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High Ki-67 Proliferative Index Predicts Disease

Specific Survival in Patients with High-Risk SoftTissue Sarcomas

Axel Hoos, M.D., Ph.D.1

Alexander Stojadinovic, M.D.1

Stephen Mastorides, M.D.2

Marshall J. Urist, B.S.2

David Polsky, M.D., Ph.D.2

Charles J. Di Como, Ph.D.2

Murray F. Brennan, M.D.1

Carlos Cordon-Cardo, M.D., Ph.D.2

1 Department of Surgery, Memorial Sloan-Ketter-

ing Cancer Center, New York, New York.

2 Department of Pathology, Memorial Sloan-Ket-

tering Cancer Center, New York, New York.

Supported by National Institutes of Health Grant

CA-47179.

The authors thank the following collaborators for

their continuous support: Denis H. Y. Leung, Ph.D.

for statistical analyses, Maria E. Dudas for immu-

nohistochemistry, James D. Woodruff, M.D., andCristina R. Antonescu, M.D. for confirmation of

histopathologic diagnoses.

Stephen Mastoridess current address: Depart-

ment of Pathology, James Haley Veterans Hospital,

Tampa, Florida.

Address for reprints: Carlos Cordon-Cardo, M.D.,

Ph.D., Department of Pathology, Memorial Sloan-

Kettering Cancer Center, New York, NY 10021;

Fax: (212)794-3186; E-mail: cordon-c@mskcc.org

Received February 26, 2001; revision received

May 8, 2001; accepted May 14, 2001.

BACKGROUND. Soft tissue sarcomas (STSs) are heterogeneous neoplasms that have

variable clinical outcome. Several clinical parameters and few molecular markers,

including Ki-67 proliferative index, have been shown to correlate with patient

prognosis. To the authors knowledge, no definitive report exists to identify one

molecular marker that can be analyzed easily in a clinical setting and that predicts

survival in a cohort of patients with high-risk STS of identical clinical characteris-

tics but variable outcome.

METHODS. The influence of clinical prognostic factors was eliminated by selecting

two patient groups with identical high-risk characteristics: large ( 10 cm), high-

grade, deep, completely resected primary extremity STS (n 47). Patients in the

first group remained disease free (no evidence of disease [NED]) after primary

tumor treatment (n 19), whereas patients in the second group subsequently died

of disease (DOD; n 28). Triplicate 0.6-mm core biopsies from defined morpho-

logic areas of paraffin embedded primary tumors were assembled on a tissue

microarray and analyzed by immunohistochemistry with the MIB-1 antibody, and

Ki-67 proliferative indices were correlated with patient outcome.

RESULTS. High Ki-67 proliferative index, defined as greater than 30% tumor cells

showing nuclear immunoreactivity, was significantly more frequent in the DOD

group than in the NED group and was associated with tumor-related mortality (P

0.02). This marker identifies an especially aggressive malignant phenotype

within a cohort of high-risk tumors that is based on well established clinical and

pathologic parameters alone and is easy to use in a clinical setting.

CONCLUSIONS. On the basis of these data and previous reports, high Ki-67 prolif-

erative index is suggested as a significant factor for predicting the prognosis of

patients with high-risk STS and should be evaluated prospectively based on clinical

trials. Cancer2001;92:86974. 2001 American Cancer Society.

KEYWORDS: immunohistochemistry, Ki-67, MIB-1, sarcoma, survival, prognosis.

Soft tissue sarcomas (STSs) are a heterogeneous group of mesen-chymal neoplasms with variable biologic behavior. Histologicgrade, tumor size, depth, and status of surgical resection margins

have been identified as clinical prognostic factors for STS.13 Because

of their clinical relevance, grade, size, and depth form the basis for the

clinical staging system of the American Joint Committee on Cancer

(AJCC) of STS4 and can be used to identify patients at high risk to die

of sarcoma. However, these prognostic variables do not explain the

biologic differences in aggressiveness between STS of similar size,

grade, and depth after complete tumor resection.

