Frequent HRAS Mutations in Malignant Ectomesenchymoma: Overlapping Genetic Abnormalities With Embryonal Rhabdomyosarcoma

Abstract

Malignant ectomesenchymoma (MEM) is an exceedingly rare pediatric sarcoma with a predilection for infants and young children and is composed of dual malignant mesenchymal and neuroectodermal components. Microscopically, MEM displays areas of rhabdomyosarcoma (RMS) with intermixed neuronal/neuroblastic foci. The molecular alterations associated with MEM and its relationship with embryonal RMS (ERMS) and malignant peripheral nerve sheath tumor (MPNST) have not yet been elucidated. In this study we used whole-transcriptome sequencing in 2 MEM index cases with available frozen tissue, followed by screening of the identified genetic abnormalities in 5 additional cases. No candidate fusion genes were detected by FusionSeq analysis; however, the mutation detection algorithms revealed HRAS and PTPRD hotspot mutations in both index cases, with 1 case harboring an additional FBXW7 mutation. As these mutation profiles have been previously described in ERMS we have tested their incidence in a control group of 7 age-matched ERMS. In addition, the gene signature of MEM was compared with that of RMS, MPNST, and neuronal lineage. All 7 MEM patients were male, with a mean age of 7.5 months (range, 0.6 to 17 mo). All except 1 occurred in the pelvic/urogenital region. Most cases showed ERMS elements, with occasional spindle or undifferentiated/round cell areas. The intermixed neuroectodermal components were mostly scattered ganglion cells, ganglioneuroma, or ganglioneuroblastoma. By Sanger sequencing, 6 of 7 (86%) MEMs had HRAS mutations, with no additional case harboring PTPRD or FBXW7 mutations. The only case lacking HRAS mutation showed neuroblastic micronodules without ganglion cells. The trimethylation at lysine 27 of histone H3 (H3K27me3) expression, typically lost in MPNST, was retained in all cases. In the control ERMS group, 5 of 7 (71%) showed RAS mutations, equally distributed among NRAS, KRAS, and HRAS genes. The expression profiling of MEM showed upregulation of skeletal muscle and neuronal genes, with no significant overlap with MPNST. Our results of common HRAS mutations and composite gene signature with RMS and neuronal/neuroblastic elements suggest a closer genetic link of MEM to RMS rather than to MPNST.

Figure 1. Microscopic features of MEM

The most common appearance consisted of discrete sharply demarcated RMS (right) and ganglioneuroma (left) components (A, MEM2). The RMS component exhibited interlacing bundles of monomorphic spindle cells (B), while the ganglioneuroma component showed ganglion cells (asterix), nerve bundles, schwannian cells, and intermixed rhabdomyoblasts (arrowheads) (C, MEM2). Few ganglion cells (arrow) were scattered within the mitotically active RMS area (inset: synaptophysin stain) (D).

Figure 2. The morphologic spectrum of the RMS and neuroectodermal components in MEM

One of the unusual patterns of RMS resembled solid variant ARMS and was composed of solid sheets of primitive round cells (so-called dense pattern of ERMS) (A, B; MEM5), which was diffusely positive for myogenin (C). The ganglioneuroblastoma areas in this case were composed of maturing neuroblastic cells with variable amount of cytoplasm, focal rosetting and neuropil stroma (D, MEM5). Some areas showed intermingled mature ganglion cells (upper) and spindled rhabdomyoblastic cells (lower) (E), while others showed pure ganglioneuroma areas (F).

Figure 3

Post-treatment MEM showed decreased cellularity and scattered rhabdomyoblasts and ganglion cells in a myxoid background (A, MEM4); an S100 stain highlights Schwannian and satellite cells (B). Another post-chemo MEM showed no treatment response, being composed of neuroblastoma islands with a micronodular pattern, within the RMS areas (C, MEM5). The neuroblastic cells have monomorphic round, hyperchromatic nuclei and form Homer-Wright rosettes (D). The desmin stain highlights the rhabdomyoblasts but not the neuroblastoma nodules (E), while synaptophysin stains the neuroblastoma nodules but not the surrounding rhabdomyoblasts (F).

Figure 4. Novel oncogenic mutations identified in MEM

(A) HRAS mutation: homozygous p.G13R mutation (left), heterozygous p.G13R mutation (middle), heterozygous p.Q61L mutation (right). (B) Distribution and frequency (%) of RAS family members mutations in MEM and RMS [RMSc, control RMS group; RMSr1, RMS meta-analysis ^,, (limited to patients 5 years of age); RMSr2, RMS from same meta-analysis (> 5 years of age)]. (C) PTPRD mutations (p.V892A and p.V847L) and protein domains (IgC, immunoglobulin-like C2 type domains; FN, fibronectin type III domain; PTPc, phosphatase catalytic domain). (D) FBXW7 mutation (p.R505H) and protein domains (D, dimerization domain; Fb, F-box domain; WD, tryptophan–aspartic acid repeat domain).

Figure 5. Overlapping gene signature of MEM and RMS

By hierarchical clustering of a large panel of sarcomas available on the RNAseq using the 279 RMS-enriched gene list, MEM grouped closely with RMS (A). Gene set enrichment analysis of combined RMS and MEM cases showed uniform enrichment of RMS signature genes, including many myogenesis genes (B).