Genetic Basis and Therapies for Vascular Anomalies

Vascular anomalies arise due to defects during early vascular development, which lead to locally, abnormally formed vessels. In general, based on their clinical and histological characteristics, they are separated into vascular malformations and vascular tumors. Vascular malformations are classified into venous, arterial, capillary, lymphatic, and combined malformations, whereas vascular tumors are separated into infantile hemangiomas (IH) and other tumors¹ (Figure 1). Most vascular malformations are present at birth or soon after birth and expand during the patient’s lifetime. However, novel lesions can also appear with time.²

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Vascular anomalies can be caused by inherited or somatic genetic mutations.^3–5 Inherited germline mutations are encountered in 11 specific vascular malformations (Figure 2). Since 2002, a somatic second-hit has been described in lesional cells generating a complete localized loss-of-function of the protein in question in the majority of them (Figure 2). In contrast, the majority of the more common noninherited, isolated vascular malformations are associated with gain-of-function somatic mutations, since the pivotal discovery of somatic TIE2 (angiopoietin-1 receptor) mutations in venous malformations (VMs) in 2009 (Figure 2).⁶

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Most mutations are detected in genes that play important roles in pathways involved in angiogenesis and lymphangiogenesis, vascular cell growth, apoptosis, and proliferation.⁷ Interestingly, many of those mutations are also found in cancers.⁷ The major pathways that are involved include angiopoietin/TIE2 (angiopoietin-1 receptor), PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin), EPHB4 (Ephrin B4)/Ras (rat sarcoma)/MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase), and TGF (transforming growth factor) b signaling. The G protein–coupled receptor signaling molecules (GNA [G protein subunit alpha] Q/GNA11/GNA14) are also often implicated.⁷

Up to now, treatment options have been limited, especially for large and extensive vascular malformations for which sclerotherapy, embolization, surgery, laser ablation, and combinations thereof, have not led to complete cures. These patients experience severe chronic pain, destruction, and poor quality of life. Due to the breakthroughs in unraveling the pathophysiological causes of vascular anomalies and the respective signaling pathways, the field has entered a new era of intensive study repurposing anticancer drugs to the treatment of vascular anomalies.

Vascular Anomalies and PI3K/AKT/mTOR Signaling (PIKopathies)

PIKopathies include the vascular anomalies in which the PI3K/AKT/mTOR signaling pathway (Figure 3) is affected. Under normal conditions, this pathway is activated after a ligand binds to its RTK (receptor tyrosine kinase; eg, EGF [epidermal growth factor] to EGFR [epidermal growth factor receptor], or angiopoietin to TIE2), to control cell proliferation, adhesion, migration, metabolism, and survival.⁸ Ligand binding leads to phosphorylation of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol-3,4,5-trisphospate through PI3K, which in turn recruits and regulates its downstream target AKT.⁹ PTEN (phosphatase and tensin homolog) negatively regulates this activity by counteracting PI3K. In vascular anomalies, various slow-flow malformations (especially venous and lymphatic malformations [LMs]) are due to mutations activating this pathway. Furthermore, mutations causing hereditary hemorrhagic telangiectasia (HHT) also seem to lead to an enhanced activation of the AKT/mTOR pathway.

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VMs and Angiopoietin/TIE2-PI3K/AKT/mTOR Signaling

VMs are often found in the skin or mucosal membranes and belong to the slow-flow lesions (Figure 1A). They are blue-colored, soft, and compressible.^10,11 Histologically, they are characterized by enlarged and distorted vein-like channels, in which a single layer of endothelial cells (ECs) is surrounded by disorganized extracellular matrix (due to eg, reduced fibronectin expression and increased proteolytic activity) and smooth muscle cells.^4,11,12

In the majority of sporadically occurring unifocal and multifocal VM (VM and multifocal venous malformation, respectively), and the blue rubber bleb nevus syndrome, as well as in inherited cutaneomucosal VMs, gain-of-function mutations in the TEK gene can be detected (Figure 2, Table). TEK encodes the endothelial receptor tyrosine kinase TIE2.⁶ TIE2 is activated by ANGPT (angiopoietin) 1, whereas ANGPT2 is able to modulate TIE2 activity depending on the context.^43,44 Ligand binding results in TIE2 activation through multimerization and cross-phosphorylation of receptor molecules, which leads to activation of the canonical PI3K/AKT/mTOR signaling pathway (Figure 3).⁴⁵

More than 20 different mutations have been described.⁴⁶ Mutations occur in the intracellular parts of the RTK (kinase domain, kinase insert domain, or carboxyl-terminal tail)^6,7,11 and result in amino acid substitutions or truncation of the inhibitory C-terminal loop.

The most frequent mutation detected in sporadic VMs is L914F^6,47 (Table). In contrast, the most common TIE2 mutation in autosomal-dominantly inherited cutaneomucosal VM is R849W (Table).^4,7,48 The latter results in a weak TIE2 autophosphorylation when overexpressed in human umbilical vein ECs (HUVECs) and needs a somatic second-hit in TEK to cause the typical multifocal and small-sized lesions.^6,13 Patients harboring multifocal venous malformation have similar small multifocal lesions as patients with inherited cutaneomucosal VM. However, they are often mosaic for the first mutation (commonly R915C) associated with a second-hit in affected areas. This somatic mutation, like the first mosaic mutation, typically introduces an additional cysteine in TIE2 (eg, Y897C)¹³ (Table). The sporadically occurring blue rubber bleb nevus syndrome is the fourth phenotype associated with TIE2 mutations. Patients display up to hundreds of widely distributed cutaneous and gastrointestinal VMs, often causing consumptive coagulopathy and chronic anemia.¹³ The number of lesions increases during life. Blue rubber bleb nevus syndrome is associated with a typical carboxy-terminal double-mutation T1105N-T1106P but also sometimes with Y897F-R915L (Table).¹³ Interestingly, the same double mutations are identified in distant lesions, whereas blood samples remain negative. This suggests a temporarily restricted circulation of mutant cells which can trigger the formation of novel lesions.

