NOTCH1: Review of its role in lymphatic development and study of seven families with rare pathogenic variants.

BACKGROUND: We developed a Next-Generation-Sequencing (NGS) protocol to screen the most frequent genetic variants related to lymphedema and a group of candidate genes. The aim of the study was to find the genetic cause of lymphedema in the analyzed patients. METHODS: We sequenced a cohort of 246 Italian patients with lymphatic malformations. In the first step, we analyzed genes known to be linked to lymphedema: 235 out of 246 patients tested negative for the most frequent variants and underwent testing for variants in a group of candidate genes, including the NOTCH1 gene, selected from the database of mouse models. We also performed in silico analysis to observe molecular interactions between the wild-type and the variant amino acids and other protein residues. RESULTS: Seven out of 235 probands, five with sporadic and two with familial lymphedema, were found to carry rare missense variants in the NOTCH1 gene. CONCLUSIONS: Our results propose that NOTCH1 could be a novel candidate for genetic predisposition to lymphedema.

Dysfunction of dermal initial lymphatics of the arm and upper body quadrant causes congenital arm lymphedema.

OBJECTIVE: The objective of this study was to explore the pathologic process underlying primary lymphedema. METHODS: Twenty-seven patients with unilateral congenital arm lymphedema who visited our clinic from January 1, 2014, to May 30, 2019, were enrolled. The patients' clinical signs and the findings of indocyanine green (ICG) lymphography, skin tissue immunohistochemical staining, and whole exome sequencing of tissue and blood were evaluated. RESULTS: Among the 27 patients, 11 were diagnosed with stage II and 16 were diagnosed with stage III lymphedema. No lymphatic vessels were visualized in the affected arm in 25 of 27 (93%) patients who underwent ICG lymphography; likewise, no lymphatics were found in the territories of axillary lymph node drainage in the trunk, irrespective of any anomalies of the axillary lymph nodes. In only two (7%) patients, an unclear lymphatic trunk gradually appeared in the dorsum of the affected hand. The number of initial lymphatics was increased in the skin specimens of all nine patients in whom lymphatics were not demonstrated by ICG lymphography. Among 14 tested patients, we found compound heterozygote variants in the PIEZO1 gene in only one (7%) patient. Two missense variants, c.4072C>T; p.Arg1358Cys and c.5033C>T; p. Ala1678Val, were identified and found to have been inherited from the father and mother, respectively. No other pathogenic or likely pathogenic variants of currently known lymphedema-related genes were identified in the remaining 13 patients. No genetic difference was found between the lymphedematous and nonedematous healthy skin tissue of the same person. CONCLUSIONS: Segmental or regional dysfunction of the dermal initial lymphatics causes congenital arm lymphedema and may have implications for clinical treatment.

Association of Lymphatic Abnormalities with Early Complications after Fontan Operation.

BACKGROUND: Increased central venous pressure is inherent in Fontan circulation but not strongly related to Fontan complication. Abnormalities of the lymphatic circulation may play a crucial role in early Fontan complications. METHODS: This was a retrospective, single-center study of patients undergoing Fontan operation from 2008 to 2015. The primary outcome was significant early Fontan complication defined as secondary in-hospital treatment due to peripheral edema, ascites, pleural effusions, protein-losing enteropathy, or plastic bronchitis. All patients received T2-weighted magnetic resonance images to assess abdominal and thoracic lymphatic perfusion pattern 6 months after Fontan completion with respect to localization, distribution, and extension of lymphatic perfusion pattern (type 1-4) and with application of an area score (0-12 points). RESULTS: Nine out of 42 patients developed early Fontan complication. Patients with complication had longer chest tube drainage (mean 28 [interquartile range [IQR]: 13-60] vs. 13 [IQR: 2-22] days, p = 0.01) and more often obstructions in the Fontan circuit 6 months after surgery (56 vs. 15%, p = 0.02). Twelve patients showed little or no abnormalities of lymphatic perfusion (lymphatic perfusion pattern type 1). Most frequently magnetic resonance imaging showed lymphatic congestion in the supraclavicular region (24/42 patients). Paramesenteric lymphatic congestion was observed in eight patients. Patients with early Fontan complications presented with higher lymphatic area score (6 [min-max: 2-10] vs. 2 [min-max: 0-8]), p = 0.001) and greater distribution and extension of thoracic lymphatic congestion (type 3-4: n = 5/9 vs. n = 1/33, p = 0.001). CONCLUSION: Early Fontan complication is related to hemodynamic factors such as circuit obstruction and to the occurrence and extent of lymphatic congestion.

