Papillary Hemangioma Harbors Somatic GNA11 and GNAQ Mutations.

Papillary hemangioma (PH) is a small, primarily dermal lesion occurring predominantly in the head and neck in both children and adults. Its signature characteristics are dilated thin-walled channels containing papillary clusters of mainly capillary-sized vessels and endothelial cytoplasmic eosinophilic inclusions. Given certain histopathologic similarities to congenital hemangioma which harbor mutations in GNAQ and GNA11 , we investigated whether similar mutations are present in PH. Seven PH specimens were studied. All presented in the first 4 years of life, with one being noted at birth. With the exception of one lesion, all were in the head and neck. Lesions were bluish and ranged in size from 0.5 to 2.8 cm. Four samples had GNA11 p.Q209L and 3 had GNAQ p.Q209L missense mutations. Mutations in GNA11 and GNAQ are associated with other types of somatic vascular lesions including capillary malformation, congenital hemangioma, anastomosing hemangioma, thrombotic anastomosing hemangioma, and hepatic small cell neoplasm. Shared mutations in GNA11 and GNAQ may account for some overlapping clinical and pathologic features in these entities, perhaps explicable by the timing of the mutation or influence of the germline phenotype.

Parkes Weber syndrome with lymphedema caused by a somatic KRAS variant.

Parkes Weber syndrome is a vascular malformation overgrowth condition typically involving the legs. Its main features are diffuse arteriovenous fistulas and enlargement of the limb. The condition has been associated with pathogenic germline variants in RASA1 and EPHB4 We report two individuals with Parkes Weber syndrome of the leg and primary lymphedema containing a somatic KRAS variant (NM_004985.5:c.35G > A; p.Gly12Asp). KRAS variants, which cause somatic intracranial and extracranial arteriovenous malformations, also result in Parkes Weber syndrome with lymphatic malformations.

EPHB4 mutation causes adult and adolescent-onset primary lymphedema.

Primary lymphedema results from the anomalous development of the lymphatic system and typically presents during infancy, childhood, or adolescence. Adult-onset primary lymphedema is rare and mutations associated with this condition have not been identified. The purpose of this investigation was to search for variants that cause adult-onset primary lymphedema. We discovered an autosomal dominant EPHB4 mutation in a patient who developed unilateral leg lymphedema at age 39 years; the same mutation affected his son who presented with the disease at 14 years of age.

Lipoblastoma phenotype contains a somatic PIK3CA mutation.

Lipoblastoma typically occurs in childhood and is associated with rearrangements of the PLAG1 gene. We present a patient with an isolated mass thought to be a lipoblastoma clinically, radiographically, and histologically. The lesion was diagnosed as a PIK3CA-adipose lesion after the tissue was negative for PLAG1 rearrangement and contained a somatic PIK3CA mutation (H1047R). Although PIK3CA variants are associated with PROS (PIK3CA-related overgrowth spectrum), this report illustrates a non-syndromic, lipoblastoma phenotype caused by a PIK3CA mutation.

Arteriovenous malformation phenotype resembling congenital hemangioma contains KRAS mutations.

Extracranial arteriovenous malformation (AVM) is most commonly caused by a somatic mutation in MAP2K1. We report two patients with vascular anomalies that had an unclear clinical diagnosis most consistent with either an AVM or congenital hemangioma. Lesions were cutaneous, reddish-purple with telangiectasias, present at birth, and had defined borders. Histopathology indicated AVM and both lesions contained somatic KRAS mutations. A rare AVM phenotype exists that shares clinical features with congenital hemangioma.

Arteriovenous Malformation MAP2K1 Mutation Causes Local Cartilage Overgrowth by a Cell-Non Autonomous Mechanism.

Extracranial arteriovenous malformation (AVM) is most commonly caused by MAP2K1 mutations in the endothelial cell. The purpose of this study was to determine if local tissue overgrowth associated with AVM is caused by direct or indirect effects of the MAP2K1 mutation (i.e., cell-autonomous or cell-non autonomous). Because cartilage does not have blood vessels, we studied ear AVMs to determine if overgrown cartilage contained AVM-causing mutations. Cartilage was separated from its surrounding tissue and isolated by laser capture microdissection. Droplet digital PCR (ddPCR) was used to identify MAP2K1 mutations. MAP2K1 (p.K57N) variants were present in the tissue adjacent to the cartilage [mutant allele frequency (MAF) 6-8%], and were enriched in endothelial cells (MAF 51%) compared to non-endothelial cells (MAF 0%). MAP2K1 mutations were not identified in the overgrown cartilage, and thus local cartilage overgrowth likely results from the effects of adjacent mutant blood vessels (i.e., cell-non autonomous).

