A gene expression-based single sample predictor of lung adenocarcinoma molecular subtype and prognosis.

Disease recurrence in surgically treated lung adenocarcinoma (AC) remains high. New approaches for risk stratification beyond tumor stage are needed. Gene expression-based AC subtypes such as the Cancer Genome Atlas Network (TCGA) terminal-respiratory unit (TRU), proximal-inflammatory (PI) and proximal-proliferative (PP) subtypes have been associated with prognosis, but show methodological limitations for robust clinical use. We aimed to derive a platform independent single sample predictor (SSP) for molecular subtype assignment and risk stratification that could function in a clinical setting. Two-class (TRU/nonTRU=SSP2) and three-class (TRU/PP/PI=SSP3) SSPs using the AIMS algorithm were trained in 1655 ACs (n = 9659 genes) from public repositories vs TCGA centroid subtypes. Validation and survival analysis were performed in 977 patients using overall survival (OS) and distant metastasis-free survival (DMFS) as endpoints. In the validation cohort, SSP2 and SSP3 showed accuracies of 0.85 and 0.81, respectively. SSPs captured relevant biology previously associated with the TCGA subtypes and were associated with prognosis. In survival analysis, OS and DMFS for cases discordantly classified between TCGA and SSP2 favored the SSP2 classification. In resected Stage I patients, SSP2 identified TRU-cases with better OS (hazard ratio [HR] = 0.30; 95% confidence interval [CI] = 0.18-0.49) and DMFS (TRU HR = 0.52; 95% CI = 0.33-0.83) independent of age, Stage IA/IB and gender. SSP2 was transformed into a NanoString nCounter assay and tested in 44 Stage I patients using RNA from formalin-fixed tissue, providing prognostic stratification (relapse-free interval, HR = 3.2; 95% CI = 1.2-8.8). In conclusion, gene expression-based SSPs can provide molecular subtype and independent prognostic information in early-stage lung ACs. SSPs may overcome critical limitations in the applicability of gene signatures in lung cancer.

Pulmonary manifestations of systemic karyomegaly.

Over 40 years ago, abnormal enlargement of the nucleus of tubular epithelial cells was reported in a rare distinct hereditary chronic interstitial nephritis, karyomegalic interstitial nephritis (KIN). Here, we report the second case of systemic karyomegaly with pulmonary manifestations and present a detailed characterization of the karyomegalic cells in lung parenchyma. A 59-year-old woman who was diagnosed with KIN developed renal failure and eventually received a renal transplant later evaluated for chronic and progressive restrictive lung disease. The KIN diagnosis prompted us to carefully examine her lung parenchyma. Karyomegalic cells were identified in the alveolar epithelium, interstitium, as well as, in the vascular wall. Viral serological and biochemical blood analyses were negative. We consider that the pulmonary manifestations of karyomegaly expands the differential diagnosis of interstitial lung disease in patients with KIN.

Development of Optimized Tissue-Factor-Targeted Peptide Amphiphile Nanofibers to Slow Noncompressible Torso Hemorrhage.

Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths. We report here on the development of injectable, targeted supramolecular nanotherapeutics based on peptide amphiphile (PA) molecules that are designed to target tissue factor (TF) and, therefore, selectively localize to sites of injury to slow hemorrhage. Eight TF-targeting sequences were identified, synthesized into PA molecules, coassembled with nontargeted backbone PA at various weight percentages, and characterized via circular dichroism spectroscopy, transmission electron microscopy, and X-ray scattering. Following intravenous injection in a rat liver hemorrhage model, two of these PA nanofiber coassemblies exhibited the most specific localization to the site of injury compared to controls (p < 0.05), as quantified using immunofluorescence imaging of injured liver and uninjured organs. To determine if the nanofibers were targeting TF in vivo, a mouse saphenous vein laser injury model was performed and showed that TF-targeted nanofibers colocalized with fibrin, demonstrating increased levels of nanofiber at TF-rich sites. Thromboelastograms obtained using samples of heparinized rat whole blood containing TF demonstrated that no clots were formed in the absence of TF-targeted nanofibers. Lastly, both PA nanofiber coassemblies decreased blood loss in comparison to sham and backbone nanofiber controls by 35-59% (p < 0.05). These data demonstrate an optimal TF-targeted nanofiber that localizes selectively to sites of injury and TF exposure, and, interestingly, reduces blood loss. This research represents a promising initial phase in the development of a TF-targeted injectable therapeutic to reduce preventable deaths from hemorrhage.

A combined gene expression tool for parallel histological prediction and gene fusion detection in non-small cell lung cancer.

