Apoptosis in leiomyosarcoma

2-a2-b2-c2-dFigs. 2a and b. TUNEL for apoptotic cells. Positive for the tumor cell nuclei at a varying degree. a: AI = 5.1;
third-recurrent tumor, 63/F. b: AI = 0.8; poorly-differentiated LMS, primary, 61/F.
Fig. 2c. Immunohistochemical stain of p53 protein. Strongly positive for the tumor cell nuclei. PI = 57,
poorly-differentiated LMS, 74/F.
Fg. 2d. Immunohistochemical stain of Ki-67. Positive for the tumor cell nuclei at a various degree; 21.5%,poorly-differentiated LMS, 74/F.

119

H. Yoshida et al.

Table 3. Comparative study on immunohistochemistry by the TUNEL method between 3
primary tumors and their recurrent/metastatic tumors
Histologicalgrading

Numberofmitoses

AIPI

Cell proliferation
Ki-67

PCNA

MCM2

Primary tumor [3]Low
2– 9
0.4 ± 0.1 0.6 ± 0.5
0.5 ± 0.2
8.4 ± 2.4
3.8 ± 2.7
Recurrent/metaIntermediate- 11–34
2.0 ± 0.7 12.3 ± 3.1 12.8 ± 3.5 22.1 ± 9.6 18.1 ± 7.9
static tumor* [9]highMean ± SD.
[ ], number of specimens.
* Recurrent period: 2–7 years.
AI, apoptotic index; MCM2, minimichrosome 2; PCNA, proliferating cell nuclear antigen; PI, p53 index;
TUNEL, terminal deoxynuleotidyl transferase-mediated dUTP-biotin nick end labeling.

higher (mean AI: 2.0 ± 0.7) in comparison withthe primary site (0.4 ± 0.1). p53-positive cells
increased significantly in cases of the recurrent/
metastatic tumors (mean PI: 12.3 ± 3.1) than inthose of the primary tumors (0.6 ± 0.5) (P < 0.05)
in the same way.

0.18 of PI, whereas 12 high-malignant LMSs
were statistically higher at a rate of 1.1 ± 0.9
and 23.9 ± 15.0, respectively (P < 0.05). Threekinds of proliferative markers of Ki-67, PCNA
and MCM2 proteins were examined to assess
the proliferating activity of the tumor cells divided into 3 categories of malignancy. The nuclear positivities of Ki-67, PCNA and MCM2
increased statistically in the high-grade malignant LMSs at a rate of 16.7 ± 6.8 in Ki-67, 33.4
± 6.8 in PCNA and 22.1 ± 10.7 in MCM2, morethan those of the low-grade ones, respectively(2.0 ± 0.6 in Ki-67, 6.8 ± 1.2 in PCNA and 3.2 ±0.8 in MCM2).

Expression of apoptotic cells between
p53-positive and -negative LMSs
Low-grade malignant LMS showed a rate of PI
under 5 as shown in Table 2, and thus we figured that p53-positive tumors were rated over 5
PI. The p53-positive LMSs were found in 12 of
29 tumors as shown in Table 4 and revealed theAI at a mean of 2.2 ± 1.6, statistically higher
than that (0.4 ± 0.1) of the p53-negative LMSs
(P < 0.05). Histologically, the p53-positive
group included 8 (66.7%) of 12 high-grade and4 (50%) of 8 intermediate malignancies, whereas 9 (100%) low-grade and 4 (50%) of 8 intermediate malignant LMSs were included in the p53negative group. The recurrence/metastasis-freeRelationship between TUNEL indicesand p53 expression
TUNEL and p53 positivities were examinedcomparatively in primary LMSs (3 cases) andtheir recurrent/metastatic tumors (9 tumors) as
shown in Table 3. TUNEL indices (AIs) of therecurrent/metastatic tumors were statistically

Table 4. AI between p53-negative and positive cases in 3 histological groups
PINumberofspecimens

p53-negative<517
p53-positive5≤
12Mean ± SD.
[ ], number of recurrent/metastatic cases.
AI, apoptotic index; PI, p53 index.

