The Pilot Award is funded by the LMI.
Year: 2024
PI Name: Dr. Maureen Cetera
Project Title: FZD6 as a novel candidate gene for Central Conducting Lymphatic Anomaly
Abstract: Central conducting lymphatic anomaly (CCLA) is a disorder characterized by conduction abnormalities of the central lymphatics that may present with non-immune fetal hydrops. Recently, mTOR, PI3K, and MEK inhibitors aimed at treating the underlying molecular causes of CCLA have been trialed. However, only 40% of affected individuals have a defined genetic cause, suggesting novel causes must be elucidated. Exome sequencing identified FZD6 as a novel candidate gene for CCLA in five families presenting with non-immune fetal hydrops, nail dysplasia, and CCLA. FZD6 is a WNT receptor involved in planar cell polarity (PCP) signaling, and in humans, loss of FZD6 function causes nail dysplasia. Previously, defects in PCP signaling due to pathogenic variants in CELSR1, FAT4, and DCHS1 have been implicated in lymphatic malformations in humans. Therefore, we hypothesize that loss of FZD6 function is the cause of the lymphatic anomalies in this cohort. In support of this, we identified a mouse strain with a loss of function mutation in Fzd6 that has mild nuchal edema, and preliminary findings revealed dilated dermal lymphatics in Fzd3-/-; Fzd6-/- mice. Using the mouse model, we will further elucidate the role of Fzd6 in lymphatic development by analyzing embryonic lymphatic morphology and function in Fzd6 mutants and controls. Further, we will elucidate the role of Fzd6 in controlling dynamic lymphatic endothelial cell behaviors through live imaging. Taken together, these studies will define the defects caused by loss of planar cell polarity signaling and confirm or refute the role of FZD6 in CCLA development.
Year: 2023
PI Name: Bryan Sisk
Project Title: Developing an Electronic Health Record Tool to Improve Patient Knowledge and Self-Management
Abstract: Complex lymphatic anomalies (CLAs) are caused by abnormal growth or development of lymphatic vessels that can lead to lifelong complications, including pain, bleeding, clotting, infections, deformity, disability, and death. Families with CLAs who receive better information about their disorders report better physical health, mental health, and ability to access needed healthcare. Yet, many families struggle to find trustworthy information, which leads them to searching the internet and social media. These patients rely on clinicians for reliable and understandable information about VMs, but most clinicians have limited knowledge about these disorders. Efforts to improve communication for patients and families affected by CLAs could lead to better menta health, physical health, and ability to navigate the healthcare system. In this study, we will work with adult patients with CLAs and parents of children with CLAs to develop a communication tool called CLA Care Companion. This tool will include push notifications, reminders, video- and text-based educational materials, and symptom surveys. Also, we will work with CLA experts to develop understandable and reliable educational materials. We will build this tool into the Epic electronic health record system. After building this tool, we will provide access to 10 families affected by CLAs. We will use surveys, interviews, and data from Epic to see if the tool is useful and acceptable to participants. We will use these results of this study to develop a large clinical trial in the future that tests whether this tool can improve communication and health outcomes for families with CLAs.
Year: 2022
PI Name: Carrie Shawber
Project Title: Assessment of proteasome inhibitors for the treatment of complex lymphatic anomalies
Abstract: Generalized lymphatic anomalies (GLAs) are due to somatic mutations in genes of the RAS/MAPK and PI3K/AKT signaling pathways. Therapies that inhibit these pathways, sirolimus, alpelisib, and trametinib have been reported to improve GLA symptoms. However, no therapy has resulted in a complete response. Using a high throughput drug screen and patient derived lymphatic endothelial cells (LECs), we identified a novel class of drugs, proteasome inhibitors (PIs) that suppressed the growth of mutant LECs significantly more than sirolimus, trametinib and alpelisib. In a xenograft model, the PI, oprozomib, suppressed the LM-like phenotype. PIs are proposed to selectively target cells with proteostasis defects for cell death, suggesting there are proteostasis defects LECs from GLA patients (GLA-LECs). Consistent with this idea, we find the GLA-LECs with mutations RAS or PI3K/AKT pathways both have increased and abnormal cytoplasmic accumulation of VECADHERIN and CD31 and a loss of their expression at the cell surface. We hypothesize that PIs target proteostasis defects downstream of PI3K hyperactivation in GLA-LECs. In Aim 1, we will determine the optimal PI that preferentially targets LECs with PIK3CA variants, and the mechanism by which PIs suppress LEC growth will be determined using lymphatic anomaly patient derived-LECs and LECs engineered to overexpress an activated form of PIK3CA identified in patients. In Aim 2, we will assess the efficacy of PIs in a mouse model in which PIK3CAH1047R is induced systemically in the lymphatic endothelium. The overall goal is to determine if PIs are a viable therapy for GLA patients and to generate preliminary data for future NIH and DOD grant submissions.
