Lymphatic endothelial cell-specific NRAS p.Q61R mutant embryos show abnormal lymphatic vessel morphogenesis.

Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.

Sirolimus treatment for intractable lymphatic anomalies: an open-label, single-arm, multicenter, prospective trial.

Introduction: Intractable lymphatic anomalies (LAs) include cystic lymphatic malformation (LM; macrocystic, microcystic, or mixed), generalized lymphatic anomaly, and Gorham-Stout disease. LAs can present with severe symptoms and poor prognosis. Thus, prospective studies for treatments are warranted. We conducted a prospective clinical trial of sirolimus for intractable LAs. Methods: This was an open-label, single-arm, multicenter, prospective trial involving five institutions in Japan. All patients with LAs received oral sirolimus once daily, and the dose was adjusted to ensure that the trough concentration remained within 5-15 ng/mL. We prospectively assessed the drug response (response rate for radiological volumetric change in target lesion), performance state, change in respiratory function, visceral impairment (pleural effusion, ascites, bleeding, pain), laboratory examination data, quality of life (QOL), and safety at 12, 24, and 52 weeks of administration. Results: Eleven patients with LAs (9 generalized lymphatic anomaly, 1 cystic LM, 1 Gorham-Stout disease) were treated with sirolimus, of whom 6 (54.5%; 95% confidence interval: 23.4-83.3%) demonstrated a partial response on radiological examination at 52 weeks of administration. No patients achieved a complete response. At 12 and 24 weeks of administration, 8 patients (72.7%) already showed a partial response. However, patients with stable disease showed minor or no reduction after 12 weeks. Adverse events, such as stomatitis, acneiform dermatitis, diarrhea, and fever, were common with sirolimus. Sirolimus was safe and tolerable. Conclusion: Sirolimus can reduce the lymphatic tissue volume in LAs and may lead to improvements in clinical symptoms and QOL.

A Case of Multifocal Lymphangioendotheliomatosis With Thrombocytopenia and Changes in Coagulopathy.

Multifocal lymphangioendotheliomatosis with thrombocytopenia is a rare disease characterized by progressive multiple vascular lesions and is accompanied by thrombocytopenia. The precise diagnosis of this disease is frequently difficult because of the heterogeneity of the clinical symptoms. We report a case of a male infant who presented with severe thrombocytopenia induced by local inflammation. In addition, enlargement of the extremities with soft tissue and bone involvement without gastrointestinal bleeding was observed. The thrombocytopenia resolved after a combination therapy of sirolimus and prednisolone. Our finding that plasma angiopoietin-2 concentrations reflected the disease status suggests its utility as a biomarker of Multifocal lymphangioendotheliomatosis with thrombocytopenia.

Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations.

Recent studies have shown that the PI3K signaling pathway plays an important role in the pathogenesis of slow-flow vascular malformations (SFVMs). Analysis of genetic mutations has advanced our understanding of the mechanisms involved in SFVM pathogenesis and may identify new therapeutic targets. We screened for somatic variants in a cohort of patients with SFVMs using targeted next-generation sequencing. Targeted next-generation sequencing of 29 candidate genes associated with vascular anomalies or with the PI3K signaling pathway was performed on affected tissues from patients with SFVMs. Fifty-nine patients with SFVMs (venous malformations n = 21, lymphatic malformations n = 27, lymphatic venous malformations n = 1, and Klippel-Trenaunay syndrome n = 10) were included in the study. TEK and PIK3CA were the most commonly mutated genes in the study. We detected eight TEK pathogenic variants in 10 samples (16.9%) and three PIK3CA pathogenic variants in 28 samples (47.5%). In total, 37 of 59 patients (62.7%) with SFVMs harbored pathogenic variants in these three genes involved in the PI3K signaling pathway. Inhibitors of this pathway may prove useful as molecular targeted therapies for SFVMs.