AI

0.4 ± 0.1
1.5 ± 0.8

120

Histological grading
Low

Intermediate

High

9 [1]
04 [2]4 [3]

48 [3]

Apoptosis in leiomyosarcoma

and bind to DNA strands for replication ofDNA under the existence of DNA polymerase.
The positivity for anti-MCM2 antibody ranged
between those of Ki-67 and PCNA.
We observed p53 positivity in 12 (41.4%) of29 LMSs, of which histological grading was
high-malignant in 8 and intermediate malignant
Discussion
in 4. In the current study, p53-immunostaining
was positive at a rate of 20 to 64% in various
In this study, we evaluated the frequency of types of soft-tissue sarcomas (Yoo et al, 1997;
apoptotic cell death in an increasing malignant Stefanou et al, 1998) and 21.6 to 43% of LMS
potential of LMS based on mitotic rate as well of soft-tissue origin (O’Reilly et al., 1997;
as expression of the proliferative markers of Ki- Konomoto et al., 1998). The mutated p53 gene
67/PCNA/MCM2 responsible of tumor growth. loses its inherent transcriptional activity, resultSecondly, the relationship between p53 over- ing in enhancement of tumor cell proliferation
expression and AIs was investigated to clarify (Endo et al., 1999). Yoo et al. (1997) suggested
the significance of apoptosis on tumor malig- the role of the p53 mutation as the pathogenesis
nancy and prognostic factor. Our results show- of soft-tissue sarcomas. Regarding the biologed a positive correlation between mitotic rate ical behavior or prognostic factor, however, theand increasing grade of histological malignancy overexpression of p53 protein was estimated ason the one hand and the proliferative markers of being diverse from the one suggesting a posiKi-67/PCNA/MCM2 on the other. These find- tive correlation between p53 protein and progings were exactly similar to those reported be- nosis (Drobijak et al., 1994; Toffoli et al., 1994;
fore by others (Drobijak et al., 1994; Yoo et al., Yoo et al., 1997) to the others that indicated no1997; Konomoto et al., 1998). MCM2 protein relation between p53 expression and biologicalis part of the MCM family which is composed behavior (O’Reilly et al., 1997; Stefanou et al.,of 6 isoforms of MCM2 to MCM7. They are 1998). We would like to emphasize here that
expressed in the G1/S phase of the cell cycle the overexpression of p53 protein could behighly valuable as a prognostic
factor taken from the findings of an(%)
increasing accumulation of p53
100protein with an intimate relation tothe grading of tumor malignancy.
p53 (–) [17]Detection and identification of
apoptotic cells was successfully carp53 (+) [12]ried out on the paraffin-embedded
50
sections by using the TUNEL method which was introduced and developed by Gavrieli et al. (1992) and
Degree of freedom = 1
Leoncini et al. (1993) and is based on
P = 0.047
detecting naturally occurring DNA
strand breaks. TUNEL-positive,48
96 (month)
apoptotic cells were easily recognized as the cells with nuclear conTime after diagnosis
densation, fragmentation separatedFig. 3. Recurrence/metastasis-free survival curves of patientsby a clear halo around the cells inwith p53-positive and -negative leiomyosarcoma (LMS). Patientswith p53-positive tumors were significantly worse than those with hematoxylin and eosin staining, in
p53-negative tumors (log-rank test: P = 0.047). [ ], number of addition to the number of normalspecimens.
looking cells as mentioned previ-Recurrence/metastasis-freesurvival curve

survival rate was compared between the p53positive and -negative groups (Fig. 3). The patients with p53-positive tumors was significantly worse than those with p53-negative tumors(log-rank test: P = 0.047).

