ORIGINAL ARTICLE
Leiomyosarcoma With Alternative Lengthening of Telomeres Is Associated With Aggressive Histologic Features, Loss of ATRX Expression, and Poor Clinical Outcome Jau-Yu Liau, MD,*w Jia-Huei Tsai, MD,*w Yung-Ming Jeng, MD, PhD,*w Jen-Chieh Lee, MD,*w Hung-Han Hsu, MS,w and Ching-Yao Yang, MD, PhDz
Abstract: Leiomyosarcoma is an aggressive soft tissue sarcoma with poor patient survival. Recently, it was shown that 53% to 62% of leiomyosarcomas use the alternative lengthening of telomeres (ALT) as their telomere maintenance mechanism. The molecular basis of this mechanism has not been elucidated. Studies of pancreatic neuroendocrine tumor have suggested that the inactivation of either a-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain–associated (DAXX) protein is associated with the ALT phenotype. In this study, we sought to determine the clinicopathologic features of leiomyosarcoma with the ALT phenotype and the possible relationship between this phenotype and ATRX/DAXX expression. Telomerase reverse transcriptase gene (TERT) promoter mutation analysis was also performed. Ninety-two leiomyosarcomas derived from the uterus, retroperitoneum/ intra-abdomen, and various other sites were analyzed. Telomere-specific fluorescence in situ hybridization revealed that 59% (51/86) of leiomyosarcomas had the ALT phenotype. Loss of ATRX expression was observed in 33% of the tumors (30/ 92), and all but 2 ATRX-deficient tumors were ALT positive. Both the ALT phenotype and loss of ATRX expression were associated with epithelioid/pleomorphic cell morphology, tumor necrosis, and poor differentiation. None of the 92 cases lost DAXX expression. No TERT promoter mutation was detected (n = 39). For survival analysis, poor differentiation, high FNCLCC grade, tumor size, and ALT phenotype were correlated with poor overall survival in univariate analysis. Tumor size and ALT phenotype remained independent prognostic
From the Departments of *Pathology; zSurgery, National Taiwan University Hospital; and wGraduate Institute of Pathology, National Taiwan University College of Medicine, National Taiwan University, Taipei, Taiwan. Conflicts of Interest and Source of Funding: Supported by grant NSC 97-2314-B-002-026-MY3, 101-2320-B-002-044-MY3, and MOST 103-2320-B-002-021- from Ministry of Science and Technology, Republic of China. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Ching-Yao Yang, MD, PhD, Department of Surgery, National Taiwan University Hospital, No 7, Chung-Shan South Road, Taipei 10051, Taiwan (e-mail:
[email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
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factors in multivariate analysis. We concluded that the ALT phenotype in the leiomyosarcoma is associated with aggressive histologic features, loss of ATRX expression, and poor clinical outcome. Key Words: leiomyosarcoma, TERT, alternative lengthening of telomeres, telomere (Am J Surg Pathol 2015;39:236–244)
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eiomyosarcomas, excluding those of uterine origin, account for 5% to 10% of all sarcomas.1 Leiomyosarcomas are aggressive neoplasms associated with poor patient survival. Previous studies have indicated that sarcomas exhibiting myogenic differentiation (smooth and skeletal muscles and myofibroblasts) are more aggressive than nonmyogenic sarcomas.2–4 Leiomyosarcomas are cytogenetically highly complex tumors without recurrent chromosomal aberrations.5,6 The current treatment modalities for leiomyosarcomas include surgical debulking, radiotherapy, and chemotherapy regimens that frequently include gemcitabine and docetaxel.7,8 However, for metastatic tumors, no clear survival benefit of chemotherapy has been proven, and most patients eventually die from disease.9,10 The outcome is particularly grave for deeply seated large tumors and tumors associated with great vessels. Further studies of