Diffuse capillary malformation with overgrowth contains somatic PIK3CA variants.

Diffuse capillary malformation with overgrowth (DCMO) is a clinical diagnosis describing patients with multiple, extensive capillary malformations (CMs) associated with overgrowth and foot anomalies. The purpose of the study was to identify somatic variants in DCMO. Skin containing CM and overgrown subcutaneous adipose tissue was collected from patients with DCMO. Exons from 447 cancer-related genes were sequenced using OncoPanel. Variant-specific droplet digital PCR (ddPCR) independently confirmed the variants and determined variant allele frequencies (VAF). One subject contained a somatic PIK3CA p.G106V variant. A second patient had a PIK3CA p.D350G variant. VAF was 27% to 29% in skin and 16% to 28% in subcutaneous adipose. Variants were enriched in endothelial cells (VAF 50%-51%) compared to nonendothelial cells (1%-8%). DCMO is associated with somatic PIK3CA variants and should be considered on the PIK3CA-related overgrowth spectrum (PROS). Variants are present in both skin and subcutaneous adipose and are enriched in endothelial cells.

Arteriovenous malformation associated with a HRAS mutation.

The majority of extracranial arteriovenous malformations (AVMs) are caused by somatic mutations in MAP2K1. We report a somatic HRAS mutation in a patient who has a facial AVM associated with subcutaneous adipose overgrowth. We performed whole exome sequencing on DNA from the affected tissue and found a HRAS mutation (p.Thr58_Ala59delinsValLeuAspVal). Mutant allelic frequency was 5% in whole tissue and 31% in isolated endothelial cells (ECs); the mutation was not present in blood DNA or non-ECs. Somatic mutations in HRAS can cause AVM.

Intramuscular fast-flow vascular anomaly contains somatic MAP2K1 and KRAS mutations.

BACKGROUND: The term "intramuscular hemangioma capillary type" (IHCT) refers to a fast-flow vascular lesion that is classified as a tumor, although its phenotype overlaps with arteriovenous malformation (AVM). The purpose of this study was to identify somatic mutations in IHCT. METHODS: Affected tissue specimens were obtained during a clinically indicated procedure. The diagnosis of IHCT was based on history, physical examination, imaging and histopathology. Because somatic mutations in cancer-associated genes can cause vascular malformations, we sequenced exons from 446 cancer-related genes in DNA from 7 IHCT specimens. We then performed mutation-specific droplet digital PCR (ddPCR) to independently test for the presence of a somatic mutation found by sequencing and to screen one additional IHCT sample. RESULTS: We detected somatic mutations in 6 of 8 IHCT specimens. Four specimens had a mutation in MAP2K1 (p.Q58_E62del, p.P105_I107delinsL, p.Q56P) and 2 specimens had mutations in KRAS (p.K5E and p.G12D, p.G12D and p.Q22R). Mutant allele frequencies detected by sequencing and confirmed by ddPCR ranged from 2 to 15%. CONCLUSIONS: IHCT lesions are phenotypically similar to AVMs and contain the same somatic MAP2K1 or KRAS mutations, suggesting that IHCT is on the AVM spectrum. We propose calling this lesion "intramuscular fast-flow vascular anomaly."

Somatic mutations in intracranial arteriovenous malformations.