Analysis of mTOR pathway expression in lymphatic malformation and related diseases.

The mammalian target of rapamycin (mTOR) inhibitor sirolimus is an effective treatment for difficult-to-treat lymphatic anomalies. However, little is known about the expression of mTOR pathway components in lymphatic anomalies. Here we investigated the expression pattern of mTOR pathway components and their phosphorylated forms (mTOR, p-mTOR, 4EBP1, p-4EBP1, S6K1 and p-S6K1) in normal lymphatic vessels and lymphatic anomalies using immunohistochemistry. We studied 18 patients of lymphatic anomalies, including lymphatic malformation (LM, n = 14), Kaposiform lymphangiomatosis (KLA, n = 2) and Kaposiform hemangioendothelioma (KHE, n = 2). Normal lymphatic vessels expressed 4EBP1, S6K1 and p-S6K1, but not p-4EBP1, mTOR or p-mTOR. The mTOR was detected in all lymphatic anomalies, whereas its activation form p-mTOR was detected in half cases of KLA and KHE but not in LM. All lymphatic anomalies expressed S6K1 and its activated form p-S6K1. The expression of 4EBP1 was also found in all lymphatic anomalies, but its activation was detected in approximately half of them. The activation of mTOR was seen in tumor (KLA and KHE) but not in malformation (LM), whereas the activation of S6K1 and 4EBP1 was seen in all and half of lymphatic anomalies, respectively.

Clinical Factors in Trunk Capillary Malformations in the Neonate: When to Suspect Other Associated Malformations? A Case-Control Study.

INTRODUCTION: Capillary malformations (CMs) can be sporadic or syndromic, in association with other underlying venous malformation (VM) or lymphatic malformation (LM). The objective of this study is to describe the clinical patterns in the neonate that allow us to differentiate sporadic CMs from those associated with other vascular malformations. MATERIALS AND METHODS: A case-control study was performed in neonates with CM located in the trunk, followed at our institution between 2008 and 2018. The patients were divided into two groups: group A (cases: CM associated with VM or LM) and group B (controls: sporadic CM without associated malformations). Demographic and clinical variables collected in the clinical history were evaluated (color, location, multifocality, bilaterality, position regarding the vascular axis, and involvement of the midline). RESULTS: Thirty-eight patients were included (18 cases and 20 controls) without differences in gender and age. In group A, the totality of patients presented CM with uniform color and lateral location (p < 0.001). In this group, bilateral and multifocal involvements were lower than in group B, without significant differences between both groups. The distribution of CMs in group A was always parallel to the vascular axis and the midline was always respected, without observing these characteristics in the group B (p < 0.001). CONCLUSION: The presence of a CM in the trunk of a neonate with uniform color, lateral location, parallel position to the vascular axis, and absence of involvement of the midline should make us suspect other underlying vascular malformations, which should be studied with complementary tests.

Sclerotherapy for lymphatic malformations of head and neck: Systematic review and meta-analysis.

