Yonago Acta medica 2001;44:115–123

Apoptotic Cell Death and p53 Expression in Leiomyosarcoma of SoftTissue Origin
Haruhiko Yoshida, Mari Watanabe*, Kohei Shomori*, Hisao Ito* and Takeshi
Minamizaki†
Department of Pathobiological Science and Technology, *First Department of Pathology and†Department of Orthopaedic Surgery, Tottori University Faculty of Medicine, Yonago 683-0826
Japan
Leiomyosarcoma (LMS) of soft-tissue origin was studied on the expression and thebiological significance of apoptosis in relation to p53 oncoprotein. Immunohistochemicalanalysis was performed initially on the paraffin-embbeded sections taken from 29
surgical tissues (20 cases) including 9 recurrent/metastatic tumors. The results are as
follows: A positive correlation was observed between the p53 indices (PIs) and theproliferative markers designated by Ki-67, PCNA and MCM2, both of which increased
significantly in the high-grade malignant LMS much more than in the low-grade one(P < 0.001). Apoptotic cells were detected by the TUNEL method and evaluated as theapoptotic index (AI). A high AI-level was shown in the high-grade malignant LMS,
especially in cases of the recurrent/metastatic sites in comparison with the tumors ofthe primary site (P < 0.05). The AI was statistically higher in the p53-positive cases ofhigh-grade malignant LMS than in the p53-negative cases of low-grade malignant LMS.
In conclusion, apoptotic activity paralleled the overexpression of p53 protein alongwith an increasing grade of malignancy and may be related intimately to the increased
malignant potential, especially to recurrence/metastasis.
Key words: apoptosis; immunohistochemistry; leiomyosarcoma; p53 protein

Leiomyosarcoma (LMS) is now known as oneof the most frequent malignant tumors occurring in the soft-tissue of adults. It has been divided into 3 categories according to location by
Enzinger and Weiss (1988), because of its clinical and biological behavior. Hashimoto et al.
(1986) emphasized a prognostic difference between superficial and deep forms of LMS, anadverse prognosis of the deep form as compared to the superficial form. Histologically, conventional LMS is classified currently into well,moderately and poorly/anaplastic differentiatedtypes depending on the degree of differentiation, but it is very difficult to evaluate the malignancy in predicting metastasis despite themultiple factors of size, cellularity, atypia andnecrosis which are known as prognostic factors.
Even at the level of mitotic activity, a serious
decision can hardly be made on the biologicalbehavior of LMS (Enzinger and Weiss, 1988).
Apoptosis is an active process of cell deathoccurring in both normal and neoplastic conditions and is characterized as the orderly participation of biological, morphological and molecular genetic events (Kerr et al., 1972, 1994;
Wyllie, 1981; Umansky, 1982). It is induced bya variety of agents including oncogenic proteins, mainly p53 protein, the bcl-2 gene family
and Fas/Fas legand among the process and regulation of apoptosis (Kerr et al., 1994). Recentstudies have disclosed 2 pathways for the expression of apoptosis (Clarke and Purdie, 1993;

Abbreviations: AI, apoptotic index; LMS, leiomyosarcoma; MCM2, minimichrosome 2; PCNA, proliferating cell nuclear antigen; PI, p53 index; TUNEL, terminal deoxynuleotidyl transferase-mediated dUTPbiotin nick end labeling115

H. Yoshida et al.

Endo et al., 1999): p53 gene-dependent (Hauptet al., 1966; Chen et al., 1996) and -independent
pathways, the latter of which can be induced by
growth factors, hormones, chemotherapeutic
agents and radiation (Clarke and Purdie, 1993;
Endo et al., 1999). In considering malignant
neoplasma, apoptosis may play an importantrole in the regulation of tumor growth inintimate relation to the p53 gene regulating thecell cycle in conjunction with cell proliferation.
The relationship between p53 gene status andapoptosis, however, has rarely been reported on
soft-tissue sarcoma (Staunton and Gaffney, 1995;
Yuki et al., 1995; Nakanishi et al., 1997; Valenti etal., 1998). We examined the relationship between
the p53 protein and apoptosis in 29 surgical
specimens from 20 cases of LMS of soft-tissue
origin which were classified histologically fromlow-grade to high-grade malignancies according
to the mitotic rate and proliferative cell markers.
In addition, we discussed the significance andevaluation of apoptotic activity as a biological andprognostic factor of LMS.

Materials and MethodsThis study was performed on 29 paraffinembedded tumor tissues from 20 cases of LMSof soft-tissue origin, all of which were removed
surgically. The histological diagnosis of thesecases were determined in addition to immunohistochemical findings of vimentin, alphasmooth muscle actin and HHF 35, and classified into 3 groups according to tumor cell differentiation from poorly to well differentiatedtypes. Mitotic activity is an another aid in evaluating tumor malignancy and thus, the numberof mitoses was counted in 10 high-power fields
selected randomly as a mitotic rate: under 10 as
low-grade, 10 to 20 as intermediate and over 20
as high-grade malignancies.
The clinical data for these cases were collected from medical records. Each case in thisstudy was analyzed according to the tumor
nodes metastases classification developed by
the Union Internationale Contra le Cancer toevaluate the relation of apoptotic activity in
LMS to their biological behavior.

