[CANCER RESEARCH 50, 4382-4387, July 15, 1990)
In Situ Evaluation of Growth Fraction Determined by Monoclonal Antibody Ki-67
and Ploidy in Surgically Resected Non-Small Cell Lung Cancers'
JoëlleSimony,2 Jean-Louis Pujol, MichèleRadal, Elisabeth Ursule, François-Bernard Michel, and Henri Pujol
Centre Régionalde Lutte contre le Cancer, Rue de la Croix Verte, 34094 Montpellier Cedex [J. S., M. R., E. U., H. P.], and Service des Maladies Respiratoires, Hôpital
l'Aiguelongue, Rue du Major Flandre, 34059 Montpellier Cedex [J-L. P., F-B. M.], France
ABSTRACT
Ploidy and growth fraction were analyzed by means of a computerassisted image processor in surgically resected non-small cell lung cancer
(NSCLC). This study was done in order (a) to evaluate the distribution
of anti-Ki-67 immunostaining and (A) to correlate this distribution to
ploidy status and pTNM stage of NSCLC. Thirty-two patients underwent
a surgical resection for primary NSCLC following complete staging.
Indirect immunoperoxidase reactions of monoclonal antibody Ki-67 were
done on frozen tissue sections. Integrated optical density and index of
stained nuclear surface were calculated by means of a computer-assisted
image processor in 120 fields of each preparation in order to quantify the
Ki-67 immunostaining. DNA content was determined by means of cytometry of Feulgen-stained cytological prints. The ploidy status was defined
for each tumor by DNA index, percentage of hypodiploid cells, and type
of DNA content histogram (near diploid, hyperdiploid, hypodiploid, and
multiplied ). Reproducibility of immunostaining quantitative analysis was
demonstrated by iterative measurements of the same slide. Intratumoral
heterogeneity of Ki-67 immunostaining induced integrated optical density
variation assessed on six nonconsecutive tissue sections from at least two
regions of the same tumor. This intratumoral variability was 15 times
lower than integrated optical density variability between tumors. The Ki67 immunostaining varied significantly according to the DNA content
histogram type ( P < 0.05, Kruskal-Wallis test); most of the specimens
with high Ki-67 immunostaining were multiploid or hypodiploid. More
over, Ki-67 immunostaining correlated to the percentage of hypodiploid
cells. Ki-67 immunostaining and ploidy status did not vary significantly
according to the tumor-nodes-metastasis stage. We conclude that (a)
quantitative analysis of Ki-67 immunostaining is a reliable evaluation of
growth fraction in NSCLC if a large number of fields are analyzed to
take into account intratumoral variability, (b) hypodiploidy and multiploidy are frequent abnormalities of DNA content, (c) Ki-67 immunostaining
is significantly higher in hypodiploid and multiploid tumors. Thus, deter
mination of growth fraction and ploidy in surgically resected NSCLC
specimens may be considered as complementary prognostic parameters
independent of the stage of the disease.
INTRODUCTION
Complete surgical resection is an efficient treatment for
limited stage I and II NSCLC3 (1,2). For stage III A NSCLC
the existence of mediastinal lymph nodes (N2) is predictive of
early relapses (3, 4). Chemotherapy and/or radiation therapy
have been proposed as adjuvant therapy of N2 NSCLC (5, 6).
However, randomized trials fail to demonstrate a real survival
benefit for patients following adjuvant therapy. Thus, reliable
and reproducible prognostic variables are needed to determine
patient subsets likely to require combined modality treatments.
Ploidy status predicts disease-free intervals and short term
survival of numerous solid tumors (7-9) including lung cancers
(10, 11). Static computer-assisted cytometry is a useful tool for
Received 10/30/89; revised 2/28/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study has been supported in part by grants from the Centre National de
la Recherche Scientifique and the Ligue Nationale Françaisecontre le Cancer.
*To whom requests for reprints should be addressed.
3 The abbreviations used are: NSCLC, non-small cell lung cancer; DI, DNA
index; IOD. integrated Optical density; PBS. phosphate buffered saline; TNM,
tumor-nodes-metastases: pTNM. pathologic tumor-node-metastases.
ploidy analysis of Feulgen-stained cytological prints of tumor
specimens (12). On the other hand, kinetic parameters used in
order to determine the growth fraction give reliable information
on stem cells and, indirectly, on effectiveness of chemotherapy
and radiation therapy (13). The Ki-67 nuclear antigen is ex
pressed throughout the cell cycle (14). Immunostaining with
monoclonal Ki-67 provides a reliable means of in situ evaluation
of the tumor growth fraction in many malignant diseases in
cluding non-Hodgkin's lymphomas (15), breast cancer ( 16), and
lung cancer (17, 18). In particular, a great variability in NSCLC
proliferative rate has been demonstrated using semiquantitative
analyses of Ki-67 immunostaining (18). Moreover, Ki-67 im
munostaining has been proposed to estimate the cell growth
fraction in surgically resected neoplastic human tissues (19).
Thus, it may be of interest to evaluate ploidy and growth
fraction in surgically resected NSCLC as complementary prog
nostic variables.
In this study a computer-assisted image processor was used
(a) to evaluate the distribution of anti-Ki-67 immunostaining
and (b) to correlate this distribution to ploidy status and pTNM
stage of NSCLC.
MATERIALS
AND METHODS
Patients
Thirty-two patients [28 men, 4 women, ages 41-74 years, 59 ±10
(SD) years] underwent a surgical resection for primary NSCLC. All
lung tumors were analyzed according to the latest WHO classification
(20) by light microscopy following hematoxylin-eosin staining of sur
gical specimens. Among them were 28 squamous cell carcinomas (15
well differentiated and 13 poorly differentiated, 3 adenocarcinomas,
and 1 adenosquamous carcinoma. Staging of NSCLC was performed
according to Ed. 4 of the TNM classification (21) and included clinical
examination, performance status assessment, chest tomodensitometry,
fiberoptic bronchoscopy, liver and adrenal gland tomodensitometry,
and bone scanning. Definite pTNM stage included data obtained from
complete mediastinal lymphadenectomy. This evaluation disclosed 17
patients with stage I disease, 6 patients with stage II, 8 patients with
stage IMA. and 1 patient with stage IV (Table 1).
Immunohistochcmistry
Tissue Preparation. The immunostaining was done on 5-^m-thick
frozen tissue sections. Sequential sections were obtained by means of
an automatic cryostat (Cryocut-Bright; Shandon, United Kingdom).
When the size of the tumor sample was sufficiently large (25 of 32
tumors), tissue preparations were performed every 5 sections in at least
2 different regions of the specimen and a total of 6 tissue preparations
were analyzed for each tumor. In other cases (7 of 32 tumors) 5
sequential tissue preparations were analyzed for each tumor.
Antisera Source. Antibody Ki-67 (Dakopatts, Glostrup, Denmark) is
a mouse IgGl monoclonal antibody raised against human proliferative
cells. The Ki-67 related antigen is expressed in G,A, GÃŽB,
S, G2, and M
cells but is absent in G0 cells (14).
Immunohistochemical Reactions. Frozen tissue sections were fixed
with cold acetone. Indirect immunoperoxidase tests were carried out
using the biotin-streptavidin-peroxidase system (Amersham, Les I lis,
France) following the three-stage procedure (22). Stage 1 sections, after
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GROWTH FRACTION AND PLOIDY OF NSCLC
Table 1 Clinical and pathological variables
StageIIIMIAIVpTNMT,
DI which represents the ratio of the cell DNA content of tumor G0/i
cells to the diploid G0/i peak (2c). Thus, DI = 1 defined near diploid
of
SQC/Ad°7/18/11/14/—V—4/
patients89242421Histology
tumors.
DNA Histogram Analysis. The DNA histograms were classified into
four types (Fig. 1): type 1, tumor G0/i cells in the near diploid region
(2c, DI = 1), with few G2M tumor cells in the tetraploid region (4c);
type 2, hyperdiploid tumor GO/I cells (DI a 1.2); type 3, hypodiploid
tumor GO/Icells (DI s 0.8), with a hypodiploid peak containing at least
20% of cells; type 4, evidence of multiploid tumor cells with multiple
aneuploid peaks, some of them in the octaploid region (8c). The
percentage of cells in the hypodiploid modal DNA (percentage of
hypodiploid cells, DI < 0.8) was calculated for each histogram.
M„T2
No
MoT,
No
M»T2
N,
M»T3
N,
M»T2
N,
M»Tj
N2
M»Tj
N2
—ÕI—I/—
N»M,No.