In vitro experiments demonstrated that all TIE2 mutations lead to ligand-independent hyperphosphorylation of the receptor and a permanent activation of the PI3K/AKT/mTOR signaling pathway (Figure 3).^4,6,11,12,49 Double mutations show a stronger phosphorylation of TIE2 compared with single mutations. HUVECs expressing TIE2-L914F secrete lower amounts of PDGF-B (platelet-derived growth factor subunit B) due to AKT-dependent phosphorylation of FOXO1 (forkhead box protein O1). This partially explains a decreased paracrine interaction between ECs and smooth muscle cells (SMC) and the relative lack of smooth muscle cells observed on histology of VMs.^49,50 Furthermore, TIE2-L914F expressing HUVECs promote SMC transition from a contractile to a synthetic phenotype.⁵⁰

A minority of sporadically occurring VMs have a somatic PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation (Figure 2, Table).^{11,12,49,51,52} The PIK3CA gene encodes the p110α catalytic subunit of PI3K, the downstream effector of TIE2. PIK3CA mutations, like the TIE2 mutations, lead to the activation of AKT (Figure 3).

Hot-spot mutations in PIK3CA include p.Glu542Lys, p.Glu545Lys (helical domain), and p.His1047Arg (kinase domain).¹² The same mutations are frequently found in cancers. They are also observed in various PIK3CA-related overgrowth syndromes (PROS), in which vascular malformations are associated with hypertrophy of soft and sometimes bony tissues.^15,53 However, non–hot-spot PIK3CA mutations seem to be more frequent in some PROS phenotypes, such as megalencephaly–capillary malformation (CM) syndrome and congenital lipomatous overgrowth with vascular anomalies, epidermal nevi and scoliosis (CLOVES) syndromes (Figure 1B, Figure 2, Table).¹⁴ Such non–hot-spot mutations seem to be more often more widely distributed in patients’ tissues (ie, giving a mosaic situation) than the hot-spot mutations.

In vitro, HUVEC overexpressing these PIK3CA mutants showed AKT activation, disruption of the cobblestone-like EC monolayer, and loss of the extracellular matrix protein fibronectin. Furthermore, ANGPT2 and PDGF-B mRNAs were downregulated. In contrast to HUVECs overexpressing mutant TIE2-L914F, HUVECs overexpressing mutant PIK3CA showed low levels of p-STAT (phosphorylated signal transducer and activator of transcription) 1 and no pERK1/2 (phosphorylated extracellular signal-regulated kinase).¹² This hints that the PI3K/AKT/mTOR pathway plays a crucial role in VMs, whether due to PIK3CA or a TIE2 mutation (Figure 3).

Lymphatic Anomalies and PI3K/AKT/mTOR Signaling

Lymphatic anomalies are divided into LM (Figure 1C) and lymphedema. LMs can be isolated or part of a syndrome, such as PROS. LM can be peripheral or central, the latter constituting the group of complicated lymphatic anomalies (CLAs). They include generalized lymphatic anomaly, Gorham-Stout Disease (GSD), kaposiform lymphangiomatosis (KLA), and central conducting lymphatic anomalies. CLAs can be associated with primary lymphedema, for which over 30 genes have been identified. These are discussed in detail in the accompanying review by Mäkinen et al.⁵⁴

The majority of LMs have a similar somatic hot-spot mutation in PIK3CA as seen in VMs (Figure 2, Table). They include p.Glu542Lys, p.Glu545Lys, and p.Glu545Gly (helical domain) as well as p.His1047Arg and pHis1047Leu (kinase domain; Table).⁵⁵ As expected, hot-spot PIK3CA mutations in isolated lymphatic ECs from LM patients were able to activate the PI3K/AKT/mTOR pathway since elevated p-AKT (phosphorylated-AKT) could be detected in Western blots.¹⁶ The hot-spot mutations can cause lethality in heterozygous state,⁵⁶ likely explaining why inherited isolated LMs have not been reported, whereas (de novo) non–hot-spot mutations are sometimes seen in patients with macrocephaly and associated features.⁵⁷

Mutation type but also timing of occurrence of the postzygotic mutation (mosaic or somatic) seem to be important. The non–hot-spot mutations are more often seen in mosaic state and in syndromic forms compared with the hot-spot mutations (P. Brouillard, et al, submitted, 2021). Moreover, the timing of activation of the PIK3CA^H1047R mutation during murine development determined the LM type. If PIK3CA^H1047R was expressed early in fetal development in lymphatic ECs, large macrocystic LMs developed, whereas expression of the mutation in postnatal lymphatic vasculature caused lymphatic hypersprouting which resulted in microcystic LMs.⁵⁸ Of interest, these effects seem to be dependent on VEGF (vascular endothelial growth factor)-C activation of the VEGFR (vascular endothelial growth factor receptor) 3 upstream of PI3K signaling.⁵⁸ See for more detail Mäkinen et al.⁵⁴

Arteriovenous Malformation and PI3K/AKT/mTOR Signaling

PTEN Hamartoma Tumor Syndrome

PTEN hamartoma tumor syndrome (PHTS) is now used for a spectrum of phenotypes including the Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, adult Lhermitte-Duclos disease, and autism spectrum disorders associated with macroencephaly.⁵⁹ In PHTS, loss-of-function PTEN mutations (Table, Figure 2) are inherited in an autosomal-dominant manner.^17,60–64 Patients harboring PHTS normally have macrocephaly, lipomas, and papillomas or trichilemmomas, associated with nonpathognomonic cutaneous and deeper soft tissue vascular lesions, which can also be characterized by fast-flow. Because PTEN inhibits the PI3K/AKT/mTOR pathway, loss-of-function mutations lead to its constitutive activation (Figure 3). Furthermore, because PTEN is an important tumor suppressor gene, PHTS patients are predisposed to several types of cancers.⁷

