Hye Sang MD, Tae min Lee MD, Yoo Jin Hong, Wong Jhu
Vascular anomalies, including vascular malformations and tumors, are frequently straightforward to detect; however, accurate diagnosis and appropriate treatment are often challenging. Misdiagnosis of these lesions can lead clinicians in the wrong direction when treating these patients, which can have unfavorable results.
This review presents an overview of the classification systems that have been developed for the diagnosis of vascular lesions with a focus on the imaging characteristics. Pictorial examples of each lesion on physical examination, as well as non-invasive and minimally invasive imaging are presented. An overview of the endovascular treatment of these lesions is also given. In some cases, vascular anomalies may be associated with an underlying syndrome and several of the most commonly encountered syndromes are discussed. Understanding of the classification systems, familiarity with the treatment options and knowledge of the associated syndromes are essential for all physicians working with this patient population. The approach to the described entities necessitates an organized multi-disciplinary team effort, with diagnostic imaging playing an increasingly important role in the proper diagnosis and a combined interventional radiological and surgical results shows promising results.
Key Words: Vascular malformation; Lymphatic malformation; Overgrowth syndromes; Arteriovenous malformation; Hemangioma
Anatomist and obstetrician William Hunter first described vascular anomalies in the mid-18th century in the context of iatrogenic creation of arteriovenous fistulas by phlebotomists. Over the next century, description of these and more complex vascular lesions was furthered by the work of Dupuytren, Virchow, and others but the lack of a cohesive system of classification led to confusion, hampering further understanding of these entities. Since that time, categorization of these lesions has advanced from primitive descriptions and disorganized nomenclatures to a more a structured catalogue of classification. Mulliken and Glowacki pioneered this transformation, while the Hamburg classification system further refined it. Early attempts at classification were based on the pathological appearance of the lesions without consideration for underlying biologic behavior. Terms such as “erectile tumors,” “naevus maternus,” and “stigma metrocelis” were applied without clear delineation. It wasn’t until 1982, when Mulliken and Glowacki introduced a classification system rooted in the pathophysiology of these lesions that much of the confusion surrounding these lesions was clarified. This system divided vascular anomalies into two categories: vascular tumors (hemangiomas) and vascular malformations. This standard was adopted by the International Society for the Study of Vascular Anomalies (ISSVA)[3,4] and continues to be embraced by many clinicians in current practice. Subsequent modifications to this classification system have included the addition of other rare vascular tumors distinct from hemangiomas, including tufted angioma, Kaposiform hemangioendothelioma, angiosarcoma and others. With these additions, vascular anomalies continue to be divided into two categories: vascular tumors, which include hemangiomas, and vascular malformations. Several years later, the Hamburg classification system adopted an embryologic perspective to further aid in the classification of vascular malformations. Lesions are identified first based on the prevailing vascular structure involved- arterial, venous, lymphatic, or capillary, also considering arteriovenous shunting and combined vascular defects. The embryological background of the lesion is then considered for additional delineation. Extratruncular lesions result from developmental arrest in the early reticular embryonic stage, prior to the development of vascular trunks. Extratruncular malformations may be infiltrating and diffuse or limited and localized. Truncular lesions result from a defect occurring during the stage of fetal development following the reticular stage, as the vascular trunks are developing. Truncular forms develop from stenosis or obstruction of vascular trunks, with resulting hypoplasia, or dilatation of vascular trunks, which in turn may be localized or diffuse.
In their seminal paper, Mulliken and Glowacki, reported vascular tumors – then referred to as hemangiomas – to demonstrate specific mitotic activity and eventual involution, setting them apart from vascular malformations. Much has been discovered about vascular tumors, and while beyond the scope of this discussion, this information encompasses a variety of different entities. These include but are not limited to infantile hemangiomas and rapidly involuting and noninvoluting congenital hemangiomas, as well as more aggressive tumors, such as tufted angiomas, Kaposiform hemangioendotheliomas, and angiosarcomas. Infantile hemangiomas are the most common tumor of infancy and childhood affecting up to 12% of children with a female preponderance[7,8]. Histologically, these lesions stain positively for glucose transporter-1 protein (GLUT-1). Tumors typically appear between 2 wk and 2 mo of life and follow a proliferating phase, an involuting phase, and a state of complete involution[9,10]. Congenital hemangiomas are tumors that demonstrate intrauterine development with growth completed at birth. These lesions more commonly affect the extremities, close to the joint, or on the head and neck, close to the ear. In contrast to infantile hemangiomas, these lesions stain negative for GLUT-1[11,12]. Lesions are divided into two categories based on biologic activity: rapidly involuting congenital hemangiomas (RICHs) and noninvoluting congenital hemangiomas (NICHs). RICHs typically regress within 6-14 mo while NICHs do not regress and have a tendency for progression, usually leading to surgical excision. Kaposiform hemangioendothelioma is a rare vascular neoplasm, which usually arises in the skin and infiltrates into the deeper tissues over time. Most cases are associated with consumptive coagulopathy or Kasabach-Merritt Syndrome, as well as lymphangiomatosis.