°SQC. squamous cell carcinoma; Ad, adenocarcinoma; M,, lung métastases
Study Design
discovered in pneumonectomy specimen.
rehydration in PBS and inhibition of endogen peroxidase, were incu
bated with 10 Mg/ml of purified monoclonal antibody for 40 min at
room temperature; in stage 2, sections were then washed in PBS and
incubated with sheep biotinylated anti-mouse IgG diluted 1:50 in PBS
for 30 min at room temperature; in stage 3, sections were finally
incubated with streptavidin-biotinylated
horseradish peroxidase at
1:200 for 30 min at room temperature. Immunohistochemical reaction
was then revealed in the dark with 3-amino-9-ethylcarbazole (Merck,
Nogent/Marne, France) using hydrogen peroxide as a substrate and
counterstained with hematoxylin. Positive and negative controls were
performed for each reaction.
Computer-assisted Image Analysis. The immunostaining quantitative
analysis was done by means of a computer-assisted image processor
(12) (Systèmed'Analyse MicrophotométriqueÃBalayage Automatique;
TITN, Grenoble, France). This microcomputer-based system is config
ured with a standard microscope (Polyvar; Richert Jung, Cambridge,
United Kingdom), a color video camera (Sony Corporation, Japan), an
image analysis processor (TSBC, TITN), and a 80286 computer (V286;
Victor, Sweden). A program developed to analyze the immunostaining
tissue sections was used (23) (Estrogen Receptor Immunocytochemical
Assay; TITN). This program quantitates intensity and distribution of
immunostaining in hematoxylin-counterstained tissue sections. Acqui
sitions by the color image processor were done through blue and red
filters. Ki-67 immunostaining was analyzed as a false red color whereas
counterstained cells were analyzed as a false blue color. For each
preparation, optical density thresholds were determined using real
microscopies image of the analyzed field as reference. Measurements
of Ki-67 immunostaining were done at x25. Fields analyzed were
randomly selected by movements of an automatic motorized plate which
scanned tumor tissue preparations on two perpendicular axes. Twenty
fields were analyzed for each section by the image analysis processor.
According to the surface of tumor tissue preparation, these 20 fields
represented from 30 to 60% of the whole section. Index of stained
nuclear surface and IOD were expressed in arbitrary units (A. U.).
Controls of immunostaining quantitative analysis reproducibility were
carried out by (a) comparison of iterative measurements done on the
same preparation and (b) comparison of measurements done on five
sequential sections of the same specimen. Evaluation of intratumoral
variation of Ki-67 immunostaining quantitative analysis was carried
out in 25 of 32 tumors by comparing the IOD measurement of 20 fields
taken from each 6 nonconsecutive sections from each tumor.
All patients had a complete staging. A surgically resected specimen
from a nonnecrotizing area of the tumor was deep frozen and cytological
prints were done. Neither chemotherapy nor radiotherapy were carried
out prior to surgical resection. Evaluation of growth fraction and cell
DNA content were done independently and without knowledge of the
pTNM stage.
Statistical analyses were done by means of nonparametric tests.
Differences between two groups were determined by means of the
Mann-Whitney U test; differences between more than two groups were
determined by means of the Kruskal-Wallis test; P < 0.05 was consid
ered as significant; Spearman rank-order correlation coefficients were
calculated. Yates correction of x2 was used for comparison between
pTNM stage and DNA content histogram type frequency distribution.
RESULTS
Ki-67 Immunostaining
Qualitative Analysis. Ki-67 immunostaining was exclusively
positive in cell nuclei. The distribution of the staining involved
nucleolus, nuclear membrane, and nucleoplasm with a great
variability (Fig. 2). The percentage of cells with nuclear Ki-67
positive varied from less than 2% to 20% (Table 2).
Quantitative Analysis. Quantitative analysis showed no sig
nificant signal in 13 tumor specimens with less than 2% Ki-67positive cells. For the remaining 19 tumor specimens, index of
stained nuclear surface ranged from 3 to 739 A. U. and the
80
60
40
30
20
10
zc
40
so
Cell DNA Content Analysis
Tissue Preparation. A cytological print from each specimen was airdried. Slides were fixed in formaldehyde-alcohol for 10 min and then
washed three times in alcohol and stained by the pararosaniline Feulgen-Schiff technique (24).
