Death-associated protein kinase (DAP kinase) alteration in soft tissue leiomyosarcoma: Promoter methylation or homozygous deletion is associated with a loss of DAP kinase expression

Death-Associated Protein Kinase (DAP Kinase) Alteration in Soft Tissue Leiomyosarcoma: Promoter Methylation or Homozygous Deletion Is Associated With a Loss of DAP Kinase Expression KEN-ICHI KAWAGUCHI, MD, YOSHINAO ODA, MD, TSUYOSHI SAITO, MD, HIDETAKA YAMAMOTO, MD, TOMONARI TAKAHIRA, MD, SADAFUMI TAMIYA, MD, YUKIHIDE IWAMOTO, MD, AND MASAZUMI TSUNEYOSHI, MD The death-associated protein kinase (DAP kinase) was initially identified as a positive mediator of programmed cell death induced by interferon-␥. To investigate the potential role and the alteration of the DAP kinase gene in soft tissue leiomyosarcoma (LMS), we first searched for homozygous deletion and promoter hypermethylation in 45 LMSs for which genomic DNA was available, using differential PCR and methylation-specific PCR, respectively. Promoter methylation was recognized in 10 of 45 cases (22%), and homozygous deletion was detected in 3 of 45 cases (7%). p53 mutation was detected in 11 of 45 LMS cases (24%). Cases with DAP kinase alteration or p53 mutation showed a close correlation with high French Federation of Cancer Centers grade or with poor prognosis (P ⴝ 0.0244, P ⴝ 0.0491, respectively). Next, to determine that DAP kinase promoter methylation or homozygous deletion is involved in the down-regulation of DAP kinase expression, we examined the expression of DAP kinase protein by immunohistochemistry. Decreased expression of DAP kinase protein was recognized in 13 of 45 LMS cases (29%). Seven of 13 cases (54%) with decreased expression of

DAP kinase protein revealed promoter methylation or homozygous deletion of DAP kinase, and the methylation status or homozygous deletion of its gene showed a close correlation with decreased DAP kinase expression (P ⴝ 0.0300). In conclusion, although DAP kinase alteration was relatively rare, DAP kinase alteration and/or p53 mutation may associate with tumor progression in soft-tissue LMSs. Furthermore, although further detailed analyses are necessary, promoter methylation or homozygous deletion status of DAP kinase may present a major alternative mechanism of a loss of or decrease in DAP kinase expression. HUM PATHOL 35:1266-1271. © 2004 Elsevier Inc. All rights reserved. Key words: death-associated protein kinase, leiomyosarcoma, promoter hypermethylation, homozygous deletion. Abbreviations: DAP kinase, death-associated protein kinase; LMS, leiomyosarcoma; FNCLCC, French Federation of Cancer Centers; AJCC, American Joint Committee on Cancer; SSCP, single-strand conformation polymorphism; PCR, polymerase chain reaction; HPFhigh-power field.

Soft tissue sarcoma represent a heterogenous group of mesenchymal malignancies, and the majority of the previous scientific studies concerning the alteration of apoptosis-related factors in soft tissue sarcomas have dealt with broad categories of different type of tumors. As a consequence, data concerning single subtype of sarcomas are limited. The death-associated protein kinase (DAP kinase) gene, located on chromosome 9p34.1, was initially isolated as a calcium/calmodulin-dependent positive mediator of INF␥–induced apoptosis and is associated with the cell cy-

toskeleton.1-3 Loss of DAP kinase expression has been reported in bladder and renal cell carcinoma,4 lung cancer,5 head and neck carcinoma,6 and B-cell malignancies,7 and it is thought to lead to immortalization of cells and development of cancers. Promoter hypermethylation of the DAP kinase CpG island was also detected in some malignancies, in which the range of methylation was variable,4,5,7,8 and the methylation status has been found to be associated with silencing of the DAP kinase gene in these malignancies. Simpson et al8 have reported that the inactivation of DAP kinase is strongly correlated with homozygous deletion or promoter hypermethylation in pituitary tumors. Furthermore, the presence of DAP kinase methylation is associated with lymph node involvement or advanced disease stage in some tumors.9-11 DAP kinase was recently characterized as an upstream regulator of p53.12 Some investigators have shown that some apoptosis-related genes, such as p53 and bcl-2, have an important role in the progression of soft tissue sarcomas.13-15 However, the frequency of DAP kinase expression and the mechanism of inactivation of its gene are not clear in soft tissue sarcomas. To elucidate the potential role or the alteration of DAP kinase, we analyzed promoter methylation and homozygous deletion in a histologically homogenous series of 45 cases of soft-tissue leiomyosarcomas (LMSs),