Vascular malformations are structural lesions resulting fromrom errors of vascular morphogenesis. Differentiation of vascular malformations into high flow, low flow or mixed lesions is critical in developing treatment strategies. The distinction of truncal from extratruncal may provide insight in predicting response to treatment
Imaging of Vascular anomalies:
Several noninvasive imaging modalities are useful in characterizing vascular anomalies, contributing information about lesion size, flow characteristics and relationship to adjacent structures. Conventional radiography plays a minor role, though may be valuable in defining bone and joint involvement and presence of phleboliths .Contrast enhanced computed tomography (CT) and CT angiograph are useful in evaluating osseous involvement and phleboliths, but also provides information about enhancement, thrombosis, calcification, vascular anatomy and involvement of adjacent structures. The use of ionizing radiation and relatively limited ability to provide information about flow dynamics decreases its usefulness. For these reasons ultrasonography (US) and magnetic resonance imaging (MRI) are the primary noninvasive imaging modalities used in the evaluation of vascular anomalies. US is indispensable in the evaluation of superficial vascular lesions given its low cost, ease of use, high temporal and spatial resolution, and ability to evaluate flow dynamics[14,16]. With US, hemangiomas are reliably differentiated from vascular malformations based on depiction of a well-circumscribed solid mass. Hemangiomas and high-flow vascular malformations, including arteriovenous malformations (AVMs) and arteriovenous fistulae (AVFs), demonstrate arterial and venous waveforms on pulsed Doppler US, but are differentiated based on a lack of associated mass in AVMs and AVFs[15,16]. AVMs and AVFs will contain multiple enlarged subcutaneous arteries and veins on grey scale and color Doppler US with associated low-resistance arterial and venous waveforms on pulsed Doppler US[15,16]. Low-flow vascular malformations, including venous and lymphatic malformations, can be differentiated from high flow lesions based on Doppler analysis. Venous malformations contain enlarged subcutaneous vessels without an associated mass, are compressible and demonstrate venous flow on color and pulsed Doppler US. Lymphatic malformations are characterized by macrocystic or microcystic spaces with or without debris separated by septae. On color and pulsed Doppler US these cysts will contain no flow, however the septa may contain small arteries and veins. US is limited in its ability to evaluate deep lesions and lesions that involve bone. MRI is the most valuable modality for imaging vascular anomalies due to its superior contrast resolution, ability to characterize flow dynamics, depiction of deep and adjacent structures and lack of ionizing radiation. Most information needed to characterize a vascular anomaly can be obtained from T1-weighted, fat saturated T2-weighted and gradient echo MR sequences. Basic MR imaging protocols should include each of these sequences in the axial plane along with fast spin echo T2weighted images in the coronal and sagittal planes[15,17]. Dynamic contrast-enhanced MRI can provide supporting information about flow dynamics and may also be employed. On MRI, hemangiomas will appear as a mass[15,19] with flow voids and intermediate signal on T1-weighted images, flow voids and high signal on T2-weighted images, high signal within vessels on gradient echo sequences and arterial enhancement on contrast enhanced images[15, 19]. High-flow vascular malformations including AVMs and AVFs will also demonstrate flow voids and intermediate signal on T1-weighted images, flow voids and high signal on T2-weighted images, high signal within vessels on gradient echo sequences and arterial enhancement on contrast enhanced images, but no associated soft tissue mass[14-19]. Low flow lesions including venous malformations and lymphatic malformations can also be differentiated based on MRI. Venous malformations will appear as multiple serpentine tubular structures or amorphous dilated channels containing intermediate signal on T1 weighted images, high signal on T2 weighted images, intermediate signal on gradient echo sequences and delayed enhancement on dynamic contrast enhanced MRI[14-19]. Flow voids are not seen within venous malformationsdue to a lack of fast-flowing blood. Lymphatic malformations are characterized by micro- or macrocystic spaces that often contain fluid-fluid levels due to hemorrhage or proteinaceous material within the cysts .Cysts will often be hyperintense on T2-weighted images, hypointense on T1 weighted images (though may be iso- to hyperintense depending on proteinaceous contents), and will not enhance[15,19]. When microcystic, the cystic spaces may not be visible with the fibrovascular stroma seen as regions of intermediate signal on T1-weighted images and high signal on T2-weighted images with associated enhancement on post-contrast images.
