Synovial sarcoma. Evaluation of prognosis with emphasis on the study of DNA ploidy and proliferation (PCNA and Ki-67) markers

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1 Anlyticl Cellulr Pthology 16 (1998) IOS Press Synovil srcom. Evlution of prognosis with emphsis on the study of DNA ploidy nd prolifertion (PCNA nd Ki-67) mrkers José M. Lopes,, Einr Hnnisdl b, Bodil Bjerkehgen c, Øyvind S. Brulnd b, Håvrd E. Dnielsen b,eriko.pettersen d, Mnuel Sobrinho-Simões ndjhnm.neslnd c Unit of Moleculr Pthology, IPATIMUP, Medicl Fculty, Porto, Portugl b Deprtments of Medicl Oncology nd Rdiotherpy, c Pthology nd d Tissue Culture, The Norwegin Rdium Hospitl nd Institute for Cncer Reserch, Montebello, Oslo, Norwy Received 17 September 1996 Revised 23 October 1997 Accepted 2 Jnury 1998 Abstrct. Controversy still exists regrding the vlidity of prmeters commonly used in the evlution of prognosis of ptients with synovil srcom (SS). Forty-nine cses of previously untreted primry SS (23 femles nd 26 mles, rnging in ge from 7 to 81, with 31 tumors locted in the lower extremity, 8 t the upper extremity nd 10 t the trunchus), without regionl lymph-node or distnt metstses were studied. We investigted the reltionship between (flow nd imge) DNA cytometry, prolifertion ctivity, clinicopthologic prmeters, nd relpse-free nd overll survivl of the ptients. The prognostic vlue of gender, ge, durtion of symptoms, loction, comprtmentliztion, size, dequcy of surgicl mrgins, residul tumor, djuvnt therpy, histologic subtype, extent of necrosis, glndulr differentition, clcifiction, nd extent of hemngiopericytic res, mitotic rte, mount of mst cells, blood vessel invsion, histologic (UICC nd NCI) grdes, DNA ploidy, percentge of cells in S nd S+G2 phses, PCNA nd Ki-67 lbeling indices (LI), nd TNM (UICC) stge of the tumors, were evluted by univrite nd multivrite (Cox hzrd model) nlyses. Short durtion of symptoms (<12 months), biphsic SS, scrcity of mst cells (<10/10 HPF), high mitotic rte ( 10/10 HPF), high histologic grde (grde 3), high PCNA-LI ( 20%), high Ki-67-LI ( 10%), DNA neuploidy, nd dvnced TNM stge (stge III) were fetures ssocited with significntly shorter relpse-free nd overll 5-yer survivl rtes in the univrite nlyses. Scrcity of mst cells, high mitotic rte, or high PCNA-LI were significnt predictors of poor survivl, in ddition to TNM stge in the multivrite nlyses. The mount of mst cells ws inversely correlted with mitotic rte nd PCNA-LI. Scrcity of mst cells, high mitotic rte, or high PCNA-LI re fctors ssocited with poor prognosis, in ddition to dvnced TNM stge in ptients with loclized SS. Keywords: Synovil srcom, mst cells, mitotic rte, PCNA, stging, prognosis, Ki-67, DNA flow cytometry, DNA imge cytometry, soft tissue tumor Adress for correspondence: Deprtment of Pthology, IPATIMUP, Medicl School of Porto, Hospitl S. João, 4200 Porto, Portugl. Fx: /98/$ IOS Press. All rights reserved

2 46 J.M. Lopes et l. / Prognosis in synovil srcom 1. Introduction Mny clinicl nd morphologicl fetures hve been pplied to the evlution of the prognosis of synovil srcom (SS), such s gender, ge t dignosis, clinicl symptoms, loction of tumor, stging of the neoplstic disese, tretment modlities, tumor size, histologic subtype, extent of glndulr differentition, mitotic rte, histologic grding, presence of rhbdoid cells, mount of mst cells, extent of clcifiction, DNA ploidy score, nd PCNA score. However, there is still contrdictory nd insufficient evidence concerning the vlidity of most of these prmeters [2,6,8,10,17,19,20,22,23,25, 29,33,34,36 39,41,43,44,47,50,52,56]. It is well estblished tht stging is the most importnt prmeter in the evlution of soft tissue srcoms. It gives vluble informtion, guiding both choice of tretment nd prognosis. Tumor size, locl spred, metstses, nd histologic grde hve ll been used in different stging systems of soft tissue srcoms [18,19,28]. The prognostic significnce of histologic subtyping of SS remins controversil. Severl uthors conclude tht there re no significnt differences between the survivl of ptients with the two most common subtypes of SS (BSS nd MSS) [6,10,17,22,23,25,33,38,39,43]. On the other hnd, some groups hve found BSS to crry more fvorble prognosis [8,56] nd others hve found tht MSS ppers to be the less ggressive vrint [36,47]. The prognostic vlues of DNA cytometry prmeters (DNA ploidy nd percentge of tumor cells in S nd S+G2 phses) hve been studied in severl series of different histologic types of soft tissue srcoms [2,17,19,38,53]. In this study, we hve performed retrospective evlution regrding the influence on the survivl of 49 ptients with primry SS of vrious clinicopthologic fetures, with prticulr emphsis on the nlysis of DNA cytometric nd prolifertive prmeters. 2. Ptients nd methods Forty-nine ner-consecutive cses of primry synovil srcom (SS) treted by surgery were selected from the files of the Deprtment of Pthology, The Norwegin Rdium Hospitl (36 cses from 1981 to 1991) nd Porto Medicl School (13 cses from 1967 to 1991). The only criterion for exclusion ws the presence of regionl lymph-node metstses or distnt metstses. Preopertive rdiotherpy ws not given to ny ptient Clinicl fetures Clinicl dt were collected from the ptients medicl records. The surgicl mrgins were clssified ccording to the criteri of Enneking et l. [18]. Mrgins obtined t the second opertion were recorded, whenever the primry tumors were treted by two sequentil surgicl procedures seprted by less thn one month, in n ttempt to chieve dequte surgicl mrgins. Follow-up ws done by clinicl exmintion every third month for the first yer, with subsequently longer intervls for the following yers. Chest X-rys nd blood profile, including liver function tests, were performed t ech visit. CT-scns of the lung were performed whenever chest X-rys indicted metstses or were inconclusive.

