Human Pathology (2015) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
Alternative lengthening of telomeres phenotype in malignant vascular tumors is highly associated with loss of ATRX expression and is frequently observed in hepatic angiosarcomas☆,☆☆ Jau-Yu Liau MD a,b , Jia-Huei Tsai MD a,b , Ching-Yao Yang MD, PhD c , Jen-Chieh Lee MD a,b , Cher-Wei Liang MD a,b , Hung-Han Hsu MS a,b , Yung-Ming Jeng MD, PhD a,b,⁎ a
Department of Pathology, National Taiwan University Hospital, Taipei 10002, Taiwan Graduate Institute of Pathology, National Taiwan University College of Medicine, Taipei 10051, Taiwan c Department of Surgery, National Taiwan University Hospital, Taipei 10002, Taiwan b
Received 18 February 2015; revised 16 May 2015; accepted 21 May 2015
Keywords: Angiosarcoma; Telomere; Alternative lengthening of telomeres; TERT; ATRX
Summary Alternative lengthening of telomeres (ALT) is a mechanism using homologous recombination to maintain telomere length and sustain limitless replicability of cancer cells. Recently, ALT has been found to be associated with inactivation of either α-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated (DAXX) protein. In this study, 119 tumors (88 angiosarcomas, 11 epithelioid hemangioendotheliomas, and 20 Kaposi sarcomas) were analyzed to determine the ALT status, its relationship to loss of ATRX/DAXX expression, and the clinicopathological features. In addition, the mutation status in the telomerase reverse transcriptase gene (TERT) promoter was also studied. Loss of ATRX expression was observed in 21% (16/77) of the primary angiosarcomas and 9% (1/11) of epithelioid hemangioendotheliomas. DAXX expression was intact in all but 2 ATRX-deficient angiosarcomas. Telomere-specific fluorescence in situ hybridization assay showed 28% (17/61) of the primary angiosarcomas were ALT positive. Remarkably, ALT was highly associated with loss of ATRX expression: all but 2 ALT-positive angiosarcomas were ATRX deficient. Notably, hepatic angiosarcomas were frequently ATRX deficient (8/13) and/or ALT positive (8/12). None of the secondary angiosarcomas were ATRX/DAXX deficient or ALT positive. The only ATRX-deficient epithelioid hemangioendothelioma was positive for ALT. Forty-seven angiosarcomas were tested for TERT promoter mutation. Despite the fact that angiosarcoma occurs most commonly in sun-damaged skin, mutation was detected in only 1 radiation-associated angiosarcoma (2%). We conclude that ALT is an important telomere maintenance mechanism in primary angiosarcomas. This feature is highly associated with loss of ATRX expression and is frequently observed in hepatic angiosarcomas. © 2015 Elsevier Inc. All rights reserved.
☆
Competing interests: All authors do not have potentially biasing relationships of a financial, professional, or personal nature to any commercial interests that would constitute a conflict of interest related to the studies reported in this manuscript. ☆☆ Funding/Support: This work is supported by Grant MOST 103-2320-B-002-021 from the Ministry of Science and Technology, Republic of China. ⁎ Corresponding author at: Department of Pathology, National Taiwan University Hospital, No 7, Chung-Shan South Rd, Taipei 10002, Taiwan. E-mail address: [email protected] (Y. -M. Jeng). http://dx.doi.org/10.1016/j.humpath.2015.05.019 0046-8177/© 2015 Elsevier Inc. All rights reserved.
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1. Introduction Angiosarcomas are aggressive soft tissue tumors exhibiting endothelial cell differentiation. They occur most commonly in the head and neck region of elderly patients, but can occur in virtually all body sites. According to the absence or presence of risk factors, such as radiation or chronic lymphedema, angiosarcomas are subclassified as primary or secondary. The pathogenesis of primary angiosarcomas is different from that of secondary tumors. Secondary angiosarcomas are characterized by MYC gene amplification [1–3]. In subsets of tumors, they also harbor alterations in other genes related to angiogenesis, such as FLT4 amplification and PTPRB and PLCG1 gene mutations [1,4]. In contrast, the pathogenesis of primary angiosarcomas remains largely unknown. To maintain limitless replicative potential and avoid the catastrophic results of telomere shortening, cancer cells seek pathways to maintain telomere length. Two main telomere maintenance mechanisms have been described: telomerase expression and alternative lengthening of telomeres (ALT) [5]. A common mechanism to activate telomerase expression is the mutations in the promoter of the telomerase reverse transcriptase (TERT) gene recently discovered in cutaneous melanomas [6,7]. The mutations (CNT or CCNTT transitions) are consistent with ultraviolet (UV) light-signature mutations and generate new binding motifs for E-twenty-six family transcription factors, which enhance the expression of the TERT gene. Approximately 10% to 15% of cancers maintain telomeres by ALT, which is dependent on homologous recombination. ALT-positive cells are characterized by marked telomere length heterogeneity and the presence of ALT-associated promyelocytic leukemia bodies, extrachromosomal T-circles and C-circles, and extrachromosomal telomere repeats [8,9]. A previous study showed that 1 of 11 angiosarcomas exhibited the ALT phenotype [10]. Recently, inactivation of either α-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated (DAXX) protein was found to be perfectly correlated with ALT in pancreatic neuroendocrine tumors, suggesting that inactivation of these proteins played crucial roles in the induction of the ALT phenotype [11,12]. In this study, we planned to determine the frequency of TERT promoter mutation and ALT in malignant vascular tumors, and the possible relationship between ALT and loss of ATRX/DAXX expression.
J. -Y. Liau et al. University Hospital. Histologic and immunohistochemical sections were reviewed to confirm the diagnoses. This study was approved by the Research Ethics Committee of the National Taiwan University Hospital, and the specimens were anonymous and analyzed blindly.
2.2. Histologic features The following histologic features were recorded when possible: mitotic activity (0-9 or ≥10 mitoses per 10 high-power fields), tumor necrosis (absent or present), and tumor differentiation. For tumor differentiation, we classified our cases as either conventional or poorly differentiated/ epithelioid angiosarcomas according to the French Fédération Nationale des Centres de Lutte Contre le Cancer system [13]. Histologic features could not be evaluated for cases with limited specimens.
2.3. Immunohistochemistry and telomere-specific fluorescence in situ hybridization ATRX (1:500; Sigma Aldrich, St Louis, MO) and DAXX (1:500; Sigma Aldrich) immunostaining and telomere fluorescence in situ hybridization (FISH) were performed as previously described [14]. A polymer–horseradish peroxidase reagent (BioGenex, San Ramon, CA) and a diaminobenzidine tetrahydroxychloride solution (BioGenex) were used for visualization. Hematoxylin was used for counterstaining. For telomere FISH, deparaffinized slides were placed in citric acid buffer at 85°C for 15 minutes, cooled in phosphate-buffered saline at room temperature, and digested with protease K for 90 to 240 seconds. The slides were then coverslipped, denatured by incubating the slides at 83°C for 5 minutes, and hybridized with a fluorescein isothiocyanate–labeled PNA probe (Panagene, Daejeon, Korea; final concentration, 200 nM) at room temperature for 2 hours. The coverslips were then removed. After washing, 4′-6-diamidino-2-phenylindole was used for counterstaining. The criteria used for interpreting the FISH result were the same as previously defined [10].
2.4. Mutation analysis
2. Materials and methods
DNA extraction, amplification, and sequencing were performed as previously described [15]. The primer sequences used to amplify the TERT promoter were also described previously [16].
2.1. Tumor samples
2.5. Statistical analysis
One hundred nineteen malignant vascular tumors (88 angiosarcomas, 11 epithelioid hemangioendotheliomas [EHEs], and 20 Kaposi sarcomas [KSs]) from various sites with available paraffin blocks were retrieved from the archives of the Department of Pathology, National Taiwan
Data analyses were conducted using SPSS 19 (IBM, Armonk, NY). Categorical variables were compared using the Pearson v2 method or Fisher exact test, when appropriate. Continuous variables were analyzed using Student t test. The survival rates were calculated using the Kaplan-Meier
ALT and loss of ATRX expression in angiosarcoma Table 1
Clinical information of the angiosarcomas
Organ
n
Mean age (y)
Sex (M/F)
Skin Liver Breast Somatic soft tissue Spleen Heart Retroperitoneum/pelvis Head and neck a Bone/joint Pleura Unknown Total
34 13 7 7 6 6 5 4 3 1 2 88
74 65 44 60 65 43 56 59 57 41 42 63
22:12 8:5 0:7 2:5 2:4 4:2 4:1 2:2 2:1 0:1 0:2 46:42
a
Included cases from the nasal cavity (n = 2), oral cavity (n = 1), and larynx (n = 1).
method, and the difference in the survival curves was analyzed using the log-rank test. All statistical results were considered significant if P b .05.
3. Results 3.1. Patients and tumor samples Table 1 summarizes the clinical information of the angiosarcoma cases. Among the 88 tumors, 77 were primary angiosarcomas occurring in various organs. Six cases were secondary angiosarcomas that developed after radiotherapy for breast cancer (n = 3), nasopharyngeal carcinoma, cervical carcinoma, or sinonasal undifferentiated carcinoma (each n = 1). Three cases were angiosarcomatous transformation in nonvascular tumors: schwannoma, malignant peripheral nerve sheath tumor, or malignant phyllodes tumor (each n = 1). All the 3 tumors were radiotherapy-naïve. Two cases were metastatic tumors of unknown origin. Sixty-eight and 20 patients presented with localized and metastatic diseases, respectively. Treatment information was available for 78 patients. Twenty-one, 41, and 11 patients received surgery alone, surgery with (neo)adjuvant chemotherapy and/or radiotherapy, and
3 chemotherapy and/or radiotherapy without surgery, respectively. Five patients did not receive treatment after biopsies. Follow-up information was available for 81 patients. The mean and median follow up time were 1.2 and 0.8 years, respectively (range, 3 days to 6.7 years), and 66 patients died of disease. Of the EHE patients, 8 were female and 3 were male. Seven cases occurred in the liver, 3 occurred in the lung/pleura, and 1 occurred in the pelvis. Two were histologically malignant. Of the KS patients, 17 were male and 3 were female. Eighteen cases occurred in the skin and 2 occurred in the oral mucosa.
3.2. Loss of ATRX and DAXX expression in angiosarcomas and EHEs Loss of ATRX expression was observed in 16 angiosarcomas (18%), 1 EHE (9%), and 0 KS (0%). All ATRX-deficient angiosarcomas were primary angiosarcomas (16/77; 21%), and none of the secondary or nonvascular tumor-associated angiosarcomas were ATRX deficient. Loss of ATRX expression was diffuse in essentially all tumor cells. Except for 2 ATRX-deficient cases (one occurred in the skin and the other occurred in the liver) that exhibited focal loss of DAXX expression, DAXX expression was preserved in all cases. None of the ATRX/DAXX-deficient tumors were postchemotherapy or postradiotherapy specimens. A bar chart of the proportion of ATRX-deficient angiosarcomas of each organ is shown in Fig. 1, and representative histologic and immunohistochemical images are shown in Figs. 2 and 3. Notably, hepatic angiosarcomas had the highest rate of loss of ATRX expression (8/13; 62%), and loss of ATRX expression was seen more frequently in hepatic than nonhepatic tumors (P b .001). Except for the location and a trend of higher mitotic activity (P = .106) in ATRX-deficient tumors, no significant differences in the clinicopathological features were noted between ATRX-deficient and ATRX-proficient angiosarcomas (Table 2). The ATRX-deficient EHE was a malignant EHE occurring in the lung (Fig. 4). It was composed of a sheet-like proliferation of epithelioid cells exhibiting nuclear pleomorphism. Areas of typical EHE with plug-like intra-alveolar
Fig. 1 Bar chart of the proportion of ATRX-deficient and ALT-positive angiosarcomas of each organ. Gray: total number; black: number of ATRX-deficient (A) and ALT-positive (B) cases. ⁎Including cases from the nasal cavity (n = 2), oral cavity (n = 1), and larynx (n = 1).
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J. -Y. Liau et al.
Fig. 2 Histology and ATRX immunohistochemistry. Hematoxylin and eosin stain of a high-grade hepatic angiosarcoma (A) with loss of ATRX expression (B). Hematoxylin and eosin stain of a high-grade angiosarcoma in the breast (C) with loss of ATRX expression (D). Nonneoplastic mammary duct and bile duct epithelial cells, inflammatory cells, and stromal cells showed strong nuclear expression and served as internal positive controls. Original magnification ×200.
Fig. 3 Histology, ATRX and DAXX immunohistochemistry, and telomere-specific FISH of angiosarcomas. A, Hematoxylin and eosin stain of a high-grade cutaneous angiosarcoma. B, Tumor cells were ATRX deficient. C, Most tumor cells were proficient for DAXX, but focal loss of DAXX expression was noted (inset). D, Large and bright telomere signals were detected by telomere-specific FISH. E, Hematoxylin and eosin stain of another high-grade cutaneous angiosarcoma. Tumor cells of this case were intact for both ATRX (F) and DAXX (G) expressions. H, FISH showed small telomere signals of similar size. A-C, E-G, and inset of C, original magnification ×200; D and H, ×1000.
ALT and loss of ATRX expression in angiosarcoma
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Table 2 Comparisons of the clinicopathological features between ATRX-deficient and ATRX-proficient and between ALT-positive and ALT-negative angiosarcomas ATRX ATRX P (−) (+) Age (y), 66.1 ± mean ± SD 9.3 Sex Male 9 Female 7 Total 16 Tumor type Primary 16 Secondary 0 Total 16 Tumor site Hepatic 8 Nonhepatic 8 Total 16 Disease at presentation Localized 14 Metastasized 2 Total 16 Necrosis Absent 2 Present 10 Total 12 Mitotic activity 0-9/10 HPF 0 ≥10/10 HPF 12 Total 12 Differentiation Conventional 2 14 Poorly differentiated/ epithelioid Total 16
ALT (+)
ALT (−)
P
62.6 ± .263 17.2 .725 37 35 72 .591 61 6 67 b.001 5 65 70 .345 54 18 72 1.000 12 49 61 .106 14 46 60 .726 14 58
64.5 ± 63.4 ± .734 8.7 17.7 .872 10 30 7 23 17 53 .317 17 44 0 5 17 49 b.001 8 4 9 47 17 51 .492 15 41 2 12 17 53 1.000 2 8 11 39 13 47 .102 0 9 14 38 14 47 .717 2 10 15 43
72
17
Abbreviation: HPF, high-power field.
53
growth of cords of epithelioid cells in a myxohyaline background could be observed. ATRX expression was lost in both components.
3.3. TERT promoter mutation TERT promoter mutation was analyzed in 47 angiosarcomas (including 21 that occurred in the skin and 5 radiation-associated tumors) and 5 EHEs. Mutation was detected in only 1 radiation-associated angiosarcoma occurring in the breast (−146CNT). This case was ATRX proficient.
3.4. Telomere-specific FISH Interpretable FISH results were obtained in 70 angiosarcomas (61 primary) and 7 EHEs. Seventeen angiosarcomas were ALT positive (17/70; 24%), and all were primary angiosarcomas (17/61; 28%). None of the ALT-positive tumors were postchemotherapy or postradiotherapy specimens. FISH results were available in 5 secondary (including the TERT promoter–mutated tumor) and 2 nonvascular tumor-associated angiosarcomas, and all were ALT negative. A bar chart of the proportion of ALT-positive angiosarcomas of each organ is shown in Fig. 1, and representative FISH images are shown in Fig. 3. Two-thirds (8/12) of the hepatic angiosarcomas were ALT positive, and ALT was observed significantly more frequently in hepatic than nonhepatic tumors (P b .001). Remarkably, for angiosarcomas, ALT was highly correlated with the expression status of ATRX. Except for 2 ATRXproficient cases, all ALT-positive cases exhibited loss of ATRX expression (P b .001). The 2 discordant cases occurred in the neck soft tissue and liver. For the 2 ATRX-deficient cases that had focal loss of DAXX expression, ALT-positive cells were observed throughout the sections and not limited to the DAXX-deficient areas. No statistically significant differences in the clinicopathological features were noted between ALT-positive and ALT-negative tumors, except for the location and a trend of higher mitotic activity (P = .102) in ALT-positive tumors (Table 2).
Fig. 4 Histology, ATRX immunohistochemistry, and telomere-specific FISH of the EHE with ATRX loss. A, Hematoxylin and eosin stain of a pulmonary EHE showing the conventional morphology with cords of epithelioid cells in a myxohyaline background extending along the alveolar spaces. B, High-grade area of the same case showed a sheet-like proliferation of epithelioid cells with nuclear pleomorphism, necrosis, and bronchial cartilage invasion. C, ATRX expression was lost in both components (upper: conventional area, lower: high-grade area). D, FISH revealed the presence of large telomere signals in both components (inset: conventional area). A-C, original magnification ×200; D and inset, ×1000.
6 Of the 7 EHEs with FISH results, only the ATRX-deficient tumor was ALT positive. Thus, for EHEs, ALT status was 100% correlated with ATRX expression. In this particular tumor, ALT-positive cells were observed in both morphologically typical and malignant areas (Fig. 4).
3.5. Survival analysis Survival analysis was performed for cutaneous and hepatic angiosarcomas, for which follow-up data were available in 32 and 9 patients, respectively. The median follow-up time for cutaneous and hepatic angiosarcoma patients were 1.3 and 0.4 years, respectively. Univariate analysis revealed no survival difference between ALT-positive and ALT-negative cutaneous (P = .813) or hepatic angiosarcomas (P = .194). Similarly, no survival difference was noted between ATRX-proficient and ATRX-deficient cutaneous (P = .877) or hepatic angiosarcomas (P = .372).
4. Discussion Telomerase activation and ALT are 2 main telomere maintenance mechanisms [5]. Most (85%-90%) cancers use telomerase to maintain telomere length. Recently, recurrent UV light-signature mutations in the promoter of the TERT gene, which encodes the catalytic subunit of telomerase, were identified in cutaneous melanomas [6,7]. These mutations are also observed frequently in several other skin tumors, such as basal cell carcinoma, squamous cell carcinoma, atypical fibroxanthoma, and primary dermal pleomorphic sarcoma, supporting the role of UV light in the pathogenesis of these tumors [16,17]. However, in our study, TERT promoter mutation was not observed in cutaneous angiosarcomas, despite the fact that they occur most commonly in the head and neck skin of elderly patients. Whether the expression of telomerase is activated by other mechanisms in angiosarcoma is still unknown. ALT is used by approximately 10% to 15% of cancers as the telomere maintenance mechanism. In our study, close to 30% of primary angiosarcomas were ALT positive. Importantly, we noted that two-thirds of hepatic angiosarcomas were ALT positive. Previous studies have found that ALT phenotype was an adverse prognostic factor in osteosarcoma, liposarcoma, and leiomyosarcoma [14,18–20]. However, no survival difference was noted between ALT-positive and ALT-negative angiosarcomas in our study. Another remarkable finding is that we observed an excellent correlation between ALT and loss of ATRX expression. ALT was first linked with the inactivation of either ATRX or DAXX in pancreatic neuroendocrine tumors [11,12]. Although loss of DAXX is more common than loss of ATRX in pancreatic neuroendocrine tumors [11], loss of ATRX is much more frequent in ALT-positive gliomas, leiomyosarcomas, and cell lines [11,14,21]. In our series, 2 ATRX-deficient angiosarcomas also exhibited focal loss of DAXX expression. The significance of this finding is unclear, but ALT-positive cells were not limited to the DAXX-deficient areas, and no
J. -Y. Liau et al. morphologic differences between DAXX-proficient and DAXX-deficient areas were appreciated. In our series, all secondary angiosarcomas were ATRX proficient and ALT negative (available in 5 cases). Secondary angiosarcomas are characterized by MYC gene amplification and protein expression [1–3]. c-Myc is a basic helix-loop-helix and leucine zipper transcription factor involved in many cellular processes such as cell proliferation, differentiation, and apoptosis. The TERT gene is one of the direct targets of c-Myc [22,23]. We speculate that telomerase activation, instead of ALT, is used to maintain telomeres in secondary angiosarcomas. In addition to angiosarcomas, we also analyzed 11 EHEs and 20 KSs. One of the EHEs was ATRX deficient and ALT positive in both conventional and morphologically malignant areas. EHE is molecularly characterized by a t(1;3) translocation and WWTR1-CAMTA3 gene fusion [24,25]. Montgomery et al [26] have suggested that ALT is not a feature of translocation-associated sarcomas. Although the number of EHE cases investigated was low, our results suggested that ALT could be used in rare cases of translocation-associated sarcomas. In our study, all KSs were ATRX/DAXX proficient. Although we did not perform FISH for the KSs, our results were consistent with those of Heaphy et al [10], who found that all 55 KSs analyzed were ALT negative. In conclusion, we demonstrate that close to 30% of primary angiosarcomas use ALT as the telomere maintenance mechanism. We also find that in angiosarcomas, the ALT phenotype is highly correlated with loss of ATRX expression, and this phenotype is frequently observed in hepatic angiosarcomas. Recently, ALT-positive cells have been shown to be sensitive to ataxia telangiectasia and Rad3-related kinase inhibitors [27]. Further study should be undertaken to determine whether patients with ALT-positive angiosarcomas benefit from Rad3-related kinase inhibitor treatment.
References [1] Guo T, Zhang L, Chang NE, Singer S, Maki RG, Antonescu CR. Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions. Genes Chromosomes Cancer 2011;50:25-33. [2] Manner J, Radlwimmer B, Hohenberger P, et al. MYC high level gene amplification is a distinctive feature of angiosarcomas after irradiation or chronic lymphedema. Am J Pathol 2010;176:34-9. [3] Mentzel T, Schildhaus HU, Palmedo G, Büttner R, Kutzner H. Postradiation cutaneous angiosarcoma after treatment of breast carcinoma is characterized by MYC amplification in contrast to atypical vascular lesions after radiotherapy and control cases: clinicopathological, immunohistochemical and molecular analysis of 66 cases. Mod Pathol 2012;25:75-85. [4] Behjati S, Tarpey PS, Sheldon H, et al. Recurrent PTPRB and PLCG1 mutations in angiosarcoma. Nat Genet 2014;46:376-9. [5] Günes C, Rudolph KL. The role of telomeres in stem cells and cancer. Cell 2013;152:390-3. [6] Horn S, Figl A, Rachakonda PS, et al. TERT promoter mutations in familial and sporadic melanoma. Science 2013;339:959-61. [7] Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L, Garraway LA. Highly recurrent TERT promoter mutations in human melanoma. Science 2013;339:957-9.
ALT and loss of ATRX expression in angiosarcoma [8] Cesare AJ, Reddel RR. Alternative lengthening of telomeres: models, mechanisms and implications. Nat Rev Genet 2010;11:319-30. [9] Royle NJ, Foxon J, Jeyapalan JN, et al. Telomere length maintenance—an ALTernative mechanism. Cytogenet Genome Res 2008; 122:281-91. [10] Heaphy CM, Subhawong AP, Hong SM, et al. Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. Am J Pathol 2011;179:1608-15. [11] Heaphy CM, de Wilde RF, Jiao Y, et al. Altered telomeres in tumors with ATRX and DAXX mutations. Science 2011;333:425. [12] Jiao Y, Shi C, Edil BH, et al. DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 2011;331:1199-203. [13] Coindre JM, Terrier P, Bui NB, et al. Prognostic factors in adult patients with locally controlled soft tissue sarcoma. A study of 546 patients from the French Federation of Cancer Centers Sarcoma Group. J Clin Oncol 1996;14:869-77. [14] Liau JY, Tsai JH, Jeng YM, Lee JC, Hsu HH, Yang CY. Leiomyosarcoma with alternative lengthening of telomeres is associated with aggressive histologic features, loss of ATRX Expression, and poor clinical outcome. Am J Surg Pathol 2015;39:236-44. [15] Liau JY, Tsai JH, Yuan RH, Chang CN, Lee HJ, Jeng YM. Morphological subclassification of intrahepatic cholangiocarcinoma: etiological, clinicopathological, and molecular features. Mod Pathol 2014;27:1163-73. [16] Scott GA, Laughlin TS, Rothberg PG. Mutations of the TERT promoter are common in basal cell carcinoma and squamous cell carcinoma. Mod Pathol 2014;27:516-23. [17] Griewank KG, Schilling B, Murali R, et al. TERT promoter mutations are frequent in atypical fibroxanthomas and pleomorphic dermal sarcomas. Mod Pathol 2014;27:502-8.
7 [18] Costa A, Daidone MG, Daprai L, et al. Telomere maintenance mechanisms in liposarcomas: association with histologic subtypes and disease progression. Cancer Res 2006;66:8918-24. [19] Ulaner GA, Huang HY, Otero J, et al. Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma. Cancer Res 2003;63:1759-63. [20] Venturini L, Motta R, Gronchi A, Daidone M, Zaffaroni N. Prognostic relevance of ALT-associated markers in liposarcoma: a comparative analysis. BMC Cancer 2010;10:254. [21] Lovejoy CA, Li W, Reisenweber S, et al. Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway. PLoS Genet 2012;8:e1002772. [22] Wu KJ, Grandori C, Amacker M, et al. Direct activation of TERT transcription by c-MYC. Nat Genet 1999;21:220-4. [23] Lebel R, McDuff FO, Lavigne P, Grandbois M. Direct visualization of the binding of c-Myc/Max heterodimeric b-HLH-LZ to E-box sequences on the hTERT promoter. Biochemistry 2007;46:10279-86. [24] Errani C, Zhang L, Sung YS, et al. A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites. Genes Chromosomes Cancer 2011; 50:644-53. [25] Tanas MR, Sboner A, Oliveira AM, et al. Identification of a diseasedefining gene fusion in epithelioid hemangioendothelioma. Sci Transl Med 2011;3:98ra82. [26] Montgomery E, Argani P, Hicks JL, DeMarzo AM, Meeker AK. Telomere lengths of translocation-associated and nontranslocationassociated sarcomas differ dramatically. Am J Pathol 2004;164:1523-9. [27] Flynn RL, Cox KE, Jeitany M, et al. Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors. Science 2015;347:273-7.
www.elsevier.com/locate/humpath
Original contribution
Alternative lengthening of telomeres phenotype in malignant vascular tumors is highly associated with loss of ATRX expression and is frequently observed in hepatic angiosarcomas☆,☆☆ Jau-Yu Liau MD a,b , Jia-Huei Tsai MD a,b , Ching-Yao Yang MD, PhD c , Jen-Chieh Lee MD a,b , Cher-Wei Liang MD a,b , Hung-Han Hsu MS a,b , Yung-Ming Jeng MD, PhD a,b,⁎ a
Department of Pathology, National Taiwan University Hospital, Taipei 10002, Taiwan Graduate Institute of Pathology, National Taiwan University College of Medicine, Taipei 10051, Taiwan c Department of Surgery, National Taiwan University Hospital, Taipei 10002, Taiwan b
Received 18 February 2015; revised 16 May 2015; accepted 21 May 2015
Keywords: Angiosarcoma; Telomere; Alternative lengthening of telomeres; TERT; ATRX
Summary Alternative lengthening of telomeres (ALT) is a mechanism using homologous recombination to maintain telomere length and sustain limitless replicability of cancer cells. Recently, ALT has been found to be associated with inactivation of either α-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated (DAXX) protein. In this study, 119 tumors (88 angiosarcomas, 11 epithelioid hemangioendotheliomas, and 20 Kaposi sarcomas) were analyzed to determine the ALT status, its relationship to loss of ATRX/DAXX expression, and the clinicopathological features. In addition, the mutation status in the telomerase reverse transcriptase gene (TERT) promoter was also studied. Loss of ATRX expression was observed in 21% (16/77) of the primary angiosarcomas and 9% (1/11) of epithelioid hemangioendotheliomas. DAXX expression was intact in all but 2 ATRX-deficient angiosarcomas. Telomere-specific fluorescence in situ hybridization assay showed 28% (17/61) of the primary angiosarcomas were ALT positive. Remarkably, ALT was highly associated with loss of ATRX expression: all but 2 ALT-positive angiosarcomas were ATRX deficient. Notably, hepatic angiosarcomas were frequently ATRX deficient (8/13) and/or ALT positive (8/12). None of the secondary angiosarcomas were ATRX/DAXX deficient or ALT positive. The only ATRX-deficient epithelioid hemangioendothelioma was positive for ALT. Forty-seven angiosarcomas were tested for TERT promoter mutation. Despite the fact that angiosarcoma occurs most commonly in sun-damaged skin, mutation was detected in only 1 radiation-associated angiosarcoma (2%). We conclude that ALT is an important telomere maintenance mechanism in primary angiosarcomas. This feature is highly associated with loss of ATRX expression and is frequently observed in hepatic angiosarcomas. © 2015 Elsevier Inc. All rights reserved.
☆
Competing interests: All authors do not have potentially biasing relationships of a financial, professional, or personal nature to any commercial interests that would constitute a conflict of interest related to the studies reported in this manuscript. ☆☆ Funding/Support: This work is supported by Grant MOST 103-2320-B-002-021 from the Ministry of Science and Technology, Republic of China. ⁎ Corresponding author at: Department of Pathology, National Taiwan University Hospital, No 7, Chung-Shan South Rd, Taipei 10002, Taiwan. E-mail address: [email protected] (Y. -M. Jeng). http://dx.doi.org/10.1016/j.humpath.2015.05.019 0046-8177/© 2015 Elsevier Inc. All rights reserved.
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1. Introduction Angiosarcomas are aggressive soft tissue tumors exhibiting endothelial cell differentiation. They occur most commonly in the head and neck region of elderly patients, but can occur in virtually all body sites. According to the absence or presence of risk factors, such as radiation or chronic lymphedema, angiosarcomas are subclassified as primary or secondary. The pathogenesis of primary angiosarcomas is different from that of secondary tumors. Secondary angiosarcomas are characterized by MYC gene amplification [1–3]. In subsets of tumors, they also harbor alterations in other genes related to angiogenesis, such as FLT4 amplification and PTPRB and PLCG1 gene mutations [1,4]. In contrast, the pathogenesis of primary angiosarcomas remains largely unknown. To maintain limitless replicative potential and avoid the catastrophic results of telomere shortening, cancer cells seek pathways to maintain telomere length. Two main telomere maintenance mechanisms have been described: telomerase expression and alternative lengthening of telomeres (ALT) [5]. A common mechanism to activate telomerase expression is the mutations in the promoter of the telomerase reverse transcriptase (TERT) gene recently discovered in cutaneous melanomas [6,7]. The mutations (CNT or CCNTT transitions) are consistent with ultraviolet (UV) light-signature mutations and generate new binding motifs for E-twenty-six family transcription factors, which enhance the expression of the TERT gene. Approximately 10% to 15% of cancers maintain telomeres by ALT, which is dependent on homologous recombination. ALT-positive cells are characterized by marked telomere length heterogeneity and the presence of ALT-associated promyelocytic leukemia bodies, extrachromosomal T-circles and C-circles, and extrachromosomal telomere repeats [8,9]. A previous study showed that 1 of 11 angiosarcomas exhibited the ALT phenotype [10]. Recently, inactivation of either α-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated (DAXX) protein was found to be perfectly correlated with ALT in pancreatic neuroendocrine tumors, suggesting that inactivation of these proteins played crucial roles in the induction of the ALT phenotype [11,12]. In this study, we planned to determine the frequency of TERT promoter mutation and ALT in malignant vascular tumors, and the possible relationship between ALT and loss of ATRX/DAXX expression.
J. -Y. Liau et al. University Hospital. Histologic and immunohistochemical sections were reviewed to confirm the diagnoses. This study was approved by the Research Ethics Committee of the National Taiwan University Hospital, and the specimens were anonymous and analyzed blindly.
2.2. Histologic features The following histologic features were recorded when possible: mitotic activity (0-9 or ≥10 mitoses per 10 high-power fields), tumor necrosis (absent or present), and tumor differentiation. For tumor differentiation, we classified our cases as either conventional or poorly differentiated/ epithelioid angiosarcomas according to the French Fédération Nationale des Centres de Lutte Contre le Cancer system [13]. Histologic features could not be evaluated for cases with limited specimens.
2.3. Immunohistochemistry and telomere-specific fluorescence in situ hybridization ATRX (1:500; Sigma Aldrich, St Louis, MO) and DAXX (1:500; Sigma Aldrich) immunostaining and telomere fluorescence in situ hybridization (FISH) were performed as previously described [14]. A polymer–horseradish peroxidase reagent (BioGenex, San Ramon, CA) and a diaminobenzidine tetrahydroxychloride solution (BioGenex) were used for visualization. Hematoxylin was used for counterstaining. For telomere FISH, deparaffinized slides were placed in citric acid buffer at 85°C for 15 minutes, cooled in phosphate-buffered saline at room temperature, and digested with protease K for 90 to 240 seconds. The slides were then coverslipped, denatured by incubating the slides at 83°C for 5 minutes, and hybridized with a fluorescein isothiocyanate–labeled PNA probe (Panagene, Daejeon, Korea; final concentration, 200 nM) at room temperature for 2 hours. The coverslips were then removed. After washing, 4′-6-diamidino-2-phenylindole was used for counterstaining. The criteria used for interpreting the FISH result were the same as previously defined [10].
2.4. Mutation analysis
2. Materials and methods
DNA extraction, amplification, and sequencing were performed as previously described [15]. The primer sequences used to amplify the TERT promoter were also described previously [16].
2.1. Tumor samples
2.5. Statistical analysis
One hundred nineteen malignant vascular tumors (88 angiosarcomas, 11 epithelioid hemangioendotheliomas [EHEs], and 20 Kaposi sarcomas [KSs]) from various sites with available paraffin blocks were retrieved from the archives of the Department of Pathology, National Taiwan
Data analyses were conducted using SPSS 19 (IBM, Armonk, NY). Categorical variables were compared using the Pearson v2 method or Fisher exact test, when appropriate. Continuous variables were analyzed using Student t test. The survival rates were calculated using the Kaplan-Meier
ALT and loss of ATRX expression in angiosarcoma Table 1
Clinical information of the angiosarcomas
Organ
n
Mean age (y)
Sex (M/F)
Skin Liver Breast Somatic soft tissue Spleen Heart Retroperitoneum/pelvis Head and neck a Bone/joint Pleura Unknown Total
34 13 7 7 6 6 5 4 3 1 2 88
74 65 44 60 65 43 56 59 57 41 42 63
22:12 8:5 0:7 2:5 2:4 4:2 4:1 2:2 2:1 0:1 0:2 46:42
a
Included cases from the nasal cavity (n = 2), oral cavity (n = 1), and larynx (n = 1).
method, and the difference in the survival curves was analyzed using the log-rank test. All statistical results were considered significant if P b .05.
3. Results 3.1. Patients and tumor samples Table 1 summarizes the clinical information of the angiosarcoma cases. Among the 88 tumors, 77 were primary angiosarcomas occurring in various organs. Six cases were secondary angiosarcomas that developed after radiotherapy for breast cancer (n = 3), nasopharyngeal carcinoma, cervical carcinoma, or sinonasal undifferentiated carcinoma (each n = 1). Three cases were angiosarcomatous transformation in nonvascular tumors: schwannoma, malignant peripheral nerve sheath tumor, or malignant phyllodes tumor (each n = 1). All the 3 tumors were radiotherapy-naïve. Two cases were metastatic tumors of unknown origin. Sixty-eight and 20 patients presented with localized and metastatic diseases, respectively. Treatment information was available for 78 patients. Twenty-one, 41, and 11 patients received surgery alone, surgery with (neo)adjuvant chemotherapy and/or radiotherapy, and
3 chemotherapy and/or radiotherapy without surgery, respectively. Five patients did not receive treatment after biopsies. Follow-up information was available for 81 patients. The mean and median follow up time were 1.2 and 0.8 years, respectively (range, 3 days to 6.7 years), and 66 patients died of disease. Of the EHE patients, 8 were female and 3 were male. Seven cases occurred in the liver, 3 occurred in the lung/pleura, and 1 occurred in the pelvis. Two were histologically malignant. Of the KS patients, 17 were male and 3 were female. Eighteen cases occurred in the skin and 2 occurred in the oral mucosa.
3.2. Loss of ATRX and DAXX expression in angiosarcomas and EHEs Loss of ATRX expression was observed in 16 angiosarcomas (18%), 1 EHE (9%), and 0 KS (0%). All ATRX-deficient angiosarcomas were primary angiosarcomas (16/77; 21%), and none of the secondary or nonvascular tumor-associated angiosarcomas were ATRX deficient. Loss of ATRX expression was diffuse in essentially all tumor cells. Except for 2 ATRX-deficient cases (one occurred in the skin and the other occurred in the liver) that exhibited focal loss of DAXX expression, DAXX expression was preserved in all cases. None of the ATRX/DAXX-deficient tumors were postchemotherapy or postradiotherapy specimens. A bar chart of the proportion of ATRX-deficient angiosarcomas of each organ is shown in Fig. 1, and representative histologic and immunohistochemical images are shown in Figs. 2 and 3. Notably, hepatic angiosarcomas had the highest rate of loss of ATRX expression (8/13; 62%), and loss of ATRX expression was seen more frequently in hepatic than nonhepatic tumors (P b .001). Except for the location and a trend of higher mitotic activity (P = .106) in ATRX-deficient tumors, no significant differences in the clinicopathological features were noted between ATRX-deficient and ATRX-proficient angiosarcomas (Table 2). The ATRX-deficient EHE was a malignant EHE occurring in the lung (Fig. 4). It was composed of a sheet-like proliferation of epithelioid cells exhibiting nuclear pleomorphism. Areas of typical EHE with plug-like intra-alveolar
Fig. 1 Bar chart of the proportion of ATRX-deficient and ALT-positive angiosarcomas of each organ. Gray: total number; black: number of ATRX-deficient (A) and ALT-positive (B) cases. ⁎Including cases from the nasal cavity (n = 2), oral cavity (n = 1), and larynx (n = 1).
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Fig. 2 Histology and ATRX immunohistochemistry. Hematoxylin and eosin stain of a high-grade hepatic angiosarcoma (A) with loss of ATRX expression (B). Hematoxylin and eosin stain of a high-grade angiosarcoma in the breast (C) with loss of ATRX expression (D). Nonneoplastic mammary duct and bile duct epithelial cells, inflammatory cells, and stromal cells showed strong nuclear expression and served as internal positive controls. Original magnification ×200.
Fig. 3 Histology, ATRX and DAXX immunohistochemistry, and telomere-specific FISH of angiosarcomas. A, Hematoxylin and eosin stain of a high-grade cutaneous angiosarcoma. B, Tumor cells were ATRX deficient. C, Most tumor cells were proficient for DAXX, but focal loss of DAXX expression was noted (inset). D, Large and bright telomere signals were detected by telomere-specific FISH. E, Hematoxylin and eosin stain of another high-grade cutaneous angiosarcoma. Tumor cells of this case were intact for both ATRX (F) and DAXX (G) expressions. H, FISH showed small telomere signals of similar size. A-C, E-G, and inset of C, original magnification ×200; D and H, ×1000.
ALT and loss of ATRX expression in angiosarcoma
5
Table 2 Comparisons of the clinicopathological features between ATRX-deficient and ATRX-proficient and between ALT-positive and ALT-negative angiosarcomas ATRX ATRX P (−) (+) Age (y), 66.1 ± mean ± SD 9.3 Sex Male 9 Female 7 Total 16 Tumor type Primary 16 Secondary 0 Total 16 Tumor site Hepatic 8 Nonhepatic 8 Total 16 Disease at presentation Localized 14 Metastasized 2 Total 16 Necrosis Absent 2 Present 10 Total 12 Mitotic activity 0-9/10 HPF 0 ≥10/10 HPF 12 Total 12 Differentiation Conventional 2 14 Poorly differentiated/ epithelioid Total 16
ALT (+)
ALT (−)
P
62.6 ± .263 17.2 .725 37 35 72 .591 61 6 67 b.001 5 65 70 .345 54 18 72 1.000 12 49 61 .106 14 46 60 .726 14 58
64.5 ± 63.4 ± .734 8.7 17.7 .872 10 30 7 23 17 53 .317 17 44 0 5 17 49 b.001 8 4 9 47 17 51 .492 15 41 2 12 17 53 1.000 2 8 11 39 13 47 .102 0 9 14 38 14 47 .717 2 10 15 43
72
17
Abbreviation: HPF, high-power field.
53
growth of cords of epithelioid cells in a myxohyaline background could be observed. ATRX expression was lost in both components.
3.3. TERT promoter mutation TERT promoter mutation was analyzed in 47 angiosarcomas (including 21 that occurred in the skin and 5 radiation-associated tumors) and 5 EHEs. Mutation was detected in only 1 radiation-associated angiosarcoma occurring in the breast (−146CNT). This case was ATRX proficient.
3.4. Telomere-specific FISH Interpretable FISH results were obtained in 70 angiosarcomas (61 primary) and 7 EHEs. Seventeen angiosarcomas were ALT positive (17/70; 24%), and all were primary angiosarcomas (17/61; 28%). None of the ALT-positive tumors were postchemotherapy or postradiotherapy specimens. FISH results were available in 5 secondary (including the TERT promoter–mutated tumor) and 2 nonvascular tumor-associated angiosarcomas, and all were ALT negative. A bar chart of the proportion of ALT-positive angiosarcomas of each organ is shown in Fig. 1, and representative FISH images are shown in Fig. 3. Two-thirds (8/12) of the hepatic angiosarcomas were ALT positive, and ALT was observed significantly more frequently in hepatic than nonhepatic tumors (P b .001). Remarkably, for angiosarcomas, ALT was highly correlated with the expression status of ATRX. Except for 2 ATRXproficient cases, all ALT-positive cases exhibited loss of ATRX expression (P b .001). The 2 discordant cases occurred in the neck soft tissue and liver. For the 2 ATRX-deficient cases that had focal loss of DAXX expression, ALT-positive cells were observed throughout the sections and not limited to the DAXX-deficient areas. No statistically significant differences in the clinicopathological features were noted between ALT-positive and ALT-negative tumors, except for the location and a trend of higher mitotic activity (P = .102) in ALT-positive tumors (Table 2).
Fig. 4 Histology, ATRX immunohistochemistry, and telomere-specific FISH of the EHE with ATRX loss. A, Hematoxylin and eosin stain of a pulmonary EHE showing the conventional morphology with cords of epithelioid cells in a myxohyaline background extending along the alveolar spaces. B, High-grade area of the same case showed a sheet-like proliferation of epithelioid cells with nuclear pleomorphism, necrosis, and bronchial cartilage invasion. C, ATRX expression was lost in both components (upper: conventional area, lower: high-grade area). D, FISH revealed the presence of large telomere signals in both components (inset: conventional area). A-C, original magnification ×200; D and inset, ×1000.
6 Of the 7 EHEs with FISH results, only the ATRX-deficient tumor was ALT positive. Thus, for EHEs, ALT status was 100% correlated with ATRX expression. In this particular tumor, ALT-positive cells were observed in both morphologically typical and malignant areas (Fig. 4).
3.5. Survival analysis Survival analysis was performed for cutaneous and hepatic angiosarcomas, for which follow-up data were available in 32 and 9 patients, respectively. The median follow-up time for cutaneous and hepatic angiosarcoma patients were 1.3 and 0.4 years, respectively. Univariate analysis revealed no survival difference between ALT-positive and ALT-negative cutaneous (P = .813) or hepatic angiosarcomas (P = .194). Similarly, no survival difference was noted between ATRX-proficient and ATRX-deficient cutaneous (P = .877) or hepatic angiosarcomas (P = .372).
4. Discussion Telomerase activation and ALT are 2 main telomere maintenance mechanisms [5]. Most (85%-90%) cancers use telomerase to maintain telomere length. Recently, recurrent UV light-signature mutations in the promoter of the TERT gene, which encodes the catalytic subunit of telomerase, were identified in cutaneous melanomas [6,7]. These mutations are also observed frequently in several other skin tumors, such as basal cell carcinoma, squamous cell carcinoma, atypical fibroxanthoma, and primary dermal pleomorphic sarcoma, supporting the role of UV light in the pathogenesis of these tumors [16,17]. However, in our study, TERT promoter mutation was not observed in cutaneous angiosarcomas, despite the fact that they occur most commonly in the head and neck skin of elderly patients. Whether the expression of telomerase is activated by other mechanisms in angiosarcoma is still unknown. ALT is used by approximately 10% to 15% of cancers as the telomere maintenance mechanism. In our study, close to 30% of primary angiosarcomas were ALT positive. Importantly, we noted that two-thirds of hepatic angiosarcomas were ALT positive. Previous studies have found that ALT phenotype was an adverse prognostic factor in osteosarcoma, liposarcoma, and leiomyosarcoma [14,18–20]. However, no survival difference was noted between ALT-positive and ALT-negative angiosarcomas in our study. Another remarkable finding is that we observed an excellent correlation between ALT and loss of ATRX expression. ALT was first linked with the inactivation of either ATRX or DAXX in pancreatic neuroendocrine tumors [11,12]. Although loss of DAXX is more common than loss of ATRX in pancreatic neuroendocrine tumors [11], loss of ATRX is much more frequent in ALT-positive gliomas, leiomyosarcomas, and cell lines [11,14,21]. In our series, 2 ATRX-deficient angiosarcomas also exhibited focal loss of DAXX expression. The significance of this finding is unclear, but ALT-positive cells were not limited to the DAXX-deficient areas, and no
J. -Y. Liau et al. morphologic differences between DAXX-proficient and DAXX-deficient areas were appreciated. In our series, all secondary angiosarcomas were ATRX proficient and ALT negative (available in 5 cases). Secondary angiosarcomas are characterized by MYC gene amplification and protein expression [1–3]. c-Myc is a basic helix-loop-helix and leucine zipper transcription factor involved in many cellular processes such as cell proliferation, differentiation, and apoptosis. The TERT gene is one of the direct targets of c-Myc [22,23]. We speculate that telomerase activation, instead of ALT, is used to maintain telomeres in secondary angiosarcomas. In addition to angiosarcomas, we also analyzed 11 EHEs and 20 KSs. One of the EHEs was ATRX deficient and ALT positive in both conventional and morphologically malignant areas. EHE is molecularly characterized by a t(1;3) translocation and WWTR1-CAMTA3 gene fusion [24,25]. Montgomery et al [26] have suggested that ALT is not a feature of translocation-associated sarcomas. Although the number of EHE cases investigated was low, our results suggested that ALT could be used in rare cases of translocation-associated sarcomas. In our study, all KSs were ATRX/DAXX proficient. Although we did not perform FISH for the KSs, our results were consistent with those of Heaphy et al [10], who found that all 55 KSs analyzed were ALT negative. In conclusion, we demonstrate that close to 30% of primary angiosarcomas use ALT as the telomere maintenance mechanism. We also find that in angiosarcomas, the ALT phenotype is highly correlated with loss of ATRX expression, and this phenotype is frequently observed in hepatic angiosarcomas. Recently, ALT-positive cells have been shown to be sensitive to ataxia telangiectasia and Rad3-related kinase inhibitors [27]. Further study should be undertaken to determine whether patients with ALT-positive angiosarcomas benefit from Rad3-related kinase inhibitor treatment.
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