From the Department of Anatomic Pathology, Pathological Sciences, and the Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Accepted for publication July 7, 2004. Supported in part by a Grant-in-Aid for Scientific Research (C; no. 15590304) from the Japan Society of the Promotion of Science, Tokyo, Japan. Address correspondence and reprint requests to Yoshinao Oda, MD, Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.07.007

1266

DAP KINASE ALTERATION IN SOFT-TISSUE LMS (Kawaguchi et al)

including the alteration of p53, which is regulated by DAP kinase. Furthermore, to examine that DAP kinase promoter methylation or homozygous deletion is involved in the down-regulation of DAP kinase expression, we examined the expression of DAP kinase protein. MATERIALS AND METHODS Tumor Samples and DNA Extraction Paraffin-embedded tissues from 29 cases and frozen materials from 16 cases, making a total of 45 LMSs of soft tissue that were registered in the soft-tissue tumor files of the Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Japan, were used for the molecular analyses. Diagnoses of all cases were based on histological examinations with hematoxylin-eosin (HE) staining, and smooth muscle differentiation was confirmed by an immunohistochemically positive reaction of desmin, musclespecific actin (HHF-35), and ␣-smooth muscle actin. In addition, the expression of c-kit, a gastrointestinal stromal tumor marker, was not recognized in any of the 45 cases. As for tumor location, the cases were divided into 2 groups: cases in which the tumors were located at intra-abdominal sites such as the retroperitoneum or abdominal cavity (abdominal type) and cases in which the tumors arose in extra-abdominal sites such as the skin, subcutis, and skeletal muscles (extra-abdominal type).16. The mitotic rate was evaluated by counting the number of mitotic figures in 10 high-power fields (HPFs). Survival data were available for 36 LMS cases, with a follow-up ranging from 14 to 258 (median, 46.0) months. Histological tumor grade was evaluated according to the grading system of the French Federation of Cancer Centers (FNCLCC).17 If possible, cases were also evaluated according to the new American Joint Committee on Cancer (AJCC) staging system. Genomic DNA was purified by standard proteinase K digestion and phenol-chloroform extraction methods. To avoid as much as possible the contamination of normal tissue, microdissection was performed for formalin-fixed, paraffin-embedded tissues. As for the frozen materials, we confirmed that the normal tissue was not contaminated in each sample, referring to their concordant HE-staining slides.

Polymerase Chain Reaction–Single-Strand Conformation Polymorphism for p53 Mutation analysis of the p53 gene was performed from exon 5 to exon 9. The primer sequences and conditions were as described elsewhere.18 Polymerase chain reaction (PCR) products were electrophoresed through 2.0% agarose gel with ethidium bromide. The DNA band was cut from the gel and purified with a SUPREC tube (TAKARA Biomedicals, Japan), and the products were reamplified for 15 cycles. Single-strand conformation polymorphism (SSCP) was performed as described elsewhere.18 To increase the quantity of DNA before sequencing, extra bands that seemed to be aberrantly migrating were excised from SSCP gel and were reamplified for 25 cycles under the same condition. The samples were analyzed for sequencing after the subsequent reaction.

Differential PCR Assay for DAP Kinase The differential PCR method for homozygous deletions of DAP kinase was based on a reported method using GAPDH

gene as an internal control.8 DNA samples were added to a PCR mix with a total volume of 25 ␮L, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.25 mM dNTP, 0.5 U Taq DNA polymerase, and 0.5 ␮M of each of the primers. The primer sequences and conditions were as described elsewhere.8 Human genomic DNA (Clontech, Palo Alto, CA), which was confirmed to have the same base sequences as those in Genbank (accession no. AF000730-734), was used as a normal control for each PCR and the subsequent reactions. We also confirmed that there was no contamination in each PCR or in the subsequent reactions by using distilled water controls. After the amplification, 10 ␮L of PCR products were electrophoresed through 3.0% agarose gel. Homozygous deletion of the lesion of the DAP kinase CpG island examined was confirmed by the absence of the expected DAP kinase PCR product in the tumor DNA.