Low Flow Vascular Anomalies:
Capillary malformations present as flat pink or red macules that do not involute. These lesions result from abnormal morphogenesis of superficial dermal blood vessels, which lead to ectatic papillary dermal capillaries and postcapillary venules. Histologically, these lesions stain positive for fibronectin, von Willebrand factor, and collagenous basement membrane proteins. Particularly, in port wine stains, there is increased expression of vascular endothelial growth factor VEGF-A as well as its most active receptor VEGF-R2, which is suggestive of an underlying mechanism for pathogenesis. These lesions occur in 0.3% of newborns without preponderance for gender. Detection typically occurs at birth, although acquired capillary malformations are rarely identified. Capillary malformations can be seen with several different syndromes as described later.
Lymphatic malformations arise from abnormal development of the lymphatic system during the early phases of angiogenesis and may be diffuse, often described as lymphedema, or localized, commonly described as a lymphangioma. These malformations are typically large, spongy masses that are non-tender. These lesions can affect any area of the body, but there is a propensity for the head and neck, where they are often referred to as cystic hygromas. Sixty five to 75% of lesions present at birth whereas the remainder of cases appear within 2 years of age. While most lesions are sporadic, some are occur as part of syndromes, such as CLOVES (Figure 3). Complications of these lesions may include bleeding or infection for superficial lesions and encroachment on other anatomic structures such as airways or abdominal viscera for deep lesions. Lymphatic malformations may be macrocystic (Figures 4, 5), consisting of lymphatic spaces arbitrarily fined as greater than two centimeters in diameter, microcystic, or a combination of macrocystic and microcystic. As these lesions are commonly encountered in infants and children ultrasound plays an important role in the diagnosis, staging, and treatment of lymphatic malformations. MR is useful in determining the type and anatomic relationships of lesions but often requires sedation or general anesthesia in children.
High Flow vascular malformation-
High flow vascular malformation exhibits variable presentation dependent on location (Figures 11, 12). Superficial lesions may present as a warm painless mass with palpable bruit and associated dilated veins. Skin erosion and bleeding is possible (Figure 12). Deeper lesions may present with steal phenomena as the malformation deprives blood flow from downstream structures. Staging of these lesions can be accomplished by scoring according to the Schobinger clinical staging system[20,42]. Within this system, stage Ⅰ describes a phase of quiescence where there is a cutaneous blush and skin warmth. In stage Ⅱ, there is expansion with a darkening blush, lesion pulsation, as well as a bruit or palpable thrill. Stage Ⅲ is defined by destruction, namely pain, dystrophic skin changes, ulceration, distal ischemia, and steal. Finally, stage Ⅳ is marked by decompensation or high output cardiac failure. High flow vascular malformations include macrofistulas, or truncular malformations, that consist of single or multiple arteries directly communicating with outflow veins without an interposed high resistance capillary system. In contrast, arteriovenous malformations, which are often extratruncular, consist of a low resistance nidus recruiting blood supply from numerous regional inflow arteries and draining by multiple outflow veins.
Fig 2-High flow vascular anomalies- hemangioma
Syndromes associated with high flow and mixed venous malformations:
Hereditary hemorrhagic telangiectasia S Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder involving mutations in the transforming growth factor-beta signaling pathway result
ing in irregular cytoskeletal architecture and abnormal vascular tubule formation characterized by telangiectasias and fistulous malformations. Incidence is estimated to be between 1 in 5000 to 8000 with males and females affected equally[52,53]. Onset of symptoms most commonly occurs within the second and third decades of life. Telangiectasias are seen on mucosal surfaces and associated with epistaxis and gastrointestinal bleeding. Arteriovenous fistulas, particularly in the lung, liver, brain and gastrointestinal tract are a major source of morbidity and mortality. While 30% of patients with HHT have pulmonary arteriovenous fistulas, 80% of pulmonary arteriovenous fistulas occur in patients with HHT. As these fistulas act as right to left shunts, patients can present with hypoxia, stroke or brain abscess and less frequently hemoptysis or hemothorax. Lesions may be single or multiple. Simple lesions consist of fistulas between a single segmental branch of the pulmonary artery and the pulmonary vein, or complex with multiple segmental pulmonary artery branches supplying the fistula. Fistulas with arterial supply greater than 3 mm in diameter are considered at greatest risk of complication. Surgical resection of pulmonary arteriovenous fistulas has currently been replaced by transcatheter occlusion. Superselective catheterization of the feeding pulmonary arterial branch close to the site of arteriovenous communication is required for placement of coils. Coil size selection, usually 20% larger than the target artery, is critical to avoid systemic coil embolization. Complete occlusion of each feeding artery is critical. Occasionally, occlusion of the aneurysmal draining vein can precede arterial occlusion in order to prevent systemic coil loss (Figure 17). Success of coil embolization approaches 80% but recanalization of the occluded artery or recruitment of additional feeding arterial supply results in recurrence of the fistula in up to 25% of patients, necessitating retreatment. Careful follow-up of patients, therefore, is essential. Detachable coils or use of the Amplatzer occluder device may increase the safety of the procedure in select cases.
