九州大学学術情報リポジトリKyushu University Institutional Repository

Leiomyosarcoma Versus Bizarre and CellularLeiomyomas of the Uterus : A Comparative StudyBased on the MIB-1 and Proliferating CellNuclear Antigen Indices, p53 Expression, DNAFlow Cytometry, and Muscle Specific Actins

尼田, 覚Graduate School of Medical Sciences, Kyushu University

https://doi.org/10.11501/3120485

出版情報：九州大学, 1996, 博士（医学）, 課程博士バージョン：権利関係：

[

International Jollrnal of Gynecological Pathology 14:134---142, Raven Press, Ltd .. New York © 1995 International Society of Gynecological Pathologist

Leiomyosarcoma Versus Bizarre and Cellular Leiomyomas

of the Uterus: A Comparative Study Based on the MIB-1 and Proliferating Cell Nuclear Antigen Indices, p53

Expression, DNA Flow Cytometry, and Muscle

Specific Actins

Satoshi Amada, M.D., Hitoo Nakano, M.D., and Masazumi Tsuneyoshi, M.D.

Summary: There is still controversy over the criteria for malignancy of smooth muscle tumors (SMTs) of the uterus. We examined 51 cellular SMTs using immunohistochemistry for MIB-1, proliferating cell nuclear antigen (PCNA), p53, HHF35, a-smooth muscle actin (SMA), and flow cytometry. Morpholog­ically, the 51 cases were classified into 24 leiomyosarcomas (LMS), two un­certain malignant potential, four bizarre leiomyomas, and 21 cellular leiomy­omas. The mean values of the MIB-1 and PCNA indices showed significant differences between LMS and benign SMTs. p53 cells were positive in eight of 241eiomyosarcomas, and 12 of 22 were aneuploid. HHF35 and a-SMA showed a diffuse positivity in almost all the benign SMTs. In contrast, I 0 of the 24 LMS were either focally positive or negative for SMA. Using a logistic regression model, at cut-off points of 3.6 on the MlB-1 index and 15.6 on the PCNA index, the LMS and the benign SMTs were classified with an overall accuracy of 92% and 82%, respectively. Moreover, by combining the MIB-1 index and a-SMA positivity, the cut-off point could be established at 0.492 on the probability scale with the highest overall accuracy of 96%. Regarding the prognosis of LMS, p53 positivity wa correlated with survival (p = 0.0357). A combination of the MIB-1 index and a-SMA was helpful in distinguishing between LMS and benign SMT. Moreover, p53 positivity was considered to be a good marker for predicting the prognosis of LMS. Key Words: Leiomyosarcoma-Ki-67-Proliferating cell nuclear antigen-p53-Fiow cytometry-Muscle-specific ac­tins.

Uterine smooth muscle tumors (SMTs) are easily diagnosed in the majority of cases. However, it is difficult to determine whether they are benign or malignant in orne cases because they often include overlapping cases of clinically benign and malignant

From the Second Department of Pathology (S.A., M.T.) and Department of Gynecology and Obstetrics (H.N.), Faculty of Medicine, Kyu hu University, Fukuoka, Japan.

Address correspondence and reprint requests to Prof. Masazumi Tsuneyoshi , Second Department of Pathology, Fac­ulty of Medicine, Kyushu Univcr ity, 3-1-1 Maidashi, Higa hi­ku, Fukuoka 812, Japan.

134

neoplasms with either aberrant or bland cytological

features ( l ,2). Although some investigators claimed to have established the criteria for malignancy in

the uterine SMTs containing morphological factors with nuclear atypia and mitotic counts (1-8), no ac­curate diagnostic criteria yet exists for malignancy. Recently, several reports disclosed correlation of the biological behavior with Ki-67 (9, 10), prolifer­

ating cell nuclear antigen (PCNA) (11-14), and the

expression of mutant p53 protein (15, 16) using an immunohistochemical assay. This study examines leiomyosarcomas and compare them with benign

SMTs (bizarre and cellular leiomyomas) in order to

LEIOMYOSARCOMA OF THE UTERUS 135

assess the correlation between their biological char­acteristics and histological findings using both im­munohistochemical techniques and flow cytometry.

