INVESTIGATION OF RELATIONSHIPS BETWEEN KI-67 SCORE, DNA INDEX, AND HISTOLOGIC GRADE IN SOFT TISSUESARCOMAS

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1 Chinese Jurnal fcancer Research 8(1): INVESTIGATION OF RELATIONSHIPS BETWEEN KI-67 SCORE, DNA INDEX, AND HISTOLOGIC GRADE IN SOFT TISSUESARCOMAS Wang Yanng ~2IE '~ Shi Daren* J[fg~A'Z Shen Zhenzhu i2~" Zhang Renyuan* ~1~{2.~ Liu Shuye ~],~ Department fsurgery, *Department fpathlgy, Cancer Hspital, Shanghai Medical University, Shanghai Prliferative activity f sft tissue sarcmas (STS) in 31 cases was estimated by histlgic grading, mittic cunt, DNA analysis by flw cytmetry, and immunhistchemical prcedures with mnclnal antibdy Ki-67. Aneuplid was fund in 12 f 16 cases (75.0%) with Grade 3, and in 4 f 15 cases (26.7%)with Grade 1, 2 (P=0.0121). Tumrs with mre than 100 Ki-67 psitive tumr cells per 10 high pwer fields (HPF) had a higher rate f aneuplid (81.3%) than thse with less than 100 Ki-67 per 10 HPF (26.7%) (P=0.0038). There were significant crrelatins between Gradeand DI (!'=0.4901, P= ), Grade and Ki-67 (r=0.4636, P= ), Ki-67 and DI (r=0.6368, P= ). The results indicate that DI and reactivity f tumr cells t Ki-67 may reflect prliferative activity and be helpful fr clinicians t judge the bilgical behaviur f tumrs mre accurately and bjectively. Supplementary t the grading f STS, DI and Ki-67 scre culd be useful as prgnstic parameters fr clinical investigatin f muitimdality therapy fr individual patients. Key wrds: Sft tissue sarcmas, Ki-67 antibdy, Flw cytmetry, Histlgic grade. Investigatins f cell kinetics have established that the assessment f cell prliferatin may be an imprtant prgnstic index fr sme tumrs, in sft tissue sarcmas (STS), the mst imprtant factr Accepted January 26, 1996 affecting patient survival is histlgicgrade whichhas been used by pathlgists t estimate the prliferative activity f the tumr and has prpsed valuable infrmatin fr clinicians t chse treatment remedy fr patients.l'2 Recently, Ki-67 expressin by immunhistlgic staining and DNA analysis by flw cytmetry have been reprted t have crrelatin with prliferative activity in a number f neplasms, including carcinmas f breast, lung, cln, as well as lymphmas and a mixed grup f sarcmas.3'4 The purpse f this study was t clarify the relatinships between mittic cunt, histlgic grade, DNA index (DI), and Ki-67 scre with an attempt t help clinicians judge the bilgical behaviur f STS mre accurately. MATERIALS TumrSelectin AND METHODS Thirty-ne patients with primary r recurrent STS lcated in extremities,trunk, and retrperitneum were selected fr the current study, they were all subjected t surgical resectin f the lesins withut receiving raditherapy r chemtherapy three mnths befre surgery. There were 21 male and 10 female patients with a mean age f 46 years (range frm 14 t 77 years) and a mean lngest tumr size f 10.4 cm (range frm 0.8 t 30 cm). Fresh tumr specimens 55

