Update August 2021

Vascular malformations, affecting approximately 1% to 1.5% of the population, comprise a spectrum of developmental patterning defects of capillaries, arteries, veins, and/or lymphatics. The majority of vascular malformations occur sporadically; however, inherited malformations exist as a part of complex congenital diseases. The malformations, ranging from birthmarks to life-threatening conditions, are present at birth, but may reveal signs and symptoms-including pain, bleeding, disfigurement, and functional defects of vital organs-in infancy, childhood, or adulthood. Vascular malformations often exhibit recurrent patterns at affected sites due to the lack of curative treatments. This review series provides a state-of-the-art assessment of vascular malformation research at basic, clinical, genetic, and translational levels.

Research into the glymphatic system reached an inflection point with steep trajectory in 2012 when it was formally recognized and named, but the historical roots for it are solid and deep, dating back to pioneers such as Cushing, Weed, and Dandy. We provide an overview of key discoveries of the glymphatic system, which promotes bulk flow of fluid and solutes throughout the brain parenchyma. We also discuss the lymphatic drainage of the central nervous system. Evidence is building that failure of the glymphatic system causes glymphedema in patients commonly managed by neurocritical care and neurosurgery specialists. We review research supporting this for decompressive craniectomy, subarachnoid hemorrhage, and normal-pressure hydrocephalus. We argue that it is time for a paradigm shift from the traditional model of cerebrospinal fluid circulation to a revised model that incorporates the glymphatic pathway and lymphatic clearance. These recent breakthroughs will inspire new therapeutic approaches to recognize, reverse, and restore glymphatic dysfunction and to leverage this pathway to deliver brain-wide therapeutics.

Lymph nodes (LNs) are tissues of the immune system that house leukocytes, making them targets of interest for a variety of therapeutic immunomodulation applications. However, achieving accumulation of a therapeutic in the LN does not guarantee equal access to all leukocyte subsets. LNs are structured to enable sampling of lymph draining from peripheral tissues in a highly spatiotemporally regulated fashion in order to facilitate optimal adaptive immune responses. This structure results in restricted nanoscale drug delivery carrier access to specific leukocyte targets within the LN parenchyma. Herein, a framework is presented to assess the manner in which lymph-derived macromolecules and particles are sampled in the LN to reveal new insights into how therapeutic strategies or drug delivery systems may be designed to improve access to dLN-resident leukocytes. This summary analysis of previous reports from our group assesses model nanoscale fluorescent tracer association with various leukocyte populations across relevant time periods post administration, studies the effects of bioactive molecule NO on access of lymph-borne solutes to dLN leukocytes, and illustrates the benefits to leukocyte access afforded by lymphatic-targeted multistage drug delivery systems. Results reveal trends consistent with the consensus view of how lymph is sampled by LN leukocytes resulting from tissue structural barriers that regulate inter-LN transport and demonstrate how novel, engineered delivery systems may be designed to overcome these barriers to unlock the therapeutic potential of LN-resident cells as drug delivery targets.

This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these proteins in functions of the healthy lymphatic system. We describe their influence on the maintenance of extracellular fluid homeostasis, immune cell trafficking to draining lymph nodes and dietary nutrient absorption by intestinal villi. Moreover, new insights into connexin mutations in primary and secondary lymphedema as well as on the implication of lymphatic connexins and pannexins in acquired cardiovascular diseases are discussed, allowing for a better understanding of the role of these proteins in pathologies linked to dysfunctions in the lymphatic system.

The lymphatic vasculature is an integral component of the cardiovascular system. It is essential to maintain tissue fluid homeostasis, direct immune cell trafficking and absorb dietary lipids from the digestive tract. Major advances in our understanding of the genetic and cellular events important for constructing the lymphatic vasculature during development have recently been made. These include the identification of novel sources of lymphatic endothelial progenitor cells, the recognition of lymphatic endothelial cell specialisation and heterogeneity, and discovery of novel genes and signalling pathways underpinning developmental lymphangiogenesis. Here, we review these advances and discuss how they inform our understanding of lymphatic network formation, function and dysfunction.

BACKGROUND: The anatomy of the lymphatic vessels in the extremities is not completely understood. The aim of this study was to elucidate the patterns of the lymphatic pathways of each lymphosome in lymphedematous legs. METHODS: We performed a retrospective study on 630 lymphosomes from 105 patients with leg lymphedema. The mean age of the subjects was 58.9 (range: 20–91) years, and the mean duration of lymphedema was 8.8 (range: 1–91) years. In indocyanine green (ICG) lymphography, we injected ICG into the multi-lymphosome: the first web space of the foot (saphenous lymphosome), lateral ankle (lateral calf lymphosome), and lateral knee (lateral thigh lymphosome). We established the saphenous, calf, and thigh (SCaT) classification based on the lymphatic location: lymphatic vessels on the medial side (type 1) and lymphatic vessels in other locations (type 2). RESULTS: In the saphenous lymphosome, 157 lymphatics (95.5%) were type 1. In the lateral calf lymphosome, 164 lymphatics (29.9%) were type 1. In the lateral thigh lymphosome, 148 lymphatics (16.9%) were type 1. The percentage of type 2 lymphatic vessels increased as the lymphoscintigraphic staging progressed. CONCLUSIONS: The lymphatic vessels in the lymphedematous legs shifted from the medial to the lateral side and finally disappeared in all lymphosomes as lymphedema worsened. We propose the SCaT classification to describe the condition of the lymphatic vessels in each lymphosome with the hope that it becomes a common staging system for sharing information on lymphedema severity among interdisciplinary medical professionals.

SIGNIFICANCE: Secondary lymphedema is a debilitating disease caused by lymphatic dysfunction characterized by chronic swelling, dysregulated inflammation, disfigurement, and compromised wound healing. Since there is no effective cure, animal model systems that support basic science research into the mechanisms of secondary lymphedema are critical to advancing the field. Recent Advances: Over the last decade, lymphatic research has led to the improvement of existing animal lymphedema models and the establishment of new models. Although an ideal model does not exist, it is important to consider the strengths and limitations of currently available options. In a systematic review adhering to the PRISMA guidelines, we present recent developments in the field of animal lymphedema models and provide a concise comparison of ease, cost, reliability, and clinical translatability. CRITICAL ISSUES: The incidence of secondary lymphedema is increasing, and there is no gold standard of treatment or cure for secondary lymphedema. FUTURE DIRECTIONS: As we iterate and create animal models that more closely characterize human lymphedema, we can achieve a deeper understanding of the pathophysiology and potentially develop effective therapeutics for patients.

