Association of PDGFRB Mutations with Pediatric Myofibroma and Myofibromatosis

Research

JAMA Dermatology | Brief Report Association of PDGFRB Mutations With Pediatric Myofibroma and Myofibromatosis

Guillaume Dachy, MD; Ronald R. de Krijger, MD, PhD; Sylvie Fraitag, MD; Ivan Théate, MD; Bénédicte Brichard, MD, PhD; Suma B. Hoffman, MD; Louis Libbrecht, MD, PhD; Florence A. Arts, PhD; Pascal Brouillard, PhD; Miikka Vikkula, MD, PhD; Nisha Limaye, PhD; Jean-Baptiste Demoulin, PhD

Supplemental content IMPORTANCE Myofibroma is the most frequent fibrous tumor in children. Multicentric myofibroma (referred to as infantile myofibromatosis) is a life-threatening disease.

OBJECTIVE To determine the frequency, spectrum, and clinical implications of mutations in the PDGFRB receptor tyrosine kinase found in sporadic myofibroma and myofibromatosis.

DESIGN, SETTING, AND PARTICIPANTS In this retrospective study of 69 patients with sporadic myofibroma or myofibromatosis, 85 tumor samples were obtained and analyzed by targeted deep sequencing of PDGFRB. Mutations were confirmed by an alternative method of sequencing and were experimentally characterized to confirm gain of function and sensitivity to the tyrosine kinase inhibitor imatinib.

MAIN OUTCOMES AND MEASURES Frequency of gain-of-function PDGFRB mutations in sporadic myofibroma and myofibromatosis. Sensitivity to imatinib, as assessed experimentally.

RESULTS Of the 69 patients with tumor samples (mean [SD] age, 7.8 [12.7] years), 60 were children (87%; 29 girls [48%]) and 9 were adults (13%; 4 women [44%]). Gain-of-function PDGFRB mutations were found in samples from 25 children, with no mutation found in samples from adults. Mutations were particularly associated with severe multicentric disease (13 of 19 myofibromatosis cases [68%]). Although patients had no familial history, 3 of 25 mutations (12%) were likely to be germline, suggesting de novo heritable alterations. All of the PDGFRB mutations were associated with ligand-independent receptor activation, and all but one were sensitive to imatinib at clinically relevant concentrations.

CONCLUSIONS AND RELEVANCE Gain-of-function mutations of PDGFRB in myofibromas may affect only children and be more frequent in the multicentric form of disease, albeit present in solitary pediatric myofibromas. These alterations may be sensitive to tyrosine kinase inhibitors. The PDGFRB sequencing appears to have a high value for diagnosis, prognosis, and therapy of soft-tissue tumors in children.

Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Jean-Baptiste Demoulin, PhD, Experimental Medicine Unit, de Duve Institute, Université Catholique de Louvain, avenue Hippocrate 75, box B1.74.05, BE-1200 Brussels, JAMA Dermatol. doi:10.1001/jamadermatol.2019.0114 Belgium (jb.demoulin Published online April 24, 2019. @uclouvain.be).

