Liposarcoma

* Context.-beta-Catenin is an adhesion molecule that also plays a role in the Wnt signaling pathway.

Objective.-To analyze beta-catenin mutation and accumulation in a series of liposarcomas and malignant fibrous histiocytomas.

Design.-beta-Catenin mutation in exon 3 was studied using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing analysis in 30 formalin-fixed, paraffin-embedded liposarcomas. The tumors included 12 dedifferentiated liposarcomas, characterized by both high-grade anaplastic components and well-differentiated liposarcoma components, plus 18 welldifferentiated liposarcomas (10 lipoma-like and 8 sclerosing-type cases). The 2 components of dedifferentiated liposarcomas were analyzed independently. beta-Catenin accumulation in the nuclei or cytoplasm and Ki-67 expression (cell-proliferation marker, MIB-1 labeling index) were examined immunohistochemically. Nine storiform-pleomorphic-type malignant fibrous histiocytomas were also studied.

Results.-Dedifferentiated liposarcomas showed mutation in 2 cases (17%) and accumulation in 5 cases (42%).

One of the 2 cases that showed mutations had a mutation in the well-differentiated component; this mutation was silent. The other case had mutations that differed between the 2 components. In well-differentiated liposarcomas, mutation was not seen in any of the cases (0/18; 0%); however, accumulation was seen frequently in the sclerosing-- type cases (5/8; 63%), but not in the lipoma-like cases (0/ 10; 0%). Malignant fibrous histiocytomas showed mutation and accumulation in 5 (56%) and 4 (44%) cases, respectively, without any exact correlation between the cases. Cases with accumulation had a higher MIB-1 labeling index than those without, among both the sclerosing-type well-differentiated liposarcomas (P

Conclusions.-Our results suggest the possible involvement of beta-catenin activation caused by beta-catenin mutation in liposarcoma and malignant fibrous histiocytoma, but the contribution would seem to be different, depending on the tumor type. beta-Catenin accumulation is also thought to be related to cell proliferation in some of the cases.

(Arch Pathol Lab Med. 2002;126:1071-1078)

beta-Catenin is one of the undercoat proteins of the adhesion molecules,1,2 and it has been identified as a coprecipitating species with the E-cadherin cell-cell adhesive complex.3 This protein also functions as a downstream transcriptional activator of the Wnt-signaling pathway, forming complexes with the DNA-binding proteins, Tcf and Lef-1.(4,5) The intracellular level of beta-catenin is regulated by proteasomal degradation through complex formation with adenomatous polyposis coli (APC) protein and by phosphorylation (by glycogen synthase kinase-3(beta) [GSK-3(beta)]) of the serine/threonine residues encoded by exon 3 of the B-catenin gene.6,7

Dysfunction of the APC gene was shown to contribute to the development of desmoid tumors, which are one of the most common extracolonic manifestations in patients with familial adenomatous polyposis8,9 as well as to sporadic desmoid tumors.10-12 Hence, the functional activation of beta-catenin caused by APC mutation is considered likely to be a key step in the formation of desmoid tumors.6 Regarding other bone and soft tissue tumors, the frequency of beta-catenin mutation in exon 3 has been reported to be 1 of 21 malignant fibrous histiocytomas (MFHs) and 1 of 7 synovial sarcomas, with no mutation in any of 7 liposarcomas. beta-Catenin accumulation was also noted in 5 of 8 MFH cases by Western blot analysis, but only 1 of these cases had beta-catenin mutation.13 In another report of a series of 115 cases of soft tissue sarcoma, including 15 Iiposarcoma cases (10 myxoid/round cell, 3 well-differentiated/dedifferentiated, 2 pleomorphic) and 22 pleomorphic-type MFH cases, stimulation of cell proliferation caused by an increased beta-catenin level was postulated, especially in high-grade sarcomas. In the series, only 1 beta-- catenin mutation (at codon 37) was seen (MFH case) in 22 examined tumors, suggesting a less important role of beta-- catenin mutation in soft tissue sarcomas.14

In 1979, Evans15 proposed the concept of dedifferentiated liposarcoma (DDLS), which is characterized by the coexistence of well-differentiated liposarcoma (WDLS) components and high-grade anaplastic components that show MFH-like features in most cases. Furthermore, this type of liposarcoma is known to have a worse prognosis compared with pure WDLS.

In this study, beta-catenin mutation and accumulation were analyzed in a series of liposarcomas comprising DDLSs and WDLSs, as well as storiform-pleomorphic-type MFHs. Cell-proliferation marker using Ki-67 expression (MIB-1 labeling index [LI]) was also investigated in these tumors immunohistochemically.

MATERIALS AND METHODS

Formalin-fixed, paraffin-embedded tissue blocks of these cases were used for immunohistochemical and molecular analysis. Twelve cases of DDLS and 18 cases of WDLS (10 lipoma-like and 8 sclerosing-type) were collected from the histopathology files at our institution. The presence of mutation was analyzed in the 2 components of DDLS independently, using a manual microdissection method.

Patients with DDLS ranged from 34 to 82 years of age (mean, 55.5 years), whereas those with WDLS ranged from 36 to 94 years (mean, 60.4 years). Dedifferentiated liposarcomas showed a male predominance of 8 men to 4 women (male-female ratio, 2:1), while WDLSs were evenly distributed (9 men and 9 women; male-female ratio, 1:1). Six cases of DDLS occurred in the retroperitoneum, 1 case occurred in the mediastinum, and the others occurred in accessible sites, including the thigh (2 cases), abdomfinal wall (1 case), groin (1 case), and lower leg (1 case). On the other hand, 5 cases of WDLS occurred in the retroperitoneum, whereas the other cases occurred in accessible sites, including the thigh (8 cases), buttock (2 cases), axilla (1 case), groin (1 case), and scrotum (1 case). Most of the tumors had been excised as an initial treatment; however, 1 tumor that occurred in the mediastinum (case D31) was inoperable because of tumor extension, therefore radiation therapy was given in that case. As for those tumors that occurred in accessible sites, wide resections were carried out. Clinical data for these cases were collected from medical records. Survival data were available for all patients with DDLS. Follow-up periods ranged from 2 to 240 months (mean, 69.3 months). Nine cases of MFH (storiform-pleomorphic type) were also selected at random and examined.

Immunohistochemical Staining

Immunohistochemical analysis was performed using antibodies against beta-catenin (beta-catenin; BD Biosciences, Franklin Lakes, NJ) and Ki-67 (MIB-1; Immunotech, Marseille, France). The histologic sections of formalin-fixed and paraffin-embedded materials were deparaffinized in xylene and rehydrated in ethanol. After dehydration, the endogenous peroxidase was blocked by methanol containing 0.3% hydrogen peroxide for 30 minutes. The sections were incubated with the primary antibody at 4 deg C overnight, followed by staining with a streptavidin-biotin-peroxidase kit (Nichirei, Tokyo, Japan). The sections were finally reacted in a 3,3'diaminobenzidine, peroxytrichloride substrate solution, then counterstained with hematoxylin. The sections were pretreated by heating in a microwave oven. The dilutions of primary antibodies were 1:200 for anti-beta-catenin and 1:100 for anti-Ki-67.

Assessment of Immunoreactivity

References

1. Takeichi M. The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development. 1988;102:639-655.

2. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991;251:1451-1455.

3. Ozawa M, Hoschutzky H, Herrenknecht K, Kemler R. A possible new adhesive site in the cell-adhesion molecule uvomorulin. Mech Dev. 1990;33:4956.

4. Barth Al, Nathke IS, Nelson WJ. Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol. 1997;9:683-690.

5. Behrens J, von Kries JP, Kuhl M, et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature. 1996;382:638-642.

6. Munemitsu S, Albert I, Souza B, Rubinfeld B, Polakis P. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Nat] Acad Sci U S A. 1995;92:3046-3050.

7. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 1996;272:1023-1026.

8. Miyaki M, Konishi M, Kikuchi-Yanoshita R, et al. Coexistence of somatic and germ-line mutations of APC gene in desmoid tumors from patients with familial adenomatous polyposis. Cancer Res. 1993;53:5079-5082.

9. Gurbuz AK, Giardiello FM, Petersen GM, et al. Desmoid tumours in familial adenomatous polyposis. Gut. 1994;35:377-381.

10. Sen-Gupta S, Van der Luijt RB, Bowles LV, Meera Khan P, Delhanty ID. Somatic mutation of APC gene in desmoid tumour in familial adenomatous polyposis. Lancet. 1993;342:552-553.

11. Palmirotta R, Curia MC, Esposito DL, et al. Novel mutations and inacti

vation of both alleles of the APC gene in desmoid tumors. Hum Mol Genet. 1995; 4:1979-1981.

12. Alman BA, Li C, Pajerski ME, Diaz-Cano S, Wolfe HJ. Increased beta-- catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors). Am] Pathol. 1997;151:329-334.

13. Iwao K, Miyoshi Y, Nawa G, Yoshikawa H, Ochi T, Nakamura Y. Frequent beta-catenin abnormalities in bone and soft-tissue tumors. Jpn J Cancer Res. 1999; 90:205-209.

14. Kuhnen C, Herter P, Muller 0, et al. Beta-catenin in soft tissue sarcomas: expression is related to proliferative activity in high-grade sarcoma. Mod Pathol. 2000;13:1005-1013.

15. Evans HL. Liposarcoma: a study of 55 cases with a reassessment of its classification. Am J Surg Pathol. 1979;3:507-523.

