations The Bradford method for protein analysis (Bradford, 976) was employed using the Bio-Rad reagent with human serum albumin (KabiVitrum, Stokholm, Sweden) as a standard in order to express antigen levels per mg of total protein. Triton X-00 up to a onentration of % did not interfere with the protein determination in pellet extrats. Statistial analysis The strength of the assoiations of tpa and tpa:pai- omplex levels determined in ytosols and pellet extrats with eah other, age and steroid hormone reeptor levels was tested with Spearman rank orrelation (r s ). The assoiations of tpa and tpa:pai- omplex with other linial variables were tested with the British Journal of Caner (999) 80(/2), Caner Researh Campaign 999

4 tpa and tpa:pai- omplexes in breast aner 289 non-parametri Wiloxon rank-sum test or the Kruskal Wallis test, inluding a Wiloxon-type test for trends aross ordered groups where appropriate. RFS and OS probabilities were alulated by the atuarial method of Kaplan and Meier (Kaplan and Meier, 958). The log-rank test for trend was used to test ordered variables. Both uni- and multivariate analyses were performed using the Cox proportional hazard model, and the assoiated likelihood ratio test was used to test for differenes. All omputations were done with the STATA statistial pakage, release 5.0 (STATA Corp., College Station, TX, USA). Two-sided P-values below 0.05 were onsidered to be statistially signifiant. RESULTS tpa and tpa:pai- omplex in ytosols and pellet extrats The antigen levels of tpa determined in the ytosols varied from 0.02 to ng mg protein (mean 6.38; median 2.40) and those in the pellet extrats varied from 0.06 to ng mg protein (mean 38.9; median 3.0). The ytosoli levels of tpa:pai- omplex ranged from 0.8 to ng mg protein (mean 2.34; median.75) and in the pellet extrats from 0.20 to (mean 9.9; median 6.57). As ompared to the ytosoli levels of the respetive antigens, the levels determined in the pellet extrats were approximately sixfold (tpa) and fourfold (tpa:pai- omplex) higher. However, the orrelations between the levels in ytosols and in the orresponding pellet extrats were statistially signifiant (r s =, P < for tpa and r s =, P < for tpa:pai- omplex). Signifiant orrelations were also found between the levels of tpa and tpa:pai- omplex in ytosols (r s = 0.30, P < ) and pellet extrats (r s =, P < ). Relation of tpa and tpa:pai- omplex to patient and tumour harateristis The levels of tpa and tpa:pai- omplex determined in ytosols and pellet extrats were related to patient and tumour harateristis (Table ). The levels of tpa and tpa:pai- omplex measured in ytosols or pellet extrats were positively related with patient s age. The ytosoli level of tpa, as well as its level in pellet extrats, was not related with menopausal status, while those of tpa:pai- omplex were signifiantly higher in tumours from post-menopausal patients. In general, tpa or tpa:pai- omplex levels were negatively related with the size of the primary tumour. In ontrast, the levels of tpa and tpa:pai- omplex, measured Table 2 Cox univariate and multivariate analyses of relapse-free and overall survival in 865 breast aner patients Fator Univariate analyses RFS Multivariate analyses RFS Univariate analyses OS Multivariate analyses OS P-value RHR (95% CI) a P-value RHR (95% CI) b P-value RHR (95% CI) a P-value RHR (95% CI) b Age and menopausal status < < < < Age/pre-menopausal d 0.59 ( ) 0.63 ( ) 0.74 ( ) 0.8 (0.57.4) Age/post-menopausal d.08 ( ) (0.85.7).49 (.27.76).36 (.5.6) Post- vs pre-menopausal e.68 ( ).38 ( ).5 ( ).9 ( ) Tumour size < 0.08 < < 2 5 m vs 2 m.56 (.24.95).22 ( ).75 ( ).8 ( ) > 5 m vs 2 m 2.43 ( ).54 ( ) 3.82 ( ) 2.6 ( ) Nodal status < < < 4 N 3 vs N 0.22 (0.93.6).5 ( ).83 ( ) 2.34 ( ) N >3 vs N (2. 