Computer-assisted Cytometry. The stoichiometric reaction was ana
lyzed using the computer-assisted image processor (Systèmed'Analyse
Microphotométrique à Balayage Automatique; TITN). For each speci
men, cell DNA content analysis was carried out on 300 randomized
malignant cells. The nuclear DNA values were computerized in order
to produce histograms. The cell DNA content was expressed in c-units
with 2c representing the mean value of normal diploid control cells
(normal hepatic tissue). Ploidy was determined for each specimen using
10
Fig. 1. DNA content histograms were classifed into four types: a, type 1,
tumor GO/Icells in the near diploid region (DI = 1); A, type 2, tumor G0/i cells in
a single hyperdiploid peak (DI = 1.6) and few G2M tumors cells in the 6c region;
c, type 3. hypodiploid tumor G(,/i cell peak (DI = 0.8); d, type 4. evidence of
multiple aneuploid tumor peaks, with DI = 1.8 for the main aneuploid peak.
4383
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GROWTH FRACTION AND PLOIDY OF NSCLC
Fig. 2. Indirect immunoperoxidase reactiv
ity of monoclonal antibody Ki-67 on poorly
differentiated squamous cell carcinoma tissue
section. Some cells demonstrate variable nu
clear staining involving nuclcoplasm and/or
nuclear matrix, x 560.
*V
800-r
Table 2 Semiquantitative analyses of immunoperoxidase reaction Ki-67 on
NSCLC tissue sections
Range of Ki-67positive cells (%)
No. of tumors
0-2
13
3-5
7
6-10
6
10-20
6
Table 3 Variations of integrated optical density measurements (a) between
iterative measurements of the same tissue section, (b) between sequential tissue
sections of the same tumor, (c) between 6 nonconsecutive tissue sections of the
same tumor, and (d) between tissue sections of different tumors.
IODMean
(A.U.)
SD
SD10cva4,525
153
1,547
3,800
9,111
2,393 x IO3 14,440 X IO3 83,010 x IO3
23,409
0.03b4,215 0.36c5,721
0.66d5,785
1.57
0 SD2, variance; CV, coefficient of variation.
IOD ranged from 121 to 36,716 A. U. [5,785 ±9,111 (SD) A.
U.]. Reproducibility of immunostaining quantitative analysis
method was demonstrated by iterative measurements of the
same slide (Table 3). Comparison of index of stained nuclear
surface versus IOD showed a linear correlation (r = 0.98, P <
0.0001; Fig. 3) demonstrating the reliability of quantitative
evaluation of Ki-67 immunostaining.
Intratumoral IOD variability was evaluated in 25 tumors by
analyzing 6 nonconsecutive tissue preparations. These addi
tional measurements were done in all except two tumors which
had more than 2% of Ki-67-positive cells and in 8 tumors which
had less than 2% Ki-67-positive cells. For tumor specimens
with less than 2% Ki-67-positive cells, the quantitative analysis
showed no significant signal in all analyzed tumor tissue prep
arations. For other tumors the procedure disclosed an intratumoral variation of IOD measurement done on 20 fields from
each section. The magnitude of this heterogeneity varied from
one tumor to the other with a higherlower IOD ratio ranging
from 1.5 to 5 (Table 4). However, this intratumoral variation
was 15 times lower than the variation between tumors with a
highenlower mean IOD ratio of 75. Thus, mean IOD of Ki-67
immunostaining was used to classify tumors.
10000
20000
30000
40000
INTEGRATED OPTICAL DENSITY
(ARBITRARY UNITS)
Fig. 3. Quantitative analyses of Ki-67 immunostaining. Correlation of inte
grated optical density to index of stained nuclear surface.
Table 4 Intratumoral variation of integrated optical density measured on six
nonconsecutive sections; samples taken from at least two different regions from 12
NSCLC
Integrated optical density (A.LJ.)
Case123456789101112HistologySQC"SQCSQCSQCSQCSQCSQCAdSQCSQCSQCSQCRange1
°SQC, squamous cell carcinoma; Ad. adenocarcinoma.