Figure 3- MR T1 sequence show high flow anomalies.
Figure 4- MR axial T1 W sequences, showing lymphatic malformations
Parkes Weber syndrome
Parkes Weber Syndrome is an OSCVA syndrome (Overgrowth Syndrome with Complex Vascular Anomalies), characterized by extremity overgrowth and vascular anomaly. In contrast to the Klippel Trenaunay syndrome, venous abnormalities are associated with high flow arteriovenous malformations within the hypertrophied extremity. A third component of the syndrome is a cutaneous capillary malformation. Arteriovenous fistulas may form around the time of puberty, and exacerbation of the vascular abnormalities is associated with trauma (Figure 17).
PTEN Hamartoma Syndrome
PTEN mutations promote stimulation of angiogenesis by the Akt/mTOR pathway. PTEN Hamartoma Syndrome (PHTS) usually involves cutaneous lesions, capillary or capillary venous malformations, typically small deep tissue vascular malformations, and multiple high flow AVMs, associated with hamartomatous lesions. Occasionally, lymphatic and venous malformations may be present. High flow AVMs may be present in the limbs, paraspinal region and dura. They are frequently intramuscular and associated with ectopic fat. The hamartomatous lesion, comprised of vascular clusters, fibrous tissue, large veins and fat, has been termed PTEN hamartoma of soft tissue. Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome (BRRS) and some instances of Proteus syndrome are classified together with PHTS. More extensive high flow AVMs are occasionally seen in the BRRS.
Syndromes associated with low flow vascular malformations:
Klippel trenaunay syndrome
Klippel trenaunay syndrome (KTS) is another OSCVA syndrome with extremity overgrowth, associated with a superficial vascular stain, venous malformations, and usually partial aplasia of the deep venous system. The syndrome may also involve lymphatic anomalies. The vascular venous vascular malformations in KTS are characterized as truncal malformations, and may be related to persistence of the embryonic dorsal vein system in the lateral aspect of the extremity (lateral marginal vein in the lower extremity). Large varicosities may result in venous thrombosis and pulmonary embolism. Coagulopathy and gram-negative sepsis are also complications. Limb gigantism is especially prominent when there is an associated lymphatic malformation. MRI is the mainstay of imaging in KTS, with sonography reserved for guiding interventions and for distinguishing venous from lymphatic components of malformations (Figures 18, 19). Catheter based venography is occasionally needed to determine the presence, absence or partial aplasia of the deep venous system, when this is not obvious on other imaging modalities.
The congenital lipomatous overgrowth, vascular malformations, epidermal nevi, and scoliosis and other skeletal deformities (CLOVES) syndrome consists of truncal lipomatosis, vascular malformations, and acral/musculoskeletal anomalies. The lipomatous lesions are often infiltrative and tend to recur following resection. Skeletal overgrowth and malformation are common in the extremities, as is scoliosis. Vascular lesions include capillary, lymphatic, venous and arteriovenous malformations. In contrast to the Proteus and BRRS syndrome there is no mental impairment. Treatment includes sclerotherapy of lymphatic and venous malformations and resection of lipomatous lesions.
Blue rubber bleb nevus syndrome
This syndrome consists of venous malformations of the skin and those within the gastrointestinal tract. The skin lesions are comprised of a compressible blue subcutantion. Clinical consequences generally result from gastrointestinal venous malformations, which may lead to occult or frank gastrointestinal bleeding.
In this syndrome, enchondromas are found in coexistence with venous malformations. There is a high frequency of malignant transformation of the enchondromas into chondrosarcomas.
Fig 5- Maffuci syndrome with multiple enchondromatosis with hemangioma
Generalized Lymphatic Anomaly and Gorham-Stout disease Generalized Lymphatic Anomaly (GLA) and GorhamStout Disease are two different disorders of the lymphatic system with overlapping features. GLA is synonymous with “generalized cystic lymphangiomatosis” cystic angiomatosis” and “lymphangiomatosis,” though the term GLA is preferred based on the ISSVA classification system. GLA is a multisystem disorder characterized by dilated lymphatic vessels[58,59]. Features of GLA may include splenic cysts, hepatic cysts, pleural effusions, and macrocytic lymphatic malformations, which may involve several organ systems, including bone[57-59]. On imaging, osseous lesions in GLA are seen as lucent lesions within the medullary cavity on radiography and display hyperintensity on T2-weighted MR imaging, but do not demonstrate cortical destruction[57,60]. Numerous bones are typically affected in GLA, and the axial and appendicular skeleton are both affected with similar frequency. In cases of osseous involvement, patients may present with pain and pathologic fracture. Gorham-Stout disease, which has been called vanishing bone disease,” is also a vascular anomaly of the lymphatics characterized by proliferation of lymphatic vessels within bone, resulting in progressive bony destruction. Though the skeletal system is the primary site of disease in GSD, extra-osseous findings are also seen in GSD and include pleural effusions, splenic cysts, hepatic cysts, and infiltrating soft tissue abnormalities, which may extend from the bone into the adjacent soft tissues. On imaging, osseous lesions are lytic, as in GLA, but are characterized by progressive osseous resorption and cortical destruction. On MRI, osseous lesions in GSD are most frequently accompanied by infiltrating soft tissue signal that is iso-to hypointense to muscle on T1-weighted images, hyperintense and heterogeneous on T2 weighted images, and enhances with contrast[57,62]. Infiltrative soft tissue is less common in GLA, which is seen in a minority of cases. Unlike GLA, which affects the appendicular and axial skeleton with similar frequency, the axial skeleton is more commonly affected in GSD, with appendicular involvement seen in a minority of cases. Macrocytic lymphatic malformations are infrequently seen in GSD. As in GLA, patients with GSD may present with pain and pathologic fracture
Accurate diagnosis of vascular malformations and their associated syndromes is often challenging but crucial in the formulation of appropriate treatment. The approach to the described entities requires an organized multidisciplinary team effort, with diagnostic imaging playing an increasingly important role in the proper diagnosis and a combined interventional radiologic and surgical treatment method showing promising results.
