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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., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number F32HD107878. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Somatic activating MAP2K1 mutations in endothelial cells (ECs) cause extracranial arteriovenous malformation (AVM). We previously reported the generation of a mouse line allowing inducible expression of constitutively active MAP2K1 (p.K57N) from the Rosa locus (R26 GT-Map2k1-GFP/+ ) and showed, using Tg-Cdh5CreER, that EC expression of mutant MAP2K1 is sufficient for the development of vascular malformations in the brain, ear, and intestines. To gain further insight into the mechanism by which mutant MAP2K1 drives AVM development, we induced MAP2K1 (p.K57N) expression in ECs of postnatal-day-1 pups (P1) and investigated the changes in gene expression in P9 brain ECs by RNA-seq. We found that over-expression of MAP2K1 altered the transcript abundance of > 1600 genes. Several genes had > 20-fold changes between MAP2K1 expressing and wild-type ECs; the highest were Col15a1 (39-fold) and Itgb3 (24-fold). Increased expression of COL15A1 in R26 GT-Map2k1-GFP/+ ; Tg-Cdh5CreER +/- brain ECs was validated by immunostaining. Ontology showed that differentially expressed genes were involved in processes important for vasculogenesis (e.g., cell migration, adhesion, extracellular matrix organization, tube formation, angiogenesis). Understanding how these genes and pathways contribute to AVM formation will help identify targets for therapeutic intervention., (© 2023. The Author(s).)

Extracranial arteriovenous malformation (AVM) is a congenital vascular anomaly causing disfigurement, bleeding, ulceration, and pain. Most lesions are associated with somatic MAP2K1 activating mutations in endothelial cells (ECs). The purpose of this study was to determine if EC expression of mutant activated MAP2K1 is sufficient to produce vascular malformations in mice. We generated mice with a ROSA26 allele containing a lox-stop-lox gene trap (GT), Map2k1 cDNA with an activating p.K57N missense mutation, an internal ribosomal entry site, and green fluorescent protein cDNA (R26 GT-Map2k1-GFP ). We expressed mutant MAP2K1 and GFP in ECs of fetal and newborn mice using Tg-Cdh5Cre or Tg-Cdh5CreER alleles. Tg-Cdh5Cre +/- ;R26 GT-Map2k1-GFP/+ animals that express mutant MAP2K1 in ECs in utero developed diffuse vascular abnormalities and died by embryonic (E) day 16.5. Tg-Cdh5CreER +/- ;R26 GT-Map2k1-GFP/+ animals in which mutant MAP2K1 expression was induced in ECs by tamoxifen at postnatal (P) day 1 developed vascular malformations in the brain, ear, and intestines by P23. The lesions consisted of abnormal networks of blood vessels containing recombined and non-recombined ECs. In conclusion, expression of MAP2K1 p.K57N is sufficient to cause vascular malformations in mice. This model can be used to study the malformation process and for pre-clinical pharmacologic studies., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Bockenheimer disease is a venous malformation involving all tissues of an extremity. Patients have significant morbidity, and treatment is palliative. The purpose of this study was to identify the cause of Bockenheimer disease to develop pharmacotherapy for the condition. Paraffin-embedded tissue from nine individuals with Bockenheimer disease obtained during a clinically indicated operation underwent DNA extraction. Droplet digital polymerase chain reaction (ddPCR) was used to screen for variants most commonly associated with sporadic venous malformations ( TEK [NM_000459.5:c.2740C > T; p.Leu914Phe], PIK3CA [NM_006218.4:c.1624G > A; p.Glu542Lys and NM_006218.4:c.3140A > G; p.His1047Arg]). ddPCR detected a TEK L914F variant in all nine patients (variant allele fraction 2%-13%). PIK3CA E542K and H1047R variants were not identified in the specimens. Sanger sequencing and restriction enzyme digestion confirmed variants identified by ddPCR. A pathogenic variant in the endothelial cell tyrosine kinase receptor TEK is associated with Bockenheimer disease. Pharmacotherapy targeting the TEK signaling pathway might benefit patients with the condition., (© 2021 Sudduth et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (© 2021 Eng et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (© 2020 Wiley Periodicals LLC.)

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., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Arteriovenous malformation (AVM) is a locally destructive congenital vascular anomaly caused by somatic mutations in MAP2K1. The mutation is isolated to endothelial cells (ECs). The purpose of this study was to determine the effects of mutant MAP2K1 on EC signaling and vascular network formation. Pathway effects were studied using both mutant MAP2K1 (K57N) human AVM tissue and human umbilical vein endothelial cells (HUVECs) engineered to overexpress the MAP2K1 (K57N) mutation. Western blot was used to determine cell signaling along the RAS/MAPK pathway. Geltrex tube formation assays were performed to assess EC vascular network formation. Cells were treated with a MAP2K1 inhibitor (Trametinib) to determine its effect on signaling and vascular tube formation. Human mutant MAP2K1-AVM ECs had similar baseline MEK1 and ERK1/2 expression with controls; however, mutant MAP2K1-AVM ECs produced significantly more phosphorylated ERK1/2 than wild-type ECs. Mutant MAP2K1 HUVECs demonstrated significantly more phosphorylated ERK1/2 than control HUVECs. Trametinib reduced the phosphorylation of ERK1/2 in mutant cells and prevented the ability of ECs to form vascular networks. AVM MAP2K1 mutations activate RAS/MAPK signaling in ECs. ERK activation and vascular network formation are reduced with Trametinib. Pharmacotherapy using MAP2K1 inhibitors may prevent the formation or progression of AVMs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Patent application filed for discovery of MAP2K1 mutation in AVMs (AKG)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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).

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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."

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal (CLOVES) anomalies and Klippel-Trenaunay (KTS) syndromes are caused by somatic gain-of-function mutations in PIK3CA, encoding a catalytic subunit of phosphoinositide 3-kinase. Affected tissue is needed to find mutations, as mutant alleles are not detectable in blood. Because some patients with CLOVES develop Wilms tumor, we tested urine as a source of DNA for mutation detection. We extracted DNA from the urine of 17 and 24 individuals with CLOVES and KTS, respectively, and screened 5 common PIK3CA mutation hotspots using droplet digital polymerase chain reaction. Six of 17 CLOVES participants (35%) had mutant PIK3CA alleles in urine. Among 8 individuals in whom a mutation had been previously identified in affected tissue, 4 had the same mutant allele in the urine. One study participant with CLOVES had been treated for Wilms tumor. We detected the same PIK3CA mutation in her affected tissue, urine, and tumor, indicating Wilms tumors probably arise from PIK3CA mutant cells in patients with CLOVES. No urine sample from a participant with KTS had detectable PIK3CA mutations. We suggest that urine, which has the advantage of being collected non-invasively, is useful when searching for mutations in individuals with CLOVES syndrome., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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., (© 2017 Wiley Periodicals, Inc.)

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.

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.

Background: Capillary malformation is a cutaneous vascular anomaly that is present at birth, darkens over time, and can cause overgrowth of tissues beneath the stain. The lesion is caused by a somatic activating mutation in GNAQ. In a previous study, we were unable to identify a GNAQ mutation in patients with a capillary malformation involving an overgrown lower extremity. We hypothesized that mutations in GNA11 or GNA14, genes closely related to GNAQ, also may cause capillary malformations., Methods: Human capillary malformation tissue obtained from 8 patients that had tested negative for GNAQ mutations were studied. Lesions involved an extremity (n = 7) or trunk (n = 1). Droplet digital PCR (ddPCR) was used to detect GNA11 or GNA14 mutant cells (p.Arg183) in the specimens. Single molecule molecular inversion probe sequencing (smMIP-seq) was performed to search for other mutations in GNA11. Mutations were validated by subcloning and sequencing amplimers., Results: We found a somatic GNA11 missense mutation (c.547C > T; p.Arg183Cys) in 3 patients with a diffuse capillary malformation of an extremity. Mutant allelic frequencies ranged from 0.3 to 5.0%. GNA11 or GNA14 mutations were not found in 5 affected tissues or in unaffected tissues (white blood cell DNA)., Conculsions: GNA11 mutations are associated with extremity capillary malformations causing overgrowth. Pharmacotherapy that affects GNA11 signaling may prevent the progression of capillary malformations.

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., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)