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OBJECTIVES: Historically, head and neck lymphatic malformations (HNLM) have been managed through surgical and interventional procedures. Sirolimus was introduced in 2016 and has aided in symptomatic control of HNLM. The study objective was to assess healthcare utilization with the introduction of sirolimus for HNLM. METHODS: An observational cohort study of LM patients treated between 2008 and 2022 at a tertiary care children's hospital was performed. 588 charts were reviewed; patients with isolated, non-syndromic HNLM and at least 2 years of follow-up were included (n = 45). Data included sirolimus use, complications, presence of tracheostomy and/or gastrostomy-tube, and number and costs of HNLM-related sclerotherapies, procedures, hospitalizations, and emergency room visits. For patients who received sirolimus, encounters two years prior to and after sirolimus initiation were recorded. For the non-sirolimus group, encounters two years after the initial clinic visit for HNLM were recorded. Statistical analysis was used to compare the groups. RESULTS: Median age at first clinic visit was 1.8 years (range 2 days-41 years). Tracheostomy was present in 43 % of sirolimus patients compared with 3 % of the non-sirolimus group (OR: 24.0, 95%CI: 1.55-1490, p = 0.02). Patients on sirolimus experienced significantly fewer sclerotherapy visits (z = 2.08, p = 0.03) compared to the non-sirolimus group. Minimal sirolimus-related side effects were reported. Total HNLM-related costs were significantly less in the sirolimus group during treatment (median $448.13, range $0-$7041.28) compared with before treatment (median $17,069.24, range $1999.16-$211,848.50, z = 2.20, p = 0.03). Median costs associated with sclerotherapy were less for the sirolimus groups during treatment compared with the non-sirolimus group (z = 1.97, p = 0.04). In the sirolimus group, costs associated with HNLM-related hospitalizations were significantly less during sirolimus treatment compared with before (z = 2.20, p = 0.03). CONCLUSION: Sirolimus has improved the clinical course for HNLM patients by decreasing number of procedures and healthcare costs, with limited side effects. Larger cohorts matching type of HNLM and age are needed to assess healthcare utilization benefits of sirolimus.
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The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods ( Ziegler et al., 2021 ). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures. Graphic abstract: Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.