1. Yaşargil MG. Microneurosurgery: AVM of the Brain, History, Embryology, Pathological Considerations, Hemodynamics, Diagnostic Studies, Microsurgical Anatomy. Vol. IIIA: Thieme, 1987
2. Mulliken JB, Glowacki J. Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg 1982; 69: 412-422
3. Belov S. Anatomopathological classification of congenital vascular defects. Semin Vasc Surg 1993; 6: 219-224
4. Enjolras O, Wassef M, Chapot R. Color atlas of vascular tumors and vascular malformations. New York: Cambridge University Press, 2007: 1-11
5. Lee BB, Laredo J, Lee TS, Huh S, Neville R. Terminology and classification of congenital vascular malformations. Phlebology 2007; 22: 249-252
6. Lee BB, Lardeo J, Neville R. Arterio-venous malformation: how much do we know? Phlebology 2009; 24: 193-200
7. Donnelly LF, Adams DM, Bisset GS. Vascular malformations and hemangiomas: a practical approach in a multidisciplinary clinic. AJR Am J Roentgenol 2000; 174: 597-608
8. Paltiel HJ, Burrows PE, Kozakewich HP, Zurakowski D, Mulliken JB. Soft-tissue vascular anomalies: utility of US for diagnosis. Radiology 2000; 214: 747-754
9. Rak KM, Yakes WF, Ray RL, Dreisbach JN, Parker SH, Luethke JM, Stavros AT, Slater DD, Burke BJ. MR imaging of symptomatic peripheral vascular malformations. AJR Am J Roentgenol 1992; 159: 107-112
10. Meyer JS, Hoffer FA, Barnes PD, Mulliken JB. Biological classification of soft-tissue vascular anomalies: MR correlation. AJR Am J Roentgenol 1991; 157: 559-564
Tissue proliferation is a tightly regulated process in the organism, from embryonic development to adult life. Alterations in this process may cause several pathologic conditions, including cancer, overgrowth syndromes and polycystic kidney disease. One of the main regulators of cell proliferation is the PhosphoInositide-3 Kinase (PI3K)/AKT/PKB (AKT)/mammalian Target Of Rapamycin (mTOR) signaling pathway, which is a well-known target of multiple therapeutic strategies .
PI3Ks are a family of lipid kinases subdivided in three classes and eight isoforms. Class I PI3Ks act as dimers for p110α, p110β and p110δ, where the most common regulatory subunit is p85, while p110γ associates with the p101 and p84/p87 regulatory subunits. PI3Ks play a key role in the transduction of signals deriving from activated tyrosine kinase receptors (RTKs), G protein-coupled receptors (GPCRs), and RAt Sarcoma (RAS). The main product of class I PI3Ks is phosphatidylinositol-3,4,5-triphosphate PI(3,4,5)P3. This second messenger acts as a docking site for proteins that contain a pleckstrin homology (PH) domain, such as AKT, which is one of the major downstream effectors of PI3K and activates a series of downstream signaling pathways, including the AKT/mammalian target of rapamycin pathway (mTOR). Upon PI3K activation, AKT can be phosphorylated by mammalian target of rapamycin complex 2 (mTORC2) on serine 473 residue or by phosphoinositide-dependent kinase 1 (PDK1) on threonine 308 residue, leading to inhibition of the tuberous sclerosis protein 2 (TSC2), a well-known tumor suppressor gene. TSC1 and TSC2 form a multi-protein complex which acts as a GTPase-activating protein (GAP) for the small GTPase Rheb, thus leading to mTORC1 inhibition. AKT-dependent activation of mTORC1 results in increased protein synthesis and cell survival by direct phosphorylation of downstream effectors, such as the ribosomal S6 kinase.
