Case report: whole exome sequencing of primary cardiac angiosarcoma highlights potential for targeted therapies

Primary cardiac tumors are very rare; the reported incidence in most autopsy and surgery series varies from 0.001 to 0.3% [1‐3]. Of tumors that arise in the heart, most are benign and approximately 25% are malignant. About 95% of the malignant tumors are sarcomas, and they comprise 10 to 15% of the total primary cardiac tumors. Sarcomas can develop in any site in the heart, but angiosarcomas, which account for a third of all sarcomas, normally originate in the right atrium near the atrioventricular groove [4, 5].

Initially, there are no noticeable symptoms for patients with cardiac angiosarcoma. When patients do present they tend to have locally advanced or metastatic disease. Most symptoms are related to intracardiac flow obstruction, pericardial effusion and tamponade, tumor embolism, and systemic or constitutional symptoms due to metastatic disease [6‐8]. Lung metastases are common at presentation [4, 9]. Additionally, metastases have been reported in liver, lymph nodes, bone, adrenal glands, and to a lesser extent the central nervous system (CNS) and spleen [4, 9]. Due to the late stage at presentation, prognosis for patients with primary angiosarcoma of the heart is poor; median overall survival varies from 6 months or less for untreated patients to 12 months for patients with incomplete tumor resection [6, 10‐12]. Tumor often invades into the adjacent tissues, making complete resection challenging, if not impossible. In some cases, cardiac transplantation for patients with primary cardiac angiosarcoma has been reported, albeit with poor outcomes, with most patients dying from recurrence within 1 year of surgery. Cardiac transplantation is not currently recommended even for patients without evidence of metastatic disease due to lack of survival benefit to patients with transplantation [13‐15].

Existing treatment strategies include resection for non-metastatic disease, chemotherapy, and radiation. There are limited data showing chemotherapy treatment algorithms for cardiac angiosarcoma. Currently used chemotherapies include traditional agents such as paclitaxel, docetaxel, and doxorubicin. Angiogenesis inhibitors, drugs that target the growth of endothelial cells, such as bevacizumab, sunitinib, and sorafenib, have been used as well. Although some therapies may extend the lifespan of patients when combined with other modalities [16], none of the traditional chemotherapies or newer agents have been shown to eradicate microscopic disease [17‐20]. Lack of efficacy in existing treatment options highlights the need for targeted cancer therapies for patients with cardiac angiosarcoma.

To date, various studies investigated specific genes and pathways altered in angiosarcoma. V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (MYC) amplified tumors are common in radiation-induced and lymphedema-associated angiosarcomas [21]. Moreover, Fms Related Tyrosine Kinase 4 (FLT4) and MYC co-amplification was observed in 25% of secondary angiosarcomas [22]. Point mutations in V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene were found in liver angiosarcoma [23]. In other reports, a primary skin angiosarcoma and liver angiosarcoma lacked expression of p16, the product of the Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) gene [24‐26]. A number of studies found high expression of mutated Tumor protein P53 (TP53) gene in the tumor cells of angiosarcoma from various tissues of origin [26‐29]. Microarray expression studies revealed upregulation of a number of vascular-specific receptor tyrosine kinases, including Tyrosine Kinase with Immunoglobulin like and EGF Like Domains 1 (TIE1), Kinase Insert Domain Receptor (KDR), SNF Related Kinase (SNRK), TEK Receptor Tyrosine Kinase (TEK), and Fms Related Tyrosine Kinase 1 (FLT1) in angiosarcomas [30]. Further examination of these five genes by full-sequencing identified 10% of angiosarcoma patients harbored mutations in the KDR gene [30]. More recently, whole exome sequencing of primary and secondary angiosarcoma demonstrated mutations in the endothelial phosphatase, Protein tyrosine phosphatase, receptor type, B (PTPRB) and Phospholipase C, gamma 1 (PLCG1), a signal transducer of tyrosine kinase activators [31]. In the current study, we used custom-designed expanded exome capture to interrogate the genome of an aggressive primary cardiac angiosarcoma case using a genome-wide sequencing approach that included analysis of the annotated coding exome along with additional probes that allowed for genome-wide copy number analysis and the structural analysis of specific regions of the genome to capture translocations and inversions. This analysis allowed for a comprehensive assessment of the genomic landscape of this aggressive angiosarcoma.

Herein we describe the results of whole exome sequencing a primary, cardiac angiosarcoma. We found a novel KDR (G681R) mutation which is a putative ligand-independent activating mutation. Additionally, we discovered a focal high-level amplification at chromosome 1q encompassing MDM4 p53 binding protein homolog (MDM4), a negative regulator of TP53. We discuss potential therapeutic therapies which may be effective in treating patients with tumors containing these mutations/amplifications.

This case was presented previously [32]. Briefly, a 56-year-old Caucasian male was transferred to our hospital for possible pericardial window by cardiothoracic surgery after a transthoracic echocardiogram revealed a large pericardial effusion. A computed tomography scan of the chest revealed numerous pulmonary nodules surrounded by ground glass densities, along with small pleural effusions, and moderate to large pericardial effusion. Approximately 1250 ml of fluid was removed by pericardiocentesis, and the fluid was negative for the presence of infection or malignancy. Whole body positron emission tomography scan revealed a large, irregularly shaped, strikingly hypermetabolic cardiac lesion of probable right atrium origin (Fig. 1a). Moreover, there were extensive hypermetabolic pulmonary nodules in a background of ground glass infiltrates (Fig. 1b). A provisional diagnosis of cardiac sarcoma was made, and the patient underwent a left video-assisted thoracotomy, pericardial window and biopsy, and left lower lobe resection. Tumor cells in the lung nodules were both epitheliod and spindle-shaped in appearance. They were immunoreactive for Cluster of Differentiation (CD)31, CD34, and Factor VIII-related antigen, findings that were consistent with an angiosarcoma (Fig. 2). Given the aggressive nature of this cancer, the patient declined treatment and proceeded with in-hospital hospice where he expired on hospital day 19. Postmortem examination showed a large tumor was present in the right atrium attached just cephalad to the tricuspid valve annulus, anteriorly and laterally (Fig. 3). The tumor extended into the right atrioventricular sulcus. The right coronary artery was compressed by the tumor in the right atrioventricular sulcus. The neoplasm extended just slightly into the right ventricular wall but was not present in the ventricular septum, left ventricular free wall, or in the left atrial wall.

In this report we characterized the genomic landscape of a rare primary cardiac angiosarcoma tumor taken postmortem from a 56 year old male patient. We have identified a number of somatic copy number events, mostly marked by amplifications, and SNV alterations in this patient’s tumor. Our approach did not uncover any translocations that could result in oncogenic fusions previously reported in primary angiosarcomas [48, 49]. Copy number coupled with the translocation analysis suggested a presence of a circular chromosome harboring the MDM4 gene. We were able to confirm our hypothesis by in silico reconstructing the discordant read pairs mapping to multiple regions on chr1 and chr8. Double minute chromosomes and homogeneously stained regions have been identified in several tumor types and are biologically complex extrachromosomal entities frequently harboring known oncogenes (i.e MYC) and/or oncogenic fusions [50]. Their origin in the nucleus is quite intriguing and is thought to arise as a result of several mechanisms including chromothripsis, a multi-step product of complex duplications and recombinations through non-homologous end joining. To our knowledge, there are no previous reports describing circular extrachromosomal DNA in primary angiosarcomas or angiosarcomas of the heart carrying MDM4 gene. Future studies investigating frequency of this occurrence in primary cardiac angiosarcomas could add clinical value.

Another amplified region in this tumor genome mapped to chrXq28. This locus encompasses the MAGE gene cluster. Genes belonging to this cluster, cancer testis antigens (CTAs), often exhibit tumor-specific expression and are currently explored as immunotherapeutic targets in sarcomas [51], other solid tumors and hematologic malignancies [52]. Several ongoing preclinical and phase I trials are investigating vaccination-induced immunotherapeutic response in sarcoma patients with advanced disease (NCT02054104, NCT01313429, and NCT013441496).

Interestingly, a recent report describes an association between MAGE-A and MDM2 oncogene, E3 ubiquitin protein ligase (MDM2)/MDM4 complex in vitro in a human cell line model [53]. Specifically, MAGE-A was shown to compete with MDM4 for binding to MDM2 leading to elevated levels of free MDM4. Additionally, the group showed a positive correlation between MAGE-A and MDM4 by immunohistochemistry in primary breast cancer. Although we cannot comment on expression or protein levels of either MDM4 or MAGE in this patient’s tumor, the co-amplification significance of multiple MAGE genes and MDM4 may merit further exploration in this tumor type.

Recently, another patient presented to our clinic with a cardiac angiosarcoma and a Foundation One genetic profile was performed on his tumor. A mutation of unknown significance in KDR was observed (N704del) in a region where other mutations, deletions, and insertions have been reported [54], including the patient described in the present study. Additionally, amplification of MDM4 was found as was seen in our initial patient. Thus, from a potential therapeutic viewpoint one could one take away several points from these whole exome sequencing results (fully realizing this is from only two patients). Firstly, the KDR-G681R mutation, which occurs within the second I-SET domain of this protein, is novel. The G681 amino acid is highly conserved throughout evolution suggesting that it is a functionally relevant residue and based on prediction algorithms is potentially functionally relevant, however this would need to be validated through in vitro and/or in vivo cell-based mechanistic approaches. Although the true functional relevance of the mutation is currently unknown, this mutation would confer the strongest therapeutic targeting potential. One could consider a vascular endothelial growth factor receptor directed therapy such as bevacizumab, pazopanib, cabozantinib, vandetanib, ziv-aflibercept, or ramucirumab. Of note is that sorafenib and sunitnib inihibited KDR activity in vitro as previously noted by Antonescu and colleagues [30]. Secondly, there was high level focal amplification at 1q32, which contains the MDM4 gene. Although there are multiple anticancer agents under clinical development which inhibit the structurally similar p53 binding protein MDM2 (e.g., the Nutlins etc.) there have not been any under development which inhibits MDM4 or MDMX (any MDM protein). But that has changed with the ongoing development of the agent ALRN-6924 an MDM2/MDMX dual inhibitor (which reactivates p53) [55]. That agent would be a potential match against the amplified MDM4 target. Although we have provided a relatively high resolution view of the genomic landscape of this tumor, further studies are needed to determine the effect of candidate cancer genes hypothesized from our analysis, and clinical validation of the frequency of these events in cardiac angiosarcomas.