Lymphatic Malformations Genetics, Mechanisms and Therapeutic Strategies

Lymphatic vessels maintain tissue fluid homeostasis by returning to blood circulation interstitial fluid that has extravasated from the blood capillaries. They provide a trafficking route for cells of the immune system, thus critically contributing to immune surveillance. Developmental or functional defects in the lymphatic vessels, their obstruction or damage, lead to accumulation of fluid in tissues, resulting in lymphedema. Here we discuss developmental lymphatic anomalies called lymphatic malformations and complex lymphatic anomalies that manifest as localized or multifocal lesions of the lymphatic vasculature, respectively. They are rare diseases that are caused mostly by somatic mutations and can present with variable symptoms based upon the size and location of the lesions composed of fluid-filled cisterns or channels. Substantial progress has been made recently in understanding the molecular basis of their pathogenesis through the identification of their genetic causes, combined with the elucidation of the underlying mechanisms in animal disease models and patient-derived lymphatic endothelial cells. Most of the solitary somatic mutations that cause lymphatic malformations and complex lymphatic anomalies occur in genes that encode components of oncogenic growth factor signal transduction pathways. This has led to successful repurposing of some targeted cancer therapeutics to the treatment of lymphatic malformations and complex lymphatic anomalies. Apart from the mutations that act as lymphatic endothelial cell-autonomous drivers of these anomalies, current evidence points to superimposed paracrine mechanisms that critically contribute to disease pathogenesis and thus provide additional targets for therapeutic intervention. Here, we review these advances and discuss new treatment strategies that are based on the recently identified molecular pathways.Peer reviewe

Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1

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Parkes Weber Syndrome: Contribution of the Genotype to the Diagnosis

Objectives: Parkes Weber syndrome (PWS) is a rare disorder that combines overgrowth, capillary malformations, and arteriovenous malformations (AVM)/arteriovenous fistulas, for which underlying activating mutations in the ras/mitogen-activated protein kinase/extracellular-signal-regulated kinase signaling pathway have been described. The clinical overlap with Klippel-Trenauny syndrome, associated with mutations in PIK3CA, is significant. This case series aimed to elaborate on the phenotypic description of PWS, to underline its clinical overlap with Klippel-Trenauny syndrome and nonsyndromic AVM, and to evaluate the contribution of genotypic characterization to the diagnosis. Methods: All patients diagnosed with PWS upon enrollment in the Bernese VAScular COngenital Malformations (VASCOM) cohort were included. The diagnostic criteria of PWS were retrospectively reviewed. A next-generation sequencing (NGS) gene panel (TSO500, Illumina) was used on tissue biopsy samples. Results: Overall, 10/559 patients of the VAScular COngenital Malformations cohort were initially diagnosed with PWS. Three patients were reclassified as nonsyndromic AVM (Kristen Rat Sarcoma Viral oncogene homolog [KRAS], KRAS+tumor protein p53, and protein tyrosine phosphatase non-receptor type 11). Finally, 7 patients fulfilled all clinical diagnostic criteria of PWS. Genetic testing was available in 5 PWS patients. Only 1 patient had the classic RASA1 mutation; another patient had mutations in G protein subunit alpha q (GNAQ) and phosphatase and tensin homolog. In a third case, a PIK3CA mutation was detected. In 2 patients, no mutations were identified. Conclusion: Overgrowth syndromes with vascular malformations are rare and their clinical overlap hampers the classification of individual phenotypes under specific syndrome labels, sometimes even despite genetic testing. To provide optimal patient care, an accurate phenotypic description combined with the identification of molecular targets for precision medicine may be more meaningful than the syndrome classification itself

Blockade of VEGF-C signaling inhibits lymphatic malformations driven by oncogenic PIK3CA mutation

Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3CA(H1047R) mutation, resulting in constitutive activation of the p110 alpha PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CA(H1047R)-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110 alpha activation determining the LM subtype. In the postnatal vasculature, PIK3CA(H1047R) promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways. Lymphatic malformation (LM) is a debilitating often incurable vascular disease. Using a mouse model of LM driven by a disease-causative PIK3CA mutation, the authors show that vascular growth is dependent on the upstream lymphangiogenic VEGF-C signalling, permitting effective therapeutic intervention.Peer reviewe

Identification of candidate regions for a novel Usher syndrome type II locus

PURPOSE: Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin. METHODS: Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes. RESULTS: Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q. CONCLUSIONS: Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q

