Wylam, Mark E.

Abstract Background Continuous positive airway pressure (CPAP) is a common mode of respiratory support used in neonatal intensive care units. In preterm infants, nasal CPAP (nCPAP) therapy is often delivered via soft, biocompatible nasal mask suitable for long-term direct-skin contact and held firmly against face. Limited sizes of nCPAP mask contribute to mal-fitting related complications and adverse outcomes in this fragile population. We hypothesized that custom-fit nCPAP masks will improve the fit with less skin pressure and strap tension improving efficacy and reducing complications associated with nCPAP therapy in neonates. Methods After IRB approval and informed consent we evaluated several methods to develop 3D facial models to test custom 3D nCPAP masks. These methods included camera-based photogrammetry, laser scanning and structured light scanning using a Bellus3D Face Camera Pro and iPhone X running either Bellus3D FaceApp for iPhone, or Heges application. This data was used to provide accurate 3D neonatal facial models. Using CAD software nCPAP inserts were designed to be placed between proprietary nCPAP mask and the model infant’s face. The resulted 3D designed nCPAP mask was form fitted to the model face. Subsequently, nCPAP masks were connected to a ventilator to provide CPAP and calibrated pressure sensors and co-linear tension sensors were placed to measures skin pressure and nCPAP mask strap tension. Results Photogrammetry and laser scanning were not suited to the neonatal face. However, structured light scanning techniques produced accurate 3D neonatal facial models. Individualized nCPAP mask inserts manufactured using 3D printed molds and silicon injection were effective at decreasing surface pressure and mask strap pressure in some cases by more than 50% compared to CPAP masks without inserts. Conclusions We found that readily available structured light scanning devices such as the iPhoneX are a low cost, safe, rapid, and accurate tool to develop accurate models of preterm infant facial topography. Structured light scanning developed 3D nCPAP inserts applied to commercially available CPAP masks significantly reduced skin pressure and strap tension at clinically relevant CPAP pressures when utilized on model neonatal faces. This workflow maybe useful at producing individualized nCPAP masks for neonates reducing complications due to malfit.

The colonization of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has a detrimental effect on the respiratory care of pediatric patients with cystic fibrosis (CF). In addition to being resistant to multiple antibiotics, S. aureus also has the ability to form biofilms, which makes the infection more difficult to treat and eradicate. In this study, we examined the ability of S. aureus strains isolated from pediatric patients with CF to form biofilms. We screened a transposon mutant library of MRSA and identified a putative cobalt transporter ATP binding domain (cbiO) that is required for biofilm formation. We discovered that deleting cbiO creating a cbiO null mutant in CFSa36 (an MRSA strain isolated from a patient with cystic fibrosis) significantly hinders the ability of CFSa36 to form biofilm. The complementation of cbiO restored the ability of the cbiO deletion mutant to generate biofilm. Interestingly, we revealed that incorporating extra copper ions to the chemically defined medium (CDM) complemented the function of cbiO for biofilm formation in a dose-dependent manner, while the addition of extra iron ions in CDM enhanced the effect of cbiO null mutation on biofilm formation. In addition, neither the addition of certain extra amounts of copper ions nor iron ions in CDM had an impact on bacterial growth. Taken together, our findings suggest that cbiO mediates biofilm formation by affecting the transportation of copper ions in the MRSA CFSa36 strain. This study provides new insights into the molecular basis of biofilm formation by S. aureus.

Current guidelines suggest screening all patients with idiopathic pulmonary arterial hypertension for genetic aberrations, particularly mutations in Bone Morphogenic Protein Receptor Type II (BMPR2), the gene most commonly implicated in the pathogenesis of PAH. Herein, we present a novel technique used to identify a pathogenic germline BMPR2 alteration in a 36‐year‐old female and family members with hereditary pulmonary arterial hypertension who each screened negative by standard cytogenetics and molecular genetics testing.

Smoking contributes to or exacerbates diseases such as asthma and COPD, where airway hyperreactivity is a key feature. Enhanced Ca 2+ in airway smooth muscle contributes to airway hyperreactivity. We previously determined that the cytokine TNFα upregulates expression of Ca 2+ regulatory molecules such as membrane bound bifunctional enzyme CD38 and Ca 2+ influx regulatory TRPC3 in human airway smooth muscle cells (HASM). We hypothesized that cigarette smoke (CS) is an inflammatory stimulus that upregulates Ca 2+ regulatory mechanisms in HASM. Western analysis showed increased expression of CD38, TRPC3 and TRPC6 in lungs from mice following 4 week in vivo CS exposure. These proteins were also upregulated in HASM cells incubated in 1 or 2% CS extract (CSE), as well as in HASM samples from patients with COPD (compared to controls). In fura‐2 loaded HASM cells, exposure to CSE enhanced histamine‐mediated Ca 2+ mobilization as well as store‐operated calcium entry: effects substantially blunted by TRPC3 siRNAs. Finally, in living lung slices of mice exposed for 4 weeks to CS, ACh‐induced airway contractility was substantially greater, compared to controls. These results indicate that CS upregulates expression and function of Ca 2+ regulatory proteins, thus contributing to airway hyperreactivity in smoking related diseases. Supported by the Annenberg Foundation (MEW), HL090595 (CMP), FAMRI (RV, YSP).

Interstitial lung diseases (ILDs) in patients with shortened telomeres have not been well characterized. We describe demographic, radiologic, histopathologic, and molecular features, and p16 expression in patients with telomeres ≤10th percentile (shortened telomeres) and compare them to patients with telomere length >10th percentile. Lung explants, wedge biopsies, and autopsy specimens of patients with telomere testing were reviewed independently by 3 pathologists using defined parameters. High-resolution computed tomography scans were reviewed by 3 radiologists. p16-positive fibroblast foci were quantified. A multidisciplinary diagnosis was recorded. Patients with shortened telomeres (N=26) were morphologically diagnosed as usual interstitial pneumonia (UIP) (N=11, 42.3%), chronic hypersensitivity pneumonitis (N=6, 23.1%), pleuroparenchymal fibroelastosis, fibrotic nonspecific interstitial pneumonia, desquamative interstitial pneumonia (N=1, 3.8%, each), and fibrotic interstitial lung disease (fILD), not otherwise specified (N=6, 23.1%). Patients with telomeres >10th percentile (N=18) showed morphologic features of UIP (N=9, 50%), chronic hypersensitivity pneumonitis (N=3, 16.7%), fibrotic nonspecific interstitial pneumonia (N=2, 11.1%), or fILD, not otherwise specified (N=4, 22.2%). Patients with shortened telomeres had more p16-positive foci (P=0.04). The number of p16-positive foci correlated with outcome (P=0.0067). Thirty-nine percent of patients with shortened telomeres harbored telomere-related gene variants. Among 17 patients with shortened telomeres and high-resolution computed tomography features consistent with or probable UIP, 8 (47.1%) patients showed morphologic features compatible with UIP; multidisciplinary diagnosis most commonly was idiopathic pulmonary fibrosis (N=7, 41.2%) and familial pulmonary fibrosis (N=5, 29%) in these patients. In conclusion, patients with shortened telomeres have a spectrum of fILDs. They often demonstrate atypical and discordant features on pathology and radiology leading to diagnostic challenges.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Introduction: Short telomere syndromes (STS) are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems. Clinical assessment of age-appropriate telomere length (TL) is performed using flow cytometry & fluorescence in-situ hybridization (flowFISH). Screening for germline variants in STS-related genes is guided by flowFISH-determined centile categories of TL, with screening recommended for TL <1st centile or 1-10th centile in lymphocytes (L) or granulocytes (G). However, the utility of genetic testing for patients with TL >10th centile and integration of clinical phenotype with flowFISH data in predictive algorithms is currently unclear. Methods: FlowFISH testing was done at reference laboratories in Vancouver (Repeat Diagnostics; Canada) & Johns Hopkins University (JHU, USA). Salient clinical features were pre-determined as, personal history of premature hair greying (onset at age < 30 years), idiopathic pulmonary fibrosis (IPF) or IPF/emphysema overlap (in smokers), cryptogenic cirrhosis or NRH, unexplained cytopenias &/or immunodeficiency, & family history of the above (in >1 1st or 2nd degree relatives). Clinical likelihood score (CLS) was assigned as low (1), intermediate (int, 2) or high (>2), based on the number of aforementioned clinical features present prior to flowFISH testing. Genetic testing was performed using either an in-house or commercial bone marrow failure-specific next generation sequencing (NGS) panel or whole exome sequencing (WES), and data for known variants affecting telomerase or telomeric function (TERT, TERC, DKC1, TINF2, NHP2, NOP10, TCAB1, NAF1, & RTEL1) was recorded. Results: One hundred forty-nine patients at our institution underwent TL assessment at Repeat diagnostics (n=38) and JHU (n=111) laboratories, respectively. Median age was 56 (range: 7-79) years; 88 (59%) being males. Significant family history was present in 40 (27%) patients, while premature greying of hair was present in 13 (9%) patients. Organ-specific clinical features included unexplained cytopenias (n=89, 60%) IPF (n=71, 48%), cryptogenic cirrhosis or NRH (n=21, 14%), & unexplained immunodeficiency (n=14, 9%). CLS stratification included low (n=74, 50%), int (n=54, 36%), & high (n=21, 14%), with higher CLS significantly correlating with lower delta TL for L (p=0.0005) but not G (p=0.3). Genetic testing was performed in 51 (35%) patients (NGS-51, WES-1) among which 13 (26%) patients had a telomere-associated variant; 5 (10%) pathogenic (pv, all TERT). CLS alone was unable to predict likelihood of finding a telomere-associated variant (p=0.4). Based on age-appropriate centile categorization of L & G TL (information for both available in 134 patients), patients were stratified into six groups (table 1). TL <1st centile in L: This group was further divided into two groups; TL<1st centile in both L & G [A1, n=7, CLS low-3 (43%), int-2 (29%), & high-2 (29%)] and TL <1st centile in L and 1-50th centile in G (A2, n=2, CLS low & high) patients. Among the 4 (57%) patients who underwent genetic (NGS-3, WES-1) testing, 3 (75%) had TERTpv in A1 subgroup and 1 A2 subgroup patient had a VUS in TERT.TL <1st centile in G, 1-10th centile in L (n=18): This group included 9 (50%) low, 8 (44%) int and 1 (6) high CLS patients, of which only 1 of 8 NGS-tested patients had TERTpv.TL 1-10th centile in L or G: This group was divided into; 1-10th centile in both L & G [C1, n=28, CLS low-3 (11%), int-14 (50%), high-11(39%)] of whom 8 (22%) underwent NGS with no pathogenic variants but 3 VUS in RTEL1, NAF1 & PARN genes, and 1-10th centile in L, >1-90th centile in G [C2, n=36, CLS low-19 (53%), 16 (44%), 1 (3%)] of whom 8 (22%) underwent NGS with 1 TERTpv and 2 VUS in TINF2TL >10th centile in L & 1-90th centile in G (n=43, 32%): CLS stratification in this group included 27 (63%) low, 12 (28%) int, 4 (9%) high. NGS testing was done in 13 (30%) patients [CLS low-9(69%), int 2(15%), high 2 (15%)], of whom only 2 (15%) had VUS in TINF2 and TERT gene, but no pathogenic variants (figure 1). Conclusion: Our study demonstrates the importance of using a flowFISH assay based predictive algorithm to screen patients with suspected STS for telomere-related genetic alternations, in comparison to a clinical likelihood score. We also demonstrate a limited role for genetic testing in patients with lymphocyte TL >10th centile, regardless of the clinical likelihood score. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