Few molecular factors have been identified to correlate with

prognosis of patients with STS, including Ki-67, among others.59

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Moreover, Ki-67 has been found to be associated with

prognosis in STS in several independent stud-

ies.5,8,1012 The Ki-67 antigen was first described in

1983 as a nuclear protein associated with cellular pro-

liferation.13 Further studies have shown that Ki-67 is

expressed from early entrance into G1-phase through-out the cell cycle and that immunohistochemical

staining correlates with the growth fraction of tu-

mors.10,14,15 However, its functional significance still

remains unclear. Recent studies suggest Ki-67 to be

involved in control and timing of mitosis.15,16

The monoclonal antibody MIB-1 frequently is

used to detect Ki-67 in the nuclei of tumor cells on the

basis of well established protocols.8,17 High Ki-67 pro-

liferative index, the fraction of tumor cell nuclei above

a defined threshold being positive for Ki-67, has been

reported to correlate with prognosis of patients with

various cancers.1824

Despite promising results with Ki-67 proliferative

index in sarcoma,5,8,11,12 no definitive study to our

knowledge has validated the clinical relevance of Ki-67

for survival in a cohort of patients with high-risk STS

of identical clinical characteristics but variable out-

come.

The main goal of the current study was to char-

acterize the potential of Ki-67 proliferative index to

discriminate patient survival in a cohort of clinical

high-risk STSs. Specifically, we analyzed two groups of

carefully selected patients with large ( 10 cm), high-

grade, deep, primary extremity sarcomas that havebeen completely resected and compared Ki-67 prolif-

erative indices of patients who remained free of dis-

ease after treatment with those of patients who sub-

sequently died of their disease.

PATIENTS AND METHODSPatients, Clinical, and Pathologic Parameters

The cohort analyzed consisted of two clinically well

characterized, matched groups of patients (n 47)

with large ( 10 cm), high-grade, deep primary ex-

tremity STSs that were completely resected. Histologic

grade was defined based on the degree of tumor dif-

ferentiation, cellularity, stroma, vascularity, necrosis,

and mitoses.25,26 All patients were treated as inpa-

tients at Memorial Sloan-Kettering Cancer Center

(MSKCC) between January 1982 and January 1998 and

were observed prospectively and entered into our sar-

coma database. Median age of the patient cohort was

64 years (range, 2787 years). Twenty-seven patients

(57%) were male, and 20 were female (43%). Median

follow-up for the patient cohort was 39 months (range,

7129 months).

On the basis of their clinical characteristics, all

patients in this study were at high risk to die of their

disease. The first group comprised 19 patients that

remained free of disease (no evidence of disease

[NED]) after primary treatment. The second group

contained 28 patients that developed local recurrence

and/or metastatic disease and subsequently died of

their disease (died of disease [DOD]). All patients pre-sented to MSKCC with primary disease only.

All patients received surgical resection of the pri-

mary tumor. Some received neoadjuvant/ adjuvant

chemotherapy/radiation at the discretion of the Mul-

tidisciplinary Soft Tissue Sarcoma Group or as part of

clinical trials. Patients were treated according to the

standard of care at MSKCC. Disease specific survival

was defined as time from primary tumor resection to

death from disease. Local recurrence was defined as

recurrence occurring more than 3 months after com-

plete resection.

Tissues, Array Construction, and Immunohistochemistry

We performed analysis on formalin fixed, paraffin em-

bedded tissue. Tissue sections of each specimen were

stained with hematoxylin and eosin and evaluated by

a reference pathologist (S.M.). All cases were reviewed

to confirm the diagnosis of sarcoma, tumor grade, and

quality of the tissue and to identify a representative

area of the specimen. From these defined areas, core

biopsies were taken with a precision instrument

(Beecher Instruments, Silver Spring, MD), as previ-

ously described.27 Tissue cores with a dimension of 0.6

mm from each specimen were punched and arrayedin triplicate on a recipient paraffin block.17 The tissue

microarray technique was chosen because it allows for

efficient analysis of multiple tumors and normal tissue

controls on one paraffin block and preserves a large

portion of the tumor block for further analysis. As

previously shown by us, triplicate 0.6-mm core biop-

sies from STS paraffin embedded tissues for the con-

struction of tissue microarrays has a 96% concordance

rate between MIB-1 immunohistochemistry per-

formed on sections from tissue microarrays and on

standard full tissue sections.17 This makes the tech-

nique feasible for the described analysis.

Five-micrometer sections of the tissue array

block were cut and placed on charged polylysine

coated slides. These sections were used for immu-

nohistochemical analysis.28 Sarcomas known to be

strongly positive for Ki-67 were used as positive

controls. Arrayed normal tissues served as negative

controls. Sections from tissue arrays were deparaf-

finized, rehydrated in graded alcohols, and pro-

cessed using the avidin-biotin immunoperoxidase

method. Briefly, sections were incubated in pre-

heated 0.05% trypsin, 0.05% CaCl2 in 0.05M Tris-HCl

(pH 7.6) for 5 minutes at 37 C and then submitted

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to antigen retrieval by microwave oven treatment

for 15 minutes in 0.01 mM citrate buffer at pH 6.0.

Slides subsequently were incubated in 5% normal horse

serum for 30 minutes followed by MIB-1 antibody incu-

bation overnight at 4 C (1:50; Immunotech, Marseille,

France). Samples then were incubated with biotinylatedanti-mouse immunoglobulins at 1:500 dilution (Vector

Laboratories, Inc., Burlingame, CA) followed by avidin-

biotin peroxidase complexes (1:25; Vector Laboratories,

Inc.) for 30 minutes. Diaminobenzidine was used as the

chromogen and hematoxylin as the nuclear counter-

stain.

Immunoreactivity was classified as a continuum

data (undetectable levels or 0% to homogeneous

staining or 100%). Slides were reviewed by several

investigators (C.C.-C., A.H., M.J.U.), and results were

scored by estimating the percentage of tumor cells

showing characteristic staining. The cutoff value usedin this study was defined as follows: high proliferative

Ki-67 index if greater than 30% tumor nuclei stained

and low Ki-67 proliferative index if less than 30% tu-

mor nuclei stained.

Statistical Analysis

Associations between clinicopathologic parameters

and laboratory data were studied using Fisher exact

test or chi-square test where appropriate.29 Survival

analysis was performed by the method of Kaplan

Meier30 and statistical significance (P 0.05) evalu-

ated by log-rank testing.

31

RESULTSClinical and Pathologic Analyses

Median age of the patient cohort was 64 years (range,

2787 years). Twenty-seven (57%) of patients were

males, and 20 were females (43%). Samples were an-

alyzed from 47 primary tumors. All lesions were large

(10 cm), deep, high-grade extremity sarcomas that

were completely resected at the time of primary sur-

gery. Patients were grouped into two categories based

on outcome alone. The first group contained 19 pa-

tients that had no evidence of disease (NED) at last

follow-up. The second group included 28 patients that

died of disease (DOD). Patients with NED did not have

any recurrences. There were no significant differences

between the study groups in terms of age, gender,

primary presentation, site, histopathology, tumor size,

grade, and depth. Table 1 shows the comparison be-

tween NED and DOD patients according to clinical

and pathologic variables.

All patients underwent complete tumor resection

at time of primary surgery. Most (94%) of patients

underwent limb-sparing resection; amputation was

required in 3 (6%) cases on the basis of significant

bone or neurovascular involvement by tumor that pre-

cluded complete functional limb-preserving resection.

The proportion of patients receiving primary adjuvant

chemoradiation therapy was similar between the two

groups (Table 2).

Median follow-up for the entire group at time of

correlating laboratory results and clinicopathologic

data was 39 months (range, 7129 months). The 3-year

disease specific survival (DSS) rate of the entire cohort

was 46%. Actuarial 5-year DSS rate was 35%. Median

disease specific survival of patients in the NED group

TABLE 1Clinicopathologic Features of High-Risk Extremity Sarcoma (n 47)

with PValues Demonstrating No Significant Differences between theTwo Groups

Variable

NED

(n 19)

DOD

(n 28) Pvalue

Male gender 0.25

9 18

Age hrs, median 0.86

64 65

Presentation 0.78

Bx/Inc. Exc. 13 19

No prior Tx 6 9

Site (extremity) 0.89

Lower 16 24

Upper 3 4

Histopathology 0.65

MFH 9 16

LMS 3 4

Lipo 3 6

Fibro 4 2

Size ( 10 cm) N/A

19 28

High grade N/A

19 28

Deep depth N/A

19 28

Bx/Inc. Exc.: prior biopsy or incomplete excision; MFH: malignant fibrous histiocytoma; LMS: leiomy-

osarcoma; Lipo: liposarcoma; Fibro: fibrosarcoma.

TABLE 2

Treatment Characteristics of High-Risk Extremity Sarcoma (n

47),Demonstrating No Differences between the DOD and NED Group

Variable

NED

(n 19)

DOD

(n 28) Pvalue

Primary procedure 0.33

Resection 18 26

Amputation 1 2

Complete resection (yes) 19 28 N/A

Adjuvant radiotherapy (yes) 14 19 0.88

Adjuvant chemotherapy (yes) 7 11 0.87

N/A: not applicable.

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was 39.1 months and of patients in the DOD group

was 17.8 months.

Ki-67 Proliferative Index and Survival in High-Risk Soft

Tissue Sarcomas

Immunohistochemical staining showed high Ki-67

proliferative index in 4 tumors (21%) of patients that

are free of disease (NED, n 19) as compared with 14

tumors (50%) of patients that died of disease (DOD, n

28). Low or no Ki-67 activity was observed in 15

sarcomas (79%) in the NED group and in 14 sarcomas

(50%) in the DOD group. Most negative cases ex-

pressed Ki-67 below the cutoff value of 30%, and only

2 cases were truly negative, 1 in the DOD and 1 in the

NED group. These two cases most likely expressed

some low level Ki-67 ( 30%) that was not detected

here because of the use of tissue microarrays.17 A

representative immunophenotype of a sarcoma from

the DOD patient group showing high nuclear expres-

sion of the Ki-67 antigen is shown in Figure 1. Patients

who suffered tumor-related deaths had a significantly

higher proportion of tumor cell immunostaining for

Ki-67 (50% DOD vs. 21% NED) than those who remain

NED (P 0.04).

Analysis of DSS revealed high Ki-67 proliferative

index to be a significant predictor of tumor-related

mortality by univariate analysis (P 0.02). The 3-year

DSS rate for patients with high Ki-67 proliferative in-

dex and low or no Ki-67 expression was 18% and 58%,

respectively (Fig. 2).

DISCUSSIONDespite various efforts to improve treatment for hu-

man STSs, a subset of patients with this disease re-

mains at high risk for tumor-related mortality. The

main reason for this is the heterogeneity of STS as

reflected by variable biologic behavior. Clinical prog-

nostic factors such as histologic grade, size, depth, and

status of surgical resection margins have been identi-

fied to better define the risk of patients to die of

disease and have been included in the AJCC classifi-

cation of STS.14 However, within the high-risk group

of patients with high-grade, large, deep sarcomas of

the extremity that are completely resected clinically

unpredictable differences in terms of patient outcome

exist.

Of the molecular markers that were analyzed with

regard to their potential to predict outcome of sar-

coma patients, the nuclear proliferation antigen Ki-67

has been suggested by several studies to be a promis-

ing candidate.5,8,1012,3234 Initial reports on 34 and 46

cases showed a correlation between Ki-67 index, mi-

totic count, cellularity, and histologic grade of STS,

suggesting it to be useful for grading and prognosti-

cation in this disease.10,11 In two studies from our

institution on larger series of patients with STS that

FIGURE 1. Representative photomicrograph of the immunophenotypes

of a high-risk soft tissue sarcoma showing high Ki-67 proliferative index

(quarter core from tissue array depicted in 400 magnification).

FIGURE 2. KaplanMeier curve for disease specific survival of patients withhigh-risk soft tissue sarcoma by Ki-67 proliferative index. High Ki-67 prolifer-

ative index was significantly associated (P 0.02) with patients who had died

of disease as compared with patients who have no evidence of disease at last

follow-up.

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were not matched with regard to clinical high-risk

parameters (n 121 and 174), the prognostic impli-

cations of Ki-67 proliferative index were investigated.

This showed a correlation between high Ki-67 prolif-

erative index, occurrence of distant metastasis, and

tumor-related mortality.5,8

This observation was con-firmed by another report on 65 patients with STS

showing the association of high Ki-67 proliferative

index with poor overall survival.12 In these studies,

Ki-67 proliferative index was defined as the percentage

of tumor cell nuclei being positive for Ki-67 in relation

to a cutoff value of 2040%.

To further characterize the clinical potential of

Ki-67 as a molecular marker for disease specific sur-

vival in patients with high-risk STS, we selected a

group of patients that remained free of disease (NED,

n 19) after primary tumor treatment and compared

the Ki-67 proliferative indices in these tumors withthose of patients who subsequently died of their dis-

ease (DOD, n 28).

The relatively small sample size of the two study

groups (n 47), attributable to the few long-term

disease free survivors with high-risk STS, is offset by

the elimination of clinically relevant prognostic factors

from the analysis, because both groups were matched

for these critical clinicopathologic variables (Table 1).

This allows for outcome analysis on the basis of Ki-67

proliferative index alone. This analysis was based on

the tissue microarray technique for efficient evalua-

tion of immunophenotypes in cancer tissues.

27

Wepreviously have validated this technique for its use in

STS. This showed 9698% concordance between trip-

licate core tissue array and full section immunohisto-

chemistry (IHC) analyses and showed no significant

differences between survival analyses based on array-

derived data compared with regular full section IHC-

derived data.17

Expression of the Ki-67 antigen in tumor nuclei in

our cohort of 47 high-risk STS showed high Ki-67

proliferative index to be a significantly more frequent

event in the DOD patient group. Clinicopathologic

analyses revealed a significant correlation between

DSS and high Ki-67 proliferative index (P 0.02) (Fig.

2). The cutoff value in our study was 30% as suggested

by the previous reports.5,8,12 This confirms the de-

scribed relevance of Ki-67 for prediction of patient

prognosis in STS and adds the important notion that

high Ki-67 proliferative index allows for identification

of patients with an especially aggressive tumor phe-

notype who cannot be identified based on well estab-

lished clinical parameters alone.

This suggests that high Ki-67 proliferative index

may allow us to select patients with high-risk sarco-

mas for systemic adjuvant therapies and, more impor-

tantly, to avoid overtreatment of patients with less

aggressive tumors within the high-risk patient group,

reflected by low Ki-67 proliferative index, and spare

them from the side effects of aggressive, often not

beneficial, adjuvant therapy.

In summary, this study further validates that highKi-67 proliferative index is an independent prognostic

marker for high-risk soft tissue sarcomas that identi-

fies patients with an especially aggressive tumor phe-

notype who cannot be identified based on well estab-

lished clinical parameters alone. On the basis of our

data and previous reports, high Ki-67 proliferative in-

dex may have potential for predicting the prognosis of

patients with high-risk STS in a clinical routine setting

and may be a useful addition to AJCC staging criteria

for STS. This needs to be prospectively confirmed on

the basis of clinical trials.