3 J.M. Lopes et l. / Prognosis in synovil srcom Pthologic fetures All vilble mteril for histologic study ws exmined without knowledge of the clinicl course of the ptients, using formlin-fixed prffin-embedded 5 µm sections (4 15 per tumor) stined with hemtoxylin nd eosin. Representtive sections from the surgicl mrgins of ech tumor were lso histologiclly evluted. The cses were clssified, ccording to the criteri of Enzinger nd Weiss [19], in biphsic (BSS, n = 19) nd monophsic (MSS, n = 30) subtypes of SS. Whenever necessry, immunohistochemicl nd ultrstructurl exmintions were performed to confirm the dignosis. In 42 cses, formlin-fixed prffin-embedded frgments were vilble for the following nlyses performed in consecutive sections of one representtive block from ech tumor: DNA flow cytometry (FCM) in 42 cses, DNA imge cytometry (ICM) in 41 cses, PCNA immunohistochemistry nd cidified toluidine blue (ph 3.0) in 40 cses, nd Ki-67 in 37 cses, depending on the representtion of tumor tissue vilble in ech section of the prffin blocks tht hd been selected. Mitotic rte ws determined by counting the most mitotic res in 10 successive high power fields (HPF; 400 mesured 0.15 mm 2 ) selected t rndom. The mount of mst cells ws evluted in 10 successive HPF cells chosen t rndom fter selecting the re displying the highest number of mst cells. The percentge of extent of tumor necrosis (bsed on gross nd microscopicl findings), s well s tht of solid/glndulr res, were evluted in ll the sections nd clssified s follows: 0; <15; 15 50; >50. Extent of clcifiction nd extent of hemngiopericytic res were clssified in four degrees: 0 (none); + (<15%); ++ (15 30%); +++ (>30%). Histologic grde of mlignncy ws independently evluted ccording to UICC [28] nd NCI [13] grding systems. Briefly, UICC grde (1 3) ws bsed on the estimtion of cellulrity, cellulr pleomorphism (e.g., nucler typi), mitotic ctivity, nd necrosis. NCI grding ws bsed on the gross nd microscopicl evlution of necrosis: <15% = grde 2, >15% = grde 3. Blood vessel invsion ws clssified s bsent or present. Residul tumor t the surgicl mrgins (R0, no residul tumor; R1, microscopicl residul tumor; R2, mcroscopicl residul tumor) nd TNM stging (I to III) of the tumors were clssified ccording to the UICC system [28] Immunohistochemistry Formlin-fixed prffin-embedded sections were stined using the vidin-biotin peroxidse complex (ABC) method [27]. Deprffinized sections were treted with 0.3% hydrogen peroxide (H 2 O 2 )in methnol for 30 min to block endogenous peroxidse. To unmsk the epitopes of PCNA nd Ki-67 the sections were microwved in <1% led solution [45] nd 10 mm citrte buffer ph 6.0 [11]. The sections were then incubted for 20 min with norml serum from the species in which the secondry ntibody ws mde. This ws done to minimize nonspecific stining. Excess norml serum ws blotted from slides before incubtion with monoclonl PCNA ntibody diluted 1 : 25 (NCL-PCNA, Novocstr Lbortory, UK) nd polyclonl Ki-67, ntibody diluted 1 : 100 (A 047, Dko, Denmrk) for h t 4 C. The sections were then incubted with 1 : 200 dilution of biotin-lbeled secondry ntibody for 30 min nd ABC (10 µg/ml of vidin nd 2.4 µg/ml of biotin-lbeled peroxidse) for 60 min (Vector, Burlingme, CA, USA). The tissue sections were stined for 5 min with 0.05% 3,3-diminobenzidine tetrhydrochloride (DAB) freshly prepred in 0.05 M tris(hydroxymethyl) minomethne (Tris) buffer t ph 7.6, contining 0.01% H 2 O 2 nd then counterstined with hemtoxylin, dehydrte, nd mounted in Ditex. All dilutions of norml ser, ntiserum, biotin-lbeled secondry ntibodies nd ABC were mde with PBS contining 5% bovine serum lbumin. All series included positive controls. Negtive