121

H. Yoshida et al.

ously by others (Lowe et al., 1994; Aihara et al.,
1994; Arai and Kino, 1995).
We compared AI and PI with histologicalgrading resulting in a positive correlation of AI
to PI which increased statistically from lowgrade to high-grade malignancy. The AI value,furthermore, was statistically much higher in
p53-positive cases of high-grade malignant
LMS than in p53-negative cases. Thus, in casesof high-grade malignant LMS, especially recurrent/metastatic tumors, a more rapid growth oftumors caused by increased proliferative activity was probably due to a loss of regulation by
p53. Apoptosis is regulated and controlled inits expression by various oncogens and suppresser genes (Kerr and Hormon, 1994). p53
gene is one of the major regulators of apoptosis
which can easily allow apoptosis in association
with MDM2 by regulating the cell cycle in themanner of growth arrest at G1/S or G2/S phaseor apoptotic cell death, but the mutative p53
gene suppresses apoptosis by a loss of transcriptional activity to cyclin-dependent kinase(Aihara et al., 1994). Ikeda et al. (1998) demonstrated that naturally occurring apoptosis
appeared to be induced predominantly via ap53-independent pathway from the observation of early and advanced gastric carcinoma.
Kawauchi et al. (2000) suggested that apoptosis
in cases of synovial sarcoma may be thought tobe caused by a multiple apoptosis-regulating
mechanism other than p53 protein. Our findings of a positive correlation between an increased amount of apoptotic cells and p53 positivity, however, is most likely to indicate that
apoptotic status was induced via a p53-dependent pathway.
There has been a positive correlation reported between apoptotic cells and higher
malignancies of non-Hodgkin’s lymphomas(Leoncini et al., 1993), prostatic carcinomas(Aihara et al, 1994) and colorectal adenomas(Arai and Kino, 1995). Tatebe et al. (1996) also
reported that apoptosis and cell proliferation
were more frequent in metastatic foci than in
primary lesions of colorectal carcinoma andsuggested that apoptosis might reflect not only
cell loss, but proliferative activity. A recent
study on synovial sarcoma indicated that apo-ptosis may be an indicator of poor prognosis(Kawauchi et al., 2000). Our results support
this hypothesis from the comparative examination of primary tumors and their recurrent/
metastatic tumors. We used the materials of
LMS originating in deep soft-tissue except for
one from organ-specific LMS of the uterus andintestine which represented rather low mitotic
activity. In fact, it is suggested that the susceptibility of undergoing apoptosis may reflect thehistological differences between tumor types(Staunton et al., 1995). Therefore, the mechanism and role of apoptosis in LMS still remain
to be elucidated.

Acknowledgments: This study was presented at the33rd Annual Musculoskeletal Tumor Meeting of theJapanese Orthopaedic Association held on 14 July,
2000 in Kumamoto. The authors thank Mr. Hisayuki
Matsumoto and Miss Yuki Torigai, both of whomare students at the College of Medical Care Technology, Tottori University.

122

References
1 Aihara M, Truong LD, Dunn JK, Wheeler TM,
Scardino PT, Thompson TC. Frequency of apoptotic bodies positively correlates with Gleason
grade in prostatic Cancer. Hum Pathol 1994;
25:797–801.
2 Arai T, Kino I. Role of apoptosis in modulationof the growth of human colorectal tubular and villous adenomas. J Pathol 1995;176:37–44.
3 Enzinger FM, Weiss SW. Soft tissue tumors. 2nded. St Lous: CV Mosby, 1988. Chapter 15. Leiomyosarcoma; p. 402–421.
4 Chen J, Wu X, Lin J, Levine AJ. mdm-2 inhibits
the G1 arrest and apoptosis functions of the p53
tumor suppressor protein. Mol Cell Biol 1996;
16:2445–2452.
5 Clarke AR, Purdie CA, Harrison DJ, Morris RG,Bird CC, Hooper ML, et al. Thymocyte apoptosis induced by p53-dependent and independentpathways. Nature 1993;362:849–852.
6 Endo K, Sakatani T, Watanabe M, Yoshida H,Nannba E, Ito H. Wild-type p53 transfer resulted
in cell cycle arrest, but not apoptosis of newly
established human malignant fibrous histiocytoma cell line. Internat J Oncol 1999;15:935–942.
7 Drobijak M, Latres E, Pollack D, Karpeh M,
Dudas M, Woodruff JM, et al. Prognositic implications of p53 nuclear overexpression and high