The formation of new vessels requires a tight synchronization between proliferation, differentiation, and sprouting. However, how these processes are differentially activated, often by neighboring endothelial cells (ECs), remains unclear. Here, we identify cell cycle progression as a regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell cycle stages, we show that venous and lymphatic precursors sprout from the cardinal vein exclusively in G1 and reveal that cell-cycle arrest is induced in these ECs by overexpression of p53 and the cyclin-dependent kinase (CDK) inhibitors p27 and p21. We further demonstrate that, in vivo, forcing G1 cell-cycle arrest results in enhanced vascular sprouting. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as a direct inducer of cell-cycle arrest in ECs and characterize the cascade of events that render “sprouting-competent” ECs. Overall, our results uncover a mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell cycle inhibitors in pathological angiogenesis and lymphangiogenesis.

Imaging of the lymphatic vasculature has gained great attention in various fields, not only because the traditional role of lymphatic vessels as a draining system in the body, but also for their implication in autoimmune diseases, organ transplant, inflammation and cancer. Thus, neolymphangiogenesis, or the generation of new lymphatics is typically an early event in the development of multiple tumor types, particularly in aggressive ones such as malignant melanoma. Still, the understanding of how lymphatic endothelial cells get activated at distal (pre)metastatic niches and their implication in therapy is still unclear. Addressing these questions is of particular interest in the case of immune modulators because endothelial cells may favor or halt inflammatory processes depending on the cellular context. Therefore, there is great interest in visualizing the lymphatic vasculature in vivo. Here, we review imaging tools and mouse models used to analyze the lymphatic vasculature during tumor progression. We also discuss therapeutic approaches based on nanomedicines to target the lymphatic system and the potential use of extracellular vesicles to track and target sentinel lymph nodes. Finally, we summarize main pre-clinical models developed to visualize the lymphatic vasculature in vivo, discussing their applications with a particular focus in metastatic melanoma.

Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.

OBJECTIVE: In the current study, we compared the distribution of blood and lymphatic vessels from paraffin-embedded tissues with those of frozen tissues of normal human and rhesus monkey. MATERIALS AND METHODS: We performed immunocytochemical staining for lymphatic and blood vessels using LYVE-1 for lymphatic vessels and von Willebrand factor (F-8) for blood vessels. RESULTS: Normal tissues included spleen, lymph node, liver, pancreas, salivary gland, colon, diaphragm, heart, lung, thyroid, adrenal gland, kidney, ovary, endometrium, and prostate. Splenic sinusoids were stained for LYVE-1 and F-8 in the frozen sections, supporting that the sinusoid is a lymphoreticular system and blood vessel in structure and function. In frozen sections, the lymphatic sinusoids were consistently positive for LYVE-1, while hepatic sinusoids were positive for LYVE-1, but not for F-8. Thus, lymphatic and blood vessels were more readily detected in frozen tissue sections than in the paraffin-embedded sections. In the endometrium, lymphatic vessels were not diffusely immunostained in paraffin-embedded sections. However, frozen sections detected cyclic changes of lymphatic vessels, growing from basalis to functionalis in the menstrual cycle. Lymphatic vessels were immunostained in many organs using frozen sections. Small pulmonary blood vessels were not immunostained by F-8 in the periphery of the bronchial vessel tree most likely these smallest blood vessels were not immunostained due to less F-8 attached to their endothelia. CONCLUSION: The present findings illustrate the differences in the immunostaining of blood vessels in sections obtained from paraffin-embedded tissues and those from frozen tissue. These new findings may be relevant for the basic histology and histopathology of lymphatic and blood vessels.

INTRODUCTION: Outcomes of surgical lymphedema treatment are currently suboptimal. Physiologic procedures including lymphovenous anastomosis (LVA) and vascularized lymph node transfer (VLNT) reestablish lymphatic flow but cannot correct fibroadipose deposition, whereas liposuction alone cannot prevent disease progression. We propose a treatment algorithm combining liposuction with LVA or VLNT that can achieve normal limb volumes and prevent disease progression in stage II-III lymphedema. METHODS: We performed a retrospective chart review of patients undergoing liposuction and physiologic lymphedema operations at our institution between January 2016 and June 2019. Patients were assigned to treatment groups according to their clinical presentation: physiologic first, followed by no further treatment (physiologic only) or liposuction (physiologic then liposuction); liposuction then physiologic; or simultaneous. Preoperative patient characteristics and sequence of operations were recorded. Compression garment usage was self-reported. Limb volumes were approximated as a truncated cone. RESULTS: Twenty-one patients met the inclusion criteria. The liposuction then physiologic group had significantly higher stage and excess limb volume at baseline, whereas the physiologic first groups had lower excess volume. While the physiologic only group had predominantly stage I disease, the patients who later required liposuction (physiologic then liposuction group) all had stage II disease. All groups achieved 82% to 106% mean excess volume reduction, and volume reduction was maintained for up to 2.4 years. Compression garment class was not reduced, but mean postoperative compression duration decreased from 12.5 to 7.5 h/d (P = 0.003). Ten of 11 patients with history of cellulitis had no further recurrence. CONCLUSION: Lymphedema represents a continuum of fluid and fibroadipose disease. Accurate staging and timely treatment with physiologic procedures and liposuction can normalize limb volume. We propose a treatment algorithm to optimize outcomes. Patients with predominantly nonpitting presentation benefit from liposuction to maximize removal of fibroadipose tissue and optimize postoperative compression, followed by LVA or VLNT to improve lymphatic drainage. Patients with primarily pitting edema are best treated with physiologic procedures initially, reserving selective liposuction as a second stage. Patients with mixed presentation are best served by single-stage combination procedures.

BACKGROUND: Lymphangiomatosis is a multisystem disorder that is rarely localized to the gastrointestinal tract. Lymphangiomatosis usually has no specific clinical presentation and is easily misdiagnosed. A case report and review of the literature on lymphangiomatosis associated with protein-losing enteropathy will help to improve the overall understanding of this disease. CASE SUMMARY: We report a case of lymphangiomatosis of the bowel and other solid organs. A 78-year-old man presented with recurrent bowel bleeding and protein-losing enteropathy, as well as cystic lesions in the spleen, liver, and kidney. Imaging examinations revealed many cystic lesions on the spleen, liver, kidney, and thickened wall of the ascending colon, as well as pleural effusion and ascites. Colonoscopy revealed a strawberry mucosa, variable spontaneous bleeding, and surface erosion located in the terminal ileum. Several cystic masses with a translucent and smooth surface as well as diffuse white spots were located in the colon. A laterally spreading tumor (LST) was located in the ascending colon. Pathology indicated highly differentiated adenocarcinoma (LST) and lymphangiomatoid dilation, and D2-40 was positive. The final diagnosis was lymphangiomatosis. The patient underwent surgery for LST and then was administered thalidomide 75–150 mg/d. His condition, however, did not improve. He eventually died 6 mo after the initial diagnosis. CONCLUSION: Lymphangiomatosis usually occurs diffusely and can involve many organs, such as the spleen, kidney, liver, lung, mesentery, and bowel. Recurrent bowel bleeding or protein-losing enteropathy is an important indicator that should alert clinicians about the possibility of this disease when it afflicts the bowel. Doctors should improve the medical understanding of lymphangiomatosis.