Bisulfite Modification and Methylation-Specific PCR for DAP Kinase Bisulfite modification was performed by using the DNA modification kit (Intergen) according to the manufacturer’s protocol. The modified DNA was added to a total volume of 20 ␮L containing 10 mM Tris-HCL (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 25 mM of dNTP, 0.25 U Taq DNA polymerase, and 0.2 ␮M of each of the primers. The primer sequences and conditions were as described elsewhere.8 As for positive control, DNA of the Raji cell line, which is known to show promoter hypermethylation of DAP kinase by MSP method, was used.7 In addition, DNA of normal skeletal muscle was used as negative control. Each of the PCR products (10␮L) was directly loaded onto 2% agarose gels, stained with ethidium bromide, and directly visualized under ultraviolet illumination.

Immunohistochemistry Immunohistochemical analysis was performed by using rabbit IgG polyclonal antibodies against DAP kinase (1:100, Sigma Ltd, UK). After antigen retrieval by a microwave oven technique, the specimens were incubated at 4°C overnight with antibodies to these proteins, followed by staining with a streptavidin-biotin-peroxidase kit (Nichirei, Tokyo, Japan) and hematoxylin counterstaining. Normal lymphoid tissues were used as an external positive control for DAP kinase. As a negative control, the primary antibodies were omitted. The extent of staining was graded as follows: 0, staining in ⬍1% of tumor cells; 1, staining in 1% to 10%; 2, staining in 10% to 50% of tumor cells; 3, staining in ⬎50% of tumor cells. The overall intensity of staining was also assessed as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. When the final score was ⬍4, we considered it to indicate decreased expression. When the tumor cells failed to stain in all areas or were stained in only some areas, we confirmed the staining of the admixed non-neoplastic cells as a positive control. The final score (range from 0 to 9) was obtained by multiplying the extent of staining with the intensity.

Statistical Analysis Fisher’s exact test was used to evaluate the association between 2 dichotomous variables. The survival correlations are illustrated with Kaplan-Meier curves, and survival analyses were performed by using the log-rank test. A P value of ⬍0.05 was considered to indicate statistical significance.

1267

HUMAN PATHOLOGY

Volume 35, No. 10 (October 2004)

TABLE 1. Clinicopathologic Parameters in 45 Cases of Leiomyosarcoma Parameter

male and 21 were female. In 27 patients, the tumors occurred in extra-abdominal sites (13 in thigh, 3 in chest or abdominal wall, 3 in buttock, 2 in upper arm, lower leg, and 1 each in the neck, axilla, forearm, groin). In contrast, in 18 patients, the tumors were located in abdominal sites (9 in retroperitoneum, 5 in mesentery, and 4 in abdominal cavity). Tumor size could be determined in 34 cases. Twenty-six tumors were ⱖ5 cm in diameter, whereas 8 cases were measured as ⬍5 cm. Histological subtype was as follows: 40 were classic type, 2 were pleomorphic type,19 2 were myxoid type,20 and 1 was epithelioid type. The mitotic rate ranged from 1 to 48 per 10 high-power fields HPFs (mean, 15.1 per 10 HPFs). Twenty-two cases had less than 9 mitoses per 10 HPFs, whereas 9 had between 10 and 19 mitoses per 10 HPFs, and 14 had more than 20 mitoses per 10 HPFs. According to the FNCLCC grading system, 13 tumors were grade 1, 21 were grade 2, and 11 were grade 3. The AJCC stage was available in 34 cases. Seven cases were considered to be AJCC stage I, 19 cases were stage II, 3 were stage III, and 5 were stage IV.

n

Age (yr) ⱖ60 ⬍60 Sex Male Female Location Extra-abdominal Abdominal Size (cm) ⱖ5 ⬍5 Unknown Mitotic rate (per 10 HPFs) ⱕ9 10–19 ⱖ20 AJCC stage I II III IV Unknown FNCLCC grading Grade 1 Grade 2 Grade 3

19 26 24 21 27 18 26 8 11 22 9 14 7 19 3 5 11

p53 Mutation in Exons 5-9

13 21 11

Abbreviations: HPFs, high-power fields; AJCC, American Joint Committee on Cancer; FNCLCC, French Federation of Cancer Centers.

p53 gene mutations were detected in 11 of the 45 LMS cases (24%) by PCR-SSCP analysis (Table 2). As for the type of base substitution, all 11 cases were missense mutations. There was no statistically significant correlation between p53 mutation and clinicopathologic parameters. Promoter Methylation and Homozygous Deletion of DAP Kinase

RESULTS Clinical and Histological Findings The clinicopathologic data are summarized in Table 1. The ages of the patients ranged from 21 to 88 years (average, 58.5 years). Twenty-four patients were

Hypermethylation of the DAP kinase gene was detected in 10 of 45 LMS cases (22%; Table 2). All 10 cases of LMS with promoter hypermethylation showed both ummethylated and methylated signals (Fig 1).

