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© 2000 American Society for Clinical Oncology p27kip1 Protein Expression Correlates With Survival in Myxoid and Round-Cell LiposarcomaFrom the Department of Laboratory Medicine and Pathology and Department of Oncology, Mayo Clinic, Rochester, MN. Address reprint requests to Ricardo V. Lloyd, MD, PhD, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First St SW, Rochester, MN 55905.
PURPOSE: The p27kip1 protein (p27) is a cyclin-dependent kinase inhibitor that has been shown to be an independent prognostic factor in a variety of human neoplasms. Low expression of p27 tends to occur in more aggressive neoplasms. The role of p27 as an independent prognostic factor in the spectrum of myxoid and round-cell liposarcomas has not been examined. MATERIALS AND METHODS: Forty-seven cases of myxoid and round-cell liposarcomas were examined. Clinicopathologic features and immunohistochemical expression of p27 and Ki-67 antigen were studied in all cases. Survival analysis was performed using the log-rank test and the Cox multivariate regression model. RESULTS: The male:female ratio was 1.4:1, and the mean age at diagnosis was 45 years. The tumors were located in the lower extremities (94%) and retroperitoneum (6%). The median tumor size was 13.5 cm. The median follow-up was 6.3 years, and the overall 5- and 10-year survival rates were 76% and 67%, respectively. Low expression of p27 was identified in 34 cases (72%) and correlated with decreased metastasis-free (P = .026) and overall survival (P = .008). In a multivariate analysis, only round-cell differentiation and low expression of p27 independently predicted decreased metastasis-free and overall survival. CONCLUSION: p27 expression predicts the clinical behavior of myxoid and round-cell liposarcomas, even in neoplasms with few or no round-cell differentiation.
THE p27kip1 PROTEIN (p27) is a cyclin-dependent kinase inhibitor that participates in the regulation of the G1/S cell-cycle checkpoint.1-3 p27 inhibits the cyclin E-CDK2 and, to a lesser extent, cyclin D-CDK4,4,5 and its expression is regulated by cell-cell contact inhibition, transforming growth factor beta and cAMP.6-8 Low p27 expression has been shown to be associated with more aggressive lesions in a variety of human neoplasms, including breast,9-13 colon,14,15 gastric,16,17 esophageal,18 lung,19-22 and prostate carcinoma23-26 and other tumors.27-35 There are a few studies that evaluate the expression of p27 in mesenchymal neoplasms,36-39 but none has addressed the value of p27 expression as a prognostic factor in liposarcomas. Dei Tos et al37 studied the expression of multiple G1/S checkpoint cell-cycle regulators in myxoid and round-cell liposarcomas, but the role of p27 as a prognostic factor was not evaluated. We investigated the expression and prognostic significance of the cell-cycle inhibitor p27 in a series of 47 completely excised myxoid and round-cell liposarcomas primarily treated at the Mayo Clinic.
Forty-seven cases of myxoid and round-cell liposarcomas primarily treated at the Mayo Clinic between 1961 and 1994 were identified. Clinical data, original pathologic and surgical reports, and follow-up information from the Mayo Clinic Cancer Registry were obtained in all cases. Only tumors with complete surgical excisions (free surgical margins) were included in this series (35 wide local excisions, eight marginal excisions, and four amputations). Twenty-nine cases were previously reported in a recently published series.40 Hematoxylin-eosin–stained archival slides were available in all cases (mean, six slides per tumor; range, one to 18 slides per tumor), and the histopathologic features addressed in each tumor included the percentage of round-cell differentiation and of spontaneous necrosis. In cases in which a preoperative radiotherapy or chemotherapy was administered, the histopathologic evaluation was performed on biopsy specimens (five cases). In these five cases, the estimation of the percentage of round-cell differentiation could not be analyzed.