MATERIALS AND METHODS

Fifty-one cases of uterine cellular SMTs were se­lected from our files of gynecological neoplasms in the Second Department of Pathology, Kyushu U ni­versity, from 1977 to 1992. Cases initially labeled as leiomyosarcoma (LMS), cellular leiomyoma (CL), bizarre leiomyoma (BL), and uncertain malignant potential category (UMP) were studied. The clinical information, including follow-up data, was obtained from both the patients' physicians and medical records. Follow-up data for all patients except for one with LMS were available. Hematoxylin and eosin preparations in the proper number for each tumor size were reviewed.

Histologic assessment

The following histologic features were semiquan­titatively evaluated. The nuclear atypia of each case was determined by dividing the range of nuclear features from mild to severe. Mildly atypical nuclei possessed regular borders and an even distribution of chromatin, similar to that of benign CL. Severely atypical nuclei were large and pleomorphic, with an irregular nuclear shape, clumped chromatin, and prominent nucleoli. Moderately atypical nuclei ranged between mild and severe. Mitoses were counted and expressed as the number of mitotic figures per I 0 high-power fields (hpf). Count was made on a binocular Olympus BH-2 microscope us­ing 40x S-Plan Apo objective and 10x/20L wide­field eyepieces while selecting the most active areas where mitoses were the most frequent. The pres­ence of coagulative tumor necrosis was examined in all the given specimens of each case.

Based on the above histological factors, including the criteria of hypercellularity, cellular atypia, mi­totic count, and the presence of tumor necrosis based on previous reports (1-8), the 51 cases were diagnosed into LMS, UMP, BL, and CL. LMS were further classified into four subgroups: (a) mod­erate to severe nuclear atypia and lO and more mi­totic figures/1 0 hpf; (b) moderate to severe nuclear atypia and five to nine mitotic figures/1 0 hpf; (c) mild nuclear atypia and 10 or more mitotic figures/ I 0 hpf; (d) mild nuclear atypia and five to nine mi­totic figures/] 0 hpf. LMS and benign SMT (BL and

CL) were used for the immunohistochemical and flow cytometric studies.

Immunohistochemistry

All sections from the formalin-fixed and paraffin­embedded specimens were studied immunohisto­chemically using avidin-biotin complex methodol­ogy (17). The primary antibodies and their dilutions are summarized in Table I. All the sections were deparaffinated with xylene and dehydrated in a graded series of alcohol. The sections for Ki-67 (MIB-1) were pretreated with boiling in citrate buffer, pH 6.0, for 30 min. All the sections were incubated in 0.3% hydrogen peroxidase in methanol for 30 min and then were exposed to the primary antibodies at given dilutions by phosphate-buffered saline at room temperature for 90 min. A subse­quent reaction was made by using an avidin-bio­tin-peroxidase kit (Nichirei, Tokyo, Japan). The sections were stained with freshly prepared diami­nobenzidine (DAB) solution and then were counter­stained with methyl green or hematoxylin.

Assessment of immunohistochemical staining

MIB-1, PCNA, and p53 staining was confined to the nuclei. The MIB-1 and PCNA indices, as the extent of MIB-1 and PCN A positivity in each case, respectively, were evaluated by determining the percentage of positive nuclei counted at high power and more than three fields that were chosen at ran­dom. A p53-positive case was defined as a case with positive cells, whereas a case that expressed spo­radic positive cells was excluded from the positive category. HHF35 and a-SMA staining were catego­rized into four grades expressed as negative (- ), focally positive (1 +; <50% of cells had a positive reaction), intermediately positive (2 +; I+ to 3 + ), and diffusely positive (3 +; almost all the tumor cells had a positive reaction).

Flow cytometry

The flow cytometric determination of tumor cell DNA content was performed on paraffin-embedded sections using an earlier published method (18, 19).

TABLE 1. Panel of antibodies

Antibody Source Dilution -- --

Ki-67 (Ml B-1) monoclonal lmmunotech S.A. 1:100 PCNA (PCIO) monoclonal DAKO 1:100 p53 (CMI) polyclonal Novocastra 1:500 HHF35 monoclonal DAKO 1:100 o.-SMA monoclonal Sigma Chemical 1:5000

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136 S. AMADA ET AL.

