Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital Hemangioma

Abstract

Congenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Glu209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Glu209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffin-embedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Glu209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors' different postnatal behaviors.

Figure 1

Representative Gross and Histological Images of RICH, NICH, and Solitary Congenital Hepatic Hemangioma from Three Participants Shown are RICH (A–C), NICH (D–F), and solitary congenital hepatic hemangioma (G–I). (A) Supraorbital purple tumor with central depression and ulceration in a 1-week-old infant (participant 8 with RICH). (B) Hematoxylin and eosin (H&E) staining of the affected tissue imaged at 100× reveals small lobules (arrows) surrounded by abundant dense fibrous tissue where lobules have involuted and only draining channels remain (asterisks). (C) H&E staining at 400× reveals lobular capillaries containing enlarged lumens and minimally prominent endothelium. (D) Raised erythematous nodule at the left nasal ala in a 5-year-old child (participant 4 with NICH). (E) H&E staining at 100× reveals a large lobule (arrows) with prominent intra- and peri-lobular channels (asterisks). (F) H&E staining at 600× shows a hobnailed endothelium (arrows) lining lobular channels surrounded by fibrous tissue. (G) Excised 5 cm hepatic mass with pallor, extensive infarction, focal calcification, and hemorrhage in a 3-month-old infant (participant 16 with hepatic RICH). (H) H&E staining at 20× reveals a highly vascular rim superiorly and an acellular infarcted and hemorrhagic zone inferiorly. (I) H&E staining at 400× shows a minimally prominent endothelium lining widened hepatic sinusoids (asterisks) with cords containing a few bile ducts (arrowheads) but devoid of hepatocytes.

Figure 2

Detection of the GNA11 c.626A>T (p.Glu209Leu) Allele in Congenital Hemangioma Specimen from Participant 3 with NICH (A) IGV screenshot indicating 12% variant allele frequency in GNA11 transcripts measured by RNA-seq. 124× indicates the depth of coverage at position 626. (B) IGV screenshot indicating 6% variant allele frequency in GNA11 measured by MIP-seq. 211× indicates the depth of coverage at position 626. (C) Water-negative control for the ddPCR assay shows no flourescence for GNA11 wild-type or variant alleles. (D) ddPCR results of human DNA from unaffected control tissue indicates the presence of only the GNA11 wild-type allele. (E) ddPCR results of congenital hemangioma indicates the presence of wild-type and variant (8%) alleles.

Figure 3

Mutant Allele Frequency in Capillary-Enriched Lobules Is Higher than That in the Surrounding Stromal Tissue within Congenital Hemangioma (A) Pre-laser capture image of NICH showing multiple dermal vascular lobules from participant 4 (staining dark blue in hematoxylin and eosin-stained section, ×20 magnification). (B) Post-laser capture image of the section depicted in (A) showing absence of virtually all vascular lobules (hematoxylin and eosin-stained section, ×20 magnification). (C) Pre-laser capture of skeletal muscle, fibrous tissue, and fat uninvolved tissue by the NICH shown in (A) and (B) (hematoxylin and eosin-stained section, ×20 magnification) that was resected en bloc. (D) Graph depicting the GNAQ c.626A>T allele frequencies in DNA extracted from the capillary enriched lobules (A), the residual tissue (B), and the adjacent muscle tissue (C) measured in triplicate by ddPCR (dots). Solid lines indicate the average of the three measurements.