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7
Prognostic Implication of Proliferative Markers MIB-1
and PClO in Esophageal Squamous Cell Carcinoma
King-Yin Lam, F.R.c.P.A.’
Simon Ying-Kit Law, F.R.c.s.’
Mike Ka-Pui So, B.SC?
Manson Fok, F.R.c.s.’
Lily Tung Ma, F.R.c.P.A.’
John Wong, F.R.C.S?
’
Department of Pathology, University of Hong
Kong.
Department of Surgery, Queen Mary Hospital,
Hong Kong.
Supported by a grant from the University of
Hong Kong (CRCGiresearch grant 33ti/046/
0065).
The authors would like to thank Mr K.H. Yeung
for his technical assistance.
Address for reprints: Dr. K.Y. Lam, Department
of Pathology, Queen Mary Hospital, Hong IKong.
Received July 3, ‘1995; revision received August
29, 1995; accepted September 22, 1995.
c 1996 American Cancer Society
BACKGROUND. Proliferative markers are related to tumor behavior. The commonly
used markers are proliferating cell nuclear antigen (PCNA) and Ki-67. The aim of
this study is to evaluate the usefulness of MIB-1 (for Ki-67) and PClO (for PCNA)
in the assessment of the clinicopathologic features and prognosis in patients with
esophageal squamous cell carcinoma.
METHODS. One hundred patients (88 males, 12 females; mean age, 63 years [range,
39 to 83 years]) with surgically resected esophageal squamous cell carcinoma (32
well differentiated, 51 moderately differentiated, and 17 poorly differentiated) were
studied. The clinicopathologic features and survival data of these patients were
noted. Representative tissue was collected from each tumor and immunohistochemical preparations for MIB-1 and PCl0 were made.
RESULTS. The percentages of cells that tested positive for PClO and MIB-1 were
much higher in tumor cells than in nonneoplastic cells. The pattern of expression
of both markers varied with the differentiation of the tumor. The results observed
with MIB-1 staining were better than those with PCIO; because MIB-1 had less
background staining, as well as stronger and more uniform positive signals compared with PC10. Thus, further investigation was performed on MIB- 1-stained
sections. The tumor cell MIB-1 scores ranged from 169 to 964 positive cells per
1000 cells (mean 598 ? 21 1; median, 636). Although it was significantly associated
with the differentiation of the tumor ( P = 0.0001), the score had no significant
relationship to the tumor size, location, or stage, or to the patients’ age and sex.
The prognosis depended on the size and stage of the lesion. In Stage I11 lesions
(n = 83), patients with MIB-1 scores below 300 had longer actual survival rates
than those with a score of 300 or above. However, the survival rates of patients in
the latter group were better if the greatest dimension of the tumor diameter was
7.5 cm or less.
CONCLUSIONS. Proliferative activity in esophageal squamous cell carcinoma, as
defined by the MIB-1 immunohistochemical method, is significantly related to
tumor differentiation. It is also potentially valuable as a prognostic marker in
addition to its use in tumor staging and size. Cancer 1996;77:7-13.
0 I996 American Cancer Socieiy.
KEYWORDS: squamous cell carcinoma, proliferating cell nuclear antigen, MIB-1, Ki67, prognosis, differentiation.
P
roliferative activity of the tumor is a useful parameter in understanding tumor behavior. A correlation between the proliferative activity
and overall prognosis has been observed in many cancers.‘ Thus, immunohistochemical determination of cell proliferation-associated antigens
has aroused the interest of pathologists in recent years. The commonly
used proliferation-associated markers include antibodies to Ki-67 and
proliferating cell nuclear antigen (PCNA).
The prognostic value of Ki-67 immunostaining in various tumors has
8
CANCER January 1,1996 / Volume 77 / Number 1
been well documented., The most widely used Ki-67
monoclonal antibody was discovered in 1983 by Gerdes
and colleagues3 and the gene for the Ki-67 has been located on chromosome
This antibody recognizes a
labile epitope on a nuclear antigen that is exclusively expressed in proliferating cells, (i.e., in the G, , S, G, phases)
and mitosis, but not in Go. The drawback in the use of
this antibody is that its application is restricted to fresh
material because the epitope does not survive routine
histopathologic fixation in formaldehyde. New antibodies
MIBMIB-1,2, 3, 5, and IND.64 have been
1 is now commercially available and can be applied to
formalin fixed and routinely processed tissues. However,
it needs microwave irradiation for retrieval of the antigenicity." The immunostaining pattern, as well as the morphologic and cell cycle distribution of MIB-1 expression,
are identical to those of Ki-67, so it can be regarded as a
true Ki-67 equivalent.'