3.4) 2.88 ( ) 3.78 ( ) 3.80 ( ) ER f < 0.64 ( ) 0.05 (0.56 ) < 0.57 ( ) ( ) PgR f ( ) ( ) < 0.59 (0.46 ) ( ) Adjuvant therapy g (0.58 ) ( ) < 0.53 ( ) ( ) tpa ytosol h h 0.8 (0.6.07) 0.99 ( ) 0.8 (0.58.). ( ) 0.7 ( ) 0.89 ( ) 0.78 ( ).04 ( ) 0.78 ( ) 0.98 (0.7.37) 0.73 ( ).04 (0.7.53) tpa pellet < 0.06 < (0.57 ) 0.86 (0.65.5) 0.76 ( ) 0.95 ( ) 0.60 ( ) ( ) 0.55 ( ) 0.73 (0.5.05) 0.55 ( ) 0.64 ( ) 0.48 ( ) 0.60 ( ) tpa:pai- omplex ytosol < <.27 ( ).26 (0.9.74).8 ( ).9 ( ).42 (.03.95).5 ( ).67 ( ).92 ( ).47 ( ).59 (.5 2.8) 2.25 ( ) 2.62 ( ) tpa:pai- omplex pellet ( ).09 ( ).0 ( ).4 ( ).09 ( ).5 ( ).20 ( ).35 ( ).05 ( ).33 ( ).24 ( ).7 ( ) a Relative hazard rate (RHR) with 95% onfidene interval (CI) of univariate analyses. b Relative hazard rate (RHR) with 95% onfidene interval (CI) of multivariate analyses (final model, 84 patients; patients with missing values for ER and PgR (n = 5), and missing information on nodal status (n = 9) not inluded) orreted for the basi model, inluding age/menopausal status, tumour size, nodal status, ER/PgR status, and adjuvant therapy; fators were added separately to the basi model. Age and menopausal status ombined. d Age in deades tested separately for pre- and post-menopausal patients. e Postmenopausal as ompared with pre-menopausal. f Cut-off points used for ER and PgR, 0 fmol mg protein. g Adjuvant therapy (yes vs no) only for node-positive patients. h P-values for the assoiated likelihood ratio test for the quarters (Q ) indiated: Q, first quarter;, seond quarter;, third quarter;, fourth quarter. The quartiles of the tpa antigen levels were suessively.2, 2.40, 4.72 ng mg (for ytosols) and 5.48, 3.0, 33.8 ng mg (for pellet extrats); the quartiles of the tpa:pai- omplex levels were suessively 0.97,.75, 3.08 ng mg (for ytosols) and 3.69, 6.57, 0.6 ng mg (for pellet extrats). Caner Researh Campaign 999 British Journal of Caner (999) 80(/2),

5 290 JH de Witte et al A tpa (pellet) B tpa:pai- omplex (ytosol) 4 (74/26) (78/26) (93/26) Q (09/27) Q (70/95) (79/97) (85/9) (9/99) P < P= 0.0 C tpa (pellet) D tpa:pai- omplex (ytosol) (52/26) (56/26) (78/26) Q (90/27) Q (4/95) (62/9) (66/97) (82/99) P < Months P< Months Figure Atuarial relapse-free and overall survival urves for breast aner patients stratified by the levels of tpa in pellet extrats (A and C) and of tpa:pai- omplex in ytosols (B and D). Patients were divided into groups aording to the quartiles (Q ) of the respetive levels as indiated in the legend to Table 2 Table 3 Cox univariate and multivariate analyses of relapse-free and overall survival in 865 breast aner patients Fator Univariate analyses RFS Multivariate analyses RFS Univariate analyses OS Multivariate analyses OS P-value RHR (95% CI) a P-value RHR (95% CI) b P-value RHR (95% CI a P-value RHR (95% CI) b PAI- ytosol < < < <.5 ( ).3 (0.94.8).23 (0.83.8).42 ( ).50 ( ).55 (.3 2.3).73 ( ).80 ( ) 2.05 ( ) 2.2 ( ) 2.6 ( ) 2.69 ( ) PAI- pellet < < <.02 (.39).08 ( ) 0.95 ( ).0 (.62).35 (.83).43 (.05.95).45 ( ).60 ( ).57 (.7 2.2).75 ( ).9 ( ) 2.9 ( ) PAI-/tPA ytosol < 0.06 < < ( ).04 (.43) 0.77 (0.52.3) 0.84 ( ).5 ( ).07 ( ).45 ( ).27 (0.88.8).63 ( ).47 ( ).85 ( ).64 ( ) PAI-/tPA pellet < 4 < <.04 (.43).22 ( ) 0.98 ( ).2 (0.8.8).45 (.06.97).33 ( ).89 ( ).68 ( ).87 ( ).82 ( ) 2.5 ( ) 2.05 ( ) a Relative hazard rate (RHR) with 95% onfidene interval (CI) of univariate analyses. b Relative hazard rate (RHR) with 95% onfidene interval (CI) of multivariate analyses orreted for the basi model, inluding age/menopausal status, tumour size, nodal status, ER/PgR status, and adjuvant therapy; fators were added separately to the basi model. P-values for the assoiated likelihood ratio test for the quarters (Q ) indiated; Q, first quarter;, seond quarter;, third quarter;, fourth quarter. The quartiles of the PAI- antigen levels were suessively.06,.62, 2.63 ng mg (for ytosols) and 3.30, 5.28, 8.6 ng mg (for pellet extrats); the quartiles of the PAI-/tPA ratios were suessively 0.27, 0.66,.65 ng mg (for ytosols) and 0.5, 0.4,.32 ng mg (for pellet extrats). British Journal of Caner (999) 80(/2), Caner Researh Campaign 999