4 48 J.M. Lopes et l. / Prognosis in synovil srcom controls included substitution of polyclonl ntibody with norml rbbit IgG, wheres negtive control for the monoclonl ntibody ws performed using mouse myelom protein of the sme subclss nd concentrtion. All control exmintions were stisfctory. Immunorection for PCNA nd Ki-67 ws considered positive whenever nucler stining ws observed, independently of the intensity of the stining. Ares with more numerously stined nuclei were selected nd used for counting t rndom. The lbeling indices were determined in every cse by counting the number of positive tumor cell nuclei mong totl of 1000 cells. The lbeling indices were expressed in percentge of positive cells DNA cytometry To minimize the mount of non-tumor tissue, ll res with tumor were outlined on blocks corresponding to the best sections, nd most of the non-tumor tissue ws removed. From ech block one 100-µm section for DNA flow cytometry (FCM) nd two 50-µm sections for DNA imge cytometry (ICM) were cut. In the end, 5-µm section ws cut for evlution of representtivity of the section nd for the estimtion of the mount of tumor tissue present. After deprffiniztion with xylol, the tissues were rehydrted in grded ethnols, rinsed in phosphte-buffered sline (PBS) nd incubted for 30 min with protese (Sigm no. 24) t 37 C. The protese ctivity ws stopped by dding 4 ml cold PBS, nd therefter the specimens were rinsed twice in 4 ml PBS before centrifugtion t 1250 g [24]. The specimens were prepred in series to control interspecimen vrition of the disintegrtion index Flow cytometry Cell suspensions were prepred by the method described by Hedley [24], with minor modifictions [21], stined with ethidium bromide. DNA FCM ws performed by the use of lbortory-built flow cytometer [35]. Briefly, the instrument ws bsed on Nikon Diphot invertoscope equipped with excittion light rnges of nm nd bem splitter t 580 nm. The output signls were sorted by multichnnel nlyzer (Nucler Dt ND 66) into histogrms with 256 chnnels Imge cytometry The isolted cells were post-fixed in buffered 4% formlin for t lest 12 h in room temperture nd centrifuged (Hettich, Tuttingen, Germny) on polylysin-coted slides t 1250 g. Feulgen Schiff stining ws pplied (5 N HCl, 60 min, 22 C) with Prrosnilin-Schiff. Hydrolysis curves were mde to ensure reproducible stining. Imges of the nuclei obtined with Zeiss photomicroscope III equipped with pln 40/0.95 objective lens nd 546-nm green filter were trnsferred to MINI-IPS imge processing unit (Kontron, Munich, Germny) using Grundig FA 76 cmer with psecon tube with fixed gin level. The imge processing unit ws equipped with 4 megbytes of imge memory, rry processor nd digitizer unit controlled by Z80 host processor. Imges were stored s pixels with 256 gry levels. The softwre ws written in FORTRAN using Kontron s subroutine librry for ccess to the rry processor. The integrted opticl density of ech nucleus ws clculted on the bsis of mesurements of opticl density nd re. Bckground opticl density ws mesured nd corrected for ech imge, ensuring stble nd reproducible mesurements. At lest 300 nuclei per specimen were mesured. Norml humn lymphocytes were used s externl stining controls of the norml diploid (2c) DNA content due to the scrcity of lymphoid cells

5 J.M. Lopes et l. / Prognosis in synovil srcom 49 observed in SS. All nuclei were rndomly selected from multiple res of the slide. Only nuclei which were structurlly intct without overlpping, nd to which no nucler frgments were ttched, were mesured with utomtic segmenttion. The reproducibility of the ICM method ws verified by renlyzing one-third of the cses, which disclosed similr results Ploidy definition FCM. The histogrms were nlyzed with reference to the DNA indices of distinguishble stemlines [26]. Histogrms with only one modl pek were regrded s DNA diploid (coefficient of vrition (CV): %, men 5.1%). A tumor ws considered to be DNA non-diploid if severl peks were distinguishble. The first pek of the histogrm ws lwys regrded s being DNA diploid. The further rnges for DNA indices (DIs) were defined s follows: DNA tetrploid: 1.80 < DI < 2.20; DNA neuploid rnge: 1.10 < DI < 1.80 nd DI 2.20; DNA multiploid: more thn one DNA non-diploid stemline. In order to identify DNA-tetrploid stemline we looked for tetrploid peks with n integrl higher thn the integrl over the S-frction of the diploid stemline. If n ppurtennt DNA-octploid G2-pek nd lso n identifible S-phse region were present, these high tetrploid peks were stted to belong to tetrploid stemline [30]. The percentge of cells in S nd G2 phses ws nlyzed from DNA histogrms by use the MULTICYCLE computer progrm (Phoenix Flow System). Since cell nuclei, nd not the whole cells, were mesured, mitotic figures were wshed out during preprtion nd were not included in the mesurements. ICM. A tumor ws considered to be in the DNA diploid rnge if only one pek ws present (CV: %, men 10.7%). If more thn one pek could be identified, the first pek of the histogrm ws considered to be in the DNA diploid rnge nd served for clcultion of the DNA ploidy nd the coefficient of vrition. When more thn 10% of the nuclei were found in the DNA tetrploid region (2 2c±2 CV) the tumor ws considered to be in the DNA tetrploid rnge. DNA polyploid tumors hd significnt pek in the multiple of 2c. A tumor ws clssified s DNA neuploid either if the DNA content of 4 or more cells exceeded the 5c vlue ccording to Böcking [5] or if prominent pek ws identified between 2c nd 4c Sttistics The Mnn Whitney rnk-sum test ws used to compre the distributions of continuous vribles. This non-prmetric method ws chosen s the vlues for the vribles did not follow norml distribution. For two-wy frequency tbles, Fisher s exct test (2-tiled) ws used. The interreltionship between continuous vribles ws estimted with the Spermn correltion coefficient [15]. The probbilities of surviving were clculted with the Kpln Meier product-limit method. The differences between the survivl curves were tested by the log-rnk test [40]. The Cox proportionl hzrds model ws used to nlyze the simultneous importnce of severl prognostic fctors [14]. The proportionl ssumption in the Cox model ws exmined with plots [15]. The Wld test ws used for the significnce test of the regression coefficients [15]. The following strtegy ws followed due to mny cndidte vribles vilble: for continuous vribles, cutoffs, reported by other investigtors, were tested in univrite survivl nlyses. The other continuous vribles were grouped into qurtiles nd univrite survivl nlyses were performed. Finlly, two-grouping ws selected from these nlyses. Only significnt vribles/cutoffs were included in the finl regression nlysis. The sttisticl softwre of BMDP (PC-90) ws used [15].