AbstractBackgroundLung transplant recipients are prone to invasive fungal infections prompting many transplant centers to use prolonged triazole antifungal prophylaxis. From a practical standpoint, it is unclear if lung transplant recipients are able to continue prolonged or lifelong prophylaxis without premature discontinuation from side effects, drug interactions, development of fungal disease, or medication cost. We examined the number of patients that are able to reach a prophylactic endpoint and understand the reasons for early termination.MethodsWe conducted a retrospective chart review of all lung and heart‐lung transplant patients at Mayo Clinic Rochester from May 1, 2002 to December 31, 2017. Type, duration, and reason for discontinuation of triazole prophylaxis were examined.ResultsDuring the study period, 193 patients underwent lung or heart‐lung transplantation. Itraconazole, voriconazole, and posaconazole were given to 180, 73, and 60 post‐transplant patients, respectively. Providers switched itraconazole to another prophylactic antifungal medication for reasons other than prophylactic completion in 61.8% (126 out of 204) of exposure episodes; this was similar with voriconazole (68.8%, 53 out of 77, P = 0.41). Posaconazole was actively discontinued significantly less often (18.3%, 11 out of 60, P < 0.05). The most common reasons for discontinuing itraconazole were malabsorption (15.5% of exposure episodes) and concern for breakthrough fungal infection (10.2%). In comparison, the most common reason for voriconazole discontinuation was side effect or intolerance (54.5% of VR exposure episodes vs 9.8% of IT exposure episodes, P < 0.05).ConclusionsItraconazole and posaconazole appeared to have fewer side effects prompting discontinuation than voriconazole, but itraconazole was discontinued more often because of malabsorption and clinical suspicion of fungal infections.