REFERENCES1. Lewis JJ, Leung DHY, Casper ES, Woodruff J, Hajdu SI,

Brennan MF. Multifactorial analysis of long-term follow-up(more than 5 years) of primary extremity sarcoma. Arch Surg1999;134:1904.

2. Brennan MF, Lewis JJ. Soft tissue sarcomas. In: Sabiston DC,editor. Textbook of surgerythe biological basis of modernsurgical practice. 15th ed. New York: Saunders, 1996:528.

3. Levine EA. Prognostic factors in soft tissue sarcoma. SeminSurg Oncol 1999;17:2332.

4. Soft tissue sarcoma. AJCC cancer staging manual. 5th ed.Philadelphia: Lippincott, Williams & Wilkins, 1998:1406.

5. Drobnjak M, Latres E, Pollack D, Karpeh M, Dudas M, Woo-druff JM, et al. Prognostic implications of p53 nuclear over-expression and high proliferation index of Ki-67 in adult soft

tissue sarcomas. J Natl Cancer Inst 1994;86:54954.6. Cordon-Cardo C, Latres E, Drobnjak M, Olivia MR, Pollack

D, Woodruff JM, et al. Molecular abnormalities of mdm2and p53 genes in adult soft tissue sarcoma. Cancer Res

1994;54:7949.7. Kim SH, Lewis JJ, Brennan MF, Woodruff JM, Dudas M,

Cordon-Cardo C. Overexpression of cyclin D1 is associatedwith poor prognosis in extremity soft tissue sarcomas. ClinCancer Res 1998;4:237782.

8. Heslin MJ, Cordon-Cardo C, Lewis JJ, Woodruff JM, BrennanMF. Ki-67 detected by MIB-1 predicts distant metastasis and

tumor mortality in primary, high grade extremity soft tissue

sarcoma. Cancer 1998;83:4907.9. Cance WG, Brennan MF, Dudas ME, Huang CM, Cordon-

Crado C. Altered expression of the retinoblastoma geneproduct in human sarcomas. N Engl J Med 1990;323:145762.

10. Kroese MC, Rutgers DH, Wils IS, Van Unnik JA, Roholl PJ.The relevance of the DNA index and proliferation rate in thegrading of benign and malignant soft tissue tumors. Cancer1990;65:17828.

11. Ueda T, Aozasa K, Tsujimoto M, Ohsawa M, Uchida A, Aoki

Y, et al. Prognostic significance of Ki-67 reactivity in softtissue sarcomas. Cancer 1989;63:1607-11.

12. Levine EA, Holzmayer T, Bacus S, Mechetner E, Mera R,Bollinger C, et al. Evaluation of newer prognostic markersfor adult soft tissue sarcoma. J Clin Oncol 1997;15:324957.

Ki-67 Proliferative Index in High-Risk Sarcoma/Hoos et al. 873

7/31/2019 Cancer-2001-Hoos-869-74

6/6

13. Gerdes J, Schwab U, Lemke H, Stein H. Production of amouse monoclonal antibody reactive with a human nuclearantigen associated with cell proliferation. Int J Cancer1983;31:1320.

14. Gerdes J, Lemke H, Baisch H, Wacker H-H, Schwab U, Stein

H. Cell cycle analysis of a cell proliferation-associated hu-

man nuclear antigen defined by the monoclonal antibodyKi-67. J Immunol 1984;133:17105.

15. Scholzen T, Gerdes J. The Ki-67 protein: from the knownand the unknown. J Cell Physiol 2000;182:31122.

16. Endl E, Gerdes J. Posttranslational modifications of theKi-67 protein coincide with two major checkpoints duringmitosis. J Cell Physiol 2000;182:37180.