Antibodies to PCNA also have been widely studied
proliferative markers in recent years because they can be
detected in formalin fixed tissues. PCNA is a 36KDa acidic
nuclear protein that functions as an auxiliary protein for
DNA polymerase-delta and has been recognized as an
endogenous histologic marker for G, IS-phase in the cell
cycle.' The most widely used antibody to PCNA is PC10.
Esophageal cancer is the sixth most frequent cause of
cancer death worldwide and ranks ninth in world cancer
incidence.'" This type of cancer is a major cause of death
in certain areas of Asia, including Hong Kong. Various
studies have attempted to find markers that act as useful
adjuncts to morphologic and clinical features to predict
the behavior of esophageal tumors.'','2 In this present
study, we examine the immunolocalization and prognostic value of MIB-1 and PCNA in 100 cases of esophageal
squamous cell carcinomas. The clinicopathologic information and survival data of all these patients are available
for assessment. To the best of our knowledge, the value
of MIB-1 has not been reported in human esophageal
malignancies.
MATERIALS AND METHODS
Data Collection
Chinese patients in Queen Mary Hospital with surgically
resected primary esophageal squamous cell carcinomas
between January 1989 and December 1993 were studied.
Tissues from the resected specimens of these patients
were prospectively collected in a fresh state and then
fixed in 10% formalin for less than 48 hours.
Macroscopically, the site and size (maximum length)
of the tumors were recorded. The histologic sections of
these primary esophageal carcinomas were reviewed and
classified using the World Health Organization (WHO)
criteria.'' Only squamous cell carcinomas were included
in the study. A total of 100 patients (88 males and 12
females) was included in the present study. The cancers
were staged according to TNM clas~ification.'~
All patients were followed regularly until death and their demographic data reviewed. The actuarial survival rate of the
patients was measured from the date of resection of the
esophageal cancer to the date of death or April 30, 1995.
Staining for Proliferative Markers
A representative block from each patient was chosen for
the study. The block used was the one in which the most
dominant histologic pattern of the carcinoma was identified (because a few carcinomas showed slight variation
in tumor differentiation in different foci of the tumor).
From the block, 5 p-thick sections were cut and mounted
on gelatin-coated glass slides and dried overnight in an
oven at 37°C. The sections were dewaxed in xylene, rehydrated through graded concentration of alcohol, and then
treated with 0.5% hydrogen peroxide at room temperature for 10 minutes to block endogenous peroxidase activity. They were washed and rinsed with Tris-buffered saline at a pH of 7.6. After pretreatment with 0.5% trypsincalcium chloride (pH = 7.8), the sections were immersed
in 10 mM sodium citrate (pH = 6) in a thermoresistant
plastic box and were processed in a microwave oven (700
W) for 9 minutes at 95"%C. Ten percent normal sheep
serum was then added at room temperature for 10 minutes. The monoclonal antibodies, MIB-1 (150, Immunotech SA, Cedex, France) and PClO (1:20, Dako, Glostrup,
Denmark) were applied and incubations performed overnight at 4°C. Immunohistochemistry was performed using the avidin-biotin complex technique with diaminobenzidine development and Mayer's hemalum counterstaining. The sections were washed in water, dehydrated
in alcohol, cleared in xylene, and mounted. Negative controls were sections treated the same as above but with
omission of the primary antibodies.
Assessment of the Proliferative Markers
The sections were first scanned at low ( X 40) and medium
power ( X 200) for all the fields with tumor and nontumor
esophageal tissues, respectively, to account for the heterogeneity of distribution. The number of cells showing
positive nuclear staining and the pattern of staining were
recorded. Necrotic tissues, stromal cells, and lymphoid
cells were not included in the recording. The distribution
of the staining of MIB-1 and PClO in the tumor and the
adjacent nonneoplastic epithelium in each case were assessed semiquantitatively and compared. The staining
was divided into categories according to the percentage
of the cells stained (0-25%, 26-50%, 51-75%, and 76100%). The sections stained with MIB-1 were then
counted at X 400 magnification. At least 1000 nuclei were
counted in each section. The MIB-1 score was presented
as number of positive nuclei per 1000 nuclei counted.
Proliferative Markers MIB-1 and PClO in Esophageal Cancerllarn et al.