2 were btained frm surgical resectin fr histpathlgic assessment f diagnsis, DNA flw cytmetric analysis, and Ki-67 determinatin. The histlgictypes fsarcmas are listed in Table 1. Table 1. Histlgic types f31 patients with sft tissue sarcmas Histlgic types N. (%) Rhabdmysarcma 5 (16.1) Malignant schwannma 5 (16.1) Fibrsarcma 5 (161 l) Malignant fibrus histcytma 4 (12.9) Lipsarcma 4 (12.9) Leimysarcma 2 (6.5) Undifferentiated sarcma 2 (6.5) Hemangipericytma 1(3.2) Synvial sarcma 1 (3.2) Unclassified 2 (6.5) Ttal 31 (100) Mittic Cunt and Histlgic Grading Sectins, cut at 5 gm, were stained with hematxylin and esin, and the number f mitsis per ten high pwer fields (HPF, magnificatin 400) was cunted. The histlgic grades were determined accrding t the criteria frussell.5 Flw CytmetricAnalysis With EPICS V flw cytmetry (Culter C., USA), a mean number f cells f every singlecell suspensin sample btained mechanically and enzymatically frm fresh tumr tissue were analyzed fr measurement f DNA cntent and DI. The DI was calculated frm the DNA histgram as the rati f G1/G0 peak f tumr cells t the G1/G0 peak f nrmal cntrlled diplidcells. In this study,lesins were classified as aneuplid ifthe D1 was greater than 1.1 and as diplid if the DI was equal t r less than 1.1. The cefficients f variatin (CV) ranged frm 3.7% t 8.2% (mean=5.1%). Ki-67 Immunhistchemical Staining Fr immunhistchemical prcedures, we used avidin-bitin-perxidase cmplex (ABC) methd.6 Crystat sectins, cut at 5 tam, were air-dried at rm temperature, fixed in acetne fr 5 minutes, rinsed in TBS (0.05 M) and air-dried. The Ki-67 mnclnal antibdy (Dakpatts, Denmark) was diluted in TBS (1:40), sectins were incubated fr 60 minutes in a mist chamber at rm temperature. Subsequently, the sectins were rinsed in TBS, incubated with bitinylated hrse anti-muse IgG (Vectr, USA diluted in TBS, 1:200) tbr 30 minutes, rinsed, incubated with ABC cmplex (Vectr, USA. diluted in TBS, 1:100) fr 60 minutes, and rinsed again in TBS. Sectins were stained tbr 5-10 minutes with freshly prepared diaminbenzidine (DAB) slutin and cunterstained with methyl green. RESULTS Mittic Cunt and Grade The mean mittic scre ranged frm 0 t 13/10 HPF (mean, 3.4_+3.1/10 HPF). The sarcmas were graded as Grade1 (G1) in 7 patients, Grade 2 (G2) in 8, and Grade 3 (G3) in 16; their mean mittic scre was 1.0/10 HPF, 3.l/10 HPF, and 4.6/10 HPF, respectively. DNA PIidy anddi Aneuplid was fund in 17 tumrs (54.8%) and dipid in 14 (45.2%). DI varied frm 0.67 t 2.4 (mean, 1.3_+0.4). The mean DI was 1.0 in sarcmas with GI, 1.3 withg2, and 1.5 with G3. DNA plidy had n significantly different distributin cnsidering sex, age, tumr size, primary r recurrent tumr, mad mitticscre Hwever, aneuplid was fund higher in lesins with G3 (75.0%) than in lesins with G1, 2 (26.7%) and the difference was significant (Table 2). Ki-67 Scre Ki-67-psitive tumr cells culd be clearly detected by their characteristic diffuse granular r glbular nuclear staining. The percentage f stained cells ften varied frm area t area. In sme cases, Ki-67 staining was restricted t certain architectural cmpnents f the tumr. The area cntaining the largest number f Ki-67-psitive cells were selected, and the numbers f psitive cells cunted in 10 HPF chsen at randm. Twenty-ne sacmas (67.7%) 56