BACKGROUND: Intracranial arteriovenous malformation (AVM) is a common cause of primary intracerebral hemorrhage in young adults. Lesions typically are sporadic and contain somatic mutations in KRAS or BRAF. The purpose of this study was to identify somatic mutations in a cohort of participants with brain AVM and to determine if any genotype-phenotype associations exist. METHODS: Human brain AVM specimens (n = 16) were collected during a clinically-indicated procedure and subjected to multiplex targeted sequencing using molecular inversion probe (MIP-seq) for mutations in KRAS, BRAF, HRAS, NRAS, and MAP2K1. Endothelial cells (ECs) were separated from non-ECs by immune-affinity purification. Droplet digital PCR (ddPCR) was used to confirm mutations and to screen for mutations that may have been missed by MIP-seq. Patient and AVM characteristics were recorded. RESULTS: We detected somatic mutations in 10 of 16 specimens (63%). Eight had KRAS mutations [G12D (n = 5), G12V (n = 3)] and two had BRAF mutations [V600E (n = 1), Q636X (n = 1)]. We found no difference in age, sex, presenting symptom, AVM location, or AVM size between patients with a confirmed mutation and those without. Nor did we observe differences in these features between patients with KRAS or BRAF mutations. However, two patients with BRAF mutations presented at an older age than other study participants. CONCLUSIONS: Somatic mutations in KRAS and, less commonly in BRAF, are found in many but not all intracranial AVM samples. Currently, there are no obvious genotype-phenotype correlations that can be used to predict whether a somatic mutation will be detected and, if so, which gene will be mutated.

Analysis of Follicle-Stimulating Hormone Receptor in Infantile Hemangioma.

INTRODUCTION: The life cycle of infantile hemangioma (IH) and secretion of follicle-stimulating hormone (FSH) are identical. We previously have shown that IH contains the FSH receptor (FSHR). The purpose of this study was to identify which cell type(s) in IH expresses FSHR. METHODS: Human proliferating IH tissues obtained during a clinically indicated surgical procedure were used. Paraffin sections and isolated cell populations (endothelial, pericyte, stem cell) were subjected to immunofluorescence for FSHR. Tissues were costained with DAPI, anti-α smooth muscle actin, or biotinylated Ulex Europaeus Agglutinin I to identify nuclei, pericytes, and endothelial cells, respectively. Whole tissue and purified single cell populations underwent polymerase chain reaction (PCR) for FSHR. Positive control specimens (ovary, sertoli cells) and negative control tissues (skin/subcutis, hepatic cells) were included. RESULTS: Immunofluorescence of 9 IHs demonstrated that FSHR was enriched in pericytes compared with endothelial cells. Follicle-stimulating hormone receptor was expressed in 6 of 6 whole tissue IHs along with the positive control via PCR. Follicle-stimulating hormone receptor was not present in the negative control samples. Four of 5 sets of pericytes expressed FSHR by PCR. Neither IH endothelial cells, IH stem cells, nor negative control cells exhibited FSHR by PCR. CONCLUSIONS: Because the secretion of FSH correlates with the growth pattern of IH, FSH might be involved in the disease process. Follicle-stimulating hormone receptor is enriched in the pericytes of IH, suggesting that this cell type may be involved in the pathogenesis of the tumor.

Association between extremity kaposiform hemangioendothelioma and lymphedema.

Kaposiform hemangioendotheliomas are pediatric vascular tumors that do not metastasize. We present a patient with a thigh kaposiform hemangioendothelioma successfully treated using a systemic corticosteroid during infancy who was diagnosed with lymphedema in the extremity 9 years later. The observation that extremity kaposiform hemangioendothelioma could possibly be associated with lymphedema has implications for the care of patients with kaposiform hemangioendothelioma.

Propranolol Treatment of Vascular Anomalies Other Than Infantile Hemangioma.

BACKGROUND: Oral propranolol has become first-line intervention for problematic infantile hemangioma (IH) that is not amenable to topical or intralesional therapies. Consensus data supporting its efficacy for other vascular anomalies does not exist. The purpose of this study was to determine the frequency and causes of propranolol use for vascular lesions other than IH. METHODS: Referrals to our Vascular Anomalies Center between 2008 and 2017 were reviewed for patients treated with propranolol at an outside institution. Patient history, photographs, imaging studies, and/or histopathology were evaluated by an interdisciplinary team to diagnose the vascular anomaly. Our center's diagnosis was compared to the referral diagnosis to categorize patients into 3 groups: Group 1 (patients were appropriately labeled with an IH); Group 2 (individuals were erroneously diagnosed with IH); and Group 3 (subjects were diagnosed with a vascular anomaly other than IH). RESULTS: Two hundred thirty-six patients met inclusion criteria. Group 1 (39%; n = 91) had an IH and were treated appropriately. Group 2 (20%; n = 49) was misdiagnosed with IH and incorrectly received propranolol. Group 3 (41%; n = 96) was given propranolol to treat another vascular anomaly. Propranolol did not have efficacy for vascular anomalies other than IH. CONCLUSIONS: Propranolol commonly is used to treat lesions other than IH; misdiagnosis of a lesion as IH is a common cause. Propranolol should be used with caution to treat other types of vascular anomalies because patients are subjected to the risks of the drug without data supporting its efficacy.