6 tpa and tpa:pai- omplexes in breast aner 29 A ombination (ytosol) B ombination (pellet) high/low (76/224) low/low (73/206) high/high (88/207) low/high (7/228) high/low (82/238) high/high (70/90) low/low (75/95) low/high (26/236) low/low: high/low: low/high: high/high: RHR [95 % CI] 0.96 [ ].8 [ ].29 [.02.89] low/low: high/low: low/high: high/high: RHR [95 % CI] 0.83 [0.6.4].64 [ ] 0.96 [ ] C ombination (ytosol) D ombination (pellet) tp-pai- high/low (55/224) low/low (48/206) high/high (74/207) low/high (99/228) tp-pai- high/low (58/238) high/high (50/90) low/low (53/95) low/high ( 4/236) low/low: high/low: low/high: high/high: RHR [95 % CI].05 [0.7.55] 2.26 [ ].70 [ ] low/low: high/low: low/high: high/high: RHR [95 % CI] 0.88 [0.6.28] 2.3 [ ].0 [ ] Months Figure 2 Atuarial relapse-free and overall survival urves for breast aner patients stratified by the ombined levels of tpa and PAI- determined in ytosols (A and C) and in pellet extrats (B and D). Patients were divided into groups with levels below ( low ) and above ( high ) the median value of the respetive tpa and PAI- levels as indiated in the legend to Table and Table 3 Table 4 Cox univariate and multivariate analyses of relapse-free and overall survival in 865 breast aner patients Fator Univariate analyses RFS Multivariate analyses RFS Univariate analyses OS Multivariate analyses OS P-value RHR (95% CI) a P-value RHR (95% CI) b P-value RHR (95% CI) a P-value RHR (95% CI) b tpa free ytosol ( ) (0.7.2) ( ) (0.6.03) tpa free pellet < 0.66 ( ) ( ) < 0.58 ( ) ( ) PAI- free ytosol <.52 (.22.89) 2.43 (.3.79) < 2.7 ( ) < 2.0 ( ) PAI- free pellet <.45 (.8.80) <.45 (.6.82) <.78 ( ) <.70 ( ) a Relative hazard rate (RHR) with 95% onfidene interval (CI) of univariate analyses. b Relative hazard rate (RHR) with 95% onfidene interval (CI) of multivariate analyses orreted for the basi model, inluding age/menopausal status, tumour size, nodal status, ER/PgR status, and adjuvant therapy; fators were added separately to the basi model. P-values for the assoiated likelihood ratio test for the median values of the levels of the free, unomplexed forms of tpa (tpa free ) and PAI- (PAI- free ). The median values in ng mg protein were:.74 for tpa free ytosol,. for tpa free pellet,.26 for PAI- free ytosol, 3.68 for PAI- free pellet. either in ytosols or pellet extrats, were not related with nodal status, exept for tpa in pellet extrats, its levels dereasing with advaned lymph node involvement. The levels of tpa determined in ytosols and pellet extrats were signifiantly lower in poorly differentiated tumours, whereas no relations were found between the levels of tpa:pai- omplex and tumour grade. Overall, higher levels of tpa and tpa:pai- omplex were measured in steroid hormone-positive tumours, the orrelation oeffiients (r s ) observed ranging between 0.0 and Relation of tpa and tpa:pai- omplex to survival In Cox univariate regression analysis, young pre-menopausal age, post-menopausal status, the size of the primary tumour, the number of positive lymph nodes, steroid hormone reeptornegativity and adjuvant therapy in node-positive patients were signifiantly assoiated with redued RFS and OS (Table 2). When divided into four groups (Q to ) by their respetive quartiles, dereasing levels of tpa in pellet extrats and inreasing levels of Caner Researh Campaign 999 British Journal of Caner (999) 80(/2),

7 292 JH de Witte et al A Free tp A (ytosol) B Free tp A (ytosol) tpa-high (8/429) PAI--low (89/394) tpa-low (58/436) PAI--high (62/388) P=3 P< C Free tp A (pellet) tpa-high (07/432) D Free tp A (pellet) PAI--low (06/409) tpa-low (69/433) PAI--high (64/408) P< P< Figure 3 Atuarial overall survival urves for breast aner patients stratified by the estimated levels of free, unomplexed tpa (A and C) and of free, unomplexed PAI- (B and D) in ytosols and in pellet extrats. Patients were divided into groups aording to the median values indiated in the legend to Table 4 tpa:pai- omplex in ytosols showed a lear trend towards a worse RFS and OS (Table 2, Figure ). The levels of tpa in ytosols and those of tpa:pai- omplex in the pellet extrats were not signifiantly related with RFS and OS. Cox multivariate regression analysis was performed to ompare the prognosti signifiane of tpa and tpa:pai- omplex with that of the lassial prognosti parameters (Table 2), these latter variables omprising a basi multivariate model. In the basi multivariate model, age, menopausal status, nodal status, ER status and adjuvant therapy were all signifiantly assoiated with both RFS and OS, nodal status being the strongest of the lassial prognosti fators. Tumour size and PgR status were only statistially signifiant in prediting OS, not RFS (Table 2). Correted for the basi multivariate model, the levels of tpa:pai- omplex in ytosols and pellet extrats were related with a poor RFS and OS, although this relationship for the pellet extrats with RFS was statistially not signifiant. The prognosti information provided by the tpa:pai- omplex appeared to be stronger in ytosols as ompared to the pellet extrats. The levels of tpa did not signifiantly add to the basi multivariate model with respet to both RFS and OS, although, in the analysis for OS, the ontribution of tpa in pellet extrats reahed statistial signifiane (P = 0.03) (Table 2). Reently, the antigen levels of PAI- were determined in the same ytosols and pellet extrats, applying the same ELISA framework (De Witte et al, 998). The (total) levels of PAI- and tpa in both types of extrats were very weakly negatively orrelated with eah other (r s = 0.073, P = 0.033, for ytosols; r s = 0.2, P =, for pellet extrats). Sine both fators have opposite effets on patient prognosis, the ratio of PAI- to tpa (PAI-/tPA) was alulated as an alternative variable to be related with survival. Both the antigen levels of PAI- and of the PAI-: tpa ratio, either in ytosols or in pellet extrats, were assoiated with the rates of relapse and death, also when orreted for the basi multivariate model (Table 3). After inluding PAI- in the basi multivariate model, tpa did not further ontribute to the prognosti information already provided by PAI-. Figure 2 shows the atuarial RFS and OS urves of patients as a funtion of the ombined effet of (total) tpa and (total) PAI- in ytosols and pellet extrats, both fators divided by their median value. Clearly, patients with low levels of tpa in their tumours, whih, in addition, ontain high levels of PAI- experiened a very poor prognosis. In turn, patients with both high tpa and low PAI- tumour levels enountered a favourable prognosis. Finally, the levels of the free, unomplexed forms of tpa and PAI- in ytosols and pellet extrats were estimated by subtration British Journal of Caner (999) 80(/2), Caner Researh Campaign 999

8 tpa and tpa:pai- omplexes in breast aner 293 of the (molar) onentrations of the tpa:pai- omplexes from the (total) onentrations of tpa and PAI-, respetively. In univariate analyses with the orresponding median values hosen to disriminate between low-risk and high-risk patients, high levels of unomplexed tpa in ytosols and pellet extrats were signifiantly assoiated with prolonged RFS and OS (Table 4, Figure 3A, C). In ontrast, high levels of unomplexed PAI- in ytosols and pellet extrats appeared to be related with a signifiantly redued RFS and OS (Table 4; Figure 3B, D). Moreover, in multivariate analyses orreted for the lassial prognosti fators, unomplexed PAI- in ytosols and in pellet extrats was found to be related with both poor RFS and OS, omparably