3 were psitively stained. The Ki-67 scres ranged frm 0 t 504/10 HPF (mean, 113/10 HPF). In lesins with G1, G2, and G3, the mean Ki-67 scres were 23.7/10 HPF, 90.3/10 HPF, and 172.8/10 HPF, respectively. Aneuplid was fund in 13 f 16 cases (81.3%) with Ki-67 scre f mre than 100/10 HPF, cmpared with 4 f 15 cases (26.7%) with Ki-67 scres f less than 100/10 HPF (P=0.0038). Table 2. Plidy distributin fvarius factrs in 31 patients with sft tissue sarcmas Factrs Aneupid Diplid Aneupid Sex Male (21) 11 (52.4) 10 (47.6) Female (10) 6 (60.0) 4 (40.0) Age >50 years (14) 7 (50.0) 7 (50.0) <50 years (17) 10 (58.8) 7 (41.2) Tumr size >10 cm (15) 8 (53.3) 7 (46.7) <10 cm (16) 9 (56.3) 7 (43.7) Primary r recurrent Primary (12) 6 (50.0) 6 (50.0) Recurrent (19) 11 (57.9) 8 (42.1) Mittic cunt _>3/10 HPF (13) 8 (61.5) 5 (38.5) <3/10 HPF (18) 9 (50.0) 9 (50.0) Histlgic grade G1, 2 (15) 4 (26.7) 11 (73.3) G3 (16) 12 (75.0) 4 (25.0) Ki-67 scre _>100/10 HPF (16) 13 (81.3) 3 (18.7) <100/10 HPF (15) 4 (26.7) 11 (73.3) Crrelatins The relatinships between mittic scre, histlgicgrade, DI, and Ki-67 scre wee evaluated by Pearsn's rank crrelatin test (Table 3). Ki-67 scre had statistically significant crrelatin with histlgicgrade (Figure 1) and DI (Figure 2). Histlgic grade significantly crrelated with mittic scre and DI. Hwever, mittic scre had n psitive crrelatin with DI and Ki-67 scre. DISCUSSION In 1977, a clinical and pathlgic staging system fr STS was prpsed by Russell et al) In this system,histlgicgrade, tumr size, and extend f the tumrs were factrs fr staging f the tumr. Histlgic grade was mainly determined accrding t frequencies f mitsis. Meanwhile, the frequencies f mitsis did nt necessarily crrelate with grade f malignancy in sme cases: STS f small rund cell type such as Ewing's sarcmas and undifferentiated sarcmas were prved t be high-grade tumrs but usually shwed lw mittic activity] Therefre, it is necessary t use mre accurate and bjective methds t evaluate the cell prliferatin in STS t estimate the prgnsis in STS and help clinicians treat individual patients. Cmplementary t cnventinal histlgic assessment, DNA cntent r DI detected by flw cytmetry has been shwn as a parameter t make a distinctin between benign and malignant tumrs and reflect the prliferative activity f malignancy) Kreicbergs, et al. fund a relatinship between tumr grade and DI in STS, and indicated that the Grade 3 57

4 sarcmas with aneuplid had a prer prgnsis than the same grade with diplid, s Our results supprt their bservatins, i.e., aneuplid was assciated with higher grades f malignancy, and plidy r DI may reflect the degree f malignancy and be an imprtant prgnstic factr in STS. Table 3. Result f spearman's rank crrelatin testfr variables fmittic cunt, histlgic grade, D1,andKi-67scre cases with STS,Ueda,et al. reprted that Ki-67 index psitively crrelated with histlgicgradeand the Ki- 67 lw index grup (less than 50/10 HPF) shwed mre favrable prgnsis than the high index grup (mre than 50/10 HPF) (P<0.005).7 In ur study, the relatinships between histlgic grade, DI, and Ki-67 were fund t be significant. The prgnstic value f these parameters will be analyzed when fllw-up times fall patients in this study are available. Variables Grade Mitsis DI Ki-67 r P Grade r P Mitsis r P <. 5,~ l O q9 0.5 ~. ~.5 a O Fig. 2. Ki-67 scre crrelated well with DNA index (r=0.6368, P=0.0001). 