the pathogenesis and cell biology of these tumors are necessary for developing more effective treatment modalities. Telomeres are repetitive hexameric TTAGGG DNA sequences at the ends of each chromosome. Telomeres shorten after each round of cell division, and the length of telomeres must be maintained to sustain limitless replicative potential. Two mechanisms of telomere maintenance have been identified: telomerase activation and alternative lengthening of telomeres (ALT).11 In most cancers (85% to 90%), telomerase is used to maintain the length of telomeres. One important mechanism of telomerase activation is the recently identified recurrent mutations in the promoter region of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of the telomerase riboprotein complex.12,13 The Am J Surg Pathol
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mutations create new binding motifs for the E-26 family of transcription factors and enhance transcription and protein expression. The lengths of telomeres are maintained through ALT in 10% to 15% of cancers. ALTpositive tumors are characterized by marked telomere length heterogeneity on Southern blotting and the presence of ALT-associated promyelocytic leukemia bodies.14,15 Brain tumors, especially astrocytomas and pediatric glioblastomas, and soft tissue sarcomas appear to use this mechanism more frequently.16–18 Among sarcomas, osteosarcoma, leiomyosarcoma, liposarcoma, and undifferentiated pleomorphic sarcoma (malignant fibrous histiocytoma) are frequently ALT positive.17,18 Studies have reported that the ALT phenotype has prognostic implications in some cancer types, such as glioblastoma, osteosarcoma, and liposarcoma.18–24 Approximately 50% to 60% of leiomyosarcomas are ALT positive, but the clinicopathologic features of these leiomyosarcomas and the cause of ALT in leiomyosarcomas have not been determined.17,18 In a recent study, 61% of pancreatic neuroendocrine tumors have been shown to be ALT positive, and this phenotype correlated perfectly with the inactivation of the either a-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain–associated (DAXX) protein.25,26 Predominant loss of ATRX has also been observed in ALT-positive gliomas and ALTpositive cell lines.27,28 ATRX and DAXX form a dimer, and they are crucial for the incorporation of histone 3.3 into telomeres and for telomere stability.29–31 Although the mechanism has not been delineated, it was supposed that the dysfunction of ATRX or DAXX leads to telomere instability, homologous recombination, and ALT. In this study, we investigated the clinicopathologic features of leiomyosarcomas with the ALT phenotype and the relationship between ALT and ATRX/DAXX expression.
MATERIALS AND METHODS Tumor Samples Ninety-two cases of leiomyosarcoma from various sites with available formalin-fixed and paraffin-embedded tissue blocks were retrieved from the archives of the Department of Pathology, National Taiwan University Hospital. Histologic and immunohistochemical sections were reviewed to confirm the diagnoses. This study was approved by the Research Ethics Committee of National Taiwan University Hospital, and the specimens were anonymous and analyzed in a blind manner.
Histologic Features The tumors were classified into 3 groups according to the predominant (> 50% tumor cells) tumor cell morphology (spindle, epithelioid, or pleomorphic). The Fe´de´ration Nationale des Centres de Lutte Contre le Cancer (FNCLCC) system was used to grade each tumor when possible. The features included: mitotic activity (0 to 9, 10 to 19, or >19 mitoses per 10 high-power fields), Copyright
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Alternative Lengthening of Telomere in Leiomyosarcoma
tumor necrosis (absent, 19/10 HPF Tumor differentiation Well/moderately Poorly Necrosis Absent Present FNCLCC grade Grade 1 and 2 Grade 3 ATRX expression Intact Lost ER expression Negative Positive ALT Negative Positive
N (%)
ATRX (+)
ATRX ()
P*
56.4 ± 13.7
50.7 ± 9.4
0.022 0.537
11 (18) 51 (82)
3 (10) 27 (90)
28 (46) 19 (31) 14 (23) 10.7 ± 6.4
18 (60) 10 (33) 2 (7) 8.5 ± 3.1
51 (82) 11 (18)
18 (60) 12 (40)
12 (23) 22 (42) 18 (35)
3 (12) 13 (52) 9 (36)
38 (73) 14 (27)
12 (48) 13 (52)
10 (19) 42 (81)
0 (0) 25 (100)
31 (60) 21 (40)
10 (40) 15 (60)
— —
— —
39 (63) 23 (37)
10 (33) 20 (67)
33 (58) 24 (42)
2 (7) 27 (93)
ALT (+)
ALT ()
52.5 ± 11.0
57.6 ± 13.4
7 (14) 44 (86)
7 (20) 28 (80)
30 (59) 14 (27) 7 (14) 9.3 ± 4.0
12 (35) 14 (41) 8 (24) 11.2 ± 7.5
34 (67) 17 (33)
32 (91) 3 (9)
5 (12) 20 (48) 17 (40)
9 (29) 13 (42) 9 (29)
21 (50) 21 (50)
25 (81) 6 (19)
1 (2) 41 (98)
9 (29) 22 (71)
17 (40) 25 (60)
21 (68) 10 (32)
24 (47) 27 (53)
33 (94) 2 (6)
24 (47) 27 (53)
22 (63) 13 (37)
— —
— —
P* 0.058 0.439
0.146
0.042 0.021
0.102
0.206 0.009
0.491
0.173
0.031
0.007
0.026
0.001
0.106
0.021
NA
< 0.001
0.008
0.149
< 0.001
NA
*Use the Fisher exact test, w2 test, and T test. NA indicates not available.
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images, and Table 2 summarizes the comparisons of clinicopathologic features between ALT-positive and ALT-negative tumors and between ATRX-intact and ATRX-deficient tumors. The ALT phenotype was associated with epithelioid/ pleomorphic cell morphology, tumor necrosis, poor differentiation, high FNCLCC grade, and loss of ATRX expression (P = 0.008, P = 0.001, P = 0.007, P = 0.021, and P < 0.001, respectively) but was not associated with mitotic activity, sex, ER expression, or tumor site (P = 0.173, 0.439, 0.149, and 0.102, respectively). A trend of statistical association between the ALT phenotype and a younger patient age was also noted (P = 0.058). Overall, the ALT phenotype was not associated with ER expression; however, of the 14 ALTpositive retroperitoneal/intra-abdominal tumors, 5 expressed ER, whereas none of the 14 ALT-negative tumors expressed ER. The difference was statistically significant (P = 0.04, Fisher exact test).
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Survival Analysis
No TERT Promoter Mutation in Leiomyosarcoma
One patient who died of postoperative complication was excluded from survival analysis. For the remaining 91 patients, the mean and median follow-up period was 3.1 and 2.2 years, respectively (range: 19 d to 12.9 y). Fortytwo patients were alive at the time of the final follow-up. The results of survival analysis are shown in Table 3. In univariate analysis using the Cox proportional-hazards regression model, poorer overall survival was associated with poor tumor differentiation, high FNCLCC grade, ALT phenotype, and tumor size (P = 0.001, 0.009, 0.025, and 0.005, respectively) but was not associated with the tumor site, age, sex, cell morphology, mitotic activity, loss of ATRX expression, or ER expression. Figure 2 shows the survival curves related to tumor differentiation, FNCLCC grade, and ALT. In multivariate analysis, tumor size and ALT phenotype were independent prognostic factors (P < 0.001 and P = 0.034, respectively), but tumor differentiation and FNCLCC were not (P = 0.130 and 0.302, respectively).
TERT promoter mutation analysis was performed in 39 cases, including 26, 10, and 3 cases of tumors derived from the uterus, retroperitoneum/intra-abdomen, and other sites, respectively. No mutation was observed.
To prevent critical telomere shortening and sustain limitless replicative potential, cancer cells maintain the
DISCUSSION
FIGURE 1. A, Hematoxylin and eosin stain of a high-grade uterine epithelioid leiomyosarcoma. The tumor cells were ATRX deficient (B), but DAXX expression was intact (C). D, Telomere-specific FISH revealed the presence of large and bright signals. E, Hematoxylin and eosin stain of a leiomyosarcoma that occurred in the thigh composed of spindle cells. ATRX (F) and DAXX (G) expressions were intact. H, Telomere-specific FISH was negative for ALT.
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Alternative Lengthening of Telomere in Leiomyosarcoma
TABLE 3. Univariate and Multivariate Analysis of Overall Survival (n = 91) Univariate
Multivariate
Features
HR (95% CI)
P
Age at diagnosis Sex Male (n = 13) Female (n = 78) Tumor site Uterus (n = 46) Retroperitoneum/intra-abdomen (n = 28) Other (n = 16) Tumor size Cell morphology Spindle (n = 68) Epithelioid/pleomorphic (n = 23) Mitotic activity 1-9/10 HPF (n = 15) 10-19/10 HPF (n = 35) > 19/10 HPF (n = 26) Tumor differentiation Well/moderately (n = 49) Poorly (n = 27) Necrosis Absent (n = 10) Present (n = 66) FNCLCC grade Grade 1 and 2 (n = 41) Grade 3 (n = 35) ATRX expression Intact (n = 62) Lost (n = 29) ER expression Negative (n = 48) Positive (n = 43) ALT Negative (n = 34) Positive (n = 51)
1.01 (0.98-1.03)
0.664
1.0 0.60 (0.25-1.42)
0.247
1.0 0.70 (0.36-1.37) 1.14 (0.52-2.52) 1.07 (1.02-1.12)
0.295 0.744 0.005
1.0 1.74 (0.94-3.22)
0.076
1.0 1.31 (0.50-3.42) 2.24 (0.88-5.74)
0.582 0.092
1.0 2.99 (1.54-5.79)
0.001
1.0 24.5 (0.30-1998)
0.154
1.0 2.47 (1.26-4.87)
0.009
1.0 1.57 (0.88-2.79)
0.125
1.0 0.96 (0.55-1.68)
0.885
1.0 2.19 (1.10-4.34)
0.025
HR (95% CI)
P
1.14 (1.07-1.22)
< 0.001
1.0 2.04 (0.81-5.16)
0.130
1.0 1.64 (0.64-4.18)
0.302
1.0 2.67 (1.08-6.60)
0.034
CI indicates confidence interval; HR, hazards ratio.
lengths of their telomeres through various mechanisms. The expression of telomerase and ALT are the 2 main telomere maintenance mechanisms.11 Most cancers (85% to 90%) express telomerase, the enzyme responsible for adding new repeated sequences to telomeres. One of the mechanisms of induction of telomerase expression is the recently discovered mutations in the TERT promoter region. Among the sarcomas, the mutations are most common in myxoid liposarcoma, atypical fibroxanthoma, and primary dermal pleomorphic sarcomas.35,36 In a large study by Killela et al,35 only 3 leiomyosarcomas were analyzed, and all of them were wild type. Our study revealed that TERT promoter mutation is not a feature of leiomyosarcoma. However, TERT promoter mutation is only one of the mechanisms of telomerase activation. Whether telomerase is activated through other mechanisms in leiomyosarcoma remains unknown. ALT is used to maintain the lengths of telomeres in approximately 10% to 15% of cancers. The ALT phenotype is characterized by marked heterogenous telomere length distribution on Southern blotting and the presence of ALT-associated promyelocytic leukemia bodies.14,15 In ALT-positive cells, homologous recombination and Copyright
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replication using DNA sequences from the same telomere, the telomere of the sister chromatid, or telomeres of other chromosomes, or extrachromosomal t-circles as templates have been proposed.15 The heterogenous telomere length can be visualized using telomere-specific FISH, because ALT-positive tumors exhibit strikingly large, clumped, and ultrabright FISH signals of variable size, whereas normal and ALT-negative cells exhibit tiny signals uniform in size. Compared with telomerase activation, ALT is a more important telomere maintenance mechanism in leiomyosarcoma. In a recent large study using telomere-specific FISH to detect ALT, Heaphy et al17 demonstrated that certain brain tumors and sarcomas were frequently ALT positive. Of the 59 leiomyosarcomas, 31 were ALT positive (53%). The result was similar to those of our study (59%) and Henson et al’s18 study (62%). Our study further characterized that ALT-positive leiomyosarcomas were associated with the epithelioid/pleomorphic cell morphology, tumor necrosis, poor differentiation, and high FNCLCC grade. We also observed that this phenotype was associated with more aggressive behavior in univariate and multivariate analyses. In the study of Henson et al,18 no www.ajsp.com |
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FIGURE 2. Survival curves by the Kaplan-Meier method. Poorer overall survival was associated with poor tumor differentiation (A), high FNCLCC grade (B), and ALT phenotype (C).
significant differences in grade or clinical outcome were noted between ALT-positive and ALT-negative sarcomas. However, the number of leiomyosarcomas included was low (n = 13). Other studies have demonstrated that the presence of a telomere maintenance mechanism, either telomerase expression or ALT, is a negative prognostic indicator in osteosarcoma and liposarcoma.22–24 By contrast, patients with ALT-positive high-grade gliomas have longer
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overall survival.18–21 However, no difference in survival between ALT-positive and ALT-negative gliomas was noted in a recent study.27 Interestingly, in gliomas the ALT phenotype may be associated with a younger patient age.18,19 In our study, patients with ATRX-deficient leiomyosarcomas were younger (P = 0.022). There was also a statistical trend of younger patient age in ALT-positive leiomyosarcomas (P = 0.058). The underlying mechanism of ALT has not been elucidated. Recently, it was found that pancreatic neuroendocrine tumors with the ALT phenotype were 100% correlated with the inactivation of either ATRX or DAXX.25,26 ATRX and DAXX form a dimer that is required for incorporating histone 3.3 into the telomeres,29–31 and both proteins are indispensable for proper telomere function. Therefore, loss of the ATRX/DAXX dimer was suggested to be an important event in ALT-positive tumors.26 However, in other tumor types, the association was not perfect, and knockdown of ATRX or DAXX did not lead to ALT in vitro.28 In gliomas, ATRX loss was correlated with ALT in most cases, but in some cases these 2 features were discordant.27 Loss of DAXX expression was common in neuroendocrine tumors37 but was rare in gliomas. In another study, loss of ATRX expression was observed in 90% of ALT-positive cell lines, suggesting that the inactivation of ATRX is a major mechanism in tumors with ALT.28 Our study revealed for the first time that ATRX expression was lost in leiomyosarcoma. In addition, we demonstrated that the loss of ATRX is highly correlated with ALT. By contrast, DAXX expression was preserved in all cases. Therefore, leiomyosarcoma was similar to glioma regarding the frequent inactivation of ATRX but not DAXX in the induction of the ALT phenotype. Only half of ALT-positive leiomyosarcomas were ATRX deficient, indicating that other mechanisms can lead to ALT in leiomyosarcoma. For the retroperitoneal/intra-abdominal tumors in our series, ER expression was observed only in ALT-positive tumors. Because a subset of leiomyosarcoma occurring in the retroperitoneum, pelvis, or abdominal wall expresses ER and is morphologically similar to uterine tumors, ER expression suggested that these tumors are phenotypically similar to uterine tumors. This was consistent with our observation that uterine tumors exhibited the highest rate of ALT, and with the fact that there are 2 distinct groups of leiomyosarcoma (ie, gynecologic and somatic types) occurring in these regions. Loss of ATRX expression, which was highly predictive of the ALT phenotype, was also associated with ER expression. When only uterine and nonuterine/nonretroperitoneal/non-intra-abdominal tumors were considered, the former exhibited a trend of more frequent loss of ATRX expression (P = 0.065). However, loss of ATRX expression was also associated with epithelioid/ pleomorphic cell morphology, poor tumor differentiation, and necrosis, and these features were more frequently observed in uterine tumors. We speculate that, although loss of ATRX expression might be more common in uterine tumors, it is likely a function of the tumor grade. Copyright