strong as total PAI-. The unomplexed form of tpa measured in pellet extrats, but not in ytosoli extrats, added signifiantly to the basi multivariate model with respet to both RFS and OS (Table 4). DISCUSSION In the present study, the prognosti impat of tpa antigen levels in a relatively large series of 865 breast aner patients was evaluated employing a newly developed ELISA. The antigen levels were determined in ytosols routinely used for steroid hormone reeptor analyses, as well as in extrats of g pellets, whih may be onsidered as inidental produts of ultraentrifugation when preparing the ytosoli frations. Its appearane in the pellet extrats may suggest the presene of a membrane-assoiated form of tpa. Indeed, reeptors for tpa have been identified on the surfaes of vasular endothelial ells (Dudani and Ganz, 996), whih, in turn, are regarded as the primary soure of tpa (Levin et al, 997). In univariate analyses, high levels of tpa in pellet extrats were signifiantly assoiated with a lower probability of developing reurrenes and of experiening an early death. However, in multivariate analyses, after orretion for the lassial prognosti fators, elevated levels of tpa in pellet extrats appeared only as a weak preditor of improved OS (P = 0.03), but not RFS (P = 0.06). These findings resemble those obtained by Jänike et al (99) who determined tpa in detergent extrats of breast aner tissue by ELISA, and showed that patients with a high tpa ontent tended to have a better prognosis than those with low, or no, detetable tpa ontent. However, the differene was not statistially signifiant. Furthermore, Duggan et al (997), applying an ELISA to breast tumour extrats prepared with detergent-ontaining buffer, determined tpa to be an independent prognosti fator in breast aner, with a alulated relative risk of relapse and of death orresponding to 0.56 and 0.47, respetively. Regarding the ytosoli extrats of breast tumour tissue, their tpa antigen levels did not have any prognosti impat on the rates of relapse or death. These findings are in apparent ontrast to a previous report by Duffy et al (988) who showed that patients with high ytosoli levels of tpa had a signifiantly longer disease-free interval and survival ompared to patients with low tpa levels. The median follow-up period, however, was onsiderably shorter (26 months). In agreement with other investigations, the ytosoli levels of tpa were positively related to steroid hormone reeptor (ER and/or PgR) status (Duffy et al, 986; Needham et al, 988; Shmitt et al, 990; Jänike et al, 99; Rella et al, 993). In this respet, the observed orrelation of high tpa antigen levels with good prognosis may be related to the fat that tpa is an oestrogen-induible enzyme (Butler et al, 983; Mira-Y- Lopez and Ossowski, 987), the presene of ER itself generally being assoiated with good prognosis in breast arinomas (Table Caner Researh Campaign 999 2). Therefore, the presene of tpa may be diretly representative of a funtional, biologially ative ER system, important for planning endorine therapy, as has been suggested by Rella et al (993). The positive linial impat of tpa on survival is also expressed by the ombined prognosti value of tpa and PAI- measured in ytosols but mainly in pellet extrats (Figure 2), exemplifying that tpa abolishes the unfavourable effet of PAI- on prognosis. An interesting finding of the present study onerns the prognosti impat of tpa:pai- omplex measured in the breast tumour ytosols on survival. In multivariate analyses, ytosoli tpa:pai- omplex provided prognosti information, whih appeared to be almost as strong as ytosoli PAI- with respet to RFS and, espeially, OS. This latter observation is in line with the supposition that the levels of tpa:pai- omplex in tumour tissue extrats may indiretly reflet the level of PAI- whih has been originally ative, sine