The mammalian vascular system consists of two networks: the blood vascular system and the lymphatic vascular system. Throughout the body, the lymphatic system contributes to homeostatic mechanisms by draining extravasated interstitial fluid and facilitating the trafficking and activation of immune cells. In the kidney, lymphatic vessels exist mainly in the kidney cortex. In the medulla, the ascending vasa recta represent a hybrid lymphatic-like vessel that performs lymphatic-like roles in interstitial fluid reabsorption. Although the lymphatic network is mainly derived from the venous system, evidence supports the existence of lymphatic beds that are of non-venous origin. Following their development and maturation, lymphatic vessel density remains relatively stable; however, these vessels undergo dynamic functional changes to meet tissue demands. Additionally, new lymphatic growth, or lymphangiogenesis, can be induced by pathological conditions such as tissue injury, interstitial fluid overload, hyperglycaemia and inflammation. Lymphangiogenesis is also associated with conditions such as polycystic kidney disease, hypertension, ultrafiltration failure and transplant rejection. Although lymphangiogenesis has protective functions in clearing accumulated fluid and immune cells, the kidney lymphatics may also propagate an inflammatory feedback loop, exacerbating inflammation and fibrosis. Greater understanding of lymphatic biology, including the developmental origin and function of the lymphatics and their response to pathogenic stimuli, may aid the development of new therapeutic agents that target the lymphatic system.

BACKGROUND: Secondary lymphedema after surgical interventions is a progressive, chronic disease that is still not completely curable. Over the past years, a multitude of surgical therapy options have been described. AIM: To summarize the single-center complications in lymph vessel (LVTx) and free vascularized lymph node transfer (VLNT). METHODS: In total, the patient collective consisted of 87 patients who were undergoing treatment for secondary leg lymphedema during the study period from March 2010 to April 2020. The data collection was performed preoperatively during consultations, as well as three weeks, six months and twelve months after surgical treatment. In the event of complications, more detailed follow-up checks were carried out. In total n = 18 robot-assisted omental lymph node transplantations, n = 33 supraclavicular lymph node transplantations and n = 36 Lymph vessel transplantations were analyzed. An exemplary drawing is shown in Figure 1. A graphical representation of patient selection is shown in Figure 2. Robotic harvest was performed with the Da Vinci Xi Robot Systems (Intuitive Surgical, CA, United States). RESULTS: In total, 11 male and 76 female patients were operated on. The mean age of the patients at study entry was: omental VLNT: 57.45 +/- 8.02 years; supraclavicular VLNT: 49.76 +/- 4.16 years and LVTx: 49.75 +/- 4.95 years. The average observation time postoperative was: omental VLNT: 18 +/- 3.48 mo; supraclavicular VLNT: 14.15 +/- 4.9 and LVTx: 14.84 +/- 4.46 mo. In our omental VLNT, three patients showed a slight abdominal sensation of tension within the first 12 postoperative days. No other donor side morbidities occurred. No intraoperative conversion to open technique was needed. Our supraclavicular VLNT collective showed 10 lift defect morbidities with one necessary surgical intervention. In our LVTx collective, 12 cases of donor side morbidity were registered. In one case, surgical intervention was necessary. CONCLUSION: Concerning donor side morbidity, robot-assisted omental VLNT is clearly superior to supraclavicular lymph node transplantation and LVTx.

BACKGROUND: Microsurgical transplantation of vascularized lymph nodes (VLNT) or lymphatic vessels (VLVT) alongside derivative lymphaticovenous procedures are promising approaches for treatment of lymphedema. However, clinically relevant training models for mastering these techniques are still lacking. Here we describe a new training model in human cadaver and validate its use as training tool for microsurgical lymphatic reconstruction. METHODS: 10 surgeons with previous exposure to microsurgery were trained in a controlled environment. Lymphatic vessel mapping and dissection in 4 relevant body regions, harvesting of five different VLNTs and one VLVT were performed in 5 fresh-frozen cadavers. The number of lymphatic vessels and lymph nodes for each VLNT were recorded. Finally, the efficacy of this model as training tool was validated using the Dundee Ready Education Environment Measure (DREEM). RESULTS: The average cumulative DREEM score over each category was 30,75 (max = 40) while individual scoring for each relevant category revealed highly positive ratings from the perspective of teaching (39,3), training 40,5 (max = 48) and self perception of the training 30,5 (max = 32) from all participants. The groin revealed the highest number of lymphatic vessels (3.2 +/- 0.29) as all other regions on the upper extremity, while the gastroepiploic VLNT had the highest number of lymph nodes (4.2 +/- 0.37). CONCLUSIONS: This human cadaver model represents a new, reproducible “all-in-one” tool for effective training in lymphatic microsurgery. Its unique diligence in accurately reproducing human lymphatic anatomy, should make this model worth considering for each microsurgeon willing to approach lymphatic reconstruction.

Lymphatic filariasis causes disfiguring and disabling lymphoedema, which is commonly and frequently exacerbated by acute dermatolymphangioadenitis (ADLA). Affected people require long-term care and monitoring but health workers lack objective assessment tools. We examine the use of an infrared thermal imaging camera as a novel non-invasive point-of-care tool for filarial lower-limb lymphoedema in 153 affected adults from a highly endemic area of Bangladesh. Temperature differences by lymphoedema stage (mild, moderate, severe) and ADLA history were visualised and quantified using descriptive statistics and regression models. Temperatures were found to increase by severity and captured subclinical differences between no lymphoedema and mild lymphoedema, and differences between moderate and severe stages. Toes and ankle temperatures detected significant differences between all stages other than between mild and moderate stages. Significantly higher temperatures, best captured by heel and calf measures, were found in participants with a history of ADLA, compared to participants who never had ADLA, regardless of the lymphoedema stage. This novel tool has great potential to be used by health workers to detect subclinical cases, predict progression of disease and ADLA status, and monitor pathological tissue changes and stage severity following enhanced care packages or other interventions in people affected by lymphoedema.

BACKGROUND: Cancer-related lymphedema will affect 10% to 50% of breast cancer survivors. Early data show that immediate lymphatic reconstruction may help prevent breast cancer lymphedema; however, the details have not been fully elucidated. The purpose of this study was to evaluate the cohort of our first 100 patients for trends in demographics, treatment, and technique. METHODS: At a tertiary care cancer center, high-risk breast cancer-related lymphedema patients underwent axillary reverse lymphatic mapping and immediate lymphatic reconstruction. After institutional review board approval, demographics, technique, and outcomes were recorded. The first 100 patients were analyzed to compare the differences between the first 50 versus the second 50 patient cohorts. RESULTS: Of the first 100 axillary reverse lymphatic mapping performed, there was a significant difference in neoadjuvant chemotherapy with 81% in the earlier cohort versus 98% in the later cohort (P = 0.01). An arborized technique was used more frequently in the second cohort (82% vs 54%, P = 0.01). The incidence of lymphedema was lower in the latter cohort (7 patients vs 1 patient, P = 0.03). The first cohort was 12.2 times more likely to develop lymphedema despite lymphatic reconstruction than the second cohort (P = 0.03). CONCLUSIONS: The data demonstrate multiple trends in the learning curve associated with immediate lymphatic reconstruction at a single institution including improvements in identifying and dissecting lymphatic structures, performing more anastomoses per patient, using the arborized technique more frequently, performing the operation with shorter operative times, and reducing the incidence of lymphedema.

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and alpha5beta1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)-ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin alpha5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

Background: This study evaluates whether dynamic contrast-enhanced magnetic resonance lymphangiography (DCMRL) and thoracic duct lymphangiography (TDL) findings in adults with lymphatic plastic bronchitis (PB) and/or nontraumatic chylothorax (NTC) support a shared pathophysiology. Materials and Results: Retrospective review of clinical and imaging findings in patients who underwent DCMRL and TDL at a single institution from March 2017 to March 2019. Categorical variables were compared with Fisher's exact test. Twenty-eight patients (median age 61 +/- 21 years, 15 women) presenting with lymphatic PB (n = 13), NTC (n = 10), or both (n = 5) were included. Lymphatic imaging demonstrated pulmonary lymphatic perfusion (PLP) in all patients. A patent thoracic duct (TD) with retrograde flow was seen in 53.4% (7/13) of patients with PB, 60% (6/10) of patients with NTC, and 20% (1/5) of patients with both (p = 0.69). An occluded TD with retrograde flow was seen in 30.8% (4/13) of patients with PB, 30% (3/10) of patients with NTC, and 80% (4/5) of patients with both (p = 0.12). Similar patterns of PLP between DCMRL and TDL were seen in 96.2% (25/26) of patients. Conclusions: DCMRL and TDL demonstrated similar findings in patients with lymphatic PB and/or NTC, supporting a common etiology. This supports the hypothesis that the clinical presentation depends on the proximity of abnormal lymphatic vessels to the pleural cavity, resulting in chylothorax, or bronchial mucosa, resulting in PB.

BACKGROUND: Lymphaticovenular anastomosis (LVA) is a minimally invasive surgical procedure used to treat lymphedema. Volumetric measurements and quality-of-life assessments are often performed to assess the effectiveness of LVA, but there is no method that provides information regarding postoperative morphological changes in lymphatic vessels and veins after LVA. Photoacoustic lymphangiography (PAL) is an optical imaging technique that visualizes the distribution of light-absorbing molecules, such as hemoglobin or indocyanine green (ICG), and provides three-dimensional images of superficial lymphatic vessels and the venous system simultaneously. In this study, we performed PAL in lymphedema patients before and after LVA and compared the images to evaluate the effect of LVA. METHODS: PAL was performed using the PAI-05 system in three patients (one man, two women) with lymphedema, including one primary case and two secondary cases, before LVA. ICG fluorescence lymphography was performed in all cases before PAL. Follow-up PAL was performed between 5 days and 5 months after LVA. RESULTS: PAL enabled the simultaneous visualization of clear lymphatic vessels that could not be accurately seen with ICG fluorescence lymphography and veins. We were also able to observe and analyze morphological changes such as the width and the number of lymphatic vessels and veins during the follow-up PAL after LVA. CONCLUSIONS: By comparing preoperative and postoperative PAL images, it was possible to analyze the morphological changes in lymphatic vessels and veins that occurred after LVA. Our study suggests that PAL would be useful when assessing the effect of LVA surgery.

Perioperative neurocognitive disorder (PND) frequently occurs in the elderly as a severe postoperative complication and is characterized by a decline in cognitive function that impairs memory, attention, and other cognitive domains. Currently, the exact pathogenic mechanism of PND is multifaceted and remains unclear. The glymphatic system is a newly discovered glial-dependent perivascular network that subserves a pseudo-lymphatic function in the brain. Recent studies have highlighted the significant role of the glymphatic system in the removal of harmful metabolites in the brain. Dysfunction of the glymphatic system can reduce metabolic waste removal, leading to neuroinflammation and neurological disorders. We speculate that there is a causal relationship between the glymphatic system and symptomatic progression in PND. This paper reviews the current literature on the glymphatic system and some perioperative factors to discuss the role of the glymphatic system in PND.

Lymphedema is a common, complex, and inexplicably underappreciated human disease. Despite a history of relative neglect by health care providers and by governmental health care agencies, the last decade has seen an explosive growth of insights into, and approaches to, the problem of human lymphedema. The current review highlights the significant advances that have occurred in the investigative and clinical approaches to lymphedema, particularly over the last decade. This review summarizes the progress that has been attained in the realms of genetics, lymphatic imaging, and lymphatic surgery. Newer molecular insights are explored, along with their relationship to future molecular therapeutics. Growing insights into the relationships among lymphedema, obesity, and other comorbidities are important to consider in current and future responses to patients with lymphedema.

Lower extremity swelling is a common condition which has a variety of etiologies and can be challenging to diagnose and manage. Swelling is usually the result of the accumulation of interstitial fluid in the subcutaneous tissues. Common etiologies include systemic, superficial, and deep venous, and lymphatic disorders. Leg swelling can occur bilaterally or unilaterally, with venous disorders being one of the most common causes of unilateral lower extremity edema.

OBJECTIVES: To characterize hepatic to systemic lymphatic connections in patients with systemic lymphatic disease using intra-hepatic lymphangiography and to compare outcomes after lymphatic intervention. METHODS: In this retrospective study, patients with intra-hepatic lymphangiography from May 2014 - April 2019 at our institution were included. Imaging review was performed and hepatic lymphatic connections and flow patterns were characterized. Clinical data were reviewed and comparisons between patients undergoing lymphatic intervention with or without abnormal hepatic lymphatics were performed. RESULTS: During the study period, 105 patients underwent intra-hepatic lymphangiography. Primary clinical presentation included ascites (19/105), chylothorax (27/105), plastic bronchitis (PB) (17/105), and protein losing enteropathy (PLE) (42/105). Five categories of hepatic lymphatic connections and flow patterns were identified (%): normal (25%, 26/105), hepatoperitoneal (12%, 13/105), hepatopulmonary (10.5%, 11/105), hepatomesenteric (7.5%, 8/105), and hepatoduodenal (41%, 43/105) with four patients having more than one abnormal pattern. A comparison between clinical presentation and imaging category revealed an increased likelihood of having ascites with hepatoperitoneal (p < .0001), chylothorax/PB with hepatopulmonary (p = .01), and PLE with hepatoduodenal (p < .001) connections. Seventy-six patients had a lymphatic intervention, 24% with normal, and 76% with abnormal liver lymphatics. There was no difference in length of hospital stay or mortality between the two groups, but there was a prolonged time to symptom resolution (p = .006) and persistent symptoms after 6 months (5% vs 44%, p = .002) in the group with abnormal liver lymphatics. CONCLUSION: We identified five liver lymphatic imaging categories with a substantial correlation to presenting lymphatic disease. Abnormal imaging patterns correlated with increased morbidity. Evaluation of liver lymphatics should be considered in patients with a systemic lymphatic disease if central lymphatic imaging is normal. KEY POINTS: * We identified five liver lymphatic imaging patterns: normal, hepatoperitoneal, hepatomesenteric, hepatopulmonary, and hepatoduodenal. * Imaging patterns were correlated with disease presentation (normal - chylothorax/PB, hepatoperitoneal - ascites/chylothorax, hepatopulmonary - chylothorax/PB, hepatoduodenal - PLE). * Abnormal imaging patterns correlated with increased morbidity.