6 50 J.M. Lopes et l. / Prognosis in synovil srcom 3. Results 3.1. Clinicl fetures The series comprised 23 femles nd 26 mles. The medin ge ws 32 yers (rnge 7 81 yers). The sites of the primry tumors were s follows: lower extremity (n = 31), upper extremity (n = 8), nd trunchus (n = 10). The thigh (n = 12) ws the most frequent single loction. Nine of the tumors were intrcomprtmentl nd 40 were extrcomprtmentl. None of the tumors ws restricted to the subcutneous comprtment. The most common initil symptom ws the presence of plpble pinless mss (25 ptients), followed by pin (15 ptients). The medin durtion of symptoms ws 10 months (rnge 1 96 months). Initil presenttion nd durtion of symptoms ws unknown in 9 cses. The medin tumor size ws 6 cm (rnge 1 25 cm). Twenty-four tumors were 5 cm or smller. All ptients were initilly treted by surgery. Adequte mrgins (wide, n = 14; nd mputtion, n = 8) were chieved in 22 cses nd indequte mrgins (intrlesionl, n = 1; nd mrginl, n = 24) in 25 cses. In two cses the surgicl mrgins were unknown. Twenty-three ptients received some form of djuvnt therpy: postopertive rdiotherpy, n = 17; djuvnt chemotherpy, n = 5; nd rdiond chemotherpy, n = 1. Locl recurrences were dignosed in 15 ptients nd distnt recurrences (lung metstses) in 29 ptients, including 11 ptients with prior locl recurrence. Synchronous (locl nd lung) recurrences were dignosed in one ptient. The relpse-free survivl rte (to locl recurrence or metstsis) ws 34% t 5 yers (95% confidence intervl (CI), 20 48%). The locl recurrence-free (metsttic relpses treted s censored observtions) nd the metstsis-free survivl rtes (locl relpses treted s censored observtions) were 69% (95% CI, 55 83%) nd 60% (95% CI, 46 74%) t 5 yers, respectively. The medin follow-up time fter initil tretment ws 51 months (rnge months). Twenty ptients were live with no evidence of disese, 5 were live with disese, 22 were ded due to the tumor, nd 2 were lost for follow-up. The overll 5-yer survivl fter initil tretment ws 64% (95% CI, 50 78%) Light microscopic fetures Seven of the BSS were of the clssicl type nd 12 showed focl biphsi. More thn 50% of solid/glndulr res were observed in 5 BSS. All MSS were of the fibrous vrint. None of the cses showed presence of rhbdoid cells. Medin mitotic rte ws 4/10 HPF (rnge 0 67/10 HPF). Fifteen cses showed mitotic rtes higher thn 10/10 HPF, nd in 8 of them it ws higher thn 15/10 HPF. Necrosis ws present in 26 cses nd occupied more thn 15% of the tumor volume in 22 cses. Focl (n = 10) or moderte (n = 3) clcifiction ws present in 13 cses. Focl (n = 3), moderte (n = 3) or extensive (n = 7) hemngiopericytic res were observed in 13 cses. The mount of mst cells (n = 40) ws 11/10 HPF (rnge 0 467/10 HPF). Usully, n ccumultion of mst cells were observed t the periphery of the tumor nd only in some cses were mst cells scttered throughout the tumor. The mount of mst cells ws higher thn 10/10 HPF in 22 cses, nd higher thn 20/10 HPF in 17. Tumor cells in blood vessels were observed in 7 cses. Focl (n = 7) or extensive (n = 1) res of tumor tissue were present t the surgicl mrgins in 8 tumors. The grdes of mlignncy obtined with the UICC nd NCI systems were concordnt (grde 2, n = 19; grde 3, n = 18) in 37 cses. The 12 discordnt cses comprised 2 tumors grde 1 by UICC

