Showing papers by "David M. Hwang published in 2019"

Microbiome networks and change-point analysis reveal key community changes associated with cystic fibrosis pulmonary exacerbations

Abstract: Over 90% of cystic fibrosis (CF) patients die due to chronic lung infections leading to respiratory failure. The decline in CF lung function is greatly accelerated by intermittent and progressively severe acute pulmonary exacerbations (PEs). Despite their clinical impact, surprisingly few microbiological signals associated with PEs have been identified. Here we introduce an unsupervised, systems-oriented approach to identify key members of the microbiota. We used two CF sputum microbiome data sets that were longitudinally collected through periods spanning baseline health and PEs. Key taxa were defined based on three strategies: overall relative abundance, prevalence, and co-occurrence network interconnectedness. We measured the association between changes in the abundance of the key taxa and changes in patient clinical status over time via change-point detection, and found that taxa with the highest level of network interconnectedness tracked changes in patient health significantly better than taxa with the highest abundance or prevalence. We also cross-sectionally stratified all samples into the clinical states and identified key taxa associated with each state. We found that network interconnectedness most strongly delineated the taxa among clinical states, and that anaerobic bacteria were over-represented during PEs. Many of these anaerobes are oropharyngeal bacteria that have been previously isolated from the respiratory tract, and/or have been studied for their role in CF. The observed shift in community structure, and the association of anaerobic taxa and PEs lends further support to the growing consensus that anoxic conditions and the subsequent growth of anaerobic microbes are important predictors of PEs.

Mesenchymal stromal cell therapy during ex vivo lung perfusion ameliorates ischemia-reperfusion injury in lung transplantation.

Abstract: BACKGROUND The application of mesenchymal stromal cell (MSC)–based therapy during ex vivo lung perfusion (EVLP) could repair injured donor lungs before transplantation. The aim of this study was to determine the efficacy of MSC therapy performed during EVLP on ischemia-reperfusion injury using a pig lung transplant model. METHODS Following 24 hours of cold storage, pig lungs were randomly assigned to 2 groups (n = 6 each), the control group without MSC vs the MSC group, where 5 × 106 cells/kg MSCs were delivered through the pulmonary artery during EVLP. After 12 hours of EVLP, followed by a 1-hour second cold preservation period, the left lung was transplanted and reperfused for 4 hours. RESULTS EVLP perfusate hepatocyte growth factor (HGF) level at 12 hours was significantly elevated in the MSC group compared with the control and was associated with a significant decrease in cell death markers, cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling–positive cells, in the MSC group. The MSC group showed significantly lower interleukin (IL)-18 and interferon gamma levels and a significantly higher IL-4 level in lung tissue at 12 hours of EVLP than the control group. After transplantation, the MSC group showed a significant increase in lung tissue HGF level compared with the control group, associated with a significantly reduced lung tissue wet-to-dry weight ratio. Lung tissue tumor necrosis factor-α level and pathological acute lung injury score were significantly lower in the MSC group than the control group. CONCLUSIONS The administration of MSCs ameliorated ischemic injury in donor lungs during EVLP and attenuated the subsequent ischemia-reperfusion injury after transplantation.

Microbiota analysis optimization for human bronchoalveolar lavage fluid.

Abstract: It is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data. Using three mock communities across a range of densities ranging from 8E+ 02 to 8E+ 09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+ 05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+ 04 and 8E+ 06 16S copies/ml. For the lowest densities, below 8E+ 03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples. Relative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+ 04 16S copies/ml.

Histogram-based models on non-thin section chest CT predict invasiveness of primary lung adenocarcinoma subsolid nodules.

Abstract: 109 pathologically proven subsolid nodules (SSN) were segmented by 2 readers on non-thin section chest CT with a lung nodule analysis software followed by extraction of CT attenuation histogram and geometric features. Functional data analysis of histograms provided data driven features (FPC1,2,3) used in further model building. Nodules were classified as pre-invasive (P1, atypical adenomatous hyperplasia and adenocarcinoma in situ), minimally invasive (P2) and invasive adenocarcinomas (P3). P1 and P2 were grouped together (T1) versus P3 (T2). Various combinations of features were compared in predictive models for binary nodule classification (T1/T2), using multiple logistic regression and non-linear classifiers. Area under ROC curve (AUC) was used as diagnostic performance criteria. Inter-reader variability was assessed using Cohen's Kappa and intra-class coefficient (ICC). Three models predicting invasiveness of SSN were selected based on AUC. First model included 87.5 percentile of CT lesion attenuation (Q.875), interquartile range (IQR), volume and maximum/minimum diameter ratio (AUC:0.89, 95%CI:[0.75 1]). Second model included FPC1, volume and diameter ratio (AUC:0.91, 95%CI:[0.77 1]). Third model included FPC1, FPC2 and volume (AUC:0.89, 95%CI:[0.73 1]). Inter-reader variability was excellent (Kappa:0.95, ICC:0.98). Parsimonious models using histogram and geometric features differentiated invasive from minimally invasive/pre-invasive SSN with good predictive performance in non-thin section CT.

Anaplastic lymphoma kinase 5A4 immunohistochemistry as a diagnostic assay in lung cancer: A Canadian reference testing center's results in population‐based reflex testing

Abstract: Background The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. Methods Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. Results Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n = 5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9 months. Conclusions ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.

Intrapulmonary Immune Regulation by Pre-Transplant Infusion of Recipient-Derived Regulatory T Cells in a Rat Model of Ex Vivo Lung Perfusion Followed by Transplantation

Abstract: Purpose Regulatory T cells (Tregs) are a potential therapeutic option in lung transplantation (LTx). We have shown that expanded recipient Tregs can be seeded pre-transplant in the donor lung graft using normothermic ex vivo lung perfusion (EVLP). Here, we examined whether this therapy can alleviate lung injury early post-transplant. Methods Study design is shown in Fig A. CD4+CD25hi Tregs were isolated from Wistar Kyoto rats (WKy, RT1l) and expanded for 7d-14d. Fischer 344 (F344 RT1lv1) heart-lung blocks were flushed and placed on EVLP. Lungs were perfused with SteenTM solution (37°C, 4h). Expanded Tregs (5-120 × 106) were infused after 1h. Control lungs received no Tregs. The right lung was used for assessment at end-EVLP, and left LTx into WKy recipients was performed after 60min cold preservation. Necropsy was performed on day 3 post-LTx. Results Wet-dry ratio was lower in Treg-treated grafts compared to controls (Fig B). Transcripts for FoxP3, IL-10, CTLA-4 and GITR were elevated in Treg-treated grafts (Fig B). Tregs isolated from the right donor lung at end-EVLP suppressed CD4+CD25− T cell proliferation in vitro (Fig C). On day 3 post-LTx, ICAM1 and CD44 (adhesion molecules) expression on both CD4+ and CD8+ T cells were lower in Treg-treated grafts compared to untreated controls (Fig D). Histologically, both Treg-treated and control grafts had minor lung injury and cellular rejection on day 3 post-LTx: acute lung injury scores (4.10 ± 1.66 and 3.88 ± 1.36), ISHLT A grades (0.60 ± 0.516 and 0.625 ± 0.744), B grades (0.400 ± 0.516 and 0.313 ± 0.458). Conclusion Expanded recipient Tregs directly delivered to the lung allograft prior to transplantation exert immune regulatory effects in vivo and in vitro. Further work is needed to optimize this pre-transplant therapeutic approach.