The cellular DNA content was measured on a FAC­Scan flow cytometer (Becton Dickinson, Sunny­vale, CA) with a 488-nm argon ion laser. Histo­grams of I x 104 cells were recorded and analyzed with Cell FIT cell-cycle analysis software (Becton Dickinson). The first G0/1 peak was assumed to be a diploid population, and DNA aneuploidy was de­fined by the presence of a well-defined second peak.

Statistical methods

A logistic regression model of the BMDP statis­tical Package Program (CBMDP Statistical Soft­ware Inc., California) on the IBM system 4381 com­puter (Armonk, New York, NY) was used to cal­culate the probabilities of a malignant or benign group (20). From this analysis, the equations and optimal thresholds for classifying the cases into ei­ther a malignant or a benign category were calcu­lated (21). The accuracy of classification was deter­mined for each individual parameter (MlB-1 and PCNA indices), and subsequently for more than one variable (adding p53, HHF35, and a-SMA sta­tus) to further improve accuracy. The survival rates of the cases with LMS were calculated by the Ka­plan-Meier method, and comparisons were made u ing the generalized Wilcoxon test. Other statisti­cal differences were evaluated with the use of either the x2 or the Wilcoxon-Mann- Whitney test.

RESULTS

Histological features

Based on the findings previously mentioned in Materials and Methods, the cellular SMTs were cia ified into 24 LMS, two UMPs, four BLs, and 21 CL . ln the 24 LMS, group A consisted of l 3 c a es; the highest mitotic count was 6 0 mitotic fig­ures/10 hpf, the average 31.8 mitotic figures/tO hpf. Eight of the 13 cases showed severe nuclear atypia, whereas the others exhibited moderate atypia. All but one showed foci of coagulative tumor necrosis. In group B, which con is ted of three cases, two of the three showed severe nuclear atypia and tumor necrosis. Another tumor showed moderate atypia and no necrosis. Group C consisted of five neo­plasms with a range of 10-16 mitotic figures/10 hpf. No cases in group C disclosed any necrosis. Group D included three tumors, all of which showed co­agulative necrosis. Two neoplasms exhibiting two and three mitotic figures/10 hpf, moderate nuclear atypia, and no tumor cell necrosi were labeled as UMPs, according to the published criteria (1 ,2).

lni J Gynecol Paihol. Vol. 14, No.2. 1995

These two UMPs were excluded from this compar­ative study (Table 2).

Four BLs exhibited diffuse severe pleomorphism with bizarre giant tumor cells but lacked any mitotic figures. Twenty-one CLs showed minimal to mild nuclear atypia and a range of zero to three mitotic figures/ I 0 hpf. Three CLs contained scattered pleo­morphic giant tumor cells. No tumor necrosis was detected in any of the BLs or CLs.

Clinical features

The age range of the patients with LMS was 38-63 years (mean 48.7). All patients with LMS were treated with a total hysterectomy as primary ther­apy. The initial laparotomy disclosed extrauterine lesions in two women of group A. Twelve women, including the two mentioned above, died of disease at 2-210 months (mean 34.4) after surgery and were from all of the four groups. One woman from group D is now being treated for a recurrent tumor. Both women with UMP are alive without any sign of dis­ease 14 and 59 months after surgery, respectively.

TABLE 2. Histolugical findings of leiomyosarcomas and UMP tumors

Age Case !yr)

Leiomyosarcomas

Atypia Mitoses (110 hpf) Necrosis

Prognosis (mo)

Moderale to se1·ere nuclear atypia and 10 and more mf/10 hpJ I 63 Severe 60 + DOD ( 19) 2 49 Severe 30 + DOD ( 5) 3 62 Severe 30 + DOD ( 48) 4 45 Severe 24 + DOD ( 41) 5 47 Severe 15 DOD ( 2) 6 50 Severe 22 + NED (117) 7 43 Severe 20 + ED ( 77) 8 59 Severe 20 + ED ( 20) 9 54 Moderate 53 + DOD ( 9)

