Modern Pathology (2013) 26, 1032–1040 1032 & 2013 USCAP, Inc All rights reserved 0893-3952/13 $32.00 Expression of angiopoietin-TIE system components in angiosarcoma Darya Buehler1, Patrick Rush1, Jason R Hasenstein2, Stephanie R Rice2, Gholam Reza Hafez1, B Jack Longley3 and Kevin R Kozak2 1Department of Pathology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 2Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA and 3Department of Dermatology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA Angiosarcoma is an aggressive malignancy of endothelial differentiation. Potential roles of the endothelial angiopoietin-tunica interna endothelial cell kinase (ANGPT-TIE) system in angiosarcoma diagnosis, pathogenesis, prognosis and treatment are undefined. To examine the expression and prognostic significance of angiopoietin-1, angiopoietin-2, TIE1 and TEK (TIE2) proteins in angiosarcoma, we immunohistochemically evaluated clinically annotated human angiosarcoma samples. Correlations of protein expression with overall survival and pathological features were explored. The cohort included 51 patients diagnosed with angiosarcoma at the age of 30–86 years (median 67). The 5-year overall survival was 45% with a median of 26 months. Moderate to strong expression of angiopoietin-1, TIE1 and TEK (TIE2) was identified in the majority of angiosarcomas and moderate to strong expression of angiopoietin-2 was observed in 42% of angiosarcomas. Increased angiopoietin-1 expression correlated with improved survival. Non-significant trends toward longer survival were also observed with increased TIE1 and TEK (TIE2) expression. Increased expression of angiopoietin-2, TIE1 and TEK (TIE2) was associated with vasoformative architecture. No differences in expression of these proteins were observed when patients were segregated by age, gender, presence or absence of metastases at diagnosis, primary tumor location, radiation association or the presence of necrosis. We conclude that components of the ANGPT-TIE system are commonly expressed in angiosarcomas. Reduced expression of these proteins is associated with non- vasoformative and clinically more aggressive lesions. Modern Pathology (2013) 26, 1032–1040; doi:10.1038/modpathol.2013.43; published online 5 April 2013 Keywords: angiosarcoma; angiopoietin; sarcoma; TEK; TIE Angiosarcoma is an aggressive soft tissue sarcoma Given the vascular differentiation of angiosarco- of endothelial differentiation. Current therapies mas, recent interest has turned to therapies that are woefully inadequate. Patients presenting with target endothelium-restricted signaling pathways. localized disease have a 5-year overall survival of Vascular endothelial growth factors (VEGFs) and o60%. Those presenting with metastatic disease their receptors are expressed in angiosarcoma.7–14 fare far worse with median survival generally o1 Moreover, activating mutations in KDR (VEGFR-2), year.1–5 The poor survival of patients with the major pro-angiogenic VEGF receptor, have been metastatic disease is largely attributable to limited reported in a small subset of angiosarcomas.15 systemic therapy options. Reported response rates Consequently, the therapeutic use of anti-VEGF with conventional cytotoxic chemotherapy are agents has been explored in this disease. The highly variable, however, they are generally similar pan-VEGF receptor tyrosine kinase inhibitor, to the disappointing response rates seen in other sorafenib, has modest single-agent activity in soft tissue sarcomas (ie, 20–30%).6 angiosarcoma.16,17 Similarly, the humanized anti- VEGF monoclonal antibody, bevacizumab, elicits objective responses in a minority of angiosarcoma 18,19 Correspondence: Professor KR Kozak, MD, PhD, Department of patients. Human Oncology, University of Wisconsin School of Medicine The ANGPT-TIE pathway is largely confined to and Public Health, Wisconsin Institute for Medical Research, vasculature and consists of two tyrosine kinase 1111 Highland Avenue, Madison, WI 53705, USA. E-mail: [email protected] receptors, TIE1 and TEK (TIE2), and three Received 12 September 2012; revised 7 January 2013; accepted 8 corresponding ligands, angiopoietins-1, -2 and -4. January 2013; published online 5 April 2013 Although considerable context-dependent altera- www.modernpathology.org ANGPT-TIE system in angiosarcoma D Buehler et al 1033 tions in function have been observed, generally, chemotherapy. After confirmation of the diagnosis, angiopoietin-1 acts as a TEK (TIE2) receptor agonist the most representative viable areas of the formalin- and angiopoietin-2 as a TEK (TIE2) receptor antago- fixed paraffin-embedded angiosarcomas were formatted nist. The TIE1 receptor has no known ligand and into a 4.5 Â 2 Â 1cm3 recipient tissue microarray appears to function primarily as a TEK (TIE2) in triplicate 1.0 mm cores using the MTA-1 receptor antagonist through interference of angio- manual tissue microarrayer (Beecher Instruments, poietin-1 receptor interaction.20 Angiopoietin-1 has Sun Prairie, WI, USA). The 11 angiosarcoma cases a key role in maintaining the integrity of existing previously reported to express TEK (TIE2) in whole- vessels and enhances endothelial cell survival, mount tissue sections were included in the tissue proliferation and migration in some settings.21–25 microarray.29 In five cases (two breast, two head and Angiopoietin-2 appears to have a critical role in neck, and one Stewart–Treves), limited tumor tissue vascular remodeling and angiogenesis.26 The necessitated use of only two tissue cores. Sections functions of the more recently described ligand, were cut (5 mm) and standard H&E and CD31 stained angiopoietin-4, are less well understood and its slides were examined to verify the presence expression is largely limited to the lung.27 of viable angiosarcoma. Little information is available regarding the expression of ANGPT-TIE pathway components in 12,15,28 human angiosarcoma samples. Recently, after Immunohistochemistry identifying TEK (TIE2) expression in 11 human angiosarcomas, we reported that TEK (TIE2) inhibi- Tissue sections (5 mm) were cut using traditional tion delayed angiosarcoma growth in two murine water bath technique and dried overnight at room models of the disease.29 To examine potential roles temperature. Slides were deparaffinized in subse- for ANGPT-TIE pathway components in angio- quent xylene and ethanol incubations followed by sarcoma diagnosis, pathogenesis, prognosis and heat induced epitope retrieval using the Lab Vision treatment, we assessed the expression of angio- PT module (Thermo Fisher Scientific, Fremont, CA, poietin-1, angiopoietin-2, TIE1 and TEK (TIE2) by USA) with Lab Vision citrate buffer pH 6.0 at 98 1C immunohistochemistry in 51 clinically annotated for 20 min without boiling. All staining was per- human angiosarcoma samples. formed at room temperature using the Lab Vision 360 automated staining system. Biocare Medical (Concord, CA, USA) reagents were used except Materials and methods where noted. Endogenous peroxidase was blocked for 5 min with Peroxidazed 1. Nonspecific protein Patients and Angiosarcoma Specimens binding was inhibited by a 10-min (angiopoietin-2, The study was approved by the Institutional TIE1, TEK (TIE2)) or 30 min (angiopoietin-1) block Review Board. Patients were identified using a with Sniper. For angiopoietin-1, nonspecific avidin surgical pathology database that spanned the years binding was blocked using the Avidin Biotin kit, 1987–2012. Candidate paraffin blocks were col- incubating 15 min for each reagent. Primary anti- lected and the angiosarcoma diagnosis was con- bodies were diluted with DaVinci Green Antibody firmed by a pathologist with specific interest in soft Diluent (angiopoietin-2, TIE1, TEK (TIE2)) or tissue and skin tumors (GRH and BJL). The presence Renaissance background reducing diluent (angio- or absence of necrosis was noted. Tumor architec- poietin-1). Slides were incubated with primary tural pattern was assessed according to Shon et al30 antibodies for 60 min as follows: angiopoietin-1 as follows: vasoformative (475% of tumor forming (goat anti-angiopoietin-1 IgG, 1:50, R&D Systems, vascular channels with identifiable lumina), non- Minneapolis, MN, USA), angiopoietin-2 (mouse vasoformative (475% of tumor demonstrating anti-angiopoietin-2 IgG, 1:50, Santa Cruz Biotech- architecturally solid epithelioid or spindle cell nology, Santa Cruz, CA, USA), TIE1 (mouse anti- morphology without vascular channels) or mixed. TIE1 IgG, 1:300, R&D Systems), TEK (goat anti-TEK Patient records were accessed for age at diagnosis, IgG, 1:50, R&D Systems). For angiopoietin-2 and sex, disease distribution at diagnosis, date of last TIE1 stains, slides were subsequently incubated for follow-up or death, primary site and tumor size. 20 min with probe and HRP-polymer from mouse Clinical notes and radiation records were reviewed. HRP-Polymer kit. For TEK (TIE2), similar incuba- Angiosarcomas were deemed radiation associated if tions were performed with the goat HRP-polymer they occurred within or adjacent to prior radiation kit. For angiopoietin-1, primary incubation was fields. followed by incubation for 15 min with biotinylated swine anti-goat IgG (1:50, Invitrogen, Carlsbad, CA, USA) followed by a 15-min streptavidin-HRP treat- Tissue Microarray Construction ment. Betazoid DAB and Mayer’s hematoxylin were each incubated for 1 min. Slides were washed with Fifty-one