A rare pediatric case of McCune-Albright syndrome with acute visual disturbance: Case report.

RATIONALE: McCune-Albright syndrome (MAS) is a rare disorder characterized by clinical findings, which includes fibrous dysplasia (FD). FD is a benign tumor that leads to increased rates of bone fracture. In some MAS cases with FD, facial deformities, severe pain, and orbital neuropathies are complicated. Aneurysmal bone cyst (ABC) is a benign bone tumor and rare complication of FD. PATIENT CONCERNS: A 9-year-old boy was admitted to our hospital because of acute visual disturbance. DIAGNOSIS AND INTERVENTIONS: The patient was clinically diagnosed as ABC complicated with MAS, and he underwent surgery. OUTCOMES: After the surgery, his sight became normal. Recurrence of ABC and visual disturbance was not observed in 3 years. Genetic analysis of a tissue sample from the ABC lesion by next-generation sequencing revealed a somatic activating GNAS mutation. LESSONS: To the best of our knowledge, this is the first case report of MAS causing optic neuropathy complicated with ABC. ABC complicated with MAS is extremely rare, but it should be considered as a possible diagnosis in patients with acute visual loss and facial swelling. In addition, our case had OAS, which is an uncommon syndrome and a rare complication in ABC with MAS, and rapid decompression of the ABC was effective in improving the patient's eyesight.

Characterization of kaposiform lymphangiomatosis tissue-derived cells.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a recently characterized systemic lymphatic anomaly. Activation of RAS/MAPK and PI3K/AKT/mTOR pathways may affect KLA pathogenesis, but the cellular basis of KLA is unclear. Abnormal-spindle endothelial cells that express lymphatic endothelial cell (LEC) markers are characteristic of KLA histopathology. This study evaluated patient-derived KLA cells to establish their morphological and biological characteristics. PROCEDURE: We established cell lines from primary KLA tissues of two patients with KLA and examined their morphological and functional characteristics, messenger RNA and protein expression profiles, gene mutations, and responses to inhibitors of the RAS/MAPK and PI3K/AKT/mTOR pathways. RESULTS: Both KLA cell lines showed spindle-shaped morphology, stained positive for podoplanin (PDPN), and exhibited impaired tube-formation properties. They expressed LEC marker PDPN and mesenchymal stem cell markers (CD90, CD105) in the absence of endothelial cell markers (CD34, CD31, VWF), per real-time polymerase chain reaction. Both mTOR inhibitor rapamycin and MEK inhibitor trametinib inhibited growth of the two cell lines. A NRAS p.Q61R variant was found in one of two independent KLA tissue samples, but not in the KLA cells (per targeted next-generation sequencing); and KLA cells with this variant had elevated AKT phosphorylation levels. ERK phosphorylation levels were undetectable in both KLA cell lines. CONCLUSIONS: Inhibition of the RAS/MAPK and PI3K/AKT/mTOR pathways may represent potential therapeutic targets in KLA. These patient-derived KLA cell lines will be useful research tools to elucidate KLA etiology, and could pave the way for basic, translational, and preclinical studies of this disease.

A pediatric case of anaplastic astrocytoma with a gliomatosis cerebri; the growth pattern and changes in serum VEGF-121 levels after bevacizumab treatment.

Gliomatosis cerebri (GC) is a rare diffusely infiltrating glial neoplasm that carries a poor prognosis. Because tumors are undetectable in most patients at early-stage of the onset, a useful diagnostic method is expected. We compared serum vascular endothelial growth factor (VEGF)-121 levels in patients with GC or glioblastoma and controls. VEGF-121 levels were significantly higher in one patient with GC and patients with glioblastoma than in controls. VEGF-121 levels decreased in a patient with GC after bevacizumab-based therapy. Thus, VEGF-121 may be useful for diagnosing GC, its disease-monitoring and understanding its etiology.

A somatic activating KRAS variant identified in an affected lesion of a patient with Gorham-Stout disease.

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.

Myelodysplastic syndromes in a pediatric patient with Cri du Chat syndrome with a ring chromosome 5.

Few hematological complications have previously been reported in association with Cri du Chat syndrome (CdCS). A case of myelodysplastic syndromes (MDS) in a pediatric patient with CdCS is herein presented. A 17-year-old female with CdCS caused by ring chromosome 5 was admitted to the hospital for investigation of a 1-month history of anemia. Based on the morphological findings of bone marrow, the patient was diagnosed with refractory cytopenia with multilineage dysplasia. The risk group was classified as intermediate-1 in the International Prognostic Scoring System (IPSS), and low in the revised IPSS. Assessment by microarray comparative genomic hybridization (CGH) identified the breakpoints of ring chromosome 5 as 46,XX,r(5)(p14.3q35.3). This revealed that the 5q terminal deletion did not include the common deleted region of MDS with del(5q). Treatment with azacitidine was initiated to control disease progression and improve quality of life. At baseline, the patient had a mean transfusion requirement of 3 units/month, which decreased to 2 units/month after six cycles of azacitidine and to 1 unit/month after 10 cycles of azacitidine. Cytopenia observed in the presented case seemed irrelevant to ring chromosome 5 which is the causative cytogenetic abnormality of CdCS, and further analyses may be needed to clarify the pathogenesis.

Identification of Homozygous Somatic DICER1 Mutation in Pleuropulmonary Blastoma.

Pleuropulmonary blastoma (PPB) is a rare, progressive, and aggressive malignant intrathoracic tumor observed during childhood. Mutations in the DICER1 gene have been considered a major etiologic factor of PPB and cause a variety of tumor types in children and young adults. We present a 3-year-old boy with type II PPB. Multimodal treatment consisting of surgery and neoadjuvant chemotherapy was effective. DICER1 mutations were examined by Sanger sequencing, microarray comparative genomic hybridization, and microsatellite markers. The results revealed that a somatic biallelic DICER1 mutation with uniparental disomy was present in the tumor tissue.

Proinflammatory Cytokine Secretion in a Patient With Recurrent Neuroblastoma Related to the Onset of Malignancy-associated Hemophagocytic Lymphohistiocytosis.

The onset of malignancy-associated hemophagocytic lymphohistiocytosis (M-HLH) may be associated with the secretion of proinflammatory cytokines from malignant cells. We here report a patient with recurrent neuroblastoma who developed hemophagocytic lymphohistiocytosis after surgery for brain metastases and relapsed after chemotherapy. The neuroblastoma cells in the recurrent tumor were positive for tumor necrosis factor-α and interleukin-6, whereas the primary site was negative. The secretion of proinflammatory cytokines from neuroblastoma cells may have been related to the pathogenesis of M-HLH in our patients. We should pay attention to possible development of M-HLH when treating advanced neuroblastoma.

Immunologic Effects of Sirolimus in Patients With Vascular Anomalies.

Emerging data have suggested that sirolimus may be a treatment option for complicated vascular anomalies (VAs). The present study aimed to investigate the immunologic effects of sirolimus treatment for 6 months in patients with VAs. Blood samples obtained from the patients enrolled in 2 multicenter studies to investigate the efficacy of sirolimus for VAs before and after sirolimus treatment for 6 months were used. Data for total white blood cell count, absolute lymphocyte count, serum immunoglobulins (Igs) levels (IgG, IgA, IgM), lymphocyte proliferation assays with mitogens including phytohemagglutinin and concanavalin A, and flow cytometric analysis of lymphocyte subsets were evaluated. A total of 18 patients with VAs receiving sirolimus treatment were included in the study. Comparisons of white blood cell, absolute lymphocyte count, IgG, IgA, IgM, and reaction rates of phytohemagglutinin and concanavalin A revealed no significant differences before and after treatment. No significant differences were observed in the absolute counts of lymphocyte subtypes before and after treatment, except for regulatory T-cell counts, which were significantly decreased after treatment. Severe infections were not observed during sirolimus treatment. The immunologic parameters assessed in the present study were hardly affected by sirolimus treatment for 6 months in patients with VAs.

Detection of NRAS mutation in cell-free DNA biological fluids from patients with kaposiform lymphangiomatosis.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) has recently been distinguished as a novel subtype of generalized lymphatic anomaly (GLA) with foci of spindle endothelial cells. All cases of KLA involve multiple organs and have an unfavorable prognosis. However, the molecular pathogenesis is unknown, and there are no useful biomarkers. In the present study, we performed genetic analysis to elucidate the cause of this disease and detect biomarkers for it. METHODS: We performed whole-exome sequencing of DNA samples from leukocytes and a biopsy specimen and analyzed cell-free DNA (cfDNA) from plasma and pleural effusion of patients to identify the NRAS c.182A > G (p.Q61R) mutation using the droplet digital polymerase chain reaction (ddPCR). RESULTS: All KLA patients (patients 1-5) had invasive and aggressive features (hemorrhagic pleural effusions, coagulation disorder, and thrombocytopenia) and characteristic findings of KLA in their pathological examinations. In whole exome sequencing for patient 1, c.182A > G missense variant (p.Q61R) in NRAS was identified in fresh frozen samples of a mass on the left chest wall at a frequency of 5% of total alleles but not in his blood leukocytes. Furthermore, the same mutation was detected in cfDNA isolated from plasma and pleural effusion by using ddPCR. ddPCR analysis of plasma/pleural effusion samples from an additional four KLA patients showed that the same mutation was detected in isolated cfDNA in three of the four, as well as in a tissue sample from one of the three plasma/effusion-positive patients that had been obtained to confirm the mutation. CONCLUSION: These results provide the first evidence that NRAS oncogenic variant was identified in DNA samples from KLA patients from not only two affected lesions but also plasma and pleural effusion.

Perampanel Inhibits Neuroblastoma Cell Proliferation Through Down-regulation of AKT and ERK Pathways.

BACKGROUND/AIM: Activation of AKT serine/ threonine kinase (AKT) predicts poor outcome in neuroblastoma, which highlights the potential of the AKT pathway as a promising target for neuroblastoma treatment. Several studies reported that blockade of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) reduces proliferation in glioblastoma or lung cancer by inhibiting AKT and extracellular signal-related kinase (ERK) pathways. In this study, we examined the effect of the AMPAR antagonist perampanel on human neuroblastoma cells. MATERIALS AND METHODS: Cell proliferation, caspase activity, and western blot assays were performed to determine the effect of perampanel on the KP-N-SI9s human neuroblastoma cell line. RESULTS: Perampanel inhibited cell proliferation without triggering apoptosis in neuroblastoma cells. Down-regulation of AKT protein levels, AKT phosphorylation, and ERK1/2 phosphorylation were also observed in neuroblastoma cells with perampanel treatment. CONCLUSION: Perampanel inhibits neuroblastoma cell proliferation through down-regulation of AKT and ERK pathways and has potential for the treatment of neuroblastoma.

Potential biomarkers of kaposiform lymphangiomatosis.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) has recently been distinguished as a novel subtype of generalized lymphatic anomaly (GLA), and is characterized by foci of spindle endothelial cells amid a background of malformed lymphatic channels. The etiology of these diseases remains unknown and diagnosis is confounded by their similar clinical findings. This study aimed to clarify differences in the clinical findings and plasma cytokine profiles of GLA and KLA patients. PROCEDURE: Clinical features data of GLA and KLA patients were obtained from a national survey. Differences in clinical findings, plasma levels of cytokines, and survival were analyzed. Plasma was obtained from healthy controls and GLA and KLA patients. Thirty-six angiogenic and lymphangiogenic factors were evaluated for cytokine concentration. RESULTS: Twenty-one patients with GLA and 11 with KLA were recruited. Mediastinal masses, hemorrhagic pericardial and pleural effusion, coagulation disorders, and thrombocytopenia were more frequent in KLA than in GLA. KLA had a significantly poorer outcome than GLA (P = 0.044). Soluble VEGFR3, angiopoietin 2, HGF, soluble HER2, tenascin C, and soluble HGFR levels were higher in KLA. Notably, soluble VEGFR3 and angiopoietin 2 levels were approximately 10-fold higher than those of other molecules measured. However, soluble VEGFR1 and soluble TIE2 were lower in KLA than in GLA and the controls. CONCLUSIONS: Patients with KLA have an unfavorable prognosis and serious symptoms (hemorrhagic pleural effusion and coagulation disorders). Our data indicate that eight angiogenic cytokines might be potential biomarkers of KLA.

The impact of sirolimus therapy on lesion size, clinical symptoms, and quality of life of patients with lymphatic anomalies.

BACKGROUND: Lymphatic anomalies (LAs) include several disorders in which abnormal lymphatic tissue invades the neck, chest, and various organs. Progressive cases may result in lethal outcomes and have proven difficult to treat. Sirolimus is showing promising results in the management of vascular anomalies. We examined the efficacy and safety of sirolimus treatment in patients with progressive LAs. METHODS: All patients with LAs treated with sirolimus from May 2015 to September 2018 were included. They received oral sirolimus once a day and the dose was adjusted so that the trough concentration remained within 5-15 ng/mL. We prospectively reviewed the response to drugs (the response rate of radiological volumetric change of the target lesion), severity scores, reported quality of life (QOL), and adverse effects at 6 months after administration. RESULTS: Twenty patients (five with cystic lymphatic malformation (LM), three with kaposiform lymphangiomatosis, three with generalized lymphatic anomaly, six with Gorham-Stout disease, and three with central conducting lymphatic anomaly) were treated with sirolimus at our institution. Fifty percent of patients (10/20) demonstrated a partial response by a radiological examination and a significant improvement in disease severity and QOL scores (P = 0.0020 and P = 0.0117, respectively). Ten patients who had no reduction in lesion size (stable disease group) showed no significant improvement in disease severity and QOL scores. Eighty percent of patients (16/20) had side effects, such as stomatitis, infection, and hyperlipidemia. CONCLUSIONS: Sirolimus impacts the reduction of the lymphatic tissue volume of LMs and could lead to improvement in clinical symptoms and QOL. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN000016580 . Registered 19 February 2015.

Fatal Progression of Gorham-Stout Disease with Skull Base Osteomyelitis and Lateral Medullary Syndrome.

Gorham-Stout disease (GSD) is a rare condition in which spontaneous, progressive resorption of bone occurs. There are no previous reports of patients with fatal progression of GSD with skull base osteomyelitis (SBO) and lateral medullary syndrome (LMS). We present the case of a 27-year-old man diagnosed with GSD with involvement of the maxillofacial bones and skull base. The patient developed SBO; LMS resulted from progressive osteolysis, and the patient died of associated brainstem stroke. Careful follow-up with special emphasis on the early detection of intracranial complications is critical in patients presenting with progressive GSD with involvement of the skull base.

Hereditary clear cell meningiomas in a single family: three-cases report.

Clear cell meningiomas (CCMs) are rare subtypes of meningiomas and usually treated by maximum safely achievable tumor resection. We here present three hereditary cases with CCMs which were confirmed by Sanger sequencing of lymphocyte DNA. Gross total resection was achieved in a 5-year-old son with a spinal CCM and a 34-year-old father with a CCM in the cerebellopontine angle (CPA). For a 14-year-old daughter with CCM in the CPA, total resection was not achieved due to its large size. Early detection by screening high-risk family with CCM is strongly recommended.