BACKGROUND: Percutaneous sclerotherapy is a commonly used modality for treatment of lymphatic malformations (LMs) of the head, face, and neck. The safety and efficacy of sclerotherapy with various agents for diverse pathologic types of LMs have not been fully established. We present the results of a systematic review and meta-analysis examining the safety and efficacy of percutaneous sclerotherapy for treatment of LMs of the head, face, and neck. METHODS: We searched PubMed, MEDLINE, and Embase from 2000 to 2018 for studies evaluating the safety and efficacy of percutaneous sclerotherapy of head, face, and neck LMs. Two independent reviewers selected studies and abstracted data. The primary outcomes were complete and partial resolution of the LM. Data were analyzed using random-effects meta-analysis. RESULTS: There were 25 studies reporting on 726 patients included. The overall rate of complete cure of any pathologic type of LM after percutaneous sclerotherapy with any agent was 50.5% (95% confidence interval, 36.6%-64.3%). Macrocystic lesions had a cure rate of 53.1% compared with cure rates of 35.1% for microcystic lesions and 31.1% for mixed lesions. Regarding agents, doxycycline had the highest cure rate (62.4%) compared with all other agents. Overall permanent morbidity or mortality was 1.2% (95% confidence interval, 0.4%-2.0%) with no deaths. I2 values were >50% for most outcomes, indicating substantial heterogeneity. CONCLUSIONS: Our systematic review and meta-analysis of 25 studies and >700 patients found that percutaneous sclerotherapy is a safe and effective modality for treatment of LMs of the head, neck, and face.

Indications for tracheostomy in children with head and neck lymphatic malformation: analysis of a nationwide survey in Japan.

PURPOSE: Airway obstruction caused by lymphatic malformation (LM) in the head and neck may require a tracheostomy. We present the results of our analysis of a nationwide survey on the indications for tracheostomy in children with head and neck LM. METHODS: We analyzed data in relation to tracheostomy based on a questionnaire about 518 children with head and neck LM without mediastinal involvement. RESULTS: Tracheostomy was performed for 43 of the 518 children. Most (32/43) of these children were younger than 1 year of age and the tracheostomy was almost always performed for airway obstruction (40/43). The lesion was in contact with the airway in 32 (72%) of these children, but in only 58 (12%) of the 473 children who were managed without tracheostomy. When the maximum circumferential area of contact was compared, only 20 (27%) of 74 patients with maximum contact of less than a half-circle required tracheostomy, whereas 11 of 13 with maximum contact of more than a half-circle required tracheostomy (P = 0.0001). Six patients without airway contact required tracheostomy because of acute swelling caused by hemorrhage, infection, or both. CONCLUSIONS: Children with head and neck LM required tracheostomy to relieve airway obstruction. Tracheostomy should be considered if the lesion is in contact with the airway and surrounds more than a half-circle, and when it causes acute swelling.

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Soft-tissue vascular malformations and tumors. Part 2: low-flow lesions. / Malformaciones vasculares y tumores de partes blandas. Parte 2: lesiones de bajo flujo.

Vascular malformations and tumors, also known as "vascular anomalies", comprise an extensive variety of lesions involving all parts of the body. Due to a lack of a complete understanding of the origin and histopathology of such lesions, this field has been traditionally obscured by the use of an unclear nomenclature. Knowledge of the classification and clinical and imaging characteristics of this group of lesions is paramount when managing these patients. The objective of this series of two articles is to review the current classification of vascular anomalies, to describe the role of imaging in their diagnosis, to summarize their distinctive histopathologic, clinical and imaging features, and to discuss the treatment options. High-flow lesions were discussed in the first article of this series. In this second article, we will focus on low-flow lesions, including complex syndromes with associated low-flow malformations.

Interstitial Transpedal MR-Lymphangiography of Central Lymphatics Using a Standard MR Contrast Agent: Feasibility and Initial Results in Patients with Chylous Effusions.

PURPOSE: To describe a novel technique for transpedal magnetic resonance lymphangiography (tMRL) with interstitial injection of a standard gadolinium-based contrast agent, and to assess its ability to visualize the central lymphatic system (CLS) in patients with chylous effusions. MATERIALS AND METHODS: Experiences with tMRL were retrospectively reviewed in seven patients with chylous effusions. High-resolution three-dimensional multi-echo (mDIXON) images of the entire torso were obtained before and after interstitial injection of an extracellular gadolinium-based contrast agent. MR examinations were assessed qualitatively by two radiologists with respect to visualization of central lymphatics, delineation of their anatomy and presence of a lymphatic pathology (chylolymphatic leakage or reflux). RESULTS: Examination was technically successful in 6/7 patients. tMRL confirmed the location of chylolymphatic leakage in 3/7, demonstrated a lymphatic malformation in 1/7, chylolymphatic reflux in 1/7, and confirmed normal chylolymphatic flow in the central lymphatics in 1/7 patients. In one patient neither CLS enhancement nor leakage were detectable. tMRL findings were considered helpful in all technically successful cases, and considerably influenced further treatment in four patients. All examinations were well tolerated without complications. No general anesthesia or conscious sedation was necessary. CONCLUSION: Transpedal MR-lymphangiography with interstitial injection of a standard extracellular gadolinium-based contrast agent is feasible in clinical routine without general anesthesia and allows for assessment of pathologic conditions of central lymphatics. KEY POINTS: · Transpedal MR-lymphangiography of central lymphatics is technically feasible.. · A standard extracellular gadolinium-based contrast agent can be used for tMRL.. · tMRL allows for evaluation of anatomy and pathologies of central lymphatics.. · tMRL is a useful tool for treatment planning in chylous effusions.. CITATION FORMAT: · Pieper CC, Schild HH. Interstitial Transpedal MR-Lymphangiography of Central Lymphatics Using a Standard MR Contrast Agent: Feasibility and Initial Results in Patients with Chylous Effusions. Fortschr Röntgenstr 2018; 190: 938 - 945.

Development of an international core outcome set for peripheral vascular malformations: the OVAMA project.

BACKGROUND: An important limitation in vascular malformation research is the heterogeneity in outcome measures used for the evaluation of treatment outcome. OBJECTIVES: To reach international consensus on a core outcome set (COS) for clinical research on peripheral vascular malformations: lymphatic (LM), venous (VM) and arteriovenous malformations (AVM). In this consensus study, we determined what domains should constitute the COS. METHODS: Thirty-six possibly relevant outcome domains were proposed to an international group of physicians, patients and the parents of patients. In a three-round e-Delphi process using online surveys, participants repeatedly rated the importance of these domains on a five-point Likert scale. Participants could also propose other relevant domains. This process was performed for LM, VM and AVM separately. Consensus was predefined as 80% agreement on the importance of a domain among both the physician group and the patient/parent group. Outcomes were then re-evaluated in an online consensus meeting. RESULTS: 167 physicians and 134 patients and parents of patients with LM (n = 50), VM (n = 71) and AVM (n = 29) participated in the study. After three rounds and a consensus meeting, consensus was reached for all three types of vascular malformations on the core domains of radiological assessment, physician-reported location-specific signs, patient-reported severity of symptoms, pain, quality of life, satisfaction and adverse events. Vascular malformation type-specific signs and symptoms were included for LM, VM and AVM, separately. CONCLUSIONS: Our recommendation is that therapeutic-efficacy studies on peripheral vascular malformations should measure at least these core outcome domains.

Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

TNFΔARE Mice Display Abnormal Lymphatics and Develop Tertiary Lymphoid Organs in the Mesentery.

Chronic inflammatory diseases are associated with a persistent and enhanced response to environmental antigens. As an adaptive response to this exaggerated immune state, affected tissue typically develops tertiary lymphoid organs. Studies of Crohn disease (CD), a chronic inflammatory disease of the intestinal tract, report tertiary lymphoid organs present within the mucosal wall, along with other lymphatic diseases, such as lymphangiogenesis and obstructed lymphatic vessels. These observations suggest that downstream mesenteric lymphatic vessels and lymph drainage into mesenteric lymph nodes may be compromised. However, information is lacking on the morphologic features and functional status of mesenteric lymphatics in CD. Using confocal imaging, PCR, flow cytometry, and functional strategies, we addressed these questions in the established TNFΔARE mouse model of CD and found that this mouse model had many lymphatic abnormalities reminiscent of human CD. These abnormalities include intestinal lymphangiectasia, mesenteric lymph node lymphadenopathy, and lymphangiogenesis in both the mesentery and mucosa. Critically, TNFΔARE mice also present mesenteric tertiary lymphoid organs and have altered lymphatic transport of dendritic cells to mesenteric lymph nodes, two features likely to actively modulate immunity. Our findings provide key insights into lymphatic remodeling in the TNFΔARE mouse model. They shed light on the involvement of these lymphatic changes in immune dysfunctions observed in CD and suggest the lymphatic system as new target for therapeutic options.