7 J.M. Lopes et l. / Prognosis in synovil srcom 51 Fig. 1. Biphsic synovil srcom displying prominent PCNA immunorectivity of the cells lining clefts nd glndulr spces. ABC, 280. nd grde 2 by NCI, 4 grde 2 by UICC nd grde 3 by NCI, nd 6 grde 3 by UICC nd grde 2 by NCI. The mitotic count ws low (<10/10 HPF) in the 6 cses in which the UICC grde ws lower thn the NCI grde, nd high ( 15/10 HPF) in the other 6 discordnt cses Immunohistochemistry In ll but one cse immunorectivity for PCNA nd Ki-67 ws reltively homogeneous; BSS showed, with both mrkers, higher number of immunorective cells in solid/glndulr res thn in the surrounding spindle cells (Figs 1 nd 2). The medin PCNA-LI (n = 40) ws 14.5% (rnge %). Eleven cses showed PCNA-LI higher thn 20%. The medin Ki-67-LI (n = 37) ws 10.7% (rnge %). Twenty cses showed Ki-67-LI higher thn 10% DNA cytometry Non-evluble histogrms (due to bd suspensions) were obtined in two cses, one by ICM nd nother by FCM. DNA ploidy descriptors (DNA diploid, n = 30; nd DNA neuploid, n = 4) were concordnt for the two methods in 34 cses. The 6 discordnt cses comprised 4 tumors tht were DNA diploid by ICM nd DNA neuploid by FCM, 1 tht ws DNA tetrploid by ICM nd DNA diploid by FCM, nd 1 tht ws DNA neuploid by ICM nd DNA tetrploid by FCM. The 4 cses tht were DNA neuploid by FCM nd DNA diploid by ICM hd DNA indices in the DNA ner-diploid rnge (Fig. 3). In the cse of DNA tetrploid by FCM, the ICM histogrm depicted 5c exceeding

8 52 J.M. Lopes et l. / Prognosis in synovil srcom Fig. 2. Biphsic synovil srcom displying Ki-67 immunorectivity lmost exclusively in cells of solid/glndulr res. ABC, 280. Fig. 3. Diploid DNA histogrm by ICM (left) nd neuploid (ner-diploid rnge) by FCM (right) of synovil srcom. rte (Fig. 4). The cse which ws DNA diploid by FCM showed DNA tetrploid popultion higher thn 10% by ICM. Whenever DNA neuploidy ws observed by ny of the two methods it ws used s such for survivl evlution. The clcultions of percentges of cells in S nd G2 phses of the cell cycle were not possible in 5 cses due to overlpping of G2 peks of DNA diploid nd DNA neuploid cells, or

9 J.M. Lopes et l. / Prognosis in synovil srcom 53 Fig. 4. Aneuploid DNA histogrm by ICM (left) nd tetrploid by FCM (right) of synovil srcom. Tble 1 Correltion between mitotic rte, Ki-67-LI nd PCNA-LI Spermn s rho Mitotic rte Ki-67-LI PCNA-LI Correltion coefficient Mitotic rte Ki PCNA p-vlue Mitotic rte Ki PCNA n Mitotic rte Ki PCNA Correltion is significnt t the 0.01 level (2-tiled). Ki-67-LI, Ki-67 lbeling index; PCNA-LI, proliferting cell nucler ntigen lbeling index. due to smll G2 peks. Medin percentge of cells in S-phse (n = 36) ws 9.1% (rnge %) nd the medin percentge of cells in G2-phse (n = 36) ws 13.8% (rnge %) Correltion between prolifertion mrkers (mitotic rte, Ki-67-LI nd PCNA-LI ) Correltion between prolifertive mrkers (mitotic rte, Ki-67-LI nd PCNA-LI) re summrized in Tble 1. All the forementioned correltions (Spermn rnk correltion coefficients) were positive nd ttined significnt level (p <0.001) Univrite nlysis The results of the univrite nlysis of clinicopthologic fetures in reltion to survivl re summrized in Tble 2. Gender of the ptients, comprtmentliztion nd loction, dimension, extent of clcifiction nd of hemngiopericytic res, percentge of glndulr res, blood vessel invsion, dequcy of surgicl

10 54 J.M. Lopes et l. / Prognosis in synovil srcom Tble 2 Univrite survivl nlysis of clinicopthologic fetures in ptients with loclized synovil srcom Fetures Groups Prmeters No. Relpse-free Overll survivl Log-rnk 5-yer Log-rnk p-vlue (%) p-vlue Gender Mle Femle 23 Age (yers) < Durtion of symptoms <12 24 < <0.05 (months) Loction Extremities Truncl 10 Comprtment Comprtmentl Extrcomprmentl 40 Dimension 5 cm >5 cm 25 Surgicl mrgin Indequte Adequte 22 Residul tumor RO R1+R2 8 Adjuvnt therpy b No Yes 23 Subtype b BSS MSS Glndulr < differentition 15 7 Necrosis Absent Present Mitotic rte <10 34 < < (10 HPF) Mst cell rte <10 18 < <0.001 (10 HPF) Clcifiction Absent Present 13 Hemngiopericytic Absent res Present 13 Vessel invsion Absent Present 7 UICC grde < < NCI grde Ploidy (FCM/ICM) Euploid Aneuploid S-phse (%) < S+G2-phse (%) <