16. Tejpar S, Nollet F, Li C, et al. Predominance of beta-catenin mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor). Oncogene. 1999;18:6615-6620.

17. Saito T, Oda Y, Tanaka K, et al. Beta-catenin nuclear expression correlates with cyclin Dl overexpression in sporadic desmoid tumours. J Pathol. 2001;195: 222-228.

18. Funayama N, Fagotto F, McCrea P, Gumbiner BM. Embryonic axis induction by the armadillo repeat domain of beta-catenin: evidence for intracellular signaling. I Cell Biol. 1995;128:959-968.

19. Molenaar M, van de Wetering M, Oosterwegel M, et al. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell. 1996;86:391-399.

20. Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275: 1787-1790.

21. Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science. 1997;275: 1784-1787.

22. He TC, Sparks AB, Rago C, et al. Identification of c-MYC as a target of the APC pathway. Science. 1998;281:1509-1512.

23. Mann B, Gelos M, Siedow A, et al. Target genes of beta-catenin-T cell-- factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci U S A. 1999;96:1603-1608.

24. Tetsu 0, McCormick F. Beta-catenin regulates expression of cyclin Dl in colon carcinoma cells. Nature. 1999;398:422-426.

25. Garcia-Rostan G, Tallini G, Herrero A, D'Aquila TG, Carcangiu ML, Rimm

DL. Frequent mutation and nuclear localization of beta-catenin in anaplastic thyroid carcinoma. Cancer Res. 1999;59:1811-1815.

26. Chan EF, Gat U, McNiff JM, Fuchs E. A common human skin tumour is caused by activating mutations in beta-catenin. Nat Genet. 1999;21:410-413. 27. Huang H, Fujii H, Sankila A, et al. Beta-catenin mutations are frequent in

human hepatocellular carcinomas associated with hepatitis C virus infection. Am PathoL 1999;155:1795-1801.

28. Oyama T, Kanai Y, Ochiai Al et al. A truncated beta-catenin disrupts the interaction between E-cadherin and alpha-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines. Cancer Res. 1994;54:6282-6287.

29. Iwao K, Nakamori S, Kameyama M, et al. Activation of the beta-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations. Cancer Res. 1998;58:1021-1026.

30. Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275: 1787-1790.

31. Ross SE, Hemati N, Longo KA, et al. Inhibition of adipogenesis by Wnt signaling. Science. 2000;289:950-953.

32. Mentzel T, Fletcher CDM. Lipomatous tumors of the soft tissues: an update. Virchows Arch. 1995;427:353-363.

33. Nakayama T, Toguchida J, Wadayama B, Kanoe H, Kotoura Y, Sasaki MS. MDM2 gene amplification in bone and soft-tissue tumors: association with tumor progression in differentiated adipose-tissue tumors. Int Cancer. 1995;64:342346.

34. Dei Tos AP, Doglioni C, Piccinin S, et al. Molecular abnormalities of the p53 pathway in dedifferentiated liposarcoma. I Pathol. 1997;181:8-13.

35. Kondo Y, Kanai Y, Sakamoto M, et al. Beta-catenin accumulation and mutation of exon 3 of the beta-catenin gene in hepatocellular carcinoma. Jpn J Cancer Res. 1999;90:1301-1309.

36. Whitehead I, Kirk H, Kay R. Expression cloning of oncogenes by retroviral transfer of cDNA libraries. Mol Cell Biol. 1995;15:704-710.

37. Young CS, Kitamura M, Hardy S, Kitajewski J. Wnt-1 induces growth, cytosolic beta-catenin, and Tcf/Lef transcriptional activation in Rat-1 fibroblasts. Mol Cell Biol. 1998;18:2474-2485.

38. Polakis P. The adenomatous polyposis coli (APC) tumor suppressor. Biochim Biophys Acta. 1997; 1332:F1 27-Fl 47.

39. Nhieu JT, Renard CA, Wei Y, Cherqui D, Zafrani ES, Buendia MA. Nuclear accumulation of mutated beta-catenin in hepatocellular carcinoma is associated with increased cell proliferation. Am J Pathol. 1999;155:703-710.

Akio Sakamoto, MD; Yoshinao Oda, MD; Toshisada Adachi, MD; Tsuyoshi Saito, MD; Sadafumi Tamiya, MD; Yukihide Iwamoto, MD; Masazumi Tsuneyoshi, MD

Accepted for publication April 22, 2002.

From the Departments of Anatomic Pathology, Pathological Sciences (Drs Sakamoto, Oda, Adachi, Saito, Tamiya, and Tsuneyoshi), and Orthopaedic Surgery (Dr Iwamoto), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Reprints: Masazumi Tsuneyoshi, MD, Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan (e-mail: masazumi@surgpath.med.kyushu-u.ac.jp).

Copyright College of American Pathologists Sep 2002
Provided by ProQuest Information and Learning Company. All rights Reserved