Paraffin-embedded archival tissues were available in all cases for immunohistochemical studies. The most representative section of the tumor was chosen for immunohistochemical evaluation, and all cell subtypes (spindle cells, lipoblasts, and round cells) were analyzed. In tumors that showed round-cell differentiation, sections with the largest percentage of those cells were selected for immunohistochemical analysis. Tissue sections were cut at 5 µm, treated with 0.1 mol/L citrate, pH 6.0, in a 800-W microwave oven for 15 minutes for antigen retrieval, and incubated with the primary antibodies overnight at room temperature. Antibodies used included monoclonal anti-p27 (Transduction Laboratory, Lexington, KY), dilution 1/1,000, and MIB-1 (Ki-67 antigen [Ki-67]; AMAC, Westbrook, ME), dilution 1/50. Immunostaining was performed with the Elite avidin biotin peroxidase kit (Vector Laboratories, Burlinghame, CA) according to manufacturer’s specifications. Positive controls included tonsil tissues for p27 and Ki-67. Negative controls consisted of substituting normal serum for the primary antibody, which resulted in no staining of the tissues. Quantification of the percentage of cells expressing p27 and Ki-67 was performed in a two-grade system: low and high expression for p27 protein were defined as less than 75% and
Association between p27 and Ki-67 expression and round-cell differentiation (categorized as high or low) was assessed with the Fisher’s exact test. Overall survival, metastasis-free survival, and local recurrence-free survival were analyzed according to the Kaplan-Meier product-limit method, and the survival curves were compared with the log-rank test.41 Multivariate analysis to access the impact of low expression of p27 (< 75%) on survival was performed using the Cox multivariate regression model after adjustment for tumor size (< 10 cm or
Clinicopathologic Features The mean age of diagnosis was 45 years (range, 20 to 72 years), and the male:female ratio was 1.4:1. The median tumor size was 13.5 cm (range, 4 to 51 cm), and the tumors were located in the lower extremities (44 cases; 94%) and retroperitoneum (three cases; 6%). The thigh was the most common single anatomic location, with 28 cases (60%). All tumors were completely excised with negative surgical margins, including the three retroperitoneal tumors. Preoperative or postoperative radiotherapy was administered in 26 cases (55%). Adjuvant chemotherapy was administered in three cases. The median follow-up was 6.3 years (range, 0.7 to 23 years), and the overall 5- and 10-year survival rates were 76% and 67%, respectively. Metastatic disease developed in 19 patients (40%), and the most common sites of metastatic involvement were the lungs, mesentery, chest wall, and bones. The 5- and 10-year metastasis-free survival rates were 65% and 56%, respectively. Local recurrence developed in 19 patients (40%), and the 5- and 10-year local recurrence-free survival rates were 61% and 48%, respectively.
Histologically, all tumors showed the typical proliferation of primitive and hyperchromatic spindled cells immersed in a myxoid matrix with a prominent plexiform vascular network (Fig 1). Mitotic activity was usually minimal. Lipoblasts were identified in 15 cases (32%) and were usually of the signet-ring cell type. Round-cell differentiation was identified in 18 (43%) of 42 cases and ranged from 5% to 100% (mean, 15%). Thirteen cases showed
Immunohistochemical Evaluation p27 nuclear expression was identified in all cases and ranged from 25% to 100% (median, 75%). All cell types, including the primitive spindled cells, lipoblasts, and round cells, showed heterogeneous expression of p27. The nuclear expression of p27 was less than 50% in nine cases (19%), between 50% and 74% in 25 cases (53%), and  75% in 13 cases (28%; Fig 3). No association between round-cell differentiation and p27 expression was found (P = .71). Four patients had tumors with a high percentage of round-cell differentiation and high p27 expression. Two of these patients developed metastases, and one died of disease. The other two patients were alive 5 and 14.9 years after the diagnosis. In contrast, 22 patients had tumors with low percentage of round-cell differentiation and low p27 expression. Five (23%) of these patients died of disease between 4 and 14 years after diagnosis, and six (27%) developed metastases between 0 and 5 years after diagnosis.
Ki-67 nuclear immunoreactivity was present in all cases and ranged from 1% to 50% (median, 10%). No association between Ki-67 and p27 expression (P = .51) or between Ki-67 and round-cell differentiation (P = .50) was identified.