Hereditary Hemorrhagic Telangiectasia

HHT (Osler-Weber-Rendu syndrome) is inherited in an autosomal-dominant manner.⁶⁵ Patients have multiple cutaneous lesions (telangiectasias) which are often associated with internal arteriovenous malformations (AVMs). These can be found in the central nervous system, lungs, liver, and other internal organs. Furthermore, one-third of the patients have recurrent nosebleeds before the age of 10 years, which commonly lead to anemia.⁶⁶ Later during life, patients develop characteristic cutaneous telangiectasias on the face, oral mucosa, and on hands.⁶⁵

Five loci are linked to HHT (Table, Figure 2).⁶⁷ In HHT1 and HHT2, loss-of-function mutations are found in endoglin (ENG)¹⁸ and activin receptor-like kinase 1 (ALK1),¹⁹ respectively. Juvenile polyposis/HHT syndrome develops due to loss-of-function mutations in MADH4 which encodes SMAD (mothers against decapentaplegic homolog) 4.²¹ Additional loci were identified on chromosome 5q31.3-32 (HHT3) and 7p14 (HHT4).^20,67 Moreover, substitutions have been identified in the GDF2 (growth/differentiation factor 2; also known as BMP9 [bone morphogenetic protein-9]) which is the ligand for ALK1 (Table).^68,69

The HHT-associated genes encode proteins that play a role in BMP signaling.⁶⁸ The ALK1 and endoglin complex is expressed on ECs. Endoglin leads to induction of BMP9/BMP10/ALK1 signaling via receptor phosphorylation and activation of transcription factors R-SMAD (receptor-regulated SMAD) 1/5/8 and SMAD4, which normally suppress EC migration and proliferation, and keep ECs in a quiescent state.⁷⁰ Loss-of-function in these genes can lead to enhanced EC migration, proliferation, and vessel formation (Figure 3). In addition, VEGF and AKT signaling seem to be enhanced. In Alk1 knockout mouse lung ECs as well as in HUVECs with ALK1 knockdown or BMP9/10 ligand blockade, AKT is activated due to inhibition of PTEN through its increased carboxy-terminal phosphorylation and translocation into the nucleus.⁶⁸ Increased VEGF occurs through downregulation of TGFB1 (tumor growth factor beta 1) signaling.⁷¹

Vascular Anomalies and RAS/Rapidly Accelerated Fibrosarcoma/MEK/ERK Signaling (RASopathies)

RASopathies include the vascular anomalies in which the RAS/RAF (rapidly accelerated fibrosarcoma)/MEK/ERK signaling is affected. The MAPK (mitogen-activated protein kinase) pathways play major roles in cellular differentiation, proliferation, apoptosis, and stress response. RAS/RAF/MEK/ERK signaling is induced by growth factors, cytokines, and G protein–coupled receptor ligands,⁷² which lead to the activation of RAS through the replacement of GDP with GTP. After binding to RAS, RAF exhibits serine/threonine-protein kinase activity and activates MEK that in turn activates ERK through phosphorylation. ERK dimerizes and is translocated to the nucleus to regulate activities of target proteins.⁷² Mutations in genes implicated in this pathway have been observed in various vascular malformations.

VMs and RAS/RAF/MEK/ERK Signaling

Verrucous VMs (VVM)—also known as verrucous hemangioma—is a rare noninherited vascular anomaly that presents as raised, deep red lesions which become hyperkeratotic over time.^22,73 Somatic activating mutations in the mitogen-activated protein kinase kinase kinase 3 (MAP3K3) gene that is a member of the MAP3K family of serine/threonine kinases have been identified in VVM tissues (Table, Figure 2).²² MAP3K3 is involved in ERK signaling as well as AKT/mTOR signaling (Figure 3).²²

Patients harboring hyperkeratotic cutaneous capillary VMs display crimson-colored lesions which can reach several centimeters in size and are covered with a hyperkeratotic dermis.^23,74 Hyperkeratotic cutaneous capillary VMs are associated with cerebral cavernous malformations (CCM).²³ Hyperkeratotic cutaneous capillary VM patients have an inherited loss-of-function mutation in Krev interaction trapped 1 (KRIT1; Table, Figure 2).^23,75 The KRIT1/CCM2/CCM3 complex is known to inhibit MAP3K3.⁷⁶ Loss-of-function of KRIT1 and thus loss of the whole CCM complex leads to activation of MAP3K3 signaling as seen in VVMs (Figure 3). The target genes including KLF2, KLF4 (Kruppel-like factor) RHO, and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif).⁷⁶

Lymphatic Anomalies and RAS/RAF/MEK/ERK Signaling

GSD, which belongs to complex LMs (CLAs), occurs sporadically. Patients show irregular lymphatic vessels in bones which are associated with osteolysis leading to entire loss of bones⁷⁷ associated with central conducting lymphatic anomalies. In one GSD patient, an activating somatic mutation in Kirsten rat sarcoma viral oncogene homolog (KRAS) could be detected (Table and Figure 1) (N. Homayun Sepehr, et al, submitted, 2021). Recently, in the aggressive lymphatic anomaly KLA, which can occur in several parts and organs of the body, a somatic activating NRAS p.Q61R variant was identified (Table, Figure 2) in 10 of 11 KLA tissues,²⁴ suggesting that RAS signaling is important for the development of KLA and GSD (Figure 3). See accompanying review by Mäkinen et al.⁵⁴

CM and RAS/RAF/MEK/ERK Signaling

About 0.3% of newborns have at birth a CM or port-wine-stain.^45,78 Lesions appear sporadically and they are manifested by flat, red-to-purple lesions⁷⁹ (Figure 1D). Somatic activating p.Arg183Gln GNAQ mutations are frequently identified in CMs (Table, Figure 2).²⁵ The loss of arginine in GNAQ leads to a reduction in hydrogen bonding between G(q) and GDP.⁸⁰ In HEK293T (human embryonic kidney 293T cells) overexpression of GNAQ pArg183Gln and pGln209Leu mutations—which are found in congenital hemangiomas—both led to the induction of ERK (Figure 3), however, with a stronger effect with the latter.²⁵