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The 2 ATRX-deficient tumors that occurred in nonvisceral soft tissue were both poorly differentiated and had tumor necrosis. Our study has several limitations. The first is that we did not perform mutational analyses of ATRX. ATRX gene mutation is the most common mechanism of loss of ATRX expression in pancreatic endocrine tumors and gliomas, and it is highly correlated with loss of ATRX expression.25,26 However, because the ATRX gene is very large, it is difficult to perform mutation analysis in paraffin-embedded samples. Additional studies using fresh tissue and deep sequencing may be required to determine the frequency of ATRX mutation and the mutation sites in leiomyosarcoma. Second, the number of nonuterine and nonretroperitoneal/non-intra-abdominal tumor cases was relatively small. Although we speculate that loss of ATRX expression and/or the ALT phenotype are related to tumor grade rather than tumor site, additional cases are necessary to prove our hypothesis. Third, our study is retrospective, the follow-up period was relatively short, and the patients did not receive uniform treatment. The prognostic implications of the ALT phenotype should be validated by independent studies. In conclusion, ALT is the telomere maintenance mechanism in approximately 60% of leiomyosarcomas, and this phenotype is correlated with aggressive histologic features, loss of ATRX expression, and poor clinical outcome. ACKNOWLEDGMENTS The authors acknowledge statistical assistance provided by the Taiwan Clinical Trial Bioinformatics and Statistical Center, Training Center, and Pharmacogenomics Laboratory (which is founded by National Research Program for Biopharmaceuticals [NRPB] at the National Science Council of Taiwan; NSC 102-2325-B-002-088) and the Department of Medical Research in National Taiwan University Hospital. We also thank the staff of the Core Labs, Department of Medical Research, National Taiwan University Hospital for technical support. REFERENCES 1. Gustafson P, Wille´n H, Baldetorp B, et al. Soft tissue leiomyosarcoma. A population-based epidemiologic and prognostic study of 48 patients, including cellular DNA content. Cancer. 1992;70: 114–119. 2. Fletcher CD, Gustafson P, Rydholm A, et al. Clinicopathologic reevaluation of 100 malignant fibrous histiocytomas: prognostic relevance of subclassification. J Clin Oncol. 2001;19:3045–3050. 3. Massi D, Beltrami G, Capanna R, et al. Histopathological reclassification of extremity pleomorphic soft tissue sarcoma has clinical relevance. Eur J Surg Oncol. 2004;30:1131–1136. 4. Deyrup AT, Haydon RC, Huo D, et al. Myoid differentiation and prognosis in adult pleomorphic sarcomas of the extremity: an analysis of 92 cases. Cancer. 2003;98:805–813. 5. Knutsen T, Gobu V, Knaus R, et al. The interactive online SKY/ M-FISH & CGH database and the Entrez cancer chromosomes search database: linkage of chromosomal aberrations with the genome sequence. Genes Chromosomes Cancer. 2005;44:52–64. 6. Wang R, Lu YJ, Fisher C, et al. Characterization of chromosome aberrations associated with soft-tissue leiomyosarcomas by twentyfour-color karyotyping and comparative genomic hybridization analysis. Genes Chromosomes Cancer. 2001;31:54–64.
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34. Ma¨kinen N, Mehine M, Tolvanen J, et al. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science. 2011;334:252–255. 35. Killela PJ, Reitman ZJ, Jiao Y, et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci USA. 2013; 110:6021–6026. 36. Griewank KG, Schilling B, Murali R, et al. TERT promoter mutations are frequent in atypical fibroxanthomas and pleomorphic dermal sarcomas. Mod Pathol. 2014;27:502–508. 37. Chen SF, Kasajima A, Yazdani S, et al. Clinicopathologic significance of immunostaining of alpha-thalassemia/mental retardation syndrome X-linked protein and death domain-associated protein in neuroendocrine tumors. Hum Pathol. 2013;44: 2199–2203.
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