Survival Analysis
Liposarcoma, one of the most common soft tissue sarcomas, is classically subclassified in five major variants: well-differentiated, myxoid, round-cell, pleomorphic, and dedifferentiated. Among these, the myxoid liposarcoma is the most common subtype and frequently occurs in the lower extremities.40,42 Round-cell differentiation is identified in approximately 50% of myxoid liposarcomas and has been shown to be a powerful predictor of a poor prognosis.40,42-44 In this regard, myxoid and round-cell liposarcoma are considered the two ends of a spectrum of the same tumor, and this concept has been confirmed with the identification of the specific translocation t(12;16)(q13;p11), which involves the CHOP gene (12q13) and the TLS (translocated in liposarcoma) gene (16p11) in both tumors.45-47 Although there are cases of pure myxoid liposarcoma, the relative proportion of this histologic subtype is overrepresented because of tissue sampling, which may not include areas of round-cell differentiation. Therefore, the identification of a prognostic factor independent of the presence of round-cell differentiation, which can be easily evaluated in small tissue samples, would be of a high clinical value. Kilpatrick et al,40 in a large study of myxoid and round-cell liposarcomas, identified only two histologic markers associated with prognosis in a multivariate analysis: round-cell differentiation ( 25%) and the presence of spontaneous necrosis. However, the latter finding was present in only four cases in that series and in none in our series, which precludes the use of this histologic marker for prognostic assessment. p27 is a member of the of the Waf1/Cip1 family of cyclin-dependent kinase inhibitors, which also includes p21 and p57.48-52 p27 binds and inhibits the cyclin E-CDK2 enzymatic complex, which causes a cell block in the G1 phase of the cell cycle.2 In addition, p27 has been shown to be involved in apoptosis modulation.53 Few studies have addressed the expression of p27 in sarcomas.36-39 In a study of cell-cycle regulators in a series of 49 localized synovial sarcomas, Antonescu et al36 showed that only p53 protein and Ki-67 expression independently predicted the outcome. p27 expression was found to have a labeling index of 69%, but its value as a prognostic factor was not discussed. Kourea et al39 studied the expression of p27 in neurofibromas and malignant peripheral-nerve sheath tumors (MPNSTs) and showed that nuclear immunostaining for p27 was present in 94% and 9% of the tumors, respectively. In MPNSTs, the absence of p27 nuclear expression was accompanied by an increased p27 cytoplasmic expression, and the latter finding correlated with a poor outcome in MPNSTs by univariate analysis.39 Only one previous report examined the expression of p27 and other protein regulators of the G1/S cell-cycle checkpoint in myxoid and round-cell liposarcomas.37 Dei Tos et al37 showed that the immunoreactivity for p27 was present in all tumors in a series of 20 cases, with the percentage of nuclear staining ranging between 70% and 90%. However, quantification and the prognostic implication of p27 expression were not evaluated in that study. We observed that low expression of p27 (< 75% nuclear staining), which was present in 72% of our cases, was an independent predictor of metastasis-free and overall survival after adjustment for multiple cofactors. p27 expression also predicted the clinical behavior when tumors that exhibited high round-cell differentiation were analyzed separately. We did not observe an inverse association relative to prognosis between p27 and Ki-67 expression, which agrees with other studies10,11,15,18,39 This finding suggests that the cell-cycle control in this type of tumor involves a more complex interaction among multiple cell-cycle regulators. Moreover, the inhibitory effects of p27 in the cell-cycle progression might not be observed with low levels of p27. Our findings imply the following: (1) p27 is a useful prognostic marker that is independent of the round-cell status of the tumor; (2) p27 evaluation can provide useful prognostic information in small biopsy samples independent of the identification of round-cell differentiation; and (3) in tumors that show round-cell differentiation, p27 can be used to stratify patients with a higher risk of disease progression. It is important to point out that p27 cannot be viewed as a substitute marker for round-cell differentiation. In contrast, the evaluation of p27 should add information to that obtained with the routine histologic evaluation. The concomitant assessment of round-cell differentiation and p27 might optimize risk stratification and treatment of patients with myxoid liposarcoma. We demonstrate for the first time the role of p27 expression as an independent prognostic factor in myxoid and round-cell liposarcoma. This finding not only expands the role of p27 expression as a prognostic marker in human cancers but also supports a potential role of this marker in other malignant tumors of mesenchymal differentiation.
Antonescu et al examined a series of 49 localized synovial sarcomas and did not find an association between p27kip1 expression and prognosis (Am J Pathol 156:977-983, 2000).
We thank V. Shane Pankratz of the Department of Health Sciences Research, Section of Biostatistics, Mayo Clinic, Rochester, MN, for contributing to this article.
1. Toyoshima H, Hunter T: p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78:67-74, 1994[Medline]
2.
Sherr CJ: Cancer cell cycles. Science 274:1672-1677, 1996
3.
Lloyd RV, Erickson LA, Jin L, et al: p27kip1: A multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am J Pathol 154:313-323, 1999 4. Soos TJ, Kiyokawa H, Yan JS, et al: Formation of p27-CDK complexes during the human mitotic cell cycle. Cell Growth Differ 7:135-146, 1996[Abstract]
5.
Blain SW, Montalvo E, Massague J: Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27kip1 with cyclin A-Cdk2 and cyclin D2-Cdk4. J Biol Chem 272:25863-25872, 1997
6.
Sherr CJ, Roberts JM: Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149-1163, 1995
7.