Apoptosis in leiomyosarcoma

proliferation index of Ki-67 in adult soft-tissuesarcomas. J Natl Cancer Inst 1994;86:549–554.
8 Hashimoto H, Daimaru Y, Tsuneyoshi M, Enjoji
M. Leiomyosarcoma of the external soft tissues.
Cancer 1986;57:2077–2088.
9 Haupt Y, Barak Y, Oren M. Cell type-specificinhibition of p53-mediated apoptosis by mdm2.
EMBO J 1996;15:1596–1606.
10 Gagvrielli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via
specific labeling of nuclear DNA fragmentation.
J Cell Biol 1992;119:493–501.
11 Ikeda M, Shomori K, Endo K, Makino T, Matsuura
T, Ito H. Frequent occurrence of apoptosis is anearly event in the oncogenesis of human gastriccarcinoma. Virchows Arch 1998;432:43–47.
12 Kawauchi S, Fukuda T, Oda Y, Saito T, Takeshita
M, Yokoyama K, et al. Prognostic significance ofapoptosis in synovial sarcoma: correlation withclinicopathologic parameters, cell proliferative
activity, and expression of apoptosis-related proteins. Mod pathol 2000;13: 755–765.
13 Kasagi N, Gomyo Y, Shirai H, Tsujitani S, Ito H.
Apoptotic cell death in human gastric carcinoma:analysis by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling. Jpn JCancer Res 1994;85:939–945.
14 Konomoto T, Fukuda T, Hayashi K, Kumazawa
J, Tsuneyoshi M. Leiomyosarcoma in soft tissue:
examination of p53 status and cell proliferating
factors in different locations. Hum Pathol 1998;
29:74–81.
15 Kerr JFK, Wyllie AH, Currie AR. Apoptosis: abasic biological phenomenon with wide-ranging
implications in tissue kinetics. Br J Cancer 1972;
26:239–257.
16 Kerr JFR, Winterford CM, Hormon BV. Apoptosis: its significance in cancer and cancer therapy.
Cancer 1994;73:2013–2026.
17 Leoncini L, Del Vacchio MT, Megha T, Barbini
P, Galieni P, Pileri S, et al. Correlations between
apoptotic and proliferative indices in malignant
non-Hodgkin’s lymphomas. Am J Pathol 1993;
142:755–763.
18 Lowe SW, Jacks T, Housman DE, Ruley HE.
Abrogation of oncogene-associated apoptosis
allows transformation of p53-deficient cells.
Proc Natl Acad Sci USA 1994;91:2026–2030.
19 Nakanishi H, Ohsawa M, Naka N, Uchida, OchiT, Aozasa K. Immunohistochemical detection ofbcl-2 and p53 proteins and apoptosis in soft tissue
sarcoma: their correlations with prognosis. Oncology 1997;54:238–244.

20 Stefanou DG, Nonni AV, Agnantis NJ,
Athnassiadou SE, Briassoulis E, Pavlidis N. p53/
MDM-2 immunohistochemical expression correlated with proliferative activity in different
types of human sarcomas: a ten-year follow-up
study. Anticancer Res 1998;18:4673–4681.
21 O’Reilly PE, Raab SS, Niemann TH, Rodgers JR,
Robinson RA. p53, proliferating cell nuclear
antigen, and Ki-67 expression in extrauterine
leiomyosarcoma. Mod Pathol 1997;10:91–97.
22 Staunton MJ, Gaffney EF. Tumor type is a determinant of susceptability to apoptosis. Am J Clin
Pathol 1995;103:300–307.
23 Tatebe S, Ishida M, Kasagi N, Tsujitani S, Kaibara
N, Ito H. Apoptosis occurs more frequently inmetastatic foci than in primary lesions of humancolorectal carcinomas: analysis by terminaldeoxynucleotidyl-transferase-mediated dUTPbiotin nick end labeling. Int J Cancer 1996;65:173–177.
24 Toffoli G, Doglioni C, Cernigoi C, Frustaci S,Perin T, Canal B, et al: p53 over-expression inhuman soft tissue sarcomas: relation to biologicalaggressiveness. Ann Oncol 1994;5:167–172.
25 Umansky SR. The genetic program of cell death.
Hypothesis and some applications: transformation, carcinogenesis, aging. J Theor Biol 1982;
97:591–602.
26 Valenti MT, Azzarello G, Vinate O, Manconi R,Balducci E, Guidolin D, et al. Differentiation,proliferation and apoptosis levels in human leiomyoma and leiomyosarcoma. J Cancer Res ClinOncol 1998;124:93–10
27 Wyllie AH. Cell death: a new classification
separating apoptosis from necrosis. In: Bowen
ID, Lockshin RA, eds. Cell death in biology andpathology. London: Chapman and Hall; 1981. p.
9–34.
28 Yoo J, Lee HK, Kang CS, Park WS, Lee JY,Shim SI. p53 gene mutation and p53 protein expression in human soft tissue sarcomas. ArchPathol Lab Med 1997;121:395–399.
29 Yuki M, Yokota K, Okuyama S. Cell proliferation and cell death (apoptosis) as indices differentiating malignant from benign gastrointestinal
myogenic tumors. Nippon Shokakibyo Gakkai
Zasshi 1995;92:206–216 (in Japanese with English abstract).

123

Received April 12, 2001; accepted May 17, 2001
Corresponding author: Prof. Haruhiko Yoshida