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yofibromas are the most frequent fibrous tumors in children.1 The presence of multiple myofibromas Key Points in an individual defines infantile myofibromatosis M Questions What is the frequency of PDGFRB mutations in (IMF). These benign tumors can appear in the skin, muscles, sporadic solitary myofibroma and multifocal myofibromatosis, and bones, and internal organs, and they often regress sponta- what are the implications for clinical management? neously. However, visceral involvement of myofibromato- Findings In this international study of 69 patient samples, we sis, sometimes referred to as generalized IMF,2 is a life- identified activating PDGFRB mutations in children and infants but 1 threatening disease. Unlike children, adults develop only not adults, and the presence of mutations was strongly associated solitary myofibromas. In 2013, the World Health Organiza- with multicentric disease. Most PDGFRB mutations were sensitive tion classified myofibroma among pericytic tumors, which to the tyrosine kinase inhibitor imatinib, suggesting that they were also include myopericytoma, based on the morphologic attractive therapeutic targets. 3 continuum between these lesions. Meaning The findings suggest that the presence of PDGFRB Recently, PDGFRB mutations were identified in a few activating mutations provides a molecular diagnostic test and a familial4,5 and sporadic cases of IMF.6 The PDGFRB gene encodes basis for targeted therapy of infantile myofibromatosis. a receptor tyrosine kinase that binds platelet-derived growth factor B (PDGFB) and platelet-derived growth factor D (PDGFD) and (13%; 4 women [44%]); 85 samples were included in the analy- is highly expressed in fibroblasts and pericytes. A recent study5 sis. None of the patients had a family history of myofibroma showed that the PDGFRB variants found in IMF were oncogenic or myofibromatosis. The Table presents patient clinical char- and sensitive to tyrosine kinase inhibitors, such as imatinib, in acteristics. We extracted DNA from fresh frozen or formalin- vitro. Recent clinical reports confirmed the safety and efficacy fixed paraffin-embedded tissue and performed targeted deep of tyrosine kinase inhibitors in 2 patients with PDGFRB sequencing of PDGFRB (coverage depth >1000×), as previ- mutations.7,8 In 1 patient, however, a PDGFRB mutation was iden- ously described.6 tified that was fully resistant to imatinib in vitro,5 suggesting that We identified PDGFRB mutations in 25 patients including primary resistance may be an issue. A limited number of patients infants and children but none in adults (Figure 1 and eTables 1 with IMF has been analyzed, and the presence of mutations in and 2 in the Supplement). The PDGFRB mutations were more adult myofibroma and in myopericytoma remains a matter of frequent in IMF (68%) compared with solitary tumors (24%) debate.9,10 The goal of the present study was to evaluate the value (Fisher exact test, P = .002). Three patients carrying PDGFRB of PDGFRB mutations as a diagnostic biomarker and therapeu- mutants had visceral involvement, including 1 patient with mul- tic target in a large group of patients. ticentric myopericytoma; none died of the disease. All mutations are reported in Figure 2.Of25patientswith PDGFRB variants, 11 (44%) carried multiple mutations. Of the Methods 11 patients, 7 (64%) had multicentric disease, with no significant association between multiple-hit status of PDGFRB and We retrospectively collected archived samples from 102 patients clinical phenotype. In 3 cases, these mutations were in cis (ie, from clinical centers in Belgium, France, the United States, and on the same allele), as detailed in the eMethods in the Supple- the Netherlands (the Dutch nationwide population-based pathol- ment. The mutations were detected at allele frequencies rang- ogy database [PALGA], Houten),11 who were diagnosed with spo- ing from 6% to 52% (mean [SD], 20.3% [12.2%]) of sequenc- radic myofibroma, myopericytoma, or myofibromatosis according reads (Figure 2). ing to the World Health Organization classification. Tumor Of the mutant cases, 17 (68%) had mutations located in the samples were analyzed by targeted deep sequencing of PDGFRB. 2 classical hotspots of oncogenic mutations of receptor tyro- Mutations were confirmed by an alternative method of sequenc- sine kinases, the juxta-membrane and the kinase domains ing and were experimentally characterized to confirm gain of (forming the core of the catalytic part of the protein). There function and sensitivity to the tyrosine kinase inhibitor imatinib was an in-frame duplication of 33 nucleotides in the juxta- (details are given in the eMethods in the Supplement). This study membrane domain. The remaining mutations were located in was approved by the medical ethics review board of the Univer- the transmembrane domain and the extracellular part of the sity of Louvain, Brussels, Belgium. Samples were retrospectively receptor, a unique finding in our experiences with sequenc- obtained from biobanks, and data were deidentified. One patient ing of PDGFRB, and an uncommon oncogenic event for other was included prospectively after diagnosis with written informed receptor tyrosine kinases. consent from a parent. Copy-number variation analysis by multiplex ligation- Statistical analysis was computed in R, version 3.5.1 dependent probe amplification experiments for 12 samples, re- (R Foundation) and Excel, 2016 version (Microsoft). Two- gardless of PDGFRB mutation status, did not reveal any am- sided P < .05 was considered to be statistically significant. plification of the PDGFRB locus.