10 54 Moderate 46 + DOD ( 23) II 38 Moderate 41 + NED ( 44) 12 43 Moderate 41 + NED ( 6) 13 45 Moderate 12 + NED (116)

Moderate to sn•ere nuclear atypia and 5-9 mf! 10 hpf 14 49 Severe 8 + DOD ( 22) 15 54 Severe 7 + DOD ( 12) 16 54 Moderate 6 DOD (210)

Mild nuclear cllypia and 10 and more mf/10 hpf 17 40 Mild 13 DOD ( 9) 18 53 Mild 16 19 47 Mild 15 NED ( 35) 20 42 Mild 14 NED ( 5) 21 43 Mild 10 NED ( 61)

Mild nuclear atypia and 5-9 mf/10 hpf 22 49 Mild 8 + DOD ( 13) 23 43 Mild 6 + AWD ( 51) 24 43 Mild 5 + NED ( 86)

UMP tumors 25 32 Moderate 3 NED ( 59) 26 43 Moderate 2 NED ( 14)

UMP. uncertain malignant potential category; mf/10 hpf, mitotic fig­ures/10 hpf: DOD, died of disease: NED, no evidence of disea e; AWD, alive with disease.

LEIOMYOSARCOMA OF THE UTERUS 137

All the patients with benign tumors have undergone either a total hysterectomy or myomectomy for the diagnosis of myoma uteri and are alive without dis­ease after surgery (follow-up period of 28-183 months, mean 70.4).

MIB-1 and PCNA indices

All tumors stained relatively homogeneously for MIB-1 (Fig. I) and PCNA (Fig. 2). The number of counted cells ranged from 600 to 2,000 at the se­lected fields in each neoplasm. There was little dif­ference in the positivity for MIB-1 or PCNA among the selected fields in each case. The MIB-1 and PCN A indices in the tumors examined were within the ranges of 0.2-47.5 and 0-85, respectively (Figs. 3 and 4). Both the mean indices of LMS and benign SMTs (BLs and CLs) were 19.2 and 1.1, and 35.2 and 6.2, respectively (Table 3). There were signifi­cant differences in both indices between LMS and benign SMTs (p < 0.0001).

p53 expression

In the 24 LMS, there were eight neoplasms pos­itive for p53, which expressed >I 0% positive cells in each high power field (Fig. 5), whereas none of the 25 benign BLs and CLs expressed a positive reaction for p53 (p = 0.0016, Table 3).

DNA ploidy pattern

Analyzable DNA histogram s were obtained from 44 of the tumors studied. The mean coefficient of variation was 5.3 (range 1.2-12.5). Twelve of the 22 LMS exhibited a DNA-aneuploid cell population, whereas all the benign SMTs showed a diploid

FIG. 1. lmmunostaining for M I B-1. In the leiomyosarcoma with moderate atypical nu­clei and 41 mitotic figure /10 hpf (case 12), 47.5% of the nuclei exhibited a positive re­action.

DNA histogram. The DNA ploidy pattern of the LMS and the benign SMTs (BLs and CLs) signifi­cantly differed between themselves (p < 0.0001, Ta­ble 3).

Muscle-specific actins

HHF35 and a-SMA were seen almost diffusely (the value exhibited 2 + or 3 +) in all the benign BLs and CLs except for two cases. In contrast, lO of 24 LMS showed either focal or negative immu­

noreactivity for HHF35 or a-SMA (0 or 1 + ). The difference of immunostaining for a-SMA was sta­tistically significant between the LMS and the be­nign SMTs (BLs and CLs) (p = 0.0004) (Table 3).

Logistic regression model

The probability of being an LMS or a benign SMT (BL or CL) was computed using a logistic regres­sion equation with either the MIB- I or PCN A in­dex. To determine the accuracy of classification into LMS and benign SMT (BL or CL), the proba­bility at different cut-off points was tested. For the MIB-1 index, at a cut-off point of 0.492 probability (MIB-1 index 3.6), 87% (21 of 24) of the LMS and 96% (24 of 25) of the benign SMTs, overall 92%, were correctly classified. Regarding the PCN A in­dex, at a cut-off point of probability of 0.525 (PCNA index 15.6), 75% (18 of 24) of LMS and 84% (21 of 25) of the benign SMTs, overall 82%, were also cor­rectly classified (Table 4).