Clurman BE, Porter P: New insights into the tumor suppression function of p27kip1. Proc Natl Acad Sci U S A 95:15158-15160, 1998 8. Koff A, Ohtsuki ME, Polyak K, et al: Negative regulation of G1 in mammalian cells: Inhibition of cyclin E-dependent kinase by TGF-ß. Science 257:1689-1694, 1993 9. Catzavelos C, Bhattacharya N, Ung YC, et al: Decreased levels of the cell cycle inhibitor p27kip1 protein: Prognostic implications in primary breast cancer. Nat Med 3:227-230, 1997[Medline] 10. Porter PL, Malone KE, Heagerty PJ, et al: Expression of cell cycle regulators p27kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 3:222-225, 1997[Medline]
11.
Tan P, Cady B, Wanner M, et al: The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinoma. Cancer Res 57:1259-1263, 1997 12. Tsuchiya A, Zhang GJ, Kanno M: Prognostic impact of cyclin dependent kinase inhibitor p27kip1 in node positive breast cancer. J Surg Oncol 70:230-234, 1999[Medline] 13. Wu J, Shen ZZ, Lu JS, et al: Prognostic role of p27kip1 and apoptosis in human breast cancer. Br J Cancer 79:1572-1578, 1999[Medline] 14. Loda M, Cukor B, Tam SW, et al: Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinoma. Nat Med 3:231-234, 1997[Medline]
15.
Thomas GV, Szigeti K, Murphy M, et al: Down-regulation of p27 is associated with development of colorectal adenocarcinoma metastases. Am J Pathol 153:681-687, 1998 16. Mori M, Mimori K, Shiraishi T, et al: p27 expression and gastric carcinoma. Nat Med 3:593, 1997[Medline] 17. Ohtani M, Isozaki H, Fujii K, et al: Impact of the expression of cyclin-dependent kinase inhibitor p27Kip1 and apoptosis in tumor cells on the overall survival of patients with non-early stage gastric carcinoma. Cancer 85:1711-1718, 1999[Medline]
18.
Singh SP, Lipman J, Goldman H, et al: Loss or altered subcellular localization of p27 in Barrett’s associated adenocarcinoma. Cancer Res 58:1730-1735, 1998
19.
Esposito V, Baldi A, DeLuca A, et al: Prognostic role of the cyclin dependent kinase inhibitor p27 in non-small cell lung cancer. Cancer Res 57:3381-3385, 1997
20.
Kawana H, Tamaru J, Tanaka T, et al: Role of p27kip1 and cyclin-dependent kinase 2 in the proliferation of non-small lung cancer. Am J Pathol 153:505-513, 1998
21.
Yatabe Y, Masuda A, Koshikawa T, et al: p27kip1 in human lung cancers: Differential changes in small and non-small cell carcinomas. Cancer Res 58:1042-1047, 1998
22.
Catazavelos C, Tsao MS, DeBoer G, et al: Reduced expression of the cell cycle inhibitor p27kip1 in non-small cell lung carcinoma: A prognostic factor independent of Ras. Cancer Res 59:684-688, 1999
23.
Tsihlias J, Kapusta LR, DeBuer G, et al: Loss of cyclin-dependent kinase inhibitor p27kip1 is a novel prognostic factor in localized human prostate adenocarcinoma. Cancer Res 58:542-548, 1998 24. Yang RM, Naitoh HJ, Murphy M, et al: Low p27 expression predicts poor disease-free survival in patients with prostate cancer. J Urol 159:941-945, 1998[Medline] 25. Cheville JC, Lloyd RV, Sebo TJ, et al: Expression of p27kip1 in prostatic adenocarcinoma. Mod Pathol 11:324-328, 1998[Medline]
26.
Cordon-Cardo C, Koff A, Drobnjak M, et al: Distinct altered patterns of p27kip1 gene expression in benign prostatic hyperplasia and prostatic adenocarcinoma. J Natl Cancer Inst 90:1284-1291, 1998 27. Sui L, Tokuda M, Ohno M, et al: The concurrent expression of p27(kip1) and cyclin D1 in epithelial ovarian neoplasms. Gynecol Oncol 73:202-209, 1999[Medline] 28. Ito Y, Matsuura N, Sakon M, et al: Expression and prognostic roles of the G1-S modulators in hepatocellular carcinoma: p27 independently predicts recurrence. Hepatology 30:90-99, 1999[Medline]
29.