5OO 4OO ~0 2OO & 8 8 ~1 ~2 G3 ~istlglc grade Fig.. 1. Ki-67 scre crrelated well with histlgic grade (r , P=0.0086). Using immunhistchemical staining with Ki-67 mnclnal antibdy t measure cell prliferatin was reprted by many authrs.4 The Ki-67 antibdy, which reacts with a nuclear antigen expressed in all phases f the cell cycle except GO, has been shwn t be mre reliable and available as a simple histlgic marker f prliferative activity in cells f varius tissues and malignant neplasms.4'9 In a study f 34 Because cells in M phase spend relatively shrter time in cell cycle, it is less reliablet judge the cell prliferatin by cunting mitsis withut taking accunt f cellularity, degree f tumr necrsis, and clinical characteristics. The mittic cunt is nt a standardized methd and is ften nt reprducible even amng the experienced pathlgists. Therefre, the mittic cunt cannt be regarded as an independent parameter f cell prliferatin and the mittic activity f a tumr can nly be interpreted in the cntext f the ther pathlgical and clinical findings] We cnsider that instead f mitsis the reactivity f tumr cells t Ki-67 and DI by flw cytmetry reflect prliferative activity f STS mre bjectively, and tgether with rutine histl0gic grading, be helpful t btain mre precise grading results in STS. Much prgress has been made in the develpment f successful treatment strategies, mainly cnservative surgery cmbined with raditherapy and chemtherapy, fr patients with high-grade STS.1~ Hwever, it is smetimes still puzzling fr clinicians t decide whether individual patient may benefit frm the treatment mdalities r nt and which ne t be 58

5 chsen frm varied remedies if he r she may. As the effect f adjuvant chemtherapy has nt been cnfirmed yet, the bilgical behaviur f tumrs needs t be judged mre precisely t cnduct the treatment n single patient. Until recently, we culd nt find any reprts abut results f treatment cnsidering Ki-67 scre and DI as criteria in clinical studies. Hence, future studies, based n grade, DI, and Ki-67 scre, are indicated t investigate multimdality therapy fr patients with STS. REFERENCES 1. GaynrJJ, Tan CC, Casper ES, et al. Refinement f clinicpathlgic staging fr lcalized sft tissue sarcma f the extremity: a study f 423 adults. J ClinOncl 1992; 10: Cllin C, Gdbld J, Hajdu S, et al. Lcalizedextremity sft tissue sarcma: an analysis f factrs affecting survival. J Clin Oncl 1987;5: Ellis CN, Burnette JJ, Sedlak R, et al. Prgnstic applicatins fdna analysis in slid malignant lesins inhumans. SurgGynecl Obstet 1991;173: Brwn DC, Gatter KC. Mnclnal antibdy Ki-67: its use in histpathlgy. Histpathlgy 1990;17: Russell WO, ChenJ, Enzinger FM, et al. A clinical and pathlgical staging system fr sft tissue sarcmas. Cancer 1977; 40: Hsu SM, Raine L, Fanger H. A cmparative study f the perxidase-antiperxidase methd and avidin-bitin cmplex methd fr studying plypeptide hrmnes with radiirmnunassay antibdies. Am J Clin Pathl 1981; 75: Ueda K, Azasa K, Tsufimt M, et al. Prgnstic significance fki-67 reactivity in sft tissue sarcmas. Cancer 1989; 63: KreicbergsA, Trbukait B, Willems J, et al. DNAflw analysis f sft tissue tumrs. Cancer 1987;59: Krese MCS, Rutgers DH, Wils IS, et al. The relevance f the DNA index and prliferatin rate in thegradingfbenign and malignant sfttissue tumrs. Cancer 1990; 65: Quinn CM,. Wright NA. The clinical assessment f' prliferatin and grwth in human tumrs: evaluatin f methds and applicatins as prgnstic variables. J Pathl 1990;160: WilliardWC, Hajdu SI, CasperES, et al. Cnlparisn f amputatin with limb-sparing peratin fr adult sft tissue sarcma f the extremity. Ann Surg 1992; 215: