Unique Aberrations in Intimal Sarcoma Identified by Next-Generation Sequencing as Potential Therapy Targets

Intimal sarcomas are rare and histologically heterogeneous tumors, commonly arising from the pulmonary arteries. They have remained challenging to treat. Few studies in the literature study the genomics of this cancer. Identifying targetable alterations is an important step in advancing the treatment of intimal sarcomas. Using data from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR GENIE) database, we cataloged genetic alterations and assessed their clinical utility from thirteen patients with intimal sarcoma. Notable copy number alterations included amplification in MDM2, CDK4, PDGFRA, and NOTCH2, as well as copy number losses in CDKN2A and CDKN2B. Actionable alterations included mutations in ATM/ATR, PTCH1, and PDGFRB. Moreover, genomic rearrangement events, specifically PDE4DIP-NOTCH2 and MRPS30-ARID2 fusions were identified. Co-occurring alterations included a NOTCH2 copy number gain in the PDE4DIP-NOTCH2 fusion positive tumor and PDGFRB mutations in both fusion-positive cases. Our study suggests that PDGFRB may be relevant in the tumorigenesis process. Including genomic profiling in the management of intimal sarcoma and potential enrollment in targeted therapy trials is warranted.


Introduction
Intimal sarcomas (IS) are rare mesenchymal tumors that involve the innermost layer of large vessels, most commonly seen in pulmonary arteries [1][2][3][4]. Diagnosing this sarcoma has proved challenging despite the criteria set by the World Health Organization (WHO), as it is largely undifferentiated and histologically variable even within the same host [1,2,[5][6][7]. This tumor has the ability to differentiate into other types of sarcomas such as leiomyosarcoma and angiosarcoma [1,3,5]. Since intimal sarcomas

Discussion
There is a large amount of heterogeneity in the presentation, histology, and genomic profile of intimal sarcomas [1][2][3][4]18]. The molecular variability and aggressive nature of this cancer makes it a challenging entity to diagnose and treat. Case reports and series have shown conventional chemotherapy with limited success, and curative radical surgery prolonging survival to a little over a year [5,[11][12][13]. To our knowledge, there are no prospective clinical trials for patients with intimal sarcoma. These patients with intimal sarcoma will need to be included in clinical trials to determine which patients would benefit from genomically-matched therapies.
Our study confirmed the recurrence of copy number gains in MDM2 and CDK4 that coincide with the location 12q12-15, and PDGFRA in 4q12, as described in prior studies. The amplification of these genes strongly suggests that PDGFR and MDM2 pathways are important for tumorigenesis in IS.
MDM2 inhibitors under investigation have demonstrated responses in early phase clinical trials in MDM2 amplified soft tissue sarcomas (with or without CDK4 co-amplification) such as liposarcoma and synovial sarcoma [19]. Our data support the use of NGS on intimal sarcomas to identify MDM2 amplification and consideration for such trials. Thirty-three percent were found to have PDGFRA amplification, but this can be explained by the choice of biopsy site due to intratumoral heterogeneity [7]. New mutations in PDGFRB found in our study may have implications for tumorigenesis and therapeutic options as well.
We also found novel somatic mutations in another receptor kinase gene such as ALK, which is known to be mutated or amplified in various other cancers such as lymphomas and non-small cell lung cancers [20]. However, the ALK E1460Q mutation is outside the tyrosine kinase domain and may not be relevant clinically. Other mutations identified include ATM/ATR, and PTCH1, a tumor suppressor that is part of the hedgehog signaling pathway involved in tumorigenesis [21,22]. A cell checkpoint kinase mutation, which is upstream from p53 in the signaling pathway, was also seen. All of these genes could be potential targets for treatment.
Additional findings of interest include fusion proteins involving NOTCH2-PDE4DIP and ARID2-MRPS30 and copy number gains in NOTCH2. Irregular NOTCH2 signaling has been associated with the initiation and progression of multiple cancers including liver, brain, and gastric cancers along with lymphomas [23]. In addition to NOTCH2, mutations in ARID2, a possible tumor suppressor, are also found in hepatocellular carcinomas [24]. The counterparts of these fusion proteins, such as MRPS30 and PDE4DIP, may also be associated with an increased risk for breast cancer and leptomeningeal disease progression, respectively [25,26]. Interestingly, both fusion proteins were found in primary tumors, but their effect on the tumorigenesis of intimal sarcomas is unknown and will require further investigation.
Limitations of this study include the sample size, which given the rarity of disease and biopsy site is likely due to intratumoral heterogeneity. Intimal sarcoma patients are currently not enrolled in clinical trials due to the location of the tumors in major vasculature. Since we noted actionable alterations in MDM2, CDK4, PDGFRA, and NOTCH2, as well as copy number losses in CDKN2A and CDKN2B, our study has clinical implications. There are now three US FDA approved CDK4/6 inhibitors for breast cancer, Palbociclib (IBRANCE ® , Pfizer Inc.), ribociclib (Kisqali ® , Novartis Pharmaceuticals Corporation), and abemaciclib (VERZENIO ™ , Eli Lilly and Company). Given the multiple aberrations seen in the CDK pathway, CDK4/6 inhibitors may be an option in clinical trials or off-label based on molecular profiling data. In addition, some of these tumors may be responsive to drugs that are already approved for the treatment of sarcomas like Pazopanib (Votrient ® ), which is a multi-kinase VEGF based inhibitor that has activity against the PDGFR pathway as well. Other novel TKIs and MDM2 antagonists should be evaluated as treatments for advanced IS. In addition, the role of ATM/ATR, NOTCH2, and PTCH1 should be studied as potential therapeutic targets. Investigators should be encouraged to enroll these patients with specific molecular aberrations to basket trials.

Materials and Methods
We queried the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) database (version 5) [27]. The GENIE registry derives existing CLIA-/ISO-certified genomic data [28]. Published records in Pubmed/Medline were reviewed for genomic data from several different platforms. Retrospective records were abstracted to appraise the benefit of using a targeted therapy approach in a large Phase 1 clinic at the University of Texas MD Anderson Cancer Center.
Ethics Statement: All AACR GENIE project data has been de-identified using the HIPAA Safe Harbor Method. Analyses were retrospective and were performed in accordance with the AACR GENIE Human Subjects Protection and Privacy policy. Institutional Review Board (IRB) details are provided in the AACR GENIE Data Guide [28].

Conclusions
Comprehensive genomic profiling may be considered for intimal sarcoma, both to confirm diagnosis and to explore treatment options on clinical trials. Clinical trial enrollment should be considered for all intimal sarcomas, and further exploration of copy number changes and genetic alterations is needed for this relatively chemotherapy-resistant disease. A global multicenter prospective registry/trial would best determine the response of this rare cancer to targeted therapies.

Funding:
The University of Texas MD Anderson Cancer Center is supported by grant CA016672 from the National Institutes of Health.