Year: 2022
PI Name: James T. Bennett
Project Title: Examination of cell free DNA and single-cell based expression profiling of chylous effusions in complex lymphatic anomalies
Abstract: The identification of somatic mutations in the PIK3CA gene in >80% of individuals with isolated lymphatic malformations is transforming their clinical management. Genetic testing is becoming a routine part of clinical care because specific molecular diagnosis leads to eligibility for targeted drug therapies. However, individuals with complex lymphatic anomalies (CLAs: generalized lymphatic anomaly (GLA), kaposiform lymphangiomatosis (KLA), Gorham-Stout disease (GSD), and central conducting lymphatic anomalies (CCLA)) have not yet benefitted from widespread genetic testing. Most mutations identified in CLA come from sequencing surgically resected tissues. Even when surgery can be safely performed, it is not always clear if the available tissue(s) should possess the mutation. Therefore, a barrier to improved molecular diagnosis of CLAs is poor tissue availability. We will overcome this by using chylous fluids as a novel diagnostic material in CLAs. Dysfunctional lymphatic drainage is a common feature of CLAs and can result in accumulation of lymph (“chylous fluid”) in spaces around the heart, lungs, and intestines. Because these fluids compromise organ function and can even lead to death, they are frequently drained and discarded. Chylous fluid contains cells from the walls of large draining lymphatic vessels- cells known to be critical in CLA development. We hypothesize that analysis of cells and free-floating DNA in this fluid will identify mutations and categorize CLA subtypes. We will sequence cell-free DNA and perform single cell-based gene expression profiling in chylous fluid from 10-12 individuals with CLA. We hope to transfer discoveries from this project into clinical diagnostics for CLA.
Year: 2022
PI Name: William Polacheck
Project Title: Development of an inducible tissue engineered model for understanding cellular dynamics in GLA
Abstract: Generalized lymphatic anomaly (GLA) is a complex lymphatic malformation (LM) characterized by the presence of diffuse or multifocal lymphangiomas infiltrating multiple organs. Symptoms generally arise from invasion and compression of the surrounding tissue and can cause significant morbidity or mortality if vital organs are affected. While somatic activating mutations of PIK3CA have been identified to be the cause of GLA, the specific mechanisms by which dysregulated PI3K signaling in a small population of endothelial cells results in tissue level vascular defects remain unclear. We hypothesize that the endothelial cytoskeleton and sites of adhesion between healthy and mutated cells are critical components in propagating effects of hyperactivated PIK3CA across the tissue. Testing this hypothesis requires a system where mutated PIK3CA can be induced in a subset of cells at a specific time, mimicking the pathophysiology of causal somatic mutations in GLA. Our proposed work will enable such studies by employing a light-inducible PIK3CA expression system in lymphatic-derived, perfusable, human microvascular networks embedded in microfluidic devices. This approach of engineering cells within an established vascular network will enable controlled induction of where and when mutant PIK3CA is expressed and real-time observation of how this induction is propagated across the tissue, resulting in unprecedented spatiotemporal maps of how somatic PIK3CA mutations lead to LM. Completion of the proposed research will result in an innovative in vitro model that will enable drug screening and toxicity studies to improve interventional approaches, while also providing key mechanistic insights into the underlying causes of GLA.