11 J.M. Lopes et l. / Prognosis in synovil srcom 55 Tble 2 (Continued) Fetures Groups Prmeters No. Relpse-free Overll survivl Log-rnk 5-yer Log-rnk p-vlue (%) p-vlue PCNA-LI < < Ki-67-LI <10 17 < TNM I II 25 < III Prmeters ssocited with short intervl-free nd overll survivl. b Postopertive rdiotherpy nd djuvnt chemotherpy were grouped together. BSS, biphsic synovil srcom; MSS: monophsic synovil srcom; HPF, high power field; FCM, DNA flow cytometry; ICM, DNA imge cytometry; PCNA-LI, proliferting cell nucler ntigen lbeling index; Ki-67-LI, Ki-67 lbeling index. mrgins, nd residul tumor were not significnt predictors of survivl (relpse-free nd overll survivl) (Tble 2). Young ge of the ptients (p = 0.04), durtion of symptoms (p <0.01), UICC grde (p <0.0001), NCI grde (p = 0.06), histologic subtype (p = 0.03), mitotic rte (p <0.001), mount of mst cells (p <0.0001), necrosis (p <0.01), PCNA-LI (p = 0.04), Ki-67-LI (p <0.0001), DNA ploidy (p = 0.04), nd TNM stge (p <0.0001) were significnt predictors of relpse-free survivl (to locl recurrence or metstsis) (Tble 2). Durtion of symptoms (p <0.05), UICC grde (p <0.0001), NCI grde (p = 0.04), histologic subtype (p = 0.04), mitotic rte (p = 0.01), mount of mst cells (p <0.001), necrosis (p <0.02), PCNA-LI (p <0.001), Ki-67-LI (p = 0.005), DNA ploidy (p = 0.007), nd TNM stge (p = ) were significnt predictors of overll survivl (Tble 2). Short durtion of symptoms (<12 months), biphsic type (BSS), scrcity of mst cells (<10/10 HPF), presence of necrosis, high mitotic rte ( 10/10 HPF), high histologic grde (grde 3), high PCNA-LI ( 20%), high Ki-67-LI ( 10%), DNA neuploidy, nd dvnced TNM stge (stge III) were fetures ssocited with significntly shorter relpse-free nd significntly shorter overll 5-yer survivl rtes. Young ge (younger thn 30 yers) ws significnt predictor of relpse-free survivl but not overll survivl. The 5-yer survivl rtes of the ptients for ech of these prmeters re given in Tble Multivrite nlysis The results of the multivrite nlysis re summrized in Tble 3. Amount of mst cells, mitotic rte, or PCNA-LI were significnt predictors of survivl in ddition to TNM stge (Fig. 5). Only one of them t ech time provided significnt dditionl informtion to TNM stging. Curiously, the dditionl prognosis informtion provided by the three prmeters seemed to be nerly the sme Correltion between mst cell count, mitotic rte, nd PCNA-LI Correltions between the significnt fetures found in the multivrite nlysis re summrized in Tble 4. The mount of mst cells ws significntly correlted with mitotic rte (p = 0.003) nd PCNA-LI (p = 0.04). Tumors with mst cell count less thn 10/10 HPF hd frequently higher mitotic rtes ( 10/10 HPF) nd high PCNA-LI ( 20%). In ddition, the medin mitotic rte nd the medin

12 56 J.M. Lopes et l. / Prognosis in synovil srcom Tble 3 Multivrite survivl nlysis (Cox model) in ptients with loclized synovil srcom using 3 lterntive models Prognostic Regression Reltive 95% CI Wld test fctors coefficient deth risk p-vlue Group 1, n = 40 TNM: III vs. I II <0.01 Mst cell rte : <10 vs <0.001 Group 2, n = 49 TNM: III vs. I II <0.05 Mitotic rte :10+ vs. < <0.05 Group 3, n = 40 TNM: III vs. I II <0.001 PCNA-LI: 20%+ vs. <20% <0.001 Per 10 high power fields. CI, confidence intervl; PCNA-LI, proliferting cell nucler ntigen lbeling index. Fig. 5. Survivl curves of ptients with synovil srcom strtified by mount of mst cells, PCNA lbeling index, nd TNM stging.

13 J.M. Lopes et l. / Prognosis in synovil srcom 57 Tble 4 Reltionship between the mount of mst cells, mitotic rte ns PCNA-LI in synovil srcom Mst cell rte p-vlue <10/10 HPF (n = 18) 10/10 HPF (n = 22) Mitotic rte <10/10 HPF /10 HPF 11 3 Medin (%) PCNA-LI <20% % 8 3 Medin (%) HPF, high power field; PCNA-LI, proliferting cell nucler ntigen lbeling index. Fisher s exct test (2-tiled); Mnn Whitney rnk-sum test. PCNA-LI were significntly higher (p = nd p = 0.03, respectively) in tumors contining few mst cells compred to tumors contining numerous mst cells. The Spermn rnk correltion coefficient between mitotic rte nd mount of mst cells ws 0.49, nd between PCNA-LI nd mount of mst cells Discussion Synovil srcom (SS) is rre soft tissue tumor, usully of high grde mlignncy. Since 1980 ll but one series on record deling with survivl rtes re bsed upon less thn 100 cses [6,8,12,20, 22,23,25,29,32 34,37,41,44,46,47,50,52,56]. The 5-yer survivl rtes rnge from 30.0 to 82.6%. The 64% 5-yer survivl rte observed in the present series of loclized SS is within the rnge of the series previously reported (Tble 5) [6,8,12, 20,22,23,25,29,32 34,37,41,44,46,47,50,52,56]. By using the TNM stging system [28] we found, s in other studies of soft tissue srcoms including SS [36,39], tht stge is powerful prognostictor of relpse-free nd overll survivl. Actully, in our series of loclized SS, ptients with tumors t TNM stges I II t presenttion hd much better prognosis thn ptients with tumors t TNM stge III. Fvorble clinicl fetures previously reported include gender [8,36,41,52], young ge [8,17,37,52], durtion of symptoms, loction of the tumors in the extremities [8,33,37,41,52], nd smll size (less thn 5 cm) of the tumors [6,17,20,37,39,41,43,52,56]. Out of the five forementioned fetures only durtion of symptoms (more thn 12 months) turned out to be fvorble fctor for relpse-free (to locl or metsttic recurrence) nd overll survivl in the univrite nlysis of our series. Ptients of young ge (less thn 30 yers) hd longer relpse-free period but not longer overll survivl. Our results re therefore consistent with those of others tht did not find significnt ssocition between ge [6,20,22,25,36,43,56], gender [6,17,22,25,43,56], tumor loction [6,25,36] nd tumor size [22,25], nd survivl of ptients with SS. In the present series, comprtmentl loction of the tumor, dequcy of surgicl mrgins, nd djuvnt tretment (rdiotherpy or chemotherpy) did not predict significntly the relpse-free nd overll survivl of ptients with SS. Also, the residul tumor t the surgicl mrgins, positive only in 8 cses (16.3%), turned out not to be prognostic mrker for overll survivl. The 5-yer relpse-free rte of our series (34%) is within the rnge of previously reported results ( %) regrding the