Arterial Spin-Labeling to Discriminate Pediatric Cervicofacial Soft-Tissue Vascular Anomalies.

BACKGROUND AND PURPOSE: Differentiating major subtypes of cervicofacial vascular lesions is crucial for appropriate management. The aim of our study was to evaluate the performance of an MR imaging arterial spin-labeling perfusion sequence in discriminating pediatric cervicofacial soft-tissue vascular anomalies. MATERIALS AND METHODS: We conducted a retrospective analysis of data from a prospectively maintained registry including pediatric patients at a tertiary pediatric center between January 2012 and January 2014. We included pediatric patients with a final diagnosis of soft-tissue vascular anomalies and an MR imaging, including an arterial spin-labeling sequence at presentation. We performed an analysis of lesion perfusion, blinded to clinical data, by using concurrent spiral 3D pseudocontinuous arterial spin-labeling (1.5T magnet; spiral matrix, 512 × 8 mm; postlabeling delay, 1025 ms). Lesional flow was recorded with calibrated intralesional ROIs. Perfusion characteristics were compared among lesion subtypes with the Mood Median test. RESULTS: Among 840 patients screened, 46 matched the inclusion criteria and were included (median age, 1.45 years; interquartile range, 0.4-5.1 years; 27 females). Hemangiomas, including infantile hemangiomas (n = 18 patients) and noninvoluting (n = 2) and rapidly involuting (n = 1) congenital types, demonstrated marked hyperperfusion (median flow, 436 mL/min/100 g; interquartile range, 212.5-603 mL/min/100 g), significantly higher than that of lymphatic malformations (median, 22.5 mL/min/100 g; interquartile range, 16-60 mL/min/100 g; P < .001) or venous malformations (median, 25 mL/min/100 g; interquartile range, 15-66.5 mL/min/100 g; P = .003). CONCLUSIONS: MR imaging arterial spin-labeling is a valuable tool for the assessment of soft-tissue vascular anomaly hemodynamics and for the classification of major lesion subtypes.

Angiopoietins as serum biomarkers for lymphatic anomalies.

Vascular anomalies can cause significant morbidity and mortality. Advances in diagnosis will be improved if noninvasive biomarkers can be identified, as obtaining a tissue biopsy can worsen the disease and precipitate complications. The goal of this study was to identify biomarkers for vascular anomaly patients to aid diagnosis and potentially give insights into pathogenesis. Blood was collected at baseline and then 6 and 12 months after treatment with the mTOR inhibitor sirolimus. Patients groups included generalized lymphatic anomaly (GLA), kaposiform lymphangiomatosis (KLA) and kaposiform hemangioendothelioma (KHE) with or without the Kasabach-Merritt phenomenon (KMP) coagulopathy. Serum was obtained from healthy controls selected to match the age and sex of the patients (21 days-28.5 years; 42% males; 58% females). Angiogenic and lymphangiogenic factors (VEGF-A, C, D, Ang-1 and Ang-2) were measured in serum using ELISA. In lymphatic anomaly patients, baseline levels of VEGF-A and VEGF-D were not different compared to controls. Angiopoietin-2 (Ang-2) levels were near controls levels in GLA patients but 10-fold greater in KLA patients and 14-fold greater in KHE patients when the KMP coagulopathy was present but not when it was absent. VEGF-C and angiopoietin-1 (Ang-1) levels were lower in KHE patients with KMP. Our analyses suggest that Ang-2 and Ang-1 can be used as biomarkers to help identify KLA and KHE patients with KMP coagulopathy with high sensitivity and specificity. After 12 months of sirolimus treatment, Ang-2 levels were lower in KLA and KHE with KMP patients compared to baseline levels and with most patients showing a clinical response. Hence, serum Ang-2 and Ang-1 levels may help in the diagnosis of patients with lymphatic anomalies and are concordant to sirolimus response.