only the ative form of PAI- is able to form omplexes with tpa (Hekman and Loskutoff, 985). Although PAI- was shown to be a strong independent prognosti parameter also in pellet extrats, tpa:pai- omplex did not have prognosti impat on RFS and was only a weak preditor of OS when measured in this type of extrats. Consequently, this latter finding may suggest that the prognosti relevane exerted by PAI- in the pellet extrats does not originate from its omplex with tpa, but may be provided by the free, unomplexed form of PAI-. Presumably, this also aounts for the prognosti signifiane of (free, unomplexed) tpa in the pellet extrats (see below). From an analytial hemial point of view, the ELISA for tpa:pai- omplex an be onsidered as being highly speifi for the omponent to be assessed. While the tpa ELISA detets both the free and omplexed forms of tpa, the tpa:pai- omplex ELISA seletively quantifies only one moleular variant of tpa, i.e. its omplex with PAI-, without being sensitive towards the separate omponents, tpa and PAI- (Grebenshikov et al, 997). Moreover, the results obtained with the tpa:pai- omplex ELISA may be regarded as being highly reliable, sine the tpa:pai- omplex standard used for quantitation of the assay results is diretly omparable to the analyte to be assessed. In ontrast, the single-hain reombinant tpa standard employed in the tpa ELISA, learly is not fully representative of the different moleular forms of tpa present in the linial samples (see below), a ompliation inherently linked to assays attempting to quantify a heterogenous omponent (Benraad et al, 996). The PAI-/tPA ratio, introdued as an alternative parameter, also provided strong prognosti information, high levels in ytosols as well as in pellet extrats prediting both redued RFS and OS. Most likely, a high PAI-:tPA ratio is representative of a large exess of (free, unomplexed) PAI- preditive of poor prognosis, while a low PAI-/tPA ratio reflets a relative exess of (free, unomplexed) tpa with a favourable effet on prognosis. This onsideration may underlie the observed assoiations of high levels of the free, unomplexed forms of both PAI- and tpa in ytosols and pellet extrats with suessively a poor and good prognosis. However, one has to realize that the levels of the free, unomplexed forms are just estimations based on subtration of tpa:pai- omplex levels from the total antigen levels of tpa and PAI-, the more so as other omplexed forms suh as tpa:pai-2 and upa:pai- might also be deteted in the ELISA for (total) tpa and (total) PAI-, respetively. Moreover, as ompared to the free, unomplexed forms, eah of the various omplexes will be deteted with different effiienies in the standard ELISAs, whih, in addition, make use of standards or alibrators (reombinant British Journal of Caner (999) 80(/2),

9 294 JH de Witte et al single-hain tpa and latent human PAI-) whih may not be ompletely representative of the linial samples to be analysed. Notwithstanding these limitations, the observed impat of the free, unomplexed forms on survival are in aordane with our expetations of a favourable (tpa) and an unfavourable (PAI-) prognosti marker. Aordingly, the development of immunoassays for the seletive detetion of speifi forms of the various omponents of the plasminogen ativation system, i.e. tpa:pai- omplex, free unomplexed tpa and/or free, unomplexed PAI-, may be a useful adjunt to the analyses of the separate omponents (tpa and/or PAI-) by ELISA, the latter assay results representing at best the total antigen ontent of the linial sample. REFERENCES Benraad ThJ, Geurts-Moespot J, Grøndahl-Hansen J, Shmitt M, Heuvel JJTM, De Witte JH, Foekens JA, Leake RE, Brünner N and Sweep CGJ (996) Immunoassays (ELISA) of urokinase-type plasminogen ativator (upa): report of an EORTC/BIOMED- workshop. 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