OBJECTIVE: Aging is one of the causes of primary lymphedema. However, the effects of aging on the lymphatic system are still not completely understood. We investigated the effects of aging on the lymphatic vessels in the lower extremities of healthy volunteers using photoacoustic imaging. METHODS: Healthy volunteers who underwent photoacoustic lymphangiography between March 2018 and January 2019 were enrolled. To visualize lymphatics, indocyanine green (ICG, 5.0 mg/mL) was injected subcutaneously into the first and fourth web spaces of the foot and under the lateral malleolus. Subsequently, near-infrared fluorescence lymphography was performed to confirm good ICG flow, and photoacoustic lymphangiography was performed on the medial side of the lower leg. Neodymium-doped yttrium aluminum garnet laser irradiation at 797 and 835 nm, the optimal wavelengths for visualizing ICG and blood, was applied. The number of lymphatic vessels shown at areas 10 cm (L10) and 20 cm (L20) cranially from the internal malleolus was counted. RESULTS: Nineteen healthy volunteers (4 males and 15 females) were enrolled in the study. Their mean age was 42.9 +/- 12.8 years. One volunteer was bilaterally imaged; 15 left lower limbs and 5 right lower limbs were imaged. The number of lymphatic vessels visualized increased with age. There were strong positive correlations between age and L10 (R = 0.729, P < .001) and between age and L20 (R = 0.570, P = .009). CONCLUSIONS: Photoacoustic imaging indicates that the number of lymphatic vessels increases with age. Lymphatic stasis resulted in visualization of not only normal drainage pathways but also nonfunctional lymphatic pathways.

Yellow nail syndrome (YNS) is a rare disease comprising the clinical triad of yellow nail discoloration, pleural effusion, and lower limb lymphedema. We encountered a difficult-to-treat case of YNS in which the diagnosis was finally made based on intranodal lymphangiography. An 84-year-old man was admitted to our hospital with pleural effusion and yellow-green discoloration of the nails, accompanied by onychomycosis and limb lymphedema. Intranodal lymphangiography revealed a slow contrast flow and narrowing of the thoracic duct, suggesting lymphatic duct dysplasia and leading to the diagnosis of YNS.

BACKGROUND: Limb lymphedema is a chronic disease with primary and secondary forms, with the latter occurring essentially after cancer treatment. In the present study, we retrospectively analyzed the cellulitis frequency and its associated risk factors for patients with primary or secondary limb lymphedema. METHODS: Information from all 1991 patients referred to a specialized lymphedema center from January to June 2018 was collected, including previous cellulitis episodes and the clinical and lymphedema characteristics. RESULTS: Of the 1846 patients whose information could be analyzed, 695 (37.6%) had experienced one or more cellulitis episodes, and 23.3% had had recurrent cellulitis. Cellulitis occurred in 39.5%, 30.5%, and 38.6% (P = .02) of the patients with secondary upper limb, secondary lower limb, and primary lower limb lymphedema, respectively. The corresponding duration of lymphedema was 106.5, 97, and 243.1 months. For secondary upper limb lymphedema, a long interval from lymphedema onset to the first consultation at our specialized center, younger age at lymphedema onset, and the use of radiotherapy were independently associated with cellulitis. However, axillary lymph node excision, the use of chemotherapy, and segmentation of the upper limb lymphedema were not associated with cellulitis. A longer interval from lymphedema onset to the first consultation and lymph node excision were associated with cellulitis in those with lower limb secondary lymphedema but higher body mass index and younger age at lymphedema onset were not. For primary lower limb lymphedema, male sex, greater body mass index, and younger age at lymphedema onset were associated with cellulitis. CONCLUSIONS: Different risk factors for cellulitis were identified for patients with lymphedema at risk. Awareness of those factors is important for physicians to recognize lymphedema promptly and refer patients to specialized centers to optimize treatment.

BACKGROUND: Lymphoedema after cancer treatment is a chronic and disabling complication that presents a significant health care burden during survivorship with limited treatment options. Vascularised lymph node transfer (VLNT) can reconstruct lymphatic flow to reduce limb volumes, but limited higher-order evidence exists to support its effectiveness. AIM: The aim of the study was to systematically review and meta-analyse the effectiveness of VLNT in reducing upper limb (UL) or lower limb (LL) volume and cellulitis episodes in patients with cancer treatment-related lymphoedema (CTRL). METHODS: PubMed, Medline (Ovid) and Embase databases were searched between January 1974 and December 2019. Full-length articles where VLNT was the sole therapeutic procedure for CTRL, reporting volumetric limb, frequency of infection episodes and/or lymphoedema-specific quality-of-life data, were included in a random-effects meta-analysis of circumferential reduction rate (CRR). Methodological quality was assessed using STROBE/CONSORT, and a novel, lymphoedema-specific scoring tool was used to assess lymphoedema-specific methodological reporting. Sensitivity analyses on the site of VLNT harvest and recipient location were performed. RESULTS: Thirty-one studies (581 patients) were eligible for inclusion. VLNT led to significant limb volume reductions in UL (above elbow pooled CRRs [CRRP] = 42.7% [95% confidence interval (CI): 36.5–48.8]; below elbow CRRP = 34.1% [95% CI: 33.0–35.1]) and LL (above knee CRRP = 46.8% [95% CI: 43.2–50.4]; below knee CRRP = 54.6% [95% CI: 39.0–70.2]) CTRL. VLNT flaps from extra-abdominal donor sites were associated with greater volume reductions (CRRP = 49.5% [95% CI: 46.5–52.5]) than those from intra-abdominal donor sites (CRRP = 39.6% [95% CI: 37.2–42.0]) and synchronous autologous breast reconstruction/VLNT flaps (CRRP = 32.7% [95% CI: 11.1–54.4]) (p < 0.05). VLNT was also found to reduce the mean number of cellulitis episodes by 2.1 episodes per year (95% CI: -2.7- -1.4) and increased lymphoedema-specific quality-of-life scores (mean difference in Lymphoedema-Specific Quality of Life (LYMQOL) “overall domain” = +4.26). CONCLUSIONS: VLNT is effective in reducing excess limb volume and cellulitis episodes in both UL and LL lymphoedema after cancer treatment. However, significant heterogeneity exists in outcome reporting, and standardisation of reporting processes is recommended.