17. Hoos A, Urist MJ,Stojadinovic A, Mastorides S, Dudas ME, KuoD, et al. Validation of tissue microarrays for immunohisto-chemical profiling of cancer specimens using the example of

human fibroblastic tumors. Am J Pathol 2001;158:124551.18. Molino A, Micciolo R, Turazza M, Bonetti F, Piubello Q,

Bonetti A, et al. Ki-67 immunostaining in 322 primary breastcancers: associations with clinical and pathological vari-ables and prognosis. Int J Cancer 1997;74:4337.

19. Chowdhury GM, Kojima K, KanayamaH, Tsuji M, KurokawaY, Kagawa S. The proliferation index of MIB-1 as a prognos-tic factor for patients with transitional cell carcinoma of theupper urinary tract. Cancer 1996;78:82733.

20. Kros JM, Hop WCJ, Godschalk JJCJ, Krishnadath KK. Prog-

nostic value of the proliferation-related antigen Ki-67 inoligodendrogliomas. Cancer 1996;78:110713.

21. Borre M, Bentzen SM, Nerstrom B, Overgaard J. Tumor cellproliferation and survival in patients with prostate cancerfollowed expectantly. J Urol 1998;159:160914.

22. Bubendorf L, Sauter G, Moch H, Schmid HG, Gasser TC,Jordan P, et al. Ki-67 labelling index: an independent pre-dictor of progression in prostate cancer treated by radicalprostatectomy. J Pathol 1996;178:43741.

23. Jansen RL, Hupperets PS, Arends JW, Joosten-Achjanie SR,Vollovics A, Schouten HC, et al. MIB-1 labelling index is an

independent prognostic marker in primary breast cancer.Br J Cancer 1998;78:4605.

24. Jensen V, Ladekarl M, Holm-Nielsen P, Melsen F, Soerensen

FB. The prognostic value of oncogenic antigen 519 (OA-519)

expression and proliferative activity detected by antibody

MIB-1 in node-negative breast cancer. J Pathol 1995;176:

34352.

25. Hajdu SI, Shiu MH, Brennan MF. The role of the pathologist

in the management of soft tissue sarcomas. World J Surg1988;12:32631

26. Wunder JS, Healey JH, Davis AM, Brennan MF. A compari-

son of staging systems for localized extremity soft tissue

sarcoma. Cancer 2000;88:272130

27. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml

P, Leighton S, et al. Tissue microarrays for high-throughput

molecular profiling of tumor specimens. Nat Med 1998;14:

8447.

28. Cordon-Cardo C, Richon VM. Expression of the retinoblas-

toma protein is regulated in normal human tissues. Am J

Pathol 1994;144:50010.

29. Mehta C, Patel N. A network algorithm for performing Fish-

ers exact test in rXc contingency tables. J Am Stat Assoc

1983;78:42734.30. Kaplan E, Meier P. Nonparametric estimation from incom-

plete observations. J Am Stat 1958;53:45781.

31. Peto R, Peto J. Asymptotically efficient rank invariant pro-

cedures. J R Stat Soc A 1972;135:185207.

32. Hoos A, Lewis JJ, Antonescu CR, Dudas ME, Leon L, Woo-

druff JM, et al. Characterization of molecular abnormalities

in human fibroblastic neoplasmsa model for genotype-

phenotype association in soft tissue tumors. Cancer Res

2001;61:31715.

33. Hasegawa T, Yokoyama R, Lee YH, Shimoda T, Beppu Y,

Hirohashi S. Prognostic relevance of a histological grading

system using MIB-1 for adult soft tissue sarcoma. Oncology

2000;58:6674.

34. Choong PF, Akerman M, Willen H, Andersson C, GustafsonP, Baldetorp B, et al. Prognostic value of Ki-67 expression in

182 soft tissue sarcomas. Proliferationa marker of metas-

tasis? APMIS 1994;102:91524.

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