When all possible combinations of more than one parameter (MIB-1 and PCNA indices, p53 and mus­cle-specific actins) were considered, the highest

lnt J Gynecol Pathol. Vol. 14. o. 2. 1995

138 S. AM ADA ET AL.

overall accuracy was achieved by combining the MIB-l index and a-SMA. At a cut-off probability of 0.492, 96% (23 of 24) of the LMS and 96% (24 of 25) of the benign SMT, 96% overall, were correctly classified (Table 4).

Survival In 23 LMS, there were no differences between

the survival and mitotic count (groups A and C vs. B and D) or nuclear atypia (groups A and B vs. C

and D). Regarding the immunohistochemical and flow cytometric factors, LMS had a tendency to correlate with survival and the p53 positivity with statistical significance (p = 0.0357, Fig. 6). How­ever, there wa no ignificant correlation between

0

-+0 8

& X Q)

-o 30 c §

c:O § � 0

§

� malignant benign

ln=c41 \n=25i

FIG. 3. Distribution of the MIB-1 index. There wa a significant difference between the malignant group (leiomyosarcomas. mean 19.2) and the benign group (bizarre and cellular leiomyo­mas, mean 1.1) (p < 0.0001).

In! J Gynecol Parhol, Vol. 14, No.2, 1995

FIG. 2. Immunostaining for PCNA. In the leiomyo arcoma with severe atypical nuclei and 20 mitotic figures/ I 0 hpf (case 8), 70% of the nuclei exhibited a positive reaction.

the other factor (MIB-1, PCNA, muscle-specific ac­tins, or DNA ploidy pattern) and the prognosis.

DISCUSSION

Several investigators have reported on the diag­nostic criteria for uterine LMS, as shown by hyper­cellularity, cellular atypia, and numerous mitotic figures (1-8). The most reliable factor of the diag­

nosis is believed to be the mitotic count (1 ,3-6). However, there is a small controversial group of the uterine SMTs with cellular atypia and only a slight amount of mitotic activity ( < 10 mitotic figures/ I 0 hpf), in which malignancy is difficult to determine.

Cll �0

� 8 X Cll u

60 -o 0 c

<( 0

z u 40 0 0...

� C>

:w C>

e> C> C>

8 Cl>

0

malignant benign \11=2-ll (11=2'\i

FIG. 4. Distribution'of the PC A index. There was a significant difference between the malignant group (leiomyosarcomas. mean 35.2) and the benign group (bizarre and cellular leiomyo­mas. mean n.:?.) (p < 0 0001).

LEIOMYOSARCOMA OF THE UTERUS 139

TABLE 3. Comparison of the immunohistochemical and flow cytometric features between leiomyosarcomas

and benign bizarre and cellular leiomyomas

LMS Benign SMT (8L and CL) p

Mean MIB-1 index (range) 19.2 (0.8--47.5) I. I (0 .2-5.0) <0.0001" Mean PCNA score (range) 35.2 (0-85) 6.2 (0-27) <0.0001" p53

+ 8 0 16 25 0.0016"

a-SMA

I+ 2+ 3+

HHF35

I+ 2+ 3+

DNA ploidy pattern Aneuploid Diploid

4 6 7 7

3 3 9 9

12 10

0

22

I 7

IIi

0 22

0.0004"

0.2415"

<0 0001"

LMS. leiomyosarcoma: SMT, smooth muscle tumor: BL. bizarre leio­myoma; CL. cellular leiomyoma.

"Wilcoxon-Mann-Whitney te�t "x" test.

In our experience, six patients who were diagnosed with LMS showed < l 0 mitotic figuresll 0 hpf, and four of them died of the disease at 12-210 months after surgery. In the groups with mild nuclear atyp­ia, two patients died from the disease while histo­

logically showing only 13 and eight mitotic figures/ 10 hpf, and one patient with six mitotic figures/10 hpf has a history of a recurrence. On the other hand, no patients with fewer than five mitotic fig­ures/10 hpf had either recurrence or a fatal course. Thus, a definition to distinguish bet ween benign and malignant SMTs might be set at five mitotic figures/ I 0 hpf if one does not need to bother about the problem of accuracy in counting mitosis as dis­cussed in the literature (22-25).