Year: 2021
PI Name: Natasha Harvey
Project title: Defining the mechanisms by which MDFIC mutations cause central conducting lymphatic anomaly (CCLA).
Abstract: Central conducting lymphatic anomaly (CCLA), characterised by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli and presenting with chylothorax, pleural effusions, chylous ascites and lymphoedema, is a severe disorder often resulting in foetal or perinatal demise. While mutations in genes including ARAF, EPHB4 and JAG1 have been documented in patients with CCLA, the genetic aetiology of CCLA remains in large part, uncharacterised. We have identified homozygous, and compound heterozygous, mutations in MDFIC, encoding the MyoD family inhibitor domain containing protein, in foetuses presenting with non-immune hydrops fetalis and children with a history of hydrops fetalis, pleural and pericardial effusions and lymphoedema, consistent with CCLA. Generation of a mouse model of the human MDFIC truncating mutation (Met131fs*) found in patients revealed that homozygous Mdfic mutant mice die perinatally exhibiting chylothorax, the accumulation of lipid rich chyle in the thoracic cavity catalysed by lymphatic vessel dysfunction. Characterisation of the lymphatic vasculature of homozygous Mdfic mutant mice revealed profoundly mis-patterned lymphatic vessels and major defects in lymphatic vessel valve development. We hypothesise that MDFIC plays crucial roles during embryonic development of the lymphatic vasculature and that the mutations identified in patients with CCLA profoundly impact protein function. The goal of this proposal is to define the mechanisms by which MDFIC governs development and function of the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches able to effectively treat this complex, devastating disease.
Year: 2021
PI Name: Dong Li
Project Title: Identification and Functional Characterization of Variants Underlying Complex Lymphatic Anomalies
Abstract: Complex lymphatic anomalies, which include a variety of diagnoses, are chronically debilitating, devastating, and often life-threatening diseases with limited treatment options. Magnetic resonance (MR) lymphangiogram has allowed for visualization of lymphatics and intervention. Our preliminary studies have revealed multiple genes converging on PI3K and MAPK pathways and modeling mutations in cellular and zebrafish systems have recapitulated the essential morphological features seen in the patients. We have found a handful of MEK/ERK inhibitors and PI3K inhibitor showed the biochemistry and morphological reversal of the effects of mutations in these genes. Taken together, we have successfully implemented precision- medicine approaches for two patients with CCLA and KLA. The absence of data on the molecular etiology, and lack of understanding of the underlying molecular mechanisms have greatly hampered further research and precision medicine focused clinical trials. Our long-term goal is to identify efficacious therapies for complex lymphatic anomalies. The objective of this application is to uncover novel/recurrent disease-causing mutations in GLA/GSD/KLA/CCLA and use in vitro and in vivo models established in our previous studies to determine optimal treatment strategies, including PI3K, mTOR, MEK, and VEGFR3 inhibitors. This proposal will test the hypothesis that sequencing of highly informative patients referred by an integrated multidisciplinary lymphatic anomalies clinic will unveil novel/recurrent variants, and these can be rapidly interrogated through our established cellular and zebrafish models to further investigate the mutation phenotype spectrum effect. The results from these experiments will provide validated pre-clinical data for molecularly implemented precision-based therapies for clinical trials.
Year: 2019
PI Name: René Haegerling
Project title: 3-dimensional visualization of the lymphatic vasculature in Gorham- Stout Disease, Generalized Lymphatic Anomaly and Kaposiform Lymphangiomatosis using VIPAR
Abstract: Over the last years, there was a lot of progress in identifying genes, which cause lymphovascular malformation in humans, but how genetic abnormalities and abnormal vascular growth cause lymphatic alterations at the cellular level, e.g. in lymphatic hyperplasia or Gorham-Stout-Disease, is still unknown. This lack of mechanistic insight is associated with the absence of suitable microscopic imaging techniques for the visualization of the vasculature as classical 2-dimensional histology is not sufficient to understand the complex lymphatic vessel architecture [1]. This has been one of the major contributing factors to the lack of detailed knowledge of the pathogenesis of lymphatic malformation. To understand the underlying pathology of Gorham-Stout-Disease (GSD), Generalized- Lymphatic-Anomaly (GLA) and Kaposiform-Lymphangiomatosis (KLA) in more detail and to overcome the limitations of classical histology, we will develop innovative and optimized immunofluorescence staining protocols for entire tissue biopsies including bony tissue and will apply VIPAR, a novel diagnostic tool for vascular malformations, on tissue biopsies from patients suffering from these diseases [1]. VIPAR, a light-sheet-microscopy-based approach for the analysis of entire tissue biopsies, is based on digital 3-dimensional reconstruction and allows the visualization of the entire vascular network. By comparing these results with control samples, we will then identify the vascular changes in 3-dimensional space. In summary, this study will allow 3D-visualization of lymphatic malformations and therefore facilitate a deep-phenotyping of patients’ tissue, which is not possible by using classical methods, e.g. 2D histology. Therefore, this study will provide novel insights into the pathogenesis of lymphatic malformations and allow verification of disease mouse models.
Year: 2019
PI Name: Fayez Safadi
Project title: The Role of Lymphatic Fluid on Bone Cellular Interaction and Function
Abstract: Optimizing a multicellular lab-on-a-chip (LOC) bone remodeling platform to systematically investigate the effects of lymphatic fluid on bone cell activity. Gorham-Stout Disease (GSD) and generalized lymphatic anomaly (GLA) are rare conditions characterized by abnormal lymphatic vessels in bone associated with bone loss. There is still much to learn from basic science research about the role of human lymphatic fluid (hLF) on bone function. In this application, we hypothesize that hLF alters bone cell differentiation and function. We propose that the bone loss associated with GSD and GLA is, at least in part, the result of specific factors in the lymphatic fluids and their interactions with bone cells resulting in bone loss. To address this, we propose two specific aims: Aim 1 will assess the effect of hLF on bone cell interactions using in vitro LOC and in vivo mouse models. Here, we will address the hypothesis that hLF alters bone cell interaction and function. Aim 2 will determine the effects of hLF on factors produced by Osteoblasts (OB) and Osteoclasts (OC) at the time of remodeling/formation. We will test the hypothesis that hLF will alter protein constituents from the conditioned media (CM) collected from OB and OC cultures exposed to hFL. Once validated, the LOC offers a reproducible and cost- effective tool to explore pathway/mechanistic interactions of hLF on bone-an attractive option for orphan disease study. Data generated from this proposal will provide new information on the role of hLF on bone cell interactions and its effects on bone formation and resorption.
Year: 2019
PI Name: Miikka Vikkula
Project title: Molecular pathogenesis of lymphatic anomalies
Abstract: Generalized lymphatic anomaly (GLA) and Gorham-Stout disease (GSD) are rare and of unknown etiology. Both are commonly identified in children and young adults, and occur sporadically, with no known hereditary predisposition. They are characterized by proliferation of lymphatic vessels, which in GSD invade bones, resulting in progressive bone destruction. The molecular mechanisms driving osteolysis and lymphangiogenesis in GLA/GSD are unknown, and there are no FDA/EMA approved therapies for treating GLA/GSD. Based on our previous work demonstrating somatic mutations in venous malformations (VMs), we hypothesized that many vascular anomalies, and other developmental disorders are caused by somatic mutations. Subsequently, somatic mutations have been identified in a number of vascular anomalies and other disorders. Therefore, we also hypothesize that GLA/GSD are caused by somatic mutations, and like in other vascular malformations, they activate oncogenic signalling pathways. One somatic NRAS mutation and four somatic PIK3CA mutations have indeed been identified in 6 GLA patients, and we have discovered a mutation in a third gene, which is undergoing functional validation in mice. However, the majority of GLA/GSD cases remain unexplained by mutations in these genes. Therefore, we propose to search for causative gene mutations and/or chromosomal alterations in GLA/GSD using deeper and wider-scale sequencing (high coverage whole-genome and whole-exome), allowing to increase sensitivity, and to analyse copy number variations and translocations.
Year: 2017
PI Name: Shoshana Greenberger
Project title: Vertebrate based model for targeting overactivation of nras pathway in GLA
Abstract: Generalized lymphatic anomaly (GLA), Gorham–Stout disease (GSD), and kaposiform lymphangiomatosis (KLA) are intractable lymphatic disorders. These are rare diseases characterized by a diffuse proliferation of lymphatic vessels in skin and internal organs. The diseases are associated with a significant morbidity and mortality rates. The etiology of these diseases remains unknown. By isolating lymphatic endothelial cells from GLA tissue (LyEC) and employing Whole Exome Sequencing (WES) to search for disease-causing genes, we identified a somatic activating mutation in neuroblastoma RAS viral oncogene homolog (NRAS) driving a Q61R amino acid substitution. This mutation is known to occur in several types of cancer including Melanoma. Herein, we propose to create a zebrafish-based GLA model system in order to validate the role of this mutation in the development of the lymphatic system, and to conduct High- throughput screening (HTS) for compounds capable of inhibiting GLA progress.
Year: 2016
PI Name: Ionela Iacobas
Project Title: Lymph-loss Markers as Predictors of Severity in Generalized Lymphatic Anomaly (GLA) and Gorham- Stout’s Disease (GSD)
Abstract: Lymphatic malformations may range from small and localized, to systemic and with high morbidity like Generalized Lymphatic Anomaly (GLA) and Gorham-Stout’s Disease (GSD). The clinical picture is progressive and the full destructive potential may not be visible from the initial presentation. Lymph-loss markers (low lymphocyte count, low albumin and low gamaglobulin – IgG) have been noticed in preliminary studies to correlate with severity of disease. They may potentially predict the accumulation of effusions (pleural, pericardial or ascites) in GLA/GSD. Lymphocyte count, albumin and IgG can all be tested via blood draws, are part of routine clinical care, not considered experimental intervention. Our study is trying to analyze if we can use these tests for risk stratification in GLA and GSD, for monitoring of condition progression and as predictors of morbidity. With the advantage of the Lymphatic Anomalies Registry from Boston Children’s Hospital and the International LGDA Registry for Lymphatic Malformations, we can obtain information regarding the association between the GLA/GSD complications and the time pattern of the lymph-loss markers. Due to the increased awareness of GSD and GLA diagnostic criteria, many patients are diagnosed early before multi-organ complications occur and it would be very important to know if for ex. having lymphopenia at diagnosis may predict the severity of disease in the future. The data will be extracted from the registries and statistically analyzed. Finally, all the results will be published and will constitute background for development of clinical practice guidelines for the management of GLA and GSD patients.
Years: 2015 – 2020
PI Name: Tim Le Cras
Project Title: Identification of biomarkers for patients with Generalized Lymphatic Anomaly (GLA), Kaposiform Lymphangiomatosis (KLA), Gorham-Stout Disease (GSD)
Years: 2014 – 2016
PI Name: Lianping Xing
Project Title: Bone cells and lymphatic endothelium interface
Year: 2014
PI Name: Eva Sevick
Project Title: Imaging lymphatic function in normal subjects and persons with lymphatic disorders
Year: 2013
PI Name: Erik Eklund
Project Title: Immunohistochemical features of lymphangiomatosis and Gorham’s disease
Years: 2012 – 2020
PI Name: Victor Martinez-Glez
Project Title: Genetic and Genomic Analysis in Patients Affected by Gorham-Stout Disease and General Lymphatic Anomaly
Years: 2012 – 2016
PI Name: Cameron Trenor
Project Title: Lymphatic Anomalies Registry
Years: 2012 – 2015
PI Name: Donald McDonald
Project Title: Transgenic mouse model to determine the mechanism and treatment of congenital pulmonary lymphangiectasia and lymphangiomatosis
Year: 2012
PI Name: Ramani Ramchandran
Project Title: Molecular crosstalk and matrix metalloproteinases in generalized lymphatic anomaly (GLA) and Gorham-Stout syndrome (GSS) patients