cancer-2001-hoos-869-74
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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: [email protected]
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.
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