Genomic mechanisms of receptor tyrosine kinase activation in myofibroma

Myofibromas are the most frequent fibrous tumors in children. The presence of multiple myofibromas in an individual defines infantile myofibromatosis (IMF). These benign tumors can appear in the skin, muscles, bones, and internal organs, and they often regress spontaneously. However, the presence of visceral lesions is life-threatening. Unlike children, adults develop only solitary myofibromas. Between 2013 and 2017, PDGFRB mutations were identified in a few familial and sporadic cases of IMF. The PDGFRB gene encodes the receptor tyrosine kinase PDGFRβ, which is highly expressed in fibroblasts and pericytes. In 2017, our laboratory demonstrated that PDGFRB mutations variants found in IMF were oncogenic and sensitive to tyrosine kinase inhibitors, such as imatinib. We brought together 85 archived myofibroma samples from 69 patients to perform targeted Ion Torrent sequencing of the PDGFRB locus at a coverage depth >1000x. Our aims were to determine the frequency, spectrum, and clinical implications of PDGFRβ mutants in sporadic IMF. Gain-of-function PDGFRB mutations were found in samples from 26 children. No mutation was found in samples from adults. We identified one of the 26 mutations a posteriori by reanalyzing some samples by whole-exome sequencing. Mutations were particularly associated with severe multicentric disease (14 of 19 myofibromatosis cases, 73%). These two findings illustrate that PDGFRB sequencing could identify a subset of patients to be carefully monitored. Although patients had no familial history, three of 26 mutations (12%) were likely to be germline, suggesting de novo inheritable alterations. All of the PDGFRB mutations were associated with ligandindependent receptor activation, and all but one were sensitive to imatinib at clinically relevant concentrations. Our findings suggest that gain-of-function mutations of PDGFRB in myofibromas affect only children and are more frequent in the multicentric form of disease, albeit present in solitary pediatric myofibromas. To analyze the genetic bases of myofibromas in a more comprehensive way, we conducted a second study in which we performed RNA sequencing on eight myofibroma samples, including two from patients with IMF. Although gene fusions are frequent and potent oncogenic drivers in soft tissue neoplasia, little is known regarding gene fusion in myofibroma. To date, only a few cases of myofibroma have been analyzed, leading to the description of gene fusions involving SRF as recurrent 3’ partner gene. We identified five different gene fusions, in six patients. The two SRF-ICA1L and SRF-CITED1 gene fusions confirmed the presence of serum-response factor fusion proteins in myofibromas. We identified a novel COL4A1-VEGFD gene fusion in two cases, one of which also carried a PDGFRB mutation. We observed a robust and specific overexpression of VEGFD by immunofluorescence on the corresponding tumor sections. Finally, we showed that the COL4A1-VEGFD fusion protein was processed to mature bioactive VEGFD. Altogether, our results unravel a new fusion gene that leads to massive production of the growth factor VEGFD under the control of the COL4A1 gene promoter. In summary, we analyzed solitary and multicentric myofibromas by performing targeted sequencing of PDGFRB locus and RNA sequencing. Our results regarding gain-of-function PDGFRB mutations and the COL4A1-VEGFD fusion gene shed light on the origin of these tumors and offer new options for diagnosis and targeted therapy.