MIB-1 (345 and 395 kDa double band in Western blot analysis) is a new antibody that reacts with the Ki-67 nuclear antigen in paraffin-embedded sec­

tions, is associated with cell proliferation, and is found throughout the cell cycle of G I, S, G2, and M phases (26,27). Few studies of MIB-1 on neoplasms have been reported (28). PCNA is a 36-kDa acidic nonhistone nuclear protein that functions as an aux­iliary protein for DNA polymerase o and is an ab­

solute requirement for DNA synthesis (29). In mes­enchymal neoplasms, a large series of PCNA im­munostaining has been performed on a few studies of hemangiopericytoma by Yu et al. (12) and syno­vial sarcoma by Oda et al. (11 ). No series of MIB-1

and PCNA immunostaining have been previously performed on the uterine SMTs. We found a signif­icant difference (p < 0.0001) in the MIB-1 and PCNA indices between the LMS (19.2 and 35.2) and benign SMTs (BLs and CLs) (I. 1 and 6.2). The mi­totic count, the most reliable diagnostic morpholog­ical factor in previous studies (1 ,3-6), shows only the number of proliferating cells in the M phase. But the proliferating cells occupy several functional

states besides mitosis. Ki-67 is found in proliferat­ing cells of the G 1, S, G2, and M phases (27), and PCNA is synthesized in the late G 1 and S phases (29). Therefore, our results of the MIB-1 and PCNA indices make it clear that the number of proliferat­ing cells is important to discriminate between LMS and benign SMTs. Indeed, at a cut-off point of 3.6 for the MlB-1 index and at 15.6 for the PCNA in­dex, the LMS and benign SMT were predicted with an overall accuracy of 92% and 82%, respectively. Hence, we conclude that the MIB-1 and PCNA in­dices are useful for the diagnosis of LMS.

The p53 gene encodes a 53-kDa nuclear phos­phoprotein involved in the negative regulation of cell growth (30,31 ). The mutations in this gene play an important role in the development of many ma­lignant tumors (32-36). Mutant p53 proteins have a longer half-life than the wild-type proteins and are easily detected by immunohistochemical methods.

Several investigators have demonstrated an over­expression of the p53 protein immunohistochemi­cally in a variety of human malignancies (15, 16). However, no large series of SMTs with p53 evalu­ation have yet been published. We found p53 pro­tein was expressed in eight of 24 LMS (33%) but in none of the benign SMTs (p = 0.0016). In addition, the p53 positivity statistically correlated with the prognosis of the LMS (p = 0.0357). Recently, mu­tant p53 has been stressed to be related to cell growth (37) and deviation from apoptosis (38,39). Because the LMS showed cell proliferation more rapid than that of SMTs as demonstrated with our MIB-1 and PCNA indices study, p53 might only be expressed in the LMS. lt is expected that the degree of cell proliferation would correlate with biological behavior of LMS (4-6,40,41 ). Although our studies did not demonstrate a correlation between the mi­totic count, MIB-1 or PCNA index, prognosis, and

p53 positivity, in our opinion the p53 expression detects cases with a poor prognosis. p53 might thu be expressed in tumors with a high cell proliferation rate that could not be detected by mitotic count or MIB-1 or PCNA index in the LMS. Therefore, the

Int J Gynecol ?athol, Vvl. 14. No. 2. /995

140 S. AMADA ET AL.

p53 expression is a good marker for predicting po­

tential cases with LMS.

A semiquantitative evaluation for muscle-specific

actins in uterine SMTs was compared with their

histological features for the first time. We examined

the characteristics of the tumor cells differentiating

toward smooth muscle while using immunostaining

( 42). In our study, benign B Ls and CLs tended to

TABLE 4. Logistic regression function

1. MlB- 1 index Equation for the probability of being LMS:

Exp(y) p(LMS) = I + Exp(y)

y = - 3.075 -t 0.8487 x MIB-l index

If p > 0.492, the case is predicted to be LMS. Otherwise. if p.::; 0.492. the case is predicted to be benign BL or CL.