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GROWTH FRACTION AND PLOIDY OF NSCLC
taking into account intratumoral variation of Ki-67 nuclear
antigen expression; (¿>)
hypodiploidy and multiploidy are fre
According to DI, the DNA content histograms were classified
quent abnormalities of DNA content; and (c) Ki-67 immuno
as follows: type 1, 9 cases; type 2, 5 cases; type 3, 9 cases; type staining is significantly higher in hypodiploid and multiploid
4, 9 cases. The frequency distribution of tumors by DI is shown
tumors and correlates to the percentage of hypodiploid cells.
in Fig. 4; for multiploid tumors, DI of major aneuploid peak
The study of cell kinetics has been proposed as a reliable
was charted. The percentage of hypodiploid cells ranged from
prognostic parameter in human neoplasms (7, 8, 25). Aneuplo0 to 72%.
idy can predict short term survival in many solid tumors (9,
26-29) including lung cancer (10, 11, 30, 31). DNA content
Correlations
studies done by means of flow cytometry analysis of single-cell
Quantitative Analysis of Ki-67 Immunostaining and Ploidy. suspension take into account thousands of cells. Static cytom
The Ki-67 immunostaining varied significantly according to etry as used in this study only analyzes a few hundred cells.
cell DNA histogram type. The mean IOD increased from type However, studies carried out in order to compare the two
1 to type 4 NSCLC specimens (P < 0.05, Kruskal-Wallis test; methods demonstrated the reliability of ploidy analysis using
static cytometry (7). DI is used to determine aneuploidy and to
Fig. 5).
A relation of Ki-67 immunostaining to the percentage of define classes of histograms. Multiploid DNA content charac
hypodiploid cells was found for the 19 NSCLC with >2% Ki- terizes tumors with more than one aneuploid subpopulation
67-positive cells. A correlation was demonstrated by quantita
(7). For multiple myeloma, it has been suggested that multiploid
tive comparison of the percentage of hypodiploid cells versus and hypodiploid DNA contents characterize a highly chemoindex of stained nuclear surface (rs = 0.47, P < 0.04; Fig. 6<z) resistent subset of the disease (32). Flow cytometry determina
tion of S, Go. i, and M phase gives an evaluation of growth
and comparison of percentage of hypodiploid cells versus IOD
(rs = 0.49, P < 0.03; Fig. 60).
fraction. However, it is very difficult to determine S phase by
pTNM Status and Kinetic Parameters. The Ki-67 immuno
means of static cytometry owing to the frequent overlap between
the aneuploid tumor and diploid nonmalignant cell populations
staining did not significantly vary according to the TNM stage.
However, mean IOD observed for T3 tumor specimens was (33). Moreover it is not possible to differentiate malignant Gì
cells from G0 cells. Thus the determination of growth fraction
lower than mean IOD of TI and T2 tumor specimens, although
this difference did not reach a significant level (respectively:
needs other means to identify the proliferative cell fraction.
157 ±352, 2111 ±2795, and 5881 ±10263 A. U.; P < 0.1,
The determination of tumor growth fraction may be a rele
Kruskal-Wallis test). The frequency distribution of the DNA
vant prognostic parameter and it gives information about stem
content histogram type did not vary significantly according to cells. Because many works demonstrate the importance of stem
pTNM staging (x2 1.54, d.f. 6, not significant; Table 5) or nodal
cell destruction as the first aim of chemotherapy and radiation
status (x2 1.79, d.f. 6, not significant; Table 6).
therapy (13), cell kinetic determination may be considered as
an important evaluation of surgically resected NSCLC. S phase
determination using labeled index (34) or 5-bromodeoxyuridine
DISCUSSION
(35) need, respectively, tritiated thymidine pulse or incubation
In this study, determination of growth fraction and ploidy in of single tumor cell suspension to incorporate labeled DNA
NSCLC demonstrates that: (a) quantitative analysis of Ki-67 metabolites. 5-Bromodeoxyuridine evaluation of S phase gives
important insight into growth fraction in humans (36). Other
immunostaining is a reliable evaluation of growth fraction when
methods have been developed to evaluate the cycling cells in
a large number of fields are included in the measurements,
the Gì,S, G2, and M phases of the cell cycle (18). Monoclonal
antibody Ki-67 has been developed against a nuclear antigen
expressed
throughout the cell cycle (14). Some reserves have
9-em
recently been made about whether or not the Ki-67 antigen is
expressed by some G0 cells (37). However, Ki-67 immunostain
ing provides an opportunity to analyze the proliferative cell
7-1V"3
fraction in frozen preserved tumor specimens (16). Quantitative
analysis of Ki-67 immunostaining has been done previously in
6-<a(X«-,
I1
breast cancer and various nonmalignant breast diseases (38).
These authors found no significant intratumoral variation, ana
B-oQJ
II
lyzing multiple preparations from the same specimen (38).
In our study we used a computer-assisted image analysis
I1
system to quantify the Ki-67 immunostaining. The reproduciDD
bility of quantitative analysis was demonstrated by (a) linear
IIII0,2
DD
correlation of two parameters of measurement (IOD and index
of stained nuclear surface) and (b) reproducibility of iterative
DOB
measurements of the same preparation. In order to detect
intratumoral variation we studied nonconsecutive sections from
B•
R
B
ll1.2
U
at least two regions of 25 of 32 tumors. This procedure dem
onstrated heterogeneity of Ki-67 immunostaining, a result in
accordance with previous studies suggesting that phenotypic
0.4 0.6 O.B 1•D2,8DNA
1,4 1.6 1.8 2 2.2 2.4 2,6
heterogeneity is a main feature of NSCLC (39-41). To take
index
into account this heterogeneity in Ki-67 immunostaining quanFig. 4. Frequency distribution of tumors by their DNA index. For multiploid
titation, the mean IOD measured on 120 fields was calculated
tumors (D), the DNA index of the main aneuploid peak is charted. •nearly
diploid and (nonmultiploid) aneuploid tumors.
for each preparation. We found that intratumoral variation of
DNA Content Analysis
*'1
3"tÃ-2-1-•II1
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GROWTH
FRACTION
40000
AND
PLOIDY
OF
NSCLC
T
-30000
^QtfT(-p>=£°P < 0.05
-25000
-1
<0^LÜ20000
Fig. 5. Relation of quantitative analysis of
Ki-67 immunostaining (IOD) to DNA content
histogram types (statistical analysis by means
of Kruskal-Wallis test).
-10000-5000-05000
mo""LU1-35000
s:
f
4
DNA CONTENT
HISTOGRAM
TYPES
Rs - 0.47
p <0.04
Rs - 0.49
p <O.OJ
U
È
^
Õ
Q(/)
Fig. 6. Relation of quantitative analysis of
Ki-67 immunostaining to the percentage of
hypodiploid cells, a. comparison of index of
stained nuclear surface versus percentage of
hypodiploid cells; b. comparison of integrated
optical density versus percentage of hypodip
loid cells (Spearman rank-order correlation).
f—
O15
o<
Qt
UJ Q3
(J
X
LJ
Q
•%
0
20
40
60
% OF HYPODIPLOID
Table 5 Distribution of DNA content histogram types according to pTNM stage"
Stage
Typel
Type 2
Type 3
1-79 (Yates correction); not significant.
Table 6 Frequency distribution of DNA content histogram types according to
nodal status"
16
232TypeS61
43
'N,NjType
2
1Type
" xj 1.54 (Yates correction); not significant.
20
40
% OF HYPODIPLOID
80
BO
CELLS
Clinical evaluation of NSCLC ploidy status disclosed a prog
nostic significance of aneuploidy (10, 11). However, aneuploidy
includes different ploidy abnormalities. In our study we found
a significant increase of the Ki-67 antigen expression from
single aneuploid tumors to multiploid tumors. It may be sug
gested that a high growth fraction with numerous cells in the
cell cycle increases the rate of ploidy abnormalities. Thus it
may be of interest to analyze the predictive prognostic value of
each subset of ploidy abnormalities. Growth fraction evaluated
by quantitative Ki-67 immunostaining and ploidy may be con
sidered as complementary prognostic parameters. Determina
tion of each factor in surgically resected NSCLC specimens is
possible and may be a useful tool for prognostic determination
and adjuvant therapy.
Type 4
I
II
IIIAandIV
NodalstatusN»
BO
CELLS
0
33
2Type
Ki-67 iniin linosi :iin ing is 15 times lower than variation between
tumors. Thus, it may be possible to classify tumor specimens
according to their Ki-67 antigen expression. There was no
correlation with pTNM stage suggesting that the two parame
ters were independent of each other.
ACKNOWLEDGMENTS
The authors wish to thank Joan Baissus for helping to prepare the
manuscript.
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GROWTH FRACTION AND PLO1DY OF NSCLC
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research.
In Situ Evaluation of Growth Fraction Determined by
Monoclonal Antibody Ki-67 and Ploidy in Surgically Resected
Non-Small Cell Lung Cancers
Joëlle Simony, Jean-Louis Pujol, Michèle Radal, et al.
Cancer Res 1990;50:4382-4387.
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