Florenes VA, Maelandsmo GM, Kerbel RS, et al: Protein expression of the cell-cycle inhibitor p27kip1 in malignant melanoma: Inverse correlation with disease free survival. Am J Pathol 153:305-312, 1998 30. Fan GK, Fujieda S, Sunaga H, et al: Expression of protein p27 is associated with progression and prognosis in laryngeal cancer. Laryngoscope 109:815-820, 1999[Medline] 31. Mineta H, Miura K, Suzuki I, et al: Low p27 expression correlates with poor prognosis for patients with oral tongue squamous cell carcinoma. Cancer 85:1011-1017, 1999[Medline] 32. Jordan RC, Bradley G, Slingerland J: Reduced levels of the cell-cycle inhibitor p27Kip1 in epithelial dysplasia and carcinoma of the oral cavity. Am J Pathol 152:585-590, 1998[Abstract] 33. Lloyd RV, Jin L, Qian X, et al: Aberrant p27kip1 expression in endocrine and other tumors. Am J Pathol 150:401-407, 1997[Abstract] 34. Erickson LA, Jin L, Wollan PC, et al: Expression of p27kip1 and Ki-67 in benign and malignant thyroid tumors. Mod Pathol 11:169-174, 1998[Medline]
35.
Pruneri G, Pignataro L, Carboni N, et al: Clinical relevance of expression of the CIP/KIP cell-cycle inhibitors p21 and p27 in laryngeal cancer. J Clin Oncol 17:3150-3159, 1999 36. Antonescu CR, Leung DH, Dudas M, et al: Cell cycle dependent molecular expression in localized synovial sarcoma: A multifactorial study with prognostic implications. Mod Pathol 12:9A, 1999 (abstr 30) 37. Dei Tos AP, Doglioni C, Muffato G, et al: S. Molecular aberrations of the G1-S checkpoint in myxoid and round cell liposarcoma. Am J Pathol 151:1531-1539, 1997[Abstract] 38. Dei Tos AP, Doglioni C, Muffato G, et al: Synovial sarcoma exhibits deregulation of the G1-S cell cycle checkpoint and of the apoptotic pathway. Mod Pathol 12:10A, 1999 (abstr 36)
39.
Kourea HP, Cordon-Cardo C, Dudas M, et al: Expression of p27kip and other cell cycle regulators in malignant peripheral nerve sheath tumors and neurofibromas: The emerging role of p27kip in malignant transformation of neurofibroma. Am J Pathol 155:1885-1891, 1999 40. Kilpatrick SE, Doyon J, Choong PFM, et al: The clinicopathologic spectrum of myxoid and round cell liposarcoma: A study of 95 cases. Cancer 77:1450-1458, 1996[Medline] 41. Klein JP, Moeschberger ML: Survival Analysis: Techniques for Censored and Truncated Data (ed 1). New York, NY, Springer-Verlag, 1997, Statistics for Biology and Health 42. Enzinger FM, Weiss SW: Liposarcoma, in Enzinger FM, Weiss SW (eds): Soft Tissue Tumors (ed 3). St Louis,Mosby, 1995, pp 431-466 43. Smith TA, Easley KA, Goldblum JR: Myxoid/round cell liposarcoma of the extremities: A clinicopathologic study of 29 cases with particular attention to extent of round cell liposarcoma. Am J Surg Pathol 20:171-180, 1996[Medline] 44. Evans HL: Liposarcoma: A study of 55 cases with reassessment of its classification. Am J Surg Pathol 3:507-523, 1979[Medline] 45. Crozat A, Aman P, Mandahl N, et al: Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature 363:640-644, 1993[Medline] 46. Rabbitts TH, Forster A, Larson R, et al: Fusion of the dominant negative transcriptor regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nat Genet 4:175-180, 1993[Medline]
47.
Knight JC, Renwick PJ, Dal Cin P, et al: Translocation t(12;16)(q13;p11) in myxoid liposarcoma and round cell liposarcoma: Molecular and cytogenetic analysis. Cancer Res 55:24-27, 1995 48. Polyak K, Lee MH, Erdjument-Bromage H, et al: Cloning of p27kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78:59-66, 1994[Medline] 49. Xiong Y, Hannon GJ, Zhang H, et al: p21 is a universal inhibitor of cyclin kinases. Nature 366:701-704, 1993[Medline] 50. Toyoshima H, Hunter T: p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell 78:67-74, 1994
51.
Matsuoka S, Edwards MC, Bai C, et al: p57/Kip2, a structurally distinct member of the p21/CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev 9:650-662, 1995
52.
Lee MH, Reynisdottir I, Massague J: Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev 9:639-649, 1995 53. Hiromura K, Pippin JW, Fero ML, et al: Modulation of apoptosis by the cyclin-dependent kinase p27kip1. J Clin Invest 103:597-604, 1999[Medline]
Submitted December 10, 1999;
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