2. PCNA index Equation for the probability of being LMS:

Exp(y) p(LMS) =

l + Exp(y)

y = - 1.675 + 0.1141 x PCNA index

If p > 0.525, the case is predicted to be LMS. Otherwise. if p ,::; 0.525, the case is predicted to be benign BL or CL.

3. MIB- 1 index and a-SMA positivity Equation for the probability of being LMS:

Exp(y) p(LMS) =

I + Exp(y)

y = 3.15 + 0.8904 X MIB-1 index 2.839 X o.-SMA positivity (0 if - . 1 if l +. 2 if 2 +, 3 if 3 +)

If p > 0.492. the case i predicted to be LMS. Otherwise, if p .::; 0.492, the case is predicted to be benign BL or CL.

lnt 1 Gynecol P11tlw/, Vol. 14, No.2, 1995

FIG. 5. Immunostaining for p53. The leio­myosarcoma exhibited p53 overexpression with the nuclei showing variable intensities (case 3).

demonstrate more diffuse immunostaining for mus­

cle-specific actins than did the LMS. Evans et al.

(43) reported desmin staining in nine uterine SMTs.

Their results were also similar to ours, in which

they described seven leiomyomas as being strongly

positive and two LMS as being negative. Thus, if

there is little or no staining with actin, an SMT

should be regarded with caution. Malignant cells

appear to be heterogenous and lose differentiation

for smooth muscle cells .

A similar analysis for the DNA ploidy patterns of

uterine SMTs was previously reported by Tsushima

et al. (44). They concluded that the DNA ploidy

pattern could not be used diagnostically to distin­

guish between malignant and benign SMTs because

(%) 100

:.J . ..

.. t ............. ;

� oL .............................. i p53 negative (n = IS)

<( : .... J ....... J. .......... . 0:: _J :; 50 > 0:: :::> (/)

p53 positive (n = 8)

Q L-----�----------�----�----� 2 3 4 5

(Years) YEARS AFTER SURGERY

FIG. 6. The urvival curves of 23 case· with leiomyosarcoma in relation to the p53 positivity. The positive p53 group had a worse survival rate compared with that of the negative p53 group (p =

0.0357). Kaplan-Meier method, generalized Wilcoxon test.

LEIOMYOSARCOMA OF THE UTERUS 141

of the abnormal DNA ploidy patterns in 12% of

benign SMTs. Our study disclosed no DNA aneu­

ploid cases in the benign SMTs, whereas 12 of 22

LMS had DNA aneuploid peaks in our series of

cellular SMTs. Therefore, although why our results

differed from those of Tsushima et al. (44) is still

unclear, the DNA ploidy patterns appear to help in

making a distinction between LMS and benign CLs

and BLs of the uterus.

In this study, the individual immunohistochemi­

cal and flow cytometric parameters that we exam­

ined proved to be useful in discriminating between

LMS and benign SMT (BL or CL). Moreover, by

combining the MIB- I index and a-SMA positivity,

96.9% of LMS and 96% of benign SMTs were clas­

sified with the highest overall accuracy (96%) using

the logi tic regression model. In view of these find­

ings, we suggest that the combination of the MIB-1

index and a-SMA positivity is clearly helpful in dis­

criminating between LMS and benign SMT.

Acknowledgment: The surgical specimens and clinical data reported herein were obtained from the following institutions: Kyushu University, National Kyushu Can­cer Center, Hamanomachi Hospital, Kyushu Welfare Pension Hospital, Fukuoka Red-Cross Hospital, Mat­suyama Red-Cross Hospital, Kitakyushu City Medical Center, Kitakyushu City Wakamatsu Hospital, Kitaky­ushu City Moji Hospital, National Nakatsu Hospital, Na­tional Beppu Hospital, Social Insurance Saga Hospital, and Kyushu Central Hospital. We thank these institutes for contributing their cases for this study. We also thank Kouhei Akazawa (Department of Medical Informatic , Faculty of Medicine, Kyushu University) for assistance with statistical analy is and BrianT. Quinn for editing the manuscript. This study was supported by a Grant-in-Aid for Scientific Research (No. 06280 I 05) from the Ministry of Education, Science and Culture, Japan.

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