Identification of novel GNAS mutations in intramuscular myxoma using next-generation sequencing with single-molecule tagged molecular inversion probes

Elise M Bekers,Uta Flucke,Suzanne Mol,Astrid Eijkelenboom,Peter Van Zwam,Emiel Ruijter,Paul Rombout,Blanca Scheijen

doi:10.1186/s13000-019-0787-3

Abstract

BackgroundIntramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas. These tumors can, especially on biopsy material, be difficult to distinguish from low-grade fibromyxoid sarcoma or low-grade myxofibrosarcoma. GNAS mutations are frequently involved in IM, in contrast to these other malignant tumors. Therefore, sensitive molecular techniques for detection of GNAS aberrations in IM, which frequently yield low amounts of DNA due to poor cellularity, will be beneficial for differential diagnosis.MethodsIn our study, a total of 34 IM samples from 33 patients were analyzed for the presence of GNAS mutations, of which 29 samples were analyzed using a gene-specific TaqMan genotyping assay for the detection of GNAS hotspot mutations c.601C > T and c602G > A in IM, and 32 samples using a novel next generation sequencing (NGS)-based approach employing single-molecule tagged molecular inversion probes (smMIP) to identify mutations in exon 8 and 9 of GNAS. Results between the two assays were compared for their ability to detect GNAS mutations with high confidence.ResultsIn total, 23 of 34 samples were successfully analyzed with both techniques showing GNAS mutations in 12 out of 23 (52%) samples. The remaining 11 samples were analyzed with either TaqMan assay or smMIP assay only. The TaqMan assay revealed GNAS mutations in 16 out of 29 samples (55%), with six samples c.601C > T (p.R201C; 38%) and ten samples c.602G > A (p.R201H; 62%) missense mutations. The smMIP assay identified mutations in 16 out of 28 samples (57%), with five samples c.601C > T (p.R201C; 31%) and seven samples c.602G > A (p.R201H; 44%) missense mutations. In addition, four samples (25%) revealed novel IM-associated mutations, including c.601C > A (p.R201S), c.602G > T (p.R201L), c.602G > C (p.R201P) and c.680A > G (p.Q227R). Combining the results of both tests, 23 out of 34 sporadic IM samples (68%) showed a GNAS mutation.ConclusionsBoth the TaqMan and the smMIP assay a show a high degree of concordance in detecting GNAS hotspot mutations in IM with comparable sensitivity. However, since the NGS-based smMIP assay permits mutation detection in whole exons of GNAS, a broader range of GNAS mutations can be identified by the smMIP approach.

Highlights

Intramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas
Histopathology and clinical information of intramuscular myxoma cases Histopathology of haematotoxylin and eosin (H&E)-stained slides confirmed that a selected set of 34 samples from 33 patients showed the classical features of IM, which were composed of uniform, sparsely distributed cytological bland spindle- or stellate-shaped cells with tapering eosinophilic cytoplasm and small nuclei embedded in an abundant myxoid stroma
One case showed prominent hypercellular areas with more collagenous stroma and was diagnosed as cellular myxoma according to the criteria defined by Nielsen et al (Fig. 1) [5, 6]

Summary

Introduction

Intramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas. These tumors can, especially on biopsy material, be difficult to distinguish from low-grade fibromyxoid sarcoma or low-grade myxofibrosarcoma. Intramuscular myxoma (IM) is a benign soft tissue neoplasm that belongs to the group of myxoid tumors characterized by a marked abundance of extracellular myxoid matrix. These tumors share several histological features, and depending on their clinical presentation and place of origin, can be subdivided into intramuscular, superficial-cutaneous, odontogenic and juxta-articular myxoma [1, 2]. Mazabraud’s syndrome and the closely related McCune-Albright syndrome, which is associated with fibrous dysplasia, café au lait macules and endocrine disorders, are caused by activating missense mutations in codon 201 of the GNAS gene [8,9,10,11,12]

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