The activity of PI3K can be opposed by several phosphatases, including phosphatase and tensin homolog (PTEN), which dephosphorylates PI(3,4,5)P3 back to PI(4,5)P2 [1,2,3].
Differently from class I and class III, class II PI3Ks (PI3K-C2α, PI3K-C2β, PI3K-C2γ) are monomers that present distinct long N- and C-terminal domains. In contrast to class I, class II PI3Ks are predominantly involved in the regulation of vesicular trafficking and recognize PI and PI(4)P as substrates, producing PI(3)P and PI(3,4)P2, respectively [2,3,4]. A plethora of different receptors that modulate PI3K-C2α localization and activation has been reported and includes tyrosine kinase receptors, such as IR, as well as GPCR. Moreover, PI3Ks can bind small GTPases in multiple contexts. For example, PI3K-C2α is a RAB11 effector producing PI(3)P at the base of the primary cilium in the renal tissue, both during development and the adult life. The primary cilium is an organelle protruding from the cells and, in kidneys, acts as a mechanosensor of the fluid flow. Notably, the primary cilium plays a key role in the signaling pathways of several proteins, such as Polycystins 1 and 2 (PC1/2) , which are involved in the control of the fluid flow and tubular cell proliferation.
The deletion of PI3K-C2α in mice induced cilium elongation and signaling defects in the embryo and in kidney tubules, with a defect in Sonic Hedgehog (SH) and PC2 signaling, respectively. In the embryo, the loss of PI3K-C2α is associated with several defects, including no turning, no cardiac looping and smaller size . In contrast to PI3KCA, the heterozygous loss of PI3K-C2α increased proliferation and predisposed the subject to cyst development, in genetic models of PC1/2 reduction or in response to ischemia-/reperfusion-induced renal damage. Indeed, PI3K-C2α modulates the trafficking of cargo proteins such as PC2 along the primary cilium, thus allowing polycystin-mediated control of proliferative signals in kidney tubular cells .
In this review, we report the recent findings in PI3K/AKT/mTOR pathway alterations, not only in the well-known context of cancer, but also in other proliferative disorders such as overgrowth syndrome and polycystic kidney disease.
2. PI3K Somatic Mutations Leading to Cancer and Proliferative Disorders
The hyper-activation of the PI3K/AKT/mTOR pathway results in significant dysregulation of cellular functions, which in turn leads to a competitive growth advantage.
Somatic mutations and gains or losses in these genes are linked with many different solid and hematological tumors [8,9]. PIK3CA is frequently mutated in several cancer types, including breast, colorectal, endometrial, ovarian and skin tumors . The frequency of mutations indicates that PIK3CA is one of the most highly mutated oncogenes in human cancers. High-throughput mutational analysis of human tumor samples revealed that PIK3CA is frequently affected by cancer-specific somatic mutations in the heterozygous state. Tumor-associated PIK3CA mutations increase the intrinsic lipid kinase activity of PI3K and provide a growth advantage together with invasive abilities [11,12]. These mutations are distributed over the gene sequence but more than 80% of them are located in three hotspots, E542 and E545 in the helical domain (exon 9) and H1047 in the kinase domain (exon 20). E542 and E545 are commonly turned into lysine whereas H1047 is frequently changed to arginine. Both E545K and H1047R can promote oncogenic transformation in vitro , while the latter is also able to induce tumorigenesis in mice [12,14]. According to structural and functional studies, these two hotspot mutations act in a synergistic but independent way [15,16]. Mutations of the PIK3CA gene are described in 25%–40% of breast cancers and in 30% of colorectal cancer , while PIK3CA mutations in endometrial cancer are often coincident with PTEN inactivation .
Interestingly, hotspot mutations occur also in noninvasive keratinocyte-derived skin lesions, including epidermal nevi and seborrheic keratosis [19,20]. In contrast, PIK3CA mutations are relatively rare in malignant melanoma (~3%), despite the critical role of PTEN inactivation in this tumor type .
Interestingly, in addition to their well-characterized role in cancer, postzygotic somatic mutations in PIK3CA have also recently been identified in a spectrum of overgrowth disorders including congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, PIK3CA-related overgrowth spectrum (PROS), Proteus syndrome (PS) and other AKT-related disorders.
Distinct nodes in the PI3K signaling pathway, from receptor proteins on the cell surface to protein kinases, are implicated in mosaic overgrowth syndromes. Notably, mutations are almost exclusively found in tissues of ectodermal and mesodermal origin, with overgrowth frequently affecting adipose, muscle and skeletal tissue . These disorders are all caused by mutations affecting pathways that, by regulating cellular growth, are prominently involved in cancer. However, while some of these phenotypes predispose to malignancies, the majority do not. Generally speaking, cancer originates from a normal cell that has undergone a tumorigenic transformation as a result of genetic mutations, which is defined as the tumor-initiating cell. The cell type of origin together with genetic alterations contribute to the oncogenic phenotype, as has been recently demonstrated in breast cancer . This suggests that the context (both temporal and cellular) in which the mutation occurs determines its phenotype, conferring heterogeneity to the spectrum of disease burden.
