Irradiation-induced angiosarcoma and anti-angiogenic therapy: A therapeutic hope?

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Research Article

Irradiation-induced angiosarcoma and anti-angiogenic therapy: A therapeutic hope? Amalia Azzaritia,n,1, Letizia Porcellia,1, Anita Mangiab, Concetta Saponarob, Anna E. Quatralea, Ondina S. Popescuc, Sabino Strippolid, Gianni Simonec, Angelo Paradisoe, Michele Guidad a

Clinical and Preclinical Pharmacology Laboratory, National Cancer Research Centre, Istituto Tumori Giovanni Paolo II, Viale O. Flacco, 65, 70124 Bari, Italy b Functional Biomorphology Laboratory, National Cancer Research Centre, Istituto Tumori Giovanni Paolo II, Viale O. Flacco, 65, 70124 Bari, Italy c Department of Pathology, National Cancer Research Centre, Istituto Tumori Giovanni Paolo II, Viale O. Flacco, 65, 70124 Bari, Italy d Medical Oncology Unit, National Cancer Research Centre, Istituto Tumori Giovanni Paolo II, Viale O. Flacco, 65, 70124 Bari, Italy e Experimental Medical Oncology, National Cancer Research Centre, Istituto Tumori Giovanni Paolo II, Viale O. Flacco, 65, 70124 Bari, Italy

article information

abstract

Article Chronology:

Angiosarcomas are rare soft-tissue sarcomas of endothelial cell origin. They can be sporadic or caused

Received 4 September 2013

by therapeutic radiation, hence secondary breast angiosarcomas are an important subgroup of patients.

Received in revised form

Assessing the molecular biology of angiosarcomas and identify specific targets for treatment is

16 December 2013

challenging. There is currently great interest in the role of angiogenesis and of angiogenic factors

Accepted 18 December 2013

associated with tumor pathogenesis and as targets for treatment of angiosarcomas. A primary cell line

Available online 31 December 2013

derived from a skin fragment of a irradiation-induced angiosarcoma patient was obtained and utilized

Keywords:

to evaluate cell biomarkers CD31, CD34, HIF-1alpha and VEGFRs expression by immunocytochemistry

Angiosarcoma

and immunofluorescence, drugs cytotoxicity by cell counting and VEGF release by ELISA immunoassay.

VEGF

In addition to previous biomarkers, FVIII and VEGF were also evaluated on tumor specimens by

VEGFR

immunohistochemistry to further confirm the diagnosis. We targeted the VEGF–VEGFR-2 axis of tumor

Caprelsa

angiogenesis with two different class of vascular targeted drugs; caprelsa, the VEGFR-2/EGFR/RET

Bevacizumab

inhibitor and bevacizumab the anti-VEGF monoclonal antibody. We found the same biomarkers

Citotoxicity

expression either in tumor specimens and in the cell line derived from tumor. In vitro experiments demonstrated that angiogenesis plays a pivotal role in the progression of this tumor as cells displayed high level of VEGFR-2, HIF-1 alpha strongly accumulated into the nucleus and the pro-angiogenic factor VEGF was released by cells in culture medium. The evaluation of caprelsa and bevacizumab cytotoxicity demonstrated that both drugs were effective in inhibiting tumor proliferation. Due to these results, we started to treat the patient with pazopanib, which was the unique tyrosine kinase inhibitor available in Italy through a compassionate supply program, obtaining a long lasting partial response. Our data

n

Corresponding author. Fax: þ39 080 5555986. E-mail address: [email protected] (A. Azzariti). 1 Equally contributed.

0014-4827/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.12.018

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suggest that the study of the primary cell line could help physicians in choosing a therapeutic approach for patient that almost in vitro shows chances of success and that the anti-angiogenetic agents are a reliable therapeutic opportunity for angiosarcomas patients. & 2014 Elsevier Inc. All rights reserved.

Introduction Angiosarcomas (AS) account for o2% of all soft tissue sarcomas and represent one of the most aggressive forms. They include a heterogeneous set of malignant mesenchymal tumors with a vascular derivation from endothelial cells of blood or lymphatic vessels [1]. Potentially arising in many anatomic sites and organs, they may be sporadic or the result of exposure to environmental conditions such as radiation, long-term lymphedema (Stewart– Treves syndrome), trauma, chronic infection or chemical agents (e.g., vinyl chloride, arsenic medication, steroids). Despite this variability, AS shares an invasiveness behavior consisting of local recurrence and an early hematogenous and lymphogenous dissemination. Clinical presentations of AS are heterogeneous and include superficial and visceral forms. Prognosis depends on the precocity of the diagnosis and the extension of the disease. For advanced disease prognosis remains poor with a progression-free survival of about 4 months and overall survival of about 8 months [2]. Regarding therapeutic options, large resection, followed if possible by adjuvant radiotherapy, is the cornerstone of curative intent treatment of localized forms [3]. For metastatic or locally advanced AS, current cytotoxic agents have a very modest impact on the disease with doxorubicin and paclitaxel providing the better results in terms of higher response rate and longer progression-free survival [4]. Recently, a strong body of biological evidence regarding the key role of angiogenesis has been accumulated in this particular type of sarcoma, supporting further investigations to explore the role of anti-angiogenic agents as an alternative therapeutic option to chemotherapy. Nevertheless, preliminary clinical experiences with antiangiogenic drugs reported modest and conflicting results [5,6]. Given the limited number of cases and the heterogeneity of the study populations, a useful interpretation of the variable efficacy of these agents may be driven by studies on primary cultures. In particular, AS tissues isolated from patients can be utilized to obtain primary cultures which allow to test the efficacy of the drug and to assess the expression of possibly predictive biomarkers. Therefore, reliable and relatively rapid pharmaco-sensitivity screening of an individual patient’s AS may allow the identification of the best regimen for personalized therapy of the patient starting from the same patient’s tumor biology. One of these drug screening tests is based on the utilization of short-term cell cultures which closely resemble the original cancers, mainly as regards features that are responsible for the pharmaco-sensitivity of the tumor. To offer insight into this process, we describe here the case of the anti-angiogenic responsiveness to the tyrosine kinase inhibitor (TKI) vandetanib/ZD6474 (Caprelsa) and the monoclonal antibody (mAb) bevacizumab (Avastins) of a primary cell line

derived from a woman with an advanced AS arising on an area irradiated for a previous breast cancer. We demonstrated that the in vitro study of the primary cell line allowed us to verify the activity of these two different therapeutic agents addressing the choice of drug with greater chances of success in AS patients.

Materials and methods Drugs and chemicals Vandetanib/ZD6474 (Caprelsa) was provided by AstraZeneca Pharmaceuticals (Macclesfield, UK.). Stock solutions were prepared at 20 mM in DMSO and stored in aliquots at –20 1C. Bevacizumab (Avastins) was commercially available. Further dilutions were made in medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50,000 UL-1 penicillin and 80 μM streptomycin.

Immunohistochemistry (IHC) The surgically excised sample was fixed in neutral 10% buffered formalin, embedded in paraffin, then cut at 4-μm thickness and stained with Haematoxylin and Eosin. In addition, 4-μm sections were incubated with CD34 (mouse monoclonal antibody, clone QBEND-10, diluted 1:50; Novocastra Lab. Ltd., UK), Factor VIII (rabbit polyclonal antibody anti-human von Willabrand factor, diluted 1:300 Dako, Denmark), vascular endothelial growth factor (VEGF) (rabbit polyclonal antibody, clone A-20, diluted 1:150; SantaCruz, USA), hypoxia inducible factor-1α (HIF-1α) (rabbit polyclonal antibody, clone H206, diluted 1:50; SantaCruz, USA) as previously described [7,8] and Ki67/MIB1 (mouse monoclonal antibody, clone MIB1, diluted 1:100; Dako, Denmark), overnight at 4 1C. The sections were then incubated with biotinylated linked secondary antibodies for 60 min at room temperature. Slides were developed with 3-amino-9-ethylcarbazole substrate-chromogen (LSAB2 System-HRP; Dako, Denmark) for VEGF and HIF-1α. For CD34, Factor VIII and Ki67/MIB1, 30 ,30 -diaminobenzidine tetrahydrochloride (Dako, Denmark) were utilized. Slides were counterstained with haematoxylin and examined by light microscopy. Each batch of staining included a negative control section treated with phosphate-buffered saline instead of primary antibodies. Tumor immunoreactivity was scored by two investigators who participated in this study. Short-term cell culture. A skin fragment from a AS patient biopsy was established in culture, after obtaining the informed consent. The tissue (100 mg) was washed twice in PBS (2.7 mM KCl, 1.5 mM KH2PO4, 0.14 M NaCl, 8.1 mM Na2HPO4  7H2O) and minced with surgical blades under aseptic conditions and layered in 60-mm-diameter dishes in high-glucose Dulbecco0 s modified Eagle0 s medium (DMEM) supplemented with 10% (v/v) fetal

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bovine serum (FBS), 1% (v/v) L-glutamine, 1% (v/v) penicillin/ streptomycin, at 37 1C in a humidified atmosphere of 5% CO2. After 7 days, the medium was changed. At day 14, the tissue fragments and the cells growing around were washed in PBS and harvested using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA. They were subcultured up to 8 months after explantation and having discarded tissue fragments. At confluence, the cells were harvested and reseeded at a 1:2 split ratio. The medium was changed every 3 days. Cultured cell immunocytochemistry (ICC). Cells were harvested and cytospin preparations were made. The specimens were subsequently air-dried overnight and fixed for 7 min in acetone at room temperature. The slides were incubated with diluted primary antibody anti-CD31, anti-CD34 and anti-HIF-1α antibody (SantaCruz, USA) in 1% PBS/BSA solution for 1 h at room temperature (RT). After 3 wash steps with PBS, cells were then incubated with EnvisionþSystem-HRP labeled polymer secondary antibody (Dako, Denmark) for 1 h. Color development was with AEC ready to use solution (Dako, Denmark) and nuclei stained with hematoxylin. Immunofluorescence (IF). Angiosarcoma cells were seeded onto glass Lab-Tek Chamber Slides (8 wells; 0.8 cm2/well) at a density of 20  104 per well (400 ml/well medium) and incubated for 2 days at 37 1C. Then, the growth medium was removed, and cell monolayers were washed twice with HBSS solution and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. Cells were washed twice with PBS and permeabilized by Triton X-100 [0.1% (w/v) in PBS for 5 min at room temperature. Nonspecific binding sites were blocked for 30 min at room temperature with PBS containing 5% rabbit/mouse serum (DakoCytomation). After serum removal, and without further washing, cells were incubated with a mouse anti-VEGFR2 monoclonal antibody FLK-1, diluted 1:20 in PBS with 4% bovine serum albumin, for 60 min at room temperature or anti-VEGFR1 rabbit polyclonal FLT-1, diluted 1:20. Both VEGFR-2 and VEGFR-1 staining were revealed by incubation with FITC-conjugated goat anti-mouse antibody and FITC-conjugated goat anti-rabbit antibody (1:50 BD Pharmigen) respectively, for 60 min at room temperature. After a final wash with water instead of PBS, the slides were mounted in DAPI I Counterstain in antifade mounting solution (Abbott) and images were obtained on a BX40 microscope (Olympus) equipped with 40  objective and with a SenSys 1401E-Photometrics charge-coupled device camera. FITC was excited using the 488 laserline. The same settings was used for both pictures to allow comparison between the two biomarkers. Control for nonspecific staining was the replacement of specific antibody with a non-specific antibody from the same class, mouse/rabbit IgG (DakoCytomation).

Analysis of VEGF release Cells, plated in 24 multiwells plates, were incubated with 10 μg/ ml Bevacizumab or 10 μM Caprelsa for 1–3 days. 200 μl of cell surnatant from each sample was utilized for VEGF release by the Quantikine—Human VEGF immunoassay (R&D Systems—Minneapolis, USA), following the manufacturer0 s instruction. The medium of AS cells was used as Blank and subtracted from each samples.

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Cell proliferation assay Cell growth inhibition was determined by counting cells, as described in Azzariti et al. [9]. Starting from the plasma concentration, 10 μg/ml Bevacizumab [10] and 2 μM Caprelsa [11], each drug was given at 2–10–50 μg/ml and 0.4–2–10 μM for 3 days, respectively.

Statistical analysis All in vitro experiments were performed in triplicate, and results have been expressed as the mean7standard deviation (SD) unless otherwise indicated.

Results Case report This is the case of a 71 year old Caucasian female with a past medical history of left breast invasive ductal carcinoma, treated by quadrantectomy and axillary lymphadenectomy combined with postoperative external beam irradiation with a dose of 50 Gy. Nine years after the initial breast cancer, the patient developed a purpleblue painless skin lesion at the site of surgical scar which evolved into an ulcerated lesion of about 1.5 cm in diameter. The histological findings of the lesion biopsy evidenced a high grade cutaneous angiosarcoma which responded to the criteria proposed by Cahan and modified by Arlen for the diagnosis of radio-induced sarcoma [12]. She therefore underwent a left mastectomy achieving an R0 resection. CT scans of the chest, abdomen and pelvis did not reveal any evidence of tumor and she remained in remission for 24 months, after which a second skin lesion of about 2 cm in diameter appeared on the pre-sternal area with evidence of a necrosis-hemorrhagic component. The lesion was completely removed, but just three months later the patient experienced a local relapse with multifocal nodes. Owing to the infiltration of the lesions into the chest wall, she was no longer considered suitable for surgical treatment. The patient was subsequently treated with local electrochemotherapy with intravenus bleomicin, obtaining a complete remission lasting for 12 months. At this time, a new CT scan evidenced a mediastinal lymph node package with skeletal and soft tissue chest wall involvement. She started systemic treatment with docetaxel and epirubicin at a dose of 50 mg/mq for both drugs, repeated every 2 weeks. Nevertheless after 4 cycles the patient showed disease progression with increased size of soft tissue chest lesions and additional lymph node involvement. Due to the interesting results from our Research Laboratory accurately described below, showing a strong inhibition of the cellular growth with the antiangiogenic drugs Caprelsa and Bevacizumab, we proposed to the patient a new therapy with an anti-angiogenic drug. We used pazopanib that was the unique TKi drug available in Italy through in a compassionate supply program.

Pathologic findings The histopathologic examination of the biopsy from the second skin lesion showed a heterogeneous high grade neoplasm made of anastomosing vascular channels lined by atypical endothelial

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Fig. 1 – Post radiotherapy angiosarcoma. (A) Solid areas anastomosing vascular channels and (B) pleomorphic hyperchromatic nuclei with numerous mitosis (thick arrows).

Fig. 2 – Immunohistochemical staining of angiosarcoma. (A) Diffuse membranous and cytoplasmic immunoreactivity of neoplastic endothelial cells for CD31. (B) Diffuse membranous and cytoplasmic immunoreactivity of neoplastic endothelial cells for CD34. (C) Focal positivity for von Willebrand factor (FVIII). (D) VEGF diffuse and strong cytoplasmic immunoreactivity of tumor cells. (E) Nuclear immunoreactivity of HIF-1α of tumor cells. (F) Ki-67 positive tumor cell nucleus.

cells (Fig. 1A), and pleomorphic hyperchromatic nuclei mixed with solid areas without necrosis (Fig. 1B). Brisk mitotic activity was evident especially in the more solid areas (Fig. 1B). The neoplastic cells were diffusely immunoreactive to CD31 (Fig. 2A), CD34 (Fig. 2B), and showed focal positivity for FVIII (Fig. 2C). VEGF protein expression was mainly observed in the cytoplasm of tumor cells (Fig. 2D). Immunostaining with HIF-1α produced cytoplasmic staining in some cells, but only cells with nuclear protein accumulation indicative of transcriptional activity were considered positive [Fig. 2E]. Ki67/MIB1 was positive in about 20% of the examined neoplastic cells (Fig. 2F).

Establishment and characterization of an angiosarcomaderived short-term cell line. The cells began to grow out of the explant after 6 days. Throughout the culture, they exhibited the same morphology. They were spindle-shaped with a morphology similar to that of transformed

fibroblasts, forming numerous multilayer nodules. The in vitro growing behavior of our isolated cells was similar to that of other fibroblasts with a doubling time of about 50 h. They have been serially passaged at a split ratio of 1:2 once a week and are still proliferating. Cells were characterized at passage 4 by analyzing the expression of CD31 and CD34 by ICC. Cells expressed both markers showing the same results obtained during the preliminary immunohistochemistry analysis for diagnosis (data not shown). This characterization confirmed that cells derived from the angiosarcoma specimen are similar to the tissue from which they derived.

Angiosarcoma cells and angiogenesis as a therapeutic target The hypothesis of the utilization of angiogenesis as a therapeutic target was investigated by characterizing cells for the expression

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Fig. 4 – VEGF release and cell growth inhibition by Caprelsa and Bevacizumab. (A) VEGF release was analyzed in surnatants from the obtained short-term AS cell culture after treatment with each drugs after 1 and 3 days. (B) Cytotoxicity of the two antiangiogenic drugs was determined as described in “Materials and methods” section.

Fig. 3 – Immunocytochemical staining for HIF-1α. (A) Nuclear localization of HIF-1α in AS cells by ICC (thick arrows), (B) VEGFR-2 and (C) VEGFR-1 expression in AS cells by IF.

and release of some angiogenic factors and by the measurement of cell proliferation after antiangiogenic drug(s) administration. We evaluated the expression of HIF-1α in cells by IHC and, as shown in Fig. 3A, angiosarcoma cells at passage 4 showed a strong staining for this marker. The presence of nuclear HIF-1α is in agreement with results obtained in tissue (Fig. 2E). Expression of VEGFR-2 and VEGFR-1 was detected on AS cells cultured for 48 h

on coverslips. VEGFR-2 was found strongly expressed on membrane, in the cytosolic compartment and in the perinuclear region (Fig. 3B) and VEGFR-1, less abundant than VEGFR-2, showed but with the same pattern of expression (see Fig. 3C). The IHC determination demonstrated a strong VEGF staining in the patient angiosarcoma tissue (Fig. 2D). In our characterization of cell features we included the evaluation of VEGF release by cells in culture media and, as reported in Fig. 4A, it was released and modulated by drug treatment as reported below. These data provide the scientific basis to investigate the efficacy of drug(s) to hit cells from angiosarcoma by blocking angiogenesis through the inhibition of the receptor VEGFR1/2 and the capture of VEGF by Caprelsa and Bevacizumab, respectively. The exposure of cells for 3 days to Caprelsa and Bevacizumab induced a strong reduction of proliferation as shown in Fig. 4B with a higher efficacy due to the exposure to the TKI. The release of VEGF after the exposure to these anti-angiogenic compounds showed that Caprelsa induced an early slight decrease and a progressive increase of this proangiogenic factor release in function of time exposure, conceivably because of the inhibition of the VEGF receptor, while the capture of VEGF after Bevacizumab administration was stable until 3 days.

Discussion As a dangerous long-term side effect of breast cancer adjuvant treatment, the incidence of secondary radio-induced angiosarcoma (AS) is likely to increase. The risk of this secondary cancer has been estimated to be around 1% with a cumulative incidence

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range from 0.6 to 0.9 per 1000 breast cancer cases over 15 years. The median latency period of development varies from 3 to 12 years [13–15]. Clinicians have been aware of this delayed complication since 1987 when Body described the first case of radioinduced breast AS. In fact, its median overall survival is between 1 and 3 years [16,17]. Regarding the treatment, problems come from some peculiar features of this sarcoma such as its high grade histology, the involvement of elderly patients, and the occurrence in previously irradiated fields [1]. Surgery is often not enough, leading to a local and distant relapse rate of 75% even when disease-free margins are present [4]. Accordingly, medical treatments remain the only option for these patients. Current cytotoxic agents have only a very modest impact, with doxorubicin and paclitaxel providing better results with a progression-free survival of about 4 months and an overall survival of about 8 months [4,18]. Recently, a body of knowledge regarding the role of VEGF and its receptor (VEGFR) in angiosarcoma, epithelioid hemangioendothelioma (EHE), and hemangiopericytoma/solitary fibrous tumor (HPC/SFT) has suggested that angiogenesis plays a crucial role in tumor progression. Thus, the inhibition of the VEGF/VEGFR pathway may be a rational and effective therapy for these neoplasms [19]. Several phase II trials have investigated the efficacy of some antiangiogenic drugs such as Bevacizumab, an anti-VEGF antibody, as well as several VEGFR tyrosine kinase inhibitors (TKIs) such as sunitinib, sorafenib, and pazopanib in various types of advanced soft tissue sarcoma, reporting controversial and not definitive results. Although response rates and progression-free survival were generally low, some patients demonstrated response or durable disease stabilization with these therapies [20]. Regarding AS, first of all experimental models of this pathology is quite limited and consequently information of its biology are scanty. Recently, Hoshina et al. have established a novel experimental angiosarcoma model that could have high impact on AS study [21]. The experimental approach utilized by authors is different from the protocol we applied in this report, i.e., they initially implanted AS tissue in mice and then, they established the cell line that utilized for in vitro and in vivo studies. They evaluated bevacizumab and sunitinib (a TKI) effectiveness without obtaining favorable evidences even if their experimental condition, such as cell starvation, addition of growth factors, etc should be taken into account to explain differences with other studies with positive outcome. A critical comparison between our experimental approach (short term culture) and xenotransplantation in mice of tumor fragments before establishment of cell lines [21] highlighted the advantages and disadvantages of the two methods. Primary cell cultures retain the characteristics of the cells in vivo but have a finite number of duplications and die due to senescence. Moreover, primary cells are a heterogeneous population of which the preparation is complex. Conversely, cell lines obtained after transplantation in mice are easy to culture and use and provide highly reproducible results. However, as Dangles-Marie reported, cells derived from the same tumor fragment using the two different protocols—direct isolation and transplantation—had no changes in morphology or gene expression but marked differences in karyotype, growth kinetics, and chemosensitivity [22]. Therefore, the choice of which method to use depends on the type of study to be carried out, and in our opinion, short term culture is more suitable to verify the

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possibility of a therapeutic approach which may help clinicians. Nevertheless, beyond several promising case reports, two large phase II trials investigated bevacizumab and sorafenib in metastatic disease [23,24]. Bevacizumab as a single agent evidenced an effective profile with a partial response of 17%, stabilization of 50% and a TTP of 6.2 months; enough to assume a potential association with chemotherapeutic drugs or radiation treatment [23]. On the contrary, patients treated with Sorafenib showed limited antitumor activity for both visceral and superficial AS with very short tumor control with a PFS of 1.8 and 3.8 months in cutaneous and visceral AS, respectively [24]. Some case reports of AS of the scalp showed successful results with sorafenib as a single agent [25] or in combination with paclitaxel [26]. Dramatic and durable efficacy of imatinib in an advanced AS has also been reported in patients without detectable KIT and PDGFRA mutations [27]. Such AS samples and cell lines showed an up-regulation of vascular specific receptor tyrosine kinase including TIE1, KDR, SNRK, TEK, and FLT1 [28]. Among these kinases, KDR showed activating mutations which conferred the most sensitivity to inhibitors such as sorafenib. The same effect seemed to exert the amplification of FLT4, a tyrosine kinase encoding the vascular endothelial growth factor receptor 3 (VEGFR3) and tunica internal endothelial cell kinase 2 (Tie2) receptor [29,30]. In this direction, we would establish a key step in planning the best available treatment for our patient heavily pre-treated with chemotherapeutic agent. In our primary AS cell line, the strong expression of VEGFR-2 together with a less abundant VEGFR-1 and the expression of HIF1α seemed to orchestrate the proliferate behavior and the release of VEGF suggesting the existence of an autocrine VEGF–VEGFR1/2 signaling in such cells and that anti-angiogenic therapy, mediated by inhibition of VEGFR-2 or VEGF capture, can be a promising therapeutic opportunity. This evidence is in agreement with Al-Salam’s results who reported that in a different AS subtype, the bilateral primary AS, the overexpression of HIF-1α, through the induction of VEGF, platelet-derived growth factor B etc., plays a central role in development of such cancer pathology [31]. Thus, HIF-1a could be considered a relevant intermediate in the cancerogenesis of AS and a wide characterization of it in different histotype of AS could enlighten this point. The hypothesis of antiangiogenic treatment in our cell model, was verified utilizing caprelsa and bevacizumab which proved to inhibit cell growth with different efficacy and to modulate the pattern of secretion in an opposing way. Whereas Caprelsa strongly inhibited cell growth enhancing the medium concentration of VEGF, Bevacizumab decreased the level of the soluble growth factor. The explanation for this opposing behavior is in the different mechanism of action of the two drugs. Bevacizumab, when binding VEGF, renders it no longer available for detection by ELISA assay, therefore a decrease in the level of this growth factor is observed in the culture medium. Conversely, Caprelsa induced a marked increase of VEGF over time, is in agreement with that already shown for a similar drug. Medinger and coauthors reported an increase in VEGF level following exposure to cediranib, another VEGFR inhibitor, explaining this as being due to either a compensation of tumorinduced hypoxia by cells or a tumor-independent VEGF increase as a stress response to the perturbation of VEGF signaling [32]. Due to these interesting results from our Laboratory, we proposed to the patient a new therapy with the TKI Pazopanib,

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the unique TKi available in Italy in a compassionate supply program. We started the therapy at the dose of 800 mg/die orally and after 1 months of therapy the patient remained in ongoing stable disease.

Conclusions In our mind, given the lack of effectiveness of standard therapy for AS patients , any attempt to identify effective systemic treatments for this fascinating group of malignancies may become important. The quick characterization of cell response to anti-angiogenic drugs may have relevant implication to suggest angiogenesis as a target for therapy for AS patients.

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[8]

[9]

[10] [11]

Competing interests [12]

The authors declare that they have no competing interests.

Authors0 contributions AA: conceived the study, coordinated the experimental design of the preclinical pharmacology study, analyzed the data and drafted the manuscript; LP: isolated the short term culture, carried out the in vitro pharmacology study and the critical revision of results; AM: carried out the biomorphological study and was involved in drafting the manuscript; CS: performed the biomorphological study; AEQ: carried out the in vitro pharmacology study; OSP: collected the angiosarcoma specimen and was involved in drafting the manuscript; SS: helped to draft the manuscript; GS: coordinated the anatomopathological characterization of the specimen for diagnosis; AP: revised the manuscript; MG: was responsible for the medical care of the patient, analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.

[13]

[14]

[15]

[16]

[17]

[18]

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