Results Discussion

Of the 69 patients with tumor samples (mean [SD] age, 7.8 [12.7] We found a broad catalog of 18 different PDGFRB mutations years), 60 were children (87%; 29 girls [48%]) and 9 were adults in myofibroma lesions, 12 of which were novel. We validated

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Table. Demographic and Clinical Characteristics of the Patients Overall and by Myofibromatosis Status

No. (%) Patients With Myofibromatosis Characteristic All Patients Multicentrica Solitary Study patients 69 19 50 Sex Male 34 (49) 9 (47) 25 (50) Female 34 (49) 10 (53) 24 (48) NA 1 (1) 0 1 (2) Country of origin Belgium 26 (41) 8 (42) 18 (36) France 10 (13) 4 (21) 6 (12) The Netherlands 32 (48) 6 (32) 26 (52) United States 1 (1) 1 (5) 0 Age at diagnosis, y <2, y 31 (45) 15 (79) 16 (32) 2-18, y 28 (41) 4 (21) 24 (48) >18, y 9 (13) 0 9 (18) NA 1 (1) 0 1 (2) Mean (SD), y 7.8 (12.7) 1 (2) 10 (12) Visceral involvement 8 (12) 8 (42) 0 Treatment Spontaneous involution 9 (13) 7 (37) 2 (4) Surgical resection 55 (80) 8 (42) 48 (96) Chemotherapy 4 (6) 4 (21) 0 Mutation status PDGFRB mutant 25 (36) 13 (68) 12 (24) PDGFRB wild type 44 (64) 6 (32) 38 (76) PDGFRB mutants Single hit 14 (20) 6 (32) 8 (16) Multiple hits 11 (16) 7 (37) 4 (8) Abbreviation: NA, not available. Recurrence 3 (4) 1 (5) 2 (4) a This subgroup includes the case of Mortality 2 (3) 2 (11) 0 multicentric myopericytoma.

Figure 1. PDGFRB Mutant Distribution by Patient Age and Myofibroma Subtype

A Multicentric myofibroma B Solitary myofibroma

20 20

PDGFRB wild type PDGFRB mutants

15 15

10 10 No. of Cases No. of Cases No.

5 5

0 0 <2 y 2-18 y >18 y <2 y 2-18 y >18 y Age Age

most variants by allele-specific polymerase chain reaction or stromal cells, this proportion was indicative of the somatic or Sanger sequencing after cloning. The latter technique also al- germline nature of the mutations. Although none of the 69 pa- lowed us to confirm the allele burden of the mutations, which tients had a family history of myofibroma or myofibromato- was consistent with the percentage of mutant reads obtained sis, our sequencing data suggested germline (likely de novo) from next-generation sequencing. Because samples con- mutations in 3 of 25 patients (12%) carrying a PDGFRB vari- tained variable amounts of adjacent normal tissue and tumor ant (eMethods in the Supplement). All 3 variants affected R561,

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Figure 2. PDGFRB Gain-of-Function Mutations in Myofibroma

N-terminus mRNA

ECD No. of Cases Imatinib 0 2 4 6 8 10 12 sensitivity

p.V506_R507insLLSVa p.A537D_I538Na p.I538_L539insRa

10 p.K559_Y562delinsNa 11 p.R561Ca

12 p.R561_Y562delinsH TMD 13 p.Y562D/Ca

JMD 14 p.Y562_R565del a 15 p.Y562_V572dup a KD N-lobe 16 p.W566R/G a 17 p.W566_V568delinsL p.Y589Da 18 KD C-lobe p.N666K/Sa p.D850V/Ya