TABLE 2. Death-Associated Protein Kinase Alteration and p53 Mutation in Soft-Tissue Leiomyosarcoma Case

Sex

Age

Location

Size (cm)

MR

FNCLCC Grade

AJCC Stage

Follow-up (Result, mo)

DAP Kinase IHC

DAP Kinase Alteration

p53 Mutation

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19

M F F M F M M M F M F M F M F M M F M

54 81 58 82 72 33 39 54 65 47 61 67 77 55 88 33 75 43 39

Thigh Retroperitoneum Mesenterium Retroperitoneum Abdominal cavity Thigh Abdominal cavity Thigh Thigh Neck Knee Knee Upper arm Lower leg Axilla Thigh Retroperitoneum Retroperitoneum Buttock

15 6 NA 9 6 5 8 7 7.5 6 4 4 10 NA 5 5 13.5 4.5 11

39 31 2 14 29 5 1 3 40 5 4 7 4 39 32 5 7 14 8

3 3 2 3 2 1 1 2 3 2 2 2 2 3 3 1 2 2 2

II II NA IV IV III II II II I II IV II NA II III II I II

DOD, 2 DOD, 44 NA DOD, 33 NA DOD, 44 NA AWD, 15 DOD, 12 AWD, 12 NA AWD, 56 NA DOD, 42 DOD, 65 DOD, 44 DOD, 4 AWD, 12 DOD, 2

N N N D D D D N D N D D D N N N N N N

Methylation Methylation Methylation HD HD Methylation Methylation Methylation HD Methylation Methylation Methylation Methylation — — — — — —

⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

Abbreviations: MR, mitotic rate per 10 high-power fields; IHC, immunohistochemistry; NA, not available; M, month; AWD, alive without disease; DOD, dead of disease HD, homozygous deletion; N; normal expression, D; Decreased expression

1268

DAP KINASE ALTERATION IN SOFT-TISSUE LMS (Kawaguchi et al)

TABLE 3. Correlation Between p53 Mutation, DAP Kinase Alteration, and Clinicopathologic Parameters in Leiomyosarcoma (n ⫽ 45) p53 Mutation and/or DAP Kinase Alteration FIGURE 1. Methylation-specific polymerase chain reaction (PCR) analysis of death-associated protein (DAP) kinase. PCR products amplified by unmethylated (U) and methylated (M) specific primers. L1 to L3 shows hypermethylation of the DAP kinase gene promoter. P, positive control; N, negative control.

Promoter hypermethylation was detected in 4 of 13 cases with decreased immunohistochemical expression of DAP kinase (31%). Homozygous deletion of the DAP kinase gene was detected in 3 of 45 LMS cases (7%; Table 2; Fig 2). All 3 cases with homozygous deletion revealed decreased expression of DAP kinase protein. A statistically significant correlation between DAP kinase alteration and/or p53 mutation and high FNCLCC grade was observed (P ⫽ 0.0244; Table 3). Furthermore, cases with DAP kinase alteration and/or p53 mutation showed a statistically significant correlation with poor prognosis (Fig 3). There was no statistical correlation between DAP kinase methylation and clinicopathologic parameters or prognosis (data not shown). Immunohistochemical Analysis of DAP Kinase To evaluate whether DAP kinase alteration is involved in a decrease of DAP kinase expression because promoter methylation or homozygous deletion of DAP kinasein 13 of 45 cases studied (29%) were detected, we examined the expression of DAP kinase protein by immunohistochemistry. Decreased expression of DAP kinase protein was recognized in 13 of 45 LMS cases (29%; Fig 4A-C). Seven of 13 cases (54%) with decreased expression of DAP kinase protein revealed promoter methylation or homozygous deletion of DAP kinase, and the methylation status or homozygous deletion of its gene showed a close correlation with decreased DAP kinase expression (P ⫽ 0.0300; Table 4). A correlation between the DAP kinase immunoreaction and clinicopathologic parameters was not observed (data not shown).