FIGURE 1. Positive staining in the nuclei of the basal and parabasal
cells of the nonneoplastic esophageal epithelium (MIB-1, x130).
9
FIGURE 2. Positive staining in the nuclei of the tumor cells in the periphery of the cell nests of a moderately differentiated squamous cell carcinoma. (MIB-1, x130).
RESULTS
Clinicopathological Features
FIGURE 3. The random positive staining in nuclei of the tumour cells
in a poorly-differentiated squamous cell carcinoma. (MIB-1, x130).
Reproducibility was determined by counting replicates
and recounting of the same case to avoid intraobserver
variation. All the counting procedures were performed by
one of the authors (Lam) in order to eliminate interobservers variations.
Statistical Analysis
All data were computerized and statistical tests were performed with the aid of the SAS statistical package. The
test used for continuous data was correlation analysis.
The Kruskal-Wallis test was used for categorical data.
The significance of various parameters on survival were
analyzed by the multivariate Cox regression. Survival
curve was plotted using the Kaplan-Meier rnethod.l5 The
tests were considered significant when their overall P values were less than 0.05.
The mean age of the 100 patients was 63 years (range, 39
to 83; standard deviation, 9). The esophageal cancers are
composed of 32% well differentiated, 51% moderately differentiated, and 17% poorly differentiated squamous cell
carcinomas. Ten percent of the tumors were in the upper
esophagus, 56% in the middle esophagus, and 34% in the
lower esophagus. The lengths of the carcinomas ranged
from 1 cm to 12 cm (mean, 5 cm). Six percent of the
patients had preoperative chemotherapy. The majority
(83%) of these patients had Stage 111 lesions (T3, N1, MO
or T4, any N, MO). The other patients had Stage I1 (13%
Stage IIA [T2/T3, NO, MO], 2% Stage IIB [Tl/T2, N1, Moll,
and Stage I V lesions (2%) (any T, any N, M l ) . Dysplasia
or carcinoma was present in either the upper or lower
resection margin in 14% of the cases.
Staining for Proliferative Markers
PClO and MIB-1 immunostaining were confined to the
nuclei in both neoplastic and nonneoplastic cells. The
mitotic figures were particularly immunoreactive and
were highlighted by these antibodies. In all nonpathologic esophageal epithelia, only the nuclei of the basal
and parabasal cells were positive for the markers (Fig. 1).
There was a heterogeneity of PClO and MIB-1 staining in
the esophageal cancers. In well and moderately differentiated squamous cell carcinomas, positive PClO or MIB1 staining was noted in the less differentiated cells in the
periphery of the tumor cell nests. The central keratinizing
areas and adjacent tumor cells were negative for the
markers (Fig. 2). Conversely, the staining was more diffuse in poorly differentiated carcinomas (Fig. 3).
In all instances, the percentages of positive cells for
either markers in nonneoplastic epithelia (< 10%) were
10
CANCER January 1,1996 I Volume 77 I Number 1
TABLE 1
Correlation of Tumor MIB-1 Score with Clinical and Pathological
Parameters in Esophageal Squamous Cell Carcinoma
Parameter
Finding
No. of
cases
Mean ? standard deviation/
median MIB-1 score
Sex
Male
Female
Upper
Middle
Lower
Well
Moderate
Poor
I1 A
I1 B
III
IV
88
12
10
56
34
32
51
li
13
2
83
2
596 5 2171799
608 i I711668
590 ? 1781590
624 5 2191674
558 ? 2051570
464 t 1961462
623 ? 1861673
776 t 1461819
577 i 2211654
741 i 91741
599 i 2131619
567 ? 2601567
Site
Differentiation
Stage
LOO
80
P value
0.91
0.25
6
>
>
.d
0.0001
0.84
40
20
0
0
less than those in the cancer cells. The staining pattern
and percentage of positive tumor cells was similar in each
case using either PClO or MIB-1 by semiquantitative
method. However, the staining in MIB-1 was easier to
interpret because it had less background staining and
more uniform and stronger positive signals when compared with PCIO. Thus, we performed the scoring based
on MIB- 1 positivity.
MIB-1 Staining and Clinicopathologic Parameters
The tumor MIB-1 scores of the whole group of patients
ranged from 169 to 964 (mean, 598 z 211; median, 636).