INTRODUCTION: 22q13.3 deletion syndrome is a well-known syndrome characterized by typical clinical findings including neonatal hypotonia, absent or severely delayed speech, intellectual disability, and other various features, and detection of a heterozygous deletion of chromosome 22q13.3 with the involvement of at least part of SHANK3. It is reported that 10% to 29% of patients with 22q13.3 deletion syndrome present lymphedema. Protein-losing enteropathy (PLE) has never been reported in 22q13.3 deletion syndrome. PATIENT CONCERNS: The patient presented to our institution for refractory hypoalbuminemia and chronic lymphedema in both legs. DIAGNOSIS: The patient manifested intellectual disability, absent speech, tooth grinding, dysmorphic face, and abnormal hands and toenails. Copy-number variation sequencing confirmed the maternal deletion in 22q13.31-q13.33 (chr22:46285592-51244566, hg19). The patient was genetically diagnosed with 22q13.3 deletion syndrome. INTERVENTIONS: Low-fat diets and medium-chain triglycerides supplements were prescribed. The patient was recommended to wear compression garments and elevate legs. OUTCOMES: The symptom of diarrhea was resolved, but hypoalbuminemia persisted. Lower extremities lymphedema was gradually becoming severe. CONCLUSIONS: Primary lymphedema and PLE can occur simultaneously in a patient with 22q13.3 deletion syndrome. The 2 phenotypes could share the same genetic etiology of congenital lymphatic abnormalities. CELSR1 deletion may play a role in lymphatic dysplasia. The case also provides additional proof of the pathogenic effect of CELSR1 on hereditary lymphedema.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is a devastating complication of breast cancer (BC) treatment. The authors hypothesized that identifying subclinical lymphedema (SCL) presents an opportunity to prevent BCRL development. They aimed to assess rates of SCL progression (relative volume change [RVC], 5–10%) to BCRL (RVC, >/ = 10%) in women undergoing axillary surgery for BC via axillary lymph node dissection (ALND) or sentinel lymph node biopsy (SLNB). METHODS: Patients treated for BC were prospectively screened at preoperative baseline and throughout the follow-up period using the perometer. The cohort was stratified according to nodal surgery (ALND or SLNB) to analyze rates of progression to BCRL. RESULTS: The study cohort included 1790 patients. Of the 1359 patients who underwent SLNB, 331 (24.4%) experienced SCL, with 38 (11.5%) of these patients progressing to BCRL. Of the 431 patients who underwent ALND, 171 (39.7%) experienced SCL, with 67 (39.2%) of these patients progressing to BCRL. Relative to the patients without SCL, those more likely to experience BCRL were the ALND patients with early SCL (< 3 months postoperatively; hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.58–4.27; p = 0.0002) or late SCL (>/ = 3 months postoperatively; HR, 3.14; 95% CI, 1.95–5.05; p < 0.0001) and the SLNB patients with early SCL (HR, 6.75; 95% CI, 3.8–11.98; p < 0.0001 or late SCL (HR, 3.02; 95% CI, 1.65–5.50; p = 0.0003). CONCLUSION: The study suggests that patients with SCL after axillary nodal surgery for BC are more likely to progress to BCRL than those who do not experience SCL. This presents a tremendous opportunity for early intervention to prevent BCRL and improve the quality of life for women treated for BC.

Background: As lymphatic vessel is a major route for solid tumor metastasis, they are considered an essential part of tumor drainage conduits. Apart from forming the walls of lymphatic vessels, lymphatic endothelial cells (LECs) have been found to play multiple other roles in the tumor microenvironment, calling for a more in-depth review. We hope that this review may help researchers gain a detailed understanding of this fast-developing field and shed some light upon future research. Methods: To achieve an informative review of recent advance, we carefully searched the Medline database for English literature that are openly published from the January 1995 to December 2020 and covered the topic of LEC or lymphangiogenesis in tumor progression and therapies. Two different authors independently examined the literature abstracts to exclude possible unqualified ones, and 310 papers with full texts were finally retrieved. Results: In this paper, we discussed the structural and molecular basis of tumor-associated LECs, together with their roles in tumor metastasis and drug therapy. We then focused on their impacts on tumor cells, tumor stroma, and anti-tumor immunity, and the molecular and cellular mechanisms involved. Special emphasis on lung cancer and possible therapeutic targets based on LECs were also discussed. Conclusions: LECs can play a much more complex role than simply forming conduits for tumor cell dissemination. Therapies targeting tumor-associated lymphatics for lung cancer and other tumors are promising, but more research is needed to clarify the mechanisms involved.

RATIONALE:: Despite significant advances in microsurgical techniques, simultaneous vascularized lymph node transfer (VLNT) and lymphovenous anastomosis (LVA) surgeries may be effective for treatment of end-stage lymphedema. This case report describes the successful treatment of end-stage lymphedema with VLNT and LVA. PATIENT CONCERNS: A 72-year-old patient with bilateral lower extremity lymphedema was referred to our lymphedema clinic. This patient had a history of lymphoma and treated with radiotherapy on right inguinal area 26 years ago. Interestingly, the patient developed lymphedema on both the right and left lower extremities although she had radiotherapy on her right inguinal area. DIAGNOSIS: According to the indocyanine green lymphography, lymphoscintigraphy, and magnetic resonance lymphangiography, the patient was diagnosed with end-stage lymphedema (International Society of Lymphology stage 3). INTERVENTION: The patient underwent simultaneous VLNT and LVA for treatment of end-stage lymphedema. OUTCOMES: Significant reduction in circumference and volume of lower extremity was achieved following simultaneous VLNT and LVA. LESSONS: Simultaneous VLNT and LVA surgeries may be effective in patients with end-stage lymphedema.

Introduction: Of the 1.38 million women who are diagnosed every year with breast cancer worldwide, 21% will develop arm lymphedema. Near-infrared fluorescence lymphatic imaging (NIRFLI) is an effective method for real-time evaluation of the lymphatic system. Reliability studies of the scoring of this NIRFLI are lacking. The aim of this study is to investigate if evaluation of the superficial lymphatic architecture and transport in patients with breast cancer-related lymphedema through NIRFLI can be performed in a reliable way. Methods and Results: The outcome parameters used to assess agreement were the presence of lymphatic transport from the injection sites, of dermal backflow patterns, of efferent lymphatic vessels, and of lymph nodes. The NIRFLI evaluations were scored before and after a break separately by two assessors. Twenty patients with lymphedema of the arm and/or hand were included. After the injection, there was weak to very strong agreement regarding the presence of transport from the injection sites (K = 0.459–1.000). The interpretation of the type of dermal backflow pattern varied from weak (WK = 0.452) to very strong agreement (WK = 1.000) between the two assessors. Agreement in the visualization of efferent lymphatic vessels was weak before and after the break (K = 0.490 and K = 0.571) and agreement regarding the presence of lymph nodes was very strong (K = 1.000). Conclusion: Overall, there was moderate to strong agreement between the assessors when evaluating the lymphatic architecture and transport through NIRFLI. The study has been registered at clinicaltrials.gov (NCT02609724).