Capillary Malformation-AVM

CM-AVM patients have small, multifocal, randomly distributed red-to brownish CM lesions that are mostly surrounded by pale halo.^81,82 CM-AVM can occur together with fast-flow lesions like arteriovenous fistula, AVM, vein of Galen aneurysmal malformation, or Parkers Weber syndrome.⁸² CM-AVM is transmitted in an autosomal-dominant manner while lesions are already visible at birth. However, they can also appear later in life.^26,82,83

CM-AVM1 is caused by RASA (RAS p21 protein activator) 1 mutations (Table, Figure 2),^26,83,84 and >40 truncating mutations in at least 100 CM-AVM families have been described.^7,26,85 In 3 patients, a second-hit on the second RASA1 allele was detected leading to a complete loss of RASA1.^85–87 Recently, distinct RASA1 low-level mosaic mutations were reported in 4 patients.⁸⁷

RASA1 encodes the RAS p21 protein activator 1 (p120RasGAP). Upon receptor tyrosine kinase activation, p120RasGAP is recruited to the cell membrane where it inhibits the RAS/MAPK/ERK signaling pathway and regulates cell growth, differentiation, proliferation, and EC network organization.⁷ Moreover, p120RasGAP has been described to interact with p190RhoGAP or FAK, both having a role in EC movement.⁸⁸ Mutations in RASA1 lead to a prolonged RAS/MAPK/ERK signaling supporting the important role of this pathway in CM-AVM1 (Figure 3). CM-AVM2 differs slightly from CM-AVM1. CM-AVM2 patients additionally show small telangiectasias around the lips and upper thorax as well as less intracerebral fast-flow lesions.²⁷ CM-AVM2 is caused by loss-of-function mutations in EPHB4 (Table, Figure 2).²⁷ EPHB4 is a transmembrane receptor, expressed on venous ECs during vascular development,⁸⁹ and its ligand, EphrinB2, is detectable on arterial ECs.⁹⁰ EPHB4 inhibits the RAS/MAPK/ERK pathway by interacting with p120RasGAP.⁹⁰ This inhibitory effect is lost in CM-AVM2 leading to a constitutive activation of RAS/MAPK/ERK signaling (Figure 3).²⁷

AVM and RAS/RAF/MEK/ERK Signaling

AVMs are the most aggressive vascular malformations since they often destroy adjacent noble structures during their development/growth. To remove the lesion completely by embolization or surgery is often not possible and remaining parts of lesions commonly lead to aggravation of AVMs. In general, AVMs can be found in any organs of the body including visceral organs or peripheral structures as well as the central nervous system (Figure 1E).

Couto et al²⁸ were able to identify a mutation in MAP2K1 (Table, Figure 2) which encodes the dual specificity mitogen-activated protein kinase MEK1 in a series of peripheral/extracranial AVMs. Somatic activating mutations in MEK1 activate the RAS/MAPK pathway (Figure 3). Furthermore, Nikolaev et al³⁰ detected somatic activating KRAS mutations (c.35G→T and c.35G→A) in brain AVMs (Table, Figure 2). ECs derived from brain AVMs showed an increased ERK activity (Figure 3), as well as an enhanced expression of angiogenesis-related genes and Notch (neurogenic locus Notch homolog protein) signaling. Furthermore, they showed an increased migratory capacity.³⁰ Moreover, Al-Olabi et al²⁹ were able to detect mutations in KRAS, BRAF (B-raf proto oncogene), and MAP2K1, supporting the role of RAS/RAF/MAPK signaling in AVMs (Table, Figure 2).

CCM and RAS/RAF/MAPK/ERK Signaling

CCM can be either transferred in an autosomal-dominant manner with incomplete penetrance or appears sporadically.⁹¹ Lesions mainly occur in the brain; however, sometimes they are also found in the spinal cord.

Loss-of-function mutations in KRIT1, malcavernin, and programmed cell death 10 (PDCD10) lead to the formation of CCMs.^31–33 Furthermore, CCM4 on 3q26.3-27.2 has been suggested as a fourth locus (Table, Figure 2).³⁴

The 3 CCM proteins built a complex in which CCM2 is found to link KRIT1 and PDCD10.⁹² Furthermore, it could be demonstrated that KRIT1 is able to interact with the ICAP1 (integrin cytoplasmic-associated protein 1). The latter suppresses activation of integrin.^93,94 ICAP1 is destabilized after loss-of-function of KRIT1 and CCM2 leading to an enhanced activation of integrin and disruption of normal tissue development.^95,96 Additionally, KRIT1 plays a role in DLL (delta-like canonical notch ligand) 4–NOTCH signaling leading to AKT activation and reduced ERK activity, which may explain why patients with KRIT1 mutations have increased levels of phosphor-ERK.⁹⁷ Since the CCM1/CCM2/CCM3 complex also interacts with MAP3K3 as well as with the small GTPase RAC1 (Ras-related C3 botulinum toxin substrate 1), loss of this complex induces MAP3K3 signaling (Figure 3), which can be seen by the upregulation of MAP3K3 target genes KLF2, KLF4, RHO, and ADAMTS.⁷⁶

Vascular Tumors and RAS/RAF/MAPK/ERK Signaling

Pyogenic Granuloma

Pyogenic granuloma (PG) is a relatively common benign vascular tumor that shows as a rapidly growing angiomatous papule or polyp. PGs can occur in the skin, mucous membranes, subcutaneously, and in the gastrointestinal tract.⁹⁸ They are often detectable within capillary malformations.^1,35 In these secondary PGs, an activating somatic mutation in GNAQ (p.Arg183Gln) was described (Table, Figure 2).³⁵ Since cooccurring CMs and PGs share the same mutation the hypothesis is that PG originates from cells of the underlying CM.³⁵ Additionally, in a small cohort of secondary PGs, a somatic mutation in BRAF (p.Val600Glu) or NRAS (p.Gln61Arg) could be identified (Table, Figure 2).³⁵ Somatic mutations in BRAF (p.Val600Glu or p.Gly464Glu), KRAS (p.Gly13Arg), GNA14, and HRAS (p.Q61R, p.E49K, p.Q61R, and p.G13S) have also been reported (Table, Figure 2).^35,37 The latter are the same as commonly seen in colon cancers.³⁶

Congenital Hemangioma

Congenital hemangioma includes rapidly involuting, partially involuting, and noninvoluting congenital hemangioma. Because congenital hemangiomas proliferate already in utero they are fully present at birth.^99–101 All 3 types do not express the GLUT (glucose transporter-1 protein) like seen in IH.¹⁰⁰ In congenital hemangiomas, mutually exclusive, mosaic missense mutations in GNAQ and GNA11 could be detected at position glutamine 209 (Table, Figure 2), which differ from those found in CMs.^38,39,102 Same missense mutations occur also in >80% of uveal melanomas where they lead to a constitutive activation of MAPK or YAP (Yes-associated protein) signaling pathways (Figure 3).³⁸

Other Signaling Pathways in Vascular Anomalies

Others pathways involved in vascular anomalies include (HGF [hepatocyte growth factor])/c-Met signaling in glomuvenous malformations (GVM) and VEGF-A/VEGFR2 signaling in IH.

VMs and HGF/c-Met Signaling

Glomuvenous Malformation

GVMs are inherited as an autosomal-dominant disorder. Patients harbor bluish-purple, cutaneous vascular lesions which are mostly found on extremities.⁴⁵ Lesions are multifocal and painful. In contrast to VMs, lesions cannot be emptied after compression.² Histological characteristics are abnormally differentiated vascular SMCs (vSMCs)—called glomus cells—which are located around dilated veins that are covered by normal-looking ECs.^40,103 Up to now, >40 different loss-of-function mutations in glomulin (GLMN/FAP68) have been discovered (Table, Figure 2).¹⁰⁴ The first somatic second hits on inherited vascular anomalies were found in 2002 for GVMs⁴⁰ (Figure 2). The most common is an acquired uniparental isodisomy of the long arm of chromosome 1,¹⁰⁵ leading to complete loss of glomulin.

Both, ECs and vSMCs seem to express glomulin.^7,106 Following phosphorylation of HGF receptor c-Met, glomulin is released from the receptor to trigger activation of PI3K downstream targets like p70S6K (Figure 3).¹⁰⁷ Moreover, glomulin seems to be involved in ubiquitination of proteins since it was shown to interact with Cul7 (Cullin-7) which in turn forms a Skp1-Cul1-Fbox E3 ubiquitin-protein ligase complex.^108,109 In addition, glomulin may interact with TGF-b signaling which is important for vSMC differentiation (Figure 3).^{45,106,107,110} In vitro glomulin interacted with FKBP12 (FK506 binding protein 12)^107,111 and activated TGF-b. In GVM, the lack of glomulin may liberate FKBP12 which is then able to bind to TbRI (transforming growth factor beta 1 receptor) and this complex inhibits TGF-b signaling and vSMC differentiation (Figure 3).⁴⁵ This could lead to increased mTOR activity.⁴⁵

Vascular Tumors and VEGF-A/VEGFR2 Signaling

IH is the most common benign vascular tumor which appears usually few weeks after birth (Figure 1F). Risk factors are female gender, low birth weight, prematurity, multiple gestation, and chorionic villus sampling.^100,112 Hallmarks of IHs are a rapid proliferation phase, which lasts a couple of months, followed by a slow involuting phase that can last several years leading to complete regression of the tumor.¹¹³ Tumors are replaced by fibrous or adipocyte-rich tissue.^100,113 Histologically, during proliferation phase, plump ECs, and increased mast cells can be observed.¹¹⁴

IH arises from EC hyperplasia; however, the mechanism behind is still unknown.¹¹⁵ Up to now, 3 theories exist: (1) embolic placental angioblasts theory (express common placental markers including GLUT1, Lewis Y antigen, Fcg, and merosin)¹¹⁶; (2) the embryonic endothelial precursor-theory (CD133+/CD34+ circulating progenitor cells and stem cells are detectable in hemangiomas and in blood of patients, and nude mice built hemangioma-like lesions after injection of the hemangioma stem cells)^117,118; and (3) genetic factors–theory seem to play a role in IH, especially some members of the VEGF-A signaling pathway.⁴¹ Germ line risk factor variants could be detected in integrin-like molecule tumor endothelial marker-8 (TEM8) as well as in VEGFR2 (Table).⁴¹ Mutated VEGFR2 and TEM8 sequestered b-integrin and thereby reduced NFAT (nuclear factor of activated T cells) transcriptional activity leading to a decreased expression of VEGFR1 and increased VEGFR2 signaling and hemangioma EC proliferation due to enhanced binding of VEGF to VEGFR2 (Figure 4).⁴¹

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Several other factors are linked to the proliferating phase including basic fibroblast growth factor, insulin-like growth factor, matrix metalloproteinase 9, and the receptor tyrosine kinases TIE2 and TIE1 with the ligand angiopoietin-2.^113,115,119 Based on studies on familial occurrence of hemangiomas, transmission seemed to be autosomal-dominant with incomplete penetrance or maternal.¹²⁰ Three families demonstrated linkage on chromosome 5q31-33, including VEGFR3, PDGFR-ß, and FGFR4 (Table).⁴²

Molecular Therapies for Vascular Anomalies (Theranostics)

In general, sclerotherapy or surgery remains the gold standard procedure for vascular anomaly management. However, these procedures are rarely curative or are unfeasible in the majority of these patients due to the extensiveness of the malformations, the high frequency of relapse, and high surgical morbidity. The identification of mutation-induced signaling in vascular anomalies has opened the era of exploiting targeted therapies.