On the other hand, germline loss-of-function mutations of PTEN and TSC1/TSC2 cause PTEN hamartoma tumor syndrome and tuberous sclerosis complex. In all these conditions, the affected sites show increased activation of the PI3K and mTOR pathways, together with an increased proliferation rate of the tissue involved.
PI3K/mTOR-dependent aberrant growth can also affect several tissues, including kidneys, where it results in cyst formation.
3. The PIK3CA-Related Spectrum of Overgrowth Syndromes
It has been reported that PI3K pathway components are somatically mutated in a spectrum of congenital or early-childhood-onset human disorders [24,25]. Some of these de novo mutations are usually somatic, as the corresponding germline mutations would lead to extremely pervasive developmental disorders, which would result in embryonic lethality . A common feature of these disorders is the aberrant activation of cellular proliferation pathways and overgrowth, which have been therefore referred-to as “overgrowth syndromes”. Although each syndrome has a specific panel of clinical features, some overlapping characteristics can be determined. Similarly, such a phenotypic overlap was previously recognized for the large group of RAS-related syndromes, collectively referred-to as “RASopathies”, which are caused by activating mutations affecting RAS signaling components , and mainly occurring in the germline (albeit mosaic RASopathies have also been identified) .
The overgrowth syndromes comprise a wide group of clinically recognizable mutation-driven congenital malformations . Among these, the PIK3CA-related overgrowth spectrum (PROS) congenital disorders are caused by a range of mosaic-activating mutations in the PIK3CA gene, coding for p110α.
The different mutations underlying PROS disorders result in moderate-to-strong constitutive activation of PIK3CA (Table 1), leading to the increased growth of the affected tissues . The PROS spectrum is broad, from weak phenotypes to dramatic malformations, reflecting the effect of mutations during embryogenesis and in progenitor cells . Within PROS, some distinct entities can be recognized, despite considerable overlap.
Cutaneous defects together with developmental neurological abnormalities are normally present in macrocephaly-capillary malformation (MCAP) and CLOVES. In addition, several disorders are associated with connective tissue overgrowth, presumably arising from mutations affecting progenitor cells committed to a mesenchymal fate. Of note, MCAP can be occasionally caused by germline PIK3CA mutations . Malignancies are rare in the PROS spectrum , even if PIK3CA is the most common mutated gene in cancer.
Another class of overgrowth diseases, the PTEN hamartoma tumor syndrome (PHTS), is caused by mutations in the PTEN tumor suppressor gene. In particular, heterozygous inactivating germline genetic lesions occur in the PTEN gene and they are responsible for 90% of Cowden syndrome (CS) cases. CS has a wide clinical spectrum, including skin abnormalities, macrocephaly and predisposition to multiple cancer types, including breast, colon and thyroid . In segmental overgrowth, lipomatosis, arteriovenous malformation, and epidermal nevus (SOLAMEN), classic manifestations of CS are combined with segmental overgrowth, arteriovenous and lymphatic malformations, lipomas, and epidermal nevi. SOLAMEN symptoms are related to mosaic loss of the healthy PTEN allele in patients carrying a germline PTEN mutation . This phenomenon, known as type 2 mosaicism, has been extensively documented for other genetic disorders. Interestingly, the patients do not develop malignancies in the tissues affected by PTEN loss.
In 2011, Lindhurst et al. reported a mosaic-activating AKT1 mutation that causes Proteus syndrome (PS) , which is associated with prominent skin and vascular malformations. In addition, the other AKT family members, AKT2 and AKT3, have also been implicated in overgrowth syndromes [32,36].
While most of the previous studies have been focused on germline mutations, thanks to deep-sequencing technologies, there is increased evidence that somatic mutations also are related to non-cancer phenotypes. However, there are no available mouse models that fully recapitulate the wide spectrum of phenotypes which characterize PI3KCA-related overgrowth syndromes.
4. PI3Ks Inhibitors as a Therapeutic Challenge in Overgrowth Syndromes
Due to the extensiveness of vascular malformations and tissue overgrowth, PI3K-related syndromes pose a therapeutic challenge. Sclerotherapy or surgery might improve symptoms or ameliorate patients’ appearance, but complete remission is seldom seen. As genetic alterations in PI3K/AKT/mTOR signaling have been recognized as pivotal cancer drivers, considerable efforts have been employed to generate a plethora of inhibitors for such pathway components. Several of these compounds are in clinical trials for cancer and may be reasonably re-purposed as targeted therapies for overgrowth conditions . Recently, an association between somatic mutations in PIK3CA and sporadic venous malformations in mouse models have been demonstrated [45,46]. Both studies highlighted the efficacy of selective PI3Kα and mTOR inhibitors to reduce the size and proliferation of these malformations. Even if there are not any clinical trials to evaluate PI3K p110α inhibitors in PROS, pre-clinical studies in both PROS and cancers bearing PIK3CA mutations have yielded promising results .