Background: Gynecological cancer-related lymphedema (GCRL) is a devastating condition that adversely influences function, health, and quality of life. We conducted a randomized-controlled clinical study as well as in vitro experiments to investigate the efficacy and safety of far infrared radiation (FIR) to treat lymphedema in patients having previously undergone surgery for gynecological tumors. Materials and Methods: Seventy-four women with GCRL, cancer free for 5 years or more, were randomly allocated into two treatment groups: standard of care with bandage treatment and treatment with FIR plus bandage. Variations of fluid, circumference of lymphedematous limbs, serum tumor markers (cancer antigen 125 [CA125]), inguinal-pelvic lymph nodes, vagina, lungs, and adverse reactions were assessed after 1 year. In vitro experiments examined the effects on cell viability, proliferation, apoptosis, and the cell cycle of fibroblast, A2780, SKOV-3, HELA, and Ishikawa cells. Results: The FIR+bandage group showed significantly decreased tissue fluid and reduced limb circumference (p < 0.05) in comparison with the control group at 1 year. There was no increase of serum CA125 in both groups, and no recurrence of neoplasia or lymphadenopathy was detected. No adverse reactions were recorded. In addition, no changes were detected after FIR treatment for fibroblast, A2780, SKOV-3, HELA, and Ishikawa cells in cell viability, proliferation, apoptosis, and cell cycle. Conclusion: FIR can be used to treat patients with GCRL following gynecological cancer treatment. Following clinical and experimental studies, we confirm that FIR is an oncologically safe treatment for lymphedema in gynecological tumor patients.

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.

Mosaic KRAS variants and other RASopathy genes cause oculoectodermal, encephalo-cranio-cutaneous lipomatosis, and Schimmelpenning-Feuerstein-Mims syndromes, and a spectrum of vascular malformations, overgrowth and other associated anomalies, the latter of which are only recently being characterized. We describe eight individuals in total (six unreported cases and two previously reported cases) with somatic KRAS variants and variably associated features. Given the findings of somatic overgrowth (in seven individuals) and vascular or lymphatic malformations (in eight individuals), we suggest mosaic RASopathies (mosaic KRAS variants) be considered in the differential diagnosis for individuals presenting with asymmetric overgrowth and lymphatic or vascular anomalies. We expand the association with embryonal tumors, including the third report of embryonal rhabdomyosarcoma, as well as novel findings of Wilms tumor and nephroblastomatosis in two individuals. Rare or novel findings in our series include the presence of epilepsy, polycystic kidneys, and T-cell deficiency in one individual, and multifocal lytic bone lesions in two individuals. Finally, we describe the first use of targeted therapy with a MEK inhibitor for an individual with a mosaic KRAS variant. The purposes of this report are to expand the phenotypic spectrum of mosaic KRAS-related disorders, and to propose possible mechanisms of pathogenesis, and surveillance of its associated findings.

Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatic vessels 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 GSD patient 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.

Vascular malformations, affecting approximately 1% to 1.5% of the population, comprise a spectrum of developmental patterning defects of capillaries, arteries, veins, and/or lymphatics. The majority of vascular malformations occur sporadically; however, inherited malformations exist as a part of complex congenital diseases. The malformations, ranging from birthmarks to life-threatening conditions, are present at birth, but may reveal signs and symptoms-including pain, bleeding, disfigurement, and functional defects of vital organs-in infancy, childhood, or adulthood. Vascular malformations often exhibit recurrent patterns at affected sites due to the lack of curative treatments. This review series provides a state-of-the-art assessment of vascular malformation research at basic, clinical, genetic, and translational levels.

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.

Gorham-Stout disease (GSD), a rare disorder of unknown etiology, is characterized by massive osteolysis that is associated with proliferation and dilation of lymphatic vessels. Variants in cancer-associated genes have been described in complex lymphatic anomalies. To explore the pathogenesis of GSD, we performed the amplicon-based deep sequencing on 50 cancer-related genes to assay affected tissues from the six patients with GSD. In one patient, a somatic activating KRAS c.182A > G variant (p.Q61R) was detected in 1% of the tissue sample. Conversely, the mutant allele was not detected in uninvolved normal skin and blood samples. Histopathology of the patient's tissue sample showed proliferation of abnormal lymphatic and blood vascular endothelial cells, osteoclasts, and activated macrophages. The activating KRAS variant is a known ‘hotspot’ variant, frequently identified in several types of human cancer. This is the first report of identifying a pathogenic variant in a patient with GSD. This finding may set the stage for elucidation of pathophysiology and the development of novel therapies for GSD.

Brain arteriovenous malformations (bAVM) are an important cause of intracranial hemorrhage (ICH), especially in younger patients. The pathogenesis of bAVM are largely unknown. Current understanding of bAVM etiology is based on studying genetic syndromes, animal models, and surgically resected specimens from patients. The identification of activating somatic mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene and other mitogen-activated protein kinase (MAPK) pathway genes has opened up new avenues for bAVM study, leading to a paradigm shift to search for somatic, de novo mutations in sporadic bAVMs instead of focusing on inherited genetic mutations. Through the development of new models and understanding of pathways involved in maintaining normal vascular structure and functions, promising therapeutic targets have been identified and safety and efficacy studies are underway in animal models and in patients. The goal of this paper is to provide a thorough review or current diagnostic and treatment tools, known genes and key pathways involved in bAVM pathogenesis to summarize current treatment options and potential therapeutic targets uncovered by recent discoveries.

Vascular and lymphatic malformations represent a challenge for clinicians. The identification of inherited and somatic mutations in important signaling pathways, including the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin), RAS (rat sarcoma)/RAF (rapidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinases), HGF (hepatocyte growth factor)/c-Met (hepatocyte growth factor receptor), and VEGF (vascular endothelial growth factor) A/VEGFR (vascular endothelial growth factor receptor) 2 cascades has led to the evaluation of tailored strategies with preexisting cancer drugs that interfere with these signaling pathways. The era of theranostics has started for the treatment of vascular anomalies. Registration: URL: https://www.clinicaltrialsregister.eu; Unique identifier: 2015-001703-32.