The VASCERN-VASCA working group diagnostic and management pathways for severe and/or rare infantile hemangiomas

The European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN), is dedicated to gathering the best expertise in Europe and provide accessible cross-border healthcare to patients with rare vascular dis-eases. Infantile Hemangiomas (IH) are benign vascular tumors of infancy that rapidly growth in the first weeks of life, followed by stabilization and spontaneous regression. In rare cases the extent, the localization or the number of lesions may cause severe complications that need specific and careful management. Severe IH may be life-threatening due to airway obstruction, liver or cardiac failure or may harbor a risk of functional impairment, severe pain, and/or significant and permanent disfigurement. Rare IHs include syndromic variants associated with extracutaneous abnormalities (PHACE and LUMBAR syndromes), and large segmental hemangiomas. There are publications that focus on evidence-based medicine on propranolol treatment for IH and consensus state -ments on the management of rare infantile hemangiomas mostly focused on PHACES syndrome. The Vascular Anomalies Working Group (VASCA-WG) decided to develop a diagnostic and management pathway for severe and rare IHs with a Nominal Group Technique (NGT), a well-established, structured, multistep, facilitated group meeting technique used to generate consensus statements. The pathway was drawn following two face-to-facePeer reviewe

The VASCERN-VASCA working group diagnostic and management pathways for lymphatic malformations

Lymphatic malformations (LMs) are developmental defects of lymphatic vessels. LMs are histologically benign lesions, however, due to localization, size, and unexpected swelling, they may cause serious complications that threaten vital functions such as compression of the airways. A large swelling of the face or neck may also be disfiguring and thus constitute a psychological strain for patients and their families. LMs are also highly immunologically reactive, and are prone to recurrent infections and inflammation causing pain as well as chronic oozing wounds.The European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN) is dedicated to gathering the best expertise in Europe. There are only few available guidelines on management and follow up of LMs, which commonly focus on very specific situations, such as head and neck LM (Zhou et al., 2011). It is still unclear, what constitutes an indication for treatment of LMs and how to follow up the patients. The Vascular Anomalies Working Group (VASCA-WG) of VASCERN decided to develop a diagnostic and management pathway for the management of LMs with a Nominal Group Technique (NGT), a well-established, structured, multistep, facilitated group meeting technique used to generate consensus statements. The pathway was drawn following 2 face-to-face meetings and multiple web meetings to facilitate discussion, and by mail to avoid the influence of most authoritative members.The VASCA-WG has produced this opinion statement reflecting strategies developed by experts and patient representatives on how to approach patients with lymphatic malformations in a practical manner; we present an algorithmic view of the results of our work.Peer reviewe

The VASCERN-VASCA Working Group Diagnostic and Management Pathways for Venous Malformations.

UNLABELLED To elaborate expert consensus patient pathways to guide patients and physicians toward efficient diagnostics and management of patients with venous malformations. METHODS VASCERN-VASCA (https://vascern.eu/) is a European network of multidisciplinary centers for Vascular Anomalies. The Nominal Group Technique was used to establish the pathways. Two facilitators were identified: one to propose initial discussion points and draw the pathways, and another to chair the discussion. A dermatologist (AD) was chosen as first facilitator due to her specific clinical and research experience. The draft was subsequently discussed within VASCERN-VASCA monthly virtual meetings and annual face-to-face meetings. RESULTS The Pathway starts from the clinical suspicion of a venous type malformation (VM) and lists the clinical characteristics to look for to support this suspicion. Strategies for subsequent imaging and histopathology are suggested. These aim to inform on the diagnosis and to separate the patients into 4 subtypes: (1) sporadic single VMs or (2) multifocal, (3) familial, multifocal, and (4) combined and/or syndromic VMs. The management of each type is detailed in subsequent pages of the pathway, which are color coded to identify sections on (1) clinical evaluations, (2) investigations, (3) treatments, and (4) associated genes. Actions relevant to all types are marked in separate boxes, including when imaging is recommended. When definite diagnoses have been reached, the pathway also points toward disease-specific additional investigations and recommendations for follow up. Options for management are discussed for each subtype, including conservative and invasive treatments, as well as novel molecular therapies. CONCLUSION The collaborative efforts of VASCERN-VASCA, a network of the 9 Expert Centers, has led to a consensus Diagnostic and Management Pathways for VMs to assist clinicians and patients. It also emphasizes the role of multidisciplinary expert centers in the management of VM patients. This pathway will become available on the VASCERN website (http://vascern.eu/)

Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a “second hit,” that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome