Complex lymphatic anomalies: Molecular landscape and medical management.

The lymphatic system is one of the most essential and complex systems in the human body. Disorders that affect the development or function of the lymphatic system can lead to multi-system complications and life-long morbidity. The past two decades have seen remarkable progress in our knowledge of the basic biology and function of the lymphatic system, the molecular regulators of lymphatic development, and description of disorders associated with disrupted lymphangiogensis. In this chapter we will touch on the clinical features of complex lymphatic anomalies, new molecular knowledge of the drivers of these disorders, and novel developmental therapeutics for lymphatic disease.

NRASQ61R mutation drives elevated angiopoietin-2 expression in human endothelial cells and a genetic mouse model.

BACKGROUND: Angiopoietin-2 (Ang-2) is increased in the blood of patients with kaposiform lymphangiomatosis (KLA) and kaposiform hemangioendothelioma (KHE). While the genetic causes of KHE are not clear, a somatic activating NRASQ61R mutation has been found in the lesions of KLA patients. PROCEDURE: Our study tested the hypothesis that the NRASQ61R mutation drives elevated Ang-2 expression in endothelial cells. Ang-2 was measured in human endothelial progenitor cells (EPC) expressing NRASQ61R and a genetic mouse model with endothelial targeted NRASQ61R. To determine the signaling pathways driving Ang-2, NRASQ61R EPC were treated with signaling pathway inhibitors. RESULTS: Ang-2 levels were increased in EPC expressing NRASQ61R compared to NRASWT by Western blot analysis of cell lysates and ELISA of the cell culture media. Ang-2 levels were elevated in the blood of NRASQ61R mutant mice. NRASQ61R mutant mice also had reduced platelet counts and splenomegaly with hypervascular lesions, like some KLA patients. mTOR inhibitor rapamycin attenuated Ang-2 expression by NRASQ61R EPC. However, MEK1/2 inhibitor trametinib was more effective blocking increases in Ang-2. CONCLUSIONS: Our studies show that the NRASQ61R mutation in endothelial cells induces Ang-2 expression in vitro and in vivo. In cultured human endothelial cells, NRASQ61R drives elevated Ang-2 through MAP kinase and mTOR-dependent signaling pathways.

Treatment practices and response in kaposiform hemangioendothelioma: A multicenter cohort study.

BACKGROUND AND OBJECTIVES: Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare vascular tumors in children historically associated with significant morbidity and mortality. This study was conducted to determine first-line therapy in the absence of available prospective clinical trials. METHODS: Patients from 17 institutions diagnosed with KHE/TA between 2005 and 2020 with more than 6 months of follow-up were included. Response rates to sirolimus and vincristine were compared at 3 and 6 months. Durability of response and response to other treatment modalities were also evaluated. RESULTS: Of 159 unique KHE/TA subjects, Kasabach-Merritt phenomenon (KMP) was present in 64 (40.3%), and only two patients were deceased (1.3%). Over 60% (n = 96) demonstrated treatment response at 3 months, and more than 70% (n = 114) by 6 months (no significant difference across groups). The vincristine group had higher radiologic response at 3 months compared to sirolimus (72.7% vs. 20%, p = .03), but there were no differences between these groups at 6 months. There were no differences in rates of recurrent or progressive disease between vincristine and sirolimus. CONCLUSIONS: In this large, multicenter cohort of 159 patients with KHE/TA, rates of KMP were consistent with historical literature, but the mortality rate (1.3%) was much lower. Overall treatment response rates were high (>70%), and there was no significant difference in treatment response or durability of disease comparing sirolimus to vincristine. Our results support individualized treatment decision plans depending on clinical scenario and patient/physician preferences. Response criteria and response rates reported here will be useful for guiding future treatment protocols for vascular tumors.

Genomic profiling informs diagnoses and treatment in vascular anomalies.

Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype-phenotype associations and guide medical therapy in individuals with vascular anomalies.

Pathogenic variants in PIK3CA are associated with clinical phenotypes of kaposiform lymphangiomatosis, generalized lymphatic anomaly, and central conducting lymphatic anomaly.

Complex lymphatic anomalies are debilitating conditions characterized by aberrant development of the lymphatic vasculature (lymphangiogenesis). Diagnosis is typically made by history, examination, radiology, and histologic findings. However, there is significant overlap between conditions, making accurate diagnosis difficult. Recently, genetic analysis has been offered as an additional diagnostic modality. Here, we describe four cases of complex lymphatic anomalies, all with PIK3CA variants but with varying clinical phenotypes. Identification of PIK3CA resulted in transition to a targeted inhibitor, alpelisib. These cases highlight the genetic overlap between phenotypically diverse lymphatic anomalies.

Kaposiform lymphangiomatosis: Diagnosis, pathogenesis, and treatment.

Kaposiform lymphangiomatosis (KLA) is a life-threatening rare disease that can cause substantial morbidity, mortality, and social burdens for patients and their families. Diagnosis often occurs long after initial symptoms, and there are few centers in the world with the expertise to diagnose and care for patients with the disease. KLA is a lymphatic anomaly and significant advancements have been made in understanding its pathogenesis and etiology since its first description in 2014. This review provides multidisciplinary, comprehensive, and state-of-the-art information on KLA patient presentation, diagnostic imaging, pathology, organ involvement, genetics, and pathogenesis. Finally, we describe current therapeutic approaches, important areas for research, and challenges faced by patients and their families. Further insights into the pathogenesis of KLA may advance our understanding of other vascular anomalies given that similar signaling pathways may be involved.

Response to sirolimus in capillary lymphatic venous malformations and associated syndromes: Impact on symptomatology, quality of life, and radiographic response.

BACKGROUND: Capillary lymphatic venous malformations (CLVM) and associated syndromes, including Klippel-Trenaunay syndrome (KTS) and congenital lipomatous overgrowth, vascular malformation, epidermal nevi, skeletal, and spinal syndrome (CLOVES), are underrecognized disorders associated with high morbidity from chronic pain, recurrent infections, bleeding, and clotting complications. The rarity of these disorders and heterogeneity of clinical presentations make large-scale randomized clinical drug trials challenging. Identification of PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha [gene]) mutations in CLVM has made targeted medications, such as sirolimus, attractive treatment options. The aim of this study was to investigate the safety and efficacy of sirolimus therapy in CLVM. PROCEDURE: A combined prospective and retrospective cohort of pediatric and young adult patients with CLVM treated with sirolimus was evaluated for disease response, including symptom improvement, quality of life (QOL), and radiologic response. Sirolimus dosing regimens and toxicities were also assessed. RESULTS: Twenty-nine patients with CLVM, including KTS and CLOVES, were included. Ninety-three percent of patients reported improved QOL, and 86% had improvement in at least one symptom. Most significantly, improvement was noted in 100% of patients with bleeding and 89% with thrombotic complications with corresponding decreases in mean D-dimer (p = .008) and increases in mean fibrinogen (p = .016). No patients had progressive disease on sirolimus. Most common side effects included neutropenia, lymphopenia, infection, and aphthous ulcers/stomatitis. No toxicities were life-threatening, and none required long-term discontinuation of sirolimus. CONCLUSION: Sirolimus appears to be effective at reducing complications and improving QOL in patients with CLVM and associated syndromes. In this patient cohort, sirolimus was well tolerated and resulted in few treatment-related toxicities.

Treatment of severe Kaposiform lymphangiomatosis positive for NRAS mutation by MEK inhibition.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a complex lymphatic anomaly involving most commonly the mediastinum, lung, skin and bones with few effective treatments. In recent years, RAS-MAPK pathway mutations were shown to underlie the pathogenesis of several complex lymphatic anomalies. Specifically, an activating NRAS mutation (p.Q61R) was found in the majority of KLA patients. Recent reports demonstrated promising results of treatment with the MEK inhibitor, Trametinib, in patients with complex lymphatic anomalies harboring gain of function mutations in ARAF and SOS1, as well as loss of function mutation in the CBL gene, a negative regulator of the RAS-MAPK pathway. We present a 9-year-old child with a severe case of KLA harboring the typical NRAS (p.Q61R) mutation detected by plasma-derived cell free DNA, responsive to trametinib therapy. METHODS: The NRAS somatic mutation was detected from plasma cfDNA using droplet digital PCR. Concurrent in-vitro studies of trametinib activity on mutant NRAS affected lymphatic endothelial cells were performed using a three-dimensional spheroid sprouting assay. RESULTS: Trametinib treatment lead to resolution of lifelong thrombocytopenia, improvement of pulmonary function tests and wellbeing, as well as weaning from prolonged systemic steroid treatment. Concurrent studies of mutant NRAS-expressing cells showed enhanced lymphangiogenic capacity along with over activation of the RAS-MAPK and PI3K-AKT-mTOR pathways, both reversed by trametinib. CONCLUSIONS: Trametinib treatment can substantially change the prognosis of patients with RAS pathway associated lymphatic anomalies. IMPACT: This is the first description of successful trametinib treatment of a patient with KLA harboring the most characteristic NRAS p.Q61R mutation. Treatment can significantly change the prognosis of patients with RAS pathway-associated lymphatic anomalies. We devised an in vitro model of KLA enabling a reproducible method for the continued study of disease pathogenesis. Mutated NRAS p.Q61R cells demonstrated increased lymphangiogenic capacity.

Sirolimus for diffuse intestinal infantile hemangioma with PHACE features: systematic review.

BACKGROUND: We report a 3-month-old female with cardiovascular anomalies and diffuse intestinal infantile hemangioma (IIH) of the small bowel suggesting possible diagnosis of PHACE syndrome (posterior fossa anomalies, hemangioma, arterial lesions, cardiac abnormalities/coarctation of the aorta, eye anomalies). The GI symptoms persisted under treatment with propranolol, whereas the addition of sirolimus led to regression of the IIH. METHODS: A systematic review was conducted using PubMed, EMBASE, and Ovid MEDLINE databases between 1982 and 2021. RESULTS: A total of 4933 articles were identified; 24 articles met inclusion criteria with 46 IIH cases. The most common GI presentations were unspecified GI bleed (40%) and anemia (38%). The most common treatments were corticosteroids (63%), surgical resection (32.6%), and propranolol (28%). Available outcomes were primarily bleeding arrest (84%). Nine cases (19.5%) were diagnosed with definite PHACE, 5 (11%) with possible PHACE, and 32 (69.5%) no PHACE. Our case presented with symptoms most consistent with those of possible PHACE and definite PHACE. No cases in this review underwent treatment with sirolimus. CONCLUSIONS: This is the first reported case of successful treatment of IIH with sirolimus. Our case, along with other patients who present with IIH and PHACE features, suggests consideration of IIH as a diagnostic criterion for PHACE syndrome. IMPACT: This is the first reported case in which sirolimus showed regression of an intestinal infantile hemangioma. This study serves to demonstrate the presentation, treatment, outcomes of intestinal infantile hemangioma, and correlation with PHACE. The potential correlation between intestinal infantile hemangioma and PHACE deserves more study in consideration of intestinal infantile hemangioma as a diagnostic criterion of PHACE.

Multifocal Kaposiform Hemangioendothelioma in a Newborn With Confirmatory Histopathology.

Kaposiform hemangioendothelioma is classified as a locally aggressive vascular tumor of childhood resulting from abnormal angiogenesis and lymphangiogenesis. Most commonly, KHE presents as a single tissue mass, ranging from an erythematous papule to a violaceous indurated tumor. Definitive diagnosis requires tissue sampling with the demonstration of ill-defined nodules and fascicles of spindle-shaped D2-40 positive endothelial cells, forming slit-like vascular channels. This newborn presented with multifocal cutaneous Kaposiform hemangioendothelioma associated with Kasabach-Merritt phenomenon confirmed on histopathology with immunostaining.

Cerebrospinal fluid leak in epidural venous malformations and blue rubber bleb nevus syndrome.

OBJECTIVE: Clinical manifestations of blue rubber bleb nevus syndrome (BRBNS) and multifocal venous malformation (MVM) vary depending on the location of the lesions. The aim of this study was to assess the risk of developing CSF leaks in patients with epidural venous malformations (VMs). METHODS: The authors retrospectively investigated the relationship between the development of a CSF leak and the presence of epidural VMs. RESULTS: Nine patients (5 females) had epidural VMs and presentation that was confirmatory or suggestive of a CSF leak: 4 had BRBNS, 4 had MVMs, and 1 had a solitary VM. Of 66 patients with BRBNS, clinical and imaging features of CSF leak were noted in 3 (4.5%) with epidural VMs at the age of 11-44 years. A fourth patient had suggestive symptoms without imaging confirmation. An epidural blood patch was ineffective in 2 patients, both with more than one source of leakage, requiring surgical repair or decompression. Symptomatic downward displacement of the cerebellar tonsils was noted in 3 patients with MVM and 1 with a solitary VM; 3 required surgical decompression. CONCLUSIONS: These findings suggest an increased risk of CSF leak in patients with epidural VM, including BRBNS, MVMs, and solitary VMs. Awareness of the association between epidural VM and CSF leakage may facilitate earlier diagnosis and therapeutic intervention.

Kaposiform Lymphangiomatosis: Pathologic Aspects in 43 Patients.

Kaposiform lymphangiomatosis is an uncommon generalized lymphatic anomaly with distinctive clinical, radiologic, histopathologic, and molecular findings. Herein, we document the pathology in 43 patients evaluated by the Boston Children's Hospital Vascular Anomalies Center from 1999 to 2020. The most frequent presentations were respiratory difficulty, hemostatic abnormalities, and a soft tissue mass. Imaging commonly revealed involvement of some combination of mediastinal, pulmonary, pleural, and pericardial compartments and most often included spleen and skeleton. Histopathology was characterized by dilated, redundant, and abnormally configured lymphatic channels typically accompanied by dispersed clusters of variably canalized, and often hemosiderotic, spindled lymphatic endothelial cells that were immunopositive for D2-40, PROX1, and CD31. An activating lesional NRAS variant was documented in 9 of 10 patients. The clinical course was typically aggressive, marked by hemorrhage, thrombocytopenia, diminished fibrinogen levels, and a mortality rate of 21%.

How we approach the use of sirolimus and new agents: Medical therapy to treat vascular anomalies.

Vascular anomalies (VAs) are a heterogeneous group of primarily congenital tumors and malformations. The International Society for the Study of Vascular Anomalies (ISSVA) has developed a standard classification of these disorders, creating a uniform approach to their diagnosis. Recent discoveries evaluating the genetic causes of VAs have revealed that they are due to mutations in cancer pathways, including the PI3K/AKT/mTOR and RAS/MAPK/MEK pathways. These discoveries have led to improved phenotype-genotype correlation and have expanded medical therapy for this group of unique disorders.

Respiratory Failure in Noonan Syndrome Treated by Microsurgical Thoracic Duct-Venous Anastomosis.

Noonan syndrome is a disorder characterized by central and peripheral lymphatic conducting anomalies, leading to chylothorax, chylous ascites, and metabolic derangement. Novel imaging methods, including dynamic contrast magnetic resonance lymphangiography and intranodal lymphangiography, have allowed for increased visualization of lymphatic pathology. Severe pulmonary insufficiency and chylothoraces developed in a 61-year-old man with Noonan syndrome. Dynamic contrast magnetic resonance lymphangiography and intranodal lymphangiography demonstrated central thoracic duct (TD) occlusion. The patient's condition significantly improved after a microsurgical TD-venous anastomosis assisted by TD catheterization for imaging guidance, resulting in decompression of the lymphatic system and resolution of the pulmonary symptoms.

Cerebrofacial vascular metameric syndrome is caused by somatic pathogenic variants in PIK3CA.

Disorganized morphogenesis of arteries, veins, capillaries, and lymphatic vessels results in vascular malformations. Most individuals with isolated vascular malformations have postzygotic (mosaic), activating pathogenic variants in a handful of oncogenes within the PI3K-RAS-MAPK pathway (Padia et al., Laryngoscope Investig Otolaryngol 4: 170-173 [2019]). Activating pathogenic variants in the gene PIK3CA, which encodes for the catalytic subunit of phosphatidylinositol 3-kinase, are present in both lymphatic and venous malformations as well as arteriovenous malformations in other complex disorders such as CLOVES syndrome (congenital, lipomatous, overgrowth, vascular malformations, epidermal anevi, scoliosis) (Luks et al., Pediatr Dev Pathol 16: 51 [2013]; Luks et al., J Pediatr 166: 1048-1054.e1-5 [2015]; Al-Olabi et al., J Clin Invest 128: 1496-1508 [2018]). These vascular malformations are part of the PIK3CA-related overgrowth spectrum, a spectrum of entities that have regionalized disordered growth due to the presence of tissue-restricted postzygotic PIK3CA pathogenic variants (Keppler-Noreuil et al., Am J Med Genet A 167A: 287-295 [2015]). Cerebrofacial vascular metameric syndrome (CVMS; also described as cerebrofacial arteriovenous metameric syndrome, Bonnet-Dechaume-Blanc syndrome, and Wyburn-Mason syndrome) is the association of retinal, facial, and cerebral vascular malformations (Bhattacharya et al., Interv Neuroradiol 7: 5-17 [2001]; Krings et al., Neuroimaging Clin N Am 17: 245-258 [2007]). The segmental distribution, the presence of tissue overgrowth, and the absence of familial recurrence are all consistent with CVMS being caused by a postzygotic mutation, which has been hypothesized by previous authors (Brinjiki et al., Am J Neuroradiol 39: 2103-2107 [2018]). However, the genetic cause of CVMS has not yet been described. Here, we present three individuals with CVMS and mosaic activating pathogenic variants within the gene PIK3CA We propose that CVMS be recognized as part of the PIK3CA-related overgrowth spectrum, providing justification for future trials using pharmacologic PIK3CA inhibitors (e.g., alpelisib) for these difficult-to-treat patients.

Parkes Weber syndrome with lymphedema caused by a somatic KRAS variant.

Parkes Weber syndrome is a vascular malformation overgrowth condition typically involving the legs. Its main features are diffuse arteriovenous fistulas and enlargement of the limb. The condition has been associated with pathogenic germline variants in RASA1 and EPHB4 We report two individuals with Parkes Weber syndrome of the leg and primary lymphedema containing a somatic KRAS variant (NM_004985.5:c.35G > A; p.Gly12Asp). KRAS variants, which cause somatic intracranial and extracranial arteriovenous malformations, also result in Parkes Weber syndrome with lymphatic malformations.

A review of mechanisms of disease across PIK3CA-related disorders with vascular manifestations.

BACKGROUND: PIK3CA-related disorders include vascular malformations and overgrowth of various tissues that are caused by postzygotic, somatic variants in the gene encoding phosphatidylinositol-3-kinase (PI3K) catalytic subunit alpha. These mutations result in activation of the PI3K/AKT/mTOR signaling pathway. The goals of this review are to provide education on the underlying mechanism of disease for this group of rare conditions and to summarize recent advancements in the understanding of, as well as current and emerging treatment options for PIK3CA-related disorders. MAIN BODY: PIK3CA-related disorders include PIK3CA-related overgrowth spectrum (PROS), PIK3CA-related vascular malformations, and PIK3CA-related nonvascular lesions. Somatic activating mutations (predominantly in hotspots in the helical and kinase domains of PIK3CA, but also in other domains), lead to hyperactivation of the PI3K signaling pathway, which results in abnormal tissue growth. Diagnosis is complicated by the variability and overlap in phenotypes associated with PIK3CA-related disorders and should be performed by clinicians with the required expertise along with coordinated care from a multidisciplinary team. Although tissue mosaicism presents challenges for confirmation of PIK3CA mutations, next-generation sequencing and tissue selection have improved detection. Clinical improvement, radiological response, and patient-reported outcomes are typically used to assess treatment response in clinical studies of patients with PIK3CA-related disorders, but objective assessment of treatment response is difficult using imaging (due to the heterogeneous nature of these disorders, superimposed upon patient growth and development). Despite their limitations, patient-reported outcome tools may be best suited to gauge patient improvement. New therapeutic options are needed to provide an alternative or supplement to standard approaches such as surgery and sclerotherapy. Currently, there are no systemic agents that have regulatory approval for these disorders, but the mTOR inhibitor sirolimus has been used for several years in clinical trials and off label to address symptoms. There are also other agents under investigation for PIK3CA-related disorders that act as inhibitors to target different components of the PI3K signaling pathway including AKT (miransertib) and PI3K alpha (alpelisib). CONCLUSION: Management of patients with PIK3CA-related disorders requires a multidisciplinary approach. Further results from ongoing clinical studies of agents targeting the PI3K pathway are highly anticipated.