Parameter Age (yr) ⱖ60 (n ⫽ 19) ⬍60 (n ⫽ 26) Sex Male (n ⫽ 24) Female (n ⫽ 21) Location Extra-abdominal (n ⫽ 27) Abdominal (n ⫽ 18) Size (cm) (n ⫽ 34) ⱖ5 (n ⫽ 26) ⬍5 (n ⫽ 8) Mitotic rate (per 10 HPFs) ⱖ20 (n ⫽ 14) ⬍20 (n ⫽ 31) AJCC stage (n ⫽ 34) I and II (n ⫽ 26) III and IV (n ⫽ 8) FNCLCC grading 1 (n ⫽ 13) 2 and 3 (n ⫽ 32)

⫹

⫺

P Value*

8 11

11 15

⬎0.9999

11 8

13 13

0.7636

11 8

16 10

⬎0.9999

13 3

13 5

0.6933

5 14

9 17

0.7460

13 3

13 5

0.6933

2 17

11 15

0.0244†

Abbreviations: DAP, death-associated protein; HPFs, high-power fields; AJCC, American Joint Committee on Cancer; FNCLCC, French Federation of Cancer Centers. *Fisher’s exact test. †Statistically significant (P ⬍ 0.05).

induced apoptosis and is associated with the cell cytoskeleton.1-3 Primary mechanisms have been postulated for inactivation of the DAP kinase gene: primarily homozygous deletion and promoter hypermethylation. Homozygous deletion of the DAP kinase gene has been reported in invasive pituitary tumors based on the method of multiplex PCR assay,8 having been observed

DISCUSSION The death-associated protein kinase (DAP kinase) gene was initially isolated as a positive mediator of INF-␥–

FIGURE 2. Differential polymerase chain reaction (PCR) for death-associated protein (DAP) kinase homozygous deletion. L4, L5, and L9 display the absence of PCR products of the DAP kinase gene.

FIGURE 3. Overall survival classified according to death-associated protein (DAP) kinase alteration or p53 mutation. Patients with DAP kinase promoter methylation or homozygous deletion and/or with p53 mutation have a poor prognosis (P ⫽ 0.0491).

1269

HUMAN PATHOLOGY

Volume 35, No. 10 (October 2004)

FIGURE 4. Immunohistochemical staining of death-associated protein (DAP) kinase proteins. (A) Immunohistochemistry showing cytoplasmic staining in the majority of tumor cells. This case had no homozygous deletion or hypermethylation. (B) A loss of DAP kinase expression was observed in the majority of tumor cells, but (C) surrounding normal tissue showed strong cytoplasmic positivity. This case showed promoter methylation of the DAP kinase gene (L6).

in 4 of 11 (36%) cases with a loss of DAP kinase expression. In the present study, although homozygous deletion was detected in only 3 of 45 LMS cases (7%), all of these cases showed decreased expression of DAP kinase. Methylation of either the 5' regulatory region or discrete regions of CG dinucleotides called CpG islands

is an important mechanism of transcriptional repression. Methylation of the 5' CpG island of DAP kinase has been described in various types of cancers, with the range of methylation varying according to the tumor type.4,5,7,8 It has been reported elsewhere that promoter hypermethylation is associated with down-regulation of DAP kinase expression in cancer cells and primary malignancies.4,5,7 In our study, the methylation status of DAP kinase alone had no correlation with decreased expression of DAP kinase protein. Simpson et al9 have described that promoter hypermethylation or homozygous deletion of DAP kinase correlates with an absence of the DAP kinase protein, indicating that the methylation status or homozygous deletion has a close association with down-regulation of the DAP kinase protein. In our study, DAP kinase methylation or homozygous deletion was detected in 7 of 13 cases (54%) with decreased expression of DAP kinase protein; our analysis also revealed a close correlation between DAP kinase methylation or homozygous deletion and immunohistochemical expression of DAP kinase. These results indicate that inactivation of the DAP kinase by promoter hypermethylation or homozygous deletion may be associated with a silencing of its expression in soft-tissue LMS. A decrease or loss of DAP kinase expression has been described in many types of carcinomas and other malignancies, and it is thought to lead to inactivation of this critical apoptotic pathway in tumorigenesis.4,5,7 DAP kinase also participates in apoptosis induced by other ligands such as TNF-␣ and Fas.21 The expression of p53 and bcl-2, as positive and negative regulators of apoptosis, respectively, has been recognized in various types of sarcomas, and these apoptosis-related factors appear to play an important role in tumor progression.13 On the other hand, it previously has been reported that expression of Fas and Fas-ligand is less frequent, indicating that the Fas system does not play an essential role in sarcomas.22,23 In our study, DAP kinase expression was relatively frequent in tumor cells (71%), and 13 of 45 LMS cases (29%) displayed decreased DAP kinase expression by immunohistochemical analysis; however, its expression status had no correlation with clinicopathologic parameters in this tumor. Further study is necessary to elucidate the role of apoptosis-related factors in sarcomas, including DAP kinase. TABLE 4. Correlation Between DAP Kinase Immunoreactivity and DAP Kinase Methylation or Homozygous Deletion (n ⫽ 45) DAP kinase IHC

