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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 p110α 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 p110α 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.

The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.

Infantile hemangioma (IH) is the most common tumor in children and a paradigm for pathological vasculogenesis, angiogenesis, and regression. Propranolol, the mainstay of treatment, inhibits IH vessel formation via a β-adrenergic receptor-independent off-target effect of its R(+) enantiomer on endothelial SOX18 - a member of the SOX (SRY-related HMG-box) family of transcription factors. Transcriptomic profiling of patient-derived hemangioma stem cells uncovered the mevalonate pathway (MVP) as a target of R(+) propranolol. Loss and gain of function of SOX18 confirmed it is both necessary and sufficient for R(+) propranolol suppression of the MVP, including regulation of sterol regulatory element-binding protein 2 (SREBP2) and the rate-limiting enzyme HMG-CoA reductase (HMGCR). A biological relevance of the endothelial SOX18-MVP axis in IH patient tissue was demonstrated by nuclear colocalization of SOX18 and SREBP2. Functional validation in a preclinical IH xenograft model revealed that statins - competitive inhibitors of HMGCR - efficiently suppress IH vessel formation. We propose an endothelial SOX18-MVP axis as a central regulator of IH pathogenesis and suggest statin repurposing to treat IH. The pleiotropic effects of R(+) propranolol and statins along the SOX18-MVP axis to disable an endothelial cell-specific program may have therapeutic implications for other vascular disease entities involving pathological vasculogenesis and angiogenesis.

Lymphoedema is the swelling of one or several parts of the body owing to lymph accumulation in the extracellular space. It is often chronic, worsens if untreated, predisposes to infections and causes an important reduction in quality of life. Primary lymphoedema (PLE) is thought to result from abnormal development and/or functioning of the lymphatic system, can present in isolation or as part of a syndrome, and can be present at birth or develop later in life. Mutations in numerous genes involved in the initial formation of lymphatic vessels (including valves) as well as in the growth and expansion of the lymphatic system and associated pathways have been identified in syndromic and non-syndromic forms of PLE. Thus, the current hypothesis is that most cases of PLE have a genetic origin, although a causative mutation is identified in only about one-third of affected individuals. Diagnosis relies on clinical presentation, imaging of the structure and functionality of the lymphatics, and in genetic analyses. Management aims at reducing or preventing swelling by compression therapy (with manual drainage, exercise and compressive garments) and, in carefully selected cases, by various surgical techniques. Individuals with PLE often have a reduced quality of life owing to the psychosocial and lifelong management burden associated with their chronic condition. Improved understanding of the underlying genetic origins of PLE will translate into more accurate diagnosis and prognosis and personalized treatment. Primary lymphoedema (PLE) refers to swelling of parts of the body (usually limbs) caused by anomalies in the development or functioning of the lymphatic system; PLE can result from genetic mutations and can be the only symptom or one of the manifestations of a syndrome.

Background Theragnostic management, treatment according to precise pathological molecular targets, requests to unravel patients’ genotypes. We used targeted next-generation sequencing (NGS) or digital droplet polymerase chain reaction (ddPCR) to screen for somatic PIK3CA mutations on DNA extracted from resected lesional tissue or lymphatic endothelial cells (LECs) isolated from lesions. Our cohort (n = 143) was composed of unrelated patients suffering from a common lymphatic malformation (LM), a combined lymphatic malformation [lymphatico-venous malformation (LVM), capillaro-lymphatic malformation (CLM), capillaro-lymphatico-venous malformation (CLVM)], or a syndrome [CLVM with hypertrophy (Klippel-Trenaunay-Weber syndrome, KTS), congenital lipomatous overgrowth-vascular malformations-epidermal nevi -syndrome (CLOVES), unclassified PIK3CA-related overgrowth syndrome (PROS) or unclassified vascular (lymphatic) anomaly syndrome (UVA)]. Results We identified a somatic PIK3CA mutation in resected lesions of 108 out of 143 patients (75.5%). The frequency of the variant allele ranged from 0.54 to 25.33% in tissues, and up to 47% in isolated endothelial cells. We detected a statistically significant difference in the distribution of mutations between patients with common and combined LM compared to the syndromes, but not with KTS. Moreover, the variant allele frequency was higher in the syndromes. Conclusions Most patients with an common or combined lymphatic malformation with or without overgrowth harbour a somatic PIK3CA mutation. However, in about a quarter of patients, no such mutation was detected, suggesting the existence of (an)other cause(s). We detected a hotspot mutation more frequently in common and combined LMs compared to syndromic cases (CLOVES and PROS). Diagnostic genotyping should thus not be limited to PIK3CA hotspot mutations. Moreover, the higher mutant allele frequency in syndromes suggests a wider distribution in patients’ tissues, facilitating detection. Clinical trials have demonstrated efficacy of Sirolimus and Alpelisib in treating patients with an LM or PROS. Genotyping might lead to an increase in efficacy, as treatments could be more targeted, and responses could vary depending on presence and type of PIK3CA -mutation.

Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function.

Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatics in bone, gradually lose bone, and can have life-threatening complications, such as chylothorax. The etiology of GSD is poorly understood, and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a patient with GSD and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.

BACKGROUNDSlow-flow vascular malformations frequently harbor activating mutations in the PI3K/AKT/mTOR cascade. Phase II trials pinpointed sirolimus effectiveness as a drug therapy. Efficacy and safety of sirolimus thus need to be evaluated in large prospective phase III trials.METHODSThe Vascular Anomaly-Sirolimus-Europe (VASE) trial, initiated in 2016, is a large multicentric prospective phase III trial (EudraCT 2015-001703-32), which evaluates efficacy and safety of sirolimus for 2 years in pediatric and adult patients with symptomatic slow-flow vascular malformations. In this interim analysis, we studied all patients enrolled up to October 2021 who received sirolimus for 12 or more months or who prematurely stopped the treatment.RESULTSThirty-one pediatric and 101 adult patients were included in this analysis; 107 completed 12 or more months of sirolimus, including 61 who were treated for the whole 2-year period. Sirolimus resulted in a clinical improvement in 85% of patients. The efficacy appeared within the first month for the majority of them. Grade 3-4 adverse events were observed in 24 (18%) patients; all resolved after treatment interruption/arrest. Sirolimus increased feasibility of surgery or sclerotherapy in 20 (15%) patients initially deemed unsuitable for intervention. Among the 61 patients who completed the 2-year treatment, 33 (54%) reported a recurrence of symptoms after a median follow-up of 13 months after sirolimus arrest. While there was no difference in efficacy, clinical improvement was faster but subsided more rapidly in PIK3CA-mutated (n = 24) compared with TIE2-mutated (n = 19) patients.CONCLUSIONSirolimus has a high efficacy and good tolerance in treatment of slow-flow vascular malformations in children and adults.TRIAL REGISTRATIONClinicalTrials.gov NCT02638389 and EudraCT 2015-001703-32.FUNDINGThe Fonds de la Recherche Scientifique (FNRS grants T.0247.19, P.C005.22, T.0146.16, and P.C013.20), the Fund Generet managed by the King Baudouin Foundation (grant 2018-J1810250-211305), the Walloon Region through the FRFS-WELBIO strategic research programme (WELBIO-CR-2019C-06), the MSCA-ITN network V.A. Cure no. 814316, the Leducq Foundation Networks of Excellence Program grant \"ReVAMP\" (LFCR grant 21CVD03), the European Union's Horizon 2020 research and innovation programme under grant agreement no. 874708 (Theralymph), the Swiss National Science Foundation under the Sinergia project no. CRSII5_193694, and a Pierre M. fellowship.

Lymphatic anomalies include a variety of developmental and/or functional defects affecting the lymphatic vessels: sporadic and familial forms of primary lymphedema, secondary lymphedema, chylothorax and chylous ascites, lymphatic malformations, and overgrowth syndromes with a lymphatic component. Germline mutations have been identified in at least 20 genes that encode proteins acting around VEGFR-3 signaling but also downstream of other tyrosine kinase receptors. These mutations exert their effects via the RAS/MAPK and the PI3K/AKT pathways and explain more than a quarter of the incidence of primary lymphedema, mostly of inherited forms. More common forms may also result from multigenic effects or post-zygotic mutations. Most of the corresponding murine knockouts are homozygous lethal, while heterozygotes are healthy, which suggests differences in human and murine physiology and the influence of other factors.

Background Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. Methods Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1 , BRCA2 , TP53 , or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. Results Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2 , in vitro expression of which caused downstream signaling pathway activation. Conclusion Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.