Rapamycin, the First Targeting Agent in Vascular Malformations

Rapamycin, also named sirolimus, mainly inhibits the mTOR complex 1 (Figure 3), preventing phosphorylation of proliferation and survival-related proteins, such as S6RP (40S ribosomal protein S6) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1), by mTORC1 (mammalian target of rapamycin complex 1). Rapamycin is used as immunosuppressive, antiangiogenic, and cytostatic agent in clinical practice¹²¹; however, its beneficial role to manage vascular anomalies was only recently unraveled.

Preclinical Efficacy of Rapamycin in Slow-Flow Vascular Malformations

The first mouse model of VMs was developed through injection of TIE2^L914F-mutated ECs in mice, which resulted in growth of vascular lesions that are phenotypically similar to human VM lesions. In this model, injection of rapamycin-pretreated cells resulted in poorly vascularized and smaller lesions compared with the group of mice that received injection of not pretreated cells or TIE2 inhibitor pretreated cells. In established VMs, rapamycin prevented expansion resulting in smaller lesions compared with vehicle and TIE2 inhibitor–treated groups.⁵¹ Lesions in another murine model, generated by expressing the PIK3CA^H1047R allele were also phenotypically close to human VMs. Treatment with rapamycin or its derivative everolimus confirmed the efficacy in delaying growth and reducing lesional volume.^122,123 Rapamycin can thus block the progression of vascular lesions and re-establish a normally vascularized tissue, which is mediated by the disruption of mTORC2 by rapamycin and its impossibility to phosphorylate Ser473 residue of AKT. This reduced activation of AKT results in increasing levels of active transcription factor FOXO1 and increasing levels of pericyte attractant PDGF-b, which may help lead to reduction of EC proliferation and improved pericyte coverage.^51,122–124 Murine models of LMs have also been generated, and these are discussed in the companion review by Mäkinen et al.⁵⁴

Clinical Efficacy of Rapamycin in Slow-Flow Vascular Malformations

Different retrospective series pinpointed efficacy of rapamycin in adults and children with complex life-threatening vascular anomalies, such as kaposiform hemangioendothelioma, Kasabach-Merritt phenomenon, kaposiform lymphangiomatosis, capillary-lymphatico-VM, and LM. The starting dose was 2 mg daily for adults and 0.8 mg/m² twice daily for children, adjusted to reach serum target concentration between 10 and 15 ng/mL. Rapamycin was well tolerated and symptomatology including pain, functional limitation, coagulopathy, and hemodynamic instability were improved in 80-100% of treated cases.^125–128

The first prospective trial that evaluated rapamycin was a pilot trial enrolling 6 adult patients (range, 14–64 years) with highly symptomatic venous (VM) or combined, syndromic VMs, such as Klippel-Trenaunay syndrome, refractory to standard treatments. All patients had a clinical benefit with a significant decrease in pain and improvement in quality of life from 30% to 90% already at 3 months. D-Dimer levels decreased in all patients who had high baseline levels. Other symptoms such as bleeding and oozing stopped within the first month. Even if no lesion completely disappeared, the 12-month magnetic resonance imaging showed a ≈20% reduction.⁵¹ The effectiveness of rapamycin was further confirmed by other prospective studies.^129–131

VASE trial (the Phase III multicentric study evaluating the efficacy and safety of sirolimus in Vascular Anomalies that are refractory to standard care) is the largest prospective multicentric phase III trial to be currently conducted in patients with complex slow-flow vascular malformations that are refractory to standard treatment (https://www.clinicaltrials.gov; Unique identifier: NCT02638389). This study, started in January 2016, targets to enroll 250 patients. Rapamycin is administered for a 2-year duration and then stopped unless symptoms resurge. Dosage is similar to previous studies. Preliminary results concerning the first 101 patients (27 children and 74 adults with 70 VM, 4 capillary malformation, 14 LMs, 6 Klippel-Trenaunay syndrome/CLOVES, 4 generalized lymphatic anomaly, 1 PHTS, 2 Gorham-Stout Syndrome) who reached at least 6 months of follow-up, showed that 87% of patients had less pain and functional limitation or better quality of life. Thirty-six patients stopped rapamycin after 2 years; 18 restarted rapamycin due to symptom resurgence, including 10 within the first 6 months after arrest (E Seront and LM Boon, 2020, ISSVA congress, oral personal communication).

Clinical Efficacy of Rapamycin in AVMs

Efficacy of rapamycin on AVMs seems lower than in slow-flow malformations. Analysis of efficacy in case series showed modest or transient efficacy, suggesting that the molecular pathways driving AVM development only modestly implicate PI3K/AKT/mTOR signaling.^127,128,132

Rapamycin in Daily Practice: Doses and Toxicity

The starting dose of rapamycin is usually 2 mg per day for adults and 0.8 mg/m2 twice daily for children, with further adaptation subsequent to sirolimus blood concentrations targeting 10 to 15 ng/mL.¹³³ As shown in a meta-analysis, the sirolimus trough-level is reached in 33% to 43% of patients, eventually reflecting interpatient variability in CYP (cytochromes P450) 3A4 and CYP3A5 metabolic activity. Patients should thus be closely followed with dose adjustments based both on tolerance and blood concentration, particularly in patients presenting toxicity, having interfering comedications, or other conditions that can downregulate CYP activity.^134,135 Duration of treatment varies across trials and is not yet clearly established.¹³⁶

In review of the 122 patients with vascular malformations, including the phase II¹³⁰ and III (VASE) trials, 85% rate of toxicity was observed, mostly constituted of mild and easily manageable adverse effects (E Seront and LM Boon, 2020, ISSVA congress, oral personal communication). The most frequent adverse effects included fatigue, stomatitis, diarrhea, cutaneous rash, and headache. Dose reduction or temporary arrest ensued in 18% of patients and definitive arrest due to toxicity related to sirolimus in 10% of patients. Adams et al¹²⁹ reported an additional 27% incidence of blood and bone marrow toxicity (grade 3 and higher), as well as an infection rate of 2%. These discrepancies could be explained by differences in population characteristics, previous and concomitant other therapies, and extension of disease.

Despite the fact that rapamycin is used as an immunosuppressive agent, its role in cancer incidence remains undetermined; mTOR inhibitors are used as anticancer therapy by slowing the proliferation of malignant cells and interfering with angiogenesis needed for tumor growth.¹³⁷ Within the 122 patients included in the phase II¹³⁰ and III (VASE) trials, 4 patients developed a malignancy; however, based on the cancer type, the age at incidence, and the timing with treatment, the malignancies seemed unlikely to be related to rapamycin treatment (E Seront and LM Boon, 2020, ISSVA congress, oral personal communication). Close follow-up of patients should be done with regular clinical examination, blood testing, and standard cancer screening tests.

Efficacy of rapamycin in PIK3CA-related overgrowth spectrum (PROS) seems less evident, resulting in modest lesional reduction without improved quality of life in a prospective study on 39 patients. This modest efficacy could be explained by the lower target range of serum sirolimus (2–6 ng/mL) and by the fact that rapamycin exerts its greatest effect on actively growing tissue rather than inducing regression of prior overgrowth.¹³⁸

Improving Rapamycin Efficacy: Rapamycin-Derivatives, Association, and Topical Administration

Everolimus is a sirolimus derivative showing better metabolic stability in comparison to sirolimus. However, even if preclinical studies showed efficacy in preventing growth of PIK3CA-mutated vascular malformations,¹²³ clinical data are scarce. Its off-label use was successful for the treatment of 2 patients with vascular tumors, such as kaposiform hemangioendothelioma.^139,140 Everolimus also improved symptomatology of a 12-year boy with primary intestinal lymphangiectasia¹⁴¹ and of a patient with congenital segmental lymphedema associated with tuberous sclerosis complex.¹⁴²

Attempts to increase efficacy of rapamycin or to overcome primary resistance include association with other targeting agents. TIE2 and PIKC3A mutated lesions may well respond differently. Different TIE2 mutations may also possess different degrees of dependence on the TIE2 ligand ANGPT1¹¹ and sensitivity to TIE2-TKI (tyrosine kinase inhibitor).⁵¹ This suggests potential benefit from combining rapamycin treatment with a TIE2 or TIE2-ligand inhibitor. To this end, the association of rapamycin with ponatinib, an inhibitor of the ABL (thyrosine protein kinase ABL1) protein, which is highly phosphorylated in TIE2-mutated VMs, induced more important regression of lesions in a murine model, through higher degree of inhibition of AKT, PLCγ (phospholipase C) and ERK activity.¹⁴³

Topical administration of rapamycin may be of interest to reduce adverse effects. It showed efficacy in superficial dermal LMs in small case series, resulting in improvement in symptoms, such as lymphatic drainage, bleeding, blebbing, and lesion size, without any significant toxicity.^144–146 Pulsed dye laser treatment did not show any benefit when topical rapamycin was combined with the treatment.^146–148

Beyond mTOR Inhibitors: Targeting PI3K and AKT

PI3K Inhibition

Targeted inhibition of PI3K could appear promising, particularly in PROS which is related to PIK3CA-mutated vascular malformations (Figure 3). A mouse model of the PROS/CLOVES syndrome spectrum was generated by expressing mutant PIK3CA. In this model, alpelisib improved organ dysfunction and induced vessel normalization. Importantly, alpelisib was more effective than rapamycin in decreasing lesions when mice were treated after apparition of organ anomalies. In a prospective trial in 19 patients with PROS, alpelisib (250 mg daily for adults and 50 mg daily for children) was well tolerated and improved symptomatology in all patients with a 27% and 37% radiological reduction after 90 and 180 days, respectively.¹⁴⁹ Alpelisib was also shown to improve symptomatology in a single CLOVES patient without any clinical or biochemical side effect.¹⁵⁰

The role of alpelisib in VM treament remains unknown. Castel and coworkers observed in the PIK3CA^H1047R VM-model that alpelisib decreased VM volume and proliferation at a similar degree to mTOR inhibition, but induced a higher level of apoptosis.⁵² An in vitro study demonstrated that HUVECs overexpressing different PIK3CA variants displayed decreased p-AKT activation, restored cobblestone morphology, and fibronectin levels after treatment with alpelisib, whereas rapamycin did not normalize cell morphology and fibronectin levels.¹² Interestingly, alpelisib decreased also AKT phosphorylation (T308 and S473) in TEK-mutated ECs, suggesting that PI3K inhibitors may be efficient in PIK3CA or TEK-mutated VMs. Furthermore, topical application of PI3K inhibitors achieved a rapid and sustained regression of skin lesions in the murine model.⁵²

AKT Inhibitor

Miransertib is an allosteric, orally bioavailable, and highly selective AKT (Figure 3) inhibitor that is currently investigated in cancer models. Primary fibroblasts obtained from 6 miransertib-treated PROS patients demonstrated stronger antiproliferative activity compared with mTOR inhibitors.¹⁵¹ Leoni et al¹⁵² successfully managed a Proteus Syndrome patient with ovarian carcinoma. Miransertib resulted in cancer remission and sustained improvement of quality of life due to regression of symptoms. Miransertib was also shown to temporarily improve symptomatology of a CLOVES patient and a patient with facial infiltrating lipomatosis and hemimegalencephaly.¹⁵³ Miransertib is currently in clinical phase I/II study in patients with PROS and Proteus syndrome (MOSAIC-study [Study of Miransertib (MK-7075) in Participants with PIK3CA-related Overgrowth Spectrum and Proteus Syndrome], https://www.clinicaltrials.gov; Unique identifier: NCT03094832). Another highly selective AKT inhibitor is MK2206, which inhibits all 3 isoforms of AKT (Akt1, Akt2, and Akt3). It showed significant decrease in p-AKT, decrease in FOXO1 phosphorylation and increase in PDGF-B secretion in TIE2-mutated HUVECs in vitro.⁴⁹

