Efficacy of systemic sirolimus in the treatment of generalized lymphatic anomaly and Gorham-Stout disease.

BACKGROUND: Generalized lymphatic anomaly (GLA) and Gorham-Stout disease (GSD) are rare complicated lymphatic malformations that occur in multiple body sites and are associated with significant morbidity and mortality. Treatment options have been limited, and conventional medical therapies have been generally ineffective. Emerging data suggest a role for sirolimus as a treatment option for complex lymphatic anomalies. PROCEDURE: Disease response was evaluated by radiologic imaging, quality of life (QOL), and clinical status assessments in children and young adults with GLA and GSD from a multicenter systematic retrospective review of patients treated with oral sirolimus and the prospective phase 2 clinical trial assessing the efficacy and safety of sirolimus in complicated vascular anomalies (NCT00975819). Sirolimus dosing regimens and toxicities were also assessed. RESULTS: Eighteen children and young adults with GLA (n = 13) or GSD (n = 5) received oral sirolimus. Fifteen patients (83%) had improvement in one or more aspects of their disease (QOL 78%, clinical status 72%, imaging 28%). No patients with bone involvement had progression of bone disease, and the majority had symptom or functional improvement on sirolimus. Improvement of pleural and pericardial effusion(s) occurred in 72% and 50% of affected patients; no effusions worsened on treatment. CONCLUSIONS: Sirolimus appears effective at stabilizing or reducing signs/symptoms of disease in patients with GLA and GSD. Functional impairment and/or QOL improved in the majority of individuals with GLA and GSD with sirolimus treatment.

A xenograft model for venous malformation.

Vascular malformations are defects caused by the abnormal growth of the vasculature. Among them, venous malformation (VM) is an anomaly characterized by slow-flow vascular lesions with abnormally shaped veins, typically in sponge-like configuration. VMs can expand over years causing disfigurement, obstruction of vital structures, thrombosis, bleeding, and pain. Treatments have been very limited and primarily based on supportive care, compression garments, sclerotherapy, and/or surgical resection. Sirolimus treatment has recently shown efficacy in some patients with complicated vascular anomalies, including VMs. Activating somatic TIE2 gene mutations have been identified in up to 60% of VMs and PIK3CA mutations have been found in another 25%. Here, we report a xenograft model of VM that reflects the patients' mutation heterogeneity. First, we established a protocol to isolate and expand in culture endothelial cells (VM-EC) from VM tissue or VM blood of nine patients. In these cells, we identified somatic mutations of TIE2, PIK3CA, or a combination of both. Both TIE2 and PIK3CA mutations induced constitutive AKT activation, while TIE2 mutations also showed high MAPK-ERK signaling. Finally, VM-EC implanted into immune-deficient mice generated lesions with ectatic blood-filled channels with scarce smooth muscle cell coverage, similar to patients' VM. This VM xenograft model could be instrumental to test the therapeutic efficacy of Sirolimus in the presence of the different TIE2 or PIK3CA mutations or to test for efficacy of additional compounds in targeting the specific mutated protein(s), thus enabling development of personalized treatment options for VM patients.

Developmental pharmacokinetics of sirolimus: Implications for precision dosing in neonates and infants with complicated vascular anomalies.

BACKGROUND: Sirolimus has recently been shown to be efficacious and tolerable in pediatric patients with complicated vascular anomalies. Nevertheless, dosing information remains very limited especially for neonates and infants. The purpose of this study was to develop an age-appropriate sirolimus starting dosing regimen based on the developmental changes in drug elimination capacity using data collected in neonates and infants. PROCEDURE: A recently developed sirolimus maturation model [Emoto et al. CPT Pharmacometrics Syst Pharmacol, 2016] was used to simulate clearance estimates using realistic age and weight covariates for age cohorts aged 0-24 months. Next, predose concentrations at steady state were generated for each age cohort of neonates and infants. Dose requirements to attain predefined target trough concentration ranges (10-15 and 5-10 ng/ml) were simulated across the different age groups. Starting doses were chosen to maximize the likelihood of achieving sirolimus-targeted concentrations. RESULTS: The trajectory of simulated sirolimus clearances increased with age and was in agreement with the previous findings in the Phase 2 study. The proposed dosing regimens covered eight age cohorts and resulted in target attainment of more than 75-95% across selected regimens. CONCLUSIONS: This study identified age-appropriate sirolimus dosing regimens for neonates and infants. The algorithm in combination with therapeutic drug management will facilitate sirolimus precision dosing in young children with vascular anomalies. A prospective evaluation is being planned.

Angiopoietins as serum biomarkers for lymphatic anomalies.

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

Response of Blue Rubber Bleb Nevus Syndrome to Sirolimus Treatment.

BACKGROUND: Blue rubber bleb nevus syndrome (BRBNS) is a rare multifocal venous malformation syndrome involving predominantly the skin and gastrointestinal tract. Traditional treatment modalities include corticosteroids, interferon-α, sclerotherapy, and aggressive surgical resection. Sirolimus has been used in several single case reports. PROCEDURE: We performed a single-institution retrospective review of four children with BRBNS, who received sirolimus as part of their treatment regimens. A diagnosis of BRBNS was based on clinical, radiologic, and pathologic criteria. RESULTS: Median age was 6.5 years (range: 2-16 years). Pathologic evaluations revealed a combined malformation with venous and lymphatic components. The novel finding of a lymphatic component was confirmed with PROX-1 immunostaining. Patients received oral sirolimus with target drug levels between 10 and 13 ng/ml. Responses to treatment were defined as stabilization/decrease in size of lesions; resolution of transfusion requirements; reduction in pain, and improvement in quality of life (QOL). Median time to response was 1.5 months (SD ± 0.96 month, range: 1-3 months). Median follow-up was 21 months (range: 18-26 months). Lesion size and characteristics improved in all patients. All patients reported decrease in pain and improvement in QOL. All three patients requiring transfusions became transfusion-independent. One patient had resolution of coagulopathy. Adverse effects of sirolimus consisted of mucositis in three patients and neutropenia in one patient. CONCLUSIONS: Sirolimus is safe and efficient for the treatment of BRBNS. Further prospective studies are needed to evaluate the long-term effectiveness of this drug. This is the first report that identifies a lymphatic component as part of BRBNS.

Efficacy and Safety of Sirolimus in the Treatment of Complicated Vascular Anomalies.

BACKGROUND AND OBJECTIVES: Complicated vascular anomalies have limited therapeutic options and cause significant morbidity and mortality. This Phase II trial enrolled patients with complicated vascular anomalies to determine the efficacy and safety of treatment with sirolimus for 12 courses; each course was defined as 28 days. METHODS: Treatment consisted of a continuous dosing schedule of oral sirolimus starting at 0.8 mg/m(2) per dose twice daily, with pharmacokinetic-guided target serum trough levels of 10 to 15 ng/mL. The primary outcomes were responsiveness to sirolimus by the end of course 6 (evaluated according to functional impairment score, quality of life, and radiologic assessment) and the incidence of toxicities and/or infection-related deaths. RESULTS: Sixty-one patients were enrolled; 57 patients were evaluable for efficacy at the end of course 6, and 53 were evaluable at the end of course 12. No patient had a complete response at the end of course 6 or 12 as anticipated. At the end of course 6, a total of 47 patients had a partial response, 3 patients had stable disease, and 7 patients had progressive disease. Two patients were taken off of study medicine secondary to persistent adverse effects. Grade 3 and higher toxicities attributable to sirolimus included blood/bone marrow toxicity in 27% of patients, gastrointestinal toxicity in 3%, and metabolic/laboratory toxicity in 3%. No toxicity-related deaths occurred. CONCLUSIONS: Sirolimus was efficacious and well tolerated in these study patients with complicated vascular anomalies. Clinical activity was reported in the majority of the disorders.

Reaming as an adjuvant to irrigation on bacterial presence and propagation: an open, cadaveric intramedullary fracture model.

OBJECTIVES: The purpose of this investigation is to evaluate the effect of intramedullary reaming on bacterial presence and propagation in an open, cadaveric intramedullary fracture model. METHODS: Twelve fresh-frozen human cadaveric femurs were osteotomized and inoculated with Staphylococcus aureus, the open, cadaveric intramedullary fracture model. Low-pressure pulsed lavage irrigation was performed to irrigate the osteotomy sites. The specimens were divided into two groups of six paired specimens: CNT, irrigation only; and REAM, irrigation coupled with intramedullary reaming. Intramedullary contents were cultured at the osteotomy site and in 1-cm increments through the distal femoral metaphysis. Mean bacterial colony-forming units were compared between groups using analysis of variance. RESULTS: A statistically significant higher bacterial colony-forming unit count was noted at the osteotomy site (bacterial presence) in the CNT group compared with the REAM group. In terms of bacterial propagation, when compared with the sterile osteotomy site, the CNT group demonstrated significant bacterial propagation only at the 1.1- to 2.0-cm increment and the REAM group demonstrated no significant propagation. In comparing bacterial propagation between the CNT and the REAM groups, no significant differences were noted at any distal increment. CONCLUSION: In this open, cadaveric intramedullary fracture model, low-pressure pulse lavage coupled with intramedullary reaming demonstrated significantly less bacterial presence at the osteotomy site compared with irrigation without reaming. Additionally, intramedullary reaming does not appear to significantly propagate bacteria into the intramedullary canal nor into the distal metaphysis. These observations might have clinical significance.

Influence of CR3 (CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human neutrophils.

CR3 (CD11b/CD18) is expressed on neutrophils, and the engagement of CR3 can promote phagocytosis. CR3 serves as the receptor for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and for the adenylate cyclase toxin (ACT), which blocks neutrophil function. The influence of CR3, FHA, and ACT on the phagocytosis of B. pertussis by human neutrophils was examined. The surface expression and function of CR3 are regulated. Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) increased CR3 surface expression, but only TNF-alpha increased the ability of neutrophils to phagocytose B. pertussis, suggesting that elevated CR3 expression alone is not sufficient to promote phagocytosis. Purified FHA and pertussis toxin also increased the surface expression of CR3 on neutrophils, while ACT and the B subunit of pertussis toxin did not affect CR3 expression. FHA-mediated attachment to CR3 can lead to phagocytosis, especially in the absence of ACT. FHA mutants failed to attach and were not phagocytosed by neutrophils. Similarly, an antibody to CR3 blocked both attachment and phagocytosis. The addition of exogenous FHA enhanced the attachment and phagocytosis of wild-type B. pertussis and FHA mutants. Mutants lacking the SphB1 protease, which cleaves FHA and allows the release of FHA from the bacterial surface, were phagocytosed more efficiently than wild-type bacteria. ACT mutants were efficiently phagocytosed, but wild-type B. pertussis or ACT mutants plus exogenous ACT resisted phagocytosis. These studies suggest that the activation and surface expression of CR3, FHA expression, and the efficiency of ACT internalization all influence whether B. pertussis will be phagocytosed and ultimately killed by neutrophils.

Phagocytosis of Bordetella pertussis incubated with convalescent serum.

Convalescent serum samples were examined for the ability to promote phagocytosis of Bordetella pertussis by human neutrophils. One sample promoted phagocytosis, and 11 of the 51 samples caused a statistically significant reduction in phagocytosis, compared with that of bacteria not incubated with serum. Phagocytosis was influenced by interactions between antibodies that promoted phagocytosis and antibodies that inhibited phagocytosis. Adenylate cyclase toxin (ACT) has been shown to block phagocytosis by neutrophils. Antibodies to ACT were removed from the sample that promoted phagocytosis, by incubation with ACT-coated paramagnetic beads, and the depleted serum no longer enhanced phagocytosis. The adhesin filamentous hemagglutinin (FHA) has been shown to mediate attachment of B. pertussis to neutrophils in a way that promotes phagocytosis. Depletion of antibodies to FHA from samples that blocked phagocytosis improved phagocytosis, compared with the no-antibody control. These results suggest that antibodies to ACT can promote phagocytosis, whereas antibodies to FHA can counteract beneficial opsonins.