14 58 J.M. Lopes et l. / Prognosis in synovil srcom Tble 5 5-yer survivl in 19 series of synovil srcom Yer Series Number of ptients 5-yer overll survivl (%) 1981 Buck et l. [15] Wright et l. [52] Vrel-Durn et l. [50] Tsuneyoshi et l. [47] Zito et l. [56] Rpjl et l. [41] Collin et l. [12] Tsujimoto et l. [46] Lck et l. [32] Golouh et l. [22] Henderson et l. [25] Brodsky et l. [6] Hshimoto et l. [23] Sntvirt [44] Ivnov-Dutescu et l. [29] Lee et l. [34] Od et l. [37] Ldenstein et l. [33] Fetch et l. [20] control of locl nd distnt disese in SS [29,34,41]. Recently, Ldenstein et l. [33] reported 74.2% 5-yer event-free survivl in multicenter tril including 31 peditric ptients with SS. Although we found in the univrite nlysis tht the BSS subtype indictes shorter relpse-free nd overll survivl, we observed, like others [6,10,17,22,23,25,33,38,39,43], tht histologic subtype ws not significnt prognostic fctor in the multivrite nlysis. In greement with others [22,37, 43,52] nd in contrst to Cgle et l. [10], we observed no reltionship between the extent of glndulr differentition nd the overll survivl. Both clcifiction [50] nd poor differentition [19] in SS re mong the morphologicl fetures reported to be relted to the prognosis. Clcifiction is frequently seen in SS [19]. Extensive clcifiction ws reported by Vrel-Durn nd Enzinger to be ssocited with good prognosis [50]. However, in our series, no reltionship between clcifiction nd survivl of the ptients ws observed. On the other hnd, none of the cses in our series showed more thn moderte extent of clcifiction. Hemngiopericytic pttern is one of the fetures described in poorly differentited SS nd is thought to be ssocited with n unfvorble prognosis [19]. None of the cses in our series ws clssified s poorly differentited, nd the extent of hemngiopericytic pttern ws not predictive for the overll survivl. We hve found, like other uthors in severl series of different types of soft tissue srcoms [49], nd Od et l. [37] in SS specificlly, tht lrge mount of mst cells is fvorble prognostic feture. The prognostic significnce of this feture ws mintined in the multivrite nlysis in our study. The frequent presence of perivsculr nd interstitil mst cells hs been described s chrcteristic feture of some benign (e.g., schwnnoms, hemngioms nd cellulr dermtofibroms) nd mlignnt (e.g., neurofibrosrcoms, synovil srcoms nd mlignnt fibrous histiocytoms) soft tissue tumors [19,49]. It hs lso been described elsewhere in other neoplsms such s brest crcinom nd cervicl crcinom. It is interesting tht, s we observed in the present study nd s hs been reported previously in severl types of soft tissue tumors, mst cells re preferentilly locted in the peripherl res of the neoplstic msses displying ctive tumor growth.