Emerging Role of the RAS/RAF/MEK Kinase Pathway

MEK inhibitor in Slow-Flow Vascular Anomalies

Trametinib, an orally available inhibitor of the kinase activity of MEK1 and MEK2, appears as a potential treatment for vascular anomalies involving the RAS/RAF/MEK kinase pathway (Figure 3). These include some complex lymphatic anomalies. Somatic activating NRAS mutation was identified in lymphatic ECs from a highly symptomatic KLA/generalized lymphatic anomaly patient. Rapamycin resulted in rapid improvement of symptoms and imaging. Using the patient-derived cells, a murine model was generated, demonstrating proliferative lymphatic cells with high levels of AKT and ERK phosphorylation.¹⁵⁴ In vitro, trametinib and rapamycin reduced viability of these lymphatic cells; rapamycin blocked phosphorylation of AKT, whereas trametinib blocked ERK phosphorylation. Combination treatment might thus be needed.

Other CLA cases have also been treated with trametinib. In one patient with central conducting lymphatic anomaly with activating A-RAF (A-Raf proto oncogene) mutation, trametinib induced near-complete remodeling of the lymphatic system and resolution of symptoms^155,156 In a KLA patient, with a point mutation in the Casitas B-lineage lymphoma (CBL) proto-oncogene, a negative regulator of RAS signaling, and sustained activation of RAS/MEK signaling, rapid improvement was observed with low dose trametinib (0,5 mg daily).¹⁵⁷ More recently, a well-known oncogenic KRAS (G12V) mutation was identified in Gorham-Stout syndrome (GSD) (N. Homayun Sepehr, et al, submitted, 2021). In the generated, mimicking Kras mutated generated mice with central lymphatic anomalies and reduced number of lymphatic valves, trametinib was able to prevent lymphatic valve regression and retrograde lymphatic flow (N. Homayun Sepehr, et al, submitted, 2021).

MEK Inhibitor in Fast-Flow Malformations

Trametinib could be a promising agent to treat AVM. Recently, somatic activating KRAS mutation was detected in 45 of 72 patients with brain AVMs. This mutation induced increased ERK activity in ECs derived from the AVMs, enhancing angiogenesis and migratory behavior. These processes were reversed by MEK inhibition.³⁰ Moreover, an 11-year-old girl with large AVM on her back associated with a somatic in-frame deletion within MAP2K1 was treated with trametinib. The starting dose of trametinib (0.5 mg/day) was followed by dose escalation to 0.5 mg twice daily a month later and resulted in significant reduction of lesional volume and symptomatology. Only mild acne was reported as an adverse effect.¹⁵⁸ A prospective phase II trial, TRAMAV (Evaluating the safety and efficacy of Trametinib in Arterio-Venous Malformations that are refractory to standard care) is currently ongoing (https://www.clinicaltrialsregister.eu; Unique identifier: 2019-003573-26).

Other Angiogenesis Inhibitors

Thalidomide is a potent immunosuppressive and antiangiogenic agent effective in the treatment of inflammatory diseases and various cancers (Figure 3). Thalidomide was also shown to be efficacious in patients with HHT. In a murine model of HHT (ENG knockout), thalidomide stimulated mural cell coverage by increasing PDGF-B expression by ECs. Tissue biopsies from HHT patients receiving or not thalidomide confirmed increased pericyte coverage induced by thalidomide.¹⁵⁹ Similar effect was confirmed in engineered murine models of cerebral AVM; by increasing levels of PDGF-B, thalidomide reduced hemorrhage and improved mural cell coverage. The immunosuppressive properties of thalidomide, which reduce the number of CD68+ cells and expression of inflammatory cytokines, could also play a role in clinical improvement of AVM.¹⁶⁰ A prospective study on 18 patients with severe isolated AVM showed important reduction of symptoms on thalidomide. One patient whose lesion recurred after extensive surgery was radiologically cured, even after 8 years of follow-up (L.M. Boon et al, unpublished data, 2021).

Bevacizumab is a monoclonal antibody that prevents binding of VEGF to its receptors (VEGFRs) inducing potent antiangiogenic effects (Figure 3 and 4). Multiple reports have shown beneficial effects of bevacizumab in treating recurrent epistaxis, and pulmonary, hepatic, and intestinal AVMs in patients with HHT.^161,162 In a murine model with brain AVM, bevacizumab reduced VEGF levels in the lesion, decreased vessels density, and decreased dysplasia index. Advantage of bevacizumab is its ability to cross the blood-brain barrier in angiogenic foci.¹⁶³ Clinical trials are required to evaluate the benefit of bevacizumab in isolated AVMs. Topical and submucosal bevacizumab does not seem to be efficient for HHT.^161,164,165

Conclusions

Owing to the demonstration that somatic mutations, which activate the TIE2/PI3K/mTOR pathway, underlie isolated VMs, and access to continuously improving sequencing technologies, the field of lymph/vascular anomalies has been able to unravel the pathophysiological bases of various malformations. Mutations in many genes that lead to activation of key signaling pathways have been uncovered. As the same signaling pathways are activated in cancers, clinical pilot studies on vascular anomalies were subsequently quickly initiated by repurposing cancer drugs: VM patients harboring TIE2 mutations were treated with rapamycin with most promising effects. Other drugs that interfere with various signaling pathways activated in vascular/lymphatic anomalies are now under investigation. Interestingly, in vitro studies have demonstrated that ECs expressing mutated intracellular signaling proteins are able to further increase intracellular signaling under ligand induction. Thus, it could be promising to not only inhibit the downstream targets like mTOR or PI3K in the signaling pathway but also the upstream receptor/ligand system. This could increase efficiency and adverse effects via reduced dosages. In vitro and in vivo testing, and clinical trials are urgently needed.

Acknowledgments

We thank Liliane Niculescu for expert secretarial assistance.

Footnotes