DAP Kinase Methylation or Homozygous Deletion

Decreased

Normal

⫹ ⫺

7 6

6* 26

Abbreviations: IHC, immunohistochemistry; DAP, death-associated protein. *Statistically significant, P ⫽ 0.0300 (decreased vs. normal IHC groups for positive DAP kinase methylation or homozygous deletion.

1270

DAP KINASE ALTERATION IN SOFT-TISSUE LMS (Kawaguchi et al)

Apoptosis in human malignancies, including sarcomas, has been described in relation to progression, tumor grade, and cell proliferative activity.24-26 In sarcomas, with regard to the apoptosis-related factors, some investigators have reported that bcl-2 expression or the bcl-2– bax ratio may be considered useful in determining the prognosis.14,15 Loss of DAP kinase expression is known to be associated with the process of metastasis.27 Previous investigators have described that the loss of DAP kinase by promoter hypermethylation is associated with tumor aggressiveness in carcinoma cell lines and primary malignancies.9-11 In the present study, although cases with DAP kinase alteration tended to have short survival, we found no significant association with poor survival in soft-tissue LMS. DAP kinase was recently characterized as an upstream regulator of p53.12 The p53 gene product can promote apoptosis in response to a variety of stimuli.28 This regulatory function of p53 may be lost by deletion and frequent mutation. In soft-tissue LMS, p53 mutation have been reported in 17% to 43% studied.16,29 Similarly, our current study showed p53 mutation in 11 of 45 cases (24%). In addition, DAP kinase alteration and/or p53 mutation had a close association with high histological grade and poor prognosis. Although further detailed analysis of a large number of cases is necessary, the alteration of DAP kinase and/or p53 mutation may have an important role in tumor progression of soft-tissue LMS. In conclusion, although DAP kinase alteration was relatively rare, DAP kinase alteration and/or p53 mutation may associate with the parameters of aggressive behavior in soft-tissue LMSs. Furthermore, promoter methylation or homozygous deletion was shown to play an important role in inactivation of the DAP kinase gene; however, further studies of DAP kinase expression related to various genetic alterations are necessary. Acknowledgments. The authors are grateful to Y. Nouzuka and N. Tateishi for their excellent technical assistance.

REFERENCES 1. Deiss LP, Feinstein E, Berissi H, et al: Identification of a novel serine/threonine kinase and a novel 15-Kd protein as potential mediators of the gamma interferon-induced cell death. Genes Dev 9:1530, 1995 2. Cohen O, Feinstein E, Kimichi A: DAP kinase is a Ca2⫹/ calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J 16:998-1008, 1997 3. Feinstein E, Druck T, Kastury K, et al: Assignment of DAP1 and DAPK-genes that positively mediate programmed cell death triggered by INF-gamma to chromosome regions 5p12.2 and 9p34.1, respectively. Genomics 29:305-307, 1995 4. Kissil JL, Feinstein E, Cohen O, et al: DAP kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: Possible implications for role as tumor suppressor gene. Oncogene 15:403-407, 1997 5. Esteller M, Sanchez-Cespedes M, Rosell R, et al: Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 59:67-70, 1999 6. Sanchez-Cespedes M, Esteller M, Wu L, et al: Gene promoter

hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 60:892-895, 2000 7. Katzenellenbogen RA, Baylin SB, Herman JG: Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. Blood 93:4347-4353, 1999 8. Simpson DJ, Clayton R, Farrell WE: Preferential loss of death associated protein kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion. Oncogene 21:1217-1224, 2002 9. Kim DH, Nelson HH, Wiencke JK, et al: Promoter methylation of DAP-kinase: Association with advanced stage in non-small cell lung cancer. Oncogene 20:1765-1770, 2001 10. Tada Y, Wada M, Taguchi K, et al: The association of deathassociated protein kinase hypermethylation with early recurrence in superficial bladder cancers. Cancer Res 62:4048-4053, 2002 11. Tang X, Khuri FR, Lee JJ, et al: Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small-cell lung cancer. Oncogene 20:1765-1770, 2001 12. Kogel D, Prehn JHM, Scheidann KH: The DAP kinase family of pro-apoptotic proteins: Novel players in the apoptotic game. Bioassays 23:352-358, 2001 13. Kawauchi S, Fukuda T, Oda Y, et al: Prognostic significance of apoptosis in synovial sarcoma: Correlation with clinicopathologic parameters, cell proliferative activity, and expression of apoptosisrelated proteins. Mod Pathol 13:755-765, 2000 14. Nakanishi H, Ohsawa M, Naka N, et al: Immunohistochemical detection of bcl-2 and p53 proteins and apoptosis in soft tissue sarcoma: Their correlations with prognosis. Oncology 54:238-244, 1997 15. Hong T, Shimada Y, Uchida S, et al: Expression of angiogenic factors and apoptotic factors in leiomyosarcoma and leiomyoma. Int J Mol Med 8:141-148, 2001 16. Konomoto T, Fukuda T, Hayashi K, et al: Leiomyosarcoma in soft tissue: Examination of p53 status and cell proliferating factors in different locations. Hum Pathol 29:74-81, 1998 17. Coindre JM, Bui NB, Bonichon F, et al: Histopathologic grading in spindle cell soft tissue sarcomas. Cancer 61:2305-2309, 1998 18. Kawaguchi K, Oda Y, Sakamoto A, et al: Molecular analysis of p53, MDM2, and H-ras genes in osteosarcoma and malignant fibrous histiocytoma of bone in patients older than 40 years. Mod Pathol 15:878-888, 2002 19. Oda Y, Miyajima K, Kawaguchi K, et al: Pleomorphic leiomyosarcoma: Clinicopathologic and immunohistochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous histiocytoma. Am J Surg Pathol 25:1030-1038, 2001 20. Rubin BP, Fletcher CD: Myxoid leiomyosarcoma of soft tissue, an underrecognized variant. Am J Surg Pathol 24:927-936, 2000 21. Cohen O, Inbal B, Kissil JL, et al: DAP-kinase participates in TNF- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol 146:141-148, 1999 22. Gaumann A, Tew DS, Mayer E, et al: Expression of apoptosisrelated proteins, p53, and DNA fragmentation in sarcomas of the pulmonary artery. Cancer 92:1237-1244, 2001 23. Fernandez-Figueras MT, Armengol P, Puig L, et al: Absence of Fas (CD95) and FasL (CD95L) immunohistochemical expression suggests Fas/FasL-mediated apoptotic signal is not relevant in cutaneous Kaposi’s sarcoma lesions. J Cutan Pathol 26:417-423, 1999 24. Soini Y, Paakko P, Lehto V-P: Histopathological evaluation of apoptosis in cancer. Am J Pathol 153:1041-1053, 1998 25. Sasatomi E, Tokunaga O, Miyazaki K: Spontaneous apoptosis in gallbladder carcinoma: Relationships with clinicopathologic factors, expression of E-cadherin, bcl-2 protooncogene, and p53 oncosuppressor gene. Cancer 78:2101-2110, 1996 26. McMenamin ME, O’Neil AJ, Gaffney EF: Extent of apoptosis in ovarian serous carcinoma: Relation to mitotic and proliferative indices, p53 expression, and survival. J Clin Pathol Mol Pathol 50: 242-246, 1997 27. Inbal B, Cohen O, Polak-Charcon S, et al: DAP kinase links the control of apoptosis to metastasis. Nature 390:180-184, 1997 28. Dabbas M, White E: Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev 7:546-554, 1993 29. Dei Tos AP, Maestro R, Doglioni C, et al: Tumor suppressor genes and related molecules in leiomyosarcoma. Am J Pathol 148: 1037-1045, 1996

1271