C-terminus 0 10 20 30 40 50 60 Allele Burden, %

Variants are reported according to protein location and affected exon. The p.N666K (C>A), p.W566_V568delinsL, and p.Y562D were identified in 4 distinct activation loop is represented in white in the C-lobe of the kinase domain. Pairs IMF lesions from the same patient. Blue bars indicate the frequency of each of mutations identified in multiple-hits samples were p.R561C + p.N666K, variant; white circles, allele burden; green, sensitive to imatinib; red, resistant to p.R561C + p.N666S, p.A537D + p.I538N, p.N666K + p.W566R, imatinib. ECD indicates extracellular domain; IMF, infantile myofibromatosis; p.W566R + p.Y589D, p.Y562_R565del + p.N666K, and R561_Y562delinsH + JMD, juxta-membrane domain; KD, kinase domain; and TMD, transmembrane p.N666K (eTable 1 and eTable 2 in the Supplement). Variants domain. p.K559_Y562delinsN and p.N666K were identified in 2 distinct a Variants were validated by an alternative method. myopericytomas from the same patient; the variants p.N666K (c.1998C>G),

the most frequently mutated residue in IMF families.4 Fur- mor types harboring kinase mutations, in which primary re- thermore, they were all associated with somatic second hits sistance is frequent. at residue N666. Healthy tissue was not available to confirm A recent study5 described the cooperation between a the germline nature of the mutations. These results suggest weak mutation affecting the juxta-membrane domain that a substantial proportion of patients may require genetic (ie, p.R561C) and a strong mutation of the kinase domain. counseling. The positive selection of 2 mutations on the same allele is We also characterized the functional outcome of the vari- unusual but has also been described for epidermal growth ants by performing luciferase reporter assays as a readout of factor receptor in lung cancer and for endothelial tyrosine mitogen-activated protein kinases and signal transducers and kinase in sporadic, multifocal venous malformations.13,14 In activators of transcription. These pathways are potent down- 2 cases, we identified different mutations in distinct lesions stream mediators of PDGFRB signaling.12 All the identified mu- from the same patient. This finding suggests an underlying tations were associated with constitutive activation of the re- mechanism predisposing mesenchymal progenitor cells to ceptor (eFigure in the Supplement), suggesting a potential PDGFRB mutation and, thereby, development of multiple causative nature and excluding passenger mutations. We then pericytic lesions, each seeded by a different mutated cell-of- showed that these oncogenic forms of PDGFRB mutations were origin. efficiently associated with inhibition by imatinib used at a clini- Of particular interest was an in-frame duplication of 33 cally relevant concentration (eFigure in the Supplement). The nucleotides in the juxta-membrane domain, to our knowl- only exception was the previously reported p.D850V mutant6 edge described here for the first time in PDGFRB, but highly affecting the kinase activation loop. This high sensitivity rate reminiscent of the internal tandem duplications described in to pharmacologic inhibitor contrasts with that of many tu- FMS-related tyrosine kinase 3 in acute myeloid leukemia.15

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The presence of a PDGFRB mutation in myopericytoma is positive results, and the results of that previous study await consistent with a recent report by Hung and Fletcher10 but con- confirmation by an independent method. trasts with the report by Agaimy et al.9 The high frequency of PDGFRB mutations in IMF, myopericytoma, and solitary lesions affecting young children suggests that these entities may Conclusions represent a morphologic continuum with common molecular bases, arguing for a refinement of the classification of these The findings suggest that gain-of-function mutations of PDG- tumors as pericytic tumors with PDGFRB mutation. FRB affect only children. They were more frequent in the multicentric form of the disease, albeit present in solitary pediat- Limitations ric myofibromas. The PDGFRB alteration status may help A limitation of our study is that we cannot rule out selection identify a specific patient subgroup to be carefully moni- bias in our case series. This may explain the discrepancy with tored. Our results shed light on the molecular basis of the peri- a previous report describing PDGFRB mutations in adult myo- cytic tumors and point to the high value of PDGFRB sequenc- fibromas using direct Sanger sequencing of polymerase chain ing for prognosis, diagnosis, and therapy of soft-tissue reaction products.9 However, this technology is prone to false- neoplasms in children.

ARTICLE INFORMATION Conflict of Interest Disclosures: Dr Dachy reported 6. Arts FA, Sciot R, Brichard B, et al. PDGFRB gain- Accepted for Publication: January 18, 2019. receiving grants and being a recipient of Aspirant of-function mutations in sporadic infantile fellowship from Fonds de la Recherche Scientifique myofibromatosis. Hum Mol Genet. 2017;26(10):1801- Published Online: April 24, 2019. during the conduct of the study. Dr Arts reported 1810. doi:10.1093/hmg/ddx081 doi:10.1001/jamadermatol.2019.0114 receiving grants and being supported by a Télévie 7. Pond D, Arts FA, Mendelsohn NJ, Demoulin JB, Author Affiliations: Experimental Medicine Unit, fellowship from Fonds de la Recherche Scientifique Scharer G, Messinger Y. A patient with germ-line de Duve Institute, Université Catholique de Louvain, during conduct of the study and serving as consul- gain-of-function PDGFRB p.N666H mutation and Brussels, Belgium (Dachy, Arts, Demoulin); tant for UCB outside the submitted work. Dr Limaye marked clinical response to imatinib. Genet Med. Department of Pathology, Princess Maxima Centre reported being a research associate at Fonds de la 2018;20(1):142-150. doi:10.1038/gim.2017.104 for Pediatric Oncology and University Medical Recherche Scientifique. Dr Demoulin reported 8. Mudry P,Slaby O, Neradil J, et al. Case report: Centre, Utrecht, Netherlands (de Krijger); receiving grants from the King Baudouin Foundation, rapid and durable response to PDGFR targeted Department of Pathology, Hôpital Necker-Enfants Fonds de la Recherche Scientifique, and Académie therapy in a child with refractory multiple infantile Malades, Assistance Publique–Hôpitaux de Paris, Royale de Médecine de Belgique during the conduct myofibromatosis and a heterozygous germline Paris, France (Fraitag); Department of Pathology, of the study. No other disclosures were reported. Institut de Pathologie et de Génétique, Gosselies, mutation of the PDGFRB gene. BMC Cancer.2017;17 Funding/Support: This work was supported by Belgium (Théate); Department of Pediatric (1):119. doi:10.1186/s12885-017-3115-x grant #16/21-073 (Communauté française de Hematology and Oncology, Cliniques Universitaires 9. Agaimy A, Bieg M, Michal M, et al. Recurrent Belgique) from Actions de Recherche Concertées, Saint-Luc, Université catholique de Louvain, somatic PDGFRB mutations in sporadic infantile/ the Simon Bauvin Fund managed by the King Brussels, Belgium (Brichard); Department of solitary adult myofibromas but not in angioleiomyo- Baudouin Foundation (Dr Demoulin), and grant Pediatrics, University of Maryland School of mas and myopericytomas. Am J Surg Pathol.2017;41 CR2015A-10R from Walloon Excellence in Medicine, Baltimore (Hoffman); Department of (2):195-203. doi:10.1097/PAS.0000000000000752 Lifesciences and Biotechnology (Dr Vikkula). Pathology, Cliniques Universitaires Saint-Luc, 10. Hung YP,Fletcher CDM. Myopericytomatosis: Role of the Funder/Sponsor: The funding Université catholique de Louvain, Brussels, Belgium clinicopathologic analysis of 11 cases with molecular organization had no role in the design and conduct of (Libbrecht); Human Molecular Genetics, de Duve identification of recurrent PDGFRB alterations in the study; collection, management, analysis, and Institute, Université Catholique de Louvain, myopericytomatosis and myopericytoma. Am J Surg interpretation of the data; preparation, review, or Brussels, Belgium (Brouillard, Vikkula); Walloon Pathol. 2017;41(8):1034-1044. doi:10.1097/PAS. approval of the manuscript; and decision to submit Excellence in Lifesciences and Biotechnology, Wal- 0000000000000862 the manuscript for publication. lonia, Belgium (Vikkula); Genetics of Autoimmune 11. Casparie M, Tiebosch AT,Burger G, et al. 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