Immunohistochemistry
An immunohistochemical study was performedby the streptavidin-biotin-complex method using the following monoclonal antibodies: antip53 nuclear protein (DO-7, 1:50, Dako, Glostrup,Denmark), anti-Ki-67 (MIB-1, 1;50, Immunotech,Marceille, France), anti-proliferating cell nuclear antigen (PCNA) (PC10, Nichirei, Tokyo,Japan) and anti-minimichrosome maintenance
2 protein (MCM2). MCM proteins are composed of 6 isoforms ranging from MCM2 toMCM7. They are expressed in the G1/S phaseof the cell cycle and bind to DNA strands for a
replication of DNA under the presence of DNApolymerase. Monoclonal antibody (2H10 clone)against MCM2 protein was produced by the immunization of oligopeptide of N-terminal fromMCM2 protein with 892 amino acids and wasreported as a proliferative marker available toexamine the grading of malignancy*. The deparaffinized sections were pretreated with citrate buffer (0.01 mol/L citric acid: pH 6.0) at92˚C for 30 min in a microwave oven (Azumaya
MI-77, Osaka, Japan) for all antibodies except
anti-PCNA antibody and then incubated at 4˚Covernight with the primary antibodies. The
positive cells were counted and evaluated as theratio of immunoreactive cells to the total number of 1,000 tumor cells.

TUNEL
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)
was employed for the detection of apoptotic
cells using an ApopTag Plus in situ apoptosis
detection kit (Oncor, Gaithersburg, MD) which
was followed by the method originally developed by Kerr et al. (1972). Briefly, the sections
were incubated with 20 µg/mL proteinase K(Behringer Mannheim/Yamanouchi, Tokyo)
for 15 min at 37˚C after blocking endogenous
peroxidase in methanol for 30 min at room temperature. Thereafter, the sections were layeredand incubated on terminal deoxynucleotidyltransferase with digoxigenin-11-dUTP in a
*Shomori K. Personal communication.

116

Apoptosis in leiomyosarcoma

moist chamber for 90 min at 37˚C. After
stopping the reaction, anti-digoxigeninperoxidase was applied to the reaction mixture
for 30 min at room temperature. The positivereaction was estimated with 3,3'-diaminobenzidine reaction at room temperature. The
positive cells were counted and evaluated as theratio of TUNEL-positive cells to a total numberof more than 1,000 tumor cells.

Table 1. Clinical data of the cases used inthis study
Age range (mean)*Sex ratio (male:female)
Primary site
Retroperitoneum and abdomen
Upper extremity
Lower extremity
Body
UterusRecurrence/metastasis*year.

35–76 (65.6)
7:13
111
41
1
5Results
The clinicopathologic data are shown in Table
1. The patients were comprised of 7 males and
13 females. The age distribution was from 35 to76 years old (mean age, 65.6 years). The primary sites of the tumors involved 12 from theretroperitoneum and abdomen, 4 from the lower
extremities, 1 from the upper extremity and 1
from the trunk in addition to 1 from the uterus.
Recurrent/metastatic cases were included in 4cases (10 specimens). Twenty nine specimensfrom 20 cases of LMS of soft-tissue origin were
histologically evaluated into 3 categories ofmalignancy based on tumor cell differentiationand the mitotic index: 9 low-grade, 8 intermediate and 12 highly malignant LMSs (Table2). The cases of low-grade malignancy appeared very clear in cell differentiation showing lessthan 10 mitotic figures per 10 high-power fields
selected randomly. The cases of intermediatemalignancy were well to moderate in cell differentiation associated with 10 to 20 mitotic
figures, and in high-grade malignancy, the tumor cells were poorly differentiated assuming

pleomorphic giant cells abundantly. The mitotic figures were easily encountered at a ratio ofover 20 per 10 high-power fields (Figs. 1a–d).

TUNEL labeling for apoptotic cells
Apoptotic cells were determined by the TUNEL
method and evaluated as the apoptotic index(AI). Apoptotic cells were distinguished ascells with condensed and fragmented nucleiseparated by a clear halo in hematoxylin andeosin stain (Fig. 1d). TUNEL-positivity wasobserved at a varying degree to the cell nuclei
with condensed and fragmented nuclei and occasionally to tumor cells which looked normal(Figs. 2a and b). Table 2 shows the AI, positivities of p53 and the proliferative markers (Ki67, PCNA and MCM2) of LMS categorized
into 3 groups according to the mitotic rate. The
immunohistochemical findings of p53 as wellas Ki-67, PCNA and MCM2 are shown in Figs.
2c and d. Nine low-grade malignant LMSs
showed 0.4 ± 0.2 of AI (mean ± SD) and 0.4 ±Table 2. Comparative studies on LMS categorized in 3 groups according to the mitotic rate
Malignancy

NumberCellofdifferspecimens entiation

AIPI

Cell proliferation
Ki-67

PCNA

MCM2

Low-grade (< 10)9Well
0.4 ± 0.2
0.4 ± 1.8 2.0 ± 0.6
6.8 ± 1.2 3.2 ± 0.8Intermediate (10–20)
8 Moderately 1.2 ± 0.5
4.6 ± 2.7 5.0 ± 3.2 16.9 ± 2.6 9.7 ± 2.1
High-grade (20 <)12
Poorly1.3 ± 0.9 23.7 ±15.0 16.7 ± 5.0 33.4 ± 6.8 22.1 ±10.7
Mean ± SD.
( ), mitotic rate/10 high-power field.
AI, apoptotic index; LMS, leiomyosarcoma; MCM2, minimichrosome 2; PCNA, proliferating cell nuclear antigen;PI, p53 index; TUNEL, terminal deoxynuleotidyl transferase-mediated dUTP-biotin nick end labeling.

117

H. Yoshida et al.

1-a1-b1-c1-dFig. 1. Histological findings of LMS. Hematoxylin and eosin stain, × 200.
a:
b:
c:
d:

Well-differentiated LMS; mitotic cells, 9/10 HPF, low-grade malignancy, 71/F.
Moderately-differentiated LMS; mitotic cells, 10/10 HPF, intermediate malignancy, 76/F.
Poorly-differentiated LMS; mitotic cells, 34/10 HPF, high-grade malignancy, 63/F.
Arrow indicates an apoptotic cell.

118