15 J.M. Lopes et l. / Prognosis in synovil srcom 59 The puttive role of mst cells in tumor growth remins to be clrified. There re experimentl studies fvoring n involvement of mst cells in tumor suppression [9]. In contrst to these studies, Roche [42] reported tht mst cells enhnce tumor prolifertion of trnsplnted srcoms in mice. We observed tht the mount of mst cells showed significnt inverse correltion with prolifertion mrkers (mitotic rte nd PCNA-LI). Moreover, the mount of mst cells ws significnt predictor of the disese-free intervl nd the overll survivl of the ptients. Thus, mst cells my be involved in suppression of tumor growth. Extent of necrosis hs been reported s n importnt prognostic fctor in series composed of different types of soft tissue srcoms [13,19,23,32] nd lso in SS [22,23,37,38,43]. Despite hving observed significnt reltionship between necrosis nd overll survivl in the univrite nlysis we, like Od et l. [37,38], filed to confirm such reltionship t the multivrite nlysis. The importnce of the mitotic rte prognostic fctor reported in the present series fter multivrite nlysis corrobortes Enzinger s sttement [19] on the existence of n inverse correltion between survivl nd mitotic ctivity in SS, nd concurs lso with the reports of other groups [10,17,20,22, 25,37,38,43,56]. Histologic grding of soft tissue srcoms is mtter of gret controversy [13,19,32,46]. There re severl grding systems using different criteri nd vrible thresholds for ech of them [13,19,28]. Our results re consistent with those of Moberger et l. [36] nd with the results recently reported by Pppo et l. [39], showing tht grding of SS is of prognostic vlue. We compred the prognostic strengths of the UICC [28] nd the NCI [13] grding systems, nd the former system ppered to be better in our study. By using the UICC grding system we showed tht the mitotic rte, which is not used in the NCI system for SS, is importnt in the grding of SS. The results we hve obtined using the UICC system comply with the existence of spectrum (from 1 to 3, in grde scle) of mlignncy in SS, s outlined by Enzinger [19]. At vrince with this, with the NCI grding system SS re lwys high grde tumors (grdes 2 3), depending on the extent of gross nd microscopicl necrosis (15% threshold). Moreover, in our multivrite nlysis, necrosis did not significntly predict the survivl probbility, thus contributing to wekening the potentil strength of the NCI grding system. We observed blood vessel invsion in 7 cses. This feture ws not ssocited with sttisticlly significnt difference on the survivl, s ws described by Rööser et l. in SS [43], nd t vrince to the results reported by other groups in series composed of different types of soft tissue srcom [19,32]. The results of DNA cytometry prmeters obtined to dte re controversil prtly becuse one tends to confound univrible nlysis with multivrite nlysis [2,17,38,53]. In our series, less thn 1/4 of the cses were DNA neuploid. This percentge is smller thn tht reported by other uthors. In fct, tking together the dt reported in severl series deling with SS, the overll percentge of DNA neuploid SS is 40% (out of 116 SS) [3,17,38]. No ssocition between prognosis nd DNA ploidy hs been observed in severl studies [19,38,53], wheres in other reports [1,2,17] DNA neuploidy hs been found to be n unfvorble prognostic fctor for ptients with soft tissue srcoms in generl. In our univrite nlysis we observed, s did Od et l. [38], n ssocition between DNA neuploidy nd n unfvorble prognosis. However, t vrince with the findings of El-Nggr et l. [17] nd Åkermn et l. [2], this ssocition ws lost in the multivrite nlysis, s ws lso described by Od et l. [38]. Moreover, the evlution of the percentge of tumor cells in S nd S+G2 phses did provide ny significnt dditionl prognostic informtion (Od et l. [38], present study).

16 60 J.M. Lopes et l. / Prognosis in synovil srcom In our series, DNA ploidy ws studied by imge (ICM) nd flow (FCM) cytometry. The concordnce rte of DNA ploidy descriptors observed in the present series fits with other reports [1]. In these studies, exmples of discordnce between DNA ploidy descriptors (DNA diploid by FCM nd DNA neuploid by ICM nd vice-vers) hve been reported. Severl explntions hve been dvnced to explin such discordnces: inherent differences in the two methods, problems t the histogrm interprettion level, differences in tissue smpling, nd intrtumor heterogeneity. In ddition, the possible effect of formlin fixtion s limiting fctor in DNA cytometry evlution should be considered, s hs been recently pointed out by Zlupski et l. in series of soft tissue srcoms studied by FCM [54]. This lst point my indeed ply mjor role in the controversil dt on record since it is known tht percentge of S-phse cells ssessed on prffin mteril tends to differ from series to series due to differences in severl procedures: fixtion, embedding nd cutting. Few studies on the prognostic vlue of PCNA expression in soft tissue srcoms hve been reported to dte. High PCNA expression ws found to be ssocited with poor prognosis in series of hemngiopericytoms [53] nd in series of different types of soft tissue srcoms [7]. No significnt ssocition between PCNA expression nd survivl ws observed in series of different types of soft tissue srcoms [4] nd in series of mlignnt fibrous histiocytom (MFH) [51]. High PCNA expression ws ssocited with better prognosis in series of rhbdomyosrcoms [31]. The PCNA lbeling index remined sttisticlly significnt prognostic fctor in our multivrite nlysis s ws reported by Od et l. [38]. There re few reported studies on the prognostic vlue of Ki-67 expression in soft tissue srcoms. In one study deling with MFH, the expression of Ki-67 ws not significntly ssocited with prognosis [55]. In other studies, high Ki-67 levels were found to be n unfvorble prognostic fctor for ptients with soft tissue srcoms [16,48]. To the best of our knowledge, this is the first report on the prognostic vlue of Ki-67 in SS. In the univrite nlysis, high Ki-67-LI ws ssocited with n unfvorble prognosis. However, this ssocition did not ttin the threshold of sttisticl significnce in the multivrite nlysis. 5. Conclusion Besides TNM (UICC) stge, our study reveled severl fctors significntly ssocited with the survivl of ptients with loclized SS in the univrite nlysis. Amount of mst cells, mitotic rte or PCNA-LI remined s significnt prognostictors, in ddition to stging, t the multivrite nlysis level. Further studies re needed to clrify the puttive reltionship between mst cell bundnce nd prolifertion mrkers. Acknowledgements We thnk Ellen Hellesylt, Metter Myre, Inger Liv Nordli, Elisbeth Mølsted nd Ruth Puntervold for expert technicl ssistnce, nd Wencke Dnielsen nd Ellen Nygenen for expert secretril ssistnce. References [1] V. Agrwll, E. Greenebum, R. Wersto nd L. Koss, DNA ploidy of spindle cell soft-tissue tumors nd its reltionship to histology nd clinicl outcome, Arch. Pthol. Lb. Med. 115 (1991),

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