Several AKT inhibitors are now in clinical trials for cancer. Notably, a phase I trial of ARQ 092, a pan-AKT inhibitor, is underway in children and adults with Proteus syndrome (ClinicalTrials.gov: NCT02594215). Allosteric inhibitors of mTOR, including sirolimus (rapamycin), everolimus, ridaforolimus, temsirolimus, are licensed therapies for a range of conditions . Notably, off-label applications of mTOR inhibitors might be successfully exploited also in non-cancer diseases. Very recently, rapamycin has been suggested as a systemic therapy to reduce vascular malformations in patients [48,49], including those carrying PTEN mutations [50,51]. A clinical trial investigating sirolimus in PROS (ClincalTrials.gov: NCT02428296) is currently underway, with expected completion in 2017. Of greatest relevance to PI3K/AKT-related disorders are dual PI3K-mTOR inhibitors, which are currently in phase I trial for cancer .
Of note, patients with somatic activation of the PI3K/AKT pathway may need life-long therapy with the inhibitor. This raises concern not only for the more common adverse effects of these inhibitors, but also for unknown effects that cannot be revealed by short-term evaluation in clinical trials, this being a critical challenge which already emerged for RAS-related syndromes. Unfortunately, data from preclinical studies in a disease model are scarce. Recently, Kinross et al. systemically overexpressed the H1047R mutation of PIK3CA, leading to increased body weight with enlarged organs due to increased cell number . In addition, Hare et al. overexpressed the PIK3CA H1047R mutation specifically in endothelial cells, leading to extensive vascular remodeling and embryonic lethality .
5. PI3Ks as a Master Controller of Proliferation in the Kidney and in Polycystic Kidney Disease
Besides cancer  and overgrowth syndromes , alterations in the PI3K/AKT/mTOR pathway are major drivers of abnormal proliferation. In the kidney, the hyper-proliferation of tubular cells induces renal cyst formation, which can be considered a sort of overgrowth syndrome and is typical of polycystic kidney disease (PKD). In this context, the increased proliferation can be caused by opposite genetic lesions occurring in PI3Ks: on one side, hyper-activation of the AKT-dependent class I PI3KCA signaling pathway, on the other side, loss of class II PI3K-C2α.
In PKD, it is well described that alterations of several components of the PI3K/AKT/mTOR pathway, already found in cancer, are implicated in the hyper-proliferation of renal tubular cells, such as TSC. TSC is a well-known tumor suppressor, which is negatively regulated by AKT and acts as a GTPase Activating Protein (GAP) for the small GTPase Rheb, thus leading to mTORC1 inhibition. TSC1 and TSC2 genes are mutated in a genetic disorder called tuberous sclerosis complex (TSC). TSC causes the development of hamartomas, which can arise in multiple organs, including brain, kidney, skin and liver. Humans and mice with loss of either TSC1 or TSC2 are prone to developing renal problems including cysts, which usually do not interfere with kidney function . However, in a small subset of patients with loss of TSC and who were affected by severe infantile polycystic kidney disease, cysts led to renal failure . It was reported that conditional knockout of TSC1 in renal tubular cells resulted in PKD . Both the hyper-activation of class I PI3KCA and the inhibition of TSC result in the mTOR-mediated increase in cell proliferation. Besides its AKT-mediated inhibition, TSC can be activated by polycystins. Polycistin-1 acts as a mechanosensor of the fluid flux and activates the calcium channel Polycistin-2. These two transmembrane proteins are encoded by PKD1 or PKD2 genes, which are mutated in autosomal-dominant polycystic kidney disease (ADPKD), a genetic disorder characterized by uncontrolled proliferation of renal cells with the consequent formation of cysts and loss of renal function [58,59]. The anti-proliferative signal of polycystins due to mTORC1 inhibition is mediated by TSC1 and TSC2 genes. Mice with concomitant loss of TSC and PC1 revealed that cyst development is closely correlated with elevated mTORC1 activity, leading to hypertrophy and increased proliferation of the renal tubular cells . In cells, the coexistence of mutation and loss of TSC alleles presented an alteration in the localization of PC1 on the plasma membrane . Re-expression of PC1 in the TSC1-mutant kidneys corrects the phenotype. On the other hand, mTORC1 acts in a negative feedback loop to downregulate PC1 expression levels. Consistently, the inhibition of mTOR increases the expression levels of polycystins and slows cyst expansion [60,61].