The scores had no significant relationship to the age of
the patients and the size of the tumors ( P = 0.64 and 0.67,
respectively, by correlation analysis). The relationship of
other clinicopathologic features to the scores is noted in
Table 1. The MIB-1 score was not related to the sex of
the patients, the location of the tumors, or the stage of the
lesions ( P = 0.91, 0.25, and 0.84, respectively, by KruskalWallis test). Conversely, the score was strongly correlated
with the differentiation of the squamous cell carcinomas
( P = 0.0001 by Kruskal-Wallis test). The median score in
the poorly differentiated squamous cell carcinomas was
much higher than the well differentiated squamous cell
carcinomas. By statistical manipulation, in order to transform the score to a variable that is normally distributed,
a value x could be calculated from the MIB- 1 scores (x =
sin^' square root of [MIB-1 score11000]). The tumors
could be classified as well differentiated squamous cell
carcinomas if they had an X value greater than or equal
to 0.9, whereas poorly differentiated squamous cell carcinomas had an x value of less than 0.9.
Survival Analysis
The mean follow-up period of the whole group was 16.5
months. Eighty-five percent of the patients had already
10
20
30
40
50
60
70
months
FIGURE 4. The survival rate of patients with Stage I1 and Stage Ill
esophageal squarnous cell carcinoma.
died by the time of completion of the study. The median
survival of the whole group was 11 months. The survival
was not related to age, sex, site, tumor differentiation, or
MIB-1 score ( P = 0.49, 0.84, 0.99, and 0.77, respectively,
by the Cox regression). Conversely, survival of patients
was related to the size ( P = 0.003) and stage ( P = 0.022)
of the lesions (Fig. 4).
In order to evaluate the relationship between MIB-1
scores and survival rates within a given stage, the tumors
in Stage 111 (n = 85) were stratified into those with scores
greater than 300 and those with scores less than or equal
to 300. Patients whose tumors had a MIB-1 score of less
than 300 (n = 11) had a median survival of 15 months,
whereas those above that score ( n = 72) had a median
survival of 9.7 months. The former group had a longer
median survival, although the results were marginally insignificant ( P = 0.068 by the Cox regression analysis). We
found the cutoff points for MIB-1 scores to start from
300, with stepwise increments of 50 to 750 in each of the
2 groups containing at least 10 patients. The value of 300
gave the largest value of the likelihood ratio statistics,
thus defining the 2 most distinguishable groups (Fig. 5).
The survival rates of patients with Stage I11 lesions
and an MIB-1 score greater than 300 could be further
stratified according to the size of the tumors. The cutoff
points were derived from the same statistical method described above. The starting point was 2.5 cm, with increments of 0.5 cm. It was observed that patients with a
maximum tumor diameter of less than or equal to 7.5 cm
had a better survival than those with a maximum tumor
Proliferative Markers MIB-1 and PClO in Esophageal CancerlLam et al.
11
100
......
MIBl s c o r e > 300
MIBl score
300
60
N
v
01
4
60
0
d
0
.a
2
40
a
v)
P
20
2o
0 0
10
20
30
40
50
60
70
months
FIGURE 5. The relationship of the MIB-1 score and the survival rate in
patients with Stage 111 esophageal squamous cell carcinomas.
diameter of greater than 7.5 cm. The former group had
a medium survival of 11.6 months, whereas the latter
group had a medium survival of 5 months ( P = 0.016 by
the Cox regression). The impact of MIB-1 score and size
on the survival rate is summarized in Figure 6. Conversely, the MIB-1 scores in Stage 111 lesions could not be
further stratified into groups if they were first stratified
into 2 groups according to the size of the tumors. Thus,
the size of the tumors was more important than the MIB1 scores in determining the behavior of the tumors in
Stage 111 esophageal squamous cell carcinomas.
For patients with Stage I1 and Stage IV lesions, the
numbers were too few to be stratified into groups for
proper statistical analysis. In addition, the results were
not significant in these patients even if we relaxed the
criteria of having at least 10 patients in each group for
the analysis. In addition, it was noted that in patients
with either Stage I1 or Stage 111 lesions, the survival was
not significantly related to the differentiation of the tumors ( P = 0.42 for Stage 2; P = 0.27 for Stage 111).