Accurate histological classification and identification of fusion genes represent two cornerstones of clinical diagnostics in non-small cell lung cancer (NSCLC). Here, we present a NanoString gene expression platform and a novel platform-independent, single sample predictor (SSP) of NSCLC histology for combined, simultaneous, histological classification and fusion gene detection in minimal formalin fixed paraffin embedded (FFPE) tissue. The SSP was developed in 68 NSCLC tumors of adenocarcinoma (AC), squamous cell carcinoma (SqCC) and large-cell neuroendocrine carcinoma (LCNEC) histology, based on NanoString expression of 11 (CHGA, SYP, CD56, SFTPG, NAPSA, TTF-1, TP73L, KRT6A, KRT5, KRT40, KRT16) relevant genes for IHC-based NSCLC histology classification. The SSP was combined with a gene fusion detection module (analyzing ALK, RET, ROS1, MET, NRG1, and NTRK1) into a multicomponent NanoString assay. The histological SSP was validated in six cohorts varying in size (n = 11-199), tissue origin (early or advanced disease), histological composition (including undifferentiated cancer), and gene expression platform. Fusion gene detection revealed five EML4-ALK fusions, four KIF5B-RET fusions, two CD74-NRG1 fusion and three MET exon 14 skipping events among 131 tested cases. The histological SSP was successfully trained and tested in the development cohort (mean AUC = 0.96 in iterated test sets). The SSP proved successful in predicting histology of NSCLC tumors of well-defined subgroups and difficult undifferentiated morphology irrespective of gene expression data platform. Discrepancies between gene expression prediction and histologic diagnosis included cases with mixed histologies, true large cell carcinomas, or poorly differentiated adenocarcinomas with mucin expression. In summary, we present a proof-of-concept multicomponent assay for parallel histological classification and multiplexed fusion gene detection in archival tissue, including a novel platform-independent histological SSP classifier. The assay and SSP could serve as a promising complement in the routine evaluation of diagnostic lung cancer biopsies.

A simplified preservation method for lungs donated after cardiac death.

UNLABELLED: The shortage of donor lungs restricts the number of lung transplantations that can be performed. However, extension of the donor pool using organs donated after cardiac death (DCD) could potentially increase the number of patients who undergo transplantation. To establish acceptance among hospital personnel and the donor's next of kin for the uncontrolled DCD procedure we proposed a simplified preservation regime for intrapleural cooling of the donor lungs. METHODS: In an uncontrolled DCD model, 12 pigs were randomized to intrapleural lung cooling using either a standard method with two bilateral chest tubes and intermittent pleural fluid exchanges, or a simplified, less-invasive method with a single bilateral chest tube and filling of the pleural space without fluid exchange. Lungs were explanted and graft function was assessed during ex vivo lung perfusion (EVLP) and by histologic examination. RESULTS: Although the mean temperature after 120 minutes of intrapleural cooling was significantly higher in the lungs cooled using the simplified method (25.9°C vs 13.5°C), this did not affect the oxygenation capacity, pulmonary vascular resistance or dynamic compliance of the lungs, as recorded during EVLP. Furthermore, no differences were found between the lungs preserved by the two methods with respect to the wet/dry ratio, levels of myeloperoxidase in bronchoalveolar lavage, or at histologic examination. CONCLUSIONS: The simplified technique for DCD lung cooling results in a higher preservation temperature but does not affect lung function during EVLP, which implies that this less invasive method can be used in the uncontrolled DCD setting. This is another step forward in the development of a simplified preservation routine for DCD.

Heparin does not improve graft function in uncontrolled non-heart-beating lung donation: an experimental study in pigs.

OBJECTIVES: Non-heart-beating donation (NHBD) has the potential to increase the number of patients treated with lung transplantation. Our study investigated, in a simulated clinical situation in the uncontrolled NHBD setting, whether or not heparin administration after death affects the donor lung function. METHODS: Twelve Swedish domestic pigs underwent ventricular fibrillation and were left untouched for 7 min followed by cardiopulmonary resuscitation with mechanical compressions for 20 min. The animals were declared dead after a 'hands-off' period of 10 min and randomized to heparin (300 IU/kg) or placebo given into a central venous catheter. In the animals receiving heparin, 2 more minutes of chest compression followed. Intrapleural cooling was initiated 1 h after death, and prevailed for 2 h. Ex vivo lung perfusion (EVLP) was performed with the Vivoline(®) system. Lung function was evaluated with blood gases at different oxygen levels, pulmonary vascular resistance (PVR), wet/dry weight ratio, macroscopic appearance and histology. RESULTS: During EVLP, there were no significant differences between groups in PaO(2) or PVR at any investigated FiO(2) level (1.0, 0.5 or 0.21). At FiO(2) 1.0 the PaO(2) in the heparin group was 64 ± 2 (range 57-73) kPa and in the non-heparin group 63 ± 4 (range 51-71) kPa. The values for PVR were 592 ± 90 (range 402-1007) and 647 ± 97 (range 426-1044), respectively. There was no significant difference between groups in wet/dry ratio or histology. CONCLUSIONS: The use of heparin is of no obvious benefit to the donor lungs in the uncontrolled NHBD situation. The exclusion of heparin will simplify lung donation from NHBDs.