Appropriate vascular function is essential for the maintenance of central nervous system homeostasis and is achieved through virtue of the blood-brain barrier; a specialized structure consisting of endothelial, mural, and astrocytic interactions. While appropriate blood-brain barrier function is typically achieved, the central nervous system vasculature is not infallible and cerebrovascular anomalies, a collective terminology for diverse vascular lesions, are present in meningeal and cerebral vasculature supplying and draining the brain. These conditions, including aneurysmal formation and rupture, arteriovenous malformations, dural arteriovenous fistulas, and cerebral cavernous malformations, and their associated neurological sequelae, are typically managed with neurosurgical or pharmacological approaches. However, increasing evidence implicates interacting roles for inflammatory responses and disrupted central nervous system fluid flow with respect to vascular perturbations. Here, we discuss cerebrovascular anomalies from an immunologic angle and fluid flow perspective. We describe immune contributions, both common and distinct, to the formation and progression of diverse cerebrovascular anomalies. Next, we summarize how cerebrovascular anomalies precipitate diverse neurological sequelae, including seizures, hydrocephalus, and cognitive effects and possible contributions through the recently identified lymphatic and glymphatic systems. Finally, we speculate on and provide testable hypotheses for novel nonsurgical therapeutic approaches for alleviating neurological impairments arising from cerebrovascular anomalies, with a particular emphasis on the normalization of fluid flow and alleviation of inflammation through manipulations of the lymphatic and glymphatic central nervous system clearance pathways.

Oncogenic RAS variants lead to constitutive overactivation and increased signal transduction into downstream pathways. They are found as somatic driver events in various types of human cancer. In a somatic mosaic status, the same RAS variants have been associated with a wide spectrum of focal or segmental tissue dysplasia and overgrowth including various types of congenital nevi, vascular malformations, and other changes (mosaic RASopathies). We present a 3-year-old male patient with segmental overgrowth of the subcutaneous fatty tissue of the right lower extremity with colocalized arteriovenous and capillary malformations and dysplastic draining veins in combination with talipes equinovarus of the right foot. In tissue biopsies of the affected extremity, we identified a mosaic KRAS variant, c.35G>A (p.Gly12Asp), while this variant was absent in the DNA extracted from a biopsy of the normal extremity. This report provides further evidence for the wide clinical and phenotypic variability associated with mosaic KRAS variants. The described pattern confirms that the combination of segmental overgrowth and vascular anomalies in the form of arteriovenous and capillary malformations is a possible manifestation of a mosaic RASopathy. The accurate genetic diagnosis is crucial for molecular-targeted therapy, which might be a future therapeutic target for mosaic RASopathies.

Cerebral cavernous malformations are acquired vascular anomalies that constitute a common cause of central nervous system hemorrhage and stroke. The past 2 decades have seen a remarkable increase in our understanding of the pathogenesis of this vascular disease. This new knowledge spans genetic causes of sporadic and familial forms of the disease, molecular signaling changes in vascular endothelial cells that underlie the disease, unexpectedly strong environmental effects on disease pathogenesis, and drivers of disease end points such as hemorrhage. These novel insights are the integrated product of human clinical studies, human genetic studies, studies in mouse and zebrafish genetic models, and basic molecular and cellular studies. This review addresses the genetic and molecular underpinnings of cerebral cavernous malformation disease, the mechanisms that lead to lesion hemorrhage, and emerging biomarkers and therapies for clinical treatment of cerebral cavernous malformation disease. It may also serve as an example for how focused basic and clinical investigation and emerging technologies can rapidly unravel a complex disease mechanism.

Sleep problems are frequently reported in infants treated with propranolol for infantile hemangiomas, possibly serving as a marker for a negative impact on central nervous system function. In this cohort study, we objectively investigate the sleep behavior of infants with infantile hemangiomas on propranolol compared to a healthy, untreated control group. Sleep of propranolol-treated infants and controls was investigated using ankle actigraphy and a 24-h diary for 7–10 days at ages 3 and 6 months. The main outcome measures were the Number of Nighttime Awakenings and Sleep Efficiency. The main secondary outcome measures included 24-hour Total Sleep, daytime sleep behavior, and parent-rated infant sleep quality and behavioral development based on the Brief Infant Sleep Questionnaire (BISQ) and the age-appropriate Ages-and-Stages Questionnaire (ASQ), respectively. Fifty-four term-born infants were included in each cohort. No group difference in any investigated parameter was seen at age 3 months. At age 6 months, the propranolol group exhibited a decrease in Sleep Efficiency and a trend towards an increased Number of Nighttime Awakenings compared to the control group. Treated infants at 6 months also had shorter daytime waking periods. 24-hour Total Sleep was unaffected by propranolol. No negative impact of propranolol on subjective sleep quality and behavioral development was noted. Conclusion: Propranolol exerts a measurable yet mild impact on objectively assessed infants' sleep measures. Behavioral developmental scores were unaffected. Our results support propranolol as first-line therapy for complicated infantile hemangiomas. What is Known: * Sleep disorders are frequently reported in infants with infantile hemangiomas treated with propranolol and often lead to treatment discontinuation. * Investigations of the sleep pattern in this patient group using objective measures are lacking. What is New: * The sleep pattern of propranolol-treated infants is assessed using actigraphy and a 24-h sleep diary and compared to healthy, untreated controls. * Propranolol leads to a decreased sleep efficiency at night and an increased demand of daytime sleep, yet effects are mild overall.

BACKGROUND: CLOVES syndrome (OMIM# 612918) is a rare overgrowth disorder resulted from mosaic gain-of-function mutations in the PIK3CA gene. All the reported CLOVES-associated PIK3CA mutations are missense mutations affecting certain residues. We aim to investigate underlying mutation and its pathogenicity in a patient with CLOVES syndrome and to evaluate the inhibitory effects of the PI3K/AKT/mTOR pathway inhibitors. RESULTS: We performed whole-exome sequencing (WES) and Sanger sequencing to detect underlying somatic mutations in the skin lesion of the patient. Quantitative real-time PCR (qRT-PCR) was employed to evaluate the mRNA abundance of PIK3CA in the patient's skin lesion. AKT phosphorylation level assessed by immunoblotting of lysates from transiently transfected cells was performed to evaluate the PIK3CA mutations and inhibitory effects of PI3K/AKT/mTOR pathway inhibitors. A somatic frameshift mutation c.3206_3207insG (p.X1069Trpfs*4) in PIK3CA was identified in the genomic DNA extracted from the vascular malformation sample of the patient. This mutation affects the canonical stop codon of PIK3CA (NM_006218.4) and is predicted to produce a prolonged protein with four additional residues. qRT-PCR demonstrated that the mRNA expression levels of the patient's affected skin tissue were comparable compared to the normal control. In vitro studies revealed that p.X1069Trpfs*4 mutant exhibited increased AKT phosphorylation significantly to that of the wildtype, which could be inhibited by PI3K/AKT/mTOR pathway inhibitors. CONCLUSIONS: We have identified the first frameshift mutation in PIK3CA that causes CLOVES syndrome, which was confirmed to overactive PI3K/AKT/mTOR pathway by transient transfection assays. We also provided more evidence of ARQ092 to be a potential therapeutic option for PROS in vitro.