DISCUSSION
Antibodies to PCNA have been widely used in recent years
to study the kinetics of various human lesions.' The expression of PCNA, as identified by PC10, has been found
to be correlated with other markers like mitotic counts,
flow cytometric analysis, nucleolar organizer region-associated proteins (AgNORs), transforming growth factor,
epidermal growth factor receptor, and bromodeoxyuridine (BrdU), as well as Ki-67.'"'* However, when com-
0
10
20
30
40
50
60
70
months
FIGURE 6. The relationship of the MIB-1 score and tumor size on the
survival rate of patients with Stage Ill esophageal squamous cell carcinomas.
pared with MIB-1, PClO suffers from certain disadvantages: (1)the long half-life of PCNA makes staining persist
in noncycling cells; (2) the staining may be positive in
DNA repair in addition to DNA synthesis; and (3) the
staining varies with antibody dilution and lacks a clear
plateau.' In the present study, we observed that staining
in MIB- 1 was easier to interpret because it had less background staining and stronger and more uniform positive
signals when compared with PC10. In addition, consistent with the findings of other authors,' the staining pattern and percentage of positivity of PClO and MIB-1 analyzed by semiquantitative method were similar. Thus, we
agree with the current thinking that MIB-1 is a more robust marker than PClO and use the MIB-1 score for asse~sment.',*,~
The limitations of clinical studies using proliferative
markers include heterogeneity in proliferation within tumors and variability in factors such as case selections,
number of cells counted, size of samples, fixation time,
etc.' We employed strict criteria in this study in order to
provide high quality data for interpretation. All the samples in the study were prospectively collected so the tissue
fixation time and the number of blocks taken were standardized and the clinical/survival data were all adequately collected. The number of patients involved in the
study was also large so as to allow more representative
statistical testing results. In addition, the choice of representative block for staining, choice of representative fields
for counting, counting of at least 1000 cells in each sec-
12
CANCER January 1,1996 / Volume 77 / Number 1
tion, and repetition of counting increased the chances of
reproducibility of the MIB-1 scores and minimized the
heterogeneity in proliferation within the tumors.
Reports of proliferative activities of esophageal squamous cell carcinomas using Ki-67 on cryostat sections
are relatively r a ~ e . ' ~ -The
' ~ number of cases reported in
these studies are usually limited. The largest group of
patients was presented by Porschen and colleague^.'^
They reported in their study of 33 German patients a
mean Ki-67 labelling index (using cryostat sections) of
35.1% (ranging from 14.2 to 64.1). The authors could find
no other data on the use of MIB-1 in esophageal squamous cell carcinomas in the English literature. In this
study of 100 patients, all tumors were positive for MIB1. The mean MIB-1 score was high (598 2 211, range, 169
to 964). In concurrence with the results of Jaskiewicz and
De G r ~ o t , 'Ki-67
~
expression was noted in the normal
epithelium, which could signify an early step of malignant
transformation. It is worth noting that the MIB-1 scores
in the nonneoplastic epithelia were much lower than in
the tumor tissue in all the cases under study.
This study showed no statistically significant relationship between MIB- 1score and clinicopathologic variables such as age, sex, tumor location, tumor size, and
TNM stage. The results were consistent with the previous
studies using Ki-67 (using cryostat sections).23,24 Conversely, the location of the positive staining in the periphery of tumor cell nests was consistent with the reports by
most other authors."^" This study also demonstrated a
highly significant correlation between the MIB-1 score
and the differentiation of the tumors. The results suggested that the score could be used as an objective measurement for grading of the squamous cell carcinoma.
Another important finding was that tumor stage and
size of esophageal cancers were important parameters
in assessing the prognosis of patients with esophageal
squamous cell carcinoma. Most of the patients with
esophageal squamous cell carcinomas in Hong Kong had
Stage I11 lesions. The tumors in this group of patients
could be stratified into two groups, those with MIB-1
scores of greater than 300 and those with scores less than
or equal to 300, in the current study by statistical evaluation. It was noted that patients in the former group had
a poorer prognosis than patients in the latter group (median survival of 9.7 months vs. 15 months). In addition,
the former group could also be further subdivided into
those with a tumor diameter of greater than 7.5 cm and
those with a tumor diameter of less than or equal to 7.5
cm, with the former group having a poor prognosis (median survival of 11.6 months vs. 5.3 months). Thus, the
MIB-1 score may be a useful adjunct in assessing tumor
behavior, but the usefulness of the scores was not as important as the stage and size of the tumors in this study.
In conclusion, our results indicate that cellular prolif-
eration in esophageal squamous cell carcinomas, as defined by MIB- 1 immunohistochemical study, correlate
with cellular differentiation and, in addition to the tumor
size, may be a good indicator in predicting the prognosis
in Stage 111 cancers.
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