Somatic PIK3CA mutations are present in multiple tissues of facial infiltrating lipomatosis.

BackgroundFacial infiltrating lipomatosis (FIL) is a congenital disorder that causes overgrowth of one side of the face. The purpose of this study was to determine whether PIK3CA mutations are present in tissues outside of the subcutaneous adipose.MethodsFIL tissues from three patients were dissected to enrich for cells from skin, subcutaneous tissue, orbicularis oris muscle, buccal fat, zygomatic bone, and mucosal neuroma. Endothelial cells within the affected tissue also were enriched using CD31 microbeads. Laser capture microdissection on formalin-fixed paraffin-embedded histologic sections was performed to collect specific cell types. DNA was extracted from each tissue and cell type, and measured for the abundance of mutant PIK3CA alleles using droplet digital PCR.ResultsWe detected mutant PIK3CA alleles in every tissue and cell type tested from each overgrown face; frequencies ranged from 1.5 to 53%. There were fewer mutant endothelial cells compared with nonendothelial cells, and the stromal cell compartment had the highest frequency of mutant cells in each tissue.ConclusionsPIK3CA mutations are not restricted to a single tissue or cell type in FIL. Overgrowth in this condition is likely due to the mutation arising in a cell that contributes to several different facial structures during embryogenesis.

Somatic MAP2K1 Mutations Are Associated with Extracranial Arteriovenous Malformation.

Arteriovenous malformation (AVM) is a fast-flow, congenital vascular anomaly that may arise anywhere in the body. AVMs typically progress, causing destruction of surrounding tissue and, sometimes, cardiac overload. AVMs are difficult to control; they often re-expand after embolization or resection, and pharmacologic therapy is unavailable. We studied extracranial AVMs in order to identify their biological basis. We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) on AVM tissue from affected individuals. Endothelial cells were separated from non-endothelial cells by immune-affinity purification. We used droplet digital PCR (ddPCR) to confirm mutations found by WES and WGS, to determine whether mutant alleles were enriched in endothelial or non-endothelial cells, and to screen additional AVM specimens. In seven of ten specimens, WES and WGS detected and ddPCR confirmed somatic mutations in mitogen activated protein kinase kinase 1 (MAP2K1), the gene that encodes MAP-extracellular signal-regulated kinase 1 (MEK1). Mutant alleles were enriched in endothelial cells and were not present in blood or saliva. 9 of 15 additional AVM specimens contained mutant MAP2K1 alleles. Mutations were missense or small in-frame deletions that affect amino acid residues within or adjacent to the protein's negative regulatory domain. Several of these mutations have been found in cancers and shown to increase MEK1 activity. In summary, somatic mutations in MAP2K1 are a common cause of extracranial AVM. The likely mechanism is endothelial cell dysfunction due to increased MEK1 activity. MEK1 inhibitors, which are approved to treat several forms of cancer, are potential therapeutic agents for individuals with extracranial AVM.

Beckwith-Wiedemann Syndrome and Primary Lymphedema of the Lower Extremity.

Beckwith-Wiedemann syndrome is the most common genetic overgrowth syndrome. Patients with Beckwith-Wiedemann syndrome may have hemihypertrophy, but their lymphatic vasculature is intact. We present a child with Beckwith-Wiedemann syndrome and lower extremity enlargement thought to be due to hemihypertrophy that was instead diagnosed with primary lymphedema. There are many causes of leg overgrowth in the pediatric population and misdiagnosis is common. While extremity enlargement secondary to hemihypertrophy may occur in 15% of patients with Beckwith-Wiedemann syndrome, progression and pitting edema only occur in primary lymphedema. This report highlights the importance of ensuring an accurate diagnosis so that patients are managed appropriately.